Suppression Of Th1 Responses Research Articles

Prior Toxoplasma Gondii Infection Ameliorates Liver Fibrosis Induced by Schistosoma Japonicum through Inhibiting Th2 Response and Improving Balance of Intestinal Flora in Mice.

Schistosomiasis is an immunopathogenic disease in which a T helper (Th) cell type 2-like response plays vital roles. Hepatic fibrosis is its main pathologic manifestations, which is the leading cause of hepatic cirrhosis. Co-infections of Schistosoma japonicum (Sj) with other pathogens are frequently encountered but are easily ignored in clinical studies, and effective therapeutic interventions are lacking. In this study, we explored the effect of Toxoplasma gondii (Tg) prior infection on Th1/Th2 response, community shifts in gut microbiome (GM), and the pathogenesis of schistosomiasis in murine hosts. Mice were prior infected with Tg before Sj infection. The effects of co-infection on Th1/Th2 response and hepatic fibrosis were analyzed. Furthermore, we investigated this issue by sequencing 16S rRNA from fecal specimens to define the GM profiles during co-infection. Tg prior infection markedly reduced the granuloma size and collagen deposit in livers against Sj infection. Prior infection promoted a shift toward Th1 immune response instead of Th2. Furthermore, Tg infection promoted the expansion of preponderant flora and Clostridiaceae was identified as a feature marker in the GM of the co-infection group. Redundancy analysis (RDA)/canonical correspondence analysis (CCA) results showed that liver fibrosis, Th1/Th2 cytokines were significantly correlated (P < 0.05) with the GM compositions. Tg infection inhibits hepatic fibrosis by downregulating Th2 immune response against Sj infection, and further promotes the GM shifts through “gut–liver axis” in the murine hosts. Our study may provide insights into potential anti-fibrosis strategies in co-infection individuals.

Regulatory T cells counteract neuropathic pain through inhibition of the Th1 response at the site of peripheral nerve injury.

The inflammatory/immune response at the site of peripheral nerve injury participates in the pathophysiology of neuropathic pain. Nevertheless, little is known about the local regulatory mechanisms underlying peripheral nerve injury that counteracts the development of pain. Here, we investigated the contribution of regulatory T (Treg) cells to the development of neuropathic pain by using a partial sciatic nerve ligation model in mice. We showed that Treg cells infiltrate and proliferate in the site of peripheral nerve injury. Local Treg cells suppressed the development of neuropathic pain mainly through the inhibition of the CD4 Th1 response. Treg cells also indirectly reduced neuronal damage and neuroinflammation at the level of the sensory ganglia. Finally, we identified IL-10 signaling as an intrinsic mechanism by which Treg cells counteract neuropathic pain development. These results revealed Treg cells as important inhibitory modulators of the immune response at the site of peripheral nerve injury that restrains the development of neuropathic pain. In conclusion, the boosting of Treg cell function/activity might be explored as a possible interventional approach to reduce neuropathic pain development after peripheral nerve damage.

CCR5 attenuates neutrophilic airway inflammation exacerbated by infection with rhinovirus

Human rhinovirus (hRV) is the most common cause of asthma exacerbation characterized by clinical and pathophysiological heterogeneity. Steroid-sensitive, Th2 type-eosinophilic asthma has been somewhat studied, but hRV-induced neutrophilic asthma exacerbation is poorly understood. Here, CCR5 was found to play a role in attenuating neutrophilic airway inflammation in hRV-induced asthma exacerbation using chicken ovalbumin (OVA)-based model. CCR5 deficiency resulted in exacerbated neutrophilic asthmatic responses in airways following hRV infection. CCR5-deficient mice showed enhanced mucus expression and altered expression of tight junction proteins in lung tissues. CCR5-deficient mice were also manifested with influx of CD45+CD11b+Siglec-F+Gr-1+ neutrophils, along with enhanced production of IL-17A, IFN-γ, IL-6, IL-1β cytokines in inflamed tissues. In contrast, CCR5-deficient mice elicited down-regulation of Th2-related cytokine proteins following hRV infection. More interestingly, the lack of CCR5 altered the equilibrium of CD4+FoxP3+ Tregs and IL-17+CD4+ Th17 in inflamed tissues. CCR5-deficient mice showed increased frequency and absolute number of IL-17-producing CD4+ Th17 cells in lung tissues compared to wild-type mice, whereas the reduced infiltration of CD4+FoxP3+ Treg cells was observed. CCR5 deficiency resulted in the skewed production of Th17 and Th1 cytokines in lymph nodes and lungs upon OVA stimulation. Likewise, CCR5-deficient mice showed enhanced expression of Th17-inducing cytokines (IL-1β, IL-6, and TNF-α) in lung tissues. These results imply that CCR5 deficiency facilitates Th17 airway inflammation during hRV-induced asthma exacerbation, along with suppressing Th2 responses. Furthermore, our results suggest that CCR5 attenuates hRV-induced neutrophilic airway inflammation through conserving the equilibrium of CD4+Foxp3+ Treg cells and IL-17+CD4+ Th17 cells in hRV-induced asthma exacerbation.

Plasmacytoid dendritic cells suppress Th2 responses induced by epicutaneous sensitization.

Epicutaneous (EC) sensitization with protein allergens is the most important sensitization route for atopic dermatitis. Plasmacytoid dendritic cells (pDCs) are characterized by massive secretion of interferon-α (IFNα). B6 mice are T helper type 1 (Th1)-prone and are representative of non-atopic humans, whereas BALB/c mice are Th2-prone and are representative of atopic humans. Here, we show that naïve BALB/c mice contain a greater number of nonactivated pDCs in peripheral lymph nodes (LNs) than do naïve B6 mice. Naïve BALB/c mice also have more of the CD8α- subset in LNs than naïve B6 mice. Moreover, in vivo depletion of pDCs during EC sensitization results in enhanced Th2 responses in BALB/c mice, but not in B6 mice. Mechanistically, when BALB/c mice undergo EC sensitization, there is an increase in pDCs entering draining LNs. These cells exhibit modest activation including comparable costimulation expression but increased cytokine expression compared with those of naïve mice. In vivo depletion of pDCs during EC sensitization significantly increases the activation of dermal dendritic cells (dDCs) suggesting a regulatory effect on these cells. To this end, a suppressive effect of pDCs on conventional dendritic cells was also demonstrated in vitro. Further, in vivo blockade of IFNα by an anti-IFNAR antibody (Ab) or in vivo reduction of IFNα production of pDCs by anti-siglec-H Ab both resulted in enhanced activation of dDCs. Collectively, our results demonstrate that pDCs suppress Th2 responses induced by EC sensitization via IFNα-mediated regulation of dDCs.

Inhibition of antigen-specific immune responses by co-application of an indoleamine 2,3-dioxygenase (IDO)-encoding vector requires antigen transgene expression focused on dendritic cells

We have previously shown that particle-mediated epidermal delivery (PMED) of plasmids encoding β-galactosidase (βGal) under control of the fascin-1 promoter (pFascin-βGal) yielded selective production of the protein in skin dendritic cells (DCs), and suppressed Th2 responses in a mouse model of type I allergy by inducing Th1/Tc1 cells. However, intranasal challenge of mice immunized with pFascin-βGal induced airway hyperreactivity (AHR) and neutrophilic inflammation in the lung. The tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) has been implicated in immune suppression and tolerance induction. Here we investigated the consequences of co-application of an IDO-encoding vector on the modulatory effect of DNA vaccination by PMED using pFascin-βGal in models of eosinophilic allergic and non-eosinophilic intrinsic airway inflammation. IDO-encoding plasmids and pFascin-βGal or pCMV-βGal were co-applied to abdominal skin of BALB/c mice without, before or after sensitization with βGal protein. Immune responses in the lung were analysed after intranasal provocation and airway reactivity was determined by whole body plethysmography. Co-application of pCMV-IDO with pFascin-βGal, but not pCMV-βGal inhibited the Th1/Tc1 immune response after PMED. Moreover, AHR in those mice was attenuated following intranasal challenge. Therapeutic vaccination of βGal-sensitized mice with pFascin-βGal plus pCMV-IDO slightly suppressed airway inflammation and AHR after provocation with βGal protein, while prophylactic vaccination was not effective. Altogether, our data suggest that only the combination of DC-restricted antigen and ubiquitous IDO expression attenuated asthma responses in mice, most probably by forming a tryptophan-depleted and kynurenine-enriched micromilieu known to affect neutrophils and T cells.

Galectin-9 Induced by Dietary Probiotic Mixture Regulates Immune Balance to Reduce Atopic Dermatitis Symptoms in Mice.

Probiotics can be an effective treatment for atopic dermatitis (AD), while their mechanism of action is still unclear. Here, we induced AD in mice with 2,4-dinitrochlorobenzene and administrated YK4, a probiotic mixture consisting of Lactobacillus acidophilus CBT LA1, L. plantarum CBT LP3, Bifidobacterium breve CBT BR3, and B. lactis CBT BL3. Then, we have validated the underlying mechanism for the alleviation of AD by YK4 from the intestinal and systematic immunological perspectives. Administration of YK4 in AD mice alleviated the symptoms of AD by suppressing the expression of skin thymic stromal lymphopoietin and serum immunoglobulin E eliciting excessive T-helper (Th) 2 cell-mediated responses. YK4 inhibited Th2 cell population through induce the proportion of Th1 cells in spleen and Treg cells in Peyer's patches and mesenteric lymph node (mLN). CD103+ dendritic cells (DCs) in mLN and the spleen were significantly increased in AD mice administered with YK4 when compared to AD mice. Furthermore, galectin-9 was significantly increased in the gut of AD mice administered with YK4. In vitro experiments were performed using bone marrow-derived DCs (BMDC) and CD4+ T cells to confirm the immune mechanisms of YK4 and galectin-9. The expression of CD44, a receptor of galectin-9, together with programmed death-ligand 1 was significantly upregulated in BMDCs following treatment with YK4. IL-10 and IL-12 were upregulated when BMDCs were treated with YK4. Cytokines together with co-receptors from DCs play a major role in the differentiation and activation of CD4+ T cells. Proliferation of Tregs and Th1 cell activation were enhanced when CD4+T cells were co-cultured with YK4-treated BMDCs. Galectin-9 appeared to contribute at least partially to the proliferation of Tregs. The results further suggested that DCs treated with YK4 induced the differentiation of naïve T cells toward Th1 and Tregs. At the same time, YK4 alleviated AD symptoms by inhibiting Th2 response. Thus, the present study suggested a potential role of YK4 as an effective immunomodulatory agent in AD patients.

Vaginal dysbiosis associated-bacteria Megasphaera elsdenii and Prevotella timonensis induce immune activation via dendritic cells

Dysbiosis of the vaginal microbiome as a result of overgrowth of anaerobic bacteria leads to bacterial vaginosis (BV) which is associated with increased inflammation in the genital mucosa. Moreover, BV increases susceptibility to sexual transmitted infections (STIs) and is associated with adverse pregnancy outcomes. It remains unclear how specific vaginal aerobic and anaerobic bacteria affect health and disease. We selected different vaginal bacteria ranging from true commensals to species associated with dysbiosis and investigated their effects on activation of dendritic cells (DCs). Commensal Lactobacilli crispatus did not induce DC maturation nor led to production of pro-inflammatory cytokines. In contrast, BV-associated bacteria Megasphaera elsdenii and Prevotella timonensis induced DC maturation and increased levels of pro-inflammatory cytokines. Notably, DCs stimulated with Prevotella timonensis suppressed Th2 responses and induced Th1 skewing, typically associated with preterm birth. In contrast, Lactobacillus crispatus and Megasphaera elsdenii did not affect Th cell polarization. These results strongly indicate that the interaction of vaginal bacteria with mucosal DCs determines mucosal inflammation and we have identified the anaerobic bacterium Prevotella timonensis as a strong inducer of inflammatory responses. Specifically targeting these inflammation-inducing bacteria might be a therapeutic strategy to prevent BV and associated risks in STI susceptibility and preterm birth.

Ablation of IL-33 suppresses Th2 responses but is accompanied by sustained mucus obstruction in the Scnn1b transgenic mouse model

Ablation of IL-33 suppresses Th2 responses but is accompanied by sustained mucus obstruction in the Scnn1b transgenic mouse model

Aberrant Th2 Immune Responses Are Associated With a Reduced Frequency of IL-35-Induced Regulatory T Cells After Allergen Exposure in Patients With Allergic Asthma.

PurposeAllergen exposure induces aberrant T helper (Th) 2 immune responses in patients with allergic asthma, but not in sensitized asymptomatic and nonallergic subjects. Interleukin (IL)-35-induced regulatory T (iTr35) cells are a new subset of regulatory T cells with immunoregulatory properties. These cells can significantly suppress Th2 responses in seasonal allergic rhinitis. However, it remains unknown whether iTr35 cells are involved in the immunoregulation of allergic asthmatic individuals after specific allergen exposure.MethodsThe iTr35 cell frequency in peripheral blood mononuclear cells (PBMCs) was measured in patients with allergic asthma as well as in asymptomatic and healthy subjects. The difference in naïve CD4+ T cell conversion to iTr35 cells in vitro during allergen stimulation was also investigated. The effects of iTr35 cells on naïve CD4+ T cell differentiation into Th2 cells, CD4+CD25− T (Teff) cell proliferation and Th2 cytokine production in vitro were assessed.ResultsSignificantly reduced iTr35 cell frequencies and IL-35 expression levels were found in asthmatic patients with Derp1 allergy compared with asymptomatic and healthy subjects. Moreover, the circulating iTr35 cell proportion and IL-35 expression level in asthmatic patients gradually decreased with disease severity. Patients with allergic asthma had reduced transformation of naïve CD4+ T cells into iTr35 cells and IL-35 production after allergen exposure compared with asymptomatic and healthy subjects. Most importantly, iTr35 cells inhibited allergen-driven differentiation of naïve CD4+ T cells into Th2 cells, Teff cell proliferation and Th2 cytokine production in an IL-35-dependent manner.ConclusionsThe results of our study suggest that iTr35 cells may play an important role in preventing Th2 responses to allergens by secreting IL-35 and that iTr35 cells may be a potential new immune regulator of allergic asthma.

Bifidobacteria adolescentis regulated immune responses and gut microbial composition to alleviate DNFB-induced atopic dermatitis in mice.

Emerging studies have reported gut microbial composition plays a key role in alleviating AD clinical symptoms during the probiotic intervention, but the correlation among clinical symptoms, immune responses and gut microbial alteration needs to be explored. Therefore, the objective was to investigate the correlation during Bifidobacterium adolescentis intervention in DNFB-induced AD mice. The mice were randomly divided into nine groups and fed B. adolescentis for 3weeks. At the end of the experiment, clinical and immune indicators were assessed. Flow cytometry was performed to explore the effect of B. adolescentis on regulatory T cells in the spleen. V3-V4 region of the 16S ribosomal RNA (rRNA) gene was sequenced to evaluate changes in the gut microbiota. Bifidobacteria adolescentis treatments reduced ear and skin thickness and suppressed eosinophils and mast cells infiltration. Th1- and Th2-type responses were regulated and the Tregs population was promoted in the spleen by B. adolescentis treatments. Bifidobacteria adolescentis increased the relative abundance of Lactobacillus but decrease Dorea and Pediococcus. Propionic and butyric acids were increased but isovaleric acid was decreased by B. adolescentis treatment. Besides, the functional modules, such as fatty acid biosynthesis, antigen processing and presentation were upregulated by B. adolescentis Ad1 treatment compared to the DNFB group. Collectively, these results imply that B. adolescentis with the role of immunomodulation promotes Treg differentiation and suppresses Th2 responses, and increases the proportion of Lactobacillus that is positively correlated to increase in propionic acid production, and thus has the potential for AD amelioration.

The Immune Micro-Environment in Diffuse Large B Cell Lymphoma Is Characterised By an Immunosuppressive Shift in T Cell Subsets

Diffuse large B-cell Lymphoma (DLBCL) is the most frequent non-Hodgkin's lymphoma with 3 molecularly distinct subtypes based on cell of origin. Genetic alterations in DLBCL, expression of checkpoint molecules and an immunosuppressive microenvironment (ME) all contribute to escape from host anti-lymphoma immunity. The clinical success of monoclonal antibodies that engage the immune system and CAR-T cellular therapy have further highlighted the importance and therapeutic potential of the immune ME in DLBCL. Here we present data from comprehensive phenotyping of cell suspensions from diagnostic DLBCL and reactive lymph node / tonsil (RLNT) biopsies by cytometry by time of flight (CyTOF), with a focus on the T-cell compartment. Cryopreserved samples from 6 DLBCL (5 LN, 1 spleen) at diagnosis and 5 RLNT (3 LN, 2 tonsil) were stained with a panel of metal-tagged antibodies and analysed by CyTOF2. Samples were acquired in 2 batches with the same RLNT (LN) sample with each to ensure staining consistency. Data were normalised, uploaded to Cytobank, gated to CD45+ CD3+ live single cells and exported for further analysis with Cytofkit in R. CD3+ events were gated further into CD4+ and CD8+ subsets, which demonstrated that CD4+ T cells were the predominant phenotype in all samples. However, there was a marked skewing of the CD4:CD8 ratio, with CD4+ T cells lower as a percentage of CD3+ T cells in the DLBCL samples (55.84 v 78.18, p=0.0173*). CD8+ T cells were higher as a percentage in DLBCL (36.22 v 16.75, p=0.03*) with no difference seen in double negative (DN) T cells. CD3+ T cells were then clustered with FlowSOM and visualised according to the tSNE algorithm. A heatmap of median marker expression intensity was generated to facilitate cluster identification. This revealed a number of differences in cluster abundance between the groups, with a significant shift in differentiation away from naïve and towards an effector memory (EM) phenotype in DLBCL. There were fewer cells in the CD27+ CD28+ CCR7+ CD45RA+ CD4+ naïve cluster in the DLBCL samples than the RLNT (p=0.0173*). Although the DLBCL samples showed an overall reduction in CD4+ T cells, the clusters of regulatory T cells (Treg: CD4+ CD25+ FOXP3+ and CD127-/low) consisted of more cells from these cases than the RLNT (p=0.0043**). Within the Treg population, the DLBCL patients had more Th1 polarised (T-bet+) Tregs and more PD-1 expressing Tregs. The Th1 Tregs predominantly secreted the suppressive cytokines IL-2, IL-10 and TGF-β on stimulation and may play a role in inhibiting Th1 responses. Conventional Th1 were not increased in DLBCL resulting in a higher Th1 Treg to Th1 ratio than in RLNT. There was a trend for RLNT samples to contribute more cells to the PD-1 high follicular helper T cell (TFH) cluster and DLBCL to the PD-1+ TIM-3+ DN cluster. The DLBCL ME had relatively more CD8+ T cells and contributed more to the CCR7- CD45RA- CD8+ EM clusters (p=0.0173*) but the CD8+ T cells in the RNLT samples tended to a naïve CCR7+ CD45RA+ PD-1- phenotype (p=0.0519). The CD8+ EM cells enriched in the DLBCL ME expressed the cytotoxic markers granzyme and perforin and responded to stimulation with degranulation (CD107a) and cytokine production (IFNγ, TNFα, TGFβ and IL-10), not suggestive of exhaustion. It is also notable that a cluster of PD-1+ TIM-3+ CD8+ EM with reduced markers of cytotoxicity, low CD107a expression and poor cytokine production after stimulation was predominantly made up of cells from DLBCL suspensions (p=0.002**). CyTOF analysis of the DLBCL ME has demonstrated a shift in the balance of T cell subsets and CD4:CD8 ratio with a relative abundance of immunosuppressive Tregs despite an overall reduction in the CD4+ population and a skew towards differentiation in CD4+ and CD8+ populations. The cytotoxic T cells in DLBCL tended to have an EM phenotype and express immune checkpoint molecules but remained capable of cytokine production. However, the production of IFNγ by these effector T cells may play a role in the development of inhibitory Tregs with a Th1 phenotype, which were enriched in these patients. A cluster of CD8+ EM cells expressing checkpoint molecules and displaying characteristics of exhaustion following stimulation was also seen in these DLBCL patients. These data provide new insights into the immunosuppressive nature of the DLBCL ME and provide a rationale for targeting the ME alongside existing therapeutic approaches, including CAR-T cells to improve outcomes. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

Tangeretin attenuates lipopolysaccharide-induced acute lung injury through Notch signaling pathway via suppressing Th17 cell response in mice

Tangeretin, a polymethoxylated flavonoid is abundant in citrus fruits, which has been reported to inhibit inflammation by inhibiting NF-κB activation and proinflammatory cytokines. Notch blockage inhibits Th17 cells response that are involved in the development of acute lung injury (ALI). This study investigated the protective effects of tangeretin on LPS-induced ALI in mice. Male C57BL/6 mice were treated with phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS and tangeretin, or LPS and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, a Notch signaling inhibitor), which were harvested at 48 h after challenged by LPS. CD4+ T cells were treated with tangeretin or DAPT and harvested after 72 h. Tangeretin notably attenuated pathological changes and decreased the wet to dry weight ratio of the mouse lungs. The total cell and neutrophil counts, tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF), myeloperoxidase activity of lung tissue were markedly reduced by tangeretin. The percentage of CD4+IL-17 + T cells in the lungs and the concentration of interleukin (IL)-17 and IL-22 in BALF were significantly down-regulated by tangeretin. As with the positive control (DAPT), tangeretin inhibited the activity of the Notch signaling pathway accompanied with the down-regulation of acid-related orphan receptor gamma t and IL-23 receptor expression. This study demonstrated that tangeretin protects against LPS-induced ALI by suppressing Th17 response at least partially, through a Notch-dependent mechanism.

Orally delivered all-trans-retinoic acid- and transforming growth factor-β-loaded microparticles ameliorate type 1 diabetes in mice

Type 1 diabetes (T1D) is a multifactorial autoimmune disease that develops as a consequence of macrophage- and T cell-dependent pancreatic β-cell death. Multiple approaches for induction of anti-inflammatory/regulatory mechanisms that would attenuate T1D have been utilized, with little or no beneficial effects. To achieve prolonged stimulation of regulatory immune cells, we orally introduced microparticles (MPs) loaded with all-trans retinoic acid (ATRA) and transforming growth factor-β (TGF-β) to C57BL/6 mice treated with multiple low doses of streptozotocin (MLDS) for T1D induction. Disease incidence was significantly lower in ATRA/TGF-β MPs-treated mice, as was the degree of immune cell infiltration into the pancreatic islets. In Peyer's patches (PP), ATRA/TGF-β MPs up-regulated tolerogenic dendritic cells (tolDCs) (CD11c+CD11b-CD103+), while the proportion of mature dendritic cells was not altered. This was accompanied by reduced Th1 and Th17 proportions and up-regulation of regulatory T cells (Tregs - CD4+CD25highFoxP3+). The immune cell composition in the pancreatic lymph nodes was similar to PP. Further, the proportion of effector Tbet+CD25med cells was decreased, while the proportion of Tbet+ Treg cells that specifically inhibit Th1 response was increased. Moreover, ATRA/TGF-β MPs treatment resulted in increased Treg proliferation and frequency of CTLA-4+PD1+ and CD39+IL-10+ Tregs, suggestive of their higher suppressive capacity. Reduced pancreatic infiltration may have been a consequence of lower cell capacity for matrix degradation. In conclusion, oral application of ATRA/TGF-β MPs ameliorated T1D through potentiation of tolDCs and Tregs, inhibition of Th1 response and prevention of the immune cell entrance into the islets.

Glucocorticoids and Glucocorticoid-Induced-Leucine-Zipper (GILZ) in Psoriasis.

Psoriasis is a prevalent chronic inflammatory human disease initiated by impaired function of immune cells and epidermal keratinocytes, resulting in increased cytokine production and hyperproliferation, leading to skin lesions. Overproduction of Th1- and Th17-cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-23, IL-17, and IL-22, is a major driver of the disease. Glucocorticoids (GCs) represent the mainstay protocol for treating psoriasis as they modulate epidermal differentiation and are potent anti-inflammatory compounds. The development of safer GC-based therapies is a high priority due to potentially severe adverse effects associated with prolonged GC use. Specific efforts have focused on downstream anti-inflammatory effectors of GC-signaling such as GC-Induced-Leucine-Zipper (GILZ), which suppresses Th17 responses and antagonizes multiple pro-inflammatory signaling pathways involved in psoriasis, including AP-1, NF-κB, STAT3, and ROR-γt. Here we review evidence regarding defective GC signaling, GC receptor (GR) function, and GILZ in psoriasis. We discuss seemingly contradicting data on the loss- and gain-of-function of GILZ in the imiquimod-induced mouse model of psoriasis. We also present potential therapeutic strategies aimed to restore GC-related pathways.

Prostaglandin E2-Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti-PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection.

Bovine leukemia virus (BLV) infection is a chronic viral infection of cattle and endemic in many countries, including Japan. Our previous study demonstrated that PGE2, a product of cyclooxygenase (COX) 2, suppresses Th1 responses in cattle and contributes to the progression of Johne disease, a chronic bacterial infection in cattle. However, little information is available on the association of PGE2 with chronic viral infection. Thus, we analyzed the changes in plasma PGE2 concentration during BLV infection and its effects on proviral load, viral gene transcription, Th1 responses, and disease progression. Both COX2 expression by PBMCs and plasma PGE2 concentration were higher in the infected cattle compared with uninfected cattle, and plasma PGE2 concentration was positively correlated with the proviral load. BLV Ag exposure also directly enhanced PGE2 production by PBMCs. Transcription of BLV genes was activated via PGE2 receptors EP2 and EP4, further suggesting that PGE2 contributes to disease progression. In contrast, inhibition of PGE2 production using a COX-2 inhibitor activated BLV-specific Th1 responses in vitro, as evidenced by enhanced T cell proliferation and Th1 cytokine production, and reduced BLV proviral load in vivo. Combined treatment with the COX-2 inhibitor meloxicam and anti-programmed death-ligand 1 Ab significantly reduced the BLV proviral load, suggesting a potential as a novel control method against BLV infection. Further studies using a larger number of animals are required to support the efficacy of this treatment for clinical application.

Estrogen signaling effects on muscle-specific immune responses through controlling the recruitment and function of macrophages and T cells

BackgroundEstrogen signaling is indispensable for muscle regeneration, yet the role of estrogen in the development of muscle inflammation, especially in the intramuscular T cell response, and the influence on the intrinsic immuno-behaviors of myofibers remain largely unknown. We investigated this issue using the mice model of cardiotoxin (CTX)-induced myoinjury, with or without estrogen level adjustment.MethodsCTX injection i.m. (tibialis anterior, TA) was performed for preparing mice myoinjury model. Injection s.c. of 17β-estradiol (E2) or estrogen receptor antagonist 4-OHT, or ovariectomy (OVX), was used to change estrogen level of animal models in vivo. Serum E2 level was evaluated by ELISA. Gene levels of estrogen receptor (ERs) and cytokines/chemokines in inflamed muscle were monitored by qPCR. Inflammatory infiltration was observed by immunofluorescence. Macrophage and T cell phenotypes were analyzed by FACS. Immunoblotting was used to assess protein levels of ERs and immunomolecules in C2C12 myotubes treated with E2 or 4-OHT, in the presence of IFN-γ.ResultsWe monitored the increased serum E2 level and the upregulated ERβ in regenerated myofibres after myotrauma. The absence of estrogen in vivo resulted in the more severe muscle inflammatory infiltration, involving the recruitment of monocyte/macrophage and CD4+ T cells, and the heightened proinflammatory (M1) macrophage. Moreover, estrogen signaling loss led to Treg cells infiltration decrease, Th1 response elevation in inflamed muscle, and the markedly expression upregulation of immunomolecules in IFN-γ-stimulated C2C12 myotubes in vitro.ConclusionOur data suggest that estrogen is a positive intervention factor for muscle inflammatory response, through its effects on controlling intramuscular infiltration and phenotypes of monocytes/macrophages, on affecting accumulation and function of Treg cells, and on suppressing Th1 response in inflamed muscle. Our findings also imply an inhibition effect of estrogen on the intrinsic immune behaviors of muscle cells.

Protein Kinase C Theta Inhibition Attenuates Lipopolysaccharide-Induced Acute Lung Injury through Notch Signaling Pathway via Suppressing Th17 Cell Response in Mice.

Acute lung injury (ALI)/acute respiratory distress syndrome is characterized by increased pulmonary inflammation, where T helper 17 (Th17) cells play an important regulatory role. Notch signaling critically regulates Th17 differentiation and is known to be linked with proximal T cell by protein kinase C theta (PKCθ). We hypothesized that PKCθ inhibition could attenuate ALI by suppressing Th17 response via the Notch signaling pathway. Male C57BL/6 mice were treated with phosphate-buffered saline (PBS), lipopolysaccharide (LPS), LPS and N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT, a Notch signaling inhibitor), or LPS and PKCθ inhibitor (PI), and the bronchoalveolar lavage fluid (BALF), blood, and lung tissues were harvested at 48h after the LPS challenge. CD4+ T cells were treated with DAPT or PI and harvested after 72h. PKCθ inhibition markedly attenuated pathological changes and decreased the wet to dry weight ratio of the mouse lungs. The total cell and neutrophil counts, tumor necrosis factor-α (TNF- α) in BALF, myeloperoxidase activity in lung tissue, and the leukocyte count in whole blood were markedly reduced by PKCθ inhibition. The concentration of interleukin (IL)-17 and IL-22 in BALF, and the percentage of CD4+IL-17A+ T cells in the lungs were significantly downregulated by PKCθ inhibition. A similar trend was observed for the expression of retinoic acid-related orphan receptor gamma t and IL-23 receptor after PKCθ inhibition accompanied with inactivation of the Notch signaling pathway in vivo and in vitro. Collectively, these data demonstrated that PKCθ inhibition protects against LPS-induced ALI by suppressing the differentiation and pathogenicity of Th17, at least partially, through a Notch-dependent mechanism.

Abstract 1313: CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas

Abstract Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells. Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with &amp;gt;100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p&amp;lt;0.05) upon antigen-specific re-stimulation in an OVA transgenic adoptive transfer model was observed, consistent with Th1-skewing. Additionally, toassess the impact of CPI-818 on Th-biasing, activated human PBMCs were cultured for 6 days with CPI-818 and inhibition of IL-4 production (average 64% inhibition, n=12) was significantly greater than IFNγ production (average 19% inhibition), supportive of Th1 skewing. To assess the potential of CPI-818 to treat human T cell lymphoproliferative disorders, the safety and efficacy of CPI-818 in companion dogs with spontaneously-occurring T cell lymphomas were evaluated. CPI-818 was dosed orally at 20 mg/kg BID for 2 weeks to 5 months. Three animals were treated: 1 with peripheral T cell lymphoma and 2 with cutaneous T cell lymphoma. Full ITK occupancy in peripheral blood was confirmed using a probe assay. Evidence of anti-tumor activity was seen in all dogs including complete and partial responses. CPI-818 was well tolerated with no change in normal lymphocyte counts. These data support evaluation of CPI-818 in clinical trials in patients with T cell malignancies. Citation Format: James W. Janc, Craig M. Hill, Patrick P. Ng, Andrew N. Hoston, Antonett Madriaga, Trang P. Dao-Pick, Kitman S. Yeung, Ryan Hudson, Anne-Marie Beausoleil, Erin Bradley, Erik Verner, Douglas H. Thamm, Richard A. Miller, Joseph J. Buggy. CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1313.

The role of Interleukin-33 in the modulation of splenic T-cell immune responses after experimental ischemic stroke

The splenic T-cell immune response to stroke has been identified as an important role in the progression of brain injury following ischemic stroke. Interleukin (IL)-33 as a novel cytokine of IL-1 family has been found to be protective for ischemic brain injury. Here, we determined the contribution of IL-33 to the T-cell immune responses in the spleen after experimental ischemic stroke. Mice were subjected to 30 min of middle cerebral artery occlusion (MCAO) for ischemic stroke induction. Recombinant mouse IL-33 (100 μg/kg) was pre-treated intraperitoneally at 30 min prior to MCAO, then the percentages of T cell subsets, related cytokines and transcription factors in the spleen tissues were measured. Intraperitoneal IL-33 pre-treatment may attenuate neurological deficit scores and infarct volumes after MCAO, which was accompanied by reduced IFN-γ+ T cells and increased Foxp3+ T cells in the spleen tissues. Meanwhile, IL-33 pre-treatment could decrease the production of IFN-γ and increase the secretion of IL-4, IL-10 and TGF-β from the spleen at 24 h after MCAO. Additionally, the mRNA level of the transcription factor T-bet was downregulated by IL-33, and the levels of GATA-3 and Foxp3 mRNA were upregulated. These results showed that the long-term protective mechanism of IL-33 in ischemic stroke may be partly associated to its modulation role for splenic T-cell immune responses through inhibiting Th1 response and promoting Treg response, suggesting that IL-33 may be a candidate treatment for human stroke via modulating the peripheral immune system following stroke.

Recombinant Virus-Like Particles Presenting IL-33 Successfully Modify the Tumor Microenvironment and Facilitate Antitumor Immunity in a Model of Breast Cancer

Recently, interleukin (IL)-33 has been closely associated witha variety of clinical cancers. IL-33 presents both protumorigenic, and less frequently, antitumorigenic functions depending on disease conditions. IL-33 signaling appears to be a possible target for the treatment of applicable tumor diseases. This study aimed to develop an effective approach to intervene in IL-33 functioning in tumors and reveal the immunotherapeutic potential of anti-IL-33 active immunization. Recombinant truncated hepatitis B virus core antigen (HBcAg), presenting mature IL-33 molecules on the surface of virus-like particles (VLPs), was prepared and used to immunize BALB/c mice in a model of murine 4T1 breast cancer. The immunization was performed through either a preventive or therapeutic strategy in two separate studies. Anti-IL-33 immunization with VLPs elicited a persistent and highly titrated specific antibody response and significantly suppressed orthotopic tumor growth and lung metastasis in both studies. The underlying mechanisms might include promoting tumor-specific Th1/CTL cellular responses and the expression of the effector molecule IFN-γ, suppressing Th2 responses, and significantly reducing the infiltration of immunosuppressive Treg cells and MDSCs into tumor tissues in the immunized mice. In conclusion, anti-IL-33 active immunization employing recombinant VLPs as an antigen delivery platform effectively modified the tumor microenvironment and promoted antitumor immunity, indicating the potential of this approach as a new and promising immunotherapeutic strategy for the treatment of cancers where IL-33 plays a definite protumorigenic role.