Anaerobic Bacterial Fermentation Products Increase Tuberculosis Risk in Antiretroviral-Drug-Treated HIV Patients

Abstract

Despite the immune-reconstitution with antiretroviral therapy (ART), HIV-infected individuals remain highly susceptible to tuberculosis (TB) and have an enrichment of oral anaerobes in the lung. Products of bacterial anaerobic metabolism, like butyrate and other short-chain fatty acids (SCFAs), induce regulatory T cells (Tregs). We tested whether SCFAs contribute to poor TB control in a longitudinal cohort of ART-treated HIV-infected South Africans. Increase in serum SCFAs was associated with increased TB susceptibility. SCFAs inhibited IFN-γ and IL-17A production in peripheral blood mononuclear cells from HIV-infected ART-treated individuals in response to M. tuberculosis antigen stimulation. Pulmonary SCFAs correlated with increased oral anaerobes, such as Prevotella in the lung, and with M. tuberculosis antigen-induced Tregs. Metabolites from anaerobic bacterial fermentation may, therefore, increase TB susceptibility by suppressing IFN-γ and IL-17A production during the cellular immune response to M. tuberculosis.

Keywords: FoxP1; FoxP3; HIV; dysbiosis; lung; short-chain fatty acids; tuberculosis.

Figure 1. SCFA are present in the lungs of HIV-infected individuals on ART; inhibiting IFN-γ and IL-17A production after M. tuberculosis antigen stimulation

A. Acetate (open triangles), Propionate (closed circles) and butyrate (open boxes) are present in BAL. Concentrations are corrected for BAL induced dilution of epithelial lining fluid (ELF). Propionate is significantly elevated in HIV-infected subjects, Mann–Whitney U test. B. In HIV infected individuals with paired samples, ELF SCFA is higher than serum in all subjects, with a median ratio of 370. C. Addition of PPD to BAL cells demonstrates induction of FoxP3 CD4⁺ cells significantly correlates with in vivo ELF acetate or propionate at the time of bronchoscopy, Spearman correlation.

Figure 2. Butyrate inhibits IFN-γ and IL-17A production after M. tuberculosis antigen stimulation and induces FoxP1 mRNA in CD4⁺ and CD8⁺ lymphocytes

A. Compared with PPD, butyrate inhibits IFN-γ and IL-17A production by PPD stimulation. Peripheral blood mononuclear cells of HIV-infected individuals on ART with latent tuberculosis were cultured in vitro without stimulation for three days (Unstim.), with PPD for three days (PPD) or with 2mM butyrate added with PPD three days (PPD+ Butyrate). Paired t test of log transformed data. B. Addition of 2mM butyrate inhibits IFN-γ and IL-17A expression in CD4⁺ and CD8⁺ lymphocytes stimulated with anti-CD3/CD28. Lymphocytes from 6 different individuals are shown. Paired t test of log transformed data. C. Addition of 2mM butyrate increases FoxP1 mRNA expression in CD4⁺ and CD8⁺ lymphocytes. Paired t test of log transformed FoxP/GAPDH.

Figure 3. Propionate-detectable individuals have increased relative abundance of oral anaerobic bacteria in BAL

A. Rarefaction curve on taxonomic 16S rRNA gene annotation demonstrates no difference in α-diversity between the taxa in the microbiomes of 10 propionate-detectable and 29 propionate-undetectable individuals. B. A principle component analysis based on UniFrac distance demonstrates a significant difference in the β-diversity between propionate-detectable and propionate-undetectable individuals (p value based on PERMANOVA). C. LEfSe demonstrates enrichment of anaerobes such as Prevotella or Veillonella in the lung microbiome of propionate-detectable individuals. The histograms on top of the panel represent relative abundance of each taxa while the histograms below represent the LDA value. The name of the enriched taxa is at the bottom of the panel. D. There is a significant inverse correlation between BAL CD4⁺ lymphocytes and log₁₀ transformed relative abundance of Prevotella or Veillonella in BAL. Spearman correlation.