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.

Purpose TILRR (Toll-like interleukin-1 receptor regulator), a variant of FREM1 (Fras-related extracellular matrix 1), is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling and inflammatory responses. It enhanced the expression of multiple genes in the NF-κB signaling pathway and promoted the production of multiple pro-inflammatory cytokines/chemokines. TILRR is an extracellular matrix protein and expressed in cells and tissues, and has never been considered to exist in the blood. The study aimed to identify circulating TILRR protein in human plasma as a biomarker of systemic inflammation. Methods and Results We developed a multiplex bead array method (Bio-Plex) using 4 monoclonal antibodies targeting different protein domains of FREM1/TILRR to investigate whether TILRR can be detected in blood plasma. The results of the multiplex bead array method were validated by Western blot analysis of affinity-purified TILRR from patient plasma samples. We subsequently analyzed 640 plasma samples from women enrolled in the Pumwani Sex Worker cohort (PSWC) (Nairobi, Kenya). Our study showed that TILRR exists in all patient plasma samples, but its quantities vary greatly among the patients, ranging from 2.38 ng/mL to 5196.79 ng/mL. The plasma TILRR below 2.38 ng/mL can only be detected by affinity purification and Western blot analysis. Conclusion Our in-house developed multiplex bead array method can successfully quantify TILRR protein in plasma samples. Because TILRR is an important modulator of many inflammation-responsive genes, it may be an inflammation biomarker in blood and play a role in modulating systemic inflammation.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachomawhich are distinguished biologically by their natural host cell infection tropism.LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculourogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmiddependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature. IMPORTANCEThe Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

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