Distinct Intestinal Microbial Signatures Linked to Accelerated Biological Aging in People with HIV

Background People with HIV (PWH), even with controlled viral replication through antiretroviral therapy (ART), experience persistent inflammation. This is partly due to intestinal microbial dysbiosis and translocation. Such ongoing inflammation may lead to the development of non-AIDS-related aging-associated comorbidities. However, there remains uncertainty regarding whether HIV affects the biological age of the intestines and whether microbial dysbiosis and translocation influence the biological aging process in PWH on ART. To fill this knowledge gap, we utilized a systems biology approach, analyzing colon and ileal biopsies, blood samples, and stool specimens from PWH on ART and their matched HIV-negative counterparts. Results Despite having similar chronological ages, PWH on ART exhibit accelerated biological aging in the colon, ileum, and blood, as measured by various epigenetic aging clocks, compared to HIV-negative controls. Investigating the relationship between microbial translocation and biological aging, PWH on ART had decreased levels of tight junction proteins in the colon and ileum, along with increased microbial translocation. This increased intestinal permeability correlated with faster intestinal and systemic biological aging, as well as increased systemic inflammation. When investigating the relationship between microbial dysbiosis and biological aging, the intestines of PWH on ART had higher abundance of specific pro-inflammatory bacterial genera, such as Catenibacterium and Prevotella. These bacteria significantly correlated with accelerated local and systemic biological aging. Conversely, the intestines of PWH on ART had lower abundance of bacterial genera known for producing short-chain fatty acids and exhibiting anti-inflammatory properties, such as Subdoligranulum and Erysipelotrichaceae, and these bacteria taxa were associated with slower biological aging. Correlation networks revealed significant links between specific microbial genera in the colon and ileum (but not in feces), increased aging, a rise in pro-inflammatory microbial-related metabolites (e.g., those in the tryptophan metabolism pathway), and a decrease in anti-inflammatory metabolites like hippuric acid and oleic acid. Conclusions We identified a specific microbial composition and microbiome-related metabolic pathways that are intertwined with both intestinal and systemic biological aging in PWH on ART. A deeper understanding of the mechanisms underlying these connections could potentially offer strategies to counteract premature aging and its associated health complications in PWH.


INTRODUCTION
The gastrointestinal (GI) tract plays a crucial role in the pathogenesis and persistence of HIV infection [1].
Several studies have indicated that HIV-related microbial translocation may be exacerbated by changes in the composition and diversity of the gut microbiome (microbial dysbiosis).This dysbiosis involves the depletion of putative, anti-in ammatory bene cial bacteria and/or the accumulation of putative, proin ammatory harmful bacteria.Among the bene cial bacteria are those that produce short-chain fatty acids (SCFAs) -key metabolites fortifying the intestinal barrier [23][24][25][26][27][28][29].In people with HIV (PWH), some dysbiosis can be attributed to behaviors like sexual activity [30], however, recent ndings show that HIVassociated dysbiosis can be independent of sexual orientation [31].This HIV-associated microbial dysbiosis is associated with greater systemic in ammation, exacerbated disease progression, and a rise in in ammation and aging-linked conditions [31].
The increased occurrence of in ammation-and age-associated comorbidities in PWH has prompted recent investigations into whether HIV accelerates the process of biological aging.Recent reports have indeed shown evidence of accelerated or heightened biological aging in the blood of PWH [32][33][34][35][36][37].However, these studies have focused on blood cells, despite the crucial role of the intestines in regulating chronic in ammation in PWH.Thus, it remains unclear whether HIV, even when controlled with ART, speci cally affects the aging process in the intestines.Additionally, it's unknown whether microbial dysbiosis and translocation contribute to the systemic acceleration of biological aging in PWH on ART.
This study aims to address these two knowledge gaps.

METHODS
Study Cohort.Ileum and colon biopsies, blood, and stool samples were collected from 25 PWH on suppressive ART and 23 age-, sex-, BMI-, and ethnicity-matched HIV-negative controls at Rush University Medical Centre.All participants provided informed written consent.Each participant lled out a detailed questionnaire about their demographics, medical history, and ART regimen.Additional details about the study cohort are available in Table 1.The study protocol was approved by the Institutional Review Board at Rush University (ORA# 19020710).
DNA and RNA Isolation.DNA and RNA from colon and ileum biopsies were isolated using the AllPrep DNA/RNA/Protein mini kit (Qiagen, catalog #80004).Brie y, the biopsies were homogenized in RLT Lysis buffer (Allprep isolation kit, Qiagen, catalog #80204) using stainless-steel beads (5 mm, Qiagen, Catalog #69989) on a Qiagen TissueLyser II.Total RNA and DNA were simultaneously puri ed from the tissue lysates following the manufacturer's protocol.On-column DNAse digestion was performed during the RNA extraction.Quanti cation of DNA and RNA was performed using a Nanodrop (ND-1000) spectrophotometer.
Quanti cation of DNA Methylation and Epigenetic Clock Calculations.300 ng of DNA per sample was bisul te-converted using the EZ DNA Methylation kit (Zymo Research) following the manufacturer's instructions.Bisul te-converted DNA samples were then randomly assigned to a chip well on the In nium HumanMethylationEPIC v1.0 BeadChip, ampli ed, hybridized onto the array, stained, washed, and imaged with the Illumina iScan SQ instrument to obtain raw image intensities.Raw Methylation EPIC array IDAT intensity data were loaded and preprocessed in the R statistical programming language (http://www.rproject.org)using the SeSAMe analysis suite R package [38].Epigenetic estimates for Horvath's multitissue predictor DNAmAge based on 353 CpG sites [39], the Horvath skin-and-blood clock based on 391 CpG sites [40], Levine DNAmPhenoAge based on 513 CpG sites [41], Hannum's clock based on 71 CpG sites [42], the Lu's telomere length predictor [43], and DNA methylation-based mortality risk assessment (GrimAge [44]) were calculated.Principal component-based epigenetic clock estimates were calculated utilizing an R script provided by Higgin-Chen et al. and 78,464 CpGs for each sample in a beta matrix [45].Mean imputation was utilized for missing values.DunedinPACE pace of aging was calculated using the publicly available Github code [46].
Telomere length quanti cation.Telomere length quanti cation in PBMCs was performed using a highthroughput (HT) Q-FISH (quantitative uorescent in situ hybridization) method developed by Life Length Technologies [47].Brie y, the cells were xed and hybridized with a uorescent Peptide Nucleic Acid (PNA) probe that recognizes three telomere repeats.After hybridization, the cells were thoroughly washed to remove any non-speci c binding, and the nucleus was stained with DAPI.The nuclei and telomeres were then imaged using a high-content screening system.The images of the nuclei and telomeres are captured by a high-content screen system (Opera Phenix, Perkin Elmer) using maximum projection image from several Z-stack individual images, in order to get a more reliable image of the telomere.The uorescent intensities translated to base pair through a standard regression curve which is generated using control cell lines with known telomere lengths.Data were analyzed using proprietary software (TAT Analyzer) to generate all TAVs.The data generated a TAV pro le with descriptive statistics of telomere length, values for each percentile of telomere length, percentages of telomere length values, percentages of cells with speci c telomere values, and dispersion parameters for each sample.
Quanti cation of Cell-associated HIV-1 DNA and RNA.Total cell-associated HIV DNA and RNA were quanti ed using a qPCR TaqMan assay with speci c LTR primers: F522-43 (5' GCC TCA ATA AAG CTT GCC TTG A 3'; HXB2522-543) and R626-43 (5' GGG CGC CAC TGC TAG AGA 3'; 626-643), coupled with a FAM-BQ probe (5' CCA GAG TCA CAC AAC AGA CGG GCA CA 3), on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems).Cell counts were normalized by qPCR using human genomic TERT for DNA and RPLP0 expression for RNA (Life Technologies), respectively.For cell-associated HIV DNA copy number determination, a reaction volume of 20µl was used, containing 10µl of 2x TaqMan Universal Master Mix II including UNG (Life Technologies), 4 pmol of each primer, 4 pmol of probe, and 5 µl of DNA.
Cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 60 cycles of 95°C for 15s and 59°C for 1 min.For cell-associated HIV RNA copy number determination, a reaction volume of 20µl was used, containing 10µl of 2x TaqMan RNA to Ct 1 Step kit (Life Technologies), 4 pmol of each primer, 4 pmol of probe, 0.5 µl reverse transcriptase, and 5µl of RNA.Cycling conditions were 48°C for 20 min, 95°C for 10 min, followed by 60 cycles of 95°C for 15s and 59°C for 1 min.External quantitation standards were prepared from DNA isolated from ACH-2 cells calibrated to the Virology Quality Assurance (VQA, NIH Division of AIDS) cellular DNA quantitation standards.For HIV RNA measurements, external quantitation standards were prepared from full-length NL4-3 virion RNA, and copy numbers were determined using the Abbott RealTime assay (Abbott Diagnostics, Des Plains, Ill), calibrated to VQA HIV-1 RNA standards.Up to 250 ng of total cellular RNA or DNA was added to each reaction well, and copy numbers were determined by extrapolation against a 7-point standard curve (1-10,000 cps) performed in triplicate.
Assessing Tight Junction Proteins in Colon and Ileum.Ileal and colonic biopsies were embedded in optimal cutting temperature (OCT) and cut into 5-µm thick sections.Subsequently, they were xed in a 1:1 acetone/methanol solution at -20ºC for 2 minutes.The slides were air-dried and then rinsed in 1X PBS for 10 minutes to rehydrate.Afterward, the slides were permeabilized at 40ºC for 5 minutes in a 0.2% Triton X-100/PBS solution.Following three washes with 1X PBS, the slides were blocked for 1 hour at room temperature using a 2% non-fat dry milk solution.The samples were stained with primary antibodies against ZO-1 (Invitrogen, catalog# 61-7300) or occludin (Invitrogen, catalog # 33-1500) diluted in 2% milk for 1 hour at 37ºC.After three washes with 1% milk for 10 minutes, secondary antibodies (Invitrogen Alexa Fluor donkey anti-rabbit 488 #A-21206 or Alexa Fluor donkey anti-mouse 488 #A-21202) were added at a dilution of 1:250 for 1 hour at 37ºC.Subsequently, the slides were washed three times in 1X PBS for 10 minutes.Sections were stained with DAPI for 3 minutes, rinsed three times in PBS followed by a quick rinse in water, and then mounted using Sigma's Fluoromount™ Aqueous Mounting Medium #F4680.Images from at least ve stained tissue elds per sample were used to determine the relative expression of each marker and to select representative images.All staining images were evaluated by two independent, blinded observers using a Zeiss Axio Observer 7 digital deconvolution immuno uorescent microscope (Zeiss, Oberkochen).All images were captured at 40x magni cation.
16S rRNA Gene Library Preparation.DNA was extracted from approximately 200 mg of stool or tissue using the Qiagen DNeasy PowerSoil Pro kit and quanti ed using the Quant-iT PicoGreen Assay Kit.Barcoded PCR primers targeting the V1-V2 region of the 16S rRNA gene were used for library generation.PCR reactions were performed in duplicate using Q5 High-Fidelity DNA Polymerase (NEB).For high microbial biomass samples such as fecal material, each PCR reaction contained 0. Read pairs were processed to identify amplicon sequence variants with DADA2 [49].Taxonomic assignments were generated by comparison to the SILVA reference database version 132 [50], using the naive Bayes classi er implemented in scikit-bio [51].Numbered taxa, such as Prevotella 2, represent distinct taxonomic groups in the SILVA taxonomy.Data les from QIIME were analyzed in the R environment for statistical computing.Linear mixed-effects models were used to estimate the mean difference between sample types.Linear models were used to estimate the mean difference between controls and PWH on ART group for each sample type.Relative abundances were log10-transformed for the tests.Only the bacteria with at least 1% mean relative abundance in at least one sample type were tested.Correlations of interest between markers and bacterial abundances were determined using Spearman correlation.When multiple tests were performed, p-values were corrected for false discovery rate using the Benjamini-Hochberg method.An inferred modelling approach, using 16S rRNA microbial relative abundances of the genus taxonomic level, allowed us to identify individual taxa and group them accordingly based on their known involvement with total SCFA-production, total butyrate-production, and putative proin ammatory-production.Assessment of these predictive microbial percent relative abundance ratios were calculated and compared between PWH and controls at all three sample sites [52,53].
Untargeted measurement of stool metabolites.About 200 mg of stool, per sample, were added to 250µL of LC-MS quality water containing 0.2 mM NaN 3 .The samples were subjected to bead beating for 30s using 0.5 mm zirconium oxide beads ceramic beads in a Bullet Blender 24 (Next Advance Inc.).750µL of methanol were added and samples were centrifuged at 16,000 xg at 4˚C for 15 min.800 µL of the supernatant were transferred to new tubes and were dried in a Speedvac (Eppendorf, model 5301).Dried samples were reconstituted in 200µL of a 1% MeOH solution in water and centrifuged at 16000 xg at 4˚C for 5 min.A 1:10 dilution of the sample was performed using a 1% MeOH solution in water.Samples were then transferred to micro-vial inserts and placed in the autosampler.A quality control (QC) sample was made by pooling 10 µL of each sample and was analyzed periodically across the metabolomics run.Metabolomic analysis of the feces extracts was performed using a liquid chromatography tandem mass spectrometry (LC-MS/MS) system using an in-house metabolite library.A Shimadzu Nexera X2 (consisting of two LC30AD pumps, a SIL30AC autosampler, a CTO20AC column oven and a CBM20A controller; Shimadzu, The Netherlands) was used to deliver a programmed gradient of water (eluent A) and methanol (eluent B), both containing 0.1% formic acid.The gradient, using a ow of 0.4 mL/min, was 0% B at 0 min, 0% B at 1.5 min, 97% B at 9.9 min, 97% B at 12.9 min, 0% B at 13.0 min and 0% B at 13.8 min.A Synergi Hydro-RP, 2.5 µm particles, 100 × 2 mm was used as column with a Phenomenex SecurityGuard Ultra C8, 2.7 µm, 5 × 2.1 mm cartridge as guard column.The column was kept at 40 ˚C and the injection volume was 2 µL.The MS was a Sciex TripleTOF 6600 (AB Sciex Netherlands B.V., The Netherlands) operated in positive and negative ESI mode, with the following conditions: ion source gas 1 50 psi, ion source gas 2 50 psi, curtain gas 30 psi, temperature 500 ˚C, acquisition range m/z 75-650, ion spray voltage 5500 V (ESI+) and − 4500 V (ESI-), and declustering potential 80 V (ESI+) and − 80 V (ESI-).
An information dependent acquisition (IDA) method was used to identify the different metabolites, with the following conditions for MS analysis: collision energy ± 10, acquisition time 80 ms and for MS/MS analysis: collision energy ± 30, collision energy spread 15, ion release delay 30, ion release width 15 and acquisition time 40 ms.The IDA switching criteria was to exclude isotopes within 4 Da for a maximum of 18 candidate ions to monitor per cycle.MS-DIAL (v4.90), with our in-house metabolite database was used to align the data and identify the different metabolites matching accurate mass, retention time and in most cases, the MS/MS fragmentation pattern against the authentic chemical standards.Only metabolites with a peak area's RSD below 30% in the QC samples and whose sample-to-blank ratio was above 5 for at least 80% of the samples within the experimental groups were kept for further data analysis.Normalization was done using the stool sample weight.
Untargeted measurement of plasma metabolites.Metabolomic analysis was conducted following previously described methods [54,55].In brief, polar metabolites were extracted using 80% methanol.A QC sample was created by pooling equal volumes of all samples and injected periodically during the sample sequence.LC-MS was performed on a Thermo Scienti c Q Exactive HF-X mass spectrometer with HESI II probe and Vanquish Horizon UHPLC system.Hydrophilic interaction liquid chromatography (HILIC) was performed at 0.

RESULTS
PWH on ART exhibit accelerated biological aging in the intestines, with rates differing from that in the blood.We collected colon and ileal biopsies, blood, and stool samples from 25 PWH on ART with a viral load of < 50 copies/ml and 23 HIV-negative controls matched by age, sex, ethnicity, and BMI (Table 1).
Using the systems biology approach illustrated in Fig. 1A [41], DNA methylation-based mortality risk assessment (PCGrimAge) [44], and Lu's telomere length predictor (PCDNAmTL) [57].For ve of these clocks, a higher value indicates an older biological age.However, for the PCDNAmTL clock, a lower value indicates older age.
Despite having a similar chronological age (Fig. 1B), the blood of PWH on ART showed an older biological age than that of HIV-negative controls.The difference ranged from + 3.1 years (using PCGrimAge) to + 7.63 years (using PCHorvath2) (Supplementary Fig. 1A).We next calculated the acceleration of biological age by regressing the outputs of the clocks against chronological age.Larger indices imply faster biological aging, except for PCDNAmTL where a smaller index denotes accelerated aging.This analysis found that the biological age of PWH on ART was accelerated between 2.59 to 7.05 years (Fig. 1C).We also employed the DunedinPACE epigenetic clock, which estimates the pace of aging [46].Higher values for this metric correlate with accelerated aging [46].Consistently, the DunedinPACE estimate was markedly higher in PWH on ART than in the controls (Fig. 1C), echoing recent studies which suggest that PWH experience accelerated biological aging in blood [32][33][34][35][36][37].
Next, we applied the same epigenetic aging clocks to DNA isolated from the ileum and colon (Supplementary Fig. 1B-C).The ileum of PWH on ART showed accelerated biological aging by four of the seven epigenetic clocks (Horvath1, Horvath2, Hannum, and PCDNAmTL; Fig. 1D), compared to controls.
Similarly, the colon of PWH on ART showed accelerated biological aging using two clocks (Horvath1 and DunedinPACE; Fig. 1E).That some clocks did not detect aging acceleration in the ileum and colon might be because many of the clocks were designed for use on blood samples.Since Horvath1 [39] was developed using tissues, we compared its age estimates and biological age acceleration across the blood, ileum, and colon samples (Fig. 1F-G).These data emphasize that the ileum, colon, and blood in PWH on ART all exhibit accelerated biological aging.However, the acceleration rate differs among tissues, suggesting that HIV accelerates aging in a tissue-speci c manner.
Epigenetic clock estimates of biological age were validated using other established and emerging markers of aging.To support the results from the epigenetic clocks, we compared these to established and emerging biomarkers of aging.As telomere length (TL) is an established aging marker [58, 59], we evaluated TL in PBMCs via HT-Q-FISH.Median TL did not differ between PWH on ART and controls (Supplementary Fig. 2A-C); however, PWH on ART had a higher percentage of cells with shorter telomeres (and a lower percentage with longer telomeres) than controls (Supplementary Fig. 2D).We then determined the correlations between biological age, as estimated by the epigenetic clocks, and measures of TL (Fig. 2A).These correlations show that higher biological age estimated by the epigenetic clocks correlate strongly with shorter TL.
In addition, new metrics for biological age have recently emerged, including the deep learning based 'in ammatory aging clock' called iAge [60].This metric is derived from the measurement of several in ammation markers in plasma, such as CXCL9 and eotaxin; these are incorporated into an in ammatory aging clock that can predict accelerated aging [60].We measured the levels of some of the markers included in iAge, as well as other in ammatory indicators pertinent to HIV infection (e.g., IL4, IL-6, MIP-1α) [61, 62] in blood using multiple cytokine arrays.Levels of several markers, including CXCL9 and eotaxin, were elevated in PWH on ART compared to controls (Fig. 2B).Higher levels of these in ammation markers correlated with accelerated biological aging (derived from the epigenetic clocks as in Fig. 1C-G), especially in blood (Fig. 2C).We also examined correlations between accelerated aging (based on Horvath1) and the levels of cell-associated HIV DNA in PBMCs, ileum, and colon, and cell-associated HIV RNA in PBMCs, as surrogates for HIV persistence.Among these, the strongest association with epigenetic age acceleration was HIV DNA levels in the ileum (Supplementary Fig. 3).These ndings validate the results obtained with the epigenetic clocks and support the conclusion that living with HIV, even with ART, accelerates biological aging in both tissues and blood, with the rate differing among them.
Intestinal permeability and microbial translocation link to accelerated biological aging.Microbial translocation and dysbiosis are increasingly hypothesized to drive systemic in ammation and thus promote in ammation-associated diseases of aging.Given that PWH on ART experience accelerated biological aging both systemically and within tissues, we explored the possibility that microbial translocation and microbial dysbiosis may drive this accelerated aging.First, we evaluated microbial translocation in PWH on ART and controls by assessing the levels of tight junction proteins (ZO-1 and occludin) in the ileum and colon using immuno uorescence and a scaling method described in Supplementary Fig. 4. Data in Fig. 3A-B show that intestinal integrity, as assessed by levels of ZO-1 and occludin, was signi cantly lower in PWH on ART compared to controls.This suggested that gut permeability was higher in PWH on ART.Consistently, markers of gut damage and microbial translocation in plasma were higher in PWH on ART compared to controls (Fig. 3C).The damage/translocation markers assessed were REG3α (intestinal stress marker Together, these data suggest that in PWH the intestinal integrity is compromised, resulting in enhanced microbial translocation. Next, we investigated the relationships between the degree of intestinal integrity or microbial translocation and the two measures of biological aging.Speci cally, we determined correlations between intestinal integrity (based on levels of tight junction proteins) or microbial translocation (based on levels of the damage/translocation markers) and either accelerated aging (calculated as in Fig. 1C-G using data from the epigenetic aging clocks in blood and tissues) or blood-based in ammatory aging markers (measured as in Fig. 2B).Correlation heat-maps (Fig. 3D) showed that intestinal integrity negatively correlated with accelerated biological aging and levels of in ammatory aging markers, while microbial translocation positively correlated with accelerated biological aging and levels of in ammatory aging markers.Moreover, the higher levels of HIV DNA and RNA in blood and/or tissues (as surrogates of HIV persistence) correlated with lower intestinal integrity (Fig. 3E-H).These ndings highlight the connections between elevated intestinal permeability and microbial translocation, accelerated aging, greater in ammation, and greater HIV persistence in the blood and intestinal tissues of PWH on ART.
Living with HIV is linked to intestinal and fecal microbial dysbiosis, notably a decrease in butyrateproducing bacteria.As we described in the preceding sections, PWH on ART have compromised intestinal integrity which may lead to accelerated biological aging both systemically and in tissues.One plausible mechanism underlying this compromised intestinal integrity is microbial dysbiosis.Microbial dysbiosis can pave the way for an increase in bacteria that produce toxic metabolites, such as those involved in tryptophan catabolism [28,69,70].It can also cause a decline in bacteria that generate metabolites considered bene cial, such as short-chain fatty acids (SCFAs) [71], notably butyrate, which are microbiome-derived metabolites known to bolster intestinal barrier integrity [72].With this context in mind, we probed the microbiome in stool, ileum, and colon samples from PWH on ART and controls using 16S rRNA sequencing.
We found that microbial alpha diversity, a hallmark of a healthy microbiome [73] as measured by various models (Richness, Shannon, and Faith), was lower in the colon of PWH on ART compared to controls (Fig. 4A); smaller non-signi cant differences were observed in feces and ileum between the groups (Supplementary Fig. 5A-B).We then assessed the relative abundance of bacteria known to produce SCFA, particularly butyrate, and the relative abundance of bacteria considered pro-in ammatory ("pathobionts"; Supplementary Table 1).The relative abundance of butyrate-producing bacteria was lower in PWH on ART than in controls (Fig. 4B).PWH on ART also tended to have a more pro-in ammatory fecal microbiome and lesser SCFA-producing fecal bacteria, but trends were not statistically signi cant (Supplementary Fig. 5C-D).
When we examined speci c bacterial genera in the feces, colon, and ileum, we found that the microbiome in these locations varied signi cantly (Fig. 4C; FDR < 0.05).Comparing PWH on ART with HIV-negative controls (Fig. 4D), we found that living with HIV on ART was associated with an enrichment of some bacterial genera and a depletion of others, in feces, colon, and/or ileum.Enriched bacterial genera include putatively pro-in ammatory bacterial genera [24] such as Catenibacterium, Prevotella 2, Allprevotella, Prevotella 9, and Enterobacteriaceae.Depleted genera included putatively anti-in ammatory bacteria [74,75] and bacteria known for their ability to produce SCFAs such as Erysipelotrichaceae UCG − 003, Alistipes, Coprococcus 3, Peptostreptococcaceae, Romboutsia, Subdoligranulum, Bacteroidales, [Ruminococcus] gauvreauii group, and Faecalibacterium.)This reinforces ndings from earlier studies [28,71], suggesting an HIV-related microbial imbalance, characterized by higher levels of potentially proin ammatory bacterial genera and lower levels of potentially anti-in ammatory bacteria.This microbial imbalance may contribute to the previously observed decrease in intestinal integrity and consequently, the accelerated biological aging in PWH on ART.
A distinct mucosal microbial signature is linked to accelerated biological aging.Given the dysbiosis observed in PWH (Fig. 4D), we next asked if this dysbiosis was related to the accelerated biological aging we had observed in PWH.Our analyses in Fig. 5A revealed that speci c bacterial genera that were enriched in colon tissue from PWH on ART (such as Catenibacterium, Prevotella 2, Allprevotella, and Prevotella 9) correlated strongly with greater accelerated aging (FDR < 10%).In contrast, other genera that were depleted in colon tissue from PWH on ART (like Erysipelotrichaceae UCG-003, Alistipes, Coprococcus 3, Romboutsia, and Subdoligranulum) correlated with slower accelerated aging.Notably, the correlations between the enriched bacteria and higher accelerated biological aging were driven by samples from PWH on ART, whereas the correlations between the depleted bacteria and slower accelerated biological aging were driven by samples from HIV-negative controls (Fig. 5B).Similar analyses using ileal (Fig. 5C), and fecal (Fig. 5D) samples did not yield any correlations with FDR < 10%, although some nominal P values were signi cant.
Beyond their associations with accelerated biological aging rates, taxa enriched in PWH on ART were linked to lower tight junction protein levels in tissues, elevated microbial translocation, and enhanced in ammation (Fig. 5E, top rows of each section).In contrast, taxa that were depleted in PWH on ART were associated with better intestinal integrity, lower microbial translocation, and lower in ammation (Fig. 5E, bottom rows of each section).Separate analyses revealed that the pro-in ammatory microbiome was associated with higher levels of HIV DNA and RNA in both blood and tissues.By contrast, the SCFAproducing bacteria, notably those producing butyrate, associated with lower levels of HIV DNA and RNA (Fig. 5F-G).These ndings suggest that certain bacterial genera, especially those from the colon, may in uence the pace of biological aging.Moreover, they shed light on the intricate relationship between microbial pro les, in ammation, HIV persistence, and the biological aging trajectory in PWH on ART.
Correlation networks reveal links between the mucosal microbiome, microbe-related metabolites, and accelerated biological aging.Building on our observations (Fig. 5) that SCFAs were associated with slower biological aging, we expanded our inquiry to other microbe-related metabolites.Recognizing that many effects of the microbiome are mediated by metabolites other than SCFAs, we conducted an untargeted metabolic analysis on stool and plasma samples from both PWH on ART and controls.Our goal was to identify additional metabolites that might bridge the microbial signature (Fig. 5) with the accelerated biological aging patterns observed.
First, we assessed a spectrum of microbiome-and gut-speci c metabolites (Supplementary Table 2).PWH on ART had elevated levels of metabolites known to be detrimental, such as L-kynurenine and quinolinic acid, both by-products of tryptophan catabolism [69,76].We con rmed this by evaluating two common measures of tryptophan catabolism, the kynurenine to tryptophan (K/T) ratio and the quinolinic acid to tryptophan (Q/T) ratio [77].Both ratios were indeed higher in PWH on ART than controls (Fig. 6A).
PWH on ART also had lower levels of metabolites associated with microbial diversity and intestinal health, like hippuric acid [78], L-ergothioneine [79], and oleic acid [80] (Fig. 6A).The metabolites enriched in PWH on ART were associated with accelerated biological aging, compromised intestinal integrity, heightened microbial translocation, and greater in ammation (Fig. 6B).Conversely, metabolites that were less abundant in PWH on ART correlated with slower biological aging, greater intestinal integrity, lower microbial translocation, and lower in ammation (Fig. 6B).
To visualize these complex interactions, we performed a network analysis, which illustrated distinct threeway interactions among microbial genera enriched in PWH on ART, elevated tryptophan catabolism metabolites, diminished bene cial gut metabolites like hippuric acid and oleic acid, and accelerated biological aging (Fig. 6C).Conversely, the network analysis also identi ed distinct connections among microbial genera depleted in PWH on ART, diminished tryptophan catabolism metabolites, abundant protective gut metabolites, and slower biological aging (Fig. 6C).These intricate relationships were most pronounced in the colon, followed by the ileum, and then the feces, underscoring the tissue-speci c microbial imprints of accelerated biological aging which were absent in the fecal microbiome.

DISCUSSION
Previous research indicates that, even under ART, HIV infection accelerates biological aging in the blood [33,36,81].Yet, the effects of HIV on biological aging in the intestines-a primary site for HIV persistence and pathogenesis-and the links between age and increased gut permeability, microbial translocation, and viral persistence are not known.In our study, we used a systems biology approach to examine colon, ileum, and blood samples from PWH on ART and HIV-negative controls.Our ndings reveal that living with HIV is associated with an acceleration or accentuation of biological aging in the ileum and colon at rates different from that of the blood.Importantly, we identi ed speci c bacterial taxa and associated microbial metabolic signatures that are linked to both intestinal and systemic biological aging.These insights pave the way for further research into the mechanisms underlying these connections and potential strategies to prevent or delay aging-related complications in PWH.
We identi ed speci c microbial signatures linked to biological aging in mucosal tissues, but not in feces.
A primary factor responsible for diminished intestinal integrity and microbial translocation is microbial dysbiosis, an imbalance in the intestinal micro ora.A healthy gut microbiome strikes a delicate balance between bene cial commensals and pathobionts.However, living with HIV tilts this equilibrium, favoring the proliferation of pathobionts and opportunistic pathogens in the gut.Although many studies have focused on the fecal microbiome, the bacteria in mucosal biopsy samples (termed the mucosalassociated microbiome) can differ signi cantly from those in feces [24,82].Furthermore, HIV-related shifts in the mucosal microbiome aren't always mirrored in fecal samples from the same individual [24].
Our ndings highlight a pronounced difference between the mucosal-associated and fecal-associated microbiomes, with only the former closely tied to accelerated biological aging.This underscores the importance of examining the microbiome across different anatomical sites.Recognizing the variations among microbiomes and their links to diverse biological conditions, like biological aging, can lead to specialized strategies.Such tactics could counter microbial dysbiosis, strengthen intestinal integrity, and prevent both intestinal and systemic in ammation, thereby slowing the accelerated aging process.
Speci c bacterial genera, including Catenibacterium, Prevotellaceae, and Enterobacteriaceae, enriched in PWH on ART, were strongly associated with accelerated biological aging.These bacterial taxa can catabolize tryptophan [28] and were correlated with elevated levels of the metabolic byproducts of tryptophan catabolism.Increased tryptophan catabolism leads to an accumulation of toxic metabolic byproducts such as kynurenine and quinolinic acid.These byproducts have been linked with adverse outcomes in chronic HIV infection [28,69,70,76,77].For example, quinolinic acid, a known neurotoxin and activator of the N-methyl-D-aspartate (NMDA) receptor, has been connected with neurological complications in HIV infection [83,84].Likewise, elevated kynurenine levels are associated with neurological de cits in the aging population [85,86].Our study consistently identi ed strong correlations among bacterial taxa capable of inducing tryptophan catabolism, the generation of these toxic metabolic byproducts, and the acceleration of biological aging at both the systemic and intestinal levels.However, more research is needed to determine the speci c bacterial species initiating tryptophan catabolism and to understand if these associations with biological aging are causative.Identifying such causal relationships could set the stage for creating therapeutic strategies to counteract the rapid onset of biological aging.For instance, inhibitors of IDO-1 (indoleamine 2,3-dioxygenase 1), like Epacadostat and Linrodostat, have been tested in cancer trials (often in tandem with immune checkpoint inhibitors) to block tryptophan depletion, might offer promising intervention routes [28].
Conversely, certain bacterial taxa, including Erysipelotrichaceae UCG-003, Coprococcus, Faecalibacterium, and Subdoligranulum, were signi cantly lower in PWH and were associated with slower rates of biological aging.Some of these bacterial species, like Erysipelotrichaceae UCG-003, have been previously associated with healthy aging [87], although their potential role in promoting healthy aging in PWH was not established prior to our study.Many of these bacteria are known for their ability to produce SCFAs, which are essential for maintaining gut health.Among SCFAs, butyrate is a primary energy source for intestinal epithelial cells and plays a pivotal role in modulating T cell responses in the gut [88].This metabolite, recognized as an HDAC inhibitor (HDACi), has strong anti-in ammatory properties, which help maintain the intestinal barrier's integrity [72].Consistently, our study revealed a marked reduction in butyrate-producing bacterial genera, such as Coprococcus, and Subdoligranulum, in both the ileum and colon; Faecalibacterium in colon; and [Ruminococcus] gauvreauii group in fecal samples from PWH on ART compared to controls.Moreover, these bacteria were positively associated with the maintenance of tight junction integrity and negatively associated with markers of in ammation, microbial translocation, and the acceleration of biological aging.These ndings underscore the potential role of SCFA production in maintaining intestinal barrier integrity and fostering the intestines' healthy aging.Such insights provide a foundation for investigating strategies to enhance SCFA levels, like the adoption of SCFA-promoting prebiotics, to potentially slow the aging process in the intestinal environment.
In addition to SCFAs, we identi ed strong associations between other microbiome-related metaboliteswhich possess well-established anti-in ammatory properties -and a decelerated rate of both intestinal and systemic aging.Notable among these are hippuric acid, L-ergothioneine, and oleic acid.Hippurate, produced through microbial activity in the colon, is often used as a marker for good gut health and increased microbial diversity [78].Similarly, L-ergothioneine serves as an indicator of a healthy gut microbiota and has shown antioxidant properties, helping counteract oxidative stress in intestinal contexts, as observed in vitro and in animal models [89,90].Oleic acid, with its anti-in ammatory properties, has been found to boost alpha diversity in older individuals with HIV when supplemented [80].Taken together, these insights indicate that living with HIV may induce changes in the gut microbiome, leading to disruptions in key, modi able, microbiome-related metabolic pathways.Such disruptions might contribute to weakened intestinal integrity, increased microbial translocation, and ongoing in ammation.
These factors could potentially contribute signi cantly to the process of both local and systemic biological aging.
Various confounding factors, including sexual orientation and practices, can in uence the intestinal microbiome's composition.For instance, Catenibacterium and Prevotella are more prevalent in men who have sex with men (MSM), regardless of their HIV status [25].Yet, microbial dysbiosis also occurs in PWH regardless of their sexual orientation or practices [31].In our study, a signi cant portion of PWH identi ed as MSM.Thus, the potential in uence of sexual practices on their gut microbiota cannot be disregarded.
Though MSM often have a more diverse microbiome [30], we observed lower alpha diversity in PWH on ART compared to controls.This points to HIV infection, rather than sexual practices, as the potential cause of this dysbiosis.However, the microbial signature of biological aging that we identi ed may not be solely driven by HIV, ART, or demographic factors in PWH but a combination thereof.Future research should carefully examine the distinct impacts of HIV, speci c ART regimens, sex/gender, sexual practices, and other potential confounding variables on the relationship between the intestinal microbiome and both intestinal and systemic biological aging.Such studies will require large and diverse participant cohorts, as well as controlled animal studies, to gain a comprehensive understanding of these intricate interactions.
Our study has several limitations, including: 1) While our human-based study cannot unequivocally demonstrate mechanistic links between the enrichment of pro-in ammatory microbial taxa, the depletion of anti-in ammatory microbial taxa, and the acceleration of biological aging, existing literature on these bacterial taxa aligns with and supports our ndings and hypotheses.Nevertheless, detailed mechanistic insights will require further research using intestinal organoids and animal models.
2) The human gut microbiome is not composed of bacteria alone; it encompasses fungi, archaea, protists, and viruses, each vital for both intestinal and systemic health.Recent studies emphasize the role of protists like Blastocystis in metabolizing tryptophan, affecting immune activation and CD4 + T cell responses [91].Also, β-glucan (a marker of fungal translocation) is associated with systemic in ammation in PWH on ART, and PWH have an altered gut virome with enriched eukaryotic viruses linked to gastrointestinal diseases [92].As such, the impact of this diverse micro ora on aging merits deeper investigation.3) We've pinpointed associations between bacterial taxa, microbial metabolites, accelerated aging, and levels of cell-associated HIV DNA in both intestines and blood.These ndings hint at intricate interactions between the gut microbiome, its metabolic activities, aging, and HIV persistence.However, it's crucial to note that the majority of cell-associated HIV DNA in PWH on ART is defective [93,94].Future work should investigate links between a pro-aging microbiome, intact HIV reservoirs in the intestines, and the relationship between active HIV persistence and faster aging.This may reveal a feedback loop where increased HIV persistence exacerbates immune dysfunction, necessitating further research.

CONCLUSIONS
Effective ART has revolutionized HIV therapeutics over the past decade and signi cantly increased the lifespan of PWH.However, this longevity has been accompanied by a high incidence of several agerelated non-AIDS associated co-morbidities such as cardiovascular and renal diseases, neurocognitive impairments, and ailments of the gut.Our study unveils previously unrecognized links between speci c intestinal microbial signatures, their metabolic activity, and accelerated biological aging in PWH on ART.
These insights pave the way for novel interventions targeting the microbiome and metabolites, aiming to strengthen the intestinal barrier, decelerate aging, and reduce in ammation-associated diseases in PWH and others with chronic in ammation stemming from a compromised intestinal barrier.
consent to participantsThe study protocols were approved by the Institutional Review Board of The Wistar Institute and Rush University (IRB protocol 19020710-IRB02).All human experimentation was conducted per the guidelines set forth by the US Department of Health and Human Services and the authors' respective institutions.

Figure 2 The
Figure 2

Figure 4 Living
Figure 4

Figure 6 Correlation
Figure 6 MS with separate runs for positive and negative polarities.Raw data were analyzed using Compound Discover 3.3 SP1 (ThermoFisher Scienti c).Accurate mass or retention time was used to identify the metabolites by utilizing an in-house database generated from pure standards or by querying the mzCloud database (www.mzCloud.org)with MS/MS spectral data.Matches with scores of 50 or greater were selected.Metabolite quanti cation utilized integrated peak areas from full MS runs.These values were corrected based on the periodic QC runs and normalized to the total signal from identi ed metabolites in each sample.
2 ml/min on a ZIC-pHILIC column (2.1 mm × 150 mm, EMD Millipore) at 45°C.All samples were analyzed by full MS with polarity switching, and the QC sample was also analyzed by data-dependent MS/