Epidermal Growth Factor Receptor Expression Licenses Type-2 Helper T Cells to Function in a T Cell Receptor-Independent Fashion.

Gastro-intestinal helminth infections trigger the release of interleukin-33 (IL-33), which induces type-2 helper T cells (Th2 cells) at the site of infection to produce IL-13, thereby contributing to host resistance in a T cell receptor (TCR)-independent manner. Here, we show that, as a prerequisite for IL-33-induced IL-13 secretion, Th2 cells required the expression of the epidermal growth factor receptor (EGFR) and of its ligand, amphiregulin, for the formation of a signaling complex between T1/ST2 (the IL-33R) and EGFR. This shared signaling complex allowed IL-33 to induce the EGFR-mediated activation of the MAP-kinase signaling pathway and consequently the expression of IL-13. Lack of EGFR expression on T cells abrogated IL-13 expression in infected tissues and impaired host resistance. EGFR expression on Th2 cells was TCR-signaling dependent, and therefore, our data reveal a mechanism by which antigen presentation controls the innate effector function of Th2 cells at the site of inflammation.

Beyond muscles: Investigating immunoregulatory myokines in acute resistance exercise - A systematic review and meta-analysis.

Myokines, released from the muscle, enable communication between the working muscles and other tissues. Their release during physical exercise is assumed to depend on immune-hormonal-metabolic interactions concerning mode (endurance or resistance exercise), duration, and intensity. This meta-analysis aims to examine the acute changes of circulating myokines inducing immunoregulatory effects caused by a bout of resistance exercise and to consider potential moderators of the results. Based on this selection strategy, a systematic literature search was conducted for resistance exercise intervention studies measuring interleukin (IL-) 6, IL-10, IL-1ra, tumor necrosis factor (TNF-) α, IL-15, IL-7, transforming growth factor (TGF-) ß1, and fractalkines (FKN) before and immediately after resistance exercise in healthy individuals. Random-effects meta-analysis was performed for each myokine. We identified a moderate positive effect of resistance exercise for IL-6 and IL-1ra. Regarding IL-15 and TNF-α, small to moderate effects were found. For IL-10, no significant effect was observed. Due to no data, meta-analyses for IL-7, TGF-ß1, and FKN could not be performed. No moderators (training status, type of exercise, risk of bias, age, sex, time of day, exercise volume, exercise intensity, exercise dose) of the results were detected for all tested myokines. Taken together, this systematic review and meta-analysis showed immediate positive effects of an acute resistance exercise session on IL-6, IL-1ra, TNF-α, and IL-15 levels.

The Neurobeachin-like 2 protein (NBEAL2) controls the homeostatic level of the ribosomal protein RPS6 in mast cells.

The Beige and Chediak-Higashi (BEACH) domain-containing, Neurobeachin-like 2 (NBEAL2) protein is a molecule with a molecular weight of 300 kDa. Inactivation of NBEAL2 by loss-of-function mutations in humans as well as deletion of the Nbeal2 gene in mice results in functional defects in cells of the innate immune system such as neutrophils, NK-cells, megakaryocytes, platelets and of mast cells (MCs). To investigate the detailed function of NBEAL2 in murine MCs we generated MCs from wild type (wt) and Nbeal2-/- mice, and deleted Nbeal2 by CRISPR/Cas9 technology in the murine mast cell line MC/9. We also predicted the structure of NBEAL2 to infer its function and to examine potential mechanisms for its association with interaction partners by using the deep learning-based method RoseTTAFold and the Pymol© software. The function of NBEAL2 was analysed by molecular and immunological techniques such as co-immunoprecipitation (co-IP) experiments, western blotting, enzyme-linked immunosorbent assay and flow cytometry. We identified RPS6 as an interaction partner of NBEAL2. Thereby, the NBEAL2/RPS6 complex formation is probably required to control the protein homeostasis of RPS6 in MCs. Consequently, inactivation of NBEAL2 leads to accumulation of strongly p90RSK-phosphorylated RPS6 molecules which results in the development of an abnormal MC phenotype characterised by prolonged growth factor-independent survival and in a pro-inflammatory MC-phenotype.

Interleukin-3 stabilizes CD124/IL-4α surface expression in mast cells via Tyk2 and STAT6.

Interleukin (IL)-4 signals can modulate mast cells, which express the IL-4Rα chain. The IL-4Rα can heterodimerise with the common γ-chain and utilizes JAK1 and JAK2 for signal transduction, while complexes of IL-4Rα with IL-13Rα1 subunit mediates signals via JAK2 and Tyk2. Here, we report that IL-3 is an essential factor for the continuous expression of the IL-4Rα chain on mast cells, which did not express the IL-13Rα1 chain. We demonstrate that the signals induced by IL-3 important for IL-4Rα expression are mediated by Tyk2 and STAT6 activation and the subsequent maintenance of HSP90 levels. In line with that, inhibition of either Tyk2, STAT6 or HSP90 impaired the IL-3-induced IL-4Rα upregulation. Consequently, the IL-3 maintained IL-4Rα surface expression via Tyk2 is essential for the costimulatory effect of IL-4 on the IL-33-induced production of IL-6 and IL-13.

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.

IL-3 is essential for ICOS-L stabilization on mast cells, and sustains the IL-33-induced RORγt+ Treg generation via enhanced IL-6 induction.

IL-33 is a member of the IL-1 family. By binding to its receptor ST2 (IL-33R) on mast cells, IL-33 induces the MyD88-dependent activation of the TAK1-IKK2 signalling module resulting in activation of the MAP kinases p38, JNK1/2 and ERK1/2, and of NFκB. Depending on the kinases activated in these pathways, the IL-33-induced signalling is essential for production of IL-6 or IL-2. This was shown to control the dichotomy between RORγt+ and Helios+ Tregs , respectively. SCF, the ligand of c-Kit (CD117), can enhance these effects. Here, we show that IL-3, another growth factor for mast cells, is essential for the expression of ICOS-L on BMMCs, and costimulation with IL-3 potentiated the IL-33-induced IL-6 production similar to SCF. In contrast to the enhanced IL-2 production by SCF-induced modulation of the IL-33 signalling, IL-3 blocked the production of IL-2. Consequently, IL-3 shifted the IL-33-induced Treg dichotomy towards RORγt+ Tregs at the expense of RORγt- Helios+ Tregs . However, ICOS-L expression was downregulated by IL-33. In line with that, ICOS-L did not play any important role in the Treg modulation by IL-3/IL-33-activated mast cells. These findings demonstrate that different from the mast cell growth factor SCF, IL-3 can alter the IL-33-induced and mast cell-dependent regulation of Treg subpopulations by modulating mast cell-derived cytokine profiles.

ATP/IL-33-triggered hyperactivation of mast cells results in an amplified production of pro-inflammatory cytokines and eicosanoids.

IL-33 and ATP are alarmins, which are released upon damage of cellular barriers or are actively secreted upon cell stress. Due to high-density expression of the IL-33 receptor T1/ST2 (IL-33R), and the ATP receptor P2X7, mast cells (MCs) are one of the first highly sensitive sentinels recognizing released IL-33 or ATP in damaged peripheral tissues. Whereas IL-33 induces the MyD88-dependent activation of the TAK1-IKK2-NF-κB signalling, ATP induces the Ca2+ -dependent activation of NFAT. Thereby, each signal alone only induces a moderate production of pro-inflammatory cytokines and lipid mediators (LMs). However, MCs, which simultaneously sense (co-sensing) IL-33 and ATP, display an enhanced and prolonged activation of the TAK1-IKK2-NF-κB signalling pathway. This resulted in a massive production of pro-inflammatory cytokines such as IL-2, IL-4, IL-6 and GM-CSF as well as of arachidonic acid-derived cyclooxygenase (COX)-mediated pro-inflammatory prostaglandins (PGs) and thromboxanes (TXs), hallmarks of strong MC activation. Collectively, these data show that co-sensing of ATP and IL-33 results in hyperactivation of MCs, which resembles to MC activation induced by IgE-mediated crosslinking of the FcεRI. Therefore, the IL-33/IL-33R and/or the ATP/P2X7 signalling axis are attractive targets for therapeutical intervention of diseases associated with the loss of integrity of cellular barriers such as allergic and infectious respiratory reactions.

IL-33-activated murine mast cells control the dichotomy between RORγt+ and Helios+ Tregs via the MK2/3-mediated IL-6 production in vitro.

In mast cells, IL-33 typically induces the activation of NF-κB, which results in the production of cytokines such as IL-6 and IL-2. Here, we demonstrate that the IL-33-induced IL-6 production in murine mast cells and the formation of RORγt+ Tregs essentially depends on the MAPKAPs, MK2, and MK3 (MK2/3) downstream of MyD88. In contrast to this, the IL-33-induced and MyD88-dependent IL-2 production in mast cells contributes to the maintenance of Helios+ Tregs . Thereby, the IL-33-induced IL-2 response and, thus, the maintenance of Helios+ Tregs are limited by an IL-6-mediated autocrine negative feedback stimulation acting on mast cells. Collectively, we present MK2/3 in IL-33-activated mast cells as a signaling node, which controls the dichotomy between RORγt+ Treg and Helios+ Treg in vitro.

The p38-MK2/3 Module Is Critical for IL-33-Induced Signaling and Cytokine Production in Dendritic Cells.

IL-33 is an IL-1 cytokine superfamily member. Binding of IL-33 to the IL-33R induces activation of the canonical NF-κB signaling and activation of MAPKs. In bone marrow-derived dendritic cells, IL-33 induces the production of IL-6, IL-13, and TNF-α. However, the signaling pathways resulting in IL-33-induced effector functions of dendritic cells are unknown. In this article, we show that the IL-33-induced cytokine production is only partly dependent on p65. Thereby, p65 mediates the production of IL-6, but not of IL-13, whereas the p38-Mapk-activated protein kinases 2/3 (MK2/3) signaling module mediates the IL-13, but not the IL-6, production. In addition, GM-CSF, which is critical for the differentiation and proliferation of bone marrow-derived dendritic cells, potentiates the p65-dependent IL-6 and the p38-MK2/3-dependent IL-13 production. Furthermore, we found that effective TNF-α production is only induced in the presence of GM-CSF and IL-33 via the p38-MK2/3 signaling module. Taken together, we found that the p38-MK2/3 signaling module is essential to mediate IL-33-induced cytokine production in dendritic cells.

The Neurobeachin-like 2 Protein Regulates Mast Cell Homeostasis.

The neurobeachin-like 2 protein (Nbeal2) belongs to the family of beige and Chediak-Higashi (BEACH) domain proteins. Loss-of-function mutations in the human NBEAL2 gene or Nbeal2 deficiency in mice cause gray platelet syndrome, a bleeding disorder characterized by macrothrombocytopenia, splenomegaly, and paucity of α-granules in megakaryocytes and platelets. We found that in mast cells, Nbeal2 regulates the activation of the Shp1-STAT5 signaling axis and the composition of the c-Kit/STAT signalosome. Furthermore, Nbeal2 mediates granule formation and restricts the expression of the transcription factors, IRF8, GATA2, and MITF as well as of the cell-cycle inhibitor p27, which are essential for mast cell differentiation, proliferation, and cytokine production. These data demonstrate the relevance of Nbeal2 in mast cells above and beyond granule biosynthesis.

MK2/3 Are Pivotal for IL-33-Induced and Mast Cell-Dependent Leukocyte Recruitment and the Resulting Skin Inflammation.

The IL-1R family member IL-33R mediates Fcε-receptor-I (FcεRI)-independent activation of mast cells leading to NF-κB activation and consequently the production of cytokines. IL-33 also induces the activation of MAPKs, such as p38. We aimed to define the relevance of the p38-targets, the MAPK-activated protein kinases 2 and 3 (MK2 and MK3) in IL-33-induced signaling and the resulting mast cell effector functions in vitro and in vivo. We demonstrate that the IL-33-induced IL-6 and IL-13 production strongly depends on the MK2/3-mediated activation of ERK1/2 and PI3K signaling. Furthermore, in the presence of the stem cell factors, IL-33 did induce an MK2/3-, ERK1/2- and PI3K-dependent production of TNF-α. In vivo, the loss of MK2/3 in mast cells decreased the IL-33-induced leukocyte recruitment and the resulting skin inflammation. Therefore, the MK2/3-dependent signaling in mast cells is essential to mediate IL-33-induced inflammatory responses. Thus, MK2/3 are potential therapeutic targets for suppression of IL-33-induced inflammation skin diseases such as psoriasis.

Membrane-bound stem cell factor is the major but not only driver of fibroblast-induced murine skin mast cell differentiation.

The maintenance and modulation of cutaneous mast cell (MC) numbers is held to be important for skin immune responses to allergens and pathogens. The increase in MC numbers in the skin is achieved by proliferation and the differentiation of precursor to mature MCs. Fibroblast-derived SCF is thought to be the major skin MC growth factor and it potently induces MC proliferation. The mechanisms of fibroblast-induced skin MC differentiation, including the role of SCF, however, remain insufficiently characterized and understood. Using cocultures of immature murine MCs and fibroblasts, we found that the adhesion of immature MCs to fibroblasts via VCAM-1 and α4 ß7 integrin is very important for subsequent differentiation, which is driven by fibroblast membrane-bound SCF and additional fibroblast-derived membrane-bound signals. Thus, our results show that fibroblast-induced MC differentiation is induced by direct cell-cell contact and involves both Kit-dependent and Kit-independent pathways. Our findings add to the understanding of how immature mast cells mature in murine skin and encourage further analyses of the underlying mechanisms, which may result in novel targets for the modulation of skin mast cell driven diseases.

Mast cell hyperplasia, B-cell malignancy, and intestinal inflammation in mice with conditional expression of a constitutively active kit.

Signaling through the receptor tyrosine kinase kit controls proliferation and differentiation of hematopoietic precursor cells and mast cells. Somatic point mutations of the receptor that constitutively activate kit signaling are associated with mastocytosis and various hematopoietic malignancies. We generated a Cre/loxP-based bacterial artificial chromosome transgenic mouse model that allows conditional expression of a kit gene carrying the kitD814V mutation (the murine homolog of the most common mutation in human mastocytosis, kitD816V) driven by the kit promoter. Expression of the mutant kit in cells of adult mice, including hematopoietic precursors, caused severe mastocytosis with 100% penetrance at young age frequently associated with additional hematopoietic (mostly B lineage-derived) neoplasms and focal colitis. Restriction of transgene expression to mature mast cells resulted in a similar mast cell disease developing with slower kinetics. Embryonic expression led to a hyperproliferative dysregulation of the erythroid lineage with a high rate of perinatal lethality. In addition, most adult animals developed colitis associated with mucosal mast cell accumulation. Our findings demonstrate that the effects of constitutive kit signaling critically depend on the developmental stage and the state of differentiation of the cell hit by the gain-of-function mutation.

ATP/IL-33-Co-Sensing by Mast Cells (MCs) Requires Activated c-Kit to Ensure Effective Cytokine Responses.

Mast cells (MCs) are sentinel cells which represent an important part of the first line of defense of the immune system. MCs highly express receptors for danger-associated molecular patterns (DAMPs) such as the IL-33R and P2X7, making MCs to potentially effective sensors for IL-33 and adenosine-triphosphate (ATP), two alarmins which are released upon necrosis-induced cell damage in peripheral tissues. Besides receptors for alarmins, MCs also express the stem cell factor (SCF) receptor c-Kit, which typically mediates MC differentiation, proliferation and survival. By using bone marrow-derived MCs (BMMCs), ELISA and flow cytometry experiments, as well as p65/RelA and NFAT reporter MCs, we aimed to investigate the influence of SCF on alarmin-induced signaling pathways and the resulting cytokine production and degranulation. We found that the presence of SCF boosted the cytokine production but not degranulation in MCs which simultaneously sense ATP and IL-33 (ATP/IL-33 co-sensing). Therefore, we conclude that SCF maintains the functionality of MCs in peripheral tissues to ensure appropriate MC reactions upon cell damage, induced by pathogens or allergens.

PI3Kγ deficiency delays the onset of experimental autoimmune encephalomyelitis and ameliorates its clinical outcome.

PI3Ks control signal transduction triggered by growth factors and G-protein-coupled receptors and regulate an array of biological processes, including cellular proliferation, differentiation, survival and migration. Herein, we investigated the role of PI3Kγ in the pathogenesis of EAE. We show that, in the absence of PI3Kγ expression, clinical signs of EAE were delayed and mitigated. PI3Kγ-deficient myelin oligodendrocyte glycoprotein (MOG)(35-55) -specific CD4(+) T cells appeared later in the secondary lymphoid organs and in the CNS than their WT counterparts. Transfer of WT CD4(+) cells into PI3Kγ(-/-) mice prior to MOG(35-55) immunisation restored EAE severity to WT levels, supporting the relevance of PI3Kγ expression in Th cells for the pathogenesis of EAE; however, PI3Kγ was dispensable for Th1 and Th17 differentiation, thus excluding an altered expression of these pathogenetically relevant cytokines as the cause for ameliorated EAE in PI3Kγ(-/-) mice. These findings demonstrate that PI3Kγ contributes to the development of autoimmune CNS inflammation.

The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells.

Members of the Toll/interleukin-1 receptor (TIR) family are of importance for host defense and inflammation. Here we report that the TIR-family member interleukin-33R (IL-33R) cross-activates the receptor tyrosine kinase c-Kit in human and murine mast cells. The IL-33R-induced activation of signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase B (PKB), and Jun NH(2)-terminal kinase 1 (JNK1) depends on c-Kit and is required to elicit optimal effector functions. Costimulation with the c-Kit ligand stem cell factor (SCF) is necessary for IL-33-induced cytokine production in primary mast cells. The structural basis for this cross-activation is the complex formation between c-Kit, IL-33R, and IL-1R accessory protein (IL-1RAcP). We found that c-Kit and IL-1RAcP interact constitutively and that IL-33R joins this complex upon ligand binding. Our findings support a model in which signals from seemingly disparate receptors are integrated for full cellular responses.

Identification of a highly efficient dual type I/II FMS-like tyrosine kinase inhibitor that disrupts the growth of leukemic cells.

Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.

A scoping review of regulatory T cell dynamics in convalescent COVID-19 patients - indications for their potential involvement in the development of Long COVID?

Background: Recovery from coronavirus disease 2019 (COVID-19) can be impaired by the persistence of symptoms or new-onset health complications, commonly referred to as Long COVID. In a subset of patients, Long COVID is associated with immune system perturbations of unknown etiology, which could be related to compromised immunoregulatory mechanisms. Objective: The objective of this scoping review was to summarize the existing literature regarding the frequency and functionality of Tregs in convalescent COVID-19 patients and to explore indications for their potential involvement in the development of Long COVID. Design: A systematic search of studies investigating Tregs during COVID-19 convalescence was conducted on MEDLINE (via Pubmed) and Web of Science. Results: The literature search yielded 17 relevant studies, of which three included a distinct cohort of patients with Long COVID. The reviewed studies suggest that the Treg population of COVID-19 patients can reconstitute quantitatively and functionally during recovery. However, the comparison between recovered and seronegative controls revealed that an infection-induced dysregulation of the Treg compartment can be sustained for at least several months. The small number of studies investigating Tregs in Long COVID allowed no firm conclusions to be drawn about their involvement in the syndrome's etiology. Yet, even almost one year post-infection Long COVID patients exhibit significantly altered proportions of Tregs within the CD4+ T cell population. Conclusions: Persistent alterations in cell frequency in Long COVID patients indicate that Treg dysregulation might be linked to immune system-associated sequelae. Future studies should aim to address the association of Treg adaptations with different symptom clusters and blood parameters beyond the sole quantification of cell frequencies while adhering to consensualized phenotyping strategies.

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma.

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.