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Antigen recognition by the T-cell receptor induces a cytosolic Ca 2+ signal that is crucial for T-cell function. The Ca 2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca 2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca 2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro , Trpm2 -/- and WT CD4 + and CD8 + T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2 -/- CD8 + T cells and unimpaired differentiation of CD4 + T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo , Trpm2 -/- and WT CD8 + T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2 -/- mice and after transfer of WT and Trpm2 -/- CD8 + T cells into infected recipients. CD4 + T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2 -/- CD4 + T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lory, Nawrocki, Corazza, Schmid, Schumacher, Bedke, Menzel, Koch-Nolte, Guse, Huber and Mittrücker.)

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca 2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kim, Yi, Kwon, Park, Kwon and Kim.)

Intestinal inflammation is a major threat to the health and growth of young animals such as piglets. As a next-generation probiotics, limited studies have shown that Akkermansia muciniphila could alleviate inflammation of intestinal epithelial cells (IECs). In this study, a TNF-α-induced inflammatory model of IPEC-J2 cells, the intestinal porcine enterocytes, was built to evaluate the effects of active or inactive A. muciniphila on the inflammation of IECs. The viability of IPEC-J2 cells was the highest when treated with active (10 8 copies/mL) or inactive (10 9 copies/mL) A. muciniphila for 7.5 h ( P < 0.01). Treated with 20 ng/mL of TNF-α and followed by a treatment of A. muciniphila , the mRNA level of proinflammatory cytokines ( IL-8, IL-1β , IL-6 and TNF-α ) was remarkably reduced ( P < 0.05) along with the increased mRNA level of tight junction proteins ( ZO-1 and Occludin , P < 0.05). Flow cytometry analysis showed that active or inactive A. muciniphila significantly suppressed the rate of the early and total apoptotic of the inflammatory IPEC-J2 cells ( P < 0.05). According to results of transcriptome sequencing, active and inactive A. muciniphila may decline cell apoptosis by down-regulating the expression of key genes in calcium signaling pathway, or up-regulating the expression of key genes in cell cycle signaling pathway. And the bacterium may alleviate the inflammation of IECs by down-regulating the expression of PI3K upstream receptor genes. Our results indicate that A. muciniphila may be a promising NGP targeting intestinal inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Luo, Lan, Xie, Li, Devillard, He, Liu, Cai, Tian, Wu, Ren, Chen, Yu, Huang, Zheng, Mao, Yu, Luo, Yan, Wang, Wang and Tang.)

Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is a naturally occurring nucleotide that has been shown to be involved in the release of Ca 2+ from intracellular stores in a wide variety of cell types, tissues and organisms. Current evidence suggests that NAADP may function as a trigger to initiate a Ca 2+ signal that is then amplified by other Ca 2+ release mechanisms. A fundamental question that remains unanswered is the identity of the NAADP receptor. Our recent studies have identified HN1L/JPT2 as a high affinity NAADP binding protein that is essential for the modulation of Ca 2+ channels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Walseth and Guse.)

Objective: Multiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators., Methods: Flow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3'-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel., Results: PsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2., Conclusion: PAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abji, Rasti, Gómez-Aristizábal, Muytjens, Saifeddine, Mihara, Motahhari, Gandhi, Viswanathan, Hollenberg, Oikonomopoulou and Chandran.)

While B cells play a significant role in the onset of type-1 diabetes (T1D), little is know about their role in those early stages. Thus, to gain new insights into the role of B cells in T1D, we converted a physiological early pancreas-infiltrating B cell into a novel BCR mouse model using Somatic Cell Nuclear Transfer (SCNT). Strikingly, SCNT-derived B1411 model displayed neither developmental block nor anergy. Instead, B1411 underwent spontaneous germinal center reactions. Without T cell help, B1411-Rag1 -/- was capable of forming peri-/intra-pancreatic lymph nodes, and undergoing class-switching. RNA-Seq analysis identified 93 differentially expressed genes in B1411 compared to WT B cells, including Irf7, Usp18 , and Mda5 that had been linked to a potential viral etiology of T1D. We also found various members of the oligoadenylate synthase (OAS) family to be enriched in B1411, such as Oas1 , which had recently also been linked to T1D. Strikingly, when challenged with glucose B1411-Rag1 -/- mice displayed impaired glucose tolerance., (Copyright © 2020 Phillips, Ke, Nham, Seidl, Freeman, Abadejos, Xiao, Nemazee, Ku and Kirak.)

Cytotoxic lymphocytes, including natural killer (NK) cells and T cells are distinguished by their ability to eliminate target cells through release of secretory lysosomes. Conventional lysosomes and secretory lysosomes are part of the pleomorphic endolysosomal system and characterized by its highly dynamic nature. Several calcium-permeable TRP calcium channels play an essential role in endolysosomal calcium signaling to ensure proper function of these organelles. In NK cells, the expression of self MHC-specific inhibitory receptors dynamically tunes their secretory potential in a non-transcriptional, calcium-dependent manner. New insights suggest that TRPML1-mediated lysosomal calcium fluxes are tightly interconnected to NK cell functionality through modulation of granzyme B and perforin content of the secretory lysosome. Lysosomal TRP channels show a subset-specific expression pattern during NK differentiation, which is paralleled with gradually increased loading of effector molecules in secretory lysosomes. Methodological advances, including organellar patch-clamping, specific pharmacological modulators, and genetically-encoded calcium indicators open up new possibilities to investigate how TRP channels influence communication between intracellular organelles in immune cells. This review discusses our current understanding of lysosome biogenesis in NK cells with an emphasis on the TRP mucolipin family and the implications for NK cell functionality and cancer immunotherapy., (Copyright © 2020 Clement, Goodridge, Grimm, Patel and Malmberg.)

Mast cells are a heterogeneous group of immune cells. The simplest and commonly accepted classification divides them in two groups according to their protease content. We have compared the action of diverse secretagogues on bone marrow derived (BMMC) and peritoneal (PMC) mast cells which represent classical models of mucosal and connective tissue type mast cells in mice. Whereas, antigen stimulation of the FcεRI receptors was similarly effective in triggering elevations of free intracellular Ca 2+ concentration ([Ca 2+ ] i ) in both BMMC and PMC, robust [Ca 2+ ] i rise following Endothelin-1 stimulation was observed only in a fraction of BMMC. Leukotriene C4 activating cysteinyl leukotriene type I receptors failed to evoke [Ca 2+ ] i rise in either mast cell model. Stimulation of the recently identified target of many small-molecule drugs associated with systemic pseudo-allergic reactions, Mrgprb2, with compound 48/80, a mast cell activator with unknown receptor studied for many years, triggered Ca 2+ oscillations in BMMC and robust [Ca 2+ ] i rise in PMCs similarly to that evoked by FcεRI stimulation. [Ca 2+ ] i rise in PMC could also be evoked by other Mrgprb2 agonists such as Tubocurarine, LL-37, and Substance P. The extent of [Ca 2+ ] i rise correlated with mast cell degranulation. Expression analysis of TRPC channels as potential candidates mediating agonist evoked Ca 2+ entry revealed the presence of transcripts of all members of the TRPC subfamily of TRP channels in PMCs. The amplitude and AUC of compound 48/80-evoked [Ca 2+ ] i rise was reduced by ~20% in PMC from Trpc1/4/6 -/- mice compared to Trpc1/4 -/- littermatched control mice, whereas FcεRI-evoked [Ca 2+ ] i rise was unaltered. Whole-cell patch clamp recordings showed that the reduction in compound 48/80-evoked [Ca 2+ ] i rise in Trpc1/4/6 -/- PMC was accompanied by a reduced amplitude of Compound 48/80-induced cation currents which exhibited typical features of TRPC currents. Together, this study demonstrates that PMC are an appropriate mast cell model to study mechanisms of Mrgprb2 receptor-mediated mast cell activation, and it reveals that TRPC channels contribute at least partially to Mrgprb2-mediated mast cellactivation but not following FcεRI stimulation. However, the channels conducting most of the Ca 2+ entry in mast cells triggered by Mrgprb2 receptor stimulation remains to be identified., (Copyright © 2020 Tsvilovskyy, Solis-Lopez, Almering, Richter, Birnbaumer, Dietrich and Freichel.)

During infection, phagocytic cells pursue homeostasis in the host via multiple mechanisms that control microbial invasion. Neutrophils respond to infection by exerting a variety of cellular processes, including chemotaxis, activation, phagocytosis, degranulation and the generation of reactive oxygen species (ROS). Calcium (Ca 2+ ) signaling and the activation of specific Ca 2+ channels are required for most antimicrobial effector functions of neutrophils. The transient receptor potential melastatin-2 (TRPM2) cation channel has been proposed to play important roles in modulating Ca 2+ mobilization and oxidative stress in neutrophils. In the present study, we use a mouse model of Listeria monocytogenes infection to define the role of TRPM2 in the regulation of neutrophils' functions during infection. We show that the susceptibility of Trpm2 -/- mice to L. monocytogenes infection is characterized by increased migration rates of neutrophils and monocytes to the liver and spleen in the first 24 h. During the acute phase of L. monocytogenes infection, Trpm2 -/- mice developed septic shock, characterized by increased serum levels of TNF-α, IL-6, and IL-10. Furthermore, in vivo depletion of neutrophils demonstrated a critical role of these immune cells in regulating acute inflammation in Trpm2 -/- infected mice. Gene expression and inflammatory cytokine analyses of infected tissues further confirmed the hyperinflammatory profile of Trpm2 -/- neutrophils. Finally, the increased inflammatory properties of Trpm2 -/- neutrophils correlated with the dysregulated cytoplasmic concentration of Ca 2+ and potentiated membrane depolarization, in response to L. monocytogenes . In conclusion, our findings suggest that the TRPM2 channel plays critical functional roles in regulating the inflammatory properties of neutrophils and preventing tissue damage during Listeria infection., (Copyright © 2020 Robledo-Avila, Ruiz-Rosado, Brockman and Partida-Sánchez.)

Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β 2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process., (Copyright © 2019 Lőrincz, Szeifert, Bartos, Szombath, Mócsai and Ligeti.)

The gut microbiota influences several biological functions including immune responses. Inflammatory bowel disease is favorably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts, and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolizes ellagic acid resulting in the formation of bioactive urolithins A, B, C, and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, whether gut metabolite UA affects the function of immune cells remains incompletely understood. T cell proliferation is stimulated by store operated Ca 2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4 + T cells with UA (10 μM, 3 days) significantly blunted SOCE in CD4 + T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4 + T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4 + T cells. UA further decreased CD4 + T cell proliferation. Thus, the gut bacterial metabolite UA increases miR-10a-5p levels thereby downregulating Orai1/STIM1/STIM2 expression, store operated Ca 2+ entry, and proliferation of murine CD4 + T cells.

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4 + T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.

Activating transcription factor-3 (ATF3) in the ER stress pathway induces cytokine production and promotes survival during gram-positive bacterial infection. IL-17A is a critical cytokine that is essential for clearance of Streptococcus pneumoniae . However, the mechanism by which ATF3 induces IL-17A production remains unknown. Here, we show that ATF3 induces IL-17A production via NLRP3 inflammasome-dependent IL-1β secretion. Survival rates were comparable in IL-17A-depleted and ATF3 KO mice but were lower than in WT mice treated with isotype control, indicating that ATF3 positively regulated IL-17A production. Indeed, ATF3 KO mice showed a marked reduction in IL-17A protein and mRNA expression compared to levels in WT mice. Moreover, mitochondrial IL-1β production by bone marrow-derived macrophages was significantly reduced in ATF3 KO mice as a result of the disruption of cellular ROS and Ca 2+ homeostasis. Accordingly, ATF3 KO mice displayed diminished survival and bacterial clearance following S. pneumoniae infection. Taken together, these data suggest a mechanism in which macrophage ATF3 promotes IL-17A production in γδ T cells to rapidly induce host defenses during early S. pneumoniae infection.

Activation of the high-affinity receptor for IgE (FcεRI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5'-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, FcγRIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon FcεRI cross-linking. Absence of FcγRIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca 2+ mobilization. Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control.