A CD38-directed, single-chain T-cell engager targets leukemia stem cells through IFN-γ-induced CD38 expression.

ABSTRACT: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs.

Histone deacetylases inhibitor chidamide synergizes with humanized PD1 antibody to enhance T-cell chemokine expression and augment Ifn-γ response in NK-T cell lymphoma.

BACKGROUND: Whether immunotherapy combined with different histone deacetylases (HDAC) inhibitors in refractory or relapsed natural killer/T-cell lymphoma (NKTCL) is superior to each agent is still lacking in head-to-head clinical trials or preclinical evidence. METHODS: NKTCL cell line xenograft models (CDX) in immunocompetent, human programmed cell death protein 1 (PD1) knock-in genetically engineered mice were used to investigate the combination effects. Different types and dosages of HDAC inhibitors were investigated. We explored the underlying mechanisms by RNA-sequencing and ChIP-sequencing. Two clinical cases treated with anti-PD1/chidamide were presented. FINDINGS: Anti-PD1/chidamide shows significant tumour rejection in two CDX models. RNA-seq and CHIP-seq revealed that chidamide is synergistic to enhance T-cell chemokine expression, augment the Ifn-γ response, and increase CD8 T-cell infiltration via histone modification. Ifn-γ neutralizing antibody can attenuate the efficacy of combination drugs. However, the anti-PD1/romidepsin failed to augment the Ifn-γ response. The expressions of Ifn-γ related gene set signatures are significantly correlated with tumour rejection in anti-PD1/chidamide. In the clinic, two NKTCL patients treated with the PD1/chidamide show promising efficacy and limited toxicity. INTERPRETATION: Anti-PD1/chidamide enhances T-cell chemokine expression and augments the IFN-γ response in preclinical NKTCL immunocompetent models. IFN-γ signatures may be good response biomarkers for the selection of potentially benefit patients. FUNDING: This study was supported by the Chinese National Major Project for New Drug Innovation (2017ZX09304015) and the Chinese Society of Clinical Oncology Research Fund (Y-BMS2019-026).

Carbon dioxide inhibits COVID-19-type proinflammatory responses through extracellular signal-regulated kinases 1 and 2, novel carbon dioxide sensors.

Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are CO2 sensors and CO2 is an efficient anti-inflammatory compound that exerts its effects through inactivating ERK1/2 in cultured endothelial cells when the CO2 concentration is elevated. CO2 is a potent inhibitor of cellular proinflammatory responses caused by H2O2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5-8% CO2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways.

The antitumor effect of the combination of aconitine and crude monkshood polysaccharide on hepatocellular carcinoma.

Aconitine, the main component in Radix Aconiti Lateralis Preparata, not only exerts the anti-tumor effect on Hepatocellular Carcinoma (HCC) but also damages on immune system. In the present study, Crude Monkshood Polysaccharide (CMP), another one natural composition component originated from the same herbal with aconitine, combined with aconitine to investigate the effects on HCC and immunity in vitro and in vivo. The combination of CMP and aconitine enhanced the ability of the immunocyte to kill the tumor cell in vitro and had an additive effect on anti-HCC in vivo. Aconitine-CMP in combination improved the spleen weights, spleen index, thymus weights, thymus index. Elevated CD4+ T and CD8+ T cells and macrophages in spleen, decreased serum IL-6 level and increased serum IFN-γ and TNF-α levels were observed in mice treated with the combination of aconitine and CMP compare with control group (P<0.05). Our results showed that the combination of aconitine and CMP exerts anti-tumor effect by directly killing tumor cells and enhancing the anti-tumor immune responses, which further implies that chemotherapy drugs combined with Chinese medicine immunopotentiator maybe a feasible and effective strategy for HCC.

Recombinant human IL-37 inhibited endometriosis development in a mouse model through increasing Th1/Th2 ratio by inducing the maturation of dendritic cells.

BACKGROUND: Endometriosis is a serious reproductive and general health consequences. Recombinant human IL-37 (rhIL-37) is an inhibitor of inflammation. METHODS: ELISA assay was performed to detect the concentration of cytokines. Flow cytometry was used to analyze cell proportion. Besides, qRT-PCR and western blotting assay were used to detect the level of gene and protein, respectively. Transwell co-culture system was used for the co-culture of dendritic cells (DCs) and CD4+T cells. RESULTS: Our data showed that rhIL-37 inhibited the development of ectopic lesions in the mice with endometriosis, increased Th1/Th2 ratio and induced DCs maturation. The co-culture system of DCs and CD4+T cells demonstrated that rhIL-37 increased Th1/Th2 cell ratio through promoting DCs maturation. Moreover, the expression of IL-4 in the DCs derived from healthy mice was inhibited by rhIL-37 treatment. rhIL-37 increased Th1/Th2 cell ratio through inhibiting IL-4 in DCs. Subsequently, our results proved that rhIL-37 promoted the maturation of DCs via inhibiting phosphorylation of STAT3. Activation of STAT3 could reverse rhIL-37-induced maturation of DCs. CONCLUSION: Overall, rhIL-37 could protect against endometriosis through increasing the ratio of Th1/Th2 cells via inducing DCs maturation and inhibiting IL-4 expression in the DCs. Furthermore, rhIL-37 induced DCs maturation by inhibiting STAT3 phosphorylation. Our data confirmed the protective effect of rhIL-37 in endometriosis. These data may provide a novel idea for the treatment of the disease.

Changes in expression of TLR-4, TGF-ß, INF-γ, TNF-α in cultured T24/83 cells of invasive bladder cancer treated with cisplatin and/or polyphenolic adjuvant melanin.

BACKGROUND: Toll-like receptor 4 (TLR4) is known to be involved in carcinogenesis and cancer progression. Changes in TLR4 expression are associated with changes in the expression of key cellular cytokines (transforming growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)), which affect cancer progression and metastasis. AIM: To study changes in the expression of TLR4, TGF-ß, TNF-α, IFN-γ genes, the level of apoptosis and cell cycle distribution in human invasive urothelial carcinoma T24/83 cells under the treatment with polyphenolic adjuvant compound of fungal origin melanin, cytotoxic drug cisplatin, and combination of both. MATERIALS AND METHODS: T24/83 cells were incubated with cisplatin (0.05 mM), melanin (5 µg/ml), or their combination. The expression level of TLR-4, TGF-ß, INF-γ, TNF-α was evaluated by the real time polymerase chain reaction. The flow cytometry was used to study cell cycle distribution, proliferative activity and level of apoptosis. Morphological analysis of the Т24/83 cells was performed as well. RESULTS: Melanin, cisplatin, and their combination downregulate TLR4 expression (2.67; 1.28; and 2.73-fold decrease, respectively) and TNF-α expression (6.5; 1.4; and 1.7-fold decrease, respectively). Melanin did not affect TGF-ß expression while cisplatin caused 13-fold downregulation of TGF-ß. The combined use of cisplatin and melanin decreased TGF-ß expression by 6.5 times. The upregulation of IFN-γ by melanin, cisplatin, and their combination was demonstrated (4.3; 6.7; and 2-fold increase, respectively). All treatment modalities increased the level of apoptosis in T24/83 cells. Melanin treatment increased significantly the proportion of fibroblast-like cells in T24/83 culture with decreased cell adhesion to the substrate. CONCLUSIONS: Melanin, cisplatin, and combination of both agents affect significantly TLR4, TNF-α, TGF-ß, INF-γ expression, cell cycle distribution and morphology in T24/83 cells suggesting their transition to less aggressive phenotype.

Repolarization of glioblastoma macrophage cells using non-agonistic Dectin-1 ligand encapsulating TLR-9 agonist: plausible role in regenerative medicine against brain tumor.

AIM OF THE STUDY: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic ß-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type. MATERIALS AND METHODS: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6). RESULTS: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1ß post treatment. CONCLUSION: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression.

Increasing studies confirm that anti-angiogenesis can increase the effectiveness of immunotherapy. In this study, we found that an angiogenesis inhibitor apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Apatinib treatment upregulated PD-L1 expression in various colon cancer cells both at the mRNA and protein levels. Further, apatinib-treated cancer cells hampered activation and IFN-γ secretion of T cells in the co-culture system, which was reversed by the anti-PD-1 antibody. Based on this, the combination of apatinib with anti-PD-1 on colon cancer growth in mice was examined. The combination treatment showed more significant inhibition on the growth of transplanted tumors in mice than single-drug treatment. Overall, our study here showed the enhancement of anti-PD-1 antitumor efficacy in a syngeneic mouse model (CT-26 cells in Balb/c) by the angiogenesis inhibitor apatinib via upregulating PD-L1 expression as well as angiogenesis inhibition, which may provide a rationale for the combination of apatinib and anti-PD-1 antibody for colorectal cancer treatment in the clinic.

Nanoparticle-cell-nanoparticle communication by stigmergy to enhance poly(I:C) induced apoptosis in cancer cells.

Nanoparticle-cell-nanoparticle communication by stigmergy was demonstrated using two capped nanodevices. The first community of nanoparticles (i.e.S(RA)IFN) is loaded with 9-cis-retinoic acid and capped with interferon-γ, whereas the second community of nanoparticles (i.e.S(sulf)PIC) is loaded with sulforhodamine B and capped with poly(I:C). The uptake of S(RA)IFN by SK-BR-3 breast cancer cells enhanced the expression of TLR3 receptor facilitating the subsequent uptake of S(sulf)PIC and cell killing.

TIGIT enhances CD4+ regulatory T-cell response and mediates immune suppression in a murine ovarian cancer model.

Ovarian cancer (OC) is the fifth-leading cause of cancer-related death in women with a pathogenesis involving activation of regulatory T cells (Tregs). The T-cell immunoglobulin and ITIM domain (TIGIT) is a well-known immune checkpoint molecule that inhibits T-cell responses. However, the role of TIGIT in OC is not comprehensively understood. In this study, we revealed crucial functions of TIGIT in the development and progression of OC. ID8 cells were used to establish a murine OC model. TIGIT expression was increased in immune cells of OC mice, particularly in CD4+ Tregs. Anti-TIGIT monoclonal antibodies (mAb) were used to block the function of TIGIT in OC mice, and we found that the anti-TIGIT treatment reduced the proportion of CD4+ Tregs, but did not affect CD4+ and CD8+ T cells or natural killer cells. Splenic CD4+ Tregs from OC mice were isolated after the anti-TIGIT treatment, and their functioning was examined. Inhibition of TIGIT lowered the degree of immunosuppression induced by CD4+ Tregs. A survival curve suggested that anti-TIGIT treatment can improve the survival rate of OC in mice. We conclude that TIGIT enhanced CD4+ Tregs response and mediated immunosuppression in the OC model. Our data suggest that inhibition of TIGIT is a potential therapeutic target in OC patients.

Study on Effects and Mechanism of Lead and High-Fat Diet on Cognitive Function and Central Nervous System in Mice.

OBJECTIVE: We sought to investigate the effects and mechanism of lead and a high-fat diet on cognitive function and the central nervous system in mice. METHODS: Eighty-four healthy male mice were randomly divided into a control group (n = 21) (fed with common diet and free drinking), a lead exposure group (n = 21) (fed with common diet and 300 mg/L lead acetate solution), a high-fat group (n = 21) (fed with high-fat diet and free drinking), and a lead + high-fat group (n = 21) (fed with high-fat diet and 300 mg/L lead acetate solution). In 10 weeks after lead exposure, the mice of all groups were tested for the cognition, learning and memory abilities, body weight, serum triglyceride (TG), low-density lipoprotein, and high-density lipoprotein, as well as for the contents of lead, interleukin 6 (IL-6), interleukin 17 (IL-17), interferon γ, advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST), and hydrogen peroxide in the brain tissues. RESULTS: Compared with the control group and the lead-exposed group, the body weights of mice in the high-fat group and the lead + high-fat group increased significantly from the sixth week of the experiment, of which the difference was statistically significant (P < 0.05). Compared with the control group and the high-fat group, the lead content in brain tissue of the lead exposure group and the lead + high-fat group increased significantly, of which the difference was statistically significant (P < 0.05). Compared with the control group, the escape latent period, triglyceride, low-density lipoprotein, IL-6, IL-17, interferon γ, and AGEs of the remaining 3 groups increased significantly, but the recognition index, passing platform times, high-density lipoprotein, and GSH-ST significantly decreased (P < 0.05); the second and third escape latent periods, IL-6, IL-17, and AGEs of lead + high-fat group, were obviously higher than the remaining 3 groups, but the passing platform times were obviously lower than the remaining 3 groups, of which the difference was statistically significant. The content of hydrogen peroxide in brain tissues had no difference among groups (P > 0.05). CONCLUSIONS: The lead and high-fat diet resulted in lipid metabolism disorders and impaired the cognitive function and central nervous system by promoting the secretion of inflammatory factors in glial cells, inducing the inflammatory reaction of brain tissue, inhibiting GSH-ST expression, and increasing AGEs content.

All-trans Retinoic Acid Counteracts Diarrhea and Inhibition of Downregulated in Adenoma Expression in Gut Inflammation.

BACKGROUND: Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated. AIMS: Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo. METHODS: Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon. RESULTS: All-trans retinoic acid (10 µM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1ß and CXCL1 and a decrease in DRA mRNA and protein levels in the colon. CONCLUSION: Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea.

Lipopolysaccharide induces steroid-resistant exacerbations in a mouse model of allergic airway disease collectively through IL-13 and pulmonary macrophage activation.

BACKGROUND: Acute exacerbations of asthma represent a major burden of disease and are often caused by respiratory infections. Viral infections are recognized as significant triggers of exacerbations; however, less is understood about the how microbial bioproducts such as the endotoxin (lipopolysaccharide (LPS)) trigger episodes. Indeed, increased levels of LPS have been linked to asthma onset, severity and steroid resistance. OBJECTIVE: The goal of this study was to identify mechanisms underlying bacterial-induced exacerbations by employing LPS as a surrogate for infection. METHODS: We developed a mouse model of LPS-induced exacerbation on the background of pre-existing type-2 allergic airway disease (AAD). RESULTS: LPS-induced exacerbation was characterized by steroid-resistant airway hyperresponsiveness (AHR) and an exaggerated inflammatory response distinguished by increased numbers of infiltrating neutrophils/macrophages and elevated production of lung inflammatory cytokines, including TNFα, IFNγ, IL-27 and MCP-1. Expression of the type-2 associated inflammatory factors such as IL-5 and IL-13 were elevated in AAD but not altered by LPS exposure. Furthermore, AHR and airway inflammation were no longer suppressed by corticosteroid (dexamethasone) treatment after LPS exposure. Depletion of pulmonary macrophages by administration of 2-chloroadenosine into the lungs suppressed AHR and reduced IL-13, TNFα and IFNγ expression. Blocking IL-13 function, through either IL-13-deficiency or administration of specific blocking antibodies, also suppressed AHR and airway inflammation. CONCLUSIONS & CLINICAL RELEVANCE: We present evidence that IL-13 and innate immune pathways (in particular pulmonary macrophages) contribute to LPS-induced exacerbation of pre-existing AAD and provide insight into the complex molecular processes potentially underlying microbial-induced exacerbations.

IL-10 in vitro could enhance IFNγ expression and suppress PD-1 expression in CD8 T cells from esophageal cancer patients.

IL-10 is commonly regarded as an immunoregulatory cytokine, but accumulating evidence suggests that IL-10 may promote CD8 T cell expansion and proliferation. In this study, tumor infiltrating (TI) and peripheral blood (PB) CD8 T cells were collected from esophageal cancer patients. Interestingly, IL-10 concentration in the tumor microenvironment increased with advancing tumor stage, while TI CD8 T cell-mediated IL-10 production decreased with advancing tumor stage. By flow cytometry, three distinctive subsets, including IL-10+IFNγ-, IL-10+IFNγ+, and IL-10-IFNγ+, could be observed in TI CD8 T cells. The former two subsets were present at much higher frequency in stage I and stage II patients than in stage III patients. IL-10+IFNγ+ TI CD8 T cells presented significantly higher IFNγ and lower PD-1 expression than the IL-10-IFNγ+ TI CD8 T cells. PB CD8 T cells, on the other hand, produced little IL-10 but potent IFNγ upon stimulation. Interestingly, intermediate level of exogenous IL-10 could significantly elevate the expression of IFNγ by PB CD8 T cells, while high level of exogenous IL-10 resulted in reduced expression of IFNγ by PB CD8 T cells. Exogenous IL-10 could not significantly reduce the frequencies of PD-1+ PB CD8 T cells, but significantly reduced the MFI of PD-1 in the PB CD8 T cells, especially in stage III patients. Together, this investigation demonstrated that IL-10 enhanced IFNγ expression and suppressed PD-1 expression in PB and TI CD8 T cells; however, the frequency of IL-10-expressing TI CD8 T cells decreased with increasing severity in esophageal cancer.

Fish Cholesterol 25-Hydroxylase Inhibits Virus Replication via Regulating Interferon Immune Response or Affecting Virus Entry.

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper (Epinephelus coioides) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker (Larimichthys crocea) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro. Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

Effect of immunosuppressive drugs on cytokine production in canine whole blood stimulated with lipopolysaccharide or a combination of ionomycin and phorbol 12-myristate 13-acetate.

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12-myristate 13-1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7-day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin-10 (IL-10), interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα) were significantly activated by PMA/I stimulation and IL-6, IL-10 and TNFα by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL-10, IFNγ and TNFα production was significantly reduced after stimulation with PMA/I compared to pre-treatment. After prednisone treatment, TNFα production was significantly reduced after stimulation with PMA/I or LPS compared to pre-treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting.

Association of synovial tissue polyfunctional T-cells with DAPSA in psoriatic arthritis.

OBJECTIVE: This study examines polyfunctional T-cells in psoriatic arthritis (PsA) synovial tissue and their associations with clinical disease and implications for therapy. METHODS: PsA synovial tissue was enzymatically/mechanically digested to generate synovial tissue single cell suspensions. Frequencies of polyfunctional CD4, CD8, T-helper 1 (Th1), Th17 and exTh17 cells, using CD161 as a marker of Th17 plasticity, were determined by flow cytometry in matched PsA synovial tissue and peripheral blood. Synovial T-cell polyfunctionality was assessed in relation to Disease Activity in PSoriatic Arthritis (DAPSA) and in synovial cell suspensions cultured with a current mode of treatment, phosphodiesterase 4 (PDE4) inhibitor. RESULTS: PsA synovial tissue infiltrating CD4+ T-cells expressed higher levels of interleukin (IL)-17A, interferon gamma (IFN-γ), GM-CSF and CD161, with parallel enrichment of Th1, Th17 and exTh17 T-helper subsets (all p<0.05). Interestingly, a significant proportion of synovial T-cell subsets were triple-positive for GM-CSF, tumour necrosis factor (-TNF), -IL-17 or IFN-γ compared with matched blood (all p<0.05). Importantly, frequencies of polyfunctional T-cells correlated with DAPSA: Th1-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p<0.01), Th17-GM-CSF+/TNF+/IL-17+ (r=0.6, p<0.057) and exTh17-GM-CSF+/TNF+/IFN-γ+ (r=0.7, p=0.0096), with no associations observed for single cytokine-producing T-cells. Following ex vivo culture of PsA synovial tissue cell suspensions, polyfunctional GM-CSF+TNFα+IL-17A+ or/IFN-γ+-producing T-cells (p<0.05), but not single cytokine-producing T-cells, were inhibited with a PDE4 inhibitor. CONCLUSION: These data demonstrate enrichment of polyfunctional T-cells in PsA synovial tissue which were strongly associated with DAPSA and ex vivo therapeutic response.

Itching in a trichophytin contact dermatitis mouse model and the antipruritic effect of antifungal agents.

BACKGROUND: Tinea is an infectious disease by dermatophytes, of which Trichophyton species accounts for the overwhelming majority of case. Tinea often causes itching with inflammation. In terms of pruritus by fungal infection, however, tinea has not been investigated sufficiently to date. AIM: To evaluate itch caused by Trichophyton infection and the effect of antifungal agents on the infection, by measuring scratch behaviour and profiles of inflammatory cytokines and chemokines. METHODS: We used a previously established mouse model of contact hypersensitivity induced by trichophytin, a crude extract from Trichophyton mentagrophytes. Scratching behaviour was recorded using a counting device that measured an electric current induced in a coil by movement of magnets that had been inserted into the hind paws of each animal. We investigated expression of various genes in lesional skin of mice and in normal human epidermal keratinocytes. We also investigated the antipruritic effects of the corticosteroid dexamethasone (DEX) and three antifungal agents: ketoconazole (KCZ), terbinafine (TBF) and liranaftate (LNF). RESULTS: Biphasic peaks of scratching were observed at 1 h and at 6-7 h during an observation period of 14 h after trichophytin induction. For lesional skin, RNA was extracted 24 h after trichophytin challenge, and increased expression was seen in the genes for interleukin (IL)-17A, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-2 and Dectin-1, whereas there was no obvious change in the genes for IL-31 and prostaglandin (PG)E2. Furthermore, KCZ inhibited histidine decarboxylase (HDC) expression in vitro and in vivo, and inhibited scratching in the very early phase. LNF inhibited expression of thymic stromal lymphopoietin (TSLP) and IL-8 in vitro, and TSLP, TNF-α, IL-1α and MIP2 in vivo, and also scratching in the early phase. TBF did not induce any significant alterations in either gene expression or scratching. DEX suppressed expression of all the chemical mediators except HDC in vitro and in vivo, and inhibited scratching. CONCLUSION: Antifungals can inhibit itching induced by fungal infection through different mechanisms.

Expression of human inducible nitric oxide synthase in response to cytokines is regulated by hypoxia-inducible factor-1.

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at -4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation.