Cytokine mRNA Profiles Research Articles

Placental infection by Salmonella enterica typhimurium in a murine model: mechanisms of pathogenesis and role of inflammatory cell death

Placental infection by Salmonella enterica typhimurium in a murine model: mechanisms of pathogenesis and role of inflammatory cell death

The enzymes regulating endothelin-1 in chorioamnionitis

s / Placenta 35 (2014) A1eA112 A65 and non-transmitting dams were compared. However, significant increases in TNFa protein (p < 0.05, t-test) were observed by ELISA in serum of CMV-transmitting dams (n1⁄43) at 21 dpi (mean 980 pg/ml), compared to non-transmitters (n1⁄43; <10 pg/ml). Conclusion: This study demonstrates that congenital CMV infection is associated with a signature inflammatory cytokine mRNA profile in the placenta, and elevated levels of TNFa in maternal blood. Elucidation of this cytokine “fingerprint” provides biomarkers for identifying pregnancies at risk for maternal-fetal CMV infection, and could be useful in tracking responses to potential therapies and for investigating pathogenesis.

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

<h3>Background</h3> We 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). <h3>Methods</h3> Tumor 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. <h3>Results</h3> Overall, 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 &lt;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 &lt;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 &lt;0.0001)). <h3>Conclusion</h3> This 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.

OP0083 Comparison of synovial tissue cytokine mrna profiles in very early arthritis patients with divergent outcomes

BackgroundIn the earliest clinically apparent phases of arthritis, it is difficult to distinguish patients who will develop RA from those whose disease will resolve. We hypothesised that synovial tissue cytokine...

OP0013 A Novel Pro-Inflammatory B Cell Population Infiltrating the Rheumatoid Synovium can be Identified by Expression of FCRL4

BackgroundThe importance of the B cell lineage in RA pathogenesis is highlighted by the clinical effectiveness of rituximab. Potential mechanisms by which B cells drive disease processes in RA include...

PB1-F2 Modulates Early Host Responses but Does Not Affect the Pathogenesis of H1N1 Seasonal Influenza Virus

In the context of infections with highly pathogenic influenza A viruses, the PB1-F2 protein contributes to virulence and enhances lung inflammation. In contrast, its role in the pathogenesis of seasonal influenza viral strains is less clear, especially in the H1N1 subtype, where strains can have a full-length 87- to 90-amino-acid protein, a truncated 57-amino-acid version, or lack the protein altogether. Toward this, we introduced the full-length 1918 PB1-F2, or prevented PB1-F2 expression, in H1N1 A/USSR/90/77, a seasonal strain that naturally expresses a truncated PB1-F2. All viruses replicated with similar efficiency in ferret or macaque ex vivo lung cultures and elicited similar cytokine mRNA profiles. In contrast, the virus expressing the 1918 PB1-F2 protein caused a delay of proinflammatory responses in ferret blood-derived macrophages, while the PB1-F2 knockout virus resulted in a more rapid response. A similar but less pronounced delay in innate immune activation was also observed in the nasal wash cells of ferrets infected with the 1918 PB1-F2-expressing virus. However, the three viruses did not differ in their virulence or clinical course in ferrets, supporting speculations that PB1-F2 is of limited importance for the pathogenesis of primary viral infection with human seasonal H1N1 viruses.

Comparison of synovial tissue cytokine mRNA profiles in very early arthritis patients with divergent outcomes

Backgroundand objectivesIn the earliest clinically apparent phases of arthritis, it is difficult to distinguish patients who will develop RA from those whose disease will resolve. The authors hypothesised that synovial...

Assessment of Molecular Signalling Mechanisms for Eosinophilia in Rottweilers

Rottweilers are predisposed to eosinophilic diseases, including hypereosinophilic syndrome. The immunopathogenesis of idiopathic eosinophilia is poorly characterised in dogs and man. Studies in people have suggested cytokines, particularly interleukin (IL)-5, play a role in instigating and perpetuating eosinophilia. This study sought to establish whether differences in gene expression, and concentration of selected, cytokines and chemokines were associated with eosinophilia in Rottweilers. Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) assays were used to quantify messenger ribonucleic acid (mRNA) encoding cytokines IL-4, -5, -10, -12p19, -12p35, -12p40, -18, interferon gamma (IFN-γ) and chemokines eotaxin-2 and -3 from peripheral blood mononuclear cell (PMBC) samples obtained from healthy dogs (breeds other than Rottweiler) with normal eosinophil blood counts (n = 5) and Rottweilers with normal (n = 6), mildly increased (n = 7) and high (n = 3) eosinophil blood counts. Quantification of plasma IFN-γ and IL-5 was performed using commercially available canine-specific enzyme-linked immunosorbent assays ELISAs. Cytokine mRNA was measurable in all samples, although eotaxin-2 and -3 were not detected. No significant differences in gene expression of any cytokine were found between groups (based on eosinophil count or breed). No significant difference in plasma IL-5 or IFN-γ concentration was present between groups. In conclusion, there were no significant differences in cytokine mRNA profiles or plasma IL-5 and IFN-γ levels between Rottweilers with increased eosinophil counts and Rottweiler and non-Rottweiler dogs with normal eosinophil counts.

Cytokine mRNA Profile of Alveolar T Lymphocytes and Macrophages in Patients with Systemic Sclerosis Suggests a Local Tr1 Response

The development of an autoimmune disease like systemic sclerosis (SSc) is suspected to be driven by an activated T lymphocyte subset, expressing a cytokine profile specific to the disease. To further characterize the type of immune reaction in SSc, we searched for a broad panel of cytokine messenger ribonucleic acids (mRNAs) in T lymphocytes and monocytes/macrophages from paired samples of bronchoalveolar lavage fluid and peripheral blood in 18 patients and 16 age- and sex-matched controls. RNA from CD3(+) T lymphocytes and CD14(+) monocytes/macrophages was examined by means of the reverse transcriptase polymerase chain reaction. SSc alveolar T lymphocytes expressed a cytokine profile suggestive of a mixed Th1/Th2 reaction, showing an increased frequency of mRNA for interleukin (IL)-10, IL-6 and interferon (IFN)γ, while IL-1β, IFNγ and tumour necrosis factor β were expressed in blood T lymphocytes in a higher percentage of patients with SSc than controls. SSc alveolar T cells expressed IL-10 mRNA more often than peripheral T cells, a phenomenon not found in controls and which may point at local IL-10 activation/response in SSc lung. Transforming growth factor β mRNA was present in all alveolar as well as peripheral blood T cell samples in patients and controls. The cytokine mRNA profile in SSc with interstitial lung disease (ILD) was similar to the profile found in SSc without ILD. Our findings point at a mixed Th1/Th2 reaction in SSc and may indicate regulatory T 1 cell activation/response in the lungs of patients with SSc.

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis

ObjectivesIn rheumatoid arthritis (RA), a complex cytokine network drives chronic inflammation and joint destruction. So far, few attempts have been made to identify the cellular sources of individual cytokines systematically....

Cytokine mRNA profiling identifies B cells as a major source of RANKL in rheumatoid arthritis

Background and objectivesCytokines are important mediators involved in chronic inflammation and tissue destruction in rheumatoid arthritis (RA). Animal models of arthritis, in vitro studies, and observations made in patients treated...

Cytokine mRNA profile of Epstein–Barr virus-stimulated highly differentiated T cells in multiple sclerosis: A pilot study

The reason why EBV-specific cellular immune responses are abnormal in multiple sclerosis (MS) patients is still missing. In this exploratory pilot study, we assessed IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ, TGF-β1 and FOXP3 mRNA expression in EBV-stimulated highly differentiated T cells (T HD) of MS patients and healthy controls (HC). We found increased levels of IFN-γ and IL-4 mRNA in CD8+ T HD cells of MS patients. All the other tested molecules were expressed similarly in MS patients and HC. Interestingly, increased IFN-γ and IL-4 suggest that the control of EBV replication may be insufficient in MS patients.

Uremia impacts renal inflammatory cytokine gene expression in the setting of experimental acute kidney injury

Inflammatory cytokines are evoked by acute kidney injury (AKI) and may contribute to evolving renal disease. However, the impact of AKI-induced uremia on proinflammatory (e.g., TNF-alpha, MCP-1, TGF-beta1) and anti-inflammatory (e.g., IL-10) cytokine gene expression remains unknown. This study was undertaken to gain some initial insights into this issue. CD-1 mice were subjected to left renal ischemia-reperfusion (I/R) in the absence or presence of uremia (+/- right ureteral transection). TNF-alpha, MCP-1, TGF-beta1, and IL-10 mRNAs, cytokine protein levels, and RNA polymerase II (Pol II) recruitment to these genes were assessed. Renal cytokine mRNA levels were also contrasted with unilateral vs. bilateral renal parenchymal damage (I/R or ureteral obstruction). Potential effects of uremia on cytokine mRNAs in the absence of parenchymal renal damage [bilateral ureteral transection (BUTx)] were sought. Finally, the impact of simulated in vitro uremia (HK-2 tubular cells exposed to peritoneal dialysate from uremic vs. normal mice) on cytokine mRNA and microRNA profiles was assessed. Uremia blunted TNF-alpha, MCP-1, and TGF-beta1 mRNA increases in all three in vivo parenchymal acute renal failure models. These results were paralleled by reductions in cytokine protein levels and Pol II recruitment to their respective genes. Conversely, uremia increased IL-10 mRNA, both in the presence and absence (BUTx) of parenchymal renal damage. The uremic milieu also suppressed HK-2 cell proinflammatory cytokine mRNA levels and altered the expression of least 69 microRNAs (P < 0.0001). We conclude that both pro- and anti-inflammatory cytokine gene expressions are influenced by uremia, with a potential predilection toward an anti-inflammatory state. Changes in gene transcription (as reflected by Pol II recruitment), and possible posttranscriptional modifications (known to be induced by microRNAs), are likely involved.

Prolongation of Rat Major Histocompatibility Complex–compatible Cardiac Allograft Survival During Pregnancy

It has been suggested that pregnancy-related hormones play a critical role in mediating selective immune tolerance during pregnancy. An understanding of why a woman's body normally does not reject the fetus is highly relevant to the prevention of transplant rejection. The hearts of female inbred F344 rats (RT-1(lvl)) were transplanted into naive Lewis (RT-1(l); nLewis) or pregnant (pLewis; Day 6, 12 and 18 of pregnancy) rats. The mean survival time (MST) of the cardiac allografts between the nLewis and pLewis rats was compared. We determined the rate of proliferation of the T cells isolated from nLewis and pLewis rats in response to concanavalin A (ConA), anti-CD3 and -CD28 antibody and alloantigen stimulation ex vivo. mRNA expression of several cytokines in these T cells was analyzed using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, the effect of estriol on the cardiac allograft was tested. The pLewis rats with transplant on Day 12 of pregnancy had the most significantly prolonged F344 cardiac graft survival (MST 13.3 days) as compared with nLewis recipients (MST 8 days). pLewis T-cell proliferation was stimulated by alloantigen and antibody but ConA was reduced, whereas Th1/Th2 cytokine mRNA profiles in the T cells were similar for nLewis and pLewis rats. Likewise, estriol also significantly prolonged survival of cardiac allografts. The results of this study demonstrate that pregnancy hormones not only appear to play a critical role in maternal acceptance of the fetus, but also have therapeutic potential for prolonging the survival of major histocompatibility complex (MHC)-compatible allografts during pregnancy.

Cytokine profiling of chemical allergens in mice: Measurement of message versus protein

Chemical respiratory allergy is an important occupational health problem, but there are currently available no validated methods for hazard identification. There has been interest for some time in the application of cytokine profiling for the characterization of chemical allergens. We have now examined whether these cytokine expression patterns are regulated at the level of mRNA and/or protein production. Mice (BALB/c strain) were exposed topically to the contact allergen 2,4-dinitrochlorobenzene (DNCB), or to the respiratory allergen trimellitic anhydride (TMA). Thirteen days after the initiation of exposure, a single cell suspension of draining (auricular) lymph node cells (LNC) was prepared. Cells were cultured for 24–120 h and supernatants analyzed for cytokine protein by cytokine bead array. In parallel experiments total RNA was prepared from freshly isolated or cultured cells and cytokine gene expression was analyzed by ribonuclease protection assay (RPA) or by real time reverse transcription-polymerase chain reaction (RT-PCR). DNCB-activated LNC secreted high levels of the type 1 cytokines interferon (IFN)-γ and IL-12 compared with TMA-stimulated LNC. The converse type 2 pattern was observed following treatment with TMA. Freshly isolated LNC from TMA-treated mice displayed a selective type 2 cytokine mRNA profile as measured by RPA. In contrast, RNA isolated from DNCB-activated LNC displayed a more mixed cytokine phenotype with relatively low levels of transcripts for both type 1 and type 2 cell products. When cytokine gene expression was measured by the more sensitive real time RT-PCR technique, more vigorous expression of IL-4 was recorded for TMA-activated LNC compared with DNCB-activated LNC but there was no evidence for elevated IFN-γ transcripts for the latter treatment. The observation that IFN-γ mRNA expression is not increased in DNCB-activated LNC despite robust secretion of this cytokine indicates that production is controlled mainly at the level of translation of previously transcribed mRNA or of protein secretion. Furthermore, the preferential cytokine profile recorded following TMA exposure was more clearly contrasting when measured at the level of protein secretion rather than at the level of mRNA expression. Experience to date suggests that the measurement of induced cytokine profiles shows promise for the hazard identification and characterization of chemical respiratory allergens, and that the end point best suited for this purpose is cytokine secretion rather than mRNA expression.

Interleukin-2 and interferon-gamma mRNA expression in canine anal furunculosis lesions and the effect of ciclosporin therapy

German Shepherd Dogs have an increased incidence of anal furunculosis (AF), which is a disease characterised by inflammation and ulceration of the perianal tissues. Ciclosporin, an immunosuppressive drug, has been successfully used to treat AF, suggesting that the pathogenesis of disease is likely to have an immune-mediated component. Previous research has shown that the cytokine mRNA profile in AF lesions is consistent with T cell-mediated inflammation. The aims of the current study were to quantify IL-2 and IFNγ mRNA expression in AF biopsies taken before and after treatment with ciclosporin and to compare cytokine expression with lesion severity. Twenty-two dogs with AF were recruited into the study and lesional biopsies were taken prior to ciclosporin therapy. Lesion severity was graded using a visual analogue scale. All dogs were evaluated after 4 weeks of ciclosporin therapy and, in 10 dogs with persistent disease, residual lesions were resected. RNA was extracted from AF-lesional tissue and control perianal tissue samples ( n = 10), which was used as the template for RT-PCR. Analysis of IL-2 and IFNγ mRNA expression was performed using real-time PCR. IL-2 and IFNγ mRNA was consistently detected in pre-treatment AF biopsies and, when quantified, this was significantly increased compared to control tissue ( P < 0.05). However, no correlation was seen between lesion severity and pre-treatment cytokine mRNA expression. In the ten paired pre- and post-treatment samples, IL-2 mRNA expression was significantly reduced in residual disease tissue following ciclosporin therapy ( P = 0.013). Treatment with ciclosporin seemed to result in decreased expression of IFNγ mRNA in AF lesions but this was not statistically significant. In six of the 10 dogs with persistent disease, T cell cytokine mRNA could still be detected in the tissues, suggesting that there was inadequate immunosuppression. The absence of a correlation between T cell cytokine expression and the severity of disease suggests that tissue destruction observed in AF might be a consequence of other inflammatory mediators or downstream effects of T cell activation.

IFN‐γ and IL‐4 have opposing effects on immunopathology and the development protective adaptive immunity against mycoplasma respiratory disease

Murine mycoplasma disease progression is dependent on host immune responses. IFNγ is critical in developing beneficial innate immunity. Lack of IFNγ impairs the ability to control infection whereas loss of IL4 does not. The purpose of this study was to discern whether the loss of either IFNγ or IL4 altered, the cytokine cascade after infection & the generation of adaptive immunity against mycoplasma. Cytokine mRNA expression was monitored in M. pulmonis infected wild-type, IFNγ & IL4 KO BALB/c mice using microarrays. The lungs of naïve IFNγ & IL4 KO mice had Th2 & Th1 dominant cytokine mRNA profiles, respectively. There was a significant increase in pro-inflammatory cytokine mRNA expression in infected IFNγ KO mice compared to WT, along with severe disease. Whereas, these mRNA levels were lower in the lungs of IL4 KO mice than the WT mice. In vitro T cell responses were consistent with mRNA profiles. To examine generation of protective immunity, nasal pulmonary immunized WT & KO mice were challenged & mycoplasma numbers determined in the nasal passages (NP) & lungs. Immunized IFNγ KO mice had severe lesions & higher numbers of mycoplasma in both NP & lungs than WT. In contrast, the IL4 KO mice were significantly better protected than the immunized WT mice. In conclusion, IFNγ is critical in reducing the development of immunopathology & mediating immune protection whereas IL4 has the opposite effect.

Cytokine Profiles in Primary and Secondary Pulmonary Granulomas of Guinea Pigs with Tuberculosis

The cytokine mRNA profiles of primary (arising from inhaled bacilli) and secondary (arising from hematogenous reseeding of the lung) granulomas from the lung lobes of bacillus Calmette-Guérin (BCG)-vaccinated and unimmunized guinea pigs challenged with virulent Mycobacterium tuberculosis by the pulmonary route were assessed in situ using laser capture microdissection (LCM) at 6 weeks after infection. The challenge dose chosen was so low that some lung lobes did not receive an implant from the airway. In unimmunized guinea pigs, some lobes contained either large, necrotic primary lesions or small, non-necrotic secondary lesions, or both. The lobes of BCG-vaccinated animals contained only non-necrotic primary tubercles, and no secondary lesions were visible. Real-time PCR analysis of the acquired RNA clearly demonstrated that primary tubercles from BCG-vaccinated guinea pigs were overwhelmed with mRNA from the anti-inflammatory cytokine, transforming growth factor (TGF)-beta, with some IFN-gamma and IL-12p40 mRNA. In contrast, primary lesions from unimmunized animals were dominated by proinflammatory TNF-alpha mRNA. The cytokine mRNA profile of secondary lesions from unimmunized animals was strikingly similar to the profile of primary lesions from BCG-vaccinated guinea pigs (i.e., a predominance of TGF-beta mRNA with some IL-12p40 and IFN-gamma mRNA), indicating that the lung lobes from which these lesions were retrieved had been naturally "vaccinated" by the time the bloodborne bacilli returned to the lung at 3 to 4 weeks after infection. Furthermore, cytokine mRNA analysis of splenic granulomas from nonvaccinated and vaccinated animals showed close resemblance to primary granulomas recovered from the lungs of the same animal, that is, high levels of TNF-alpha mRNA in unimmunized animals, and mostly TGF-beta mRNA in BCG-vaccinated guinea pigs. Taken together, these data indicate that mycobacteria returning to the lungs of unimmunized guinea pigs 3 to 4 weeks after infection induce a local cytokine response that is fundamentally different from the response to inhaled bacilli and is reminiscent of the primary response in a vaccinated animal.

After several years of witchhunting, can calcium-based phosphate binding be released on probation?

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Cutaneous Gene Expression by DNA Microarray in Murine Sclerodermatous Graft-Versus-Host Disease, a Model for Human Scleroderma

The molecular mechanisms governing skin fibrosis in murine sclerodermatous graft-versus-host disease (Scl GVHD) are not known. We used Affymetrix DNA microarrays representing >14,000 mouse genes to characterize global gene expression in skin during development of Scl GVHD in lethally irradiated BALB/c (H-2d) mice transplanted with B10.D2 (H-2d) bone marrow and spleen cells. These mice develop skin thickening, whereas control mice transplanted with syngeneic BALB/c (H-2d) bone marrow and spleen cells do not develop disease. We found consistent differences between mice with Scl GVHD and controls in cytokine messenger RNAs (mRNAs) for both Th1-like (IFN-gamma) and Th2-like (IL-6, Il-10, and IL-13) inflammatory patterns. mRNAs for chemokines CCL2, CCL5, CCL17, IFN-gamma inducible chemokines (CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC), and for growth factors such as platelet-derived growth factor-c, connective tissue growth factor, fibroblast growth factor 1, epidermal growth factor, nerve growth factor-beta, vascular endothelial growth factor (VEGF)-alpha, and VEGF-beta were elevated, similar to human scleroderma. mRNAs for cell adhesion molecules, such as L-selectin (selectin lymphocyte), P-selectin (selectin platelet), E-selectin (selectin endothelium), and vascular cell adhesion molecule 1, were also upregulated. In separate experiments, we confirmed the increased synthesis of IFN-gamma and IL-2, unchanged IL-10, and absence of tumor necrosis factor-alpha, and IL-4 proteins by flow cytometry of isolated skin T cells. These constellations of immunologic changes provide a "fingerprint" for fibrosing autoimmune disease. They are useful to understand the pathogenesis of Scl GVHD, to identify markers for early diagnosis of disease, and to devise more effective strategies for intervention in early scleroderma and Scl GVHD.