Mouse Mast Cell Protease Research Articles

Evaluation of the suitability of β-lactoglobulin and whey protein in a BALB/c mouse model of cow's milk allergy

Cow's milk allergy (CMA) is common in infants and young children, which can adversely affect their growth and quality of life. Currently, research on CMA is mainly carried out in mouse models due to ethical constraints in population studies, but the determination of the optimal modelling protocol remains controversial. Here, we aim to evaluate the applicability of β-lactoglobulin (BLG) and whey protein from multiple perspectives in the construction of CMA models in BALB/c mice. To this end, the mice were sensitized by oral gavage of allergens, and then challenged by BLG or whey protein orally administered as well as whey intradermally in the ear, respectively. The allergic reactions were determined by the evaluation of anaphylactic symptoms, levels of specific immunoglobulins and Th2 cytokines, and intestinal tissue histopathology. Overall, mice in the BLG group showed more severe allergic symptoms and decreased body temperature than the other groups. The stronger allergic reactions to BLG in mice were also proved in higher levels of mouse mast cell protease-1 (mMCP-1), allergen specific antibodies, and Th2 cytokines as well as more serious intestinal damage than whey protein. These results suggest that BALB/c mice vary in the susceptibility to different cow's milk allergens, and the single component represented by BLG instead of whey protein, may be a more appropriate and effective allergen for the construction of BALB/c mouse model of CMA.

IBD functions as a double-edged sword for food allergy in BALB/c mice model.

Inflammatory bowel disease (IBD) and food allergy (FA) increase in tandem, but the potential impact of IBD on FA remains unclear. We sought to determine the role of IBD on FA. We first assessed the changes of FA-related risk factors in dextran sulphate sodium salt (DSS) induced colitis mice model. Then, we evaluated the role of IBD on FA in mice. FA responses were determined using a clinical allergy score, body temperature change, serum antibody levels, cytokines level and mouse mast cell protease 1 (MMCP-1) concentration. Accumulation of regulatory T cells was tested using flow cytometry. Intestinal changes were identified by histology, immunohistochemistry, gene expression and gut microbial community structure. In DSS-induced colitis mice model, we found the intestinal damage, colonic neutrophil infiltration, and downregulation of splenic Th2 cytokines and Tregs in mesenteric lymph nodes (MLN). Moreover, we also found that IBD can alleviate the FA symptoms and lead to the significant downregulation of Th2 cytokines, serum IgE and MMCP-1. However, IBD exacerbates intestinal injury and promotes the gene expression levels of IL-33 and IL-5 in the small intestine, damages the intestinal tissue structure and aggravates intestinal dysbiosis in FA. IBD functions as a double-edged sword in FA. From the perspective of clinical symptoms and humoral immune responses, IBD can reduce FA response by downregulating Th2 cytokines. But from the perspective of the intestinal immune system, IBD potentially disrupts intestinal tolerance to food antigens by damaging intestinal tissue structure and causing intestinal dysbiosis.

Safflower yellow in Carthami Flos is responsible for Xuebijing Injection-induced immediate hypersensitivity reaction through activating complement C3

ObjectiveXuebijing Injection (XBJI) is mainly used for treating sepsis in China, and even COVID-19 recently. This study aimed to clarify the molecular mechanism(s) and identify the potential “common culprit(s)” for XBJI-caused immediate hypersensitivity reaction (IHR) which is the main type of its adverse reactions. MethodsAntiserum against XBJI was prepared by intraperitoneal immunization in combination with aluminum adjuvant for five weeks. Antagonistic experiments were performed by using several antagonists against different mediators in Evans Blue leakage model. Propranolol-pretreated mice were used to determine the capacity of XBJI to trigger systemic IHR. Serum total IgE (tIgE) and mouse mast cell protease 1 (MCPT-1) levels, complement activation, and the levels of supernatant inflammatory mediators were determined by ELISAs. Lipopolysaccharide (LPS)-activated RAW264.7 macrophages were used for evaluating the anti-inflammatory activity of XBJI, while human mast cells (LAD2) were used for assessing the effect of XBJI on mast cell degranulation. ResultsContinuous treatment (i.p.) with XBJI along with aluminum adjuvant did not elevate the levels of serum tIgE and MCPT-1. In vitro, XBJI could not directly cause the degranulation of LAD2 cells. It induced a robust Evans Blue leakage after the first injection in mouse paw. Mechanism study demonstrated that antagonists for histamine H1/H2 receptors and complement C3a receptor counteracted XBJI-induced IHR. XBJI also directly activated complement C3 in human serum. Through screening five herbs of XBJI and the constituents, only safflower yellow (SY) in Carthami Flos was able to induce IHR. The discolored-XBJI not only did not induce IHR locally and systemically, but also could suppressing the production of proinflammatory mediators in LPS-activated RAW264.7 macrophages. ConclusionXBJI failed to induce immune IHR, but potently triggered non-immune IHR through direct activating complement C3 to provoke histamine release. SY in Carthami Flos was the underlying “common culprit” responsible for XBJI-caused IHR. The anti-inflammatory action of XBJI can be retained after decolorization. Our study provides a scientific basis for not only preventing and treating XBJI-caused IHR clinically, but also improving its production process.

Lycopene alleviates food allergy by modulating the PI3K/AKT pathway in peanut-sensitized BALB/c mice

Food allergies, which lead to life-threatening acute symptoms, are considered an important public health problem. Therefore, it is essential to develop efficient preventive and treatment measures. We developed a crude peanut protein extract (PPE)–induced allergy mouse model to investigate the effects of lycopene on peanut allergy. Mice were divided into four groups: 5 mg/kg lycopene, 20 mg/kg lycopene, no treatment, and control groups. Serum inflammatory factors were detected using enzyme-linked immunosorbent assay. In addition, pathology and immunohistochemistry analyses were used to examine the small intestine of mice. We found that lycopene decreased PPE-specific immunoglobulin E (IgE) and IL-13 levels in the serum, relieved small intestine inflammation, attenuated the production of histamine and mouse mast cell protease-1, and downregulated PI3K and AKT1 expression in the small intestine tissues of mice allergic to peanuts. Our results suggest that lycopene can ameliorate allergy by attenuating the PI3K/AKT pathway and the anaphylactic reactions mediated by PPE-specific IgE.

Insights into sensitizing and eliciting capacity of gastric and gastrointestinal digestion products of shrimp (Penaeus vannamei) proteins in BALB/c mice

Shrimp (Penaeus vannamei) proteins have been shown an allergenic potential; however, little information is available on the sensitizing and eliciting capacity of shrimp protein digestion products. In this study, a BALB/c mice model was used to explore the allergenicity of shrimp protein sample (SPS) and their gastric and gastrointestinal digestion products (GDS/GIDS). As compared with the SPS groups, the GDS/GIDS groups caused lower specific immunoglobulins (IgE/IgG1) levels (P < 0.05), but higher than the control groups, indicating that the digestion products sensitized the mice. Meanwhile, spleen index, mouse mast cell protease-1 (mMCP-1) concentration and proportion of degranulated mast cells were significantly reduced in the GDS/GIDS groups (P < 0.05); simultaneously, allergic symptoms, vascular permeability and histopathological changes of tissues were alleviated. Nevertheless, the allergenicity of digestion products cannot be eliminated and still cause systemic allergic reactions in mice. The study showed that the digestion products of shrimp still had high sensitizing and eliciting capacity.

Therapeutic administration of mouse mast cell protease 6 improves functional recovery after traumatic spinal cord injury in mice by promoting remyelination and reducing glial scar formation.

Traumatic spinal cord injury (SCI) most often leads to permanent paralysis due to the inability of axons to regenerate in the adult mammalian central nervous system (CNS). In the past, we have shown that mast cells (MCs) improve the functional outcome after SCI by suppressing scar tissue formation at the lesion site via mouse mast cell protease 6 (mMCP6). In this study, we investigated whether recombinant mMCP6 can be used therapeutically to improve the functional outcome after SCI. Therefore, we applied mMCP6 locally via an intrathecal catheter in the subacute phase after a spinal cord hemisection injury in mice. Our findings showed that hind limb motor function was significantly improved in mice that received recombinant mMCP6 compared with the vehicle-treated group. In contrast to our previous findings in mMCP6 knockout mice, the lesion size and expression levels of the scar components fibronectin, laminin, and axon-growth-inhibitory chondroitin sulfate proteoglycans were not affected by the treatment with recombinant mMCP6. Surprisingly, no difference in infiltration of CD4+ T cells and reactivity of Iba-1+ microglia/macrophages at the lesion site was observed between the mMCP6-treated mice and control mice. Additionally, local protein levels of the pro- and anti-inflammatory mediators IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFNγ, and MCP-1 were comparable between the two treatment groups, indicating that locally applied mMCP6 did not affect inflammatory processes after injury. However, the increase in locomotor performance in mMCP6-treated mice was accompanied by reduced demyelination and astrogliosis in the perilesional area after SCI. Consistently, we found that TNF-α/IL-1β-astrocyte activation was decreased and that oligodendrocyte precursor cell (OPC) differentiation was increased after recombinant mMCP6 treatment in vitro. Mechanistically, this suggests effects of mMCP6 on reducing astrogliosis and improving (re)myelination in the spinal cord after injury. In conclusion, these data show for the first time that recombinant mMCP6 is therapeutically active in enhancing recovery after SCI.

Effects of hazelnut protein isolate-induced food allergy on the gut microenvironment in a BALB/c mouse model.

Hazelnuts are reported as among the nuts that cause severe allergic reactions. However, few systematic studies exist on the changes in the gut microenvironment following hazelnut allergy. This study focused on the effects of hazelnut allergy on the duodenum, jejunum, ileum and colon microenvironment in vivo. We established a hazelnut protein isolate (HPI)-allergic mouse model, which was distinguished by the visible allergy symptoms, dropped temperatures and enhanced allergic inflammatory factor levels in serum, such as HPI-specific immunoglobulin E (sIgE), sIgG2a, interleukin-4, histamine, mouse mast cell protease-1, TNF-α, monocyte chemotactic protein-1 and lipopolysaccharide. For HPI sensitized mice, aggravated mast cell degranulation, severe morphologic damage and inflammatory cell infiltration were observed in the duodenum, jejunum, ileum, and colon, while goblet cell numbers were reduced in the duodenum, jejunum and ileum. Secretory IgA of the jejunum and tight junctions of the duodenum and jejunum were decreased significantly after HPI sensitization. There was no remarkable difference in the pH values of small intestinal contents, but the pH values of colonic contents were elevated, which was due to the decreased short-chain fatty acids (mainly acetate, propionate and butyrate) in the colon. The antioxidant capacity of both large and small intestinal contents declined after HPI sensitization, as evidenced by the increased malondialdehyde and decreased superoxide dismutase activity. HPI sensitization induced gut microbiota dysbiosis with decreased α diversity and altered β diversity in colonic contents. Spearman correlation analysis indicated that the increased characteristic genera, namely Bacteroides, Lactobacillus, Alloprevotella, Erysipelatoclostridium, Parabacteroides, and Helicobacter, played potentially synergistic roles in promoting allergy and gut microenvironment dysregulation.

PSAT091 The Impact of Pregnancy and Aging on the Female Mouse Adrenal Transcriptome

Abstract Introduction Mouse adrenal glands contain a zona glomerulosa (mineralocorticoid production), zona fasciculata (glucocorticoid production), and a remnant of the fetal adrenal cortex (termed the x-zone). Instead of involuting at birth, as seen in humans, the x-zone expands postnatally and is predicted to function in the metabolism of progesterone and 11-deoxycorticosterone via its high expression of the enzyme, 20α-hydroxysteroid dehydrogenase (Akr1c18). Moreover, mouse adrenals have a clear x-zone sex-divergence at the time of puberty when the x-zone regresses in males. In females, the x-zone regresses during the first pregnancy and with aging. Sexual dimorphism unrelated to the x-zone has been documented in mouse adrenals, including differentially expressed sex-chromosome and sex-steroid driven genes, making the definition of x-zone specific transcripts more challenging. Objective Use RNA sequencing (RNAseq) to better define the x-zone transcriptome by comparing virgin mice to post-pregnant and aged females. Methods Adrenals from four 25-week-old (wk) virgin, four 25 wk post-pregnant, and four 50 wk female mice were used for RNAseq. Reads were aligned to the mm38 assembly with STAR. Expression was quantified with featureCounts. Normalization was performed with edgeR using the TMM method. Unwanted sources of variation were removed with sva. Statistical comparison was performed using limma. Statistically significant (FDR-adjusted, P&amp;lt;0.05) expression between groups allowed characterization of differentially expressed genes (DEGs). Results RNAseq allowed the analysis of adrenal gland RNA transcripts and identified 17,829 genes that met read count thresholds. 246 DEGs were found between virgin and post-pregnant females with 93 showing higher expression in virgin adrenals and 153 with higher expression in post-pregnant female adrenals. The most altered genes included Akr1c18 (fold change (FC) = 174.06; higher in virgin adrenals) and the proton transporter, Otop1 (FC = 20.82; higher in post-pregnant adrenals). When comparing 25 wk to 50 wk adrenals, 269 DEGs were found with 136 showing higher expression in 25 wk adrenals and 133 higher in the 50 wk adrenals. The most altered genes were Akr1c18 (FC = 34.17; higher in 25 wk adrenals) and the mouse mast cell protease, Mcpt2 (FC = 14.42; higher in 50 wk adrenals). The 25 wk virgin mice had 22 transcripts that were higher and 41 that were lower than found in both post-pregnant and 50 wk animals. Within this list are the established x-zone markers: Akr1c18 and the phosphoinositide 3-kinase, Pik3c2g. Discussion Clear differences in transcript levels were observed between virgin mice and those who had underwent pregnancy or aged to 50 wk. Both aged and post-pregnant mice showed lower expression of known x-zone transcripts and RNAseq analysis provided a tentative list of additional x-zone markers. Using female mice with differing x-zone status reduced the number of sexually dimorphic genes and allowed the characterization of genes related to the x-zone. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.

Maternal obesity exacerbates the responsiveness of offspring BALB/c mice to cow’s milk protein-induced food allergy

Food allergy has become a significant public health problem affecting a large number of people worldwide. Maternal obesity causes inflammation and alters the immune system of offspring, which may exacerbate their food allergy. The aim of this study was to determine whether offspring mice born to obese mothers would have more serve reactions to cow’s milk protein-induced food allergy, and further investigate the underlying mechanisms. Female offspring BALB/c mice of mothers with normal and high-fat diets were sensitized with β-lactoglobulin (BLG), respectively. Maternal obesity increased the serum immunoglobulin E and mouse mast cell protease levels, though did not have significant influence on anaphylactic symptom score, core temperature and diarrhea rate of offspring mice after BLG sensitization. Furthermore, maternal obesity led to a lower level of occludin mRNA expression in BLG -sensitized mice. The mice born to obese mothers exhibited increased mRNA expression levels of GATA-3, interleukin (IL)-4 and IL-10 in jejunum after BLG sensitization, indicating maternal obesity intensified Th2-type biased immune responses. In conclusion, maternal obesity exerted exacerbating effects on the responsiveness of their offspring to cow’s milk protein sensitization.

Oral administration of Forsythiasides-rich extract from Forsythiae fructus decreases the IgE production through suppressing Th2 response in shrimp protein-sensitized BALB/c mice

BackgroundIgE-mediated allergic diseases are characterized by an overreaction of type 2 response which promotes B cell differentiation and IgE production via Th2 cytokines. Forsythiae Fructus, a well-known traditional Chinese medicine, has been clinically used for relieving IgE-mediated type I allergy by continuous treatment for several days. PurposeWe aimed to investigate the effect of continuous oral administration with Forsythiasides-rich extract (FRE) from Forsythiae Fructus on type 2 response and the underlying mechanism in shrimp protein (SP)-sensitized mice. Study design and methodsWe established a SP-sensitized mice model and investigated effect of FRE both in vivo and ex vivo. Serum total IgE (tIgE), specific IgE (sIgE), mouse mast cell protease 1 (mMCP-1) and supernatant Th2 cytokines (IL-4, IL-5 and IL-13) were determined by ELISA. The cell viability was examined by MTS assay. ResultsContinuous oral administration of FRE could significantly lower the levels of serum tIgE, sIgE and mMCP-1 in SP-sensitized mice. Ex vivo study indicated that a single treatment of FRE could potently suppress the release of Th2 cytokines (IL-4, IL-5 and IL-13) in isolated mesenteric lymph nodes cells and splenocytes of SP-sensitized mice in a concentration-dependent manner. ConclusionFRE can suppress Th2 cytokines (IL-4, IL-5 and IL-13) to decrease the production of IgE in SP-sensitized mice. In view of its another ability to stabilize mast cells, Forsythiae Fructus is reasonably suitable for treating IgE-mediated allergic diseases.

Co-fermented cow milk protein by Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 attenuates its allergic immune response in Balb/c mice.

Milk protein is one of the major food allergens. As an effective processing method, fermentation may reduce the potential allergenicity of allergens. This study aimed to evaluate the therapeutic potential of co-fermented milk protein using Lactobacillus helveticus KLDS 1.8701 and Lactobacillus plantarum KLDS 1.0386 in cow milk protein allergy (CMPA) management. This study determined the secondary and tertiary structures of the fermented versus unfermented proteins by Fourier-transform infrared spectroscopy and surface hydrophobicity to evaluate its conformational changes. Our results showed that different fermentation methods have significantly altered the conformational structures of the cow milk protein, especially the tertiary structure. Further, the potential allergenicity of the fermented cow milk protein was assessed in Balb/c mice, and mice treated with the unfermented milk and phosphate-buffered saline were used as a control. We observed a significant reduction in allergenicity via the results of the spleen index, serum total IgE, specific IgE, histamine, and mouse mast cell protease 1 in the mice treated with the co-fermented milk protein. In addition, we analyzed the cytokines and transcription factors expression levels of spleen and jejunum and confirmed that co-fermentation could effectively reduce the sensitization of cow milk protein by regulating the imbalance of T helper (Th1/Th2 and Treg/Th17). This study suggested that changes of conformational structure could reduce the potential sensitization of cow milk protein; thus, fermentation may be a promising strategy for developing a method of hypoallergenic dairy products.

Bisdemethoxycurcumin attenuates OVA-induced food allergy by inhibiting the MAPK and NF-κB signaling pathways.

Bisdemethoxycurcumin (BDMC) is an important ingredient derived from turmeric in addition to curcumin. It has been reported that BDMC can be used to treat mast cell-mediated allergic diseases. In the present study, a food allergy (FA) murine model sensitized by intraperitoneal injection followed by oral challenge with ovalbumin (OVA) was established. BDMC was orally administered at 100 and 200 mg/kg for 11 days in the challenge phase to treat OVA-induced FA mice. FA symptoms such as diarrhea score, anaphylactic symptom score and rectal temperature were recorded. Intestinal tissue was also observed by hematoxylin and eosin staining. In addition, other allergic indicators were also analyzed by ELISA and western blot analysis. The present study demonstrated that BDMC could suppress the decreases in rectal temperature, diarrhea and anaphylactic symptoms in FA mice. BDMC could also ameliorate the inflammation of intestinal tissues in FA mice. BDMC not only decreased the production of OVA-specific immunoglobulin (OVA-sIg)E, IgG1, histamine, mouse mast cell protease-1, diamine oxidase, cytokines (IL-4, IL-5 and IL-13) but increased cytokines interferon-γ production. The protein expression results showed that the levels of Gata-3 were decreased but T-bet levels were increased. Furthermore, compared with the OVA group, phosphorylated (p)-p38, p-JNK, p-ERK and p-NF-κBp65 levels were decreased and p-IκBα level was increased. In conclusion, the results showed that BDMC possessed a protective effect on FA. Furthermore, BDMC was able to regulate the T-helper cells (Th)1/Th2 immune balance and inhibit the activation of MAPK and NF-κB pathways in FA mice.

House dust mite exposure enhances immune responses to ovalbumin-induced intestinal allergy

House dust mites (HDM) are one of the important factors of airway allergic diseases, HDM allergens can be detected in the human gut mucosa, which induces local inflammation and increases intestinal epithelial permeability. This study tests a hypothesis that HDM contribute to the development of OVA (ovalbumin)-induced intestinal allergy. The serum levels of IgE against HDM in patients with food allergy were detected with UniCAP100 (Pharmacia, Uppsala, Sweden); a mouse model of food allergy was developed with OVA and HDM as the specific antigens. Compared to healthy controls, patients with food allergy have higher levels of serum HDM-specific IgE. Compared to food allergy alone groups, the levels of HDM-specific IgE in patients with food allergy and asthma or allergic rhinitis were significantly higher. In mouse models, we found that HDM/OVA induced allergy-like symptoms, lower body temperature, and lower body weight. The levels of IgE, IgG1, mMCP-1 (mouse mast cell protease-1), IL-4 and IL-5 in the HDM and HDM + CT (cholera toxin) groups were higher than the control groups, and the levels of IgE, IgG1, IL-4 and IL-5 in the HDM, OVA and HDM + OVA groups were higher than the control groups. The pathological changes of intestinal tissues in the HDM and HDM + CT/the HDM, OVA and HDM + OVA groups were more severe, more eosinophil infiltration than the control groups. Moreover, exposure to HDM induced intestinal barrier dysfunction, and facilitated the development of intestinal allergy in mice. In conclusion, HDM exposure enhances immune responses to OVA-induced food allergy.

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi.

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4 -/-) mice and Mcpt4 +/+ C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4 -/- mice compared with Mcpt4 +/+ controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4 -/- mice. Relative to infected Mcpt4 +/+ mice, ileal MC accumulation in Mcpt4 -/- mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4 +/+ but not Mcpt4 -/- mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-α, IL-12p40 and IL-3 were observed in Mcpt4 -/- mice, while levels of IL-2, IL-10 and MIP1β (CCL4) were increased over the same period in Mcpt4 +/+ mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4 -/- mice and type-2 response in Mcpt4 +/+ mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4 -/- parasitemia with TNF-α and IFN-γ, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4 +/+ mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4 -/- mice compared with infected Mcpt4 +/+ mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-α and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility.

MMCP7, a Mouse Ortholog of δ Tryptase, Mediates Pelvic Tactile Allodynia in a Model of Chronic Pelvic Pain.

Chronic prostatitis/Chronic pelvic pain syndrome (CP/CPPS) is a condition that affects a large number of men and has unknown etiology. We have previously demonstrated the presence of elevated levels of mast cell tryptase in expressed prostatic secretions (EPS) of CP/CPPS patients. In a murine model of CP/CPPS, we showed tryptase and its cognate receptor PAR2 as critical to the development of pelvic pain and lower urinary tract symptoms. Here, we extend these observations to demonstrate that an isoform of tryptase called delta (δ)-tryptase, is elevated in the EPS of patients with CP/CPPS and is correlated with pelvic pain symptoms. Using an Escherichia coli (CP1) -induced murine model of CP/CPPS, we demonstrated a differential response in C57BL/6J and NOD/ShiLtJ mice, with C57BL6/J mice being resistant to an increase in pelvic tactile allodynia, despite having equivalent levels of activated mast cells in the prostate. Activated tryptase+ve mast cells were observed to be in closer apposition to PGP9.5+ve nerve fibers in the prostate stroma of NOD/ShiLtJ in comparison to C57BL/6J mice. The mouse ortholog of δ-tryptase, mouse mast cell protease 7 (mMCP7) has been reported to be unexpressed in C57BL/6J mice. We confirmed the absence of mMCP7 in the prostates of C57BL/6J and its presence in NOD/ShiLtJ mice. To evaluate a role for mMCP7 in the differential allodynia responses, we performed direct intra-urethral instillations of mMCP7 and the beta (β)-tryptase isoform ortholog, mMCP6 in the CP1-infection model. mMCP7, but not mMCP6 was able to induce an acute pelvic allodynia response in C57BL/6J mice. In-vitro studies with mMCP7 on cultured mast cells as well as dissociated primary neurons demonstrated the ability to induce differential activation of pain and inflammation associated molecules compared to mMCP6. We conclude that mMCP7, and possibility its human ortholog δ-tryptase, may play an important role in mediating the development of pelvic tactile allodynia in the mouse model of pelvic pain and in patients with CP/CPPS.

Interleukin-18 Receptor α Modulates the T Cell Response in Food Allergy.

PurposeThe prevalence of food allergy, triggered by T-helper type 2 (Th2) cell-mediated inflammation, is increasing worldwide. Interleukin (IL)-18 plays an important role in inflammatory diseases by binding with the IL-18 receptor. IL-18/IL-18 receptor α (IL-18Rα) is a cofactor for immunoglobulin E (IgE) production and Th2 cell development. Studies have not investigated the association between the IL-18/IL-18Rα signaling pathway and food allergy. Here, we investigated the role of IL-18Rα in food allergy induction and development.MethodsWild-type (WT) and IL-18Rα-null mutant (IL-18Rα−/−) C57BL/6 mice were sensitized and challenged using ovalbumin (OVA) for food allergy induction. Food allergy symptoms, T cell-mediated immune responses, and signal transducer and activator of transcription (STAT)/suppressors of cytokine signaling (SOCS) pathways were analyzed in mice.ResultsIL-18Rα expression was increased in WT mouse intestines after OVA treatment. Food allergy-induced IL-18Rα−/− mice showed attenuated systemic food allergic reactions, OVA-specific IgE and mouse mast cell protease-1 production, inflammatory cell infiltration, and T cell activation. Ex vivo experiments showed that cell proliferation and Th2 cytokine production were lower in IL-18Rα−/− mouse splenocytes than in WT mouse splenocytes. IL-18Rα blockade in WT splenocytes attenuated cell proliferation and Th2 cytokine production. Moreover, STAT3 phosphorylation was reduced in IL-18Rα−/− mice, and SOCS3 and SOCS1 activation were diminished in IL-18Rα−/− intestinal T cells.ConclusionsIL-18Rα regulates allergic reactions and immune responses by regulating T cell responses in food allergies. Moreover, IL-18Rα is involved in the STAT/SOCS signaling pathways. Targeting IL-18Rα signaling might be a novel therapeutic strategy for food allergy.

Mucosal Mast Cell-Specific Gene Expression Is Promoted by Interdependent Action of Notch and TGF-β Signaling.

Rodent mast cells are classified into two major subsets, mucosal mast cells (MMCs) and connective tissue mast cells. MMCs arise from mast cell progenitors that are mobilized from the bone marrow to mucosal tissues in response to allergic inflammation or helminth infection. TGF-β is known as an inducer of MMC differentiation in mucosal tissues, but we have previously found that Notch receptor-mediated signaling also leads to the differentiation. Here, we examined the relationship between Notch and TGF-β signaling in MMC differentiation using mouse bone marrow-derived mast cells (BMMCs). We found that the coexistence of Notch and TGF-β signaling markedly upregulates the expression of MMC markers, mouse mast cell protease (mMCP)-1, mMCP-2, and αE integrin/CD103, more than Notch or TGF-β signaling alone, and that their signals act interdependently to induce these marker expressions. Notch and TGF-β-mediated transcription of MMC marker genes were both dependent on the TGF-β signaling transducer SMAD4. In addition, we also found that Notch signaling markedly upregulated mMCP-1 and mMCP-2 expression levels through epigenetic deregulation of the promoter regions of these genes, but did not affect the promoter of the CD103-encoding gene. Moreover, forced expression of the constitutively active Notch2 intracellular domain in BMMCs showed that Notch signaling promotes the nuclear localization of SMADs 3 and 4 and causes SMAD4-dependent gene transcription. These findings indicate that Notch and TGF-β signaling play interdependent roles in inducing the differentiation and maturation of MMCs. These roles may contribute to the rapid expansion of the number of MMCs during allergic mucosal inflammation.

Immunotherapy With Recombinant Alt a 1 Suppresses Allergic Asthma and Influences T Follicular Cells and Regulatory B Cells in Mice.

Background Alternaria is a major source of asthma-inducing allergens. Allergen-specific immunotherapy improves the progression of allergic asthma. The current treatment is based on crude Alternaria extracts. Alt a 1 is the predominant allergen in Alternaria. However, the treatment efficacy of recombinant Alt a 1 (rAlt a 1) in an asthmatic animal model and its influence on Tfh and Breg cells are unknown.ObjectiveTo explore the therapeutic treatment effects of rAlt a 1 on the progress of an asthmatic mouse model and its effect on Tfh and Breg cells.MethodsWe synthesized and purified rAlt a 1. Alternaria-sensitized and challenged mice received subcutaneous immunotherapy (SCIT) with four different rAlt a 1 dosages (5, 50, 100, and 150 µg) or PBS only. Finally, lung and airway inflammation, mouse mast cell protease 1 (MMCP-1), serum immunoglobulin responses, Tfh and Breg cell levels, and the correlation between asthmatic features (inflammation grades and IL-4 and IL-10 levels) and these two cell types were measured after Alternaria rechallenge.ResultsHigh purity and allergenic potency of rAlt a 1 protein were obtained. Following treatment with four different rAlt a 1 dosages, both lung and airway inflammation ameliorated, including lung pathology, serum MMCP-1 levels, inflammatory cell numbers, and cytokine levels in bronchoalveolar lavage fluid (BALF). Additionally, rAlt a 1-SCIT increased the expression of Alternaria-sIgG1, rAlt a 1-sIgG1, rAlt a 1-sIgG2a, and rAlt a 1-sIgG2b in serum. Moreover, the number and percentage of CXCR5+PD-1+Tfh cells were increased in the PC control, while they decreased in the rAlt a 1-SCIT groups. Meanwhile, the absolute numbers and proportions of Breg cells were evaluated after administration of rAlt a 1. A positive correlation was observed between CXCR5+PD-1+Tfh cells and inflammation grades (r = 0.50, p = 0.01), as well as a slightly strong positive relationship with IL-4 (r = 0.55, p = 0.005) and IL-10 (r = 0.58, p = 0.003) levels; Breg cells showed an opposite correlation with the grades of inflammation (r = -0.68, p = 0.0003), along with a negative correlation to IL-4 (r = -0.61, p = 0.001) and IL-10 (r = -0.53, p = 0.008) levels.ConclusionsWe verified that treatment with rAlt a 1 can alleviate asthma progression and further have a regulatory effect on Tfh and Breg cells in an Alternaria-induced asthmatic mouse model.

Compound Kushen Injection Induces Immediate Hypersensitivity Reaction Through Promoting the Production of Platelet-Activating Factor via de Novo Pathway.

Compound Kushen Injection (CKI) is a bis-herbal formulation extracted from Kushen (Radix Sophorae Flavescentis) and Baituling (Rhizoma Heterosmilacis Yunnanensis). Clinically, it is used as the adjuvant treatment of cancer. However, with the increased application, the cases of immediate hypersensitivity reactions (IHRs) also gradually rise. In this study, we investigated the underlying mechanism(s) and active constituent(s) for CKI-induced IHRs in experimental models. The obtained results showed that CKI did not elevate serum total IgE (tIgE) and mouse mast cell protease 1 (MMCP1) after consecutive immunization for 5 weeks, but could induce Evans blue extravasation (local) and cause obvious hypothermia (systemic) after a single injection. Further study showed that alkaloids in Kushen, especially matrine, were responsible for CKI-induced IHRs. Mechanism study showed that various platelet-activating factor (PAF) receptor antagonists could significantly counter CKI-induced IHRs locally or systemically. In cell system, CKI was able to promote PAF production in a non-cell-selective manner. In cell lysate, the effect of CKI on PAF production became stronger and could be abolished by blocking de novo pathway. In conclusion, our study identifies, for the first time, that CKI is a PAF inducer. It causes non-immunologic IHRs, rather than IgE-dependent IHRs, by promoting PAF production through de novo pathway. Alkaloids in Kushen, especially matrine, are the prime culprits for IHRs. Our findings may provide a potential approach for preventing and treating CKI-induced IHRs.

Effects of enzymatic hydrolysis on the allergenicity of natural cow milk based on a BALB/c mouse model

Cow milk allergy is one of the most prevalent food allergies worldwide, particularly in infants and children. To the best of our knowledge, minimal research exists concerning the antigenicity of cow milk (CM). This study was performed to evaluate the allergenicity of enzymatically hydrolyzed cow milk (HM) in a BALB/c mouse model. The mice were randomly divided into 5 groups (n = 12/group), which were sensitized with phosphate-buffered saline, CM, and HM (Alcalase-, or Protamex-, or Flavorzyme-treated cow milk; Novo Nordisk; AT, PT, FT, respectively), respectively, using cholera toxin as adjuvant on d 0, 7, 14, 21. On d 28, the test mice were orally challenged with phosphate-buffered saline, CM, and HM (AT, PT, or FT) alone. Anaphylactic symptoms were monitored in the mice. Antibody, cytokine, histamine, and mouse mast cell protease-1 (mMCP-1) levels were measured using enzyme-linked immunosorbent assays. In addition, the numbers of T helper (Th)1 and Th2 cells, as well as the proportions of CD4+CD25+Foxp3+ Treg cells, in mouse spleens were detected using flow cytometry. Statistical significance was determined by one-way ANOVA. The results revealed significant differences between CM- and HM-challenged mice. Among these, the clinical scores of HM-challenged mice (AT, 1.50; PT, 2.00; FT, 1.92) were lower than those of CM-challenged mice (positive control, 2.83), but body weight and temperature of HM-challenged mice were higher than those of CM-challenged mice. In addition, significant reductions of allergen-specific IgE, IgG, histamine, and mMCP-1 were showed in HM-challenged mice, especially for histamine, ranging from 171.42 ng/mL to 214.94 ng/mL. Remarkable reductions of IL-4, IL-5, and IL-13 levels, as well as elevations of interferon-γ and IL-10 levels in the spleens of HM-challenged mice were also detected. Moreover, the number of Th2 cells decreased in the HM-challenged mice, to 2.36% (AT), 1.79% (PT), and 4.03% (FT), respectively, whereas the numbers of Th1 cells (AT, 6.30%; PT, 6.70%; FT, 6.56%) and the proportions of CD4+CD25+Foxp3+Tregs (AT, 8.86%; PT, 9.21%; FT, 9.16%) increased significantly. Our findings indicate that exposure to HM was sufficient to induce a shift toward a Th1 response, thereby reducing potential allergenicity. Importantly, these results will lay a theoretical foundation for the development of hypoallergenic CM products.