Role of translocated bacterial flagellin in monocyte activation among individuals with chronic HIV-infection

Jenny Svärd1, Dominic Paquin-Proulx1, Kajsa Noyan2, Marcus Buggert2, Babilonia Barqasho2, Anders Sönnerborg1,2, Piotr Nowak1; Department of Medicine Huddinge, Karolinska Institutet1; Department of Laboratory Medicine, Karolinska Institutet2
Affiliates: 1Department of Medicine Huddinge, Karolinska Institutet, 2Department of Laboratory Medicine, Karolinska Institutet

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Background
Microbial translocation (MT); the leakage of bacterial products through a weakened gut-blood barrier, is widely believed to contribute to the chronic and systemic immune activation characteristic of HIV-1 infection. However, the golden standard MT marker lipopolysaccharide (LPS) displays high variability between reports. The presence of elevated levels of circulating anti-flagellin antibodies among HIV-infected subjects (Nowak et al Int J Microbiol 2012) indicate that bacterial flagellin may also translocate and contribute to systemic immune activation, through activation of Toll-like receptor 5 (TLR5) on antigen-presenting cells such as monocytes. This study was designed to assess flagellin and anti-flagellin plasma levels in HIV-infected subjects, their correlation to other markers of MT and immune activation, the effect of highly active antiretroviral therapy (HAART), and their relationship to TLR5 responsiveness.

Methods
Blood samples were collected from four groups: 1) 23 HAART-naïve HIV-positive subjects; 2) 24 HIV- positive subjects with samples prior to initiation of HAART as well as after 5-12 months on therapy; 3) 21 HIV- negative subjects (“negative controls”); and 4) 27 individuals with diarrhea (“positive controls”). Plasma levels of flagellin, anti-flagellin IgG, total IgG were measured by ELISA and compared to established plasma markers of microbial translocation (LPS, LBP) and monocyte activation (sCD14, sCD163). To assess TLR5 responsiveness, PBMCs from HIV-positive and HIV-negative individuals were exposed to flagellin in vitro, followed by flow cytometry analysis of cell surface activation markers (CD14, CD163, HLA-DR) and cytokine production (TNF, IL-1beta, IL-6, IL-8).

Results
Patients with diarrhea had significantly higher plasma levels of both flagellin and anti-flagellin (normalized to total IgG), whereas among HIV-positive patients these levels did not differ significantly from HIV-negative controls. Nevertheless, plasma flagellin correlated to plasma anti-flagellin and anti-flagellin was associated with other markers of microbial translocation (LPS: P = 0.007, LBP: P = 0.045) and markers of monocyte activation (sCD14: P < 0.001, sCD163: P = 0.003). Flow cytometry analysis revealed elevated levels of IL-6/IL-8 producing monocytes among HIV-infected subjects with high circulating anti-flagellin levels (r = 0.504, P = 0.028). Following flagellin stimulation in vitro, PBMCs from patients with high circulating anti-flagellin levels displayed an attenuated induction of IL-1β/IL-8 (r = -0.566, P = 0.014) as well as a less pronounced TLR5 downregulation (r = 0.612, P = 0.007).

Conclusions
Our results suggest that although levels of flagellin and anti-flagellin in HIV infection were not significantly different to HIV-negative controls, correlation to other plasma markers of MT and monocyte activation as well as to cytokine production in monocytes indicate that translocated bacterial flagellin contributes to systemic immune activation in HIV-1 infection. Furthermore, it appears that chronic exposure to flagellin reduces cell surface expression of TLR5 and results in a hyporesponsive state in monocytes.