Research ArticleHIV

Cotrimoxazole reduces systemic inflammation in HIV infection by altering the gut microbiome and immune activation

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Science Translational Medicine  03 Apr 2019:
Vol. 11, Issue 486, eaav0537
DOI: 10.1126/scitranslmed.aav0537
  • Fig. 1 Systemic inflammation is lower among HIV-positive children randomized to continue daily oral cotrimoxazole prophylaxis.

    Geometric mean concentrations of (A) CRP, (B) IL-6, (C) sCD14, and (D) TNFα in plasma of HIV-positive children who had been receiving ART and cotrimoxazole for ≥96 weeks and were then randomized to stop (orange circles) or continue (green squares) cotrimoxazole. Randomized groups were compared across time points using generalized estimating equations (GEE) and at individual time points using standard regression models (normal distribution for log-transformed values), adjusted for center and baseline concentrations (global P; A to D). (E) Serum protein concentrations at week 48 post-randomization; horizontal bars indicate means. Comparisons between groups by Mann-Whitney U test; *P < 0.05, **P < 0.01.

  • Fig. 2 Cotrimoxazole effects on systemic inflammation are not solely due to differences in HIV disease progression, symptomatic infections, or nutritional status.

    (A) Percentage of children with viral load of <80 copies/ml; (B) geometric mean percentage of CD4+ T cells; mean proportions of children with caregiver-reported (C) cough, (D) fever, (E) vomiting/nausea, and (F) abdominal pain; geometric mean (G) weight-for-age and (H) height-for-age Z-scores in children randomized to continue versus stop cotrimoxazole prophylaxis (n per group is shown under each graph). Randomized groups were compared by GEE across all time points post-randomization (global P) and at individual time points using standard regression models (binomial distribution for viral load; normal distribution for log-transformed values) adjusted for recruitment center; *P < 0.05.

  • Fig. 3 Continuation of cotrimoxazole suppresses the abundance and function of VGS in stool samples from HIV-positive children.

    NMDS plots of the Bray-Curtis dissimilarity index for stool samples from 72 HIV-positive Zimbabwean children randomized to stop (orange) versus continue (green) cotrimoxazole at (A) week 84 and (B) week 96 post-randomization. Red crosses indicate individual bacterial species irrespective of randomized group; VGS species that consistently differed between randomized groups are labeled. Randomized groups were compared by permutation tests. (C) Effect size plots of relative abundance ratios (±95% CI) for all Streptococcus spp. and their Pfam and mevalonate pathway–associated genes (KEGG EC) and metabolic pathways (all bacterial species) that significantly differed between randomized groups at both weeks 84 and 96 in FDR-adjusted zero-inflated beta regression. Identities for Pfam and KEGG EC were established using HUMANn2 against the UniRef90 database. Relative abundance ratio of <1.0 indicates lower relative abundance in children who continued versus stopped cotrimoxazole. Vertical line indicates null value. Size of square is inversely proportional to P value. Percentage of samples positive for any of the four VGS or individual species according to (D) MetaPhlAn and (E) PanPhlAn at week 84 (continue, n = 36; stop, n = 36) and week 96 (continue, n = 33; stop, n = 35).

  • Fig. 4 Intestinal inflammation in HIV-positive children is associated with gut-resident VGS that are suppressed by continuation of cotrimoxazole.

    Myeloperoxidase at (A) week 84 and (B) week 96 in stool samples from HIV-positive Zimbabwean children randomized to stop versus continue cotrimoxazole. Randomized groups compared by Mann-Whitney U test; *P < 0.05, horizontal lines indicate median. (C) Effect size plot showing average change in myeloperoxidase per 1% change in relative abundance (±95% CI) for all Streptococcus spp. and their Pfam and mevalonate pathway–associated genes (KEGG EC) and metabolic pathways (all bacterial species) that significantly differed between randomized groups at both weeks 84 and 96 in FDR-adjusted zero-inflated beta regression (Fig. 3C). Identities for Pfam and KEGG EC were established using HUMANn2 against the UniRef90 database. Average change of >1.0 indicates increase in myeloperoxidase with increased abundance. Vertical line indicates null value. Size of square inversely proportional to P value.

  • Fig. 5 Cotrimoxazole inhibits in vitro proinflammatory cytokine responses to bacterial and fungal antigens.

    Tukey boxplots of (A) TNFα and (B) IL-6 concentrations in supernatants from whole-blood cultures without antigen (no stimulus), with HKST, LPS, or zymosan. Cultures were treated with media with no drug and no DMSO (ND), with low-dose cotrimoxazole (CTX[Low]; 2 μg/ml of trimethoprim and 50 μg/ml of sulfamethoxazole), high-dose cotrimoxazole (CTX[High]; 8 μg/ml of trimethoprim and 200 μg/ml of sulfamethoxazole) or volume-matched drug diluent controls (DMSO[Low], DMSO[High]). Proportions of monocytes (left), CD4+ (center), and CD8+ T cells (right) (C) producing TNFα and (D) expressing HLA-DR after 6 hours of PBMC culture with HKST or SEB. Gray bars indicate HIV-negative (n = 8); red indicates HIV-positive ART-treated (n = 6); and blue indicates HIV-positive ART-naïve group (n = 10). Cytokine concentrations in cotrimoxazole-treated cultures are indicated by darker shading. Drug treatments compared within groups by Friedman tests with post hoc uncorrected Dunn’s tests; *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 6 Cotrimoxazole reduces in vitro IL-8 production by gut epithelial cells under inflammatory conditions.

    (A) Light microscopy of a confluent Caco-2 monolayer (scale bar, 200 μm) and diagram showing transwell culture model. (B) Percentage lactose dehydrogenase activity relative to lysed cells (%LDH) of Caco-2 cells cultured for 24 hours with titrated concentrations of cotrimoxazole (CTX; black bars) or volume-matched DMSO control (gray bars); %LDH compared to untreated controls and between volume-matched pairs of cotrimoxazole and DMSO by adjusted Tukey’s test; ***P < 0.001. (C) Daily TEER in transwell Caco-2 cultures without drug (white circles), cotrimoxazole (1 mg/ml; black circles), or DMSO (gray circles) relative to transwells without Caco-2 cells (no cells; white triangles); mean ± SEM, n = 3 separate experiments. Dotted line indicates culture confluence (TEER ≥800 ohm). (D) Epithelial cell functions (ΔTEER, %LDH, % apical-to-basal passage of Lucifer Yellow (LY) dye relative to transwells without Caco-2 cells, and IL-8 concentration in apical supernatants) of confluent Caco-2 monolayers treated with CTX (1 mg/ml) or DMSO since seeding and then incubated with media alone (no stimulus) or IL-1β for 24 hours; mean ± SEM, n = 3 separate experiments. Cotrimoxazole and DMSO treatment compared by two-tailed t tests; **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/11/486/eaav0537/DC1

    Materials and Methods

    Fig. S1. Cotrimoxazole alters circulating CD4+ T cell phenotype in HIV infection.

    Fig. S2. Fecal bacterial species that differ between HIV-positive ART-treated Zimbabwean children randomized to continue versus stop cotrimoxazole prophylaxis.

    Fig. S3. Protein families that differ between stool samples from HIV-positive ART-treated Zimbabwean children randomized to continue versus stop cotrimoxazole prophylaxis.

    Fig. S4. Fecal biomarkers of enteropathy that were unaffected by continuing versus stopping cotrimoxazole prophylaxis.

    Fig. S5. Associations between all fecal bacterial species that differed between HIV-positive children randomized to continue versus stop cotrimoxazole prophylaxis and fecal myeloperoxidase.

    Fig. S6. Associations between all fecal Pfam that differed between HIV-positive children randomized to continue versus stop cotrimoxazole prophylaxis and fecal myeloperoxidase.

    Fig. S7. Optimization of in vitro blood leukocyte activation and cotrimoxazole treatment conditions.

    Fig. S8. HIV-positive adults have greater systemic inflammation, monocyte, and T cell activation than HIV-negative adults.

    Fig. S9. Flow cytometry gating strategy for analysis of monocyte and T cell intracellular cytokine responses.

    Table S1. Characteristics of HIV-negative and HIV-positive UK adult volunteers.

    Table S2. Details of fluorophore-conjugated antibody combinations used for flow cytometry analysis of PBMC from HIV-negative and HIV-positive adults.

    Data file S1. Primary data.

    References (6468)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Cotrimoxazole alters circulating CD4+ T cell phenotype in HIV infection.
    • Fig. S2. Fecal bacterial species that differ between HIV-positive ART-treated Zimbabwean children randomized to continue versus stop cotrimoxazole prophylaxis.
    • Fig. S3. Protein families that differ between stool samples from HIV-positive ART-treated Zimbabwean children randomized to continue versus stop cotrimoxazole prophylaxis.
    • Fig. S4. Fecal biomarkers of enteropathy that were unaffected by continuing versus stopping cotrimoxazole prophylaxis.
    • Fig. S5. Associations between all fecal bacterial species that differed between HIV-positive children randomized to continue versus stop cotrimoxazole prophylaxis and fecal myeloperoxidase.
    • Fig. S6. Associations between all fecal Pfam that differed between HIV-positive children randomized to continue versus stop cotrimoxazole prophylaxis and fecal myeloperoxidase.
    • Fig. S7. Optimization of in vitro blood leukocyte activation and cotrimoxazole treatment conditions.
    • Fig. S8. HIV-positive adults have greater systemic inflammation, monocyte, and T cell activation than HIV-negative adults.
    • Fig. S9. Flow cytometry gating strategy for analysis of monocyte and T cell intracellular cytokine responses.
    • Table S1. Characteristics of HIV-negative and HIV-positive UK adult volunteers.
    • Table S2. Details of fluorophore-conjugated antibody combinations used for flow cytometry analysis of PBMC from HIV-negative and HIV-positive adults.
    • References (6468)

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    Other Supplementary Material for this manuscript includes the following:

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