Th2-mediated Inflammation Research Articles

Exploiting the Gut Microbiota to Boost Immunotherapy and Switch the Trajectory of Multiple Myeloma

Clonal proliferation of neoplastic plasma cells in the bone marrow (BM) and organ dysfunction characterizes multiple myeloma (MM), a treatable but incurable disease. Although asymptomatic smoldering MM (SMM) often precedes full blown MM, most patients affected by SMM are only offered active observation, which increases their frustration and anxiety. In transgenic Vk*MYC mice developing de novo MM, we found a direct link between gut microbiota composition and T helper 17 (Th17) lymphocytes, which migrated from the gut to the BM during asymptomatic MM (Early-MM), and favored the expansion of neoplastic plasma cells, thus fueling progression to symptomatic disease (Late-MM) [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Similarly, in SMM patients, higher levels of BM Interleukin-17 (IL-17) predicted accelerated disease [Calcinotto A. et al. Nat Commun (2018) 9(1):4832]. Interestingly, Prevotella melaninogenica ( P.m.), a human commensal, limited MM aggressiveness in mice challenged with Vk*MYC-derived MM cells (t-Vk*MYC) by restraining Th17 cell expansion [Calcinotto et al. Nat Commun (2018) 9(1):4832]. Because the gut microbiota also contributes to the therapeutic efficacy of immune checkpoint blockade (ICB) and human neoplastic plasma cells express PD-L1, we hypothesized that modulation of the gut microbiota by P.m. could limit Th17 cell expansion in mice affected by Early-MM, thus fully exploiting the therapeutic potential of anti-PD-L1 against MM. Clinical trials combining anti-PD-1/PD-L1 antibodies, dexamethasone and immune modulatory drugs in MM were halted by US FDA because of unfavorable benefit-risk profile and life-threatening immune related adverse events (irAEs). Because IL-17 is involved in irAEs [Johnson et al. Cancer Immunol Res (2019) 7 (6): 860-865], we also hypothesized treatment with P.m. could restrain ICB-induced expansion of Th17 cells while unleashing CD8-mediated antitumor immunity. Early-MM Vk*MYC mice and t-Vk*MYC, the latter mimicking aggressive MM, were treated with P.m. and/or anti-PD-L1 and disease progression was monitored by paraprotein quantification in blood. Anemia provided clinical evidence of symptomatic disease. At sacrifice, gut, spleen and BM were analyzed by flow cytometry. In vitro assays were conducted to clarify the mechanism by which the gut microbiota influenced the induction of Th17 cells. To mimic cutaneous irAE, the skin of treated mice was exposed to imiquimod, which induces Th17-mediated inflammation [van der Fits et al. J Immunol (2009 )182(9): 5836-5845]. Body weight loss, skin inflammation, and inflammatory infiltrate into secondary lymphoid organs were taken as measure of imiquimod-induced toxicity. In Vk*MYC mice, all neoplastic plasma cells expressed PD-L1. Treatment of Early-MM Vk*MYC mice with anti-PD-L1 antibodies delayed progression to Late-MM, thus extending previous findings on the use of ICB in this model to anti-PD-L1 antibodies and Early-MM [Guillerey et al. J Clin Invest (2015 )125(5): 2077-2089]. Treatment with P.m. exerted a similar effect on disease progression, suggesting that a treatment as simple as taking a probiotic has an impact on MM evolution. When P.m. was combined with anti-PD-L1 antibodies, evolution to Late-MM was further delayed or even blocked. Single and combined treatments also reduced MM aggressiveness in t-Vk*MYC mice. Mechanistically, P.m. limited the expansion of gut-born Th17 cells and their accumulation in the BM of treated mice without dampening the antitumor cytotoxic response elicited by the ICB. Thus, only the combined treatment provided the most favorable CD8/Th17 and CD8/T regulatory cell ratios. Ex vivo, P.m.-conditioned BM DCs showed a limited propensity to produce Th17-polarizing cytokines. In vitro, stimulation of both human and mouse DCs with P.m. reduced polarization of naïve T cells toward a Th17 phenotype. Skin application of imiquimod in C57BL/6 mice induced psoriasis-like lesions and expansion of Th17 cells both in draining lymph nodes and spleen. The contextual administration of anti-PD-L1 antibodies worsened imiquimod-induced inflammation. An ongoing experiment will clarify if mice receiving P.m. and ICB experience reduced skin lesions. Taken together, our data support the development of microbiota-based strategies in combination with ICB to treat full-blown MM and to prevent progression of asymptomatic SMM to full-blown disease.

A clinically-relevant STING agonist restrains human TH17 cell inflammatory profile

The STING signaling pathway has gained attention over the last few years due to its ability to incite antimicrobial and antitumoral immunity. Conversely, in mouse models of autoimmunity such as colitis and multiple sclerosis, where TH17 cells are implicated in tissue inflammation, STING activation has been associated with the attenuation of immunogenic responses. In this line, STING was found to limit murine TH17 pro-inflammatory program in vitro. Here we demonstrate that 2′3′-c-di-AM(PS)2(Rp,Rp), a STING agonist that has been undergoing clinical trials for antitumor immunotherapy, activates the STING signalosome in differentiating human TH17 cells. Of particular interest, 2′3′-c-di-AM(PS)2(Rp,Rp) reduces IL-17A production and IL23R expression by human TH17 cells while it favors the generation of regulatory T (Treg) cells. These findings suggest that STING agonists may be promising approaches for treating human TH17-mediated chronic inflammation.

Aminooxy acetic acid suppresses Th17-mediated psoriasis-like skin inflammation by inhibiting serine metabolism.

Background: Psoriasis is a common chronic inflammatory skin disease characterized by an external red rash that is caused by abnormal proliferation and differentiation of keratinocytes and immune T cells. This study aimed to elucidate the role of aminooxy acetic acid (AOA) in alleviating psoriasis from the perspective of immunology and metabolomics. Therefore, contributing to the development of new drugs as candidates for psoriasis treatment. Methods: To investigate the symptom-alleviating effects and the related mechanisms of AOA on the treatment of psoriasis, we used a 12-O-tetradecanoylphorbol-13-acetate-induced psoriasis-like skin mouse model and interleukin (IL)-17-stimulated human keratinocytes. Results: The results showed that AOA ameliorated psoriasis-related symptoms and decreased inflammation-associated antimicrobial peptides and T-helper 17 (Th17)-associated cytokines in a mouse model of psoriasis. Furthermore, AOA inhibited the activation of mechanistic target of rapamycin (mTOR) by suppressing serine metabolism-related genes. Importantly, mTOR inhibition ameliorated psoriatic disease by affecting the differentiation of various T cells and normalizing the Th17/regulatory T (Treg) cell balance. In addition, IL-17-stimulated human keratinocytes showed the same results as in the in vivo experiments. Conclusion: Taken together, these results suggest that targeting the serine metabolism pathway in the treatment of psoriasis is a novel strategy, and that AOA could be utilized as a novel biologic to treat psoriasis.

Exclusive Enteral Nutrition Alleviates Th17-Mediated Inflammation via Eliminating Mechanical Stress-Induced Th17-Polarizing Cytokines in Crohn's-like Colitis.

Exclusive enteral nutrition (EEN) with a liquid diet is the only established dietary treatment for Crohn's' disease (CD). However, the mechanism of action of EEN in CD is unclear. T helper 17 (Th17) immune response plays a critical role in CD. We hypothesized that EEN alleviates Th17 response by eliminating mechanical stress-induced expression of Th17-polarizing cytokines. A rat model of Crohn's-like colitis was established by intracolonic instillation of TNBS (65 mg/kg in 250 µL of 40% ethanol). Control rats were treated with saline. We characterized immunophenotypes and molecular changes of the colon in control and colitis rats with and without EEN treatment. Th17 differentiation was determined using coculture assays. TNBS instillation induced transmural inflammation with stenosis in the inflammation site and a marked increase of Th17-polarizing cytokines interleukin (IL)-6 and osteopontin and the Th17 cell population in the mechanically distended preinflammation site (P-site). EEN treatment eliminated mechanical distention and the increase of IL-6, osteopontin, and Th17 response in the P-site. IL-6 and osteopontin expression was found mainly in the muscularis externa. Mechanical stretch of colonic smooth muscle cells in vitro induced a robust increase of IL-6 and osteopontin. When naïve T cells were cultured with conditioned media from the P-site tissue or stretched cells, Th17 differentiation was significantly increased. Inhibition of IL-6, but not deletion of osteopontin, blocked the increase of Th17 differentiation. Mechanical stress induces Th17-polarizing cytokines in the colon. EEN attenuates Th17 immune response by eliminating mechanical stress-induced IL-6 in Crohn's-like colitis.

The vitamin B5/coenzyme A axis: A target for immunomodulation?

Coenzyme A (CoA) serves as a vital cofactor in numerous enzymatic reactions involved in energy production, lipid metabolism, and synthesis of essential molecules. Dysregulation of CoA-dependent metabolic pathways can contribute to chronic diseases, such as inflammatory diseases, obesity, diabetes, cancer, and cardiovascular disorders. Additionally, CoA influences immune cell activation by modulating the metabolism of these cells, thereby affecting their proliferation, differentiation, and effector functions. Targeting CoA metabolism presents a promising avenue for therapeutic intervention, as it can potentially restore metabolic balance, mitigate chronic inflammation, and enhance immune cell function. This might ultimately improve the management and outcomes for these diseases. This review will more specifically focus on the contribution of pathways regulating the availability of the CoA precursor Vitamin B5/pantothenate in vivo and modulating the development of Th17-mediated inflammation, CD8-dependent anti-tumor immunity but also tissue repair processes in chronic inflammatory or degenerative diseases.

The cytokine profile in obesity and asthma in children

Background. Childhood obesity is one of the pressing problems in modern healthcare, since it is associated with a high risk of non-communicable diseases, such as bronchial asthma (BA). The aim. To determine the features of cytokine profiles in children with and without BA, depending on body weight and visceral fat area.Materials and methods. At the first stage, 506 Tomsk schoolchildren underwent anthropometry with the calculation of the body mass index (BMI) and measurement of the visceral fat area (VFA) using the InBody 770 analyzer. Fiftyone (51) children from the first stage were included in the second clinical and diagnostic stage. The children were divided into four clinical groups: “Obesity” (n = 17), “Visceral Obesity” (n = 7), “Asthma” (n = 15), and “Healthy Children” (n = 12). In all study participants, the levels of interleukin (IL)-6, IL-8, IL-4, IL-10, and immunoglobulin (Ig) E in the blood serum were determined by the multiplex assay (MagPix and Luminex 200 c analyzers). Statistical data analysis was carried out using the Statistica 10.0 software package and the 4.2.2 version of R.Results. The levels of IL-10 in the “Asthma” (p < 0.006) and “Obesity” (p < 0.008) groups were significantly higher than in the “Visceral Obesity” group. Significantly higher levels of IL-8 were found in patients with asthma (p < 0.003) and obesity (p < 0.003) compared to the “Visceral Obesity” group. Higher concentrations of IL-6 were found in the “Asthma” (p < 0.001) and “Obesity” (p < 0.028) groups compared to the “Visceral Obesity” group.Conclusion. Similar upward changes in IL-6, IL-8, and IL-10 in children with asthma and obesity without a history of asthma may explain the contribution of obesity to a risk of asthma in children, possibly through excessive production of these proinflammatory cytokines that contribute to the implementation of Th2-mediated allergic inflammation.

SENP2 restrains the generation of pathogenic Th17 cells in mouse models of colitis

The molecular mechanisms contributing to the regulation of Th17-mediated inflammation remain underexplored. We here report a SUMO-specific protease (SENP)2-mediated pathway induced in pathogenic Th17 cells that restricts the pathogenesis of inflammatory colitis. SENP2 regulates the maturation of small ubiquitin-like modifiers (SUMO) and recycles SUMO from the substrate proteins. We find higher levels of SENP2 in pathogenic Th17 cells. By deleting Senp2 in T-cell lineages in mice, we demonstrate that the lack of Senp2 exacerbates the severity of experimental colitis, which is linked to elevated levels of GM-CSF+IL-17A+ pathogenic Th17 cells and more severe dysbiosis of the intestinal microbiome. Adoptive transfer experiments demonstrate the cell-autonomous effect of Senp2 in restraining Th17 differentiation and colitis. The enzymatic activity of SENP2 is important for deSUMOylation of Smad4, which reduces Smad4 nuclear entry and Rorc expression. Our findings reveal a SENP2-mediated regulatory axis in the pathogenicity of Th17 cells.

Clinical translation of anti-inflammatory effects of Prevotella histicola in Th1, Th2, and Th17 inflammation.

EDP1815 is a non-colonizing pharmaceutical preparation of a single stain of Prevotella histicola isolated from the duodenum of a human donor. We report here preclinical and clinical studies showing that the action of EDP1815, an orally delivered and gut restricted single strain of commensal bacteria can regulate inflammatory responses throughout the body. Supported by evidence for anti-inflammatory activity in three preclinical mouse models of Th1-, TH2-, and Th17-mediated inflammation, EDP1815 was tested clinically in three Phase 1b studies in patients with psoriasis, patients with atopic dermatitis, and healthy volunteers in a KLH skin challenge model. Preclinically, EDP1815 was efficacious in all three mouse models of inflammation, showing reduction in skin inflammation as well as related tissue cytokines. In the Phase 1b studies, EDP1815 was found to be well tolerated by participants, with a safety profile comparable to placebo, including no severe or consistent side-effects reported, and no evidence of immunosuppression with no opportunistic infection occurring in these studies. In psoriasis patients, signs of clinical efficacy were seen after 4 weeks of treatment, which continued beyond the treatment period in the higher-dose cohort. In atopic dermatitis patients, improvements were seen throughout the key physician-and patient-reported outcomes. In a healthy-volunteer study of a KLH-induced skin inflammatory response, consistent anti-inflammatory effects were seen in two cohorts through imaging-based measures of skin inflammation. This is the first report demonstrating clinical effects from targeting peripheral inflammation with a non-colonizing gut-restricted single strain of commensal bacteria, providing proof of concept for a new class of medicines. These clinical effects occur without systemic exposure of EDP1815 or modification of the resident gut microbiota, and with placebo-like safety and tolerability. The breadth of these clinical effects of EDP1815, combined with its excellent safety and tolerability profile and oral administration, suggests the potential for a new type of effective, safe, oral, and accessible anti-inflammatory medicine to treat the wide range of diseases driven by inflammation.Clinical Trial Registration: EudraCT # 2018-002807-32; EudraCT # 2018-002807-32; NL8676; https://clinicaltrials.gov/ct2/show/NCT03733353; http://www.trialregister.nl.

Prohibitin is Upregulated on Donor T cells in Murine Acute Graft-versus-Host Disease and Promotes Th17 Response

Abstract Introduction: Acute graft-versus-host disease (GVHD) is a T cell-mediated disorder and the main cause of non-relapse mortality in allogeneic hematopoietic cell transplantation recipients. Here, we investigate PHB in Th17-mediated inflammation and as a therapeutic target for acute GVHD. Methods: Irradiated mice received T cell depleted bone marrow cells and allogeneic or syngeneic splenocytes. At day 25 post-transplant, blood, spleen, liver, and colon were harvested and analyzed for surface PHB, IFNγ, and IL-17 on donor T cells by flow cytometry. Splenocytes were cultured in the presence of PHB pharmacological inhibitor rocaglamide (RocA) and analyzed for PHB and IL-17 expression in donor T cells. Naïve murine CD4 T cells were cultured in Th17 polarizing conditions with RocA. CRISPR/Cas9-mediated knockout of PHB was performed followed by evaluation of Il17a, Il17f, and Il22 gene expression by quantitative PCR. Results: Surface expression of PHB is increased in murine allogeneic donor T cells in liver, spleen, colon, and peripheral blood in acute GVHD. PHB-expressing splenic T cells secrete IL-17, which is reduced with treatment with a PHB inhibitor in ex vivo experiments. PHB inhibition of murine CD4 T cells in vitro impairs differentiation into the Th17 subtype resulting in reduced IL-17 secretion and downregulation of Il17a, Il17f, and Il22 genes. Knockout of PHB reduces transcription of Il17 and Il22 genes. Conclusions: These findings show relevancy of PHB in Th17-mediated inflammation and suggest a potential role for PHB in acute GVHD. To our knowledge, this study is the first to investigate PHB in acute GVHD, and our findings will offer greater support for clinical use of PHB inhibition to treat this fatal disease. Supported by grants from AAI (KS, PR), ACS (PR; RSG-22-053-01-IBCD), and NIH NCI (PR; R01 CA252469).

PP4 deficiency contributes to DEP exacerbated epithelial barrier dysfunction and airway inflammation in asthmatic patients

Abstract Diesel exhaust particles (DEP) emitted from motor vehicle or heavy-duty industries is thought to be the major source of air pollutants that lead to the rapid increase and worsening of allergic disease, such as asthma. In asthma, exposure to DEP has been linked to aberrant epithelial barrier integrity, which leads to the promotion of allergen delivery into the subepithelia and induced alarmins (IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)) release to promote Th2-mediated inflammation. However, it is still not known the detail mechanism of airway epithelial barrier in response to DEP. In this study, we fund that PP4 expression was decreased in DEP-challenged human bronchial epithelial cells and air-pollution animal model. Moreover, silencing PP4 decreased E-cadherin and barrier integrity through and activated Src kinase and increased expression of Slug. Sequentially, transactivating the EGFR phosphorylation led to increased alarmin synthesis. Overexpression of PP4 wild type, but not phosphatase mutant, prevented DEP-induced loss of airway epithelial E-cadherin expression and decreased alarmin release, suggested that phosphatase activity is critical to maintain epithelial integrity and alarmin production. Primary human bronchial epithelial cells (HBECs) from mild and severe asthmatic patients showed a significantly low TJ integrity in ALI cultures in response to DEP, compared with HBECs from healthy subjects. Our data demonstrate that exposure to DEP causes a significant reduction in expression of PP4 leading to decreased epithelial barrier function might accelerate the allergen-induced airway inflammatory response in asthma. Supported by grants from (MOST 110-2320-B-038-065-MY3) from the Ministry of Science and Technology of Taiwan.

Simvastatin Reduces NETosis to Attenuate Severe Asthma by Inhibiting PAD4 Expression.

Patients with severe asthma respond poorly to corticosteroids, and their care accounts for more than 60% of the total costs attributed to asthma. Neutrophils form neutrophil extracellular traps (NETs), which play a crucial role in severe asthma. Statins have shown anti-inflammatory effects by reducing NETosis. In this study, we investigate if simvastatin can attenuate severe asthma by reducing NETosis and the underlying mechanism. Mice were concomitantly sensitized with ovalbumin (OVA), house dust mite (HDM), and lipopolysaccharide (LPS) during sensitization to establish a mouse model of severe asthma with neutrophil predominant inflammation (OVA+LPS mice) and treated with or without simvastatin. In inflammatory response, proportions of Th2, Th17, and Treg cells in lung tissue were detected by flow cytometry, and the levels of cytokines, dsDNA, and MPO-DNA in bronchoalveolar lavage fluid (BALF) were analyzed by ELISA. Citrullinated histone H3 (CitH3) and peptidyl arginine deiminase 4 (PAD4) in lung tissue were determined by Western blot and immunofluorescence imaging. PAD4 mRNA was determined by quantitative PCR (qPCR). HL-60 cells were differentiated into neutrophil-like cells by 1.25% DMSO. The neutrophil-like cells were treated with or without LPS, and simvastatin was then stimulated with PMA. CitH3 and PAD4 expressions were determined. Sensitization with OVA, HDM, and LPS resulted in neutrophilic inflammation and the formation of NETs in the lungs. Simvastatin treatment reduced the inflammation score, cytokine levels, total cells, and neutrophil counts in the BALF and reduced proportions of Th2 and Th17 but increased Treg cells in lungs of OVA+LPS mice. Simvastatin-treated OVA+LPS mice show reduced NET formation in BALF and lung tissue compared to control mice. Adoptive transfer of neutrophils was sufficient to restore NETosis and neutrophilic inflammation in simvastatin-treated OVA+LPS mice. Simvastatin reduced PAD4 mRNA and protein expression in lung tissues and neutrophils isolated from lungs of OVA+LPS mice and consequent NET formation. In vitro, simvastatin reduced LPS-induced PAD4 upregulation and NETosis in HL-60-differentiated neutrophil-like cells. Furthermore, PAD4-overexpressed lentiviral transduction was sufficient to restore PAD4 protein expression and NETosis in simvastatin-treated HL-60-differentiated neutrophil-like cells. Simvastatin reduces Th17-mediated neutrophilic inflammation and airway hyperreactivity by reducing PAD4 expression and inhibiting NETosis in a mouse model of severe asthma. Severe asthmatic patients with high levels of circulating NETs or sputum NETs may show improved responses to statin treatment.

Lead optimization of aryl hydrocarbon receptor ligands for treatment of inflammatory skin disorders

Therapeutic aryl hydrocarbon receptor (AHR) modulating agents gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR’s known ligand promiscuity, we generated novel AHR modulating agents by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. SGA388 showed the highest agonist activity with potent normalization of keratinocyte hyperproliferation, restored expression of skin barrier proteins and dampening of chemokine expression by keratinocytes upon Th2-mediated inflammation in vitro. The topical application of SGA360f and SGA388 reduced acute skin inflammation in vivo by reducing cyclooxygenase levels, resulting in less neutrophilic dermal infiltrates. The minimal induction of cytochrome P450 enzyme activity, lack of cellular toxicity and mutagenicity classifies SGA360f and SGA388 as novel potential therapeutic AHR ligands and illustrates the potential of medicinal chemistry to fine-tune AHR signaling for the development of targeted therapies in dermatology and beyond.

Dupilumab improves clinical symptoms in children with Netherton syndrome by suppressing Th2-mediated inflammation.

Netherton syndrome is a rare, life-threatening autosomal recessive genetic disorder with no effective treatment yet. Skin barrier dysfunction caused by SPINK5 gene mutations is a hallmark of the disease. Antigen penetration through the defective skin and nonspecific inflammation provide a pro-T helper 2 (Th2) immune microenvironment in the disease. Therefore, Th2 cytokines are considered to be candidate therapeutic targets. To evaluate the clinical responses of patients with Netherton syndrome to dupilumab, an IL-4Rα antagonist, and identify changes in the Th1/2/17 pathway activity, skin barrier defect protein LEKTI expression after treatment. Four children with severe Netherton syndrome (aged 2 y to 4 y and 6m) who were treated with dupilumab from January to June 2022 were evaluated at baseline, and at 4, 8, 12, 16, and 20 weeks after the start of dupilumab administration. Treatment response was assessed using the Eczema Area and Severity Index (EASI), the Numerical Rating Scale (NRS), the Dermatology Life Quality Index (CDLQI), and the Dermatitis Family Impact-questionnaire (DFI). Blood eosinophil counts, serum IgE levels and inflammatory cytokines were measured. The immunotyping of Th1/2/17 cells was performed by flow cytometry and cytokine expressions in T cell subsets were analyzed by single-cell RNA sequencing. In addition, expression of the LEKTI in skin lesions was evaluated by immunohistochemical analysis. All four patients experienced clinical improvement, with significantly reduced EASI scores (by 75.0-83.9%) and NRS (by 87.5-90.0%) from baseline to 20 weeks of treatment. Improved quality of life scores were also seen for all patients, as measured by CDLQI and DFI. Serum IgE levels also fell by 75.6-86.9%. The serum Th2 cytokines IL-4, IL-5 and IL-13 were found at low level, with no significant changes during the treatment. However, Th2 cytokines expressed by T cells, especially IL-4, decreased at single-cell level after treatment (P = 0.029). The baseline percentage of Th2 cells (among total CD3+CD4+ T cells) was significantly higher in patients than that in healthy controls (HC) (P < 0.0001); this percentage fell from 8.25% ± 0.75% to 4.02% ± 0.62% after 20 weeks dupilumab treatment. There was no noticeable change in LEKTI protein expression in skin lesions pre- and post-treatment. Two patients reported mild ocular adverse effects, but there were no severe adverse events. Dupilumab may be an effective and safe treatment option in a subset of pediatric patients with Netherton syndrome, especially in improving itch and the quality of life. These effects were achieved in part by suppression of the Th2-mediated inflammation.

Epigenetic drug screen identified IOX1 as an inhibitor of Th17-mediated inflammation through targeting TET2.

Targeting helper T cells, especially Th17cells, has become a plausible therapy for many autoimmune diseases. Using an invitro culture system, we screened an epigenetics compound library for inhibitors of IFN-γ and IL-17 expression in murine Th1 and Th17 cultures. This identified IOX1 as an effective suppressor of IL-17 expression in both murine and human CD4+ T cells. Furthermore, we found that IOX1 suppresses Il17a expression directly by targeting TET2 activity on its promoter in Th17cells. Using established pre-clinical models of intraocular inflammation, treatment with IOX1 invivo reduced the migration/infiltration of Th17cells into the site of inflammation and tissue damage. These results provide evidence of the strong potential for IOX1 as a viable therapy for inflammatory diseases, in particular of the eye. This study was supported by the National Key Research and Development Program of China 2021YFA1101200 (2021YFA1101204) to LW and XW; the National Natural Science Foundation of China 81900844 to XH and 82171041 to LW; the China Postdoctoral Science Foundation 2021M700776 and the Scientific Research Project of Guangdong Provincial Bureau of Traditional Chinese Medicine 20221373 to YZ; and the National Institute for Health Research (NIHR) Biomedical Research Centre at Moorfields Eye Hospital NHS (National Health Service) Foundation Trust and University College London Institute of Ophthalmology, UK (DAC, LPS, PJPL, MS, ADD and RWJL). The views expressed are those of the authors and not necessarily those of the NIHR or the UK's Department of Health and Social Care.

Intranasal administration of nucleus-deliverable GATA3-TMD alleviates the symptoms of allergic asthma

Although the T helper 2 (Th2) subset is a critical player in the humoral immune response to extracellular parasites and suppression of Th1-mediated inflammation, Th2 cells have been implicated in allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. GATA binding protein 3 (GATA3) is a primary transcription factor that mediates Th2 differentiation and secretion of Th2 cytokines, including IL-4, IL-5, and IL-13. Here, a nucleus-deliverable form of GATA3-transcription modulation domain (TMD) (ndG3-TMD) was generated using Hph-1 human protein transduction domain (PTD) to modulate the transcriptional function of endogenous GATA3 without genetic manipulation. ndG3-TMD was shown to be efficiently delivered into the cell nucleus quickly without affecting cell viability or intracellular signaling events for T cell activation. ndG3-TMD exhibited a specific inhibitory function for the endogenous GATA3-mediated transcription, such as Th2 cell differentiation and Th2-type cytokine production. Intranasal administration of ndG3-TMD significantly alleviated airway hyperresponsiveness, infiltration of immune cells, and serum IgE level in an OVA-induced mouse model of asthma. Also, Th2 cytokine secretion by the splenocytes isolated from the ndG3-TMD-treated mice substantially decreased. Our results suggest that ndG3-TMD can be a new therapeutic reagent to suppress Th2-mediated allergic diseases through intranasal delivery.

The Role of T Helper Type 2 (Th2) Cytokines in the Pathogenesis of Eosinophilic Granulomatosis with Polyangiitis (eGPA): an Illustrative Case and Discussion.

Purposeof ReviewThe pathogenesis of eosinophilic granulomatosis with polyangiitis (eGPA) is driven largely by CD4 + type 2 helper T cells (Th2), B cells, and eosinophils. Interleukin (IL)-4 and IL-13 are critical cytokines in Th2 cell–mediated inflammation; however, inhibition of IL-4 and IL-13 does not reduce serum eosinophil counts and has even been associated with hypereosinophilia. This review explores the role of IL-4, IL-5, and IL-13 in Th2-mediated inflammation to consider the potential clinical consequences of inhibiting these individual cytokines in eGPA.Recent FindingsTreatments for eosinophilic granulomatosis with polyangiitis (eGPA) are rapidly evolving through using biologic therapies to modulate the Th2 inflammatory response via eosinophil inhibition. While IL-4, IL-5, IL-13, and IL-25 can all affect eosinophils, only IL-5 inhibition has demonstrated therapeutic benefit to-date. In this review, we report a clinical vignette of a patient with adult-onset asthma who developed severe manifestations of eGPA after switching from mepolizumab (an IL-5 inhibitor) to dupilumab (an inhibitor of IL-4 and IL-13).SummaryBy understanding the role of IL-4, IL-5, and IL-13 in Th2-mediated vasculitis, we can start to understand how eGPA might respond differently to focused cytokine inhibition.

Androgen receptor signaling decreases glutaminolysis in Th17 cells to reduce airway inflammation in asthma

Abstract Women have higher rates of severe asthma compared to men. Severe asthma has increased Th2-mediated eosinophilic and/or Th17-mediated neutrophilic airway inflammation. We showed androgens, signaling through the androgen receptor (AR), decreased Th2- and Th17-mediated airway inflammation, and that Th17 and Th2 rely on glutaminolysis during allergic airway inflammation. Therefore, we hypothesized that AR signaling decreases glutaminolysis in Th2 and Th17 cells, resulting in decreased airway inflammation. Th2 and Th17 were differentiated in vitro from wild-type male and ArTfm male mice, with a nonfunctional AR. Mitochondrial metabolism and glycolysis were determined by measuring oxygen consumption rate (OCR), reactive oxygen species (ROS), and the extracellular acidification rate (ECAR). In Th17 cells, AR signaling decreased OCR, while increasing mitochondrial ROS, suggesting decreased glutaminolysis. Further, both 5α-DHT (0.1nM) and CB839 (a glutaminase inhibitor, 0.5μM) reduced OCR in Th17 cells from male mice. AR signaling had no effect on ECAR in Th17 cells or OCR and ECAR in Th2 cells. To induce eosinophilic and neutrophilic airway inflammation, house dust mite (HDM) was intranasally administered to ArfloxedCD4-Cre and Arfloxed male mice and markers of glycolysis and glutaminolysis were determined by flow cytometry. AR signaling decreased airway neutrophils, the number of Th17 lung cells, and GLUD1, a glutaminolysis marker, expression in T cells, but had no effect on eosinophils or Th2 cells. Combined, these data showed that AR signaling decreased glutaminolysis in Th17, but not Th2, cells. Understanding these pathways may provide new potential therapeutic targets in women with difficult-to-treat asthma. Supported by grants from NIH (R01 HL12254, R01 HL136664, T32 GM007347)

TCF-1 controls Treg functions that regulate inflammation, CD8 T-cell cytotoxicity, and severity of colon cancer

Abstract The transcription factor TCF-1 is essential for the development and function of T regulatory (Treg) cells, however its function is poorly understood. Here, we show that TCF-1 primarily suppresses transcription of genes that are co-bound by Foxp3. Single-cell RNA-seq analysis identified effector- and central-memory Treg-cells with differential expression of Klf2 and memory and activation markers. TCF-1 deficiency did not change the core Treg transcriptional signature, but promoted alternative signaling pathways whereby Treg-cells became activated and gained gut-homing and TH17 characteristics. TCF-1-deficient Treg-cells strongly suppressed T-cell proliferation and cytotoxicity, but were compromised in controlling CD4+ T-cell polarization and inflammation. In mice with polyposis, Treg cell-specific TCF-1 deficiency promoted tumor growth. Consistently, tumor-infiltrating Treg cells of colorectal cancer patients showed lower TCF-1 expression and increased TH17 expression signatures compared to adjacent normal tissue and circulating T-cells. Thus, Treg cell-specific TCF-1 expression differentially regulates TH17-mediated inflammation and T-cell cytotoxicity, and can determine colorectal cancer outcome. Supported by NIH R01 AI 108682 (FG &amp; KK), NIH RO1 AI 147652 (FG), NIH R35GM138283 (MK), and Praespero Innovation Award Alberta, Canada (FG &amp; KK)

T cell‐intrinsic miR‐155 is required for Th2 and Th17‐biased responses in acute and chronic airway inflammation by targeting several different transcription factors

Asthmatic airway inflammation is divided into two typical endotypes: Th2-mediated eosinophilic and Th1- or Th17-mediated neutrophilic airway inflammation. The miRNA miR-155 has well-documented roles in the regulation of adaptive T-cell responses and innate immunity. However, no specific cell-intrinsic role has yet been elucidated for miR-155 in T cells in the course of Th2-eosinophilic and Th17-neutrophilic airway inflammation using actual in vivo asthma models. Here, using conditional KO (miR155ΔCD4 cKO) mice that have the specific deficiency of miR-155 in T cells, we found that the specific deficiency of miR-155 in T cells resulted in fully suppressed Th2-type eosinophilic airway inflammation following acute allergen exposure, as well as greatly attenuated the Th17-type neutrophilic airway inflammation induced by repeated allergen exposure. Furthermore, miR-155 in T cells appeared to regulate the expression of several different target genes in the functional activation of CD4+ Th2 and Th17 cells. To be more precise, the deficiency of miR-155 in T cells enhanced the expression of c-Maf, SOCS1, Fosl2 and Jarid2 in the course of CD4+ Th2 cell activation, while C/EBPβ was highly enhanced in CD4+ Th17 cell activation in the absence of miR-155 expression. Conclusively, our data revealed that miR-155 could promote Th2 and Th17-mediated airway inflammation via the regulation of several different target genes, depending on the context of asthmatic diseases. Therefore, these results provide valuable insights into actual understanding of specific cell-intrinsic role of miR-155 in eosinophilic and neutrophilic airway inflammation for the development of fine-tune therapeutic strategies.

CD44 is critical for the enhancing effect of hyaluronan in allergen-specific sublingual immunotherapy in a murine model of chronic asthma.

Allergen-specific sublingual immunotherapy (SLIT) is a potentially effective disease-modification treatment for patients with allergic asthma. Because CD44 signaling enhances regulatory T (Treg) cell-induction, administering CD44 ligands such as hyaluronan (HA) with allergen-specific SLIT may enhance the therapeutic effects. We evaluated the role of CD44 in Treg cell-induction in T helper type 2 (Th2)-mediated chronic airway inflammation using CD44-/- mice and the efficacy of HA on SLIT in a Dermatophagoides farinae (Df)-induced murine model of chronic asthma. Th2 responses and Treg cell induction were evaluated in CD44-/- mice. We devised a new SLIT model of Df-induced chronic asthma utilizing HA as an adjuvant. The effects of HA added to the new SLIT model were evaluated by the early asthmatic response (EAR) and airway hyperresponsiveness (AHR), eosinophilic airway inflammation, and serum Df-specific IgE levels. Th2-mediated chronic eosinophilic airway inflammation was worse in CD44-/- mice compared with Df-sensitized wild-type (WT) mice. HA enhanced the effect of Df-induced Treg cells in a CD44-dependent manner. Sublingual Df treatment in combination with HA, but not alone, normalized EAR and AHR, and significantly reduced the serum IgE levels and the bronchoalveolar lavage fluid (BALF) eosinophil number. HA also induced Treg cells in a Df-sensitized spleen cell culture in a CD44-dependent manner. The treatment-enhancing effects of HA in this SLIT model were diminished in CD44-/- mice. CD44 is a key contributor to Treg cell induction and critical for the enhancing effects of HA in a Df-induced murine model of chronic asthma.