Latent Tuberculosis Infection Is Associated with an Enrichment of Short-Chain Fatty Acid-Producing Bacteria in the Stool of Women Living with HIV.

Latent tuberculosis infection (LTBI) is common in people living with HIV (PLHIV) in high-TB-burden settings. Active TB is associated with specific stool taxa; however, little is known about the stool microbiota and LTBI in PLHIV. We characterised the stool microbiota of PLHIV with [interferon-γ release assay (IGRA)- and tuberculin skin test (TST)-positive] or without (IGRA- and TST-negative) LTBI (n = 25 per group). The 16S rRNA DNA sequences were analysed using QIIME2, Dirichlet-Multinomial Mixtures, DESeq2, and PICRUSt2. No α- or ß-diversity differences occurred by LTBI status; however, LTBI-positive people were Faecalibacterium-, Blautia-, Gemmiger-, and Bacteroides-enriched and Moryella-, Atopobium-, Corynebacterium-, and Streptococcus-depleted. Inferred metagenome data showed that LTBI-negative-enriched pathways included several metabolite degradation pathways. Stool from LTBI-positive people demonstrated differential taxa abundance based on a quantitative response to antigen stimulation. In LTBI-positive people, older people had different ß-diversities than younger people, whereas in LTBI-negative people, no differences occurred across age groups. Amongst female PLHIV, those with LTBI were, vs. those without LTBI, Faecalibacterium-, Blautia-, Gemmiger-, and Bacteriodes-enriched, which are producers of short-chain fatty acids. Taxonomic differences amongst people with LTBI occurred according to quantitative response to antigen stimulation and age. These data enhance our understanding of the microbiome's potential role in LTBI.

Latent tuberculosis infection is associated with an enrichment of short chain fatty acid producing bacteria in the stool of women living with HIV.

Background: Latent tuberculosis infection (LTBI) is common in people living with HIV (PLHIV) in high TB burden settings. Active TB is associated with specific stool taxa; however, little is known about the stool microbiota and LTBI, including in PLHIV. Method: Within a parent study that recruited adult females with HIV from Cape Town, South Africa into predefined age categories (18-25, 35-60 years), we characterised the stool microbiota of those with [interferon-γ release assay (IGRA)- and tuberculin skin test (TST)-positive] or without (IGRA- and TST- negative) LTBI (n=25 per group). 16S rRNA DNA sequences were analysed using QIIME2, Dirichlet Multinomial Mixtures, DESeq2 and PICRUSt2. Results: No α- or ß-diversity differences occurred by LTBI status; however, LTBI-positives were Faecalibacterium-, Blautia-, Gemmiger-, Bacteroides-enriched and Moryella-, Atopobium-, Corynebacterium-, Streptococcus-depleted. Inferred metagenome data showed LTBI-negative-enriched pathways included several involved in methylglyoxal degradation, L-arginine, putrescine, 4-aminobutanoate degradation and L-arginine and ornithine degradation. Stool from LTBI-positives demonstrated differential taxa abundance based on a quantitative response to antigen stimulation (Acidaminococcus-enrichment and Megamonas-, Alistipes-, and Paraprevotella-depletion associated with higher IGRA or TST responses, respectively). In LTBI-positives, older people had different ß-diversities than younger people whereas, in LTBI-negatives, no differences occurred across age groups. Conclusion: Amongst female PLHIV, those with LTBI had, vs. those without LTBI, Faecalibacterium, Blautia, Gemmiger, Bacteriodes-enriched, which are producers of short chain fatty acids. Taxonomic differences amongst people with LTBI occurred according to quantitative response to antigen stimulation and age. These data enhance our understanding of the microbiome's potential role in LTBI.

Mycobacterium tuberculosis resisters despite HIV exhibit activated T cells and macrophages in their pulmonary alveoli.

To understand natural resistance to Mycobacterium tuberculosis ( Mtb ) infection, we studied people living with HIV (PLWH) in an area of high Mtb transmission. Given that alveolar leukocytes may contribute to this resistance, we performed single cell RNA-sequencing of bronchoalveolar lavage cells, unstimulated or ex vivo stimulated with Mtb . We obtained high quality cells for 7 participants who were TST & IGRA positive (called LTBI) and 6 who were persistently TST & IGRA negative (called resisters). Alveolar macrophages (AM) from resisters displayed more of an M1 phenotype relative to LTBI AM at baseline. Alveolar lymphocytosis (10%-60%) was exhibited by 5/6 resisters, resulting in higher numbers of CD4 + and CD8 + IFNG -expressing cells at baseline and upon Mtb challenge than LTBI samples. Mycobactericidal granulysin was expressed almost exclusively by a cluster of CD8 + T cells that co-expressed granzyme B, perforin and NK cell receptors. For resisters, these poly-cytotoxic T cells over-represented activating NK cell receptors and were present at 15-fold higher numbers in alveoli compared to LTBI. Altogether, our results showed that alveolar lymphocytosis, with increased numbers of alveolar IFNG -expressing cells and CD8 + poly-cytotoxic T cells, as well as activated AM were strongly associated with protection from persistent Mtb infection in PLWH.

Neutrophil extracellular trap formation and gene programs distinguish TST/IGRA sensitization outcomes among Mycobacterium tuberculosis exposed persons living with HIV.

Persons living with HIV (PLWH) have an increased risk for tuberculosis (TB). After prolonged and repeated exposure, some PLWH never develop TB and show no evidence of immune sensitization to Mycobacterium tuberculosis (Mtb) as defined by persistently negative tuberculin skin tests (TST) and interferon gamma release assays (IGRA). This group has been identified and defined as HIV+ persistently TB, tuberculin and IGRA negative (HITTIN). To investigate potential innate mechanisms unique to individuals with the HITTIN phenotype we compared their neutrophil Mtb infection response to that of PLWH, with no TB history, but who test persistently IGRA positive, and tuberculin positive (HIT). Neutrophil samples from 17 HITTIN (PMNHITTIN) and 11 HIT (PMNHIT) were isolated and infected with Mtb H37Rv for 1h and 6h. RNA was extracted and used for RNAseq analysis. Since there was no significant differential transcriptional response at 1h between infected PMNHITTIN and PMNHIT, we focused on the 6h timepoint. When compared to uninfected PMN, PMNHITTIN displayed 3106 significantly upregulated and 3548 significantly downregulated differentially expressed genes (DEGs) (absolute cutoff of a log2FC of 0.2, FDR < 0.05) whereas PMNHIT demonstrated 3816 significantly upregulated and 3794 significantly downregulated DEGs following 6h Mtb infection. Contrasting the log2FC 6h infection response to Mtb from PMNHITTIN against PMNHIT, 2285 genes showed significant differential response between the two groups. Overall PMNHITTIN had a lower fold change response to Mtb infection compared to PMNHIT. According to pathway enrichment, Apoptosis and NETosis were differentially regulated between HITTIN and HIT PMN responses after 6h Mtb infection. To corroborate the blunted NETosis transcriptional response measured among HITTIN, fluorescence microscopy revealed relatively lower neutrophil extracellular trap formation and cell loss in PMNHITTIN compared to PMNHIT, showing that PMNHITTIN have a distinct response to Mtb.

Phenotype Definition for "Resisters" to Mycobacterium tuberculosis Infection in the Literature-A Review and Recommendations.

Tuberculosis (TB) remains a worldwide problem. Despite the high disease rate, not all who are infected with Mycobacterium Tuberculosis (Mtb) develop disease. Interferon-γ (IFN-γ) specific T cell immune assays such as Quantiferon and Elispot, as well as a skin hypersensitivity test, known as a tuberculin skin test, are widely used to infer infection. These assays measure immune conversion in response to Mtb. Some individuals measure persistently negative to immune conversion, despite high and prolonged exposure to Mtb. Increasing interest into this phenotype has led to multiple publications describing various aspects of these responses. However, there is a lack of a unified "resister" definition. A universal definition will improve cross study data comparisons and assist with future study design and planning. We review the current literature describing this phenotype and make recommendations for future studies.

An observational study identifying highly tuberculosis-exposed, HIV-1-positive but persistently TB, tuberculin and IGRA negative persons with M. tuberculosis specific antibodies in Cape Town, South Africa.

BACKGROUND: Mycobacterium tuberculosis (Mtb) infection is inferred from positive results of T-cell immune conversion assays measuring Mtb-specific interferon gamma production or tuberculin skin test (TST) reactivity. Certain exposed individuals do not display T-cell immune conversion in these assays and do not develop TB. Here we report a hitherto unknown form of this phenotype: HIV-1-positive persistently TB, tuberculin and IGRA negative (HITTIN). METHODS: A community-based case-control design was used to systematically screen and identify adults living with HIV (HIV+), aged 35-60 years, who met stringent study criteria, and then longitudinally followed up for repeat IGRA and TST testing. Participants had no history of TB despite living in TB hyper-endemic environments in Cape Town, South Africa with a provincial incidence of 681/100,000. Mtb-specific antibodies were measured using ELISA and Luminex. FINDINGS: We identified 48/286 (17%) individuals who tested persistently negative for Mtb-specific T-cell immunoreactivity (three negative Quantiferon results and one TST = 0mm) over 206±154 days on average. Of these, 97·2% had documented CD4 counts<200 prior to antiretroviral therapy (ART). They had received ART for 7·0±3·0 years with a latest CD4 count of 505·8±191·4 cells/mm3. All HITTIN sent for further antibody testing (n=38) displayed  Mtb-specific antibody titres. INTERPRETATION: Immune reconstituted HIV+ persons can be persistently non-immunoreactive to TST and interferon-γ T-cell responses to Mtb, yet develop species-specific antibody responses. Exposure is evidenced by Mtb-specific antibody titres. Our identification of HIV+ individuals displaying a persisting lack of response to TST and IGRA T-cell immune conversion paves the way for future studies to investigate this phenotype in the context of HIV-infection that so far have received only scant attention.

Neutrophils: Innate Effectors of TB Resistance?

Certain individuals are able to resist Mycobacterium tuberculosis infection despite persistent and intense exposure. These persons do not exhibit adaptive immune priming as measured by tuberculin skin test (TST) and interferon-γ (IFN-γ) release assay (IGRA) responses, nor do they develop active tuberculosis (TB). Genetic investigation of individuals who are able to resist M. tuberculosis infection shows there are likely a combination of genetic variants that contribute to the phenotype. The contribution of the innate immune system and the exact cells involved in this phenotype remain incompletely elucidated. Neutrophils are prominent candidates for possible involvement as primers for microbial clearance. Significant variability is observed in neutrophil gene expression and DNA methylation. Furthermore, inter-individual variability is seen between the mycobactericidal capacities of donor neutrophils. Clearance of M. tuberculosis infection is favored by the mycobactericidal activity of neutrophils, apoptosis, effective clearance of cells by macrophages, and resolution of inflammation. In this review we will discuss the different mechanisms neutrophils utilize to clear M. tuberculosis infection. We discuss the duality between neutrophils' ability to clear infection and how increasing numbers of neutrophils contribute to active TB severity and mortality. Further investigation into the potential role of neutrophils in innate immune-mediated M. tuberculosis infection resistance is warranted since it may reveal clinically important activities for prevention as well as vaccine and treatment development.

Genetic Resistance to Mycobacterium tuberculosis Infection and Disease.

Natural history studies of tuberculosis (TB) have revealed a spectrum of clinical outcomes after exposure to Mycobacterium tuberculosis, the cause of TB. Not all individuals exposed to the bacterium will become diseased and depending on the infection pressure, many will remain infection-free. Intriguingly, complete resistance to infection is observed in some individuals (termed resisters) after intense, continuing M. tuberculosis exposure. After successful infection, the majority of individuals will develop latent TB infection (LTBI). This infection state is currently (and perhaps imperfectly) defined by the presence of a positive tuberculin skin test (TST) and/or interferon gamma release assay (IGRA), but no detectable clinical disease symptoms. The majority of healthy individuals with LTBI are resistant to clinical TB, indicating that infection is remarkably well-contained in these non-progressors. The remaining 5-15% of LTBI positive individuals will progress to active TB. Epidemiological investigations have indicated that the host genetic component contributes to these infection and disease phenotypes, influencing both susceptibility and resistance. Elucidating these genetic correlates is therefore a priority as it may translate to new interventions to prevent, diagnose or treat TB. The most successful approaches in resistance/susceptibility investigation have focused on specific infection and disease phenotypes and the resister phenotype may hold the key to the discovery of actionable genetic variants in TB infection and disease. This review will not only discuss lessons from epidemiological studies, but will also focus on the contribution of epidemiology and functional genetics to human genetic resistance to M. tuberculosis infection and disease.