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Trends Microbiol. Author manuscript; available in PMC 2023 April 01.
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Published in final edited form as:

Trends Microbiol. 2022 April ; 30(4): 390–402. doi:10.1016/j.tim.2021.09.002.

Emerging roles of the complement system in host-pathogen

interactions
Sanjaya K. Sahu1,#, Devesha H. Kulkarni2,#, Ayse N. Ozanturk1, Lina Ma1, Hrishikesh S.
Kulkarni*,1,3
1Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington
University School of Medicine
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2Division of Gastroenterology, Department of Medicine, Washington University School of

Medicine
3Department of Molecular Microbiology, Washington University School of Medicine.

Abstract
The complement system has historically been entertained as a fluid-phase, hepatically-derived
system, which protects the intravascular space from encapsulated bacteria. However, there has
been an increasing appreciation for its role in protection against non-encapsulated pathogens.
Specifically, we have an improved understanding of how pathogens are recognized by specific
complement proteins, as well as how they trigger and evade them. Additionally, we have an
improved understanding of locally-derived complement proteins, many of which promote host
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defense. Moreover, intracellular complement proteins have been identified that facilitate local
protection and barrier function despite pathogen invasion. Our review aims to summarize these
advances in the field, as well as provide an insight into the pathophysiological changes occurring
when the system is dysregulated in infection.

Keywords
complement activation; pneumonia; colitis; innate immunity; autophagy; inflammasomes

Expanding the role of the complement system from a single cell to complex
organisms
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The complement system is an evolutionarily conserved arm of innate immunity, which

forms one of the first lines of host defense against pathogens [1]. Comprising over 60
proteins, the system can be rapidly activated by pathogens via three pathways [the classical
(CP), lectin (LP), or alternative (AP)] to generate enzymes that facilitate cleavage of its

*
Correspondence: hkulkarn@wustl.edu (H.S. Kulkarni).
#contributed equally
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 Sahu et al. Page 2

components, enabling rapid amplification of the cascade on a surface (Key Figure 1, Box
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1) [1]. This cascade results in the release of peptides/proteins that facilitate not only
chemotaxis/vasodilation (e.g., C3a, C5a) but also opsonization (e.g., C3b) and formation
of the membrane attack complex (MAC), which perturbs the cell membrane, and potentiates
lysis and death [2,3]. MAC-mediated bacterial killing requires surface-bound enzymes (i.e.,
convertases) for cleaving C5 locally to form C5b, which attracts components C6 and C7
to anchor the complex to the bacterial cell envelope, and subsequently form MAC pores
with C8 and C9 [4,5]. However, there is an increasing appreciation that the complement
system may have originated and functioned intracellularly [6]. This perspective is based on
both phylogenetic analyses [7], as well as observations in immune and non-immune cells
that complement proteins affect cell survival, inflammation and metabolic reprogramming
[8–11]. Certain complement-like proteins have been reported in the simplest form of
multicellular organisms such as sponges and sea urchins [7]. Moreover, their ability to
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opsonize bacteria has been observed in invertebrates [12]. Thus, an early opsonic (and
subsequently lytic) system appears to have evolved via gene duplications resulting in
complement gene families (e.g., C3/C4/C5, Bf/C2) [7]. Some of these proteins were
secreted, and ‘patrolled’ the interstitial space [13]. The system has thus evolved from simple,
multicellular organisms without tissues or organs, to complex systems, such as in humans, to
facilitate homeostasis and promote recovery — especially in the context of an infection [7].
However, uncontrolled activation of this system may result in tissue damage [14,15]. Hence,
at every step, there is a system of fluid-phase and membrane inhibitors to ensure the system
functions optimally (Box 2). At the same time, pathogens have evolved an ability to subvert
the complement system and persist in the host, thereby propagating injury.

Over the last few years, there have been multiple advances in the understanding of how
the complement system protects the host from pathogens: in the fluid phase, locally at sites
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of infection, as well as in the intracellular space. Moreover, important discoveries have

occurred in terms of distinct interactions between pathogens and components of the cascade
[16,17] and how local complement synthesis modulates infection pathogenesis (Box 3 and
4) [8,9]. Importantly, we now have a better understanding of how dysregulated activation
of the system by pathogens drives a hyperinflammatory response [15,18,19]. In this review,
we aim to account for these discoveries to provide a perspective on how the complement
system can be appropriately harnessed to control infection and mitigate tissue injury. Given
that there are existing reviews on how pathogens evade complement [20,21], this topic will
not be discussed in detail as a part of this review.

Improved understanding of interactions between pathogens and the

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complement system
A variety of components on pathogen surfaces are recognized by pattern recognition
molecules (PRMs) that engage distinct arms of the complement system (Key Figure
1). Bacteria, such as Klebsiella, activate complement via interactions between virulence
factors such as lipopolysaccharide (LPS), O-antigen, outer membrane proteins (OMPs)
and the bacterial capsular polysaccharide (CPS), which contains sugars (e.g., D-mannose,
L-rhamnose) thereby triggering the LP [22]. However, recent epidemiological studies have

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shown that many multidrug-resistant strains of Klebsiella develop CPS modifications,

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thereby impairing LP-mediated CPS recognition. For example, CPS impairs complement
activation via increased thickness or variation in the polysaccharide motifs. Deletion of
genes involved in CPS production such as rfaH and hrtA improves complement-mediated
bacterial recognition and killing [23].

Different components of the LP such as lectins and ficolins serve as PRMs for bacteria,
thereby facilitating protection. Ficolin-2, for example, binds to enteroaggressive Escherichia
coli (EAEC) and enhances serum bactericidal activity [24,25]. More recently, newer PRMs
such as collectins have been identified as playing key roles in recognizing bacteria. For
example, collectin kidney 1 (CL-K1) recognizes and binds to Streptococcus pneumoniae,
thereby activating the LP and mitigating pneumonia severity [26]. C1q/TNF-related protein
6 (CTRP6) has been identified as a PRM that binds to EAEC and Pseudomonas aeruginosa,
facilitates collectin-11 recruitment, and triggers complement activation [27]. Collectin-11
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also binds to Enterococcus fecalis, along with C1q, and activates the LP and CP [28]. Other
PRMs such as pentraxin-3 (PTX3) also promote host defense in the setting of urinary tract
infection [29] and shigellosis [30], in addition to protecting against pneumonia [31], not
by directly activating the LP, but rather by recognizing microbial moieties and interacting
with LP components. For example, PTX3 enhances the innate immune response by
directly binding to bacteria such as Shigella flexneri and Pseudomonas aeruginosa, thereby
enhancing serum bactericidal activity [30]. These observations suggest an increasing number
of LP components can recognize bacterial motifs and promote complement-mediated host
defense. Some of these components have additional protective effects independent of their
ability to engage with the complement cascade [31]. For example, PTX3 enhances the
production of antibodies against microbial capsular polysaccharides through a neutrophil-
regulated pathway [32]. These caveats need to be considered while optimally developing
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therapeutics to control infection and mitigate tissue injury.

Similar events occur in fungi, in whom MAC-mediated lysis is not the predominant method
by which complement controls infection. In the case of Aspergillus fumigatus, certain
components of the conidial cells wall (e.g., RodAp) activate the AP, while β-(1,3)-glucan
and galactomannan activate the CP and LP [33]. Similarly, chitin in the cell wall of
Aspergillus binds to ficolin-1 and activates the LP [34]. Additionally mannose-associated
serine proteases (MASP-1 and -3), which are key enzymes in LP activation, directly bind
to Aspergillus in different growth stages (e.g., conidia, germ tubes, and hyphae), and
recruit MBL and ficolins to activate complement via MASP-2 [35]. A plausible hypothesis
currently put forth is that — similar to pathogens invading the bloodstream — the AP
recognizes components on the conidia, initiating complement activation, and subsequently,
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components of the CP and LP continue to trigger the cascade, which is amplified by the AP
[36].

How viruses interact with the complement system is even more nuanced. Although viruses
also trigger complement activation via the three pathways, they can also interact with
membrane inhibitors, which facilitate cell entry [20]. In many cases, viruses also bind to
fluid-phase inhibitors, allowing them to evade complement deposition [14]. Moreover, some
of these fluid-phase inhibitors interact with viruses and modulate infection without affecting

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complement activation. For example, while it is well-known that influenza A virus (IAV) can
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activate CP, LP and AP [20], both CP and AP are required for efficient direct neutralization
of the H1N1 strain, demonstrating lack of redundancy [37]. Moreover, Factor H, directly
binds to IAV envelope glycoproteins, affects strain-specific IAV infectivity (H1N1 > H3N2),
and also serves as an entry inhibitor for H1N1 [38].

Recently, both convertase-dependent and –independent control of viral infection by

the complement system has been reported. In context of coronavirus infections, MBL
binds to structural proteins (e.g., spike glycoprotein) of SARS-CoV via a critical N-
linked glycosylation site, thereby preventing viral entry [39]. This understandably, led to
investigations on whether complement activation protects against SARS-CoV-2 early on, but
is also responsible for the deleterious consequences of severe SARS-CoV-2 infection (i.e.,
COVID-19) at later time points. It has since been demonstrated that PRMs such as MBL,
ficolin-2 and collectin-11 bind to SARS-CoV-2 structural proteins, inducing LP-mediated
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complement activation [40]. The N protein of SARS-CoV-2 activates complement by

MASP-2-mediated cleavage of C2 [41] by directly binding to MASP-2 [40]. Moreover, the
spike proteins (S1 and S2) bind to heparan sulfate and activate the AP in a virus-free in vitro
system, and this effect appears to be specific to SARS-CoV-2 spike proteins, compared to
the N protein, or the HCoV-OC43 S proteins [42]. At this time, we still need to understand
whether SARS-CoV-2 uses any of the complement proteins as evasion mechanisms, and
why in some patients, complement activation results in a dysregulated immune response in
vivo (see ‘Outstanding Questions’) [15].

In context of adenoviral infections, host-pathogen interactions can be either dependent

or independent of C3 [43]. C1q and C4 mediate antibody-dependent viral neutralization
independent of C2 and C3, but do not affect viral attachment or internalization [44].
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Rather, CP activation results in C4b deposition, which prevents capsid disassembly,

endosomal escape, cytosolic entry and nuclear trafficking. Interestingly, CP-mediated viral
neutralization works synergistically with tripartite motif-containing protein 21 (TRIM21)
to prevent adenoviral infection in vivo. Additionally, interactions of adenoviruses with the
complement system have also provided important insights to better develop vectors both
for vaccines as well as for gene therapy [45]. While a majority of recent studies have
focused on how viruses bind to PRMs, thereby activating complement, it is critical to note
that pathogens have been using certain complement receptors for cell entry. CD46, which
otherwise regulates complement activation, is used by a majority of human adenoviruses for
cellular entry. While many adenoviruses use the knob domain of the fiber capsid protein to
interact with sialic acid-containing glycans on cells, most human adenovirus D species bind
to CD46 using their hexon protein rather than the fiber, a noncanonical mechanism for cell
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entry [46]. Given that CD46 is present on a majority of cells including antigen-presenting
cells, it is thus a potentially novel target for vaccine delivery.

Pathophysiological implications of alterations in the complement cascade

Both genetic and acquired deficiencies of complement components increase the risk of
recurrent bacterial infections [47]. A recent retrospective French cohort study indicated
that complement deficiencies reveal themselves in adults as infectious episodes [48].

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Most of the patients presented before the age of 25 years, although patients in the age
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group of 66-75 years also presented with complement deficiencies. In this study, the most
common complement deficiency was in the common final pathway, with the main clinical
feature being Neisseria meningitidis meningitis. Other infections included pneumonia,
osteo-articular infection, otitis and salpingitis. Nearly 50% of the cohort had sublethal severe
sepsis or septic shock. Deficiencies in functional components of the LP have also long been
considered as predisposing to infection, as many of them recognize pathogen-associated
molecular patterns (PAMPs) on bacteria, fungi and viruses [49]. For example, certain
single nucleotide polymorphisms of the MBL2 gene result in decreased functional MBL
levels, which increases the susceptibility of pneumonia in premature babies and infants
[49]. However, the attributable risk of LP component deficiencies becomes complicated
with increasing age. Part of this discrepancy may be attributable to the redundancy of
LP components with other mechanisms of host defense taking over (e.g., antibodies) as
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well as the ability of pathogens to subvert binding to LP components (e.g., Streptococcus

pneumoniae) [49]. These attributes may explain findings from a recent study that showed
that patients referred for an immunological evaluation did not have a higher prevalence
of LP component deficiencies compared to healthy individuals [50]. On the other hand,
mannose-binding lectin (MBL)-deficient haplotypes have been associated with an increased
risk of post-transplant infection in solid-organ transplant recipients (e.g., liver) [51]. Hence,
whether the therapeutic immunosuppression unmasks LP component deficiencies, thereby
predisposing to recurrent infection, is a question that needs to be answered (see ‘Outstanding
Questions’).

Multiple conditions other than genetic deficiencies also contribute to acquired deficiencies
of complement proteins and increase infection risk. One example are therapies targeting
excessive complement activation [52–54]. Another example are comorbidities, such as
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end-stage liver disease and kidney disease, such as nephrotic syndrome [55,56]. Since
the liver is the primary source of plasma C3 and C4, patients with cirrhosis develop
acquired hypocomplementemia [21,55,57]. These patients are predisposed to infections
with Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli (among other
pathogens), which can result in pneumonia, peritonitis and septicemia [55]. As a result,
acquired complement deficiencies are now recognized as contributing to cirrhosis-associated
immune dysfunction (CAID), which can predispose these patients to infection, and is
associated with a worse prognosis [58]. For example, low circulating C3 and C4 levels
are risk factors for mortality or liver transplantation (independently of liver function) in
these patients [55,57]. Which of the complement pathways are involved in predisposing to
infection is an area of active investigation.
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In the case of Klebsiella pneumoniae (KP) pneumonia, complement-mediated protection

depends on the extent to which the strains can modify their capsule (either through excess
CPS, or changes in polysaccharide content or structure) or the O-antigen of LPS to evade
complement activation [22]. Although multiple cell wall components are recognized by
the LP, protection appears to be primarily AP-mediated [59]. Specifically, C3- and Factor
B-deficient mice had higher splenic bacterial burden after acute pneumonia with both KP2, a
hypervirulent reference KP strain, and KPC5, a clinical isolate with muted virulence that is

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resistant to all tested antibiotics [59]. Both C3- and Factor B-deficient mice also had higher
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pulmonary and systemic inflammation when compared to wildtype mice. Correspondingly,

a large prospective cohort study of critically ill patients identified that increased AP
hemolytic activity (AH50) but not CP hemolytic activity (CH50), was associated with
decreased 30-day mortality, independent of chronic liver disease [59]. Elevated AH50 was
associated with increased levels of Factor B and properdin, a reduced risk of having a
“hyperinflammatory” subphenotype, and fewer bloodstream infections. This may explain
how complement components play a protective role in the context of an acute phase
response to bacterial pneumonia. Additionally, this ability of the AP to reduce bacterial
burden is being investigated as an adjunctive treatment for multidrug-resistant infection [60].

On the other hand, increased complement activation has been implicated in certain diseases
associated with a systemic hyperinflammatory response, such as sepsis [61] and viral
infections [62], including SARS-CoV-2 [15,18]. For example, it may help control viral
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replication early on in the course of influenza [63] but contributes to lung injury at
later time points [64,65]. Dengue virus infection is associated with an increased vascular
permeability and plasma leakage, which has in part been attributed to a dysregulated
complement response [66] as well as the ability of the virus to evade complement activation
[67]. Although the viral proteins directly bind to LP components to both prevent cellular
entry of the virus and facilitate opsonization, secreted components (i.e., non-structural
protein NS1) not only protect the virus from MBL-mediated neutralization, but can also
contribute to endothelial dysfunction [68]. Similar results have also been seen in Zika
virus infection in vitro, wherein an imbalance between in AP components resulted in AP-
mediated complement activation, and cellular injury [14]. Based on such studies, strategies
to induce inhibitors, such as Factor H, to prevent over-activation of complement and reduce
inflammation offer promise (see ‘Outstanding Questions’).
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Experimental models have shown that coronavirus infection augments local

proinflammatory cytokine production and tissue injury via complement activation
[69,70]. Both SARS-CoV- and MERS-CoV-mediated disease can have a wide range of
manifestations in patients, from asymptomatic cases to acute respiratory distress syndrome
(ARDS) and eventual respiratory arrest. Although a detailed description of the role of
complement in COVID-19 has been covered elsewhere [71], we have recently shown that
circulating levels of C5b-9 are higher in those who die from critical illness in the setting
of SARS-CoV-2 infection compared to those who die in the setting of non-SARS-CoV-2
critical illness [15]. Increased activation of the AP as well as the LP have been implicated,
likely due to certain structural proteins of the virus that bind and activate these pathways
[41,42]. These pathways and their effectors have thus opened up avenues for modulating
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complement activation in SARS-CoV-2 infection [18]. Of note, complement activation

resulting in virus-induced multiorgan failure is not new [14,68]. Hence, we need to utilize
refined approaches to appropriately target this pathway and minimize side-effects.

Role of intracellular complement in host defense

An important discovery in the field has been the identification of an intracellular
complement system (termed the ‘complosome’) in both immune and non-immune cells,

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where it is critical for cell function, homeostasis and survival [72]. How these intracellular
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proteins are relevant in host-pathogen interactions is just beginning to be understood.

Investigators have shown that intracellular complement signaling and function is dependent
on cell type and location (Box 3). In myeloid cells, intracellular C3 is induced during
methicillin-resistant Staphylococus aureus (MRSA) ear infection via interactions between
LFA1 and ICAM1 that occur during diapedesis, and ICAM1 is critical for the metabolic
reprogramming occurring in both myeloid cells, as well as CD4+ T cells [73]. Furthermore
thapsigargin, a calcium depleting agent, increased the interaction between intracellular C3
and GRP94 (a hallmark of the unfolded protein response), facilitating their co-secretion
from stressed M2 macrophages [74]. In neutrophils, intracellular C5aR1 is induced by
TSLP in a mouse model of MRSA skin infection, and is required for TSLP-mediated
killing of bacteria, as observed in C5aR1-deficient mice [75], TSLP also increases C5a
secretion from neutrophils, which is unusual given that neutrophils are usually not thought
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to be sources of C5 or C5a, but rather respond to it as an anaphylatoxin. Key questions

arising from this work include how C5 is induced, generated and processed intracellularly
(either directly by infection, or indirectly e.g., primarily via TSLP), and whether C5aR1
engagement in neutrophils is primarily occurring in an autocrine manner.

Moreover, C3 may also act as a sensor for intracellular pathogens, activating cell-intrinsic
immunity when a C3-bound pathogen enters the cells. In case of Francisella tularensis,
coating of bacteria with C3 (resulting in iC3b formation), and the cytoplasmic entry of
bacteria-bearing C3 peptides is required for the induction of macrophage death (independent
of bacterial burden) [76]. In case of adenoviral infection, C3 binds to the capsid/envelope
of Adenovirus type 5 vector (AdV) and activates NF-κB signaling independent of PRMs
but in a MAVS-dependent manner [16]. To confirm the role of surface-bound C3 that
activates intracellular signaling, serum was treated with cobra venom factor (CVF), which
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cleaves and degrades C3. No NF-κB activation occurred when cells were infected with
AdV incubated with CVF-treated serum, but was restored when recombinant C3 protein was
added to C3-deficient serum. Thus, C3 carried by pathogens from serum into the cells is
sensed intracellularly to induce an immune response [16]. This brings up a key question, as
to what is the role of intracellular complement during infection, and whether the signaling is
same or different depending on the location and the source. Moreover, is this any different in
the context of bacterial compared to viral infections (see ‘Outstanding Questions’)?

In this manner, our understanding about the roles of these intracellular complement proteins
is expanding not just in immune cells, but also in non-immune cells. In the context of
bacterial infection, C3 binds to the surface of Listeria monocytogenes during infection of
intestinal epithelial cells and facilitates the clearance of pathogens by inducing autophagy
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through interaction with ATG16L1 (Figure 2) [17]. Absence of C3 inhibited autophagy,

which increased bacterial load in C3-deficient mice, as well as in the intestinal epithelial cell
line when incubated with C3-deficient serum. Shigella flexneri and Salmonella typhimurium
were able to inhibit this C3-ATG16L1 interaction by shedding off the coated C3 on their
surface by omptins, a family of outer membrane proteases that can cleave C3 (PgtE in
Salmonella, IcsP in Shigella), escaping autophagy-mediated growth restriction. Similar
facilitation of autophagy by intracellular C3 has been observed in pancreatic beta cells [11].

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Previously, epithelial cells had been identified as a potential source of complement [1].
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For example, epithelial cells of the airway, intestine and cervix were all shown to produce
complement proteins, which played key roles in interacting with pathogens at the barrier
surfaces [8,17,77]. However, the relevance of this system to epithelial cell function,
and the relevance of epithelial-cell derived complement to host defense remain to be
deciphered. Thus, a major development in understanding of this role has occurred by
investigating it at barrier surfaces, not only within the intestinal epithelium [17,78,79],
but also at the respiratory epithelium [8,16,18,19]. It is now recognized that in the lung
epithelium, intracellular complement activation may either be protective or deleterious,
depending on the type and the time course of the stimuli. Our group demonstrated that
airway epithelial cells synthesize, store and secrete C3 [8]. The secretion is polarized
apically, which is potentially relevant to host defense in diseases such as cystic fibrosis.
We also showed that C3 protects airway epithelial cells from death via internalization
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of C3, which occurs when it conformationally changes to C3(H2O) [10]. On the other
hand, the deleterious consequences of epithelial-cell derived complement are increasingly
appreciated, especially in the context of SARS-CoV-2 infection. Upregulation of certain
complement genes (e.g. C3, CFB) in different airway epithelial cell culture systems, as well
as local C3a production is associated with increased inflammation and poor outcomes in
COVID-19 [18]. CD46, C3aR and IFN-α/β signaling genes closely tracked with disease
severity in Type 1 and Type 2 pneumocytes, which subsequently led to a finding that
STAT1 and RELA strongly bound to the promoter regions of C3 and CFB, thus potentially
regulating their expression. Cell-permeable complement inhibitors or ruxolitinib (a JAK1/2
kinase inhibitor) normalized SARS-CoV-2 driven complement hyperactivation [18]. Thus, it
appears that early host response genes, such as those belonging to the complement system
as well as interferon response genes are upregulated in SARS-CoV-2 infection; and certain
components such as STAT1 and RELA subsequently perpetuate a feedforward loop that
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amplifies inflammation. An intriguing question arises whether intracellular complement

activation directly or indirectly affects NF-κB activation, which would have implications
on the epithelial response to other infections. Parallels are drawn with the gut epithelium,
wherein intracellular complement proteins may be protective in certain instances [17,79], but
their chronic upregulation may be deleterious [78].

On a related note, SARS-CoV-2 induces C3 expression in infected and neighboring primary

human alveolar epithelial (HAE) cells, and SARS-CoV-2 spreads from infected to non-
infected cells via C3-containing nanotubes [19]. MAC formation was detected in SARS-
CoV-2-infected HAEs and was associated with loss of epithelial integrity. Additionally, C5a
in itself disrupted epithelial integrity, suggesting both direct and indirect effects of viral
infection. Antagonism of C3aR and C5aR (presumably C5aR1) reduced viral titers and
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also reduced basolaterally secreted C3a-desArg, and proinflammatory cytokines from HAE
cultures. C5aR (compared to C3aR) antagonism of HAE was also more effective in reducing
infectivity. This work demonstrated that interruption of local complement activation
reduces viral infection and the hyperinflammatory response, and maintains airway epithelial
integrity. Thus, while constitutively active intracellular complement proteins appear to be
necessary for barrier function, how best to interrupt their uncontrolled activation needs to be
better understood.

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Concluding Remarks
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The complement system has a long-standing role in host defense against infection. With an
increasing understanding of local (and intracellular) complement activation, there is now an
increasing appreciation for how this system plays a key part in linking innate and adaptive
immune responses, how it facilitates autocrine and paracrine activation of both immune
and non-immune cells, and how organism-specific interactions with complement proteins
affect the cellular phenotype. Importantly, complement also appears to be patrolling the
intracellular space, in a way conceptually similar to how it has been known to guard the
intravascular space from pathogens. Thus, understanding these intricacies and interactions
will help us to optimally modulate the system (see Outstanding Questions), with the goal of
maintaining and restoring barrier integrity, organ function, and thus promoting homeostasis
in the setting of infectious stressors.
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Acknowledgements
The authors would like to thank Dr. John P. Atkinson for his thoughtful comments on the manuscript, and the
respective agencies that fund related work. H.S.K. is funded by the National Institutes of Health (K08HL148510),
Department of Defense (W81XWH2010143), the Children’s Discovery Institute (PD-FR-2020-867) and the
Rheumatic Diseases Research Resource-Based Center (P30AR073752). D.H.K. is funded by the Crohn’s and
Colitis Foundation (CCF 610605) and the Longer Life Foundation.

GLOSSARY
AH50 screening assay to test functional activity of the alternative
pathway

ATG16L1 Autophagy Related 16 Like 1 is a protein encoded by the

ATG16L1 gene, which interacts with other proteins and
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lipids, thus helping it recognie membranes and activating

autophagolysosome formation.

ARDS acute respiratory distress syndrome, a form of severe acute

lung injury that results in impaired oxygen transport from
the alveolar space into the blood.

CAID cirrhosis-associated immune dysfunction, a condition

associated with both increased systemic inflammation and
immunodeficiency in patients with end-stage liver disease.

CH50 screening assay to test functional activity of the classical

pathway
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CD46 a membrane inhibitor of complement that binds to C3b,

and can also affect intracellular complement signaling.

CVF cobra venom factor, a reagent used to induce a state of

relative C3 deficiency by binding to Factor B, thereby
cleaving and consuming C3.

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GRP94 an HSP90-like endoplasmic reticulum protein that is

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involved in the unfolded protein response (UPR).

HMGB1 High mobility group box 1 protein, which binds to RAGE

(receptor for advanced glycation end products).

ICAM1 intercellular adhesion molecule 1, a transmembrane protein

present on endothelial cells and leukocytes.

LFA1 Lymphocyte function-associated antigen 1, an integrin

present on lymphocytes.

MAC membrane-attack complex, or C5b-9, formed upon

activation of the complement cascade.

MAPK mitogen-activated protein kinase

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MAVS mitochondrial antiviral-signaling protein, located in outer

membranes of organelles such as mitochondria, play an
important role in innate immunity via signal transduction.

NLRP3 NOD-, LRR- and pyrin domain-containing protein 3, an

intracellular sensor that can detect components of microbes
and damaged cells.

PRR pattern recognition receptors, germline-encoded host

sensors that can detect pathogen-associated molecular
patterns (PAMPs) or damage-associated molecular patterns
(DAMPs).
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PTX3 pentraxin-3, a component of the pentraxin superfamily that

can activate the complement cascade via the classical and
lectin pathways.

SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, the virus

responsible for the COVID-19 (coronavirus disease-19)
pandemic.

TSLP thymic stromal lymphoprotein, a cytokine expressed

primarily by non-hematopoietic cells that skews naive T
cells to a Th2 phenotype.
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REFERENCES
1. Kulkarni HS, et al. (2018) The complement system in the airway epithelium: An overlooked host
defense mechanism and therapeutic target? J. Allergy Clin. Immunol 141, 1582–1586.e1 [PubMed:
29339260]
2. Heesterbeek DA, et al. (2019) Bacterial killing by complement requires membrane attack complex
formation via surface-bound C5 convertases. EMBO J. 38, e99852 [PubMed: 30643019]
3. Heesterbeek DAC, et al. (2019) Complement-dependent outer membrane perturbation sensitizes
Gram-negative bacteria to Gram-positive specific antibiotics. Sci. Rep 9, 3074 [PubMed: 30816122]

Trends Microbiol. Author manuscript; available in PMC 2023 April 01.

 Sahu et al. Page 11

4. Menny A, et al. (2018) CryoEM reveals how the complement membrane attack complex ruptures
lipid bilayers. Nat. Commun 9, 5316 [PubMed: 30552328]
Author Manuscript

5. Doorduijn DJ, et al. (2020) Bacterial killing by complement requires direct anchoring of membrane
attack complex precursor C5b-7. PLoS Pathog. 16, e1008606 [PubMed: 32569291]
6. Elvington M, et al. (2016) Evolution of the complement system: from defense of the single cell to
guardian of the intravascular space. Immunol. Rev 274, 9–15 [PubMed: 27782327]
7. Nonaka M (2014) Evolution of the complement system. Subcell. Biochem 80, 31–43 [PubMed:
24798006]
8. Kulkarni HS, et al. (2019) Intracellular C3 Protects Human Airway Epithelial Cells from Stress-
associated Cell Death. Am. J. Respir. Cell Mol. Biol 60, 144–157 [PubMed: 30156437]
9. Arbore G, et al. (2016) T helper 1 immunity requires complement-driven NLRP3 inflammasome
activity in CD4+ T cells. Science 352, aad1210 [PubMed: 27313051]
10. Elvington M, et al. (2017) A C3(H20) recycling pathway is a component of the intracellular
complement system. J. Clin. Invest 127, 970–981 [PubMed: 28192370]
11. King BC, et al. (2019) Complement Component C3 Is Highly Expressed in Human Pancreatic
Islets and Prevents β Cell Death via ATG16L1 Interaction and Autophagy Regulation. Cell Metab.
Author Manuscript

29, 202–210 [PubMed: 30293775]

12. Zhu Y, et al. (2005) The ancient origin of the complement system. EMBO J. 24, 382–394
[PubMed: 15616573]
13. Losada-Barragán M, et al. (2019) Proteomic profiling of splenic interstitial fluid of malnourished
mice infected with Leishmania infantum reveals defects on cell proliferation and pro-inflammatory
response. J. Proteomics 208, 103492 [PubMed: 31434010]
14. Cabezas S, et al. (2018) Dengue Virus Induces Increased Activity of the Complement Alternative
Pathway in Infected Cells. J. Virol 92, e00633–18 [PubMed: 29743365]
15. Ma L, et al. (2021) Increased complement activation is a distinctive feature of severe SARS-CoV-2
infection. Sci. Immunol 6, eabh2259 [PubMed: 34446527]
16. Tam JCH, et al. (2014) Intracellular sensing of complement C3 activates cell autonomous
immunity. Science 345, 1256070 [PubMed: 25190799]
17. Sorbara MT, et al. (2018) Complement C3 Drives Autophagy-Dependent Restriction of Cyto-
invasive Bacteria. Cell Host Microbe 23, 644–652.e5 [PubMed: 29746835]
Author Manuscript

18. Yan B, et al. (2021) SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation. Sci.
Immunol 6, eabg0833 [PubMed: 33827897]
19. Posch W, et al. (2021) C5aR inhibition of non-immune cells suppresses inflammation and
maintains epithelial integrity in SARS-CoV-2-infected primary human airway epithelia. J. Allergy
Clin. Immunol 147: 2083–2097.e6 [PubMed: 33852936]
20. Agrawal P, et al. (2020) The imitation game: a viral strategy to subvert the complement system.
FEBS Lett. 594, 2518–2542 [PubMed: 32506518]
21. Lewis LA and Ram S, (2020) Complement interactions with the pathogenic Neisseriae: clinical
features, deficiency states, and evasion mechanisms. FEBS Lett. 594, 2670–2694 [PubMed:
32058583]
22. Gonzalez-Ferrer S, et al. (2021) Finding Order in the Chaos: Outstanding Questions in Klebsiella
pneumoniae Pathogenesis. Infect. Immun 89, e00693–20 [PubMed: 33558323]
23. Short FL, et al. (2020) Genomic Profiling Reveals Distinct Routes To Complement Resistance in
Klebsiella pneumoniae. Infect. Immun 88, e00043–20 [PubMed: 32513855]
Author Manuscript

24. Man-Kupisinska A, et al. (2018) Interaction of Mannose-Binding Lectin With Lipopolysaccharide

Outer Core Region and Its Biological Consequences. Front. Immunol 9, 1498 [PubMed:
30008719]
25. Adler Sørensen C, et al. (2018) The Lectin Complement Pathway Is Involved in Protection Against
Enteroaggregative Escherichia coli Infection. Front. Immunol 9, 1153 [PubMed: 29896194]
26. Hwang I, et al. (2017) Collectin Kidney 1 Plays an Important Role in Innate Immunity against
Streptococcus pneumoniae Infection. J. Innate Immun 9, 217–228 [PubMed: 28068663]

Trends Microbiol. Author manuscript; available in PMC 2023 April 01.

 Sahu et al. Page 12

27. Kirketerp-Møller N, et al. (2020) C1q/TNF-Related Protein 6 Is a Pattern Recognition Molecule

That Recruits Collectin-11 from the Complement System to Ligands. J. Immunol 204: 1598–1606.
Author Manuscript

[PubMed: 32041782]
28. Ali YM, et al. (2019) Enterococcus faecalis Escapes Complement-Mediated Killing via
Recruitment of Complement Factor H. J. Infect. Dis 220, 1061–1070 [PubMed: 31058287]
29. Jaillon S, et al. (2014) The humoral pattern recognition molecule PTX3 is a key component of
innate immunity against urinary tract infection. Immunity 40, 621–632 [PubMed: 24745336]
30. Ciancarella V, et al. (2018) Role of a fluid-phase PRR in fighting an intracellular pathogen: PTX3
in Shigella infection. PLoS Pathog. 14, e1007469 [PubMed: 30532257]
31. Asgari F, et al. (2021) The Long Pentraxin PTX3 Controls Klebsiella Pneumoniae Severe
Infection. Front. Immunol 12, 666198 [PubMed: 34093560]
32. Chorny A, et al. (2016) The soluble pattern recognition receptor PTX3 links humoral innate
and adaptive immune responses by helping marginal zone B cells. J. Exp. Med 213, 2167–2185
[PubMed: 27621420]
33. Wong SSW, et al. (2020) Differential Interactions of Serum and Bronchoalveolar Lavage Fluid
Complement Proteins with Conidia of Airborne Fungal Pathogen Aspergillus fumigatus. Infect.
Author Manuscript

Immun 88, e00212–20 [PubMed: 32571987]

34. Jensen K, et al. (2017) M-ficolin is present in Aspergillus fumigatus infected lung and modulates
epithelial cell immune responses elicited by fungal cell wall polysaccharides. Virulence 8, 1870–
1879 [PubMed: 28060571]
35. Rosbjerg A, et al. (2021) MASP-1 and MASP-3 Bind Directly to Aspergillus fumigatus and
Promote Complement Activation and Phagocytosis. J. Innate Immun DOI: 10.1159/000514546
36. Parente R, et al. (2020) The complement system in Aspergillus fumigatus infections and its
crosstalk with pentraxins. FEBS Lett. 594, 2480–2501 [PubMed: 31994174]
37. Rattan A, et al. (2017) Synergy between the classical and alternative pathways of complement is
essential for conferring effective protection against the pandemic influenza A(H1N1) 2009 virus
infection. PLoS Pathog. 13, e1006248 [PubMed: 28301559]
38. Murugaiah V, et al. (2020) Complement-Independent Modulation of Influenza A Virus Infection by
Factor H. Front. Immunol 11, 355 [PubMed: 32269562]
39. Zhou Y, et al. (2010) A single asparagine-linked glycosylation site of the severe acute respiratory
Author Manuscript

syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through

multiple mechanisms. J. Virol 84, 8753–8764 [PubMed: 20573835]
40. Ali YM, et al. (2021) Lectin Pathway Mediates Complement Activation by SARS-CoV-2 Proteins.
Front. Immunol 12, 666198 [PubMed: 34093560]
41. Kang S, et al. (2021) A SARS-CoV-2 antibody curbs viral nucleocapsid protein-induced
complement hyperactivation. Nat. Commun 12, 2697 [PubMed: 33976229]
42. Yu J, et al. (2020) Direct activation of the alternative complement pathway by SARS-CoV-2 spike
proteins is blocked by factor D inhibition. Blood 136, 2080–2089 [PubMed: 32877502]
43. Allen RJ and Byrnes AP, (2019) Interaction of adenovirus with antibodies, complement, and
coagulation factors. FEBS Lett. 593, 3449–3460 [PubMed: 31660588]
44. Bottermann M, et al. (2019) Complement C4 Prevents Viral Infection through Capsid Inactivation.
Cell Host Microbe 25, 617–629.e7 [PubMed: 30926239]
45. Gentile CM, et al. (2019) Genetic strategy to decrease complement activation with adenoviral
therapies. PLoS One 14, e0215226 [PubMed: 31026285]
Author Manuscript

46. Persson BD, et al. (2021) Human species D adenovirus hexon capsid protein mediates cell entry
through a direct interaction with CD46. Proc. Natl. Acad. Set U. S. A 118, e2020732118
47. El Sissy C, et al. (2019) Clinical and Genetic Spectrum of a Large Cohort With Total and Sub-total
Complement Deficiencies. Front. Immunol 10, 1936 [PubMed: 31440263]
48. Audemard-Verger A, et al. (2016) Infections Revealing Complement Deficiency in Adults: A
French Nationwide Study Enrolling 41 Patients. Medicine (Baltimore) 95, e3548 [PubMed:
27175654]

Trends Microbiol. Author manuscript; available in PMC 2023 April 01.

 Sahu et al. Page 13

49. Świerzko AS and Cedzyński M, (2020) The Influence of the Lectin Pathway of Complement
Activation on Infections of the Respiratory System. Front. Immunol 11, 585243 [PubMed:
Author Manuscript

33193407]
50. Jørgensen CM, et al. (2019) Pattern Recognition Molecules of the Lectin Pathway-Screening of
Patients with Suspected Immunodeficiency. J. Clin. Immunol 39, 668–677 [PubMed: 31377972]
51. Fernández-Ruiz M, et al. (2019) Impact of MBL2 gene polymorphisms on the risk of infection
in solid organ transplant recipients: A systematic review and meta-analysis. Am. J. Transplant 19,
1072–1085 [PubMed: 30378749]
52. McNamara LA, et al. (2017) High Risk for Invasive Meningococcal Disease Among Patients
Receiving Eculizumab (Soliris) Despite Receipt of Meningococcal Vaccine. Morb. Mortal. Wkly.
Rep 66, 734–737
53. Ladhani SN, et al. (2019) Invasive meningococcal disease in patients with complement
deficiencies: a case series (2008-2017). BMC Infect. Dis 19, 522 [PubMed: 31200658]
54. Crew PE, et al. (2019) Disseminated Gonococcal Infections in Patients Receiving Eculizumab: A
Case Series. Clin. Infect. Dis 69, 596–600 [PubMed: 30418536]
55. Glargaard S, et al. (2018) Prognostic value of lectin pathway molecules and complement proteins
Author Manuscript

in ascitic fluid and blood in patients with liver cirrhosis. Scand. J. Gastroenterol 53, 64–69
[PubMed: 28982257]
56. Zhang H, et al. (2021) Risk Factors for Poor Prognosis of Severe Infection in Children With
Idiopathic Nephrotic Syndrome: A Double-Center, Retrospective Study. Front. Pediatr 9, 656215
[PubMed: 34336733]
57. Li Q, et al. (2020) Lower level of complement component C3 and C3a in the plasma means
poor outcome in the patients with hepatitis B virus related acute-on-chronic liver failure. BMC
Gastroenterol. 20, 106 [PubMed: 32293297]
58. Irvine KM, et al. (2019) Causes and Consequences of Innate Immune Dysfunction in Cirrhosis.
Front. Immunol 10, 293 [PubMed: 30873165]
59. Bain W, et al. (2020) Increased Alternative Complement Pathway Function and Improved Survival
during Critical Illness. Am. J. Respir. Crit. Care Med 202, 230–240 [PubMed: 32374177]
60. Abd El-Aziz AM, et al. (2019) Bacteriophage Therapy Increases Complement-Mediated Lysis of
Bacteria and Enhances Bacterial Clearance After Acute Lung Infection With Multidrug-Resistant
Author Manuscript

Pseudomonas aeruginosa. J. Infect. Dis 219, 1439–1447 [PubMed: 30476337]

61. Fattahi F, et al. (2017) Complement-induced activation of MAPKs and Akt during sepsis: role in
cardiac dysfunction. FASEB J. 31, 4129–4139 [PubMed: 28572445]
62. Pinto AK, et al. (2014) Deficient IFN signaling by myeloid cells leads to MAVS-dependent
virus-induced sepsis. PLoS Pathog. 10, e1004086 [PubMed: 24743949]
63. Zhang K, et al. (2017) The innate immunity of guinea pigs against highly pathogenic avian
influenza virus infection. Oncotarget 8, 30422–30437 [PubMed: 28418930]
64. Song N, et al. (2018) C5a receptor1 inhibition alleviates influenza virus-induced acute lung injury.
Int. Immunopharmacol 59, 12–20 [PubMed: 29621732]
65. Monsalvo AC, et al. (2011) Severe pandemic 2009 H1N1 influenza disease due to pathogenic
immune complexes. Nat. Med 17, 195–199 [PubMed: 21131958]
66. Chan KWK, et al. (2019) A T164S mutation in the dengue virus NS1 protein is associated with
greater disease severity in mice. Sci. Transl. Med 11, eaat7726 [PubMed: 31243154]
67. Thiemmeca S, et al. (2016) Secreted NS1 Protects Dengue Virus from Mannose-Binding Lectin-
Mediated Neutralization. J. Immunol 197, 4053–4065 [PubMed: 27798151]
Author Manuscript

68. Puerta-Guardo H, et al. (2019) Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial
Dysfunction Reflecting Disease Tropism. Cell Rep. 26, 1598–1613.e8 [PubMed: 30726741]
69. Gralinski LE, et al. (2018) Complement Activation Contributes to Severe Acute Respiratory
Syndrome Coronavirus Pathogenesis. mBio 9, e01753–18 [PubMed: 30301856]
70. Jiang Y, et al. (2019) Complement Receptor C5aR1 Inhibition Reduces Pyroptosis in hDPP4-
Transgenic Mice Infected with MERS-CoV. Viruses 11, 39
71. Java A, et al. (2020) The complement system in COVID-19: friend and foe? JCI Insight 5, e140711

Trends Microbiol. Author manuscript; available in PMC 2023 April 01.

 Sahu et al. Page 14

72. Liszewski MK, et al. (2013) Intracellular complement activation sustains T cell homeostasis and
mediates effector differentiation. Immunity 39, 1143–1157 [PubMed: 24315997]
Author Manuscript

73. Kolev M, et al. (2020) Diapedesis-Induced Integrin Signaling via LFA-1 Facilitates Tissue
Immunity by Inducing Intrinsic Complement C3 Expression in Immune Cells. Immunity 52,
513–527.e8 [PubMed: 32187519]
74. Chaumonnot K, et al. (2021) The HSP GRP94 interacts with macrophage intracellular complement
C3 and impacts M2 profile during ER stress. Cell Death Dis. 12, 114 [PubMed: 33483465]
75. West EE, et al. (2016) A TSLP-complement axis mediates neutrophil killing of methicillin-
resistant Staphylococcus aureus. Sci. Immunol 1, eaaf8471 [PubMed: 28783679]
76. Brock SR and Parmely MJ, (2017) Complement C3 as a Prompt for Human Macrophage Death
during Infection with Francisella tularensis Strain SCHU S4. Infect. Immun 85, e00424–17
[PubMed: 28739830]
77. Edwards JL, et al. (2002) A co-operative interaction between Neisseria gonorrhoeae and
complement receptor 3 mediates infection of primary cervical epithelial cells. Cell Microbiol.
4, 571–584 [PubMed: 12390350]
78. Sünderhauf A, et al. (2017) Regulation of epithelial cell expressed C3 in the intestine - Relevance
Author Manuscript

for the pathophysiology of inflammatory bowel disease? Mol. Immunol 90, 227–238 [PubMed:
28843904]
79. Ohno M, et al. (2020) Lipopolysaccharide O structure of adherent and invasive Escherichia coli
regulates intestinal inflammation via complement C3. PLoS Pathog. 16, e1008928 [PubMed:
33027280]
80. Ekdahl KN, et al. (2019) Is generation of C3(H20) necessary for activation of the alternative
pathway in real life? Mol. Immunol 114, 353–361 [PubMed: 31446306]
81. Irmscher S, et al. (2018) Kallikrein Cleaves C3 and Activates Complement. J. Innate Immun 10,
94–105 [PubMed: 29237166]
82. Lausen M, et al. (2020) Opsonophagocytosis of Chlamydia pneumoniae by Human Monocytes and
Neutrophils. Infect. Immun 88, e00087–20 [PubMed: 32284372]
83. Ramlall V, et al. (2020) Immune complement and coagulation dysfunction in adverse outcomes of
SARS-CoV-2 infection. Nat. Med 26, 1609–1615 [PubMed: 32747830]
84. Bastaert F, et al. (2018) Pseudomonas aeruginosa LasB Subverts Alveolar Macrophage Activity by
Author Manuscript

Interfering With Bacterial Killing Through Downregulation of Innate Immune Defense, Reactive
Oxygen Species Generation, and Complement Activation. Front. Immunol 9, 1675 [PubMed:
30083156]
85. Herrmann JB, et al. (2018) Complement C5a Receptor 1 Exacerbates the Pathophysiology of N.
meningitidis Sepsis and Is a Potential Target for Disease Treatment. mBio 9, e01755–17
86. Müller-Redetzky H, et al. (2020) Neutralizing Complement C5a Protects Mice with Pneumococcal
Pulmonary Sepsis. Anesthesiology 132: 795–807 [PubMed: 32101978]
87. Liszewski MK, et al. (2017) Complement Dysregulation and Disease: Insights from Contemporary
Genetics. Annu. Rev. Pathol 12, 25–52 [PubMed: 27959629]
88. Wygrecka M, et al. (2017) Antihistone Properties of C1 Esterase Inhibitor Protect against Lung
Injury. Am. J. Respir. Crit. Care Med 196, 186–199 [PubMed: 28005404]
89. Ozeki T, et al. (2021) C1 inhibitor mitigates peritoneal injury in zymosan-induced peritonitis. Am.
J. Physiol. Renal Physiol 320, F1123–F1132 [PubMed: 33818127]
90. Ermert D, et al. (2019) The hijackers guide to escaping complement: Lessons learned from
pathogens. Mol. Immunol 114, 49–61 [PubMed: 31336249]
Author Manuscript

91. Xu G, et al. (2018) Importance of the Complement Alternative Pathway in Serum Chemotactic
Activity During Sepsis. Shock 50, 435–441 [PubMed: 29040213]
92. Luo L, et al. (2019) Anaphylatoxins Enhance Recruitment of Nonclassical Monocytes via
Chemokines Produced by Pleural Mesothelial Cells in Tuberculous Pleural Effusion. Am. J.
Respir. Cell. Mol. Biol 60, 454–464 [PubMed: 30422670]
93. Shivshankar P, et al. (2020) In response to complement anaphylatoxin peptides C3a and C5a,
human vascular endothelial cells migrate and mediate the activation of B-cells and polarization of
T-cells. FASEB J. 34, 7540–7560 [PubMed: 32301538]

Trends Microbiol. Author manuscript; available in PMC 2023 April 01.

 Sahu et al. Page 15

94. Narni-Mancinelli E, et al. (2017) Complement factor P is a ligand for the natural killer cell-
activating receptor NKp46. Sci. Immunol 2, eaam9628 [PubMed: 28480349]
Author Manuscript

95. Miyabe Y, et al. (2019) Atypical complement receptor C5aR2 transports C5a to initiate neutrophil
adhesion and inflammation. Sci. Immunol 4, eaav5951 [PubMed: 31076525]
96. Mueller-Ortiz SL, et al. (2019) The Second Receptor for C5a, C5aR2, Is Detrimental to Mice
during Systemic Infection with Listeria monocytogenes. J. Immunol 203, 2701–2711 [PubMed:
31597707]
97. Zhang T, et al. (2020) The C5a/C5aR2 axis promotes renal inflammation and tissue damage. JCI
Insight 5, e134081
98. Yu S, et al. (2019) The complement receptor C5aR2 promotes protein kinase R expression and
contributes to NLRP3 inflammasome activation and HMGB1 release from macrophages. J. Biol.
Chem 294, 8384–8394 [PubMed: 30971430]
99. Laumonnier Y, et al. (2017) Novel insights into the expression pattern of anaphylatoxin receptors
in mice and men. Mol. Immunol 89, 44–58 [PubMed: 28600003]
100. Li XX, et al. (2020) C5aR2 Activation Broadly Modulates the Signaling and Function of Primary
Human Macrophages. J. Immunol 205, 1102–1112 [PubMed: 32611725]
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Box 1:
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Assembly, activation and amplification of the complement cascade

The complement cascade can be activated by three pathways – the classical pathway
(CP), the lectin pathway (LP) and the alternative pathway (AP) (Figure 1). The CP is
triggered when C1q, a sensing protein, binds to antigen-antibody complexes. However,
C1q also can trigger the CP independent of antigen-antibody complexes by binding to
pathogen surfaces, C-reactive protein (CRP), pentraxin-3 (PTX3), amyloid fibrils, among
others. Thus, the absence of these triggers may increase the risk of infection. The LP
is activated when certain carbohydrates on pathogen surfaces bind to pattern-recognition
molecules such as mannose-binding lectin (MBL), ficolins and collectins. The AP is
constitutively active with a steady turnover in the circulation (when C3 forms (C3H2O))
[80], but can be triggered by pathogens, when C3(H2O) binds to Factor B and facilitates
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the formation of the AP-convertase. Such convertases cleave proteins central to the
cascade (e.g., C3, C5), thereby amplifying it (Figure 1). Factor D, a serine protease,
facilitates the cleavage of Factor B to Ba and Bb, and thus, activates the AP. However,
there is an increasing appreciation of Factor D-independent AP activation [15,18,81].
Properdin acts as positive regulator of the AP by stabilizing the AP convertase, but can
also initiate the AP, either directly or indirectly.

C3 is cleaved by the C3 convertase to form C3a, which binds to the C3aR (a G-protein
coupled receptor) and C3b. The C3a-C3aR interaction facilitates vasodilation, chemotaxis
of myeloid cells, and cell survival [72]. C3b serves as the major opsonin [82], interacts
with Bb to form the C3 convertase thereby amplifying the AP, and also forms the
C5 convertase (C3bBbC3b). The C5 convertase cleaves C5 to form C5a and C5b.
C5a binds to C5aR1 and C5aR2 [detailed in Box 4]. C5b facilitates the assembly
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of other complement proteins (C6-9) to form the membrane attack complex (MAC),
often needed for killing of bacteria [2]. Cleavage of C5 can be convertase-dependent or
independent; the latter is an example of the crosstalk between the complement, clotting
and coagulation systems [83]. Historically, genetic deficiencies of MAC components
were associated with invasive Neisseria infections, as were acquired defects (i.e.,
treatment with anti-C5 therapies) [52–54]. More recently, data suggest that deficiency of
complement components increase the risk of infection by various bacteria [47,48,55,84].
Importantly, amplification of the complement system is not always protective. Thus, the
system needs to be tightly controlled, both spatially (i.e. right targets) and temporally
(i.e., right time/duration) [85,86].
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Box 2.
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Inhibition of the complement cascade

Although the activation of the complement system is designed to protect the host from
pathogens and facilitate the clearance of debris, dysregulated activation of the system
can result in end-organ dysfunction [87]. Hence, there are a series of fluid-phase and
membrane inhibitors that keep the activation checked (Figure 1). A detailed overview of
these inhibitors is beyond the purview of this review, but has been covered elsewhere
[87]. Briefly, fluid-phase inhibitors include C1 esterase inhibitor (C1INH), Factor H,
Factor I, C4BP (C4-binding protein), and vitronectin. C1INH binds to and irreversibly
inactivates C1r and C1s proteases in the C1 complex of CP, and MASP-1 and MASP-2
proteases in LP. Factor I cleaves C3b and C4b; this requires the participation of cofactors
(discussed below). Factor H is one such cofactor for Factor I-mediated cleavage and
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also accelerates the decay of C3bBb (the AP convertase). C4BP (C4 binding protein)
inhibits both CP and LP activation. It can also bind to C4b, accelerates the decay of
C3-convertase (C4bC2a) and is a cofactor for Factor I to cleave C4b. Vitronectin hinders
the insertion of C5b-7 into the cell membrane, and thus, inhibits the formation of the
lytic MAC pore. It also binds to circulating terminal complement complexes. Membrane
inhibitors include CD46, CR1, CD55 and CD59. Both CD46 and CR1 serve as cofactors
for Factor I-mediated cleavage of C3b and C4b, although CR1 is the only member of the
complement inhibitor family that can generate C3d. CD55 recognizes C3b and C4b and
prevents the formation of the convertase as well as decays the convertase. CD59 prevents
MAC formation by preventing C8 and C9 from inserting into the C5b-7 polymer. Some
of these inhibitors can be harnessed to reduce end-organ dysfunction in experimental
models [88,89]. In certain cases, such as C1INH, the protective effects extend beyond
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complement regulation, as it also inhibits proteases in the fibrinolytic, clotting, and kinin
pathways [88]. However, these inhibitors (especially Factor H) can also be hijacked by
pathogens to subvert complement activation [20,90].
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Box 3.
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Effect of local complement activation on immune cells.

Products generated via the complement cascade modulate the behavior of immune
cells. For example, C3a and C5a are powerful anaphylatoxins. They promote the
chemotaxis of neutrophils and monocytes by binding to C3aR and C5aR1 [91]. In
tuberculous pleural effusion, C3a and C5a stimulated production of IL-1β, IL-17, and
IL-27 in monocytes, and stimulated pleural mesothelial cells to secrete CCL2, CCL7,
and CX3CL1, which recruited CD14+CD16+ monocytes to the pleural cavity [92]. C3a
and C5a-activated endothelial cells promoted the activation of B-lymphoblasts with
increased Fas Ligand (CD95L), CD69, and IL1R1 expression, as well as increased
IL-1α, IL-6 and IL-8 production, and skewed T-lymphocytes towards a Th1 subtype
[93]. Multiple complement receptors (e.g., CR1, CR2) are present on lymphocytes and
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follicular dendritic cells, and bind to cleavage products such as C3b, C3dg and iC3b,
modulating effector functions. Recently, newer roles for some of these ligand-receptor
interactions have been identified, and the roles of some of these receptors have also
been better elucidated, especially in the context of infection. For example, properdin
(a positive regulator of the AP) has been reported as a ligand for the natural killer
cell-activating receptor NKp46, playing a key role in facilitating NKp46+ group 1 ILCs
in protecting against Neisseria meningitidis infection [94].

Not only does complement activation modulate immune cell effector function by
engaging cell surface receptors, but it also does so via its intracellular activation. Cells
produce and internalize C3, which is constitutively cleaved by intracellular proteases
(such as cathepsins) to promote differentiation and survival. For example, in CD4+ T
cells, C3 is processed into active C3a and C3b by cathepsin L [72]. Active C3a bind
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to the C3aR of the same cells in an autocrine manner and helps in T-cell survival by
regulating mTOR phosphorylation [72]. The generation of intracellular C3a promotes
naive CD4 T cells to differentiate to a Th1 effector phenotype [72]. In addition to the
C3a-C3aR axis interacting with mTOR, intracellular C3b activates CD46 and regulates
the metabolic events like glycolysis, oxidative phosphorylation, and amino acid uptake
[72]. CD46 activation also induces C5 expression in CD4+ T cells, which interacts with
C5aR1 to promote NLRP3 inflammasome activation, autocrine IL1β production and Th1
induction [9]. How these intracellular proteins are relevant to infection resilience remains
to be elucidated.
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Box 4.
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Emerging roles of C5aR1 and C5aR2 in infection.

Once the complement cascade is activated and an enzyme (e.g., either a C5 convertase,
or thrombin) is available to cleave C5 to C5a and C5b, the assumption had historically
been that C5a binds to C5aR1 to exert its proinflammatory functions, while C5aR2
was considered a decoy receptor. However, there is an increasing appreciation in the
field that C5aR2 is no longer a decoy receptor. First, experimental models suggest
that C5aR2 expressed on the endothelium is required for neutrophil adhesion, and
facilitates rapid C5a transport into the vasculature [95]. Second, C5aR2 dampens NF-κB-
mediated production of IFN-γ and IL-12p70 from mouse splenocytes in a model of
Listeria monocytogenes infection. This increases splenic and hepatic bacterial burden,
enhances proinflammatory cytokines in the circulation, liver and spleen (i.e., G-CSF,
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IL-6, and MCP-1), worsens liver damage, and increases necrosis and lymphocyte
apoptosis in the spleen [96]. Third, both C5aR1 and C5aR2 contribute to inflammasome
induction and p38-induced MAPK and Akt activation in cardiomyocytes, resulting in
cardiac dysfunction in experimental peritoneal sepsis [61]. Fourth, C5aR2 also increases
renal inflammation and tubular damage in models of uropathogenic Escherichia coli
infection via increased HMGB1 release and inflammasome activation [97]. These in vivo
findings are paralleled in vitro, wherein in murine macrophage cultures, the C5a-C5aR2
interaction facilitates HMGB1 release and NLRP3 activation [97,98]. However, there are
inherent differences between mice and humans in the downstream effects of C5aR1 and
C5aR2 engagement [99]. For example, selective C5aR2 activation in human macrophages
was found to dampen C5aR1-and C3aR-induced ERK signaling, as well as downregulate
cytokine production induced by various Toll-like receptors, C-type lectin receptors, and
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the cytosolic DNA sensor stimulator of IFN genes (STING) [100]. Similarly, in human
CD4+ T cells, NLRP3 assembly and inflammasome activation is negatively regulated
by surface-expressed C5aR2 [9]. Thus, a better understanding of how the interactions
between C5a, C5aR1 and C5aR2 affect host-pathogen interactions will help advance our
field, given that drugs are actively being designed and used to inhibit C5 (or its cognate
receptors) in the setting of severe infection (see ‘Outstanding Questions’).
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HIGHLIGHTS
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• Novel pattern recognition molecules trigger the complement cascade, and in

certain cases, may downregulate components of the system with the goal of
evading the host immune response.

• Genetic and acquired deficiencies of certain complement components are

associated with a higher risk of bacterial infections.

• Extrahepatic complement proteins modulate host defense especially in the

context of intracellular bacteria.

• Intracellular complement proteins, derived from both biosynthesis and uptake,

play critical roles in cellular survival, pathogen burden, and effector function.

• Structural proteins on the surface of viruses such as SARS-CoV-2

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contribute to increased complement activation, which is associated with a

hyperinflammatory response and worse outcomes.
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OUTSTANDING QUESTIONS
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Improved understanding of interactions between pathogens and the complement

system
• How can the knowledge regarding the structural basis for complement evasion
by bacteria be utilized to overcome multidrug-resistant infection?

• Are SARS-CoV-2 variants able to evade the complement system?

• Is excessive complement activation in severe SARS-CoV-2 infection a

consequence of the virus interacting with complement proteins?

Pathophysiological implications of alterations in the complement cascade

• How do we best incorporate complement analytes into prognostic factors for

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critically ill patients with infection (e.g., pneumonia, sepsis)?

• Should haplotyping of complement activation be considered in assessing risk

of post-transplant infections?

• Can the acquired downregulation of complement inhibitors be overcome

to reduce end-organ dysfunction induced by emerging viruses and other
pathogens?

Effect of local complement activation on immune cells

• Are the effects of locally-derived complement proteins at barrier surfaces

distinct from that of circulating complement proteins?

• How can experimental models of C5aR1 and C5aR2 modulation accurately

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represent their effects in humans?

• How can the balance between C5aR1 and C5aR2 be optimally targeted to
enhance effector functions of immune cells, while minimizing tissue injury?

Role of intracellular complement in host defense

• How does intracellular C3a and C5a generation and function differ in each
cell type?

• Which in vivo model systems accurately reflect the role of CD46 in key
cellular processes, especially in response to infection?

• Is intracellular complement signaling the same or different depending on

its subcellular location and its source? Moreover, are there key differences
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pertinent to the intracellular complement system in the context of bacterial

versus viral infections?

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Key Figure 1. The complement cascade.

The complement cascade is a family of over 60 proteins, which are activated via three
pathways, the classical pathway (CP), lectin pathway (LP) and alternative pathway (AP)
via assembly of different proteins. Each of these three pathways are triggered by certain
components on the surface of pathogens, which facilitate assembly of molecules, that
subsequently get cleaved by proteases. The proteases for each pathway are highlighted
in green. In certain cases, a component can activate more than one pathway [e.g.
pentraxin-3 (PTX3), lipopolysaccharide (LPS)]. These three pathways converge to form the
C3 convertase (C4bC2a for CP and LP; C3bBb for AP), which cleaves C3 to C3a and C3b.
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Both C3b and C4b covalently bind to a target. C3b can also bind to Factor B to subsequently
form the AP convertase, thereby amplifying the cascade. Subsequently, C3b also contributes
to the formation of a C5 convertase (C4bC2aC3b for CP and LP; C3bBbC3b for AP), which
cleaves C5 to C5a and C5b. However, convertase-independent C5 cleavage can also occur
in setting of acute inflammation via proteases such as thrombin (not shown). C5b interacts
with C6, C7, C8 and C9 to form the membrane attack complex (MAC), which perturbs
cell membranes, thereby promoting cell lysis. At each step, there are membrane- and
fluid-phase inhibitors that prevent the uncontrolled activation of the cascade. Abbreviations:

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 Sahu et al. Page 23

Ab: antibody; C4BP: C4-binding protein; CPS: capsular polysaccharide; CRP: C-reactive
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protein; MASP: mannose-binding protein-associated serine protease; PTX3: pentraxin-3.

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Figure 2. Differences in intracellular complement signaling in setting of bacterial and viral

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infection.
C3 binds to bacteria such as Listeria, Salmonella and Shigella. Upon intracellular
entry, C3 interacts with ATG16L1, and the bacteria are targeted for autophagy, initially
via formation of an autophagosome, followed by autophagolysosomal processing. In
comparison, Salmonella and Shigella express omptins, which can cleave C3 form the
surfaces of these bacteria. As a result, they escape autophagy and persist in the cell. Viruses
are also coated with C3, and are brought into the cell and packaged into endosomes. Sensing
of coated-C3 targets them for proteasomal-mediated degradation as well as engagement with

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 Sahu et al. Page 25

mitochondrial antiviral signaling (MAVS) adaptor proteins, which promote proinflammatory

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cytokine production, thereby restricting viral replication. However, if viruses have 3C

proteases that can cleave C3 (e.g., enteroviruses), they can escape these innate immune
responses. Created with BioRender.com.
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Trends Microbiol. Author manuscript; available in PMC 2023 April 01.