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

Cotrimoxazole curbs inflammation

Prophylactic antibiotics are used in HIV-endemic areas and have been shown to reduce mortality and morbidity. Using samples from a clinical trial where HIV-positive children were randomized to continue or stop cotrimoxazole, Bourke et al. examined potential mechanisms behind these protective effects. They observed decreases of certain types of Streptococcus in the gut and decreased systemic inflammatory markers in children who continued treatment. Treatment of primary samples from HIV-infected adults or a gut epithelial cell line revealed that cotrimoxazole dampened inflammatory cytokine production. Their results showcase how this antibiotic interacts with the gut microbiome and the immune system to alter the inflammatory response with beneficial outcomes.

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.

INTRODUCTION

In 2017, 36.9 million people were living with HIV and 940,000 died from AIDS-related illnesses (1). To reduce mortality and morbidity (2, 3), World Health Organization (WHO) guidelines recommend long-term cotrimoxazole prophylaxis for all people living with HIV in areas with a high prevalence of malaria and/or severe bacterial infections (4). However, it is unclear how cotrimoxazole reduces mortality and morbidity, given the high rates of antimicrobial resistance and selection for resistant pathogens with long-term use (2). There is therefore a need to better understand the effect of cotrimoxazole on HIV pathogenesis.

Systemic inflammation is independently associated with mortality in HIV infection (57). Cotrimoxazole might plausibly confer benefits by reducing inflammation, either indirectly by targeting pathogens or directly by modulating cells that produce proinflammatory mediators. Animal models suggest that antibiotics confer anti-inflammatory benefits (8), and observational studies of HIV-positive adults suggest that cotrimoxazole can reduce plasma inflammatory biomarkers (9, 10). Data from randomized trials and low-income settings are lacking, and no studies have evaluated the effects of cotrimoxazole on proinflammatory pathways in HIV-positive individuals.

HIV drives a chronic enteropathy, characterized by loss of villous architecture, increased gut permeability, mucosal CD4+ T cell depletion (11), leukocyte infiltration (1214), and microbial translocation (15, 16), accompanied by increased pathogen carriage and an altered microbiome (17, 18); together, these changes contribute to systemic inflammation. Cotrimoxazole prophylaxis could influence intestinal inflammation through antibiotic effects on enteropathogens and/or the microbiome or via direct effects on mucosal leukocytes and gut epithelial cells (19, 20). Among HIV-positive Ugandan adults, cotrimoxazole had limited effects on the gut microbiome (21); however, the effects of cotrimoxazole have not been assessed in a randomized trial or in children.

Cotrimoxazole comprises two folate pathway inhibitors, trimethoprim and sulfamethoxazole. The hypothesis that cotrimoxazole can directly inhibit proinflammatory immune cell activation was first posited in 1970, after the observation that intramuscular trimethoprim prolonged skin graft retention in mice (22). However, subsequent in vitro studies of the direct effects of cotrimoxazole on immune cells have yielded conflicting results (2326), and none have assessed its anti-inflammatory effects in HIV-positive individuals. Cotrimoxazole treatment of rats affects absorption across the gut epithelium (19), suggesting that cotrimoxazole may influence gut barrier function, a critical regulator of cross-talk between the circulation and gut-resident microorganisms.

Thus, cotrimoxazole prophylaxis confers long-term clinical benefits in HIV infection, which are not entirely explained by its antibiotic effects (2, 3). Inconsistent evidence suggests that cotrimoxazole may have anti-inflammatory properties, but data are inconclusive. We therefore capitalized on a randomized trial of continuing versus stopping cotrimoxazole in HIV-positive children in sub-Saharan Africa to test the hypothesis that cotrimoxazole reduces systemic inflammation. We then explored mechanistic pathways through which this may occur, using clinical data, stored specimens, and in vitro models.

RESULTS

Cotrimoxazole reduces systemic inflammation in HIV-positive children

We have previously shown that randomization to continue versus stop cotrimoxazole prophylaxis reduced hospitalization or death among HIV-positive children on long-term antiretroviral therapy (ART) in the Antiretroviral research for Watoto trial (ARROW) in Uganda and Zimbabwe (27). Because the inflammatory biomarkers C-reactive protein (CRP) and interleukin-6 (IL-6) were independently associated with mortality in ARROW (5), we hypothesized that the benefits of cotrimoxazole might be partly mediated through reductions in systemic inflammation. CRP, IL-6, soluble CD14 (sCD14), and tumor necrosis factor–α (TNFα) were quantified in longitudinal plasma samples from children randomized to continue (n = 144) versus stop (n = 149) cotrimoxazole (Fig. 1).

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.

Biomarkers were similar between groups at baseline (Fig. 1, A to D), but subsequent CRP concentrations from week 24 until the end of follow-up were lower in children randomized to continue cotrimoxazole (global P = 0.006; Fig. 1A). IL-6 was also significantly lower among children continuing cotrimoxazole, particularly at early time points (global P = 0.010; week 12, P = 0.014; week 24, P = 0.003; Fig. 1B). There was no evidence of global differences between groups in sCD14 (Fig. 1C) or TNFα (Fig. 1D). Serum albumin was significantly higher (median, 42 g/liter versus 41 g/liter; P = 0.041), and total protein was significantly lower (median, 76 g/liter versus 78 g/liter; P = 0.038) in children continuing cotrimoxazole at week 48 (Fig. 1E), consistent with less systemic inflammation. Collectively, these results show that cotrimoxazole reduces systemic inflammation in HIV-positive children.

To estimate the clinical implications of these findings, we used relative risk estimates of adverse outcomes (death, new, or recurrent WHO clinical stage 4 events or poor immunological response to ART) associated with baseline (i.e., pre-ART) concentrations of CRP and IL-6 in ARROW (5). Stopping cotrimoxazole led to 1.65-fold higher CRP [stop (2.71 mg/liter) versus continue (1.64 mg/liter); Fig. 1A] and 1.18-fold higher IL-6 [stop (5.36 pg/ml) versus continue (4.54 pg/ml); Fig. 1B] at week 24, corresponding to an increased relative risk of adverse clinical outcomes among children stopping cotrimoxazole of 13% [95% confidence interval (CI), 4 to 24%] and 11% (95% CI, 4 to 18%), respectively, within 24 weeks (5). Relative differences in CRP peaked at week 48 [1.92-fold increase; stop (2.86 mg/liter) versus continue (1.49 mg/liter); Fig. 1A], corresponding to an 18% (95% CI, 6 to 32%) increased risk of adverse clinical outcomes. Thus, differences in CRP and IL-6 with continued cotrimoxazole are important for long-term survival, health, and immune restoration among HIV-positive children.

Reduced systemic inflammation is not solely due to less clinical disease

Lower systemic inflammation with long-term cotrimoxazole could be due to reductions in HIV disease progression or clinical illness (2, 27). However, there was no evidence of global differences in the proportion of children with viral suppression (<80 HIV RNA copies/ml; Fig. 2A) or in CD4+ T cell percentages (%CD4; Fig. 2B) between randomized groups. There was also no evidence for global differences in caregiver-reported cough (Fig. 2C), fever (Fig. 2D), nausea/vomiting (Fig. 2E), or abdominal pain (Fig. 2F). Too few children had persistent, bloody, or moderate-to-severe diarrhea, difficult/fast breathing, and/or weight loss for comparison between groups.

HIV-positive children frequently have malnutrition; antibiotics (including cotrimoxazole) have been shown to improve growth (28) and slow weight loss (29). We therefore compared anthropometry between randomized groups, reasoning that differences in systemic inflammation might be explained by underlying wasting or stunting. We found no evidence of differences in weight-for-age (Fig. 2G) or height-for-age Z-scores (Fig. 2H). Thus, effects of cotrimoxazole on systemic inflammation were not explained by differences in HIV disease progression, symptomatic infections, or malnutrition between groups.

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.

Cotrimoxazole alters circulating CD4+ T cell phenotypes in HIV-positive children

Although cotrimoxazole continuation had no impact on total CD4+ T cell counts, we hypothesized that CD4+ T cell phenotypes would differ between randomized groups because systemic inflammation is associated with T cell activation and proliferation (30, 31, 5). T cell immunophenotyping in a subset of Ugandan ARROW participants (stop, n = 48; continue, n = 47; fig. S1A) revealed no evidence of differences between randomized groups in the proportions of total CD4+ T cells expressing the activation marker Human Leukocyte Antigen–DR isotype (HLA-DR) or the proliferation marker Ki67 (fig. S1, B and C). Children continuing cotrimoxazole had higher percentages of recent thymic emigrant-like (RTE) cells [CD4+CD45RA+CD31+ T cells]—an indicator of thymic output (32)—than children stopping prophylaxis (fig. S1B). There was no evidence of differences in proportions of naïve (CD4+CD45RA+CD31) or effector memory (CD4+CD45RACD31) T cells or in the expression of HLA-DR on any CD4+ T cell subpopulations (fig. S1C). However, children continuing cotrimoxazole had lower percentages of proliferating (Ki67+) RTE and naïve T cells, particularly at later time points after randomization (fig. S1D). Thus, cotrimoxazole continuation led to some changes in circulating T cells consistent with reduced systemic inflammation (5).

Cotrimoxazole suppresses abundance and function of gut-resident Streptococci

The gut microbiome is disrupted by HIV infection, which contributes to local and systemic inflammation (17, 33). We hypothesized that continuing cotrimoxazole would drive sustained subclinical differences in gut pathogens and commensals. We conducted whole-metagenome shotgun sequencing of total fecal DNA from children randomized to continue (n = 36 at week 84; n = 33 at week 96) versus stop cotrimoxazole (n = 36 at week 84; n = 35 at week 96). Randomized groups did not differ in species-level diversity [Shannon indices: 13.1 (continue) versus 14.3 (stop), P = 0.27; 13.5 (continue) versus 14.8 (stop), P = 0.72] or evenness [Pileou’s index: 0.59 (continue) versus 0.60 (stop), P = 0.605; 0.60 (continue) versus 0.61 (stop), P = 0.883] at week 84 or 96. Bacterial community composition was also similar between groups (Fig. 3, A and B). However, false discovery rate (FDR)–adjusted zero-inflated beta regression analysis of individual microbiome characteristics identified 7 bacterial species (Alistipes onderdonkii, Eggerthella lenta, Clostridium bartlettii, Haemophilus parainfluenzae, Streptococcus mutans, Streptococcus parasanguinis, and Streptococcus vestibularis; fig. S2) and 11 protein families (Pfam; fig. S3) mapping to S. parasanguinis, Streptococcus salivarius, and H. parainfluenzae that were consistently less abundant at both time points in the continue group versus the stop group (relative abundance ratio, < 1). The differentially abundant Streptococci are all within the viridans group of Streptococci (VGS) and largely fell in the quadrant of the nonmetric multidimensional scaling (NMDS) ordination plot where the extremes of the treatment groups lay (Fig. 3, A and B). The relative abundance of Enterobacteriaceae, which includes gastrointestinal pathogens (e.g., Salmonella, Escherichia coli, and Shigella) that are frequently resistant to cotrimoxazole (34, 35), was not affected by continuation of cotrimoxazole at week 84 (relative abundance ratio, 0.65; adjusted P = 0.108) and was increased in those continuing versus stopping cotrimoxazole at week 96 (4.48; adjusted P < 0.001).

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).

To understand the effect of cotrimoxazole on microbiome function, we quantified the abundance of full sets of genes involved in metabolic pathways across bacterial taxa. Only mevalonate pathway I, which influences neutrophil and monocyte recruitment and function, was consistently different between groups at both time points. Mevalonate pathway–associated genes were significantly less abundant in stool samples from children continuing cotrimoxazole (week 84, adjusted P = 0.042; week 96, adjusted P = 0.019; Fig. 3C). Of the enzyme-encoding genes within mevalonate pathway I, those with identity to S. parasanguinis {Kyoto Encyclopedia of Genes and Genomes Enzyme Commission (KEGG EC) (five enzymes): 1.1.1.88 [hydroxymethylglutaryl–coenzyme A (HMG-CoA) reductase], 2.3.3.10 (HMG-CoA synthase), 2.7.1.36 (mevalonate kinase), 4.1.1.33 (diphosphomevalonate decarboxylase), and 5.3.3.2 (isopentenyl-diphosphate delta-isomerase); adjusted P < 0.05 at both time points} and S. salivarius [KEGG EC (two enzymes): 1.1.1.88 and 5.3.3.2; adjusted P < 0.05 at both time points] were significantly less abundant in the continue group (Fig. 3C), suggesting that continuation of cotrimoxazole reduces VGS metabolic function in the gut.

To confirm this metagenomic signature of VGS suppression by cotrimoxazole, we conducted high-resolution mapping of metagenome sequencing reads to Streptococci pangenome datasets using PanPhlAn software, which has a lower false-positive rate for species identification and better discrimination between samples containing the same versus different bacterial genomes than initial mapping using MetaPhlAn (36). Of the 140 stool samples sequenced (both groups at weeks 84 and 96), PanPhlAn identified 29 samples positive for any Streptococci (nine species present: S. salivarius, S. parasanguinis, S. mutans, S. vestibularis, S. australis, S. infantarius, S. oligofermentans, S. pasteurianus, and S. sanguinis) and, of these, 20 samples were positive for at least one of the four VGS species identified using MetaPhlAn (7 at week 84 and 13 at week 96). PanPhlAn identified a lower percentage of VGS-positive samples on account of its higher species-level resolution (Fig. 3, D and E). Six samples from children continuing and 14 samples from children stopping cotrimoxazole were confirmed VGS-positive across both time points, corroborating VGS suppression by cotrimoxazole. Individual VGS species were present less often in children continuing cotrimoxazole (Fig. 3E). Together, these findings show that continuing compared to stopping long-term cotrimoxazole does not affect global microbiome community composition but does drive specific alterations in gut microbiome structure and function, with suppression of VGS and associated reductions in VGS mevalonate pathway genes.

Cotrimoxazole-induced changes in Streptococci reduce intestinal inflammation

We next tested whether these microbiome changes influenced HIV enteropathy. We compared levels of fecal inflammatory markers at weeks 84 and 96 after randomization to continue (n = 37) or stop (n = 38) cotrimoxazole. At week 84, fecal myeloperoxidase was significantly lower in children continuing versus stopping cotrimoxazole (median, 1694 ng/ml versus 3178 ng/ml; P = 0.022; Fig. 4A), but there was no evidence of differences in neopterin, α-1-antitrypsin, or regenerating family member 1 β (REG1β) between groups (P > 0.15; fig. S4A). At week 96, myeloperoxidase did not significantly differ between randomized groups (1262 ng/ml versus 1473 ng/ml, P = 0.093; Fig. 4B), and there was no evidence of differences in neopterin, α-1-antitrypsin, or REG1β (P > 0.15; fig. S4B). Because myeloperoxidase is an abundant peroxidase enzyme in monocytes and neutrophils that perpetuates granulocyte activation (37) and both cell types home to the gut mucosa during HIV infection (13, 14), these observations suggest that cotrimoxazole reduces innate immune cell activity in the gut.

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.

Of the bacterial species suppressed by cotrimoxazole, S. mutans, S. vestibularis, S. parasanguinis, and H. parainfluenzae were positively associated with myeloperoxidase levels at week 96 (Streptococcus spp. are summarized in Fig. 4C; analysis of all species is shown in fig. S5) after adjustment for age, sex, and cotrimoxazole group. Myeloperoxidase was also positively associated with Pfam that were differentially abundant according to cotrimoxazole treatment: five with identity to S. parasanguinis, two to S. salivarius, two to H. parainfluenzae, and one to Eubacterium bioforme at week 96 (Pfam with identify to Streptococcus spp. are summarized in Fig. 4C; analysis of all Pfam is shown in fig. S6). Overall, mevalonate pathway I abundance was significantly associated with higher myeloperoxidase at week 96 (adjusted P < 0.001; Fig. 4C). Of the mevalonate pathway I enzymes that differed between randomized groups, only those with identity to S. parasanguinis (five enzymes, adjusted P < 0.001) and S. salivarius (two enzymes, adjusted P < 0.01) had a significant positive association with myeloperoxidase (Fig. 4C). We therefore show that all VGS components suppressed by cotrimoxazole (Fig. 3C) were positively associated with myeloperoxidase (Fig. 4C), suggesting that reduced VGS abundance and function contribute to lower intestinal inflammation among children continuing cotrimoxazole.

Cotrimoxazole blunts proinflammatory cytokine responses in vitro

Having established that cotrimoxazole reduces both systemic and intestinal inflammation, we next investigated whether cotrimoxazole has direct immunomodulatory properties. To isolate any direct effects of cotrimoxazole on immune cells from its impact on enteropathy and the microbiome, we optimized an in vitro model of whole-blood cytokine responses to bacterial and fungal antigens: heat-killed Salmonella typhimurium (HKST), which activates immune cells via Toll-like receptor 2 (TLR2), TLR4, and TLR5; purified E. coli lipopolysaccharide (LPS), which engages TLR4; and the Saccharomyces cerevisiae cell wall component zymosan, which engages TLR2 and dectin-1. Antigens engaging pattern recognition receptors were chosen to reflect microbial translocation, which drives systemic inflammation and immune activation in HIV infection (7, 15, 18, 33). The cotrimoxazole dose was chosen to reflect maximum [high-dose; trimethoprim (8 μg/ml) and sulfamethoxazole (200 μg/ml)] and minimum [low-dose; trimethoprim (2 μg/ml) and sulfamethoxazole (50 μg/ml)] serum concentrations in HIV-positive patients taking cotrimoxazole (38). Laboratory cotrimoxazole preparations were confirmed to have antibiotic activity (fig. S7A), and doses did not reduce leukocyte viability in culture (fig. S7, B to D).

Because the inflammatory milieu can affect immune cell responses, we obtained blood samples from three groups of UK adults (HIV-positive ART-treated, n = 6; HIV-positive ART-naïve, n = 10; and HIV-negative, n = 8; table S1), with distinct baseline inflammatory profiles (fig. S8). There was no difference between groups in spontaneous cytokine production in 24 hours of unstimulated cultures (Fig. 5).

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.

High-dose cotrimoxazole significantly reduced HKST-, LPS-, and zymosan-induced TNFα (Fig. 5A) and IL-6 (Fig. 5B) production relative to control treatment with drug diluent alone [dimethyl sulfoxide (DMSO)] in ≥1 group. This was particularly evident for HKST- and LPS-induced TNFα and LPS- and zymosan-induced IL-6, which were significantly lower across all three clinical groups. LPS- and zymosan-induced TNFα and zymosan-induced IL-6 were also significantly reduced by low-dose cotrimoxazole in the HIV-positive ART-naïve group (Fig. 5, A and B). These observations confirm our hypothesis that cotrimoxazole directly modulates proinflammatory immune cell activation by pathogen antigens, both in HIV-positive and HIV-negative individuals, independently of its effects on the microbiome or intestinal inflammation.

To determine the immune cell types modulated by cotrimoxazole, we evaluated intracellular TNFα production and surface expression of HLA-DR by monocytes and T cells during 6 hours of peripheral blood mononuclear cell (PBMC) culture with or without high-dose cotrimoxazole (see fig. S9 for gating strategy and table S2 for antibodies). Cotrimoxazole reduced the proportion of TNFα+ monocytes after HKST stimulation relative to control-treated cultures in the HIV-negative group but not in the HIV-positive groups (Fig. 5C). Cotrimoxazole did not alter HKST-induced up-regulation of HLA-DR by monocytes (Fig. 5D). Cotrimoxazole also had no effect on the proportion of TNFα+ or HLA-DR+ CD4+ or CD8+ T cells after polyclonal stimulation with staphylococcal enterotoxin B (SEB; Fig. 5, C and D). Thus, although cotrimoxazole reduces proinflammatory cytokine production by blood leukocytes and TNFα production by monocytes specifically, it did not directly reduce monocyte maturation or T cell activation.

Cotrimoxazole reduces IL-8 production by gut epithelial cells

The gut epithelium provides a barrier between the microbiota and mucosal immune cells, responds to TLR ligands, and produces leukocyte chemoattractants under inflammatory conditions; direct effects of cotrimoxazole on epithelial cell function could contribute to its anti-inflammatory effects. To isolate direct effects of cotrimoxazole on the epithelial barrier from its impact on leukocytes or the microbiome, we used transwell cultures of the Caco-2 human colonic epithelial cell line as a well-established model of gut epithelium. We induced epithelial inflammation with IL-1β and evaluated the effect of cotrimoxazole on four epithelial functions: epithelial integrity [trans-epithelial resistance (TEER)], epithelial cell death [%lactose dehydrogenase (LDH) activity], apical-to-basal translocation of a fluorescent dye (%Lucifer Yellow passage, a proxy for gut-to-circulation microbial translocation), and production of the neutrophil chemoattractant IL-8 (Fig. 6A). We used high cotrimoxazole concentrations for these experiments to reflect the concentration found in the gut lumen after oral dosing, after first titrating cotrimoxazole in Caco-2 cultures to identify a dose that did not differ in cytotoxicity from DMSO controls (1 mg/ml; Fig. 6B).

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.

Cotrimoxazole treatment throughout Caco-2 growth did not significantly alter the rate of monolayer confluence (mean TEER/plate, >800 ohm; Fig. 6C), ΔTEER, %LDH activity, or %Lucifer Yellow passage under inflammatory conditions (1, 10, or 100 μg/ml of IL-1β for 24 hours; Fig. 6D). However, cotrimoxazole-treated monolayers produced significantly less IL-8 than control-treated cultures when the inflammatory stimulus was highest [IL-1β (100 μg/ml), P = 0.003; Fig. 6D]. Together, these experiments suggest that cotrimoxazole directly inhibits IL-8 production by gut epithelial cells, which may contribute to reduced neutrophil recruitment to the intestinal mucosa under inflammatory conditions.

DISCUSSION

Inflammation drives morbidity and mortality in HIV infection. There is therefore interest in using anti-inflammatory agents with ART to improve clinical outcomes (3942). Long-term cotrimoxazole prophylaxis is recommended for children and adults living with HIV in settings with high prevalence of malaria or invasive bacterial infections, although global coverage remains poor (4, 43). We show here that cotrimoxazole reduces systemic inflammation in ART-treated children in sub-Saharan Africa and demonstrate several underlying mechanisms, including antibiotic effects on the gut microbiome and direct anti-inflammatory effects on leukocytes and gut epithelial cells. Synergy between antibiotic and anti-inflammatory pathways may explain the sustained clinical benefits of cotrimoxazole (3, 27) and provides an additional rationale for increasing cotrimoxazole coverage in sub-Saharan Africa.

Using samples from the ARROW trial, we show definitively, using the randomized stop-versus-continue design, that systemic inflammatory biomarkers (CRP and IL-6) are reduced by cotrimoxazole. Pre-ART levels of CRP and IL-6, but not TNFα or sCD14, predicted mortality, WHO stage 4 clinical events, and poor CD4 reconstitution in ARROW; a twofold increase in CRP or IL-6 was independently associated with 19 and 54% increased risk, respectively (5). On the basis of these predictions, the reductions in CRP and IL-6 among children continuing cotrimoxazole would reduce the relative risk of adverse outcomes by 13 and 11%, respectively. HIV-positive children have lower absolute mortality risk after starting ART; however, our estimates highlight that the additive anti-inflammatory benefits of continuing cotrimoxazole are clinically meaningful. Previous studies suggest that systemic inflammatory mediators are better predictors of poor clinical outcomes than T cell activation among HIV-positive people in resource-limited settings (44). Our assessment of circulating immune cell activation was limited to HLA-DR expression on CD4+ T cells, which did not differ between randomized groups. However, we observed lower percentages of proliferating naïve CD4+ T cells among children continuing cotrimoxazole, which we interpret as beneficial, because elevated CD4+ T cell proliferation without a corresponding increase in total counts leads to depletion of the naïve T cell pool (45).

We went on to explore potential explanatory mechanisms. Systemic inflammation in HIV infection is partly driven by enteropathogen carriage and chronic enteropathy (1113, 15). Using stool samples from a subset of ARROW children, we demonstrated that VGS were less abundant at weeks 84 and 96 after randomization in those continuing cotrimoxazole. Because speciation of VGS is challenging, we confirmed these differences using high-resolution mapping of metagenome sequencing reads to Streptococcal pangenome databases (36). Cotrimoxazole effects on VGS are particularly notable because the global microbiome community composition did not differ between randomized groups, likely because all children had received cotrimoxazole for a median of 2 years before randomization (27). VGS are a heterogeneous group of bacteria, which can be both commensal and pathogenic (46). They are found throughout the healthy human gut (47, 48) and are enriched in stool samples from children with stunting (49), a form of chronic malnutrition associated with systemic inflammation (50). VGS express several immune stimulatory antigens that may drive intestinal inflammation and potently trigger innate immune cell cytokine production in vitro (51). In contrast to changes in VGS, we found no evidence for suppression of Enterobacteriaceae, which include pathogens causing severe bacterial infections in sub-Saharan Africa (34, 35). Our microbiome analyses focused on later time points after randomization, due to stool sample availability; there may plausibly be additional cotrimoxazole-driven changes at earlier time points and at other anatomic sites.

Children randomized to continue cotrimoxazole had lower fecal myeloperoxidase, an antimicrobial peroxidase enzyme abundant in neutrophils, and a biomarker of enteropathy (52). Of the cotrimoxazole-affected VGS, S. mutans, S. parasanguinis, and S. vestibularis were positively associated with myeloperoxidase, suggesting that subclinical antibiotic effects of cotrimoxazole on VGS reduce intestinal inflammation. This does not appear to be a universal characteristic of antibiotic treatment because suppression of gut-resident Gram-positive bacteria with vancomycin in rhesus macaques subsequently infected with simian immunodeficiency virus did not reduce IL-6 or CD4+ T cell activation in mesenteric lymph nodes (53). It is likely that timing of treatment, baseline microbiome, ART history, intercurrent infections, and antibiotic specificity influence the relationship between antibiotic prophylaxis, gut microbiome, and enteropathy.

Functional analysis of ARROW stool samples identified a metagenomic signature of mevalonate metabolism, predominantly mapping to VGS, which was positively associated with fecal myeloperoxidase and suppressed by cotrimoxazole. The mevalonate pathway is one of two metabolic processes that produce isoprenoids, naturally occurring organic precursors in eukaryote cholesterol and prokaryote cell wall peptidoglycan (a TLR2 ligand) synthesis (54). Several in vitro studies indicate that inhibition of mevalonate pathway enzymes impairs innate leukocyte recruitment and proinflammatory cytokine responses, providing a precedent for how inhibiting VGS mevalonate metabolism might influence HIV enteropathy. For example, inhibiting farnesyl pyrophosphate synthesis reduces neutrophil priming by IL-8 (55) and inhibiting HMG-CoA reductase reduces monocyte-derived IL-6 and IL-8 (56) and neutrophil trans-epithelial migration (39, 42). HMG-CoA reductase with identity to S. parasanguinis and S. salivarius was among the cotrimoxazole-suppressed mevalonate pathway enzymes identified.

Leukocytes are an abundant source of proinflammatory cytokines. Levels of circulating microbial products that could trigger these pathways are elevated during HIV infection, including the TLR4 ligand LPS (10, 15). We developed an in vitro model of leukocyte activation by TLR ligands to isolate direct anti-inflammatory effects of cotrimoxazole from its antibiotic effects, using blood samples from HIV-negative and HIV-positive UK adults not receiving cotrimoxazole. Although this cohort differed in age, geographic location, likely HIV clade, and comorbidities compared to children in ARROW, these in vitro experiments provide proof of concept that physiologically relevant cotrimoxazole doses consistently inhibited whole-blood TNFα and IL-6 production elicited via TLR2, TLR4, and TLR5. Collectively, these findings suggest that modulation of innate proinflammatory cytokine production is a property of cotrimoxazole per se, affects multiple innate signaling pathways, and occurs independently of its antibiotic effects, HIV-driven inflammation, or ART exposure. Intracellular cytokine staining suggested that monocyte rather than T cell cytokine production was the most affected by cotrimoxazole. Our demonstration of direct modulation of proinflammatory cytokine production by human leukocytes clarifies a long-standing theory that cotrimoxazole modulates immune responses in mice via an undefined mode of action (22), for which subsequent in vitro models have yielded opposing conclusions for innate and adaptive immune cells (2326). Although these immunomodulatory effects were quantitatively subtle, our relative risk estimates in ARROW indicate that even small reductions in inflammatory markers may improve clinical outcomes (5). The pharmacology of cotrimoxazole-mediated immunosuppression, its interaction with TLR signaling, and its potential therapeutic value in other inflammatory disorders are yet to be established.

Cotrimoxazole reduced production of the neutrophil chemoattractant IL-8 by gut epithelial cells in vitro. This is a putative pathway through which cotrimoxazole could directly contribute to reduced neutrophil recruitment and myeloperoxidase production in the gut mucosa. Cotrimoxazole did not alter epithelial characteristics associated with barrier function in vitro; however, it remains possible that cotrimoxazole alters these pathways in vivo by affecting gut barrier components such as mucus (19) and tight junction proteins (53), which we did not model. Primary epithelial cells and biopsies, which would better mimic trans-epithelial transport in vivo, were not available from ARROW. Because VGS express abundant TLR2 ligands and the Caco-2 cell line has limited TLR2 expression (57), alternative epithelial models are required to explore interrelationships between cotrimoxazole, VGS metabolism, and epithelial barrier function.

Our study raises the possibility that antibiotics other than cotrimoxazole may confer anti-inflammatory benefits that contribute to their impact at scale, including the recent finding of reduced child mortality after mass administration of azithromycin in sub-Saharan Africa (58). Accessory benefits from antibiotics are important considerations in the debate around antimicrobial stewardship, particularly in settings where antimicrobial resistance is already high and in conditions such as HIV, where chronic inflammation combines with intercurrent infection to exacerbate clinical outcomes. Whether cotrimoxazole has clinical benefits for HIV-positive people in high-income settings, where long-term cotrimoxazole prophylaxis is not currently recommended and ART alone does not fully prevent pathology, warrants further study. Recognition of its anti-inflammatory benefits should drive renewed efforts for universal cotrimoxazole coverage to improve clinical outcomes for all people living with HIV in sub-Saharan Africa.

MATERIALS AND METHODS

Study design

The study objectives were to determine whether cotrimoxazole has anti-inflammatory effects and to elucidate underlying mechanisms. Experimental work comprised the following: (i) analysis of longitudinal blood samples [using ELISA (enzyme-linked immunosorbent assay) and flow cytometry] and stool samples (using ELISA and whole-metagenome sequencing) collected from HIV-positive Ugandan and Zimbabwean children randomized to continue versus stop open-label cotrimoxazole in the ARROW trial (27) until 16 March 2012; and (ii) in vitro cotrimoxazole treatment using blood samples from UK adults (ELISA and flow cytometry) and epithelial cell line (Caco-2) cultures. Full details are in Supplementary Materials and Methods.

Within ARROW, children/adolescents (median age, 7.9 years; interquartile range, 4.6 to 11.1) who had been receiving ART and once-daily cotrimoxazole prophylaxis (200 mg of sulfamethoxazole/40 mg of trimethoprim, 400 mg of sulfamethoxazole/80 mg of trimethoprim, or 800 mg of sulfamethoxazole/160 mg of trimethoprim for body weight 5 to 15, 15 to 30, or >30 kg, respectively) for >96 weeks at four sites in Uganda and Zimbabwe were randomized to stop (n = 382) or continue (n = 386) cotrimoxazole (27, 59). Children with a history of Pneumocystis jirovecii pneumonia were excluded (27). Children (98%) enrolled into ARROW during the last 6 months of recruitment were also included in an immunology substudy; additional assays were conducted for these children and for a random 23% sample of all remaining nonimmunology substudy children (5). This study included children with available baseline plasma of sufficient volume to measure inflammatory biomarkers (stop, n = 149; continue, n = 144). Stool samples were collected at weeks 84 and 96 after randomization from a subgroup of children in Zimbabwe to assay intestinal inflammation. Total DNA was extracted from 150 mg of stool for whole-metagenome sequencing (stop, n = 36; continue, n = 36).

Blood was collected from 8 HIV-uninfected adults, 6 HIV-positive adults on ART for ≥2 years, and 10 HIV-positive ART-naïve adults (table S1) who were not taking cotrimoxazole for 24 hours of whole-blood culture and 6 hours of PBMC culture with bacterial and fungal antigens. Proinflammatory cytokine responses were compared between parallel cultures treated with cotrimoxazole and volume-matched diluent without drug (DMSO).

Caco-2 monolayers were grown in transwell cultures as a gut epithelium model. Epithelial functions (integrity, cell death, translocation across the epithelium, and chemokine production) were quantified after 24 hours of stimulation with IL-1β and compared between cultures treated with cotrimoxazole or DMSO throughout growth, run in triplicate. Transwell cultures were repeated three times using separate Caco-2 passages. Data from individual transwells were excluded if monolayers were subconfluent. Primary data are reported in data file S1.

Ethics

ARROW (ISRCTN Registry no. ISRCTN24791884) was approved by Research Ethics Committees in Uganda, Zimbabwe, and the United Kingdom. Written informed consent from all caregivers and assent from participants (where appropriate) were obtained (27, 59). Approval for UK donor recruitment was provided by the National Health Service Research Authority (IRAS project ID: 209553; Research Ethics Council reference: 17/WM/0018) and the Research Ethics Committee of Queen Mary University of London. All participants provided written informed consent.

Statistical analysis

For ARROW data, fold change in geometric means between randomized groups was compared for continuous variables at each time point using standard regression models and globally across all time points using GEE (normal distribution for log-transformed values), both with adjustment for recruitment center and baseline values, and assuming variation in treatment effect by time point. Proportions of children with HIV viral load of <80 copies/ml were compared between randomized groups at each time point using exact tests and globally across all time points using GEE (binomial distribution) with adjustment for recruitment center and assuming variation in treatment effect by time point. Relative risk projections for CRP and IL-6 differences between randomized groups were calculated from the output of models based on enrolment (i.e., pre-ART and pre-cotrimoxazole) biomarker levels in the ARROW immunology subcohort (5). GEE and exact tests were conducted in Stata version 15.1 (StataCorp LLC). Concentrations of fecal inflammatory markers and serum protein (Shapiro-Wilk test for normality, P < 0.05) were compared between randomized groups using Mann-Whitney U tests in GraphPad Prism version 7.02.

For microbiome sequencing data, differences in species relative abundance and diversity between randomized groups were evaluated at each time point by intention-to-treat analysis using linear regression models fitted against natural log-transformed inverse Shannon species-level a diversity indices. Species-level beta diversity was evaluated using the Bray-Curtis dissimilarity index and visualized using NMDS. Differences in relative abundance of species, Pfam, metabolic pathways, and enzymes (microbiome characteristics) were evaluated at each time point by intention-to-treat analysis using separate zero-inflated beta regression models fitted against relative abundances for each microbiome characteristic. Cotrimoxazole treatment effect was the ratio of relative microbiome characteristic abundance in continue group versus stop group. P values were adjusted for multiple comparisons to maintain the FDR significance level (α = 0.05) (60). Only differentially abundant microbiome characteristics with consistent significant differences between groups at both weeks 84 and 96 were interpreted as causally related to cotrimoxazole continuation. Rank-based regression models were fitted against fecal myeloperoxidase concentration adjusted for age, sex, and randomized group, with FDR adjustment for multiple comparisons. Microbiome analyses were conducted in R version 3.3.2. Vegan (61) was used to calculate Shannon diversity, Bray-Curtis dissimilarity, and NMDS. Gamlss was used for zero-inflated beta regression (62). Rfit was used for rank-based regression (63).

For UK adults, continuous variables were compared between groups using unpaired Kruskall-Wallis tests. Comparisons between drug treatments were only conducted for responses that were significantly up-regulated in antigen-stimulated cultures without drug treatment versus unstimulated cultures without drug treatment (paired Wilcoxon test, P < 0.05). Comparisons between drug treatments used Friedman tests with post hoc pairwise comparisons via uncorrected Dunn’s test; post hoc tests were only conducted where the global test was statistically significant. Caco-2 readouts (TEER, ΔTEER, %LDH activity, %Lucifer Yellow passage, and IL-8; Shapiro-Wilk test for normality, P > 0.05) were compared between cotrimoxazole- and DMSO-treated cultures using paired two-tailed t tests. All analyses were conducted using GraphPad Prism version 7.02.

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.

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REFERENCES AND NOTES

Acknowledgments: We thank participants, caregivers, and staff from all study centers; ViiV Healthcare/GlaxoSmithKline who donated ART and funded ARROW viral loads; Ministries for Health in Uganda and Zimbabwe who provided cotrimoxazole for ARROW; M. Govha, S. Rukobo, M. Kihembo, L. Nakiire, and H. Poulsom for laboratory work in ARROW; A. Waters, S. Murphy, and J. Hand for assistance with UK participant recruitment; and A. McKnight who hosted UK laboratory work. Funding: This work was supported by the Wellcome Trust (grant nos. 093768/Z/10/Z and 108065/Z/15/Z to A.J.P. with subcontract to A.R.M.; and 206225/Z/17/Z to C.D.B., cofunded by The Royal Society); Canadian Institutes of Health Research (grant to E.K.G.); Medical Research Council (MRC; grant nos. G0300400 to A.J.P., V.M., M.B.-D., A.K., N.K., D.M.G., and A.S.W. and G1001190 to A.J.P., V.M., M.B.-D., A.K., N.K., D.M.G., and A.S.W.); Department for International Development under MRC/DFID Concordat agreement and EDCTP2 programme supported by the European Union; MRC Clinical Trials Unit at UCL (grant no. MC_UU_12023/26 to A.J.S., M.J.S., D.M.G., and A.S.W.). Author contributions: C.D.B. conceptualized and implemented in vitro work, analyzed data, prepared figures, recruited donors, and led manuscript preparation; E.K.G. conceptualized and implemented microbiome analysis, prepared figures, and led manuscript preparation; G.P., A.S., and C.B. implemented ARROW biomarker assays; L.T. recruited UK donors with oversight from J.R.D. and C.D.B.; L.B. and Y.K. assisted C.D.B. with whole-blood culture and Caco-2 assays; N.C. supported Caco-2 experiments; D.M.G. and A.S.W. conceptualized and managed ARROW; N.K., A.J.P., A.S.W., D.M.G., M.J.S., and A.J.S. conceptualized and managed ARROW immunology work; M.B.-D., V.M., J.L., and A.K. undertook ARROW clinical management; K.M. managed ARROW assays in Zimbabwe; M.G. and H.M.G. assisted E.K.G. with microbiome assays; C.P. supported UK-based experiments; T.J.E. and A.R.M. conducted PanPhlAn analyses; A.S.W. and A.J.S. conducted ARROW statistical analysis and prepared figures; A.J.P. and A.R.M. conceptualized the study and had primary responsibility for the manuscript. All authors read and contributed to the manuscript and approved submission. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.
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