Immune Responses in The Liver

IY36CH09_Kubes ARI 27 December 2017 13:12
Annual Review of Immunology
Immune Responses in the Liver

Paul Kubes1,2 and Craig Jenne1,3
1
Calvin, Phoebe & Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary,
Alberta, T2N 4N1, Canada; email: pkubes@ucalgary.ca, cnjenne@ucalgary.ca
2
Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, T2N
Access provided by University of Reading on 01/12/18. For personal use only.
4N1, Canada
Annu. Rev. Immunol. 2018.36. Downloaded from www.annualreviews.org
3
Department of Microbiology, Immunology and Infectious Diseases, University of Calgary,
Calgary, Alberta, T2N 4N1, Canada
Annu. Rev. Immunol. 2018. 36:9.1–9.31 Keywords

The Annual Review of Immunology is online at liver, sentinel, inflammation, tolerance, innate, adaptive
immunol.annualreviews.org
https://doi.org/10.1146/annurev-immunol- Abstract
051116-052415
The liver is a key, frontline immune tissue. Ideally positioned to detect
Copyright c 2018 by Annual Reviews. pathogens entering the body via the gut, the liver appears designed to de-
All rights reserved
tect, capture, and clear bacteria, viruses, and macromolecules. Containing
the largest collection of phagocytic cells in the body, this organ is an impor-
tant barrier between us and the outside world. Importantly, as portal blood
also transports a large number of foreign but harmless molecules (e.g., food
antigens), the liver’s default immune status is anti-inflammatory or immuno-
tolerant; however, under appropriate conditions, the liver is able to mount
a rapid and robust immune response. This balance between immunity and
tolerance is essential to liver function. Excessive inflammation in the absence
of infection leads to sterile liver injury, tissue damage, and remodeling; in-
sufficient immunity allows for chronic infection and cancer. Dynamic inter-
actions between the numerous populations of immune cells in the liver are
key to maintaining this balance and overall tissue health.
9.1
Review in Advance first posted on

January 12, 2018. (Changes may
still occur before final publication.)
For the king of Babylon stood at the parting of the way, at the head of the two ways, to use divination:
he made his arrows bright, he consulted with images, he looked in the liver.
—Ezekiel 21:21
INTRODUCTION
Waging war on bacteria or viruses can occur only with a functioning liver. Aside from a few
encapsulated bacteria, the liver appears to be the most dominant organ with respect to infections.
The coordinated response to infection is a purposeful act perpetrated by the hepatic immune
system. A patient with liver failure will die primarily of infection. Therefore, as the adaptive
immune system recognizes and begins to mount a response over days, the liver responds in seconds,
holding the fort until the cavalry arrives. In this review, we first summarize innate immunity in
the liver and the growing body of evidence that this organ takes the hit for all the other organs
in an attempt to cheat death. This hepatic immune response depends upon a combination of
unique architecture, resident occupancy of critical immune cells, immune surveillance, and rapid
recruitment. We summarize how the adaptive immune system helps to eradicate and/or isolate
the invading pathogen. We discuss how certain pathogens have developed unique ways to evade
the immune system in the liver. We also examine the notion of immune tolerance in the liver and
what that really means. Finally, we discuss some of the more prevalent noninfectious liver diseases.
IMMUNE SENTINEL: LIVER ANATOMY AND BLOOD SUPPLY

The liver is a unique organ in that it is supplied by both arterial and venous blood. The anatomical
organization of the liver allows these two blood supplies to mix and to bath the various structures
and cells in the liver with their contents. Oxygen-rich arterial blood enters the liver via the hepatic
artery; however, this is a minor source of the blood supplied to the liver. The majority of blood
entering the liver is supplied by the portal vein. Portal blood is rich in both nutrients and pathogen-
derived molecules [for example, lipopolysaccharide (LPS)] (1, 2). Damage to or dysfunction of the
gut epithelium can result in the translocation of whole, intact pathogens from the gut into the portal
blood (3). Whereas some translocated pathogens drain via the lymphatics to the mesenteric lymph
node, where these pathogens appear to prime the adaptive immune response, portal blood flows
directly to the liver, bypassing classic immune sentinel tissues such as the spleen and lymph nodes
(4). The absence of systemic infection when this translocation occurs is a testament to the ability
of the liver to screen and immunologically filter the blood. These observations clearly illustrate
the capacity of the liver to remove gut-derived molecules and pathogens from the circulation.
The architecture of the liver is first and foremost permissive for immune function within
blood vessels (Figure 1). Although the liver is critical for the production of proteins, for the
metabolism of nutrients, and for the clearance of toxins, it is also designed to maximize immune
cell pathogen interactions within the bloodstream. The critical positioning of the liver downstream
of the gut, where venous blood drains, is suggestive of important functions such as eradication of
any translocated bacteria. Bacteria that cross the epithelial barrier would encounter the intestinal
interstitium and, as such, could potentially move via the lymphatics through the mesenteric lymph
node, raising the possibility that the liver may not be positioned downstream of the intestines for
immunological reasons. Indeed, a recent study failed to detect any bacteria in portal blood under
basal conditions (5), although this finding does not necessarily mean that the liver does not play a
critical role in immunity following intestinal injury and bacterial breach.
A portion of blood flow leaves the heart, enters the hepatic artery, and provides oxygen to
the liver. However, the remaining 80% of blood flow derives from many of the gastrointestinal
9.2 Kubes · Jenne

Liver sinusoids Hepatic vein

• Slow blood flow • Lipoproteins (LDL, HDL, etc.)
• Reduce pressure • Complement, acute-phase proteins
• Maximize contact between • Cytokines to modulate systemic immunity
blood and Kupffer cells/LSECs
Kupffer cells
• Pathogen capture/clearance
• Phagocytosis
• Cell recruitment
• FcR, CR, scavenger cytokine LSECs
production • Line the sinusoids
• Antigen presentation • Cell recruitment
• Maintenance of tolerance • Cytokine production
• Antigen presentation
• Tolerance
Hepatocytes
Portal vein Hepatic artery

• Gut-derived pathogens • Antibodies
• Microbe-derived molecules • Systemic infection
(e.g., LPS) • Proteins for clearance
• Nutrients, lipids
Figure 1
Anatomical organization of the liver as an immune organ. The liver is supplied with blood by both the hepatic artery and the portal vein
(bottom). These sources may carry potential pathogens; microbe-derived molecules (e.g., pathogen-associated molecular patterns);
nutrients; and old, oxidized, or damaged molecules for clearance. As the blood enters the liver, it is forced into the honeycomb of the
sinusoids, where flow rate and pressure drop. This reduction in flow rate maximizes the exposure of the blood to the Kupffer cells and
liver sinusoidal endothelial cells (LSECs). Both Kupffer cells and LSECs filter the blood, removing pathogens and molecules. Under
the appropriate conditions, these cells can activate and initiate the immune response through the production of cytokines and the
recruitment of additional immune cells. Between the liver sinusoids are columns of hepatocytes, the principal cell type responsible for
the production of clotting factors, complement proteins, and acute-phase proteins. Following filtering, the blood exits the liver through
draining venules, eventually coalescing into the hepatic vein. Abbreviations: CR, complement receptor; FcR, Fc receptor; HDL,
high-density lipoprotein; LDL, low-density lipoprotein; LPS, lipopolysaccharide.
www.annualreviews.org • Liver Immunity 9.3

organs and also enters the liver, where it mixes with arterial blood and flows through the many
thousands of sinusoids, which are capillary-like vessels, in the liver (6). Although this process seems
to slow the blood flow considerably, individual red blood cells are not readily visible by intravital
microscopy. Nevertheless, the velocity of flow through the vessels is approximately half that of
other capillary beds, maximizing the likelihood that a given molecule or pathogen will be “seen”
by the immune system (6). Indeed, concentrations of pathogen-derived molecules, such as LPS,
drop 100-fold between portal blood and peripheral venous blood (2). The velocity of flow still
poses a challenge to immune cells in that flowing bacteria must be captured as they flow through
the liver. The predominant cell to do this task is the liver macrophage.
LIVER-RESIDENT IMMUNITY
Kupffer Cells
The largest population of macrophages reside in the liver as resident Kupffer cells (KCs)
(Figure 2). KCs are liver-resident immobile macrophages and represent ∼35% of the non-
parenchymal cells in liver and 90% of all tissue macrophages (7). These very large intravascular
cells fill the sinusoidal lumens, scanning for any foreign material in blood that skims along the
surfaces of these cells. KCs have the incredible capacity to discriminate between the millions of
iNKTcells CD4+ T cells

• Liver-resident, patrolling lymphocytes • Multiple subtypes (Th1, Th2, Th17)
• Produce both pro- and anti- • Immunomodulatory
inflammatory cytokines • Few resident cells; rapidly recruited
• Modulate local and systemic immunity during liver infection/injury
CD8+ T cells Hepatocytes
• Cytotoxic • Present antigen (MHC-I and -II and CD1d)
• Release inflammatory cytokines • Maintain immunotolerance
• Clear viral infections • Produce acute-phase and complement
• Few resident cells; rapidly recruited proteins
during liver infection/injury
Monocytes
Neutrophils • Multiple subtypes (CCR2+ pro-
• Few resident cells; rapidly recruited 50 µm inflammatory, CX3CR1+ reparative)
during liver infection/injury • Produce a variety of cytokines
• One of the principal phagocyte types Kupffer cells • Involved in damage mitigation and
responsible for pathogen clearance • Intravascular liver-resident macrophages tissue repair
• Release NETs • Express a number of immune receptors • Differentiate into macrophages
• Cause collateral damage to the liver (Fc, CR), PRRs (TLRs), and scavenger
• Involved in tissue repair? receptors
• Have key role in pathogen detection,
capture, and clearance
• Present antigen via MHC-I and -II and CD1d
• Maintain immune hyporesponsiveness/
immunotolerance
• Produce a wide variety of cytokines
Figure 2
A picture of liver immunity. A representative image of immune cell populations in the liver as captured by
intravital microscopy. Invariant natural killer T (iNKT) cells are visualized by the CXCR6-GFP reporter
construct genetically engineered into the mouse imaged (bright green). Hepatocytes are visible due to natural
autofluorescence under illumination with 488-nm light (dark green). CD8+ (cyan) and CD4+ (blue) T cells
were labeled by anti-CD8 and anti-CD4 antibodies administered intravenously prior to imaging.
Neutrophils ( purple) were labeled by anti-Ly6G antibodies, whereas monocytes are identified as CD11b+ ,
F4/80− , and Ly6G− ( yellow). Finally, Kupffer cells were labeled using anti-F4/80 antibodies (red ).
Abbreviations: CR, complement receptor; NET, neutrophil extracellular trap; PRR, pattern recognition
receptor; TLR, Toll-like receptor.
9.4 Kubes · Jenne

red blood cells, platelets, and immune cells that flow by and to select the few pathogens that
may have breached a barrier and entered the bloodstream. Such discrimination is done in part
through opsonins that tag the pathogen as foreign; these opsonins include complement and more
specifically C3b and inactivated C3b. However, complement receptors such as CR1, CR2, CR3,
and CR4, although capable of grabbing stationary bacteria, are completely incapable of tethering
their ligands under shear conditions. Indeed, the receptor on KCs that can bind C3b and iC3b
under shear, the complement receptor of the immunoglobulin superfamily (CRIg) (8), is unique
and not found on most other cells. In fact, CRIg shares some, but not all, of the physical char-
acteristics of other shear-dependent receptors known to use catch bonds. These receptors, such
as selectins, have the capacity to function more effectively as the shear is increased until a point
is reached at which the ligand is released. In the case of CRIg, it seems to catch extremely well
under shear, and once it binds to C3b under shear, it does not release (8). High expression of
CRIg is largely restricted to the liver, where it plays the critical role in capturing bacteria from
flowing blood (8). Spleen cells can also capture bacteria from the bloodstream; however, CRIg
on the spleen macrophage plays a minor or no role in capturing bacteria (9). In the liver, CRIg
is critical for the capture and clearance of C3b-labeled and iC3b-labeled bacterial and viral tar-
gets from the circulation (10, 11). Recently, CRIg was also shown to be a pattern recognition
receptor (PRR) for LTA, which is found on the majority of gram-positive bacteria. This finding
suggests that although the complement-CRIg pathway is a dominant clearance mechanism for
gram-negative bacteria, complement is unnecessary for gram-positive bacteria, as CRIg can bind
directly to bacterial LTA under shear conditions (12). Bacterial particles are also cleared by the
liver; smaller immune complexes are cleared predominantly by sinusoidal endothelium, whereas
larger complexes are cleared by KCs (13, 14).
Another major family of opsonin receptors are the Fc receptors; however, whether they are able
to capture antibody-opsonized bacteria similar to CRIg remains to be determined. Significantly,
a proposed mechanism for the therapeutic anti-CD20 antibody, which helps remove B cells from
blood, is thought to require Fc receptors on KCs (15). By contrast, an IgG antibody against
Psl on Pseudomonas did not improve KC catching (16). Nevertheless, we recently observed IgM-
dependent capturing of bacteria, but the identity of that receptor remains unknown.
The ontogeny of KCs is now becoming clearer. Although KCs were initially thought to come
from bone marrow as monocytes and to differentiate into these highly specialized tissue-resident
macrophages (17), lineage-tracing experiments have suggested that KCs, like microglia in the
brain and pulmonary macrophages in the lung, come from the yolk sac and likely replicate locally
in the liver (18). However, massive eradication of KCs with either chlodronate or other approaches
suggests that, under these conditions in which there may be few or no macrophages left to prolif-
erate, a CX3 CR1-positive monocyte or monocyte precursor will seed the liver and slowly take on
many of the properties of the original KCs (19, 20). Proteomics and genomics data suggest that by
30–35 days the new liver macrophage is indistinguishable from the original cell in terms of func-
tion but may or may not retain certain unique transcriptomic signatures (19, 21). Whether these
unique signatures would lead to distinct immune responses is not clear, but another study showed
that catching bacteria effectively and mounting a tolerogenic or appropriate immune response to
acetaminophen took up to 5 weeks to restore to normal levels (20). This finding suggests that an
organism may be vulnerable for a period of time if new macrophages are bone marrow derived.
However, these studies involve massive KC depletion, and whether KCs repopulate an injury
site after limited KC insults is not entirely clear. Bleriot et al. (22) reported that infection by Listeria
monocytogenes induced the early necroptotic death of KCs, which was followed by replacement of
monocytes that started as type 1 inflammatory cells but, due to interleukin (IL)-4, became more
type 2, returning the liver to homeostasis. In a type 2 immune response to helminth infection,

by contrast, peritoneal and pleural tissue-resident macrophages proliferated locally in an IL-4-

dependent manner, without any recruitment of monocyte-derived cells; however, this work did
not look at KCs (23). If indeed a KC is immobile, it is hard to imagine how a KC might enter an
area devoid of macrophages, although a daughter cell might have the capacity to crawl.
Significantly, KCs do not appear to be a single homogeneous population of cells. As such, dif-
ferences in development or life span may simply reflect different subpopulations of KCs. Indeed,
using an unbiased cytometry by time of flight (CyTOF) approach, researchers observed two dis-
tinct KC populations, one positive and one negative for CD11c, and this distinction was confirmed
with imaging (20). However, no differences in distribution or ability to capture bacteria were noted
(20). Some studies have suggested that KCs are relatively short lived and are replaced within 1–
2 weeks, whereas others have reported long-lived KCs (on a timescale of 3 months to 1 year) after
pulse labeling or transplantation (summarized in Reference 7). In specific pathogen-free facilities,
where most experimental mice are kept, there may be limited turnover of macrophages, whereas
in the wild, where exposure to pathogens is more common, a more rapid turnover of KC may
occur. Along the same lines, in modern-day humans constantly exposed to drugs and alcohol but
perhaps fewer pathogens, KC turnover may also be more frequent. Whatever role the microbiome
may have in KC turnover, one study found that KCs from germ-free mice had functional systemic
bacterial capturing, suggesting that KCs do not depend on instructions from microbiota for their
development and function (20).
In addition to expressing CRIg, KCs express an array of scavenger receptors, Toll-like receptors
(TLRs), complement receptors, and antibody receptors, all of which are molecules that allow KCs
to detect, bind, and internalize pathogens and associated molecules (see below). Furthermore,
these receptors, in part, drive activation of KCs, which leads to the production of cytokines and
chemokines and allows this cell type to function as an immune sentinel, alerting other components
of the immune system to the presence of harmful pathogens.
Pathogen Evasion of Kupffer Cells

Under most circumstances, KCs capturing bacteria from the bloodstream results in the killing
of the pathogen. For example, Escherichia coli and various other commensal bacteria are rapidly
killed, as are Pseudomonas aeruginosa, Bacillus cereus, Borrelia burgdorferi, and others. Encapsulated
bacteria such as Streptococcus pneumonia are capable of evading the liver but are captured by splenic
marginal zone and red pulp macrophages (10). Removal of the capsule bestows the KCs with the
ability to capture S. pneumonia, suggesting that the capsule is capable of functioning as an evasion
mechanism (10). Not surprisingly, humans that lack a spleen are 50 times more susceptible to
S. pneumonia infections (24). KCs also capture L. monocytogenes, but elimination of the pathogen
requires the recruitment of neutrophils to the liver and then the capture of L. monocytogenes by
neutrophils that were bound to the KC surface (25). In this example, although the KCs were not
directly responsible for the killing of the pathogen, they had an essential role in pathogen capture
and in the recruitment of additional immune cells to the liver. A limitation in this work is the
short time frame (10 min) that was examined, which might not have been sufficient time for KCs
to phagocytose the pathogen. In addition, the extracellular location of the pathogen was judged
on the basis of sensitivity to gentamicin, which is unlikely to be the most robust method. In fact,
other work disagrees with L. monocytogenes not being phagocytosed and killed by liver macrophages
but, instead, remaining bound extracellularly. Indeed, other researchers have demonstrated that
L. monocytogenes inside KCs leads to the demise of the cells (22), and our own imaging has confirmed
the internalization but then rapid expulsion of these pathogens, not dissimilar to what has been
described in epithelial cells (26). Regardless of the exact route of evasion, it is clear that KCs have
difficulty containing and eradicating L. monocytogenes.
9.6 Kubes · Jenne

Staphylococcus aureus is another bacterial strain that is able to evade the KC killing machinery.
However, in this particular case, the pathogen is apparently captured and phagocytosed efficiently
by KCs (27). In a small percentage of KCs, the bacteria appear to survive the phagolysosome and to
ultimately lyse the macrophages, leading to dissemination. Whether the bacteria overtly lyse the
KCs to escape into the bloodstream or whether the KCs lyse to expose the pathogen remains un-
clear. The percentage of KCs that suffer this fate is only approximately 10%, with the other 90% of
KCs that capture bacteria being able to kill the pathogen primarily by the NADPH oxidase system.
Importantly, in the absence of this defense mechanism, both mice and humans suffer liver abscesses.
The addition of IFN-γ did not improve bacterial capture by KCs, whereas the addition of IL-4
reduced the ability of KCs to sequester bacteria (28, 29). This finding suggests that the basal state
of capturing bacteria in the bloodstream is set at an optimal level that cannot be improved upon;
however, the responses can be downmodulated. Although KCs are normally in a tolerogenic state
with respect to responses to LPS and other activating molecules; this tolerogenic phenotype does
not appear to affect bacterial trapping.
Other Functions of Kupffer Cells

Pathogens are not the only target cell bound and cleared by KCs. KCs can bind and phago-
cytose activated and apoptotic neutrophils (30). In fact, a prevalent view in the literature is that
approximately a third of senescent neutrophils are removed via the liver. Indeed, inhibition of KC-
mediated phagocytosis resulted in neutrophilia and the accumulation of activated neutrophils in
the spleen and lungs (30). Depending on the receptors used to bind and internalize the neutrophils
(Fc receptors versus integrin-CD36 complexes), this immune cell clearance can activate the KCs
to produce either pro- or anti-inflammatory cytokines (31). Furthermore, chilled or activated
platelets bind and are cleared by KCs (32). Clearance of activated host cells, and by extension the
process of limiting the potential of these cells to produce inflammatory mediators, is an important
mechanism for regulating inflammation, limiting collateral host cell damage, and initiating tissue
resolution (7). Our imaging reveals that platelets do interact with KCs and are taken up (following
desialylation), whereas a similar phenomenon for neutrophils has not been seen; however, this
result could be related to imaging being limited to the more superficial areas of the liver.
Aside from pathogen capture and clearance, KCs are important antigen-presenting cells
(APCs), expressing MHC-I, MHC-II, and costimulatory molecules needed for activation of T
cells. However, under basal conditions KCs are poor activators of the adaptive immune response
(33). As is the case with liver sinusoidal endothelial cells (LSECs), continual exposure to LPS
from intestines appears to dampen the ability of KCs to activate lymphocytes (33), preventing the
generation of immunity to common gut-derived antigen present in the portal circulation (34).
Although KCs are poor stimulators of T cell activation under basal conditions, the presence of
other pathogen-associated molecules (TLR3 and TLR9 ligands) or inflammatory cytokines can
modulate KCs, converting these normally tolerogenic cells into potent APCs capable of robust
activation of T cells (33, 35). Additionally, KCs are extremely effective at activating the invariant
natural killer T (iNKT) cells that live and patrol the sinusoids of the liver. Indeed, upon capture
of bacteria, antigens can be presented to iNKT cells via CD1d found in abundance on KCs (36).
These processes occur entirely within the vasculature of the liver.
Natural Killer Cells

The liver contains the largest population of natural killer (NK) cells in the body. Between one-
third and one-half of all lymphocytes within the liver can be defined as NK cells (37, 38). These
innate lymphocytes, normally rare in most other tissue, play a key role in screening for infection

and tissue pathology (39). Whereas most other immune cells identify targets based on either
foreignness (e.g., presence of PAMPs) or the presence of a specific antigen (as in the case of B
cells and T cells), NK cells screen targets for the absence of self. Oncogenesis or infection with
a wide variety of pathogens results in the downregulation of antigen-presenting molecules, such
as MHC-I, in an effort to hide from the immune system. NK cells use this absence of self to
target cells for clearance and to facilitate cellular activation and cytokine production (e.g., IFN-
γ) (37). NK cells express a balance of activating and inhibitory receptors. These receptors bind
self-molecules on various cells within the tissue. An absence of normal self-molecules results in
a failure to engage the inhibitory receptors, leading to a shift in the balance between activation
and inhibition. This loss of balance between activating and inhibitory signals results in NK cell
activation and target killing through the release of cytotoxic granules containing molecules such
as perforin and granzyme. Interestingly, although NK cells are innate lymphocytes, an increasing
number of studies in recent years suggest they are able to generate memory. Although not as
specific as classic lymphocyte memory, NK cell memory does result in more rapid and stronger
responses to secondary infections with a given viral pathogen (40–42). This innate memory has
greatly expanded our understanding of the role of NK cells in immune surveillance and identifies
their importance in tissues such as the liver, where frequent, reoccurring exposure to common
viral vectors (arriving via the gut) is expected.
Invariant Natural Killer T Cells

It is becoming clear that iNKT cells contribute to most immune responses in the liver. In general,
iNKT cells respond to lipid antigens produced by either the host or a microbe. These cells
are defined by their T cell receptor (TCR), which recognizes a limited number of glycolipid
antigens presented by CD1d (43, 44). iNKT cells express an αβ TCR that depends on somatic
recombination and selection in the thymus. The TCR is made of the TCRα chain (Vα14-Jα18 in
mice and Vα24-Jα18 in humans), which pairs with a limited repertoire of TCRβ chains (Vβ2, Vβ7,
Vβ8.1, Vβ8.2, or Vβ8.3 in mice and Vβ11 in humans) (45, 46). Another important distinction
between mice and humans is the many more iNKT cells found in rodents (47), suggesting that
these cells may be far more important in the latter animals. There is huge variability in the numbers
of iNKT cells in humans, and iNKT cell numbers can increase following stimulation. These T
cells are typically detected by glycolipid-loaded CD1d tetramers by using flow cytometry. α-
Galactosylceramide (α-GalCer) was the first identified and most potent glycolipid antigen that
led to specific activation of iNKT cells. The recognition of α-GalCer-loaded CD1d tetramer is
highly conserved by mouse and human iNKT cells despite the restricted T cell repertoire (48).
In addition to iNKT cells (type I NKT cells), there is a second subset of NKT cells (type II NKT
cells) that are CD1d restricted and recognize the self-lipid sulfatide, but type II NKT cells do not
recognize α-GalCer (49, 50) or other microbial antigens.
iNKT cells form in the thymus as CD4− CD8− double-negative precursors, develop into
CD4+ CD8+ double-positive cells, and randomly rearrange their TCRs. TCRs that recognize
self-lipids on thymocytes enter the iNKT cell lineage. Positive selection depends on low-affinity
interactions with self-lipids presented on CD1d by thymocytes (51). iNKT cells were thought to
only emigrate from the thymus and seed the liver and other peripheral tissues at stage 2, followed
by differentiation into mature iNKT cells (52). However, a recent study demonstrated that an
alternative developmental pathway exists in which iNKT cells could develop from the double-
negative stage in the thymus and distribute only to the liver, spleen, and lung (53). In the liver, these
cells highly express CCR5 and CXCR3 receptors, which are key chemokine receptors providing
direction to iNKT cells in certain inflammatory responses.
9.8 Kubes · Jenne

An important characteristic of iNKT cells is that they have effector potential and can rapidly
become activated after stimulation through the TCR by lipid antigens without the need for cos-
timulation (54). iNKT cells originating from the double-negative stage elicit strong antitumor
cytotoxicity (53). A strong signal through the TCR and a weak cytokine signal through cytokine
receptors is sufficient for the activation and to exert cytotoxic effector functions similar to those
of conventional cytotoxic T cells (55). This finding suggests that iNKT cells are involved in im-
mediate innate immune responses. α-GalCer-activated iNKT cells secrete copious amounts of
different proinflammatory and anti-inflammatory cytokines that can modulate the microenviron-
ment. Although this robust response involving both proinflammatory and anti-inflammatory cy-
tokinesis true for α-GalCer, one of the most potent glycolipids, most iNKT cell TCR–stimulating
glycolipids likely cause a more selective production of Th1, Th2, or Th17 cytokines. Although
subsets of iNKT cells—including iNKT1 (producing mainly IFN-γ), iNKT2 (producing IL-4),
and iNKT17 (producing IL-17)—have been gaining acceptance (56–58), the paradigm is some-
what hazier than what has been reported for conventional T cells. For example, even though liver
iNKT cells are thought to predominantly produce IFN-γ and are of the Th1 phenotype, numer-
ous studies clearly show they can produce IL-4; in partial hepatectomy, iNKT cells producing
IL-4 were observed to be beneficial for regrowth of the liver (59). By contrast, if liver iNKT cells
were activated with α-GalCer, iNKT cells instead produced IFN-γ, which inhibited liver regen-
eration (60). Similar findings were also observed in CCl4 -induced liver injury (61). Interestingly,
α-GalCer can induce a mild form of liver injury that is entirely IFN-γ dependent (62). Finally,
in a model of stroke in which sympathetic innervation of the liver induces immunosuppression,
iNKT cells produced IL-10 and IL-4 (63).
There may be some plasticity between the different iNKT cell subtypes. For example, both
iNKT1 and iNKT17 cells produce IL-4 (in addition to the iNKT2 subset), suggesting that the
subsets are not composed of a uniform population of cells (64) or that a single iNKT cell has
the capacity to produce different cytokines dependent on the stimulus. Indeed, the α-GalCer
analog OCH results in IL-4 production, in contrast to α-GalCer, which results in iNKT cells
predominantly producing IFN-γ (65). Finally, cytokine production by iNKT cells also depends on
the type of APC (66); for example, α-GalCer-pulsed hepatocytes that were cocultured with iNKT
cells elicited IL-4 production, but not IFN-γ production (67), and resulted in IFN-γ production
if macrophages or dendritic cells (DCs) were the APC. In addition, the cytokine milieu together
with the type of APC can also affect what cytokines the iNKT cells produce. A final worthy
attribute is the speed with which cytokines are produced. Some cytokines are released in less than
2 h, a response that may be due to the presence of preformed transcripts for these molecules (68),
making these cells the potential orchestrator of the type of immunity that ensues.
The liver houses the most iNKT cells in the body, and imaging has been critical to elucidating
function. These cells continuously patrol the sinusoids crawling along the endothelium in a random
fashion with, as well as against, the flow of blood. Although a lineage-specific antibody that would
bind and allow tracking of these cells in vivo is not readily available, the CXCR6–green fluorescent
protein (GFP) reporter mouse has been used for this purpose (69). It is worth a cautionary note
that only 60–70% of liver CXCR6-GFP+ cells are CD1d-restricted iNKT cells (69). Therefore,
additional experiments may be required to show responsiveness to α-GalCer or expression of
the appropriate TCR. However, neither of these approaches works well for imaging and the
tracking of the cells because both of them activate iNKT cells. Finally, in tissues such as the
intestines and lymph nodes, the iNKT cells make up less than 10% of CXCR6-positive cells (P.
Kubes, unpublished observations). Nevertheless, some important information has been derived
from these mice, and it is now clear that in the intestines and joints iNKT cells are found resident
in the tissues rather than patrolling the vasculature (11).

This scenario begs the question of why in the liver iNKT cells are found in the hepatic sinusoids,
and not in the tissue. One possibility is to screen for perturbations, affect systemic immune status,
and release immunomodulatory molecules as needed. Indeed, poststroke, when the brain increases
sympathetic drive to the periphery, iNKT cells in the liver, but not those in the blood or the
spleen, respond by shutting down the production of the inflammatory cytokine IFN-γ and by
increasing IL-10 levels (63). Another example of iNKT cell involvement in immunity at distal
sites is during hypersensitivity reactions in the skin, where it is postulated that an endogenous
glycolipid enters the blood and activates iNKT cells to synthesize abundant amounts of IL-4,
leading to antibody production by B cells (70, 71). In this way, the liver iNKT cell, acting as a
sentinel, can detect a distant tissue insult and, as a result, modulate the global immune status of the
animal.
Interestingly, iNKT cells, but not NK cells, monocytes, or neutrophils, continually crawl along
the sinusoid walls under basal conditions, independent of the direction of blood flow, scanning for
cognate ligand. Upon activation, these cells cease crawling and firmly adhere in the vasculature
(36, 69). The adhesion has been correlated closely with the activation and production of cytokines.
Whereas α-GalCer causes iNKT cells to immediately stop, presumably due to the binding of this
glycolipid to CD1d on endothelial cells and macrophages within the vasculature, cytokines like
IL-12 and IL-18 are also capable of causing these immune cells to arrest (72). Although these
pharmacological experiments demonstrate that these molecules are arrest stimuli for iNKT cells,
the scenario is slightly different for pathogens and dead cells. In the case of pathogens, KCs rapidly
capture bacteria in the bloodstream and process them for glycolipid presentation to iNKT cells.
In addition, these macrophages release iNKT cell chemoattractants, causing the iNKT cells to
not stop instantly where they may be but rather to crawl to the KCs, where large aggregates of
iNKT cells can be seen (36). These iNKT cells then produce IFN-γ for antimicrobial activity. In
the case of dead cells, endogenous glycolipids are released and presented to iNKT cells by various
cell types that express CD1d. In this manner, iNKT cells are presented with self-antigen only
around the dead cells where they produce IL-4 and not IFN-γ (73). In this situation, hepatocytes
begin to proliferate, and if necessary, vasculature regrows and the local response is more anti-
inflammatory.
RECRUITMENT OF IMMUNE CELLS

As important as resident immune cells are in liver immune responses, many responses require the
recruitment of immune cells and specifically neutrophils and monocytes from the bloodstream.
In most vascular beds, neutrophil recruitment into inflamed tissues requires a series of sequential
steps, mediated by the interaction of receptor-ligand interactions on leukocytes and the vascu-
lar endothelium (74). The first step requires that cells flowing freely in the blood tether to the
endothelium. This step is thought to capture the neutrophil to the surface of the vessel wall.
Selectins, including P-selectin and E-selectin, on endothelium bind to selectin ligands, including
PSGL-1, on the neutrophil (75). These interactions hold the neutrophil in place for a brief period,
and as additional selectin-ligand interactions occur, the cells begin to roll along the endothelial
surface (76). However, these interactions have been described in postcapillary venules, and not in
capillaries. By contrast, in the liver more than 85% of neutrophil interactions occur in capillar-
ies or sinusoids, which are more narrow structures that do not express selectins; consistent with
this observation, rolling of neutrophils in sinusoids seems to be a very rare event (as assessed by
intravital microscopy) (77). Not surprisingly, there is little role for these adhesion molecules
in neutrophil recruitment in the hepatic microcirculation, as documented numerous times
(77–79).
9.10 Kubes · Jenne

CD44 and Hyaluronan: Signature Adhesion Molecules in the Liver

Among the numerous nonselectin molecules that can support cell recruitment are molecules such
as CD44 and its ligand, hyaluronan (HA); receptors siglec-9 and siglec-10 and their ligand, vascular
adhesion protein 1 (VAP-1) (80, 81); and VLA4 and its ligand, VCAM-1. In a quantitative radioim-
munolabeling approach, HA was expressed at much higher levels in the liver than in any other
organ, and such expression is greater than that of any other adhesion molecule in the liver (77).
HA is a member of the glycosaminoglycan family of extracellular matrix molecules, formed from
a basic structural unit of repeating disaccharides of N-acetylglucosamine and N-glucuronic acid.
Numerous imaging studies have reported that the luminal surface of liver sinusoidal endothelium
is densely coated with HA (82). This may be the result of HA scavenging receptors being found
on sinusoidal endothelium. Unlike other vascular beds, in which endothelial CD44 is required
to anchor HA to the vessel wall, LSECs express a variety of other scavenger receptors, including
Lyve-1 (83) and stabilin (84), to capture circulating HA on the endothelial surface. Moreover,
endothelial CD44 is not needed to support neutrophil adhesion, suggesting other endothelial re-
ceptors bind HA in the liver (77). In addition to promoting HA endocytosis and clearance from the
bloodstream, the liver may use HA as an adhesion molecule in sinusoids (85). Although no studies
to date have investigated the functional role of HA scavenger receptors in neutrophil adhesion,
HA has been shown to be a key ligand for neutrophil adhesion within liver sinusoids (77).
Expression of CD44 on immune cells, in contrast to LSECs, is important for their adhesion
within the liver. Under basal conditions, however, neutrophils do not adhere to HA in liver
sinusoids (77), a finding that differs from those of other studies that have reported that lymphocytes
could roll on HA in vitro (86). A potential explanation is that CD44 is a type I transmembrane
glycoprotein encoded by a single gene but expressed in more than 20 isoforms as a result of
alternative splicing as well as posttranslational modifications (85). This process leads to different
isoforms and diverse functions on different cell types. Stimulation of the vasculature with LPS or
E. coli was sufficient to induce profound neutrophil recruitment in liver sinusoids; such recruitment
could be reduced by 50–70% by blocking CD44 or removing HA with hyaluronidase (87). Menezes
and colleagues (88) performed an in-depth functional analysis of the CD44-HA interaction by
using confocal intravital microscopy in mouse models of endotoxemia and gram-negative bacterial
sepsis. In addition to mediating initial adhesion, CD44 was required for subsequent stages of
recruitment. The neutrophils did not crawl, perhaps in part because CD11b was shed from the
surface as a result of IL-10 production.
Numerous groups have reported that expression and activation of CD44 are necessary on
lymphocytes, monocytes, and other cell types but that activation does not appear to be required for
neutrophils. In fact, neutrophil recruitment was dependent on changes in HA function at the level
of the vascular endothelium (77). Although endothelium has the ability to augment surface expres-
sion of HA under inflammatory conditions (89), structural modification of HA by an HA-binding
protein was critical. Serum-derived HA-associated protein (SHAP), an HA-binding protein,
modulates neutrophil adhesion in liver sinusoids in response to LPS (87). SHAP is structurally
composed of the heavy chains of inter-α-trypsin inhibitor, a proteoglycan that circulates in blood,
and binds bikunin (a serine protease inhibitor) (90). At sites of inflammation, SHAP, through
a transesterification reaction, is transferred to HA through catalysis by tumor necrosis factor–
stimulated gene 6 (TSG-6). This process yields a covalently bound SHAP-HA complex (90) that
avidly binds neutrophils to the sinusoidal endothelium through alterations of HA macromolecular
structure (86). SHAP-HA complexes form only during inflammation (87). Mice lacking the SHAP
precursor (IαI mice) show 50% less neutrophil adhesion in liver relative to wild-type animals
(87). SHAP binding coalesces HA polymers into multicell-length cables (91). This development

may reduce degradation or internalization of HA, allowing it to mediate neutrophil adhesion.

Whether CD44 needs also to be altered is not clear, although these clusters of HA polymers
may induce high-avidity interactions with neutrophil CD44. In addition to being observed in
inflamed liver, SHAP-HA complexes were seen in the synovial fluid of patients with rheumatoid
arthritis (86) and in colon tissue from patients with active inflammatory bowel disease (91).
Mac-1 and ICAM-1

Although CD44 on leukocytes accounts for at least 50% of cell recruitment to inflamed liver,
there is clearly a role for other molecules. Some investigators have reported a roughly equivalent
role for monocyte infiltration when Mac-1–ICAM-1 and CD44-HA interactions were blocked
(92). In the case of monocytes, CD44 and Mac-1 function cooperatively to induce adhesion or in
a sequential manner, with CD44 mediating initial tethering and adhesion and Mac-1 supporting
subsequent intravascular crawling toward the afflicted target. In neutrophils, evidence does not
support cooperativity, as following LPS stimulation, Mac-1 appeared to be shed from the surface
of neutrophils, allowing CD44 to predominate (88). By contrast, when a chemokine was presented
to the neutrophils, the cells used CD11b, and not CD44. Other studies failed to see an additive ef-
fect, demonstrating a key role for neutrophil CD44 in models of infection and a role for neutrophil
CD11b in sterile models of inflammation (88). A key phenotypic difference was that neutrophils
crawled in response to chemokines and, in the case of focal sterile injury, toward the afflicted
tissue, whereas in the cases of bacteria and LPS, neutrophils tended to arrest and not crawl.
Numerous studies have shown that activation of neutrophils within the liver via LPS, bacteria, or
viruses led to platelet adhesion to the neutrophils, inducing neutrophil extracellular traps (NETs;
discussed below) (93, 94).
A similar role for CD44-HA interactions has been noted for the recruitment of other cell
types to inflamed liver. Monocyte recruitment to L. monocytogenes–infected liver was reduced by
more than 50% by anti-CD44 antibody (92). Cytotoxic T lymphocyte recruitment in a mouse
model of viral hepatitis was also dependent upon CD44-HA interactions (95), with the caveat that
endothelial CD44 was most important. This observation remains unexplained. It is also worth
mentioning that CD44-HA interactions have been implicated in the trafficking of hematopoietic
stem cells to the liver in primary biliary cirrhosis and alcoholic liver disease and in cancer cell
metastasis to the liver (85).
A number of recent studies have described a critical role for CD44-HA interactions in the
pathogenesis of fatty liver disease, the leading cause of chronic liver disease in the United States
and a growing problem worldwide (96). CD44-deficient mice have markedly attenuated hepatitis
in a mouse model of nonalcoholic steatohepatitis (NASH), induced by administration of a high-fat
diet (97, 98). Leukocytes harvested from lithogenic diet–fed mice display significant upregulation
of their HA-binding capacity (97). This correlated with diminished leukocyte infiltration into the
livers of CD44-deficient animals. Using a similar model of lithogenic diet–induced hepatic steato-
sis, Kang et al. (98) observed that CD44-deficient mice displayed reduced leukocyte infiltration
into the liver, in addition to broader defects in steatosis development, adipose tissue inflamma-
tion, and insulin resistance. Therefore, in addition to supporting leukocyte infiltration into the
liver and the development of NASH, CD44 may be a keystone in the systemic pathogenesis of
obesity-associated diseases and the metabolic syndrome.
VLA4 and VAP-1

Certain immune cells, including iNKT cells, Th1 cells, and Th2 cells, do not use either Mac-1
or CD44. Th1 cells depend upon the integrin VLA4, which mediates rolling, adhesion, and
9.12 Kubes · Jenne

crawling on various cell types and binds VCAM-1, a molecule that is abundantly expressed on
inflamed sinusoidal endothelium (99). By contrast, Th2 cells appear to use VAP-1, a 170-kDa
homodimeric glycoprotein expressed on endothelial cells that functions as both an amine oxidase
producing oxidants and an adhesion molecule for lymphocytes (99). In Th2 cell recruitment,
the structure of VAP-1, and not its oxidase activity, appeared to mediate adhesion. In humans,
sinusoids constitutively express VAP-1, which is upregulated during an immune response (100).
Patients with liver disease have elevated serum levels of soluble VAP-1 (101). The intrahepatic
enzyme activity of VAP-1 was elevated in primary sclerosing cholangitis (PSC) relative to immune-
mediated disease controls and nondiseased liver. The adhesion of gut-tropic α4β7+ lymphocytes
to hepatic endothelial cells in vitro under flow was attenuated by 50% following administration of
the VAP-1 inhibitor semicarbazide. Importantly, VAP-1 is an amine oxidase and as such functions
beyond a static adhesion molecule. VAP-1 expression is increased in PSC and promotes adhesion
of gut-tropic lymphocytes to the liver sinusoidal endothelium, and the circulating soluble form of
VAP-1 is responsible for oxidase activity predicted clinical outcome in patients (102). Although
VAP-1 can function structurally as an adhesion molecule, its enzymatic activity can also play a role
in modifying and regulating cellular recruitment. Studies have shown that VAP-1 can generate
hydrogen peroxide and that this product can serve to further enhance neutrophil recruitment in
the kidney (103).
In three murine nonalcoholic fatty liver disease (NAFLD) models, including a high-fat diet,
a Western lifestyle model, and a methionine-choline-deficient (MCD) diet, there was reduced
inflammation, steatosis, and fibrosis in the absence or blockade of VAP-1. In the MCD model, a
reduction of these parameters was also observed if the VAP-1 was catalytically inactive and not
producing oxidants, suggesting the importance of the oxidase rather than that VAP-1 is necessarily
an adhesion molecule. Finally, in a model of carbon tetrachloride, fibrosis and immune cell influx
were also reduced in the absence of VAP-1 and suggest that targeting VAP-1 has therapeutic
potential for various chronic fibrotic liver diseases (104).
CCR2
Monocyte recruitment appears to be significant in numerous disease states in the liver. The
major issue is that inflammatory monocytes convert to macrophages that have been associated
with injury. CCR2-high, Ly6C-high inflammatory monocytes are recruited from bone marrow
to inflamed liver. These cells are increased in numerous human liver diseases, and their absence
in CCR2−/− mice results in reduced inflammation in, for example, ischemia/reperfusion injury
(105), acetaminophen-induced toxicity (106), and other hepatitis models (107). A second monocyte
subset which is Ly6C low, CCR2 negative, and CX3 CR1 high, but these monocytes appear to be
found patrolling in the kidney, skin, and mesenteric blood vessels under basal conditions (108,
109). They have been implicated in eradication of virally infected endothelial cells in the kidney
and in the recruitment of neutrophils to sites of skin infection. In the liver, their patrolling behavior
has yet to be investigated, and in some forms of inflammation, they seem to transition from Ly6C-
high, CCR2-high inflammatory monocytes to the more patrolling-like phenotype (110). Whether
this is the natural progression toward a Ly6C-negative macrophage remains to be determined.
This latter macrophage modifies the injured tissue to allow for healing (107).
In more chronic models of liver disease such as NAFLD and alcoholic liver disease, there is
a pressing need to identify new ways of clinical intervention. In particular, in fatty liver diseases,
which affects one-third of the population and is becoming an overwhelming burden, hepato-
cytes are killed, and damage-associated molecular patterns (DAMPs) activate the recruitment of
monocytes and macrophages. Indeed, CCR2-high monocytes are recruited to the liver in these

metabolic disorders, and these cells convert to specialized macrophages that may induce or reduce
inflammation (111, 112). There is consensus in the literature that KCs are likely critical to the
initial inflammation associated with the various models of fatty liver disease (107). These disease
states ultimately lead to a liver fibrotic state, and again there is significant information that both
KCs and monocyte-recruited macrophages contribute to this progression (107, 113). A multina-
tional clinical trial (CENTAUR) is under way to evaluate the anti-inflammatory and antifibrotic
potential of a dual CCR2/CCR5 antagonist, cenicriviroc, in patients with NASH (114).
Other Forms of Leukocyte Recruitment

Classically, most recruitment of immune cells to the liver is thought to occur via the vasculature.
Moreover, macrophages are recruited as monocytes to sites of infection and inflammation. There-
fore, the infiltration of mature macrophages to inflammatory sites in liver has seemed unimag-
inable. However, recent work in a model of focal liver injury revealed profound infiltration of
mature F4/80-positive macrophages that appeared to cover the injury site as early as 1 h after the
injury (115). These cells could not be seen migrating to the site via the sinusoids, suggesting a
novel portal for the recruitment of mature macrophages. These macrophages were also positive
for a particular transcription factor, GATA6, which was specific for large peritoneal macrophages
but not KCs or monocytes. Indeed, although more than 20,000 publications have been published
about peritoneal macrophages, their function in this cavity is not well explained. Indeed, in this
recent study, peritoneal macrophages were demonstrated to infiltrate injured tissue due to the
release of ATP, which activated macrophage CD44 with HA exposed on the liver surface (115).
These macrophages actively took on an M2-like phenotype and aided the dismantling of dead
cells to help in repair. In models of liver injury such as carbon tetrachloride, macrophages not
only infiltrated the top layer but also were able to migrate deep into the tissues to affect repair.
Although these findings are preliminary, other organs in the peritoneal cavity and other cavities in
the body (e.g., the pericardium) also have these macrophages, and they too may affect repair. It is
unclear whether the small peritoneal macrophages (GATA6 negative) also infiltrate into infectious
sites from the peritoneal cavity.
ADAPTIVE IMMUNE RESPONSES IN THE LIVER

Although not a classical secondary lymphoid organ, the liver represents a unique environment for
the development and function of the adaptive immune response. Despite the relative wealth of
APCs in the liver, and the ability of this tissue to rapidly recruit diverse populations of immune
cells, the generation of an integrated adaptive immune response in the liver is, in general, not
an efficient and straightforward process. Much of this immune paralysis is due to the anatomical
location and physiological function of the liver. Situated as a frontline filter and sentinel for
pathogens and PAMPs entering the body from the intestine via the portal vein, the liver is also
often one of the first points of contact for other antigens entering the body, including those
derived from healthy commensal microbiota and food (1–3). As such, the immune response in
the liver has to strike a delicate balance between tolerance for the nonthreats and immunity to
pathogens. Importantly, although capable of activating a robust T cell response, the APC, under
baseline conditions, appears to have a default response of immunotolerance, driving the expansion
of cognate T lymphocytes but not supporting the licensing of cytotoxic effector functions. This
response is perhaps not entirely surprising, given the potential damage done to the organism if
a vigorous immune response is mounted against a routine food-derived antigen or against the
normal gut microbiota; hence, the liver appears to err on the side of immune tolerance. This
hypoimmune environment is perhaps best exemplified by the relative success of liver transplants
9.14 Kubes · Jenne

and the exploitation of the liver environment by some viruses [e.g., hepatitis B virus (HBV),
hepatitis C virus (HCV)], leading to chronic infection (116–121).
Immune Hyporesponsiveness
Liver immune hyporesponsiveness can be attributed to the complex interactions between the vari-
ous liver-resident immune sentinels and other peripheral leukocyte populations. Under basal con-
ditions, most liver-resident cells (e.g., KCs, LSECs, DCs, hepatocytes) functionally suppress the
adaptive immune response, maintaining a general state of immune nonresponsiveness (122–128).
This immune suppression can be directly attributed to the absence of costimulatory molecules,
to the relatively low expression of MHC, and to the constitutive expression of anti-inflammatory
cytokines such as IL-10 (125, 128, 129). Not only does a lack of costimulation blunt the induc-
tion of functional T cell–mediated immunity, but the liver also possesses other mechanisms that
actively impede the generation of cytotoxic T cells.
One such mechanism involves the expression of molecules such as PD-L1 (B7-H1) by LSECs
and actively induces T cell tolerance (130–132). Whereas canonical T cell activation involves
the simultaneous recognition of a peptide-MHC complex by the TCR and the engagement of
costimulatory molecules such as B7.1 or B7.2 on the APC by CD28 on the T cell, the engagement
of PD-L1 on LSECs with its receptor, PD-1, on the T cell results in attenuated or alternative
T cell activation and in tolerance (130). Interactions between T cells and LSECs expressing a
cognate antigen induce rapid T cell proliferation, indicating efficient T cell activation; however,
the resulting effector T cells fail to produce effector cytokines (e.g., IFN-γ) and have reduced
cellular cytotoxicity (130, 132). These findings suggest that, although LSECs can support T cell
activation, the default pathway is to induce the production of nonlicensed T cells that are unable
to kill targets in an antigen-specific fashion.
Liver Sinusoidal Endothelial Cells

Importantly, LSECs are not restricted to simply presenting endogenous antigens to CD8+ T cells.
LSECs can also function as true APCs and are very efficient at cross-presentation of exogenous
antigen within the context of both MHC-I and MHC-II (131–133). In this fashion, LSECs can
activate and tolerize both CD4+ and CD8+ T cells to blood- and gut-derived antigen. This cross-
presentation ability within the context of PD-L1 expression suppresses the immune response
to gut-derived pathogens; however, this same mechanism also impacts the ability of the host
to generate an efficient effector T cell response to systemically administered therapeutics (e.g.,
vaccines or immunomodulatory compounds to treat infections such as HBV) (132, 133). This
antigen presentation by LSECs, together with the expression of immunosuppressive molecules
such as PD-L1, represents a key mechanism for the rapid generation of T cell tolerance, acting
immediately upon antigen presentation by an APC or a target cell and preceding the generation
of more conventional regulatory T cells. In the presence of PD-L1, CD8+ T cells are activated by
APCs; however, these do not become “licensed to kill” and as such do not develop into functional
cytotoxic cells, instead assuming an anergic like phenotype (35).
Hepatocytes
In addition to LSECs, hepatocytes, under the correct conditions, can serve as APCs within the
liver. Although the hepatocyte’s primary roles are metabolism, protein production (including
immune proteins such as acute-phase proteins and complement), and toxin neutralization, these

parenchymal cells are able to detect pathogens and present antigen to the adaptive immune system.
Hepatocytes make up approximately 80% of, and express a wide variety of, immune receptors
(e.g., PRRs, MHC, and costimulatory and adhesion molecules) (134, 135). Although some of
these receptors play a role in hepatocyte-mediated immunity (e.g., inhibition of viral replication,
production and release of inflammatory cytokines and acute-phase proteins), others activate and
coordinate the adaptive immune response (136).
Hepatocytes express MHC-I, antigen-processing machinery, and costimulatory molecules and,
under inflammatory conditions, can be induced to express MHC-II (124, 137–139). Both in vitro
and in vivo studies have shown that hepatocytes can present antigen directly to naive T cells
(122, 123, 140, 141). Electron microscopy has shown that hepatocytes and naive T cells physically
interact in an ICAM-1 and MHC-dependent fashion (139). More recently, the Iannacone group
has shown by using intravital imaging in live animals that CD8+ T cells crawl along the liver
vasculature, actively patrolling and extending cell processes through the fenestrated sinusoidal
endothelium to scan the underlying hepatocytes (142). Upon recognition of an antigen, these cells
then switch from crawling to firm adhesion and deliver a lethal hit to the hepatocyte presenting
the cognate antigen. Although hepatocytes can directly support T cell activation and immunity
against intracellular infections (e.g., viruses), given the surrounding environment rich in other,
more “professional” APCs (e.g., KCs, DCs, LSECs), it remains unclear to what extent hepatocytes
functionally contribute to generating immune responses to exogenous antigen.
Dendritic Cells
KCs, LSECs, and hepatocytes are not the only APC types found within the liver vasculature. The
liver also contains a number of populations of DCs and macrophages (33, 143, 144). Although these
professional APCs are more classically associated with T cell activation in other tissues such as the
spleen and lymph nodes, within the liver microenvironment, these cells also tolerize by default.
DCs enter the liver as immature cells via the portal vein. Once in the liver, the DCs continue to
mature as they move from the portal circulation to the central vein or as they transmigrate through
the LSECs to enter the space of Disse, eventually accessing the liver lymphatics (145, 146). Liver
DCs can be broadly divided into two categories: myeloid DCs (mDCs), which are CD11c high,
and plasmacytoid DCs (pDCs), which are CD123+ . Additionally, in the mouse, a population of
CD11c+ CD8+ lymphoid-related DCs and a population of CD11c+ NK1.1+ cytotoxic DCs have
been identified in the liver, although it is not entirely clear whether this population of cells is
entirely distinct from activated NK cells (126, 143, 147, 148).
This diverse spectrum of DC populations allows for the induction of a variety of T cell responses
following antigen exposure. A number of studies have shown that, despite efficient internalization
and presentation of exogenous antigen, both the mDC and pDC populations appear to be poor
activators of T cells relative to DCs from other tissues (e.g., skin) (126, 127, 148, 149). This
reduced capacity to activate T cells is in part due to cytokine secretion and costimulatory molecule
expression. Whereas skin-derived mDCs release IL-12 upon activation, helping to drive T cell
stimulation, liver-derived mDCs produce IL-10 and indoleamine 2,3-dioxygenase (IDO), potent
anti-inflammatory molecules that attenuate T cell responses (126, 127, 148, 150). Additionally,
pDCs in the liver constitutively express PD-L1, a molecule that induces antigen-specific tolerance
upon recognition by its receptor on the surfaces of T cells, further limiting the ability of liver DCs
to generate an effective adaptive immune response to pathogens (148, 151).
In contrast to the mDCs and pDCs in the liver, CD11c+ CD8+ DCs support robust T cell
activation, leading to the generation of the Th1-type responses associated with the production
of IL-12 and TNF-α (148, 151). Additionally, CD11c+ NK1.1+ cytotoxic DCs both directly lyse
9.16 Kubes · Jenne

cellular targets and present antigen to naive T cells, resulting in efficient activation. These NK1.1+
DCs also produce large quantities of cytokines, such as IFN-γ, that promote the development and
activation of cytotoxic T cells (147, 152). This variety of DC cell subsets helps maintain the balance
between tolerance and immunity in the liver. Shifts in the number and activity of individual DC
populations in response to the liver microenvironment (e.g., presence of cytokines or PAMPs) tip
the scales either toward immunosuppression or toward robust, cell-mediated immune responses.
The inhibition of immunosuppressive signals in the liver (e.g., blockade of IL-10) can also help
tip the scales, promoting the development of DCs that are able to support enhanced antigen
presentation, costimulatory molecule expression, and robust T cell activation (153).
Kupffer Cells as Antigen-Presenting Cells for Adaptive Immunity

Perhaps the best recognized of the APCs in the liver are the KCs, which are the largest macrophage
population in the body, representing 80–90% of all tissue-resident macrophage (7). Although non-
motile, KCs live directly within the bloodstream, residing within the lumen of the liver sinusoids,
and as such are ideally positioned to sample and capture antigens and pathogens from the blood.
As discussed above, following antigen/pathogen capture, KCs act as important APCs, expressing
MHC-I, MHC-II, and costimulatory molecules required for T cell activation. Despite this clear
ability to support the activation of adaptive immunity, under basal conditions, KCs are largely
responsible for the immune hyporesponsiveness of the liver (33). Similar to what is observed in
LSECs, continual exposure to low levels of gut-derived PAMPs, such as LPS, dampens the ability
of KCs to activate lymphocytes (33, 34, 129). This failure to activate T cells appears to be by de-
sign, preventing the generation of immunity to common gut-derived antigen present in the portal
circulation (125). Although KCs are poor stimulators of T cell activation under basal conditions,
the presence of other pathogen-associated molecules (e.g., TLR3 and TLR9 ligands) or inflam-
matory cytokines can modulate the KCs, converting these normally tolerogenic cells into potent
APCs capable of robust T cell activation (33, 154). Again, in contrast to T cell activation, KCs are
extremely effective at activating the iNKT cells that live in and patrol the sinusoids of the liver.
VIRAL INFECTION OF THE LIVER

Although the initial immune response to viral infection is often robust—driven and supported
by a rapid innate immune response that is followed by the rapid expansion of pathogen-specific
CD8+ T cells—early T cell exhaustion or depletion frequently leads to the establishment of a
chronic infection (119, 142, 155, 156). This outcome is particularly likely when the infectious
organism targets nonimmune cells such as hepatocytes, as opposed to infection of other cell types
such as KCs by myxoma virus (MYXV). In the case of MYXV, rapid and robust innate immunity
leads to clearance of the infection within 24 h (94), whereas infection of hepatocytes by HBV can
result in lifelong disease (119, 157). Perhaps the most important question is why, once infection
is established within the hepatocyte, the adaptive immune system is no longer able to clear the
infection. Although some of the answer lies in the ability of specific viruses to modulate or hide
from the immune system (e.g., the inhibition of nuclear translocation of STAT1 by HBV), host
regulation of the adaptive immune response plays a large role.
Acute viral infection of the liver results in antigen presentation by a number of cell types,
including KCs, LSECs, and hepatocytes. This antigen presentation triggers the activation and
expansion of specific T cells (both CD4+ and CD8+ ) and can lead to the formation of tertiary
immune structures known as intrahepatic myeloid cell aggregates for T cell population expan-
sion (iMATEs) (35). These nonperfused follicle-like structures consist of inflammatory myeloid

cells (monocyte-derived NKp46− Ly6G− CD11b+ F4/80+ cells) and CD8+ T cells. Production of
TNF-α and expression of OX40L, CD80, and CD86 support expansion of cytotoxic T lympho-
cytes (CTLs) within these iMATEs but not elsewhere in the liver parenchyma (35). Importantly,
although present following many acute viral infections, these iMATEs, in the absence of appro-
priate proinflammatory signals, quickly involute and cease to support CD8+ T cell activation and
expansion, leading to collapse of the CTL response (35). However, if appropriate signals such as
the TLR9 ligand CpG DNA are provided, these iMATEs and the associated CTL response are
sustained, leading to the clearance of even chronic viral infections (35).
Both CD4+ and CD8+ T cells play central roles in the control and clearance of liver viral
infection. In HCV infection, high numbers of HCV-specific CD4+ T cells are associated with an
acute resolving infection (157). This resolution of viral infection is largely due to the ability of the
CD4+ T cell population to shape and drive the CTL responses mediated by CD8+ T cells. In HCV
infection, a number of different subsets of CD4+ T cells have been reported. Specifically, Th1 cells
are responsible for the production of IL-2, driving CD4+ and CD8+ T cell expansion, whereas
Th17 cells produce IL-17 and IL-21 (157, 158). High levels of IL-17 and IL-21 are associated
with a self-limiting acute viral infection. Loss of the Th1 cells and their associated IL-2 production
lead to collapse of the CD4+ and CD8+ cell numbers and to the establishment of a chronic viral
infection (158). In contrast to the immunomodulation provided by CD4+ T cell subsets, CD8+
T cells are primarily involved in the direct clearance of virally infected liver cells. Production of
high levels of IFN-γ and direct cytotoxic activity result in the killing of HCV-infected liver cells
and in the generation of hepatic lesions (121, 156, 158). The efficacy of the CTL response can be
directly measured through the quantification of liver-specific enzymes (ALT, AST) in the serum
as a marker of cytotoxic clearance of infected hepatocytes (35, 159). Interestingly, during the
transition from acute to chronic viral infection, the number of CD8+ T cells within the liver does
not change, suggesting that the lack of immunological control of HCV infection is instead due to
the functional impairment of the CD8+ T cells (157). Repeated exposure to low doses of HCV
results in reduced IFN-γ production by liver CD8+ T cells. Additionally, expression of PD-1
and CTLA-4 on HCV-specific T cells during chronic infection renders these cells susceptible to
tolerizing signals (151, 158). Inhibition of both PD-1 and CTLA-4 in chronic HCV infection can
lead to reactivation of the CD8+ population and to elevated IFN-γ production and viral clearance.
IMMUNE RESPONSE TO CANCER IN THE LIVER

The liver is one of the most common sites for cancer in the body. Partly due to the liver’s
immunological state and partly due to its anatomy, this organ is a frequent target of both primary
and secondary (metastasizing) tumors. With respect to primary tumors, hepatocellular carcinoma
(HCC) represents the largest burden of liver cancer and is the most common cause of cancer-
related death in the world (160–162).
HCC often arises secondarily to situations of chronic liver inflammation (e.g., NASH, alcoholic
liver disease, HBV infection, HCV infection) (120, 121, 159–161). It is believed that chronic liver
inflammation, regardless of the source, leads to continual cellular stress and damage, resulting in
an increased likelihood of carcinogenesis. In many instances, HCC develops after the initiation
of severe liver damage and tissue remodeling and scaring (e.g., cirrhosis). The exception to this
general observation is the appearance of HCC secondary to HBV infection. In patients with
chronic HBV infection, the occurrence of HCC can precede cirrhosis and is closely associated
with viral load as measured by the presence of viral proteins and DNA in the patient’s blood (161).
Additionally, some viral proteins, such as HBV-encoded protein X, directly inflict damage to the
genome of the patient, mutating both tumor suppressor genes and oncogenes (163).
9.18 Kubes · Jenne

In addition to viral proteins, various noncoding viral RNAs have been associated with the
modulation of the host cell cycle; such modulation leads to increased cellular replication; reduced
apoptosis; and increased epithelial-mesenchymal transition, a key step in the generation of HCC
tumors (163). In addition to viral RNAs, a number of host noncoding RNAs have been shown to be
important in both tumor suppression and tumor growth. These host RNAs become dysregulated
during states of chronic inflammation (e.g., due to viral infection, NASH), leading to the devel-
opment of HCC (164). Furthermore, the general metabolic syndrome (marked by obesity and
insulin resistance) associated with NAFLD and NASH has been linked to immune dysregulation
and tumorigenesis (111, 165–167).
Importantly, these inflammatory processes (e.g., due to viral infection, alcohol, NASH) are not
isolated mechanisms but rather can synergistically trigger tumorigenesis. Studies have shown that
dual infection with HBV and HCV results in a significantly greater risk of HCC than does infection
with either virus alone (161). These data suggest a cumulative effect of infectious disease and
inflammation on the mutagenesis of host liver cells and the development of HCC. Interestingly,
although elimination of these chronic inflammatory insults (e.g., clearance of HCV infection by
antiviral treatment) results in a dramatic reduction in the occurrence of HCC, it does not eliminate
the development of cancer. Studies have shown the appearance of HCC in patients more than a
decade after “curing” HCV infections, as measured by sustained virological response (SVR), which
is defined as the absence of detectable HCV RNA 24 weeks after cessation of treatment (168, 169).
In these patients, HCC was associated with the presence of fibrosis or cirrhosis, suggesting that,
despite viral clearance, long-term reprogramming of the liver microenvironment may persist,
making this tissue susceptible to later tumorigenesis events (161, 168–170). Interestingly, the
choice of drugs used to treat HCV may impact the development of HCC. HCV treatment has
transitioned from interferon-based treatments to direct-acting antivirals (DAAs). This transition
has been driven by higher efficacy in treating HCV and by reduced toxicity; however, emerging
evidence suggests that patients achieving SVR following treatment by DAAs may have a higher
incidence of HCC than do those patients achieving SVR following interferon treatment (168).
These findings clearly illustrate our need to better understand the pathways involved in linking
chronic viral infection to the development of HCC.
Once the initial tumorigenesis event has occurred, the generally tolerogenic and immunosup-
pressive environment of the liver protects these neotumors, allowing for growth, further epithelial-
mesenchymal transition, and the development of true tumor microenvironments. These microen-
vironments further suppress immunity (e.g., expression of PD-L1, recruitment of myeloid-derived
suppressor cells, production of anti-inflammatory cytokines) and protect the cancer cells. Tradi-
tionally, once established, HCC is treated by surgical resection, liver transplantation, restriction
of tumor blood supply [transarterial chemoembolization (TRACE)], or either radiofrequency ab-
lation or microwave ablation (as reviewed in Reference 161). More recently, systemic attempts
to modulate the host immune response have been tried, with varying success. Administration of
the tyrosine kinase inhibitor sorafenib significantly increased patient survival time, but to date
sorafenib has not been used to cure (161, 170). Additionally, the administration of an engineered
oncolytic vaccinia virus, JX-594, also increased survival time (171). Similar to sorafenib, the JX-594
virus failed to produce long-term cures, but this technology represents an exciting step forward in
the treatment of advanced cancers. Not only do oncolytic viruses directly target the tumor itself,
but this form of therapy also stimulates the host immune response, an important approach when
one considers the overall immunosuppressive environment of the liver (171).
Not only does the immune environment of the liver make this tissue a frequent target for
the development of primary tumors, but the liver is also a frequent site of metastases for tumors
originating in other tissues. Perhaps the best-studied example is the involvement of the liver in

colorectal cancer (CRC) metastasis. Although the presence of an immunotolerogenic environment

may explain why metastases thrive in the liver, it does not explain why the liver appears targeted by
the metastatic cells originating from the colon. This apparent targeting of the liver is most likely the
result of a number of factors working in synergy to deliver the metastatic cells to this tissue. First,
the anatomical location and structure of the liver place it directly downstream of the colon (via
the portal circulation), making it the first tissue encountered by metastatic cells in the circulation
(172–174). Second, the reduced blood pressure and convoluted vasculature of the liver sinusoids
enhance trapping of carcinoma cells within this tissue. This capture or lodging of cancer cells in
the liver vasculature is further enhanced by the expression of vascular adhesion molecules by some
cancers. Interactions between selectins and selectin ligands mediate tumor cell adherence within
the liver in an animal model, a process greatly enhanced in the presence of inflammation (175–177).
Interestingly, a significant proportion of CRC metastasis to the liver appears secondarily to
surgical tumor resection. It has been postulated that the release of microbes, or microbe-derived
molecules such as LPS, into the circulation may drive inflammation in the liver, promoting the
trapping, in the liver, of cells released during surgery (176). Experimentally, this phenomenon
has been demonstrated to involve NETs (178). Introduction of LPS into the bloodstream of a
mouse results in the substantial production of NETs within the liver vasculature. These NETs are
composed of a sticky web of decondensed nuclear DNA released into the extracellular environment
and are decorated with a number of cytotoxic and antimicrobial proteins (e.g., histones, neutrophil
elastase, defensins, lysozymes) (179). During the normal immune response, NETs enhance capture
of pathogens (both bacterial and viral) from the blood and limit dissemination (93, 94); however,
in the context of CRC metastasis, NETs appear to ensnare cancer cells, promoting their retention
in the liver (178). A better understanding of how systemic inflammation and surgery modulate the
liver microenvironment may lead to improved therapeutic strategies for the resection of primary
tumors such as CRC without the significant complication of metastasis to the liver.
STERILE INJURY AND TISSUE REPAIR IN THE LIVER

A major issue regarding the role of various immune cells in sterile injury is the definition of sterile
injury. If one considers traumatic injury, including lacerations, resection of lobes of the liver, or
even acute toxicity, no tissue heals better and faster than the liver. Removing or impairing the
function of various immune cells in this context inevitably leads to delayed or impaired healing,
highlighting the importance of the immune system in repair. Indeed, it is becoming clear that these
types of injuries lead to the rapid recruitment of platelets, neutrophils, monocytes, macrophages,
and potentially various other immune cells that perform very critical healing functions. In a focal
necrosis model of liver injury that enabled observation of the recruitment of different cell types,
platelets are the first to arrive at the injury, adhering to injured blood vessels that have either
slightly modified glycocalyx or more profound exposure of the basement membrane (180). These
platelets pave the injured site, where they have the capacity to release many growth factors to help
in the healing process. Indeed, packed platelets are used to help improve healing of wounds. The
paving of platelets along injured endothelium can also serve another important function of allow-
ing for the recruitment of neutrophils. These cells begin to enter the site as early as one hour after
the injury and immediately begin to remove debris and dead cells and dismantle the vasculature (P.
Kubes, unpublished observations), and preventing the recruitment of these cells delays the deposi-
tion of new collagen and the ability to regrow vasculature. Inflammatory monocytes surround the
injury site, but only a few enter immediately (110). In fact, most of these cells remain around the
periphery of the injury together with iNKT cells. The latter sample the environment and, presum-
ably through CD1d-dependent self-antigen presentation, make high quantities of IL-4, but not
9.20 Kubes · Jenne

IFN-γ. IL-4 is critical for monocyte switching toward a more M2 (CCR2-low, Ly6C-low,
CX3 CR1-high) monocyte that does not yet express Mertk or F4/80 (110). However, within
the injury site, there is very active hepatocyte proliferation that is also mediated by the iNKT
cell–produced IL-4 (73). Clearly, the iNKT cells, which are potent producers of IFN-γ during
infection, can detect noninfectious sterile injury, presumably through self-antigens, and can help
convert inflammation to repair. Interestingly, throughout this process, Kupffer cells, which are im-
mobile cells, cannot enter the injury; however, peritoneal macrophages do enter the site and help to
disassemble dead cells though a trogocytotic process that coats the surface of the injury with a thick
DNA mesh (115). It is intriguing to consider that DNA and histones have antimicrobial properties
that might provide a protective barrier. Disruption of any of these processes delays healing.
By contrast, many liver models of disease are not traumatic per se but rather involve processes
that are lifestyle induced. Conditions related to chronic alcohol use, high-fat diet, chronic tox-
ins such as repeated carbon tetrachloride, clinical operations such as ischemia/reperfusion (e.g.,
liver transplant), and inappropriate stimulation of immunity with concanavalin A activate the im-
mune system to mount an inflammatory response that leads to tissue damage. Unlike in trauma,
for which evolution has had the opportunity to design an optimal healing response, in these ia-
trogenic situations, there is no program to promote healing, especially when there is recurrent
exposure. In ischemia/reperfusion of the liver, neutrophils appear to infiltrate the tissue and to
release oxidants and proteases, and depletion of these cells or prevention of their recruitment or
their effector functions always leads to less inflammation and less tissue dysfunction. There are
nearly 800 studies in this regard, and investigators have examined the role of chemokines such as
CXCR2; chemoattractants such as fMLP, PAF, and LTB4; adhesion molecules such as integrins
and selectins; and oxidants as well as proteases (181). However, almost all of these studies looked
at the first 24 h and most looked at the first 6 h, and not at later time points, so little can be said
about repair per se. One study did examine the recovery phase at 96 h in wild-type and CXCR2-
deficient mice, and although CXCR2 ligands were shown to recruit neutrophils, the repair phase
was associated with CXCR2 on hepatocytes (182). Thus, the jury is out on the role of neutrophils
in repair in this model. Another complication is that whole-liver ischemia/reperfusion leads to
ischemia of the intestines, leading to further liver injury, and as such it is not easy to dissociate
the effects of ischemia/reperfusion of the liver versus the intestines (183). This linkage between
the liver and intestine becomes particularly relevant when one sees protection with, for example,
antiselectin therapy if indeed the intestine and not liver microcirculation has selectin-dependent
neutrophil recruitment, suggesting that inhibiting cellular recruitment to the gut can protect an
ischemic liver.
Similarly, in acute acetaminophen-induced liver injury, the main reason for liver transplan-
tation, there is also a role for neutrophils in the early injury, but whether repair is affected by
neutrophils is unclear (184). Neutrophils are also present in acute alcoholic hepatitis, primary bil-
iary cirrhosis, PSC, alcoholic liver disease, NASH, and carbon tetrachloride–induced liver injury,
consistent with the view that neutrophils are an underlying cause of most liver injury. However,
the results are not consistent. In carbon tetrachloride–induced liver injury, neutrophils have been
shown to be both important and irrelevant. In alcohol-induced liver injury, the model becomes a
major factor. Neutrophil numbers remain unchanged after chronic ethanol feeding but increase
markedly after chronic and binge ethanol feeding (185, 186). Depletion of neutrophils did improve
liver damage induced by chronic and binge ethanol. Alcohol likely induces increases in intestinal
permeability, which increases at least LPS and other immunostimulants in addition to bacterial
translocation. As such, alcohol-induced liver injury could be associated with these other factors,
including the microbiome. In NASH, a devastating condition leading to fatty liver and ultimately
liver cancer, a role for neutrophils is difficult to assess due to the length of time during which one

would have to deplete neutrophils. Nevertheless, Zang et al. (187) depleted neutrophils in mice
for 1 and 2 weeks and observed a major reduction in ALT levels in mice fed an MCD diet that
leads to fatty liver disease. However, by 4 weeks, neutrophil depletion was no longer of benefit,
and overall inflammation was worse by 8 weeks. Neutrophils clearly must perform an important
repair function that is potentially masked by inappropriate activation of these cells.
SUMMARY
The liver is a frontline immune organ specifically designed to detect and clear potential pathogens
from the blood while maintaining a general state of immune hyporesponsiveness. This default pro-
graming of immunotolerance prevents unwanted inflammatory responses directed against harm-
less food antigens or the normal, background levels of microbe-derived molecules that may enter
the bloodstream from the gut (Figure 3). However, if the level or context of microbial products
changes, the liver can rapidly switch from immune hyporesponsiveness to generate a robust in-
flammatory response and efficient adaptive immunity. Likewise, in the case of acute liver injury,
Inflammation and/or immunity Tolerance and/or hypoimmunity
• Acute Inflammation
Homeostatic
• Neutrophil recruitment • Lack of response against

food antigens
• Activation of resident lymphocytes
• Tolerance of baseline microbe-
• Pathogen clearance derived products
• Recruitment and differentiation • Facilitation of organ
of reparative monocytes transplantation
• Resolution
• Chronic inflammation • Failure to initiate/sustain an

effective immune response
• Differentiation of cells into
Pathogenic
proinflammatory monocytes/ • Cellular exhaustion

macrophages • Establishment of chronic viral
• Sustained activation of resident infections
and recruited lymphocytes • Failure to clear de novo
• Tissue damage and remodeling tumorigenesis events
• Fibrosis and organ dysfunction • Creation of an environment
supportive of metastases
Figure 3
Liver immunity: a response regulated by balance. The immune response in the liver is typically tolerogenic
or hypoimmune (top right). This environment ensures that there are no untold responses to common food
antigens or to low levels of microbe-derived molecules that enter the body daily through the portal
circulation. In instances in which a pathogen enters the liver, a rapid and acute immune response ensues,
leading to self-limiting inflammation, pathogen clearance, and eventual repair/restoration of the tissue to a
functional level. In instances in which inflammation is not limiting but rather is chronic, substantial cellular
recruitment and tissue damage may result. In the presence of sustained inflammation, this damage is often
repaired inappropriately, resulting in tissue remodeling, fibrosis, and organ dysfunction. Likewise, too little
immunity can also be bad. Failure to initiate an appropriate immune response in the presence of viral
infection or cancer can result in the development of chronic disease, tissue dysfunction, and organ failure.
9.22 Kubes · Jenne

inflammation is initiated to help support tissue repair. This balance between immunotolerance
and effective immune responses is mediated by the interactions occurring between the numerous
populations of immune cells that reside within, and are recruited to, the liver. Moreover, this
balance is essential to the normal functioning and homeostasis of this tissue. In situations in which
inappropriate immune responses are initiated or inflammation is sustained, balance is lost, and
liver pathology (e.g., NAFLD, NASH) is observed. Likewise, failure to initiate an efficient im-
mune response, when needed, can result in the establishment of chronic viral infections or failure
to clear cancer cells. The ability of the liver to maintain balance between acute immune responses
and tolerance is essential to overall health and underscores the importance of this tissue as a key
immune organ.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS
We thank Dr. Seok-Joo Kim and Rachelle Davis for providing Figure 3.
LITERATURE CITED
1. Berg RD. 1995. Bacterial translocation from the gastrointestinal tract. Trends Microbiol. 3:149–54
2. Lumsden AB, Henderson JM, Kutner MH. 1988. Endotoxin levels measured by a chromogenic assay in
portal, hepatic and peripheral venous blood in patients with cirrhosis. Hepatology 8:232–36
3. Son G, Kremer M, Hines IN. 2010. Contribution of gut bacteria to liver pathobiology. Gastroenterol.
Res. Pract. 2010:453563. https://doi.org/10.1155/2010/453563
4. Paulos CM, Wrzesinski C, Kaiser A, Hinrichs CS, Chieppa M, et al. 2007. Microbial translocation
augments the function of adoptively transferred self/tumor-specific CD8+ T cells via TLR4 signaling.
J. Clin. Investig. 117:2197–204
5. Balmer ML, Slack E, de Gottardi A, Lawson MA, Hapfelmeier S, et al. 2014. The liver may act as
a firewall mediating mutualism between the host and its gut commensal microbiota. Sci. Transl. Med.
6:237ra66
6. Oda M, Yokomori H, Han JY. 2003. Regulatory mechanisms of hepatic microcirculation. Clin. Hemorheol.
Microcirc. 29:167–82
7. Bilzer M, Roggel F, Gerbes AL. 2006. Role of Kupffer cells in host defense and liver disease. Liver Int.
26:1175–86
8. Helmy KY, Katschke KJ Jr., Gorgani NN, Kljavin NM, Elliott JM, et al. 2006. CRIg: a macrophage
complement receptor required for phagocytosis of circulating pathogens. Cell 124:915–27
9. Deniset JF, Surewaard BG, Lee WY, Kubes P. 2017. Splenic Ly6Ghigh mature and Ly6Gint immature
neutrophils contribute to eradication of S. pneumoniae. J. Exp. Med. 214:1333–50
10. He JQ, Katschke KJ Jr., Gribling P, Suto E, Lee WP, et al. 2013. CRIg mediates early Kupffer cell
responses to adenovirus. J. Leukoc. Biol. 93:301–6
11. Lee WY, Sanz MJ, Wong CH, Hardy PO, Salman-Dilgimen A, et al. 2014. Invariant natural killer T
cells act as an extravascular cytotoxic barrier for joint-invading Lyme Borrelia. PNAS 111:13936–41
12. Zeng Z, Surewaard BG, Wong CH, Geoghegan JA, Jenne CN, Kubes P. 2016. CRIg functions as
a macrophage pattern recognition receptor to directly bind and capture blood-borne gram-positive
bacteria. Cell Host Microbe 20:99–106
13. Johansson AG, Sundqvist T, Skogh T. 2000. IgG immune complex binding to and activation of liver cells.
An in vitro study with IgG immune complexes, Kupffer cells, sinusoidal endothelial cells and hepatocytes.
Int. Arch. Allergy Immunol. 121:329–36

14. Kosugi I, Muro H, Shirasawa H, Ito I. 1992. Endocytosis of soluble IgG immune complex and its
transport to lysosomes in hepatic sinusoidal endothelial cells. J. Hepatol. 16:106–14
15. Montalvao F, Garcia Z, Celli S, Breart B, Deguine J, van RN, Bousso P. 2013. The mechanism of
anti-CD20-mediated B cell depletion revealed by intravital imaging. J. Clin. Investig. 123:5098–103
16. Thanabalasuriar A, Surewaard BG, Willson ME, Neupane AS, Stover CK, et al. 2017. Bispecific antibody
targets multiple Pseudomonas aeruginosa evasion mechanisms in the lung vasculature. J. Clin. Investig.
127:2249–61
17. Gale RP, Sparkes RS, Golde DW. 1978. Bone marrow origin of hepatic macrophages (Kupffer cells) in
humans. Science 201:937–38
18. Schulz C, Gomez PE, Chorro L, Szabo-Rogers H, Cagnard N, et al. 2012. A lineage of myeloid cells
independent of Myb and hematopoietic stem cells. Science 336:86–90
19. Scott CL, Zheng F, De Baetselier P Martens L, Saeys Y, et al. 2016. Bone marrow–derived monocytes
give rise to self-renewing and fully differentiated Kupffer cells. Nat. Commun. 7:10321
20. David BA, Rezende RM, Antunes MM, Santos MM, Freitas Lopes MA, et al. 2016. Combination of mass
cytometry and imaging analysis reveals origin, location, and functional repopulation of liver myeloid cells
in mice. Gastroenterology 151:1176–91
21. Beattie L, Sawtell A, Mann J, Frame TC, Teal B, et al. 2016. Bone marrow–derived and resident liver
macrophages display unique transcriptomic signatures but similar biological functions. J. Hepatol. 65:758–
68
22. Bleriot C, Dupuis T, Jouvion G, Eberl G, Disson O, Lecuit M. 2015. Liver-resident macrophage necrop-
tosis orchestrates type 1 microbicidal inflammation and type-2-mediated tissue repair during bacterial
infection. Immunity. 42:145–58
23. Jenkins SJ, Ruckerl D, Thomas GD, Hewitson JP, Duncan S, et al. 2013. IL-4 directly signals tissue-
resident macrophages to proliferate beyond homeostatic levels controlled by CSF-1. J. Exp. Med.
210:2477–91
24. Holdsworth RJ, Irving AD, Cuschieri A. 1991. Postsplenectomy sepsis and its mortality rate: actual
versus perceived risks. Br. J. Surg. 78:1031–38
25. Gregory SH, Sagnimeni AJ, Wing EJ. 1996. Bacteria in the bloodstream are trapped in the liver and
killed by immigrating neutrophils. J. Immunol. 157:2514–20
26. Czuczman MA, Fattouh R, van Rijn JM, Canadien V, Osborne S, et al. 2014. Listeria monocytogenes
exploits efferocytosis to promote cell-to-cell spread. Nature 509:230–34
27. Surewaard BG, Deniset JF, Zemp FJ, Amrein M, Otto M, et al. 2016. Identification and treatment of
the Staphylococcus aureus reservoir in vivo. J. Exp. Med. 213:1141–51
28. MacParland SA, Tsoi KM, Ouyang B, Ma XZ, Manuel J, et al. 2017. Phenotype determines nanoparticle
uptake by human macrophages from liver and blood. ACS Nano 11:2428–43
29. Bleriot C, Dupuis T, Jouvion G, Eberl G, Disson O, Lecuit M. 2015. Liver-resident macrophage necrop-
tosis orchestrates type 1 microbicidal inflammation and type-2-mediated tissue repair during bacterial
infection. Immunity. 42:145–58
30. Shi J, Gilbert GE, Kokubo Y, Ohashi T. 2001. Role of the liver in regulating numbers of circulating
neutrophils. Blood 98:1226–30
31. Fadok VA, Bratton DL, Henson PM. 2001. Phagocyte receptors for apoptotic cells: recognition, uptake,
and consequences. J. Clin. Investig. 108:957–62
32. Grozovsky R, Hoffmeister KM, Falet H. 2010. Novel clearance mechanisms of platelets. Curr. Opin.
Hematol. 17:585–89
33. You Q, Cheng L, Kedl RM, Ju C. 2008. Mechanism of T cell tolerance induction by murine hepatic
Kupffer cells. Hepatology 48:978–90
34. Knolle P, Schlaak J, Uhrig A, Kempf P, Meyer zum Buschenfelde KH, Gerken G. 1995. Human Kupffer
cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge. J. Hepatol. 22:226–29
35. Huang LR, Wohlleber D, Reisinger F, Jenne CN, Cheng RL, et al. 2013. Intrahepatic myeloid-cell
aggregates enable local proliferation of CD8+ T cells and successful immunotherapy against chronic
viral liver infection. Nat. Immunol. 10:574–83
36. Lee WY, Moriarty TJ, Wong CH, Zhou H, Strieter RM, et al. 2010. An intravascular immune response
to Borrelia burgdorferi involves Kupffer cells and iNKT cells. Nat. Immunol. 11:295–302
9.24 Kubes · Jenne

37. Crispe IN. 2009. The liver as a lymphoid organ. Annu. Rev. Immunol. 27:147–63
38. Doherty DG, O’Farrelly C. 2000. Innate and adaptive lymphoid cells in the human liver. Immunol. Rev.
174:5–20
39. Notas G, Kisseleva T, Brenner D. 2009. NK and NKT cells in liver injury and fibrosis. Clin. Immunol.
130:16–26
40. O’Sullivan TE, Sun JC, Lanier LL. 2015. Natural killer cell memory. Immunity 43(4):634–45
41. Sun JC, Madera S, Bezman NA, Beilke JN, Kaplan MH, Lanier LL. 2012. Proinflammatory cytokine
signaling required for the generation of natural killer cell memory. J. Exp. Med. 209(5):947–54
42. Sun JC, Beilke JN, Lanier LL. 2009. Adaptive immune features of natural killer cells. Nature
457(7229):557–61. Erratum. 2009. Nature 457(7233):1168
43. Bendelac A, Savage PB, Teyton L. 2007. The biology of NKT cells. Annu. Rev. Immunol. 25:297–336
44. Bendelac A, Rivera MN, Park SH, Roark JH. 1997. Mouse CD1-specific NK1 T cells: development,
specificity, and function. Annu. Rev. Immunol. 15:535–62
45. Matsuda JL, Naidenko OV, Gapin L, Nakayama T, Taniguchi M, et al. 2000. Tracking the response of
natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192:741–54
46. Dascher CC, Brenner MB. 2003. Evolutionary constraints on CD1 structure: insights from comparative
genomic analysis. Trends Immunol. 24:412–18

47. Kenna T, Golden-Mason L, Porcelli SA, Koezuka Y, Hegarty JE, et al. 2003. NKT cells from normal
and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells.
J. Immunol. 171:1775–79
48. Brossay L, Chioda M, Burdin N, Koezuka Y, Casorati G, et al. 1998. CD1d-mediated recognition of
an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution.
J. Exp. Med. 188:1521–28
49. Arrenberg P, Halder R, Dai Y, Maricic I, Kumar V. 2010. Oligoclonality and innate-like features in the
TCR repertoire of type II NKT cells reactive to a beta-linked self-glycolipid. PNAS 107:10984–89
50. Blomqvist M, Rhost S, Teneberg S, Lofbom L, Osterbye T, et al. 2009. Multiple tissue-specific isoforms
of sulfatide activate CD1d-restricted type II NKT cells. Eur. J. Immunol. 39:1726–35
51. Bendelac A. 1995. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes.
J. Exp. Med. 182:2091–96
52. Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. 2002. A thymic precursor to the NK T cell lineage.
Science 296:553–55
53. Dashtsoodol N, Shigeura T, Aihara M, Ozawa R, Kojo S, et al. 2017. Alternative pathway for the
development of Vα14+ NKT cells directly from CD4− CD8− thymocytes that bypasses the CD4+ CD8+
stage. Nat. Immunol. 18:274–82
54. Gumperz JE, Miyake S, Yamamura T, Brenner MB. 2002. Functionally distinct subsets of CD1d-
restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195:625–36
55. Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, et al. 1998. Natural killer–like nonspecific tumor cell
lysis mediated by specific ligand-activated Vα14 NKT cells. PNAS 95:5690–93
56. Constantinides MG, Bendelac A. 2013. Transcriptional regulation of the NKT cell lineage. Curr. Opin.
Immunol. 25:161–67
57. Lee PT, Benlagha K, Teyton L, Bendelac A. 2002. Distinct functional lineages of human Vα24 natural
killer T cells. J. Exp. Med. 195:637–41
58. Lee YJ, Wang H, Starrett GJ, Phuong V, Jameson SC, Hogquist KA. 2015. Tissue-specific distribution
of iNKT cells impacts their cytokine response. Immunity 43:566–78
59. DeAngelis RA, Markiewski MM, Kourtzelis I, Rafail S, Syriga M, et al. 2012. A complement-IL-4
regulatory circuit controls liver regeneration. J. Immunol. 188:641–48
60. Yin S, Wang H, Bertola A, Feng D, Xu MJ, et al. 2014. Activation of invariant natural killer T cells impedes
liver regeneration by way of both IFN-γ- and IL-4-dependent mechanisms. Hepatology 60:1356–66
61. Park O, Jeong WI, Wang L, Wang H, Lian ZX, et al. 2009. Diverse roles of invariant natural killer T
cells in liver injury and fibrosis induced by carbon tetrachloride. Hepatology 49:1683–94
62. Biburger M, Tiegs G. 2005. α-Galactosylceramide-induced liver injury in mice is mediated by TNF-α
but independent of Kupffer cells. J. Immunol. 175:1540–50

63. Wong CH, Jenne CN, Lee WY, Leger C, Kubes P. 2011. Functional innervation of hepatic iNKT cells
is immunosuppressive following stroke. Science 334:101–5
64. Georgiev H, Ravens I, Benarafa C, Forster R, Bernhardt G. 2016. Distinct gene expression patterns
correlate with developmental and functional traits of iNKT subsets. Nat. Commun. 7:13116
65. Anantha RV, Mazzuca DM, Xu SX, Porcelli SA, Fraser DD, et al. 2014. T helper type 2–polarized
invariant natural killer T cells reduce disease severity in acute intra-abdominal sepsis. Clin. Exp. Immunol.
178:292–309
66. Bai L, Constantinides MG, Thomas SY, Reboulet R, Meng F, et al. 2012. Distinct APCs explain the
cytokine bias of α-galactosylceramide variants in vivo. J. Immunol. 188:3053–61
67. Trobonjaca Z, Leithauser F, Moller P, Schirmbeck R, Reimann J. 2001. Activating immunity in the
liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-γ release by liver NKT
cells. J. Immunol. 167:1413–22
68. Stetson DB, Mohrs M, Reinhardt RL, Baron JL, Wang ZE, et al. 2003. Constitutive cytokine mRNAs
mark natural killer (NK) and NK T cells poised for rapid effector function. J. Exp. Med. 198:1069–76
69. Geissmann F, Cameron TO, Sidobre S, Manlongat N, Kronenberg M, et al. 2005. Intravascular immune
surveillance by CXCR6+ NKT cells patrolling liver sinusoids. PLOS Biol. 3:e113
70. Campos RA, Szczepanik M, Lisbonne M, Itakura A, Leite-de-Moraes M, Askenase PW. 2006. Invariant
NKT cells rapidly activated via immunization with diverse contact antigens collaborate in vitro with B-1
cells to initiate contact sensitivity. J. Immunol. 177:3686–94
71. Campos RA, Szczepanik M, Itakura A, Kahira-Azuma M, Sidobre S, et al. 2003. Cutaneous immunization
rapidly activates liver invariant Vα14 NKT cells stimulating B-1 B cells to initiate T cell recruitment
for elicitation of contact sensitivity. J. Exp. Med. 198:1785–96
72. Velazquez P, Cameron TO, Kinjo Y, Nagarajan N, Kronenberg M, Dustin ML. 2008. Cutting edge:
Activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of
liver sinusoids. J. Immunol. 180:2024–28
73. Liew PX, Lee W-Y, Kubes P. 2017. iNKT cells orchestrate a switch from inflammation to resolution of
sterile liver injurty. Immunity. 47:752–65.e5
74. Petri B, Phillipson M, Kubes P. 2008. The physiology of leukocyte recruitment: an in vivo perspective.
J. Immunol. 180:6439–46
75. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. 2007. Getting to the site of inflammation: the leukocyte
adhesion cascade updated. Nat. Rev. Immunol. 7:678–89
76. Beste MT, Hammer DA. 2008. Selectin catch-slip kinetics encode shear threshold adhesive behavior of
rolling leukocytes. PNAS 105:20716–21
77. McDonald B, McAvoy EF, Lam F, Gill V, de la Motte C, et al. 2008. Interaction of CD44 and hyaluronan
is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids. J. Exp. Med. 205:915–
27
78. Fox-Robichaud A, Kubes P. 2000. Molecular mechanisms of tumor necrosis factor α–stimulated leuko-
cyte recruitment into the hepatic circulation. Hepatology 31:1123–27
79. Wong J, Johnston B, Lee SS, Bullard DC, Smith CW, et al. 1997. A minimal role for selectins in the
recruitment of leukocytes into the inflamed liver microvasculature. J. Clin. Investig. 99:2782–90
80. Kivi E, Elima K, Aalto K, Nymalm Y, Auvinen K, et al. 2009. Human Siglec-10 can bind to vascular
adhesion protein-1 and serves as its substrate. Blood 114:5385–92
81. Aalto K, Autio A, Kiss EA, Elima K, Nymalm Y, et al. 2011. Siglec-9 is a novel leukocyte ligand for vascular
adhesion protein-1 and can be used in PET imaging of inflammation and cancer. Blood 118:3725–33
82. Ichida T, Sugitani S, Satoh T, Matsuda Y, Sugiyama M, et al. 1996. Localization of hyaluronan in human
liver sinusoids: a histochemical study using hyaluronan-binding protein. Liver 16:365–71
83. Jackson DG. 2004. Biology of the lymphatic marker LYVE-1 and applications in research into lymphatic
trafficking and lymphangiogenesis. APMIS 112:526–38
84. Falkowski M, Schledzewski K, Hansen B, Goerdt S. 2003. Expression of stabilin-2, a novel fasciclin-
like hyaluronan receptor protein, in murine sinusoidal endothelia, avascular tissues, and at solid/liquid
interfaces. Histochem. Cell Biol. 120:361–69
85. McDonald B, Kubes P. 2015. Interactions between CD44 and hyaluronan in leukocyte trafficking. Front.
Immunol. 6:68
9.26 Kubes · Jenne

86. Zhuo L, Kanamori A, Kannagi R, Itano N, Wu J, et al. 2006. SHAP potentiates the CD44-mediated
leukocyte adhesion to the hyaluronan substratum. J. Biol. Chem. 281:20303–14
87. McAvoy E, McDonald B, Kubes P. 2007. The adhesion molecules CD44 and hyaluronan, not physical
trapping, are responsible for neutrophil sequestration in inflamed liver sinusoids [abstract]. J. Immunol.
178(1 Suppl.):S189–90
88. Menezes GB, Lee WY, Zhou H, Waterhouse CC, Cara DC, Kubes P. 2009. Selective down-regulation
of neutrophil Mac-1 in endotoxemic hepatic microcirculation via IL-10. J. Immunol. 183:7557–68
89. Mohamadzadeh M, DeGrendele H, Arizpe H, Estess P, Siegelman M. 1998. Proinflammatory stimuli
regulate endothelial hyaluronan expression and CD44/HA-dependent primary adhesion. J. Clin. Investig.
101:97–108
90. Day AJ, de la Motte CA. 2005. Hyaluronan cross-linking: a protective mechanism in inflammation?
Trends Immunol. 26:637–43
91. de la Motte CA, Hascall VC, Drazba J, Bandyopadhyay SK, Strong SA. 2003. Mononuclear leukocytes
bind to specific hyaluronan structures on colon mucosal smooth muscle cells treated with polyinosinic
acid:polycytidylic acid: Inter-α-trypsin inhibitor is crucial to structure and function. Am. J. Pathol.
163:121–33
92. Shi C, Velazquez P, Hohl TM, Leiner I, Dustin ML, Pamer EG. 2010. Monocyte trafficking to hep-
atic sites of bacterial infection is chemokine independent and directed by focal intercellular adhesion
molecule-1 expression. J. Immunol. 184:6266–74
93. McDonald B, Urrutia R, Yipp BG, Jenne CN, Kubes P. 2012. Intravascular neutrophil extracellular
traps capture bacteria from the bloodstream during sepsis. Cell Host Microbe 12:324–33
94. Jenne CN, Wong CH, Zemp FJ, McDonald B, Rahman MM, et al. 2013. Neutrophils recruited to sites
of infection protect from virus challenge by releasing neutrophil extracellular traps. Cell Host Microbe
13:169–80
95. Kimura K, Nagaki M, Saio M, Moriwaki H, Kakimi K. 2009. Role of CD44 in CTL-induced acute liver
injury in hepatitis B virus transgenic mice. J. Gastroenterol. 44:218–27
96. Younossi ZM, Stepanova M, Afendy M, Fang Y, Younossi Y, et al. 2011. Changes in the prevalence of the
most common causes of chronic liver diseases in the United States from 1988 to 2008. Clin. Gastroenterol.
Hepatol. 9:524–30
97. Egan CE, Daugherity EK, Rogers AB, Abi Abdallah DS, Denkers EY, Maurer KJ. 2013. CCR2 and
CD44 promote inflammatory cell recruitment during fatty liver formation in a lithogenic diet fed mouse
model. PLOS ONE 8:e65247
98. Kang HS, Liao G, DeGraff LM, Gerrish K, Bortner CD, et al. 2013. CD44 plays a critical role in regu-
lating diet-induced adipose inflammation, hepatic steatosis, and insulin resistance. PLOS ONE 8:e58417
99. Bonder CS, Norman MU, Swain MG, Zbytnuik LD, Yamanouchi J, et al. 2005. Rules of recruitment
for Th1 and Th2 lymphocytes in inflamed liver: a role for α-4 integrin and vascular adhesion protein-1.
Immunity 23:153–63
100. Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams DH. 2002. Vascular adhesion protein-1
mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells. J. Immunol.
169:983–92
101. Kurkijarvi R, Adams DH, Leino R, Mottonen T, Jalkanen S, Salmi M. 1998. Circulating form of human
vascular adhesion protein-1 (VAP-1): increased serum levels in inflammatory liver diseases. J. Immunol.
161:1549–57
102. Trivedi PJ, Tickle J, Vesterhus MN, Eddowes PJ, Bruns T, et al. 2017. Vascular adhesion protein-1
is elevated in primary sclerosing cholangitis, is predictive of clinical outcome and facilitates recruitment
of gut-tropic lymphocytes to liver in a substrate-dependent manner. Gut. In press. https://doi.org/
10.1136/gutjnl-2016-312354
103. Tanaka S, Tanaka T, Kawakami T, Takano H, Sugahara M, et al. 2017. Vascular adhesion protein-1
enhances neutrophil infiltration by generation of hydrogen peroxide in renal ischemia/reperfusion injury.
Kidney Int. 92:154–64
104. Weston CJ, Shepherd EL, Claridge LC, Rantakari P, Curbishley SM, et al. 2015. Vascular adhesion
protein-1 promotes liver inflammation and drives hepatic fibrosis. J. Clin. Investig. 125:501–20

105. Zhang J, Xu P, Song P, Wang H, Zhang Y, et al. 2016. CCL2-CCR2 signaling promotes hepatic
ischemia/reperfusion injury. J. Surg. Res. 202:352–62
106. Mossanen JC, Krenkel O, Ergen C, Govaere O, Liepelt A, et al. 2016. Chemokine (C-C motif ) receptor
2–positive monocytes aggravate the early phase of acetaminophen-induced acute liver injury. Hepatology
64:1667–82
107. Krenkel O, Tacke F. 2017. Liver macrophages in tissue homeostasis and disease. Nat. Rev. Immunol.
17:306–21
108. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, et al. 2007. Monitoring of blood vessels and
tissues by a population of monocytes with patrolling behavior. Science 317:666–70
109. Carlin LM, Stamatiades EG, Auffray C, Hanna RN, Glover L, et al. 2013. Nr4a1-dependent Ly6Clow
monocytes monitor endothelial cells and orchestrate their disposal. Cell 153:362–75
110. Dal-Secco D, Wang J, Zeng Z, Kolaczkowska E, Wong CH, et al. 2015. A dynamic spectrum of mono-
cytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury. J. Exp. Med.
212:447–56
111. Reid DT, Reyes JL, McDonald BA, Vo T, Reimer RA, Eksteen B. 2016. Kupffer cells undergo funda-
mental changes during the development of experimental NASH and are critical in initiating liver damage
and inflammation. PLOS ONE 11:e0159524

112. Wang M, You Q, Lor K, Chen F, Gao B, Ju C. 2014. Chronic alcohol ingestion modulates hepatic
macrophage populations and functions in mice. J. Leukoc. Biol. 96:657–65
113. Lefebvre E, Moyle G, Reshef R, Richman LP, Thompson M, et al. 2016. Antifibrotic effects of the
dual CCR2/CCR5 antagonist cenicriviroc in animal models of liver and kidney fibrosis. PLOS ONE
11:e0158156
114. Friedman S, Sanyal A, Goodman Z, Lefebvre E, Gottwald M, et al. 2016. Efficacy and safety study
of cenicriviroc for the treatment of non-alcoholic steatohepatitis in adult subjects with liver fibrosis:
CENTAUR Phase 2b study design. Contemp. Clin. Trials 47:356–65
115. Wang J, Kubes P. 2016. A reservoir of mature cavity macrophages that can rapidly invade visceral organs
to affect tissue repair. Cell 165:668–78
116. Durand F, Francoz C. 2017. The future of liver transplantation for viral hepatitis. Liver Int. 37(Suppl.
1):130–35
117. Wong YC, McCaughan GW, Bowen DG, Bertolino P. 2016. The CD8 T-cell response during tolerance
induction in liver transplantation. Clin. Transl. Immunol. 5:e102
118. Fahrner R, Dondorf F, Ardelt M, Settmacher U, Rauchfuss F. 2016. Role of NK, NKT cells and
macrophages in liver transplantation. World J. Gastroenterol. 22:6135–44
119. Protzer U, Maini MK, Knolle PA. 2012. Living in the liver: hepatic infections. Nat. Rev. Immunol.
12:201–13
120. Maini MK, Gehring AJ. 2016. The role of innate immunity in the immunopathology and treatment of
HBV infection. J. Hepatol. 64:S60–70
121. Dustin LB, Cashman SB, Laidlaw SM. 2014. Immune control and failure in HCV infection: tipping the
balance. J. Leukoc. Biol. 96:535–48
122. Qian S, Wang Z, Lee Y, Chiang Y, Bonham C, Fung J, Lu L. 2001. Hepatocyte-induced apoptosis of
activated T cells, a mechanism of liver transplant tolerance, is related to the expression of ICAM-1 and
hepatic lectin. Transplant. Proc. 33:226
123. Bertolino P, McCaughan GW, Bowen DG. 2002. Role of primary intrahepatic T-cell activation in the
‘liver tolerance effect’. Immunol. Cell Biol. 80:84–92
124. Wahl C, Bochtler P, Chen L, Schirmbeck R, Reimann J. 2008. B7-H1 on hepatocytes facilitates priming
of specific CD8 T cells but limits the specific recall of primed responses. Gastroenterology 135:980–88
125. Knolle PA, Uhrig A, Hegenbarth S, Loser E, Schmitt E, et al. 1998. IL-10 down-regulates T cell
activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake
via the mannose receptor and lowered surface expression of accessory molecules. Clin. Exp. Immunol.
114:427–33
126. Pillarisetty VG, Shah AB, Miller G, Bleier JI, DeMatteo RP. 2004. Liver dendritic cells are less immuno-
genic than spleen dendritic cells because of differences in subtype composition. J. Immunol. 172:1009–17
9.28 Kubes · Jenne

127. Tokita D, Sumpter TL, Raimondi G, Zahorchak AF, Wang Z, et al. 2008. Poor allostimulatory function
of liver plasmacytoid DC is associated with pro-apoptotic activity, dependent on regulatory T cells.
J. Hepatol. 49:1008–18
128. Sana G, Lombard C, Vosters O, Jazouli N, Andre F, et al. 2013. Adult human hepatocytes promote
CD4+ T cell hyporesponsiveness via interleukin-10 producing allogeneic dendritic cells. Cell Transplant.
23:1127–42
129. Knolle PA, Germann T, Treichel U, Uhrig A, Schmitt E, et al. 1999. Endotoxin down-regulates T cell
activation by antigen-presenting liver sinusoidal endothelial cells. J. Immunol. 162:1401–7
130. Diehl L, Schurich A, Grochtmann R, Hegenbarth S, Chen L, Knolle PA. 2008. Tolerogenic matura-
tion of liver sinusoidal endothelial cells promotes B7-homolog 1–dependent CD8+ T cell tolerance.
Hepatology 47:296–305
131. Berg M, Wingender G, Djandji D, Hegenbarth S, Momburg F, et al. 2006. Cross-presentation of
antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+
T cell tolerance. Eur. J. Immunol. 36:2960–70
132. Limmer A, Ohl J, Wingender G, Berg M, Jungerkes F, et al. 2005. Cross-presentation of oral antigens
by liver sinusoidal endothelial cells leads to CD8 T cell tolerance. Eur. J. Immunol. 35:2970–81
133. Limmer A, Ohl J, Kurts C, Ljunggren HG, Reiss Y, et al. 2000. Efficient presentation of exogenous
antigen by liver endothelial cells to CD8+ T cells results in antigen-specific T-cell tolerance. Nat. Med.
6:1348–54
134. Racanelli V, Rehermann B. 2006. The liver as an immunological organ. Hepatology 43:S54–62
135. Seki E, Brenner DA. 2008. Toll-like receptors and adaptor molecules in liver disease: update. Hepatology
48:322–35
136. Zhang X, Meng Z, Qiu S, Xu Y, Yang D, et al. 2009. Lipopolysaccharide-induced innate immune
responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-
independent pathways. Cell Microbiol. 11:1624–37
137. Franco A, Barnaba V, Natali P, Balsano C, Musca A, Balsano F. 1988. Expression of class I and class II
major histocompatibility complex antigens on human hepatocytes. Hepatology 8:449–54
138. Chen M, Tabaczewski P, Truscott SM, Van Kaer L Stroynowski I. 2005. Hepatocytes express abundant
surface class I MHC and efficiently use transporter associated with antigen processing, tapasin, and low
molecular weight polypeptide proteasome subunit components of antigen processing and presentation
pathway. J. Immunol. 175:1047–55
139. Warren A, Le Couteur DG Fraser R, Bowen DG, McCaughan GW, Bertolino P. 2006. T lymphocytes
interact with hepatocytes through fenestrations in murine liver sinusoidal endothelial cells. Hepatology
44:1182–90
140. Balam S, Romero JF, Bongfen SE, Guillaume P, Corradin G. 2012. CSP—a model for in vivo presentation
of Plasmodium berghei sporozoite antigens by hepatocytes. PLOS ONE 7:e51875
141. Bertolino P, Bowen DG, McCaughan GW, Fazekas de St GB. 2001. Antigen-specific primary activation
of CD8+ T cells within the liver. J. Immunol. 166:5430–38
142. Guidotti LG, Inverso D, Sironi L, Di LP, Fioravanti J, et al. 2015. Immunosurveillance of the liver by
intravascular effector CD8+ T cells. Cell 161:486–500
143. Crispe IN. 2011. Liver antigen-presenting cells. J. Hepatol. 54:357–65
144. Parker GA, Picut CA. 2005. Liver immunobiology. Toxicol. Pathol. 33:52–62
145. Sato T, Yamamoto H, Sasaki C, Wake K. 1998. Maturation of rat dendritic cells during intrahepatic
translocation evaluated using monoclonal antibodies and electron microscopy. Cell Tissue Res. 294:503–14
146. Kudo S, Matsuno K, Ezaki T, Ogawa M. 1997. A novel migration pathway for rat dendritic cells from
the blood: hepatic sinusoids–lymph translocation. J. Exp. Med. 185:777–84
147. Pillarisetty VG, Katz SC, Bleier JI, Shah AB, DeMatteo RP. 2005. Natural killer dendritic cells have
both antigen presenting and lytic function and in response to CpG produce IFN-γ via autocrine IL-12.
J. Immunol. 174:2612–18
148. Doherty DG. 2016. Immunity, tolerance and autoimmunity in the liver: a comprehensive review.
J. Autoimmun. 66:60–75
149. Goddard S, Youster J, Morgan E, Adams DH. 2004. Interleukin-10 secretion differentiates dendritic
cells from human liver and skin. Am. J. Pathol. 164:511–19

150. Gao X, Wang S, Fan Y, Bai H, Yang J, Yang X. 2010. CD8+ DC, but Not CD8− DC, isolated from
BCG-infected mice reduces pathological reactions induced by mycobacterial challenge infection. PLOS
ONE 5:e9281
151. Heymann F, Tacke F. 2016. Immunology in the liver—from homeostasis to disease. Nat. Rev. Gastroen-
terol. Hepatol. 13:88–110
152. Chen L, Calomeni E, Wen J, Ozato K, Shen R, Gao JX. 2007. Natural killer dendritic cells are an
intermediate of developing dendritic cells. J. Leukoc. Biol. 81:1422–33
153. Kingham TP, Chaudhry UI, Plitas G, Katz SC, Raab J, DeMatteo RP. 2007. Murine liver plasmacytoid
dendritic cells become potent immunostimulatory cells after Flt-3 ligand expansion. Hepatology 45:445–
54
154. Abo T, Kawamura T, Watanabe H. 2000. Physiological responses of extrathymic T cells in the liver.
Immunol. Rev. 174:135–49
155. Inverso D, Iannacone M. 2016. Spatiotemporal dynamics of effector CD8+ T cell responses within the
liver. J. Leukoc. Biol. 99:51–55
156. Benechet AP, Iannacone M. 2016. Determinants of hepatic effector CD8+ T cell dynamics. J. Hepatol.
66:228–33
157. Wang L, Wang K, Zou ZQ. 2015. Crosstalk between innate and adaptive immunity in hepatitis B virus
infection. World J. Hepatol. 7:2980–91
158. Shuai Z, Leung MW, He X, Zhang W, Yang G, et al. 2016. Adaptive immunity in the liver. Cell. Mol.
Immunol. 13:354–68
159. Zamor PJ, deLemos AS, Russo MW. 2017. Viral hepatitis and hepatocellular carcinoma: etiology and
management. J. Gastrointest. Oncol. 8:229–42
160. Cholankeril G, Patel R, Khurana S, Satapathy SK. 2017. Hepatocellular carcinoma in non-alcoholic
steatohepatitis: current knowledge and implications for management. World J. Hepatol. 9:533–43
161. Balogh J, Victor D III, Asham EH, Burroughs SG, Boktour M, et al. 2016. Hepatocellular carcinoma: a
review. J. Hepatocell. Carcinoma 3:41–53
162. Ghouri YA, Mian I, Rowe JH. 2017. Review of hepatocellular carcinoma: epidemiology, etiology, and
carcinogenesis. J. Carcinog. 16:1
163. Zhang B, Han S, Feng B, Chu X, Chen L, Wang R. 2017. Hepatitis B virus X protein–mediated non-
coding RNA aberrations in the development of human hepatocellular carcinoma. Exp. Mol. Med. 49:e293
164. Jin K, Li T, Sanchez-Duffhues G, Zhou F, Zhang L. 2017. Involvement of inflammation and its related
microRNAs in hepatocellular carcinoma. Oncotarget 8:22145–65
165. Ahmed A, Wong RJ, Harrison SA. 2015. Nonalcoholic fatty liver disease review: diagnosis, treatment,
and outcomes. Clin. Gastroenterol. Hepatol. 13:2062–70
166. van der Poorten D, Milner KL, Hui J, Hodge A, Trenell MI, et al. 2008. Visceral fat: a key mediator of
steatohepatitis in metabolic liver disease. Hepatology 48:449–57
167. Kubes P, Mehal WZ. 2012. Sterile inflammation in the liver. Gastroenterology 143:1158–72
168. Baumert TF, Juhling F, Ono A, Hoshida Y. 2017. Hepatitis C–related hepatocellular carcinoma in the
era of new generation antivirals. BMC Med. 15:52
169. Ponziani FR, Mangiola F, Binda C, Zocco MA, Siciliano M, et al. 2017. Future of liver disease in the era
of direct acting antivirals for the treatment of hepatitis C. World J. Hepatol. 9:352–67
170. Tahmasebi BM, Carloni V. 2017. Tumor microenvironment, a paradigm in hepatocellular carcinoma
progression and therapy. Int. J. Mol. Sci. 18:405
171. Fukuhara H, Ino Y, Todo T. 2016. Oncolytic virus therapy: a new era of cancer treatment at dawn.
Cancer Sci. 107:1373–79
172. Jones RP, Kokudo N, Folprecht G, Mise Y, Unno M, et al. 2016. Colorectal liver metastases: a critical
review of state of the art. Liver Cancer 6:66–71
173. Clark AM, Ma B, Taylor DL, Griffith L, Wells A. 2016. Liver metastases: microenvironments and
ex-vivo models. Exp. Biol. Med. 241:1639–52
174. Vatandoust S, Price TJ, Karapetis CS. 2015. Colorectal cancer: metastases to a single organ. World J.
Gastroenterol. 21:11767–76
175. Itatani Y, Kawada K, Inamoto S, Yamamoto T, Ogawa R, et al. 2016. The role of chemokines in
promoting colorectal cancer invasion/metastasis. Int. J. Mol. Sci. 17:643
9.30 Kubes · Jenne

176. McDonald B, Spicer J, Giannais B, Fallavollita L, Brodt P, Ferri LE. 2009. Systemic inflammation
increases cancer cell adhesion to hepatic sinusoids by neutrophil mediated mechanisms. Int. J. Cancer
125:1298–305
177. Spicer JD, McDonald B, Cools-Lartigue JJ, Chow SC, Giannias B, et al. 2012. Neutrophils promote
liver metastasis via Mac-1-mediated interactions with circulating tumor cells. Cancer Res. 72:3919–27
178. Cools-Lartigue J, Spicer J, McDonald B, Gowing S, Chow S, et al. 2013. Neutrophil extracellular traps
sequester circulating tumor cells and promote metastasis. J. Clin. Investig. 123:3446–58
179. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, et al. 2004. Neutrophil extracellular
traps kill bacteria. Science 303:1532–35
180. Slaba I, Wang J, Kolaczkowska E, McDonald B, Lee WY, Kubes P. 2015. Imaging the dynamic platelet-
neutrophil response in sterile liver injury and repair in mice. Hepatology 62:1593–605
181. Woolbright BL, Jaeschke H. 2017. The impact of sterile inflammation in acute liver injury. J. Clin.
Transl. Res. 3:170–88
182. Kuboki S, Shin T, Huber N, Eismann T, Galloway E, et al. 2008. Hepatocyte signaling through CXC
chemokine receptor-2 is detrimental to liver recovery after ischemia/reperfusion in mice. Hepatology
48:1213–23
183. Kubes P, Payne D, Woodman RC. 2002. Molecular mechanisms of leukocyte recruitment in postischemic
liver microcirculation. Am. J. Physiol. Gastrointest. Liver Physiol. 283:G139–47
184. Jaeschke H, McGill MR, Williams CD. 2013. Pathophysiological relevance of neutrophils in ac-
etaminophen hepatotoxicity. Hepatology 57:419
185. Bertola A, Park O, Gao B. 2013. Chronic plus binge ethanol feeding synergistically induces neutrophil
infiltration and liver injury in mice: a critical role for E-selectin. Hepatology 58:1814–23
186. Gao B, Xu MJ, Bertola A, Wang H, Zhou Z, Liangpunsakul S. 2017. Animal models of alcoholic liver
disease: pathogenesis and clinical relevance. Gene Expr. 17:173–86
187. Zang S, Wang L, Ma X, Zhu G, Zhuang Z, et al. 2015. Neutrophils play a crucial role in the early stage
of nonalcoholic steatohepatitis via neutrophil elastase in mice. Cell Biochem. Biophys. 73:479–87


Immune Responses in The Liver

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Immune Responses in The Liver

Uploaded by

Copyright:

Available Formats

IY36CH09_Kubes ARI 27 December 2017 13:12

Annual Review of Immunology

Immune Responses in the Liver

Annu. Rev. Immunol. 2018. 36:9.1–9.31 Keywords

Review in Advance first posted on

IMMUNE SENTINEL: LIVER ANATOMY AND BLOOD SUPPLY

9.2 Kubes · Jenne

Review in Advance first posted on

Liver sinusoids Hepatic vein

Portal vein Hepatic artery

www.annualreviews.org • Liver Immunity 9.3

Review in Advance first posted on

iNKTcells CD4+ T cells

9.4 Kubes · Jenne

Review in Advance first posted on

www.annualreviews.org • Liver Immunity 9.5

Review in Advance first posted on

by contrast, peritoneal and pleural tissue-resident macrophages proliferated locally in an IL-4-

Pathogen Evasion of Kupffer Cells

9.6 Kubes · Jenne

Review in Advance first posted on

Other Functions of Kupffer Cells

Natural Killer Cells

www.annualreviews.org • Liver Immunity 9.7

Review in Advance first posted on

Invariant Natural Killer T Cells

9.8 Kubes · Jenne

Review in Advance first posted on

www.annualreviews.org • Liver Immunity 9.9

Review in Advance first posted on

RECRUITMENT OF IMMUNE CELLS

9.10 Kubes · Jenne

Review in Advance first posted on

CD44 and Hyaluronan: Signature Adhesion Molecules in the Liver

www.annualreviews.org • Liver Immunity 9.11

Review in Advance first posted on

may reduce degradation or internalization of HA, allowing it to mediate neutrophil adhesion.

Mac-1 and ICAM-1

VLA4 and VAP-1

9.12 Kubes · Jenne

Review in Advance first posted on

www.annualreviews.org • Liver Immunity 9.13

Review in Advance first posted on

Other Forms of Leukocyte Recruitment

ADAPTIVE IMMUNE RESPONSES IN THE LIVER

9.14 Kubes · Jenne

Review in Advance first posted on

Liver Sinusoidal Endothelial Cells

www.annualreviews.org • Liver Immunity 9.15

Review in Advance first posted on

9.16 Kubes · Jenne

Review in Advance first posted on

Kupffer Cells as Antigen-Presenting Cells for Adaptive Immunity

VIRAL INFECTION OF THE LIVER

www.annualreviews.org • Liver Immunity 9.17

Review in Advance first posted on

IMMUNE RESPONSE TO CANCER IN THE LIVER

9.18 Kubes · Jenne

Review in Advance first posted on

www.annualreviews.org • Liver Immunity 9.19

Review in Advance first posted on

colorectal cancer (CRC) metastasis. Although the presence of an immunotolerogenic environment