Immunoregulatory and Anti-cancer Activities of Combination Treatment of Novel Four-Herb Formula and Doxorubicin in 4T1-Breast Cancer Bearing Mice.

OBJECTIVE: To investigate the in vivo immunomodulatory and anti-tumor mechanisms of the combined treatment of novel Four-Herb formula (4HF) and doxorubicin in triple-negative breast cancer (TNBC). METHODS: Murine-derived triple-negative mammary carcinoma cell line, 4T1 cells, was cultured and inoculated into mouse mammary glands. Sixty-six mice were randomly assigned into 6 groups (n=11 in ench): naïve, control, LD 4HF (low dose 4HF), HD 4HF (high dose 4HF), LD 4HF + D (low dose and doxorubicin), and D (doxorubicin). Apart from the naïve group, each mouse received subcutaneous inoculation with 5 × 105 4T1 cells resuspended in 100 µL of normal saline in the mammary fat pads. Starting from the day of tumor cell inoculation, tumors were grown for 6 days. The LD and HD groups received daily oral gavage of 658 and 2,630 mg/kg 4HF, respectively. The LD 4HF+D group received daily oral gavage of 658 mg/kg 4HF and weekly intraperitoneal injection of doxorubicin (5 mg/kg). The D group received weekly intraperitoneal injections of doxorubicin (5 mg/kg). The treatment naïve mice received daily oral gavage of 0.2 mL double distilled water and 0.1 mL normal saline via intraperitoneal injection once a week. The control group received daily oral gavage of 0.2 mL double-distilled water. The treatment period was 30 days. At the end of treatment, mice organs were harvested to analyze immunological activities via immunophenotyping, gene and multiplex analysis, histological staining, and gut microbiota analysis. RESULTS: Mice treated with the combination of 4HF and doxorubicin resulted in significantly reduced tumor and spleen burdens (P<0.05), altered the hypoxia and overall immune lymphocyte landscape, and manipulated gut microbiota to favor the anti-tumor immunological activities. Moreover, immunosuppressive genes, cytokines, and chemokines such as C-C motif chemokine 2 and interleukin-10 of tumors were significantly downregulated (P<0.05). 4HF-doxorubicin combination treatment demonstrated synergetic activities and was most effective in activating the anti-tumor immune response (P<0.05). CONCLUSION: The above results provide evidence for evaluating the immune regulating mechanisms of 4HF in breast cancer and support its clinical significance in its potential as an adjunctive therapeutic agent or immune supplement.

Adjuvant activities of immunostimulating natural products: Astragalus membranaceus (Fisch.) Bge. and Coriolus versicolor in BNT162b2 vaccination against COVID-19 infection.

The global pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been developing all over the world for more than 3 years. In late 2020, several variants of concern of SARS-CoV-2 virus emerged, with increased viral fitness and transmissibility by mutations of the spike proteins of the viral particle, denting hopes of the use of early-generation vaccines for a widespread protective immunity against viral infection. The use of adjuvants may enhance the immune responses of the conventional application of the COVID-19 vaccine. We have shown that the water extract of 2 ß-glucan-enriched immunostimulating natural products, Astragalus membranaceus (Fisch.) Bge. (AM) and Coriolus versicolor (CV), could induce innate immunity-related cytokines from human monocytes (CCL5, interleukin [IL]-6, IL-10, and tumor necrosis factor α) and monocyte-derived dendritic cells (IL-1ß, IL-10, IL-12, and tumor necrosis factor α). Using BALB/c mice, orally administrated AM and CV (1,384 and 742 mg/kg/d) for 4 d after vaccination, respectively, could enhance (1) the immunoglobulin G binding activities of BNT162b2 vaccination against ancestral and Delta SARS-CoV-2 spike proteins by 5.8- and 4.3-fold, respectively; (2) the immunoglobulin G3 subclass production of BNT162b2 vaccination against ancestral and variant SARS-CoV-2 spike proteins; and (3) the in vitro antibody-neutralizing activities of BNT162b2 vaccinated mice. In conclusion, combining AM and CV was effective in acting as an oral adjuvant with the messenger RNA vaccine BNT162b2 to improve the antigen binding activities against SARS-CoV-2 ancestral and variant SARS-CoV-2 spike proteins, probably via trained immunity of macrophages and dendritic cells.

Prostein expression on circulating tumor cells as a prognostic marker in metastatic castration-resistant prostate cancer.

BACKGROUND: Identification of emerging molecular biomarkers on circulating tumor cells (CTCs) represents an attractive feature of liquid biopsy that facilitates precision and tailored medicine in the management of metastatic castration-resistant prostate cancer (mCRPC). Prostein is an androgen-regulated transmembrane protein with high prostate specificity. Prostein-positive circulating tumor cell (CTC) was recently suggested to have diagnostic potential; however, no study has been conducted to evaluate its prognostic value in mCRPC. METHODS: CTCs from mCRPC patients were enumerated using the CellSearch System. Prostein-positive CTCs were identified by immunostaining results. The relationships between prostein expression on CTCs and PSA response rate, PSA progression-free survival (PSA-PFS), radiographic progression-free survival (PFS), and overall survival (OS) were tested by Fisher's exact test or evaluated using Kaplan-Meier and multivariate Cox analyses. RESULTS: Prostein-positive CTCs were identified in 31 of 87 baseline samples from mCRPC patients and 16 of 51 samples collected at the first follow-up visit. PSA response rates were significantly lower in baseline prostein-positive patients (0%, 0/31) than in prostein-negative patients (19.6%, 11/56) (p = 0.007). The 31 prostein-positive patients had significantly shorter PSA-PFS (p < 0.001), radiographic PFS (p < 0.001), and OS (p = 0.018), compared to the 56 prostein-negative patients at baseline. The association with PSA-PFS maintained its significance (p = 0.028) in multivariate analyses. Analyzing prostein expression at the first follow-up as well as the conversion of prostein expression from baseline to follow-up samples not only confirmed the association with PSA-PFS, but also demonstrated prognostic significance with OS. CONCLUSION: Our study provides the first evidence to support the potential of prostein expression on CTCs to serve as a novel prognostic marker in mCRPC patients. Future large-scale prospective studies are needed to validate our findings.

Pretest Genetic Education Video Versus Genetic Counseling for Men Considering Prostate Cancer Germline Testing: A Patient-Choice Study to Address Urgent Practice Needs.

PURPOSE: Germline testing (GT) for prostate cancer (PCA) is now central to treatment and hereditary cancer assessment. With rising demand for and shortage of genetic counseling (GC), tools to deliver pretest informed consent across practice settings are needed to improve access to GT and precision care. Here, we report on Evaluation and Management for Prostate Oncology, Wellness, and Risk (EMPOWER), a patient-choice study for pretest video-based genetic education (VBGE) versus GC to inform urgent practice needs. PATIENTS AND METHODS: Men with PCA or at risk for PCA (family history of PCA) were eligible and could choose pretest VBGE or GC. Outcomes included decisional conflict for GT, change in genetics knowledge, satisfaction, and intention to share results with family and/or providers. Descriptive statistics summarized results with counts and percentages for categorical variables and mean ± standard deviation for continuous variables. Data were compared with Fisher's exact, chi-squared, or Wilcoxon two-sample tests. Mean change in genetics knowledge was compared with t tests. The significance level was set a priori at .05. RESULTS: Data on the first 127 participants were analyzed. Characteristics were White (85.8%), bachelor's degree (66.9%), and PCA diagnosis (90.6%). The majority chose VBGE (71%) versus GC (29%; P < .001). No differences were observed in decisional conflict for GT or satisfaction. Cancer genetics knowledge improved in both groups without significant difference (+0.9 VBGE, +1.8 GC, P = .056). Men who chose VBGE had higher intention to share GT results (96.4% VBGE v 86.4% GC, P = .02). Both groups had high rates of GT uptake (VBGE 94.4%, GC 92%). CONCLUSION: A substantial proportion of men opted for pretest VBGE, with comparable patient-reported outcomes and uptake of GT. The results support the use of pretest video to address the critical GC shortage in the precision era.

Effects of topical application of a tri-herb formula on inflammatory dry-skin condition in mice with oxazolone-induced atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammatory and pruritic skin disease, affecting 10-20% of the population worldwide. Paeonia suffruticosa Andrews (Paeoniaceae) (Cortex Moutan) and Mentha haplocalyx Briq. (Labiatae) (Herba Menthae) have shown beneficial effects on AD. Calendula officinalis L. (Asteraceae) is commonly used for treating skin rashes and wounds. PURPOSE: In the present study, a three-herbs formula including Cortex Moutan and Herba Menthae, together with C. officinalis at 1:1:1 weight ratio was used as a topical agent and its therapeutic effects on AD was investigated. METHODS: In vitro effects of individual herbs and three-herbs formula (0.125-1 mg/ml) were examined using cytokine release assay on human mast HMC-1 cells, inflammation test on murine macrophage RAW cells and human keratinocyte (HaCaT) cells, and migration scratch assay on human umbilical vein endothelial cells (HUVEC). The contributing functional pathway of three-herbs formula in AD was explored using Western Blot assay in HMC-1 cells. Oxazolone-induced AD-like mice model was also used to investigate the in vivo therapeutic effect of the topical application of the three-herbs formula. RESULTS: Herba Menthae, Cortex Moutan, and three-herbs formula significantly reduced the production of IL-6 and tumor necrosis factor (TNF)-α in HMC-1 cells, inhibited the expression of IL-6, IL-8 and CCL2 in TNF-α/IFN-γ stimulated HaCaT cells, and suppressed the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Moreover, Herba Menthae and three-herbs formula significantly suppressed CCL2 and TNF-α production in LPS-induced RAW 264.7 cells. C. officinalis and three-herbs formula promoted wound healing in HUVEC. For intracellular mechanisms, three-herbs formula inhibited the expressions of molecules in STAT1 and STAT3-dependent pathways. In vivo model showed that topical application of three-herbs formula on challenged ear reduced ear swelling and mice scratching frequencies. H&E and toluidine blue staining of the challenged ear tissue demonstrated that three-herbs formula reduced the epidermal thickness and mast cell infiltration, respectively. CONCLUSION: The three-herbs formula of Cortex Moutan, Herba Menthae and C. officinalis at 1:1:1 (w/w) exhibited anti-inflammatory effect and promotion of cell migration in vitro. It also alleviated ear redness, swelling, epidermal thickness and inflammation of the OXA-induced AD mice. These findings suggest a potential beneficial role of the topical application of the three-herbs formula for treatment of AD.

Interleukin-38 ameliorates poly(I:C) induced lung inflammation: therapeutic implications in respiratory viral infections.

Interleukin-38 has recently been shown to have anti-inflammatory properties in lung inflammatory diseases. However, the effects of IL-38 in viral pneumonia remains unknown. In the present study, we demonstrate that circulating IL-38 concentrations together with IL-36α increased significantly in influenza and COVID-19 patients, and the level of IL-38 and IL-36α correlated negatively and positively with disease severity and inflammation, respectively. In the co-cultured human respiratory epithelial cells with macrophages to mimic lung microenvironment in vitro, IL-38 was able to alleviate inflammatory responses by inhibiting poly(I:C)-induced overproduction of pro-inflammatory cytokines and chemokines through intracellular STAT1, STAT3, p38 MAPK, ERK1/2, MEK, and NF-κB signaling pathways. Intriguingly, transcriptomic profiling revealed that IL-38 targeted genes were associated with the host innate immune response to virus. We also found that IL-38 counteracts the biological processes induced by IL-36α in the co-culture. Furthermore, the administration of recombinant IL-38 could mitigate poly I:C-induced lung injury, with reduced early accumulation of neutrophils and macrophages in bronchoalveolar lavage fluid, activation of lymphocytes, production of pro-inflammatory cytokines and chemokines and permeability of the alveolar-epithelial barrier. Taken together, our study indicates that IL-38 plays a crucial role in protection from exaggerated pulmonary inflammation during poly(I:C)-induced pneumonia, thereby providing the basis of a novel therapeutic target for respiratory viral infections.

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.

The Active Compounds and Therapeutic Mechanisms of Pentaherbs Formula for Oral and Topical Treatment of Atopic Dermatitis Based on Network Pharmacology.

To examine the molecular targets and therapeutic mechanism of a clinically proven Chinese medicinal pentaherbs formula (PHF) in atopic dermatitis (AD), we analyzed the active compounds and core targets, performed network and molecular docking analysis, and investigated interacting pathways. Information on compounds in PHF was obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and target prediction was performed using the Drugbank database. AD-related genes were gathered using the GeneCards and Online Mendelian Inheritance in Man (OMIM) databases. Network analysis was performed by Cytoscape software and protein-protein interaction was analyzed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). The Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources were applied for the enrichment analysis of the potential biological process and pathways associated with the intersection targets between PHF and AD. Autodock software was used to perform protein compound docking analysis. We identified 43 active compounds in PHF associated with 117 targets, and 57 active compounds associated with 107 targets that form the main pathways linked to oral and topical treatment of AD, respectively. Among them, quercetin, luteolin, and kaempferol are key chemicals targeting the core genes involved in the oral use of PHF against AD, while apigenin, ursolic acid, and rosmarinic acid could be used in topical treatment of PHF against AD. The compound-target-disease network constructed in the current study reveals close interactions between multiple components and multiple targets. Enrichment analysis further supports the biological processes and signaling pathways identified, indicating the involvement of IL-17 and tumor necrosis factor signaling pathways in the action of PHF on AD. Our data demonstrated the main compounds and potential pharmacological mechanisms of oral and topical application of PHF in AD.

IL-37 Ameliorating Allergic Inflammation in Atopic Dermatitis Through Regulating Microbiota and AMPK-mTOR Signaling Pathway-Modulated Autophagy Mechanism.

Interaction between eosinophils and dermal fibroblasts is essential for provoking allergic inflammation in atopic dermatitis (AD). In vitro co-culture of human eosinophils and dermal fibroblasts upon AD-related IL-31 and IL-33 stimulation, and in vivo MC903-induced AD murine model were employed to investigate the anti-inflammatory mechanism of IL-1 family cytokine IL-37 in AD. Results showed that IL-37b could inhibit the in vitro induction of AD-related pro-inflammatory cytokines IL-6 and TNF-α, and chemokines CXCL8, CCL2 and CCL5, increase autophagosome biogenesis-related LC3B, and decrease autophagy-associated ubiquitinated protein p62 by regulating intracellular AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signaling pathway. In CRISPR/Cas9 human IL-37b knock-in mice, IL-37b could significantly alleviate MC903-stimulated ear tissue swelling, itching sensation and the level of circulating cytokine IL-6 and ear in situ expression of AD-related TNF-α, CCL5 and transforming growth factor-ß. Moreover, IL-37b could significantly upregulate Foxp3+ regulatory T cells (Treg) in spleen and ear together with significantly increased serum Treg cytokine IL-10, and decrease eosinophil infiltration in ear lesion. IL-37b knock-in mice showed a distinct intestinal microbiota metabolic pattern upon MC903 stimulation. Furthermore, IL-37b restored the disordered gut microbiota diversity, through regulating the in vivo autophagy mechanism mediated by intestinal metabolite 3-methyladenine, adenosine monophosphate, 2-hydroxyglutarate, purine and melatonin. In summary, IL-37b could significantly ameliorate eosinophils-mediated allergic inflammation via the regulation of autophagy mechanism, intestinal bacterial diversity and their metabolites in AD. Results therefore suggest that IL-37 is a potential anti-inflammatory cytokine for AD treatment.

Anti-inflammatory mechanisms of the novel cytokine interleukin-38 in allergic asthma.

We elucidated the anti-inflammatory mechanisms of IL-38 in allergic asthma. Human bronchial epithelial cells and eosinophils were cocultured upon stimulation with the viral RLR ligand poly (I:C)/LyoVec or infection-related cytokine TNF-α to induce expression of cytokines/chemokines/adhesion molecules. House dust mite (HDM)-induced allergic asthma and humanized allergic asthma NOD/SCID murine models were established to assess anti-inflammatory mechanisms in vivo. IL-38 significantly inhibited induced proinflammatory IL-6, IL-1ß, CCL5, and CXCL10 production, and antiviral interferon-ß and intercellular adhesion molecule-1 expression in the coculture system. Mass cytometry and RNA-sequencing analysis revealed that IL-38 could antagonize the activation of the intracellular STAT1, STAT3, p38 MAPK, ERK1/2, and NF-κB pathways, and upregulate the expression of the host defense-related gene POU2AF1 and anti-allergic response gene RGS13. Intraperitoneal injection of IL-38 into HDM-induced allergic asthma mice could ameliorate airway hyperreactivity by decreasing the accumulation of eosinophils in the lungs and inhibiting the expression of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid (BALF) and lung homogenates. Histological examination indicated lung inflammation was alleviated by reductions in cell infiltration and goblet cell hyperplasia, together with reduced Th2, Th17, and innate lymphoid type 2 cell numbers but increased proportions of regulatory T cells in the lungs, spleen, and lymph nodes. IL-38 administration suppressed airway hyperreactivity and asthma-related IL-4 and IL-5 expression in humanized mice, together with significantly decreased CCR3+ eosinophil numbers in the BALF and lungs, and a reduced percentage of human CD4+CRTH2+ Th2 cells in the lungs and mediastinal lymph nodes. Together, our results demonstrated the anti-inflammatory mechanisms of IL-38 and provided a basis for the development of a regulatory cytokine-based treatment for allergic asthma.

High-Throughput Immunological Analysis of Dictamni Cortex: Implication in the Quality Control of Herbal Medicine.

Quality inconsistency of herbal medicine is an obstacle that limits the extensive use and study of traditional Chinese medicine. Differences in environmental conditions and processing methods of herbal medicine often result in varying clinical outcomes in patients. Standard chemical markers used for the quality control (QC) of herbal medicine are usually the most abundant and characteristic components, which may not be therapeutically relevant or cannot comprehensively reflect the biological quality of the herbs. In view of this, a novel QC method for better assessment of herbal medicine has been developed via bioactivities analysis. Immunological activities of Dictamni Cortex, a typical herbal medicine for the treatment of various inflammatory diseases, from different geographical locations in China, were evaluated. Upon in vitro treatment of their water and ethanol extracts, distinct patterns of inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-6, IL-12p70, IL-1ß, and chemokine CXCL8 were released from the lipopolysaccharides- and/or phytohaemagglutinin-stimulated human peripheral blood mononuclear cells (PBMC). Thus, in addition to the commonly used morphological, chemical, or DNA markers, the novel high-throughput profiling of inflammatory cytokines and chemokines of PBMC upon treatment with herbal extracts could be an important reference to help for the quality control of herbal medicine in the future.

TNF-α-induced protein 3 is a key player in childhood asthma development and environment-mediated protection.

BACKGROUND: Childhood asthma prevalence is significantly greater in urban areas compared with rural/farm environments. Murine studies have shown that TNF-α-induced protein 3 (TNFAIP3; A20), an anti-inflammatory regulator of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, mediates environmentally induced asthma protection. OBJECTIVE: We aimed to determine the role of TNFAIP3 for asthma development in childhood and the immunomodulatory effects of environmental factors. METHODS: In a representative selection of 250 of 2168 children from 2 prospective birth cohorts and 2 cross-sectional studies, we analyzed blood cells of healthy and asthmatic children from urban and rural/farm environments from Europe and China. PBMCs were stimulated ex vivo with dust from "asthma-protective" farms or LPS. NF-κB signaling-related gene and protein expression was assessed in PBMCs and multiplex gene expression assays (NanoString Technologies) in isolated dendritic cells of schoolchildren and in cord blood mononuclear cells from newborns. RESULTS: Anti-inflammatory TNFAIP3 gene and protein expression was consistently decreased, whereas proinflammatory Toll-like receptor 4 expression was increased in urban asthmatic patients (P < .05), reflecting their increased inflammatory status. Ex vivo farm dust or LPS stimulation restored TNFAIP3 expression to healthy levels in asthmatic patients and shifted NF-κB signaling-associated gene expression toward an anti-inflammatory state (P < .001). Farm/rural children had lower expression, indicating tolerance induction by continuous environmental exposure. Newborns with asthma at school age had reduced TNFAIP3 expression at birth, suggesting TNFAIP3 as a possible biomarker predicting subsequent asthma. CONCLUSION: Our data indicate TNFAIP3 as a key regulator during childhood asthma development and its environmentally mediated protection. Because environmental dust exposure conferred the anti-inflammatory effects, it might represent a promising future agent for asthma prevention and treatment.

Anti-Allergic Inflammatory Activity of Interleukin-37 Is Mediated by Novel Signaling Cascades in Human Eosinophils.

IL-1 family regulatory cytokine IL-37b can suppress innate immunity and inflammatory activity in inflammatory diseases. In this study, IL-37b showed remarkable in vitro suppression of inflammatory tumor necrosis factor-α, IL-1ß, IL-6, CCL2, and CXCL8 production in the coculture of human primary eosinophils and human bronchial epithelial BEAS-2B cells with the stimulation of bacterial toll-like receptor-2 ligand peptidoglycan, while antagonizing the activation of intracellular nuclear factor-κB, PI3K-Akt, extracellular signal-regulated kinase 1/2, and suppressing the gene transcription of allergic inflammation-related PYCARD, S100A9, and CAMP as demonstrated by flow cytometry, RNA-sequencing, and bioinformatics. Results therefore elucidated the novel anti-inflammation-related molecular mechanisms mediated by IL-37b. Using the house dust mite (HDM)-induced humanized asthmatic NOD/SCID mice for preclinical study, intravenous administration of IL-37b restored the normal plasma levels of eosinophil activators CCL11 and IL-5, suppressed the elevated concentrations of Th2 and asthma-related cytokines IL-4, IL-6, and IL-13 and inflammatory IL-17, CCL5, and CCL11 in lung homogenate of asthmatic mice. Histopathological results of lung tissue illustrated that IL-37b could mitigate the enhanced mucus, eosinophil infiltration, thickened airway wall, and goblet cells. Together with similar findings using the ovalbumin- and HDM-induced allergic asthmatic mice further validated the therapeutic potential of IL-37b in allergic asthma. The above results illustrate the novel IL-37-mediated regulation of intracellular inflammation mechanism linking bacterial infection and the activation of human eosinophils and confirm the in vivo anti-inflammatory activity of IL-37b on human allergic asthma.

Sophora flavescens protects against mycobacterial Trehalose Dimycolate-induced lung granuloma by inhibiting inflammation and infiltration of macrophages.

The immune system responds to Mycobacterium tuberculosis (MTB) infection by forming granulomas to quarantine the bacteria from spreading. Granuloma-mediated inflammation is a cause of lung destruction and disease transmission. Sophora flavescens (SF) has been demonstrated to exhibit bactericidal activities against MTB. However, its immune modulatory activities on MTB-mediated granulomatous inflammation have not been reported. In the present study, we found that flavonoids from Sophora flavescens (FSF) significantly suppressed the pro-inflammatory mediators released from mouse lung alveolar macrophages (MH-S) upon stimulation by trehalose dimycolate (TDM), the most abundant lipoglycan on MTB surface. Moreover, FSF reduced adhesion molecule (LFA-1) expression on MH-S cells after TDM stimulation. Furthermore, FSF treatment on TDM-activated lung epithelial (MLE-12) cells significantly downregulated macrophage chemoattractant protein (MCP-1/CCL2) expression, which in turn reduced the in vitro migration of MH-S to MLE-12 cells. In addition, FSF increased the clearance of mycobacterium bacteria (Mycobacterium aurum) in macrophages. FSF mainly affected the Mincle-Syk-Erk signaling pathway in TDM-activated MH-S cells. In TDM-induced mouse granulomas model, oral administration with FSF significantly suppressed lung granulomas formation and inflammation. These findings collectively implicated an anti-inflammatory role of FSF on MTB-mediated granulomatous inflammation, thereby providing evidence of FSF as an efficacious adjunct treatment during mycobacterial infection.

Activation of Eosinophils Interacting with Bronchial Epithelial Cells by Antimicrobial Peptide LL-37: Implications in Allergic Asthma.

The role of antimicrobial peptide LL-37 in asthma exacerbation is unclear. Microbial infection, which is the most common inducer of asthma exacerbation, is accompanied by elevated LL-37. The present study found that co-culture of eosinophils and bronchial epithelial cell line BEAS-2B significantly enhanced intercellular adhesion molecule-1 on both cells and CD18 expression on eosinophils upon LL-37 stimulation. IL-6, CXCL8 and CCL4 were substantially released in co-culture in the presence of LL-37. LL-37 triggered the activation of eosinophils interacting with BEAS-2B cells in a P2X purinoceptor 7/epidermal growth factor receptor-dependent manner. Eosinophils and BEAS-2B cells differentially contribute to the expression of cytokines/chemokines in co-culture, while soluble mediators were sufficient to mediate the intercellular interactions. Intracellular p38-mitogen-activated protein kinase, extracellular signal-regulated kinase and NF-κB signaling pathways were essential for LL-37-mediated activation of eosinophils and BEAS-2B cells. By using the ovalbumin-induced asthmatic model, intranasal administration of mCRAMP (mouse ortholog of LL-37) in combination with ovalbumin during the allergen challenge stage significantly enhanced airway hyperresponsiveness and airway inflammation in sensitized mice, thereby implicating a deteriorating role of LL-37 in allergic asthma. This study provides evidence of LL-37 in triggering asthma exacerbation via the activation of eosinophils interacting with bronchial epithelial cells in inflammatory airway.

Anti-Inflammatory Activities of Pentaherbs Formula, Berberine, Gallic Acid and Chlorogenic Acid in Atopic Dermatitis-Like Skin Inflammation.

Atopic dermatitis (AD) is a common allergic skin disease, characterized by dryness, itchiness, thickening and inflammation of the skin. Infiltration of eosinophils into the dermal layer and presence of edema are typical characteristics in the skin biopsy of AD patients. Previous in vitro and clinical studies showed that the Pentaherbs formula (PHF) consisting of five traditional Chinese herbal medicines, Flos Lonicerae, Herba Menthae, Cortex Phellodendri, Cortex Moutan and Rhizoma Atractylodis at w/w ratio of 2:1:2:2:2 exhibited therapeutic potential in treating AD. In this study, an in vivo murine model with oxazolone (OXA)-mediated dermatitis was used to elucidate the efficacy of PHF. Active ingredients of PHF water extract were also identified and quantified, and their in vitro anti-inflammatory activities on pruritogenic cytokine IL-31- and alarmin IL-33-activated human eosinophils and dermal fibroblasts were evaluated. Ear swelling, epidermis thickening and eosinophils infiltration in epidermal and dermal layers, and the release of serum IL-12 of the murine OXA-mediated dermatitis were significantly reduced upon oral or topical treatment with PHF (all p < 0.05). Gallic acid, chlorogenic acid and berberine contents (w/w) in PHF were found to be 0.479%, 1.201% and 0.022%, respectively. Gallic acid and chlorogenic acid could suppress the release of pro-inflammatory cytokine IL-6 and chemokine CCL7 and CXCL8, respectively, in IL-31- and IL-33-treated eosinophils-dermal fibroblasts co-culture; while berberine could suppress the release of IL-6, CXCL8, CCL2 and CCL7 in the eosinophil culture and eosinophils-dermal fibroblasts co-culture (all p < 0.05). These findings suggest that PHF can ameliorate allergic inflammation and attenuate the activation of eosinophils.

Aberrant Expression of Bacterial Pattern Recognition Receptor NOD2 of Basophils and Microbicidal Peptides in Atopic Dermatitis.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease, associated with basophil infiltration into skin lesions and Staphylococcus aureus (S. aureus)-induced inflammation. Pattern recognition receptors (PRRs), including microbicidal peptide human neutrophil α-defensins (HNP) and dermcidin, can exert immunomodulating activity in innate immunity and skin inflammation. We investigated the plasma concentration of HNP and dermcidin, the expression of bacterial toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptors of basophils and plasma concentration and ex vivo induction of AD-related inflammatory cytokines and chemokines using ELISA and flow cytometry, in AD patients and control subjects. Plasma concentrations of HNP, dermcidin and AD-related Th2 chemokines CCL17, CCL22 and CCL27 were significantly elevated in AD patients compared with controls (all p < 0.05). Plasma concentrations of CCL27 and CCL22 were found to correlate positively with SCORing atopic dermatitis (SCORAD), objective SCORAD, % area affected, lichenification and disease intensity, and CCL27 also correlated positively with pruritus in AD patients (all p < 0.05). Protein expressions of NOD2 but not TLR2 of basophils were significantly down-regulated in AD patients compared with controls (p = 0.001). Correspondingly, there were lower ex vivo % inductions of allergic inflammatory tumor necrosis factor-α, IL-6 and CXCL8 from peripheral blood mononuclear cells upon NOD2 ligand S. aureus derived muramyl dipeptide stimulation in AD patients comparing with controls. The aberrant activation of bacterial PRRs of basophils and anti-bacterial innate immune response should be related with the allergic inflammation of AD.