Expression Of Regulatory Cytokines Research Articles

Abstract 5626: Multiomic spatial profiling of the tumor immune microenvironment at single cell resolution

Abstract Background: It has been well established that the tumor microenvironment (TME), which comprises cancer cells, stromal cells, and surrounding extracellular matrix, plays a critical role in cancer development, progression, and control. The immunological components within tumors, known as the tumor immune microenvironment (TiME), have also been implicated in tumor development, recurrence, and metastasis. Effective strategies for cancer immunotherapies will require a deep understanding of the factors that shape both the TME and TiME. Here, we describe a spatial multiomics approach that utilizes RNAscope™ ISH technology paired with high-plex whole-slide spatial phenotyping with the PhenoCycler™-Fusion platform. This two-step approach is compatible with human FFPE tissues and enables researchers to characterize the spatial biology of the TiME more accurately by detecting RNA and protein markers on serial sections. The resulting multiomic data more accurately reveal the interplay between TME and TiME by giving insight into cell lineages, surrounding structures, as well as secreted chemokines and cytokines that exist within the TME ecosystem. Methods: We performed ultrahigh-plex spatial phenotyping on the PhenoCycler-Fusion on FFPE tumor tissue sections, using an antibody panel that is designed for immune cell phenotyping, evaluation of immune contexture and proliferation across the TME. Using serial sections from the same tissue blocks, we then ran the RNAscope HiPlex v2 assay automated on the PhenoCycler-Fusion system. This assay consisted of a 12-plex immuno-oncology panel of RNA target probes, which were selected to detect macrophages, chemokines, and cytokines within tumors. We used Phenoplex software to analyze the protein and RNA datasets and to compute cell phenotypes and spatial associations. Results and Conclusions: In this proof-of-concept study, we demonstrate the utility of multiomic spatial profiling on the PhenoCycler-Fusion platform. Analysis of the resulting multiplex imaging data not only revealed the structural organization of cells within the TME, but also activation states of immune cells. Together, this information provides a more complete functional map of immune cells within the TME and TiME and thereby enriches our understanding of tumor biology that may be deterministic of immunotherapy responsiveness. This work paves the way for future research that will rely on deep spatial phenotyping with protein biomarkers coupled with accurate quantification of the expression of regulatory cytokines, chemokines, growth factors, or non-coding RNAs that only RNA probes can detect. Citation Format: Niyati Jhaveri, HaYeun Ji, Anushka Dikshit, Jessica Yuan, Emerald Doolittle, Steve Zhou, Maithreyan Srinivasan, Bassem B. Cheikh, Fabian Schneider, James Mansfield, Julia Kennedy-Darling, Oliver Braubach. Multiomic spatial profiling of the tumor immune microenvironment at single cell resolution. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5626.

Probiotics, prebiotics, and symbiotics in the treatment of obesity: A new vision

Obesity is a chronic non-communicable disease whose prevalence has doubled during the last three decades, becoming, along with its metabolic complications, a worldwide public health problem. Recently, the alteration of the intestinal microbiota balance, known as dysbiosis, has been identified as a possible pathophysiological process that promotes weight gain in individuals who suffer from it. Dietary intervention with prebiotics, probiotics, or symbiotics aimed at correcting intestinal dysbiosis in obese patients can provide health benefits by facilitating weight loss and maintenance, improving constipation, glucose, and lipid levels, as well as immunity and expression of regulatory cytokines, thus contributing to the reduction of the chronic inflammatory process of intestinal cells.

Mettl14-mediated m6A modification enhances the function of Foxp3+ regulatory T cells and promotes allograft acceptance.

N6-methyladenosine (m6A), the most prevalent form of internal mRNA modification, is extensively involved in Treg cells differentiation and function. However, the involvement of m6A in functional Treg cells for transplantation tolerance remains to be elucidated. By using an experimental transplantation mouse model, we found that m6A levels in Treg cells were altered during the induction of transplant tolerance by performing a dot blotting assay. Subsequently, we used the heterogenic Treg-specific Mettl14 knockout mice (Foxp3-Mettl14f/+ cKO) to reduce METTL14 expression and performed islets allograft transplantation. Our result revealed that reduced expression of METTL14 prevented Treg cells expansion and promoted the infiltration of CD4+ and CD8+ T cells around the allograft, which led to rapid allograft rejection in Foxp3-Mettl14 f/+ cKO mice. The expression of regulatory cytokines including IL-10 and TGF-β was significantly decreased in Foxp3-Mettl14 f/+ cKO mice, and the suppressive function of Treg cells was also abrogated. In addition, an analysis of RNA-seq data revealed that the SOCS family (SOCS1, SOCS2 and SOCS3) is the subsequent signaling pathway affected by the METTL14 mediated m6A modification in Treg cells to modulate the suppressive function after transplantation. Taken together, our study showed for the first time that the METTL14-mediated m6A modification is essential for the suppressive function of Treg cells in transplantation and may serve as a regulatory element of Treg cell-based therapy in transplant medicine.

The imbalance between Type 17 T-cells and regulatory immune cell subsets in psoriasis vulgaris.

Psoriasis vulgaris is a common inflammatory disease affecting 7.5 million adults just in the US. Previously, psoriasis immunopathogenesis has been viewed as the imbalance between CD4+ T-helper 17 (Th17) cells and regulatory T-cells (Tregs). However, current paradigms are rapidly evolving as new technologies to study immune cell subsets in the skin have been advanced. For example, recently minted single-cell RNA sequencing technology has provided the opportunity to compare highly differing transcriptomes of Type 17 T-cell (T17 cell) subsets depending on IL-17A vs. IL-17F expression. The expression of regulatory cytokines in T17 cell subsets provided evidence of T-cell plasticity between T17 cells and regulatory T-cells (Tregs) in humans. In addition to Tregs, other types of regulatory cells in the skin have been elucidated, including type 1 regulatory T-cells (Tr1 cells) and regulatory dendritic cells. More recently, investigators are attempting to apply single-cell technologies to clinical trials of biologics to test if monoclonal blockade of pathogenic T-cells will induce expansion of regulatory immune cell subsets involved in skin homeostasis.

Restoring the Balance between Pro-Inflammatory and Anti-Inflammatory Cytokines in the Treatment of Rheumatoid Arthritis: New Insights from Animal Models.

Rheumatoid arthritis (RA) and other autoimmune inflammatory diseases are examples of imbalances within the immune system (disrupted homeostasis) that arise from the effects of an accumulation of environmental and habitual insults over a lifetime, combined with genetic predispositions. This review compares current immunotherapies—(1) disease-modifying anti-rheumatic drugs (DMARDs) and (2) Janus kinase (JAK) inhibitors (jakinibs)—to a newer approach—(3) therapeutic vaccines (using the LEAPS vaccine approach). The Ligand Epitope Antigen Presentation System (LEAPS) therapies are capable of inhibiting ongoing disease progression in animal models. Whereas DMARDs ablate or inhibit specific proinflammatory cytokines or cells and jakinibs inhibit the receptor activation cascade for expression of proinflammatory cytokines, the LEAPS therapeutic vaccines specifically modulate the ongoing antigen-specific, disease-driving, proinflammatory T memory cell responses. This decreases disease presentation and changes the cytokine conversation to decrease the expression of inflammatory cytokines (IL-17, IL-1(α or β), IL-6, IFN-γ, TNF-α) while increasing the expression of regulatory cytokines (IL-4, IL-10, TGF-β). This review refocuses the purpose of therapy for RA towards rebalancing the immune system rather than compromising specific components to stop disease. This review is intended to be thought provoking and look forward towards new therapeutic modalities rather than present a final definitive report.

Lactobacillus fermentum (MTCC-5898) alleviates Escherichia coli-induced inflammatory responses in intestinal epithelial cells by modulating immune genes and NF-κB signalling.

Dietary intervention using probiotic bacteria has emerged as a promising preventive strategy in addressing foodborne infections or gastrointestinal disorders. This study investigated the immunomodulatory effects of Lactobacillus fermentum (MTCC-5898) on Escherichia coli-induced inflammatory responses in intestinal epithelial cells. The immune response of intestinal cells (Caco-2) in the presence of probiotic Lact. fermentum was determined during exclusion, competition and displacement of E.coli as the inflammatory agent. To achieve this objective, transcriptional modulation of key immune-related genes (cytokines, pattern recognition receptors and NF-κB), release of cytokines and nuclear translocation of the NF-κB subunit p-65 were studied. Expression of pro-inflammatory cytokines IL-8, TNF-α, IFN-ϒ and IL-23 was high in E.coli-exposed intestinal cells. However, overexpression of these E.coli-induced pro-inflammatory cytokines was prevented by Lact. fermentum during exclusion and competition assays. It also modulated the transcriptional expression of regulatory cytokines (IL-10 and TGF-β), pattern recognition receptors (TLR-2 and TLR-4) and genes associated with master inflammatory regulators (NF-κB and SIGIRR) to reduce E.coli-induced inflammation. The protective effect of Lact. fermentum was further confirmed by suppression of nuclear translocation of cytoplasmic NF-κB subunit (p-65). Lactobacillus fermentum alleviated E.coli-induced inflammatory responses by modulating the NF-κB signalling besides pro-inflammatory and regulatory cytokines expression. Lactobacillus fermentum holds significant promise as a potent nutraceutical that prevents and manages inflammatory gut-associated dysfunctions.

Immunoregulatory effects of indole-3-carbinol on monocyte-derived macrophages in systemic lupus erythematosus: A crucial role for aryl hydrocarbon receptor

Macrophages are versatile phagocytic cells in immune system with immunoregulatory functions. However, the removal of apoptotic cells by macrophages is disturbed in systemic lupus erythematosus (SLE). Aryl hydrocarbon receptor (AhR) is a ligand-activated cytoplasmic receptor and transcription factor with diverse effects on immune response. Indole-3-carbinol (I3C) is an AhR agonist which has been implicated as a beneficial factor in regulating inflammation and cytokine expression in murine models of SLE. However, the molecular mechanisms are not thoroughly studied. Here, we aimed to investigate the ex vivo effects of I3C on polarization of monocyte-derived macrophages (MDMs) in SLE patients and the expression of regulatory cytokines upon AhR activation. MDMs from 15 newly diagnosed SLE patients and 10 normal subjects were induced by Jurkat apoptotic bodies (JABs) and treated with I3C. I3C enhanced the nuclear accumulation of AhR among MDMs of SLE patients and altered the expression of AhR target genes including CYP1A1, IL1- β, IDO-1 and MRC-1. The imbalanced expression of pro- and anti- inflammatory cytokines (IL-10, IL-12, TGFβ1, TNFα, IL-23, IL-6 and IFN-γ) was compensated in response to I3C. AhR activation was also associated with the overexpression of M2 markers (CD163) and downregulation of M1 markers (CD86). Thus, macrophages are activated alternatively in response to I3C. The obtained data indicate that I3C-mediated AhR activation possess immunoregulatory effects on macrophages of SLE patients by exerting an obvious downregulation in the expression of pro-inflammatory and overexpression of anti-inflammatory cytokines. Therefore, AhR could be targeted and further investigated as a choice of anti-inflammatory therapies for autoimmune disorders such as SLE.

Lactobacillus casei BL23 regulates Treg and Th17 T-cell populations and reduces DMH-associated colorectal cancer.

Chronic intestinal inflammation alters host physiology and could lead to colorectal cancer (CRC). We have previously reported beneficial effects of the probiotic strain of Lactobacillus casei BL23 in different murine models of intestinal inflammation. In addition, there is an emerging interest on the potential beneficial effects of probiotics to treat CRC. We thus explored whether L. casei BL23 displays protective effects on CRC. Mice were subcutaneously injected with 1,2-dimethylhydrazine (DMH) weekly during 10weeks and orally administered with L. casei BL23 in the drinking water until the 10th week. Multiple plaque lesions in the large intestine were observed macroscopically and counted and intestinal tissues were also histologically analyzed. Finally, T-cell populations and cytokine production were evaluated after co-incubation of L. casei BL23 with spleen cells from non-treated mice to determine the immuno-modulatory effects of this bacterium. Our results show that oral treatment with this probiotic bacterium modulates host immune responses and significantly protect mice against DMH-induced CRC. This protection may be associated with the modulation of regulatory T-cells towards a Th17-biased immune response accompanied by the expression of regulatorycytokines (IL-6, IL-17, IL-10 and TGF-β), as demonstrated in L. casei BL23-treated splenocytes, but also with the colonic expression of IL-22 observed in vivo on L. casei BL23-treated mice; suggesting the induction of a fine-tune Th17-biased response. Altogether our results reveal the high potential of L. casei BL23 to treat CRC and opens new frontiers for the study of immunomodulatory functions of probiotics.

Oral tolerance correlates with high levels of lymphocyte activity

Oral tolerance is defined as an inhibition of specific immune responsiveness to a previously ingested antigen. Paradoxically, we found an increased lymphocyte activity in tolerant mice alongside the specific inhibition. Orally-tolerant mice presented higher number of immunoglobulin secreting cells (ISC) in spleen and bone marrow; showed a greater variety of Ig classes being produced: IgM and IgA in the spleen and IgG and IgM in the bone marrow. ISC from immunized mice produced mainly IgG. Despite having the same number of regulatory and activated T cells in the spleen after immunization, these cells appeared earlier in tolerant mice, right after the primary immunization. Also, tolerant mice showed a prompt expression of regulatory cytokines (TGF-β and IL-10) and a transient expression of effector cytokines (IL-2 and IFN-γ). Thus, in addition to an inhibited specific responsiveness, orally-tolerant mice displayed an early and widespread mobilization of activated and regulatory lymphocytes.

In SituExpression of Regulatory Cytokines by Heart Inflammatory Cells in Chagas' Disease Patients with Heart Failure

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. The immune system plays an important role in the reduction of parasite load, but may also contribute to the development of lesions observed during the chronic phase of the disease. We analyzed cytokines produced by inflammatory heart cells in 21 autopsy samples obtained from patients with Chagas' disease divided according to the presence or absence of heart failure (HF). Left ventricular sections were analyzed by immunohistochemistry using antibodies against human IL-4, IFN-γ, TGF-β, TNF-α, and NOS2. In situ mRNA expression was quantified by a Low Density Array. The number of IFN-γ-positive cells was significantly higher than IL-4 positive cells. TNF-α, TGF-β and NOS2 were detected in 65%, 62% and 94% of samples respectively. There was an association between TNF-α-producing cells and the presence of HF. Subjects with HF presented higher levels of STAT4 mRNA, whereas FoxP3 and STAT6 levels were similar in the two groups. A Th1 cytokine pattern predominated in the cardiac inflammatory cell infiltrate of Chagas' disease patients associated with HF. High degree of fibrosis was associated with low NOS2 expression. These results support the idea that Th1 immune responses are involved in heart lesions of Chagas' disease patients.

Tumor Necrosis Factor-α and Muc2 Mucin Play Major Roles in Disease Onset and Progression in Dextran Sodium Sulphate-Induced Colitis

The sequential events and the inflammatory mediators that characterize disease onset and progression of ulcerative colitis (UC) are not well known. In this study, we evaluated the early pathologic events in the pathogenesis of colonic ulcers in rats treated with dextran sodium sulfate (DSS). Following a lag phase, day 5 of DSS treatment was found clinically most critical as disease activity index (DAI) exhibited an exponential rise with severe weight loss and rectal bleeding. Surprisingly, on days 1-2, colonic TNF-α expression (70-80-fold) and tissue protein (50-fold) were increased, whereas IL-1β only increased on days 7-9 (60-90-fold). Days 3-6 of DSS treatment were characterized by a prominent down regulation in the expression of regulatory cytokines (40-fold for IL-10 and TGFβ) and mucin genes (15-18 fold for Muc2 and Muc3) concomitant with depletion of goblet cell and adherent mucin. Remarkably, treatment with TNF-α neutralizing antibody markedly altered DSS injury with reduced DAI, restoration of the adherent and goblet cell mucin and IL-1β and mucin gene expression. We conclude that early onset colitis is dependent on TNF-α that preceded depletion of adherent and goblet cell mucin prior to epithelial cell damage and these biomarkers can be used as therapeutic targets for UC.

Pathophysiology of IgG4-Related Disease

It is still a mystery what is the role of immunoglobulin G4 (IgG4) in IgG4-related disease, or which kinds of immune responses are involved in the pathogenesis. Possible involvement of autoimmunity is supported by the fact that the patients commonly have antinuclear antibodies or hyper γ-globulinemia. The expression of human leukocyte DR antigen in epithelia of the affected organs, and possible reactivity of serum IgG4 of the patients with tissue specimens also support an autoimmune etiology. On the other hand, the Th2-dominant cytokine expression profile resembles allergic disorders rather than classical autoimmune diseases. Another unique point is that many regulatory T-cells infiltrate into the inflamed sites, being associated with the active expression of regulatory cytokines. IL-10 and TGF-β might be the key molecules involved in the two major histological findings of IgG4-related disease: IgG4 class switch and diffuse fibrosis, because IL-10 and TGF-β have a potent function in directing B cells to produce IgG4 and a strong fibrogenic function, respectively. The function of IgG4 in this disorder is still unknown. Unique characters of IgG4 might suggest that IgG4 is not a pathogenetic antibody, but acts as an anti-inflammatory factor by binding to other types of IgG. Recent proteomics studies have discovered possible autoantigens and autoantibodies. Several hypotheses have been proposed so far, but the consensus remains to be reached. Immunopathology in IgG4-related disease is not typical for known disease entities. Keywords: IgG4, autoimmune pancreatitis, cytokine, regulatory T cells, pathology, Systemic lupus erythematosus, Hypophysitis, Chronic sclerosing dacryoadenitis, Chronic sclerosing sialadenitis, Inflammatory pseudotumor, Sclerosing cholangitis

Inflammatory mediators promote production of shed LRP1/CD91, which regulates cell signaling and cytokine expression by macrophages

LRP1 is a type-1 transmembrane receptor that mediates the endocytosis of diverse ligands. LRP1 β-chain proteolysis results in release of sLRP1 that is present in human plasma. In this study, we show that LPS and IFN-γ induce shedding of LRP1 from RAW 264.7 cells and BMMs in vitro. ADAM17 was principally responsible for the increase in LRP1 shedding. sLRP1 was also increased in vivo in mouse plasma following injection of LPS and in plasma from human patients with RA or SLE. sLRP1, which was purified from human plasma, and full-length LRP1, purified from mouse liver, activated cell signaling when added to cultures of RAW 264.7 cells and BMMs. Robust activation of p38 MAPK and JNK was observed. The IKK-NF-κB pathway was transiently activated. Proteins that bind to the ligand-binding clusters in LRP1 failed to inhibit sLRP1-initiated cell signaling, however an antibody that targets the sLRP1 N terminus was effective. sLRP1 induced expression of regulatory cytokines by RAW 264.7 cells, including TNF-α, MCP-1/CCL2, and IL-10. These results demonstrate that sLRP1 is generated in inflammation and may regulate inflammation by its effects on macrophage physiology.

Identification of a potent immunostimulatory oligodeoxynucleotide from Streptococcus thermophilus lacZ

Immunostimulatory sequences of oligodeoxynucleotides (ODNs), such as CpG ODNs, are potent stimulators of innate immunity. Here, we identified a strong immunostimulatory CpG ODN, which we named MsST, from the lac Z gene of Streptococcus (S.) thermophilus ATCC19258, and we evaluated its immune functions. In in vitro studies, MsST had a similar ability as the murine prototype CpG ODN 1555 to induce inflammatory cytokine production and cell proliferation. In mouse splenocytes, MsST increased the number of CD80+CD11c+and CD86+CD11c+ dendritic cells and CD4+CD25+ regulatory T cells. We also analyzed the effects of MsST on the expression of regulatory cytokines by real-time quantitative PCR. MsST was more potent at inducing interleukin-10 expression than the ODN control 1612, indicating that MsST can augment the regulatory T cell response via Toll-like receptor 9, which plays an important role in suppressing T helper type 2 responses. These results suggest that S. thermophilus, whose genes include a strong Immunostimulatory sequence-ODN, is a good candidate for a starter culture to develop new physiologically functional foods and feeds.

Therapeutic potential of helminth soluble proteins in TNBS-induced colitis in mice

The hygiene hypothesis suggests an inverse relationship between the incidence of parasitic infections and chronic inflammatory bowel diseases (IBD). We investigated the therapeutic potential of Schistosoma mansoni and Ancylostoma caninum soluble proteins on experimental colitis in mice. Colitis was induced by intrarectal administration of 10 mg trinitrobenzene sulfonic acid (TNBS) in 30% ethanol. Six hours after TNBS injection, mice were treated intraperitoneally with helminth proteins. Three days later, colonic inflammation was scored based on 5 inflammatory parameters: clinical disease activity, macroscopic and microscopic inflammation score, extent of inflammation, and myeloperoxidase (MPO) activity. To determine immunological pathways induced by S. mansoni proteins we measured cytokine profiles of T-lymphocytes from colon, mesenteric lymph nodes (MLN), and spleen by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Control mice showed no signs of inflammation, whereas all inflammatory parameters were significantly increased in mice with colitis. Treatment of mice with colitis with S. mansoni or A. caninum proteins decreased the macroscopic inflammation score, extent of inflammation, and MPO activity. Immunologically, induction of colitis significantly increased expression of IFN-gamma mRNA in the inflamed colon. Treatment with S. mansoni proteins caused a decrease of proinflammatory cytokines (IFN-gamma, IL-17) in colon and MLN, whereas the production of regulatory cytokines (IL-10, TGF-beta) increased significantly in colon tissue. Treatment with proteins of S. mansoni and A. caninum ameliorated TNBS-induced colitis in mice. S. mansoni proteins increased mRNA expression of regulatory cytokines while suppressing expression of proinflammatory cytokines. Therefore, we suggest a therapeutic potential for helminth proteins in the treatment of IBD.

Escherichia coli inoculation of porcine mammary glands affects local mRNA expression of Toll-like receptors and regulatory cytokines

The expression of mRNAs for the Toll-like receptors (TLRs) TLR2 and TLR4, pro- and anti inflammatory cytokines and their receptors was evaluated in mammary gland biopsy material collected from sows intramammarily inoculated with Escherichia coli strain O127 at parturition. Quantitative real-time RT-PCR analysis showed increased mRNA levels for TLR2, the proinflammatory cytokines interleukin IL-1β and tumor necrosis factor-alpha TNF-α, and the anti-inflammatory cytokine IL-10 in the inoculated mammary glands 24 h after inoculation. Increased mRNA levels of the proinflammatory cytokine IL-6 were only observed in the inoculated mammary glands of sows that developed clinical signs of mastitis. In contrast, the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-β1) mRNA was unaltered, as was mRNA expression for the IL-1 receptor type I (IL-1R1). Furthermore, IL-1β and IL-10 mRNA expression was higher in the inoculated mammary glands of sows that developed clinical signs of mastitis compared with sows that remained clinically healthy. Notably, sows that developed clinical signs of mastitis had significantly lower pre-inoculation levels of IL-1β mRNA than sows that remained clinically healthy. These findings suggest that development of coliform mastitis is associated with the level of local expression of regulatory cytokines in response to intramammary E. coli inoculation and infection.

Allergen challenge decreases mRNA expression of regulatory cytokines in whole blood of high‐IgE beagles

The kinetics of cytokine expression was evaluated in whole blood from high-IgE beagles previously sensitized to house dust mites (HDM) and known to develop clinical signs compatible with atopic dermatitis (AD) upon allergen exposure. Six high-IgE beagles were environmentally challenged daily for 3 h on three consecutive days with a HDM solution. Clinical signs were evaluated before, during, and after the conclusion of the challenge (days 0, 2, 4 and 17) and expression analyses of Th2 (IL-4 and IL-13) and regulatory (IL-10 and TGF-beta) cytokine mRNA were undertaken on blood samples at each time point using real-time polymerase chain reaction. Multiple comparison used to detect significant differences in clinical scores and expression levels of cytokine mRNA revealed that the clinical scores on days 2 and 4 were higher than those on days 0 and 17 (P < 0.05) but no temporal differences in the expression levels of IL-4 and IL-13 mRNA. Expression of TGF-beta mRNA was, however, significantly lower on day 4 (P < 0.05) and the expression of IL-10 mRNA on days 4 and 17 was significantly lower than those on days 0 and 2 (P < 0.05). The results indicate that allergen challenge decreases mRNA expression of regulatory cytokines in whole blood without enhanced mRNA expression of Th2 cytokines and suggest aberrant regulatory T-cell function in the immunopathogenesis of AD in high-IgE beagles.

Expression of IL-12 and T helper cell 1 cytokines in the fluid of paranasal sinus mucoceles

Objective The objective of this study was to assess the expression of regulatory cytokines and T helper cell (Th)1/Th2 cytokines in paranasal sinus mucoceles. Materials and methods Fluid samples of 12 paranasal sinus mucoceles were assessed by enzyme-linked immunosorbent assay for concentrations of regulatory cytokines (interleukin [IL]-10 and IL-12), Th1 cytokines (IL-2 and interferon gamma), and Th2 cytokines (IL-4 and IL-5). Results IL-12 was detected in all samples, whereas IL-10 was detected in only one case. The concentration of IL-12 tended to correlate with that of interferon gamma and was significantly and positively correlated with that of IL-2. Conclusions Th1 cytokines and the Th1 regulatory cytokine IL-12, but not IL-10, potentially play a key role in the pathogenesis of paranasal sinus mucoceles. Together with our recent report showing that lipopolysaccharide is highly detected in mucocele fluid, the data from this study suggest that the Th1 response induced by lipopolysaccharide may affect the immunological inflammation in the epithelium of paranasal sinus mucoceles.

Decrease and dysfunction of dendritic cells correlate with impaired hepatitis C virus‐specific CD4+ T‐cell proliferation in patients with hepatitis C virus infection

Through the production of stimulatory and suppressive cytokines, dendritic cells (DCs) regulate virus-specific immune responses that are crucial to virus eradication. To explore a possible role of DCs in the persistence of hepatitis C virus (HCV) infection, in this study we analysed peripheral blood DCs (PBDCs) in patients with chronic hepatitis C (CHC) compared with those in both healthy seronegative (HSN) controls and a group of subjects who had spontaneously resolved infection, defined as healthy HCV-seropositive (HSP), and we evaluated the relationships between PBDCs and HCV-specific CD4(+) T-cell reactivity. The number of PBDCs, their immunophenotype and expression of regulatory cytokines were evaluated by flow cytometry on whole-blood samples. HCV-specific CD4(+) T-cell activation, proliferation and cytokine production were evaluated in cultures of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with HCV peptides. We found that PBDCs from CHC subjects were numerically reduced and showed lower interleukin-12 (IL-12) and higher IL-10 expression than those from HSN controls. PBDCs from HSP subjects were similar to those from HSN controls. HCV-specific CD4(+) T-cell proliferation was less frequent and vigorous in CHC than in HSP patients and was directly related to the number of PBDCs and their IL-12 production but inversely related to their IL-10 production. Taken together, these results seem to suggest that cytokines of DC origin contribute to the regulation of HCV-specific immunity in CHC patients and indicate that PBDCs may represent a novel non-invasive tool for immune monitoring of these patients.

Cytokine and Chemokine Responses Associated with Clearance of a Primary Salmonella enterica Serovar Typhimurium Infection in the Chicken and in Protective Immunity to Rechallenge

Infection of poultry with Salmonella enterica serovar Typhimurium poses a significant risk to public health through contamination of meat from infected animals. Vaccination has been proposed to control infections in chickens. However, the vaccines are currently largely empirical, and our understanding of the mechanisms that underpin immune clearance and protection in avian salmonellosis is not complete. In this study we describe the cytokine, chemokine, and antibody responses and cellular changes in primary and secondary infections of chickens with Salmonella serovar Typhimurium. Infection of 1-week-old chickens induced early expression of a macrophage inflammatory protein (MIP) family chemokine in the spleen and liver, followed by increased expression of gamma interferon accompanied by increased numbers of both CD4(+) and CD8(+) T cells and the formation of granuloma-like follicular lesions. This response correlated with a Th1-mediated clearance of the systemic infection. Primary infection also induced specific immunoglobulin M (IgM), IgG, and IgA antibody responses. In contrast to previously published studies performed with newly hatched chicks, the expression levels of proinflammatory cytokines in the gastrointestinal tract were not greatly increased following infection. However, significant expression of the anti-inflammatory cytokine transforming growth factor beta4 was detected in the gut early in infection. Following secondary challenge, the birds were fully protected against systemic infection and showed a high level of protection against gastrointestinal colonization. Rapid expression of the MIP family chemokine and interleukin-6 was detected in the guts of these birds and was accompanied by an influx of lymphocytes. Increased levels of serum IgA-specific antibodies were also found following rechallenge. These findings suggest that cellular responses, particularly Th1 responses, play a crucial role in immune clearance in avian salmonellosis and that protection against rechallenge involves the rapid recruitment of cells to the gastrointestinal tract. Additionally, the high levels of inflammatory response found following Salmonella serovar Typhimurium infection of newly hatched chicks were not observed following infection of older birds (1 week old), in which the expression of regulatory cytokines appeared to limit inflammation.