Gene Expression Of Proinflammatory Chemokines Research Articles

Time-Restricted Feeding Ameliorates Methionine-Choline Deficient Diet-Induced Steatohepatitis in Mice.

Non-alcoholic steatohepatitis (NASH) is an inflammatory form of non-alcoholic fatty liver disease (NAFLD), closely associated with disease progression, cirrhosis, liver failure, and hepatocellular carcinoma. Time-restricted feeding (TRF) has been shown to decrease body weight and adiposity and improve metabolic outcomes; however, the effect of TRF on NASH has not yet been fully understood. We had previously reported that inositol polyphosphate multikinase (IPMK) mediates hepatic insulin signaling. Importantly, we have found that TRF increases hepatic IPMK levels. Therefore, we investigated whether there is a causal link between TRF and IPMK in a mouse model of NASH, i.e., methionine- and choline-deficient diet (MCDD)-induced steatohepatitis. Here, we show that TRF alleviated markers of NASH, i.e., reduced hepatic steatosis, liver triglycerides (TG), serum alanine transaminase (ALT) and aspartate aminotransferase (AST), inflammation, and fibrosis in MCDD mice. Interestingly, MCDD led to a significant reduction in IPMK levels, and the deletion of hepatic IPMK exacerbates the NASH phenotype induced by MCDD, accompanied by increased gene expression of pro-inflammatory chemokines. Conversely, TRF restored IPMK levels and significantly reduced gene expression of proinflammatory cytokines and chemokines. Our results demonstrate that TRF attenuates MCDD-induced NASH via IPMK-mediated changes in hepatic steatosis and inflammation.

Inflammatory Immune Responses Trigger Rejection of Allogeneic Fibroblasts Transplanted into Mouse Skin

Fibroblasts, or their homolog stromal cells, are present in most tissues and play an essential role in tissue homeostasis and regeneration. As a result, fibroblast-based strategies have been widely employed in tissue engineering. However, while considered to have immunosuppressive properties, the survival and functionality of allogeneic fibroblasts after transplantation remain controversial. Here, we evaluated innate and adaptive immune responses against allogeneic fibroblasts following intradermal injection into different immune-deficient mouse strains. While allogeneic fibroblasts were rejected 1 week after transplantation in immunocompetent mice, rejection did not occur in immunodeficient γ chain–deficient NOD-SCID (NSG) mice. T-cell- and B-cell-deficient RAG1 knockout mice showed greater loss of fibroblasts by day 5 after transplantation compared with NSG mice (P ≤ 0.05) but prolonged persistence compared with wild-type recipient (P ≤ 0.005). Loss of fibroblasts correlated with the expression of proinflammatory chemokine genes and infiltration of myeloid cells in the transplantation site. Depletion of macrophages and neutrophils delayed rejection, revealing the role of innate immune cells in an early elimination of fibroblasts that is followed by T-cell-mediated rejection in the second week. These findings indicate that the application of allogeneic fibroblasts in tissue engineering products requires further improvements to overcome cell rejection by innate and adaptive immune cells.

Single-Cell Profiling Reveals Contribution of Tumor Extrinsic and Intrinsic Factors to BCMA-Targeted CAR-T Cell Efficacy in Multiple Myeloma

▪Background: BCMA CAR-T cell therapy has shown great promise in relapsed/refractory multiple myeloma (RRMM) patients, even though there is unpredictable variability in the duration and depth of response. The mechanisms behind these divergent outcomes and relapse are not well understood and heterogeneity of MM patients at the level of both tumor genomics and tumor microenvironment (TME) likely contributes to this important knowledge gap. To explore this question, we performed a longitudinal high resolution single cell genomic and proteomic analysis of bone marrow (BM) and peripheral blood (PB) samples in MM patients treated with BCMA CAR-T.Methods: Longitudinal comprehensive immune phenotyping of 3.5 million peripheral blood mononuclear cells (PBMC, CD45+CD66b-) from 11 BCMA CAR-T (idecabtagene vicleucel, ide-cel) patients was achieved via mass cytometry (CyTOF) with a panel of 39 markers. In addition, a total of 45,161 bone marrow mononuclear cells (BMMC) were analyzed from 6 patients before initiation of ide-cel therapy and at relapse by unbiased mRNA profiling via single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Downstream analysis was performed using the CATALYST and Seurat R packages, respectively. Immune cell populations are reported as % of PBMC and CD138- BMMC respectively, unless noted otherwise. Reported p values correspond to non-parametric tests or paired t test where applicable.Results: We compared baseline immune cell populations in the PB and the TME (BM) with regards to depth of CAR-T response. In PB, good responders (≥VGPR) had a higher proportion of CD8+ T cells (37% in good vs 11% in poor responders (<VGPR), p=0.08) and a lower proportion of CD14+ monocytes (30% vs 61%, p=0.28) and NK cells (2% vs 6%, p=0.08). In the TME, a similar trend was confirmed for CD8+ T cells and CD14+ monocytes. (Fig. 1A)Longitudinal analysis of PBMCs revealed phenotypic changes coinciding with CAR-T expansion; CD14+ monocytes declined from week 0 to week 4 after CAR-T infusion (40% vs 13%, p=0.04), while (non-CAR) CD8+ T cells expanded from week 0 to week 4 (32% vs 43%, p=0.15). The non-CAR CD8+ T cell expansion is characterized by differentiation towards a CD8+ effector-memory phenotype (EM, CCR7-CD45RA-) (73% vs 92% of CD8+ T cells, p=0.005). (Fig. 1B) BM samples at CAR-T relapse showed reversal of this shift: CD14+ monocyte levels remain constant or are slightly elevated, while non-CAR CD8+ T cells decrease at relapse.scRNA-seq of BMMC revealed significant gene expression changes between screening and relapse tumor samples, suggesting tumor-intrinsic factors of CAR-T response. For example, when comparing the pre and post tumor samples of a patient with durable response (PFS 652 days), we observed a significant upregulation of gene expression of pro-inflammatory chemokines (CCL3, CCL4), anti-apoptotic genes (MCL-1, FOSB, JUND), and NF-kB signaling genes (NFKBIA) in post tumor. Gene Set Enrichment Analysis (GSEA) of differentially expressed genes showed significant enrichment for TNFA signaling via NF-kB Hallmark Pathway (p.adj = 0.04). We observed similar statistically significant findings between other screening and relapse samples within our cohort, as well as upon comparison of baseline samples of poor vs good responders. (Fig. 1C, D) Thus, our data suggest that anti-apoptotic gene expression could be one of the tumor intrinsic mechanisms of CAR-T therapy resistance. Notably, we did not observe loss of BCMA expression in any tumor samples.Conclusion: Single cell immune profiling and transcriptomic sequencing highlights changes in the PB, TME and within the tumor, which in concert may influence CAR-T efficacy. Our integrated data analysis indicates general immune activation after CAR-T cell infusion that returns to baseline levels at relapse. Specifically, the expansion of non-CAR cytotoxic CD8+ EM T cells provides a rationale for co-administration of IMiDs to enhance CAR-T efficacy. Significant up-regulation of anti-apoptotic genes at baseline in poor responders, and at relapse in good responders, suggest a novel tumor-mediated escape mechanism. Targeting the MCL-1/BCL-2 axis may augment CAR-T efficacy by sensitizing tumor cells and enhancing the effect of CAR-T killing. We will confirm these findings in a longitudinal cohort of BMMC/PBMC CITE-seq patients (n=23) and will present results at the conference. [Display omitted] DisclosuresSebra: Sema4: Current Employment. Parekh: Foundation Medicine Inc: Consultancy; Amgen: Research Funding; PFIZER: Research Funding; CELGENE: Research Funding; Karyopharm Inv: Research Funding.

Toll-like receptor-2 mediates systemic inflammation in gentamicin-induced rat nephrotoxicity.

Gentamicin is an aminoglycoside antibiotic commonly administrated to patients with Gram-negative infections. Gentamicin induced nephrotoxicity by functional and structural impairment. Toll-like receptors (TLRs) as key mediators in the innate and adaptive immune system response involved in gentamicin-induced nephrotoxicity. The present study aimed to investigate the gene expression of TLR2 and pro-inflammatory cytokines in the renal tissues and buffy coat of the whole blood in gentamicin-treated rats. Twenty adult male Sprague Dawley rats weighing 180-200 were randomly divided into gentamicin (100mg/kg, i.p) and control groups (n=10). After 10days, the serum creatinine (Cr) levels and blood urea nitrogen (BUN) were measured. The mRNA levels of TLR2, tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and monocyte chemoattractant peptide (MCP)-1 were investigated in the renal tissue and buffy coat by qRT-PCR. Kidney histological analysis performed by hematoxylin-eosin (H&E) staining. Functional disturbance is characterized by a significant increase in the serum levels of Cr and BUN in the gentamicin group. Renal tissue slides of the gentamicin group indicated severe glomerular and tubular damage including lobulation of the glomerular tuft, Bowman's space enlargement, acute tubular necrosis, and proximal tubular destruction. The mRNA levels of IL-1β, TNF-α, MCP-1, and TLR2 increased in the buffy coat, but all of them except TLR2 decreased in the renal tissues in the gentamicin group compared with controls. Gentamicin administration induced relative systemic inflammation, which may be related to an increase in the mRNA levels of TLR2 results in gene expression of pro-inflammatory chemokines and cytokines including IL-1β, TNF-α, and MCP-1 in immune cells.

Abstract CT229: A phase II and biomarker study of an irreversible pan-human EGF receptor (HER) tyrosine kinase inhibitor, dacomitinib, in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M-SCCHN)

Abstract Background: Dacomitinib is an oral irreversible tyrosine kinase inhibitor of EGFR, HER2 and HER4 signaling. The goals of this study were to investigate the clinical activity, safety and predictive biomarkers of dacomitinib in R/M-SCCHN. Methods: Patients with R/M-SCCHN were eligible if they had progressed on platinum-based chemotherapy or considered platinum-intolerant, and were treated with dacomitinib 45mg/day. The primary endpoint was objective response rate (ORR) by RECIST v1.1; Secondary endpoints included progression-free survival rate (PFS), overall survival (OS) and safety. We charaterized somatic mutations using the Ion AmpliSeq™ platform (2,800 mutations from 50 genes), gene copy number (86 genes) and gene expression (230 genes) using NanoString nCounter® and CDKN2A (p16INK4A) expression by immunohistochemistry, and investigated their relationship with clinical outcomes. Results: A total of 48 patients were enrolled and evaluable for efficacy and toxicity. Patient and disease characteristics included median age (61 years; range, 30-82); male (85%); ECOG performance status 0/1/2 (4%/75%/21%); locoregional/distant metastatic/both (31/23/46%); oral cavity/larynx/oropharynx primary (38%/19%/23%); prior chemotherapy regimens 0/1/≥2 (8%/52%/40%). Ten patients (21%) had partial responses and 31 patients (65%) had stable diseases. With a median 8.4 months of follow up, the median PFS and OS were 3.9 months (95% CI, 2.9-5.0) and 8.2 months (95% CI, 5.8-10.3). Adverse events (AEs) were mostly grade 1-2 and manageable. The most common AEs were skin toxicities (paronychia, 65%; acneiform dermatitis, 44%) and diarrhea (52%). Results from the biomarker analyses were available in 28 patients for AmpliSeq™, 31 patients for gene copy number, and 12 patients for gene expression. Mutations in PIK3CA and PTEN was significantly associated with shorter PFS (2.5 vs 5.4 months, P=0.013). There were trends toward higher ORR (23% vs 0) for patients with wild PIK3CA and PTEN, with unusually long durations of response (5.1, 5.4, 5.8, 13.1, and 18.9 months, respectively). Increased gene expression of proinflammatory chemokines and inflammatory response modulators (IL6, IL8, PTGS2, PLA2G2A) was significantly associated with shorter PFS (1.9 vs 6.8 months, P=0.001). Patients with low gene expression of proinflammatory chemokines also tend to have higher ORR (40% vs 0), with unusually long durations of response (18.9 and 5.4 months, respectively). Conclusions: Dacomitinib demonstrated promising efficacy with manageable toxicity in platinum-failed R/M-SCCHN patients. Screening of PI3K pathway mutation and proinflammatory chemokines may help identify which R/M-SCCHN patients are likely to gain a benefit from dacomitinib. Citation Format: Han Sang Kim, Hyung Joo Kwon, Myung-Ju Ahn, Byung Woog Kang, Jong-Mu Sun, Dok-Hyun Yoon, Keon Uk Park, Se-Hoon Lee, Yoon Woo Koh, Se Hun Kim, Eun Chang Choi, Hye Ryun Kim, Hyo Song Kim, Joo Hang Kim, Byoung Chul Cho. A phase II and biomarker study of an irreversible pan-human EGF receptor (HER) tyrosine kinase inhibitor, dacomitinib, in patients with recurrent and/or metastatic squamous cell carcinoma of head and neck (R/M-SCCHN). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT229. doi:10.1158/1538-7445.AM2014-CT229

Sterol Intermediates of Cholesterol Biosynthesis Inhibit Hair Growth and Trigger an Innate Immune Response in Cicatricial Alopecia

Primary cicatricial alopecia (PCA) is a group of inflammatory hair disorders that cause scarring and permanent hair loss. Previous studies have implicated PPARγ, a transcription factor that integrates lipogenic and inflammatory signals, in the pathogenesis of PCA. However, it is unknown what triggers the inflammatory response in these disorders, whether the inflammation is a primary or secondary event in disease pathogenesis, and whether the inflammatory reaction reflects an autoimmune process. In this paper, we show that the cholesterol biosynthetic pathway is impaired in the skin and hair follicles of PCA patients. Treatment of hair follicle cells with BM15766, a cholesterol biosynthesis inhibitor, or 7-dehydrocholesterol (7-DHC), a sterol precursor, stimulates the expression of pro-inflammatory chemokine genes. Painting of mouse skin with 7-DHC or BM15766 inhibits hair growth, causes follicular plugging and induces the infiltration of inflammatory cells into the interfollicular dermis. Our results demonstrate that cholesterologenic changes within hair follicle cells trigger an innate immune response that is associated with the induction of toll-like receptor (TLR) and interferon (IFN) gene expression, and the recruitment of macrophages that surround the hair follicles and initiate their destruction. These findings reveal a previously unsuspected role for cholesterol precursors in PCA pathogenesis and identify a novel link between sterols and inflammation that may prove transformative in the diagnosis and treatment of these disorders.

Ambivalent roles of early growth response 1 in inflammatory signaling following ribosomal insult in human enterocytes

NF-κB expression and activity are strictly regulated in gut epithelia to prevent overstimulation of pro-inflammatory responses following exposure to commensal bacteria. The effects of epithelial EGR-1 on responses to bacterial NF-κB-activating lipopolysaccharide (LPS) in intestinal epithelial cells under ribosomal stress were assessed. This was done to determine the potential of EGR-1 as a modulator of epithelial NF-κB signaling. Nuclear translocation of phosphorylated p65 protein was observed in the cells exposed to LPS although chemokine expression was marginally affected. In contrast, simultaneous exposure to LPS and ribosomal insults prevented epithelial NF-κB activation while chemokine expression was enhanced. The effect of EGR-1, another pro-inflammatory signaling mediator, was monitored to determine the involvement of this factor on chemokine production in response to this co-treatment. Similar to the previously reported ribosomal stress response, EGR-1 expression was elevated by ribosomal insults alone and positively affected gene expression of pro-inflammatory chemokines in the intestinal epithelial cells. However, EGR-1 suppression led to super-induction of chemokines by simultaneous treatment with LPS and ribosomal insult, indicating that EGR-1 is a negative modulator of chemokine gene expression. Particularly, mucosal ribosomal insult-triggered EGR-1 mediated PPARγ induction, which counteracted NF-κB activation by LPS. It can be thus concluded that EGR-1 regulates pro-inflammatory NF-κB activation by LPS via PPARγ although EGR-1 is a positive mediator of chemokine expression following ribosomal insult in intestinal epithelial cells.

Pulsed acoustic cellular expression as a protective therapy against I/R injury in a cremaster muscle flap model

BackgroundTissue ischemia and reperfusion (I/R) affects blood flow restoration and oxygen delivery to the damaged tissues contributing to tissue morbidity and microcirculatory compromise. Pulsed acoustic cellular expression (PACE) technology is known to support tissue neovascularization. The aim of this study was to test PACE conditioning mechanism of action on microcirculatory hemodynamics in ischemia–reperfusion injury model. Methods34 rat cremaster muscle flaps were monitored under intravital microscopy system in 4 experimental groups: 1) non-ischemic controls (n=10), 2) 5h ischemia without conditioning (n=8), 3) pre-ischemic (5h) PACE conditioning (n=8), 4) post-ischemic (5h) PACE conditioning (n=8). Standard microcirculatory hemodynamics of RBC velocity, vessel diameters and functional capillary perfusion were recorded for 2h after I/R. Immunohistochemistry assessed expression of proangiogenic factors: VEGF and vWF, whereas real-time PCR assessed proangiogenic (VEGF, eNOS) and proinflammatory factors (iNOS; chemokines: CCL2, CXCL5 and chemokine receptor CCR2). ResultsPre-ischemic PACE conditioning (group 3) resulted in increased RBC velocity of second (A-2) and third order arterioles (A-3) and venule (V-1) by 40%, 15% and 24% respectively comparing to ischemic group without conditioning (p<0.05). Post-ischemic PACE conditioning (group 4) revealed: 1) increase in RBC velocity in second (A-2) and third order arterioles (A-3) by 65% and 31% respectively comparing to ischemia without conditioning (group 2), 2) 33% increase in first order arterioles diameter (A-1) (p<0.05) compared to ischemic controls, 3) 21% increase in number of functional capillaries compared to ischemia without conditioning (group 2) (P<0.05). Immunostaining assays showed that PACE postconditioning up-regulated proangiogenic factors vWF and VEGF protein expression. This correlated with increased gene expression of VEGF (up to 180%). In contrast, gene expression of proinflammatory factors (iNOS, CCL2, CXCL5) decreased compared to ischemic controls. Pre-ischemic PACE conditioning decreased gene expression of proinflammatory chemokines (CCL2 and CXCL5), compared to ischemic controls without conditioning. ConclusionsAs expected 5h ischemia resulted in deterioration of microcirculatory hemodynamics confirmed by decreased vessels diameters and RBC velocities. This was alleviated by pre- and post-ischemic PACE conditioning which improved functional capillary density and stimulated angiogenesis as confirmed by up-regulated VEGF expression. Furthermore, post-ischemic PACE conditioning correlated with decreased expression of early proinflammatory factors (iNOS, CCL2, CXCL5). Both types of PACE conditioning ameliorated deleterious effect of ischemia–reperfusion injury on microcirculatory hemodynamics of muscle flaps.

Modulation of Gene Expression Related to Toll-Like Receptor Signaling in Dendritic Cells by Poly(γ-Glutamic Acid) Nanoparticles

Poly(gamma-glutamic acid) (gamma-PGA) nanoparticles (NPs) have previously been reported as an efficient antigen delivery system with adjuvant activity. In this study, the gene expression in murine bone marrow-derived dendritic cells (DCs) treated with gamma-PGA NPs was examined by oligonucleotide microarray analysis and compared with that in cells treated with other adjuvants. The gene expression of proinflammatory chemokines, cytokines, and costimulatory molecules was upregulated considerably in DCs treated with gamma-PGA NPs. The upregulation pattern was similar to that in DCs treated with lipopolysaccharide (LPS) but not to that in DCs treated with unparticulate gamma-PGA. The activation of DCs by gamma-PGA NPs was confirmed by real-time reverse transcriptase PCR (RT-PCR) analysis of genes related to Toll-like receptor (TLR) signaling. The effect of gamma-PGA NPs on DCs was not annihilated by treatment with polymyxin B, an inhibitor of LPS. Furthermore, the immunization of mice with gamma-PGA NPs carrying ovalbumin (OVA) as an antigen significantly induced antigen-specific CD8(+) T cells and antigen-specific production of interleukin-2, tumor necrosis factor alpha, and gamma interferon from the cells. Such activities of gamma-PGA NPs were more potent than those obtained with immunization with OVA plus aluminum hydroxide or OVA plus complete Freund's adjuvant. These results suggest that gamma-PGA NPs induce a CD8(+) T-cell response by activating innate immunity in a fashion different from that of LPS. Thus, gamma-PGA NPs may be an attractive candidate to be developed further as a vaccine adjuvant.

Differential gene expression of proinflammatory chemokines and cytokines in lungs of ascites-resistant and -susceptible broiler chickens following intravenous cellulose microparticle injection

Intravenous injection of microparticles (MPs) is a tool to reveal susceptibility to pulmonary hypertension (PH) syndrome (PHS, ascites) in broilers. After injection MPs get lodged in pulmonary arterioles and cause localized inflammation. To examine the expression of chemokines/cytokines during the MP-induced pulmonary inflammatory response, lungs were collected from 4-week-old broilers (6/line/time point) from the PHS-resistant (RES) and -susceptible (SUS) broilers before (0 h) and after (2, 6, 12, 24, and 48 h) MP injection and analyzed using quantitative RT-PCR. In both lines, expression of interleukin-1β (IL-1β), IL-6, IL-8, and K60 increased from 0 to 6 h, reached peak levels at 6 and 12 h, and decreased thereafter, whereas IL-4 and interferon gamma (IFN-γ) expression remained elevated past 12 h. Lungs from the RES line broilers had higher expression ( P < 0.05) of IL-1β and IL-6 at 2, 6, and 12 h; higher IL-8 at 6 and 12 h; higher K60 at 6, 12, and 24 h; higher IL-4 at 12, 24, and 48 h and higher IFN-γ expression at 6 and 48 h post-MP injection than SUS line broilers. Higher expression of chemokines/cytokines in RES compared to SUS line lungs may explain the ability of RES line broilers to effectively counteract the MP-induced PH and resolve the vascular occlusion.

Timing of Expression of Inflammatory Mediators in Skeletal Muscles from Mice Acutely Infected with the RA Strain of Trypanosoma cruzi

Objective: Chagas’ disease is caused by persistent Trypanosoma cruzi infection in muscle cells that ultimately results in chronic inflammation and tissue destruction. The goal of this study was to determine the expression of different chemokines and their receptors, as well as proinflammatory cytokines and inducible nitric oxide synthase, in muscles from mice acutely infected with T. cruzi. Methods: Histological, semiquantitative reverse transcriptase polymerase chain reaction and immunohistochemical studies were performed on skeletal muscle and myocardium of BALB/c mice infected with T. cruzi, RA strain. Results: Early induction of muscular mRNA expression for CCL5/CCR5 and CXCL9/CXCR3, as well as for iNOS, IFN-γ, TNF-α and MIF, was demonstrated accompanied by progressive increases in parasitism and leukocyte recruitment. Protein overexpression for MIF and CCL5/CCR5 was also verified in the infected muscles. Conclusions: In muscles from acutely T. cruzi RA-infected mice, upregulated gene expression of proinflammatory chemokines, chemokine receptors, cytokines and iNOS is associated with the severity of parasite burden and myopathic alterations. Compared to the heart, striated muscles displayed differential timing of expression of several inflammatory mediators throughout acute infection. Our findings suggest that enhanced early production of these factors could contribute to T. cruzi-dependent inflammatory damage to skeletal muscles.

Intrarenal gene expression of proinflammatory chemokines and cytokines in chronic proteinuric glomerulopathies

Proteinuria has been recently shown to be an independent risk factor for the progression of chronic nephropathies, but the actual mechanisms by which urinary protein load damages renal tissue in humans remain unsolved. Using real-time RT-PCR method we evaluated intrarenal mRNA expression of various cytokines and chemokines in patients with biopsy-proven IgA nephropathy (IgAN, n=11), membranous nephropathy (MN, n=6) and focal and segmental glomerulosclerosis (FSGS, n=6) who exhibited proteinuria over 0.5 g/day. There was a significant positive correlation between the proteinuria extent and the intrarenal RANTES (regulated upon activation normal T cell expressed and secreted) mRNA expression in patients with IgAN, a similar trend was also observed in patients with MN and FSGS. There were no clear relationships between the proteinuria and intrarenal mRNA expression of tumor necrosis factor alpha, transforming growth factor beta1 and monocyte chemoattractant peptide-1. There were no differences in the pattern of cytokine mRNA expression between different glomerulopathies. In conclusion, our results support the hypothesis that lymphocytes, macrophages and their products provoke tissue injury in response to proteinuria independently of the nature of renal diseases in man.

Microarray analysis identifies a set of CXCR3 and CCR2 ligand chemokines as early IFNbeta-responsive genes in peripheral blood lymphocytes in vitro: an implication for IFNbeta-related adverse effects in multiple sclerosis.

BackgroundA substantial proportion of multiple sclerosis (MS) patients discontinue interferon-beta (IFNβ) treatment due to various adverse effects, most of which emerge at the early phase after initiation of the treatment and then diminish with time. At present, the molecular mechanism underlying IFNβ-related adverse effects remains largely unknown. The aim of this study is to identify a comprehensive list of early IFNβ-responsive genes (IRGs) in peripheral blood mononuclear cells (PBMC) that may play a key role in induction of adverse effects.MethodsTotal RNA of PBMC exposed to 50 ng/ml recombinant human IFNβ for 3 to 24 hours in vitro was processed for cDNA microarray analysis, followed by quantitative real-time RT-PCR analysis.ResultsAmong 1,258 genes on the array, IFNβ elevated the expression of 107 and 87 genes, while it reduced the expression of 22 and 23 genes at 3 and 24 hours, respectively. Upregulated IRGs were categorized into conventional IFN-response markers, components of IFN-signaling pathways, chemokines, cytokines, growth factors, and their receptors, regulators of apoptosis, DNA damage, and cell cycle, heat shock proteins, and costimulatory and adhesion molecules. IFNβ markedly upregulated CXCR3 ligand chemokines (SCYB11, SCYB10 and SCYB9) chiefly active on effector T helper type 1 (Th1) T cells, and CCR2 ligand chemokines (SCYA8 and SCYA2) effective on monocytes, whereas it downregulated CXCR2 ligand chemokines (SCYB2, SCYB1 and IL8) primarily active on neutrophils.ConclusionIFNβ immediately induces a burst of gene expression of proinflammatory chemokines in vitro that have potential relevance to IFNβ-related early adverse effects in MS patients in vivo.

Effect of Leptin on the Expression of Chemokine Genes in THP-1 Cells

Background: Leptin is a 16-KDa non-glycosylated peptide hormone synthesized almost exclusively by adipocytes. The well-known function of leptin is regulation of food intake and energy expenditure. Leptin also plays a regulatory role in immune and inflammatory process including cytokine production. The purpose of this study was to investigate the effect of leptin on the expression of several chemokine genes(RANTES, IL-8, MCP-1, IP-10, Mig, MIP-, MIP-, and GRO-) in THP-1 cells. Materials and Methods: Total RNA of THP-1 cells were prepared by Trizol method, and then stimulated with the leptin(250 ng/) or LPS(100 ng/). We examined the expression patterns of various chemokine mRNAs in THP-1 cell lines by RT-PCR and Northern blot. Results: Leptin did not induce the expression of chemokine mRNAs in THP-1 cells. The expression patterns of RANTES, IL-8, MCP-1, IP-10, and Mig mRNAs in THP-1 cells stimulated with leptin and LPS simultaneously was almost same to the patterns of LPS alone-induced chemokine mRNAs. RANTES mRNA expression was independent on the concentrations of leptin. Although leptin did not have strong effect on the expression of RANTES, IL-8, MCP-1, IP-10, Mig, MIP-, MIP-, and GRO- mRNAs in THP-1 cells, leptin could induce the expression of long isoform of leptin receptor(OB-RL) mRNA, and its expression was elevated in simultaneous stimulation of leptin and LPS. Conclusion: These data suggest that leptin is able to induce OB-RL in THP-1 cells, however, leptin has little effect on the expression of pro-inflammatory chemokine genes.