Blockade of CD80/CD86-CD28 co-stimulation augments the inhibitory function of peptide antigen-specific regulatory T cells.

Mixed lymphocyte culture under the blockade of CD80/CD86-CD28 co-stimulation induces anergic (completely hyporesponsive) T cells with immune suppressive function (inducible suppressing T cells: iTS cells). Previously, iTS cell therapy has demonstrated outstanding benefits in clinical trials for organ transplantation. Here, we examined whether peptide antigen-specific iTS cells are inducible. DO 11.10 iTS cells were obtained from splenocytes of BALB/c DO 11.10 mice by stimulation with OVA peptide and antagonistic anti-CD80/CD86 mAbs. When DO 11.10 iTS or Foxp3- DO 11.10 iTS cells were stimulated with OVA, these cells produced IL-13, but not IL-4. DO 11.10 iTS cells decreased IL-4 and increased IL-13 production from OVA-stimulated naïve DO 11.10 splenocytes. When Foxp3+ DO 11.10 iTS cells were prepared, these cells significantly inhibited the production of IL-4 and IL-13 compared with freshly isolated Foxp3+ DO 11.10 T cells. Moreover, an increase in the population expressing OX40, ICOS, and 4-1BB suggested activation of Foxp3+ DO 11.10 iTS cells. Thus, blockade of CD80/CD86-CD28 co-stimulation during peptide antigen stimulation augments the inhibitory function of Foxp3+ regulatory T cells, and does not induce anergic Foxp3- conventional T cells. Peptide-specific Foxp3+ regulatory iTS cells could be useful for the treatment of allergic and autoimmune diseases without adverse effects.

HUNK as a key regulator of tumor-associated macrophages in triple negative breast cancer.

Triple-negative breast cancer (TNBC) lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). TNBC tumors are not sensitive to endocrine therapy, and standardized TNBC treatment regimens are lacking. TNBC is a more immunogenic subtype of breast cancer, making it more responsive to immunotherapy intervention. Tumor-associated macrophages (TAMs) constitute one of the most abundant immune cell populations in TNBC tumors and contribute to cancer metastasis. This study examines the role of the protein kinase HUNK in tumor immunity. Gene expression analysis using NanoString's nCounter PanCancer Immune Profiling panel identified that targeting HUNK is associated with changes in the IL-4/IL-4 R cytokine signaling pathway. Experimental analysis shows that HUNK kinase activity regulates IL-4 production in mammary tumor cells, and this regulation is dependent on STAT3. In addition, HUNK-dependent regulation of IL-4 secreted from tumor cells induces polarization of macrophages into an M2-like phenotype associated with TAMs. In return, IL-4 induces cancer metastasis and macrophages to produce epidermal growth factor. These findings delineate a paracrine signaling exchange between tumor cells and TAMs regulated by HUNK and dependent on IL-4/IL-4 R. This highlights the potential of HUNK as a target for reducing TNBC metastasis through modulation of the TAM population.

Differentiation and Regulation of Bovine Th2 Cells In Vitro.

Bovine Th2 cells have usually been characterized by IL4 mRNA expression, but it is unclear whether their IL4 protein expression corresponds to transcription. We found that grass-fed healthy beef cattle, which had been regularly exposed to parasites on the grass, had a low frequency of IL4+ Th2 cells during flow cytometry, similar to animals grown in feedlots. To assess the distribution of IL4+ CD4+ T cells across tissues, samples from the blood, spleen, abomasal (draining), and inguinal lymph nodes were examined, which revealed limited IL4 protein detection in the CD4+ T cells across the examined tissues. To determine if bovine CD4+ T cells may develop into Th2 cells, naïve cells were stimulated with anti-bovine CD3 under a Th2 differentiation kit in vitro. The cells produced primarily IFNγ proteins, with only a small fraction (<10%) co-expressing IL4 proteins. Quantitative PCR confirmed elevated IFNγ transcription but no significant change in IL4 transcription. Surprisingly, GATA3, the master regulator of IL4, was highest in naïve CD4+ T cells but was considerably reduced following differentiation. To determine if the differentiated cells were true Th2 cells, an unbiased proteomic assay was carried out. The assay identified 4212 proteins, 422 of which were differently expressed compared to those in naïve cells. Based on these differential proteins, Th2-related upstream components were predicted, including CD3, CD28, IL4, and IL33, demonstrating typical Th2 differentiation. To boost IL4 expression, T cell receptor (TCR) stimulation strength was reduced by lowering anti-CD3 concentrations. Consequently, weak TCR stimulation essentially abolished Th2 expansion and survival. In addition, extra recombinant bovine IL4 (rbIL4) was added during Th2 differentiation, but, despite enhanced expansion, the IL4 level remained unaltered. These findings suggest that, while bovine CD4+ T cells can respond to Th2 differentiation stimuli, the bovine IL4 pathway is not regulated in the same way as in mice and humans. Furthermore, Ostertagia ostertagi (OO) extract, a gastrointestinal nematode in cattle, inhibited signaling via CD3, CD28, IL4, and TLRs/MYD88, indicating that external pathogens can influence bovine Th2 differentiation. In conclusion, though bovine CD4+ T cells can respond to IL4-driven differentiation, IL4 expression is not a defining feature of differentiated bovine Th2 cells.

JAK/STAT Inhibition Normalizes Lipid Composition in 3D Human Epidermal Equivalents Challenged with Th2 Cytokines.

Derangement of the epidermal barrier lipids and dysregulated immune responses are key pathogenic features of atopic dermatitis (AD). The Th2-type cytokines interleukin IL-4 and IL-13 play a prominent role in AD by activating the Janus Kinase/Signal Transduction and Activator of Transcription (JAK/STAT) intracellular signaling axis. This study aimed to investigate the role of JAK/STAT in the lipid perturbations induced by Th2 signaling in 3D epidermal equivalents. Tofacitinib, a low-molecular-mass JAK inhibitor, was used to screen for JAK/STAT-mediated deregulation of lipid metabolism. Th2 cytokines decreased the expression of elongases 1, 3, and 4 and serine-palmitoyl-transferase and increased that of sphingolipid delta(4)-desaturase and carbonic anhydrase 2. Th2 cytokines inhibited the synthesis of palmitoleic acid and caused depletion of triglycerides, in association with altered phosphatidylcholine profiles and fatty acid (FA) metabolism. Overall, the ceramide profiles were minimally affected. Except for most sphingolipids and very-long-chain FAs, the effects of Th2 on lipid pathways were reversed by co-treatment with tofacitinib. An increase in the mRNA levels of CPT1A and ACAT1, reduced by tofacitinib, suggests that Th2 cytokines promote FA beta-oxidation. In conclusion, pharmacological inhibition of JAK/STAT activation prevents the lipid disruption caused by the halted homeostasis of FA metabolism.

The interleukin-4/interleukin-13 pathway in type 2 inflammation in chronic rhinosinusitis with nasal polyps.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is predominantly a type 2 inflammatory disease associated with type 2 (T2) cell responses and epithelial barrier, mucociliary, and olfactory dysfunction. The inflammatory cytokines interleukin (IL)-4, IL-13, and IL-5 are key mediators driving and perpetuating type 2 inflammation. The inflammatory responses driven by these cytokines include the recruitment and activation of eosinophils, basophils, mast cells, goblet cells, M2 macrophages, and B cells. The activation of these immune cells results in a range of pathologic effects including immunoglobulin E production, an increase in the number of smooth muscle cells within the nasal mucosa and a reduction in their contractility, increased deposition of fibrinogen, mucus hyperproduction, and local edema. The cytokine-driven structural changes include nasal polyp formation and nasal epithelial tissue remodeling, which perpetuate barrier dysfunction. Type 2 inflammation may also alter the availability or function of olfactory sensory neurons contributing to loss of sense of smell. Targeting these key cytokine pathways has emerged as an effective approach for the treatment of type 2 inflammatory airway diseases, and a number of biologic agents are now available or in development for CRSwNP. In this review, we provide an overview of the inflammatory pathways involved in CRSwNP and describe how targeting key drivers of type 2 inflammation is an effective therapeutic option for patients.

LncRNA TUG1 mediates microglial inflammatory activation by regulating glucose metabolic reprogramming.

Microglia are natural immune cells in the central nervous system, and the activation of microglia is accompanied by a reprogramming of glucose metabolism. In our study, we investigated the role of long non-coding RNA taurine-upregulated gene 1 (TUG1) in regulating microglial glucose metabolism reprogramming and activation. BV2 cells were treated with Lipopolysaccharides (LPS)/Interferon-γ (IFN-γ) to establish a microglial activation model. The glycolysis inhibitor 2-Deoxy-D-glucose (2-DG) was used as a control. The expression levels of TUG1 mRNA and proinflammatory cytokines such as Interleukin-1ß (IL-1ß), Interleukin -6, and Tumor Necrosis Factor-α mRNA and anti-inflammatory cytokines such as IL-4, Arginase 1(Arg1), CD206, and Ym1 were detected by RT-qPCR. TUG1 was silenced using TUG1 siRNA and knocked out using CRISPR/Cas9. The mRNA and protein expression levels of key enzymes involved in glucose metabolism, such as Hexokinase2, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Lactate dehydrogenase, Glucose 6 phosphate dehydrogenase, and Pyruvate dehydrogenase (PDH), were determined by RT-qPCR and Western blotting. The glycolytic rate of microglial cells was measured using Seahorse. Differential metabolites were determined by metabolomics, and pathway enrichment was performed using these differential metabolites. Our findings revealed that the expression of TUG1 was elevated in proinflammatory-activated microglia and positively correlated with the levels of inflammatory factors. The expression of anti-inflammatory cytokines such as IL-4, Arg1, CD206, and Ym1 were decreased when induced with LPS/IFN-γ. However, this decrease was reversed by the treatment with 2-DG. Silencing of GAPDH led to an increase in the expression of TUG1 and inflammatory factors. TUG1 knockout (TUG1KO) inhibited the expression of glycolytic key enzymes and promoted the expression of oxidative phosphorylation key enzymes, shifting the metabolic profile of activated microglia from glycolysis to oxidative phosphorylation. Additionally, TUG1KO reduced the accumulation of metabolites, facilitating the restoration of the tricarboxylic acid cycle and enhancing oxidative phosphorylation in microglia. Furthermore, the downregulation of TUG1 was found to reduce the expression of both proinflammatory and anti-inflammatory cytokines under normal conditions. Interestingly, when induced with LPS/IFN-γ, TUG1 downregulation showed a potentially beneficial effect on microglia in terms of inflammation. Downregulation of TUG1 expression inhibits glycolysis and facilitates the shift of microglial glucose metabolism from glycolysis to oxidative phosphorylation, promoting their transformation towards an anti-inflammatory phenotype and exerting anti-inflammatory effects in BV2.

3-carene supresses inflammatory cytokine interleukin-4, interleukin-5 and interleukin-13 in a murine model of asthma.

Asthma is a common airway disease associated with allergic inflammation. Environmental factors, such as pollens, pollution, insect-borne antigens, or commercial chemicals, cause this disease. The common symptoms of this airway allergic reaction are increasing mucus, narrowing of the airway wall, coughing, and chest tightness. Medications, such as steroids, alleviate the disease but with severe side effects. Several studies have reported the anti-inflammatory effects of tree-based essential oil components, particularly 3-carene. Therefore, this study used 3-carene to determine if it alleviates asthmatic symptoms in the murine model. First, BALB/c mice were sensitized to an ovalbumin and aluminium hydroxide mixture on day 7th and 14th. From days 21st to 23rd, the mice were challenged with 3-carene and budesonide. The lung trachea, plasma, and bronchiolar lavage fluid (BAL fluid) were collected on day 24. The 3-carene treatment suppressed the cytokine gene expression, such as interleukin-4 (IL-4), IL-5, and IL-13, reducing the lung epithelial cell thickness in the asthmatic model. These results suggest that essential oil 3-carene has an anti-asthmatic effect.

Mesenchymal Stem Cells from the Deciduous Tooth Pulp Lose their Ability to Suppress the Differentiation of Dendritic Cells during Long-Term Culturing.

A culture of cells expressing markers of mesenchymal stem cells (MSC) (CD73, CD90, CD44, CD29, and CD49b), but not hematopoietic cell markers, and capable of multilineage differentiation was isolated from the deciduous tooth pulp. Co-culturing with immature dendritic cells in the presence of LPS did not reveal an ability of the MSC to suppress the maturation of dendritic cells. On the contrary, co-culturing of MSC with monocytes in the presence of granulocyte-macrophage CSF and IL-4 led to complete suppression of monocyte differentiation into dendritic cells. However, long-term culturing of MSC from dental pulp showed that by the passage 11, they almost completely lose their suppressor ability. These results indicate that the immunological properties of MSC can change during culturing without changing their phenotypic markers. This should be taken into account when creating biomedical cell products.

[The effects and mechanisms of Gefitinib on airway inflammation and airway remodeling in C57BL/6 mice with asthma].

Objective: To investigate the effects of the epidermal growth factor receptor(EGFR) inhibitor Gefitinib on airway inflammation and airway remodelling in asthmatic C57BL/6 mice, and to analyze its possible mechanisms. Methods: Male C57BL/6 mice, aged 6-8 weeks, were randomly assigned into five groups: Group A (control group), Group B (asthma group), Group C (asthma+20 mg/kg gefitinib group), Group D (asthma+40 mg/kg gefitinib group), and Group E (40 mg/kg gefitinib group), with seven mice per group. Mice were sensitized by intraperitoneal injection of a mixture of 0.2 ml solution containing OVA and Al(OH)3 [20 µg OVA+2 mg Al(OH)3 dissolved in 0.2 ml of physiological saline] at Day 0 and 14. Starting from Day 25 to 31, Group B, C, and D were challenged with nebulization of 1% OVA solution (8 ml) to induce asthma, once a day for approximately 40 minutes, with continuous aerosolization for 7 days. Group C and D were given 0.2 ml of Gefitinib dissolved in 0.5% carboxymethylcellulose sodium (CMCNa) by gavage half an hour before challenging, and Group E was simultaneously given with 0.2 ml of Gefitinib dissolved in 0.5% CMCNa only. Group A and B were given an equivalent volume of 0.5% CMCNa by gavage. After 24 h of final challenge, the bronchoalveolar lavage fluid (BALF) was prepared for the determination of total cell count and eosinophil count. The levels of total immune globulin E (IgE) in serum and interleukin (IL)-4, IL-5 and IL-13 in BALF and lung tissue homogenates were measured by ELISA. The mRNA expression levels of IL-4, IL-5, IL-13 in lung were measured. Immunohistochemistry and Western blot experiments were used to detect the expression levels of EGFR in lung tissues. Results: In Group B, the level of total IgE in serum, total cell count, eosinophil count, the levels of IL-4, IL-5, IL-13 in BALF and the phosphorylation of EGFR and its downstream activation in lung were higher than those in Group A (all P<0.05). The levels of total IgE in serum [(261.32±44.38) ng/ml, (194.09±52.39) ng/ml vs (1 023.70±105.51) ng/ml], total cell count [(23.70±4.08)×105/ml, (14.92±4.06)×105/ml vs (35.36±6.30)×105/ml], eosinophil count [(108.00±13.69)×104/ml, (67.00±17.28)×104/ml vs (147.86±20.06)×104/ml], IL-4 [(36.42±4.48) pg/ml, (30.45±8.12) pg/ml vs (58.72±7.17) pg/ml], IL-5 [(16.20±4.62) pg/ml, (13.38±5.14) pg/ml vs (23.46±5.38) pg/ml], IL-13 [(18.45±7.28) pg/ml, (14.33±7.70) pg/ml vs (104.12±24.66) pg/ml] in BALF of Group C and D were lower than those in Group B (all P<0.05). The levels of IL-4, IL-5, and IL-13 as well as their mRNA levels in the lung tissue of Group C and D were lower than those in Group B (all P<0.05). In Group C and D, the positive expression rate of phosphorylated epidermal growth factor receptor (p-EGFR) in lung tissue [(40.53±6.80)%, (23.60±4.42)% vs (70.78±5.36)%], p-EGFR/EGFR (61.68±7.48, 51.13±5.19 vs 105.90±11.66), phosphorylated extracellular regulated protein kinase (p-Erk)/extracellular regulated protein kinase (Erk) (75.28±7.11, 47.54±4.83 vs 98.76±4.71), and phosphorylated protein kinase B (p-Akt)/protein kinase B (Akt) (96.24±5.40, 68.52±2.73 vs 103.30±4.52) was lower than those of Group B (all P<0.05). There was no statistically significant difference in the relevant indicators between Group A and E (all P>0.05). Conclusion: Gefitinib may alleviate airway inflammation and airway remodeling in asthmatic mice by inhibiting EGFR phosphorylation and affecting the activation of downstream Erk and Akt.

Modelling atopic dermatitis in healthy human skin for the characterization of topical compounds.

Suitable human models for the development and characterization of topical compounds for inflammatory skin diseases such as atopic dermatitis are not readily available to date. We describe here the development of a translational model involving healthy human skin mimicking major aspects of AD and its application for the characterization of topical Janus kinase inhibitors. Full thickness human abdominal skin obtained from plastic surgery stimulated in vitro with IL4 and IL13 shows molecular features of AD. This is evidenced by STAT6 phosphorylation assessed by immunohistochemistry and analysis of skin lysates. Broad transcriptome changes assessed by AmpliSeq followed by gene set variation analysis showed a consistent upregulation of gene signatures characterizing AD in this model. Topical application of experimental formulations of compounds targeting the JAK pathway to full thickness skin normalizes the molecular features of AD induced by IL4 and IL13 stimulation. The inhibitory effects of topical JAK inhibitors on molecular features of AD are supported by pharmacokinetic analysis. The model described here is suited for the characterization of topical compounds for AD and has the potential to be extended to other inflammatory skin diseases and pathophysiological pathways.

The Possible Roles of IL-4/IL-13 in the Development of Eosinophil-Predominant Severe Asthma.

Bronchial asthma is characterized by airway inflammation, airway hyperresponsiveness, and reversible airway obstruction. Eosinophils contribute to the pathogenesis of airway disease mainly by releasing eosinophil-specific granules, lipid mediators, superoxide anions, and their DNA. Type-2 cytokines such as interleukin (IL)-4 and IL-13 also play roles in the development of bronchial asthma. Among these cytokines, IL-4 is involved in T-cell differentiation, B-cell activation, B-cell differentiation into plasma cells, and the production of immunoglobulin E. Although IL-13 has similar effects to IL-4, IL-13 mainly affects structural cells, such as epithelial cells, smooth muscle cells, and fibroblasts. IL-13 induces the differentiation of goblet cells that produce mucus and induces the airway remodeling, including smooth muscle hypertrophy. IL-4 and IL-13 do not directly activate the effector functions of eosinophils; however, they can induce eosinophilic airway inflammation by upregulating the expression of vascular cell adhesion molecule-1 (for adhesion) and CC chemokine receptor 3 ligands (for migration). Dupilumab, a human anti-IL-4 receptor α monoclonal antibody that inhibits IL-4 and IL-13 signaling, decreases asthma exacerbations and mucus plugs and increases lung function in moderate to severe asthma. In addition, dupilumab is effective for chronic rhinosinusitis with nasal polyps and for atopic dermatitis, and IL-4/IL-13 blocking is expected to suppress allergen sensitization, including transcutaneous sensitization and atopic march.

Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

Introduction: During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. Methods: High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. Results: The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1ß and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. Discussion: We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

Type II Interleukin-4 Receptor Activation in Basal Breast Cancer Cells Promotes Tumor Progression via Metabolic and Epigenetic Modulation.

Interleukin-4 (IL4) is a Th2 cytokine that can signal through two different receptors, one of which-the type II receptor-is overexpressed by various cancer cells. Previously, we have shown that type II IL4 receptor signaling increases proliferation and metastasis in mouse models of breast cancer, as well as increasing glucose and glutamine metabolism. Here, we expand on those findings to determine mechanistically how IL4 signaling links glucose metabolism and histone acetylation to drive proliferation in the context of triple-negative breast cancer (TNBC). We used a combination of cellular, biochemical, and genomics approaches to interrogate TNBC cell lines, which represent a cancer type where high expression of the type II IL4 receptor is linked to reduced survival. Our results indicate that type II IL4 receptor activation leads to increased glucose uptake, Akt and ACLY activation, and histone acetylation in TNBC cell lines. Inhibition of glucose uptake through the deletion of Glut1 ablates IL4-induced proliferation. Additionally, pharmacological inhibition of histone acetyltransferase P300 attenuates IL4-mediated gene expression and proliferation in vitro. Our work elucidates a role for type II IL4 receptor signaling in promoting TNBC progression, and highlights type II IL4 signaling, as well as histone acetylation, as possible targets for therapy.

Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling.

Background: Periostin (POSTN) is a critical extracellular matrix protein in various tumor microenvironments. However, the function of POSTN in thyroid cancer progression remains largely unknown. Methods: Postn and Rag1 knock-out mice and orthotopic mouse models were used to determine the role of POSTN on papillary thyroid tumor progression. Immunofluorescence, cell co-culture, fluorescence in situ hybridization, chromatin immunoprecipitation assay, recombinant protein and inhibitor treatment were performed to explore the underlying mechanisms of POSTN-promoted papillary thyroid tumor growth. Results: POSTN is up-regulated in papillary thyroid tumors and negatively correlates with the overall survival of patients with thyroid cancer. Cancer-associated fibroblast (CAF)-derived POSTN promotes papillary thyroid tumor growth in vivo and in vitro. POSTN deficiency in CAFs significantly impairs CAF-promoted papillary thyroid tumor growth. POSTN promotes papillary thyroid tumor cell proliferation and IL-4 expression through integrin-FAK-STAT3 signaling. In turn, tumor cell-derived IL-4 induces the activation of CAFs and stimulates POSTN expression by activating STAT6. We reveal the crucial role of CAF-derived POSTN and tumor cell-derived IL-4 in driving the development of papillary thyroid tumors through the POSTN-integrin-FAK-STAT3-IL-4 pathway in tumor cells and IL-4-STAT6-POSTN signaling in CAFs. Conclusion: Our findings underscore the significance of POSTN and IL-4 as critical molecular mediators in the dynamic interplay between CAFs and tumor cells, ultimately supporting the growth of papillary thyroid tumors.

Accelerating diabetic wound healing by ROS-scavenging lipid nanoparticle-mRNA formulation.

Current treatment options for diabetic wounds face challenges due to low efficacy, as well as potential side effects and the necessity for repetitive treatments. To address these issues, we report a formulation utilizing trisulfide-derived lipid nanoparticle (TS LNP)-mRNA therapy to accelerate diabetic wound healing by repairing and reprogramming the microenvironment of the wounds. A library of reactive oxygen species (ROS)-responsive TS LNPs was designed and developed to encapsulate interleukin-4 (IL4) mRNA. TS2-IL4 LNP-mRNA effectively scavenges excess ROS at the wound site and induces the expression of IL4 in macrophages, promoting the polarization from the proinflammatory M1 to the anti-inflammatory M2 phenotype at the wound site. In a diabetic wound model of db/db mice, treatment with this formulation significantly accelerates wound healing by enhancing the formation of an intact epidermis, angiogenesis, and myofibroblasts. Overall, this TS LNP-mRNA platform not only provides a safe, effective, and convenient therapeutic strategy for diabetic wound healing but also holds great potential for clinical translation in both acute and chronic wound care.

Basophil-Derived IL-4 and IL-13 Protect Intestinal Barrier Integrity and Control Bacterial Translocation during Malaria.

Our previous work demonstrated that basophils regulate a suite of malaria phenotypes, including intestinal mastocytosis and permeability, the immune response to infection, gametocytemia, and parasite transmission to the malaria mosquito Anopheles stephensi. Given that activated basophils are primary sources of the regulatory cytokines IL-4 and IL-13, we sought to examine the contributions of these mediators to basophil-dependent phenotypes in malaria. We generated mice with basophils depleted for IL-4 and IL-13 (baso IL-4/IL-13 (-)) and genotype controls (baso IL-4/IL-13 (+)) by crossing mcpt8-Cre and Il4/Il13fl/fl mice and infected them with Plasmodium yoelii yoelii 17XNL. Conditional deletion was associated with ileal mastocytosis and mast cell (MC) activation, increased intestinal permeability, and increased bacterial 16S levels in blood, but it had no effect on neutrophil activation, parasitemia, or transmission to A. stephensi. Increased intestinal permeability in baso IL-4/IL-13 (-) mice was correlated with elevated plasma eotaxin (CCL11), a potent eosinophil chemoattractant, and increased ileal MCs, proinflammatory IL-17A, and the chemokines MIP-1α (CCL3) and MIP-1ß (CCL4). Blood bacterial 16S copies were positively but weakly correlated with plasma proinflammatory cytokines IFN-γ and IL-12p40, suggesting that baso IL-4/IL-13 (-) mice failed to control bacterial translocation into the blood during malaria infection. These observations suggest that basophil-derived IL-4 and IL-13 do not contribute to basophil-dependent regulation of parasite transmission, but these cytokines do orchestrate protection of intestinal barrier integrity after P. yoelii infection. Specifically, basophil-dependent IL-4/IL-13 control MC activation and prevent infection-induced intestinal barrier damage and bacteremia, perhaps via regulation of eosinophils, macrophages, and Th17-mediated inflammation.

Nasal mucosal fibroblasts produce IL-4 to induce Th2 response.

Th2 polarization is essential for the pathogenesis of allergic rhinitis (AR). Th2 polarization's mechanism requires further understanding. IL-4 is the primary cytokine involved in Th2 response. Fibroblasts play a role in immune regulation. This study aims to elucidate the role of nasal mucosal fibroblast-derived IL-4 in the induction of Th2 responses. Nasal mucosal tissues were obtained from surgically removed samples from patients with nasal polyps, whether with or without AR. Fibroblasts were isolated from the tissues by flow cytometry cell sorting, and analyzed by RNA sequencing (RNAseq). The data from RNAseq showed that nasal fibroblasts expressed genes of GATA3, CD80, CD83, CD86, STAT6, IL2, IL4, IL5, IL6, IL13 and costimulatory factor. The data were verified by RT-qPCR. The level of gene activity was positively correlated with those of AR-related cytokines present in nasal secretions. Nasal fibroblasts release IL-4 upon activation. Nasal fibroblasts had the ability to transform naive CD4+ T cells into Th2 cells, which can be eliminated by inhibiting IL-4 receptor or CD28 in CD4+ T cells. To sum up, nasal mucosal fibroblasts produce IL-4, which can induce Th2 cell development. The data implicate that nasal fibroblasts are involved in the pathogenesis of nasal allergy.

Acupuncture at "Die E acupoint" alleviates inflammatory reaction via inhibiting TLR4/MyD88/NF-κB signaling in rats with allergic rhinitis. / 基于Tollæ ·å—ä½“4/é«“æ ·åˆ†åŒ–å› å­88/æ ¸è½¬å½•å› å­κB信号通路探讨针刺"è¶è ­ç©´"æ”¹å–„å˜åº”æ€§é¼»ç‚Žå¤§é¼ å ç–«ç‚Žæ€§ååº”çš„æœºåˆ¶.

OBJECTIVES: To observe effects of acupuncture at "Die E acupoint" on the protein expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear transcription factor κB (NF-κB), transcription factor T-bet (T-bet), and GATA-binding protein-3 (GATA-3) in the nasal mucosa and the serum contents of related inflammatory cytokines in rats with allergic rhinitis, so as to explore the mechanism of acupuncture in treating allergic rhinitis. METHODS: Twenty-four healthy SD rats were randomly divided into blank, model, acupuncture, and sham acupuncture groups, with 6 rats in each group. The rat model of allergic rhinitis was established by using ovalbumin induction. The rats in the acupuncture group received bilateral acupuncture at the "Die E acupoint" with a depth of 15-20 mm, while the rats in the sham acupuncture group received only sham acupuncture (light and shallow acupunture of the skin at the "Die E acupoint" ). Both interventions were performed once daily for a total of 6 days. Behavioral scores of rats in each group were recorded. Pathological changes of nasal mucosa were observed by H.E. staining. Serum contents of IgE, ovalbumin-specific IgE (OVA-sIgE), interferon(IFN)-γ, interleukin(IL)-4, IL-10 and IL-17 were measured by ELISA and the protein expression levels of T-bet, GATA-3, TLR4, MyD88 and NF-κB p65 in the nasal mucosa were detected by Western blot. RESULTS: After modeling, compared with the blank group, rats in the model group showed increased behavioral scores, serum IgE, OVA-sIgE, IL-4, and IL-17 contents, and nasal mucosal GATA-3, TLR4, MyD88, and NF-κB p65 protein expression levels (P<0.05), whereas the contents of serum IFN-γ, IL-10 and the protein expression level of T-bet in the nasal mucosa were decreased (P<0.05). Comparison between the EA and model groups showed that acupuncture intervention can decrease the behavioral scores of rats with allergic rhinitis, the contents of serum IgE, OVA-sIgE, IL-4, IL-17, and the protein expression levels of GATA-3, TLR4, MyD88, and NF-κB p65 in the nasal mucosa (P<0.05), and up-regulate the contents of serum IFN-γ, IL-10, and the nasal mucosal T-bet protein expression level. Sham acupuncture did not have a significant modulating effect on the above indicators. Inflammatory infiltration of nasal mucosa was seen in the model group and sham acupuncture, and the inflammatory reaction was milder in the acupuncture group. CONCLUSIONS: Acupuncture at "Die E acupoint" can alleviate the symptoms of allergic rhinitis and suppress the inflammation of nasal mucosa in rats, which may be related to inhibiting the TLR4/MyD88/NF-κB signaling and balancing the levels of cytokines of Th1/Th2 and Treg/Th17, and T-bet/GATA-3.

Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

A single infusion of engineered long-lived and multifunctional T cells confers durable remission of asthma in mice.

Asthma, the most prevalent respiratory disease, affects more than 300 million people and causes more than 250,000 deaths annually. Type 2-high asthma is characterized by interleukin (IL)-5-driven eosinophilia, along with airway inflammation and remodeling caused by IL-4 and IL-13. Here we utilize IL-5 as the targeting domain and deplete BCOR and ZC3H12A to engineer long-lived chimeric antigen receptor (CAR) T cells that can eradicate eosinophils. We call these cells immortal-like and functional IL-5 CAR T cells (5TIF) cells. 5TIF cells were further modified to secrete an IL-4 mutein that blocks IL-4 and IL-13 signaling, designated as 5TIF4 cells. In asthma models, a single infusion of 5TIF4 cells in fully immunocompetent mice, without any conditioning regimen, led to sustained repression of lung inflammation and alleviation of asthmatic symptoms. These data show that asthma, a common chronic disease, can be pushed into long-term remission with a single dose of long-lived CAR T cells.