Direct biomarkers of microbial translocation as predictors of immune activation in adult Zambians with environmental enteropathy and hepatosplenic schistosomiasis.

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Kaonga, Patrick
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The University of Zambia
Background: Microbial translocation is a poorly understood consequence of several disorders such as environmental enteropathy (EE) and hepatosplenic schistosomiasis (HSS) disease. Direct biomarkers of microbial translocation such lipopolysaccharide, 16S rRNA gene and Toll-like receptor ligands may predict immune activation. This study evaluated whether direct biomarkers of microbial translocation correlate and predict immune activation in adult Zambians with EE and HSS disease. The public health importance of biomarkers is that they can be used to predict individuals with EE or HSS who are likely to develop chronic immune activation or are at risk. The biomarkers may be used for early diagnosis and stratify for any treatment or intervention. Methods: An unmatched case-control study was conducted in participants with EE (n=67) recruited from Misisi compound, Lusaka, Zambia, with two comparison groups, HSS participants (n=86) from The University Teaching Hospital and healthy controls (n=41). Plasma lipopolysaccharide (LPS) was measured by Limulus Amoebocyte Lysate Assay, plasma 16S rRNA gene copy number was quantified by quantitative real-time PCR, Toll-like receptor ligands (TLRLs) activity by QUANTI-Blue detection medium, plasma biomarkers of host response (C-reactive protein, soluble CD14, soluble CD163 and lipopolysaccharide-binding protein) to microbial translocation were measured by ELISA and cytokines (TNF-α, IL-6, IL-10, IL-4, IL-2, IFN-γ and IL-17) from cell culture supernatant by Cytometric Bead Array. Results: Plasma lipopolysaccharide levels were elevated in EE group with median 378.9 (IQR, 82.7 - 879.5) EU/ml compared to participants with HSS with median 213.1 (IQR, 77.2 - 358.3) EU/ml; p=0.03 or healthy controls with median 202.3 (IQR, 43.2 - 251.1) EU/ml; p=0.01. The 16S rRNA copy number were significantly elevated in the EE group with median 2651 (IQR, 529 – 8779) copies/μl compared to the levels in participants with HSS with median 387 (IQR, 165 – 1990) copies/μl; p<0.001) or healthy controls with median 193 (IQR, 132 – 455) copies/μl; p<0.001. TLRLs activity was significantly higher in the EE group with median 0.49 (IQR, 0.0 - 0.8) OD units than in participants with HSS with median 0.13 (IQR, 0.0 - 0.8) OD units; p=0.01 or the healthy controls with median 0.02 (IQR, 0.0 - 0.12) OD units; p=0.004. Participants with HSS had higher TLRLs activity compared to healthy controls p=0.02. In multivariate multiple regression models LPS, 16S rRNA copy number, and TLRL activity were independent predictors of cytokines while controlling for baseline characteristics. In the EE group, a good model fit was obtained (R2 = 0.526, F = 47.53, p < 0.001) which predicted TNF-α, IL-6, and IL-10. In the HSS group, a less impressive but still significant fit was obtained (R2 = 0.382, F = 22.43, p = 0.002 which predicted 16S rRNA and TLRLs. In healthy controls, no satisfactory model was obtained (R2 = 0.040, F =1.03, p = 0.38). Conclusions: Direct biomarkers of microbial translocation were higher in EE and HSS participants compared to healthy controls. The biomarkers seems to correlate and predict immune activation in individuals with EE and HSS infection. This data support the model that proposes that biomarkers of microbial origin in the gastrointestinal tract move across a vii compromised intestinal barrier leading to heightened immune activation in conditions with intestinal barrier dysfunction. The study recommends measurement of microbial translocation using these biomarkers. They are cheaper, practical and non-invasive.
Thesis of of Doctor of Philosophy in Immunology