Probiotic supplementation reduces inflammatory profiles but does not prevent oral immune perturbations during SIV infection

HIV/SIV infections lead to massive loss of mucosal CD4 + T cells and breakdown of the epithelial mucosa resulting in severe microbial dysbiosis and chronic immune activation that ultimately drive disease progression. Moreover, disruption of one of the most understudied mucosal environments, the oral cavity, during HIV-induced immunosuppression results in significant microbial and neoplastic co-morbidities and contributes to and predicts distal disease complications. In this study we evaluated the effects of oral probiotic supplementation (PBX), which can stimulate and augment inflammatory or anti-inflammatory pathways, on early SIV infection of rhesus macaques. Our study revealed that similar to the GI mucosae, oral CD4 + T cells were rapidly depleted, and as one of the first comprehensive analyses of the oral microflora in SIV infection, we also observed significant modulation among two genera, Porphyromonas and Actinobacillus, early after infection. Interestingly, although PBX therapy did not substantially protect against oral dysbiosis or ameliorate cell loss, it did somewhat dampen inflammation and T cell activation. Collectively, these data provide one of the most comprehensive evaluations of SIV-induced changes in oral microbiome and CD4 + T cell populations, and also suggest that oral PBX may have some anti-inflammatory properties in lentivirus infections.


Scientific Reports
| (2021) 11:14507 | https://doi.org/10.1038/s41598-021-93918-x www.nature.com/scientificreports/ the mechanisms are not entirely clear for oral diseases in HIV/SIV infections, SIV-induced dysbiosis of the oral microbiome does result in increased production of cytokines and other soluble inflammatory mediators in the oral cavity, while simultaneously suppressing antimicrobial functions and epithelial development 27 . So called innate lymphoid cells (ILC) are identifiable by their lack of common lineage markers (those identifying T cells, B cells, NK cells, and myeloid cells), and can be distinguished by high expression of the IL-7 receptor, CD127 [28][29][30][31] . ILC3 in particular play a major role in modulating gut homeostasis, but can be disrupted by changes in the inflammatory microenvironment, infectious disease, and shifts in the microbiome 32 . HIV and SIV infections drive a perturbation of ILC3 resulting in loss of intestinal barrier integrity, increased inflammation and potential dysregulation of the local microflora. Specifically, others and we have shown that acute SIV infection causes depletion of ILC3 in the GI mucosae [33][34][35] . In PLWH, ILCs are depleted but partially restored by ART 36 . Much like had been shown in SIV-infected macaque models loss of ILC3 is associated with microbial translocation. Neither HIV nor SIV infection directly depletes ILC3, but rather changes in the cytokine milieu and local microbiotic signals lead to increased apoptosis 33,34,36 . Importantly, we have previously shown that probiotic therapy improves IL-23 production and as a result expands ILC3 numbers in naïve macaques 37 .
It has been shown that HIV alters human microbiome including oral bacteria communities 38 . Periodontal disease, a common oral inflammatory infection in HIV infected patients, has been shown to be associated with general dysbiosis in the oral cavity, as well as with HIV disease progression 39 . Dysbiosis in the oral cavity and changes to the oral bacterial communities might play a role in the sustained host proinflammatory responses to the virus. Restorative therapies that could improve oral and GI function, inflammation, and microbial health could be of significant benefit to PLWH. Indeed, previous studies by our groups have demonstrated that therapy with probiotic, beneficial bacteria can improve mucosal health and dampen inflammation in healthy macaques as well as in the context of SIV infection 37,40,41 . Thus, we hypothesized that probiotic supplementation prior to SIV infection and during acute infection may enhance the oral microbiome and prevent pathogenic consequences of SIV. In this study we evaluated the effects of pre-and acute administration of oral probiotic therapy on viral kinetics, inflammation, CD4 + T cell loss, microbiome, and associated lymphoid tissues in SIV-infected rhesus macaques.

Results and discussion
Early probiotic administration during SIV infection. To determine the effects of probiotic (PBX) administration pre-SIV and during acute SIV infection, we evaluated 12 Indian origin rhesus macaques, 6 receiving oral PBX and 6 receiving oral vehicle control. Animals were given PBX for a total of 42 days: 28 days pre-virus challenge and for a subsequent 14 days following challenge (Fig. 1a). Macaques were intrarectally chal-  www.nature.com/scientificreports/ lenged with SIVmac251 as described previously 42 , and buccal, colorectal, and lymph node biopsies in addition to blood draws were collected as indicated. No significant differences were observed in plasma viral loads longitudinally (Fig. 1b,c), nor were any differences observed in peak viral loads between the two groups ( Fig. 1d). However, previous studies have also reported that the benefits of probiotic therapy in HIV and SIV are uncoupled from viremia which is generally not affected 40 .
T cell responses during PBX treatment of SIV-infected macaques. To assess broad antiviral T cell responses we performed longitudinal ELISPOT assays on PBMC as well as tissues at necropsy. Interestingly, PBX-treated animals consistently had lower levels of both GAG (fourfold at day 112) and ENV (ninefold at Day 112) ELISPOT responses (Fig. 2a,b). Further, the total magnitude of the ELISPOT response (as measured POL, ENV, GAG) in control animals was significantly greater at necropsy in all tissues measured (PBMC, spleen, oral lymph nodes, and tonsil) (Fig. 2c).
Since SIV infection has a significant impact on memory CD4 + T cell populations, as well as CD8 + T cell activation, we next used polychromatic flow cytometry to evaluate these cell subsets in detail in PBX-treated animals. As expected, the frequency of total CD4 + T cells in circulation declined early following SIV challenge concomitant with an increase in the frequency of CD8 + T cells (Fig. 3a). Similar trends were observed in colorectal tissue where the shift was even more robust (Fig. 3b), in line with previous studies 43,44 . Remarkably, this study demonstrated for the first time an acute and sustained loss of CD4 + T cells in the buccal mucosa following SIV infection (Fig. 3c). However, little to no difference was observed in CD4 + and CD8 + T cell dynamics in any tissue when comparing PBX-treated to control animals, including lymph nodes (Fig. 3d). There was also little difference between the two groups when specifically examining CD28 + CD95 + central memory CD4 + T cells in any tissue (Fig. 4a). However, PBX treatment reduced Ki67 expression (measured at day 42 PBX/Day 14 post-challenge) in both bulk and CM CD4 + T cells in blood and colonic tissue (Fig. 4b). Interestingly, proliferation was not altered in the bucca by PBX. Although it is difficult to speculate, this could be due to differences in local flora, immune cell composition, but also likely due to time of exposure. The probiotics move more rapidly through the oral mucosae than they do through distal mucosae, so this could reduce some effects. Nonetheless, these data suggest that while PBX may not protect CD4 + T cell loss, it may reduce T cell activation, which is well in line with previous observations 37 . Thus, PBX could be a beneficial augmentive therapy to protect CD4 + T cells and reduce overall cellular activation.

Effects of probiotic treatment on ILC3 populations during SIV infection. We and others have
previously shown that HIV and SIV infection can modulate ILC3 populations in the mucosae. Further, our www.nature.com/scientificreports/ groups have demonstrated that probiotic supplementation can actually expand ILC3 numbers in some animals and could possibly function as a putative rheostat mechanism of action for controlling inflammation during lentivirus infections. In this study we quantified the frequency of Lin-NKp44 + NKG2A/C-ILC3, as described   34,45 , in longitudinal colorectal and buccal biopsies. Interestingly, little change was observed in ILC3 frequencies during the probiotic supplementation period (day 0 to day 28) in this study (Fig. 5). Although some    14 (colorectal) or day 28 (oral) following probiotic supplementation, these changes were not significant. It is unclear why ILC3 might not be expanded in this context, but previous work from our laboratory has shown that colorectal ILC3 are less likely to be increased compared to those in the jejunum and there is significant animal-to-animal variation 37 . Overall, this study suggested in contrast to our previous pilot work that PBX may only marginally impact SIV-induced changes in ILC3 frequencies, indicating that while PBX may impact T cell responses, it is unlikely to protect against loss of ILC3.

Soluble measures of inflammation and immune activation.
To further address the impact that PBX and ILC3 modulation might be having on global inflammation during SIV infection we compared soluble inflammatory mediators in the circulation by Luminex. Animals treated with PBX had a distinctly lower inflammatory profile on day 28 (day of challenge) as indicated by both analyte expression (heatmaps) and PCA, suggesting that PBX treatment alone was sufficient to decrease soluble mediators of inflammation (Fig. 6a, Supplementary Fig. 2, Supplementary Table 2). This reduced inflammatory profile persisted from day 42 (day 14 post-challenge), and even persisted in some animals up to day 112 (day 84 post-challenge. However, PBX therapy did not appear to have any effect on microbial translocation products (Fig. 6b) which is similar to HIV studies showing improved immune function but no consistent decline in circulating bacterial products 46,47 .

Probiotic supplementation partially modulates oral microbiome during SIV infection.
To study the effects of PBX on the microbiome, we used 16 s rRNA gene sequencing to profile bacterial communities in the buccal and colonic mucosa. No notable differences in community richness, evenness, Shannon diversity or over all principal components analysis were observed between groups in either oral or gut samples, consistent with previous studies of probiotic supplementation. The most abundant bacteria in the oral mucosa of all animals were of Firmicutes phyla, with lesser representation of Bacteroidetes, Proteobacteria, Fusobacteria, and Actinobacteria (not shown). The colorectal mucosa top phlya was Epsilonbacteraeota followed by Bacteroidetes and Spirochaetes (not shown). At the phyla level, no notable differences between groups or across time points were observed in either site. At the genus level for the oral mucosa, a high abundance of Streptococcus and Veillonella was observed in all samples, followed by minor abundances of Alloprevotella, Carnobacterium, Actin-   (Fig. 7a). The top genera for colorectal samples were dominated by Helicobacter with an abundance of fifty-five percent followed by Prevotella, Treponema, Alloprevotella, Ruminococcaceae, Camplobacter with abundances starting at nine percent (Fig. 7b). We performed differential abundance analysis on the PBX and placebo groups across all timepoints in the oral mucosae. In the placebo group, Porphyromonas and Actinobacillus were found to be significant between day -21 and 0 (p = 0.012 and 0.021), day -21 and day 28 (p = 0.028), and day 28 and day 42 (p = 0.004 and 0.001) (Fig. 7c,d). In the PBX group, Porphyromonas and Actinobacillus were significant between day -21 and day 28 (p = 0.04) and day 42 and day 112 (p = 0.037 and p = 0.017) (Fig. 7d). However, notable differences were not observed between groups, but it does appear that the changes observed were dampened in the probiotics group compared to the placebo group. Thus, while probiotics did not have a substantial impact in modulation of the microbiome, these changes reflect possible dampening of SIV-mediated dysbiosis in the oral microbiome. It is also important to note that this study used a general PBX formulation. Future studies using formulations more specific to individual sections of the GI tract could have more targeted impacts.

Animals, probiotic supplementation and SIV challenge. Sixteen experimentally naive age-matched
Indian-origin rhesus macaques (Macaca mulatta) were analyzed in this study. All animals were colony-housed at Biomere, Inc. and were free of simian retrovirus type D and simian T-lymphotropic virus type 1. Animals were challenged intrarectally with SIVmac251 as described previously 42  Blood and tissue processing. Blood samples were collected in EDTA-treated tubes, and peripheral mononuclear cells (PBMCs) were isolated using standard density gradient centrifugation over lymphocyte separation media (MP Biomedicals, Solon, OH) and any contaminating red blood cells were lysed using hypotonic ammonium chloride solution. Processing of tissues was carried out using protocols optimized in our laboratory 27,30,41 . www.nature.com/scientificreports/ Briefly, colon and buccal tissue were cut into 1 cm 2 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 bilayer (35%/60%) isotonic Percoll density gradient. Lymph nodes and spleen were trimmed of excess tissue 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 while remaining cells were cryopreserved in a DMSO solution and stored in liquid nitrogen vapor. Antibodies and flow cytometry. Flow cytometry staining of mononuclear cells was carried out for cell surface and intracellular molecules (Supplementary Table 1). Briefly, samples were incubated with the LIVE/ DEAD Aqua amine dye (Invitrogen, Carlsbad, CA), then washed before staining with surface antibodies. After staining, cells were permeabilized using Thermo Fisher Scientific Fix & Perm buffer kit (ThermoFisher Scientific, Waltham, MA) and incubated with the intracellular staining antibodies. Isotype-matched controls were included in all assays. All acquisitions were made on an LSRII (BD Biosciences) and analyzed using FlowJo (9.9.6).

Viral load quantification.
Quantified RNA was used for RT-PCR against a conserved region of gag using gene-specific primers 48 . In the first step, RNA was reverse-transcribed followed by treatment with RNase for 20 min at 37 °C. Next, cDNA was amplified using 7300 ABI Real-Time PCR system (applied Biosystems) according to the manufacturer's protocol.
DNA extraction, 16S rRNA gene sequencing and data analysis. We extracted DNA from cryopreserved buccal and colorectal biopsies using the PowerFecal DNA Isolation Kit (Qiagen, Valencia, CA). We then prepared sequencing libraries as described by the Earth Microbiome Project and sequenced them using the Illumina MiSeq Sequencer (Illumina, San Diego, CA). 515F-806R primers were used to sequence the V3-V4 region of the 16 s SSU rRNA as previously described 49 . 16 s sequencing reads were demultiplexed, trimmed and then processed using QIIME2 (version 2020.2) 50 . Operational taxonomic units (OTUs) were clustered at 99% similarity using the DADA2 method and assigned taxonomy with the Silva 132 classifier for taxonomic determination. All sequence reads and operational taxonomic unit (OTU) observations were included in our analyses, in order to maximize the observed diversity of the bacterial communities and was done utilizing R (v3.6.0) packages. Alpha diversity (richness, evenness (Pielou)), and Shannon diversity) was calculated by the microbiome package, beta diversity was calculated with pairwise sample dissimilarity and unweighted unifrac ordination analysis by means of principal coordinates analysis (PCoA) using the vegan package for Ordination, Diversity and Dissimilarities. Statistical analyses were completed using the Pairwise Adonis package. Differential abundance analysis was completed using DESeq2. Beta diversity and taxonomic plots were created utilizing the phyloseq and ggplot. Alpha diversity and differential abundance plots were created in Prism 8 version 8.4.3 (GraphPad Software).

Statistical analyses.
Statistical analyses were carried out using Prism Version 8.3.0 (GraphPad Software).
Unpaired, nonparametric, Mann-Whitney U tests, Wilcoxon, and t tests were used where indicated, and P < 0.05 were assumed to be significant. Samples were grouped and heatmaps were generated in Prism versions 7.0d, 8.3.0, or 8.4.3 (GraphPad Software; www. graph pad. com/ scien tific-softw are/ prism). For beta diversity, statistical significance was assessed using the pairwise permutational multivariate analysis of variance (PERMANOVA; Adonis) test in R. For taxonomic differential abundance DESeq2 was used and accounted for sequencing depth and multiple comparisons using the Benjamini and Hochberg method to control the false discovery rate (FDR) and adjusted p values of < 0.05 were considered significant.
Multiplex cytokine analysis of plasma. Plasma samples were analyzed for rhesus macaque cytokines and chemokines by Luminex using established protocols for non-human primates. Evaluation of analytes IL-6, IL-12p70, IL-17, TNF-α, IL-2, G-CSF, CXCL13, MIP-1β, IP-10, IL-1Ra, Perforin, IFN-α, IFN-γ, MCP-1, MIG, Il-23 and MIF were included in this assay. Only analytes quantifiable above the limit of detection in plasma are presented. Log2 fold change for Luminex data was calculated per rhesus macaque in this study. Briefly, each rhesus macaque timepoint log2 fold change was calculated using the following formula: NaN or Inf values were replaced with NA. Log2 fold change was calculated in R v4.0.0 (https:// www.R-proje ct. org) and heatmaps were generated with pheatmap v. 1.0.12 (https:// CRAN.R-proje ct. org/ packa ge= pheat map). Row clustering was performed on cytokine log2 fold changes with default parameters. Column clustering was not performed.