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

Abstract

Long-term cotrimoxazole prophylaxis reduces mortality and morbidity in HIV infection, but the mechanisms underlying these clinical benefits are unclear. Here, we investigate the impact of cotrimoxazole on systemic inflammation, an independent driver of HIV mortality. In HIV-positive Ugandan and Zimbabwean children receiving antiretroviral therapy, we show that plasma inflammatory markers were lower after randomization to continue (n = 144) versus stop (n = 149) cotrimoxazole. This was not explained by clinical illness, HIV progression, or nutritional status. Because subclinical enteropathogen carriage and enteropathy can drive systemic inflammation, we explored cotrimoxazole effects on the gut microbiome and intestinal inflammatory biomarkers. Although global microbiome composition was unchanged, viridans group Streptococci and streptococcal mevalonate pathway enzymes were lower among children continuing (n = 36) versus stopping (n = 36) cotrimoxazole. These changes were associated with lower fecal myeloperoxidase. To isolate direct effects of cotrimoxazole on immune activation from antibiotic effects, we established in vitro models of systemic and intestinal inflammation. In vitro cotrimoxazole had modest but consistent inhibitory effects on proinflammatory cytokine production by blood leukocytes from HIV-positive (n = 16) and HIV-negative (n = 8) UK adults and reduced IL-8 production by gut epithelial cell lines. Collectively we demonstrate that cotrimoxazole reduces systemic and intestinal inflammation both indirectly via antibiotic effects on the microbiome and directly by blunting immune and epithelial cell activation. Synergy between these pathways may explain the clinical benefits of cotrimoxazole despite high antimicrobial resistance, providing further rationale for extending coverage among people living with HIV in sub-Saharan Africa.

Figure 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 timepoints using generalized estimating equations and at individual timepoints using standard regression models (normal distribution for log-transformed values), adjusted for center and baseline concentrations (global p; A-D); *p<0.05, **p<0.01 ***p<0.001. (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 ***p<0.001.

Figure 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 <80 copies/mL; (B) geometric mean percentage 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 shown under each graph). Randomized groups were compared by generalized estimating equations across timepoints (global p) and at individual timepoints using standard regression models (binomial distribution for viral load; normal distribution for log-transformed values) adjusted for recruitment center; *p<0.05, **p<0.01 ***p<0.001.

Figure 3. Continuation of cotrimoxazole suppresses the abundance and function of viridans group Streptococci in stool samples from HIV-positive children.

Non-metric multidimensional scaling 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 labelled. Randomized groups were compared by permutation tests. (C) Effect size plots of relative abundance ratios (±95% confidence interval) for all Streptococcus spp. and their protein families (Pfam) and mevalonate pathway-associated genes (KEGG EC), and metabolic pathways (all bacterial species) that significantly differed between randomized groups at both week-84 and week-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 <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 analysis at week-84 (continue n=36, stop n=36) and week-96 (continue n=33, stop n=35)

Figure 4. Intestinal inflammation in HIV-positive children is associated with gut-resident viridans group Streptococci 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 plots showing average change in myeloperoxidase per 1% change in relative abundance (±95% confidence interval) for all Streptococcus spp. and their protein families (Pfam) and mevalonate pathway-associated genes (KEGG EC), and metabolic pathways (all bacterial species) that significantly differed between randomized groups at both week-84 and week-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 >1.0 indicates increase in myeloperoxidase with increased abundance. Vertical line indicates null value. Size of square inversely proportional to p-value.

Figure 5. Cotrimoxazole inhibits in vitro pro-inflammatory 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 heat-killed Salmonella typhimurium (HKST), lipopolysaccharide (LPS); or zymosan. Cultures were treated with low-dose cotrimoxazole (CTX_[Low]: 2 μg/mL trimethoprim, 50 μg/mL sulfamethoxazole), high-dose cotrimoxazole (CTX_[High]: 8 μg/mL trimethoprim, 200 μg/mL sulfamethoxazole) or volume-matched controls (DMSO_[Low], DMSO_[High]). Proportions of monocytes (left), CD4+ (center) and CD8+ T-cells (C) producing TNFα and (D) expressing HLA-DR after 6h PBMC culture with HKST or staphylococcal enterotoxin B (SEB). Grey bars indicate HIV-negative (n=8); red indicate HIV-positive ART-treated (n=6); and blue indicate HIV-positive ART-naïve group (n=10). Cytokine concentrations in cotrimoxazole-treated cultures are indicated by darker shading. Drug treatments compared within groups by Freidman tests with post-hoc uncorrected Dunn’s tests; *p<0.05, **p<0.01, ***p<0.001.

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

(A) Light microscopy of confluent Caco-2 monolayer (200 μm scale bar) and diagram showing transwell culture model. (B) Percentage lactose dehydrogenase activity relative to lysed cells (%LDH) of Caco-2 cultured for 24h with titrated concentrations of cotrimoxazole (CTX; black bars) or volume-matched DMSO control (grey 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 trans-epithelial resistance (TEER) in transwell Caco-2 cultures without drug (white circles), 1 mg/mL cotrimoxazole (black circles) or DMSO (grey circles) relative to transwells without Caco-2 (no cells; white triangles); mean ±SEM, n=3 separate experiments. Dotted line indicates culture confluence (TEER≥800Ω). (D) Epithelial cell functions (Δ TEER, % LDH, % apical-to-basal passage of Lucifer Yellow dye relative to transwells without Caco-2 cells, and IL-8 concentration in apical supernatants) of confluent Caco-2 monolayers treated with 1 mg/mL CTX or DMSO since seeding, then incubated with media alone (no stimulus) or IL-1β for 24h; mean ±SEM, n=3 separate experiments. Cotrimoxazole and DMSO-treatment compared by 2-tailed t-tests; *p<0.05, **p<0.01