Toll-like Interleukin -1 Receptor Regulator (TILRR) Protein, a Major Modulator of Inflammation, is Expressed in Normal Human and Macaque Tissues and PBMCs.

Purpose: TILRR is a modulator of genes in the NF-κB inflammation pathway. It regulates inflammation-responsive genes, the secretion of inflammatory mediators, and the migration of immune cells. Because inflammation drives the pathogenesis of many infectious and inflammatory diseases, it is important to know the expression of TILRR protein in tissues and cells. This study examined TILRR protein expression in healthy adult human and macaques' tissues and PBMCs (peripheral blood mononuclear cells). Methods and Results: Tissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, rectum, vagina, cervix, uterus, and penis) and PBMCs from humans and macaques were lysed in RIPA (radioimmunoprecipitation assay) lysis buffer. The TILRR protein was examined by fluorescent Western blot analysis. The relative fluorescence units (rfu) of TILRR protein expression were quantified by Image Studio software (LI-COR). The results showed that adult healthy female (n=1) rectal and cervicovaginal tissues expressed a higher level of TILRR protein than the other tissues (trachea, lungs, stomach, small intestine [ileum], cecum, colon, uterus, and penis) examined. Like humans, the lungs, colon, and rectal tissues of healthy adult female cynomolgus monkeys (Macaca fascicularis) (n=2) expressed the TILRR protein. In addition, PBMCs of healthy adult women (n=4), adult female cynomolgus monkeys (Macaca fascicularis) (n=4), and adult male and female rhesus monkeys (Macaca mulatta) (n=4) showed a similar expression level of TILRR protein (p= 0.2858). TILRR protein was not detected in most of the human cell lines examined, except in Jurkat cells. Conclusion: Our study for the first time showed that TILRR protein is expressed in healthy adult human and monkey tissues and PBMCs. The TILRR protein in these tissues and PBMCs may play a role in the inflammatory response of these tissues and cells in response to infectious pathogens.

High level of plasma TILRR protein is associated with faster HIV seroconversion.

BACKGROUND: TILRR (Toll-like Interleukin-1 Receptor Regulator) is a modulator of many genes in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. It promotes the production of inflammatory mediators and the migration of immune cells. Recently, we showed that TILRR protein circulates in human blood. Thus, it could influence systemic inflammation. Systemic and mucosal inflammations increase the susceptibility to HIV infection. In this study, we analyzed the TILRR protein levels of the archived plasma samples of women enrolled in the Pumwani cohort to determine whether the plasma TILRR protein levels before seroconversion are correlated with differential risk of HIV seroconversion. METHODS: TILRR protein of 941 archived HIV negative plasma samples from 390 women who were HIV negative at the cohort enrollment was quantified with an in-house developed multiplex bead array method. Proinflammatory cytokines/chemokines were measured using a 14-plex bead array method. Spearman rank correlation analysis was used to determine the correlation between plasma TILRR protein and proinflammatory cytokines/chemokines. Kaplan-Meier survival analysis was conducted to evaluate whether the median plasma TILRR protein levels correlate with increased risk of HIV seroconversion. FINDINGS: The level of plasma TILRR protein was positively correlated with plasma IL-1ß (rho: 0.2593, p<0.0001), MCP-1 (rho: 0.2377, p<0.0001), and IL-17A (rho: 0.1225, p=0.0216). Women with median plasma TILRR protein levels ≥100 ng/ml seroconverted significantly faster than women with plasma TILRR protein levels <100 ng/ml (log-rank= 100.124, p<0.0001; relative risk= 3.72 and odds ratio= 15.29). Furthermore, the factors causing genital inflammation, such as STIs (sexually transmitted infections), vaginal discharge, and genital ulcers were not statistically significantly different among women with different median plasma TILRR protein levels. INTERPRETATION: The high plasma TILRR protein levels are highly correlated with several plasma proinflammatory cytokines/chemokines. High median plasma TILRR protein (≥100 ng/ml) strongly predicted an increased risk of HIV seroconversion. Reducing plasma TILRR protein levels may reduce the risk of HIV acquisition. FUNDING: The study was funded by an operating grant from the Canadian Institutes of Health Research (CIHR), operating grant-PA: CHVI Vaccine Discovery and Social Research (http://www.cihr-irsc.gc.ca/e/193.html), and National Microbiology Laboratory of Canada.

Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies.

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood.

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.

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.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars.

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 trachoma-which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital 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 plasmid-dependent 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.IMPORTANCE The 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.

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.

A genetic polymorphism of FREM1 is associated with resistance against HIV infection in the Pumwani sex worker cohort.

A subgroup of women enrolled in the Pumwani sex worker cohort remain seronegative and PCR negative for human immunodeficiency virus type 1 despite repeated exposure through high-risk sex work. Studies have shown that polymorphisms of genes involved in antigen presentation and viral restriction factors are associated with resistance to HIV infection. To discover other possible genetic factors underlying this HIV-resistant phenotype, we conducted an exploratory nonbiased, low-resolution, genome-wide single-nucleotide polymorphism (SNP) analysis comparing 60 HIV-resistant women to 48 HIV-infected controls. The SNP minor allele rs1552896, in an intron of FREM1, was significantly associated with the resistant phenotype (P = 1.68 × 10(-5); adjusted P = 2.37 × 10(-4); odds ratio [OR], 9.51; 95% confidence interval [CI], 2.82 to 32.05). We expanded the sample size by genotyping rs1552896 in the Pumwani cohort and comparing 114 HIV-resistant women to 609 HIV-infected controls and confirmed the association (P = 1.7 × 10(-4); OR, 2.67; 95% CI, 1.47 to 4.84). To validate the association in a second cohort, we genotyped 783 women enrolled in a mother-child health study and observed the minor allele of rs1552896 enriched in HIV-uninfected women (n = 488) compared to HIV-infected enrollees (n = 295) (P = 0.036; OR, 1.69; 95% CI, 0.98 to 2.93). Quantitative reverse transcription-PCR showed that FREM1 mRNA was highly expressed in tissues relevant for HIV-1 infection, and immunohistochemical analysis revealed that FREM1 protein is expressed in the ectocervical mucosa of HIV-resistant women. The significant association of rs1552896 with an HIV-resistant phenotype, together with the expression profile of FREM1 in tissues relevant to HIV infection, suggests that FREM1 is a potentially novel candidate gene for resistance to HIV infection.

High-throughput homogeneous immunoassay readily identifies monoclonal antibody to serovariant clostridial neurotoxins.

High-throughput screening can create the potential ability to screen large numbers of monoclonal antibodies (mAb) in a short time period. A major bottleneck in the hybridoma method for mAb development has historically been the inability to sift through large numbers of hybridoma culture supernatants to identify clones secreting mAbs of the desired specificity. Herein, we develop a homogeneous fluorometric microvolume assay technology (FMAT) and compare it to conventional ELISA screening techniques for monoclonal antibody against soluble protein toxin fragments of the Clostridium botulinum types A, B and E neurotoxin (BoNT) proteins. In total 8,744 hybridoma clones were screened to identify 29 stable hybridomas to neurotoxin binding domain; six of these would have been missed by ELISA alone. Screening of hybridoma supernatants on days 1 and 4 following cloning from semi-solid HAT agarose reveals that FMAT provides a reliable method for screening hybridoma clones to purified protein toxins. The homogeneous FMAT utilizes far less reagent (antigen and hybridoma supernatant) allowing for simultaneous screening against multiple serovariant antigens early in the hybridoma cloning cycle. This reduces costs for reagents and labour by lowering numbers of clones being maintained with undesired specificity. Furthermore, this assay easily accommodates replicate screening which facilitates identification of cross-reactivity to neurotoxin serotypes, thus readily identifying mAb to serovariant antigens. These findings have broad application in accelerating mAb development to serovariant cell-surface or bead bound targets without arraying devices. In summary, FMAT provides a reliable method for the screening of mAbs against C. botulinum neurotoxins.