Furler 2019

AIDS Research and Human Retroviruses
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© Mary Ann Liebert, Inc.
DOI: 10.1089/AID.2019.0156
1
Running Title: Histoarchitectural Deterioration of Lymphoid
Tissues in HIV-1 Infection and in Aging
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.
Robert L. Furler1,*, Kevin L. Newcombe1, Perla del Rio2, Gustavo Reyes-Terán2, Christel H.
Uittenbogaart3, and Douglas F. Nixon1
1 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, 413

E 69th St., Belfer Research Building, New York, NY 10021, USA.
Histoarchitectural Deterioration of Lymphoid Tissues in HIV-1 Infection and in Aging (DOI: 10.1089/AID.2019.0156)
2 Departmento de Investigación en Enfermedades Infecciosas, Instituto Nacional de

Downloaded by Karolinska Institutet University Library from www.liebertpub.com at 09/07/19. For personal use only.
Enfermedades Respiratorias “Ismael Cosío Villegas”, CDMX, Mexico.

3 Departments of Microbiology, Immunology and Molecular Genetics, Medicine,
Pediatrics, UCLA AIDS Institute and the Jonsson Comprehensive Cancer Center,
University of California, Los Angeles, 615 Charles E. Young Drive South, BSRB2, Los
Angeles, CA 90095.
* Correspondence: (p) 646-962-2478; rlf2001@med.cornell.edu
Manuscript keywords: HIV-1, Aging, Lymph Node, Thymus, Bone Marrow, Extracellular
Matrix
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Abstract
Impaired immunity is a common symptom of aging and advanced Human

Immunodeficiency Virus type 1 (HIV-1) disease. In both diseases, a decline in lymphocytic

function and cellularity leads to ineffective adaptive immune responses to opportunistic
infections and vaccinations. Furthermore, despite sustained myeloid cellularity there is a
background of chronic immune activation and a decrease in innate immune function in
aging. In HIV-1 disease, myeloid cellularity is often more skewed than in normal aging, but
similar chronic activation and innate immune dysfunction typically arise. Similarities
between aging and HIV-1 infection have led to several investigations into HIV-1 mediated
aging of the immune system. In this article, we review various studies that report
alterations of leukocyte number and function during aging and compare those alterations
to those observed during progressive HIV-1 disease. We pay particular attention to
changes within lymphoid tissue microenvironments and how histoarchitectural changes

seen in these two diseases affect immunity. As we review various immune compartments
including peripheral blood as well as primary and secondary lymphoid organs, common
themes arise that help explain the decline of immunity in the elderly and in HIV-1 infected
individuals with advanced disease. In both conditions lymphoid tissues often show signs of
histoarchitectural deterioration through fat accumulation and/or fibrosis. These structural
changes can be attributed to a loss of communication between leukocytes and the
surrounding stromal cells that produce the extracellular matrix components and growth
factors necessary for cell migration, cell proliferation, and lymphoid tissue function.
Despite the common general impairment of immunity in aging and HIV-1 disease,
deterioration of immunity is caused by distinct mechanisms at the cellular and tissue levels
in these two diseases.
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Leukocyte cellularity and function is drastically altered throughout the normal aging
process. The developing immune system found in the very young and the declining
immune system in the elderly lack focused functionality and thus leave these populations
vulnerable to infections and cancers not typically found in individuals between extremes of
age. In addition to aging, immune dysfunction can be caused by radiation, chemotherapy,
or through infectious agents like Human Immunodeficiency Virus type 1 (HIV-1).
Similarities of immune impairment caused by HIV-1 and aging were immediately evident at
the beginning of the epidemic when opportunistic infections normally found in the elderly
were being reported in younger adults.
Does the degeneration of effective immunity seen in aging and advanced HIV-1
disease have the same root cause? There is intense interest in this question, especially
with an increasing population of aging HIV-1 positive individuals. Answering this question
is difficult since we still do not have a clear mechanistic understanding of the natural aging
process. However, anatomical and physiological signs and symptoms of aging are
numerous, including bone and muscle atrophy, cardiovascular disease, and susceptibility
to infection. At the cellular and tissue level, aging has been attributed to intracellular
changes including metabolic alterations, DNA damage, and telomere attrition.
Extracellular characteristics of tissue including changes in extracellular matrix composition
and organization, tissue hardening, and fibrosis are also often seen during aging. In this
article, we will compare cellular properties within the peripheral blood of aging or HIV-1
infected individuals and then shift our focus to changes in the extracellular
microenvironments of lymphoid compartments that occur during aging and HIV-1 disease.
Are there common cellular and histoarchitectural changes that occur in the immune
compartments during aging and HIV-1 infection that influence the diminished immunity
seen in both?
Although both aging and HIV-1 disease cause dysregulated innate and adaptive
immune responses, these two diseases have distinct mechanisms for degenerative
immunity. Myeloid and lymphoid cell population numbers and function are altered
differently in aging and progressive HIV-1 disease. These differences at the cellular and
tissue levels that both lead to impaired immunity still share a key similarity. Depletion of
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one or more subsets of leukocytes causes a disruption of a delicate relationship between
leukocytes and surrounding stromal cells within lymphoid compartments. These
populations provide growth factors and vital survival cues to each other that help build the
tissue architecture required to build an effective immune response. Restoring the
interdependent relationship between leukocytes and stromal cells may reverse the
impairment of immunity in aging and in HIV-1 disease.
Aging and HIV-1 Mediated Alterations of the Peripheral Blood

A precipitous decline in absolute numbers of peripheral blood lymphocytes,

decreased myeloid cell function, chronic immune activation, and pro-inflammatory
cytokine production are common manifestations in both aging and HIV-1 disease. In aging,
there is a dramatic decline in peripheral blood B cells, a gradual decline in naïve
lymphocytes, and an increasingly dysfunctional innate immune system. In HIV-1 disease,
CD4+ T cell depletion leads to abnormal activation and dysfunction of innate and adaptive
immunity. Differences between peripheral blood myeloid and lymphoid populations in
aging and HIV-1 infection are discussed below.
Aging has a profound effect on both innate and adaptive immune function;
however, the decreases in absolute number of peripheral blood subsets are predominantly
found in lymphocyte populations. As an individual ages, the absolute numbers of
circulating neutrophils and other myeloid cells do not change significantly1; however, they
tend to produce increased levels of pro-inflammatory cytokines and are impaired in
effector functions like phagocytosis. This decline in innate immunity leads to increased
opportunistic infection in the elderly1-3. Hearps et al. investigated the peripheral blood of
146 healthy adults and saw an age-related alteration in monocyte phenotype and function.
Aged monocytes were less capable of phagocytosis, produced more pro-inflammatory
cytokines like TNF-, and also had shortened telomeres4. In contrast to myeloid subsets,
lymphocyte numbers declined greatly during the aging process. Valiathan et al. measured
the percentage and absolute number of peripheral blood lymphocyte subsets of 191
individuals separated into five different groups based on age, ranging from infants within
their first year of life to the elderly, defined as being 70 to 92 years of age in their study5.
They reported the age-related decrease in peripheral blood lymphocytes was primarily due
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to drastic decreases in CD19+ B cell numbers and to a lesser extent in CD4+ and CD8+ T
cells. The absolute count of B cells dropped from its peak of 1,375+/-141 cells/mm3 in
childhood to 198+/-34 cells/mm3 in the elderly. Decreased lymphocyte number and

function corresponded to reduced responsiveness to vaccination in the elderly.
Although aging and HIV-1 disease both lead to a decline in immune function, there
are differences in both peripheral blood myeloid and lymphoid cell alterations. In
advanced HIV-1 disease various hematological abnormalities occur which can affect
myeloid subsets. Parinitha and Kulkarni6 analyzed blood samples from 250 HIV-1 infected
individuals and saw anemia in 91.4%, leukopenia in 26.8%, lymphopenia in 80%, and
thrombocytopenia in 21.7% of individuals with CD4+ T cell counts less than 200 cells/mm3.
Granulocytopenia and decline in myeloid dendritic cells (mDC, CD11c+) and plasmacytoid
dendritic cells (pDC, CD11c-) occurred when CD4+ T cell counts declined below 200
cells/mm3 and HIV-1 viral loads increased7. The average peripheral blood mDC counts
dropped from 6978 cells/mL to 2298/mL and pDC counts decreased from 9299 cells/mL to
1640 cells/mL between uninfected controls and individuals with viral loads greater than
105 HIV-1 RNA copies/mL7. Following ART, mDC numbers returned to baseline; however,
the pDC number did not return to baseline and their function remained suppressed8.
Unlike in aging, there is a profound decline of peripheral blood myeloid cells in advanced
HIV-1 disease.
Similar to aging, lymphocyte subsets are significantly altered in HIV-1 disease.

However, there are differences in which lymphocyte subsets are most affected. Although
CD4+ T cell loss is most profound in HIV-1 infection, other lymphocyte subsets are
functionally altered. Kalayjian et al. investigated common T cell correlates of HIV-1
infection and aging9. They measured proliferative capacity, exhaustion markers, and
functionality of CD4+ and CD8+ T cells in two adult age groups (≤ 30 years old and ≥45
years old). Naïve CD8+ T cells lost CD28 expression with increasing age. Although they saw
decreased T cell proliferative capacity, increased apoptosis, and decreased delayed-type
hypersensitivity responses in the HIV-1 infected groups, they were not able to associate
these with age. Gianesin found reduced T-cell Receptor Excision Circles (TRECs) and
CD8+CD45RA+CD31+ recent thymic emigrants in the peripheral blood of the HIV-1 infected
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children compared to age-matched controls. Furthermore, they found that higher
percentages of CD8+ T cells in the HIV-1 infected group express markers for senescence
(CD28-CD57+), activation (CD38+HLA-DR+), and exhaustion (PD1+) compared to uninfected

controls10. Cao et al. stratified HIV-1 infected individuals based on disease progression
rate and reported that fast disease progression was associated with increased loss of CD28
on all T cells, a selective decrease in CD31 on CD4+ T cells, and an increase of CD57 on CD8+
T cells11. In contrast, Lee et al. found that although there was an increase in CD8+CD28-
cells, there was only an increase in CD8+CD57+ cells in aging, not during HIV-1 infection12.
Antiretroviral therapy is reported to restore CD45RA+CD31+ T cells to age appropriate

levels; however, the CD45RA+CD31- cells remain at decreased levels13. It is the
CD45RA+CD31- cell population that is thought to maintain the numbers of naïve CD4+ T
cells during aging without HIV infection14. A decrease in naïve T cells is one common
outcome between aging and HIV-1 disease; however, the ablation of the CD4+ T cell subset
is more attributable to HIV-1 infection.
Since the beginning of the HIV-1 epidemic it was known that B cell number and
functionality drastically change in HIV-1 infection. An historical perspective on B cell
dysfunction in HIV-1 infection in the presence or absence of ART is well summarized by
Moir and Fauci15. Similar to aging, chronic HIV-1 infection leads to decreased peripheral B
cell number and functional impairment of class-switch recombination16. HIV-1 positive
individuals have reduced antibody titers to previously exposed antigens and have impaired
development of robust protective humoral immunity following vaccination compared to
HIV-1 negative age-matched controls17.
Aging and HIV-1 infection both lead to impaired immunity, but there are
differences in leukocyte subset depletion. Despite these differences, immune impairment
in both conditions may be linked to chronic inflammation and leukocyte telomere attrition.
Similar to aging, HIV-1 infection leads to decreasing numbers of naïve lymphocytes and
chronic inflammation along with increased production of proinflammatory cytokines like
TNF-IFN-γ, CXCL10, soluble CD163, soluble CD14, neopterin, and CD16+ monocytes18, 19.
Telomere shortening is also seen in both aging and HIV-1 infection13, 20. In HIV-1 disease
the greatest period of PBMC telomere shortening occurs during acute infection21.
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Gianesin et al. assessed the impact of HIV-1 infection on accelerated aging in children and
reported shortened telomere length within PBMC of 71 HIV-1 infected children compared
to 65 HIV-exposed uninfected children, and 56 HIV negative children. Their findings

suggest that ART may dampen this shortening effect10. Telomere length was also
measured in adult PBMC, CD4+ T cells, and CD8+ T cells to assess the effect of HIV-1
infection on aging. Bestilny et al. reported at least a five-fold increase in cellular aging due
to HIV-1 infection, as measured by telomere decrease in the HIV-1 infected cohort. They
indicate that a 37 year-old HIV-1 infected individual has similar telomere length as an
uninfected 75 year-old21. T cells, especially CD8+ T cells, are most profoundly affected by
telomere shortening in HIV-1 infection20, 21. Telomere shortening rate is significant in the
elderly, but not nearly as profoundly affected as those with late-stage HIV-1 infection21.
In summary, HIV-1 infection and normal aging both cause functional declines in
adaptive and innate immunity. There is a large decrease in functional B cells in the
periphery and a decline in circulating naïve lymphocytes. Myeloid cell function and innate
immunity decrease as persistent chronic inflammation occurs. Despite these similarities,
there are differences in circulating leukocyte subsets in normal aging and HIV-1 infection.
In aging, there is a drastic reduction of peripheral B cells whereas HIV-1 infection severely
reduces the circulating CD4+ T cell numbers. As for myeloid subsets, HIV-1 infection affects
the numbers of circulating myeloid cells more than the normal aging process. Normal
aging of the immune system and HIV-1 infection both impair immunity, but not by the
same mechanism. As only a small number of lymphoid cells are present in the peripheral
blood while the majority of lymphoid cells are located in lymphoid tissues such as lymph
nodes and mucosal associated lymphoid tissues (MALT), we now shift our focus from
circulating leukocyte composition to age and HIV-1 mediated alterations of lymphoid
tissue cellularity and histoarchitecture.
Aging and HIV-1 Mediated Alterations of the Bone Marrow

The bone marrow is a critical developmental niche for CD34+ hematopoietic stem
cells (HSC) that give rise to adult blood including the myeloid and lymphoid cells required
for innate and adaptive immunity1, 2, 22-25
. In the adult human, hematopoiesis occurs
primarily in the red bone marrow of flat and irregular bones, but it is known that the
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progenies of the HSC are skewed in both aging and HIV-1 disease. How is the bone
marrow affected during the aging process and does HIV-1 infection mimic these age-
related changes?
Fetal hematopoiesis begins in the liver and then migrates to the bone marrow,
where the majority of adult hematopoiesis occurs26. In aging humans, the leukocyte
progenitor cell composition within the bone marrow changes over time. Similar to the
peripheral blood subsets, bone marrow myeloid lineage cells retain their production levels
while common lymphoid and early B cell progenitors decline in number22, 27. The aged
bone marrow niche has been linked to peripheral myeloid lineage skewing and
phenotypically different myeloid neoplasias seen in the elderly28, 29.
Bone marrow alterations in HIV-1 disease also mirror the various conditions seen in
the peripheral blood but are more pronounced than those seen in normal aging. Several
early studies reported a variety of hematological abnormalities in the bone marrow of HIV-
1 infected individuals. A report of bone marrow abnormalities in 160 HIV-1 infected
individuals showed that of the 107 individuals who were diagnosed with AIDS, 93.12%
were anemic. The most significant difference between the AIDS and non-AIDS groups was
evidence of myeloid dysplasia, primarily in granulocytes30. Tripathi et al. reported that
erythroid dysplasia was found in the bone marrow of over 50% of HIV infected individuals
and one-third of them had abnormal granulocytic and megakaryocytic development and
elevated levels of plasma cells31, 32
. Another analysis of 102 individual bone marrow
aspirates showed indications of hypercellularity, dysplasia, plasmacytosis, and lymphoid
infiltrates in AIDS individuals, which correlated with anemia and granulocytopenia33. Sun
et al. examined bone marrow biopsies of 20 individuals diagnosed with AIDS and 39
individuals with asymptomatic HIV-1 infection. Anemia and leukopenia were associated
more with an AIDS diagnosis while thrombocytopenia was more common in the group
with less advanced disease34. Delacrétaz et al. examined bone marrow biopsies of 18 HIV-
1 infected individuals between December 1981 and December 198635. Although various
bone abnormalities were found, 17 out of 18 individuals had myelodysplasia including
dysmegakaryocytopoiesis (n=16), dyserythropoiesis (n=15), and dysgranulopoiesis (n=6).
Nearly half of the biopsies showed signs of lymphocytic clusters that appeared to be B cells
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while over half of the specimens had increased macrophages, a known target for HIV-1
infection.
Are the various bone marrow alterations seen in advanced HIV-1 disease due to
direct infection? HIV-1 infection inhibited in vitro hematopoiesis from bone marrow
histocultures36; however, there is ongoing debate whether CD34+ HSC can be infected by
HIV-137-40. Other cells within the bone marrow, including CD4+ monocyte precursors may
harbor HIV-1. Sun et al. detected HIV-1 nucleic acids via a cDNA probe in various cells
including mononucleated lymphocytes and histiocytes. However endothelial cells,

interdigitating reticulum cells, nucleated red blood cells, and immature myeloid cells were
also reported to possibly contain HIV-1 nucleic acids34. Infection of these monocyte
precursors or accessory stromal cells may alter important leukocyte to stromal cell
communication and skew the myeloid compartment function during chronic HIV-1
infection41, 42. Aside from immediate cytopathic effects of HIV-1, bone marrow fibrosis is
also seen in AIDS and other diseases which may contribute to dysfunctional
hematopoiesis.
The bone marrow has an optimized microarchitecture of stiff bony trabeculae,

mesenchymal cells, adipocytes, vasculature, and growth factors for the homeostatic
balance of the HSC population and continuous production of red and white blood cells43-45.
The stromal cells produce several extracellular matrix (ECM) proteins including collagens
type III and IV, fibronectin, hyaluronan, laminin, and tenascin, each with their own role in
supporting resident cell functions22, 43, 44
. The ECM provides a necessary scaffold for
attachment, migration, and acts as a depot for survival and growth factors (Figure 1).
Irregular production or organization of the ECM can lead to niche dysfunction and
impaired hematopoiesis. Overgrowth of certain types of ECM fibers including collagen
type I within a tissue is termed fibrosis and is often a marker for disease. How do age and
HIV-1 infection related fibrosis affect hematopoiesis in the bone marrow niche?
Kuter et al. associated two different types of bone marrow fibrosis, reticulin and
collagenous, with severity of disease46. Reticulin fibrosis is composed of
glycosaminoglycans along with individual or loose fibrils of collagen III surrounding a core
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of collagen I. Reticulin fibrosis appeared to be reversible and often seen in nonmalignant
diseases and to a lesser severity in a majority of healthy controls46. On the other hand,
collagen fibrosis in the bone marrow was shown to be linked to more severe irreversible
disease conditions. Kuter et al. defined collagen fibrosis as an abundance of collagen I
within the bone marrow. Collagen I has a thicker fiber diameter than reticulin fibrosis and
increased collagen density may impede cell migration, communication, and tissue
function46.
Severe bone marrow collagen fibrosis is not a typical phenotype of the normal
aging or HIV-1 infected bone marrow46. However, O’Malley et al. reported moderate to
severe reticulin fibrosis within the bone marrow of the majority 35 HIV-1 infected
individuals in their study47. As in previous studies, the reticulin fibrosis was associated
with low-affinity nerve growth factor receptor positive adventitial reticular stromal cells
(ARC)46, 47, and ARC quantities inversely correlated with peripheral CD4+ T cells counts.
Despite this correlation, the authors did not find a significant correlation between the CD4+
T cells count and the observed degree of bone marrow fibrosis47. Delacrétaz et al. also
found mild bone marrow reticulin fibrosis in 15 of 18 individuals in their study35. In seven
of the individuals, they found niche deterioration including hypoplasia, fat atrophy, and
gelatinous infiltration. The ECM production of both types of fibrosis is associated with
TGF-1 signaling46 in bone marrow stromal cells including ARC, perisinusoidal adventitial
cells, periarterial adventitial cells, endosteal cells, and adipocytes. Disruption of stromal
cell function and ECM production by alteration of leukocyte numbers and phenotype may
be the underlying common cause of histoarchitectural changes found in the aging and HIV-
1 infected bone marrow.
In both aging and HIV-1 infection, the bone marrow compartment mirrored the
peripheral blood leukocyte alterations; however, aging and HIV-1 infection differed in
specific subset alterations. Although bone marrow lymphoid progenitors were the primary
subset of cells that declined greatly in normal aging, multiple myeloid and lymphoid
subsets are affected in progressive HIV-1 disease. While reticulin fibrosis was seen in both
conditions, this was reported to be reversible compared to the collagenous fibrosis seen in
bone marrows of leukemias and other hyperproliferative disorders. Aging and HIV-1
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infection both skew hematopoiesis in the bone marrow; however, advanced HIV-1
infection causes an increased number of leukocyte abnormalities in the bone marrow
compared to the normal aging process.
Aging and HIV-1 Mediated Alterations of the Thymus

The thymus is the primary site of normal T cell development during fetal and
neonatal life. A human thymic rudiment with T cell precursors is present as early as 7-8
weeks of gestation48 and a fully formed thymus consists of several lobules with cortical and
medullary areas which contain developing thymocytes, epithelial cells, macrophages,

dendritic cells and other hematopoietic cells including B cells. T cell progenitor migration
into the thymus, intrathymic T cell development and emigration of mature naïve T cells to
the periphery are all involved in maintenance of cellular immunity49.
Several murine studies suggest there is an interdependent relationship between

the thymic stroma and the developing thymocytes, each providing vital cytokines. Cortical
and medullary thymic epithelial cells (TECs) are of endodermal origin and differ in their
functions and expression of cell surface markers. Recent data show that cortical as well as
medullary epithelial cells are heterogeneous50. The heterogeneity of the various epithelial
subpopulations provides the proper developmental cues for functional and self-tolerant T
cells50. It is important to note that TECs are essential for T cell development and
reciprocally, developing thymocytes are critical for the maintenance of a TECs and a
functioning thymus51.
T cell precursors from fetal liver or bone marrow adhere to the thymic vascular
epithelium and cross the perivascular epithelial cell layer to enter the thymus52, 53
.
Integrins such as VLA-4/α4β1 and VLA-5/α5β1 are expressed on thymocytes and used to
adhere to and migrate along thymic epithelium and fibronectin ECM54, 55. The developing
thymocytes are guided by chemokine gradients produced by the thymic stroma56-58,
including TEC-produced CXCL1259, 60. As the thymocytes migrate throughout the cortex
and medulla, they are provided pro-survival cues from cytokines like Interleukin-7 (IL-7),
which is produced by the thymic stroma61. IL-7 administration has been proposed as a
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potential therapeutic approach to thymus reconstitution following aging and HIV-1
infection62.
T cell development is characterized by differentiation and proliferation of

thymocytes. Loss and acquisition of cell surface markers characterize various stages of T
cell differentiation in the thymus49. Positive selection and generation of CD4+ and CD8+
single positive cells require engagement of CD3/TCRαβ complex on the CD4+CD8+ double
positive thymocytes at low affinity with self-peptide-MHC complexes on the cortical

thymic epithelial cells. Thymic medullary epithelial cells and hematopoietic cells
presenting self-antigens are essential for negative selection of T cells by inducing the
deletion of cells with high affinity to self-antigens thereby preventing autoimmunity. After
positive and negative selection, only about 95% of thymocytes reach the stage where they
can exit the thymus. The most mature thymocyte subset, which is ready to leave the
thymus, displays the following phenotype: CD3+/highCD45RA+CD27+CD31+ , expresses either

CD4 or CD8, and the Sphingosine 1 Phosphate Receptor 1 (S1P-R1), but not CD6963, 64.
A decline in thymus function related to thymic volume is observed in aging65.

Mackall et al. showed a decline in T cell regeneration related to age after chemotherapy66;
however, normal thymus tissue continues to be present and functional late in adult life67.
With age, thymic structures become less ordered and functional, and thymic output
decreases consistently. It is also important to understand other major factors that may
affect the function and degradation of the thymus, such as HIV-1 infection.
A powerful method to identify differences between typical age-dependent thymic

involution and the manner in which the thymus of an HIV-1-infected individual changes
over the course of infection is to compare the thymi of age-matched individuals 68, 69. With
a cohort of non-treated HIV-1 seropositive adults (n = 99, median age 39 years), as well as
one of HIV-1 seronegative adults (n = 32, median age 32 years), McCune et al. used non-
contrast chest CT imaging to show evidence that support that younger individuals were
more likely to retain thymic tissue than older counterparts, regardless of infection status70.
These conclusions were made following the establishment of a thymic index of the cohort
which compared age, serostatus, years passed since seroconversion, and CD4+ T cell
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counts. The investigators added that in the HIV-1 seropositive subjects, a higher thymic
index was associated with higher CD4+ cell counts.
The thymus is severely affected by CCR5–tropic and CXCR4-tropic HIV isolates71.

CCR5-tropic HIV-1 isolates impair T cell development although the percentage of
thymocytes expressing the HIV-1 co-receptor CCR5 is much less than those expressing
CXCR458, 72, 73
. However, despite low overall expression in the thymus, CCR5 is still
expressed on the more mature thymocytes resulting in a significant impact of T cell

reconstitution by CCR5 tropic HIV-1 isolates58, 72, 73. Infection of the thymus in utero and in
early life by HIV-1 isolates pathogenic for T cell precursors was reported to have
repercussions not only in situ, but also affect the overall course of disease in children73, 74.
Involvement of the thymus in pediatric AIDS was also suggested by histological studies
showing thymic involution during HIV-1 infection of thymocytes in fetuses and children,
and by anomalies in peripheral T cell subset distribution in HIV-1 infected children75-77.

Together, these findings suggest that HIV-1 has a deleterious effect upon the thymus of
infected individuals, generally showing greater thymic involution than uninfected
individuals of similar age, though the impact of this involution at these ages is of varying
importance.
Involution of the thymus, whether by aging or HIV-1-associated destruction, is a

serious structural and functional change that leads to long-lasting effects, contributing to
the onset of immune senescence. The lack of a site for extensive T cell proliferation and
maturation effectively acts as a bottleneck for the overall efficacy of the adaptive immune
system and has a cascading effect upon secondary lymphoid compartments. Fewer mature
T lymphocytes means decreased overall immunity, resulting in an increased probability of
serious infection, not to mention other housekeeping tasks, such as lymphatic and
circulatory filtration and surveillance. However, it is important to also note that while this
process of atrophy is currently understood to be an inevitable byproduct of aging, there
exists the potential for the identification of strategies to mitigate, halt, or even reverse the
involution of the thymus. It has been well documented in the young that malnutrition
leads to rapid involution of the thymus, but the reintroduction of proper nutrition allows
for rapid regeneration of the tissue78. With the use of ART, HIV-1-infected individuals may
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recover significant functional thymic mass after its degradation by effects of the virus.
Douek et al. measured thymic output in HIV-1 infected individuals and saw a decline in
thymic emigrants in aged as well as HIV-1 infected individuals; however, ART could rapidly
reverse this decrease in thymic output in HIV-1 infected individuals79. As not all HIV-1
infected individuals recover their CD4+ T cell levels while on ART, Rb-Silva et al. developed
a mathematical model to predict immune reconstitution in HIV-1 infected individuals
started on ART based on thymic function80.
However, as normal thymus tissue continues to be present and functional late in

adult life67, the impact of aging on the thymus does not completely reflect the structure
and function of the thymus in HIV-1 infection even in the era of ART therapy.
Aging and HIV-1 Mediated Alterations of the Lymph Nodes

The general structure of the lymph node is similar to the thymus, with an inner
medullary region and an outer cortical region containing hyperproliferative cells

surrounded by a fibrous capsule. Between the cortical region and the capsule is a sinus
that transports draining lymph, antigens, and leukocytes from the afferent lymphatics and
sites of inflammation81, 82. Lymphocytes, primarily T and B cells, along with nutrients from
the blood enter the secondary lymphoid tissues through specialized cells called High
Endothelial Venules (HEVs). If an infection is present in the body, an incoming flow of
lymphocytes and nutrients from the blood will intersect with an incoming flow of antigen
and antigen presenting cells (APC) from the afferent lymphatics. This intersection provides
all the necessary components to build an adaptive immune response.
Similar to other immune compartments, the architecture of the lymph node is built
through intercellular communication between lymphocytes and stromal cells. Stromal
cells within the lymph node including fibroblastic reticular cells (FRC) are critical in creating
the matrix and reticulum where leukocytes interact. FRC also produce IL-7, a pro-survival
signal for naïve T cells83, and require lymphotoxin- (LT) signaling from the infiltrating T
cells84, emphasizing the delicate relationship between lymphocytes and stromal cells
within lymphoid compartments. The cortical reticulum where B cell follicles reside is a
more open and loose mesh network compared to the medullary T cell zone which is more
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compact and has a higher density of fibrous networks85 composed of ECM proteins like
collagen I and III, fibronectin, and tenascin86 (Figure 2).
The age-related decline in ECM-producing stromal cells contributes to changes in

lymph node morphology and impairment of adaptive immunity in the elderly. Thompson
et al. provide a concise summary of how these stromal cells deteriorate with age83.
Lymphatic endothelial cells become more permeable and result in decreased antigen flow
from the tissues into the lymph node. Furthermore, aged HEVs become less permeable
and hinder lymphocyte entry into the lymph nodes. Decreased flow of antigens, APC, and
lymphocytes into the lymph node dampen adaptive immune responses and levels of
lymphocyte-derived stromal cell growth factors, causing further degeneration of lymph
node histoarchitecture. The follicular dendritic cells (FDC) that present antigen and
immune complexes within the cortical B cell follicles and the fibroblastic reticular cells
(FRC) that produce the medullary reticulum and provide a network for the interaction of T
cells and dendritic cells also decrease with age.
Several studies have looked at lymph node architecture and deterioration

throughout the aging process. In 1947, Denz described age-associated morphological
differences in over 300 lymph nodes from 150 autopsies85. Hadamitzky et al. studied age-
related histoarchitectural changes in 41 superficial inguinal lymph nodes from individuals
ranging in age from 17 to 98 years old87. Much of this early work was recapitulated in later
studies, including Tsakraklides et al. in their 2,250 axillary lymph node study and Luscieti et
al. who also found age-related and location-specific structural changes in human lymph
nodes88. The general consensus is that lymph node histoarchitecture varies based on
underlying disease, lymph node location in the body, and exposure to antigens.
The most pronounced age-related morphological change found by Luscieti et al.

and others was the decrease in germinal center size and number88, 89. Cortical germinal
centers do not develop until 2-6 months after birth85, concomitant with foreign antigen
exposure88, 89, and maximum lymph node size is reached in children by 7-10 years of age 85.
Development of germinal centers are more prominent in spherical cervical, mesenteric,
and lumbar lymph nodes that are found deep within the body and peripheral to the
Page 16 of 37
16
mucosa as opposed to flattened superficial axillary, popliteal, or cubital lymph nodes85, 88.
The discrepancy in germinal center number and size is thought to be associated with
amount of antigen exposure between mucosal and superficial locations. Following

puberty, the superficial lymph nodes begin to show signs of degeneration, including
increased fibrosis, fatty tissue accumulation (lipomatosis), and decreased cellularity (Figure
3). Contrary to superficial lymph nodes, deep nodes such as CLN or MLN in individuals 80
years and older contained germinal centers, although their activity and number appear to
decrease with age85.

Several of the studies indicate that lipomatosis and fibrosis occur independently.
Fat accumulation was rarely seen in lymph nodes from the youngest groups but was
consistently found in those from the older groups87. Lipomatosis was not often seen in
mesenteric lymph nodes with continuous antigen exposure, but could readily be seen in
superficial axillary and inguinal nodes85. Although fibrosis was more severe and prominent
in the older age groups, mild fibrosis was still seen in the youngest cohort, indicating
fibrosis is not solely dependent on age87. Denz reported that beginning at age 30 the
medullary reticulum begins to decrease in cellularity and increase in collagenous material
with limited if any lipomatosis85, 89, 90
. Fibrosis only occurred following medullary
lymphocyte decline and subsequent compaction of reticulum fibers, yet the fibrosis did
not impair medullary cell proliferation during active inflammation.
Similar to aging, HIV-1 infection causes substantial deterioration of lymph node

architecture including germinal center alterations and fibrosis. Several studies have
investigated HIV-1 mediated effects on lymph node structure and function. Paiva et al.
evaluated cervical lymph node biopsies of 31 adult HIV-1 infected individuals with or
without AIDS prior to ART91. Schacker et al. compared inguinal lymph node histology of 33
individuals, 24 of which were HIV-1-positive and ART-naïve while 9 were from HIV-1-
negative individuals92. In another study, Schacker et al. analyzed inguinal lymph node
fibrosis of 11 HIV-1 positive individuals before and after ART administration93.
A general theme emerges from these studies. Chronic antigen exposure leads to
prominent follicular development and lymphadenopathy early in infection. Elevated
Page 17 of 37
17
concentrations of HIV-1 antigens can be found in the lymph nodes at concentrations
greater than 300pg/mL and is thought to drive the follicular hyperplasia present during
chronic infection91, 94, 95. As the disease progresses, follicular lysis and involution occur in
the germinal centers. Atrophy of cortical germinal centers may be due to disruption of
intercellular communication between follicular dendritic cell (FDC), CD4+ T follicular helper
cells (TFH), and B cells. A similar reduction in the FRC network, IL-7 production, and naïve T
cell infiltration of the medulla is also seen during HIV-1 infection81, 84.
Decreased stromal cell survival and function may also be due to decreased
lymphocyte entry into the lymph node91, 92
. In biopsies from AIDS patients, increased
fibrosis was seen in the medulla and the basement membrane surrounding HEVs92 (Figure
4). Increased collagen-I and fibronectin fibrosis is also seen in the lymph nodes of
nonhuman primate animal models infected with simian immunodeficiency virus (SIV)81, 96.
Like the fibrosis seen in other tissues, lymph node fibrosis seen in HIV-1 and SIV infection
may be induced by TGF-1 signaling initiated by surrounding Tregs81, 84, 96
, which can
stimulate myofibroblastic production of ECM components97.
Aging and HIV-1 disease, through different mechanisms, both lead to deterioration
of lymph node structure and function and subsequent impairment of adaptive immunity81.
After sufficient tissue deterioration and loss of lymphocytes in both aging and HIV-1,
lymph nodes become effectively non-functional, aside from passing lymph to other nodes
87
.
Aging and HIV-1 Mediated Alterations of Mucosal Associated Lymphoid Tissue
Mucosal Associated Lymphoid Tissues (MALT) are critical in developing appropriate

adaptive immune responses against mucosal pathogens. Along the mucosa, antigen can
be transported from the mucosal lumen through M cells to specialized lymphoid inductive
sites that generally contain follicles, interfollicular areas, subepithelial dome regions, and
overlying epithelium98. These regions have distinct T and B cell zones, similar to other
secondary lymphoid organs. Although T cells receive inductive signals at these locations,
they are thought to migrate to the mesenteric lymph nodes for further differentiation.
The differentiated T cells that reside within the inductive sites are effector cells called
Page 18 of 37
18
lamina propria lymphocytes99, 100
. Detailed analysis of age-related alterations of these
dynamically organized mucosal sites of immune effector induction are highly studied in
animal models, but data in humans is comparatively scarce101.
MALT effector sites are distributed throughout the mucosa and are the sites of IgA
secretion into the mucosal lumen98. These tissues can be found throughout the mucosal
areas and include tonsils and adenoids (Waldeyer’s ring), Bronchial ALT (BALT), Larynx ALT
(LALT), Nose ALT (NALT), and Gut ALT (GALT). GALT includes Peyer’s patches, lamina
propria of the small intestines, and follicular aggregates of the stomach and large
intestines. The majority of these mucosal sites are not typically developed in the fetus and
only become populated by lymphocytes following exposure to antigen and microbes after
birth98, 101, 102. Derbetin et al. reported that 40% of children studied had developed BALT
and NALT while 80% developed LALT all within the first year of life101, 102. In adulthood,
healthy humans typically lose BALT while 56% of healthy adults still maintain LALT, while
NALT presence is still unknown101. Beharka et al. investigated how age affects MALT by
phenotyping intraepithelial lymphocytes (IEL), lamina propria lymphocytes (LPL), and
PBMC in addition to measuring immunoglobulin A (IgA) levels in the saliva and serum of
young (20-40 years old) versus older (65 years or older) individuals (n=17). They did not
find a significant change in salivary IgA, but reported an increase in serum IgA with age103.
Although there are limited data on age-related changes of MALT in humans, several
reports on Peyer’s Patches and GALT in aging rodents mirror what happens in human
lymph nodes. With age, there is a decrease in naïve CD4+ T cells and follicular dendritic
cells within GALT, which led to decreased germinal center development and altered gut
IgA levels104, 105. The decrease in lymphocytes lead to a decrease in LT signaling, stromal
cell survival, and organization of MALT, once again emphasizing the interdependent
relationship between lymphocytes and stromal cells in developing these secondary
lymphoid tissues106.
Chronic antigen stimulation and proinflammatory cytokines found in the mucosa

provide an ideal niche for HIV-1 replication. Deterioration of the MALT in progressive HIV-1
disease has been associated with chronic inflammation. Several groups have reported the
Page 19 of 37
19
rapid depletion of CD4+ T cells in GALT during acute HIV-1 infection96, 100, 107. Estes et al.
examined peripheral blood, inguinal lymph nodes and terminal ileum biopsies from 35
HIV-1 infected and 11 HIV-1 uninfected individuals to see if morphological changes within
these tissue correlate with peripheral CD4+ T cell counts and disease status before and
after 6 months of ART107. They reported that fibrosis within the GALT prevents optimal
reconstitution of naïve and central memory CD4+ T cells in the Peyer’s Patches, even after
treatment107. SIV-infected rhesus macaques also have significant loss of Th17 CD4+ T cells
in the GALT108, 109, which has been linked to increased microbial translocation and chronic
inflammation in SIV and HIV-1 infections110.
Although additional human studies are needed to determine the effects of aging
and HIV-1 infection on MALT structure and function, both conditions may impair this
immune compartment in similar ways to peripheral lymph nodes by disrupting
communication between lymphocytes and surrounding stromal cell (Figure 5).
Conclusions
How do tissue microenvironments change in immune compartments in an aging
individual and how does this compare to alterations of immune microenvironments seen
during HIV-1 disease? Although both conditions lead to impairment of innate and adaptive
immunity in the face of chronic inflammation, there are distinct differences between the
two. In the peripheral blood and bone marrow of aging individuals there is often a
myeloid skewing due to loss of naïve lymphocytes and drastic reduction in peripheral B
cells. On the other hand, HIV-1 infection depletes CD4+ T cells in addition to causing a
variety of hematological abnormalities in the peripheral blood and lymphoid tissues that
impact both the myeloid and lymphoid lineages.
Gradual deterioration of other lymphoid structures including the thymus, lymph

nodes, and MALT are seen in aging. Destruction of these cellular microenvironments is
even more prominent in advanced HIV-1 disease. Increased fibrosis, lipomatosis, and
subsequent disorganization of secondary lymphoid tissues ensues111-114. Changes in
lymphoid tissue structure during aging and HIV-1 infection may be caused by a disruption
of a delicate relationship between lymphocytes and the surrounding stromal cells. The
Page 20 of 37
20
interdependent relationship between lymphocytes and the surrounding stromal cells,
including fibroblastic reticular cells and follicular dendritic cells in the lymph node,
adventitial reticular cells in the bone marrow, and epithelial cells within the thymus
deteriorates with age and is exacerbated in HIV-1 infection.
Acknowledgements
The authors wish to thank Dr. Mauricio González-Navarro, Dr. Maria Fernanda
Torres-Ruiz, and Dr. Yara Andrea Luna-Villalobos in obtaining the cervical and inguinal
lymph nodes as well as Dr. Thomas Troost for supplying tonsillar tissue samples for these
studies. We also gratefully acknowledge Dr. Beth Jamieson for her valuable comments and
thoughtful review. This publication resulted in part from research supported by NIAID
award AI126617, co-funded by NIDA, NIMH, NINDS. The content is solely the responsibility
of the authors and does not necessarily represent the official views of the NIH.
Disclosures
The authors have no disclosures or conflicts of interest to declare.

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107. Estes J, Baker JV, Brenchley JM, et al. Collagen deposition limits immune
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Page 33 of 37
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Figure Legends
Figure 1: Leukocyte migrating along extracellular matrix in human tonsil tissue. The
nucleus of the leading edge of a migrating cell (arrow) can be seen on the left of the image.
Migrating cells adhere to extracellular matrix (ECM) proteins and neighboring cells.
Lymphoid tissue architecture is dependent on ECM proteins like the collagen seen in this
image (asterisk) and the stromal cells that produce ECM. Disruption of communication
between leukocytes and stromal cells in lymphoid tissues leads to aberrant deterioration
or excessive buildup of ECM proteins, which can hinder tissue function. Scale bar = 1 μm.
Page 34 of 37
34
Figure 2: Polarized cell adherent to adjacent collagen bundles in lymphoid tissue. This
migrating lymphocyte (arrow) makes several contacts to the ECM proteins like the collagen
bundles seen here (asterisk). Proper density and placement of these architectural
components within lymphoid tissues are critical for leukocyte migration and function.
Matrix degeneration, fibrosis, and lipomatosis of lymphoid tissues have previously been
associated with various diseases including aging and progressive HIV-1 disease. Scale bar =
2 μm.
Page 35 of 37
35
Figure 3: Age and active HIV-1 replication affect lymph node histoarchitecture.
Hematoxylin and eosin staining of sectioned lymph nodes from HIV-1 infected individuals
show how antigen exposure and age may affect tissue structure. (A & B) Follicular
hyperplasia can be seen in a 22 year-old individual with ongoing HIV-1 replication (226
CD4+ T cells/μL peripheral blood; 391,929 copies HIV-1 RNA/mL). B cell follicles are
indicated by asterisks immediately below pink fibrous capsule and are found in lymph
nodes with active antigen exposure. (C) Lipomatosis lymph node degeneration is
prevalent in this lymph node of a 52 year-old HIV-1 infected individual on ART and
suppressed viremia (410 CD4+ T cells/μL peripheral blood; undetectable viral load). Fat
accumulation can are clear areas denoted by asterisks. (D) The same aged lymph node
shows increased collagen fibrosis (pink fibers denoted by asterisks). Clear B cell follicles
are not seen in this lymph node possibly due to decreased HIV-1 antigen burden following
antiretroviral therapy.
Page 36 of 37
36
Figure 4: The edge of this blood vessel found within human lymphoid tissue shows several
erythrocytes (asterisk) in the lumen of the blood vessel. In the bottom of the micrograph,
extracellular matrix can be seen below the endothelial cell layer (arrow). Increased fibrosis
within the basement membrane of high endothelial venules is thought to decrease
lymphocyte homing to lymphoid tissues in both progressive HIV-1 disease and aging. Scale
bar = 2 μm.
Page 37 of 37
37
Figure 5: Cell communication in lymphoid tissues is critical in maintaining tissue structure

and function. In this micrograph of human tonsils, several cells can be seen projecting
membrane protrusions to communicate with each other (arrow). Signaling between

leukocytes and stromal cells is critical for both lymphoid tissue architecture and leukocyte
function and survival. Depletion of leukocyte subsets or stromal cells in aging and HIV-1
infection disrupts this interdependent relationship and alters the structure and function of
lymphoid tissues. Scale bar = 2 μm.

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AIDS Research and Human Retroviruses

1 Division of Infectious Diseases, Department of Medicine, Weill Cornell Medicine, 413

2 Departmento de Investigación en Enfermedades Infecciosas, Instituto Nacional de

Enfermedades Respiratorias “Ismael Cosío Villegas”, CDMX, Mexico.

* Correspondence: (p) 646-962-2478; rlf2001@med.cornell.edu

Impaired immunity is a common symptom of aging and advanced Human

Immunodeficiency Virus type 1 (HIV-1) disease. In both diseases, a decline in lymphocytic

changes within lymphoid tissue microenvironments and how histoarchitectural changes

Aging and HIV-1 Mediated Alterations of the Peripheral Blood

A precipitous decline in absolute numbers of peripheral blood lymphocytes,

childhood to 198+/-34 cells/mm3 in the elderly. Decreased lymphocyte number and

Similar to aging, lymphocyte subsets are significantly altered in HIV-1 disease.

(CD28-CD57+), activation (CD38+HLA-DR+), and exhaustion (PD1+) compared to uninfected

Antiretroviral therapy is reported to restore CD45RA+CD31+ T cells to age appropriate

is more attributable to HIV-1 infection.

to 65 HIV-exposed uninfected children, and 56 HIV negative children. Their findings

Aging and HIV-1 Mediated Alterations of the Bone Marrow

including mononucleated lymphocytes and histiocytes. However endothelial cells,

The bone marrow has an optimized microarchitecture of stiff bony trabeculae,

compared to the normal aging process.

Aging and HIV-1 Mediated Alterations of the Thymus

medullary areas which contain developing thymocytes, epithelial cells, macrophages,

Several murine studies suggest there is an interdependent relationship between

T cell development is characterized by differentiation and proliferation of

positive thymocytes at low affinity with self-peptide-MHC complexes on the cortical

thymus, displays the following phenotype: CD3+/highCD45RA+CD27+CD31+ , expresses either

A decline in thymus function related to thymic volume is observed in aging65.

A powerful method to identify differences between typical age-dependent thymic

The thymus is severely affected by CCR5–tropic and CXCR4-tropic HIV isolates71.

expressed on the more mature thymocytes resulting in a significant impact of T cell

and by anomalies in peripheral T cell subset distribution in HIV-1 infected children75-77.

Involution of the thymus, whether by aging or HIV-1-associated destruction, is a

However, as normal thymus tissue continues to be present and functional late in

Aging and HIV-1 Mediated Alterations of the Lymph Nodes

medullary region and an outer cortical region containing hyperproliferative cells

The age-related decline in ECM-producing stromal cells contributes to changes in

Several studies have looked at lymph node architecture and deterioration

The most pronounced age-related morphological change found by Luscieti et al.

amount of antigen exposure between mucosal and superficial locations. Following

decrease with age85.

Similar to aging, HIV-1 infection causes substantial deterioration of lymph node

Aging and HIV-1 Mediated Alterations of Mucosal Associated Lymphoid Tissue

Mucosal Associated Lymphoid Tissues (MALT) are critical in developing appropriate

animal models, but data in humans is comparatively scarce101.

Chronic antigen stimulation and proinflammatory cytokines found in the mucosa

inflammation in SIV and HIV-1 infections110.

communication between lymphocytes and surrounding stromal cell (Figure 5).

Gradual deterioration of other lymphoid structures including the thymus, lymph

The authors have no disclosures or conflicts of interest to declare.

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6. Parinitha S, Kulkarni M. Haematological changes in HIV infection with correlation to

8. Chehimi J, Campbell DE, Azzoni L, et al. Persistent decreases in blood plasmacytoid

distinct from that of CMV and aging. PLoS One. 2014;9(2):e89444.

compartment during aging. J Immunol. Feb 1 2008;180(3):1499-1507.

16. Cagigi A, Nilsson A, Pensieroso S, Chiodi F. Dysfunctional B-cell responses during

pathogenesis. AIDS. Jul 1996;10(8):F17-22.

Hematol. Oct 2014;100(4):317-325.

Clinical Implications. Microbiol Spectr. Oct 2016;4(5).

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deficiency virus (HIV). Br J Haematol. Jun 1987;66(2):251-256.

marrow in human immunodeficiency virus infection. Virchows Arch A Pathol Anat