IL-12p40 mRNA Expression Research Articles

Oral supplementation with Bifidobacterium infantis and 2′-fucosyllactose revives gut microbiota perturbation and intestinal and immune developmental delay following early-life antibiotic challenge in BALB/c mice

Probiotics and synbiotics can mitigate the negative health consequences of early-life antibiotic exposure. This study aimed to determine whether supplementation with Bifidobacterium longum ssp. infantis 79 (B79) or synbiotics composed of B79 and 2'-fucosyllactose (2'-FL) could mitigate the negative impact of ceftriaxone exposure in early-life. We found that antibiotic-treated mice exhibited lower body weight, crypt depth, short-chain fatty acid content, and α-diversity indices at weaning, while they increased the relative abundance of opportunistic pathogens (such as Enterococcus and Staphylococcus) and decreased the relative abundance of intestinal commensal bacteria. Supplementation with B79 and 2'-FL revived these antibiotic-induced negative effects and reduced the mRNA expression of IL-6, IL-12p40, and TNF-α in the spleen at weaning. Moreover, B79 and 2'-FL supplementation persistently improved crypt depth, propionic acid synthesis, and IgG and sIgA production and revived the gut microbiota structure and composition in adulthood. In conclusion, our study suggests that early-life supplementation with B79 alone or in combination with 2'-FL can mitigate ceftriaxone-induced negative effects on the gut microbiota and intestinal and immune development of mice, and these improvements can partially last into adulthood.

Expression of STAT3, IL-27p28, and IL-12p35 Is Deregulated and Linked to Autoimmune Markers in Chronic Spontaneous Urticaria.

Chronic spontaneous urticaria (CSU) is a common inflammatory disorder characterized by wheals, angioedema, or both, for more than 6 weeks. Autoimmunity is held to be one of the most frequent causes, but little is known about the expression and relevance of autoimmunity-driving genes in CSU, such as STAT3, STAT1, IL-27p28, and IL-12p35. To investigate CSU patients or the expression of STAT3, STAT1, IL-27p28, and IL-12p35 and possible links to clinical features. Gene expression levels, in 26 CSU patients and 19 healthy controls (HCs), were determined by quantitative real-time PCR. CSU patients were assessed for total IgE and IgG-anti-TPO, markers of autoimmune CSU. Patients with CSU showed significantly higher expression of STAT3, but not STAT1, and 17 and 10 of 26 CSU patients had elevated STAT3 expression and STAT3/STAT1 ratios, respectively, as compared to only 1 of 19 HCs. High STAT3 expression and STAT3/STAT1 ratios were linked to low IgE and elevated IgG-anti-TPO. As compared to HCs, CSU patients had markedly lower and correlated IL-27p28 and IL-12p35 mRNA expression levels. Low IL-27p28 and IL-12p35 expression levels were linked to higher STAT3/STAT1 ratios and low IgE. STAT3 upregulation, higher STAT3/STAT1 ratios, and IL-27p28 and IL-12p35 downregulation cluster with features of autoimmune CSU. The role of STAT3 as a potential pathogenic driver of autoimmune CSU and target of treatment should be explored further.

Biofilm-derived membrane vesicles exhibit potent immunomodulatory activity in Pseudomonas aeruginosa PAO1.

Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesiclesin Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharideof native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patternsin lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.

D-alanine Inhibits Murine Intestinal Inflammation by Suppressing IL-12 and IL-23 Production in Macrophages.

Free D-amino acids, which have different functions from L-amino acids, have recently been discovered in various tissues. However, studies on the potential interactions between intestinal inflammation and D-amino acids are limited. We examined the inhibitory effects of D-alanine on the pathogenesis of intestinal inflammation. We investigated serum D-amino acid levels in 40 patients with ulcerative colitis and 34 healthy volunteers. For 7 days [d], acute colitis was induced using dextran sulphate sodium in C57BL/6J mice. Plasma D-amino acid levels were quantified in mice with dextran sulphate sodium-induced colitis, and these animals were administered D-alanine via intraperitoneal injection. IFN-γ, IL-12p35, IL-17A, and IL-23p19 mRNA expression in the colonic mucosa was measured using real-time polymerase chain reaction [PCR]. In vitro proliferation assays were performed to assess naïve CD4+ T cell activation under Th-skewing conditions. Bone marrow cells were stimulated with mouse macrophage-colony stimulating factor to generate mouse bone marrow-derived macrophages. Serum D-alanine levels were significantly lower in patients with ulcerative colitis than in healthy volunteers. Dextran sulphate sodium-treated mice had significantly lower plasma D-alanine levels than control mice. D-alanine-treated mice had significantly lower disease activity index than control mice. IFN-γ, IL-12p35, IL-17A, and IL-23p19 mRNA expression levels were significantly lower in D-alanine-administered mice than in control mice. D-alanine suppressed naïve T cell differentiation into Th1 cells in vitro, and inhibited the production of IL-12p35 and IL-23p19 in bone marrow-derived macrophages. Our results suggest that D-alanine prevents dextran sulphate sodium-induced colitis in mice and suppresses IL-12p35 and IL-23p19 production in macrophages.

Immunotherapy Enhancement by Targeting Extracellular Tumor pH in Triple-Negative Breast Cancer Mouse Model.

Triple-negative breast cancer (TNBC), as one of the most aggressive forms of breast cancer, is characterized by a poor prognosis and a very low rate of disease-free and overall survival. In recent years, immunotherapeutic approaches targeting T cell checkpoint molecules, such as cytotoxic lymphocyte antigen-4 (CTLA-4), programmed death1 (PD-1) or its ligand, programmed death ligand 1 (PD-L1), have shown great potential and have been used to treat various cancers as single therapies or in combination with other modalities. However, despite this remarkable progress, patients with TNBC have shown a low response rate to this approach, commonly developing resistance to immune checkpoint blockade, leading to treatment failure. Extracellular acidosis within the tumor microenvironment (also known as the Warburg effect) is one of the factors preventing immune cells from mounting effective responses and contributing to immunotherapy treatment failure. Therefore, reducing tumor acidity is important for increasing cancer immunotherapy effectiveness and this has yet to be realized in the TNBC environment. In this study, the oral administration of sodium bicarbonate (NaHCO3) enhanced the antitumor effect of anti-PD-L1 antibody treatment, as demonstrated by generated antitumor immunity, tumor growth inhibition and enhanced survival in 4T1-Luc breast cancer model. Here, we show that NaHCO3 increased extracellular pH (pHe) in tumor tissues in vivo, an effect that was accompanied by an increase in T cell infiltration, T cell activation and IFN-γ, IL2 and IL12p40 mRNA expression in tumor tissues, as well as an increase in T cell activation in tumor-draining lymph nodes. Interestingly, these changes were further enhanced in response to combined NaHCO3 + anti-PD-L1 therapy. In addition, the acidic extracellular conditions caused a significant increase in PD-L1 expression in vitro. Taken together, these results indicate that alkalizing therapy holds potential as a new tumor microenvironment immunomodulator and we hypothesize that NaHCO3 can enhance the antitumor effects of anti-PD-L1 breast cancer therapy. The combination of these treatments may have an exceptional impact on future TNBC immunotherapeutic approaches by providing a powerful personalized medicine paradigm. Therefore, our findings have a great translational potential for improving outcomes in TNBC patients.

Increased gut permeability and intestinal inflammation precede arthritis onset in the adjuvant-induced model of arthritis

BackgroundIntestinal inflammation, dysbiosis, intestinal permeability (IP), and bacterial translocation (BT) have been identified in patients with spondyloarthritis but the time at which they appear and their contribution to the pathogenesis of the disease is still a matter of debate.ObjectivesTo study the time-course of intestinal inflammation (I-Inf), IP, microbiota modification BT in a rat model of reactive arthritis, the adjuvant-induced arthritis model (AIA).MethodsAnalysis was performed at 3 phases of arthritis in control and AIA rats: preclinical phase (day 4), onset phase (day 11), and acute phase (day 28). IP was assessed by measuring levels of zonulin and ileal mRNA expression of zonulin. I-inf was assessed by lymphocyte count from rat ileum and by measuring ileal mRNA expression of proinflammatory cytokines. The integrity of the intestinal barrier was evaluated by levels of iFABP. BT and gut microbiota were assessed by LPS, soluble CD14 levels, and 16S RNA sequencing in mesenteric lymph node and by 16S rRNA sequencing in stool, respectively.ResultsPlasma zonulin levels increased at the preclinical and onset phase in the AIA group. Plasma levels of iFABP were increased in AIA rats at all stages of the arthritis course. The preclinical phase was characterized by a transient dysbiosis and increased mRNA ileal expression of IL-8, IL-33, and IL-17. At the onset phase, TNF-α, IL-23p19, and IL-8 mRNA expression were increased. No changes in cytokines mRNA expression were observed at the acute phase. Increased CD4+ and CD8+ T cell number was measured in the AIA ileum at day 4 and day 11. No increase in BT was observed.ConclusionThese data show that intestinal changes precede the development of arthritis but argue against a strict “correlative” model in which arthritis and gut changes are inseparable.

In vitro effects of monophosphoryl lipid A and Poly I:C combination on equine cells.

Toll-like receptor (TLR) agonists have been used as adjuvants to modulate immune responses in both animals and humans. The objective of this study was to evaluate the combined effects of the TLR 4 agonist monophosphoryl lipid A (MPL) and the TLR 3 agonist polyinosinic:polycytidylic acid (Poly I:C) on equine peripheral blood mononuclear cells (PBMCs), monocyte-derived dendritic cells (MoDCs), and bone marrow-derived mesenchymal stromal cells (BM-MSCs). The PBMCs, MoDCs, and BM-MSCs collected from three mixed breed horses were treated with MPL, Poly I:C, and their combination. The mRNA expression of interferon gamma (IFN-γ), interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-12p40, tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), and monocyte chemoattractant protein-1 (MCP-1) was determined using real-time polymerase chain reaction. The combination of MPL and Poly I:C significantly upregulated immunomodulatory responses in equine cells/ without cytotoxicity. The combination induced greater mRNA expression of pro-inflammatory cytokines IFN-γ and IL-6 than MPL or Poly I:C stimulation alone in PBMCs. In addition, the combination induced significantly higher mRNA expression of IL-1β, IL-6, and IL-12p40 in MoDCs, and IL-8, MCP-1, and VEGF in BM-MSCs compared to stimulation with a single TLR agonist. The combination of MPL and Poly I:C can be used as a potential adjuvant candidate for vaccines to aid in preventing infectious diseases in horses.

Characterization of Porcine Monocyte-Derived Macrophages Cultured in Serum-Reduced Medium.

Simple SummaryThe aim of this study was to provide an improved in vitro model of porcine monocyte-derived macrophages (MDMs) based on reduced-serum culture. The partial or complete absence of serum in media is suitable for experimental studies focused on molecules that are naturally present in serum or in cultures used for subsequent protein purification. Moreover, the benefit of serum-free or reduced-serum supplements follows the current trend of reducing FBS use in biological research. In order to confirm that a reduced-serum MDM culture is suitable for in vitro functional studies of porcine macrophages, we assessed their morphology, yield after differentiation, surface marker expression, respiratory burst, and phagocytic activity, as well as the induction of cytokine gene expression.The aim of this study was to establish a cell culture system for the generation of porcine monocyte-derived macrophages (MDMs) under reduced-serum conditions. Cultures based on either the Nu-Serum™ Growth Medium Supplement (NUS) or a conventional fetal bovine serum (FBS) were compared, which included the assessment of FBS from two different providers (FBS1 and FBS2). The data obtained confirmed the significant impact of culture conditions on in vitro-generated MDMs. The MDMs cultured under reduced-serum conditions showed increased levels of IL-1β and CD86 mRNA and a proinflammatory cytokine profile, characterized by the increased mRNA expression of IL-23p19, CXCL10, and CCL5. Phagocytic and respiratory burst activities were not adversely affected. Surprisingly, the difference between the two FBSs was much more pronounced than the effect of the reduced-serum supplement. The FBS1 culture conditions gave rise to macrophages with higher surface levels of CD14, CD16, and CD163, a lower CD80 mRNA expression, and an increased induction of IL-10 gene expression. In contrast, none of these trends were observed in macrophage cultures supplemented with FBS2. Instead, the FBS2 culture showed increased levels of IL-1b and CD86 mRNA. In conclusion, reduced-serum culture is a useful tool for in vitro porcine MDM generation, in line with the current research trend of reducing FBS use in biological research.

Gelsolin Attenuates Neonatal Hyperoxia-Induced Inflammatory Responses to Rhinovirus Infection and Preserves Alveolarization.

Prematurity and bronchopulmonary dysplasia (BPD) increase the risk of asthma later in life. Supplemental oxygen therapy is a risk factor for chronic respiratory symptoms in infants with BPD. Hyperoxia induces cell injury and release of damage-associated molecular patterns (DAMPs). Cytoskeletal filamentous actin (F-actin) is a DAMP which binds Clec9a, a C-type lectin selectively expressed on CD103+ dendritic cells (DCs). Co-stimulation of Clec9a and TLR3 induces maximal proinflammatory responses. We have shown that neonatal hyperoxia (a model of BPD) increases lung IL-12+Clec9a+CD103+ DCs, pro-inflammatory responses and airway hyperreactivity following rhinovirus (RV) infection. CD103+ DCs and Clec9a are required for these responses. Hyperoxia increases F-actin levels in bronchoalveolar lavage fluid (BALF). We hypothesized that the F-actin severing protein gelsolin attenuates neonatal hyperoxia-induced Clec9a+CD103+ DC-dependent pro-inflammatory responses to RV and preserves alveolarization. We exposed neonatal mice to hyperoxia and treated them with gelsolin intranasally. Subsequently we inoculated the mice with RV intranasally. Alternatively, we inoculated normoxic neonatal mice with BALF from hyperoxia-exposed mice (hyperoxic BALF), RV and gelsolin. We analyzed lung gene expression two days after RV infection. For in vitro studies, lung CD11c+ cells were isolated from C57BL/6J or Clec9agfp-/- mice and incubated with hyperoxic BALF and RV. Cells were analyzed by flow cytometry. In neonatal mice, gelsolin blocked hyperoxia-induced Il12p40, TNF-α and IFN-γ mRNA and protein expression in response to RV infection. Similar effects were observed when gelsolin was co-administered with hyperoxic BALF and RV. Gelsolin decreased F-actin levels in hyperoxic BALF in vitro and inhibited hyperoxia-induced D103lo DC expansion and inflammation in vivo. Gelsolin also attenuated hyperoxia-induced hypoalveolarization. Further, incubation of lung CD11c+ cells from WT and Clec9agfp-/- mice with hyperoxic BALF and RV, showed Clec9a is required for maximal hyperoxic BALF and RV induced IL-12 expression in CD103+ DCs. Finally, in tracheal aspirates from mechanically ventilated human preterm infants the F-actin to gelsolin ratio positively correlates with FiO2, and gelsolin levels decrease during the first two weeks of mechanical ventilation. Collectively, our findings demonstrate a promising role for gelsolin, administered by inhalation into the airway to treat RV-induced exacerbations of BPD and prevent chronic lung disease.

Glycyrrhizin Attenuates Salmonella Typhimurium-Induced Tissue Injury, Inflammatory Response, and Intestinal Dysbiosis in C57BL/6 Mice.

Salmonellae are one of the most important foodborne pathogens, which threaten the health of humans and animals severely. Glycyrrhizin (GL) has been proven to exhibit anti-inflammatory and tissue-protective properties. Here, we investigated the effects of GL on tissue injury, inflammatory response, and intestinal dysbiosis in Salmonella Typhimurium-infected mice. Results showed that GL or gentamicin (GM) significantly (P < 0.05) alleviated ST-induced splenomegaly indicated by the decreased spleen index, injury of liver and jejunum indicated by the decreased hepatocytic apoptosis, and the increased jejunal villous height. GL significantly (P < 0.05) increased secretion of inflammatory cytokines (IFN-γ, IL-12p70, IL-6, and IL-10) in spleen and IL-12p40 mRNA expression in liver. Meanwhile, GL or GM pre-infection treatments significantly (P < 0.05) decreased ST-induced pro-inflammatory cytokine (IFN-γ, TNF-α, and IL-6) expression in both spleen and liver and increased (P < 0.05) anti-inflammatory cytokine IL-10 secretion in spleen. Furthermore, GL or GM pre-infection treatment also regulates the diversities and compositions of intestinal microbiota and decreased the negative connection among the intestinal microbes in ST-infected mice. The above findings indicate that GL alleviates ST-induced splenomegaly, hepatocytic apoptosis, injury of jejunum and liver, inflammatory response of liver and spleen, and intestinal dysbacteriosis in mice.

Effect of chemically modified tetracycline-8 (CMT-8) on hematology, blood chemistry, cytokines and peripheral blood lymphocyte subsets of healthy dogs

Tetracyclines are antibiotics widely used in human and veterinary medicine. Effects on the immune system and inflammatory response, including effects on blood leukocytes proliferation and function and in cytokines synthesis, have been described. Chemically modified tetracyclines (CMT) have lost their antimicrobial activity, but maintain these other properties. This study analyzes the effect of chemically modified tetracycline-8 (CMT-8) on the evolution of complete blood count, blood chemistry, the mRNA expression of selected cytokines and peripheral blood lymphocyte subpopulations distribution in healthy dogs. CMT-8 at a dose of 10 mg/kg once daily was administered per os to six healthy dogs. A control group of five healthy dogs, living in the same conditions than dogs treated with CMT-8, received placebo with an identical therapeutic regimen. When given at the doses used in this study, no side effects of CMT-8 were detected, suggesting a good tolerance and a limited toxicity of the drug. Dogs treated with CMT-8 showed a gradual increase in mean corpuscular hemoglobin. The administration of CMT-8 in healthy dogs did not affect blood mRNA expression of IFN-γ, TNFα, IL-4, IL-6, IL-10, IL-12 p40 and IL-13. However, the lymphocytes expressing class II MHC on their surface decreased during the first two weeks of CMT-8 treatment and subsequently increased for the next three months. Considering the absence of antimicrobial properties of the drug, the effects of CMT-8 detected in this study seem to be unrelated to the classical antimicrobial activity attributed to tetracyclines.

Colonic mucosal and cytobrush sample cytokine mRNA expression in canine inflammatory bowel disease and their correlation with disease activity, endoscopic and histopathologic score.

Canine inflammatory bowel disease (IBD) is a group of chronic gastrointestinal disorders, the pathogenesis of which remains elusive, but it possibly involves the interaction of the intestinal immune system with luminal microbiota and food-derived antigens. Mucosal cytokines profiles in canine IBD have been investigated mainly in small intestinal disease, while data on cytokine profiles in large intestinal IBD are limited. The objective of this study was to measure colonic mucosal and cytobrush sample messenger (m)RNA expression of interleukin (IL)-1β, IL-2, IL-12p40, IL-23p19, tumor necrosis factor-alpha (TNF-α) and chemokine C‐C motif ligand (CCL28) in dogs with IBD and healthy controls using quantitative real-time polymerase chain reaction (PCR), and assess their correlation with clinical disease activity, endoscopic and histopathologic score. Dogs with IBD had a significantly increased mRNA expression of IL-1β, IL-23p19 and CCL28 in the colonic mucosa, compared to healthy controls. None of the selected cytokines had significantly different mRNA expression in the colonic cytobrush samples between the two groups or between the colonic mucosa and cytobrush samples of dogs with IBD. Finally, there was a statistically significant correlation of clinical disease activity with endoscopic activity score and fibrosis and atrophy of the colonic mucosa in dogs with large intestinal IBD. IL-1β, IL-23p19 and CCL28 could play a role in the pathogenesis of canine large intestinal IBD. Colonic cytokine expression does not correlate with clinical disease activity and/or endoscopic score. However, clinical signs reflect the severity of endoscopic lesions.

Neonatal porcine blood derived dendritic cell subsets show activation after TLR2 or TLR9 stimulation

The present study investigated the innate immune response in vitro to determine porcine neonate responses with Toll-like receptor (TLR)2 ligand (Pam3Cys) or TLR9 ligand (CpG) and compared these with adults. We identified the same phenotypically defined dendritic cell (DC) subsets and DC proportions in porcine neonate and adult blood by flow cytometry, which were plasmacytoid DCs (pDCs): CD14−CD4+CD172a+CADM1-) and conventional DCs (cDCs), being further divided into a cDC1 (CD14−CD4−CD172alowCADM1+) and a cDC2 (CD14−CD4−CD172a+CADM1+) subset. With neonatal cells, the TLR2 ligand induced a stronger TNF expression in monocytes and pDCs, and a stronger CD80/86 upregulation in cDC1, when compared to adult cells. Furthermore, in neonatal mononuclear cells TLR9 ligand was more potent at inducing IL12p40 mRNA expression. These results indicate clear responses of porcine neonatal antigen presenting cells after TLR2 and TLR9 stimulation, suggesting that corresponding ligands could be promising candidates for neonatal adjuvant application.

Reprint of “Dual blockade of the pro-inflammatory chemokine CCL2 and the homeostatic chemokine CXCL12 is as effective as high dose cyclophosphamide in murine proliferative lupus nephritis”

Induction therapy of proliferative lupus nephritis still requires the use of unselective immunosuppressive drugs with significant toxicities. In search of more specific drugs with equal efficacy but fewer side effects we considered blocking pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) and homeostatic chemokine stromal cell-derived factor-1 (SDF-1/CXCL12), which both contribute to the onset and progression of proliferative lupus nephritis yet through different mechanisms. We hypothesized that dual antagonism could be as potent on lupus nephritis as the unselective immunosuppressant cyclophosphamide (CYC). We estimated serum levels of CCL2 and CXCL12 in patients with SLE (n=99) and compared the results with healthy individuals (n=21). In order to prove our hypothesis we used l-enantiomeric RNA Spiegelmer® chemokine antagonists, i.e. the CCL2-specific mNOX-E36 and the CXCL12-specific NOX-A12 to treat female MRL/lpr mice from week 12 to 20 of age with either anti-CXCL12 or anti-CCL2 alone or both. SLE patients showed elevated serum levels of CCL2 but not of CXCL12. Female MRL/lpr mice treated with dual blockade showed significantly more effective than either monotherapy in preventing proteinuria, immune complex glomerulonephritis, and renal excretory failure and the results are at par with CYC treatment. Dual blockade reduced leukocyte counts and renal IL-6, IL-12p40, CCL-5, CCL-2 and CCR-2 mRNA expression. Dual blockade of CCL2 and CXCL12 can be as potent as CYC to suppress the progression of proliferative lupus nephritis probably because the respective chemokine targets mediate different disease pathomechanisms, i.e. systemic autoimmunity and peripheral tissue inflammation.

307 Identification of resolvin E1, an omega-3 poly-unsaturated fatty acids-derived lipid mediator, as an inhibitor for psoriatic dermatitis by downregulating migration of IL-17A-producing cutaneous γδ T cells and IL-23 production from dendritic cells

The potential of omega-3 poly-unsaturated fatty acids (PUFAs) as a therapeutic target for psoriasis is a long-disputed question, since various epidemiological studies have suggested that intake of omega-3 PUFAs have ameliorating effect on the development of psoriasis. However, its actual significance and the molecular mechanisms have not been explored. In this study, we examined the effect of resolvin E1 (RvE1), a metabolite derived from omega-3 PUFAs, on psoriatic dermatitis using an imiquimod-induced mouse psoriasis model. RvE1 potently suppressed the inflammatory cell infiltration and epidermal hyperplasia in the psoriatic skin. In addition, RvE1 decreased the mRNA expression of key cytokines including IL-23 and IL-17A, which are produced by cutaneous dendritic cells (DCs) and γδ T cells, respectively. Consistently, RvE1 inhibited IL-23p19 and IL-23p40 mRNA expression in bone marrow-derived DCs in vitro. Furthermore, an in vivo cell migration assay using Kaede (a photo-convertible protein)-transgenic mice revealed that RvE1 significantly inhibited migration of both cutaneous DCs and γδ T cells from skin to draining lymph nodes (dLNs), an essential step for γδ T cell expansion in dLNs and their subsequent recirculation into skin to accelerate the psoriatic dermatitis. We have also revealed that this suppressive effect of RvE1 on γδ T cell migration was mediated by RvE1’s antagonistic function on BLT1, a receptor of leukotriene B4. Taken together, these results indicate that RvE1 exerts anti-inflammatory effects on psoriatic dermatitis in two manners; the attenuation of γδ T cell migration from skin to dLNs and the inhibition of IL-23 production from DCs. Our results have clarified a novel mechanism of omega-3 PUFA-mediated amelioration of psoriasis, and suggest a potential of RvE1 as a therapeutic target for psoriasis.

Molecular, biochemical characterization and assessment of immunogenic potential of cofactor-independent phosphoglycerate mutase against Leishmania donovani: a step towards exploring novel vaccine candidate.

Despite immense efforts, vaccine against visceral leishmaniasis has yet not been developed. Earlier our proteomic study revealed a novel protein, cofactor-independent phoshoglycerate mutase (LdiPGAM), an important enzyme in glucose metabolism, in T helper cells type 1 (Th1) stimulatory region of soluble Leishmania donovani antigen. In this study, LdiPGAM was biochemically and molecularly characterized and evaluated for its immunogenicity and prophylactic efficacy against L. donovani. Immunogenicity of recombinant LdiPGAM (rLdiPGAM) was initially assessed in naïve hamsters immunized with it by analysing mRNA expression of inducible nitric oxide (NO) synthase (iNOS) and other Th1/T helper cells type 2 cytokines, which revealed an upregulation of Th1 cytokines along with iNOS. Immunogenicity of rLdiPGAM was further evaluated in lymphocytes of treated Leishmania-infected hamsters and peripheral blood mononuclear cells of Leishmania patients in clinical remission by various parameters, viz. lymphoproliferation assay and NO production (hamsters and patients) and levels of various cytokines (patients). rLdiPGAM induced remarkable Lymphoproliferative response and NO production in treated Leishmania-infected hamsters as well as in patients and increase in interferon gamma (IFN-γ), interleukin-12 (IL-12p40) responses in Leishmania patients in clinical remission. Vaccination with rLdiPGAM exerted considerable prophylactic efficacy (73%) supported by increase in mRNA expression of iNOS, IFN-γ and IL-12p40 with decrease in transforming growth factor beta and interleukin-10. Above results indicate the importance of rLdiPGAM protein as a potential vaccine candidate against visceral leishmaniasis.

Nickel Sulfate Promotes IL-17A Producing CD4+ T Cells by an IL-23-Dependent Mechanism Regulated by TLR4 and Jak-STAT Pathways

Allergic contact dermatitis, caused by nickel, is a delayed-type hypersensitivity reaction, and 14.5% of the general population may be affected in Europe. Among a wide range of cytokines, the IL-12 family has unique structural and immunological characteristics. Whereas IL-12p70 promotes T helper (Th) 1 cell polarization, IL-23 promotes Th17 cell development and both have been isolated from nickel-allergic patients. In this work, we were interested in understanding the mechanism behind nickel-induced Th17 cell development. We showed that nickel induced an early production of IL-23 in human monocyte-derived dendritic cells along with an increase in the expression of il-23p19 and il-12p40 mRNA. However, the production of a significant level of IL-12p70 required an additional signal such as IFN-γ. Moreover, nickel-treated monocyte-derived dendritic cells induced an increase in the percentage of IL-17A+ CD4+ T cells, an effect reduced by IL-23 neutralization. We then investigated the molecular mechanism of IL-23 production. Our results showed that toll-like receptor 4, p38 mitogen-activated protein kinase, and NF-κB were involved in IL-23 production induced by nickel. However, Jak-signal transducer and activator of transcription activation seems to maintain the IL-23/IL-12p70 balance by limiting IL-23 production and promoting Th1 polarization. These results indicate that nickel-induced Th17 cell development is dependent on the production of IL-23 by human monocyte-derived dendritic cells via toll-like receptor 4, p38 mitogen-activated protein kinase, NF-κB, and Jak-signal transducer and activator of transcription pathways.

CD46 activation on monocyte-derived dendritic cells

Abstract Objective CD46 is an important immune modulatory receptor that influences the expression of certain cytokines in different cell types. This function has been proposed to depend on expression of specific isoforms of CD46. The common isoforms are named BC1, BC2, C1 and C2, and activation of T cells induces a switch towards dominance of BC2 and C2. However, the effect of CD46 activation on the cytokine production in dendritic cells (DCs) has so far been inconclusive and the relative distribution of CD46 isoforms in monocytes and DCs is unknown. Methods Monocytes were isolated from peripheral mononuclear blood cells and differentiated to dendritic cells (moDC). The maturation of moDCs was confirmed by flow cytometry using a panel of maturation markers. Monocytes and moDCs were stimulated with LPS in presence or absence of CD46 activation and the production of various cytokines were analyzed by real-time PCR. The monocytes and moDCs were furthermore analyzed for their expression of different CD46 isoforms by an isoform-specific real-time PCR assay. Results Upregulation of CD40, CD80, and CD83 on the surface of moDCs demonstrated the maturation of monocytes to moDCs. Activation of CD46 in moDCs during LPS stimulation was found to reduce the mRNA expression of IL12p40 and CXCL10 while no effect of CD46 activation on these cytokines was observed in monocytes. Examination of CD46 isoforms revealed a dominant expression of the isoforms BC2/C2 in moDCs upon differentiation from monocytes. Conclusion The results presented here suggest that CD46 activation reduces the pro-inflammatory potential of LPS stimulation in moDCs but not in monocytes. These functional changes are associated with a switch towards dominant expression of BC2/C2 isoforms.

Immune response after autologous hematopoietic stem cell transplantation in type 1 diabetes mellitus

BackgroundThis study explored the details of the immune response after autologous hematopoietic stem cell transplantation (AHSCT) treatment in type 1 diabetes mellitus.MethodsPeripheral blood mononuclear cells (PBMCs) from 18 patients with type 1 diabetes mellitus were taken at baseline and 12 months after AHSCT or insulin-only therapy. The lymphocyte proliferation, mRNA expression and secretion of pro-inflammatory and anti-inflammatory cytokines belonging to T-helper type 1 (Th1), T-helper type 17 (Th17) and regulatory T (Treg) cells in PBMC culture supernatants were assessed.ResultsCompared with patients receiving insulin-only treatment, the patients receiving AHSCT treatment showed better residual C-peptide secretion, lower anti-GAD titers and less exogenous insulin dosages after 12 months of follow-up. AHSCT treatment was associated with significantly reduced Th1 and Th17 cell proportions as well as decreased IFN-γ, IL-2, IL-12p40 and IL-17A levels in the PBMC culture supernatants (all P < 0.05). Although there was no significant Treg cell expansion after AHSCT treatment, we observed increased IL-10, TGF-β and Foxp3 mRNA expression and increased TGF-β levels. However, we found no significant changes in the T-cell subpopulations after insulin treatment, except for higher IL-12p40 mRNA expression and a lower proportion of Treg cells.ConclusionsAHSCT treatment was associated with decreased expansion and function of Th1 and Th17 cells, which may explain the better therapeutic effect of AHSCT compared with the traditional intensive insulin therapy.Trial registrationClinicaltrials.gov NCT00807651. Registered 18 December 2008.

Ginger extract modulates the expression of IL-12 and TGF-β in the central nervous system and serum of mice with experimental autoimmune encephalomyelitis.

The main function of IL-12 is differentiation of naive T cells intoTh1 cells and TGF-β is a powerful immunoregulatory cytokine. The immunomodulatory and anti-inflammatory properties of ginger have also been reported in some studies. The aim of this study was to evaluate the effects of ginger extract on the expression of IL-12 and TGF-β in a model of experimental autoimmune encephalomyelitis (EAE). EAE was induced in C57BL/6 mice by immunization with myelin oligodendroglial glycoprotein emulsified in complete Freund's adjuvant. The mice were administered intra-peritoneally with ginger extracts or PBS, from day +3 to +30. On day 31, mice were scarified and the expression of IL-12 and TGF-β mRNA in the spinal cord were determined by using real time-PCR. The serum levels of cytokines were measured by ELISA. In PBS-treated EAE mice, the expression of IL-12 P35 and IL-12 P40 mRNA in the CNS and the mean serum levels of IL-12 were significantly higher than those of healthy group (p<0.001). In ginger-treated EAE mice, the expression of IL-12 mRNA and its serum levels were significantly lower as compared to PBS-treated EAE mice. No significant difference was observed between PBS-treated EAE mice and healthy group regarding the expression of TGF-β mRNA. In ginger (300 mg/kg)-treated EAE group, the expression of TGF-β mRNA and its serum levels were significantly higher in comparison to PBS-treated EAE mice (p<0.01 and p<0.05, respectively). These results indicated that ginger extract modulates the expression of IL-12 and TGF-β in CNS and serum of EAE mice.