Mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) via breastfeeding is widely considered responsible for half of new infections of children with HIV (1
), including approximately 180,000 newly infected infants in 2017 alone (2
). As breastfeeding comes with a myriad of benefits and as advocating for formula feeding is not without its own hurdles and repercussions, understanding the interplay between the oral immune response and virus could play an important role in limiting transmission (3
). The oral immune network is continually exposed to diverse commensal microbial communities, airborne allergens/antigens, and foods and must react accordingly (4
). This dynamic environment requires tight orchestration between resident immune and epithelial cells to maintain a balance between successful immune surveillance and toleration of harmless antigens and commensals (5
). HIV/simian immunodeficiency virus (SIV) infection disrupts this delicate balance, altering the immune cell frequencies and phenotypes and resulting in a more extensively proinflammatory environment prone to opportunistic diseases (6
NK cells represent a heterogeneous cell population and an integral part of the innate immune response against viral infections. NK cells mediate protection by rapidly killing infected cells and affecting the adaptive immune response through the secretion of cytokines (8
). The rapid protection afforded by NK cells suggests a capacity to limit perinatal transmission from infected mother to newborn. In vitro
experiments have demonstrated that, through the copious secretion of the CC chemokines CCL3, CCL4, and CCL5 (ligands for CCR5), NK cells are able to suppress entry of the virus into target cells by competitive inhibition (9
). Studies in HIV-infected children have shown elevated expression of NK cell activating and stimulatory receptors (10
). NK cells are capable of performing potent cytolytic functions and secreting proinflammatory cytokines and represent a subset of group 1 innate lymphoid cells (ILC). ILC constitute a heterogenous population of innate immune cells. Although they lack somatic rearrangement of antigen receptors, the members of this diverse family of tissue-resident, transcriptionally poised effector lymphocytes are capable of rapidly responding to tissue damage-associated danger signals and microbially induced signals by producing cytokines that promote pathogen killing (12
). Group 3 ILC (ILC3), a subset of the innate lymphoid cell family, play a critical role in the maintenance of tolerance and immune homeostasis in the gastrointestinal (GI) tract of mammals (13
). They are characterized by their expression of the transcriptional factor retinoic acid-related orphan receptor gamma isoform t (RORγt) and of aryl hydrocarbon receptor (AHR) and by their lack of machinery to directly sense microbial patterns (i.e., those associated with Toll-like receptors [TLR]) (14
). ILC3 are able to rapidly respond to tissue damage-associated danger signals and microbe-induced signals via prolific cytokine secretion, especially secretion of interleukin-17 (IL-17) and IL-22 (13
). IL-22 plays critical roles in prevention of inappropriate immune responses to microbial and environmental antigens, which can most clearly be described in the maintenance of healthy host interactions with commensal bacteria at mucosal surfaces (15
). IL-17 has been shown to synergize with IL-22 to augment production of microbial peptides (16
ILC3’s central role as healthy gut mediators can be knocked out of balance by a variety of factors, including genetic and environmental factors and, notably, viral infection (15
). Previous studies showed that HIV/SIV infections in particular cause massive loss of ILC3 and drive disruptions of tolerance and intestinal barrier integrity (17
). HIV-1 infection is linked with translocation of commensal bacteria and breakdown of the epithelium, leading to systemic inflammation (15
). In support of this, we and others have previously shown that acute SIV infection in adult macaques causes rapid depletion of ILC3 in the GI tract (19
). Notably, HIV/SIV infection does not seem to directly cause depletion of ILC3 but instead alters the survival signals of ILCs, leading to a dramatic increase in apoptosis (21
). Interestingly, ILC3 also undergo a functional shift during infection, moving from a tissue maintenance phenotype toward a proinflammatory profile with increased secretion of gamma interferon (IFN-γ), macrophage inflammatory protein 1 beta (MIP-1β), and tumor necrosis factor alpha (TNF-α) and decreased secretion of IL-17 and IL-22 (22
). Similarly, HIV/SIV viremia also induces functional and phenotypic changes in NK cells (9
). Previous evidence indicated that lentiviral infection in pediatric populations actuated a reduction in cytotoxic function, as measured by the level of the degranulation marker CD107a, but an increase in the frequency of NK cells expressing activating receptors (24
The use of nonhuman primates (NHP) in experimental infection studies has been paramount in studies of HIV/SIV (25
). In an effort to better understand the mechanics of postnatal HIV transmission in human infants, members of our laboratory performed a comprehensive study of ILC3 and NK cells in SIV/SHIV-infected infant rhesus macaques (RM). The development of next-generation simian-human immunodeficiency viruses (SHIV) enables a more precise investigation of ontological differences in immune responses directed toward HIV between infants and adults. SHIV.C.CH505, a SHIV with enhanced CD4 binding and replication in RM, and SHIV-1157ipd3N4, an exclusively R5-tropic mucosally transmissible SHIV that induces high peak viral RNA loads, as well the traditionally used SIVmac251
strain, were chosen for this study as infection models (26
To the best of our knowledge, this report presents the first comprehensive analysis of the impact of SIV/SHIV infection on ILC3 and NK cells in the oral mucosae and GI tissues of infant RM. We observed a substantial depletion of ILC3 in the colon of infected animals that contrasted with an expansion of both ILC3 and NK cells in various compartments of the oropharyngeal mucosae. This expansion can be partially explained by alterations to trafficking and chemokine receptors, specifically, CD62L, CD103, and CXCR3. Furthermore, we found that ILC3 and NK cells displayed tissue-specific functional repertoires that were perturbed by infection.
Previously, we reported that ILC3 were depleted in the colon during both acute and chronic SIV infections of adult RM but that that response was followed by partial expansion in the oral mucosa (22
). Since those initial findings were published, others have corroborated our results in the GI mucosae of SIV-infected RM and persons living with HIV (PLWH) (33
). Multiple analyses revealed that this loss was due in part to increased concentrations of local inflammatory cytokines (19
). We saw similar results in orally infected infant RM; however, due to the limited sample availability typical of infant studies, we were unable to confirm whether the full mechanism found in adults was recapitulated (Fig. 3A
). This expansion in select tissues in the oral mucosa can also be partially explained by alterations in trafficking and in the chemokine signature induced by infection. Although the data were not statistically significant, we saw specific increases in CD62L and CXCR3 expression. Increases in CXCR3 levels result in recruitment of a variety of immune cells to sites of inflammation, as it binds to the chemokine CXCL10. Both CXCR3 and CXCL10 are upregulated in HIV and SIV infections and may redirect ILC3 and NK cell migration and accumulation (35
Since their initial description, the role of ILC3 in maintaining mucosal homeostasis and epithelial integrity has been repeatedly demonstrated (13
). Thus, it is important to understand how lentiviral infection impacts their capacity to fulfill those functions. Indeed, our data in infant RM support previous results reflecting this role, as we found that ILC3 from the SubLN, tonsil, and colon all produced significant amounts of IL-17 and IL-22, tissue-signaling cytokines that support protection and regeneration of the epithelial barrier and also protect the cells from opportunistic pathogens (36
). Not surprisingly, infection abrogated ILC3 homeostatic functions, with results showing an antimicrobial functional profile, secretion of higher levels of MIP-1β, IFN-γ, and upregulation of CD107a (Fig. 7A
). Indeed, our data also revealed a systemic loss of IL-22 production, but not IL-17 production, by ILC3 in the tonsil and colon of infected RM. This disparity supports our finding of sustained RORγt expression in ILC3 during infection, as IL-17 expression is directly mediated by RORγt protein binding to IL-17 gene promoter and/or regulatory elements (37
). Meanwhile, IL-22 expression is also dependent on the transcription factor AHR, whose ligands include environmental toxins and endogenous ligands, which indicates that SIV/SHIV infection might disrupt the microenvironment in which sampling of ILC3 occurred in specific tissues (38
). Interestingly, and similarly to our findings in adult RM showing that ILC3 dysregulation is compartmentalized (22
), ILC3 in the SubLN did not demonstrate this change in function.
In partial contrast to our previous findings in adult RM (22
), we found a significant expansion of the NK cell population in the tonsil and SubLN in SHIV/SIV-infected infants (Fig. 3B
). However, this increased magnitude did not appear to be associated with protection as there was no relationship between NK cell frequencies in tissues and either peak viral load or viral load at necropsy (data not shown). Of note, this increased magnitude could have been a result of an altered trafficking repertoire, as an increase of CD103 expression has been shown previously to play an important role in the retention of NK cells and CTL in the oral mucosa (39
). Interestingly, Woodberry and colleagues had shown that CD103 expression was linked with a higher reactivity, corroborating earlier findings obtained in our laboratory indicating that infection increased frequencies of activating receptors on NK cells (22
). Similarly to ILC3, NK cells from the SubLN, tonsil, and colon displayed compartmentalized functions. Colon NK cells exhibited a higher propensity for a cytotoxic phenotype than those from the oral mucosa, and that propensity was maintained during infection, although NK cells from all tissues exhibited a competent four-function response (TNF-α, IFN-γ, MIP-1β, and CD107a) (Fig. 8A
In summary, our study revealed that acute SIV/SHIV infection has significant effects on the frequency, phenotype, and function of innate cells in the oral and gut mucosa. As ILC3 and NK cells are critical populations in the protection and maintenance of the oral mucosa, understanding how lentiviral infection perturbs their functional niches can provide a better understanding of how opportunistic diseases/coinfections may exploit these changes the oral mucosae during HIV infection. This is particularly relevant in infants infected with HIV, where the immune system is not fully functional and the effects are further compounded by the presence of the virus itself. Whether this is attributable to an altered microbiome, chronic inflammation, or some other mechanism is underdetermined and warrants further study into the underlying consequences of oral innate perturbation and its clinical significance.
MATERIALS AND METHODS
Animals and SHIV/SIV infections.
This study utilized samples from previous studies, and no animals were acquired specifically for the analyses described here. The animals were housed and all experimental procedures were performed at the California National Primate Research Center (CNPRC), a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). All animal care was performed in compliance with the Guide for the Care and Use of Laboratory Animals provided by the Institute for Laboratory Animal Research (2011) and the Weatherall report entitled “The use of nonhuman primates in research.” The studies were approved by the Institutional Animal Care and Use Committee of the University of California, Davis (protocols 18650, 18655, and 19779). The macaques were kept in indoor housing in stainless steel cages (Lab Product, Inc.), were exposed to a 12-h light/12-h dark cycle (65 to 75°F, 30% to 70% room humidity), and received enrichment. Animals had free access to water and initially received infant formula; the infant formula was gradually replaced with commercial chow (high protein diet; Ralston Purina Co.) and fresh fruit and vegetable supplements. When necessary, macaques were immobilized with ketamine HCl (Parke-Davis) at approximately 10 mg/kg of body weight and injected intramuscularly after overnight fasting. Blood samples were collected using venipuncture. Animals were euthanized with an overdose of pentobarbital, followed by necropsy with extensive tissue collection.
A total of 35 rhesus macaques (Macaca mulatta
) were analyzed in this study, including 20 naive animals, 4 SHIV.C.CH505.375H.dCT-infected animals, 6 SHIV1157ipd3N4-infected animals, and 5 SIVmac251-infected animals. The animals were of Indian origin and from the type D retrovirus-free, SIV-free, and STLV-1-free colony of the California National Primate Center (CNPRC; Davis, CA). Animals were kept under conditions that complied with American Association for Accreditation of Laboratory Animal Care standards and the Guide for the Care and Use of Laboratory Animals (41
The generation of SHIV.C.CH505.375H.dCT and SHIV1157ipd3N4 was previously described (27
). SHIV.C.CH505.375H.dCT and SHIV1157ipd3N4 challenge stocks (provided by George M. Shaw, University of Pennsylvania, and Ruth Ruprecht, Southwest National Primate Research Center, respectively) and SIVmac251
were used to orally challenge infant macaques. A summary of animals, viruses, and virologic outcomes in this study is provided as Table 1
. All animals were euthanized at between 18 and 42 weeks of age. Tissues were collected from colon, tonsil, and submandibular, submental, cervical, and retropharyngeal lymph nodes.
Processing of tissues was carried out using protocols optimized in our laboratory (28
). Briefly, colon tissue was cut into 1-cm2
sections and then incubated in 5 mM EDTA for 30 min before undergoing mechanical and enzymatic disruption. Samples were then gently pushed through filters before lymphocytes were isolated via the use of a bilayer (35%/60%) isotonic Percoll density gradient. Oral lymph nodes were trimmed of excess tissue and then mechanically disrupted. Manual cell counts were performed for each sample using trypan blue. Two million cells from each sample were then used for real-time flow cytometry staining, and the remaining cells were cryopreserved in a dimethyl sulfoxide (DMSO) solution and stored in liquid nitrogen vapor.
Antibodies and flow cytometry.
Flow cytometry staining of mononuclear cells was carried out for cell surface molecules (see Table S1 in the supplemental material). Briefly, thawed samples were incubated with LIVE/DEAD Aqua amine dye (Invitrogen, Carlsbad, CA) and were then washed before staining with surface antibodies was performed. After staining, the cells were permeabilized using a Thermo Fisher Scientific Fix & Perm buffer kit (Thermo Fisher Scientific, Waltham, MA) and incubated with the intracellular staining antibodies. Isotype-matched controls were included in all assays. All acquisitions were made on an LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo (10.5.3). Intracellular expression of the transcription factor RORγt was evaluated using the FoxP3 buffer set protocol (BD, Bedford, MA).
We analyzed multiple functions of ILC3 and NK cells ex vivo following mitogen stimulation (cell activation cocktail; BioLegend, San Diego, CA). Mononuclear cells were stimulated with cell activation cocktail, and anti-CD107a, GolgiPlug (brefeldin A) (Becton, Dickinson, Franklin Lakes, NJ), and GolgiStop (monensin) (Becton, Dickinson) were added directly into each of the tubes; unstimulated samples served as controls. All samples were then cultured for 12 h at 37°C in 5% CO2. After incubation, samples were stained for surface and intracellular markers to delineate cell phenotype and function. The percentage of positive cells was calculated by subtracting the baseline cytokine expression in control samples.
Tissue sections (5 μm) were seqentially cut and stained for CD3 and IL-17 as previously described (44
). In situ
hybridization was used to detect RORc mRNA via the use of a 1-Plex ViewRNA ISH tissue assay kit and SIVmac251
or beta actin (positive control) ViewRNA probe sets and a ViewRNA chromogenic signal amplification kit (all from Thermo Fisher, Waltham, MA) (44
). The slides were imaged with a Zeiss AxioObserver microscope and an AxioCam MRm camera. Composite overlays of CD3/IL-17-stained slides with ISH slides were prepared using Zen Lite v2.3 software (Zeiss). Quantitative measurements were obtained using ImageJ software (https://imagej.net/
). Cells were assessed for CD3, IL-17, and RORc mRNA and enumerated by hand using the Cell Counter Plugin for ImageJ (K. De Vos; https://imagej.nih.gov/ij/plugins/cell-counter.html
Viral load quantification.
Following initial challenge, virological analysis of weekly plasma samples was performed using reverse transcription-PCR (RT-PCR) for SIV/SHIV RNA as previously described (47
) but with manual RNA extraction due to limited volumes. The limit of detection was 15 copies/ml. Samples showing transient low viremia followed by SIV/SHIV RNA-negative time points were retested to confirm the initial PCR results. Data are reported as the number of SIV/SHIV RNA copy equivalents per milliliter of plasma.
Statistical analyses were carried out using Prism Version 7.0d (GraphPad Software) and SPICE Version 6.0 (48
). Unpaired, nonparametric, Mann-Whitney U
tests and permutation tests were used where indicated, and P
values of <0.05 were assumed to be significant. t
-distributed stochastic neighbor embedding) analysis was performed using CytoDRAV (https://github.com/ReevesLab/CytoDRAV