The Potential Role of FREM1 and Its Isoform TILRR in HIV-1 Acquisition through Mediating Inflammation.

FREM1 (Fras-related extracellular matrix 1) and its splice variant TILRR (Toll-like interleukin-1 receptor regulator) have been identified as integral components of innate immune systems. The potential involvement of FREM1 in HIV-1 (human immunodeficiency virus 1) acquisition was suggested by a genome-wide SNP (single nucleotide polymorphism) analysis of HIV-1 resistant and susceptible sex workers enrolled in the Pumwani sex worker cohort (PSWC) in Nairobi, Kenya. The studies showed that the minor allele of a FREM1 SNP rs1552896 is highly enriched in the HIV-1 resistant female sex workers. Subsequent studies showed that FREM1 mRNA is highly expressed in tissues relevant to mucosal HIV-1 infection, including cervical epithelial tissues, and TILRR is a major modulator of many genes in the NF-κB signal transduction pathway. In this article, we review the role of FREM1 and TILRR in modulating inflammatory responses and inflammation, and how their influence on inflammatory responses of cervicovaginal tissue could enhance the risk of vaginal HIV-1 acquisition.

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

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-1ß, 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.

Development of monoclonal antibodies to interrogate functional domains and isoforms of FREM1 protein.

FREM1 was first identified as an extracellular matrix protein that is essential for the formation of the epithelial basement membrane during embryonic development. Recent studies have shown that FREM1 also modulates innate immunity through its isoform 2 splice variant protein, known as Toll-like/interleukin-1 receptor regulator (TILRR). TILRR is a co-receptor that enhances pro-inflammatory IL-1R1 signal transduction. Our previous study identified the minor allele of a SNP, rs1552896, in the intronic region of FREM1 gene to be associated with natural resistance to HIV-1 infection in a subgroup of Kenyan sex workers in the Pumwani cohort. To study the role of FREM1 and its variants in differential susceptibility to HIV-1 infection, we generated a panel of 17 monoclonal antibodies against two recombinant proteins of FREM1, rspD and rspF. Epitope mapping using overlapping pin peptides showed that the monoclonal antibody (MAb) panel interrogated seven unique regions across five different domains, including the C-type lectin domain disulfide bond and the TILRR GAG serine attachment site. Utility of three selected FREM1 MAbs were demonstrated by FACS and immunohistochemical detection of FREM1 in 293F kidney embryonic cells, HeLa 229 cervical cells, and Sup-T1 cells. Thus, these monoclonal antibodies could be used to study the functional domains of FREM1 and its isoforms.