Cytokine mRNA Profiles Research Articles

Peripheral Blood Mononuclear Cell Cytokine mRNA Profiles in Acute Respiratory Infection Patients.

Respiratory infections, collectively, are one of the World's most common and serious illness groups. As recent observations have shown, the most severe courses of acute respiratory infection, often leading to death, are due to uncontrolled cytokine production (hypercytokinemia). The study involved 364 patients with respiratory illness being treated in clinics in St. Petersburg (Russia) in 2018-2019 and 30 healthy controls. Cytokine analysis was carried out in the acute phase of illness (2-3 days from onset of initial symptoms) and in the stage of recovery (days 9-10). The research presented is devoted to the assessment of mRNA expression of specific cytokines (interleukin [IL]-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, tumor necrosis factor-α [TNF-α], and interferon-λ) and MxA in whole blood leukocytes, by means of real-time polymerase chain reaction. In 70% of patients, bacterial or viral pathogens were identified, with influenza viral infections (types A and B) prevailing. Significant increases in the expression of IL-18, TNF, and IL-10 were observed, relative to controls, only with influenza viral infections. We have shown a difference in IL-6 mRNA expression in patients with bacterial or viral pathogens. No statistically significant difference was found in white blood cells IL-4 expression levels between patients and healthy controls. Investigation of the nuances of systemic cytokine production, in response to specific viral and bacterial pathogens, makes it possible to assess the risks of developing hypercytokinemia during respiratory infection with agents circulating in the human population and to predict the pathogenicity and virulence of circulating threats.

Enterococcus faecalis shifts macrophage polarization toward M1-like phenotype with an altered cytokine profile

ABSTRACT Background: The macrophage is an innate immune defense cell involved in pathogen recognition and clearance. Aim: In view of the diversity of the macrophage phenotype and function, the present study investigated how Enterococcus faecalis infection affects the differentiation, phenotype and cytokine profile of macrophages. Methods: Murine bone marrow-derived stem cells were co-cultured with E. faecalis before and after differentiation. Macrophage M0 polarization towards M1 or M2 was initiated at day 6 by addition of LPS and INF-γ, or IL-4 and IL-13, respectively. Results: E. faecalis did not inhibit macrophage differentiation and were identified within macrophages. Viability of the macrophages infected with E. faecalis prior to differentiation was enhanced, evidenced by apoptosis inhibition, as was expression of CD38 and IRF5 proteins, indicators of M1-like polarization. These M1-like macrophages expressed an aberrant cytokine mRNA profile, with reduction in inflammatory cytokines IL-1β and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-β1 were detected, compared with the control groups. This atypical M1-like phenotype was retained even upon stimulation with growth factors that normally trigger their development into M2 macrophages. Conclusions: These findings suggested that E. faecalis infection of bone marrow-derived stem cells during differentiation into macrophages induces an atypical M1-like phenotype associated with intracellular bacterial survival.

Pirfenidone Alleviates Features of Well-Established Chronic Pancreatitis in Mouse Models

Abstract Introduction/Objective Chronic pancreatitis (CP) is a fibro-inflammatory disease of pancreas with no targeted therapy and is considered irreversible. Antifibrotic agent pirfenidone is FDA approved for idiopathic pulmonary fibrosis. However, exact molecular mechanism of its action is not clear. The aim of this study was to evaluate pirfenidone as a therapeutic agent for CP. Methods Caerulein-CP was induced in C57BL/6 mice by caerulein injections (50ug/kgx7, i.p., hourly x twice weekly x10 weeks). At 11 weeks, animals were randomized and assigned to either saline or pirfenidone group (400 mg/kg/d by oral gavage for 5 weeks). Mice were euthanized at 17 weeks. L-arginine induced CP was induced by i.p. injections of L-arginine (4.5g/kg x2 hourly, once a week x 4) and treatment was started after 5 weeks of start. Mice were sacrificed at early time-points after starting treatment. Single-cell suspension of pancreata were used for flow- cytometry. Pancreatic atrophy, histology, fibrosis and cytokine mRNA profile were evaluated. In vitro studies were done on stellate cells. Results The treated caerulein-CP mice had improvement in pancreas/mouse weight ratio, (7.03±0.41 vs. 4.75±0.28; p<0.0001). Histology scores and fibrosis markers were reduced. Pancreatic atrophy and histology scores showed significant improvement by day 14 of treatment in L-arginine CP. Flow cytometry showed that by day 7 of treatment there was significant reduction in macrophage infiltration (1.09 ± 0.18 % vs 3.26 ± 0.4 %; p<0.001) and pro-fibrotic M2 macrophage markers [IL-4 (1.5 ± 0.1 % vs 2.8 ± 0.2%; p=0.007)], while M1 marker (MHC II) did not change. mRNA levels of pro-inflammatory and pro-fibrotic cytokines decreased, and of anti-inflammatory cytokines increased. In vitro study on stellate cells showed reduction in mRNA levels of pro-fibrotic and pro-inflammatory cytokines as well as fibrosis markers in treatment group. Conclusion Pirfenidone ameliorates well-established CP in mouse models by altering immune cells.

Enhanced local and systemic inflammatory cytokine mRNA expression in women with endometriosis evokes compensatory adaptive regulatory mRNA response that mediates immune suppression and impairs cytotoxicity.

Endometriosis is a disease characterized by ectopic implantation of endometrium and impaired immune responses. To explore its pathogenic mechanisms, we studied the local and systemic cytokine mRNA profiles and their role in the immunity of patients with endometriosis and healthy controls. mRNA for eleven cytokines defining cytotoxic Th1, humoral Th2, regulatory Tr1/Th3, and inflammatory cytokine profiles was characterized locally in endometriotic tissue and endometrium, and systemically in PBMCs from women with endometriosis and healthy controls, using real-time qRT-PCR. In addition, immunohistochemical stainings with monoclonal antibodies were performed looking for T regulatory cells in endometriotic lesions. We found a downregulation of mRNA for cytokines mediating cytotoxicity and antibody response and an upregulation of inflammatory and T-regulatory cytokines in the endometriotic tissues and endometrium from the patients with endometriosis, suggesting enhanced local inflammation and priming of an adaptive regulatory response. Consistent with those findings, there was an abundancy of T regulatory cells in the endometriotic lesions. The ectopic implantation seen in endometriosis could be possible as a consequence of increased inflammation and priming of adaptive T regulatory cells, resulting in impaired cytotoxicity and enhanced immune suppression.

Cytokine mRNA and protein expression by cell cultures of epithelial ovarian cancer-Methodological considerations on the choice of analytical method for cytokine analyses.

To get a comprehensive picture of cytokine expression in health and disease is difficult, cytokines are transiently and locally expressed, and protein analyses are burdened by biological modifications, technical issues, and sensitivity to handling of samples. Thus, alternative methods, based on molecular techniques for cytokine mRNA analyses, are often used. We compared cytokine mRNA and protein expression to evaluate whether cytokine mRNA profiles can be used instead of protein analyses. In kinetic experiments, cytokine mRNA and protein expression of IL-1β, IL-6, IL-8, TNF-α, and TNF-β/LTA were studied using real-time RT-qPCR and Luminex® microarrays in the ovarian cancer cell lines OVCAR-3, SKOV-3 and the T-cell line Jurkat, after activation of transcription by thermal stress. In addition, we analyzed IL-6 and IL-8 mRNA and protein in a small number of ovarian cancer patients. Ovarian cancer cells can express cytokines on both mRNA and protein level, with 1-4hours' time delay between the mRNA and protein peak and a negative Spearman correlation. The mRNA and protein expression in patient samples was poorly correlated, reflecting previous studies. Cytokine mRNA and protein expression levels show diverging results, depending on the material analyzed and the method used. Considering the high sensitivity and reproducibility of real-time RT-qPCR, we suggest that cytokine mRNA profiles could be used as a proxy for protein expression for some specific purposes, such as comparisons between different patient groups, and in defining mechanistic pathways involved in the pathogenesis of cancer and other pathological conditions.

Efficacy of fungal immunomodulatory protein to promote swine immune responses against porcine reproductive and respiratory syndrome virus infection

Fungal immunomodulatory protein (FIP) is one of the bioactive compounds of edible mushrooms, which has been shown to trigger type 1 T helper (Th1) pathway activation in research with mice. This study was designated to assess immunomodulatory effects of recombinant FIP-Flammulina velutipes (rFIP-fve) on swine and the protective efficacy against PRRSV infection. In the in vitro evaluations, rFIP-fve significantly triggered up-regulation of IL-2 and IFN-γ mRNA in porcine PBMCs and stimulated natural killer cytotoxicity. Porcine pulmonary alveolar macrophages (PAMs) treated with rFIP-fve showed prolonged life times, up-regulation of both MHC I and II molecules and enhanced abilities to present antigen. In the in vivo trial, two doses of 2 mg rFIP-fve significantly reduced drops in the CD4/CD8 ratio after PRRSV challenge, and the cytokine mRNA profile of PBMC revealed a tendency of IFN-γ up-regulation and a decrease in IL-10 in the rFIP-treated group. Moreover, administration of rFIP-fve also decreased the PRRSV viremia with 1 log10 in titer (p = 0.07) and alleviated the severity of clinical signs after PRRSV challenge. Conclusively, these results illustrate the in vitro and in vivo immunological changes of rFIP-fve administered to pigs and reveal its potential to be used as an immunomodulatory therapeutic against PRRSV infection.

Canine multicentric lymphoma exhibits systemic and intratumoral cytokine dysregulation

Non-Hodgkin Lymphoma (NHL) is among the most common neoplasias in dogs and humans. Owing to remarkable similarities with its human counterpart, the canine lymphoma (cNHL) model has been proposed as a powerful framework for rapid and clinically relevant translation of novel immunotherapies. However, the establishment of cNHL as a predictive preclinical model has been hampered by the limited characterization of the canine immune system. Cytokines are key players of the interaction between tumor and its microenvironment. In human NHL, multiple cytokines have been linked to the development of lymphoma and are relevant biomarkers for treatment response and prognosis. In contrast, few studies have investigated cytokines in cNHL. Within this context, this study aimed to investigate cytokine regulation in cNHL.A multicentric cNHL biobank was successfully constructed. Cytokine mRNA profiles in tumor tissue and circulating PBMC were analyzed by qRT-PCR and compared to a healthy control group. Specific primers were used to evaluate Th1, Th2 and Th17 responses. Systemic cytokine concentrations were measured using a commercial canine multiplex assay which included IL-2, IL6, IL-10 and TNF-α, and compared to a healthy control group. Our results demonstrated a dysregulation of cytokine mRNA expression, representative of the tumor microenvironment and systemic response in cNHL. Intratumoral cytokine response revealed a significant downregulation of humoral and Th1 responses. The systemic response demonstrated a distinct mRNA pattern, however immunosuppression also prevailed. Cytokine serum quantification showed a significant increase of IL-10 concentration in cNHL. Significant differences in hematological parameters were described and a correlation between IL-6 protein serum levels and neutrophil count was shown. Finally, data analysis demonstrated that baseline pretreatment IFN-γ tissue mRNA levels were correlated to survival outcome, predicting a favorable response to chemotherapy.Altogether, these results revealed that cNHL presents a local and systemic dysregulation in cytokine response. By confirming and extending previous research, our work contributed for the evaluation of potential cytokine candidates for diagnostic, prognostic purposes and therapeutic intervention, therefore adding value to comparative oncology.

CHRONIC EOSINOPHILIC PNEUMONIA IN AN AIR-BRUSH PAINTER WITH POOR RESPONSE TO SYSTEMIC STEROIDS

SESSION TITLE: Medical Student/Resident Occupational and Environmental Lung Diseases SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Air-brush paints contain low molecular weight chemicals that can cause occupational asthma, respiratory sensitization and hypersensitivity pneumonitis; however, its relationship to Chronic Eosinophilic Pneumonia (CEP) has never been reported. Hereby, we describe a case of CEP in a patient who was exposed to air-brush paints for a long time. CASE PRESENTATION: A 48-year-old male, who worked as an air-brush painter for 20 years, presented with exertional dyspnea and cough productive of clear sputum for 4 weeks. He also had pleuritic chest pain, low grade fever, chills, malaise, and 10 lbs. weight loss. He had 12 pack-year smoking history and quit 2 years ago. Chest exam revealed bilateral basilar coarse crackles. White Blood Cell (WBC) count showed mild leukocytosis with Eosinophils (E) 5%, absolute E count 500/μL, elevated CRP and negative Procalcitonin. Computed Tomography (CT) of the chest (Figure 1) showed bilateral lower lobes infiltrates and ground glass opacities. After 2 days, his WBC count jumped to 17,000 with E% 7.5 and absolute E count 1300/μL with worsening oxygen requirement. Bronchoscopy was not performed due to his high oxygen requirement. However, because of patient’s progressive symptoms and despite being at high risk, open lung biopsy was done for definitive diagnosis. Pathological examination (Figure 2) revealed CEP with organizing pneumonia pattern. He was then started on high-dose systemic steroids with gradual tapering but adequate clinical recovery was not achieved. His hospital course was also complicated by pneumothorax and trapped lung too, and he continued to have high oxygen requirement. A repeat CT of the chest (Figure 3) showed worsening of lung infiltrates and fibrosis. DISCUSSION: Our patient was exposed to air-brush paints for 20 years and presented with CEP; thus, we believe it is warranted to investigate the relationship between CEP and occupational exposures, such as air-brush painting. Generally, patients with CEP have excellent response to systemic steroids and avoidance of the hypothetical inciting factor; however, our patient had poor response to systemic steroids. We believe that his prolonged exposure to air-brush paints and the additional pathology of organizing pneumonia might have contributed to the unsatisfactory response to systemic steroids, prolonged hypoxia, and the overall worse prognosis. CONCLUSIONS: We are presenting a unique association between CEP and prolonged exposure to air-brush paints. In this case, the patient had poor response to systemic steroids too, which was likely due to prolonged occupational exposure in addition to the evolution of progressive fibrosis and organizing pneumonia. Reference #1: Plitnick LM, Loveless SE, Ladics GS, Holsapple MP, Smialowicz RJ, Woolhiser MR, et al. Cytokine mRNA profiles for isocyanates with known and unknown potential to induce respiratory sensitization. Toxicology 2005; 207: 487-99. Reference #2: Di Stefano F, Di Giampaolo L, Verna N, Di Gioacchino M. Occupational eosinophilic bronchitis in a foundry worker exposed to isocyanate and a baker exposed to flour. Thorax 2007; 62: 368-70. Reference #3: Carrington CB, Addington WW, Goff AM, Madoff IM, Marks A, Schwaber JR, et al. Chronic eosinophilic pneumonia. New Engl J Med 1969; 280: 787-98. DISCLOSURES: No relevant relationships by Ravindra Bharadwaj, source=Web Response No relevant relationships by Manish Patel, source=Web Response No relevant relationships by Nethuja salagundla, source=Admin input No relevant relationships by AHMED TAHA, source=Web Response No relevant relationships by Thien Vo, source=Web Response

Enhanced Th1 and inflammatory mRNA responses upregulate NK cell cytotoxicity and NKG2D ligand expression in human pre-eclamptic placenta and target it for NK cell attack.

Pre-eclampsia (PE), a severe human pregnancy disorder, is associated with exaggerated systemic inflammation, enhanced cytokine production, and increased shedding of microvesicles leading to endothelial dysfunction, coagulopathy, and extensive placenta destruction. The cause of PE is still unclear. Evidence suggests that its origin lies in the placenta and that the maternal immune system is involved. A shift in cytokine production in PE pregnancy promotes NK cell activation, suggested to be important in PE pathogenesis. In line with this suggestion, we studied NK cell cytotoxicity in peripheral blood of PE patients and controls and the mRNA expression of cytokines and of the NKG2D receptor and its ligands MICA/B and ULBP1-3 in PE- and normal placenta. The cytotoxic capacity of peripheral blood NK cells was analyzed using K562 target cells. The cytokine mRNA profiles and the mRNA expression of the NKG2D receptor and its ligands MICA/B and ULBP 1-3 in PE placenta were assessed and compared to those in normal placenta using real-time quantitative RT-PCR. The cytotoxicity of peripheral blood NK cells was upregulated in PE cases. Further, we found an enhanced inflammatory cytokine mRNA response combined with a dysregulated regulatory response and a significant mRNA overexpression of NKG2D receptor and its ligands MICA/B and ULBP in PE placenta. The destruction of chorionic villi observed in PE placenta might be conveyed by an enhanced local cytotoxic response through the NKG2D receptor-ligand pathway, which in turn might be promoted by an intense inflammatory response not counteracted by regulatory cytokine responses.

Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity.

ObjectivesRecurrent respiratory papillomatosis (RRP) is a relatively rare, chronic disease caused by Human Papilloma Virus (HPV) 6 and 11, and characterized by wart‐like lesions in the airway affecting voice and respiratory function. The majority of HPV infections are asymptomatic and resolve spontaneously, however, some individuals are afflicted with persistent HPV infections. Failure to eliminate HPV 6 and 11 due to a defect immune responsiveness to these specific genotypes is proposed to play a major role in the development of RRP.MethodsWe performed a phenotypic characterization of peripheral blood mononuclear cells (PBMC) collected from 16 RRP patients and 12 age‐matched healthy controls, using immunoflow cytometry, and monoclonal antibodies against differentiation and activation markers. The cytokine mRNA profile of monocytes, T helper‐, T cytotoxic‐, and NK cells was assessed using RT‐qPCR cytokine analysis, differentiating between Th1‐, Th2‐, Th3/regulatory‐, and inflammatory immune responses.ResultsWe found a dominance of cytotoxic T cells, activated NK cells, and high numbers of stressed MIC A/B expressing lymphocytes. There was an overall suppression of cytokine mRNA production and an aberrant cytokine mRNA profile in the activated NK cells.ConclusionThese findings demonstrate an immune dysregulation with inverted CD4+/CD8+ ratio and aberrant cytokine mRNA production in RRP patients, compared to healthy controls.

Assessment of Cytokine mRNA Expression Profiles in Tumor Microenvironment and Peripheral Blood Mononuclear Cells of Patients with High-grade Serous Carcinoma of the Ovary

Objective: Tumor establishment, metastatic spreading and poor survival in ovarian cancer is strongly associated with progressive derangement of the patient’s immune system. Accumulating evidence suggests that immune impairment is influenced by the production and presence of cytokines in the tumor microenvironment. Methods: Cytokine mRNA profiles in tumor tissue and peripheral blood mononuclear cells (PBMC) were analyzed in patients with high grade serous carcinoma (HGSC) of the ovary and compared it to patients with benign ovarian conditions and controls with normal ovaries. Cytokine assessment was done by real-time quantitative RT-PCR and specific primers and probes for 12 cytokines-IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-15, TNF-α, TNF-β/LTA, TGF-β1, and GM-CSF chosen to distinguish between cytotoxic Th1, humoral Th2, regulatory Th3/Tr1 and inflammatory responses. Results: The cytokine mRNA response in the HGSC patients was significantly up regulated compared to patients with benign ovarian conditions and normal ovary controls confirming the immunogenicity of HGSC and implying immune recognition and reaction locally in the tumor microenvironment and systemically in the peripheral blood.There was an up-regulation of inflammatory and inhibitory cytokine mRNA promoting tumor progression, T-regulatory cell priming and T-regulatory cell-mediated immune suppression. In contrast, there was an inability to mount the crucially important IFN gamma response needed for upregulation of the cytotoxic anti-tumor response in the local microenvironment. In addition, systemic IL-4- mediated Th2 response prevailed in the peripheral blood deviating the systemic defense towards humoral immunity. Conclusions: Taken together, these results suggest local and systemic cytokine cooperation promoting tumor survival, progression and immune escape. Our study confirms and extends previous investigations and contributes to the evaluation of potential cytokine candidates for diagnostic cytokine mRNA profiles and for future therapeutic interventions based on cytokine inhibition.

Dynamics of cytokine mRNA expression and fecal biomarkers in school-children undergoing a double-blind placebo-controlled food challenge series

BackgroundThere is need for prognostic markers for symptomatic food allergy since current diagnostic methods are insufficient and/or time and labor consuming. ObjectiveTo estimate the cytokine mRNA profiles in peripheral blood mononuclear cells (PBMC) before and after a double-blind placebo-controlled food challenge series in schoolchildren with suspected allergy to milk, egg or cod and in healthy controls. Analyses of fecal inflammatory biomarkers before and after the challenge were included. MethodsTwelve-year-old children from a population-based cohort reporting complete avoidance of milk, egg, cod or wheat due to perceived hypersensitivity were clinically examined and those with suspected food allergy were evaluated with a 3-session double-blind placebo-controlled food challenge (n=18). Seven healthy controls participated in a double-blind challenge with egg. Before and after the challenge series, the cytokine mRNA expression was quantified for 13 cytokines discriminating between humoral Th2-, cytotoxic Th1-, regulatory Th3/Tr1- and inflammatory responses. Fecal calprotectin and eosinophil-derived neurotoxin (EDN) were also analyzed in children with suspected food allergy before and after the challenge series. ResultsPre challenge, children with suspected food allergy had higher IL-13andTNF-α expression and lower IFN-γ and IL-15 expression compared to healthy controls (all p<0.05). Children with challenge-proven food allergy had increased IL13andIL-10 expression compared to the levels seen in negative challenges (p<0.05). Post challenge, IL-1β and IL-6 mRNA levels were elevated in the food allergic children compared to controls (p<0.05). Fecal calprotectin and EDN levels were higher in challenge-proven food allergy compared to a negative challenge although not statistically significantly. Conclusion & clinical relevanceIncreased baseline mRNA levels of the Th2-related cytokine IL-13 and the regulatory cytokine IL-10 predicted a positive food challenge outcome. These cytokines in combination with fecal calprotectin and EDN might serve as future prognostic markers for symptomatic, IgE-mediated food allergy but need further validation in a larger patient cohort.

Identification of neutrophil-derived proteases and angiotensin II as biomarkers of cancer cachexia.

Background:Cachexia is a metabolic disorder characterised by muscle wasting, diminished response to anti-cancer treatments and poor quality of life. Our objective was to identify blood-based biomarkers of cachexia in advanced cancer patients. Hence, we characterised the plasma cytokine and blood cell mRNA profiles of patients grouped in three cohorts: patients with cachexia, pre-cachexia (no cachexia but high CRP levels: ⩾5 mg l−1) and no cachexia (no cachexia and CRP: <5 mg l−1).Methods:A total of 122 newly diagnosed cancer patients with seven cancer types were studied prior to their initial therapy. Plasma levels of 22 cytokines were quantified using the bio-plex technology. mRNAs isolated from whole blood and expression profiles were determined by the chip array technology and Ingenuity Pathway Analysis (IPA) software.Results:In comparison with non-cachectic individuals, both pre-cachectic and cachectic patients showed an increase (⩾1.5-folds) in mRNA expression of neutrophil-derived proteases (NDPs) and significantly elevated angiotensin II (Ang II) (P=0.005 and P=0.02, respectively), TGFβ1 (P=0.042 and P<0.0001, respectively) and CRP (both P<0.0001) in the plasma. Moreover, cachectic patients displayed a significant increase in IL-6 (P=0.005), IL-8 (P=0.001) and absolute neutrophil counts (P=0.007).Conclusions:Ang II, TGFβ1, CRP and NDP are blood biomarkers for cancer cachexia. These findings contribute to early diagnosis and prevention of cachexia.

Abstract 18331: Endothelial Microrna-155 Promotes Myocardial Microvascular Permeability and Inflammatory Cell Adhesion in Experimental Septic Cardiomyopathy

Introduction: Septic cardiomyopathy remains a leading cause of death in critically ill patients. Myocardial edema and inflammatory cell recruitment to cardiac interstitial space are proposed mechanisms underlying sepsis-induced contractile dysfunction. Hypothesis: Evaluate the role of endothelial microRNA-155 (miR-155) in experimental septic cardiomyopathy. Methods: Experimental sepsis was induced using LPS injection in WT (n=20) and miR-155-/- (KO; n=20) male mice. Cardiac function was evaluated through echocardiography. Expression of NOS2, NOS3, VASP phosphorylation and myocardial nitrite/nitrate content was evaluated. Cardiac microvascular (MV) permeability was assessed with evans blue-albumin conjugate leaking to extravascular space and myocardial water content. Mouse cardiac MV endothelial cells (MCMVEC; CD31+Sca-1+CD45-) were isolated with FACS. Quantification of miR-155, cytokine mRNA profile and adhesion molecules (ICAM1, VCAM1 and E-selectin) was performed. Human cardiac MV endothelial cells (HCMVEC) were transfected with anti-miR-155 or scramble LNA probe and incubated with LPS (100ng/mL) or vehicle for 24h. Evaluation of cytokine mRNA profile, NOS isoforms, VASP phosphorylation and adhesion molecules was performed. Monocyte adhesion assay was performed using Dil-stained THP1 monocytic cells. Results: LPS exposure increased miR-155 expression in the myocardium, MCMVEC and HCMVEC. KO mice presented with attenuated LPS-induced cardiac dysfunction, MV permeability, NOS2 expression and VASP phosphorylation, compared with WT. KO MCMVEC presented with attenuated LPS-induced cytokine expression and adhesion molecules, compared with WT cells. Mir-155 inhibition in HCMVEC prevented LPS-induced overexpression of cytokines and adhesion molecules, nitric oxide production and monocyte adhesion, through SOCS1-mediated inhibition of STAT3 and NF-kB signaling pathways. Conclusions: Endothelial miR-155 is upregulated in experimental septic cardiomyopathy. MiR-155 knockout attenuates LPS-induced myocardial dysfunction, edema and MV permeability. In MCMVEC and HCMVEC, miR-155 inhibition blunted LPS-induced nitric oxide production, cytokine and cell adhesion molecules overexpression.

F4+ ETEC infection and oral immunization with F4 fimbriae elicits an IL-17-dominated immune response.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of post-weaning diarrhea (PWD) in piglets. Porcine-specific ETEC strains possess different fimbrial subtypes of which F4 fimbriae are the most frequently associated with ETEC-induced diarrhea in piglets. These F4 fimbriae are potent oral immunogens that induce protective F4-specific IgA antibody secreting cells at intestinal tissues. Recently, T-helper 17 (Th17) cells have been implicated in the protection of the host against extracellular pathogens. However, it remains unknown if Th17 effector responses are needed to clear ETEC infections. In the present study, we aimed to elucidate if ETEC elicits a Th17 response in piglets and if F4 fimbriae trigger a similar response. F4+ ETEC infection upregulated IL-17A, IL-17F, IL-21 and IL-23p19, but not IL-12 and IFN-γ mRNA expression in the systemic and mucosal immune system. Similarly, oral immunization with F4 fimbriae triggered a Th17 signature evidenced by an upregulated mRNA expression of IL-17F, RORγt, IL-23p19 and IL-21 in the peripheral blood mononuclear cells (PBMCs). Intriguingly, IL-17A mRNA levels were unaltered. To further evaluate this difference between systemic and mucosal immune responses, we assayed the cytokine mRNA profile of F4 fimbriae stimulated PBMCs. F4 fimbriae induced IL-17A, IL-17F, IL-22 and IL-23p19, but downregulated IL-17B mRNA expression. Altogether, these data indicate a Th17 dominated response upon oral immunization with F4 fimbriae and F4+ ETEC infection. Our work also highlights that IL-17B and IL-17F participate in the immune response to protect the host against F4+ ETEC infection and could aid in the design of future ETEC vaccines.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-015-0264-2) contains supplementary material, which is available to authorized users.

Lamina propria macrophage phenotypes in relation to Escherichia coli in Crohn's disease.

BackgroundAbnormal handling of E. coli by lamina propria (LP) macrophages may contribute to Crohn’s disease (CD) pathogenesis. We aimed to determine LP macrophage phenotypes in CD, ulcerative colitis (UC) and healthy controls (HC), and in CD, to compare macrophage phenotypes according to E. coli carriage.MethodsMucosal biopsies were taken from 35 patients with CD, 9 with UC and 18 HCs. Laser capture microdissection was used to isolate E. coli-laden and unladen LP macrophages from ileal or colonic biopsies. From these macrophages, mRNA was extracted and cytokine and activation marker expression measured using RT-qPCR.ResultsE. coli-laden LP macrophages were identified commonly in mucosal biopsies from CD patients (25/35, 71 %), rarely in UC (1/9, 11 %) and not at all in healthy controls (0/18). LP macrophage cytokine mRNA expression was greater in CD and UC than healthy controls. In CD, E. coli-laden macrophages expressed high IL-10 & CD163 and lower TNFα, IL-23 & iNOS irrespective of macroscopic inflammation. In inflamed tissue, E. coli-unladen macrophages expressed high TNFα, IL-23 & iNOS and lower IL-10 & CD163. In uninflamed tissue, unladen macrophages had low cytokine mRNA expression, closer to that of healthy controls.ConclusionIn CD, intra-macrophage E. coli are commonly found and LP macrophages express characteristic cytokine mRNA profiles according to E. coli carriage. Persistence of E. coli within LP macrophages may provide a stimulus for chronic inflammation.

Cytokine mRNA profiling and the proliferative response of bovine peripheral blood mononuclear cells to Mycoplasma bovis

Mycoplasma bovis is known as a significant pathogen and cause of large economic losses in beef and dairy calves worldwide. Numerous factors appear to play an important role in the development of disease during infection with M. bovis, e.g., inhibition of immune cell proliferation and induction of lymphocyte apoptosis. However, the mechanisms involved in M. bovis infections have not been explored and remain incompletely understood. We investigated the major cytokine mRNA expression in bovine PBMC stimulated with M. bovis, for comparison, Staphylococcus aureus and Escherichia coli, which are the representative mastitis-causing pathogens. Here we demonstrated that live M. bovis significantly induced tumor necrosis factor alpha (TNF-α), interleukin 12p40 (IL-12), and interferon gamma (IFN-γ) mRNA expression in bovine peripheral blood mononuclear cells (PBMC) at a multiplicity of infection (MOI) of 1000 but not at an MOI of 10 and 100. Live M. bovis at MOIs of 1, 10, and 100 induced significant bovine PBMC proliferative responses compared with unstimulated bovine PBMC. Furthermore, we showed that the cultural supernatant of M. bovis induced a significant increase in TNF-α, IL-6, and IL-10 mRNA expression in bovine PBMC. Our results suggest that M. bovis weakly affects the cellular integrity of bovine PBMC and induces clear proliferative responses and associated cytokine production in them. However, large numbers of live M. bovis are required to induce an immune response in bovine PBMC.

Abstract 20517: MicroRNA-155: A New Player in Sepsis-Induced Cardiac Dysfunction

Introduction: Cardiac dysfunction is an important outcome predictor in patients with septic shock and lacks effective and targeted therapies. MicroRNA-155 (miR-155) is a multifunctional microRNA with important target genes in inflammation, immunity and cardiovascular system. Hypothesis: The present study assessed the role of miR-155 in experimental and human sepsis-induced cardiac dysfunction. Methods: Experimental sepsis was induced using endotoxin injection (LPS) or cecal ligation and puncture (CLP) models. Genetic loss (miR-155 KO) and pharmacological inhibition (LNA-anti-miR; AM) of miR-155 were evaluated. Cardiac function was assessed through echocardiography; mortality analysis was also performed. Quantification of myocardial miR-155, pro-inflammatory cytokine mRNA profile, miR-155 targets, apoptosis, cellular adhesion proteins and activation of pro-inflammatory intracellular signaling pathways were assessed. In another experiment, myocardial miR-155 expression, cellular localization and associations between its levels and other clinical variables were evaluated using post-mortem samples from septic shock (SS) patients. Results: Myocardial miR-155 levels were increased in experimental and human septic myocardium. This was associated with a decreased ejection fraction (EF) and cardiac output (CO) and increased diastolic LV diameter (LVDd) in septic WT animals. Knockout and anti-miR-155 treated animals and presented with preserved EF, CO and LVDd, compared with WT; this was accompanied by ~50% mortality reduction. Experimental sepsis induced downregulation of SOCS1 and CD47, myocardial cytokine upregulation, increased apoptosis and adhesion protein expression as well as pro-inflammatory kinase and transcription factor activation, all of which were attenuated in KO and AM. In human myocardium, miR-155 expression was positively associated with 24h antemortem period plasma troponin I levels. Conclusions: miR-155 is upregulated in experimental and human septic myocardium. Loss or inhibition of miR-155 attenuated sepsis-induced cardiac dysfunction, blunted proinflammatory activation and reduced mortality. This suggests miR-155 as a potential target in human sepsis-associated cardiac dysfunction.

Detailed characterization of tumor infiltrating lymphocytes in two distinct human solid malignancies show phenotypic similarities.

BackgroundWe examined the phenotype and function of lymphocytes collected from the peripheral blood (PBL) and tumor (TIL) of patients with two different solid malignancies: colorectal cancer liver metastases (CRLM) and ovarian cancer (OVC).MethodsTumor and corresponding peripheral blood were collected from 16 CRLM and 22 OVC patients; immediately following resection they were processed and analyzed using a multi-color flow cytometry panel. Cytokine mRNA from purified PBL and TIL CD4+ T cells were also analyzed by qPCR.ResultsOverall, we found similar changes in the phenotypic and cytokine profiles when the TIL were compared to PBL from patients with two different malignancies. The percentage of Treg (CD4+/CD25+/FoxP3+) in PBL and TIL was similar: 8.1% versus 10.2%, respectively in CRLM patients. However, the frequency of Treg in primary OVC TIL was higher than PBL: 19.2% versus 4.5% (p <0.0001). A subpopulation of Treg expressing HLA-DR was markedly increased in TIL compared to PBL in both tumor types, CRLM: 69.0% versus 31.7% (p = 0.0002) and OVC 74.6% versus 37.0% (p <0.0001), which suggested preferential Treg activation within the tumor. The cytokine mRNA profile showed that IL-6, a cytokine known for its immunosuppressive properties through STAT3 upregulation, was increased in TIL samples in patients with OVC and CRLM. Both TIL populations also contained a significantly higher proportion of activated CD8+ T cells (HLA-DR+/CD38+) compared to PBL (CRLM: 30.2% vs 7.7%, (p = 0.0012), OVC: 57.1% vs 12.0%, (p <0.0001)).ConclusionThis study demonstrates that multi-color flow cytometry of freshly digested tumor samples reveals phenotypic differences in TIL vs PBL T cell sub-populations. The TIL composition in primary and metastatic tumors from two distinct histologies were remarkably similar, showing a greater proportion of activated/suppressive Treg (HLA-DR+, CD39+, CTLA-4+ and Helios+) and activated cytotoxic T cells (CD8+/HLA-DR+/CD38+) when compared to PBL and an increase in IL-6 mRNA from CD4 TIL.Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-014-0038-9) contains supplementary material, which is available to authorized users.

Establishment of a vascular endothelial cell-reactive type II NKT cell clone from a rat model of autoimmune vasculitis.

We previously generated a rat model that spontaneously developed small vessel vasculitis (SVV). In this study, a T cell clone reactive with rat vascular endothelial cells (REC) was established and named VASC-1. Intravenous injection of VASC-1 induced SVV in normal recipients. VASC-1 was a TCRαβ/CD3-positive CD4/CD8 double-negative T cell clone with expression of NKG2D. The cytokine mRNA profile under unstimulated condition was positive for IL-4 and IFN-γ but negative for IL-2 and IL-10. After interaction with REC, the mRNA expression of IL-2, IL-5 and IL-6 was induced in VASC-1, which was inhibited by blocking of CD1d on the REC surface. Although the protein levels of these cytokines seemed to be lower than the detection limit in the culture medium, IFN-γ was detectable. The production of IFN-γ from the VASC-1 stimulated with LPS-pre-treated REC was inhibited by the CD1d blockade on the REC. These findings indicated VASC-1 as an NKT cell clone. The NKT cell pool includes two major subsets, namely types I and II. Type I NKT cells are characterized by expression of semi-invariant TCRs and the potential to bind to marine sponge-derived α-galactosylceramide (α-GalCer) loaded on CD1d; whereas, type II NKT cells do not manifest these characteristics. VASC-1 exhibited a usage of TCR other than the type I invariant TCR α chain and did not bind to α-GalCer-loaded CD1d; therefore, it was determined as a type II NKT cell clone. The collective evidence suggested that REC-reactive type II NKT cells could be involved in the pathogenesis of SVV in rats.