IFN-γ mRNA Levels Research Articles

Intrauterine sensitization of ovalbumin in the third trimester increases the risk of food allergy in progeny.

Intrauterine sensitization caused by food allergens plays an important role in the food allergy development in progeny. The aim of our study was to determine the critical period of intrauterine sensitization during pregnancy. Female mice were exposed to ovalbumin (OVA) during different trimesters of pregnancy. Lymphocytes from their offspring were isolated and cultured, and proliferation was evaluated by CCK-8 assay. The levels of IFN-γ and IL-4 in serum were measured using ELISA. In addition, the expressions of IFN-γ and IL-4 mRNAs and proteins were detected by real-time PCR and western blot. The mice were divided into the first trimester pregnancy (FTP1 and FTP2) group, the second trimester pregnancy (STP1 and STP2) group, and the third trimester pregnancy (TTP1 and TTP2) group based on the stages of pregnancy in which their mothers were exposed to OVA and their ages. The OVA-specific lymphocyte proliferation of the TTP1 group was statistically significantly greater that in the FTP1 and STP1 groups. The serum level of IFN-γ in the TTP1 group was significantly decreased, and the serum level of IL-4 in the TTP1 group was significantly increased compared with the levels in the FTP1 and STP1 groups. The mRNA and protein expression levels of IFN-γ in the TTP1 group were significantly decreased and the mRNA and protein expression levels of IL-4 in this group were significantly increased compared with the levels in the FTP1 and STP1 groups. Our results suggest that OVA-induced intrauterine sensitization in the third trimester may increase the risk of food allergy after birth.

Costimulation blockade by combining CTLA4Ig with anti-CD40L mAb markedly inhibits the inflammatory response of experimental autoimmune myocarditis

The aim of this study was to investigate the effect of costimulation blockade with cytotoxic T-lymphocyte-associated-antigen 4-immunoglobulin (CTLA4Ig) and anti-CD40L monoclonal antibody (anti-CD40L mAb) on an experimental autoimmune myocarditis (EAM) mouse model. Characteristics of myocardial tissue were observed by hematoxylin and eosin (H&E) staining. The messenger RNA (mRNA) levels of CTLA4, CD40L, IFN-γ, and IL-4 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Serum concentrations of IFN-γ and IL-4 were determined by ELISA. After immune intervention, the inflammatory score, mRNA levels of CTLA4 and CD40L, and IFN-γ level were decreased. Furthermore, these parameters in the combinational intervention group (blockade by CTLA4Ig and anti-CD40L mAb) were significantly decreased, compared to the single intervention group (blockade by CTLA4Ig or anti-CD40L mAb). However, after costimulation, blockade serum IL-4 levels were increased. Therefore, costimulation blockade by combination CTLA4Ig and anti-CD40L mAb could more effectively inhibit the inflammatory response of EAM than single use of CTLA4Ig or anti-CD40L mAb.

Hypermethylation of IFN-γ in oral cancer tissues.

The study aimed to evaluate the methylation pattern of the interferon-gamma (IFN-γ) gene in oral cancer tissues compared with normal and benign oral disease tissues. The oral tissues were gained from the patients of 85 cases of oral squamous cell carcinoma (OSCC), 47 cases of oral dysplastic lesions, and 53 normal biopsies. IFN -γ methylation in oral tissues was verified through methylation-specific polymerase chain reaction (PCR) and DNA sequencing analyses, and the expression levels of IFN-γ messenger RNA (mRNA) and protein were detected using real-time reverse transcription (RT)-PCR and enzyme-linked immunosorbent assays, respectively. IFN-γ was localized in macrophages from oral tissues and detected via immunostaining. IFN-γ mRNA and protein expression levels were evidently decreased in oral cancer tissues, whereas the IFN-γ methylation rate was significantly higher in malignant tumors than in benign and normal tissues (normal, 22.6%; benign, 38.3%; and cancer, 55.3%; P<0.05). Furthermore, the expression of IFN-γ mRNA was significantly downregulated in oral tumors with methylation compared with tumors without methylation, as determined by real-time RT-PCR (4.76-fold difference; P<0.05). Likewise, mRNA expression was downregulated by 6.79-fold in oral epithelial dysplasia tissues with methylation compared with those without methylation (P<0.01). Co-immunostaining to detect MAC2 and IFN-γ demonstrated that macrophages comprised the main source of IFN-γ in oral tissues. IFN-γ methylation demonstrated a significant association with the clinical stage, histopathology grade, and primary tumor. Aberrant IFN-γ promoter methylation may be involved in the process of tumorigenesis of oral cancer. IFN-γ hypermethylation during the process of oral carcinogenesis could be useful for the clinical diagnosis and treatment for OSCC.

Suppressive Effect of a Thioamide-Related Compound SH-2251 on a Murine Allergic Rhinitis Model

Background: SH-2251 inhibits IL-5 production and type2 airway inflammation in a murine bronchial asthma model. However, the therapeutic effect of SH-2251 on allergic rhinitis (AR) has not been tested. We investigated whether or not SH-2251 could improve the nasal symptoms and inflammation in the nasal mucosa of a murine model of AR.Methods: AR was induced via intraperitoneal injection of ovalbumin (OVA) followed by daily intranasal challenge with OVA. SH-2251 (10 mg/kg) or vehicle (DMSO+Tween80) was administered orally once a day in the same period as the nasal challenge. Nasal symptoms were evaluated by counting the number of sneezing and nasal rubbing events on days 14, 17, 21, 28 and 35. On day 35, the levels of Il4, Il5, Il13, Ifnγ and Il1rl1 mRNA in the nasal mucosa were examined. The histological findings were examined on day 36.Results: The numbers of sneezing and nasal rubbing events were significantly decreased by the administration of SH-2251. Both eosinophil infiltration and the hickness of the nasal mucosa were improved in the SH-2251- administered mice compared that in vehicle-treated control mice. The mRNA expression of Il5, Il13 and Il1rl1 in the nasal mucosa was significantly decreased in the SH-2251-treated group compared to the vehicle group.Conclusion: These results suggest that SH-2251 may be a new therapeutic candidate for AR. In addition, SH-2251 might improve the pathogenesis of type2 chronic inflammation by targeting the IL-33 signaling via the inhibition of Il1rl1 expression.

A Proinflammatory Effect of the β-Glucan from Pleurotus cornucopiae Mushroom on Macrophage Action.

PCPS from P. citrinopileatus mushroom extract is a β-1,6-glucan possessing a proinflammatory effect on innate immune cells. The PCPS stimulated THP-1 macrophages to secrete significant levels of TNF. Moreover, the mRNA expressions of TNF and IL-1β were significantly enhanced by PCPS treatment. However, the PCPS did not induce to express both IL-12 and IL-10 mRNA in the macrophages. Next, the P. cornucopiae extract (containing mainly PCPS) treatment against mice showed significant increases in TNF and IL-1β mRNA expressions in the peritoneal macrophages of them. In this study, the expression levels of IFNγ mRNA in the spleen were almost the same between the extract- (PCPS-) treated group and control group. However, the expression of IL-4 mRNA showed a lower level in the extract-treated group than that in the control. Our results suggested that the PCPS could induce proinflammatory action in the immune response. In addition, the proinflammatory effect of the PCPS on THP-1 was enhanced by 5′-GMP-Na, while it was reduced by vitamin D2. These two compounds are majorly contained in the P. citrinopileatus mushroom. Therefore, these results suggested that the P. citrinopileatus mushroom might contain other immune regulative compounds, such as vitamin D2, as well as PCPS.

Boost of Immune Responses Against NY-ESO-1 Following Local Radiation Therapy in Patients with Multiple Myeloma: A Potential Contribution to Tumor Immunosurveillance

IntroductionLocal radiation therapy (RT) is commonly employed to treat bone lesions and/or extramedullary manifestations of multiple myeloma (MM) or solitary bone plasmacytoma (SBP). Besides providing sufficient control of local tumor growth, radiation therapy may also contribute to broader anti-tumor immunity by generating off-site cellular immune responses. Here, we put a focus on tumor-associated antigens (TAAs) well known for their expression in MM such as Wilms' Tumor Protein 1 (WT1), New York esophageal squamous cell carcinoma 1 (NY-ESO-1), human telomerase reverse transcriptase (hTERT), mucin 1 (MUC1), and preferentially expressed antigen of melanoma (PRAME) and assessed CD8-mediated immune responses before and during radiation.MethodsWe prospectively recruited HLA-A*02:01-positive patients with multiple myeloma or solitary plasmacytoma requiring local radiation therapy for bone and extramedullary lesions in this scientific, non-interventional study, supported by a grant of the Wilhelm Sander Foundation, Germany, and approved by our local ethical committee. All patients provided written informed consent. Immune responses against HLA-A*02:01-restricted peptides of WT1, NY-ESO-1, hTERT, MUC1, and PRAME were assessed at three different time points: prior to RT, end of RT and six to eight weeks after RT. CD8+ T lymphocytes were isolated from peripheral blood and stimulated with irradiated, peptide-pulsed T2 cells (174 x CEM.T2; ATCC® CRL-1992™). After extraction of total RNA, Interferon gamma (IFNγ) mRNA expression was analyzed by RT-qPCR. IFNγ mRNA levels were normalized to CD8 mRNA levels. IFNγ mRNA expression of cells stimulated with the irrelevant melanoma antigen Glycoprotein 100 (gp100) served as calibrator. An expression level of 2.0 or more as compared to gp100 was considered as a positive result. Furthermore, an intracellular cytokine staining assay to detect IFNγ release in peptide-stimulated cells was employed to confirm PCR results on a protein level. Peptide-pulsed T2 cells also served as targets in CD107a degranulation assays.ResultsTo date, 19 patients including 16 MM and three SBP patients received fractionated local RT with or without concomitant systemic therapy. 13 patients were previously untreated; three patients received prior allogeneic and five prior autologous hematopoietic stem cell transplantation. At baseline, positive immune responses were frequently detected against NY-ESO-1 (10 out of 17 assessed patients; 58.8%) but interestingly not against hTERT, WT1, MUC1 or PRAME (see Figure). Consequently, baseline immune responses against NY-ESO-1 were significantly higher (p<0.001) as compared to all other TAAs investigated (Kruskal Wallis test). These median response levels against NY-ESO-1 further increased in the course of radiation therapy and were confirmed by flow cytometry on a protein level. The functionality and specificity of these responses could be demonstrated in degranulation assays suggesting the ability to deliver efficient anti-tumor immunity.ConclusionsImmunity against NY-ESO-1 was preexisting in the majority of patients and was undergoing marked expansion under local radiotherapy. This phenomenon was not found for any other TAA investigated suggesting that NY-ESO-1 carries a unique immunogenicity justifying most recent attempts to employ NY-ESO-1 as a target for genetically engineered T lymphocytes. Finally, this tumor-specific immunity is likely to contribute to immunosurveillance of multiple myeloma additionally supported by immune modulatory drugs applied for treatment such as IMiDs and proteasome inhibitors. [Display omitted] DisclosuresKnop:Takeda: Consultancy. Einsele:Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Speakers Bureau. Mielke:MSD: Consultancy, Other: Travel grants; Gilead: Other: Travel grants; Novartis: Consultancy; Celgene: Other: Travel grants, Speakers Bureau; JAZZ Pharma: Speakers Bureau.

A novel atheroprotective role of MF59-like adjuvant when co-administered with CETP vaccine in rabbit model of atherosclerosis.

Objective(s):In this study, for the first time, MF59 adjuvant was used to develop a cholesteryl ester transfer protein (CETP) vaccine. The efficacy of the vaccine was compared with the efficacy of CETP vaccine formulated with Alum/CpG, the formulation that its immunogenicity has been already demonstrated in rabbit and mice.Materials and Methods:Tetanus toxoid- CETP peptide (TT-CETP) was mixed with Alum/CpG or MF59-like and administered subcutaneously for total five times in rabbit model of atherosclerosis. Anti-TT-CETP specific antibody, CETP activity in sera and mRNA level of cytokine IL-4 and IFN-γ in peripheral mononuclear cells were determined. Therapeutic response was also examined by tracking serum lipoprotein levels and pathologic observation of atherosclerotic lesions at aortic site.Results:More anti-TT-CETP antibody was found in Alum/CpG vaccinated rabbits compared to buffer (P<0.001). Antibody induced by MF59-like formulation was not significantly higher than buffer. CETP activity and lipoprotein levels were not significantly different between vaccinated and control rabbits. The mRNA level of IL-4 was significantly lower than buffer while, IFN-γ gene expression was significantly higher in both vaccinated groups. Atherosclerosis thickness grade of aorta was dramatically lower than buffer (P<0.01) in both vaccinated groups.Conclusion:It is concluded that MF59-adjuvanted CETP vaccine showed anti-atherosclerosis properties, but the protective effect could not be directly attributed to the immune response induced by anti TT-CETP antibody and CETP inhibition. Further studies are needed to explain the anti-atherosclerosis properties of MF59 in the presence of TT-CETP peptide.

Ginseng Stem-and-Leaf Saponin (GSLS)-Enhanced Protective Immune Responses Induced by Toxoplasma gondii Heat Shocked Protein 70 (HSP70) Against Toxoplasmosis in Mice.

Toxoplasma gondii is an obligate intracellular protozoan parasite and is able to infect birds and mammals including humans. In order to find effective antigen-adjuvant combinations that can boost the immunogenicity and protection of antigen vaccines against toxoplasmosis, we examined the protective efficacy in mice immunized with recombinant protein HSP70 when co-administered with ginseng stem-and-leaf saponins (GSLS) isolated from Panax ginseng . All immunized mice produced significantly high levels of specific antibodies against rTgHSP70, and splenocytes from mice presented strong proliferative immune responses. Vaccinated mice displayed a significantly increased percentage of CD4+ and CD8+ T cells, indicating a strong immune response was triggered. The cellular and humoral immune responses were enhanced, which could be reflected of the increased mRNA levels of IFN-γ and IL-4, respectively. Immunization with rTgHSP70 and GSLS prolonged survival time of the treated mice compared to the controls, which died within 6 days after challenge with the virulent T. gondii RH strain. Our data demonstrate that by addition with GSLS, rTgHSP70 induced a strong immune response and provided partial protection against T. gondii ; therefore GSLS could be used as a promising vaccine adjuvant against acute toxoplasmosis.

Effect of ginseng polysaccharides on NK cell cytotoxicity in immunosuppressed mice.

The aim of the present study was to investigate the effects of Ginseng polysaccharides (GPS) on natural killer (NK) cell cytotoxicity in immunosuppressed mice. Cyclophosphamide (Cy) was used to construct an immunosuppressed mouse model. The mice in each group were submitted to gavages with 200 or 400 mg/kg GPS every day for 10 days. Magnetic-activated cell sorting was used to isolate spleen NK cells, and the NK cell cytotoxicity, blood distribution, expression levels of perforin and granzyme, and the mRNA expression levels of interferon (IFN)-γ were detected. Compared with the normal control group, the cytotoxicity and proportion of NK cells in the blood, and the expression levels of perforin, granzyme and IFN-γ mRNA in the Cy model group were significantly reduced (P<0.05). In addition, compared with the Cy model group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin and granzyme in the NK cells in the Cy + low-dose GPS and Cy + high-dose GPS groups were significantly increased (P<0.05). However, the mRNA expression levels of IFN-γ in the NK cells did not significantly change (P>0.05). Compared with the normal control group, the cytotoxicity and proportion of NK cells in the whole blood, and the expression levels of perforin in the Cy + low-dose GPS and the Cy + high-dose GPS groups were significantly lower (P<0.05). However, the expression levels of granzyme in the NK cells was not significantly different, as compared with the normal control group (P>0.05). These results suggested that GPS promotes NK cell cytotoxicity in immunosuppressed mice by increasing the number of NK cells in the whole blood and upregulating the expression of perforin and granzyme. Thus, the present study investigated the molecular mechanism underlying NK cell activation by GPS, the research showed that GPS have a wide application prospects in the treatment of cancer and immunodeficiency diseases.

Addition of maraviroc to antiretroviral therapy decreased interferon-γ mRNA in the CD4+ T cells of patients with suboptimal CD4+ T-cell recovery.

The CCR5 antagonist, maraviroc (MVC), is associated with an enhanced CD4+ T-cell response independent of virological suppression; however, its mechanism of action has not been elucidated. In this study, we confirmed the effect of MVC on CD4+ T-cell count recovery in immunological non-responders, and compared the conventional combination antiretroviral therapy (cART) with MVC-intensified cART. We also investigated the effect of MVC on interferon-γ (IFN-γ) production in CD4+ T cells invitro and invivo, and evaluated the relationship between the mRNA level of IFN-γ and the degree of CD4+ T-cell count recovery. Invitro analysis indicated that MVC significantly decreased mRNA levels of IFN-γ in HIV-Tat stimulated CD4+ T cells from healthy donor peripheral blood mononuclear cells. Of the 18 HIV-infected patients treated with MVC-intensified cART, 12 had a significantly increased CD4+ T-cell count after 24 weeks of additional treatment with MVC. In patients exhibiting a response in CD4+ T-cell counts, mRNA levels of IFN-γ in CD4+ T cells were lower than those in patients showing a non-response at baseline and at week 24, while mRNA levels of IFN-γ decreased in both groups at 24 weeks. In conclusion, MVC decreased the mRNA level of IFN-γ in CD4+ T cells invitro and invivo, especially in patients whose CD4+ T-cell count increased significantly. We also found that the lower baseline IFN-γ mRNA level and the larger decreased rate of IFN-γ mRNA in CD4+ T cells were associated with a good response to MVC regarding CD4+ T-cell recovery.

Deficiency of LIGHT signaling pathway exacerbates Chlamydia psittaci respiratory tract infection in mice

LIGHT, a costimulatory member of the immunoglobulin superfamily (Ig SF), can greatly impact T cell activation. The role of the LIGHT signaling pathway in chlamydial infection was evaluated in mice following respiratory tract infection with Chlamydia psittaci. Compared with wild type (WT) mice, LIGHT knockout (KO) mice showed significant reduction of body weight, much lower survival rate, higher bacterial burden, prolonged infection time courses and more severe pathological changes in lung tissue. The mRNA levels of IFN-γ, TNF-α, IL-17 and IL-12 in the lung tissue of LIGHT KO mice were significantly lower than those in WT mice. While there was no obvious difference in the percentages of CD4+ and CD8+ T cells in the spleens of the two groups of mice, there was a markedly elevated percentage of CD4+ CD25+ FoxP3+ Treg cells in LIGHT KO mice. Together, these results demonstrate that the LIGHT signaling pathway is not only required for inflammatory cytokine production as part of the host response to chlamydial infection, but also influences the differentiation of CD4+ CD25+ FoxP3+ Treg cells, both of which may be essential for control of C. psittaci respiratory tract infection.

The effect of perioperative analgesic drugs omnopon and dexketoprofen on the functional activity of immune cells in murine model of tumor surgery.

We aimed to investigate the effect of perioperative analgesia with nonselective cyclooxygenase-2 inhibitor dexketoprofen and opioid drug omnopon on the functional activity of immune cells in tumor excision murine model. Lewis lung carcinoma cells were transplanted into hind paw of C57/black mice. On the 23th day tumor was removed. Analgesic drugs were injected 30 min before and once a day for 3 days after the surgery. Biological material was obtained a day before, 1 day and 3 days after the tumor removal. IFN-γ, IL-4, IL-10 and TGF-β mRNA levels in splenic cells were assessed by quantitative real-time RT-PCR. Cytotoxic activity of splenocytes was estimated by flow cytometry. We found that in splenocytes of mice received opioid analgesia IL-10 mRNA level was increased 2.3 times on day one after the surgery compared to preoperative level (P < 0.05), while in dexketoprofen group this parameter did not change. IFN-γ gene expression level on day 3 after tumor removal was 40% higher in splenocytes of dexketoprofen treated mice as compared with omnopon treated animals (P < 0.05). Cytotoxic activity of splenocytes on day 3 postsurgery was (62.2 ± 2.4)% in dexketoprofen against (50.2 ± 3.3)% in omnopon group. In conclusion, perioperative analgesia with cyclooxygenase inhibitor dexketoprofen in contrast to opioid analgesia with omnopon preserves higher functional activity of murine immune cells in the experimental model of tumor surgery.

Toxoplasma gondiiInfection Potentiates Cognitive Impairments of Alzheimer's Disease in the BALB/c Mice

This study tests the hypothesis that in chronic Toxoplasma gondii infection communication among immune cells promotes neuroinflammation through cytokine networks and potentiate cognitive impairments in BALB/c mice with Alzheimer's disease (AD). The animal model of Toxoplasma infection was established by the intraperitoneal inoculation of 20-25 tissue cysts from the Tehran strain of T. gondii . We injected amyloid-beta 1-42 peptide (Aβ1-42, 1 and 2 μl) into the hippocampus of BALB/c mice to establish an animal model of AD. The behavioral experiments such as spatial learning and memory were performed using the Morris water maze test. The mRNA levels of TNF-α, IL-1β, IFN-γ, and inducible nitric oxide synthase (iNOS) were examined by real-time PCR. We found that T. gondii infection caused AD-like symptoms and impaired learning and memory functions of the infected BALB/c mice. We also found that in Toxoplasma infection + Aβ1-42 (1 μl) group, T. gondii infection could potentiate AD in infected mice receiving subdoses of Aβ1-42 (1 μl) and caused considerable impairment in learning and memory functions similar to AD group. Comparison of the results demonstrated that mRNA levels of IL-1β, TNF-α, IFN-γ, and iNOS significantly (P < 0.001) increased in T. gondii + Aβ1-42 (1 μl) in comparison with the other tested groups. The obtained results showed that chronic T. gondii infection communication among immune cells promotes neuroinflammation through cytokine networks and induces pathological progression of AD in the mice brain, whereas the presence of neuroanatomical Toxoplasma tissue cysts in the brain could also affect the behavioral functions in T. gondii -infected mice.

Gingival Toll-like receptor and cytokine messenger RNA levels in equine periodontitis and oral health.

Equine periodontitis is a common and painful condition. However, the disease often goes unnoticed by owners and is thus a major welfare concern. The aetiopathogenesis of the condition remains poorly understood and has been investigated in few studies. The innate immune system is known to play an important role in human periodontitis, but its role in equine periodontitis has not been examined. To quantify the messenger (m)RNA levels of Toll-like receptors (TLRs) and cytokines in gingival tissue from orally healthy horses and those affected by periodontitis. Observational study. Gingival tissue samples were taken post-mortem from 13 horses with no clinical signs of oral disease and 20 horses with periodontitis. mRNA levels of TLR2, TLR4 and TLR9 and cytokines interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-4, IL-6, IL-10, IL-12, IL-17 and interferon-γ (IFN-γ) were determined using quantitative real-time PCR. The statistical significance of results was assessed using appropriate t tests. mRNA levels of all TLRs and cytokines were upregulated in equine periodontitis. Significant increases in mRNA levels of TLR2, TLR9, IL-4, IL-10, IL-12 (P≤0.05) and IFN-γ (P≤0.01) were observed for both unweighted and age-weighted analyses of diseased gingival tissue samples compared with healthy gingival samples. In comparisons of samples of periodontitis lesions with healthy gingival control samples from the same horse, significant increases in mRNA levels of TLR4, TLR9, IL-10, IFN-γ (P≤0.05), TLR2, IL-1β and IL-12p35 (P≤0.01) were observed. This study has provided an initial insight into the involvement of the immune system in equine periodontitis. Increased mRNA levels of TLR2, TLR4 and TLR9 indicate substantial microbial challenge in diseased gingival tissue. A mixed Th1/Th2/Th17 cytokine response is produced in equine periodontitis. Further studies are required to more fully characterise the role of the innate immune system in this disease.

Multi-glycoside of Tripterygium wilfordii Hook.f. ameliorates imiquimod-induced skin lesions through a STAT3-dependent mechanism involving the inhibition of Th17-mediated inflammatory responses.

Multi-glycoside of Tripterygium wilfordii Hook. f. (GTW) possesses anti-inflammatory and immunosuppressive properties, and has been used as a traditional treatment for psoriasis for many years, although the underlying immunological mechanisms remain poorly understood. The T helper (Th)17 cell response is considered to play a major role in the pathogenesis of psoriasis. Th17 cells are implicated in the mechanism of pathogenesis of imiquimod (IMQ)-induced skin inflammation. Using a mouse model, we demonstrated that GTW protected mice from developing psoriasis-like lesions induced by topical IMQ administration. This protection was associated with significantly decreased mRNA levels of Th17 cytokines such as interleukin (IL)-17A, IL-17F and IL-22 in mouse skin samples as well as fewer IL-17-secreting splenic CD4+ lymphocytes in IMQ-exposed mice. There were no significant effects on the proportion of CD4+ interferon (IFN)-γ+ T cells, CD4+IL-4+ T cells and CD4+CD25+Foxp3+ Treg cells in the spleen cells. Taken together with the unchanged mRNA levels of Th1 cytokine IFN-γ, Th2 cytokine IL-4 and Treg cytokine IL-10 in IMQ-exposed mouse skin following GTW administration, our findings suggest that the immunosuppressive effect of GTW in psoriasis is exerted mainly on Th17 cells, rather than on Th1, Th2 or Treg cells. Furthermore, we showed that GTW suppressed Th17 function through the inhibition of STAT3 phosphorylation. These results have the potential to pave the way for the use of GTW as an agent for the treatment of psoriasis.

Oral imazalil exposure induces gut microbiota dysbiosis and colonic inflammation in mice

The fungicide imazalil (IMZ) is used extensively in vegetable and fruit plantations and as a post-harvest treatment to avoid rot. Here, we revealed that ingestion of 25, 50 and 100 mg IMZ kg−1 body weight for 28 d induced gut microbiota dysbiosis and colonic inflammation in mice. The relative abundance of Bacteroidetes, Firmicutes and Actinobacteria in the cecal contents decreased significantly after exposure to 100 mg kg−1 IMZ for 28 d. In feces, the relative abundance in Bacteroidetes, Firmicutes and Actinobacteria decreased significantly after being exposed to 100 mg kg−1 IMZ for 1, 14 and 7 d, respectively. High throughput sequencing of the V3-V4 region of the bacterial 16S rRNA gene revealed a significant reduction in the richness and diversity of microbiota in cecal contents and feces of IMZ-treated mice. Operational taxonomic units (OTUs) analysis identified 49.3% of OTUs changed in cecal contents, while 55.6% of OTUs changed in the feces after IMZ exposure. Overall, at the phylum level, the relative abundance of Firmicutes, Proteobacteria and Actinobacteria increased and that of Bacteroidetes decreased in IMZ-treated groups. At the genus level, the abundance of Lactobacillus and Bifidobacterium decreased while those of Deltaproteobacteria and Desulfovibrio increased in response to IMZ exposure. In addition, it was observed that IMZ exposure could induce colonic inflammation characterized by infiltration of inflammatory cells, elevated levels of lipocalin-2 (lcn-2) in the feces, and increased mRNA levels of Tnf-α, IL-1β, IL-22 and IFN-γ in the colon. Our findings strongly suggest that ingestion of IMZ has some risks to human health.

IL-33 signaling is essential to attenuate viral-induced encephalitis development by downregulating iNOS expression in the central nervous system.

BackgroundViral encephalitis is a common cause of lethal infections in humans, and several different viruses are documented to be responsible. Rocio virus is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the CNS compartmentalized immune response. Interleukin 33 (IL-33), a member of the IL-1 family, is an immunomodulatory cytokine that is highly expressed in the CNS. However, the role of IL-33 on viral encephalitis remains unclear. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during Rocio virus infection.MethodsWild-type (WT), ST2 (ST2−/−), and nitric oxide synthase-deficient mice (iNOS−/−) and Stat6 (Stat6−/−)-deficient mice were infected with different concentrations of the Rocio virus by intraperitoneal route, the cytokine mRNA level in CNS was analyzed by qPCR, and cellular immunophenotyping was performed on infected mice by the flow cytometry of isolated CNS mononuclear cells.ResultsWe have shown that the mRNA expression of IL-33 and ST2 receptors is increased in the CNS of Rocio virus-infected WT mice and that ST2−/− mice showed increased susceptibility to infection. ST2 deficiency was correlated with increased tissue pathology, cellular infiltration, and tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA levels and higher viral load in the CNS, compared with wild-type mice. The increased Th1 cytokine levels released in the CNS acted on infiltrating macrophages, as evidenced by flow cytometry characterization of cellular infiltrates, inducing the expression of iNOS, contributing to brain injury. Moreover, iNOS−/− mice were more resistant to Rocio virus encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology.ConclusionsWe provide evidences of a specific role for IL-33 receptor signaling in nitric oxide induction through local IFN-γ modulation, suggesting that nitric oxide overproduction might have an important role in the progression of experimental viral encephalitis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0628-1) contains supplementary material, which is available to authorized users.

L-Arginine supplementation inhibits the growth of breast cancer by enhancing innate and adaptive immune responses mediated by suppression of MDSCs in vivo.

BackgroundL-Arg is involved in many biological activities, including the activation of T cells. In breast cancer patients, L-Arg is depleted by nitric oxide synthase 2 (NOS2) and arginase 1 (ARG-1) produced by myeloid-derived suppressor cells (MDSCs). Our aim was to test whether L-Arg supplementation could enhance antitumor immune response and improve survivorship in a rodent model of mammary tumor.MethodsTumor volumes in control and L-Arg treated 4 T1 tumor bearing (TB) BALB/c mice were measured and survival rates were recorded. The percentages of MDSCs, dendritic cells (DCs), regulatory T cells (Tregs), macrophages, CD4+ T cells, and CD8+ T cells were examined by flow cytometry. Additionally, levels of IL-10, TNF-α, and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA) and nitric oxide (NO) levels were measured by the Griess reaction. IFN-γ, T-bet, Granzyme B, ARG-1 and iNOS mRNA levels were examined by real-time RT-PCR.ResultsL-Arg treatment inhibited tumor growth and prolonged the survival time of 4 T1 TB mice. The frequency of MDSCs was significantly suppressed in L-Arg treated TB mice. In contrast, the numbers and function of macrophages, CD4+ T cells, and CD8+ T cells were significantly enhanced. The IFN-γ, TNF-α, NO levels in splenocytes supernatant, as well as iNOS, IFN-γ, Granzyme B mRNA levels in splenocytes and tumor blocks were significantly increased. The ARG-1 mRNA level in tumor blocks, the frequency of Tregs, and IL-10 level were not affected.ConclusionL-Arg supplementation significantly inhibited tumor growth and prolonged the survival time of 4 T1 TB mice, which was associated with the reduction of MDSCs, and enhanced innate and adaptive immune responses.

CD200 Inhibits Inflammatory Response by Promoting KATP Channel Opening in Microglia Cells in Parkinson's Disease.

BackgroundAs the second most common neurodegenerative disorder after Alzheimer’s disease (AD), Parkinson’s disease (PD) principally impacts the motor system in approximately 7 million patients worldwide. The present study aimed to explore the effects of cluster of differentiation (CD200) on adenosine triphosphate-sensitive potassium (KATP) channels and inflammatory response in PD mice.Material/MethodsWe created an in vivo PD model by intraperitoneal injection of 30 mg/kg/day 1-Methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride (MPTP. HCL) for 5 consecutive days, and we created an in vitro PD model by injection of 100 μM 1-methyl-4-phenylpyridinium ion (MPP+) in primary microglia cells. Expression level of CD200/CD200R, inwardly rectifying potassium (Kir6.1/6.2), and sulfonylurea receptor (Sur1/2) were detected by Western blot (WB). Immunohistochemistry (IHC) was utilized to assess CD11b (microglia marker) and tyrosine hydroxylase (TH, a marker reveals dopamine level in neurons) expression levels. An in vitro PD model was applied to detect the influence of CD200 on ATP and inflammatory factors released from microglia. Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β mRNA levels were explored by realtime quantitative polymerase chain reaction (RT-QPCR), and their protein levels were identified by enzyme-linked immunosorbent assay (ELISA).ResultsWB exhibited time-dependent down-regulation of CD200/CD200R in cerebra of PD mice compared to control mice, with Kir 6.1 and SUR 2 expressed mainly in microglia. IHC showed that CD11b reached a peak at the 1st day after MPTP treatment, followed by time-dependent reduction, and TH decreased noticeably after MPTP induction. RT-QPCR demonstrated that compared with controls, IFN-γ, TNF-α, and IL-1β mRNA levels were significantly elevated at MPTP-1d, was reduced at MPTP-3d, and then returned to baseline at MPTP-7d. IHC showed that MPP+ significantly elevated microglia release of ATP. Similar to the effect of pinacidil (K+ channel opener), CD200 remarkably depressed MPP+-induced ATP release. ELISA showed that MPP+ significantly increased IFN-γ, TNF-α, and IL-1β release, and CD200 and pinacidil remarkably suppressed this elevation.ConclusionsOur results show a novel role of CD200 in promoting opening of the KATP channel, inhibiting microglia activation and release of ATP, as well as inflammatory factors, thus protecting dopaminergic (DA) neurons against damage and alleviating PD.

Relationship of baseline tumoral IFNγ mRNA and PD-L1 protein expression to overall survival in durvalumab-treated NSCLC patients.

3036 Background: Biomarkers may help to identify non-small cell lung cancer (NSCLC) patients (pts) more likely to have improved outcomes to anti-PD-L1 therapy. From 112 durvalumab (D)-treated pts, we previously reported that high pretreatment levels of PD-L1 protein and IFNγ mRNA expression in tumor biopsies were associated with higher objective response rates. We extend this analysis with mature survival outcome data that includes additional available pts; in addition, we examined changes in gene expression from on-treatment tumors in a subset of pts. Methods: CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating D in pts with advanced NSCLC or other solid tumors. As of 14 DEC 2015, 298 previously treated NSCLC pts received 10 mg/kg Q2W of D ≤12 mos with a median 16.1 mos follow up. Expression profiling of 100 immune-associated genes using Fluidigm Biomark on frozen biopsies and immunostaining for tumoral PD-L1 on fresh or archival biopsies was conducted. 157 pts (55% squamous; 54% ≥2 prior therapy lines) had matched baseline mRNA and PD-L1 data of sufficient quality. Samples with detectable levels of IFNγ mRNA were considered IFNγ+. Pts with ≥25% tumor cells stained for PD-L1 at any intensity were scored PD-L1+. Of the 157 pts, 32 pts had pre- and post-treatment tumors evaluable for 100-gene mRNA analysis. Results: Pts who were IFNγ+/PD-L1+ had a trend of longer overall survival compared to those who were IFNγ-/PD-L1-, even after adjusting for gender, age, prior therapy lines, histology, ECOG, and smoking (Table 1). Following treatment with D, the transcript most highly induced was IFNγ (~2-fold; p<0.016). Conclusions: NSCLC pts with elevated IFNγ mRNA and PD-L1 protein expression in baseline tumor biopsies may have longer survival with D treatment. D treatment induces IFNγ gene expression within the tumor microenvironment. Clinical trial information: NCT01693562.OS by IFNγ and PD-L1 status. # Pts (# events) Median in months (95% CI) Adjusted HRa; p IFNγ+/PD-L1+ 43 (11) NR (8.8, NA) 0.40; 0.016 IFNγ+/PD-L1‒ 20 (8) NR (6.5, NA) 0.38; 0.021 IFNγ‒/PD-L1+ 42 (16) 9.7 (5.1, NA) 0.64; 0.18 IFNγ‒/PD-L1‒ 52 (34) 5.9 (4.1, 10.2) NR=Not Reached; NA=Not Applicable; a Univariate log-rank p<0.05 for all adjusted p <0.05 (not shown)