Mast Cell-deficient Mice Research Articles

Mast cells promote choroidal neovascularization in a model of age-related macular degeneration

‘Wet’ age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (KitW−sh/W−sh) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in ‘wet’ AMD.

TRPV1 and mast cell involvement in repeated variate stress-induced urinary bladder dysfunction in adult female mice.

The etiology of interstitial cystitis/bladder pain syndrome (IC/BPS) is unknown but likely multifactorial. IC/BPS symptoms can be exacerbated by psychological stress, but underlying mechanisms remain to be defined. Transient receptor potential vanilloid 1 (TRPV1) channels, expressed on nerve fibers, have been implicated in bladder dysfunction and colonic hypersensitivity with stress in rodents. Histamine/H1R activation of TRPV1+ nerves increases bladder afferent fiber sensitivity to distension. TRPV1 channels are also expressed on mast cells, previously implicated in contributing to IC/BPS etiology and symptoms. We have examined the contribution of TRPV1 and mast cells to bladder dysfunction after repeated variate stress (RVS). RVS increased (P ≤ 0.05) serum and fecal corticosterone expression and induced anxiety-like behavior in wild-type (WT) mice. Intravesical instillation of the selective TRPV1 antagonist capsazepine (CPZ) rescued RVS-induced bladder dysfunction in WT mice. Trpv1 knockout (KO) mice did not increase voiding frequency with RVS and did not exhibit increased serum corticosterone expression despite exhibiting anxiety-like behavior. Mast cell-deficient mice (B6.Cg-Kitw-sh) failed to demonstrate RVS-induced increased voiding frequency or serum corticosterone expression, whereas control (no stress) mast cell-deficient mice had similar functional bladder capacity to WT mice. TRPV1 protein expression was significantly increased in the rostral lumbar (L1-L2) spinal cord and dorsal root ganglia (DRG) in WT mice exposed to RVS, but no changes were observed in lumbosacral (L6-S1) spinal segments or DRG. These studies demonstrated TRPV1 and mast cell involvement in RVS-induced increased voiding frequency and suggest that TRPV1 and mast cells may be useful targets to mitigate stress-induced urinary bladder dysfunction.NEW & NOTEWORTHY Using pharmacological tools and transgenic mice in a repeated variate stress (RVS) model in female mice, we demonstrate that transient receptor potential vanilloid 1 (TRPV1) and mast cells contribute to the increased voiding frequency observed following RVS. TRPV1 and mast cells should continue to be considered as targets to improve bladder function in stress-induced bladder dysfunction.

The decrease in peripheral blood basophils in a mouse model of IgE-induced inflammation involves their migration to lymph nodes

BackgroundDuring the active phase of urticaria, a decrease in peripheral blood basophils, known as basopenia, is observed. We previously reported that basopenia occurs as a result of basophils migrating to the skin in a contact dermatitis model where a Th2 response is induced with oxazolone. ObjectiveAlthough there is currently no established model for urticaria, given that urticaria is an IgE-mediated immediate-type allergic reaction, we aimed to determine whether an IgE-mediated model could reproduce the decrease in basophils in peripheral blood observed during the active phase of urticaria. MethodsMice were pretreated with 2,4,6-trinitrophenylhaptene (TNP)-specific IgE and basophil dynamics were examined following stimulation with TNP-ovalbumin. Mast cell-deficient WBB6F1-KitW/KitW-v mice were used to investigate the role of mast cells in this IgE-mediated model. ResultsFollowing stimulation, we observed immediate ear swelling and basopenia after 0.5 hours. However, the number of basophils observed in the skin lesions was low, while a higher number of basophils were observed in the antigen-draining lymph nodes (LN). In mast cell-deficient mice, no increase in basophils in the LN was observed, reflecting reduced antigen influx into the LN, but basophils remained in the skin. ConclusionsIn the IgE-mediated mouse model, basopenia was observed, which coincided with the induction of inflammation in the skin. The migration of basophils to the LN in this model suggests that the systemic immune system, including the LN, should be considered when exploring the pathogenesis of urticaria in humans.

Mrgprb2-mediated mast cell activation exacerbates Modic changes by regulating immune niches

Modic changes are radiographic features associated with microfracture, low-virulence organism infection and chronic inflammation with inflammatory cell infiltration in the vertebral endplate region. Mast cells, as innate immune cells similar to macrophages, are present in painful degenerated intervertebral discs. However, the involvement and mechanisms of mast cells in the development of Modic changes remain unclear. Herein, we found increased mast cell infiltration in samples from patients with Modic changes and in mouse models of Modic changes. To clarify the role of mast cells in the progression of Modic changes, we used mast cell-deficient (KITW-SH/W-SH) mice to construct a model of Modic changes and found that the severity of Modic changes in KITW-SH/W-SH mice was significantly lower than that in WT mice. These findings were further supported by the use of a mast cell-specific activator (compound 48/80) and a stabilizer (cromolyn). Furthermore, we found that mast cells were not activated via the classic IgE pathway in the Modic change models and that Mrgprb2 is the specific receptor for mast cell activation reported in recent studies. Then, we utilized Mrgprb2 knockout mice to demonstrate that Mrgprb2 knockout inhibited mast cell activation and thus reduced the degree of Modic changes. Transcriptomic sequencing revealed aberrant PI3K-AKT and MAPK pathway activation in the Mrgprb2-deficient mast cells. Additionally, Mrgpbrb2-activated mast cells regulate immune niches by recruiting macrophages, promoting M1 polarization and reducing M2 polarization, thereby promoting the progression of Modic changes. These findings suggest that mast cells may serve as a novel therapeutic target for addressing Modic changes.

Mast cell-derived interleukin-4 mediates activation of dendritic cell during toll-like receptor 2-mediated inflammation.

During an innate inflammation, immune cells form distinct pro- and anti-inflammatory regions around pathogen-containing core-regions. Mast cells are localized in an anti-inflammatory microenvironment during the resolution of an innate inflammation, suggesting antiinflammatory roles of these cells. High-content imaging was used to investigated mast cell-dependent changes in the regional distribution of immune cells during an inflammation, induced by the toll-like receptor (TLR)-2 agonist zymosan. The distance between the zymosan-containing core-region and the anti-inflammatory region, described by M2-like macrophages, increased in mast cell-deficient mice. Absence of mast cells abolished dendritic cell (DC) activation, as determined by CD86-expression and localized the DCs in greater distance to zymosan particles. The CD86- DCs had a higher expression of the pro-inflammatory interleukins (IL)-1β and IL-12/23p40 as compared to activated CD86+ DCs. IL-4 administration restored CD86 expression, cytokine expression profile and localization of the DCs in mast cell-deficient mice. The IL-4 effects were mast cell-specific, since IL-4 reduction by eosinophil depletion did not affect activation of DCs. We found that mast cells induce DC activation selectively at the site of inflammation and thereby determine their localization within the inflammation. Overall, mast cells have antiinflammatory functions in this inflammation model and limit the size of the pro-inflammatory region surrounding the zymosan-containing core region.

Mast cells help organize the Peyer's patch niche for induction of IgA responses.

Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFβ-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.

Immunomodulatory contribution of mast cells to the regenerative biomaterial microenvironment

Bioactive immunomodulatory biomaterials have shown promise for influencing the immune response to promote tissue repair and regeneration. Macrophages and T cells have been associated with this response; however, other immune cell types have been traditionally overlooked. In this study, we investigated the role of mast cells in the regulation of the immune response to decellularized biomaterial scaffolds using a subcutaneous implant model. In mast cell-deficient mice, there was dysregulation of the expected M1 to M2 macrophage transition typically induced by the biomaterial scaffold. Polarization progression deviated in a sex-specific manner with an early transition to an M2 profile in female mice, while the male response was unable to properly transition past a pro-inflammatory M1 state. Both were reversed with adoptive mast cell transfer. Further investigation of the later-stage immune response in male mice determined a greater sustained pro-inflammatory gene expression profile, including the IL-1 cytokine family, IL-6, alarmins, and chemokines. These results highlight mast cells as another important cell type that influences the immune response to pro-regenerative biomaterials.

Preliminary studies on the role of mast cells in the phenotype and immune activation of type 2 inflammatory cytokine release of atopic dermatitis

Objective: To investigate the role of mast cells in atopic dermatitis (AD) phenotype and the immune activation of type 2 inflammatory cytokine release. Methods: Nine AD skin samples were obtained from the Department of Dermatology, the Second Affiliated Hospital of Xi'an Jiaotong University, and nine healthy skin control samples were obtained from the surgical excision of excess normal skin by orthopedic surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, which were subjected to toluidine blue staining and fluorescence staining to clarify the mast cell degranulation activation status of the AD skin lesions. We investigated whether MC903 could directly activate mast cells in vivo through the toe swelling and exudation assay in wild-type mice; we constructed the MC903-AD model using wild-type and KitW-sh/W-sh mast cell-deficient mice in order to investigate whether mast cells affected the phenotype, histopathology, and the level of type 2 inflammatory factors in AD mice; we extracted mouse peritoneal mast cells and the ability of MC903 to activate mast cells to release inflammatory mediators in vitro was explored by calcium imaging, tryptase and β-aminohexokinase release assays, and MCP-1 and CXCL-2 release assays. Results: The number of degranulated mast cells in an activated state was increased in skin lesions of AD patients compared to healthy controls, with (5.40±1.14) and (2.20±0.84), respectively (P<0.001). KitW-sh/W-sh mast cell-deficient AD mice had an attenuated phenotype with ADI scores of (5.50±1.05), compared to wild-type AD mice with (10.00±0.89) (P<0.001). The release of type 2 inflammatory factors in wild-type AD mice was higher than those in KitW-sh/W-sh mast cell-deficient AD mice, with IL-4 levels of (29.50±1.87) and (15.33±1.86) pg/mg (P<0.001), IL-13 levels were (6.32±0.25) and (3.93±0.22) pg/mg (P<0.001), IL-31 levels were (9.73±0.38) and (6.89±0.27) pg/mg (P<0.001), and TSLP levels were (206.00±4.43) and (99.00±4.86) pg/mg (P<0.001), respectively. MC903 could cause mast cell activation in wild-type mice, leading to increased swelling and exudation in the toes of mice, and MC903 could activate mast cells in vitro, leading to increased degranulation and release of inflammatory factors such as MCP-1 and CXCL-2. Conclusions: The number of activated mast cells was increased in skin lesions of AD patients than in healthy controls. KitW-sh/W-sh mast cell-deficient AD mice showed significantly reduced phenotype, histopathology, and type 2 inflammatory factor levels compared with wild-type AD mice. MC903 activates mast cells in vivo and in vitro. Mast cells play a key role in AD phenotype and immune activation.

The mast cell stimulator compound 48/80 causes urothelium-dependent increases in murine urinary bladder contractility.

Mast cells and degranulation of preformed inflammatory mediators contribute to lower urinary tract symptoms. This study investigated pathways by which the mast cell stimulator compound 48/80 alters urinary bladder smooth muscle contractility via mast cell activation. We hypothesized that 1) mast cell degranulation causes spontaneous urinary bladder smooth muscle contractions and 2) these contractions are caused by urothelium-derived PGE2. Urothelium-intact and -denuded urinary bladder strips were collected from mast cell-sufficient (C57Bl/6) and mast cell-deficient (B6.Cg-Kitw-sh) mice to determine if compound 48/80 altered urinary bladder smooth muscle (UBSM) contractility. Electrical field stimulation was used to assess the effects of compound 48/80 on nerve-evoked contractions. Antagonists/inhibitors were used to identify prostanoid signaling pathways activated or if direct activation of nerves was involved. Compound 48/80 caused slow-developing contractions, increased phasic activity, and augmented nerve-evoked responses in both mast cell-sufficient and -deficient mice. Nerve blockade had no effect on these responses; however, they were eliminated by removing the urothelium. Blockade of P2 purinoreceptors, cyclooxygenases, or G protein signaling abolished compound 48/80 responses. However, only combined blockade of PGE2 (EP1), PGF2α (FP), and thromboxane A2 (TP) receptors inhibited compound 48/80-induced responses. Thus, the effects of compound 48/80 are urothelium dependent but independent of mast cells. Furthermore, these effects are mediated by druggable inflammatory pathways that may be used to manage inflammatory nonneurogenic bladder hyperactivity. Finally, these data strongly suggest that great care must be taken when using compound 48/80 to determine mast cell-dependent responses in the urinary bladder.NEW & NOTEWORTHY Urothelial cells are first responders to noxious contents of the urine. Our study demonstrates that the urothelium is not only a barrier but also a modulator of urinary bladder smooth muscle phasic activity and contractility independent of immune cell recruitment in response to an inflammatory insult.

Mast cells selectively produce inflammatory mediators and impact the early response to Chlamydia reproductive tract infection.

Chlamydia trachomatis (C. trachomatis) is a Gram-negative obligate intracellular bacterium that causes reproductive tract complications in women, including ectopic pregnancies and tubal factor infertility. We hypothesized that mast cells, which are common at mucosal barriers, may contribute to responses to Chlamydia infection and aimed to define human mast cell responses to C. trachomatis. Human cord blood-derived mast cells (CBMCs) were exposed to C. trachomatis to assess bacterial uptake, mast cell degranulation, gene expression, and production of inflammatory mediators. The role of formyl peptide receptors and Toll-like receptor 2 (TLR2) were investigated using pharmacological inhibitors and soluble TLR2. Mast cell-deficient mice and littermate controls were used to examine the in vivo role of mast cells in influencing the immune response to Chlamydia infection in the female reproductive tract. C. trachomatis bacteria were taken up by human mast cells but did not replicate efficiently inside CBMCs. C. trachomatis-activated mast cells did not degranulate but maintained viability and exhibited cellular activation with homotypic aggregation and upregulation of ICAM-1. However, they significantly enhanced the gene expression of IL1B, CCL3, NFKB1, CXCL8, and IL6. Inflammatory mediators were produced, including TNF, IL-1β, IL-1RA, IL-6, GM-CSF, IL-23, CCL3, CCL5, and CXCL8. Endocytic blockade resulted in reduced gene expression of IL6, IL1B, and CCL3, suggesting C. trachomatis induced mast cell activation in both extracellular and intracellular locations. The IL-6 response to C. trachomatis was reduced when CBMCs were treated with C. trachomatis coated with soluble TLR2. Mast cells derived from TLR2-deficient mice also demonstrated a reduced IL-6 response to C. muridarum. Five days following C. muridarum infection, mast cell-deficient mice showed attenuated CXCL2 production and significantly reduced numbers of neutrophils, eosinophils, and B cells in the reproductive tract when compared with mast cell-containing littermates. Taken together, these data demonstrate that mast cells are reactive to Chlamydia spp. through multiple mechanisms that include TLR2-dependent pathways. Mast cells also play an important role in shaping in vivo immune responses in Chlamydia reproductive tract infection through both effector cell recruitment and modification of the chemokine microenvironment.

Increased activity of lacrimal gland mast cells are associated with corneal epitheliopathy in aged mice

The lacrimal gland undergoes significant structural and functional deterioration with aging. Marked with increased inflammation and fibrosis, the aged lacrimal gland is unable to perform its protective function. As a result, the ocular surface becomes highly susceptible to various ocular surface pathologies, including corneal epitheliopathy. We and others have previously shown that mast cells mediate tissue inflammation by recruiting other immune cells. However, despite their well-known characteristics of secreting various inflammatory mediators, whether mast cells contribute to the immune cell aggregation and activation, and acinar dystrophy of the aged lacrimal gland has not been investigated. Here, we demonstrate the role of mast cells in age-related lacrimal gland pathophysiology using mast cell-deficient (cKitw-sh) mice. Our data demonstrated a significant increase in mast cell frequencies and immune cell infiltration in the lacrimal gland of aged mice. Interestingly, mast cell deficiency resulted in a substantial reduction in inflammation and preservation of lacrimal gland structure, suggesting that mast cells mediate the aging process of the lacrimal gland.

Compound 48/80 increases murine bladder wall compliance independent of mast cells

A balance between stiffness and compliance is essential to normal bladder function, and changes in the mechanical properties of the bladder wall occur in many bladder pathologies. These changes are often associated with the release of basic secretagogues that in turn drive the release of inflammatory mediators from mast cells. Mast cell degranulation by basic secretagogues is thought to occur by activating an orphan receptor, Mas-related G protein-coupled receptor B2 (Mrgprb2). We explored the effects of the putative mast cell degranulator and Mrgprb2 agonist Compound 48/80 on urinary bladder wall mechanical compliance, smooth muscle contractility, and urodynamics, and if these effects were mast cell dependent. In wild-type mice, Mrgprb2 receptor mRNA was expressed in both the urothelium and smooth muscle layers. Intravesical instillation of Compound 48/80 decreased intermicturition interval and void volume, indicative of bladder overactivity. Compound 48/80 also increased bladder compliance while simultaneously increasing the amplitude and leading slope of transient pressure events during ex vivo filling and these effects were inhibited by the Mrgprb2 antagonist QWF. Surprisingly, all effects of Compound 48/80 persisted in mast cell-deficient mice, suggesting these effects were independent of mast cells. These findings suggest that Compound 48/80 degrades extracellular matrix and increases urinary bladder smooth muscle excitability through activation of Mrgprb2 receptors located outside of mast cells. Thus, the pharmacology and physiology of Mrgprb2 in the urinary bladder is of potential interest and importance in terms of treating lower urinary tract dysfunction.

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion.

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.

Skin Treatment with Detergent Induces Dermatitis with H1-Antihistamine-Refractory Itch and Upregulates IL-4 and Th17/Th22 Cytokine Gene Expression in C57BL/6 Mice

Introduction: Repeated skin contact to detergents causes chronic irritant contact dermatitis (ICD) associated with itch sensation and eczema. However, the mechanisms of detergent-induced ICD are poorly understood. Here, we established a new murine model of detergent-induced ICD with H1-antihistamine-refractory itch. Methods: Ear skin of wild-type and mast cell-deficient mice on the C57BL/6 genetic background was treated with a detergent, sodium dodecyl/lauryl sulfate (SDS), daily for approximately 2 weeks with or without administration of an H1-antihistamine, fexofenadine. Skin inflammation, barrier dysfunction, and itching were analyzed. Quantitative PCR for earlobe gene expression and flow cytometry analysis for draining lymph node cells were conducted. Results: SDS treatment induced skin inflammation with ear swelling, increased transepidermal water loss, and hind-paw scratching behaviors in the wild-type and mast cell-deficient mice. The peak value of scratching bouts was retained for at least 48 h after the last SDS treatment. H1-antihistamine administration showed no or little reduction in the responses. SDS treatment upregulated gene expression for a Th2 cytokine IL-4 and Th17/Th22 cytokines, IL-17A, IL-17F, and IL-22, and increased cell numbers in draining lymph nodes of CD4⁺ T, CD8⁺ T, and γδT cells with enhanced expression of GATA3, RORγt, T-bet, or FOXP3 compared with untreated mice. Conclusions: The present study showed that SDS treatment of ear skin in C57BL/6 mice induces mast cell-independent skin inflammation with H1-antihistamine-refractory itch and suggested a possible Th cytokine- and/or lymphocyte-mediated regulation of the model. The model would be useful for elucidation of mechanisms for inflammation with H1-antihistamine-refractory itch in detergent-induced ICD.

Mast cells promote viral entry of SARS-CoV-2 via formation of chymase/spike protein complex

The pulmonary pathological findings associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) result from the release of multiple proinflammatory cytokines, which causes the subsequential damage of the lungs. The current study was undertaken to investigate the responses of mast cells to viral inoculation and their contribution to host defenses from the point of view of viral entry. Pseudovirions, in which the spike glycoprotein of SARS-CoV-2 was incorporated, triggered activation of mast cells, and a mast cell-derived chymase, MCP2, formed a complex with spike protein, which promoted protease-dependent viral entry. According to the quantification results of viral entry, 10 μM quercetin, a mast cell stabilizer, potentially potently inhibited 41.3% of viral entry, while 100 μM chymostatin, which served as a chymase inhibitor, suppressed 52.1% of viral entry, compared to non-treated cells. Study using mast cell-deficient mice showed that the absence of mast cells may influence early viral loading in the upper respiratory tract, which consequently increases the risk of viral invasion into the lower respiratory system. Furthermore, mast cell-deficient mice exhibited ongoing infection in the late phase post-viral inoculation, while clearance of virus-positive cells was observed in wild-type mice. In conclusion, mast cells act as a multifaceted immune modulator that is equipped with both protective effects and pathogenic influences on viral entry of SARS-CoV-2. The utility of mast cell stabilizers and chymase inhibitors in the treatment of SARS-CoV-2-induced acute respiratory syndrome should be optimized regarding the infection stage and the risk of cytokine storm.

Mechanism of Phosgene Oxime Induced Skin Toxicity in C57BL/6 Mouse Model

Phosgene Oxime (CX) is a potent chemical warfare agent. It is grouped with vesicants; however, its effect resembles of an urticant or nettle agent. CX has faster penetration compared to other vesicating chemical warfare agents and causes erythema, urticaria, blanching, itching hives, necrosis, and systemic toxic effects upon exposure. The mechanism of CX induced toxicity is not known and there are no approved therapies. We have shown that dermal CX exposure in SKH‐1 hairless mice causes clinical lesions, epidermal cell death, mast cell degranulation, infiltration of immune cells, and increase in the level of DNA damage and inflammatory markers. In this study we examined the mechanism of CX‐induced inflammatory response in C57BL/6 mice. Dorsal skin of mice was exposed to neat CX for 0.5 or 1.0 min using two 12 mm vapor caps at MRI Global. CX exposure caused decrease in heart rate and breath rate with significant mortality (50%) after 1.0 min CX exposure. CX exposure caused erythema, and edema at the exposure site and led to increase in epidermal thickness. Significant mast cell degranulation was observed within 2h of CX exposure in male and female mice at both the exposure durations. The increase in mast cell degranulation was associated with increased tryptase and chymase levels in the mouse skin. CX exposure also led to increased expression of inflammatory mediators, cyclooxygenase‐2 (COX‐2), matrix metallopeptidase‐9 (MMP‐9), myeloperoxidase (MPO), and MAP kinases JNK. Inflammatory cytokines and chemokines were analyzed in the skin tissue lysates using cytokine array. CX exposure caused increases in several pro‐inflammatory cytokines and chemokines including IL‐6 and TNF‐α, and a decrease in anti‐inflammatory cytokines IL‐4 and IL‐10. Together, these results suggest mast cells as important contributors of CX induced dermal inflammation and toxicity. Further studies are being carried out using WT (C57BL/6) and mast cell deficient mice to evaluate the role of mast cells in CX‐induced toxicity.

Mast Cells Drive Systemic Inflammation and Compromised Bone Repair After Trauma.

There is evidence that mast cells contribute to inflammation induced by hemorrhagic shock, severe tissue injury or sepsis. Mast cells are highly responsive to alarm signals generated after trauma, and release many inflammatory mediators including interleukin-6, a key mediator of posttraumatic inflammation. An overwhelming posttraumatic inflammation causes compromised bone healing; however, the underlying cellular and molecular mechanisms are poorly understood. Recently, we found that mast cells trigger local and systemic inflammation after isolated fracture leading to uneventful bone repair. Here, we investigated whether mast cells critically contribute to trauma-induced compromised bone healing. Male Mcpt5-Cre+ R-DTA mice, which lack connective tissue type mast cells, and their mast cell-competent Cre− littermates underwent a femur fracture with/without thoracic trauma. Posttraumatic systemic and local inflammation and bone repair were assessed 3 h and 21 d post injury. Both, the systemic and pulmonary inflammation was significantly increased in mast cell-competent mice upon combined trauma compared to isolated fracture. In mast cell-deficient mice, the increase of inflammatory mediators in the circulation induced by the severe trauma was abolished. In the bronchoalveolar lavage fluid, the trauma-induced increase of inflammatory cytokines was not reduced, but the neutrophil invasion into the lungs was significantly diminished in the absence of mast cells. Locally in the fracture hematoma, mast cell-competent mice displayed reduced inflammatory mediator concentrations after combined trauma compared to isolated fracture, which was abolished in mast cell-deficient mice. Notably, while combined trauma resulted in compromised bone repair in mast cell-competent mice, indicated by significantly reduced bone and increased cartilage fracture callus contents, this was abolished in Mcpt5-Cre+ R-DTA mice. Therefore, mast cells contribute to trauma-induced compromised bone repair and could be a potential target for new treatment options to improve fracture healing in multiply injured patients.

Mast cell modulates tumorigenesis caused by repeated bowel inflammation condition in azoxymethane/dextran sodium sulfate-induced colon cancer mouse model

Mast cells infiltrate the inflammatory microenvironment and regulate the production of many pro-inflammatory cytokines and mediators of inflammatory cell production to promote tumor development and growth in intestinal lesions. Currently, there are insufficient studies of the mediators and signaling pathways regulated by mast cells that influence the pathogenesis of colon cancer in inflamed colon tissue. This study aimed to confirm the role of mast cells in the incidence and growth of colitis-associated colon cancer (CAC) and to identify inflammation-mediated factors and signaling pathways related to tumor development. CAC was induced by the administration of azoxymethane (AOM) and dextran sodium sulfate (DSS) in mast cell-deficient (WBB6F1/J-W/WV) and mast cell–sufficient control (WBB6F1_+/+) mice. The results confirmed that mast cell-deficient mice exhibited less tumor formation than normal mice under the same conditions, and down-regulated expression of pro-inflammatory cytokines and mediators. Mast cells play an important role in tumor formation by regulating pro-inflammatory cytokines and inflammatory mediators in CAC, indicating that they can act as new targets for the prevention and treatment of CAC.

Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation.

Allergens have been identified as potential triggers in patients with atopic dermatitis (AD). Patients with AD are highly sensitive to cockroach allergen. The underlying mechanism, however, remains undetermined. Here, we established a cockroach allergen–induced AD-like mouse model, and we demonstrate that repeated exposure to cockroach allergen led to aggravated mouse skin inflammation, characterized by increased type 2 immunity, type 2 innate lymphoid cells (ILC2s), and mast cells. Increased mast cells were also observed in patients with AD. Mast cell–deficient mice (KitW-sh/W-sh) showed diminished skin inflammation, suggesting that mast cells are required in allergen-induced skin inflammation. Furthermore, DC immunoreceptor (DCIR) is upregulated in skin mast cells of patients with AD and mediates allergen binding and uptake. DCIR–/– mice or reconstituted KitW-sh/W-sh mice with DCIR–/– mast cells showed a significant reduction in AD-like inflammation. Both in vitro and in vivo analyses demonstrate that DCIR–/– mast cells had reduced IgE-mediated mast cell activation and passive cutaneous anaphylaxis. Mechanistically, DCIR regulates allergen-induced IgE-mediated mast cell ROS generation and oxidation of calmodulin kinase II (ox-CaMKII). ROS-resistant CaMKII (MM-VVδ) prevents allergen-induced mast cell activation and inflammatory mediator release. Our study reveals a DCIR/ROS/CaMKII axis that controls allergen-induced mast cell activation and AD-like inflammation.

Mucosal Serotonin Reuptake Transporter Expression in Irritable Bowel Syndrome Is Modulated by Gut Microbiota Via Mast Cell–Prostaglandin E2

Increased colonic serotonin (5-HT) level and decreased serotonin reuptake transporter (SERT) expression in irritable bowel syndrome (IBS) may contribute to diarrhea and visceral hypersensitivity. We investigated whether mucosal SERT is modulated by gut microbiota via a mast cell-prostaglandin E2 (PGE2) pathway. C57Bl/6 mice received intracolonic infusion of fecal supernatant (FS) from healthy controls or patients with diarrhea-predominant irritable bowel syndrome (IBS-D). The role of mast cells was studied in mast cell-deficient mice. Colonic organoids and/or mast cells were used for invitro experiments. SERT expression was measured by quantitative polymerase chain reaction and Western blot. Visceromotor responses tocolorectal distension and colonic transit were assessed. Intracolonic infusion of IBS-D FS in mice caused an increase in mucosal 5-HT compared with healthy control FS, accompanied by ∼50% reduction in SERT expression. Mast cell stabilizers, cyclooxygenase-2 inhibitors, and PGE2 receptor antagonist prevented SERT downregulation. Intracolonic infusion of IBS-D FS failed to reduce SERT expression in mast cell-deficient (W/Wv) mice. This response was restored by mast cell reconstitution. The downregulation of SERT expression evoked by IBS FS was prevented by lipopolysaccharide (LPS) antagonist LPS from Rhodobacter sphaeroides and a bacterial trypsin inhibitor. Invitro LPS treatment caused increased cyclooxygenase-2 expression and PGE2 release from cultured mouse mast cells. Intracolonic infusion of IBS-D FS in mice reduced colonic transit, increased fecal water content, and increased visceromotor responses to colorectal distension. Ondansetron prevented these changes. Fecal LPS acting in concert with trypsin in patients with IBS-D stimulates mucosal mast cells to release PGE2, which downregulates mucosal SERT, resulting in increased mucosal 5-HT. This may contribute to diarrhea and abdominal pain common in IBS.