In this study we show that the gut microbiome of 25 aging ART-controlled PWH compared to 3 independent groups of paired non-HIV infected controls, presents major alterations linked with insulin resistance and a dysmetabolic profile. First, a core set of metagenomic species, some of which belonging to the Prevotella genus, were robustly enriched in PWH vs. each of the 3 matched control groups. These markers are also related with worsened insulin resistance profiles. This signature does not seem to be explained by MSM sexual preferences alone as tested based on stratified analyses by this variable. Second, depleted butyrate-producing lineages of the microbiome could also be responsible for worsened metabolic profile and insulin resistance in PWH individuals, consistent across MSM sexual preferences. This defect is probably related to the HIV infection, even though all patients were controlled with ART modification. And third, machine learning methods uncovered robust predictive signatures, consistent with univariate analyses, which generalized relatively well in two additional external cohorts. This indicates the specific impact of the HIV infection in the microbiome ecosystem.
An initial limitation of this study was that only PWH were recruited, so to characterize the specific microbial signatures in the microbiome of PWH we capitalized on the availability of quantitative metagenomic profiles from three studies (MetaHit, MicroObese, MetaCardis). These external cohorts included individuals from different countries (French in MicroObese and MetaCardis; Danish in MetaHit), with variations in corpulence profiles (non-obese in MetaCardis and MetaHit; moderately obese and obese in MicroObes), and with metagenomic data generated with different sequencing technologies (SOLiD in MicroObese, Illumina in MetaHit, IonTorrent in MetaCardis). These as well as other technical and clinical/lifestyle covariates (stool collection methods, library preparation protocol, nutritional profiles) could have a major impact on the metagenomic profiles and derived signatures associated to study groups in quantitative metagenomic studies [47–49].
The reference databases may also play a major role in quantitative metagenomic profiling [50, 51], although important effort is put to harmonize metagenomic data from different studies into common reference space [52]. Moreover, different library preparation methods and sequencing technologies may have an important effect on the final abundance profiles. All these different aspects would be impossible to de-confound in this study if a single control group was included. Indeed, the integration of our PWH data with the metagenomic data in three control groups on the same reference space allowed to identify a core set of taxonomic and functional signatures that characterizes the microbiome of PWH with robust variations across all three control groups. Moreover, we showed that with different machine learning approaches (Predomics and Random Forest models) it was possible to correctly classify the samples of PWH vs. healthy controls in two independent external studies (80–82% AUC).
Among these signatures we observed a strong enrichment of Prevotella lineages in PWH that is reflected at both MGS and enterotype levels and that constitutes a signature that has already been identified as enriched in the microbiome of PWH in other studies [11] but that has been also shown as potentially cofounded by the sexual preferences [12]. Here we lack information for this variable in the control groups, but stratified analyses on PWH by sexual preference shows a non-significant tendency towards higher enrichment of Prevotella enterotype in PWH of the MSM group (Supplemental Fig. 2) that would be in line with previous reports. However, we also observe that the increase of several core MGS of the Prevotella genus in the PWH was robust to sexual preferences (Fig. 2B, Supplemental Fig. 4).
In addition to these well-known signatures, our study identified other MGS that were highly specific of the microbiome of PWH and robust to MSM status like notably CAG00726: Succinivibrio sp000431835, an MGS that was predominantly absent from the three control groups and that showed the highest importance in the classification models of PWH vs. different control groups. In this context, a longitudinal study of PWH with 16S profiling showed drastic increase in the Succinivibrio genus under conventional ART therapy [53], and a 16S-based cross-sectional study showed also that Succinivibrio genus abundance was specifically associated to treatment- naïve PWH specifically infected by HIV-C subtype [54]. On the other side of the balance, the depletion of butyrate-producing firmicutes lineages of the Bacillota_A phyla, notably from the Faecalibacterium genus and evolutionary close lineages like Gemmiger and Ruthenibacterium[55], that were described in previous studies characterizing the microbiome of PWH [11, 14] were also reproduced in our cohort. This includes MGS like CAG00755: Faecalibacterium SP900539945, CAG00463: Faecalibacterium prausnitzii_I or CAG00208: Gemmiger qucibialis included in the core MGS consistently depleted in PWH in comparison to the three control groups and robust to MSM preferences (Fig. 2). But we also observed the near complete absence of CAG01272: Adlercreutzia hattorii in the microbiome of PWH individuals (MSM and no-MSM). This lineage was recently characterized as a close relative (93% ANI values) of Adlercreutzia equolifaciens[56], a bacterium with anti-inflammatory properties both in vitro and in vivo in a humanized mouse model of NAFLD whose abundance decrease with the severity of hepatic disease[57]. In the context of PWH, 16S metabarcoding studies have previously shown a depletion of Adlercreutzia genus in women with HIV infection with or at high risk of HIV having developed carotid artery atherosclerosis [58], as well as a consistent depletion in women with HIV with and without T2D in comparison with non-HIV and non-T2D controls [17]. Here, thanks to the availability of shotgun data and precise taxonomic annotation based on the Genome Taxonmy DataBase[32], we report to our knowledge the first evidence of the near complete absence Adlercreutzia hattorii in the microbiome of PWH, which could be associated to the inflammatory profile induced by HIV infection.
Our study strongly suggests that insulin resistance can be partly related to the HIV status and partly to sexual preference in MSM, a highly represented population in Caucasian PWH in Western countries, but this latter point has not been precisely addressed. First, a high level of insulin resistance has been commonly reported in PWH as compared to the general population. In the general population, the average HOMA-IR was 1.1 in individuals with normal BMI and low body fat and of 1.6 in those with high body fat [6]. The HOMA-IR value was markedly higher in the Modena cohort in Italy: among 2000 ART-controlled PWH, 68% men, aged in median 45 years old and with a median BMI of 23 kg/m2, median HOMA-IR was 2.4 (IQR 1.4-4) and half patients were considered as insulin-resistant (HOMA-IR > 2) [5]. In the TANGO trial including more than 700 ART-controlled PWH, 92% men, 79% Caucasian, with a BMI of 26, median HOMA-IR was 2.6 with about 70% being insulin-resistant [59]. Second, the role played by the sexual preference in the level of insulin resistance in PWH has not been directly explored. Interestingly, in MSM from the MACS cohort, HOMA-IR was high in non-HIV infected MSM aged 44.9 years at 2.9 for a BMI of 26.3 kg/m2, and was significantly increased in MSM PWH aged 40.6 years at 3.3 for a significantly lower BMI at 25.4 kg/m2 [7], strongly suggesting that sexual preference in MSM could enhance insulin resistance. Accordingly, a high level of HOMA-IR was not found in the corresponding WIHS cohort of women infected or not by HIV, with an HOMA-IR of 2.0 for a BMI of 29.4 kg/m2 in non-infected women and a HOMA-IR of 1.9 for a BMI of 26.2 kg/m2 for women PWH [60]. Thus, elevated HOMA-IR in MSM PWH could result both from sexual preference and from HIV infection.
Here, in 25 PWH with a mean BMI of 25 kg/m2 we observed a very high level of insulin-resistance (mean HOMA-IR 5.1) that is not clearly explained by BMI or metabolic disorders. Moreover, HOMA-IR was higher in the MSM vs the non-MSM group. We propose that altered microbiota, with increased Prevotella, which could be influenced by sexual preference (we observed a non-significant potential enrichment of Prevotella enterotype in PWH-MSM group; Supplemental Fig. 3), and decreased species producing butyrate, which could be linked to HIV infection, could both play a role in the insulin-resistant profile observed in PWH.
The relationship between gut dysbiosis and insulin resistance has been poorly addressed in PWH. A decreased level of butyrate-producing bacteria has been reported in several studies [10, 61] being independent on sexual preference [13, 61] with no reference to insulin resistance. Two studies evaluated gut microbiota in PWH with or without metabolic syndrome, in the absence of non-infected controls. They reported that Faecalibacterium was reduced in those with a metabolic syndrome [14] and was related to the liver fat index [15]. In patients with diabetes, one study evaluating women with HIV and diabetes reported a marginal decrease in Faecalibacterium as compared to non-diabetics (p = 0.07)[17]. However, in another study evaluating PWH and non-infected controls, with or without diabetes, the overall capacity for butyrate metabolism as predicted by a PICRUSt analysis did not differ between the groups [16]. Conversely, such bacteria have been negatively linked to prediabetes and also with diabetes in non-infected individuals (for a review see [62]) in most studies but a positive relationship between Faecalibacterium and HOMA-IR has been recently reported in non-diabetic subjects with overweight or obesity [63]. In the present study, we present the clear link between their major reduction in PWH as compared to the three control groups and higher insulin resistance. This is translated at the functional level into an overall depletion of butyrate production potential in the microbiome of PWH, independent of sexual preferences. In this context, a recent study has shown that this depletion in the butyrate production potential of the microbiome of PWH was not reflected into systemic nor fecal levels of butyrate determined by metabolomics experiments, but that a depletion of propionate derived from microbiome lactate consumption was better reflected at both metagenomic and metabolomic level as signature of ART-treated PWH, with levels that decreases in PWH preceding morbidity and mortality [61]. Interestingly, among the functional modules significantly depleted in PWH vs. the three control groups we observed a signature of lactate consumption, robust to MSM preferences only in comparisons vs. the MicroObes control cohort (MF0080: Lactate consumption II; Fig. 3). We also report an association between several modules linked to amino-acid degradation and insulin sensitivity, although such an association has been previously reported in non-diabetic overweight or obese subjects [63].
Regarding microbial diversity, we observed a tendency to a decreased microbial gene richness in PWH vs the MetaCardis and MetaHit group, but a significant decrease was only observed vs the MicroObes group. We cannot exclude that potential biases associated with different sequencing technologies could influence these results, even if data was properly downsized to same equal sequencing depth and normalized. In previous studies with PWH, discordant results have been reported regarding microbial diversity, being either decreased or not [9]. This discrepancy could be also related to sexual preference, MSM individuals presenting higher diversity compared with non-MSM individuals [12, 13]. Our stratified analyses of microbial gene richness between MSM and non-MSM individuals revealed no significant difference between both groups. The results on diversity appear to be counterbalanced between sexual preference and HIV infection.
We observed that the module accounting for LPS production was higher in PWH as compared to controls. However, when considering the MSM and non-MSM groups, LPS production was higher in the MSM group but not in the non-MSM group as compared to the 3 control groups. This suggests that higher LPS production, in favor of a pro-inflammatory profile, could be linked to the Prevotella enrichment in the MSM group, in good accordance with previous studies reporting the pro-inflammatory profile of Prevotella [10]. As well, we do not observe a strong association between tested inflammatory markers (IL-6, CRP) and MGS abundances in our group of PWH.
We were also able to analyze the evolution of gut microbiome according to ART. In fact, we analyzed 7 ART-controlled PWH first when talking ART including a protease inhibitor and second after a mean duration of 17 weeks when taking a dual therapy including an integrase strand transfer inhibitor (RAL) and a CCR5 inhibitor (MVC). While this shift was associated with a major modification in the profile of genes expressed in subcutaneous adipose tissue [64], with a profile indicating higher insulin resistance and decreased T lymphocytes activation, we did not observe major differences in the gut microbiome profile. In particular, the level of butyrate-producing bacteria was not modified, in accordance with the unmodified HOMA-IR index. While several studies compared gut dysbiosis before and after ART initiation and indicated the persistence of dysbiosis [9, 10], only a few studies evaluated gut microbiome modifications according to the nature of ART. Lower microbiota richness as compared to HIV-negative controls was observed in PWH receiving protease inhibitors while the richness was similar in those receiving integrase strand transfer inhibitors [65], but the sexual preference was not taken into consideration. The effect of maraviroc on gut microbiota has not been previously evaluated in PWH. Importantly, we analyzed the same subjects twice. Nevertheless, the low number of PWH in our study precludes the identification of small differences related to ART.