Type 3 innate lymphoid cell depletion is mediated by TLRs in lymphoid tissues of simian immunodeficiency virus–infected macaques

Abstract

Innate lymphoid cells (ILCs) type 3, also known as lymphoid tissue inducer cells, plays a major role in both the development and remodeling of organized lymphoid tissues and the maintenance of adaptive immune responses. HIV/simian immunodeficiency virus (SIV) infection causes breakdown of intestinal barriers resulting in microbial translocation, leading to systemic immune activation and disease progression. However, the effects of HIV/SIV infection on ILC3 are unknown. Here, we analyzed ILC3 from mucosal and systemic lymphoid tissues in chronically SIV-infected macaques and uninfected controls. ILC3 cells were defined and identified in macaque lymphoid tissues as non-T, non-B (lineage-negative), c-Kit⁺IL-7Rα⁺ (CD117⁺CD127⁺) cells. These ILC3 cells highly expressed CD90 (∼63%) and aryl hydrocarbon receptor and produced IL-17 (∼63%), IL-22 (∼36%), and TNF-α (∼72%) but did not coexpress CD4 or NK cell markers. The intestinal ILC3 cell loss correlated with the reduction of total CD4⁺ T cells and T helper (T_h)17 and Th22 cells in the gut during SIV infection (P < 0.001). Notably, ILC3 could be induced to undergo apoptosis by microbial products through the TLR2 (lipoteichoic acid) and/or TLR4 (LPS) pathway. These findings indicated that persistent microbial translocation may result in loss of ILC3 in lymphoid tissues in SIV-infected macaques, further contributing to the HIV-induced impairment of gut-associated lymphoid tissue structure and function, especially in mucosal tissues.—Xu, H., Wang, X., Lackner, A. A., Veazey, R. S. Type 3 innate lymphoid cell depletion is mediated by TLRs in lymphoid tissues of simian immunodeficiency virus–infected macaques.

Innate lymphoid cells (ILCs) represent a phenotypically and functionally heterogeneous population of lymphocytes that has important roles in lymphoid tissue formation during embryonic and adult life, maintenance and protection of epithelial barriers against infection, regulation of adaptive immune responses, maintenance of gut-associated organized lymphoid tissue (GALT), and restoration of GALT following injury to mucosal barriers (1–8). In contrast to T and B cells, ILCs do not express antigen-specific receptors. All ILCs require the transcriptional regulator inhibitor of DNA binding 2 for development (9). However, ILCs have been recently categorized into 3 distinct groups on the basis of their cytokine production, similar to the classification scheme for various T helper (T_h) cell subsets (5, 10). ILC1 cells express the transcription factor T-bet and produce the T_h1-type cytokine IFN-γ, and this subset is IL-7Rα⁻ and includes conventional NK cells and most intraepithelial lymphocytes, which mediate protection of mucosal surfaces against intracellular infections (11–13). ILC2 cells produce T_h2 cytokines IL-5 and IL-13, express the transcriptional factor guanine adenine thymine adenine sequence-binding protein 3, and play important roles in immunity to helminth infections and in the pathogenesis of allergic reactions such as asthma (14–16). ILC3 cells are an important source of the T_h17 cytokines IL-17A and IL-22 and express the retinoic acid receptor-related orphan receptor γ. ILC3 cells are implicated in mucosal immunity to extracellular bacteria, regulation of adaptive immune cell homeostasis, maintenance of intestinal memory CD4⁺ T cells, providing help to marginal zone B cells, and in the organogenesis and maintenance of secondary lymphoid tissues (7, 17–20).

HIV/simian immunodeficiency virus (SIV)-induced immunodeficiency is characterized by a marked loss of intestinal CD4⁺ T cells, severe disruption of GALT, and increased intestinal permeability resulting in microbial translocation, which in turn causes systemic immune activation resulting in more rapid disease progression (21, 22). Little is known regarding the role of ILCs in HIV/SIV infection in mucosal immunity and microbial translocation. However, it is clear that subsets of these cells play critical roles in the development and maintenance of the intestinal barrier and the development and maintenance of mucosal immune tissues and responses, suggesting that ILCs may play a major role in HIV pathogenesis.

We and others have previously shown that CD3⁻CD8^high lymphocytes in the intestine represent a heterogeneous population including NK cells and other ILCs (2, 23, 24). In this study, we found that ILC3 subsets, defined by c-Kit⁺IL-7α⁺ lineage-negative cells, are distinct from CD3⁻CD8^high cells, as indicated by ILC3 containing considerable CD3⁻CD8⁻ cells (∼70%) in macaques. Evidence indicates that dietary deficiency of vitamin A or its active metabolite, retinoic acid, in mice may result in a decrease in ILC3 and its cytokines (IL-17 and IL-22), corresponding with an increase in ILC2, and IL-5 and IL-13, which is accompanied by an enhanced susceptibility to infection and disease due to Citrobacter rodentium (25). These findings suggest that various ILC types in heterogeneous ILCs possess distinct functions; their alterations in the balance may result in changes of immune competence in mucosal lymphoid tissues.

ILC3 cells express c-Kit and IL-7Rα/CD127, which are important for the development and survival of ILCs (1, 26, 27). ILC3 cells are also required for formation of Peyer’s patches and cryptopatches in the intestine and anlagen formation in the fetus, and they also direct the formation of isolated lymphoid follicles by recruiting dendritic cells and B cells and promoting T_h cell survival and IgA production by B cells (28, 29). Unlike T_h17 cells, ILC3 cells are not induced by the microbiota but are programmed and present in tissues prior to birth (30). In contrast to ILC3 cells, the IL-22–producing NKp46⁺ cells express low levels of c-Kit and IL-7Rα, albeit with the ability to produce IL-22. These are found in the intestinal lamina propria and within intestinal villi (31–33).

To better understand the characteristics of ILCs in the systemic and mucosal lymphoid tissues in AIDS, here, we define ILC3 cells as lineage-negative, c-Kit⁺ IL-7Rα⁺, CD45⁺ lymphocytes and examined their anatomic distribution, characterization, and changes in tissues in SIV-infected rhesus macaques. Type 3 ILCs were largely restricted to mucosal and lymphoid tissue compartments and are distinct from conventional NK cells in that they preferentially produce IL-17, IL-22, and TNF-α. Furthermore, ILC3 cells in lymphoid tissues were markedly depleted in chronically SIV-infected macaques, and their loss directly correlated with reduction of other intestinal integrity-associated T_h17/T_h22 cells during SIV infection. Also, our data indicated that the ILC3 cells could be induced to undergo apoptosis by microbial products through TLR-dependent pathways. These findings suggest that persistent gut microbial translocation during HIV/SIV infection might promote loss of ILC3 in lymphoid tissues and contribute to impairment of ILC3 function and corresponding damage of lymphoid tissue, favoring persistent immune activation of HIV disease infection.

MATERIALS AND METHODS

Ethics statement

All animals in this study were housed at the Tulane National Primate Research Center in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International standards. All studies were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee under protocol numbers 3562 and 3663. Animal housing and studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals [#000594; National Institutes of Health (NIH), Bethesda, MD, USA] and with the recommendations of the Weatherall Report on the use of nonhuman primates in research. All clinical procedures, including administration of anesthesia and analgesics, were carried out under the direction of a laboratory animal veterinarian. All procedures were performed under anesthesia using ketamine, and all efforts were made to minimize stress, improve housing conditions, and to provide enrichment opportunities (e.g., objects to manipulate in cage, varied food supplements, foraging and task-oriented feeding methods, and interaction with caregivers and research staff). Tulane University complies with NIH policy on animal welfare, the Animal Welfare Act, and all other applicable federal, state, and local laws.

Animals and virus

A total of 72 Indian-origin rhesus macaques (Macaca mulatta) from 0 to 17 yr old were utilized to examine ILCs in blood and/or tissues. Numbers of tissues examined for individual analyses are described in the figure legends. All animals were housed at the Tulane National Primate Research Center in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International standards. All studies were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee. Of these, 41 animals were uninfected controls, and others (>1 yr old; n = 9) were chronically infected with SIVmac251 defined as animals infected for >3 mo with no overt clinical signs of disease (chronic asymptomatic). To examine cells from blood and tissues such as intestine, spleen, and lymph node, etc., macaques were euthanized for tissue collection from uninfected controls, or chronically SIV-infected macaques.

Cell isolation and processing

Mononuclear cells from peripheral blood, spleen, lymph nodes, and intestinal tissues were isolated and processed. Briefly, total peripheral blood mononuclear cells were isolated from EDTA-treated venous blood by density gradient centrifugation with Lymphocyte Separation Medium (MP Biomedicals, Santa Ana, CA, USA) as per manufacturer’s instruction. Tissues were collected from the jejunum and colon within minutes of euthanasia and processed immediately for cell suspensions using enzymatic digestion as previously described (34).

Phenotyping

Flow cytometry for surface and intracellular staining was performed using standard protocols (35). Cells were stained with CD3 (SP34), CD4 (L200), CD8 (SK1), CD14 (M5E2), CD16 (3G8), CD20 (L27), CD34 (563), CD45 (TU116), CD56 (NCAM16.2), CD90 (5E10), c-Kit (EP10; Biocare Medical, Concord, CA, USA), IL-7Rα (R34.34; Beckman Coulter, Brea, CA, USA), NKp46 (BAB281; Beckman Coulter), NKG2A (Z199; Beckman Coulter), FcεRI (CAR1; Miltenyi Biotec, Bergisch Gladbach, Germany), TNF-α (MAB11), IL-2 (MQ1-17H12), IL-17 (CZ8-23G1; Miltenyi Biotec), IL-22 (IL22JOP; eBioscience, Inc., San Diego, CA, USA), TLR2 (TL2.1; InvivoGen, San Diego, CA, USA), TLR4 (HTA125; AbD Serotec, Raleigh, NC, USA), Annexin V, and Live/Dead Fixable Aqua Dead Cell Stain kit (Invitrogen/Life Technologies, Grand Island, NY, USA). All antibodies and reagents were purchased from BD Biosciences Pharmingen (San Diego, CA, USA) unless otherwise noted. Samples were resuspended in BD Stabilizing Fixative (BD Biosciences, San Jose, CA, USA) and acquired on a BD fluorescence-activated cell sorting (Facs)Verse or BD Fortessa flow cytometer (Becton Dickinson, San Jose, CA, USA). Data were analyzed with FlowJo software (Treestar, Ashland, OR, USA).

Multicolor confocal microscopy

Paraffin-embedded sections were deparaffinized and unmasked using high-temperature antigen retrieval, which consisted of heating slides in a steam bath chamber (Black & Decker FlavorScenter SteamerPlus, Latham, NY, USA) with 0.01 M citrate buffer (pH 6.0) for 20 min, cooling, and washing twice in PBS. For confocal microscopy, 3-color fluorescent immunostaining was performed on formalin-fixed tissues. In brief, sections were sequentially incubated with primary antibodies to c-Kit (clone EP10; Biocare Medical), aryl hydrocarbon receptor (AhR; clone RPT1; Thermo Scientific, Waltham, MA, USA), and CD20 (2H7; BD Biosciences Pharmingen), then washed and incubated with appropriate secondary antibodies: Alexa Fluor 488 (green), Alexa Fluor 568 (red), or Alexa 633 (blue) (Molecular Probes, Eugene, OR, USA). Finally, slides were mounted with fluorescent mounting medium (EP10; Dako, Inc., Carpinteria, CA, USA) and visualized by a confocal microscope. Confocal microscopy was performed using a Leica TCS SP2 confocal microscope equipped with 3 lasers (Leica Microsystems, Exton, PA, USA). Individual optical slices representing 0.2 μm and 32–62 optical slices were collected at 512 × 512 pixel resolution. NIH Image (version 1.62) and Adobe Photoshop (version 7.0; San Jose, CA, USA) were used to assign colors to the channels collected.

LPS levels

To determine plasma LPS levels, the plasma was diluted by 20% endotoxin-free water and then heated to 70°C for 10 min to inactivate plasma proteins. For LPS detection in intestinal samples, the mucosa is scraped from the underlying muscle layer with a glass slide, placed in 1.5 ml Eppendorf tubes with buffer [10 mM Tris-HCl and 1 mM EDTA (pH 7.4)] at 20 mg/ml, and centrifuged at 12,000 g for 10 min at 4°C. The supernatants were collected for later examination. LPS was quantified by Limulus Amebocyte assay (Cambrex, East Rutherford, NJ, USA) according to the manufacturer’s protocol.

Cell stimulation and detection of cell apoptosis

Lymphocytes (10⁶) from jejunum were stimulated in vitro with 0.1 μM phorbol 12-myristate-13-acetate and 0.5 μg/ml ionomycin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37°C. Cells were cultured for an additional 4 h in the presence of 5 μg/ml Brefeldin A (Sigma-Aldrich), then stained for cell surface markers, fixed in 2% paraformaldehyde, permeabilized with Cytofix/Cytoperm solution (BD Biosciences), and intracellularly costained with fluorochrome-labeled antibodies for the cytokines.

For assessing apoptosis of ILC3 cells, intestine lymphocytes (10⁶) were stimulated in vitro with TLR9 ligand (10 μg/ml, ODN BW006), TLR2 ligand [1 μg/ml, purified lipoteichoic acid (LTA)], TLR4 ligand (50 ng/ml, ultrapure LPS), or LPS plus LPS-RS ultrapure (1 μg/ml, LPS antagonist; all from InvivoGen) for 24 h. Parallel samples were cultured with medium alone as controls. All cells were harvested and stained for surface markers and with Annexin V. Cell samples were acquired with a BD FacsVerse cytometer. Data were analyzed with FlowJo software.

Statistics

Graphic presentation and statistical analysis of the data were performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). Comparisons between groups were analyzed by a 1-way ANOVA and a nonparametric Mann-Whitney U test. Values of P < 0.05 were considered statistically significant. Correlations between samples were calculated and expressed using the Spearman rank correlation coefficient.

RESULTS

Definition and distribution of ILC3 cells in tissues in uninfected rhesus macaques

To define ILC3 cells in macaques by flow cytometry, we first gated on CD45⁺ lineage-negative (CD3⁻CD14⁻CD20⁻) lymphocytes and then examined c-Kit⁺IL-7Rα⁺ (ILC3 cells) in blood and various lymphoid tissues. ILC3 cells were essentially all CD45⁺ lymphocytes, compared with CD45^dim epithelial cells (data not shown). Although c-Kit is also expressed on hematopoietic progenitor cells (CD34⁺) and mast cells (FcεRI⁺), ILC3 cells were shown here to be both CD34⁻ and FcεRI⁻ when gating through Lin⁻ cells (Fig. 1A), indicating that ILC3 cells are distinct from stem cells and mast cells. ILC3 cells were predominantly distributed in various lymphoid tissues, including spleen, lymph nodes, and intestinal and vaginal mucosal tissues (Fig. 1B). Organized lymphoid tissues and mucosal tissues contained substantial numbers of ILC3 cells, significantly higher than found in peripheral blood (P < 0.05) (Fig. 1C). Notably, percentages of ILC3 cells in intestinal tissues were higher in neonatal and infant macaques (<6 mo) and gradually decreased with age in juvenile and adult macaques (Fig. 1D), consistent with evidence indicating that ILC3 cells are present in tissues prior to birth and play critical roles for lymphoid tissue formation (30). These findings suggest that ILC3 cells preferentially reside in lymphoid tissues and are involved in organogenesis in macaques.

Figure 1.

ILC3 cells in peripheral blood and various lymphoid tissues in uninfected, normal rhesus macaques. A) Gating strategy for defining ILC3 cells as Lin⁻CD45⁺c-Kit⁺IL-7Rα⁺ lymphocytes (lyms). FSC-H, forward-scatter height; SSC-A, side-scatter area. B) Representative dot plot of ILC3 cells in blood and various lymphoid tissues, gated Lin⁻CD45⁺ lymphocyte populations. Mes LN, mesenteric lymph node; PMBC, peripheral blood mononuclear cell. C) Distribution of ILC3 cells in blood, spleen, lymph nodes, and mucosal tissues in adult macaques (n = 9). D) Correlation of ILC3 cells with age in jejunum in normal rhesus macaques (n = 41). ILC3 cells are gated through Lin⁻, CD45⁺ cells (B–D).

Phenotyping and cytokine production of ILC3 cells in uninfected macaques

ILCs are comprised of conventional NK cells (ILC1), ILC2, and ILC3 cells with distinct phenotypes and cytokine-producing properties. Here, we examined the expression of conventional NK cell markers and cytokine production in intestinal ILC3 cells in healthy macaques. The AhR is a sensor of plant-derived dietary antigens and/or microflora in the gut, which is believed to promote receptor-related orphan receptor γ-positive ILC3 function, particularly in the initial formation of lymphoid follicles in neonates (anlagen) and maintenance of intestinal immunity in adults (36–39). Here, we examined the expression of AhR by confocal microscopy and show that it is expressed intracellularly in ILC3 but not CD20⁺ B cells, as indicated by coexpression of c-Kit and AhR in lymph nodes of uninfected macaques (Fig. 2A). These findings further support the notion that coexpression of c-Kit and IL-7Rα in macaque lymphoid tissues is indicative of ILC3 cells. As shown in Fig. 2B, D, ILC3 (c-Kit⁺IL-7Rα⁺) cells expressed little to no CD4 but highly expressed CD8 and CD90, consistent with descriptions in humans (1). Many ILC3 cells (mean 17.3%) also expressed NKp46, possibly representing a proportion of IL-22–producing NKp46⁺ cells. In comparison, the typical NK cell markers, including CD56, CD16, and NKG2A, were not expressed on ILC3 cells. To identify cytokine production in ILC3, the lymphocytes isolated from intestinal lamina propria were stimulated with phorbol 12-myristate-13-acetate plus ionomycin. As indicated in Fig. 2C, E, ILC3 cells were the major producers of IL-17, IL-22, and TNF-α, consistent with a previous report in mice (31). These results indicate that ILC3 cells in macaques can be defined by c-Kit⁺IL-7Rα⁺ expression to distinguish them from conventional NK-like cell populations.

Figure 2.

Phenotyping and cytokine-producing ability of ILC3 in lymphoid tissues of normal, uninfected rhesus macaques. A) ILC3 cells are confirmed in situ by coexpression of c-Kit (red) and AhR (green) but do not coexpress CD20 (blue) in macaque lymph nodes. Note that AhR is localized in the nucleus and cell plasma. B) Expression of CD4 and conventional NK cell-associated molecules on intestinal ILC3 subsets. C) Cytokine production of ILC3 following mitogen stimulation of intestinal lamina propria lymphocytes from uninfected macaques. D) Levels of NK markers on intestinal ILC3 cells in uninfected macaques (n = 10). E) Mean levels of cytokine production by intestinal ILC3 (n = 10). Note that ILC3 cells do not express CD4 or conventional NK cell markers and represent a distinct population of ILCs.

Profound loss of ILC3 cells occurs in lymphoid tissues in chronically SIV-infected rhesus macaques

To investigate the effects of HIV/SIV infection on ILC3 cells, we comparatively examined changes in percentages of ILC3 cells in peripheral blood and various lymphoid tissues in uninfected and SIV-infected animals (all >1 yr old). The results showed that ILC3 cells were significantly reduced in the jejunum lamina propria (5.4 ± 1.12%) and lymph nodes (6.1 ± 1.32%) in chronically SIV-infected rhesus macaques, compared with jejunum lamina propria (18.53 ± 2.3%) and lymph nodes (18.49 ± 3.6%) in uninfected animals (P < 0.0001). However, no reductions of ILC3 cells were observed in the blood between infected and uninfected groups (Fig. 3A). Because ILC3 cells are predominantly distributed in lymphoid tissues and are critical in the formation of lymphoid follicles and maintenance of intestinal integrity and function, the profound loss of ILC3 in SIV infection suggests that this may play a role in the loss of intestinal barrier integrity and mucosal immunity in HIV infection and AIDS.

Figure 3.

Loss of ILC3 cells in lymphoid tissues in chronically SIV-infected macaques. A) ILC3 cells were depleted in the jejunum and lymph nodes but not peripheral blood in asymptomatic chronic SIV infection, compared with uninfected controls. The loss of ILC3 positively correlated with reductions of CD4⁺ T cells (B), T_h17 (C), and T_h22 cells (D) in the intestine during SIV infection. Uninfected, n = 9; chronic, n = 9. All animals are over 1 yr of age.

Because we found a marked loss of ILC3 cells in the lymphoid tissues after SIV infection, we sought to determine whether this loss correlated with the overall CD4⁺ T-cell loss or losses of other lymphoid tissue integrity-associated CD4⁺ T cells (T_h17 and T_h22) in the intestine during SIV infection. The data showed that the loss of ILC3 cells positively correlated with reductions of the total CD4⁺ T cells (R², 0.78; P < 0.0001), T_h17 (R², 0.68; P < 0.0001), and T_h22 cells (R², 0.77; P < 0.0001) in intestinal mucosal tissues (Fig. 3B–D). Thus, despite the absence, or very low expression, of CD4, these cells are also depleted in SIV infection. Because HIV/SIV infection results in massive losses of intestinal CD4⁺ T cells, particularly IL-17/IL-22–producing cells, and damages the intestinal mucosal barrier (40, 41), the loss of ILC3 cells may be an additional contributing factor to the disruption of intestinal integrity and disease progression in AIDS.

Exposure to microbial products results in loss of ILC3 cell viability

Because ILC3 cells do not express CD4 (Fig. 2) and, thus, are not likely targets for SIV/HIV infection, we explored potential mechanisms by which the loss of ILC3 cells occurs. Specifically, we hypothesized that excessive stimulation by microbial products associated with microbial translocation induced by SIV infection could contribute to the ILC3 loss in lymphoid tissues. Thus, we examined levels of LPS in blood and intestine, expression of TLR2 and TLR4 on intestinal ILC3 cells, and finally investigated the effects of microbial products on ILC3 cell apoptosis upon TLR ligand stimulation in vitro. Here, we show that levels of peripheral and intestinal LPS significantly increased in the asymptomatic chronic stage, compared with the uninfected state (Fig. 4A), and ILC3 cells in intestine express TLR2 and TLR4 on their surface in healthy macaques (Fig. 4B). Furthermore, exposure to microbial products such as LTA or LPS that bind TLR2 or TLR4 ligands, respectively, could significantly induce apoptosis of intestinal ILC3 cells after 24 h stimulation in vitro, compared with medium controls and TLR9 ligand (ODN BW006) treatment. In contrast, TLR4 antagonist (LPS-RS) blocked the effects of LPS on TLR4-mediated ILC3 apoptosis (Fig. 4C, D). These data suggest that microbial products in HIV/SIV infection that breach the epithelial barrier due to loss of CD4⁺ T cells in general may promote the loss of ILC3 cells in the gastrointestinal tract, further disrupting mucosal immune structure and function (Fig. 4E), leading to even more damage to the mucosal barrier resulting in systemic immune activation and disease progression.

Figure 4.

ILC3 apoptosis mediated by microbial products through TLR. A) Levels of LPS in plasma (n = 9) and mucosal intestinal tissues (n = 6) in uninfected and chronically SIV-infected macaques. SIVmac, SIVmac251. B) Representative histogram of TLR2 and TLR4 expression on ILC3 cells in the jejunum. max, maximum. C and D) Histogram and statistics of ILC3 cell apoptosis induced by 24 h stimulation in vitro to microbial products: TLR9 ligand, ODN BW006; TLR2 ligand, LTA; and TLR4 ligand, LPS, or LPS plus TLR4 antagonist, LPS-RS. The results are from 4 different uninfected adult macaques, and all experiments were performed in triplicate. *P < 0.05, compared with medium control or between LPS and LPS-RS treatment. E) Schematic of possible mechanisms of ILC3 loss in GALT in SIV infection. Microbial products breaching the epithelium may result in exposure of underlying ILC3 cells to TLR ligands, resulting in ILC3 apoptosis and subsequent damage to organized lymphoid tissue structure and function, and further impairment of innate and humoral immune responses to HIV infection.

DISCUSSION

ILC3 cells play an important role in lymphoid tissue organogenesis, remodeling, and mucosal immunity in general. Here, we define ILC3 cells as Lin⁻CD45⁺c-Kit⁺IL-7Rα⁺ lymphocytes that are predominantly distributed in lymphoid tissues and distinct from conventional NK cells and CD3⁻CD8⁺ cells in rhesus macaques. These ILC3 cells lack CD4 and typical NK-associated molecules, but they produce considerable levels of IL-17 and IL-22, which are known to contribute to the maintenance of lymphoid tissue structure and mucosal barriers. HIV infection is associated with loss of intestinal barrier function, resulting in bacterial translocation, which drives systemic immune activation and disease progression (21, 22), but the early events involved in loss of mucosal barrier function remain uncertain. This study shows that SIV infection results in losses of ILC3 cells in lymphoid tissues, which may be a consequence of microbial products entering mucosal tissues and apoptosis through TLR pathways.

As a marker of ILC3 cells, c-Kit is also expressed on pluripotent hematopoietic progenitor cells (CD34⁺) and mast cell progenitors (FcεRI⁺) (42, 43). To exclude granulocytes (which include mast cells), ILC3 cells are first gated through lymphocytes that are negative for T, B, and monocyte markers. As shown in Fig.1A, remaining lineage-negative lymphocytes coexpressing c-Kit and IL-7Rα are negative for CD34 and FcεRI. Interestingly, these ILC3 cells are found in various lymphoid tissues, but not in peripheral blood. Furthermore, these cells are abundant at birth but gradually decrease with age until animals reach adulthood (Fig. 1C, D). These findings are consistent with reports that ILC3 cells, unlike T_h17 cells, develop in prenatal life, suggesting a critical role in lymphoid tissue development.

ILCs are a heterogeneous population that includes NK (ILC1), ILC2, and ILC3 cells. ILC3 cells in macaques, as lymphoid tissue initiators for colonization of secondary lymphoid development (18, 44), express little to no CD4 and other NK markers but produce IL-2, IL-22, and TNF-α (Fig. 2B–E). Notably, ILC3 cells are significantly and consistently reduced in lymphoid tissues including lymph nodes and GALT during persistent SIV infection, and their loss positively correlates with reductions of total CD4⁺ T cells, including the T_h17 and T_h22 cells in the intestine during SIV infection (Fig. 3). The loss of T_h17 and T_h22 cells has been correlated with the breakdown of mucosal integrity, microbial translocation, and chronic immune activation (22, 40, 41). Because ILC3 cells play critical roles in formation of lymphoid tissue, adaptive immunity, regulation of mucosal microbiota via AhR, restoration of secondary lymphoid tissues after injury, and maintenance of intestinal memory CD4 T cells (3, 17, 18, 45), their losses undoubtedly contribute to compromised mucosal immunity in HIV infection and AIDS, including failure of humoral immune responses.

Importantly, macaque ILC3 cells do not express CD4, consistent with humans (1), indicating that they are not direct targets for HIV/SIV infection. However, because persistent HIV/SIV infection results in disruption of GALT and increases intestinal permeability and microbial translocation, we hypothesized that increased exposure of intestinal ILC3 to microbial products alone may result in loss of these cells. As shown in Fig. 4A, levels of LPS in peripheral blood and intestine significantly increased in persistent SIV infection, and the intestinal ILC3 cells express both TLR2 and TLR4 (Fig. 4B), which specifically recognize microbial products including LTA and LPS, respectively. However, ILC3 cells induced significant apoptosis once exposed to TLR2 and/or TLR4 ligands in vitro compared with medium controls and TLR9 ligand treatment (Fig. 4C, D). Combined, these data suggest that TLR2 and TLR4 binding mediates apoptosis of intestinal ILC3 cells after exposure to microbial products that might be translocated into mucosal and lymphoid tissues during HIV/SIV infection. In the intestinal mucosa, ILC3 cells are in close proximity to the epithelial barrier and are likely among the first cells to encounter microbial products once the epithelial barrier is compromised. Although direct evidence showing that the loss of ILC3 correlates with intestinal mucosal damage in SIV-infected rhesus macaques is lacking, studies in mice suggest that once ILC3 cells are lost, the integrity and innate immunity of mucosal tissues will be further compromised (18, 46, 47), and our data showed that ILC3 cells positively correlated with intestinal T_h22 cells, which are closely associated with mucosal integrity (40, 48). Therefore, preservation or restoration of ILC3 cells during HIV infection may facilitate restoration of lymphoid tissues, and both innate and adaptive immune responses, and mitigate microbial translocation and the systemic immune activation that leads to disease progression in SIV/HIV (49).

Glossary

AhR

aryl hydrocarbon receptor

GALT

gut-associated lymphoid tissue

ILC

innate lymphoid cell

LTA

lipoteichoic acid

SIV

simian immunodeficiency virus

T_h

T helper