Splenic Th17 Research Articles

Human Milk Oligosaccharides in Combination with Galacto- and Long-Chain Fructo-Oligosaccharides Enhance Vaccination Efficacy in a Murine Influenza Vaccination Model.

Early-life nutrition significantly impacts vaccination efficacy in infants, whose immune response to vaccines is weaker compared to adults. This study investigated vaccination efficacy in female C57Bl/6JOlaHsd mice (6 weeks old) fed diets with 0.7% galacto-oligosaccharides (GOS)/long-chain fructo-oligosaccharides (lcFOS) (9:1), 0.3% human milk oligosaccharides (HMOS), or a combination (GFH) for 14 days prior to and during vaccination. Delayed-type hypersensitivity (DTH) was measured by assessing ear swelling following an intradermal challenge. Influvac-specific IgG1 and IgG2a levels were assessed using ELISAs, while splenic T and B lymphocytes were analyzed for frequency and activation via flow cytometry. Additionally, cytokine production was evaluated using murine splenocytes co-cultured with influenza-loaded dendritic cells. Mice on the GFH diet showed a significantly enhanced DTH response (p < 0.05), increased serological IgG1 levels, and a significant rise in memory B lymphocytes (CD27+ B220+ CD19+). GFH-fed mice also exhibited more activated splenic Th1 cells (CD69+ CXCR3+ CD4+) and higher IFN-γ production after ex vivo restimulation (p < 0.05). These findings suggest that GOS/lcFOS and HMOS, particularly in combination, enhance vaccine responses by improving memory B cells, IgG production, and Th1 cell activation, supporting the potential use of these prebiotics in infant formula for better early-life immune development.

Geniposide modulates GSK3β to inhibit Th17 differentiation and mitigate endothelial damage in intracranial aneurysm.

Intracranial aneurysm (IA) is a common cerebrovascular disease. Immune system disorders and endothelial dysfunction are essential mechanisms of its pathogenesis. This study aims to explore the therapeutic effect and mechanism of Geniposide (Gen)on IA, which has a protective impact on endothelial cells and cardiovascular and cerebrovascular diseases. IA mouse models were administered intraperitoneal injections of geniposide for 2 weeks following elastase injection into the right basal ganglia of the brain for intervention. The efficacy of Gen in treating IA was evaluated through pathological testing and transcriptome sequencing analysis of Willis ring vascular tissue. The primary mechanism of action was linked to the expression of GSK3β in Th17 cells. The percentage of splenic Th17 cell differentiation in IA mice was significantly inhibited by Gen. GSK3β/STAT3, and other pathway protein expression levels were also significantly inhibited by Gen. Additionally, TNF-α and IL-23 cytokine contents were significantly downregulated after Gen treatment. These results indicated that Gen significantly inhibited the percentage of Th17 cell differentiation, an effect that was reversed upon overexpression of the GSK3B gene. Furthermore, Gen-treated, Th17 differentiation-inducing cell-conditioned medium significantly up-regulated the expression of tight junction proteins ZO-1, Occludin, and Claudin-5 in murine aortic endothelial cells. Administering the GSK3β inhibitor Tideglusib to IA mice alleviated the severity of IA disease pathology and up-regulated aortic tight junction protein expression. In conclusion, Gen inhibits Th17 cell differentiation through GSK3β, which reduces endothelial cell injury and up-regulates tight junction protein expression.

Glycyrrhiza Glabra Extract Modulates Type 1 T Helper (TH1) and Regulatory T Cell-Related Immune Responses in an Animal Model of Breast Cancer.

It is well-known that TH1 and Treg cells exert anti- and pro-tumorigenic activity, respectively. Thus, TH1 cell suppression together with Treg cell hyperactivation contribute to tumor development. Glycyrrhiza glabra (G. glabra) has various immunomodulatory and anti-tumorigenic properties. To explore the impacts of G. glabra extract on different parameters related to TH1 and Treg cells using a breast cancer (BC) model. Four groups of Balb/C mice bearing 4T1 cell-induced BC were treated intraperitoneally with either saline or G. glabra extract at dosages of 50, 100 and 150 mg/kg (G. glabra-50, G. glabra-100, and G. glabra-150, respectively). After sacrificing animals on day 26, the frequency of splenic TH1 and Treg cells, the levels of serum IFN-γ, TGF-β, and IL-12, and intra-tumoral expressions of granzyme-B, T-bet, and FOXP3 were assessed. Compared to untreated tumor control (UTC) group, treatment with G. glabra-50, G. glabra-100, or G. glabra-150 increased the survival rate, percentage of TH1 cells, and T-bet expression. Conversely, they reduced the percentage of Treg cells, and serum TGF-β levels. In comparison to the UTC group, treatment with G. glabra-50 and G. glabra-150 increased the serum IL-12 levels. Treatment with G. glabra-100 and G. glabra-150 boosted granzyme-B expression. Treatment with G. glabra-150 elevated IFN-γ levels, while treatment with G. glabra-50 decreased the FOXP3 expression. IL-12 levels were higher in mice treated with G. glabra-150 compared to those treated with G. glabra-100. Treatment of mice with BC using G. glabra extract improved survival rate, reduced tumor growth, and modulated T cell-mediated immune responses.

Suppression of Inflammation-induced Abdominal Aortic Aneurysm Formation by Induction of Elastin Tolerance

Abdominal aortic aneurysm (AAA) is a vascular disease process whereby the aorta expands to apoint where rupture may occur. This serious condition is diagnosed in approximately 200,000 people in the United States per year and accounts for over 15,000 deaths annually. The only medical intervention proven to reduce the risk of AAA rupture is surgical repair; however, such repair is associated with high risk of death, reduced quality of life, and high expense. AAA is caused by the weakening of the artery wall due to inflammation-induced destruction of its structural components. Our clinical data shows increased levels of circulating elastin degradation products in patients with AAA, especially smokers, compared to risk factor matched controls. This observation led us to hypothesize that an immune reaction to elastin fragments initiates the inflammatory cascade in the aorta that leads to AAA formation. To test this hypothesis, C57BL/6 mice were injected with poly(lactide-co-glycolide) nanoparticle-encapsulated IL-10 to induce immune tolerance or nanoparticle-encapsulated control ovalbumin. Injection of elastin fragments was performed 7 days later to induce an immune response. AAA of the infrarenal aorta was induced by topical application of elastase during laparotomy procedure 14 days after nanoparticle injection. Aorta diameter was measured 16 days post-operatively with Microfil. Immunologic changes were evaluated by cytokine analysis, Tr1/Th17 cell ratio inperipheral blood, and splenic Th17 and Tr1 response to elastin. Based on prior work, we expect that induction of elastin tolerance using poly(lactide-co-glycolide) nanoparticle-encapsulated IL-10 will suppress abdominal aortic aneurysm expansion and promote an anti-inflammatoryenvironment characterized by increased Tr1/Th17 cell ratio, increased levels of anti-inflammatory cytokines, and decreased pro-inflammatory cytokine expression.

Obesity and diet independently affect maternal immunity, maternal gut microbiota and pregnancy outcome in mice.

Maternal obesity poses risks for both mother and offspring during pregnancy, with underlying mechanisms remaining largely unexplored. Obesity is associated with microbial gut dysbiosis and low-grade inflammation, and also the diet has a major impact on these parameters. This study aimed to investigate how maternal obesity and diet contribute to changes in immune responses, exploring potential associations with gut microbiota dysbiosis and adverse pregnancy outcomes in mice. Before mating, C57BL/6 mice were assigned to either a high-fat-diet (HFD) or low-fat-diet (LFD) to obtain obese (n=17) and lean (n=10) mice. To distinguish between the effects of obesity and diet, 7 obese mice were switched from the HFD to the LFD from day 7 until day 18 of pregnancy ("switch group"), which was the endpoint of the study. T helper (Th) cell subsets were studied in the spleen, mesenteric lymph nodes (MLN) and Peyer's patches (PP), while monocyte subsets and activation status were determined in maternal blood (flow cytometry). Feces were collected before and during pregnancy (day 7,14,18) for microbiota analysis (16S rRNA sequencing). Pregnancy outcome included determination of fetal and placental weight. Obesity increased splenic Th1 and regulatory T cells, MLN Th1 and PP Th17 cells and enhanced IFN-γ and IL-17A production by splenic Th cells upon ex vivo stimulation. Switching diet decreased splenic and PP Th2 cells and classical monocytes, increased intermediate monocytes and activation of intermediate/nonclassical monocytes. Obesity and diet independently induced changes in the gut microbiota. Various bacterial genera were increased or decreased by obesity or the diet switch. These changes correlated with the immunological changes. Fetal weight was lower in the obese than the lean group, while placental weight was lower in the switch than the obese group. This study demonstrates that obesity and diet independently impact peripheral and intestinal immune responses at the end of pregnancy. Simultaneously, both factors affect specific bacterial gut genera and lead to reduced fetal or placental weight. Our data suggest that switching diet during pregnancy to improve maternal health is not advisable and it supports pre/probiotic treatment of maternal obesity-induced gut dysbiosis to improve maternal immune responses and pregnancy outcome.

Lupus mice derived mesenchymal stromal cells: Beneficial or detrimental on SLE disease outcome

BackgroundSystemic Lupus Erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies against nuclear genes, deposition of immune complexes, and autoimmune T cells, through which, tissue damage would ultimately occur. Furthermore, loss of immune tolerance and imbalance of Th1/Th2 cells in addition to Th17/Treg are contributed to the pathogenesis of SLE. Mesenchymal stromal cells (MSCs) infusion is a potential therapy for SLE disease. Despite a majority of SLE patients achieving clinical remission after allogeneic MSC infusion from healthy individuals, SLE patients have less benefited from autologous MSC infusion, justifying the probable compromised function of SLE patients-derived MSCs. In this study, we aim to further investigate the potential immunoregulatory mechanisms in which mesenchymal stromal cells derived from pristane-induced lupus mice, following injection into healthy and lupus mice, exert their possible effects on the lupus process. Method40 female Balb/c mice aged 3 weeks were purchased and randomly divided into six groups. First, lupus disease was induced into the lupus groups by intraperitoneal injection of pristane and then the mice were surveyed for 6 months. The body weight, anti-dsDNA autoantibody levels, serum creatinine, and Blood Urea Nitrogen (BUN) levels were measured in two-month intervals. After 6 months, the group of lupus mice was sacrificed, and lupus MSCs were isolated. Two months later, cultured lupus MSCs were intravenously injected into two groups of healthy and lupus mice. After two months, the mice were euthanized and the kidneys of each group were examined histologically by hematoxylin & eosin (H&E) staining and the immunofluorescence method was also performed to evaluate IgG and C3 deposition. The frequency of splenic Th1, Th2, Th17, and Treg cells was measured by flow cytometry. Moreover, the cytokine levels of IFN-γ, IL-4, IL-17, and TGF-β in sera were measured by ELISA method. ResultsOur results showed that the induction of lupus disease by pristane in Balb/c mice caused the formation of lipogranuloma, increased levels of anti-dsDNA autoantibodies, and impaired renal function in all pristane-induced lupus groups. In addition, the injection of lupus mesenchymal stromal cells (L-MSC) into healthy and lupus mice led to a further rise in anti-dsDNA serum levels, IgG and C3 deposition, and further dysfunction of mice renal tissue. Also, the flow cytometry results implicated that compared to the control groups, splenic Th1, Th2, and Th17 inflammatory cell subtypes and their secreted cytokines (IFN-γ, IL-4, and IL-17) in the sera of healthy and lupus mice were increased after the intake of L-MSC. Additionally, the splenic Treg cells were also significantly increased in the lupus mice receiving L-MSC. However, a decrease in serum levels of TGF-β cytokine was observed in healthy and lupus mice following L-MSC injection. In contrast, the lupus mice receiving healthy mesenchymal stem cells (H-MSC) manifested opposite results. ConclusionIn a nutshell, our results suggest that although allogeneic MSCs are encouraging candidates for SLE treatment, syngeneic MSCs may not be eligible for treating SLE patients due to their defects in regulating the immune system in addition to their capability in promoting inflammation which would consequently worsen the SLE disease status.

WLJP-025p, a homogeneous Lonicera japonica polysaccharide, attenuates atopic dermatitis by regulating the MAPK/NFκB/AP-1 axis via Act1

Atopic dermatitis (AD) is usually treated with steroids, but long-term use is not an effective cure because side effects and disease aggravation. Therefore, more effective and safer treatments are needed. Using dexamethasone as the positive control, the mechanism of action of water-extracted homogeneous honeysuckle Lonicera japonica polysaccharide (WLJP-025p) to alleviate AD was studied. Mice were administered 2,4-dinitrochlorobenzene in their bare back and right ear to mimic an AD model.The efficacy of WLJP-025p in AD was assessed by measuring right ear thickness and skin lesion scores, pathological observation (haematoxylin–eosin and toluidine blue staining), and serum IgE and IL-1β concentrations. The expression of relevant genes and proteins in the serum and back skin was detected using RT-qPCR, ELISA, western blotting, and immunofluorescence. Molecular docking and dynamic simulation of WLJP-025p and Act1 were performed. WLJP-025p considerably alleviated skin hyperplasia and pathological abnormalities in AD mice and inhibited the expression of Act1, Nucleus-P65, Nucleus-AP-1, and MAPK-related proteins in skin tissues. WLJP-025p formed a stable conformation with Act1, inhibited splenic Th17 differentiation, IL-17 release, and upregulated the expression of related skin barrier proteins. In conclusion, WLJP-025p affects the inflammation regulation via the MAPK/NFκB/AP-1 axis by binding to Act1, promotes the recovery of epithelial barrier function, and alleviates AD in mice.

The Effect of Antibiotics Treatment on the Maternal Immune Response and Gut Microbiome in Pregnant and Non-Pregnant Mice

The gut microbiota are involved in adaptations of the maternal immune response to pregnancy. We therefore hypothesized that inducing gut dysbiosis during pregnancy alters the maternal immune response. Thus, pregnant mice received antibiotics from day 9 to day 16 to disturb the maternal gut microbiome. Feces were collected before, during and after antibiotic treatment, and microbiota were measured using 16S RNA sequencing. Mice were sacrificed at day 18 of pregnancy and intestinal (Peyer’s patches (PP) and mesenteric lymph nodes (MLN)) and peripheral immune responses (blood and spleen) were measured using flow cytometry. Antibiotic treatment decreased fetal and placental weight. The bacterial count and the Shannon index were significantly decreased (Friedman, followed by Dunn’s test, p &lt; 0.05) and the bacterial genera abundance was significantly changed (Permanova, p &lt; 0.05) following antibiotics treatment as compared with before treatment. Splenic Th1 cells and activated blood monocytes were increased, while Th2, Th17 and FoxP3/RoRgT double-positive cells in the PP and MLNs were decreased in pregnant antibiotics-treated mice as compared with untreated pregnant mice. In addition, intestinal dendritic cell subsets were affected by antibiotics. Correlation of immune cells with bacterial genera showed various correlations between immune cells in the PP, MLN and peripheral circulation (blood and spleen). We conclude the disturbed gut microbiota after antibiotics treatment disturbed the maternal immune response. This disturbed maternal immune response may affect fetal and placental weight.

Vaccination with celecoxib-treated dendritic cells improved cellular immune responses in an animal breast cancer model

PurposeProstaglandin E2 (PGE2), a product of cyclooxygenase (COX) pathway of arachidonic acid, exerts inhibitory impacts on dendritic cell (DC) activity to repress anti-tumor immune responses. Therefore, targeting COX during DC vaccine generation may enhance DC-mediated antitumor responses. We aimed to investigate the impacts of DC vaccine treated with celecoxib (CXB), a selective COX2 inhibitor, on some T cell-related parameters. Materials and methodsBreast cancer (BC) was induced in BALB/c mice, and then they received DC vaccine treated with lipopolysaccharide (LPS-mDCs), LPS with a 5 ​μM dose of CXB (LPS/CXB5-mDCs) and LPS with a 10 ​μM dose of CXB (LPS/CXB10-mDCs). The frequency of splenic Th1 and Treg cells and amounts of IFN-γ, IL-12 and TGF-β production by splenocytes, as well as, the expression of Granzyme-B, T-bet and FOXP3 in tumors were determined using flow cytometry, ELISA, and real-time PCR, respectively. ResultsCompared with untreated tumor group (T-control), treatment with LPS/CXB5-mDCs and LPS/CXB10-mDCs decreased tumor growth (P ​= ​0.009 and P ​< ​0.0001), escalated survival rate (P ​= ​0.002), increased the frequency of splenic Th1 cells (P ​= ​0.0872, and P ​= ​0.0155), increased the IFN-γ (P ​= ​0.0003 and P ​= ​0.0061) and IL-12 (P ​= ​0.001 and P ​= ​0.0009) production by splenocytes, upregulated T-bet (P ​= ​0.062 and P ​< ​0.0001) and Granzyme-B (P ​= ​0.0448 and P ​= ​0.4485), whereas decreased the number of Treg cells (P ​= ​0.0014, and P ​= ​0.0219), reduced the amounts of TGF-β production by splenocytes (P ​= ​0.0535 and P ​= ​0.0169), and reduced the expression of FOXP3 (P ​= ​0.0006 and P ​= ​0.0057) in comparison with T-control group. ConclusionsOur findings show that LPS/CXB-treated DC vaccine potently modulated antitumor immune responses in a mouse BC model.

T follicular helper cells contribute to pathophysiology in a model of neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are inflammatory autoimmune disorders of the CNS. IgG autoantibodies targeting the aquaporin-4 water channel (AQP4-IgGs) are the pathogenic effector of NMOSD. Dysregulated T follicular helper (Tfh) cells have been implicated in loss of B cell tolerance in autoimmune diseases. The contribution of Tfh cells to disease activity and therapeutic potential of targeting these cells in NMOSD remain unclear. Here, we established an autoimmune model of NMOSD by immunizing mice against AQP4 via in vivo electroporation. After AQP4 immunization, mice displayed AQP4 autoantibodies in blood circulation, blood-brain barrier disruption, and IgG infiltration in spinal cord parenchyma. Moreover, AQP4 immunization induced motor impairments and NMOSD-like pathologies, including astrocytopathy, demyelination, axonal loss, and microglia activation. These were associated with increased splenic Tfh, Th1, and Th17 cells; memory B cells; and plasma cells. Aqp4-deficient mice did not display motor impairments and NMOSD-like pathologies after AQP4 immunization. Importantly, abrogating ICOS/ICOS-L signaling using anti-ICOS-L antibody depleted Tfh cells and suppressed the response of Th1 and Th17 cells, memory B cells, and plasma cells in AQP4-immunized mice. These findings were associated with ameliorated motor impairments and spinal cord pathologies. This study suggests a role of Tfh cells in the pathophysiology of NMOSD in a mouse model with AQP4 autoimmunity and provides an animal model for investigating the immunological mechanisms underlying AQP4 autoimmunity and developing therapeutic interventions targeting autoimmune reactions in NMOSD.

Panax notoginseng saponins (PNS) attenuate Th17 cell differentiation in CIA mice via inhibition of nuclear PKM2-mediated STAT3 phosphorylation

Context Rheumatoid arthritis (RA) is an autoimmune disease with aberrant Th17 cell differentiation. Panax notoginseng (Burk.) F. H. Chen (Araliaceae) saponins (PNS) have an anti-inflammatory effect and can suppress Th17 cell differentiation. Objective To investigate mechanisms of PNS on Th17 cell differentiation in RA, and the role of pyruvate kinase M2 (PKM2). Materials and methods Naive CD4+T cells were treated with IL-6, IL-23 and TGF-β to induce Th17 cell differentiation. Apart from the Control group, other cells were treated with PNS (5, 10, 20 μg/mL). After the treatment, Th17 cell differentiation, PKM2 expression, and STAT3 phosphorylation were measured via flow cytometry, western blots, or immunofluorescence. PKM2-specific allosteric activator (Tepp-46, 50, 100, 150 μM) and inhibitor (SAICAR, 2, 4, 8 μM) were used to verify the mechanisms. A CIA mouse model was established and divided into control, model, and PNS (100 mg/kg) groups to assess an anti-arthritis effect, Th17 cell differentiation, and PKM2/STAT3 expression. Results PKM2 expression, dimerization, and nuclear accumulation were upregulated upon Th17 cell differentiation. PNS inhibited the Th17 cells, RORγt expression, IL-17A levels, PKM2 dimerization, and nuclear accumulation and Y705-STAT3 phosphorylation in Th17 cells. Using Tepp-46 (100 μM) and SAICAR (4 μM), we demonstrated that PNS (10 μg/mL) inhibited STAT3 phosphorylation and Th17 cell differentiation by suppressing nuclear PKM2 accumulation. In CIA mice, PNS attenuated CIA symptoms, reduced the number of splenic Th17 cells and nuclear PKM2/STAT3 signaling. Discussion and conclusions PNS inhibited Th17 cell differentiation through the inhibition of nuclear PKM2-mediated STAT3 phosphorylation. PNS may be useful for treating RA.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

A polysaccharide from Pleurotus citrinopileatus mycelia enhances the immune response in cyclophosphamide-induced immunosuppressed mice via p62/Keap1/Nrf2 signal transduction pathway

The manuscript aimed to study the immunoregulatory activity and the mechanism of the polysaccharide (CMP) from Pleurotus citrinopileatus mycelia. The mice were divided into normal group, model group, different dosage of CMP (50, 100 and 200 mg/kg, respectively) groups and levamisole hydrochloride treated group. The results showed that, compared with the model group, CMP could significantly improve the auricle swelling rate, half hemolysis value and phagocytic index in mice. The indices of immune organs were raised, and tissue damage of spleen was relieved. Splenic Th1 cells were decreased, while Th2 cells were increased, furthermore the proliferation of splenic lymphocytes and the cytotoxicity of NK cells were increased. The levels of interleukin-12 (IL-12), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in spleen were decreased, while interleukin-4 (IL-4) and interleukin-10 (IL-10) were increased. In serum and spleen, the levels of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities were increased, while the level of malondialdehyde (MDA) was decreased. And the levels of Immunoglobulin were also increased. Western blot showed that CMP had immunoregulatory activity by activating Nrf2, Keap1, p62, HO-1, and NQO1 in the p62/Keap1/Nrf2 signaling pathway. The study proved that CMP could be used as a biological Immune regulating agent.

Echinocystic Acid Ameliorates Arthritis in SKG Mice by Suppressing Th17 Cell Differentiation and Human Rheumatoid Arthritis Fibroblast-Like Synoviocytes Inflammation

Echinocystic acid (EA), a pentacyclic triterpene, exhibits anti-inflammatory, antioxidant, and analgesic activities to counteract pathological effects in various diseases. Here, we aimed to determine the immunomodulatory effect of EA on zymosan-induced arthritis in SKG mice and how it would influence Th17 differentiation and human rheumatoid arthritis fibroblast-like synoviocytes inflammation. Our results showed that EA (10 and 25 mg/kg) attenuated arthritis symptoms, including high arthritis scores, infiltrating inflammatory cells, synovial hyperplasia, bone erosion, and the high levels of proinflammatory cytokines, such as TNF-α, interleukin (IL)-6, and IL-1β in paw tissues, and reduced the number of splenic Th17 cells. Mechanistically, we found that in vitro treatment of EA inhibited both IL-6- and transforming growth factor-β (TGF-β)-induced Th17 cell differentiation by suppressing the phosphorylation of signal transducers and transcriptional activators, especially STAT3. In line with the in vivo result, EA significantly reduced the protein and mRNA expression of IL-6 and IL-1β in human RA-FLA cells, MH7A cells. Furthermore, the production of both cytokines was confirmed with the downregulation of mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) signaling pathways under the stimulation of TNF-α. In conclusion, these findings revealed that EA was capable of amelioration of arthritic disorders in SKG mice through inhibiting Th17 cell differentiation and synovial fibroblast inflammation, supporting that EA is a promising therapeutic candidate for treating RA patients.

A novel GRK2 inhibitor alleviates experimental arthritis through restraining Th17 cell differentiation

T helper type 17 (Th17) cell which is induced by interleukine-6 (IL-6)-signal transducers and activators of transcription 3 (STAT3) signaling is a central pro-inflammatory T cell subtype in rheumatoid arthritis (RA) and could be significantly reduced by paeoniflorin-6′-O-benzene sulfonate (CP-25) treatment with unclear mechanisms. This study was aimed to found out the mechanism of CP-25 in hampering Th17 cells differentiation in arthritic animals thus explore more therapeutic targets for RA. In mice with collagen-induced arthritis (CIA), both circulating and splenic Th17 subsets were expanded with increased STAT3 phosphorylation and decreased Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1)-β-arrestin2 (arrb2)-STAT3 interaction in CD4+ helper T (Th) cells. Either CP-25 or paroxetine (PAR), an established G protein coupled receptor kinase 2 (GRK2) inhibitor treatment effectively relieved the joints inflammation of CIA mice with substantially reduced Th17 cell population through inhibiting STAT3 and restoring the SHP1-arrb2-STAT3 complex. Knockout of arrb2 exacerbated the clinical manifestations of collagen antibody-induced arthritis with upregulated Th17 cells. In vitro studies revealed that depletion of arrb2 or inhibition of SHP1 promoted Th17 cell differentiation. Moreover, stimulation of adenosine A3 receptor (A3AR) simultaneously promoted Th17 cell differentiation via accelerating abbr2-A3AR binding, which could be prevented through inhibiting GRK2 phosphorylation by CP-25 or PAR, or genetically reducing GRK2. This work has demonstrated that CP-25 or PAR treatment recovers the SHP1-arrb2-STAT3 complex which prevents STAT3 activation in Th cells through reducing arrb2 recruitment to A3AR by inhibiting GRK2 phosphorylation, leading to the reduction in Th17 cell differentiation and arthritis attenuation.

Oral Pathobionts Promote MS-like Symptoms in Mice

Dysbiotic oral microbiota has been associated with multiple sclerosis. However, the role and mechanism of oral microbiota in the development of multiple sclerosis are still elusive. Here, we demonstrated that ligature-induced periodontitis (LIP) aggravated experimental autoimmune encephalomyelitis (EAE) in mice, and this was likely dependent on the expansion of T helper 17 (Th17) cells. LIP increased the splenic richness of Enterobacter sp., which was able to induce the expansion of splenic Th17 cells and aggravate EAE in mice. LIP also led to enrichment of Erysipelotrichaceae sp. in the gut and increased Th17 cells in the large intestinal lamina propria of EAE mice. Fecal microbiota transplantation from EAE mice with LIP also promoted EAE symptoms. In conclusion, periodontitis exacerbates EAE, likely through ectopic colonization of oral pathobionts and expansion of Th17 cells.

Protective effect of Schistosoma japonicum eggs on TNBS-induced colitis is associated with regulating Treg/Th17 balance and reprogramming glycolipid metabolism in mice.

Inflammatory bowel diseases (IBDs) have been classified as modern refractory diseases. However, safe, well-tolerated, and effective treatments for IBDs are still lacking. Therefore, there is an urgent need to develop novel therapeutic targets with fewer undesirable adverse reactions. A growing body of research has shown that infection with live helminths or exposure to defined helminth-derived components can downregulate pathogenic inflammation due to their immunoregulatory ability. Here we were to explore the protective role of Schistosoma japonicum eggs on murine experimental colitis caused by trinitrobenzene sulfonic acid (TNBS) and the underlying mechanism. Frequencies of splenic Treg and Th17 cells were detected by flow cytometry. Protein and mRNA expressions of Foxp3 and RORγt were investigated by Western Blot and quantitative real-time polymerase chain reaction (qPCR), respectively. Concentrations of transforming growth factor-beta1 (TGF-β1), interleukin-10 (IL-10) and IL-17A were assessed with ELISA. Expression levels of genes related to glycolipid metabolism were measured with qPCR. The results showed that pre-exposure to S. japonicum eggs contributed to the relief of colitis in the TNBS model, evidenced by improved body weight loss, reversing spleen enlargement and colon shortening, and decreased histology scores. Compared with the TNBS group, the TNBS+Egg group had increased Treg immune response, accompanied by decreased Th17 immune response, leading to the reconstruction of Treg/Th17 balance. In addition, a ratio of Treg/Th17 was correlated negatively with the histological scores in the experiment groups. Furthermore, the regulation of Treg/Th17 balance by S. japonicum eggs was associated with inhibiting the glycolysis pathway and lipogenesis, along with promoting fatty acid oxidation in the TNBS+Egg group. These data indicate that S. japonicum eggs have a protective effect against TNBS-induced colitis, which is related to restoring Treg/Th17 balance and regulating glucose and lipid metabolism.

Water-extracted Lonicera japonica polysaccharide attenuates allergic rhinitis by regulating NLRP3-IL-17 signaling axis

In this study, the immunomodulatory effects and mechanism of action of a novel water-extracted Lonicera japonica polysaccharide (WLJP) on allergic rhinitis (AR) was investigated. For the efficacy of WLJP, behavioral symptoms (rubbing and sneezing), serum inflammatory factors, pathological damage, splenic T cell differentiation, gut microbiota imbalance, and protein analysis of the nasal mucosa and colon were assessed. WLJP and the NLRP3 inhibitor, CY-09, were co-evaluated in the AR model established using LPS + IFN-γ-induced THP-1 cells. The WLJP group showed decreased serum inflammatory factors, eosinophils, goblet cells, NLRP3 inflammasomes, splenic Th17 cell differentiation, and expression of IL-17, p-p65, and gut NLRP3 in the nasal mucosa while maintaining gut microbiota balance, repairing the mechanical barrier, and significantly improving AR behavioral symptoms. In vitro interaction analysis showed a significant interaction between CY-09 and WLJP. In conclusion, WLJP improves AR by repairing the gut barrier and inhibiting NLRP3 inflammasome-driven inflammation and the Th17 immune response.

Oral dextran sulfate sodium administration induces peripheral spondyloarthritis features in SKG mice accompanied by intestinal bacterial translocation and systemic Th1 and Th17 cell activation

BackgroundSpondyloarthritis (SpA) is an autoimmune and autoinflammatory musculoskeletal disease characterised by systemic enthesitis. Recent research has focused on subclinical inflammatory bowel disease (IBD) in SpA pathogenesis. SKG mice, harbouring the Zap70 W163C mutation, increase autoreactive Th17 cells intrinsically, and in a conventional environment, they exhibit spontaneous arthritis with fungal factors. Under SPF conditions, they show SpA features, including enteritis, after peritoneal injection of β-1,3-glucan. This study aimed to clarify whether oral dextran sulfate sodium (DSS) administration, utilised in IBD model mice, can provoke SpA features in SKG mice under SPF conditions, focusing on the relationship between gut microorganisms and SpA pathogenesis.MethodsBALB/c and SKG mice were administered oral DSS, and their body weights, arthritis, and enthesitis scores were recorded. In another cohort, antibiotics (meropenem and vancomycin) or an anti-fungal agent (amphotericin B) was administered orally before DSS administration. The splenic Th1 and Th17 cell populations were examined before and after DSS administration using flow cytometry. Furthermore, the amount of circulating bacterial DNA in whole blood was measured by absolute quantitative polymerase chain reaction (qPCR), and the number and characteristics of bacterial species corresponding to these circulating DNA were analysed by next-generation sequencing (NGS).ResultsAnkle enthesitis as a peripheral SpA feature was elicited in half of DSS-administered SKG mice, and none of the BALB/c mice. Pre-administration of antibiotics suppressed enthesitis, whilst an anti-fungal agent could not. Th1 and Th17 cell levels in the spleen increased after DSS administration, and this was suppressed by pre-administration of antibiotics. SKG mice have a larger amount of bacterial DNA in whole blood than BALB/c mice before and 1 day after the initiation of DSS administration. The number of bacterial species in whole blood increased after DSS administration in BALB/c and SKG mice. Some genera and species significantly specific to the DSS-treated SKG mouse group were also detected.ConclusionOral DSS administration alone elicited peripheral enthesitis in SKG mice with bacterial translocation accompanied by increased splenic Th1 and Th17 cell levels. Pre-administration of antibiotics ameliorated these DSS-induced SpA features. These findings suggest that intestinal bacterial leakage plays a pivotal role in SpA pathogenesis.

Cimetidine and Ibuprofen Modulate T Cell Responses in a Mouse Model of Breast Cancer.

Cimetidine and ibuprofen exhibit immunomodulatory effects as an antagonist of histamine H2 receptor, and a cyclooxygenase inhibitor, respectively. Here, the effects of cimetidine and ibuprofen on some effector T cell-related parameters were investigated using a breast cancer (BC) model. BC was established in Balb/c mice using the 4T1 cell line. On day 10 after tumor induction, the BC-bearing mice were classified into four groups and treated with PBS, cimetidine (20 mg/kg), ibuprofen (20 mg/kg) or a combination of “cimetidine + ibuprofen” via intraperitoneal injection (daily from days 11 to 30). The mice were sacrificed on day 31 and the frequency of splenic Th1 and Treg cells, plasma IFN-γ and TGF-β levels, and intra-tumoral T-bet, GATA3, FOXP3 and RORγt expressions were detected using flowcytometry, ELISA and real-time-PCR, respectively. In untreated cancerous mice, the percentage of splenic Th1 cells and plasma IFN-γ levels were lower (P<0.003 and P<0.01, respectively), whereas the percentage of splenic Treg cells and plasma TGF-β levels were higher than in healthy mice (P<0.04 and P<0.005, respectively). Treatment of BC-bearing mice with cimetidine, ibuprofen or both drugs promoted the frequency of Th1 cells (P<0.05, P<0.007 and P<0.005, respectively) as well as IFN-γ levels (P<0.004, P<0.0001 and P<0.03, respectively), while reduced the frequencies of Treg cells (P<0.02, P<0.03 and P<0.01, respectively), TGF-β levels (P<0.006, P<0.02 and P<0.002, respectively), intra-tumoral expression of FOXP3 (P<0.006, P<0.005 and P<0.005, respectively), and intra-tumoral expression of RORγt (P<0.04, P<0.03 and P<0.05, respectively) compared with untreated BC mice. The “cimetidine + ibuprofen”-treated mice displayed greater T-bet expression than the un-treated mice (P<0.006). Cimetidine and/or ibuprofen-treated BC-bearing mice exhibited reduced intra-tumoral expression of GATA3 compared with the untreated BC mice, but the differences were not significant. Cimetidine and ibuprofen correct some effector T cell-related parameters in cancerous mice. Immunotherapeutic potentials cimetidine and ibuprofen in cancers need investigations.