© 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610

RESEARCH ARTICLE SPECIAL ISSUE: CELL BIOLOGY OF THE IMMUNE SYSTEM Loss of Ca2+ entry via Orai–TRPC1 induces ER stress, initiating immune activation in macrophages Viviane Nascimento Da Conceicao1, Yuyang Sun1, Emily K. Zboril1, Jorge J. De la Chapa2 and Brij B. Singh1,*

ABSTRACT interacts with proteins at the plasma membrane Ca2+ influx channels Activation of cellular stresses is associated with inflammation; (Liao et al., 2008; Shaw and Feske, 2012a). 2+ however, the mechanisms are not well identified. Here, we provide SOCE in immune cells is mediated by the highly Ca -selective 2+ 2+ evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) Ca release-activated Ca (CRAC) channel and is essential for stress in primary macrophages and in murine macrophage cell line the proper immune response (Shaw and Feske, 2012a; Vaeth et al., 2+ Raw 264.7, in which the unfolded protein response is initiated to 2015). An increase in [Ca ]i is initiated by agonist binding to cell- modulate cytokine production, thereby activating the immune surface receptors, which generates second messenger IP3, leading to 2+ 2+ response. Stressors that initiate the ER stress response block store- the release of Ca stored in the ER. Release of ER Ca allows ER dependent Ca2+ entry in macrophages prior to the activation of the protein STIM1 to aggregate, and also to interact with Orai1 (Feske unfolded protein response. The endogenous Ca2+ entry channel is et al., 2006; Liao et al., 2008) and transient receptor potential dependent on the Orai1–TRPC1–STIM1 complex, and the presence canonical 1 (TRPC1) channels (Asanov et al., 2015; Liao et al., 2+ of ER stressors decreased expression of TRPC1, Orai1 and STIM1. 2008), thus increasing the [Ca ]i that is essential for cellular Additionally, blocking Ca2+ entry with SKF96365 also induced functions (Parekh and Putney, 2005; Putney et al., 2017). ER stress, promoted cytokine production, activation of autophagy, Importantly, immune cell activation is also dependent on SOCE, increased caspase activation and induced apoptosis. Furthermore, and loss of Orai1 or STIM1 has been shown to lead to an impaired ER stress inducers inhibited cell cycle progression, promoted immune function (Shaw and Feske, 2012b). Another mechanism that the inflammatory M1 phenotype, and increased phagocytosis. also affects the innate and adaptive immune response is ER stress, Mechanistically, restoration of Orai1–STIM1 expression inhibited with chronic ER stress contributing to abnormal physiological the ER stress-mediated loss of Ca2+ entry that prevents ER stress and processes involved in disease pathogenesis and progression (Islam inhibits cytokine production, and thus induced cell survival. These et al., 2006; Kaufman, 1999; Kim et al., 2013; Todd et al., 2008). results suggest an unequivocal role of Ca2+ entry in modulating ER Moreover, abnormal immune activation caused by chronic ER stress stress and in the induction of inflammation. is also associated with autoimmune and inflammatory disorders such as diabetes, atherosclerosis, myositis and inflammatory bowel KEY WORDS: SOCE channels, Ca2+ modulation, TRPC1, ER stress, disease (Arruda and Hotamisligil, 2015). Thus, establishing the Immune activation mechanism(s) by which ER stress induces immune activation is essential for research into potential treatments for these diseases. INTRODUCTION The ER not only plays an important role in Ca2+ signaling but is The endoplasmic reticulum (ER) is a vital organelle responsible for also essential for protein synthesis and proper protein folding and various cellular functions, such as lipid and protein synthesis and modification (Braakman and Hebert, 2013). More than half of regulation of Ca2+ homeostasis (Braakman and Hebert, 2013; Parekh newly synthesized proteins translocate to the lumen of the ER, for and Putney, 2005). Importantly, cellular stress provokes the release folding and targeting to various cellular organelles or to be of pro-inflammatory cytokines that stimulate the inflammatory transported to the surface of the cell. (Görlach et al., 2006; response (Liu et al., 2017; Pinton et al., 2008); however, the Kaufman, 1999; Lawless and Greene, 2012; Zhang and Kaufman, mechanisms by which ER stress leads to immune activation are not 2008). Ca2+ is required for the chaperone activity of many ER well known. In non-excitable cells, change in intracellular Ca2+ proteins, and loss of this vital function induces ER stress. In 2+ 2+ ([Ca ]i) levels directly related to the release of Ca stores from the addition, genetic or chemical inhibition can also activate the ER ER, is due to activation of the store-operated Ca2+ entry (SOCE) stress response, through a common signaling mechanism. ER stress mechanism (Tojyo et al., 2014). Once ER Ca2+ stores are depleted, triggers a cascade of signaling networks referred to as the unfolded stromal interaction molecule 1 (STIM1), which acts as an ER sensor, protein response (UPR) to reduce stress and restore homeostasis. The UPR activates the cellular response that leads to the transcription of 1Department of Periodontics, School of Dentistry, University of Texas Health San molecular chaperones to alleviate this stress response. Importantly, Antonio, San Antonio, TX 78229, USA. 2Department of Comprehensive Dentistry, three major ER proteins, inositol-requiring enzyme 1α (IRE1α,also School of Dentistry, University of Texas Health San Antonio, San Antonio, TX 78229, known as ERN1), the PKR-like ER kinases (PERK), and activating USA. transcription factor 6α (ATF6α) have been shown to bind to *Author for correspondence ([email protected]) misfolded or unfolded proteins to initiate the UPR. Activation of the UPR is necessary to reduce stress; however, insufficient clearance of B.B.S., 0000-0003-0535-5997 these misfolded proteins or prolonged activation of the pathways that This is an Open Access article distributed under the terms of the Creative Commons Attribution induce ER stress result in apoptotic cell death (Chen et al., 2015; License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. Endo et al., 2006; Ron and Walter, 2007). ER stress activates the inflammatory processes, especially in

Received 5 August 2019; Accepted 25 October 2019 macrophages, which are the sentinels of the immune systems Journal of Cell Science

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(Ho et al., 2012). These specialized cells, called first responders, production, which regulates inflammation. These results not play major roles in the maintenance of tissue integrity or tissue only help in understanding the physiology of ER stress-related damaging signals, host defense and inflammation. Disturbances in disorders but could help in the development of novel therapeutic this delicate balance cause the cells to be more susceptible to modalities for ER stress-related disorders by improving our activation of inflammatory processes, leading to immune diseases comprehension of the relationship between ER stress and the and disorders (Franken et al., 2016; Gautier et al., 2012; Lavin et al., UPR processes. 2015). Macrophages are key components of the innate immune system and are crucial parts of a system that senses and responds to RESULTS tissue invasion by infectious microorganisms and tissue injury Treatment with tunicamycin or brefeldin A induces ER through various scavenger, pattern recognition and phagocytic stress, affecting cell proliferation of macrophages receptors (Lavin et al., 2015). Although the exact mechanism for the ER stress response could trigger an inflammatory response, activation of macrophages under ER stress is not entirely therefore causing metabolic issues in the cell (Cao and Kaufman, understood, Ca2+ could be the vital link. In addition, switching of 2014; Grootjans et al., 2016). Thus, we initiated our study by the macrophages to the pro-inflammatory M1 phenotype is the key assessing the response to well-known ER stress inducers to chronic immune activation. tunicamycin (Tuni), a known glycosylation suppressor, and The aim of this study was to identify the pathways that involve brefeldin A (BFA), an inhibitor of Golgi complex transportation ER stress and lead to immune activation. Our data show that Ca2+ in the macrophage cell line. Raw 264.7 cells were incubated at homeostasis is vital to macrophages activation. We report for the different concentrations of both Tuni and BFA (5 µM and 10 µM) first time that ER stress inducers lead to the loss of Ca2+ entry, for 6 and 12 h. After treatment, proteins were extracted, and western which corresponds to a decrease in ER Ca2+ levels, and precede the blots were performed, using β-actin as positive control. Importantly, unfolded protein response. Loss of ER and intracellular Ca2+ was cells treated with 5 µM Tuni for 12 h showed an increase in the primarily due to the decrease in expression of Orai1, TRPC1 and expression of ER stress markers, whereas, treatment for 6 h did STIM1. Orai1 is a pore-forming component of ICRAC, and STIM1 not show a similar increase in CHOP (also known as DDIT3) or is an ER-Ca2+ sensor, which activates SOCE. TRPC1 and Orai1, other ER stress proteins (Fig. S1A). Expression of molecular together with STIM1, produce Ca2+ signals that consequently chaperone GRP94 (also known as HSP90B1), was significantly regulate cell functions. We further report that the endogenous Ca2+ increased, along with increased expression of PDI (also known entry channel in macrophages cells is dependent on a TRPC1– as P4HB), IRE1α and CHOP proteins in the presence of Tuni at 12 h Orai1 complex, and that loss of TRPC1–Orai1, function induces of treatment (Fig. 1A,B). Increasing Tuni concentration, abnormal UPR and the induction of ER stress. Furthermore, loss however, showed a dose-dependent increase in the expression of of Ca2+ entry increased the pro-inflammatory M1 phenotype, ER stress markers, whereas no change in the actin level was induced cytokine production, and increased phagocytosis. observed (Fig. 1A,B; Fig. S1A). Consistent with these results, Taken together, these results suggest that loss of Ca2+ signaling when exposed to BFA Raw 264.7 cells also showed an increase initiates ER stress, and that restoration of Orai1–STIM1 complex in ER stress markers, where an increase in the expression of function is essential for inhibiting ER stress and cytokine CHOP, GRP94 and other ER stress proteins was observed in

Fig. 1. Tunicamycin and brefeldin A treatment induces ER stress-causing cell death in macrophages. (A,C) Representative immunoblot images showing the expression of ER stress markers PDI, CHOP, IREα and GRP94, and of actin, in Raw 264.7 macrophages pretreated with 5 μMor10μM tunicamycin (Tuni) (A), or BFA (C) for 12 h. (B,D) Quantification of densitometric values (mean±s.d.) from bands as shown in A and C. (E,G) Representative immunoblot images showing the expression of ER stress markers in bone marrow-derived macrophages (BMDMs) pretreated with 10 μM Tuni (E) or BFA (G) for 12 h. (F,H) Quantification of densitometric values (mean±s.d.) from bands as shown in E and G. (I,J) Quantification (mean±s.d.) of cell survival (I) and caspase activity (J) of Raw 264.7 cells pretreated with 10 μM BFA or Tuni for 12 h. **P<0.01, ***P<0.001 by one-way ANOVA test. The data shown are representative of three independent experiments. Journal of Cell Science

2 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 cells treated with a higher concentration of BFA for 12 h (Fig. 1C,D; (Fig. 1J). Analyzed together, these results show that when ER stress Fig. S1B). is induced in macrophages, it causes upregulation of known markers To further confirm our cell line results, we isolated mouse of the UPR, which leads to chronic activation of caspase 3, and primary macrophages to investigate whether ER stress markers are thereby triggers apoptosis. also increased upon the addition of Tuni and BFA. Our data again showed that when we treated primary bone marrow-derived Treatment with tunicamycin or brefeldin A decreases ER and macrophages (BMDMs) with either Tuni or BFA, expression cytosolic Ca2+ of ER stress markers was increased within 12 h of treatment. To establish the mechanism by which ER stress is initiated, we (Fig. 1E–H). Importantly, treating for a longer time (16 h) evaluated Ca2+ signaling in BMDMs. Addition of thapsigargin (Tg) significantly decreased the cell number of primary macrophage (1 µM in Ca2+-free buffer), a SERCA pump blocker that depletes 2+ 2+ 2+ (data not shown), suggesting that both Tuni and BFA lead to a ER Ca , caused a small increase in intracellular Ca ([Ca ]i) chronic unfolded protein response, resulting in cell death. Thus, we levels (first peak) in BMDMs (Fig. 2A–H). In the presence of 1 mM 2+ 2+ also evaluated the consequence of this ER stress and tested cell external Ca , cells showed a significant increase in [Ca ]i levels viability where cells were exposed to Tuni or BFA treatments. (second peak), indicating the presence of store-mediated Ca2+ entry Interestingly, our data showed that 6 h of Tuni or BFA treatment did (Fig. 2A–H). Importantly, cells treated with Tuni (10 µM for 6 h) to not induce cell death and no significant difference in cell viability induce ER stress were shown to have a significant reduction in was observed (data not shown). In contrast, a significant increase in SOCE without any change in internal ER Ca2+ release (Fig. 2A,B). cell death was observed after prolonged treatment (12 h) with In contrast, when Tuni treatment (10 µM) continued for 12 h, not 10 µM of either Tuni or BFA (Fig. 1I). Additionally, incubating for only was Ca2+ entry significantly decreased, but also the internal ER 24 h with either Tuni or BFA further increased cell death and the Ca2+ level (Fig. 2C,D). Consistent with these results, addition of majority of cells were dead within 24 h of treatment (data are BFA (10 µM for 6 h), also revealed a significant decrease in Ca2+ shown). To identify how ER stress induces cell death, caspase 3 entry; whereas 12 h treatment led to a decrease in both ER Ca2+ activity was measured, and was shown to be significantly increased level and Ca2+ entry, respectively (Fig. 2E–H). To establish the in cells that were treated with 10 µM (12 h) of either Tuni or BFA molecular identity of the Ca2+ influx channel in macrophages,

Fig. 2. Tunicamycin and brefeldin A treatment decreased the Ca2+ currents in murine macrophages. (A,C,E,G) Ca2+ tracing was performed in Raw 264.7 cells following treatment with 10 μM Tuni for 6 h (A) or 12 h (C), or 10 μM BFA for 6 h (E) or 12 h (G). Representative traces of the analog plots of the fluorescence ratio (340/380) from an average of 40–60 cells are shown. (B,D,F,H) Quantification (mean±s.d.) of fluorescence ratio (340/380) under conditions as shown in A,C,E,G, as labeled in the figure. (I) Whole-cell patch recording showed that bath application of 1 µM Tg induced an inward-rectifying current in Raw 264.7 cells. (J,K) Average IV curves (J) and current density (K) from 6–9cellsat−80 mV under conditions as shown in I. *P<0.05 by one-way ANOVA test; NS, non-significant. Journal of Cell Science

3 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 electrophysiological recordings of membrane currents were ER stress. These results are consistent with a previous study wherein performed. The addition of Tg induced an inward current, which membrane depolarization was not enough to generate cytosolic was partially inward-rectifying in nature and reversed between 0 Ca2+ signals in immune cells (Demaurex and Nunes, 2016), and −5 mV (Fig. 2I,J). The current properties observed were mixed suggesting that Ca2+ signaling is mostly via the SOCE mechanism. as previously observed with TRPC1 and Orai1 channels (Liu et al., To further confirm these findings, we studied the expression of 2004; Selvaraj et al., 2012; Shi et al., 2012; Yuan et al., 2007). SOCE components on primary BMDMs (Fig. 3E–H) treated with Moreover, in the presence of Tuni or BFA, Tg-induced Ca2+ Tuni or BFA (10 µM each) for 12 h. Confirming our cell line results, currents were significantly inhibited in Raw 264.7 cells (Fig. 2I–K). the expression of TRPC1, Orai1 and STIM1 was also decreased. Taken together, these results suggest that in macrophages, the Additionally, electrophysiology was performed on BMDMs addition of ER stress inducers (Tuni, BFA) leads to a loss of SOCE (Fig. 3I,J), and in further support of our cell line data, a decrease that further decreases ER Ca2+ levels, thereby leading to ER stress. in Ca2+ currents was observed when primary cells were treated with Importantly, Ca2+ entry was dependent on TRPC1 and Orai1 Tuni or BFA. The findings of this study so far have established a channels, and Tuni- and BFA-induced loss of Ca2+ entry precedes direct relationship between Ca2+ levels and ER stress on the the expression of ER stress markers. regulation of cell survival in macrophages.

Ca2+ channels are downregulated by treatment with SKF96365 treatment induces ER stress in tunicamycin or brefeldin A macrophages cells To further understand how Ca2+ influx is inhibited in macrophages, We next evaluated the physiological effect of blocking SOCE we decided to investigate the expression of the Ca2+ channel channels on ER stress. SKF96345 (SKF), a well-established Ca2+ proteins themselves. Raw 264.7 cells were again exposed to Tuni or entry blocker that blocks both TRPC1 and Orai1 channels BFA for 6 or 12 h and the expression of SOCE channel proteins was (Sukumaran et al., 2018), was used. Importantly, exposing Raw observed. Importantly, cells pretreated with either 5 or 10 µM of 264.7 cells to SKF (10 µM for 12 h) resulted in a significant Tuni resulted in a significant downregulation of TRPC1 and Orai1 decrease in Tg-induced Ca2+ currents (Fig. 4A,B). Consistent with (Fig. 3A,B). In addition, activity of STIM1, which modulates both the loss of SOCE, a significant increase in levels of ER stress TRPC1 and Orai1, was also significantly decreased, with no markers such as CHOP, GRP94 and PDI, but not actin, was observed changes in actin levels (Fig. 3A,B). Similar results were observed in cells that were pretreated with SKF (10 µM for 12 h) also observed with BFA, with significant decreases in TRPC1, (Fig. 4C,D). We next evaluated whether SKF treatment also Orai1 and STIM1 expression levels (Fig. 3C,D). Importantly, cells decreased the expression of SOCE channel proteins as observed treated with lower doses of either Tuni or BFA for 6 h did not show a in Fig. 3. Importantly, no change in TRPC1, STIM1 or Orai1 significant decrease in TRPC1, STIM1 or Orai1 protein levels expression was observed upon SKF treatment (Fig. 4E,F), (Fig. S1C,D). Taken together, these results suggest that STIM1 and suggesting that the loss of Ca2+ entry was the reason for the Orai1 are important Ca2+ entry channels in macrophages, along with induction of the ER stress response. Furthermore, we observed that the TRPC1 channel, as they are all decreased during the induction of when macrophages were treated with SKF, it also resulted in a

Fig. 3. Tunicamycin and brefeldin A treatment decrease Ca2+ channel expression in murine macrophages. (A,C) Representative immunoblots showing the decreased expression of Ca2+ entry channels proteins TRPC1, Orai1 and STIM1 in Raw 264.7 macrophages pretreated with 5 μMor10μM Tuni (A) or BFA (C) for 12 h. (B,D) Quantification of densitometeric values (mean±s.d.) from bands as shown in A and C. (E,G) Representative immunoblot images showing the decreased expression of Ca2+ entry channels in BMDMs pretreated with 10 μM Tuni (E) or BFA (G) for 12 h. (F,H) Quantification of densitometric values (mean±s.d.) from bands as shown in E and G. (I,J) Whole-cell patch recording showing average IV curves (I) and current intensity (J) from 6–9 cells at −80 mV of BMDMs pretreated with 10 μM BFA or Tuni for 12 h. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA test; NS, non-significant. The data shown are representative of three independent experiments. Journal of Cell Science

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Fig. 4. ER stress is induced by SKF96365 pretreatment on the murine macrophage cell line. (A,B) Whole-cell patch recording showing average IV curves (A) and current intensity (B) from 6–9 cells at −80 mV in Raw 264.7 macrophages pretreated with 10 μM SKF96365 (SKF) for 12 h. (C) Representative immunoblot images showing the induction of ER stress as seen in the increased expression of marker proteins in Raw 264.7 macrophages pretreated with 10 μM SKF for 12 h. (D) Quantification of densitometric values (mean±s.d.) from bands as shown in C. Immunoblots representing the expression of Ca2+ entry channel proteins TRPC1, TRPC3, Orai1 and STIM1 in Raw 264.7 macrophages pretreated with 10 μM SKF for 12 h. (F) Quantification of densitometric values (mean±s.d.) from bands as shown in E. (G,H) Quantification (mean±s.d.) of cell survival (G) and caspase activity (H) of Raw 264.7 cells pretreated with 10 μM SKF for 12 h. **P<0.01, ***P<0.001 by one-way ANOVA test; NS, non-significant. The data shown are representative of three independent experiments. significant decrease in cell survival (Fig. 4G), along with an Treatment with tunicamycin, brefeldin A or SKF96365 increase in caspase 3 activity (Fig. 4H). These data showed that an enhances the secretion of cytokines and causes cell increase in ER stress is directly related to inhibiting SOCE channel cycle arrest activity, which leads to a decrease in ER Ca2+ levels and induces cell Following our findings, we focused on the effects of the treatments death in macrophages. on the production of inflammatory cytokines under conditions of ER stress. It has been established that cytokines play critical roles Treatment with tunicamycin, brefeldin A or SKF96365 in host defense against pathogens; however, when produced in induces cell autophagy and apoptosis high demand, they may also be responsible for pathological It has been established that cell death starts once ER stress is highly inflammation (Brozzi et al., 2015; Smith, 2018; Vaeth et al., elevated in the cell. ER stress and autophagy appear to be initially 2015). Both Raw 264.7 and BMDM cells were treated with 10 µM involved in actions to protect the cell, but induce cell death under of Tuni, BFA or SKF96365 for 12 h and cytokine levels were prolonged stress conditions (Rashid et al., 2015). Our data show that analyzed. We observed that release of the major cytokines treatment with Tuni, BFA or SKF induces caspase activity, which interleukin-6 (IL-6), interleukin 1 beta (IL-1β) and tumor necrosis leads to cell death. To establish the correlation between the factor alpha (TNF-α) were all significantly increased (Fig. 6A; increase in ER stress and autophagy, we decided to analyze the Fig. S2A). Similarly, intracellular levels of these cytokines were also expression of known autophagy and apoptosis markers LC3B increased in the presence of all three treatment agents. Moreover, (active component), beclin1, Bcl-2, caspase 3, caspase 9 and expression of proteins involved in three UPR pathways, IRE1α (data Bax in Raw 264.7 cells. Importantly, an increase in the not shown), PERK and ATF-6 (Hotamisligil, 2010), along with the expression of autophagy markers, and of caspase 3, caspase 9 activation of NF-κB, were increased in the presence of these and Bax, was observed (Fig. 5A,B). Confocal images of cells stressors (Fig. 6B,C). It has previously been established that all three treated with the ER stress inducers Tuni or BFA, or with Ca2+ UPR pathways impact the activation of NF-κB, which is transported channel inhibitor SKF, again showed an increase in autophagic to the nucleus in response to immune signaling, where it triggers vesicles (as observed with staining for LC3B or beclin1) and an inflammatory cytokines such as IL-6 and TNF-α. (Hayden and increase in ER stress protein CHOP (Fig. 5C). Taken together, Ghosh, 2008; Smith, 2018). these data suggest that loss of Ca2+ channels, which is directly Cell cycle progression was evaluated next, where Raw 264.7 responsible for an increase in ER stress, causes a chain reaction cells were treated with various ER stress inducers, and cell cycle that triggers an increase in autophagy, resulting in apoptosis of distribution was analyzed by measuring the DNA content using a macrophages. flow cytometer. The results of the cell cycle analysis clearly Journal of Cell Science

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Fig. 5. Tunicamycin, brefeldin A and SKF96365 treatment induce autophagy and apoptosis in Raw 264.7 macrophages. (A) Representative immunoblot images showing expression of markers for the induction of autophagy and apoptosis in Raw 264.7 macrophages treated with 10 µM Tuni, BFA or SKF for 12 h. (B) Quantification of densitometric values (mean±s.d.) from bands as shown in A. (C) Representative microscopy images of Raw 264.7 macrophages stained for LC3B (green), CHOP (red) and DAPI (blue) after cells were exposed to 10 µM Tuni, BFA or SKF for 12 h. **P<0.01; ***P<0.001 by one-way ANOVA test. The data shown are representative of three independent experiments. Scale bars: 20 µm. showed that addition of Tuni or BFA inhibited cell cycle increase in the number of cells in the G1 phase, with a progression, which is consistent with our cell viability data. subsequent decrease in the number of cells in the S and G2 phase Importantly, Tuni and BFA treatments induced a significant (Fig. 6D,E), indicating inhibition of cell cycle progression.

Fig. 6. ER stress induces macrophages to produce mature pro-inflammatory cytokines. (A) Raw 264.7 macrophages were treated with DMSO or 10 µM of ER stress inducers Tuni, BFA or SKF for 12 h. Levels of pro-inflammatory cytokines IL-6, TNFα and IL-1β in cell supernatants were measured using ELISA. LPS was used as a positive control. (B) Representative immunoblots showing expression levels of UPR pathway proteins and NF-κB in cell extracts from Raw 264.7 macrophages treated with ER stress-inducing drugs or DMSO. (C) Quantification of densitometric values (mean±s.d.) from bands as shownin B. (D) Typical DNA content frequency histograms representing Raw 264.7 cells treated with DMSO, Tuni or BFA. The cells were stained with PI and fluorescence was measured. (E) Percentage of cells in each cell phase for the treatment groups as shown in D. (F) Representative immunoblots showing expression levels of cell cycle proteins in cell lysates from Raw 264.7 macrophages treated with 10 µM Tuni or BFA for 12 h. (G) ELISA results showing increased expression of HMGB1, Hsp70, and histone 3 when Raw 264.7 macrophages were treated with DMSO, Tuni, BFA or SKF. Data are representative of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA test; NS, non-significant. The data shown are representative of three independent experiments. Journal of Cell Science

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To further corroborate our cell cycle findings, western blotting ER stress inducers promote the M1 phenotype and stimulate demonstrated that expression of proteins associated with the cell phagocytic activity cycle, such as cyclin D1, phosphorylated Rb (Rb is also known Studies have shown that the phagocytosis of pathogens by as RB1) and phosphorylated p53 (also known as TP53), was macrophages is a crucial function of innate immune responses decreased (Fig. 6F). Studies have shown that cyclin D1, along (Santoni et al., 2018; Hirayama et al., 2017). To further understand with Rb, are limiting for progression of the cell cycle, and hence the effects of ER stress inducers on the activation of macrophages their inhibition results in G1 cell cycle arrest (Brewer and Diehl, and consequently on their polarization, cells were again treated with 2000). These data agree with studies which showed that the UPR ER stress inducers and their macrophage function was evaluated. is responsible for the coordination of the induction of ER Importantly, our data showed that ER stress activates macrophages, chaperones, while at the same time decreasing the proteins and leads to an increase in numbers of M1 type macrophages. synthesis, promoting growth arrest in the G1 phase of the cell Stimulation of Raw 264.7 cells with IFNγ for 24 h in the presence of cycle (Brewer and Diehl, 2000; Han et al., 2013). Damage- Tuni, BFA or SKF revealed a significant increase in the expression associated molecular patterns (DAMPs) are known to play a role of known M1 marker iNOS (also known as NOS2) (Fig. 7A,B). in inflammatory response and influence adaptive immunity by Consistent with this, a subsequent decrease in the M2 phenotype activating several types of innate immune machinery, including (evaluated using marker arginase-1) was observed (Fig. 7A,B). For inflammasomes (Land, 2015). Furthermore, it has been shown further confirmation of macrophage polarization, we evaluated the previously that HSP70 is responsible for the activation of expression of iNOS and arginase-1 when Raw 264.7 cells were monocytes (Pockley et al., 2008). To evaluate the expression treated with Tuni, BFA or SKF without stimulation with IFNγ, of DAMPs, we chose to investigate the following representatives which again led to an increase in iNOS expression and a decrease in of the group: high-mobility group box 1 (HMGB1), heat-shock arginase-1 when compared to the cells treated with DMSO protein 70 (HSP70) and histone 3. Importantly, increased (Fig. 7C). We also used other classic M1/2 markers, such as expression of all the DAMPs was observed when we treated CD80low/CD206high, which again showed that the M2 phenotype Raw 264.7 cells with Tuni, BFA or SKF (Fig. 6G), suggesting was decreased in the presence of ER stress inducers (Fig. S2B). that loss of Ca2+ entry leads to the secretion of DAMPs that To verify how ER stress influences macrophage function, increase immune function. phagocytic activity in the Raw 264.7 macrophage cell line was

Fig. 7. ER stress induces macrophage polarization and controls macrophage phagocytic action. (A) Validation of M1 and M2 polarization using flow cytometry. Raw 264.7 macrophages were treated with 10 μM Tuni, BFA or SKF for 12 h and stained for iNOS and arginase-1, which are specific surface markers of M1 and M2 macrophages, respectively. (B) Percentages of iNOS- and arginase-1-positive cells for each treatment as shown in A. (C) Representative immunoblots showing expression levels of iNOS and arginase-1 in cell extracts from Raw 264.7 macrophages treated with 10 μM Tuni, BFA or SKF for 12 h. Quantification of densitometric values (mean±s.d.) from bands are shown below. (D) Flow cytometric evaluation of latex beads (IgG–FITC complex) in Raw 264.7 cells treated with Tuni, BFA or SKF (10 μM for 12 h) and analyzed for FITC fluorescence. LPS was used as a positive control. (E) Quantification of numbers (mean±s.d.) of FITC-positive cells as shown in D. (F) Raw 264.7 macrophages were incubated with FITC-conjugated latex beads for 2 h hours following treatment with Tuni, BFA or SKF (10 μM for 12 h), and phagocytic activity was measured using fluorescent microscopy. The mean±s.d. phagocytic index was calculated for each treatment group (percentage of cells with engulfed particles multiplied by the mean number of particles engulfed per cell). *P<0.05, **P<0.01,

***P<0.001 by Student’s t-test; NS, non-significant. The data shown are representative of three independent experiments. Journal of Cell Science

7 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 evaluated. Phagocytosis assays were performed using flow Importantly, silencing of TRPC1, Orai1 or STIM1 further increased cytometry or microscopy with FITC-labeled latex beads, which cytokine levels (Fig. S4B). Taken together, these results clearly revealed a significant increase in levels of phagocytosis, as well as show that Orai1- and/or STMI1-mediated SOCE is critical for Ca2+ total FITC count, in cells pretreated with Tuni, BFA or SKF signaling in non-excitable cells and is essential for ER stress- (Fig. 7D–F). Importantly, treatment with LPS, which was used as a induced abnormal activation of immune cells and cell survival. positive control, also led to an increase in phagocytosis. In conclusion, our data suggest that ER stress promotes the M1 DISCUSSION phenotype, which induces increased cytokine levels and activation Incidence of chronic inflammation is associated with several diseases of phagocytosis. such as rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis (Hunter, 2012). Consequently, scientific inquiry Overexpression of Orai1 and STIM1 allows recovery of the into the mechanisms underlying prolonged immune activation has macrophage cell line by decreasing ER stress protein levels gained interest. Although an array of diseases are linked to alterations After observing the importance of SOCE in modulating ER stress, in Ca2+ homeostasis, the study of Ca2+ signaling in modulating we next evaluated whether overexpression of Orai1 and/or STIM1 ER stress has been underrepresented. Evidence suggests that in 2+ 2+ would inhibit ER stress and induce cell survival. Raw 264.7 cells macrophages, [Ca ]i is mediated by the store-operated Ca entry overexpressing Orai1 and STIM1 showed a decrease in ER stress (SOCE) mechanism (Chauhan et al., 2018; Tian et al., 2016). SOCE markers when treated with Tuni or BFA (Fig. 8A). Densitometric was first identified as a major component of non-excitable cells values for individual blots showing the expression of the proteins (Parekh and Putney, 2005), but further research has identified are given in Fig. S3. Accordingly, overexpression of Orai1 and SOCE within a multitude of tissue types (Woo et al., 2018; Prakriya STIM1 restored levels of Ca2+ entry that had been decreased by and Lewis, 2015). It has further been determined that STIM1 and Tuni or BFA treatment (Fig. 8B–G). Cell survival assays also Orai1 proteins are critical mediators of SOCE and might also revealed a significant decrease in Tuni- or BFA-induced cell death modulate immune cell functions (Shaw and Feske, 2012b; Vaeth in cells overexpressing Orai1 and STMI1 (Fig. 8H). Importantly, a et al., 2015). Thus, we focused on identification of the endogenous significant decrease in pro-inflammatory cytokines IL-6, IL-1β and Ca2+ entry channel and establishing its role in macrophage function, TNFα (Fig. 8I; Fig. S4A) was observed in both Raw 264.7 and specifically in ER stress conditions. primary macrophages, indicating that restoration of Ca2+ entry In our study, we identify that TRPC1 and Orai1 are major could prevent ER stress-induced cytokine release. To further regulators of ER-stress induced immune activation. We initially establish whether loss of TRPC1, Orai1 or STIM1 expression showed that in macrophages, when Ca2+ entry is inhibited by the would also induce pro-inflammatory cytokine release, we also action of stress-inducing agents, it directly leads to the activation of investigated the release of pro-inflammatory cytokines in cells ER stress and promotes cell death. Importantly, increased where the expression of TRPC1, Orai1 or STIM1 was inhibited. expression of CHOP, which is a multifunctional transcription

Fig. 8. Overexpression of calcium channel proteins Orai1 and STIM1 rescues macrophages from ER stress and cell death. (A) Representative immunoblots showing expression of ER stress and apoptosis markers in control (mock) Raw 264.7 cells or cells overexpressing Orai1 and STIM1, pretreated with Tuni or BFA (10 μM for 12 h). (B–G) Whole-cell patch recording showing average IV curves (B,D,F) and current intensity (C,E,G) of 6–9 cells at −80 mV under conditions as indicated in Raw 264.7 macrophages. (H) Quantification (mean±s.d.) of cell survival in control Raw 264.7 cells or cells overexpressing Orai1 and STIM1, pretreated with 10 µM Tuni or BFA for 12 h. (I) ELISA results showing mean±s.d. IL-6 levels in supernatants of Raw 264.7 macrophages treated with

DMSO, Tuni or BFA. *P<0.05, ***P<0.001 by one-way ANOVA test; NS, non-significant. The data shown are representative of three independent experiments. Journal of Cell Science

8 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 factor in the ER stress response and plays a major role in the MATERIALS AND METHODS promotion of cell death, and increased activation of IRE1α, a known Cell culture, primary cells and reagents protein involved in the regulation of the UPR (Li et al., 2014; Sano The murine Raw 264.7 cell line was obtained from Sigma-Aldrich, and Reed, 2013), were observed. Interestingly, addition of either tested for mycoplasma contamination and maintained at 37°C with 5% ’ ’ Tuni or BFA, which initiate ER stress, albeit through different CO2 in Dulbecco s modified Eagle s medium (DMEM) supplemented mechanisms, decreased SOCE protein components, suggesting that with 10% fetal bovine serum (Gibco) with streptomycin and penicillin. As the cells were from a commercial source we did not authenticate this might be the common pathway. Activation of the UPR releases κ them prior to our study. Tunicamycin (Tuni), brefeldin A (BFA), a cascade of events that promote the phosphorylation of NF B and thapsigargin and SKF96365 (SKF) were purchased from Sigma-Aldrich consequently move it to the nucleus. This in turn activates the and used at the indicated concentrations. For the primary cells, bone expression of pro-inflammatory cytokines such as TNFα, IL-6 and marrow-derived macrophages (BMDMs) were isolated from the femurs IL-1β, which activate downstream mediators of inflammation of C57BL/6 mice (6 weeks old, male) (Jackson’s laboratory and bred in (Hotamisligil, 2010). We showed that when the UPR is our animal facility). The animal protocol was approved by the activated, it promotes ER stress and increases cell death. Our University of Texas Health San Antonio IACUC committee. Femurs results provide evidence that the loss of Ca2+ influx induces ER were flushed out with PBS using a sterile needle. The BMDM cells were stress levels in macrophages. Moreover, we showed that SOCE cultivated in DMEM medium (Life Technologies) containing L929 channels are essential for Ca2+ entry and for cell survival, which conditioned media. After 5 days, fully differentiated BMDMs had formed on the bottom of the culture plates. The isolated macrophages can be relevant in autoimmune diseases. Alongside TRPC1, 2+ were then incubated at 37°C in DMEM medium supplemented with 10% Orai1 and STMI1 were shown to have an important role in Ca FBS and 1% penicillin/streptomycin solution (Trouplin et al., 2013; homeostasis. Basic macrophage functions such as phagocytosis Weischenfeldt and Porse, 2008). and macrophage polarization were also affected by the activation of ER stress. Overexpression of Orai1 and STIM1 Macrophages are responsible for detecting, engulfing and Orai1 and STIM1 adenovirus were purchased from AbmGood (Richmond, destroying pathogens and apoptotic cells. They are also the first BC, Canada), and were amplified in HEK 293 cells (ATCC) using the line of defense that phagocytose pathogens and initiate the cellular manufacturer’s protocol to obtain the crude viral lysate. For overexpression immune response (Freeman and Grinstein, 2014; Martin et al., experiments, 100 μl of the crude viral lysate encoding Orai1 or STIM1 was 2014). Interestingly, Ca2+ is critical for the engulfment of added to 75% confluent cells in a 6-well dish. Gene transduction phagocytes, and impaired Ca2+ influx could be responsible for the was evaluated 24 h after transduction using western blotting. For silencing experiments respective siRNA (siOrai1, assay ID: 288184; iTRPC1, assay failure of the anti-inflammatory response, which could lead to ID: 187432; both Thermo Fisher Scientific) was used, and the transfection inflammation and autoimmunity (Gronski et al., 2009). Any change was performed with Lipofectamine RNAiMAX Reagent (Thermo Fisher 2+ in Ca influx could result in abnormal function and affect the Scientific) following the manufacturer’s protocol. Cells were seeded to be regulation of immune functions, such as the activation of pro- 70% confluent, and the Lipofectamine reagent was diluted to an optimal inflammatory cytokines TNF-α, IL-6 and IL-1β, which are involved concentration (25 pmol per well in a 6-well dish) in Opti-MEM medium in the upregulation of inflammatory reactions. Consequently, (Gibco). We added the Lipofectamine to the diluted siRNA in a 1:1 ratio and decreases in expression of the Ca2+ channel proteins could be incubated for 5 min at room temperature, after the stipulated time the associated with an abnormal immune response. complex was layered on the cells for 5 h, followed by adding fresh media. Our data show that loss of function of TRPC1 and/or Orai1 Transfected cells were then used for individual experiments. causes a decrease in Ca2+ influx, leading to autophagy and cell Cell viability assay death. These findings are consistent with previous studies showing 2+ Cell viability was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5- that intracellular Ca is widely recognized as a key regulator of diphenyltetrasolium bromide (MTT) (Sigma-Aldrich) according to the autophagy (Parys et al., 2012) and apoptosis (Sano and Reed, 2013; manufacturer’s protocol. Primary BMDMs were seeded in 96-well plates at Yoshino et al., 2017). Therefore, a decrease in the expression of a density of 0.5×105 cells/well in DMEM supplemented with 10% FBS, and Ca2+ channels in macrophages can be strongly associated with cell streptomycin and penicillin. The cultures were incubated for 24 h at 37°C 2+ death, as demonstrated by our results. Moreover, Ca signaling with 5% CO2, followed by the addition of fresh medium prior to the regulates cell cycle progression in various cell types. Ca2+ transients experiment being performed. Cell viability was measured using the MTT are detected at wakening from quiescence, at the G1/S shift, during method. 10 μl of MTT reagent (5 mg/ml MTT in PBS) was added to each the S-phase and after mitosis (Chen et al., 2016). The activation of well and incubated in a CO2 incubator for 4 h. The resulting formazan dye μ SOCE channels indeed regulates cell-cycle protein levels and was extracted with 100 l of 0.01 N HCl in isopropanol, and within an hour, the absorbance was measured on an ELISA plate reader with a test controls the G1/S cell cycle transition. Loss of Ca2+ influx prevents wavelength of 570 nm and a reference wavelength of 630 nm. Cell viability the progression of cells into the cell cycle, causing cell cycle arrest of experimental cultures was expressed as a percentage of the control that could further lead to apoptosis. Consistent with those studies, culture. we demonstrated that macrophages under ER stress show a loss of SOCE, which further decreases the expression of cell cycle proteins Calcium measurement and electrophysiology associated with cell cycle arrest. Consistent with these results, For fluorescence measurements, cells were incubated with 2 μM Fura-2 2+ overexpression of Orai1 and/or STIM1 seems to restore Ca (Molecular Probes) for 45 min and then washed twice with Ca2+-free SES influx in these cells. We also observed an increase in cell buffer [in mM, NaCl, 145; KCl, 5; MgCl2, 1; HEPES, 10; glucose, 10; pH survival, as it is characterized by a decrease in ER stress protein 7.4 (NaOH); reagents from Sigma-Aldrich]. Cells were monitored with a expression, and observed a reverse in the induction of apoptosis charge-coupled device (CCD) camera-based imaging system. and the production of pro-inflammatory cytokines. Taken Images of multiple cells collected at each excitation wavelength together, our results could indicate that expression of SOCE were processed using C*IMAGING Systems - SimplePCI (Compix channels Orai1 and/or TRPC1 plays a major role in modulating Inc.) to provide ratios of Fura-2 fluorescence from excitation at ER stress and, consequently, inducing inflammation by immune 340 nm to that from excitation at 380 nm (F340/F380) as previously activation via the UPR pathway. described (Sun et al., 2014). For patch-clamp experiments, Journal of Cell Science

9 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610 coverslips with cells were transferred to the recording chamber and values for the murine cell line Raw 264.7 and bone marrow-derived perfused with an external Ringer’s solution with the following macrophages serum samples examined were above the limit of detection and within the reportable range of each assay. composition: 145 mM NaCl, 5 mM KCl, 1 mM MgCl2,1mM CaCl2, 10 mM HEPES, 10 mM glucose, pH 7.4 (NaOH). The patch pipette had resistances between 3–5 m after filling with the standard Confocal microscopy For immunofluorescence staining, the cells were first fixed, then stained for intracellular solution that contained the following: 145 mM cesium the detection of specific antibodies, blocked with 10% donkey serum in methanesulfonate, 8 mM NaCl, 10 mM MgCl2, 10 mM HEPES, phosphate-buffered saline, and probed for 1–2 h with anti-CHOP (Cell 10 mM EGTA, pH 7.2 (CsOH). The maximum peak currents were Signaling, L63F7), anti-LC3B (Cell Signaling, D11) and anti-Beclin1(Cell calculated at a holding potential of −80 mV. The I/V curves were Signaling, D40C5) antibodies, all at 1:1000 at room temperature. Following made using a ramp protocol where current density was evaluated at incubation with primary antibodies, the cells were washed and reacted with various membrane potentials and plotted. fluorophore-conjugated respective secondary antibodies for 1 h at room temperature. Thereafter, the slides were washed again, and coverslip Caspase 3 activity mounted using VECTASHIELD HardSet Mounting Media with DAPI Caspase 3 activity was measured using the Abcam Caspase 3 assay kit. (Vector Laboratories, Inc.). Images were acquired at 63× magnifications 1×106 cells were isolated using the cell lysis buffer provided and the liquid using an Olympus FV-1000 confocal/MPE laser-scanning microscope. fraction was used to analyse the caspase 3 activity as per the manufacturer’s instructions. The protocol is based on the formation of the chromophore Cell cycle analysis p-nitroaniline (p-NA) by cleavage from the labeled substrate DEVD-pNA. Flow cytometry was used to assess whether chemical treatments arrested Levels of p-NA can be quantified using a spectrophotometer reading Raw 264.7 cells at any stage of the cell cycle. At the time of analysis, the absorbance at 400 or 405 nm. cells were centrifuged, washed in PBS, fixed with cold methanol for two hours at 4°C and stained with a freshly made solution containing 0.1 mg/ml Protein extractions and western blotting propidium iodide (PI) (Sigma-Aldrich) and 0.2 mg/ml ribonuclease A in Whole-cell protein lysates were prepared in lysis buffer (10 mM Tris, PBS. All samples were incubated overnight in the dark at 4°C. Cell-cycle 140 mM NaCl, 1% NP-40, 0.5% SDS, and protease inhibitors, pH 8.0), distribution was determined using a FACSCalibur flow cytometer (BD protein concentrations were determined using the Bradford reagent assay Biosciences) and data analyzed with FlowJo software on DNA instrument (Bio-Rad Laboratories), and 25–50 µg of proteins were resolved on settings (linear FL2) on low. NuPAGE Novex 4–12% BisTris gels, transferred to PVDF membranes. The membrane was blocked with skim milk in PBST solution and incubated Macrophage polarization with primary antibodies as listed below. After washing with PBST, Raw 264.7 cells were exposed to IFNγ (20 ng/ml, Peprotech) 24 h prior to secondary antibodies were applied and detected using the Clarity Western treatment with Tuni, BFA or SKF96365 to generate the M1 inflammatory ECL Substrate and Clarity Max Western ECL Substrate (Bio-Rad phenotype. Stimulated cells were removed from wells and incubated in anti- Laboratories). Analysis and results were corrected for protein loading by mouse BD Fc Block CD16/CD32 (BD Biosciences) for 30 min on ice in normalization for β-actin expression. Densitometric analysis was performed FACS buffer (PBS with 3% FBS), then surface-stained with PE-conjugated using ImageJ software and results were corrected for protein loading by anti-iNOS (Cell Signaling Technology, D6B6S), anti-arginase-1 (Cell normalization for β-actin expression as previously described (Singh et al., Signaling Technology, D43EM), anti-CD80 (Abcam, ab134120) and anti- 2004). Blot stripping was performed whenever necessary, using Restore CD206 (Abcam, ab64693) antibodies for 15 min at 4°C. Cells were washed PLUS Western Blot Stripping Buffer (Thermo Fisher). The blots were three times with FACS buffer, then fixed with paraformaldehyde (4%) for washed with PBS Tween 20, immersed in stripping buffer and incubated 40 min at 4°C and washed three times in PBS. For intracellular staining, cells for 5–15 min at room temperature, then the stripping buffer was removed, were permeabilized by adding ice-cold 100% methanol slowly to the cold the blot washed again and then blocked for another 30 min before cells with gentle vortexing, to a final concentration of 90% methanol. incubation with a new antibody. The following primary antibodies Cells were then incubated for 30 min at 4°C prior to staining with anti- were used for western blot analysis (all at 1:1000): anti-Orai1 (Thermo arginase-1 primary antibody for 1 h at 4°C, cells were then washed three Fisher, PA5-26378), anti-TRPC1 (Alamone, ACC-010), anti-STIM1 times by means of centrifugation at 400 g for 5 min with FACS buffer, (D88E10), anti-CHOP (L63F7), anti-PDI (C81H6), anti-GRP94 and then incubated in the dark with FITC- and PE-labeled secondary (D6X2Q), anti-NFκB p65 (p65) (8242S), anti-pNFκB p65 (pp65) antibody [FITC-conjugated goat anti-rabbit-IgG (Abcam, ab6717) and (3033S), anti-Bax (D2E11), anti-IREI1α (14C10), anti-Bcl2 (D17C4), PE-conjugated goat anti-rabbit-IgG (Abcam, ab97024), both used at 1:50 anti-ATF6 (D4Z8V), anti-PERK (C33E10), anti-pPERK (16F8), anti- dilution] 30 min at 4°C. Cells were washed three times by means of TNFα (D2D4), anti-IL-6 (D5W4V), anti-IL-1β (D6D6T), anti-HMGB1 centrifugation at 400 g for 5 min, then analyzed using a BD LSR II flow (D3E5), anti-Histone 3 (D1H2), anti-HSP70 (4872S), anti-Rb (D20), cytometer (BD Biosciences) and FlowJo software version 9.0. anti-pRB (D20B12), anti-Cyclin D1 (92G2), anti-p53 (1C12), anti-pp53 (Ser15), anti-LC3B (D11), anti-caspase 3 (9662S), anti-caspase 9 (C9) and Phagocytosis assay anti-β-actin (4970S) (all from Cell Signaling Technology). HRP-conjugated After treatment, Raw 264.7 cells were assessed for phagocytic activity using goat anti-rabbit IgG (7074S) and goat anti-mouse IgG (7076S) (both from the Phagocytosis Assay Kit (Cayman Chemical). Cells were incubated with Cell Signaling Technology) were used as secondary antibodies. latex beads with rabbit IgG–FITC conjugates (1:100) for 4 h followed by a 15-min incubation with Hoechst 33258 (Thermo Fisher) to quench non- Measurement of cytokines levels phagocytosed bead fluorescence. We used LPS (100 ng/ml, Sigma-Aldrich) IL-6, TNF-α and IL-1β levels in the supernatant were measured using the as a positive control, cells were tested following incubation for 3 h after appropriate enzyme-linked immunosorbent assay (ELISA) kit (Life treatments. Thereafter, the slides were washed, and coverslips mounted Technologies) following the manufacturer’s instructions, using standard using VECTASHIELD HardSet Mounting Media with DAPI (Vector diluent buffers designed for use with mouse serum. We used Laboratories, Inc.). Five images were acquired at 63× magnifications from lipopolysaccharides (LPS, 100 ng/ml, Sigma-Aldrich) as a positive each well using an Olympus FV-1000 confocal/MPE laser-scanning control, cells were tested following incubation for 3 h after treatments. microscope. For the flow cytometry part of the assay, after incubation HMGB1, histone 3 and HSP70 levels were measured by direct ELISA using cells were washed and analyzed using a BD LSR II flow cytometer. The a self-coating protocol, as previously described (Zhang et al., 2012). All phagocytic index was calculated using the formula: phagocytic samples were measured on a single 96-well plate for each cytokine, the index=[(total number of engulfed cells/total number of counted cultured medium was centrifuged at 800 g for 5 min at 4°C and the macrophages)×(number of macrophages containing engulfed cells/total supernatant was used as the sample. Based on that criterion, all cytokine number of counted macrophages)]×100. Journal of Cell Science

10 RESEARCH ARTICLE Journal of Cell Science (2020) 133, jcs237610. doi:10.1242/jcs.237610

Statistical analysis lipopolysaccharide-induced inflammation. J. Immunol. 176, 6245-6253. doi:10. Data analysis was performed using a one-way ANOVA or Student’s t-test on 4049/jimmunol.176.10.6245 GraphPad Prism 8.0. Ca2+ measurement and electrophysiology were Feske, S., Gwack, Y., Prakriya, M., Srikanth, S., Puppel, S.-H., Tanasa, B., Hogan, P. G., Lewis, R. S., Daly, M. and Rao, A. (2006). A mutation in Orai1 analyzed using Origin 9.0 software (OriginLab). Experimental values are P P P causes immune deficiency by abrogating CRAC channel function. Nature 441, expressed as means±s.d. -values are represented as * <0.05, ** <0.01 179-185. doi:10.1038/nature04702 and ***P<0.001. Franken, L., Schiwon, M. and Kurts, C. (2016). Macrophages: sentinels and regulators of the immune system. Cell. Microbiol. 18, 475-487. doi:10.1111/cmi. Acknowledgements 12580 The University of Texas Health San Antonio Flow Cytometry Facility is Freeman, S. A. and Grinstein, S. (2014). Phagocytosis: receptors, signal supported by UTHSCSA, NIH-NCI P30 CA054174-20 and UL1 TR001120. integration, and the cytoskeleton. Immunol. Rev. 262, 193-215. doi:10.1111/imr. Images were generated in the Core Optical Imaging Facility which is supported 12212 by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA Gautier, E. L., Shay, T., Miller, J., Greter, M., Jakubzick, C., Ivanov, S., Helft, J., P01AG19316. Chow, A., Elpek, K. G., Gordonov, S. et al. (2012). Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. 13, 1118-1128. doi:10.1038/ni.2419 Competing interests Görlach, A., Klappa, P. and Kietzmann, T. (2006). The endoplasmic reticulum: The authors declare no competing or financial interests. folding, calcium homeostasis, signaling, and redox control. Antioxid Redox Signal. 8, 1391-1418. doi:10.1089/ars.2006.8.1391 Author contributions Gronski, M. A., Kinchen, J. M., Juncadella, I. J., Franc, N. C. and Ravichandran, Conceptualization: V.N.D.C., B.B.S.; Methodology: V.N.D.C., Y.S.; Formal analysis: K. S. (2009). An essential role for calcium flux in phagocytes for apoptotic cell V.N.D.C., Y.S., E.K.Z., J.J.D.l.C., B.B.S.; Investigation: V.N.D.C., Y.S., E.K.Z., engulfment and the anti-inflammatory response. Cell Death Differ. 16, 1323-1331. J.J.D.l.C.; Writing - original draft: V.N.D.C.; Writing - review & editing: B.B.S. doi:10.1038/cdd.2009.55 Grootjans, J., Kaser, A., Kaufman, R. J. and Blumberg, R. S. (2016). The Funding unfolded protein response in immunity and inflammation. Nat. Rev. Immunol. 16, This work was funded by grant support from the National Institutes of Health National 469-484. doi:10.1038/nri.2016.62 Institute of Dental and Craniofacial Research (R01DE017102; R01DE022765) Han, C., Jin, L., Mei, Y. and Wu, M. (2013). Endoplasmic reticulum stress inhibits awarded to B.B.S. The funders had no further role in study design, data analysis, cell cycle progression via induction of p27 in melanoma cells. Cell. Signal. 25, and/or interpretation of the data. Deposited in PMC for immediate release. 144-149. doi:10.1016/j.cellsig.2012.09.023 Hayden, M. S. and Ghosh, S. (2008). Shared principles in NF-kappaB signaling. Supplementary information Cell 132, 344-362. doi:10.1016/j.cell.2008.01.020 Hirayama, D., Iida, T., and Nakase, H. (2018) The Phagocytic Function of Supplementary information available online at Macrophage-Enforcing Innate Immunity and Tissue Homeostasis. Int. J. Mol. Sci. http://jcs.biologists.org/lookup/doi/10.1242/jcs.237610.supplemental 19, 92. doi:10.3390/ijms19010092 Ho, H.-J., Huang, D.-Y., Ho, F.-M., Lee, L.-T. and Lin, W.-W. (2012). Inhibition of Peer review history lipopolysaccharide-induced inducible nitric oxide synthase expression by The peer review history is available online at endoplasmic reticulum stress. Cell. 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