IL-4 mRNA Expression Research Articles

Huangbai Liniment Ameliorates Skin Inflammation in Atopic Dermatitis

Atopic dermatitis (AD), also known as atopic eczema, is one of the most common skin diseases and is characterized by allergic skin inflammation, redness, and itchiness and is associated with a hyperactivated type 2 immune response. The leading causes of AD include an imbalance in the immune system, genetic predisposition, or environmental factors, making the development of effective pharmacotherapies complex. Steroids are widely used to treat AD; however, they provide limited efficacy in the long term and can lead to adverse effects. Thus, novel treatments that offer durable efficacy and fewer side effects are urgently needed. Here, we investigated the therapeutic potential of Huangbai Liniment (HB), a traditional Chinese medicine, using an experimental AD mouse model, following our clinical observations of AD patients. In both AD patient and the mouse disease model, HB significantly improved the disease condition. Specifically, patients who received HB treatment on local skin lesions (3–4 times/day) showed improved resolution of inflammation. Using the 1-Chloro-2,4-dinitrobenzene (DNCB)-induced AD model in BALB/c mice, we observed that HB profoundly alleviated severe skin inflammation and relieved the itching. The dermatopathological results showed markedly reversed skin inflammation with decreased epidermal thickness and overall cellularity. Correspondingly, HB treatment largely decreased the mRNA expression of proinflammatory cytokines, including IL-1β, TNF-α, IL-17, IL-4, and IL-13, associated with declined gene expression of IL-33, ST2, and GATA3, which are connected to the type 2 immune response. In addition, HB restored immune tolerance by promoting regulatory T (TREG) cells and inhibiting the generation of TH1, TH2, and TH17 cells in vitro and in the DNCB-induced AD mouse model. For the first time, we demonstrate that HB markedly mitigates skin inflammation in AD patients and the DNCB-induced AD mouse model by reinvigorating the T cell immune balance, shedding light on the future development and application of novel HB-based therapeutics for AD.

​Selecting the Best Combination of Adjuvant and the Delivery Vehicle for Optimizing the Th1 and Th2 Biased Responses of NDV Live Vaccine in Indigenous Aseel Chicken

Background: This study aimed at the evaluation of the efficacy of TLR ligands (viz. Poly I:C, Lipopolysaccharide and CpG:ODN) as vaccine adjuvants in combination with a suitable carrier (oil-in-water emulsion, normal saline solution, or alum) by studying the expression profile of cytokines belonging to Th1 and Th2 biased immune systems. Methods: Whole blood was collected from 108 birds that were divided into 9 different experimental groups: 3 TLR ligands each with 3 delivery vehicles. Th1 (IFN-gamma) and Th2 (IL4) biased cytokines were detected by qPCR followed by ELISA. Both the results showed the highest level of IL4 (Th1 biased) with LPS and the highest level of IFNg (Th1 biased) with CpG-ODN. Uneven TLR expression was detected from day 3 onwards till day 21 but ligands and delivery vehicles administered along with NDV vaccine did not show significant influence on the immune response. Result: It is evident that the TLR-3, 4 and 9 expressions increase following exposure to the respective ligands. Hence, TLR ligands can be used as reliable adjuvants in NDV vaccine. The expression of the Th2 biased cytokine IL4 mRNA and Th1 biased cytokine Interferon-gamma was found to be the highest on day 3 in the LPS+NSS group and on day 21 in CpG-ODN+Alum group, respectively. The findings of the real-time PCR were supported to some by the ELISA.

Airway Exposure to Polyethyleneimine Nanoparticles Induces Type 2 Immunity by a Mechanism Involving Oxidative Stress and ATP Release

Polyethyleneimine (PEI) induced immune responses were investigated in human bronchial epithelial (hBE) cells and mice. PEI rapidly induced ATP release from hBE cells and pretreatment with glutathione (GSH) blocked the response. PEI activated two conductive pathways, VDAC-1 and pannexin 1, which completely accounted for ATP efflux across the plasma membrane. Moreover, PEI increased intracellular Ca2+ concentration ([Ca2+]i), which was reduced by the pannexin 1 inhibitor, 10Panx (50 μM), the VDAC-1 inhibitor, DIDS (100 μM), and was nearly abolished by pretreatment with GSH (5 mM). The increase in [Ca2+]i involved Ca2+ uptake through two pathways, one blocked by oxidized ATP (oATP, 300 μM) and another that was blocked by the TRPV-1 antagonist A784168 (100 nM). PEI stimulation also increased IL-33 mRNA expression and protein secretion. In vivo experiments showed that acute (4.5 h) PEI exposure stimulated secretion of Th2 cytokines (IL-5 and IL-13) into bronchoalveolar lavage (BAL) fluid. Conjugation of PEI with ovalbumin also induced eosinophil recruitment and secretion of IL-5 and IL-13 into BAL fluid, which was inhibited in IL-33 receptor (ST2) deficient mice. In conclusion, PEI-induced oxidative stress stimulated type 2 immune responses by activating ATP-dependent Ca2+ uptake leading to IL-33 secretion, similar to allergens derived from Alternaria.

The IL-33-ILC2 pathway protects from amebic colitis

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2–/– mice but not RAG2−/−γc−/− mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2−/− mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2−/−γc−/− mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

Marked up-regulation of anti inflammatory cytokine gene expression in the peripheral blood mononuclear cells of postpartum cows with endometritis

This study was designed to determine the mRNA expression profile of inflammatory and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMC) of endometritic and non-endometritic cows to find out a possible marker for diagnosis of endometritis. Cows (21) including 12 endometritic, and 9 normal (non-endometritic) that did not develop uterine diseases postpartum (pp) were selected. Expression profile (mRNA) of cytokines, viz. IL-1β, TNFα, IL-8 and IL-4 in endometritic cows relative to non-endometritic was studied in PBMC isolated from the blood of the cows collected within 4 to 5 weeks pp. Fold difference (n-fold) in cytokine gene expression in the PBMC of endometritic cows relative to normal was calculated using relative quantification method (2-ΔΔCt). The relative fold change in gene expression for IL-1 was significantly lower in endometritic animals (0.31) than in cows that did not develop endometritis. Significant up-regulation was noted for the proinflammatory cytokine TNFα (9.53 fold and anti-inflammatory cytokine IL-4 (90.09) in cows suffering from endometritis relative to nonendometritic cows. Up-regulation of chemokine IL-8 gene (6.25 fold; was also observed in endometritic compared to non-endometritic cows. Highest and significant up-regulation of mRNA expression for IL-4 followed by TNFα in PBMC of endometritic cows recorded in the study may indicate the development of endometritis in postpartum cows.

Perivascular clusters of Th2 cells and M2 macrophages in allergic contact dermatitis to methylchloroisothiazolinone and methylisothiazolinone.

Methylisothiazolinone (MI) and Methylchloroisothiazolinone (MCI) are among the most common skin sensitizers, yet the immunological events that occur during MCI/MI allergic contact dermatitis (ACD) are still poorly understood. To analyse dendrocytes, macrophage subtypes and T cells in skin during the elicitation phase of MCI/MI ACD. Thirteen patients with positive patch test reactions to MCI/MI (ACD group) and 11 individuals with negative patch test results were selected. Skin biopsies were only performed at 48hours of patch testing. Immunohistochemistry was conducted to assess T cells, dendrocytes (Factor XIIIa), M1 (p-Stat1, CD68) and M2 (c-Maf, CD163) macrophages. Transcriptional analyses were performed for cytokines and related factors, and further compared to atopic dermatitis samples (n=4). Immunofluorescence assays addressed T cells location, along with IL-4 or IL-13, within the skin. MCI/MI elicited dermal dendrocytes and macrophages, pronouncedly the M2subtype. T cells, majorly CD4+ T cells, accumulated in the perivascular areas. Similarly, abundant IL-4 protein was detected in these areas. There was an upregulation of IL-4 and IL-13mRNA expression, a mild increase in IFNG mRNA levels and a down-regulation of RORC in the ACD group. Immunofluorescence revealed dermal clusters of T cells co-localized with IL-4. M2macrophages and Th2 cells participate in the immunopathogenesis of MCI/MI ACD. Dermal dendrocytes and M2macrophages may assist the formation of CD4+ T cells perivascular clusters. These findings render a mechanistic insight into the MCI/MI reaction. Further analysis at different timepoints of patch testing is required to fully comprehend this ACD kinetics.

Deficient inflammasome activation permits an exaggerated asthma phenotype in rhinovirus C-infected immature mice.

Compared to other RV species, RV-C has been associated with more severe respiratory illness and is more likely to occur in children with a history of asthma or who develop asthma. We therefore inoculated six-day-old mice with sham, RV-A1B, or RV-C15. Inflammasome priming and activation were assessed, and selected mice treated with recombinant IL-1β. Compared to RV-A1B infection, RV-C15 infection induced an exaggerated asthma phenotype, with increased mRNA expression of Il5, Il13, Il25, Il33, Muc5ac, Muc5b and Clca1, increased lung lineage-negative CD25+CD127+ST2+ ILC2s, increased mucous metaplasia, and increased airway responsiveness. Lung vRNA, induction of pro-inflammatory type 1 cytokines, and inflammasome priming (pro-IL-1β and NLRP3) were not different between the two viruses. However, inflammasome activation (mature IL-1β and caspase-1 p12) was reduced in RV-C15-infected mice compared to RV-A1B-infected mice. A similar deficiency was found in cultured macrophages. Finally, IL-1β treatment decreased RV-C-induced type 2 cytokine and mucus-related gene expression, ILC2s, mucous metaplasia and airway responsiveness, but not lung vRNA level. We conclude that RV-C induces an enhanced asthma phenotype in immature mice. Compared to RV-A, RV-C-induced macrophage inflammasome activation and IL-1β are deficient, permitting exaggerated type 2 inflammation and mucous metaplasia.

Effector memory CD4+T cells in mesenteric lymph nodes mediate bone loss in food-allergic enteropathy model mice, creating IL-4 dominance.

Intestinal inflammation can be accompanied by osteoporosis, but their relationship, mediated by immune responses, remains unclear. Here, we investigated a non-IgE-mediated food-allergic enteropathy model of ovalbumin (OVA) 23-3 mice expressing OVA-specific T-cell-receptor transgenes. Mesenteric lymph nodes (MLNs) and their pathogenic CD4+T cells were important to enteropathy occurrence and exacerbation when the mice were fed an egg-white (EW) diet. EW-fed OVA23-3 mice also developed bone loss and increased CD44hiCD62LloCD4+T cells in the MLNs and bone marrow (BM); these changes were attenuated by MLN, but not spleen, resection. We fed an EW diet to F1 cross offspring from OVA23-3 mice and a mouse line expressing the photoconvertible protein KikGR to track MLN CD4+T cells. Photoconverted MLN CD44hiCD62LloCD4+T cells migrated predominantly to the BM; pit formation assay proved their ability to promote bone damage via osteoclasts. Significantly greater expression of IL-4 mRNA in MLN CD44hiCD62LloCD4+T cells and bone was observed in EW-fed OVA23-3 mice. Anti-IL-4 monoclonal antibody injection canceled bone loss in the primary inflammation phase in EW-fed mice, but less so in the chronic phase. This novel report shows the specific inflammatory relationship, via Th2-dominant-OVA-specific T cells and IL-4 production, between MLNs and bone, a distant organ, in food-allergic enteropathy.

MiR-155 regulates Th9 differentiation in children with methicillin-resistant Staphylococcus aureus pneumonia by targeting SIRT1

Th9 is a subset of CD4+ T cells that mainly secrete IL-9. Th9/IL-9 participates in immune response during Staphylococcus aureus and methicillin-resistant Staphylococcus aureus pneumonia (MRSA) infection. Here, we collected bronchoalveolar lavage fluid (BALF) from 30 children with MRSA pneumonia (MRSA group) and 10 children with bronchial foreign bodies (Control group). RT-PCR, ELISA and flow cytometry were used to detect the expression of miR-155 and IL-9 in BALF and the number of Th9 cells. CD4+ T cells isolated from BALF of MRSA and Control group were transfected with miR-155 mimic or inhibitor, and then induced Th9 cell differentiation. The results showed that the expression of miR-155 and IL-9 were significantly increased in BALF and Th9 cell of MRSA group, as well as the number of Th9 cells. miR-155 mimic upregulated IL-9 mRNA expression, IL-9 secretion and increased number of Th9 cells. On the contrary, miR-155 inhibitor inhibited IL-9 mRNA expression, IL-9 secretion and decreased number of Th9 cells. The dual luciferase assays demonstrated miR-155 can target binding to SIRT1 3′UTR. Moreover, overexpression of SIRT1 could reverse the effect of miR-155 mimic on IL-9 expression level, Th9 cell number and transcription factors PU.1 and IRF4 expression. In conclusion, miR-155 regulates Th9 differentiation in children with MRSA by targeting SIRT1.

Airway Epithelial Cells Drive Airway Smooth Muscle Cell Phenotype Switching to the Proliferative and Pro-inflammatory Phenotype.

The increased mass of airway smooth muscle (ASM) in the airways of asthmatic patients may contribute to the pathology of this disease by increasing the capacity for airway narrowing. Evidence for the airway epithelium as a participant in ASM remodeling is accruing. To investigate mechanisms by which airway epithelial cells induce ASM cell (ASMC) proliferation, we have employed a co-culture model to explore markers of ASMC proliferative phenotype. Co-culture with epithelial cells led to incorporation of bromodeoxyuridine into ASMCs, indicating augmented proliferation and an associated increase in mRNA of the pro-proliferative co-transcription factor Elk1. Although the mitogen heparin-binding epidermal growth factor (HB-EGF) was augmented in the co-culture supernatant, the ASMC epidermal growth factor receptor (EGFR), an effector of HB-EGF induced proliferation, did not mediate epithelial-induced proliferation. The co-culture increased the expression of ASMC mRNA for the pro-inflammatory cytokines IL-6 and IL-8 as well as the pro-proliferative microRNA miR-210. The transcriptional repressor Max-binding protein (Mnt), a putative target of miR-210, was transcriptionally repressed in co-cultured ASMCs. Together, these data indicate that the airway epithelium-induced proliferative phenotype of ASMCs is not driven by EGFR signaling, but rather may be dependent on miR210 targeting of tumor suppressor Mnt.

Group 2 innate lymphoid cells promote TNBC lung metastasis via the IL-13-MDSC axis in a murine tumor model

Group 2 innate lymphoid cells (ILC2s) are reportedly associated with the progression of many tumors. However, the role of ILC2s in triple‐negative breast cancer (TNBC) lung metastasis remains unclear. In this study, we found that ILC2s may be a key element in the process of TNBC lung metastasis since the adoptive transfer of pulmonary ILC2s increased the numbers of metastatic lung nodules and reduced the survival of tumor-bearing mice. ILC2-promoted 4 T1 lung metastasis appears to be related to ILC2-derived IL-13. An expansion of IL-13-producing ILC2s and an elevated expression of IL-13 mRNA in pulmonary ILC2s were determined in tumor-bearing mice, in parallel with an increase in the levels of local IL-13 by ILC2 transfer. The neutralization of IL-13 reduced the increased pulmonary metastatic nodules and improved the decreased survival rate caused by ILC2-adoptive transfer. Interestingly, adoptive transfer of ILC2s elevated IL-13Ra1 expression in myeloid-derived suppressor cells (MDSCs). Treatment of ILC2-transferred tumor-bearing mice with anti-IL-13 antibodies significantly diminished the number of pulmonary MDSCs and inhibited MDSC activation. Moreover, when pulmonary MDSCs were cocultured with ILC2s in the presence of an anti-IL-13 mAb, the number and activation of MDSCs were reduced. Depletion of MDSCs may promote the proliferation of CD4+ T cells and CD8+ T cells, but reduce the expansion of regulatory T cells (Tregs) in the lungs of ILC2-transferred tumor-bearing mice. Our results suggest that pulmonary ILC2s may promote TNBC lung metastasis via the ILC2-derived IL-13-activated MDSC pathway.

Systemic inflammatory markers in neuropathic pain, nerve injury, and recovery.

The role that inflammation plays in human nerve injury and neuropathic pain is incompletely understood. Previous studies highlight the role of inflammation in the generation and maintenance of neuropathic pain, but the emerging evidence from the preclinical literature for its role in the resolution of neuropathic pain remains to be explored in humans. Here, we use carpal tunnel syndrome (CTS) as a human model system of nerve injury and neuropathic pain to determine changes in serum cytokine protein levels and gene expression levels before (active stage of disease) and after carpal tunnel decompression surgery (recovery). Fifty-five CTS patients were studied and 21 healthy age and gender matched participants served as controls. In the active stage of the disease (CTS before surgery vs healthy controls), PTGES2 mRNA was decreased in patients (adjusted p=0.013), while TGF-β and CCL5 protein levels were increased (adjusted p=0.016 and p=0.047 respectively). In the resolution phase (CTS before surgery vs after surgery), IL-9 mRNA was increased after surgery (adjusted p=0.014) and expression of IL-6 mRNA and IL-4 protein levels were increased before surgery (adjusted p=0.034 and p=0.002 respectively). IL-9 mRNA expression negatively correlated with several (neuropathic) pain scores. In contrast, protein levels of IL-4 positively correlated with pain scores. In conclusion, we demonstrate specific dysregulation of systemic cytokine expression both in the active and resolution phases of nerve injury and neuropathic pain. IL-9 represents an interesting candidate associated with resolution of nerve injury and neuropathic pain.

RUNX1 gene expression changes in the placentas of women smokers.

The placenta can be affected by environmental factors, such as exposure to cigarette smoke. This exposure in the fetal context is considered a risk factor for the development of short-term postnatal diseases, such as asthma. Asthma is an inflammatory disease characterized by predominant acquisition of CD4 T lymphocytes (TLs) of the Th2 type. Transcription factors such as GATA binding protein 3 (GATA3) and STAT6 actively participate in the differentiation of virgin TLs towards the Th2 profile, while transcription factors such as STAT1, T-Box transcription factor 21 (T-BET), RUNX1 and RUNX3 participate in their differentiation towards the Th1 profile. The objective of the current study was to evaluate the impact of exposure to cigarette smoke on the gene expression of STAT1, T-BET, GATA3, IL-4, RUNX1 and RUNX3 during the gestation period, and to determine whether the expression levels of these genes are associated with changes in global methylation. STAT1, GATA3, RUNX1 and RUNX3 protein and mRNA expression levels in the placental tissue of women smokers and non-smoking women were determined via immunohistochemistry and quantitative PCR (qPCR) respectively. Additionally, T-BET and IL-4 mRNA expression levels were determined by qPCR. On the other hand, global methylation was determined via ELISA. In the present study, significant increases were observed in RUNX1 transcription factor expression in placentas from women smokers when compared with placentas of non-smoking women. Similarly, significant increases in the expression of GATA3, IL-4 and RUNX3 mRNA were observed. The changes in gene expression were not associated with changes in the global methylation levels. Finally, a higher frequency of low-birth-weight infants were identified in cases of exposure to cigarette smoke during pregnancy when compared with infants not exposed to cigarette smoke during pregnancy. Thus, the data of the present study contributed to the understanding of the genetic and clinical impacts of exposure to cigarette smoke during pregnancy and its importance in maternal and fetal health.

Ameliorative effects of eosinophil deficiency on immune response, endoplasmic reticulum stress, apoptosis, and autophagy in fungus-induced allergic lung inflammation

BackgroundRespiratory fungal exposure is known to be associated with various allergic pulmonary disorders. Eosinophils have been implicated in tissue homeostasis of allergic inflammation as both destructive effector cells and immune regulators. What contributions eosinophils have in Aspergillus fumigatus (Af)-induced allergic lung inflammation is worthy of investigating.MethodsWe established the Af-exposed animal asthmatic model using eosinophil-deficient mice, ∆dblGATA1 mice. Airway inflammation was assessed by histopathological examination and total cell count of bronchoalveolar lavage fluid (BALF). The protein level in BALF and lung mRNA level of type 2 cytokines IL-4, IL-5, and IL-13 were detected by ELISA and qRT-PCR. We further studied the involvement of endoplasmic reticulum (ER) stress, apoptosis, and autophagy by western blots, qRT-PCR, immunofluorescence, TUNEL, or immunohistochemistry. RNA-Seq analysis was utilized to analyze the whole transcriptome of Af-exposed ∆dblGATA1 mice.ResultsHematoxylin and eosin (HE) staining and periodic acid–Schiff staining (PAS) showed that airway inflammation and mucus production were alleviated in Af-challenged ∆dblGATA1 mice compared with wild-type controls. The protein and mRNA expressions of IL-4, IL-5, and IL-13 were reduced in the BALF and lung tissues in Af-exposed ∆dblGATA1 mice. The results demonstrated that the significantly increased ER stress markers (GRP78 and CHOP) and apoptosis executioner caspase proteases (cleaved caspase-3 and cleaved caspase-7) in Af-exposed wild-type mice were all downregulated remarkably in the lungs of ∆dblGATA1 mice with Af challenge. In addition, the lung autophagy in Af-exposed ∆dblGATA1 mice was found elevated partially, manifesting as higher expression of LC3-II/LC3-I and beclin1, lower p62, and downregulated Akt/mTOR pathway compared with Af-exposed wild-type mice. Additionally, lung RNA-seq analysis of Af-exposed ∆dblGATA1 mice showed that biological processes about chemotaxis of lymphocytes, neutrophils, or eosinophils were enriched but without statistical significance.ConclusionsIn summary, eosinophils play an essential role in the pathogenesis of Af-exposed allergic lung inflammation, whose deficiency may have relation to the attenuation of type 2 immune response, alleviation of ER stress and apoptosis, and increase of autophagy. These findings suggest that anti-eosinophils therapy may provide a promising direction for fungal-induced allergic pulmonary diseases.

MO690HIGHER EXPRESSION OF TOLL LIKE RECEPTOR AND CELL ADHESION MOLECULES ARE ASSOCIATED WITH RECURRENT/RELAPSE PERITONITIS AND MORTALITY IN PATIENT ON CAPD

Abstract Background and Aims CAPD is an established modality of treatment for patients with end stage renal disease (ESRD). Peritonitis is a leading cause of technique failure and death in patients on CAPD. Studies on expressions of host genetic factors like TLRs, CAMs, Pro and Anti-inflammatory cytokines and their link to peritonitis and other co-morbidity and functional status are lacking throughout the world. Hence the present study was done to determine the expressions of TLR2 and TLR4, CAMs and inflammatory cytokines in patients on CAPD. Method A total of 110 ESRD patients categorized into 3 groups: 1- CAPD patients (n=45), group 2- CAPD with peritonitis patients (n=35), and group 3- CAPD patients with recurrent/relapsing peritonitis. (n=30) from department of Nephrology, Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGIMS), Raebareli Road, Lucknow were included in the study. Blood sample was collected for analysis of cytokines and Cell adhesion molecules level and RNA extraction. Dialysate (only from patients undergoing PD with or without peritonitis)- whole drained bag; 50-100 ml for microscopy and culture for bacterial and fungal pathogens using automated BACTEC culture system in patients with peritonitis and supernatant was collected for cytokine ELISA. Statistical analysis: One-way ANOVA (Two tailed) and non-parametric student t-test used for statistical analysis of differences between the group. Results mRNA expression of TLR 2 and TLR 4 expression was higher in peritonitis and recurrent/relapsing peritonitis group as compared to CAPD patient without peritonitis group. mRNA expression of TNF-alpha, IL-1B and IFN-gamma was higher in peritonitis group of patients compared to all other group of patients. mRNA expression of IL-10 and IL-4 was also highest in Peritonitis group. Level of IL-1β and TNF-alpha was observed to be elevated in peritonitis and recurrent/relapse peritonitis patients compared to CAPD in sera and dialysate. IL-12 level in Sera was also elevated in peritonitis patients but its level in dialysate was highest in CAPD without peritonitis as compared to peritonitis and recurrent/relapse. IL-10 and IL-4 was observed to be elevated in peritonitis group followed by CAPD with recurrent/relapse peritonitis in sera whereas in dialysate level of IL-4 was higher in both peritonitis group and Recurrent groups of patients but IL-10 level was highest in peritonitis group but less in recurrent/relapse. mRNA Expression levels of adhesion molecule ICAM-1 and LFA-1 were observed to be higher in Peritonitis and Recurrent groups as compared to the CAPD without peritonitis; whereas, in sera the level of ICAM-1 was higher in peritonitis patients. However, in dialysate its level was observed to be almost similar. Level of LFA-1 was observed to be higher in peritonitis group in dialysate but in sera its level was higher in Recurrent group with peritonitis. Conclusion It is likely that CAMs are also involved in the pathogenesis of CAPD peritonitis, its morbidity and mortality. TLRs activation by bacterial molecules leads to the induction of chemokines and cytokines through the activation of NF-ķB pathway and may be responsible for atherosclerosis, morbidity, and mortality in ESRD patients. Elevated level of ICAM-1, IL-1beta, TNF-a and IFN-G may be responsible for chronic inflammation in PD patients.

P051 Macrophages as a source of Notch Ligands in Crohn’s disease: implications in fibrosis

Abstract Background Fibrosis constitute the main complications associated to Crohn’s disease (CD). Notch signalling has been implicated in lung, kidney, liver and cardiac fibrosis. Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their microenvironment. The aim of the present study is to analyze the role of Notch ligands derived from macrophages in the complications of CD. Methods We have analyzed: the mRNA expression of cytokines and Notch ligands in CD patients with fistulizing and stenting pattern, the mRNA and protein expression of macrophage markers and Notch ligands in macrophages treated with the main cytokines present in CD patients (IFNγ-, IL10-, IL4, TNFα-U937 treated cells), the mRNA expression of fibrosis markers of DLL4-HT29 treated cells. Results are expressed as fold induction (mean±SEM, n≥4). Statistical analysis was performed with one-way ANOVA followed by Newman-Keuls test. Correlations were analyzed with the Spearman coefficient. Results The mRNA expression of IFNγ, TNFα, IL4, IL6 and IL10 was significantly higher in intestinal samples from B2- and B3-CD patients (11.4±1.6, 6.9±1.3, 16.4±3.9, 4.1±0.4, and 5.0±1.0 respectively for B2, and 14.2±1.8, 9.6±2.6, 21.5±4.1, 6.3±1.0 and 9.2±1.7 respectively for B3) than in controls (1.0±0.09, 1.1±0.2, 1.2±0.1, 1.1±0.1 and 1.0±0.1 respectively). The mRNA expression of DLL3 and DLL4 was significantly higher in intestinal samples from B2 (6.1±1.3 and 9.3±2.4, respectively) than in B3 CD patients (4.4±1.0 and 1.8±0.7, respectively) and in controls (0.9±0.2 and 0.7±1.2 respectively). IFNγ-U937 treated cells increased significantly the mRNA expression of DLL3 and DLL4 (2,9±0,6 N=5 N=4 and 3,7±1,1 N=4, respectively) respect vehicle; IL1β increased significantly the expression of DLL4 (1,8±0,01 N=4) and TNFα increased significantly the expression of DLL3 (7,1±2,2 N=4), respect vehicle. DLL4-HT29 treated cells increased significantly fibrosis markers (VIMENTIN (4,6±0,6 N=6), α-SMA (20,9±8,2 N=6), SNAIL1 (1,8±0,4 N=6), SNAIL2 (8,3±1,3 N=6), ZEB1 (8,2±4,3 N=6) and ZEB2 (4,3±0,2 N=6), respect vehicle. Conclusion Macrophages may act as a source of Notch ligands who could act as fibrosis mediators in CD patients with a stenting (B2) behavior. The microenvironment rich in IFNγ could activate the fibrosis process in epithelial cells by favoring the expression of DLL4 in macrophages.

Comparison of Cytokines in Skin Biopsies and Tape Strips from Adults with Atopic Dermatitis

Skin biomarkers for disease severity and treatment response in atopic dermatitis (AD) are needed. Biopsies cause scarring and tape stripping represents an alternative minimally invasive method for stratum corneum sampling. In this study, we examined the gene expression of cytokines in skin biopsies and cytokines in stratum corneum tape strips collected from adults with AD. We collected punch biopsies and tape strips from healthy controls (n = 6) and subjects with AD (n = 12) at baseline and after 2 weeks of topical treatment with mometasone furoate 0.1% cream. We found that IFN-γ, IL-13, and IL-10 mRNA (biopsies) and IL-1β protein expression levels (tape strips) were significantly increased in lesional AD skin compared to healthy control skin. Treatment with topical corticosteroid led to a significant decrease in mRNA levels for IL-13 and IL-4R but no significant differences in cytokine protein levels measured in tape strips. Finally, we found no significant correlations between cytokine levels in tape strips and mRNA levels in skin biopsies.

Th17/Treg cell imbalance plays an important role in respiratory syncytial virus infection compromising asthma tolerance in mice

Mucosal tolerance is induced early in life and is an important mechanism of protection from diseases, such as asthma. Respiratory syncytial virus (RSV) is a main cause of bronchiolitis and pneumonia in infants. Clinical studies have found that there is a strong association between RSV infection in infancy and later development of asthma, but the underlying mechanisms are unclear. A mouse model of immune tolerance induced by oral feeding of ovalbumin(OVA) was successfully established in our previous studies. We found that RSV infection could break the oral immune tolerance state.RSV infection increased the mRNA expression of IL-17A and IL-17A/Foxp3(the transcription factor forkhead box P3) in OT mice, but the mRNA expression of IL-4 and other T helper(Th)2 cytokines did not change significantly. As detected by flow cytometry analysis, RSV infection elevated Th17 cell levels and correspondingly decreased Regulatory T(Treg) cell levels in the hilar lymph nodes (HLNs) and mesenteric lymph nodes (MLNs), but there were no significant differences in the spleen or peripheral blood.We hypothesized that an imbalance in Th cells played an important role in RSV infection compromising asthma tolerance.RSV infection disrupted asthma tolerance by increasing the Th17/Treg ratio rather than the Th1/Th2 ratio'.Therefore, altering the Th17/Treg ratio has been identified as a potential therapeutic target in asthma caused by RSV or another virus.

CDX2 regulates interleukin-33 gene expression in intestinal epithelial cells (LS174T).

Dysregulation of interleukin‐33 (IL‐33) has been implicated in the pathogenesis of several autoimmune and inflammatory diseases, but few studies have examined transcriptional regulation of the IL33 gene. In the intestines, gene regulation is controlled by a transcription factor network of which the intestinal‐specific transcription factor CDX2 is a key component. In this study, we investigated whether CDX2 regulates IL33 mRNA expression. We examined IL33 mRNA expression in primary colonic epithelial cells from healthy humans and epithelial cell lines, revealing high expression levels in primary colonic and LS174T cells. Combining genomics data (ChIP‐seq, RNA‐seq) and IL33 promoter analyses in LS174T cells revealed intronic enhancer activity in the IL33 gene that is dependent on CDX2 expression. Western blotting and qRT‐PCR confirmed that IL33 expression is upregulated in a CDX2 concentration‐dependent manner, thereby providing the first evidence that CDX2 regulates the expression of IL33.

The role of sphingosine 1‐phosphate type 2 receptor on IL‐4/IL‐13‐stimulated STAT6 phosphorylation in phorbol12‐myristate13‐acetate‐treated THP‐1 cells

Background/Purpose We previously observed reduced activation of STAT6 in broncho-alveolar lavage fluids (BALF) cells from bleomycin-administered sphingosine 1-phosphate receptor type 2 (S1P2) knockout mice compared to BALF cells from bleomycin-administered wild-type mice (PLoS One, 2018). In this study, we examined the role of S1P2 on STAT6 activation using THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) as a model for macrophages (MΦs). Methods THP-1 cells were cultured in RPMI-1640 containing 10% fetal bovine serum. THP-1 cells were treated with PMA to differentiate into adherent MΦs. S1P2knockout THP-1 cells were established using CRISPR-Cas9 gene editing system. Quantification of mRNA expression was performed by quantitative RT-PCR using specific primers. Jak/STAT6 activation was performed by Western blot analysis using antibodies against tyrosine-phosphorylated Jak1, Jak2 and STAT6. Results Quantitative PCR analysis showed that THP-1 cells expresses S1P2. We examined the responses of PMA-treated THP-1 MΦs to stimulation with IL-4/IL-13 in the presence of S1P. IL-4/IL-13 stimulation induced a strong increase in STAT6 phosphorylation in PMA-treated THP-1 MΦs. The major signaling pathway of S1P2 is a Rho–Rho kinase pathway. Pretreatment of PMA-treated THP-1 MΦs with the S1P2 inhibitor JTE-013 and the Rho kinase inhibitor Y27632 inhibited IL-4/IL-13–induced increase in STAT6 phosphorylation. PMA-treated S1P2knockout THP-1 MΦs also showed a lower extent of STAT6 phosphorylation in response to IL-4/IL-13 stimulation than PMA-treated wild-type THP-1 MΦs. mRNA expression of IL-4/IL-13 receptors was not different between wild-type and S1P2knockout THP-1 MΦs. No difference in mRNA and protein expression of STAT6 was observed between wild-type and S1P2knockout THP-1 MΦs. The phosphorylation of Jak1 and Jak2 in S1P2knockout THP-1 MΦs was diminished compared with wild-type THP-1 MΦs. Finally, S1P2 knockout in THP-1 MΦs attenuated IL-4/IL-13-induced increase in the mRNA expression of the M2 marker arginase 1. Conclusion These results suggested that S1P2/Rho kinase pathway is necessary for full activation of STAT6 by IL-4 or IL-13 in MΦs and could be a drug target for Th2-associated diseases.