Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells This information is current as Martin Vaeth, Isabelle Zee, Axel R. Concepcion, Mate of September 23, 2021. Maus, Patrick Shaw, Cynthia Portal-Celhay, Aleena Zahra, Lina Kozhaya, Carl Weidinger, Jennifer Philips, Derya Unutmaz and Stefan Feske J Immunol 2015; 195:1202-1217; Prepublished online 24 June 2015; Downloaded from doi: 10.4049/jimmunol.1403013 http://www.jimmunol.org/content/195/3/1202 Supplementary http://www.jimmunol.org/content/suppl/2015/06/23/jimmunol.140301 http://www.jimmunol.org/ Material 3.DCSupplemental References This article cites 87 articles, 41 of which you can access for free at: http://www.jimmunol.org/content/195/3/1202.full#ref-list-1 Why The JI? Submit online. by guest on September 23, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Ca2+ Signaling but Not Store-Operated Ca2+ Entry Is Required for the Function of Macrophages and Dendritic Cells Martin Vaeth,*,1 Isabelle Zee,*,1 Axel R. Concepcion,* Mate Maus,* Patrick Shaw,* Cynthia Portal-Celhay,† Aleena Zahra,† Lina Kozhaya,*,† Carl Weidinger,* Jennifer Philips,* Derya Unutmaz,*,† and Stefan Feske* Store-operated Ca2+ entry (SOCE) through Ca2+ release–activated Ca2+ (CRAC) channels is essential for immunity to infection. CRAC channels are formed by ORAI1 proteins in the plasma membrane and activated by stromal interaction molecule (STIM)1 and STIM2 in the endoplasmic reticulum. Mutations in ORAI1 and STIM1 genes that abolish SOCE cause severe immunodefi- ciency with recurrent infections due to impaired T cell function. SOCE has also been observed in cells of the innate immune Downloaded from system such as macrophages and dendritic cells (DCs) and may provide Ca2+ signals required for their function. The specific role of SOCE in macrophage and DC function, as well as its contribution to innate immunity, however, is not well defined. We found that nonselective inhibition of Ca2+ signaling strongly impairs many effector functions of bone marrow–derived macrophages and bone marrow–derived DCs, including phagocytosis, inflammasome activation, and priming of T cells. Surprisingly, however, macrophages and DCs from mice with conditional deletion of Stim1 and Stim2 genes, and therefore complete inhibition of SOCE, showed no major functional defects. Their differentiation, FcR-dependent and -independent phagocytosis, phagolysosome fusion, http://www.jimmunol.org/ cytokine production, NLRP3 inflammasome activation, and their ability to present Ags to activate T cells were preserved. Our findings demonstrate that STIM1, STIM2, and SOCE are dispensable for many critical effector functions of macrophages and DCs, which has important implications for CRAC channel inhibition as a therapeutic strategy to suppress pathogenic T cells while not interfering with myeloid cell functions required for innate immunity. The Journal of Immunology, 2015, 195: 1202–1217. 2+ 2+ a influx across the plasma membrane through Ca 1,4,5-triphosphate (IP3), opening of IP3 receptor channels in the release–activated Ca2+ (CRAC) channels is an important membrane of the endoplasmic reticulum (ER), and release of Ca2+ C signaling pathway for the activation of immune cells (1). from ER stores. Engagement of other ITAM motif–containing by guest on September 23, 2021 In T lymphocytes, where this pathway is best characterized, immunoreceptors and G protein–coupled receptors on immune CRAC channels are activated after TCR stimulation that results in cells also activates PLCg and PLCb, respectively, and leads to IP3 the activation of phospholipase C (PLC)g, production of inositol production (1). Ca2+ release from the ER results in a transient 2+ 2+ increase in intracellular Ca concentrations ([Ca ]i) and a de- crease in ER Ca2+ concentrations. The latter causes the opening of *Department of Pathology, New York University School of Medicine, New York, NY 10016; and †Department of Medicine, New York University School of Medicine, store-operated CRAC channels in the plasma membrane via the New York, NY 10016 activation of stromal interaction molecule (STIM)1 and STIM2, 1M.V. and I.Z. contributed equally to this work. which are single-pass transmembrane proteins located in the ER 2+ Received for publication December 3, 2014. Accepted for publication May 26, 2015. membrane. Dissociation of Ca from the ER luminal part of This work was supported by National Institutes of Health Grants AI097302 (to S.F.), STIM proteins results in their translocation to ER–plasma mem- AI065303 (to D.U.), and AI087682 (to J.P.) and by postdoctoral fellowships from the brane junctions and binding to ORAI1, the pore-forming subunit National Multiple Sclerosis Society (to P.S.), the Alfonso Martin Escudero Founda- tion (to A.R.C.), and the Deutsche Forschungsgemeinschaft (Grant We 5303/1-1 to of the CRAC channel (2–4). Opening of ORAI1 results in store- 2+ 2+ C.W. and Grant VA 882/1-1 to M.V.). operated Ca entry (SOCE), thus called because this form of Ca 2+ Address correspondence and reprint requests to Prof. Stefan Feske, Department of influx is regulated by the ER Ca concentrations (5). SOCE has Pathology, Experimental Pathology Program, New York University School of Med- been demonstrated not only in T cells but also in many other types icine, 550 First Avenue, Smilow 316, New York, NY 10016. E-mail address: [email protected] of immune cells, including B cells, mast cells, macrophages, dendritic cells (DCs), and neutrophils (1). The online version of this article contains supplemental material. CRAC channels play an important role for immunity to infection, Abbreviations used in this article: AIHA, autoimmune hemolytic anemia; 2-APB, 2-aminoethoxydiphenylborate; AUC, area under the curve; BCG, bacillus Calmette– as patients with inherited mutations in ORAI1 and STIM1 genes Gue´rin; BMDC, bone marrow–derived dendritic cell; BMDM, bone marrow–derived that abolish SOCE suffer from SCID-like disease (6–8), which 2+ 2+ macrophage; [Ca ]i, intracellular Ca concentration; CASR, calcium-sensing re- necessitates hematopoietic stem cell transplantation. These ceptor; CRAC, Ca2+ release–activated Ca2+; DC, dendritic cell; ER, endoplasmic reticulum; FlaA, Legionella pneumophila flagellin; IP3, inositol 1,4,5-triphosphate; patients have recurrent and chronic infections with viruses, bac- LAMP, lysosomal-associated membrane protein; LFn, N-terminal domain of Bacillus teria, and fungal pathogens that have been attributed to impaired anthracis lethal factor; MFI, mean fluorescence intensity; MOI, multiplicity of in- fection; mRBC, mouse RBC; MSU, monosodium urate; NLRP, NOD-like receptor T cell function because of severely impaired proliferation and family, pyrin domain–containing; PA, recombinant anthrax protective Ag; PLC, cytokine production of patient T cells in vitro. T cell–specific 2+ phospholipase C; PRR, pattern recognition receptor; SOCE, store-operated Ca deletion of Stim1 gene expression in mice impairs immunity to entry; STIM, stromal interaction molecule; TG, thapsigargin; WT, wild-type. Mycobacterium tuberculosis (9), and deletion of both Stim1 and + Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 Stim2 compromises antiviral immunity due to impaired CD4 and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1403013 The Journal of Immunology 1203 CD8+ T cell responses (10). In contrast to the well-documented abnormalities in the development of macrophages and DCs in vivo function of CRAC channels in T cells, their role in innate immune or the differentiation of bone marrow–derived macrophages responses is not well defined, and it is unclear whether defects in (BMDMs) and bone marrow–derived DCs (BMDCs) in vitro. myeloid cells contribute to the immunodeficiency of ORAI1- and Despite abolished SOCE, murine macrophages showed normal STIM1-deficient patients. FcR-dependent and independent phagocytosis and maturation of In macrophages, intracellular Ca2+ was shown to regulate sev- phagolysosomes. Furthermore, the production of cytokines in re- eral cell functions such as the production of TNF-a and NO (11, sponse to various pattern recognition receptor (PRR) ligands and 12). FcR-dependent and -independent phagocytosis by macro- the activation of the NLRP3 and NLRC4 inflammasomes were phages is associated with intracellular Ca2+ transients (13–16). normal in STIM1/STIM2-deficient DCs and macrophages. Lastly, Whether phagocytosis requires cytosolic Ca2+ signals, however, is the Ag-presenting function and the subsequent activation of cog- controversial and various studies buffering extra- and intracellular nate T cells remained intact in SOCE-deficient BMDCs. In con- Ca2+ have come to different conclusions