IL-33 and its receptor ST2 play crucial roles in tissue repair and homeostasis. However, their involvement in optic neuropathy due to trauma and glaucoma remains unclear. Here, we report that IL-33 and ST2 were highly expressed in the mouse optic nerve and retina. Deletion of IL-33 or ST2 exacerbated retinal ganglion cell (RGC) loss, retinal thinning, and nerve fiber degeneration following optic nerve (ON) injury. This heightened retinal neurodegeneration correlated with increased neurotoxic astrocytes in Il33^-/- mice. In vitro, rIL-33 mitigated the neurotoxic astrocyte phenotype and reduced the expression of pro-inflammatory factors, thereby alleviating the RGC death induced by neurotoxic astrocyte-conditioned medium in retinal explants. Exogenous IL-33 treatment improved RGC survival in Il33^-/- and WT mice after ON injury, but not in ST2^-/- mice. Our findings highlight the role of the IL-33/ST2 axis in modulating reactive astrocyte function and providing neuroprotection for RGCs following ON injury.
Animals ; Interleukin-33/genetics* ; Interleukin-1 Receptor-Like 1 Protein/genetics* ; Optic Nerve Injuries/pathology* ; Retinal Ganglion Cells/pathology* ; Astrocytes/pathology* ; Mice ; Mice, Knockout ; Mice, Inbred C57BL ; Neuroprotection/physiology*

High mobility group box 1 (HMGB1), when released extracellularly, plays a pivotal role in the development of spinal cord synapses and exacerbates autoimmune diseases within the central nervous system. In experimental autoimmune encephalomyelitis (EAE), a condition that models multiple sclerosis, the levels of extracellular HMGB1 and interleukin-33 (IL-33) have been found to be inversely correlated. However, the mechanism by which IL-33 deficiency enhances HMGB1 release during EAE remains elusive. Our study elucidates a potential signaling pathway whereby the absence of IL-33 leads to increased binding of P300/CBP-associated factor with HMGB1 in the nuclei of astrocytes, upregulating HMGB1 acetylation and promoting its release from astrocyte nuclei in the spinal cord of EAE mice. Conversely, the addition of IL-33 counteracts the TNF-α-induced increase in HMGB1 and acetylated HMGB1 levels in primary astrocytes. These findings underscore the potential of IL-33-associated signaling pathways as a therapeutic target for EAE treatment.
Animals ; Encephalomyelitis, Autoimmune, Experimental/metabolism* ; Astrocytes/metabolism* ; Interleukin-33/metabolism* ; HMGB1 Protein/metabolism* ; Acetylation ; Mice, Knockout ; Mice, Inbred C57BL ; p300-CBP Transcription Factors/metabolism* ; Mice ; Spinal Cord/metabolism* ; Cells, Cultured ; Female ; Signal Transduction

Atopic dermatitis (AD) is a prevalent inflammatory skin disorder in which patients experience recurrent eczematous lesions and intense itching. The colonization of Staphylococcus aureus (S. aureus) is correlated with the severity of the disease, but its role in AD development remains elusive. Using single-cell RNA sequencing, we uncovered that keratinocytes activate a distinct immune response characterized by induction of Il24 when exposed to methicillin-resistant S. aureus (MRSA). Further experiments using animal models showed that the administration of recombinant IL-24 protein worsened AD-like pathology. Genetic ablation of Il24 or the receptor Il20rb in keratinocytes alleviated allergic inflammation and atopic march. Mechanistically, IL-24 acted through its heterodimeric receptors on keratinocytes and augmented the production of IL-33, which in turn aggravated type 2 immunity and AD-like skin conditions. Overall, these findings establish IL-24 as a critical factor for onset and progression of AD and a compelling therapeutic target.
Dermatitis, Atopic/genetics* ; Interleukins/metabolism* ; Animals ; Methicillin-Resistant Staphylococcus aureus/immunology* ; Mice ; Keratinocytes/microbiology* ; Humans ; Interleukin-33/immunology* ; Inflammation/microbiology* ; Staphylococcal Infections/microbiology* ; Disease Models, Animal ; Hypersensitivity/microbiology* ; Mice, Inbred C57BL

The aim of this study was to compare the immune responses of C57BL/6 mice immunized with two pathogens, foot-and-mouth disease virus (FMDV) and Senecavirus A (SVA), and to provide clues for revealing the regulatory mechanisms of acquired immunity. Inactivated and purified FMDV and SVA antigens were used to immunize C57BL/6 mice respectively, and the mice immunized with PBS were taken as the control. The percentages of Th1 and Th2 cells in the spleen lymphocytes of mice in each group were analyzed by flow cytometry at 14 and 28 days after immunization. RNA-Seq was performed for the spleen. Mouse macrophages were stimulated with the antigens in vitro to examine the expression of the differentially expressed genes (DEGs) screened out. The results showed that 14 days after immunization, there was no significant difference in the magnitude of the Th1/Th2 immune response elicited by the FMDV and SVA antigens. After 28 days, the magnitudes of the Th1 and Th2 immune responses elicited by the SVA antigen were higher than those elicited by the FMDV antigen. RNA-Seq revealed two common DEGs, Rsad2 and Tspan8, between the two immunization groups, which indicated that the two genes may be involved in the activation of the Th1/Th2 immune responses by FMDV and SVA antigens. FMDV and SVA antigens stimulated macrophages to secrete interleukin (IL)-12 and IL-33 in vitro, and the expression of Tspan8 and Rsad2 was consistent with the RNA-Seq results. The expression of Rsad2 was regulated by type I interferons (IFNα, IFNβ). In this study, we obtained the DEGs involved in the immune responses to the two antigens in mouse spleen, which provides a molecular basis for investigating the immune response mechanisms induced by FMDV and SVA.
Animals ; Foot-and-Mouth Disease Virus/genetics* ; Mice ; Spleen/cytology* ; Mice, Inbred C57BL ; Antigens, Viral/genetics* ; Transcriptome ; Th1 Cells/immunology* ; Immunization ; Viral Vaccines/immunology* ; Th2 Cells/immunology* ; Foot-and-Mouth Disease/immunology* ; Interleukin-33/genetics* ; Female ; Macrophages/immunology* ; Picornaviridae

OBJECTIVES: To investigate the changes and significance of type 2 innate lymphoid cells (ILC2), interleukin-33 (IL-33), interleukin-25 (IL-25), thymic stromal lymphopoietin (TSLP), interleukin-5 (IL-5), and interleukin-13 (IL-13) in peripheral blood of preterm infants with bronchopulmonary dysplasia (BPD). METHODS: A total of 76 preterm infants with a gestational age of <32 weeks and a length of hospital stay of ≥14 days who were admitted to the Department of Pediatrics of the Affiliated Hospital of Jiangsu University from September 2020 to December 2021 were enrolled. According to the diagnostic criteria for BPD, they were divided into a BPD group with 30 infants and a non-BPD group with 46 infants. The two groups were compared in terms of the percentage of ILC2 and the levels of IL-33, IL-25, TSLP, IL-5, and IL-13 in peripheral blood on days 1, 7, and 14 after birth. RESULTS: The BPD group had significantly lower birth weight and gestational age than the non-BPD group (P<0.05). On days 7 and 14 after birth, the BPD group had significantly higher levels of ILC2, IL-33, TSLP, and IL-5 than the non-BPD group (P<0.05), and these indices had an area under the curve of >0.7 in predicting the devolpment of BPD (P<0.05). Multivariate logistic regression analysis showed that after adjusting for gestational age and birth weight, peripheral blood IL-33, TSLP and IL-5 on days 7 and 14 after birth were closely related to the devolpment of BPD (P<0.05). CONCLUSIONS Early innate immune activation and upregulated expression of related factors may be observed in preterm infants with BPD. ILC2, IL-33, TSLP, and IL-5 may be used as biological indicators for early diagnosis of BPD.
Child ; Humans ; Infant ; Infant, Newborn ; Birth Weight ; Bronchopulmonary Dysplasia/pathology* ; Cytokines ; Immunity, Innate ; Infant, Premature ; Interleukin-13 ; Interleukin-33 ; Interleukin-5 ; Lymphocytes/pathology* ; Thymic Stromal Lymphopoietin

Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell^TM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
Animals ; Mice ; Bleomycin/metabolism* ; Cell Differentiation ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Fibroblasts ; Interleukin-33/pharmacology* ; Mice, Inbred C57BL ; Myofibroblasts/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Pulmonary Fibrosis ; Signal Transduction

Objective To investigate the effect of calcitonin gene-related peptide (CGRP) on the regulation of group 2 innate lymphoid cells (ILC2) in the peripheral blood of patients with allergic rhinitis (AR). Methods Peripheral blood mononuclear cells (PBMCs) were extracted from normal healthy individuals and AR patients, then stimulated with CGRP, interleukin 33 (IL-33) and CGRP combined with IL-33 for 3 days, with blank stimulus as control. The percentage of ILC2 in the four groups was measured by flow cytometry. After being sorted, ILC2 was given to CGRP, IL-33 and CGRP combined with IL-33 stimulation for 3 days, with blank stimulus as control. The percentage of IL-5 and IL-13 positive cells in ILC2 was detected by flow cytometry, and the levels of IL-5 and IL-13 in ILC2 supernatant were measured by ELISA. Results The percentage of ILC2 in the peripheral blood of AR patients was significantly higher than that of the control group. The levels of IL-5⁺ILC2 and IL-13⁺ILC2 were significantly increased by IL-33 single stimulation after culturing PBMCs. After adding IL-33 combined with CGRP stimulation, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 in PBMCs were significantly reduced; after CGRP single stimulation, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 in PBMCs were further decreased. After ILC2 was sorted and cultured, the levels of IL-5⁺ILC2 and IL-13⁺ILC2 showed significant increase after IL-33 single stimulation. The levels of IL-5⁺ILC2 and IL-13⁺ILC2 were decreased by IL-33 and CGRP co-stimulation, and they were further reduced after CGRP single stimulation. Compared to IL-33 single stimulation, IL-5 and IL-13 levels dropped significantly due to the IL-33 and CGRP co-stimulation. The levels of IL-5 and IL-13 were further reduced by CGRP single stimulation. Conclusion CGRP inhibits the proliferation and activation of peripheral blood ILC2 in AR and exert anti-inflammatory effects in AR.
Humans ; Calcitonin Gene-Related Peptide/pharmacology* ; Leukocytes, Mononuclear ; Immunity, Innate ; Interleukin-33/pharmacology* ; Interleukin-13 ; Lymphocytes ; Interleukin-5/pharmacology* ; Rhinitis, Allergic ; Cell Proliferation

Objective: To investigate the effects of combined blockade of interleukin-33 (IL-33) and inducible co-stimulatory molecule (ICOS) on carbon tetrachloride-induced chronic liver fibrosis and imbalance of T helper lymphocyte subsets in mice. Methods: There were 40 BALB/c mice in each model and control group. Flow cytometry was used to determine the proportion of Th1/Th2/Th17 cells in the splenic lymphocyte suspension of mice, the expression levels of interferon γ, IL-4, and IL-17 in the splenic lymphocyte suspension of liver fibrosis mice after combined blockade of IL-33 and ICOS, and the pathological changes of liver histopathology in mice with liver fibrosis. Two independent sample t-test was used to compare data between groups. Results: Compared with the non-blocking group, the proportion of Th2 and Th17 cells in the IL-33/ICOS blocking group was significantly down-regulated (Th2: 65.96% ± 6.04% vs. 49.09% ± 7.03%; Th17: 19.17% ± 4.03% vs. 9.56% ± 2.03%), while the proportion of Th1 cells and Th1/Th2 ratio were up-regulated (Th1: 17.14% ± 3.02% vs. 31.93% ± 5.02%; Th1/Th2: 0.28 ± 0.06 vs. 0.62 ± 0.23), and the difference was statistically significant (t = 5.15, 6.03, 7.14, 4.28, respectively, with P < 0.05). After entering the chronic inflammation stage of liver fibrosis in mice (10 weeks), compared with the non-blocking group, the expression levels of IL-4 and IL-17 in the blockade group were significantly down-regulated [IL-4: (84.75 ± 14.35) pg/ ml vs. (77.88 ± 19.61) pg/ml; IL-17: (72.38 ± 15.13) pg/ml vs. (36.38 ± 8.65) pg/ml], while the expression of interferon γ was up-regulated [(37.25 ± 11.51) pg/ml vs. (77.88 ± 19.61) pg/ml], and the difference was statistically significant (t: IL-4: 4.71; IL-17: 5.84; interferon γ: 5.05, respectively, with P < 0.05). Liver histopathological results showed that hepatic necrosis, hepatic lobular structural disorder, and fibrous tissue hyperplasia were significantly lower in the blockade group than those in the non-blocking group at 13 weeks of liver fibrosis. Conclusion: Combined blockade of the ICOS signaling pathway and IL-33 can regulate Th2 and Th17 polarization, down-regulate the inflammatory response, and inhibit or prevent the occurrence and progression of fibrosis.
Mice ; Animals ; Interferon-gamma/metabolism* ; Interleukin-17/metabolism* ; Interleukin-33/metabolism* ; Cytokines/metabolism* ; Carbon Tetrachloride ; Th2 Cells ; Interleukin-4/metabolism* ; Liver Cirrhosis/pathology* ; Th1 Cells ; Th17 Cells/pathology* ; Immunity

OBJECTIVE: To observe the clinical efficacy of scalp acupuncture for spastic cerebral palsy (CP), and to explore its possible mechanism based on brain white matter fiber bundles, nerve growth related proteins and inflammatory cytokines. METHODS: A total of 90 children with spastic CP were randomly divided into a scalp acupuncture group and a sham scalp acupuncture group, 45 cases in each group. The children in the two groups were treated with conventional comprehensive rehabilitation treatment. The children in the scalp acupuncture group were treated with scalp acupuncture at the parietal temporal anterior oblique line, parietal temporal posterior oblique line on the affected side, and parietal midline. The children in the sham scalp acupuncture group were treated with scalp acupuncture at 1 cun next to the above point lines. The needles were kept for 30 min, once a day, 5 days a week, for 12 weeks. Before and after treatment, the diffusion tensor imaging (DTI) indexes of magnetic resonance (FA values of corticospinal tract [CST], anterior limb of internal capsule [ICAL], posterior limb of internal capsule [ICPL], genu of internal capsule [ICGL], genu of corpus callosum [GCC], body of corpus callosum [BCC] and splenium of corpus callosum [SCC]), serum levels of nerve growth related proteins (neuron-specific enolase [NSE], glial fibrillary acidic protein [GFAP], myelin basic protein [MBP], ubiquitin carboxy terminal hydrolase-L1 [UCH-L1]) and inflammatory cytokines (interleukin 33 [IL-33], tumor necrosis factor α [TNF-α]), cerebral hemodynamic indexes (mean blood flow velocity [Vm], systolic peak flow velocity [Vs] and resistance index [RI], pulsatility index [PI] of cerebral artery), surface electromyography (SEMG) signal indexes (root mean square [RMS] values of rectus femoris, hamstring muscles, gastrocnemius muscles, tibialis anterior muscles), gross motor function measure-88 (GMFM-88) score, modified Ashworth scale (MAS) score, ability of daily living (ADL) score were observed in the two groups. The clinical effect of the two groups was compared. RESULTS: After treatment, the FA value of each fiber bundle, Vm, Vs, GMFM-88 scores and ADL scores in the two groups were higher than those before treatment (P<0.05), and the above indexes in the scalp acupuncture group were higher than those in the sham scalp acupuncture group (P<0.05). After treatment, the serum levels of NSE, GFAP, MBP, UCH-L1, IL-33, TNF-α as well as RI, PI, MAS scores and RMS values of each muscle were lower than those before treatment (P<0.05), and the above indexes in the scalp acupuncture group were lower than those in the sham scalp acupuncture group (P<0.05). The total effective rate was 95.6% (43/45) in the scalp acupuncture group, which was higher than 82.2% (37/45) in the sham scalp acupuncture group (P<0.05). CONCLUSION Scalp acupuncture could effectively treat spastic CP, improve the cerebral hemodynamics and gross motor function, reduce muscle tension and spasticity, and improve the ability of daily life. The mechanism may be related to repairing the white matter fiber bundles and regulating the levels of nerve growth related proteins and inflammatory cytokines.
Child ; Humans ; Cerebral Palsy/therapy* ; Interleukin-33 ; Diffusion Tensor Imaging/methods* ; Scalp ; Muscle Spasticity ; Tumor Necrosis Factor-alpha ; Acupuncture Therapy ; Cytokines

Objective To create a recombinant rabies virus overexpressing IL-33 and to clarify the effect of IL-33 overexpression on the phenotypic characteristics of recombinant virus in vitro. Methods The IL-33 gene was obtained and amplified from the brain of a highly virulent strain of rabies infected mouse. It was then inserted between the G and L genes of the parental virus LBNSE genome by reversing genetic manipulation and rescuing a recombinant virus overexpressing IL-33. BSR cells or mouse NA cells were infected with recombinant rabies virus (rLBNSE-IL33) and the parental strain LBNSE. Sequencing and fluorescent antibody virus neutralization assay was employed to detect the stability of recombinant virus at multiplicity of infection=0.01. Viral titres focal forming units (FFU) were detected to plot multi-step growth curves (multiplicity of infection=0.01). Cytotoxicity assay kit was used to detect cellular activity. ELISA was adopted to identify the IL-33 in the supernatant of infected cells of different multiplicity of infection. Results Rescued rLBNSE-IL33 overexpressing IL-33 remained stable for at least 10 consecutive generations and had virus titers of approximately 10⁸ FFU/mL. rLBNSE-IL33 was able to express IL-33 at high levels in a dose-dependent manner, but no high expression of IL-33 was detected in the supernatant of cells infected by LBNSE. Examination of the titers of rLBNSE-IL33 and the parental strain LBNSE in BSR and NA cells over 5 days showed no significant differences and similar kinetic properties in growth. Overexpression of IL-33 had no significant effect on the proliferation and activity of infected cells. Conclusion Overexpression of IL-33 does not significantly affect the phenotypic characteristics of recombinant rabies virus in vitro.
Animals ; Cricetinae ; Mice ; Cell Line ; Interleukin-33/genetics* ; Rabies virus/genetics* ; Phenotype