Baicalein alleviates cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway.

BACKGROUND: The experimental autoimmune myocarditis (EAM) model is valuable for investigating myocarditis pathogenesis. M1-type macrophages and CD4+T cells exert key pathogenic effects on EAM initiation and progression. Baicalein (5,6,7-trihydroxyflavone, C15H10O5, BAI), which is derived from the Scutellaria baicalensis root, is a primary bioactive compound with potent anti-inflammatory and antioxidant properties. BAI exerts good therapeutic effects against various autoimmune diseases; however, its effect in EAM has not been thoroughly researched. PURPOSE: This study aimed to explore the possible inhibitory effect of BAI on M1 macrophage polarisation and CD4+T cell differentiation into Th1 cells via modulation of the JAK-STAT1/4 signalling pathway, which reduces the secretion of pro-inflammatory factors, namely, TNF-α and IFN-γ, and consequently inhibits TNF-α- and IFN-γ-triggered apoptosis in cardiomyocytes of the EAM model mice. STUDY DESIGN AND METHODS: Flow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (q-PCR), and western blotting were performed to determine whether BAI alleviated M1/Th1-secreted TNF-α- and IFN-γ-induced myocyte death in the EAM model mice through the inhibition of the JAK-STAT1/4 signalling pathway. RESULTS: These results indicate that BAI intervention in mice resulted in mild inflammatory infiltrates. BAI inhibited JAK-STAT1 signalling in macrophages both in vivo and in vitro, which attenuated macrophage polarisation to the M1 type and reduced TNF-α secretion. Additionally, BAI significantly inhibited the differentiation of CD4+T cells to Th1 cells and IFN-γ secretion both in vivo and in vitro by modulating the JAK-STAT1/4 signalling pathway. This ultimately led to decreased TNF-α and IFN-γ levels in cardiac tissues and reduced myocardial cell apoptosis. CONCLUSION: This study demonstrates that BAI alleviates M1/Th1-secreted TNF-α- and IFN-γ-induced cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway.

Anti-allergic effect of vitamin C through inhibiting degranulation and regulating TH1/TH2 cell polarization.

BACKGROUND: Food allergy has become a global public health problem. This study aimed to explore the possible anti-allergic effect of vitamin C (VC). A rat basophilic leukemia (RBL)-2H3 cell degranulation model was used to assess the effect of VC on degranulation in vitro, and an ovalbumin (OVA)-induced BALB/c mouse allergy model was used to assess the anti-allergy effect of VC in vivo. RESULTS: In vitro, VC significantly attenuated the release of ß-hexosaminidase, tryptase and histamine, and also reduced cytokine production (interleukins 4 and 6, tumor necrosis factor α) significantly (P < 0.05), with the inhibitory effect demonstrating a positive correlation with VC dose. In vivo, compared with the OVA group, the levels of serum immunoglobulins E and G1 of the VC low-dose (VCL) group (50 mg kg-1) and high-dose (VCH) group (200 mg·kg-1) were significantly reduced (P < 0.05). Furthermore, the plasma histamine level was also significantly decreased (P < 0.05). Moreover, TH2 cell polarization in mice of the VCL and VCH groups was significantly inhibited (P < 0.05), promoting the TH1/TH2 cell polarization balance. Additionally, VC treatment enhanced the expression of CD80 (P < 0.05) in spleen and small intestine tissues, while significantly inhibiting the expression of CD86 (P < 0.05); notably, high-dose VC treatment was more effective. CONCLUSION: VC exerted an anti-allergic effect through inhibiting degranulation and regulating TH1/TH2 cell polarization balance. © 2024 Society of Chemical Industry.

Repeated immunization with ATRA-containing liposomal adjuvant transdifferentiates Th17 cells to a Tr1-like phenotype.

In many autoimmune diseases, autoantigen-specific Th17 cells play a pivotal role in disease pathogenesis. Th17 cells can transdifferentiate into other T cell subsets in inflammatory conditions, however, there have been no attempts to target Th17 cell plasticity using vaccines. We investigated if autoantigen-specific Th17 cells could be specifically targeted using a therapeutic vaccine approach, where antigen was formulated in all-trans retinoic acid (ATRA)-containing liposomes, permitting co-delivery of antigen and ATRA to the same target cell. Whilst ATRA was previously found to broadly reduce Th17 responses, we found that antigen formulated in ATRA-containing cationic liposomes only inhibited Th17 cells in an antigen-specific manner and not when combined with an irrelevant antigen. Furthermore, this approach shifted existing Th17 cells away from IL-17A expression and transcriptomic analysis of sorted Th17 lineage cells from IL-17 fate reporter mice revealed a shift of antigen-specific Th17 cells to exTh17 cells, expressing functional markers associated with T cell regulation and tolerance. In the experimental autoimmune encephalomyelitis (EAE) mouse model of MS, vaccination with myelin-specific (MOG) antigen in ATRA-containing liposomes reduced Th17 responses and alleviated disease. This highlights the potential of therapeutic vaccination for changing the phenotype of existing Th17 cells in the context of immune mediated diseases.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

The Characterization of the Th1/Th2 Ratio in Moderate-Severe Alzheimer's Disease Patients and Its Response to an Aloe Polymannose-Based Dietary Supplement.

BACKGROUND: Alzheimer's disease (AD) is a leading killer of Americans, imparting a tremendous societal toll. Relationships between immune function and inflammation with cognition are well-established in AD, but the Th1/Th2 ratio of immune function is unknown. Describing the Th1/Th2 ratio and its relationship with cognition may shed light on the disease's clinical context. How the Th1/Th2 ratio responds to dietary supplementation is another unknown question in this population. OBJECTIVE: The objectives of the study were to: 1) characterize the Th1/Th2 ratio according to IL-2/IL-10, IFN-γ/IL-10, IL-2/IL-4, IFN-γ/IL-4, IL-2/TNF-α, and IFN-γ/TNF-α in subjects with moderate-to-severe AD and in comparison to healthy adults; 2) investigate the effect of an aloe polymannose multinutrient complex (APMC) dietary supplement on the Th1/Th2 ratios over 12 months; and 3) compare the changes in the Th1/Th2 ratios with the changes in cognition from baseline to 12 months. METHODS: Subjects consumed 2.5 g of the APMC four times per day for 12 months, and they were assessed on cognition and cytokines at baseline and 12 months. RESULTS: The Th1/Th2 ratios in AD patients were significantly higher than the healthy controls, and five of the six ratios decreased from baseline to 12 months follow-up (other than IL-2/TNF-α). Several significant relationships were noted between the changes in Th1/Th2 ratios with cognitive assessments. CONCLUSIONS: Our results showed an overall rebalancing of the Th1/Th2 ratio in response to APMC, these changes were related to improved cognition in subjects with moderate-to-severe AD, and the APMC supplement was safely tolerated.

Association of Nonpuerperal Mastitis with Cytokines Related to Helper T Cells TH1/TH2 and TH17/Treg.

Objective: To investigate the association of nonpuerperal mastitis with cytokines related to the helper T cells TH1/TH2 and TH17/Treg and associated immune balance. Methods: From 2016 to 2021, we included 40 patients with non-puerperal mastitis who underwent surgery at China-Japan Friendship Hospital and compared them with 40 control patients with benign non-infectious breast disease. Hematoxylin-eosin staining detects inflammatory infiltrates of breast tissue. The expression of interferon γ and interleukin 4 in breast tissue was detected by immunofluorescence imaging, and the relative protein expression of TH1/TH2 and TH17/Treg cell-associated cytokines in CD4+ T cells was detected by western blotting. CD4+ T cells were isolated by fluorescence-activated cell sorting for detection of the relative protein expression of interferon γ and interleukin 4 in CD4+ T cells. Results: Hematoxylin-eosin staining showed that the nonpuerperal mastitis group had significantly greater inflammatory infiltration than the control group. Immunofluorescence images showed the relative fluorescence intensity of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative fluorescence intensity of interleukin 4 did not significantly differ between the 2 groups (P = .0686). Western blotting revealed that the relative protein expression of interferon γ, interleukin 2, and interleukin 17 was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 (P = .0512), interleukin 10 (P = .3088), and transforming growth factor ß (P = .0653) did not significantly differ between the 2 groups. Flow cytometry of isolated CD4+ T cells showed the relative protein expression of interferon γ was significantly higher in the nonpuerperal mastitis group than in the control group (P < .001), but the relative protein expression of interleukin 4 did not significantly differ between the 2 groups (P = .0680). Conclusion: The expression of the TH1 cytokines interferon γ and interleukin 2 and the TH17 cytokine interleukin 17 was significantly higher in patients with nonpuerperal mastitis, while the TH2 cytokine interleukin 4 and the Treg cytokines interleukin 10 and transforming growth factor ß were expressed at lower levels. This study provides new research ideas for the treatment of mastitis.

Total Flavonoids of Astragalus Inhibit Activated CD4[Formula: see text] T Cells and Regulate Differentiation of Th17/Th1/Treg Cells in Experimental Autoimmune Encephalomyelitis Mice by JAK/STAT and NF[Formula: see text]B Signaling Pathways.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by CD4[Formula: see text] T cell-mediated immune cell infiltration and demyelination in the central nervous system (CNS). The subtypes of CD4[Formula: see text] T cells are T helper cells 1 (Th1), Th2, Th17, and regulatory T cells (Treg), while three other types of cells besides Th2 play a key role in MS and its classic animal model, experimental autoimmune encephalomyelitis (EAE). Tregs are responsible for immunosuppression, while pathogenic Th1 and Th17 cells cause autoimmune-associated demyelination. Therefore, suppressing Th1 and Th17 cell differentiation and increasing the percentage of Treg cells may contribute to the treatment of EAE/MS. Astragali Radix (AR) is a representative medicine with immunoregulatory, anti-inflammatory, antitumor, and neuroprotective effects.The active ingredients in AR include astragalus flavones, polysaccharides, and saponins. In this study, it was found that the total flavonoids of Astragus (TFA) could effectively treat EAE in mice by ameliorating EAE motor disorders, reducing inflammatory damage and demyelination, inhibiting the proportion of Th17 and Th1 cells, and promoting Tregs differentiation by regulating the JAK/STAT and NF[Formula: see text]B signaling pathways. This novel finding may increase the possibility of using AR or TFA as a drug with immunomodulatory effects for the treatment of autoimmune diseases.

Magnolol as STAT3 inhibitor for treating multiple sclerosis by restricting Th17 cells.

OBJECTIVE: Multiple sclerosis (MS) is an immune disease in the central nervous system (CNS) associated with Th17 cells. Moreover, STAT3 initiates Th17 cell differentiation and IL-17A expression through facilitating RORγt in MS. Here, we reported that magnolol, isolated from Magnolia officinalis Rehd. Et Wils, was regarded as a candidate for MS treatment verified by both in vitro and in vivo studies. METHODS: In vivo, experimental autoimmune encephalomyelitis (EAE) model in mice was employed to evaluate the alleviation of magnolol on myeloencephalitis. In vitro, FACS assay was employed to evaluate the effect of magnolol on Th17 and Treg cell differentiation and IL-17A expression; network pharmacology-based study was applied to probe the involved mechanisms; western blotting, immunocytochemistry, and luciferase reporter assay was used to further confirm the regulation of magnolol on JAK/STATs signaling pathway; surface plasmon resonance (SPR) assay and molecular docking were applied to manifest affinity with STAT3 and binding sites; overexpression of STAT3 was employed to verify whether magnolol attenuates IL-17A through STAT3 signaling pathway. RESULTS: In vivo, magnolol alleviated loss of body weight and severity of EAE mice; magnolol improved lesions in spinal cords and attenuated CD45 infiltration, and serum cytokines levels; correspondingly, magnolol focused on inhibiting Th17 differentiation and IL-17A expression in splenocyte of EAE mice; moreover, magnolol selectively inhibited p-STAT3(Y705) and p-STAT4(Y693) of both CD4+ and CD8+ T cells in splenocyte of EAE mice. In vitro, magnolol selectively inhibited Th17 differentiation and IL-17A expression without impact on Treg cells; network pharmacology-based study revealed that magnolol perhaps diminished Th17 cell differentiation through regulating STAT family members; western blotting further confirmed that magnolol inhibited p-JAK2(Y1007) and selectively antagonized p-STAT3(Y705) and slightly decreased p-STAT4(Y693); magnolol antagonized both STAT3 nucleus location and transcription activity; magnolol had a high affinity with STAT3 and the specific binding site perhaps to be at SH2 domain; overexpression of STAT3 resulted in failed inhibition of magnolol on IL-17A. CONCLUSION: Magnolol selectively inhibited Th17 differentiation and cytokine expression through selectively blocking of STAT3 resulting in decreased the ratio of Th17/Treg cells for treating MS, suggesting that the potential of magnolol for treating MS as novel STAT3 inhibitor.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells.

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 ß (IL-1 ß), 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 ß and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide (P<0.01), inhibited the secretion of TNF-α, IL-1 ß, 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.

Selenium Supplementation Alter the Frequency of Th17 but not Th1/Th2 Lymphocytes in Diffuse Large B Cell Lymphoma Patients.

BACKGROUND: Selenium (Se) is a micronutrient, which has recently been proven to have a positive effect on the immune system of cancer patients, but the underlying mechanism is not clearly defined. In this randomized controlled trial, we evaluated the effect of three-month Se supplementation on the profile of CD4+ T-helper subsets including IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 cells in sixteen diffuse large B cell lymphoma (DLBCL) patients at stable remission phase who consumed Se (Se+) compared to the fourteen control patients who did not receive Se (Se-). METHODS: The frequency of IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 lymphocytes was determined using a four-color flow cytometry method. RESULTS: The results revealed that three-month Se supplementation significantly decreased the proportion of CD4+IL-17+ Th17 lymphocytes but not IFN-γ+/IL-4- Th1 and IFN-γ-/IL-4+ Th2 subtypes in DLBCL patients at stable remission. Change in the percentage of IFN-γ+/IL-4- Th1, IFN-γ-/IL-4+ Th2, and CD4+IL-17+ Th17 cells did not significantly differ between Se+ and Se- groups. No positive correlation was observed between changes in different Th subpopulations in both Se+ and Se- groups. CONCLUSIONS: Taken together, three-month Se supplementation can reduce the proportion of CD4+IL-17+ Th17 cells in DLBCL patients at stable remission phase. Larger population and longer follow-up of patients is necessary to specify the clinical significance of Se supplementation on the popularity of T-helper cells in DLBCL patients.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells / 中国结合医学杂志

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.

Artemisia gmelinii Extract Alleviates Allergic Airway Inflammation via Balancing TH1/TH2 Homeostasis and Inhibiting Mast Cell Degranulation.

A new terminology "combined allergic rhinitis and asthma syndrome (CARAS)" was introduced to describe patients suffering from both allergic rhinitis (AR) and asthma. The pathogenesis of allergic airway inflammation has been well known, with the main contribution of TH1/TH2 imbalance and mast cell degranulation. Artemisia gmelinii has been used as an herbal medicine with its hepaprotective, anti-inflammatory, and antioxidant properties. In this study, the effect of A. gmelinii extracts (AGE) on the ovalbumin (OVA)-induced CARAS mouse model was investigated. AGE administration significantly alleviated the nasal rubbing and sneezing, markedly down-regulated both OVA-specific IgE, IgG1, and histamine levels, and up-regulated OVA-specific IgG2a in serum. The altered histology of nasal and lung tissues of CARAS mice was effectively ameliorated by AGE. The AGE treatment group showed markedly increased levels of the TH1 cytokine interleukin (IL)-12 and TH1 transcription factor T-bet. In contrast, the levels of the TH2 cytokines, including IL-4, IL-5, IL-13, and the TH2 transcription factor GATA-3, were notably suppressed by AGE. Moreover, AGE effectively prevented mast cell degranulation in vitro and mast cell infiltration in lung tissues in vivo. Based on these results, we suggest that AGE could be a potential therapeutic agent in OVA-induced CARAS by virtue of its role in balancing the TH1/TH2 homeostasis and inhibiting the mast cell degranulation.

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

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

Improving adjuvanticity of crude polysaccharides from cultivated Artemisia rupestris L. for influenza vaccine by promoting long-term immunity and TH1/TH2 response with dose-sparing effect.

ETHNOPHARMACOLOGICAL RELEVANCE: Influenza virus vaccines (IVV) with balanced TH1/TH2 responses are critical for controlling seasonal influenza. Emerging evidences suggest that herbal polysaccharides can induce potent TH1 or mixed TH1/TH2 responses. AIM OF STUDY: The study aims to determine the efficacy and safety of crude polysaccharides from cultivated Artemisia rupestris L. (CPCAR) as an adjuvant for IVV. MATERIALS AND METHODS: CPCAR was prepared with hot extraction and ethanol precipitation method and primary physico-chemical characters were tested. Mice were vaccinated by subcutaneous route with IVV formulated with different dose of CPCAR to detecting the elicited TH1/TH2 responses and long-term immune responses with dose-sparing sparing effect. RESULTS: IVV formulated with CPCAR without LPS contamination could augment balanced TH1/TH2 responses, as indicated by early IgG response, hemagglutination inhibition (HAI) antibodies, effector T-cells, and cytotoxic T lymphocytes (CTL). Moreover, CPCAR elicited long-term IgG, HAI antibodies, memory T cells, and balanced CD4/CD8 responses within 168 days after vaccination. Compared with IVV alone, a low or high dose of IVV formulated with CPCAR improved the levels of IgG, IgG1, and IgG2a and enhanced memory T cells and balanced CD4/CD8 responses, displaying a 10-fold dose-sparing effect. As determined by IgE response and monitoring results of weekly body weight and daily symptoms after vaccination, anaphylaxis or adverse effect was not observed. CONCLUSIONS: Collectively, the study demonstrated the potential of CPCAR as an aqueous polysaccharide adjuvant for IVV to induce rapid and balanced TH1/TH2 responses and long-lasting immunity with dose-sparing effect.

Anti-Th17 and anti-Th2 responses effects of hydro-ethanolic extracts of Aframomum melegueta, Khaya senegalensis and Xylopia aethiopica in hyperreactive onchocerciasis individuals' peripheral blood mononuclear cells.

Hyperreactive onchocerciasis (HO) is characterized by a severe skin inflammation with elevated Th17-Th2 combined responses. We previously demonstrated the anthelminthic activity of Aframomum melegueta (AM), Xylopia aethiopica (XA) and Khaya senegalensis (KS) used by traditional healers to treat helminthiasis in the endemic area of Togo. However, their effect on severe onchocerciasis is poorly investigated. The present study aimed to investigate the anti-Th17 and anti-Th2 effects of hydro-ethanolic extracts of AM, XA and KS during HO. Onchocerca volvulus-infected individuals were recruited in the Central region of Togo in 2018. Isolated peripheral blood mononuclear cells (PBMCs) from both generalized onchocerciasis (GEO) and HO forms were activated with anti-CD3 and anti-CD28 monoclonal antibodies in the presence or absence of the hydro-ethanolic extracts of AM, XA and KS as well as their delipidated, deproteinized and deglycosylated fractions. After 72 hours, cytokines were assayed from cell culture supernatants. Then, flow cytometry was used to investigate the effects of the extracts on cell activation, proliferation, intracellular cytokines and T cells transcription factors. The production of both Th17 and Th2 cytokines IL-17A and IL-5 were significantly inhibited upon T-cell receptor (TCR) activation in the presence of the hydro-ethanolic extracts of AM, XA and KS in HO individuals' PBMCs in vitro. AM and XA inhibited CD4+RORC2+IL-17A+ and CD4+GATA3+IL-4+ cell populations induction. This inhibition was not Th1 nor Treg-dependent since both IFN-γ and IL-10 were also inhibited by the extracts. AM and XA did not interfere with T cell activation and proliferation for their inhibitory pathways. Lipid and protein compounds from AM and XA were associated with the inhibition of IL-17A. This study showed that in addition to their anthelminthic effects, hydro-ethanolic extracts of Aframomum melegueta, Xylopia aethiopica and Khaya senegalensis could downregulate both Th17 and Th2 responses and prevent the severe skin disorder observed.

Identification of Sorafenib as a Treatment for Type 1 Diabetes.

Th1 cell activation is considered a key mediator of the pathogenesis of type 1 diabetes. Targeting IL-12-induced Th1 cell differentiation seems to be an effective way to block the development of type 1 diabetes. However, given the critical function of Th1 in the immune system, the potential side effects hinder the application of anti-Th1 therapy in the treatment of type 1 diabetes. To identify safe anti-Th1 treatment(s), we screened the FDA-approved tyrosine kinase inhibitor (TKI) drug library using an IL-12-induced Th1 differentiation cell model. We found that among the TKIs with little effect on T cell viability, sorafenib is the top contender for the inhibition of Th1 differentiation. Treatment of NOD mice with sorafenib significantly impeded the development of type 1 diabetes and ameliorated insulitis, which coincided with a specifically decreased accumulation of Th1 cell population in the pancreas but not in peripheral immune organs. Mechanistically, sorafenib indirectly inhibited janus kinase 2 (JAK2) activity and blocked IL-12-induced phosphorylations of JAK2 and signal transducer and activator of transcription 4 (STAT4). Since sorafenib is classified as an FDA-approved drug, it serves as a preliminary lead point for additional experimentation and may be a promising therapy for type 1 diabetes in humans.

The Purinergic Receptor P2X4 Promotes Th17 Activation and the Development of Arthritis.

Purinergic signaling plays a major role in T cell activation leading to IL-2 production and proliferation. However, it is unclear whether purinergic signaling contributes to the differentiation and activation of effector T cells. In this study, we found that the purinergic receptor P2X4 was associated with human Th17 cells but not with Th1 cells. Inhibition of P2X4 receptor with the specific antagonist 5-BDBD and small interfering RNA inhibited the development of Th17 cells and the production of IL-17 by effector Th17 cells stimulated via the CD3/CD28 pathway. Our results showed that P2X4 was required for the expression of retinoic acid-related orphan receptor C, which is the master regulator of Th17 cells. In contrast, inhibition of P2X4 receptor had no effect on Th1 cells and on the production of IFN-γ and it did not affect the expression of the transcription factor T-bet (T-box transcription factor). Furthermore, inhibition of P2X4 receptor reduced the production of IL-17 but not of IFN-γ by effector/memory CD4+ T cells isolated from patients with rheumatoid arthritis. In contrast to P2X4, inhibition of P2X7 and P2Y11 receptors had no effects on Th17 and Th1 cell activation. Finally, treatment with the P2X4 receptor antagonist 5-BDBD reduced the severity of collagen-induced arthritis in mice by inhibiting Th17 cell expansion and activation. Our findings provide novel insights into the role of purinergic signaling in T cell activation and identify a critical role for the purinergic receptor P2X4 in Th17 activation and in autoimmune arthritis.

Time-dependent dual beneficial modulation of interferon-γ, interleukin 5, and Treg cytokines in asthma patient peripheral blood mononuclear cells by ganoderic acid B.

Th2 cytokines play a dominant role in the pathogenesis of allergic asthma. Interferon gamma (IFN-γ), a Th1 cytokine, links to therapeutic mechanisms of allergic asthma. Interleukin (IL)-10, a regulatory cytokine, is involved in the induction of immune tolerance. We previously demonstrated that Anti-Asthma Simplified Herbal Medicine Intervention (ASHMI) suppressed Th2 and increased IFN-γ in patients with asthma and in animal models, but its bioactive compound is unknown. Ganoderic acid beta (GAB) was isolated from Ganoderma lucidum (one herb in ASHMI). Human peripheral blood mononuclear cells (PBMCs) from adult patients with asthma were cultured with GAB or dexamethasone (Dex) in the presence of environmental allergens. The cytokine levels of IL-10, IFN-γ, IL-5, transcription factors T-bet, Foxp-3, and GATA3 were measured. Following 3-day culture, GAB, but not Dex, significantly increased IL-10 and IFN-γ levels by allergic patients' PBMCs. Following 6-day treatment, GAB inhibited IL-5 production, but IL-10 and IFN-γ remained high. Dex suppressed production of all three cytokines. GAB suppressed GATA3 and maintained Foxp-3 and T-bet gene expression, while Dex significantly suppressed GATA3 and T-bet expression. GAB simultaneously increased IL-10, IFN-γ associated with induction of T-bet and Foxp3, while suppressing IL-5, which was associated with suppression of GATA3, demonstrating unique beneficial cytokine modulatory effect, which distinguishes from Dex's overall suppression.

Arsenic Trioxide Cooperate Cryptotanshinone Exerts Antitumor Effect by Medicating Macrophage Polarization through Glycolysis.

Hepatocellular carcinoma (HCC) is an often-fatal malignant tumor with high lethality. Despite advances and significant efficacy in monotherapy, cancer therapy continues to pose several challenges. Novel combination regimens are an emerging strategy for anti-HCC and have demonstrated to be effective. Here, we propose a potential combination for HCC treatment named arsenic trioxide cooperate cryptotanshinone (ACCS). A remarkable synergistic therapeutic effect has been achieved compared with drugs alone in both in vivo and in vitro experiments. Mechanism study indicated that ACCS exerts its therapeutic actions by regulating macrophage-related immunity and glycolysis. ACCS potentiates the polarization of M1 macrophages and elevates the proportion of M1/M2 to remodel tumor immunity. Further molecular mechanism study revealed that ACCS intensifies the glucose utilization and glycolysis in the macrophage by increasing the phosphorylation of AMPK to activating the AMPK singling pathway. In conclusion, ACCS is a highly potential combination regimen for HCC treatment. The therapeutic potential of ACCS as a candidate option for anticancer drugs in restoring the balance of immunity and metabolism deserves further investigation.

Preclinical evaluation of a SARS-CoV-2 mRNA vaccine PTX-COVID19-B.

Safe and effective vaccines are needed to end the COVID-19 pandemic. Here, we report the preclinical development of a lipid nanoparticle­formulated SARS-CoV-2 mRNA vaccine, PTX-COVID19-B. PTX-COVID19-B was chosen among three candidates after the initial mouse vaccination results showed that it elicited the strongest neutralizing antibody response against SARS-CoV-2. Further tests in mice and hamsters indicated that PTX-COVID19-B induced robust humoral and cellular immune responses and completely protected the vaccinated animals from SARS-CoV-2 infection in the lung. Studies in hamsters also showed that PTX-COVID19-B protected the upper respiratory tract from SARS-CoV-2 infection. Mouse immune sera elicited by PTX-COVID19-B vaccination were able to neutralize SARS-CoV-2 variants of concern, including the Alpha, Beta, Gamma, and Delta lineages. No adverse effects were induced by PTX-COVID19-B in either mice or hamsters. Based on these results, PTX-COVID19-B was authorized by Health Canada to enter clinical trials in December 2020 with a phase 2 clinical trial ongoing.