Chemokine Gene Expression Research Articles

Therapeutic treatment of a novel selective JAK3/JAK1/TBK1 inhibitor, CS12192, in rat and mouse models of rheumatoid arthritis

Rheumatoid arthritis (RA) is a representative autoimmune disease characterized by chronic inflammation and joint destruction. Although biological inhibitors such as TNF-α and IL-6 antibodies have achieved success in clinical therapy, small molecule inhibitors against the Janus kinases (JAKs) involved in the signaling pathways of various cytokine receptors have gained more attraction as safe and efficacious options. In this study, we identified CS12192 as a novel selective JAK3/JAK1/TBK1 inhibitor and investigated its pharmacological effects on the experimental arthritis models in rat and mouse. We found that CS12192 showed a more selective inhibitory activity on JAK3, and to a less extent on JAK1 and TBK1, that were verified by decreased activation of p-STATs and p-IRF3 as well as down-regulation of IFN gene expression in the cultured cells with relevant stimuli. Furthermore, oral treatment with CS12192 dose-dependently ameliorated the disease severity, hind paw swelling, body weight loss, and bone destruction in rat models of adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA). In a mouse CIA model, CS12192 also attenuated the disease severity, which was correlated with the suppressed CD4+ T cell activation and Th17 function, as well as the reduced cytokine levels in sera and pro-inflammatory cytokine and chemokine gene expression in joint tissue. Corroboratively, RANKL-induced osteoclast formation was inhibited by CS12192. Thus, these results suggest that CS12192 as a novel selective JAK inhibitor has therapeutic potential for the treatment of RA and may provide a new strategy for the control of autoimmune diseases.

Impact of chemokine C\u2013C ligand 27, foreskin anatomy and sexually transmitted infections on HIV-1 target cell availability in adolescent South African males

We compared outer and inner foreskin tissue from adolescent males undergoing medical male circumcision to better understand signals that increase HIV target cell availability in the foreskin. We measured chemokine gene expression and the impact of sexually transmitted infections (STIs) on the density and location of T and Langerhans cells. Chemokine C–C ligand 27 (CCL27) was expressed 6.94-fold higher in the inner foreskin when compared with the outer foreskin. We show that the density of CD4+CCR5+ cells/mm2 was higher in the epithelium of the inner foreskin, regardless of STI status, in parallel with higher CCL27 gene expression. In the presence of STIs, there were higher numbers of CD4+CCR5+ cells/mm2 cells in the sub-stratum of the outer and inner foreskin with concurrently higher number of CD207+ Langerhans cells (LC) in both tissues, with the latter cells being closer to the keratin surface of the outer FS in the presence of an STI. When we tested the ability of exogenous CCL27 to induce T-cell migration in foreskin tissue, CD4 + T cells were able to relocate to the inner foreskin epithelium in response. We provide novel insight into the impact CCL27 and STIs on immune and HIV-1 target cell changes in the foreskin.

P3113Functional deficiency in activating fcgamma receptor protects against aortic abdominal aneurysm in mice

Abstract Background and aim Abdominal aortic aneurysm (AAA) is a degenerative disorder characterized by a localized and permanent dilation of the aorta. Pathological features of AAA include proteolysis, vascular smooth muscle cells (VSMC) apoptosis, oxidative stress and inflammation. Previous studies have demonstrated the role of innate and adaptive immunity in the initiation and progression of AAA. However, the specific mechanisms of humoral, antibody-mediated, immune response elicited by self and non-self-antigens are not completely known. IgG Fc receptors (FcgammaR) play an important role in the initiation and regulation of many immunological and inflammatory processes, thus providing a link between humoral and cellular immune responses. In this work, we focus on IgG immune response against antigens exposed in the damaged vessel, analysing the specific role played by FcgammaR in the pathogenesis of AAA. Methods and results AAA was induced by aortic elastase perfusion in wild-type (WT) C57BL/6 mice (n=24 males, 12 weeks old). Compared with healthy aortas, AAA tissue showed IgG and IgM deposition and increased expression levels of activating (IA, IIIA and IVA) and inhibitory (IIB) FcgammaR (5-, 3-, 10, and 2-fold increases; p<0.005) at day 14 post-elastase perfusion. To explore the functional contribution of activating FcgammaR to AAA formation, parallel experiments were performed in mice deficient in gamma-chain (gamma-KO), the common signalling subunit of FcgammaRI, III and IV. Compared with WT mice, gamma-KO mice (n=21) exhibited lower IgG and IgM deposits (21±4% and 28±12% vs WT, respectively; p<0.02) and decreased AAA lesions with reduced aortic expansion (% vs WT: aortic wall thickness, 67±3%; aortic diameter, 55±3%; p<0.0001). AAA lesions from gamma-KO mice showed less disruption of elastin layers (Verhoeff-van Gieson staining) and reduced loss of medial VSMC (α-actin immunofluorescence). Inflammatory markers such as leukocyte content (CD68+ macrophages, Ly6G+ neutrophils, CD45R+ B cells, and CD3+ T lymphocytes) and the gene expression of chemokines (MCP-1, RANTES), adhesion molecules (ICAM-1), cytokines (TNFα, IFNg, IL-10) and metalloproteinases (MMP9) were all significantly lower in AAA lesions from gamma-KO mice than those from WT mice. In vitro, cross-linking of FcgammaR with fibrinogen-containing immune complexes triggers the gene expression of proinflammatory cytokines and chemokines in both VSMC and bone marrow-derived macrophages. Conclusion Activating FcgammaR participate in AAA formation by promoting inflammatory cell recruitment, and cytokine and matrix-degrading protease expression. Therefore, modulation of FcgammaR-dependent responses could be a promising therapeutic option for the treatment of AAA. Acknowledgement/Funding MINECO/FEDER (SAF2015-63696-R), ISCIII (FIS/FEDER PI17/01495), Spanish Society of Arteriosclerosis, La Caixa (HR17-00247), Conchita Rabago Foundation

1908P - Tobacco smoking is associated with the immune suppressive microenvironment in head and neck squamous cell carcinoma (HNSCC)

BackgroundHead and neck squamous cell carcinoma (HNSCC) patients with a significant tobacco smoking history have a poor prognosis compared to never smokers. We hypothesize the poor prognosis in tobacco smokers with HNSCC is, at least in part, due to ongoing suppression of immune response and that understanding of deregulated signals in each step may provide insights to improve clinical outcomes of HNSCC patients. MethodsWe characterized the tumor immune microenvironment (TIM) of HNSCC in a retrospective cohort of 177 current, former, and never smokers. Tumors were subjected to analysis of CD3, CD8, FOXP3, PD-1, PD-L1, and pan-cytokeratin by multiplex immunofluorescence, whole exome sequencing, and RNA sequencing. The immune markers were measured in the tumor core (TC), tumor margin (TM), and stroma (S). ResultsOur data indicate that current smokers have significantly lower numbers of CD8+ cytotoxic T-cells and PD-L1+ cells compared to never- and former-smokers. While tumor mutation burden and mutant-allele tumor heterogeneity score do not associate with smoking status, gene-set enrichment analyses reveal significant suppression of IFN-α and IFN-γ response pathways in current smokers. Gene expression of canonical IFN-response chemokines of the CXCL9, CXCL10, CXCL11/CXCR3 axis are lower in current smokers than in former smokers suggesting decreased immune cell migration to the tumor site. ConclusionsThese results indicate that active tobacco use in HNSCC has an immunosuppressive effect through inhibition of tumor infiltration of cytotoxic T-cells likely as a result of suppression of IFN response pathways. Our study highlights the importance of understanding the interaction between smoking and TIM in light of emerging immune modulators for cancer management and the importance of smoking cessation during treatment with immunotherapy. Legal entity responsible for the studyThe authors. FundingThe James and Esther King Biomedical Research Grant. DisclosureC.H. Chung: Honoraria (self): bristol myers squibb; Honoraria (self): astrazeneca; Honoraria (self): Lilly; Honoraria (self): CUE Biopharma. J. Conejo-Garcia: Honoraria (self): Compass Therapeutics; Honoraria (self): Anixa Biosciences. R. Slebos: Spouse / Financial dependant: Bristol Myers Squibb; Spouse / Financial dependant: AstraZeneca ; Spouse / Financial dependant: CUE Biopharma; Spouse / Financial dependant: Lilly Oncology. All other authors have declared no conflicts of interest.

A Comprehensive Profile of Chemokine Gene Expression in the Tissues of the Female Reproductive Tract in Mice

ABSTRACTHomeostatic leukocyte trafficking into and within the female reproductive tract (FRT) contributes to fertility and reproductive health. It is unclear how this process is regulated in the anatomically distinct reproductive tissues, or whether the genes involved are affected by cyclical changes in reproductive hormones. In tissues such as skin and intestine, mouse studies have defined evolutionarily conserved molecular mechanisms for tissue-specific homing, interstitial positioning, and leukocyte egress. Chemokine family members are invariably involved, with the chemokine expression profile of a tissue regulating leukocyte content. Reproductive tissues (ovary, vagina, cervix, uterine horn) of 8 week old virgin female C57BL/6 mice (n = 20) were collected, and expression of mRNA for leukocyte markers and chemokines conducted by qPCR. Lymphocytic and myeloid cell populations within the uterus, cervix, bone marrow and PALN from virgin C57BL/6 mice were determined by flow cytometric analysis. Variation in leukocyte content between reproductive tissues is evident, with the uterus and cervix containing complex mixtures of lymphocytes and myeloid cells. Twenty-six chemokine genes are expressed in the FRT, many by several component tissues, some preferentially by one. Most striking are Xcl1 and Ccl28, which are restricted to the uterus. Ccl20 and genes encoding CXCR2 ligands are primarily transcribed in cervix and vagina. Ovary shows the lowest expression of most chemokine genes, with the notable exception of Ccl21 and Ccl27. We also identify eight chemokines in the vagina whose expression fluctuates substantially across the oestrous cycle. These data reveal complex chemokine networks within the FRT, and provide a framework for future studies of homeostatic leukocyte trafficking into and within these tissues.Abbreviations: BM: bone marrow; DC: dendritic cell; DN: double negative; FRT: female reproductive tract; FSC: forward scatter; NK: natural killer; PALN: para-aortic lymph node; SSC: side scatter; Tregs: regulatory T cells

Production of Membrane Vesicles by Enterococcus faecium Cultured With or Without Subinhibitory Concentrations of Antibiotics and Their Pathological Effects on Epithelial Cells.

Enterococcus faecium is a clinically important pathogen associated with opportunistic infection and multi-drug resistance. E. faecium has been shown to produce membrane vesicles (MVs), but MV production by E. faecium under antibiotic stress conditions and the pathogenic traits thereof have yet to be determined. This study investigated the production of MVs in E. faecium ATCC 700221 cultured with sub-minimum inhibitory concentrations (MICs) of vancomycin or linezolid and determined their pathologic effects on colon epithelial Caco-2 cells. E. faecium ATCC 700221 cultured with 1/2 MIC of vancomycin or linezolid produced 3.0 and 1.5 times more MV proteins than bacteria cultured without antibiotics, respectively. Totals of 438, 461, and 513 proteins were identified in MVs from E. faecium cultured in brain heart infusion broth (MVs/BHI), BHI broth with 1/2 MIC of vancomycin (MVs/VAN), or BHI broth with 1/2 MIC of linezolid (MVs/LIN), respectively. Intact MVs/BHI induced cytotoxicity and the expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells in a dose-dependent manner, but proteinase K-treated MVs significantly suppressed these pro-inflammatory responses. MVs/LIN were more cytotoxic toward Caco-2 cells than MVs/BHI and MVs/VAN, whereas MVs/VAN stimulated more pro-inflammatory cytokine gene expression in Caco-2 cells than MVs/BHI and MVs/LIN. Overall results indicated that antibiotics modulate the biogenesis and proteomes of MVs in E. faecium at subinhibitory concentrations. MVs produced by E. faecium cultured under antibiotic stress conditions induce strong host cell responses that may contribute to the pathogenesis E. faecium.

Abstract 496: Inhibition of the Rna Binding Protein Hur Protects Against Cardiac Ischemia/reperfusion Injury by Reducing Inflammatory Gene Expression

Despite medical advances, cardiac ischemia/reperfusion (I/R) injury remains a leading cause of morbidity and a huge economic burden in the United States. RNA binding proteins are becoming recognized as potential mediators of cardiac physiology and pathology, but the role of HuR, an RNA binding protein highly expressed in myocytes, in acute cardiac I/R injury is unknown. HuR has been shown in other tissues to be a critical post-transcriptional mediator of pro-inflammatory chemokine and cytokine gene expression, and we have shown HuR to be activated (nuclear-to-cytoplasmic translocation) in cardiomyocytes 2 hours post-ischemia/reperfusion injury. To address the functional role of HuR in I/R, cardiomyocyte-specific HuR deletion mice (iCM-HuR -/- ) were subjected to 30 minutes of LAD (left anterior descending) coronary artery ligation followed by 24 hours of reperfusion. In parallel, a separate group of wild-type mice were subjected to 30 minutes ischemia with a pharmacological inhibitor of HuR given just prior to reperfusion. Analysis of infarct size showed a smaller infarct with both HuR genetic deletion (~10% decrease in infarct size compared to control, N=3, P<0.05) and pharmacological inhibition (~11% decrease in infarct size compared to vehicle, N=4, P=0.069). Similarly, HuR inhibition significantly reduced cell death, caspase-3 activity, and inflammatory gene expression in an in vitro model of simulated I/R using neonatal rat ventricular myocytes. HuR inhibition results in a significant blunting of IL-6 and TNF-alpha gene expression two hours post-reperfusion in vivo , suggesting that HuR activity is necessary for the early induction of inflammatory gene expression networks. In addition, it appears that HuR inhibition attenuates macrophage infiltration following ischemia/reperfusion injury. In conclusion, our results suggest that inhibition of HuR is protective against cardiac I/R injury through a reduction in inflammatory gene expression.

Responses of human mast cells and epithelial cells following exposure to influenza A virus

As a part of innate immune defense, the role of mast cells during viral replication has been incompletely understood. In this study, we characterized and compared the responses of the human mast cell line, LAD2, and human lung epithelial cell line, Calu-3, against three influenza A virus strains; A/PR/8/34 (H1N1), A/WS/33 (H1N1) and A/HK/8/68 (H3N2). We found that there were strain-dependent mast cell responses, and different profiles of cytokine, chemokine and antiviral gene expression between the two cell types. All three strains did not induce histamine or β-hexosaminidase release in LAD2. A/HK/8/68 induced release of prostaglandin D2 in LAD2, whereas A/PR/8/34 and A/WS/33 did not. We found that, among those examined, only CCL4 (by A/PR/8/34) was statistically significantly released from LAD2 cells. Furthermore, there was increased mRNA expression of viral recognition receptors (RIG-I and MDA5) and antiviral protein, viperin, but levels and kinetics of the expression were different among the cell types, as well as by the strains examined. Our findings highlight the variability in innate response to different strains of influenza A virus in two human cell types, indicating that further investigation is needed to understand better the role of mast cells and epithelial cells in innate immunity against influenza A viruses.

Sonic Hedgehog signaling limits atopic dermatitis via Gli2-driven immune regulation.

Hedgehog (Hh) proteins regulate development and tissue homeostasis, but their role in atopic dermatitis (AD) remains unknown. We found that on induction of mouse AD, Sonic Hedgehog (Shh) expression in skin and Hh pathway action in skin T cells were increased. Shh signaling reduced AD pathology and the levels of Shh expression determined disease severity. Hh-mediated transcription in skin T cells in AD-induced mice increased Treg populations and their suppressive function through increased active transforming growth factor–β (TGF-β) in Treg signaling to skin T effector populations to reduce disease progression and pathology. RNA sequencing of skin CD4+ T cells from AD-induced mice demonstrated that Hh signaling increased expression of immunoregulatory genes and reduced expression of inflammatory and chemokine genes. Addition of recombinant Shh to cultures of naive human CD4+ T cells in iTreg culture conditions increased FOXP3 expression. Our findings establish an important role for Shh upregulation in preventing AD, by increased Gli-driven, Treg cell–mediated immune suppression, paving the way for a potential new therapeutic strategy.

SUN-303 DIETARY MANIPULATION OF THE GUT MICROBIOTA REDUCES DIABETIC KIDNEY INJURY IN MICE

Diabetic nephropathy (DN) is the leading cause of chronic kidney disease (CKD) globally. High dietary fibre intake has been associated with decreased inflammation and mortality in CKD. Short-chain fatty acids (SCFA) derived from gut microbial fermentation of dietary fibre are thought to regulate gut and systemic inflammation. Here we evaluated the effect of dietary modification, or supplementation with the three main SCFAs (acetate, propionate, butyrate) on the development of DN in a mouse model of streptozotocin (STZ) induced diabetes. Diabetes was induced using a low dose STZ protocol in C57BL/6 mice. Three weeks after STZ injection, diabetic mice were switched to a zero-fibre (ZF) or high-fibre (HF) diet, whilst control diabetic mice remained on the normal diet. For mechanistic studies, two-weeks after STZ, groups of diabetic mice received daily intra-peritoneal injections of either sodium acetate (SA), propionate (SP), butyrate (SB) 200mg/kg or saline for 2 weeks, then switched to oral SCFAs (SA 150mM, SP 100mM, SB 50mM, or pH and sodium-matched water) in their drinking water for the duration of the study. Blood, urine and kidneys were collected after 12 weeks. All STZ-treated mice developed diabetes within two weeks. Diet altered the gut microbiome in diabetic mice, with significant preservation of diversity evident in HF compared to ZF mice. As compared to ZF and normal diets, HF fed mice had higher faecal SCFA levels (>2-fold, p<0.05) and serum acetate concentrations (>3-fold, p<0.001). HF fed mice were protected from DN, with a reduction in albuminuria of 50% (p<0.01), reduced kidney to body weight ratio (1.33 vs 2.4, p<0.0001), glomerular hypertrophy (22±17.5 vs 35.5±15.3 (x104 μm3), p<0.0001), glomerular cellularity (24.9±1.7 vs 36.6±1.7, p<0.0001), interstitial fibrosis (p<0.0001) and podocyte injury (p<0.0001) compared to diabetic controls on normal or ZF diets. Treatment of diabetic mice with SA, SP or SB achieved similar degrees of protection to the HF-diet, with less glomerular hypertrophy (p<0.01), podocyte injury (p<0.05), interstitial fibrosis (p<0.001) and interstitial macrophage accumulation (p<0.01), as compared to diabetic controls. Albuminuria was also reduced by over 50% (p<0.05). mRNA expression of cytokines (IL6, TGF-β), chemokine (CCL2) and pro-fibrotic (fibronectin) genes in the kidney were attenuated in SA treated diabetic mice versus controls (p<0.05). Treatment with dietary fibre supplementation provides protection against development of DN through modulation of the gut microbiota with increased gastrointestinal release and systemic distribution of SCFAs. SCFA treatment was similarly protective, supporting SCFAs as a key mediator of the beneficial effects of the HF diet. Manipulation of the gastrointestinal microbiota through diet may be a novel strategy in prevention and management of diabetic nephropathy.

Abstract 520: Alteration of chemokine gene expression in PC-3 cells treated with NSAIDs

Abstract Background: The role of chronic inflammation for developing prostate cancer and its progression to metastasis is supported by increasing evidence. Among the pro-inflammatory regulators that seem to play a critical function in prostate tumorigenesis, the most implicated have been the members of the CXC chemokines and their receptors, which have been deemed as significant players in several of the critical steps in tumorigenesis and/or metastasis. CXC chemokines have been classified into two subclasses, ELR+ and ELR−. Most of the ELR+ CXC chemokines are angiogenic, while most of the ELR- are anti-angiogenic. Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with reduced risk of several cancers, including prostate cancer (PCa). The protective effect of NSAIDs has been attributed to inhibition of cancer metastasis. The aim of this work was to determine the expression profiles of the CXC chemokines in the metastatic prostatic cancer cell line PC-3 compared to the non-tumorigenic PWR-1E cell line and to evaluate the effects of NSAIDs in the expression patterns of these molecular components. Methods: Differences in gene expression were determined between highly metastatic PC-3 cells and the non-tumorigenic PWR-1E by qRT-PCR and ELISA for a panel of 13 genes encoding CXC chemokines: ELR+ (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8) and ELR- (CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, and CXCL14). Changes in the expression of these genes in response to treatment with NSAIDs (ibuprofen and nimesulide) were also determined. Results: Highly metastatic PC-3 cells showed high expression levels of six ELR+ CXC chemokines: CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8; there was no detectable expression for CXCL7. In contrast, expression of ELR- CXC chemokines (CXCL9, CXCL10, CXCL11, and CXCL14) was not detected in either PC-3 or PWR-1E cells. The expression levels of CXCL4 and CXCL12 were comparable in the two cell lines. Treatment of PC-3 cells with ibuprofen and nimesulide affected the expression pattern of the genes evaluated in these cells. Ibuprofen treatment caused decreased expression of ELR+ CXCL6 in PC-3 cells; this effect was not seen with nimesulide treatment. Ibuprofen and Nimesulide treatments caused an increased expression of ELR+ CXCL5 and CXCL8 chemokines. The expression levels of the remaining CXC chemokines: CXCL1, CXCL2, CXCL3, CXCL4, and CXCL12 were not affected by treatment with the NSAIDs assayed. Conclusions: Highly metastatic PC-3 cells had increased expression levels of ELR+ CXC chemokines compared to non-tumorigenic PWR-1E cells. NSAIDs (Nimesulide and ibuprofen) caused differential changes in the pattern of CXC chemokine expression of PC-3 cells. Pharmacological effects of NSAIDs in prostate cancer warrants further investigation at the clinical settings. Additional studies are required to determine the clinical usefulness of NSAIDs in prostate cancer. Citation Format: Niradiz Reyes, Juan Rebollo, Raj Tiwari, Jan Geliebter. Alteration of chemokine gene expression in PC-3 cells treated with NSAIDs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 520.

Abstract 5314: Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer

Abstract Background: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Of the patients diagnosed with HNSCC, previous findings show that SMAD4 loss is more common in recurrent HNSCC patients compared with newly diagnosed cases and is strongly associated with poor patient survival and metastases. SMAD4 is a tumor suppressor gene and is a central mediator of the transforming growth factor β (TGF-β) signaling pathway and acts as a co-SMAD which binds receptor-regulated SMADs such as SMAD2 and 3. SMAD2/3/4 complex is important for regulating downstream gene transcription that is involved in tumor invasion and metastasis. Purpose: Since the TGF-β pathway is highly dysregulated in HNSCC, we investigate in this study how SMAD4 downregulation alters TGF-β pathway signaling and the tumor microenvironment. Results: Using isogenic HNSCC cell lines, we visualized using immunofluorescence staining that levels of activated phospho-SMAD2 in the nucleus are correlated with expression of SMAD4. Genome-wide screening using Gene Set Enrichment Analysis (GSEA) further revealed changes in gene expression during SMAD4 knockdown in HNSCC cells. From GSEA results, the 20 most significantly altered Gene Ontology (GO) pathways were determined. It was shown that majority of these GO pathways are associated with regulating neutrophil migration and chemotaxis with an upregulation of CXC chemokine gene expression, specifically with CXCL1. In contrast, anti-tumor chemokines, such as CXCL9 and CXCL11, were downregulated during loss of SMAD4 expression. Using an orthotopic mouse model we show that in vivo, decreased expression of SMAD4 promotes tumor take and growth. Conclusion: Collectively, these results suggest that HNSCC tumors with SMAD4 loss may have poor prognosis attributed to immunosuppressive functions of chemokine expression that acts on tumor-associated neutrophils. Citation Format: Jude Masannat, Xuefeng Wang, Biwei Cao, FeiFei Song, Ritu Chaudhary, Janis de la Iglasia, Ciaara Gorgoglione, Robbert Slebos, Christine Chung. Smad4 loss upregulates chemokine expression and promotes tumor inflammation and growth in head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5314.

Korean red ginseng water extract alleviates atopic dermatitis-like inflammatory responses by negative regulation of mitogen-activated protein kinase signaling pathway in vivo

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Korean red ginseng is a Korean traditional medicine. In this study, we estimated the effects of Korean red ginseng water extract (RGE) in the 1-chloro-2,4-dinitrobenzene (DNCB)-induced BALB/c mouse model which develops AD-like lesions. After RGE administration (100, 200, and 400 mg/kg) to DNCB-induced mice there were improvements in the dermatitis score and skin pH, a decrease in trans-epidermal water loss, and improved skin hydration. RGE also significantly inhibited eosinophil infiltration, increased filaggrin protein levels, and decreased serum IgE levels, epidermal thickness, mast cell infiltration, and ceramidase release. Compared with that in DNCB-induced mice, RGE effectively decreased the mRNA expression levels of interleukin-6 (IL-6), thymic stromal lymphopoietin (TSLP), and tumor necrosis factor-α (TNF-α), as well as the protein level of thymus and activation-regulated chemokine (TARC). These inhibitory RGE effects are mediated by inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Furthermore, we confirmed that RGE suppresses interferon-γ (IFN-γ) and TNF-α-induced expression of macrophage-derived chemokine (MDC) and TARC genes in human keratinocyte (HaCaT) cells. Taken together, these results demonstrate that RGE may exert anti-atopic related to responses by suppression the expression of inflammatory mediators, cytokines, and chemokines via downregulation of MAPK signaling pathways, suggesting that RGE may be an effective therapeutic approach for prevention of AD-like disease.

Effects of dietary exposure to chlorpyrifos on immune cell populations and inflammatory responses in mice with dextran sulfate sodium-induced colitis

This study investigated the effects of chlorpyrifos (CPF) on immune-cell populations and intestinal inflammation using a mouse model of inflammatory bowel disease induced by dextran sulfate sodium (DSS). C57BL/6 mice were randomly assigned to five groups with one normal control (NC) and four DSS-treated groups. Mice in the NC group were given distilled water, whereas the DSS-treated groups received distilled water containing 3% DSS for 6 days to induce colitis. The NC and disease control (DC) groups were fed a control semipurified diet, while the remaining groups were exposed to CPF in the AIN-93 diet at doses of 1, 2.5, or 5 mg/kg/day throughout the study. Results showed that dietary exposure to CPF in colitic mice significantly increased circulating classical monocytes and upregulated gene expressions of chemokines in the colon compared to the NC group. Meanwhile, CPF exposure groups had lower plasma cholinesterase activities and higher percentages of circulating neutrophils than those of the DC group. A shorten length, tissue edema, and lipid peroxidation of the colon were also observed in all CPF-exposed mice. These findings suggest that dietary exposure to CPF affected immune-cell populations and inflammatory responses, which led to more severe tissue injury in mice with DSS-induced colitis.

Single-cell transcriptomes of murine bone marrow stromal cells reveal niche-associated heterogeneity.

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non‐overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long‐term maintenance of immune cells.

Different epithelial cell response to membrane vesicles produced by Listeria monocytogenes cultured with or without salt stress

We have previously shown that Listeria monocytogenes, a causative agent of listeriosis, can produce membrane vesicles (MVs) during in vitro culture. The aim of this study was to investigate the ability of MVs from L. monocytogenes cultured with or without salt stress to induce cytotoxicity and pro-inflammatory responses in colon epithelial Caco-2 cells. MVs were purified from wild-type L. monocytogenes 10403S strain and an isogenic ΔsigB mutant strain. MVs from both wild-type and ΔsigB mutant strains increased viability of Caco-2 cells regardless of salt stress. Both MVs from wild-type and ΔsigB mutant strains stimulated expression of pro-inflammatory cytokine and chemokine genes in Caco-2 cells. Expression levels of pro-inflammatory cytokine genes in cells treated with MVs from bacteria cultured without salt stress were significantly higher than those in cells treated with MVs from bacteria cultured with salt stress. However, expression levels of chemokine genes in cells treated with MVs from bacteria cultured with salt stress were significantly higher than those in cells treated with MVs from bacteria cultured without salt stress. In addition, expression levels of interleukin (IL)-1β and IL-8 genes were partially inhibited by either lysozyme-treated MVs or ethylenediaminetetraacetic acid-treated MVs compared to those after treatment with intact MVs. Our results suggest that salt stress can affect the production of L. monocytogenes MVs, thus causing different pro-inflammatory responses in host cells.

Comprehensive molecular profiling of metastatic melanoma to predict response to monotherapy and combination immunotherapy.

9511 Background: Several factors have been proposed as biomarkers for response to PD1 therapy, including tumor mutational burden (TMB), immune gene expression, PD-L1 expression and TILs, while few specific mechanisms of resistance have been identified. The relative importance of these factors or detailed examination of biomarkers of response to combination immunotherapy have yet to be explored. Methods: Cutaneous metastatic melanoma (MM) patients (pts) treated with anti-PD-1 (PD1) +/- anti-CTLA-4 (CTLA4) were selected. Pre-treatment tumors underwent whole genome sequencing (WGS), RNA sequencing (RNAseq) and immunohistochemistry (IHC; TILs and PD-L1). Results: Tumors from 77 pts treated with PD1 (n = 53) or PD1+CTLA4 (n = 24) underwent WGS. Higher TMB (p = 0.0001), lower structural variant (SV) burden (p = 0.001) and higher neoantigen load (p = 0.001) were associated with response. There was no difference in the expression of specific genes reported to confer resistance (JAK1/2, PTEN or BAF/PBAF complex members) or response (SERPINB3/4, ARID) in responders vs non-responders. RNAseq was performed on 53/77 samples; IFN and TCR signalling pathways were enriched in responders. Cytolytic activity (CYT, p = 0.002), T cell proportion estimated by CIBERSORT (p = 0.002) and confirmed by IHC (p = 0.033), and PD-L1 expression (IHC, p = 0.026) were also higher in responders. Multivariate analysis including DNA (TMB, SV count), RNA (six gene IFN expression signature - IFNG.6; effector T cell gene expression signature; chemokine gene expression; CYT), IHC (PD-L1, TILs) and clinical factors (sex, age, RECIST Sum of diameters, LDH) identified TMB and IFNG.6 as independent predictors of response (AUC = 0.83). 15 outliers with discordant molecular features and clinical outcomes had varying profiles, including 5 non-responders with high TMB, but low IFNG.6 expression, suggestive of a failure of immune activation. Conclusions: Comprehensive clinical and genomic analysis demonstrated that TMB and IFNG expression independently predict response, suggesting defects in both immune recognition or activation in non-responders.

Nonsteroidal Anti-inflammatory Drugs Modulate Gene Expression of Inflammatory Mediators in Oral Squamous Cell Carcinoma.

Most patients with head and neck cancer receive nonsteroidal anti-inflammatory drugs concomitant with oncogenic treatment in order to control cardiovascular diseases and chronic inflammatory processes. Inflammation is closely related to neoplastic development and the release of inflammatory cytokines and chemokines represents a crucial event in this relationship. The aim of the present study was to evaluate the effect of acetylsalicylic acid (ASA) and celecoxib treatment in the gene expression pattern of cytokines and chemokines in squamous cell carcinoma (OSCC) cell lines. Cells were treated with plasmatic concentrations of ASA and celecoxib and were submitted to cell viability assay and immunoenzymatic assay to investigate interleukin 6 (IL6) production. Treated cells were collected and a gene expression array was performed using the reverse transcriptase-quantitative polymerase chain reaction. Both treatments provoked a discrete inhibitory effect on cell viability and modulated IL6 production. The mRNA expression of several cytokines, chemokines, chemokine receptors, and other chemotaxis-related genes were modulated after treatment with ASA and celecoxib. Plasmatic doses of ASA and celecoxib altered the expression of IL6 and the gene expression of chemokines (ligands and receptors) and cytokines in a dose- and time-dependent manner.

Putative Periodontal Pathogens, Filifactor Alocis and Peptoanaerobacter Stomatis, Induce Differential Cytokine and Chemokine Production by Human Neutrophils.

Periodontitis is a highly prevalent infectious disease that affects ~ 50% of the adults in the USA alone. Two Gram-positive anaerobic oral bacteria, Filifactor alocis and Peptoanaerobacter stomatis, have emerged as important periodontal pathogens. Neutrophils are a major component of the innate host response in the gingival tissue, and the contribution of neutrophil-derived cytokines and chemokines plays a central role in disease progression. The pattern of cytokines and chemokines released by human neutrophils upon stimulation with newly appreciated periodontal bacteria compared to the keystone oral pathogen Porphyromonas gingivalis was investigated. Our results showed that both F. alocis and P. stomatis triggered TLR2/6 activation. F. alocis induced significant changes in gene expression of cytokines and chemokines in human neutrophils compared to unstimulated cells. However, except for IL-1ra, neutrophils released lower levels of cytokines and chemokines in response to F. alocis compared to P. stomatis. Furthermore, bacteria-free conditioned supernatant collected from neutrophils challenged with P. stomatis, but not from P. gingivalis or F. alocis, was chemotactic towards both neutrophils and monocytes. Elucidating stimuli-specific modulation of human neutrophil effector functions in the context of dysbiotic microbial community constituents provides valuable information for understanding the pathogenesis of periodontal diseases.

Roles of Toll-like Receptors in Regulating Fibroblastic Reticular Cells Pathobiology during Sepsis

Abstract Fibroblastic reticular cells (FRCs), a subpopulation of stromal cells in lymphoid organs and fat-associated lymphoid clusters (FALCs) in peritoneal fat, play immune-regulatory roles in the host defense. However, the mechanisms underlying the regulation of FRC pathobiology, especially in response to Toll-like receptor (TLR) signaling is unclear. Therefore, we aimed to understand the roles of TLR signaling in FRC pathobiology during sepsis. FALC cells were isolated from mesenteric adipose tissue in wild-type mice before and after cecal ligation and puncture (CLP). The transcriptomic profile of FALC cells were assessed using single cell RNA sequencing. Notably, FRCs were a major population of FALC cells (50 +/− 10.2 % in control, 30 +/− 8.6% in CLP). One population of FRCs were identified in control mice, and two populations of FRCs were identified in CLP mice. According to gene set enrichment analysis, FRCs after CLP are enriched in gene expression associated with inflammatory response and chemotaxis. Interestingly, activation of TLR4 (LPS) and TLR3 (Poly I:C) signaling in mesenteric adipose tissue derived FRCs significantly increased the expression of chemokines known to attract neutrophils (Cxcl2 and Cxcl5), monocytes (Cxcl3), and lymphocytes (Cxcl13 and Ccl20), while activation of TLR9 (ODN1518) significantly decreased these chemokine gene expressions. Importantly, we found similar regulatory effects of TLR3, 4, and 9 on chemokine production in human adipose tissue-derived FRCs. Together, our results indicate that TLRs plays critical roles in regulating chemokine production by FRCs. These data form a knowledge basis upon which to design new therapeutic strategies of FRC-based treatments for sepsis.