Variation in blood microbial lipopolysaccharide (LPS) contributes to immune reconstitution in response to suppressive antiretroviral therapy in HIV

Abstract

Background: In HIV infection, even under long-term antiretroviral therapy (ART), up to 20% of HIV-infected individuals fail to restore CD4+ T cell counts to the levels similar to those of healthy controls. The mechanisms of poor CD4+ T cell reconstitution on suppressive ART are not fully understood.

Methods: Here, we tested the hypothesis that lipopolysaccharide (LPS) from bacteria enriched in the plasma from immune non-responders (INRs) contributes to blunted CD4+ T cell recovery on suppressive ART in HIV. We characterized plasma microbiome in HIV INRs (aviremic, CD4+ T cell counts < 350 cells/μl), immune responders (IRs, CD4+ T cell counts > 500 cells/μl), and healthy controls. Next, we analyzed the structure of the lipid A domain of three bacterial species identified by mass spectrometry (MS) and evaluated the LPS function through LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs. In comparison, we also evaluated plasma levels of proinflammatory cytokine and chemokine patterns in these three groups. At last, to study the causality of microbiome-blunted CD4+ T cell recovery in HIV, B6 mice were intraperitoneally (i.p.) injected with heat-killed Burkholderia fungorum, Serratia marcescens, or Phyllobacterium myrsinacearum, twice per week for total of eight weeks.

Findings: INRs exhibited elevated plasma levels of total microbial translocation compared to the IRs and healthy controls. The most enriched bacteria were Burkholderia and Serratia in INRs and were Phyllobacterium in IRs. Further, unlike P. myrsinacearum LPS, B. fungorum and S. marcescens LPS induced proinflammatory responses and CD4+ T cell apoptosis in PBMCs, and gene profiles of bacteria-mediated cell activation pathways in THP-1 cells in vitro. Notably, LPS structural analysis by mass spectrometry revealed that lipid A from P. myrsinacearum exhibited a divergent structure consistent with weak toll-like receptor (TLR) 4 agonism, similar to the biological profile of probiotic bacteria. In contrast, lipid A from B. fungorum and S. marcescens showed structures more consistent with canonical TLR4 agonists stemming from proinflammatory bacterial strains. Finally, intraperitoneal (i.p.) injection of inactivated B. fungorum and S. marcescens but not P. myrsinacearum resulted in cell apoptosis in mesenteric lymph nodes of C57BL/6 mice in vivo.

Interpretation: These results suggest that the microbial products are causally associated with INR phenotype. In summary, variation in blood microbial LPS immunogenicity may contribute to immune reconstitution in response to suppressive ART. Collectively, this work is consistent with immunologically silencing microbiome being causal and targetable with therapy in HIV.

Funding: This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID; R01 AI128864, Jiang) (NIAID; P30 AI027767, Saag/Health), the Medical Research Service at the Ralph H. Johnson VA Medical Center (merit grant VA CSRD MERIT I01 CX-002422, Jiang), and the National Institute of Aging (R21 AG074331, Scott). The SCOPE cohort was supported by the UCSF/Gladstone Institute of Virology & Immunology CFAR (P30 AI027763, Gandhi) and the CFAR Network of Integrated Clinical Systems (R24 AI067039, Saag). The National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR001450 (the pilot grant, Jiang). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Keywords: HIV; Immune non-responders; Immune responders; Lipid A; Lipopolysaccharide.

Figure 1

Systemic microbial translocation and distinct circulating microbial profiles in INRs. Plasma levels of LPS (A), sCD14 (B), and bacterial 16S rDNA (C) from the three study groups. (D) The Gini Simpson diversity index (α-diversity) was used to compare the diversity of the plasma-circulating microbial community within INRs and IRs or healthy controls. (E) PCoA was conducted based on the unweighted UniFrac distance to determine the beta diversity of the plasma microbial community. One-way ANOVA was used, followed by Tukey's post hoc test. The statistical significance of the beta diversity was tested using the Multivariate Welch t-test. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Differentially abundant bacterial taxa from circulating microbiome. Dots represent ASVs assigned to the indicated bacterial (column label). The y-axis indicates the relative abundance of ASVs in healthy individuals or IRs compared to INRs. Bacterial ASVs with significant differences between healthy control versus INRs (A) and IRs versus INRs (B) were graphed by log2 fold-change (y-axis). Different colors show various genus bacteria and their affiliated phylum bacteria. (C) Heatmap showing the relative abundance of the 12 bacteria that differed the most (y-axis) by sample (x-axis) when INRs were compared with IRs or healthy controls. The gradient key indicates percent abundance.

Figure 3

Determination of lipid A structures. (A–C) MALDI-MS and MS/MS analysis of lipid A from S. marcescens (A), B. fungorum (B), and P. myrsinacearum (C). Representative major structures with predicted mass of the deprotonated ion are shown. Evidence of hydroxylation shown as delta m/z 16 and aminoarabinose as delta m/z 131.

Figure 4

Gene expression profiles of THP-1 cells after stimulation with different bacterial LPS. (A) Volcano plots of differential expression genes (DEGs, threshold fold change > 1.5 and FDR < 0.05) in THP-1 cells stimulated with LPS from each bacterium compared with unstimulated THP-1 cells. Red dots indicate upregulated DEGs and blue dots indicate downregulated DEGs. The horizontal and vertical dark green lines indicate fold-change (log2) thresholds and adjusted p-value (log10). (B) Heatmap of fold changes (log2) in gene expression for B. fungorum, S. marcescens, or P. myrsinacearum stimulated THP-1 cells compared to unstimulated THP-1 cells. (C) The most altered GO pathways enriched in coherently changed genes in THP-1 cells stimulated by B. fungorum or S. marcescens versus P. myrsinacearum. (D) The expression of various genes encoding products in selected pathways showing increased (red) or decreased (blue) expression in THP-1 cells treated with B. fungorum (B. f) or S. marcescens (S. m) compared to P. myrsinacearum (P. m).

Figure 5

Plasma cytokines in INRs in vivo were consistent with B. fungorum and S. marcescens-induced cytokines in vitro. (A) The extracted LPS from B. fungorum, S. marcescens, and P. myrsinacearum were used to stimulate peripheral blood mononuclear cells (PBMC) from healthy subjects; LPS from E. coli 055:B5 was chosen as a positive control. The LPS induced multiplex cytokines in the cell culture supernatants were measured. (B) Plasma levels of cytokines in INRs compared with IRs or healthy controls. One-way ANOVA followed by Tukey's post hoc test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

B. fungorum and S. marcescens, the bacteria enriched in INRs, impaired CD4+ T cell function. 6-week-old healthy C57BL/6 mice were injected with PBS, heat-killed B. fungorum, S. marcescens, or P. myrsinacearum twice a week for eight weeks by i.p. route (n = 5 per group). The mice were sacrificed on day three after the last injection (day 1). (A) The percentage of apoptotic cells and dead cells in the mesenteric lymph nodes. One-way ANOVA followed by Tukey's post hoc test, *p < 0.05, **p < 0.01. (B, C) The portion of functional memory CD4+ T cells (B) and functional naïve CD4+ T cells (C) from spleen. The spleen CD4+ T cells were stimulated using a leukocyte activation cocktail after 18 h and measured cytokine production using flow cytometry. One-way ANOVA followed by Dunnett's multiple comparisons test. *p < 0.05, ^#p < 0.01.