Microbiota composition and systemic inflammation in HIV+ patients of different ethnicities

Introduction Thirty years after the discovery of the HIV virus and the advances in the treatment of HIV infections, AIDS remains one of the top priorities for public health. The last years has seen a rapid expansion in the understanding of how the gut microbiota (GM), the community of bacteria, viruses and fungi that colonized human gastrointestinal tract, influences immunity development and function. The huge amount of metagenomics data generated in recent years has revolutionized the field of the human microbiota, showing it as a complex network with a critical impact on both human biology and pathophysiology1,2. Moreover, evidence from numerous studies confirm that the chronic immune activation is a major driver of HIV disease progression and mortality among HIV-infected patients5,6,7,8. Considering the key role of specific communities of gut microbes to local and systemic inflammatory diseases, and that systemic inflammation is believed one of the primary contributors to morbidity and mortality in both untreated and antiretroviral-treated HIV-infected patients, the GM role in HIV infection has gathered great interest9. The Tuscany, according to the latest data published by the Istituto Superiore di Sanità, continues to have a higher incidence rate than the national one and ranks fifth among the regions. Among the cases diagnosed in Tuscany in the period 2018-20, 187 (34.1% of the total) concern the immigrant population: the most frequent foreign nationalities are Brazil, Peru and Nigeria11. Aim The aim of the present study was to further characterize the role of microbiota-immunity axis (MIA) in patients infected by HIV-1. First of all, we compared oral and fecal microbiota of treatment-naïve patients with healthy controls (HC) to evaluate if HIV infection was associated with a change in the microbiota composition. Thereafter, we investigated the fecal microbial composition, serum and fecal microbial metabolites, and serum cytokine profile of treatment-naïve patients before starting ART (Anti-Retroviral Treatment) and after reaching virological suppression (HIV RNA < 50 copies/mL) next 24 weeks of ART. Finally, in order to define the role of microbiota and their metabolites in immune reconstitution we compared patients that reached a CD4+ reconstitution (CD4/CD8 ratio ≥ 1) and patients that didn’t reach CD4+ (CD4/CD8 ratio < 1) reconstitution despite ART. Methods We enrolled 12 treatment-naïve HIV-infected patients receiving ART and 12 healthy Controls (sex and age matched). Fecal and oral microbiota composition was assessed through next generation sequencing. In addition, a comprehensive analysis of a blood broad-spectrum cytokines’ panel was performed through a multiplex approach. At the same time, serum and fecal free fatty acid (FFA) levels were assessed through gas chromatography-mass spectrometry. Results and discussion First of all, in order to understand the impact of HIV on the microbiota-immunity axis we compared the oral and fecal microbiota and the systemic inflammation of HIV infected patients and healthy controls. Our data highlight a decrease of the Bacteroidia and an increase of Negativicutes class in the stool of HIV+ patients. In addition, our results showed that HIV infected patients had low levels of Ruminococcaceae family, that plays both protective and disruptive roles within the GM community, such as the production of anti-inflammatory SCFA (Short Chain Fatty Acids). We also carried out a longitudinal analysis to compare the gut and oral microbiota before and after "viral suppression" (T24). Our results showed modest changes in the GM composition after ART. Considering the microbiota role in influencing the immunity through the bacterial metabolites, we evaluated the SCFA levels in blood and stool samples. We observed significant change of two serum SCFA after the ART, or propionic and butyric acids were increased in “viral suppression” condition. To evaluate the inflammatory status after ART, we measured a panel of selected multifunctional cytokines (totally 27) in serum and we observed a decrease of IP-10 after the treatment, confirming the downregulation of this chemokine production in HIV patients during ART. In addition, we documented a significant increased trend of IL-8 levels with suppressed viral load after 24 weeks of ART. Finally, in order to investigate the microbiota role in the immunity reconstitution, we divided our patients in two distinct cohorts: patients with CD4/CD8 ratio < 1 (with insufficient reconstitution of CD4+ T cells’ amount), despite achieving virologic suppression after 24 weeks of ART, and those with CD4/CD8 ≥ 1, which reached a robust reconstitution of CD4+ T cells’ number. Regarding the oral microbiota, we obtained an interesting result at Phylum level since we observed a high abundance of Proteobacteria in salivary samples of IR (Immune Responders) patients respect to INR (Immune No Responders). In conclusion, our findings offered a new perspective on the effects of HIV infection and especially on the intricate relationship between microbiota, immune system and HIV. 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