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-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 (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.

INTRODUCTION
FREM1, a Fras Related Extracellular Matrix 1 protein, originates from epithelial and mesenchymal cells (1,2). It is widely expressed in the developing embryo in the region of epithelial/mesenchymal interaction (3) and the basement membrane zone of hair follicles (4). It is also highly expressed in the cervix, kidney and small intestine compared to other tissues in human (5). A significant number of studies have shown that mutations in the FREM1 and its splice variants are associated with MOTA (Manitoba-oculo-tricho-anal) syndrome (6)(7)(8)(9)(10)(11), BNAR (Bifid nose, anorectal and renal agenesis) syndrome (10,12), prenatal hydrocephalus and shortened limbs (13), metopic craniosynostosis (14), and congenital diaphragmatic hernia (15,16) in human and mice. Recent study demonstrated that FREM1 is also associated with facial morphology in human (17). Previously, we identified FREM1 as a novel candidate gene involved in HIV-1 resistance/susceptibility in the Pumwani Sex worker cohort (5). Of over 15 FREM1 splice variants, one has been identified as a toll-like interleukin-1 receptor regulator (TILRR). TILRR was described as a novel regulatory component, which functions as an IL-1R1 co-receptor (18,19). Recently, it has been shown that TILRR is responsible for the development of cardiovascular disease via aberrant activation of inflammatory genes (20). Structurally, TILRR contains three CSPG (chondroitin sulfate proteoglycan) domains and a RGD (arginine-glycine-aspartic acid) domain that interact with collagen, integrin, and fibronectin (4). It also has Calx-β, C-type lectin (LecC) domains, and two GAG (glycosaminoglycan) attachment sites that bind to IL-1R1 (Figure 1) (5). It has been demonstrated that TILRR amplifies innate immune and inflammatory responses after binding to IL-1R1 and TLR complexes by enhancing the recruitment of MYD88 (Myloid differentiation primary response 88) in the Rasdependent NFκB signal transduction pathway (19,21). However, it is not known whether TILRR interacts with or regulates other genes in the NFκB signal transduction and pro-inflammatory responses. The NFκB and pro-inflammatory responses are very important in initiating innate immune responses to pathogens, including HIV infection, and in linking innate and adaptive immune responses. Therefore, it is important to understand the effect of TILRR on genes in the NFκB signal transduction pathway. We hypothesized that TILRR regulates genes in the NFκB signal transduction pathway, directly involved in immune activation and inflammatory responses.
In this study, we used two epithelial cell lines that do not express TILRR mRNA to investigate the effect of TILRR overexpression on mRNA of genes in the NFκB signaling pathway and its effect on several soluble immune mediators. We evaluated the mRNA expression of 84 genes related to the NFκB signaling using PCR arrays and the secretion of 13 soluble immune mediators using a multiplex bead array system.

Cell Lines and Culture Condition
Our previous study showed that FREM1 mRNA is highly expressed in human cervical cells (5). To study the effect of FREM1 variant TILRR expression on cervical cells we used two model cervical epithelium cell lines, HeLa and VK2/E6E7. RNA-seq analysis and qRT-PCR analysis showed that these two cell lines do not express TILRR mRNA under the cell culture conditions we used in the study. Thus, we can use them to overexpression TILRR to study the effect on other inflammatory response related genes.

Plasmid Constructs, Reagents and Transfection
Both the TILRR-plasmid (GeneCopoeia, Catalog# EX-I2135-68) and Empty vector-plasmid control (GeneCopoeia, catalog# EX-NEG-68) containing a CMV promoter, an ampicillin marker, and a puromycin marker, were used for transfecting cells ( Figure S1). PmaxGFP (Lonza, Walkersville, MD, USA) was used as a standard enhanced GFP (Green fluorescence protein) control vector to monitor the transfection efficiency by Flow Cytometry and Confocal Microscopy. EndofectinMax (GeneCopoeia, Catalog# EFM1004-01) transfection reagent was used for the lipid based transfection of cells. Approximately 2.5 × 10 5 cells/ml were plated into each well of a 12-well culture plate in complete DMEM growth medium (HeLa) or complete Keratinocyte-SFM (1X) (VK2/E6E7) a day before transfection, and incubated at 37 • C with 5% CO 2 for 24 h. Once the cells reached 80-90% confluency, the medium was replaced with antibiotic free fresh growth medium. Co-transfection was performed using different concentration of either TILRRplasmid (vector+TILRR) (0.25, 0.5, 1.0, or 2.0 µg per well) or empty vector-plasmid (empty vector control) (0.25, 0.5, 1.0, or 2.0 µg per well) in combination with PmaxGFP-plasmid DNA (0.05, 0.1, 0.2, or 0.4 µg per well, respectively; 1:5 ratio) with 2 µl of EndofectinMax transfection reagent following to the protocol recommended by the manufacturer. After incubation at 37 • C with 5% CO 2 for 24 h the effect of TILRR overexpression on mRNA expression of 4 genes was analyzed. The optimized concentration of TILRR-plasmid (1.0 µg/well) or empty vector (1.0 µg/well) in combination with PmaxGFP vector (0.2 µg/well; 1:5 ratio) was used to transfect HeLa and VK2/E6E7 cells with 2 µl of EndofectinMax transfection reagent to analyze the effect of overexpression of TILRR on mRNA expression of NFκB signal transduction pathway related genes and pro-inflammatory cytokine/chemokine(s).
We also quantified overexpression of TILRR protein in transfected HeLa and parental cells by FACS analysis (BD Accuri C6, BD Biosciences). We stained the cells according to the BD Biosciences (California, USA) protocol. Briefly, 5 × 10 5 HeLa cells from each of the experimental conditions were prepared and washed with 1x PBS containing 2% FCS (fetal calf serum), then incubated with 50 µl Alexa Fluor 647 labeled in-house developed mabs (F218G1 and F218G5) (2 µg/ml, diluted in 1x PBS containing 3% BSA) for 30 min at 4 • C in dark (APEX Antibody Labeling kit, Invitrogen, Catalog# A10475). After washing (PBS containing 2% FCS), 100 µl BD permeabilizing solution (BD Biosciences, catalog# 554714) was added. After 10 min permeabilization, the cells were washed twice with 1x Perm/Wash buffer (BD Biosciences, catalog# 554714), and then 50 µl of Alexa Fluor 647 labeled mabs cocktail (F218G1 and F218G5) (2 µg/ml, diluted in 1x Perm/Wash buffer) was further added and incubated for 30 min at 4 • C in dark. Finally, the cells were resuspended in PBS containing 2% FCS after two times washes with 1x Perm/Wash buffer and analyzed with BD Accuri C6. In parallel, the cells were also stained with isotype control mab (F400G3S) (2 µg/ml) labeled with Alex Fluor 647. FlowJo Software (Treestar, USA) was used for analysis. Cell viability by FACS was measured using Live/Dead Fixable Red Dead Cell stain (Life Technologies, Catalog# L34971) following the company's recommended protocol.

Collection of Conditioned Media for Cytokine/Chemokine(s) Assay
The HeLa and VK2/E6E7 cells were transfected with TILRRplasmid or empty vector-plasmid control as described in the method above. Twenty-four hours after transfection the cells were treated with puromycin dihydrochloride (Gibco, Catalog# A11138-03) for 24 h to remove untransfected cells. The cells were then incubated in serum free DMEM (HeLa) or Keratinocyte SFM (1X) (VK2/E6E7). In parallel experiments, the cells were also incubated with human interleukin-1β (IL-1β; 1 nM) (Sigma-Aldrich, Catalog# I9401) in serum free HeLa and VK2/E6E7 cells medium. The cell culture medium was collected at 1, 3, 6, 15, and 24 h for cytokine/chemokine(s) analysis.
RNA Extraction, Purification, Quantification, Quality Analysis and cDNA Synthesis RNA was extracted from cells under different experimental conditions using RLT buffer from RNeasy Mini Kit (Qiagen, Catalog# 74104). Extracted and purified RNA from 5 × 10 5 cells/experimental condition using RNAeasy Mini Kit according to the manufacturer's instructions. The purified RNA was quantified using a NanoDrop 1000 Spectrophotometer (Thermofisher Scientific, USA), and the A260:A230 ratio of the isolated RNA was >1.7 and their A260:A280 ratio was between 1.8 and 2.0. RNA quality was also assessed with 2100 Agilent (R) Bio-analyzer (Agilent Technologies, USA) using an RNA 6000 Nano LabChip (R) kit (Agilent Technologies, Catalog# 5067-1511), and verified the quality with sharp bands/peaks for both the 18S and 28S ribosomal RNAs. The RIN was ≥7.0 for each sample. The cDNA was synthesized using RT 2 first strand kit (Qiagen, Catalog# 330404) with 500 ng purified RNA per reaction as recommended by the manufacturer's protocol.

RT 2 qPCR Primer Assay and RT 2 Profiler PCR Array
Real time quantification of TILRR overexpression was done using a commercial RT 2 qPCR primer assay (Qiagen, Catalog# PPH11469A-200). NFκB signaling pathway expression was quantified using RT 2 profiler qPCR array (Qiagen, Catalog# PAHS-025Z) and RT 2 SYBR (R) Green ROX qPCR Mastermix (Qiagen, Catalog# 330523). We also performed RT 2 qPCR primer assay for 4 selected immune responsive genes, CCL5 (Catalog# PPH00703B-200), CXCL8 (Catalog# PPH00568A-200), IL-6 (Catalog# PPH00560C-200) and TNFα (Catalog# PPH00341F-200), to measure the mRNA transcript expression with similar Mastermix as mentioned above. All primers were purchased from Qiagen. We used 1 µl of cDNA in 25 µl reaction volume. Amplification of cDNA performed in 40 cycles, consisting of initial 1 cycle at 95 • C for 10 min followed by 40 cycles, each cycle run at 95 • C for 15 s followed by 60 • C for 1 min. After 40 cycles, we also performed dissociation curve for all 84 genes and threshold was manually corrected at 0.4. Data were exported and finally organized in Microsoft Office Excel sheet and analyzed by GeneGlobe Data Analysis Centre (Qiagen). Applied BioSystem 7900 HT Fast Real time PCR 96-well standard block (ThemoFisher Scientific, USA) was used for all qRT-PCR analysis.

Western Blot
A previously published method was used with slight modifications (22). Briefly, SDS-PAGE was conducted using NuPAGE Bis-Tris mini gel electrophoresis protocol. Approximately 1 × 10 6 cells were lysed with 50 µl RIPA lysis and extraction buffer (Thermo Fisher Scientific, Catalog# 89900), then passed through a QIAshredder column (Qiagen, Catalog# 79654) by centrifugation at 15,000 g for 2 min. The lysate was then prepared and loaded into a NuPAGE 4-12% Bis-Tris 1.0 mm × 10well gel (Thermo Fisher Scientific, Catalog# NP0321BOX). Several monoclonal antibodies were used to detect the TILRR protein including F218, F208, F217, F244, F220, and F237 previously developed in our lab (23). The monoclonal antibodies were diluted in antibody buffer (wash buffer containing 0.5% skimmed milk) to give a concentration of 1 µg/ml for each antibody, and then incubated overnight with membranes at 4 • C with shaking. Then, the secondary antibody, goat anti mouse IgG-HRP (Santa Cruz Biotechnology, Catalog# sc-2005) was diluted at 1:5,000 in antibody buffer and incubated with membrane for 1 h at room temperature with shaking. Chemiluminescent detection was performed on a ChemiDoc XRS instrument using Quantity One 4.6.9 software (Bio-Rad). The level of TILRR protein expression was defined as ratio of the band intensity of TILRR to that of GAPDH, and finally normalized to parental cells.  (Table S5 for detailed information). The primary antibodies (mouse) for each cytokine/chemokine were coupled to 1.25 × 10 6 Bio-Plex Pro TM Magnetic COOH Beads (BioRad, Catalog# MC10053-01) using BioPlex (R) Amine coupling kit (BioRad, Catalog# 171-406001) according to the supplier's instructions. The assay was performed based on Bio-Plex Pro TM assays protocol (BioRad). Briefly, the antibody coupled beads were vortexed and combined at 1:600 dilutions in assay buffer (Bio-Plex Pro TM reagent kit, BioRad, Catalog# 171-304070M To generate standard curve, we added 50 µl of 4-fold standard dilutions in 6-wells in duplicates and the correlation coefficient (R 2 ) was being calculated in each experiment to see the linearity of the standard curve. Data were generated by Bio-Plex Manager 6.1 software.

Statistical Analysis
RT 2 Primer qPCR assay and RT 2 profiler PCR array data were analyzed using GeneGlobe Data Analysis Centre (Qiagen) (https://www.qiagen.com/us/shop/genes-and-pathways/dataanalysis-center-overview-page/). The mRNA transcript fold changes in threshold cycle (CT) were calculated relative to the CT level of empty vector control. Fold change 1.0 assigned as a baseline control and threshold cycle (CT) assays were performed in three independent experimental replicates.  Figure S2). (L) Confirmation of the TILRR mRNA transcripts overexpression in both cells using RT 2 qRT-PCR primer assay and data presented as log10 fold change, which was normalized against HPRT1 housekeeping gene. Student t-test with 95% CI performed for the statistical analysis using GraphPad prism version 7.03, all p < 0.05 were reported and indicated using an asterisks' *p < 0.05, and ****p < 0.0001.
All mRNA transcript data were normalized against HPRT1 housekeeping gene. Graphical presentation of fold change was organized by GraphPad Prism software, version 7.03 (GraphPad Software, Inc. USA). The cytokine/chemokine(s) data were also analyzed by GraphPad Prism version 7.03 and presented as relative to the concentration of empty vector control, and show mean ± SEM of three independent experiments. The final statistical comparisons conducted using student t-test with 95% CI, all p < 0.05 were reported and indicated using an asterisks' * p < 0.05, * * p < 0.01, * * * p < 0.001, and * * * * p < 0.0001.

TILRR Overexpression in Transfected Cells
To assess the effect of TILRR overexpression on genes in the NFκB inflammatory pathway, we overexpressed TILRR in HeLa (a cervical epithelial cell-line) and VK2/E6E7 (a normal human vaginal mucosal epithelial cell-line) cells. We transiently transfected HeLa (Figures 2A,B) and VK2/E6E7 (Figures 2E,F) cells with a TILRR expression plasmid that includes a CMV promoter and Puromycin selection marker. Confocal microscopy image analysis showed that cells containing plasmids with either TILRR plus puromycin selection marker (vector+TILRR) or only puromycin marker (empty vector control) were alive after 24 h puromycin dihydrochloride selection as shown by the cells attached to the culture plate with active pseudopods and intact morphology (Figures 2A,B,E,F). Whereas, within the same period of time under puromycin selection the non-transfected cells or the cells that only contain PmaxGFP vector were died, showing complete loss of pseudopodia and detachment from the culture plate with distracted morphology (Figures 2C,D,G,H). Flow cytometry quantification of GFP expressing cells showed that the transfection efficiency was between 87.0 and 90.9% of empty vector control-and TILRR (vector+TILRR)-transfected HeLa cells, respectively (Figures 2I,J).
Western blot analysis of transfected cells showed that cells transfected with TILRR expressed significantly higher amount of TILRR protein compared to parental-HeLa cells and cells transfected with empty vector (p < 0.05) (Figure 2K; full length original blots presented in Figure S2). RT 2 qPCR Primer analysis showed that cells transfected with TILRR containing plasmid significantly overexpressed the TILRR mRNA compared to non-transfected parental control and empty vector (p < 0.0001; Figure 2L). Confocal microscopy imaging analysis further confirmed the overexpressed TILRR protein in HeLa cells (Figures 3A-D and Figures S3A-D) compared to the respective controls (Figures 3E,F and Figures S3E-L). We also confirmed the TILRR protein expression in transfected HeLa cells by flow cytometry analysis using Alexa Fluor 647 labeled monoclonal antibodies (F218G1 and F218G5) recognizing epitopes in TILRR. The mean fluorescence intensity (MFI) of TILRR transfected cells showed significantly higher expression of TILRR compared to the parental control, isotype control and empty vector transfected control (Figure 3G, detailed gating strategy provided in Figure S4). These data indicated that TILRR transfected HeLa and VK2/E6E7 cells overexpressed TILRR.  Figure 4A) and in VK2/E6E7 cells (Figure 4B). HeLa cells transfected with 0.25-2.0 µg of TILRR-plasmid DNA significantly increased mRNA of all 4 genes in a dose dependent manner except TNFα, which was only significantly increased with the dose of 0.5 µg or above ( Figure 4A; Table S1). In the case of VK2/E6E7 cells (Figure 4B), the effect of different amount of TILRR-plasmid DNA transfection was only observed for the mRNA of CXCL8 and IL-6 ( Figure 4B and Table S2). The data showed that one microgram of plasmid DNA worked best in upregulating the selected inflammation responsive genes (GeneCopoeia recommended protocol). Thus, we used 1.0 µg of plasmid DNA for all subsequent experiments in this study.

Overexpression of TILRR Significantly Influenced the mRNA Level of Genes in NFκB Signal Transduction Pathway
Next, we investigated the effect of TILRR overexpression on the 84 genes in the NFκB signal transduction pathway. We hypothesized that upon interaction with IL-1R1 receptor, TILRR would influence the downstream signaling events by regulating mRNA transcript of genes in the NFκB signal transduction pathway. To test this, we investigated the effect of overexpression of TILRR on a panel of 84-genes (Tables S3, S4) that are directly related to NFκB signaling pathway, immune activation and inflammatory responses using the Human NFκB pathway RT 2 profiler PCR array.
TILRR overexpression enhanced the expression of 8 NFκB transcription factors (Figure 7C)  The expression of immune responsive genes directly associated with the NFκB signaling pathway was also examined. We observed significant induction of mRNA transcripts for 7 immuno-regulatory genes when TILRR was overexpressed ( Figure 7E) 1.42 ± 0.14, p = 0.0155) was enhanced in TILRR transfected HeLa and VK2/E6E7 cells, respectively. CSF3 (G-CSF) and CCL5 (RANTES) were only significantly regulated in HeLa cells.
Finally, we examined the effect of TILRR on the expression of genes that are involved in cytoplasmic sequestration or release of NFκB complex proteins (Figure 7F). The expression of three out of six genes evaluated was significantly up-regulated in HeLa cell line, including CHUK (IKKa), IKBKB (IKKβ), and FIGURE 5 | Heat map presentation of up-and down-regulated genes in NFκB pathway for the presence of TILRR. Log2 fold changes of gene expression in HeLa and VK2/E6E7 cells generated by RStudio (https://www.rstudio.com). Gradient red color indicates the up-regulated genes; gradient green represents the down-regulated genes. Solid black means baseline, which represents the fold change 1 or log2 = 0 (control). "X" axis showing the triplicate biological samples for each cell line, "Y" axis (right side) represents the 84 tested genes categorized into different groups. Legend on the upper left side shows the scale of log2 fold change.
IKBKE (IKKε). Only the expression of CHUK was up-regulated in VK2/E6E7 cells. Thus, TILRR has direct influence on genes involved in formation of transcription factors NFκB1 (p50) and NFκB2 (p52) that subsequently translocate to the nucleus and potentiate signal transduction.
We further tested the effect of TILRR overexpression in the presence of IL-1β on selected genes directly involved in immune activation and inflammatory response in HeLa and VK2/E6E7 cells. TILRR transfected HeLa and VK2/E6E7 cells were incubated with or without IL-1β in parallel experiments and the expression of mRNA transcript was quantified using RT 2 qPCR Primer assay. The results showed that TILRR overexpression, in the presence or absence of added IL-1β, significantly increased the expression of 4 immune and inflammation responsive genes in HeLa and VK2/E6E7 cells (Figure 8).
We next examined the level of cytokine/chemokine(s) production in culture supernatants of TILRR-overexpressed VK2/E6E7 cells. Similar to the HeLa cells, in the absence of IL-1β, there was a gradual increase of cytokine/chemokine(s) production in VK2/E6E7 cell culture supernatants at different time points compared to the empty vector-transfected cells (Figure 10). Unlike the HeLa cells, we observed that after 1 h incubation with serum free media, only IP-10 (p = 0.0109) was significantly increased in TILRR overexpressed VK2/E6E7 cell supernatants. However, after longer incubations time (3-, 6-, 15-, and 24-h incubation), the levels of IL-6 (p = 0.0061, 0.0013, To determine whether TILRR overexpression augments the production of pro-inflammatory cytokine/chemokine(s) in the presence of IL-1β, we analyzed the effect of TILRR in the presence of IL-1β in cell culture supernatants of HeLa and VK2/E6E7 cells. The analysis showed that in HeLa cells the level of these cytokine/chemokine(s) increased in a time-dependent manner with the presence of TILRR and added IL-1β in comparison In the case of VK2/E6E7 cells, we observed a trend of increase of pro-inflammatory cytokine/chemokine(s) production in the presence of TILRR and IL-1β compared to the empty vector control in serum free media (Figure 10). Unlike the HeLa cells, only IP-10 (p = 0.0004) was significantly higher with the TILRR overexpression and added IL-1β in VK2/E6E7 cell supernatants after 1 h incubation. Similar to HeLa cells, after longer incubation time (3-and 24-h incubation) the levels of IL-6 (p = 0.0058 and 0.0439), IL-8 (CXCL8) (p = 0.0011 and 0.0045), IP-10 (p = 0.0362 and 0.0004) and RANTES (p = 0.0281 and 0.0329) were significantly increased in VK2/E6E7 cell culture supernatants. The significant increase of IL-8 (CXCL8) (p = 0.0049), IP-10 (p = 0.0384) and RANTES (p = 0.0021) was also observed after 6h incubation and the higher level of IL-6 (p = 0.0022), IP-10 (p = 0.0004), RANTES (CCL5) (p = 0.0012) and MIP-1β (p = 0.0332) was also observed after 15h incubation. However, the production of CSF2 (GM-CSF), IFNγ, IL-10, IL17A, MCP-1, MIP-1α, and TNFα was not detected following addition of IL-1β in the VK2/E6E7 cell culture supernatant (data not shown). Altogether, these data suggested that TILRR, in the presence or absence of added IL-1β, can modulate the production of pro-inflammatory cytokine/chemokines during inflammatory process and microbial infection.

DISCUSSION
Previous studies identified a variant of FREM1 as a co-receptor of IL-1R1, and its association with IL-1R1 enhances the recruitment of MYD88, controls the induction of Ras GTPase and amplifies the activation of NFκB and inflammatory responses (18,19). This variant of FREM1 was named as TILRR (Toll-like/IL-1 receptor regulator) (19). In this study, we conducted extensive analysis of genes influenced by TILRR overexpression in two cell lines, human cervical epithelial (HeLa) cells and human normal vaginal mucosal (VK2/E6E7) cells, using a PCR array system that can simultaneously interrogate the expression of 84 genes in the NFκB signal transduction pathway and RT 2 qPCR Primer assay for selected immune and inflammation responsive genes.
The data from our study showed that TILRR overexpression significantly regulated the expression of immune and inflammation responsive genes in a dose dependent manner. Our study also showed that overexpression of TILRR up-regulated the expression of many genes in the NFκB signaling pathway, far more than previously reported. In addition to the expression FIGURE 9 | TILRR overexpression in HeLa cells increased the production of Pro-inflammatory cytokine/chemokine(s) in the presence or absence of added IL-1β. HeLa (5 × 10 5 cells/well) cells were co-transfected with either pEZ-TILRR-M68 (1.0 µg/5 × 10 5 cells) or pEZ-NEG-M68 (1.0 µg/5 × 10 5 cells) with PmaxGFP (0.2 µg/5 × 10 5 cells) vector as explained in materials and methods section. In parallel, cells were incubated with or without the addition of IL-1β (1 nM) in serum free DMEM (HeLa) media and then the cultured media were collected (see methods). Thirteen different inflammatory cytokines were measured using an in-house developed multiplex cytokine/chemokine(s) bead assay with BioPlex 200 (BIORAD). The data represent the relative level of vector + TILRR, in the presence or absence of IL-1β, compared to the empty vector control. The sample measurements below the detection limit were assigned as zero. The data represent mean of three independent experiments (mean ± SEM). Statistical comparisons conducted using student t-test with 95% CI, all p < 0.05 were reported and indicated using an asterisks' *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. of IL-1R1, MYD88, TRAF6 reported previously (19), among the 84 genes involved in the NFκB signaling pathway, TILRR overexpression significantly up-regulated the expression of many genes in HeLa and VK2/E6E7 cells. Among the significantly up-regulated genes, some have critical roles in NFκB activation, and innate and adaptive immune responses ( Table S6). The effects of TILRR on these NFκB signaling related genes and inflammation mediated genes demonstrated the importance of TILRR in immune regulation and inflammatory responses.
NFκB signal transduction pathway (Figure 7) and significantly augmented the mRNA transcript expression of several immune responsive genes when together with IL-1β in serum free media (Figure 8). Thus, TILRR appears to not only be a co-receptor of the IL-1R1, but also have a direct effect on genes in the NFκB signal transduction and inflammation pathway.
This study also showed that as the result of TILRR overexpression the production of several inflammatory cytokine/chemokine(s) secretion was also increased. Overexpression of TILRR increased IL-6, IL-8 (CXCL8), IP-10 (CXCL10), MCP-1, and RANTES (CCL5) in the HeLa cell culture supernatants. The IL-6, IL-8 (CXCL8), IP-10 (CXCL10), MIP-1β, and RANTES (CCL5) in VK2/E6E7 cell culture supernatants were also increased. The increase in protein level of these mediators is consistent with the increase in mRNA transcript level expression. It shows that TILRR influences the production of the inflammatory mediators, although the mechanisms need to be explored in future studies. These pro-inflammatory cytokine/chemokine(s) have been reported as multifunctional, local exudation inducer, potent activator of nuclear localization of NFκB, and enhancer of inflammation (29)(30)(31)(32)(33)(34)(35). Regulation of these pro-inflammatory cytokine/chemokine(s) by TILRR suggests that TILRR may be a direct regulator in the NFκB signal transduction and inflammatory responses.
In humans, cervical epithelial cells express higher amounts of FREM1 mRNA when compared to other tissues (5). Using HeLa and VK2/E6E7 cells as an in vitro system in this study may help to understand the influence of TILRR, a variant of FREM1, on the inflammatory responses in the epithelial mucosal barrier. Mucosal epithelial cells not only serve as a physical barrier, but also act as the first line of defense against infection. Breaches in the epithelial lining increase the risk of inflammation and infection (36,37).
Our study is the first to show that TILRR overexpression regulates the expression of many genes in the NFκB signal transduction pathway. TILRR could be an important mediator of NFκB signaling pathway and plays a major role in regulating innate immune and inflammatory responses and may play an important role in microbial infection and disease pathogenesis.