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.

The Correlation Between IFNG Gene Methylation and Th1|Th2 Cell Balance in ROU and the Interventional Study of Jiaweidaochi Powder.

The pathogenesis of recurrent oral ulcers (ROU) is complex, with a long duration of illness and challenging to cure. According to traditional Chinese medicine (TCM),"heat accumulation in the heart-spleen" is one of the main causative factors. Jiaweidaochi powder (JWDCP) is based on the ancient Chinese medicine formula JWDCS, with the addition of Tongcao and gypsum and the removal of Mu Tong. It is generally used to treat "heat accumulation in the heart-spleen." Previous studies have demonstrated that it effectively reduces recurrence rates and is anti-inflammatory in modulating immunity. The ROU rats' model for JWDCP intervention treatment had been established, and histological tests revealed that JWDCP has a therapeutic effect on the pathological changes in the oral mucosa. In addition, the methylation levels of peripheral blood IFNG gene were detected by bisulfite sequencing PCR (BSP), and the methylation levels of the IFNG promoter region in the model group and each dose group were lower than those in the control group. However, no significant methylation differences were observed. Furthermore, the results of enzyme-linked immunosorbent assay (ELISA) and RNA quantitative polymerase chain reaction showed that JWDCP could reduce IFN-γ and IL-4 protein concentrations, with high GATA-3 mRNA production, T-bet mRNAproduction was upgraded, elevated IL-4 mRNA levels, and reduced IFN-γ mRNA levels after treatment (P < 0.001). The expression of transcription factor T-betmRNA and GATA-3 gene mRNA was accompanied by changes in IFN-γmRNA and IL-4mRNA, demonstrating that Th2 type differentiation in RAS suppresses the body's immunity and that the imbalance of transcription factor expression further leads to Th1/Th2 drift. JWDCP is likely to reduce the protein concentration by regulating the imbalance of transcription factors and enhancing antioxidant capacity, thus achieving therapeutic effects. Treatment of recurrent oral ulcer models is not sufficient to reset IFNG methylation levels, correlating with the refractoriness of ROU, further confirming the complexity of epigenetic mechanisms and that epigenetic alterations in specific mediators may persist locally.

Gypenoside A from Gynostemma pentaphyllum Attenuates Airway Inflammation and Th2 Cell Activities in a Murine Asthma Model.

Our previous study found that oral administration of Gynostemma pentaphyllum extract can attenuate airway hyperresponsiveness (AHR) and reduce eosinophil infiltration in the lungs of asthmatic mice. Gypenoside A is isolated from G. pentaphyllum. In this study, we investigated whether gypenoside A can effectively reduce asthma in mice. Asthma was induced in BALB/c mice by ovalbumin injection. Asthmatic mice were treated with gypenoside A via intraperitoneal injection to assess airway inflammation, AHR, and immunomodulatory effects. In vitro, gypenoside A reduced inflammatory and oxidative responses in inflammatory tracheal epithelial cells. Experimental results showed that gypenoside A treatment can suppress eosinophil infiltration in the lungs, reduce tracheal goblet cell hyperplasia, and attenuate AHR. Gypenoside A significantly reduced Th2 cytokine expression and also inhibited the expression of inflammatory genes and proteins in the lung and bronchoalveolar lavage fluid. In addition, gypenoside A also significantly inhibited the secretion of inflammatory cytokines and chemokines and reduced oxidative expression in inflammatory tracheal epithelial cells. The experimental results suggested that gypenoside A is a natural compound that can effectively reduce airway inflammation and AHR in asthma, mainly by reducing Th2 cell activation.

Fructus Amomi extract attenuates nasal inflammation by restoring Th1/Th2 balance and down-regulation of NF-κB phosphorylation in OVA-induced allergic rhinitis.

Fructus Amomi Cardamomi (FA) is the mature fruit of Amomum villosum Lour (family Zingiberaceae) and is commonly used in Chinese traditional medicine to treat various gastrointestinal disorders. FA's possible benefits as an allergic rhinitis (AR) treatment, however, have not been examined. We used an ovalbumin (OVA)-induced AR mouse model to identify any anti-allergic effects associated with the administration of 200 mg/kg FA or dexamethasone (Dex) 2.5 mg/kg by oral administration. The results of our testing confirm that FA ameliorated nasal symptoms and alleviated nasal epithelium swelling, reduced the goblet cell hyperplasia and eosinophil cell infiltration in the nasal epithelium, and inhibited lung tissue inflammation and Dex as well. Significantly decreased Th2 cytokine (interleukin (IL)-1ß, IL-4, and IL-5) expression, and a correspondingly significant increase in Th1 cytokine (IL-12, interferon (IFN)-γ) production, was observed in nasal lavage fluid (NALF) taken from mice that received FA or Dex treatment. FA also reduced the presence of OVA-specific immunoglobulin (Ig) E, OVA-specific IgG1, and histamine levels in serum, and inhibited mast cell degranulation in vitro. In addition, these effects were involved with the reduction in NF-κB phosphorylation. These results suggest that FA restores Th1/Th2 balance and inhibits NF-κB phosphorylation and mast cell degranulation, thereby achieving a notable anti-inflammatory effect. Accordingly, it has the potential to be used as an efficacious therapeutic treatment for AR.

Artemisia annua water extract attenuates DNCB-induced atopic dermatitis by restraining Th2 cell mediated inflammatory responses in BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia annua L. (A. annua) is a traditional Chinese medicine that has been used since ancient times to treat malaria, eczema, dermatomycosis, jaundice, and boils. Modern pharmacological studies show that it has immunosuppressive and anti-inflammatory effects. However, the mechanism of A. annua in the treatment of atopic dermatitis (AD) remains unclear. AIM OF THE STUDY: This study was aimed to investigate the effect of A. annua water extract (AWE) on 2,4-dinitrochlorobenzene (DNCB)-induced AD mouse model and tried to explore its possible underlying mechanisms. MATERIALS AND METHODS: AD was induced in BALB/c mice by the topical repeated application of DNCB. Oral drug intervention of AWE and dexamethasone (DEX, positive control) began from the 7th day and continued for 13 consecutive days. The clinical skin score, ear thickness and the weight of ear and spleen were assessed. The ear tissue were stained with toluidine blue and hematoxylin and eosin (H&E) to detect inflammatory cell infiltration. IgE, terleukin (IL)-4 and IL-13 levels in the serum and IgE level in splenocytes were quantified by enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of IL-4, IL-6, IL-13, IL-17, tumor necrosis factor (TNF)-α and thymic stromal lymphopoietin (TSLP) were measured by quantitative real time polymerase chain reaction. The phosphorylation levels of mitogen-activated protein kinases (MAPKs)-p38 and nuclear factor (NF)-κB in ear tissue were detected by Western blot. RESULTS: Results demonstrated that AWE treatment significantly attenuated the AD-like symptoms in DNCB-induced BALB/c mice, including the skin dermatitis severity and ear edema. Further study disclosed that AWE treatment suppressed the expressions of IgE, IL-4, IL-6, IL-13, IL-17, TNF-α and TSLP at mRNA and protein levels. Moreover, AWE showed inhibitory effect on the phosphorylation of p38 MAPK and NFκB in ear tissues of AD mice. CONCLUSIONS: Collectively, our results suggested that AWE suppressed DNCB-induced AD in mice probably by restraining Th2 type inflammatory response. These findings might pave the road for the potential clinical application of AWE for AD treatment.

Short-Chain Fatty Acids Promote Immunotherapy by Modulating Immune Regulatory Property in B Cells.

The dysfunction of regulatory B cells (Breg) may result in immune inflammation such as allergic rhinitis (AR); the underlying mechanism is not fully understood yet. Short-chain fatty acids, such as propionic acid (PA), have immune regulatory functions. This study is aimed at testing a hypothesis that modulates PA production alleviating airway allergy through maintaining Breg functions. B cells were isolated from the blood obtained from AR patients and healthy control (HC) subjects. The stabilization of IL-10 mRNA in B cells was tested with RT-qPCR. An AR mouse model was developed to test the role of PA in stabilizing the IL-10 expression in B cells. We found that the serum PA levels were negatively correlated with the serum Th2 cytokine levels in AR patients. Serum PA levels were positively associated with peripheral CD5+ B cell frequency in AR patients; the CD5+ B cells were also IL-10+. The spontaneous IL-10 mRNA decay was observed in B cells, which was prevented by the presence of PA through activating GPR43. PA counteracted the effects of Tristetraprolin (TTP) on inducing IL-10 mRNA decay in B cells through the AKT/T-bet/granzyme B pathway. Administration of Yupinfeng San, a Chinese traditional medical formula, or indole-3-PA, induced PA production by intestinal bacteria to stabilize the IL-10 expression in B cells, which promoted the allergen specific immunotherapy, and efficiently alleviated experimental AR. In summary, the data show that CD5+ B cells produce IL-10. The serum lower PA levels are associated with the lower frequency of CD5+ B cells in AR patients. Administration with Yupinfeng San or indole-3-PA can improve Breg functions and alleviate experimental AR.

Yiqi Jiemin decoction alleviates allergic rhinitis in a guinea pig model by suppressing inflammation, restoring Th1/Th2 balance, and improving cellular metabolism.

We investigated the mechanisms underlying the therapeutic effects of Yiqi Jiemin decoction (YJD), a traditional Chinese medicine (TCM), in the ovalbumin (OVA)-induced allergic rhinitis (AR) model in guinea pigs. YJD significantly decreased infiltration of mast cells and eosinophils into the nasal mucosa of AR model guinea pigs. YJD also increased expression of TGF-ß in the nasal mucosa, restored the balance of Th1/Th2 immune cell responses, and decreased serum levels of various pro-inflammatory mediators, including histamine (HA), neuropeptide Y (NPY), acetylcholine (ACH), norepinephrine and immunoglobulin E (IgE). Metabolic analyses using liquid chromatography coupled with high-resolution mass spectrometry revealed that YJD improved cellular metabolism in AR model guinea pigs and increased serum levels of glycocholic acid while decreasing levels 1-palmitoyl lysophosphatidic acid. RNA-sequencing analysis identified BPIFB2 as a potential diagnostic biomarker and therapeutic target for AR. Functional enrichment analyses showed that YJD significantly inhibited cytokine secretion pathways in AR model guinea pigs. These findings demonstrate that YJD protects against OVA-induced AR in guinea pigs by suppressing inflammation in the nasal mucosa, restoring Th1/Th2 balance, and improving cellular metabolism.

Bovine Holo-Beta-Lactoglobulin Cross-Protects Against Pollen Allergies in an Innate Manner in BALB/c Mice: Potential Model for the Farm Effect.

The lipocalin beta-lactoglobulin (BLG) is a major protein compound in cow's milk, and we detected it in cattle stable dust. BLG may be a novel player in the farm protective effect against atopic sensitization and hayfever. In previous studies, we demonstrated that only the ligand-filled holo-form of BLG prevented sensitization to itself. Here, we investigated whether holo-BLG could, in an innate manner, also protect against allergic sensitization to unrelated birch pollen allergens using a murine model. BALB/c mice were nasally pretreated four times in biweekly intervals with holo-BLG containing quercetin-iron complexes as ligands, with empty apo-BLG, or were sham-treated. Subsequently, mice were intraperitoneally sensitized two times with apo-BLG or with the unrelated birch pollen allergen apo-Bet v 1, adjuvanted with aluminum hydroxide. After subsequent systemic challenge with BLG or Bet v 1, body temperature drop was monitored by anaphylaxis imaging. Specific antibodies in serum and cytokines of BLG- and Bet v 1-stimulated splenocytes were analyzed by ELISA. Additionally, human peripheral blood mononuclear cells of pollen allergic subjects were stimulated with apo- versus holo-BLG before assessment by FACS. Prophylactic treatment with the holo-BLG resulted in protection against allergic sensitization and clinical reactivity also to Bet v 1 in an unspecific manner. Pretreatment with holo-BLG resulted in significantly lower BLG-as well as Bet v 1-specific antibodies and impaired antigen-presentation with significantly lower numbers of CD11c+MHCII+ cells expressing CD86. Pretreatment with holo-BLG also reduced the release of Th2-associated cytokines from Splenocytes in BLG-sensitized mice. Similarly, in vitro stimulation of PBMCs from birch pollen allergic subjects with holo-BLG resulted in a relative decrease of CD3+CD4+ and CD4+CRTh2 cells, but not of CD4+CD25+CD127- Treg cells, compared to apo-BLG stimulation. In conclusion, prophylactic treatment with holo-BLG protected against allergy in an antigen-specific and -unspecific manner by decreasing antigen presentation, specific antibody production and abrogating a Th2-response. Holo-BLG therefore promotes immune resilience against pollen allergens in an innate manner and may thereby contribute to the farm protective effect against atopic sensitization.

Efficacy and action mechanisms of a Chinese herbal formula on experimental models of atopic dermatitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Atopic dermatitis (AD) is a skin inflammatory disease characterized by erythema, eruption, lichenification and pruritus. Shi Zhen Formula (SZF), an empirical Chinese herbal preparation, has clinical efficacy in relieving the symptoms of AD patients. However, the underlying molecular mechanisms of SZF remained unclear. AIM OF THE STUDY: We aimed to investigate the anti-AD effects of SZF and elucidate its underlying molecular mechanisms using in vitro and in vivo models of AD. MATERIALS AND METHODS: High-performance liquid chromatography analysis was performed for quality control of SZF extract. The anti-inflammatory effect of SZF was investigated through evaluating the levels of nitric oxide (NO), chemokines and pro-inflammatory cytokines in the lipopolysaccharide (LPS) stimulated RAW264.7 cells. AD-like skin lesions in female BALB/c mice were induced by 2,4-dinitrochlorobenzene (DNCB). SZF (3.15, 6.30 and 9.45 g/kg) and dexamethasone (5 mg/kg) were administered by gavage daily for 15 consecutive days. The body weight, skin thickness, skin dermatitis severity and scratching behaviors were recorded throughout the study. Histological analysis, reverse transcription-quantitative polymerase chain reaction (RT-PCR), western blot (WB) and ELISA analysis were used to illuminate the molecular targets associated with the anti-AD effects of SZF. RESULTS: SZF markedly decreased the epidermal thickening and infiltration of mast cells in the ears and dorsal skin of the 2,4-dinitrochlorobenzene (DNCB)-treated mice. SZF not only suppressed the levels of immunoglobulin E (IgE), histamine, thymic stromal lymphopoietin (TSLP) and IL-4 in the serum but also suppressed the over-production of IL-4 and IL-6 and gene expressions of IL-4, IL-13, IL-31 and TSLP in the dorsal skin. Moreover, SZF improved epidermal barrier by increasing the protein expressions of filaggrin, involucrin and loricrin and inhibited the activation of NF-κB p65 pathway in the dorsal skin of the DNCB-treated mice. CONCLUSION: SZF alleviates DNCB induced AD-like skin lesions in mice through regulating Th1/Th2 balance, improving epidermal barrier and inhibiting skin inflammation. Our research findings provide scientific footing on the use of this Chinese herbal formula for the treatment of AD.

The Immuno-Modulatory Activities of Pentaherbs Formula on Ovalbumin-Induced Allergic Rhinitis Mice via the Activation of Th1 and Treg Cells and Inhibition of Th2 and Th17 Cells.

Allergic rhinitis (AR) is a highly prevalent allergic disease induced by immunoglobulin (Ig) E-mediated hypersensitivity reaction at the nasal epithelium against inhaled allergens. Previous studies have demonstrated that Pentaherbs formula (PHF), a modified herbal formula comprising five herbal medicines (Flos Lonicerae, Herba Menthae, Cortex Phellodendri, Cortex Moutan and Rhizoma Atractylodis), could suppress various immune effector cells to exert anti-inflammatory and anti-allergic effects in allergic asthma and atopic dermatitis. The present study aimed to further determine the anti-inflammatory activities of PHF in an ovalbumin (OVA)-induced AR BALB/c mouse model. Nasal symptoms such as sneezing and nose rubbing were recorded and the serum total IgE and OVA-specific IgG1, as well as interleukin (IL)-4, IL-5, IL-10, IL-13, chemokines CXCL9 CXCL10, and tumor necrosis factor (TNF)-α concentrations in nasal lavage fluid (NALF) were measured during different treatments. Effects of PHF on the expression of inflammatory mediators in the sinonasal mucosa were quantified using real-time QPCR. PHF was found to suppress allergic symptoms, infiltration of inflammatory cells, and hyperplasia of goblet cells in the nasal epithelium of the OVA-induced AR mice. PHF could reduce OVA-specific IgG1 level in serum, and TNF-α and IL-10 in nasal lavage fluid (NALF), significantly up-regulate the splenic regulatory T (Treg) cell level, increase the Type 1 helper T cell (Th1)/Type 2 helper T cell (Th2) ratio, and reduce the Th17 cells (all p < 0.05). PHF could also alleviate in situ inflammation in sinonasal mucosa of OVA-induced AR mice. In conclusion, oral treatment of PHF showed immuno-modulatory activities in the OVA-induced AR mice by regulating the splenic T cell population to suppress the nasal allergy symptoms and modulating inflammatory mediators, implicating that PHF could be a therapeutic strategy for allergic rhinitis.

Tilianin attenuates HDM-induced allergic asthma by suppressing Th2-immune responses via downregulation of IRF4 in dendritic cells.

BACKGROUND: Acacetin 7-O-ß-D-glucoside (tilianin) is a major constituent of Agastache rugosa, a traditional medicine that has long been used for the treatment of gastrointestinal disorders. Tilianin has a wide variety of pharmacological properties such as cardioprotective, neuroprotective, and anti-atherogenic activities. We recently discovered that tilianin has the ability to suppress MUC5AC expression in vitro. In addition, we have established an in vivo model of allergic asthma using house dust mite (HDM) that can be applied to tilianin. PURPOSE: We investigated the effects of tilianin on airway inflammation in a HDM-induced asthma mouse model and associated mechanisms. METHODS: Tilianin was treated in splenocytes cultured in Th0 condition and HDM-stimulated bone marrow-derived dendritic cells (BMDCs), and their mRNA expression and cytokines production were determined by quantitative real-time PCR and ELISA. To evaluate the effects of tilianin in an allergic asthma model, mice were sensitized and challenged with HDM. Tilianin was administered prior to challenge by oral gavage and airway hyper-reactivity (AHR) to methacholine, inflammatory cell infiltration, cytokine levels, and airway remodeling were assessed. RESULTS: Tilianin inhibited the production of Th2-related cytokines in splenocytes, which play pivotal roles in allergic airway inflammation. When treated in HDM-stimulated BMDCs, tilianin decreased Th2-skewing cytokine IL-33 and transcription factor IRF4. On the contrary, tilianin increased Th1-skewing regulators, IL-12 and IRF1. In an HDM-induced asthmatic mouse model, tilianin attenuated AHR and airway inflammation. Tilianin suppressed the expression of Th2-related cytokines, IL-13 and IL-33 in lung tissues. As seen in HDM-stimulated BMDCs, tilianin also downregulated the expression of the transcription factor IRF4 but not IRF1. CONCLUSION: Taken together, these results suggest that tilianin attenuates HDM-induced allergic airway inflammation by inhibiting Th2-mediated inflammation through the selective inhibition of the IRF4-IL-33 axis in dendritic cells.

Aqueous Pyrostegia venusta (Ker Gawl.) Miers extract attenuates allergen-induced asthma in a mouse model via an antioxidant mechanism.

Objective:Pyrostegia venusta (Ker-Gawl.) Miers (Bignoniaceae) is a perennial invasive vine, distributed worldwide. In folk medicine, its parts are used for the treatment of inflammatory respiratory diseases. Extracts of P. venusta have antioxidant, antimicrobial, and antinociceptive properties. The aim of this study was to evaluate the effects of two extracts (aqueous and hydroethanolic) of P. venusta in the treatment of asthma in an animal model.Methods: Balb/c mice were sensitized twice with ovalbumin (OVA) intraperitoneally (ip), one week apart, and after one week, challenged with OVA intranasally on four alternate days. Mice were treated ip with 300 mg/kg of aqueous or hydroethanolic extracts for seven consecutive days. Control groups received saline on the same days. Bronchial hyperresponsiveness, production of Th1 and Th2 cytokines, lung and airway inflammation, and antioxidant activity in lung tissue were assessed.Results: Treatment with aqueous extract significantly decreased bronchial hyperresponsiveness, measured by total and tissue resistance and elastance. The administration of hydroethanolic extract did not reduce bronchial hyperresponsiveness. In addition, both extracts significantly reduced total cell and eosinophil counts in bronchoalveolar lavage. Both extracts did not change significantly IL-4, IL-5, IL-9, IL-13, IFN-gamma, and TGF-beta levels. Of note, only the aqueous extract significantly increased the total antioxidant activity and reduced lung inflammation.Conclusion: Aqueous extract of P. venusta reduced bronchial hyperresponsiveness, lung and airway inflammation, probably via an antioxidant mechanism. These results demonstrate that P. venusta may have potential for asthma treatment.

Kombucha ameliorates experimental autoimmune encephalomyelitis through activation of Treg and Th2 cells.

Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS). Kombucha is produced by the fermentation of sugared tea with a symbiotic culture of bacteria and yeasts. This research was designed to reveal the therapeutic impact and the molecular and cellular processes determining the effect of kombucha on MS alleviation in an experimental autoimmune encephalomyelitis (EAE). The EAE was induced using myelin oligodendrocyte glycoprotein (MOG35-55) peptide emulsified in CFA and injected subcutaneously over two flank areas in C57BL/6 mice. In addition, pertussis toxin was injected intraperitoneally and repeated 48 h later. Treatment groups were received three different doses of kombucha (K1: low dose, K2: medium dose and K3: high dose) to obtain a maximum protection. Clinical scores and other criteria were followed daily for the 25 days. At the end of the course, T-helper-related cytokines (IFN-γ, IL-17, IL-4, and TGF-ß) were measured through ELISA. Moreover, nitric oxide (NO) concentration in spinal cord tissue was detected. The severity of disease on the peak of disease in K1, K2, and K3 groups were 3.4 ± 0.18 and 2.6 ± 0.18 and 2 ± 0.14 respectively, compared to the CTRL group with 4.5 ± 0.19 (p < 0.001). Kombucha increased production of interleukin IL-4 (K1 = 95 ± 5, K2 = 110 ± 10, K3 = 115 ± 5 and CTRL = 65 ± 5; p < 0.05) and TGF-ß (K1 = 1750 ± 80, K2 = 2050 ± 65, K3 = 2200 ± 75 and CTRL = 850 ± 85; p < 0.001) but concurrently resulted in a remarkable reduction in the production of IFN-γ (K1 = 950 ± 70, K2 = 890 ± 65, K3 = 850 ± 85 and CTRL = 3850 ± 115; p < 0.001) and IL-17 (K1 = 1250 ± 75, K2 = 1050 ± 90, K3 = 970 ± 80 and CTRL = 6450 ± 125; p < 0.001). Moreover, NO concentration in spinal cord tissue in the treatment groups was significantly less than the control group (K1: 35.42 ± 2.1, K2 = 31.21 ± 2.2, K3 = 28.24 ± 2.6 and CTRL = 45.25 ± 2.7; p < 0.05). These results supported that kombucha could reduce the severity of disease in an EAE model through motivating polarization of CD4+ T cells by induction of IL-4 and TGF-ß as well as inhibition of IFN-γ and IL-17.

Selenium deficiency induced necroptosis, Th1/Th2 imbalance, and inflammatory responses in swine ileum.

Selenium (Se) deficiency has a significant impact on the swine breeding industry by inducing digestive system damage and diarrhea. However, the molecular mechanism remains unclear. Our objectives were to investigate if different amounts of necroptosis, inflammatory responses, and T helper cell 1/T helper cell 2 (Th1/Th2) imbalances were induced by Se deficiency in intestinal porcine jejunal epithelial cells (IPEC-J2) and swine ileum tissue. Therefore, Se-deficient models were successfully established both in vitro and in vivo. In the current study, the cell morphological observation results showed that Se deficiency seriously affected the growth and differentiation of IPEC-J2 cells. Moreover, the necroptosis staining and histomorphology observation results showed that the number of necroptotic cells increased significantly, and the ileal tissue exhibited abnormal structures, including necroptotic features and inflammatory cell infiltration, in the Se-deficient group. Furthermore, Se deficiency resulted in accelerated cell necroptosis by increasing (p < .05) the expression of genes related to the tumor necrosis factor-α pathway at both the protein and messenger RNA (mRNA) levels compared to the control group. Moreover, the relative mRNA and protein expression of the inflammatory genes and their responses to dietary Se deficiency were consistent with the resultant Th1/Th2 imbalances in vitro and in vivo. Taken together, the results suggested that Se deficiency caused necroptosis, inflammatory responses, and abnormal expression of cytokines in swine ileum tissue. These findings might help us to explain the damage induced by Se deficiency to the digestive system of swine.

Diosmin restores the skin barrier by targeting the aryl hydrocarbon receptor in atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is an inflammatory chronic skin disease that is characterized by the dysfunction or lack of skin barrier proteins. Recent studies have proposed that the pharmacological upregulation of skin barrier proteins is an effective treatment for AD. Aryl hydrocarbon receptor (AhR) is a transcription factor that positively regulates the expression of skin barrier proteins upon its activation. PURPOSE: This study aimed to identify AhR agonists from phytochemicals and investigate its effect on skin barrier restoration as well as its mechanisms of action in AD. STUDY DESIGN: A publicly available assay database and HaCaT cells stably transduced with a luciferase gene driven by an AhR-target gene promoter (CYP1A1) were used to screen for the activity of AhR agonists from phytochemicals. Normal human epidermal keratinocytes (NHEKs) and a human skin equivalent (HSE) model were used to investigate the effect of AhR agonists on skin restoration and its underlying mechanisms. METHODS: A Gaussia luciferase assaywas performed to screen for AhR agonist activity. Western blotting, qRT-PCR analysis, immunofluorescence, drug affinity responsive target stability assay, and siRNA-mediated AhR knockdown were performed in NHEKs. Hematoxylin and eosin staining was performed to measure epidermal thickness in the HSE model. RESULTS: Diosmin, a potential AhR agonist derived from natural products, upregulated the expression of skin barrier proteins (filaggrin and loricrin) and their upstream regulator (OVOL1) in NHEKs. Diosmin treatment also increased epidermal thickness in the HSE model. In addition, incubating NHEKs with diosmin restored the expression of skin barrier proteins and mRNAs that were suppressed by Th2 cytokines and inhibited STAT3 phosphorylation that was induced by Th2 cytokines. Diosmin also upregulated the expression of NQO1, a negative regulator of STAT3. Immunofluorescence results showed that diosmin stimulated AhR nuclear translocation, and the drug affinity responsive target stability assay revealed that this phytochemical directly bound to AhR. Furthermore, AhR knockdown abolished diosmin-induced filaggrin and loricrin expression. CONCLUSION: These results suggest that diosmin is a potential treatment for AD that targets AhR.

Immuno-modulatory effects of methanolic extract of Ferula szowitsiana on isolated human Th1/Th2/Treg cytokines levels, and their genes expression and nitric oxide production.

BACKGROUND: Anti-inflammatory and anti-oxidants activities of Ferula szowitsiana L. (F. szowitsiana) were shown in ancient texts and assayed by modern studies. However, immunomodulatory properties of the plant are poorly understood. METHODS: The effects of F. szowitsiana extract (10, 40 and 160 µg/ml), dexamethasone and vehicle were investigated on nitric oxide (NO) level, cell proliferation, and cytokines (IL-4, IL10 and IFN-γ) expression at gene and protein levels in non-stimulated and phytohaemagglutinin-stimulated human lymphocytes (n = 15 in each group). RESULTS: Cell proliferation, cytokines secretion, NO production and levels of genes expression were significantly inhibited but IFN-γ/IL-4 and IL-10/IL-4 ratios (T helper 1/Th2 and Treg/Th2 balances respectively) were increased by dexamethasone and all three concentrations of the extract compared to control group in stimulated lymphocytes (P < 0.001 for all cases). The effect of three concentrations of the extract in all experiments was significantly lower than dexamethasone (P < 0.001 for all cases). CONCLUSION: The extract of F. szowitsiana concentration-dependently decreased NO level but increased Th1/Th2 and Treg/Th2 ratios toward Th1 and Treg. These results suggest the therapeutic potential of the plant's extract in inflammatory diseases with dominant Th2 polarization such as asthma or cancers.

Sojadodamgangki-tang attenuates allergic lung inflammation by inhibiting T helper 2 cells and Augmenting alveolar macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Sojadodamgangki-tang (SDG) is a traditional East-Asian herbal medicine mainly composed of Pinellia ternate (Thunb.) Makino, Perilla frutescens (L.) Britt and 10 kinds of medicinal herbs. It has been used to treat asthma and mucus secretion including lung and bronchi. AIM OF THE STUDY: The aim of this study was to investigate the anti-inflammatory effects of Sojadodamgangki-tang (SDG) on allergic lung inflammation in vitro and in vivo as well as the underlying mechanisms. MATERIALS AND METHODS: We used an ovalbumin (OVA)-induced murine allergic airway inflammation model. Five groups of 8-week-old female BALB/C mice were divided into the following groups: saline control group, the vehicle (allergic) group that received OVA only, groups that received OVA and SDG (200 mg/kg or 400 mg/kg), and a positive control group that received OVA and Dexamethasone (5 mg/kg). In vitro experiments include T helper 2 (TH2) polarization system, murine macrophage cell culture, and human bronchial epithelial cell line (BEAS-2B) culture. RESULTS: SDG administration reduced allergic airway inflammatory cell infiltration, especially of eosinophils, mucus production, Th2 cell activation, OVA-specific immunoglobulin E (IgE), and total IgE production. Moreover, the activation of alveolar macrophages, which leads to immune tolerance in the steady state, was promoted by SDG treatment. Interestingly, SDG treatment also reduced the production of alarmin cytokines by the human bronchial epithelial cell line BEAS-2B stimulated with urban particulate matter. CONCLUSION: Our findings indicate that SDG has potential as a therapeutic drug to inhibit Th2 cell activation and promote alveolar macrophage activation.

Vitamin A deficiency exacerbates extrinsic atopic dermatitis development by potentiating type 2 helper T cell-type inflammation and mast cell activation.

BACKGROUND: Vitamin A deficiency (VAD) has been hypothesized to play a role in the pathophysiology of atopic dermatitis (AD). OBJECTIVE: We sought to verify whether VAD can exacerbate AD development, and explore the possible pathophysiologic mechanism. METHODS: We detected serum vitamin A (VA) concentration in different phenotypes of AD infants (intrinsic AD, iAD and extrinsic AD, eAD), and established ovalbumin (OVA) percutaneous sensitized AD model and passive cutaneous anaphylaxis (PCA) model on VAD and vitamin A supplementation (VAS) model in wild-type mice (C57BL/6) and established AD model on both normal VA (VAN) and VAD feeding mast cell deficiency mice (ckitw-sh/w-sh ). RESULTS: The average serum VA concentration of eAD was significantly lower than that of iAD, as well as healthy controls. In OVA-induced C57BL/6 mouse AD model, compared with VAN group, VAD mice manifested significantly more mast cells accumulation in the skin lesions, more severe Th2-mediated inflammation, including higher serum IgG1 and IgE levels, more IL-4, IL-13 mRNA expression in OVA-sensitized skin, and lower Th1 immune response, including lower serum IgG2a and IFN-γ mRNA expression in the skin. But there was no significant difference in the expression of IL-17 mRNA between OVA-treated skin of VAN and VAD mice. However, in OVA-induced ckitw-sh/w-sh mouse AD model, we did not find any significant differences in the above measurements between VAD and VAN group. In PCA model, VAD mice showed remarkable more blue dye leakage than that in VAN mice. Compared with VAD group, the above-mentioned inflammatory measurements in VAS group and VAN group were similar in OVA-induced AD model mice. CONCLUSIONS AND CLINICAL RELEVANCE: VAD can exacerbate extrinsic AD by augmenting Th2-mediated inflammation and mast cell activation. Therapeutic VAS can rescue VAD-aggravated eAD. It may provide a new strategy for future prevention or treatment of atopic dermatitis.

Lessons Learned from Experimental Human Model of Zinc Deficiency.

Zinc is an essential element for humans, and its deficiency was documented in 1963. Nutritional zinc deficiency is now known to affect over two billion subjects in the developing world. Conditioned deficiency of zinc in many diseases has also been observed. In zinc-deficient dwarfs from the Middle East, we reported growth retardation, delayed sexual development, susceptibility to infections, poor appetite, and mental lethargy. We never found a zinc-deficient dwarf who survived beyond the age of 25 y. In an experimental model of human mild zinc deficiency, we reported decreased thymulin (a thymopoietic hormone) activity in Th1 cells, decreased mRNAs of IL-2 and IFN-gamma genes, and decreased activity of natural killer cells (NK) and T cytotoxic T cells. The effect of zinc deficiency on thymulin activity and IL-2 mRNA was seen within eight to twelve weeks of the institution of zinc-deficient diet in human volunteers, whereas lymphocyte zinc decreased in 20 weeks and plasma zinc decreased in 24 weeks after instituting zinc-deficient diet. We hypothesized that decreased thymulin activity, which is known to proliferate Th1 cells, decreased the proliferation differentiation of Th1 cells. This resulted in decreased generation of IL-2 and IFN-gamma. We observed no effect in Th2 cell function; thus, zinc deficiency resulted in an imbalance of Th1 to Th2 function resulting in decreased cell-mediated immunity. Zinc therapy may be very useful in many chronic diseases. Zinc supplementation improves cell-mediated immunity, decreases oxidative stress, and decreases generation of chronic inflammatory cytokines in humans. Development of sensitive immunological biomarkers may be more sensitive than an assay of zinc in plasma and peripheral blood cells for diagnosis of marginal zinc deficiency in human.

Potential classification of chemical immunologic response based on gene expression profiles.

Occupational immune diseases are a serious public health burden and are often a result of exposure to low molecular weight (LMW) chemicals. The complete immunological mechanisms driving these responses are not fully understood which has made the classification of chemical allergens difficult. Antimicrobials are a large group of immunologically-diverse LMW agents. In these studies, mice were dermally exposed to representative antimicrobial chemicals (sensitizers: didecyldimethylammonium chloride (DDAC), ortho-phthalaldehyde (OPA), irritants: benzal-konium chloride (BAC), and adjuvant: triclosan (TCS)) and the mRNA expression of cytokines and cellular mediators was evaluated using real-time qPCR in various tissues over a 7-days period. All antimicrobials caused increases in the mRNA expression of the danger signals Tslp (skin), and S100a8 (skin, blood, lung). Expression of the TH2 cytokine Il4 peaked at different timepoints for the chemicals based on exposure duration. Unique expression profiles were identified for OPA (Il10 in lymph node, Il4 and Il13 in lung) and TCS (Tlr4 in skin). Additionally, all chemicals except OPA induced decreased expression of the cellular adhesion molecule Ecad. Overall, the results from these studies suggest that unique gene expression profiles are implicated following dermal exposure to various antimicrobial agents, warranting the need for additional studies. In order to advance the development of preventative and therapeutic strategies to combat immunological disease, underlying mechanisms of antimicrobial-induced immunomodulation must be fully understood. This understanding will aid in the development of more effective methods to screen for chemical toxicity, and may potentially lead to more effective treatment strategies for those suffering from immune diseases.