Mast Cell Cytokine Production Research Articles

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions.

Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

Lysophosphatidylinositol Promotes Chemotaxis and Cytokine Synthesis in Mast Cells with Differential Participation of GPR55 and CB2 Receptors

Mast cells (MCs) are the main participants in the control of immune reactions associated with inflammation, allergies, defense against pathogens, and tumor growth. Bioactive lipids are lipophilic compounds able to modulate MC activation. Here, we explored some of the effects of the bioactive lipid lysophosphatidylinositol (LPI) on MCs. Utilizing murine bone marrow-derived mast cells (BMMCs), we found that LPI did not cause degranulation, but slightly increased FcεRI-dependent β-hexosaminidase release. However, LPI induced strong chemotaxis together with changes in LIM kinase (LIMK) and cofilin phosphorylation. LPI also promoted modifications to actin cytoskeleton dynamics that were detected by an increase in cell size and interruptions in the continuity of the cortical actin ring. The chemotaxis and cortical actin ring changes were dependent on GPR55 receptor activation, since the specific agonist O1602 mimicked the effects of LPI and the selective antagonist ML193 prevented them. The LPI and O1602-dependent stimulation of BMMC also led to VEGF, TNF, IL-1α, and IL-1β mRNA accumulation, but, in contrast with chemotaxis-related processes, the effects on cytokine transcription were dependent on GPR55 and cannabinoid (CB) 2 receptors, since they were sensitive to ML193 and to the specific CB2 receptor antagonist AM630. Remarkably, GPR55-dependent BMMC chemotaxis was observed towards conditioned media from distinct mouse and human cancer cells. Our data suggest that LPI induces the chemotaxis of MCs and leads to cytokine production in MC in vitro with the differential participation of GPR55 and CB2 receptors. These effects could play a significant role in the recruitment of MCs to tumors and the production of MC-derived pro-angiogenic factors in the tumor microenvironment.

The Heat Shock Protein 90 (HSP90) Is Required for the IL-33-Induced Cytokine Production in Mast Cells (MCs).

The alarmin interleukin-33 (IL-33) is released upon cell stress and damage in peripheral tissues. The receptor for IL-33 is the Toll/Interleukin-1 receptor (TIR)-family member T1/ST2 (the IL-33R), which is highly and constitutively expressed on MCs. The sensing of IL-33 by MCs induces the MyD88−TAK1−IKK2-dependent activation of p65/RelA and MAP-kinases, which mediate the production of pro-inflammatory cytokines and amplify FcεRI-mediated MC-effector functions and the resulting allergic reactions. Therefore, the investigation of IL-33-induced signaling is of interest for developing therapeutic interventions effective against allergic reactions. Importantly, beside the release of IL-33, heat shock proteins (HSPs) are upregulated during allergic reactions. This maintains the biological functions of signaling molecules and/or cytokines but unfortunately also strengthens the severity of inflammatory reactions. Here, we demonstrate that HSP90 does not support the IL-33-induced and MyD88−TAK1−IKK2-dependent activation of p65/RelA and of mitogen-activated protein (MAP)-kinases. We found that HSP90 acts downstream of these signaling pathways, mediates the stability of produced cytokine mRNAs, and therefore facilitates the resulting cytokine production. These data show that IL-33 enables MCs to perform an effective cytokine production by the upregulation of HSP90. Consequently, HSP90 might be an attractive therapeutic target for blocking IL-33-mediated inflammatory reactions.

Analysis of Lymphoma-Related Genes with Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Enrichment.

Lymphoma is a serious malignant tumor that contains more than 70 different types and seriously endangers the body's lymphatic system. The lymphatic system is the regulatory center of the immune system and is important in the immune response to foreign antigens and tumors. Studies showed that multiple genetic variants are associated with lymphoma but determining the pathogenic mechanisms remains a challenge. In the present study, we first applied the Gene Ontology (GO) and KEGG pathway enrichment analyses of lymphoma-associated and lymphoma-nonassociated genes. Next, the Boruta and max-relevance and min-redundancy feature selection methods were performed to filter and rank features. Then, features preselected and ranked using the incremental feature selection method were applied for the decision tree model to identify the best GO terms and KEGG pathways and extract classification rules. Results indicate that our predicted features, such as B-cell activation, negative regulation of protein processing, negative regulation of mast cell cytokine production, and natural killer cell-mediated cytotoxicity, are associated with the biological process of lymphoma, consistent with those of recent publications. This study provides a new perspective for future research on the molecular mechanisms of lymphoma.

Fluoxetine-mediated suppression reveals a role for P2X receptors in mast cell function and allergic disease

Abstract There is a clinical need for new treatment options addressing allergic disease because current approaches are often suboptimal. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have been shown to possess anti-inflammatory properties, but the mechanism of action is unclear. Because mast cells are critical effectors of allergic inflammation, we tested SSRI effects on IgE-induced mast cell function. We show that the SSRI fluoxetine suppresses IgE-mediated degranulation and cytokine production in mouse and human mast cells. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchoresponsiveness and inflammation. IgE crosslinkage elicited rapid ATP release from mast cells, suggesting ATP-P2X receptor signaling may potentiate IgE-driven responses. We subsequently found that fluoxetine inhibits ATP-induced mast cell function. We propose that fluoxetine acts by blocking an ATP-P2X autocrine or paracrine loop that augments mast cell-mediated inflammation. These data support the potential repurposing of fluoxetine in allergic disease. Supported by NIH/NIAID R01138495 R01164710

MiR-155 Is a Positive Regulator of FcεRI-Induced Cyclooxygenase-2 Expression and Cytokine Production in Mast Cells.

MicroRNA-155 (miR-155) has been implicated in IgE-dependent allergic disease including asthma and atopic dermatitis. A few roles for miR-155 have been described in mast cells and some specifically related to IgE receptor signaling, but it is not completely understood. Here, we demonstrate by miRNA seq profiling and quantitative RT-PCR that miR-155 expression is significantly increased in human skin-derived mast cells (SMCs) and mouse bone marrow-derived mast cells (BMMCs) following FcεRI crosslinking with antigen. We demonstrate that FcεRI-induced expression of cyclooxygenase-2 (COX-2) was significantly inhibited in miR-155 knockout (KO) BMMCs whereas arachidonate-5-lipoxygenase (ALOX-5) expression and leukotriene C4 (LTC4) biosynthesis, and degranulation were unaffected. FcεRI-induced cytokine production (TNF, IL-6, and IL-13) from miR-155 KO BMMCs was also significantly diminished. Correspondingly, Akt phosphorylation, but not protein expression, was inhibited in the absence of miR-155 whereas p38 and p42/44 were unaffected. Interesting, lipopolysaccharide (LPS)-induced cytokine production was increased in miR-155 KO BMMCs. Together, these data demonstrate that miR-155 specifically targets the FcεRI-induced prostaglandin and cytokine pathways, but not the leukotriene or degranulation pathways, in mast cells. The data further suggest that miR-155 acts indirectly by targeting a repressor of COX-2 expression and a phosphatase that normally blocks Akt phosphorylation. Overall, this study reveals the role of miR-155 as a positive regulator of mast cell function.

Synergistic Cytokine Production by ATP and PGE2 via P2X4 and EP3 Receptors in Mouse Bone-Marrow-Derived Mast Cells.

ATP is an important intercellular messenger in the extracellular space. In mast cells (MCs), ATP stimulates the ionotropic P2X4 receptor (P2X4R), resulting in enhanced degranulation and exacerbation of acute allergic reactions. In this study, we investigate whether ATP regulates inflammatory cytokine production in MCs. Gene expression was analyzed by quantitative RT-PCR, and cytokine production was measured using ELISA. The stimulation of mouse bone-marrow-derived MCs (BMMCs) with ATP alone had little effect on cytokine secretion. However, the co-stimulation with prostaglandin (PG) E2 resulted in a marked increase in the secretion of various cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and IL-13, accompanied by an increase in their mRNA levels. The effects of ATP were inhibited by P2X4R antagonists and diminished in BMMCs derived from P2X4R-deficient mice, suggesting that P2X4R mediated the reaction. The effects of PGE2 were mimicked by an EP3 receptor (EP3R) agonist and blocked by an EP3R antagonist. The synergistic cytokine mRNA elevations induced by ATP and PGE2 were blocked by nuclear factor-κB and Ca2+-calcineurin signaling inhibitors. Altogether, these results suggest that combining P2X4R and EP3R signaling enhances acute degranulation and the subsequent cytokine secretion, exacerbating allergic inflammation.

How to Prevent and Mitigate Hypersensitivity Reactions to Biologicals Induced by Anti-Drug Antibodies?

Biologicals are widely used therapeutic agents for rheumatologic diseases, cancers, and other chronic inflammatory diseases. They are characterized by complex structures and content of variable amounts of foreign regions, which may lead to anti-drug antibodies (ADA) development. ADA onset may limit the clinical usage of biologicals because they may decrease their safety. In fact they are mainly associated with immediate hypersensitivity reactions (HSRs). Development of ADAs is reduced by concomitant immunosuppressive treatment, while it is increased by longer intervals between drug administrations; thus, regular infusion regimens should be preferred to reduce HSRs. Once ADAs have formed, some procedures can be implemented to reduce the risk of HSRs. ADAs may belong to different isotype; the detection of IgE ADA is advisable to be assessed when high and early ADAs are detected, in order to reduce the risk of severe HRs. In patients who need to reintroduce the biological culprit, as alternative therapies are not available, drug desensitization (DD) may be applied. Desensitization should be conceptually dedicated to patients with an IgE-mediated HSR; however, it can be performed also in patients who had developed non-IgE-mediated HSRs. Although the underlying mechanisms behind successful DD has not been fully clarified, the DD procedure is associated with the inhibition of mast cell degranulation and cytokine production. Additionally, some data are emerging about the inhibition of drug-specific immune responses during DD.

Signal-transducing adaptor protein-2 has a nonredundant role for IL-33-triggered mast cell activation

Signal-transducing adaptor protein (STAP)-2 is one of the STAP family adaptor proteins and ubiquitously expressed in a variety types of cells. Although STAP-2 is required for modification of FcεRI signal transduction in mast cells, other involvement of STAP-2 in mast cell functions is unknown, yet. In the present study, we mainly investigated functional roles of STAP-2 in IL-33-induced mast cell activation. In STAP-2-deficient, but not STAP-1-deficient, mast cells, IL-33-induced IL-6 and TNF-α production was significantly decreased compared with that of wild-type mast cells. In addition, STAP-2-deficiency greatly reduced TLR4-mediated mast cell activation and cytokine production. For the mechanisms, STAP-2 directly binds to IKKα after IL-33 stimulation, leading to elevated NF-κB activity. In conclusion, STAP-2, but not STAP-1, participates in IL-33-induced mast cells activation.

Immunomodulatory effects of Brominated Flame Retardants on Cytokine Production in Mast cells

Polybrominated diphenyl ethers (PBDEs), extensively used as flame-retardants have been classified as persistent environmental pollutants. PBDEs have also been shown to cause neurotoxicity, immunotoxicity, and endocrine disruption; however, the exact mechanisms are largely unknown. In this study, we propose to investigate PBDE-induced immunotoxicity in mast cells. We examined the effects of two of the most commonly occurring brominated diphenyl ether (BDE) congeners, BDE-47 and -99, and a metabolite of BDE-47, 6-OH-BDE-47 on MC/9 mast cells. MC/9 cells treated with BDE-47, -99, and 6-OH-BDE-47 (0.25, 0.5, 5, 20 and 40μM, 24h) were stimulated with IgE/antigen (1μg/ml), and levels of IL-6 and TNF-α were measured by ELISA. Our results indicate that PBDEs inhibit the IgE-mediated cytokine release in MC/9 cells in a concentration-dependent manner. Interestingly, exposure to BDE-47 and 6-OH-BDE47 decreased IL-6 release in a concentration-dependent manner; however, BDE-99 decreased IL-6 release only at concentrations above 20 μM. These results demonstrate that tetra-brominated BDE-47 and its 6-OH metabolite can suppress cytokine release with increasing concentrations, while penta-brominated BDE-99 congener inhibits IL-6 release only at higher concentrations. We observed a similar decrease in TNF-α release when tge MC/9 cells were exposed to the BDE-47, -99, and 6-OH-BDE-47. Mast cell degranulation measured by β-hexoaminidase also showed that BDE congeners decreased degranulation in a concentration-dependent manner in IgE stimulated mast cells. In order to test effects on cell viability, we exposed the MC/9 cells to increasing concentrations of the BDE congeners and measured cytotoxicity at 24 and 48h by MTS assay. We found that tetra-brominated BDE-47 exhibited more potent cytotoxicity (EC50 µM =42.38 at 24h and 32.33 at 48h) in comparison to the penta-brominated BDE-99 (EC50 µM =48.41 and 35.04 at 48h) in the MC/9 cells. However, the 6-OH metabolite of BDE-47 was more cytotoxic in comparison to its parent compound BDE-47 as well as penta-brominated BDE-99 (EC50 µM =25.82 at 24h and 20.16 at 48h). We are further characterizing the development of apoptosis in the MC9 cells in the presence of BDE congeners. Our results suggest that PBDE congeners can modulate cytokine release in mast cells, and the extent of inhibition is dependent on the bromination of PBDEs. These results address the need to better understand the molecular and cellular mechanisms by which PBDE exposure can mediate immunotoxicity.

Inhibition of cholesterol pathway intermediates attenuates IL-33 mediated mast cell cytokine production

Abstract Asthma is a growing problem in developed countries, affecting over 24 million individuals in the US alone. Allergic asthma is denoted by a Th2 response that supports IgE-mediated mast cell function and is enhanced by the cytokine IL-33. Although corticosteroids and other treatments are available to help control asthma, many patients have persistent symptoms. In order to meet this unmet need, we investigated the utility of repurposing statin drugs. Statins decrease serum cholesterol by inhibiting HMG-CoA reductase. These drugs have anti-inflammatory effects largely ascribed to loss of isoprenyl lipids downstream of HMGCR. We previously found that statins could decrease IgE-mediated mast cell function. Our current data demonstrate that HMGCR inhibition by simvastatin or isoprenylation blockade with the novel compound FGT-2734 attenuates IL-33 induced mast cell cytokine production in vitro. Statin efficacy for the treatment of allergic asthma has been inconsistent in clinical trials. However, our data suggest that inhibiting isoprenylated signaling molecules may provide a feasible therapeutic target in mast cell-driven disease, and supports the use of statins as a potential adjunct or alternative treatment for individuals with asthma.

The Use of Bruton's Tyrosine Kinase Inhibitors to Treat Allergic Disorders.

Purpose of reviewStudies show that inhibitors of Bruton’s tyrosine kinase (BTKis), currently FDA-approved for the treatment of B cell malignancies, can prevent IgE-mediated reactions through broad inhibition of the FcεRI signaling pathway in human mast cells and basophils. This review will summarize recent data supporting the use of these drugs as novel therapies in various allergic disorders.Recent findingsRecent studies have shown that BTKis can prevent IgE-mediated degranulation and cytokine production in primary human mast cells and basophils. Two oral doses of the second-generation BTKi acalabrutinib can completely prevent moderate passive systemic anaphylaxis in humanized mice and even protect against death during severe anaphylaxis. Furthermore, two doses of ibrutinib can reduce or eliminate skin prick test responses to foods and aeroallergens in allergic subjects. BTKis in development also show efficacy in clinical trials for chronic urticaria. Unlike other therapies targeting IgE, such as omalizumab, BTKis appear to have rapid onset and transient effects, making them ideal candidates for intermittent use to prevent acute reactions such as IgE-mediated anaphylaxis.SummaryThese studies suggest that BTKis may be capable of preventing IgE-mediated anaphylaxis, paving the way for future trials in food allergy and urticaria.

Association of Mast Cell Burden and TIM-3 Expression with Recalcitrant Chronic Rhinosinusitis with Nasal Polyps.

Previous work showed that higher polyp mast cell load correlated with worse postoperative endoscopic appearance in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Polyp epithelial mast cells showed increased expression of T-cell/transmembrane immunoglobulin and mucin domain protein 3 (TIM-3), a receptor that promotes mast cell activation and cytokine production. In this study, CRSwNP patients were followed post-operatively to investigate whether mast cell burden or TIM-3 expression among mast cells can predict recalcitrant disease. Nasal polyp specimens were obtained via functional endoscopic sinus surgery (FESS) and separated into epithelial and stromal layers via enzymatic digestion. Mast cells and TIM-3-expressing mast cells were identified via flow cytometry. Mann-Whitney U tests and Cox proportional hazard models assessed whether mast cell burden and TIM-3 expression were associated with clinical outcomes, including earlier recurrence of polypoid edema and need for treatment with steroids. Twenty-three patients with CRSwNP were studied and followed for 6 months after undergoing FESS. Higher mast cell levels were associated with earlier recurrence of polypoid edema: epithelial HR = 1.283 (P = .02), stromal HR = 1.103 (P = .02). Percent of mast cells expressing TIM-3 in epithelial or stromal layers was not significantly associated with earlier recurrence of polypoid edema. Mast cell burden and TIM-3+ expression were not significantly associated with need for future treatment with steroids post-FESS. Mast cell load in polyp epithelium and stroma may predict a more refractory postoperative course for CRSwNP patients. The role of TIM-3 in the chronic inflammatory state seen in CRSwNP remains unclear.

PI3Kγ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Bruton's tyrosine kinase inhibition effectively protects against human IgE-mediated anaphylaxis.

No known therapies can prevent anaphylaxis. Bruton's tyrosine kinase (BTK) is an enzyme thought to be essential for high-affinity IgE receptor (FcεRI) signaling in human cells. We tested the hypothesis that FDA-approved BTK inhibitors (BTKis) would prevent IgE-mediated responses including anaphylaxis. We showed that irreversible BTKis broadly prevented IgE-mediated degranulation and cytokine production in primary human mast cells and blocked allergen-induced contraction of isolated human bronchi. To address their efficacy in vivo, we created and used what we believe to be a novel humanized mouse model of anaphylaxis that does not require marrow ablation or human tissue implantation. After a single intravenous injection of human CD34+ cells, NSG-SGM3 mice supported the population of mature human tissue-resident mast cells and basophils. These mice showed excellent responses during passive systemic anaphylaxis using human IgE to selectively evoke human mast cell and basophil activation, and response severity was controllable by alteration of the amount of allergen used for challenge. Remarkably, pretreatment with just 2 oral doses of the BTKi acalabrutinib completely prevented moderate IgE-mediated anaphylaxis in these mice and also significantly protected against death during severe anaphylaxis. Our data suggest that BTKis may be able to prevent anaphylaxis in humans by inhibiting FcεRI-mediated signaling.

IgE-induced mast cell function is suppressed by antagonizing isoprenylation or Ras activation

Abstract Mast cells are tissue resident innate immune cells activated by IgE and other ligands. Upon activation they release histamine, cytokines, chemokines, proteases, and lipid mediators evoking allergic symptoms. New ways of targeting mast cells could greatly benefit allergic disease therapy. We previously found that statin drugs reduced IgE-mediated inflammation in vitro and in vivo by inhibiting production of the isoprenoid lipid geranylgeranyl pyrophosphate. However, this suppression was dependent on genetic background. To overcome this limitation, we tested a dual farnesyl and geranylgeranyl transferase inhibitor (FGTI 2734). Using mouse mast cells, we show that FGTI 2734 reduced IgE-mediated degranulation and cytokine production similar to statin effects. Unlike statins, FGTI 2734 did not show a significant difference in IC50 based on genetic background. Furthermore, FGTI 2734 suppressed IgE-mediated systemic anaphylaxis in vivo. Ras GTPases are important for mast cell function and among the signaling proteins modified by isoprenylation. We show that Pan-Ras inhibitors can mimic statin and FGTI 2734 effects on cytokine production in mouse mast cells. These data indicate that dual farnesyl and geranylgeranyl transferase inhibition may be effective in suppressing IgE-induced inflammation, possibly due by disrupting Ras-mediated signals.

MALT1 Protease Plays a Dual Role in the Allergic Response by Acting in Both Mast Cells and Endothelial Cells.

The signaling protein MALT1 plays a key role in promoting NF-κB activation in Ag-stimulated lymphocytes. In this capacity, MALT1 has two functions, acting as a scaffolding protein and as a substrate-specific protease. MALT1 is also required for NF-κB-dependent induction of proinflammatory cytokines after FcεR1 stimulation in mast cells, implicating a role in allergy. Because MALT1 remains understudied in this context, we sought to investigate how MALT1 proteolytic activity contributes to the overall allergic response. We compared bone marrow-derived mast cells from MALT1 knockout (MALT1-/-) and MALT1 protease-deficient (MALTPD/PD) mice to wild-type cells. We found that MALT1-/- and MALT1PD/PD mast cells are equally impaired in cytokine production following FcεRI stimulation, indicating that MALT1 scaffolding activity is insufficient to drive the cytokine response and that MALT1 protease activity is essential. In addition to cytokine production, acute mast cell degranulation is a critical component of allergic response. Intriguingly, whereas degranulation is MALT1-independent, MALT1PD/PD mice are protected from vascular edema induced by either passive cutaneous anaphylaxis or direct challenge with histamine, a major granule component. This suggests a role for MALT1 protease activity in endothelial cells targeted by mast cell-derived vasoactive substances. Indeed, we find that in human endothelial cells, MALT1 protease is activated following histamine treatment and is required for histamine-induced permeability. We thus propose a dual role for MALT1 protease in allergic response, mediating 1) IgE-dependent mast cell cytokine production, and 2) histamine-induced endothelial permeability. This dual role indicates that therapeutic inhibitors of MALT1 protease could work synergistically to control IgE-mediated allergic disease.

Induction of IκBζ Augments Cytokine and Chemokine Production by IL-33 in Mast Cells.

IκBζ (encoded by the Nfkbiz) is a member of the nuclear IκB family, which is involved in the expression of secondary response genes based on signals from TLR or IL-1R. ST2L, an IL-33R, is a member of the IL-1R family and abundantly expressed in tissue-resident immune cells, such as mast cells and innate lymphoid cells; however, its downstream signaling pathway remains unelucidated. In this study, we examined the role of IκBζ in ST2L-mediated cytokine and chemokine production in mast cells. Murine bone marrow cells were differentiated ex vivo into bone marrow-derived mast cells (BMMCs). The treatment of BMMCs with IL-33 transiently induced robust IκBζ expression. Of the 40 cytokines and chemokines examined using a cytokine and chemokine array, the concentrations of IL-6, IL-13, CCL2, CCL3, and TNF-α in the supernatant were augmented by IL-33. The deletion of IκBζ in BMMCs resulted in a significant reduction of the production of these mediators and the expression of their mRNA. NF-κB p50 but not p65 translocated to the nucleus by IL-33 and was not affected by the deletion of IκBζ. However, induction of IκBζ and the resultant cytokine and chemokine productions were significantly inhibited by pretreatment with an NF-κB inhibitor. The deletion of IκBζ did not affect the phosphorylation of ERK, p38 MAPK, or JNK by IL-33, and the treatment with inhibitors of these mitogen-activated kinases failed to abolish the expression of Nfkbiz Our findings suggest that IκBζ augments IL-33-dependent cytokine and chemokine production in BMMCs through the action of NF-κB.

Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

BackgroundHuntington’s disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases.MethodsBone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed.ResultsR6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice.ConclusionsOur data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

Resveratrol inhibits IL-33\u2013mediated mast cell activation by targeting the MK2/3\u2013PI3K/Akt axis

Interleukin-33 (IL-33)/ST2–mediated mast cell activation plays important roles in the pathophysiology of allergic diseases. Hence, pharmacologically targeting the IL-33/ST2 pathway in mast cells could help to treat such diseases. We found that resveratrol inhibits IL-33/ST2–mediated mast cell activation. Resveratrol suppressed IL-33–induced IL-6, IL-13, and TNF-α production in mouse bone marrow–derived mast cells (BMMCs), mouse fetal skin–derived mast cells, and human basophils. Resveratrol also attenuated cytokine expression induced by intranasal administration of IL-33 in mouse lung. IL-33–mediated cytokine production in mast cells requires activation of the NF-κB and MAPK p38–MAPK-activated protein kinase-2/3 (MK2/3)–PI3K/Akt pathway, and resveratrol clearly inhibited IL-33–induced activation of the MK2/3–PI3K/Akt pathway, but not the NF-κB pathway, without affecting p38 in BMMCs. Importantly, resveratrol inhibited the kinase activity of MK2, and an MK2/3 inhibitor recapitulated the suppressive effects of resveratrol. Resveratrol and an MK2/3 inhibitor also inhibited IgE-dependent degranulation and cytokine production in BMMCs, concomitant with suppression of the MK2/3–PI3K/Akt pathway. These findings indicate that resveratrol inhibits both IL-33/ST2–mediated and IgE-dependent mast cell activation principally by targeting the MK2/3–PI3K/Akt axis downstream of p38. Thus, resveratrol may have potential for the prevention and treatment of broad ranges of allergic diseases.