Toll-like Interleukin 1 Receptor Regulator Is an Important Modulator of Inflammation Responsive Genes

Abstract

TILRR (Toll-like interleukin-1 receptor regulator), a transcript variant of FREM1, is a novel regulatory component, which stimulates innate immune responses through binding to IL-1R1 (Interleukin-1 receptor, type 1) and TLR (Toll-like receptor) complex. However, it is not known whether TILRR expression influences other genes in the NFκB signal transduction and pro-inflammatory responses. Our previous study identified FREM1 as a novel candidate gene in HIV-1 resistance/susceptibility in the Pumwani Sex worker cohort. In this study, we investigated the effect of TILRR overexpression on expression of genes in the NFκB signaling pathway in vitro . The effect of TILRR on mRNA expression of 84 genes related to NFκB signal transduction pathway was investigated by qRT-PCR. Overexpression of TILRR on pro-inflammatory cytokine/chemokine(s) secretion in cell culture supernatants was analyzed using Bioplex multiplex bead assay. We found that TILRR overexpression significantly influenced expression of many genes in HeLa and VK2/E6E7 cells. Several cytokine/chemokine(s), including IL-6, IL-8 (CXCL8), IP-10, MCP-1, MIP-1β, and RANTES (CCL5) were significantly increased in the cell culture supernatants following TILRR overexpression. Although how TILRR influences the expression of these genes needs to be further studied, we are the first to show the influence of TILRR on many genes in the NFκB inflammatory pathways. The NFκB inflammatory response pathways are extremely important in microbial infection and pathogenesis, including HIV-1 transmission. Further study of the role of TILRR may identify the novel intervention targets and strategies against HIV infection.

TILRR Promotes Migration of Immune Cells Through Induction of Soluble Inflammatory Mediators

“…Because THP-1 (monocytes) and MOLT-4 (lymphocytes) cells express HIV-1 receptor/co-receptors CD4, CCR5 and CXCR4 essential for R5-and X4-tropic HIV-1 strains to infect the host (Dejucq et al, 1999;Dejucq, 2000;Konopka and Duzgunes, 2002;Miyake et al, 2003;Melo et al, 2014;Huang et al, 2016), and these cells are widely used as in vitro model for HIV-1 infection (Ushijima et al, 1991;Dejucq et al, 1999;Konopka and Duzgunes, 2002;Blanco et al, 2004;Cassol et al, 2006;Guo et al, 2014;Lodge et al, 2017), we therefore utilized these cell lines as a model for in vitro cell migration assay. HeLa cells (NIH, Catalog# 153) were maintained as described in our earlier study (Kashem et al, 2019). Briefly, the cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, Catalog# D5796) supplemented with 10% FBS (Gibco, Catalog# 12483-020) and 1% Antibiotic-Antimycotic (Gibco, Catalog# 15240062).…”

“…We overexpressed the TILRR in HeLa cells as described previously (Kashem et al, 2019). In brief, approximately 2.5 × 10 5 cells/ml was plated into each well of a 12-well culture plate containing complete DMEM growth medium a day before transfection.…”

“…Our group also showed that FREM1 is highly expressed in human epithelial tissues and immune cells, such as cervical tissues (Luo et al, 2012), CD4+ and CD8+ T cells, B cells, monocytes and natural killer (NK) cells (Omange et al, under revision). Recently, we have shown that TILRR modulates expression of many inflammation responsive genes and production of pro-inflammatory cytokines/chemokines such as IL-6, IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, MIP-1β/CCL4 and RANTES/CCL5 in cervico-vaginal epithelial cells (Kashem et al, 2019). The inflammatory cytokines/chemokines secreted by the TILRR-overexpressed cervical epithelial cells could influence immune cell migration to tissues.…”

“…In fact, several studies have shown that chemo-attractants including IL-8/CXCL8, IP-10/CXCL10, MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MIP-3α/CCL20, and RANTES/CCL5 produced by female genital epithelium induced rapid influx of circulating immune cells, resulting in increased risk of HIV acquisition (Mueller and Strange, 2004;Wira et al, 2005;Li et al, 2009;Masson et al, 2015;Arnold et al, 2016). Because cervical epithelial cells express FREM1 (Luo et al, 2012), and TILRR overexpression increases the production of pro-inflammatory mediators by cervical epithelial cell lines (Kashem et al, 2019), we hypothesized that the cytokines/chemokines produced by the cervical epithelial cell line may influence the migration and tissue infiltration of immune cells. In this study, we investigated the effect of supernatants of TILRR-overexpressed cervical epithelial cells (HeLa cells) on the migration of immune cells using two cell lines, THP-1 and MOLT-4, by Transwell assay and a novel microfluidic device that can record cell migration images.…”

Cervico-Vaginal Inflammatory Cytokine and Chemokine Responses to Two Different SIV Immunogens

“…Cytokines/chemokines were quantified using a custom 14-plex assay based on a previously published Bio-Plex assay method with some modifications (25,27). Bio-Plex R Amine Coupling Kit (Bio-Rad, Canada) was used to couple 20 µg of capture antibody for each cytokine/chemokine to 1.25 × 10 7 Bio-Plex Pro TM fluorescently-dyed magnetic COOH Beads, diluted to a stock concentration of 5 × 10 6 beads/ml.…”