Objective To explore the phenotypic conversion of regulatory T cells (Tregs) in the lungs of mice with bronchopulmonary dysplasia (BPD)-affected mice. Methods A total of 20 newborn C57BL/6 mice were divided into air group and hyperoxia group, with 10 mice in each group. The BPD model was established by exposing the newborn mice to hyperoxia. Lung tissues from five mice in each group were collected on postnatal days 7 and 14, respectively. Histopathological changes of the lung tissues was detected by HE staining. The expression level of surfactant protein C (SP-C) in the lung tissues was examined by Western blot analysis. Flow cytometry was performed to assess the proportion of FOXP3⁺ Tregs and RORγt⁺FOXP3⁺ Tregs in CD4⁺ lymphocytes. The concentrations of interleukin-17A (IL-17A) and IL-6 in lung homogenate were measured by using ELISA. Spearman correlation analysis was used to analyze the correlation between FOXP3⁺Treg and the expression of SP-C and the correlation between RORγt⁺FOXP3⁺ Tregs and the content of IL-17A and IL-6. Results The hyperoxia group exhibited significantly decreased levels of SP-C and radical alveolar counts in comparison to the control group. The proportion of FOXP3⁺Tregs was reduced and that of RORγt⁺FOXP3⁺Tregs was increased. IL-17A and IL-6 concentrations were significantly increased. SP-C was positively correlated with the expression level of RORγt⁺FOXP3⁺ Tregs. RORγt⁺FOXP3⁺ Tregs and IL-17A and IL-6 concentrations were also positively correlated. Conclusion The number of FOXP3⁺ Tregs in lung tissue of BPD mice is decreased and converted to RORγt⁺ FOXP3⁺ Tregs, which may be involved in hyperoxy-induced lung injury.
Animals ; Mice ; Mice, Inbred C57BL ; Bronchopulmonary Dysplasia ; T-Lymphocytes, Regulatory ; Interleukin-17 ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; Hyperoxia ; Interleukin-6 ; Forkhead Transcription Factors ; Lung

Objective To explore the significance of interleukin-17C(IL-17C)-mediated follicular helper T cell (Tfh) differentiation in atopic dermatitis (AD) model. Methods BALB/c mice were divided into control group, AD model group, low-dose MOR106 (anti-IL-17C huIgG1)(MDR106-L)treatment group and high-dose MOR106 (MOR106-H) treatment group, 8 mice in each group. Except for the control group, all the other groups were treated with 2, 4- dinitrochlorobenzene (DNCB) to establish AD models. The low-dose and high-dose MOR106 groups were treated with 5 mg/kg or 10 mg/kg MOR106 respectively. The differentiation of Tfh cell subsets in peripheral blood of mice was analyzed by flow cytometry, and the expression of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signal pathway protein in skin tissue was detected by Western blot analysis. Results Compared with the control group, the dermatitis severity score, mass difference between two ears, spleen mass and spleen index of DNCB group increased significantly, while those of MOR106-L group and MOR106-H group decreased significantly. Compared with the control group, the Tfh subgroup of AD mice showed deregulated differentiation, resulting in a significant increase in the percentage of CD4⁺CXCR5⁺IFN-γ⁺Tfh1 cells, CD4⁺CXCR5⁺IL-17A⁺Tfh17 and CD4⁺CXCR5⁺IL-21⁺Tfh21 cells, and a significant decrease in the percentage of CD4⁺CXCR5⁺IL-10⁺Tfh10 cells and CD4⁺CXCR5⁺FOXP3⁺Tfr cells in peripheral blood. The protein levels of phosphorylated JAK2(p-JAK2) and p-STAT3 were significantly increased. MOR106 effectively reversed these changes of Tfh1, Tfh10, Tfh17, Tfh21 and Tfr cells in peripheral blood of AD mice. Compared with AD group, the levels of p-JAK2 and p-STAT3 protein in low-dose and high-dose MOR106 treatment groups decreased significantly. Conclusion MOR106 can reduce the inflammatory response of AD mice by blocking JAK2/STAT3 signaling pathway and inhibiting the differentiation of Tfh cells mediated by IL-17C.
Animals ; Mice ; Dermatitis, Atopic/drug therapy* ; Interleukin-17 ; T Follicular Helper Cells ; Janus Kinase 2 ; Dinitrochlorobenzene ; Inflammation ; Cell Differentiation ; Signal Transduction

Humans ; Arthritis, Psoriatic/drug therapy* ; Interleukin-17 ; Interleukin Inhibitors ; Antirheumatic Agents/therapeutic use* ; Tumor Necrosis Factor-alpha/therapeutic use* ; Interleukin-23/therapeutic use*

INTRODUCTION: There is a paucity of information on the cytokine, complement, endothelial activation, and coagulation profiles of multisystem inflammatory syndrome in adults (MIS-A), a rare but serious complication following recovery from SARS-CoV-2 infection. We aim to examine the immune biomarker and coagulation profiles in association with the clinical presentation and course of MIS-A. METHOD: The clinical features of MIS-A patients admitted to our tertiary hospital were documented. Their levels of interleukin (IL)-1β, IL-6, IL-10, IL-17, IL-18, interferon-α (IFN-α), IFN-γ, interferon gamma-induced protein 10 (IP-10), tumour necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, complement activation product (complement 5a [C5a]), and endothelial biomarker intercellular adhesion molecule-1 (ICAM-1) levels were assayed. The haemostatic profile was assessed with standard coagulation testing and thromboelastography. RESULTS: Three male patients were diagnosed with MIS-A at our centre from January to June 2022 with a median age of 55 years. All had tested positive for SARS-CoV-2 12-62 days prior to MIS-A presentation, with gastrointestinal and cardiovascular systems as the most commonly involved. Levels of IL-6, IL-10, IL-18, IP-10 and MCP-1 were raised whereas IL-1β, IFN-α, IFN-γ, IL-17 and TNF-α remained normal. Markedly elevated levels of C-reactive protein (CRP), ferritin and ICAM-1 were present in all. C5a was elevated in 2 patients. A hypercoagulable state was demonstrated by raised levels of D-dimer, factor VIII, von Willebrand factor antigen, and ristocetin cofactor with corresponding raised parameters in thromboelastography in the 2 patients who had their coagulation profile assessed. CONCLUSION MIS-A patients demonstrate activation of pro-inflammatory cytokines, endotheliopathy, complement hyperactivation and hypercoagulability.
Humans ; Adult ; Male ; Middle Aged ; COVID-19/complications* ; Interleukin-10 ; Interleukin-18 ; Intercellular Adhesion Molecule-1 ; Interleukin-17 ; Chemokine CXCL10 ; Interleukin-6 ; SARS-CoV-2 ; Connective Tissue Diseases ; Hemostatics

OBJECTIVES: To study the expression of interleukin-17A (IL-17A) in the serum of children with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD) and its clinical significance. METHODS: A total of 143 children with KD who were hospitalized in Wuhan Children's Hospital, Tongji Medical College, Huazhong University of Science and Technology, from June 2021 to June 2022 were enrolled in this prospective study, among whom 115 had IVIG-sensitive KD and 28 had IVIG-resistant KD. After matching for sex and age, 110 children with acute respiratory infectious diseases (fever time ≥5 days but without KD) were enrolled as the control group. The enzyme-linked immunosorbent assay was used to measure the serum level of IL-17A. The levels of white blood cell count (WBC), neutrophil count (NE), platelet count, erythrocyte sedimentation rate, and C-reactive protein (CRP) were measured. The receiver operating characteristic curve was plotted to analyze the value of WBC, NE, CRP, and IL-17A in the prediction of IVIG-resistant KD. The multivariate logistic regression analysis was used to evaluate the predictive factors for resistance to IVIG in children with KD. RESULTS: Before IVIG treatment, the KD group had a significantly higher serum level of IL-17A than the control group (P<0.05), and the children with IVIG-resistant KD had a significantly higher serum level of IL-17A than those with IVIG-sensitive KD (P<0.05). The receiver operating characteristic curve analysis showed that WBC, NE, CRP, and IL-17A had an area under the curve of 0.718, 0.741, 0.627, and 0.840, respectively, in the prediction of IVIG-resistant KD. With serum IL-17A ≥44.06 pg/mL as the cut-off value, IL-17A had a sensitivity of 84% and a specificity of 81% in the prediction of IVIG-resistant KD. The multivariate logistic regression analysis showed that a high serum level of IL-17A was a predictive factor for resistance to IVIG in children with KD (OR=1.161, P=0.001). CONCLUSIONS Serum IL-17A levels are elevated in children with IVIG-resistant KD, and serum IL-17A level (≥44.06 pg/mL) may have a predictive value for resistance to IVIG in children with KD.
Humans ; Child ; Infant ; Aged, 80 and over ; Immunoglobulins, Intravenous/therapeutic use* ; Mucocutaneous Lymph Node Syndrome/drug therapy* ; Interleukin-17 ; Clinical Relevance ; Prospective Studies ; C-Reactive Protein/analysis* ; Retrospective Studies

OBJECTIVES: Ozone is widely applied to treat allergic skin diseases such as eczema, atopic dermatitis, and contact dermatitis. However, the specific mechanism remains unclear. This study aims to investigate the effects of ozonated oil on treating 2,4-dinitrochlorobenzene (DNCB)-induced allergic contact dermatitis (ACD) and the underling mechanisms. METHODS: Besides the blank control (Ctrl) group, all other mice were treated with DNCB to establish an ACD-like mouse model and were randomized into following groups: a model group, a basal oil group, an ozonated oil group, a FcεRI-overexpressed plasmid (FcεRI-OE) group, and a FcεRI empty plasmid (FcεRI-NC) group. The basal oil group and the ozonated oil group were treated with basal oil and ozonated oil, respectively. The FcεRI-OE group and the FcεRI-NC group were intradermally injected 25 µg FcεRI overexpression plasmid and 25 µg FcεRI empty plasmid when treating with ozonated oil, respectively. We recorded skin lesions daily and used reflectance confocal microscope (RCM) to evaluate thickness and inflammatory changes of skin lesions. Hematoxylin-eosin (HE) staining, real-time PCR, RNA-sequencing (RNA-seq), and immunohistochemistry were performed to detct and analyze the skin lesions. RESULTS: Ozonated oil significantly alleviated DNCB-induced ACD-like dermatitis and reduced the expressions of IFN-γ, IL-17A, IL-1β, TNF-α, and other related inflammatory factors (all P<0.05). RNA-seq analysis revealed that ozonated oil significantly inhibited the activation of the DNCB-induced FcεRI/Syk signaling pathway, confirmed by real-time PCR and immunohistochemistry (all P<0.05). Compared with the ozonated oil group and the FcεRI-NC group, the mRNA expression levels of IFN-γ, IL-17A, IL-1β, IL-6, TNF-α, and other inflammatory genes in the FcεRI-OE group were significantly increased (all P<0.05), and the mRNA and protein expression levels of FcεRI and Syk were significantly elevated in the FcεRI-OE group as well (all P<0.05). CONCLUSIONS Ozonated oil significantly improves ACD-like dermatitis and alleviated DNCB-induced ACD-like dermatitis via inhibiting the FcεRI/Syk signaling pathway.
Animals ; Mice ; Dinitrochlorobenzene/metabolism* ; Skin/metabolism* ; Cytokines/metabolism* ; Interleukin-17/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Dermatitis, Allergic Contact/pathology* ; Dermatitis, Atopic/chemically induced* ; Signal Transduction ; RNA, Messenger/metabolism* ; Mice, Inbred BALB C

OBJECTIVE: To investigate the correlation of inducible co-stimulatory molecules (ICOS) with mesenteric vascular endothelial- mesenchymal transition (EndMT) and sclerosis in spontaneously hypertensive rats (SHR). METHODS: Twenty 4-week-old WKY rats and 20 SHRs of the same strain were both randomly divided into 4 groups for observation at 4, 6, 10 and 30 weeks of age. ICOS expression frequency in rat spleen CD4⁺T cells was analyzed using flow cytometry, and the expressions of ICOS, VE-cad, α-SMA and Col3 mRNA in rat mesentery were detected by RT-PCR. The distributions of ICOS, IL-17A and TGF-β in rat mesentery were detected by immunohistochemistry. The levels of IL-17A and TGF-β in rat plasma were measured using ELISA. The morphological changes of rat mesenteric vessels were observed with Masson staining. Spearman or Pearson correlation analyses were used to evaluate the correlation between ICOS expression and the expressions of the markers of vascular EndMT and sclerosis. RESULTS: Compared with the control WKY rats, the SHRs began to show significantly increased systolic blood pressure and ICOS expression frequency on CD4⁺T cells at 6 weeks of age (P < 0.05). In the SHRs, the mRNA and protein expressions of ICOS, α-SMA, Col3, IL-17A and TGF-β in the mesentery were significantly higher than those in control group (P < 0.05), while the mRNA and protein expressions of VE-cad started to reduce significantly at 10 weeks of age (P < 0.05). The plasma levels of IL-17A and TGF-β were significantly increased in SHRs since 6 weeks of age (P < 0.05) with progressive worsening of mesenteric vascular sclerosis (P < 0.05). ICOS mRNA and protein expression levels in the mesenteric tissues of SHRs began to show positive correlations with α-SMA and Col3 expression levels and the severity of vascular sclerosis at 6 weeks of age (P < 0.05) and a negative correlation with VE-cad expression level at 10 weeks (P < 0.05). CONCLUSION ICOS play an important pathogenic role in EndMT and sclerosis of mesenteric vessels in essential hypertension by mediating related immune responses.
Rats ; Animals ; Rats, Inbred SHR ; Rats, Inbred WKY ; Hypertension ; Interleukin-17 ; Sclerosis/pathology* ; Transforming Growth Factor beta ; Mesentery/pathology* ; RNA, Messenger/metabolism* ; Blood Pressure

OBJECTIVE: To examine the anti-inflammatory effect of grape seed extract (GSE) in animal and cellular models and explore its mechanism of action. METHODS: This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17 and the inflammatory factors such as tumor necrosis factor- α (TNF- α), interleukin-1 β (IL-1 β), IL-6, IL-12, IL-17 and interferon-γ (IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay (ELISA), immunofluorescence staining and Western blot, respectively. RESULTS: GSE reduced the secretion of TNF-α, IL-1 β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days (P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors (P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4 (TLR4) and Rho-associated kinase (ROCKII, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors (P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45⁺CD11b⁺ and CD45⁺CD4⁺ cells, and weakened the differentiation of Th1 and Th17 (P<0.05). Moreover, it reduced the secretion of inflammatory factors (P<0.01), and prevented the activation of microglia (P<0.05). CONCLUSION GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.
Mice ; Animals ; Encephalomyelitis, Autoimmune, Experimental/pathology* ; Grape Seed Extract/therapeutic use* ; Interleukin-17 ; Interleukin-1beta ; Tumor Necrosis Factor-alpha/metabolism* ; Interleukin-6/metabolism* ; Th1 Cells ; Mice, Inbred C57BL ; Interferon-gamma/therapeutic use* ; Th17 Cells/metabolism* ; Interleukin-12/therapeutic use* ; Cytokines/metabolism*

Spinal cord injury (SCI) causes motor, sensory, and autonomic dysfunctions. The gut microbiome has an important role in SCI, while short-chain fatty acids (SCFAs) are one of the main bioactive mediators of microbiota. In the present study, we explored the effects of oral administration of exogenous SCFAs on the recovery of locomotor function and tissue repair in SCI. Allen's method was utilized to establish an SCI model in Sprague-Dawley (SD) rats. The animals received water containing a mixture of 150 mmol/L SCFAs after SCI. After 21 d of treatment, the Basso, Beattie, and Bresnahan (BBB) score increased, the regularity index improved, and the base of support (BOS) value declined. Spinal cord tissue inflammatory infiltration was alleviated, the spinal cord necrosis cavity was reduced, and the numbers of motor neurons and Nissl bodies were elevated. Enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (qPCR), and immunohistochemistry assay revealed that the expression of interleukin (IL)‍-10 increased and that of IL-17 decreased in the spinal cord. SCFAs promoted gut homeostasis, induced intestinal T cells to shift toward an anti-inflammatory phenotype, and promoted regulatory T (Treg) cells to secrete IL-10, affecting Treg cells and IL-17⁺ γδ T cells in the spinal cord. Furthermore, we observed that Treg cells migrated from the gut to the spinal cord region after SCI. The above findings confirm that SCFAs can regulate Treg cells in the gut and affect the balance of Treg and IL-17⁺ γδ T cells in the spinal cord, which inhibits the inflammatory response and promotes the motor function in SCI rats. Our findings suggest that there is a relationship among gut, spinal cord, and immune cells, and the "gut-spinal cord-immune" axis may be one of the mechanisms regulating neural repair after SCI.
Animals ; Rats ; Interleukin-17 ; Rats, Sprague-Dawley ; Recovery of Function ; Spinal Cord Injuries/drug therapy* ; T-Lymphocytes, Regulatory ; Receptors, Antigen, T-Cell, gamma-delta/immunology*

OBJECTIVE: To explore the effect of interleukin-17A (IL-17A) on liver and kidney injury and prognosis in septic mice. METHODS: A total of 84 SPF male C57BL/6 mice were randomly divided into sham operation group (Sham group), cecal ligation and puncture (CLP) induced sepsis model group (CLP group), and IL-17A intervention group. IL-17A intervention group were then divided into five subgroups according to the dose of IL-17A (0.25, 0.5, 1, 2, 4 μg). Mice in the IL-17A intervention group were intraperitoneally injected with the corresponding dose of IL-17A 100 μL immediately after surgery. The other groups were intraperitoneally injected with 100 μL phosphate buffer solution (PBS). The survival rate of mice was observed at 7 days, and peripheral blood and liver, kidney and spleen tissues were collected. According to the 7-day survival, another 18 mice were randomly divided into Sham group, CLP group, and 1 μg IL-17A intervention group. Peripheral blood samples were collected at 12 hours and 24 hours after CLP, and the mice were sacrificed to obtain liver, kidney, and spleen tissues. The behavior and abdominal cavity of each group were observed. The levels of peripheral blood liver and kidney function indexes and inflammatory factors were detected. The histopathological changes of liver and kidney were observed under light microscope. The peripheral blood and spleen tissues were inoculated in the medium, the number of bacterial colonies was calculated, and the bacterial migration of each group was evaluated in vitro. RESULTS: Except for the Sham group, the 7-day survival rate of mice in the 1 μg IL-17A intervention group was the highest (75.0%), so this condition was selected as the intervention condition for the subsequent study. Compared with Sham group, the liver and kidney functions of CLP group were significantly damaged at each time point after operation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and serum creatinine (SCr) reached the peak at 24 hours after operation, and the liver and kidney pathological scores reached the peak at 7 days after operation, the levels of inflammatory cytokines interleukin (IL-17A, IL-6, IL-10) reached the peak at 12 hours after operation, and tumor necrosis factor-α (TNF-α) reached the peak at 24 hours after operation. In addition, a large number of bacteria proliferated in the peripheral blood and spleen, which reached the peak on day 7. Compared with the CLP group, exogenous administration of 1 μg IL-17A significantly delayed the rising trend of each index in the early stage of sepsis [24-hour ALT (U/L): 166.95±5.20 vs. 271.30±6.11, 24-hour AST (U/L): 599.42±7.25 vs. 1 013.27±3.37, 24-hour BUN (mg/L): 815.4±26.3 vs. 1 191.2±39.4, 24-hour SCr (μmol/L): 29.34±0.87 vs. 60.75±3.83, 7-day liver pathological score: 2.50 (2.00, 3.00) vs. 9.00 (8.50, 9.00), 7-day kidney pathological score: 1.00 (1.00, 2.00) vs. 5.00 (4.50, 5.00), 12-hour IL-17A (ng/L): 105.21±0.31 vs. 111.28±1.37, 12-hour IL-6 (ng/L): 83.22±1.01 vs. 108.88±0.99, 12-hour IL-10 (ng/L): 731.54±3.04 vs. 790.25±2.54, 24-hour TNF-α (μg/L): 454.67±0.66 vs. 576.18±0.76, 7-day peripheral blood colony count (CFU/mL): 600 (400, 600) vs. 4 200 (4 200, 4 300), 7-day spleen tissue colony count (CFU/g): 4 600 (4 400, 4 600) vs. 23 400 (23 200, 23 500), all P < 0.05]. CONCLUSIONS Appropriate dose (1 μg) of exogenous IL-17A can reduce the lethal inflammatory response induced by CLP and improve the ability of bacterial clearance, thereby alleviating liver and kidney injury and improving the 7-day survival rate of septic mice.
Animals ; Male ; Mice ; Interleukin-10 ; Interleukin-17/pharmacology* ; Interleukin-6 ; Kidney/physiopathology* ; Liver/physiopathology* ; Mice, Inbred C57BL ; Prognosis ; Sepsis ; Tumor Necrosis Factor-alpha