Catharina MISSAILIDIS [1], Nikolaj SØRENSEN [2] , Senait ASHENAFI [6], Wondwossen AMOGNE [3], Endale Kassa [3], Amsalu BEKELE [3], Meron GETACHEW [3], Nebiat GEBRESELASSIE [4], Abraham ASEFFA [4], Getachew ADERAYE [3], Jan ANDERSSON [5,6], Susanna BRIGHENTI [6], Peter BERGMAN [1]

Affiliates

[1] Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Stockholm, Sweden. [2] Clinical Microbiomics, Copenhagen, Denmark [3] Department of Internal Medicine, Faculty of Medicine, Black Lion University Hospital and Addis Ababa University, Addis Ababa, Ethiopia. [4] Armauer Hansen Research Institute (AHRI), Addis Ababa, Ethiopia. [5] Department of Medicine, Division of Infectious Diseases, Karolinska University Hospital Huddinge, Stockholm, Sweden. 6 Center for Infectious Medicine (CIM), F59, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden.

Abstract

Background
Dysregulation of the gut immune barrier function contributes to chronic immune activation in HIV-1. Vitamin D and phenylbutyrate are nutritional compounds that can possess modulatory effects on gut homeostasis where one key mechanism involves induction of the antimicrobial peptide LL-37 from epithelial and immune cells. Moreover, vitamin D stabilizes epithelial barrier function and may have regulatory effects on the intestinal microbiota composition.
In this study, we investigated the effect of vitamin D and phenylbutyrate supplementation on gut-derived inflammatory markers, microbial metabolites and gut microbiome.

Methods
A well-characterized cohort of treatment-naïve HIV-1-infected individual (n=167) were included from a double-blind, randomized and placebo-controlled trial of daily supplementation with 5000 IU vitamin D and 500 mg phenylbutyrate for 16 weeks. Baseline and per-protocol plasma samples at week 16 were analysed for sCD14, LL-37, kynurenine/tryptophan-ratio, TMAO, choline, betaine, and methionine. The microbiome was determined through 16s rDNA from colonic biopsies.

Results
Treatment significantly increased vitamin D-levels in plasma (p<0.0001) but no effects were observed on sCD14, the kynurenine/tryptophan-ratio, TMAO, choline or methionine levels. Subgroup analyses of vitamin D insufficient subjects found a significant increase of LL-37 in the treatment group (p=0.014), whereas vitamin D+phenylbutyrate failed to significantly impact the estimate of LL-37 in multiple regression analysis (p=0.089). Further, no effects on microbiome was found in number of operational taxonomic units (OTUs) (p=0.71), Shannon microbial diversity index (p=0.82) or in principal component analyses (p=0.79).

Conclusion
Daily supplementation with vitamin D and phenylbutyrate for 4 months did not modulate levels of gut-derived inflammatory markers or microbial composition. We suggest that the lack of effect may relate to persistent viral replication, where phenylbutyrate, by virtue of its role as a histone deacetylase inhibitor, may have reactivated latent HIV. We propose further studies of vitamin D supplementation in HIV-infected individuals upon initiation of ART to better evaluate the modulatory effects on gut immune barrier function.