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By Ana Catarina Pereira, Nuno Madeira, Sofia Morais, António Macedo, Maria Teresa Cruz, and Cláudia M. F. Pereira

Excerpt from the article published in Cells 11, no. 3: 433, 27 January 2022, DOI: https://doi.org/10.3390/cells11030433

Editor’s Highlights

• Activation of the the Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome has been implicated in a wide range of diseases.
• The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is a pump that belongs to the P-type ATPase family, which uses the energy produced by ATP hydrolysis for the active transport of Ca²⁺ ions from the cytosol into the sarcoplasmic reticulum or endoplasmic reticulum (ER) lumen, in the case of muscle or non-muscle cells, respectively. 
• Inhibition of SERCA activity has been closely associated with the depletion of Ca²⁺ levels in ER reservoirs and, subsequently, the disruption of ER functions, namely the activity of the Ca²⁺-dependent ER chaperons.
• NLRP3 inflammasome is not activated by SERCA inhibition in human monocytes, either in primary monocytes or in the THP-1 monocytic cell line.
• Human monocytes trigger adaptive strategies through remodeling of mitochondrial morphology towards fusion in order to preserve metabolism and energy production, a mechanism called mitochondrial hyperfusion, preventing mitochondrial dysfunction in response to ER stress caused by SERCA inhibition, namely ROS accumulation, membrane depolarization, and fission, thus avoiding NLRP3 inflammasome activation and, subsequently, the release of the pro-inflammatory cytokine IL-1β.
• Therefore, human monocytes can counteract early ER stress due to SERCA inhibition by modulating mitochondrial morphology and function in order to preserve energy production and cell survival and to prevent sterile inflammation induced by NLRP3 inflammasome activation.
• The ER transmembrane chaperone Sigma-1 receptor (Sigma-1R) seems to be fundamental to trigger and fit anti-stress responses, since it can interact with multiple proteins and exhibit pleotropic effects.

Abstract

Sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is a crucial component of the cellular machinery responsible for Ca²⁺ homeostasis. The selective inhibition of SERCA by thapsigargin (TG) leads to perturbations in Ca²⁺ signaling, which can trigger endoplasmic reticulum (ER) stress. The unfolded protein response (UPR) pathway is activated in response to ER stress and induces an adaptive response to preserve cell survival or committee cells to programmed death, depending on stress duration and/or level. Early stages of ER stress stimulate mitochondrial metabolism to preserve survival but under chronic ER stress conditions, mitochondrial dysfunction is induced, which, in turn, can enhance inflammation through NLRP3 inflammasome activation. This study was aimed at investigating the role of SERCA inhibition on NLRP3 inflammasome activation in human monocytes, which was evaluated in primary monocytes isolated from healthy individuals and in the THP-1 human monocytic cell line. Findings obtained in both THP-1 and primary monocytes demonstrate that SERCA inhibition triggered by TG does not activate the NLRP3 inflammasome in these innate immune cells since IL-1β secretion was not affected. Results from THP-1 monocytes showing that SERCA inhibition increases mitochondrial Ca²⁺ content and fusion, in the absence of changes in ROS levels and membrane potential, support the view that human monocytes counteract ER stress that arises from inhibition of SERCA through modulation of mitochondrial morphology towards mitochondria fusion, thus preventing NLRP3 inflammasome activation. Overall, this work contributes to a better understanding of the molecular mechanisms that modulate the activity of the NLRP3 inflammasome leading to sterile inflammation, which are still poorly understood.

1. Introduction

Endoplasmic reticulum (ER) is the main intracellular Ca²⁺ store and the major site for protein synthesis, folding and maturation in eukaryotic cells [1]. The sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is a pump that belongs to the P-type ATPase family, which uses the energy produced by ATP hydrolysis for the active transport of Ca²⁺ ions from the cytosol into the sarcoplasmic reticulum or endoplasmic reticulum (ER) lumen, in the case of muscle or non-muscle cells, respectively. Based on its molecular mechanism of action, SERCA maintains a low cytosolic Ca²⁺concentration playing a pivotal role in the regulation of Ca²⁺ homeostasis, which is crucial for cell signaling and survival [2,3]. Indeed, Ca²⁺ is a ubiquitous intracellular messenger able to coordinate various cellular functions; however, at high concentrations, Ca²⁺ becomes cytotoxic [4,5]. Inhibition of SERCA activity has been closely associated with the depletion of Ca²⁺ levels in ER reservoirs and, subsequently, the disruption of ER functions, namely the activity of the Ca²⁺-dependent ER chaperons, which leads to the accumulation of unfolded proteins in the ER lumen, triggering ER stress [5,6,7]. Thapsigargin (TG) is a specific SERCA inhibitor described as a known ER stressor and therefore as an activator of the unfolded protein response (UPR) cascade, which consists of a set of transcriptional and translational events triggered in response to cellular stress [8,9].

Regarding ER stress signaling, BiP/GRP78 chaperone, which is critical for ER protein quality control, is associated with the three ER stress sensors of the UPR: protein kinase R-like ER kinase (PERK), inositol-requiring enzyme 1 alpha (IRE1α), and transcription factor 6 (ATF6), keeping them inactive during resting conditions. Upon ER stress, BiP/GRP78 chaperone binds to unfolded proteins accumulated in the ER lumen, which leads to dissociation of BiP/GRP78 protein from the ER stress sensors, culminating in UPR activation [10,11]. Depending on the level of cell stress, UPR orchestrates an adaptive response able to restore ER homeostasis and avoid cell death, or can trigger UPR-mediated apoptosis [9,12,13].

In addition to the UPR, cells also have other signaling mechanisms that act as strategic responses to help them deal with stressful conditions. The ER transmembrane chaperone Sigma-1 receptor (Sigma-1R) seems to be fundamental to trigger and fit anti-stress responses, since it can interact with multiple proteins and exhibit pleotropic effects [14,15]. For instance, Sigma-1R interacts with BiP/GRP78 chaperone, forming a complex at MAM surface. Upon ER Ca²⁺ depletion, which can arise from chronic ER stress, Sigma-1R dissociates from BiP/GRP78 in order to regulate Ca²⁺ flux from the ER into mitochondria via IP3 receptors (IP3Rs). In addition, Sigma-1R also influences bioenergetics and cellular survival through modulation of ER Ca²⁺ signaling, [16,17]. Recently, Sigma-1R was found particularly enriched in specialized ER-subdomains, called mitochondria-associated membranes (MAMs), which are contact sites resulting from the physical interaction of ER and mitochondria, thus suggesting its role in regulating ER-mitochondria communication [16].

Chronic/severe ER stress often triggers inflammatory responses, culminating in systemic metabolic dysfunction [18]. Over the last few years, multiprotein complexes called NLRP3 inflammasomes have been identified as central players in innate immune responses [19]. They are composed by a cytosolic pattern-recognition receptor, the Nod-like receptor pyrin domain containing 3 (NLRP3), the enzyme caspase-1, and the adaptor protein termed apoptosis-associated speck-like protein containing a CARD (ASC). The self-association of all these components leads to inflammasome activation, which is characterized by caspase-1-dependent maturation of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). NLRP3 is the best-characterized inflammasome and has been defined as a driver of inflammation in response to mitochondrial damage. Classic NLRP3 activating stimuli include reactive oxygen species (ROS), release of mitochondrial DNA (mtDNA), cardiolipin externalization, release of cathepsins into the cytosol after lysosomal destabilization, and alterations in Ca²⁺ homeostasis [20,21,22]. Mitochondrial dysfunction is intrinsically linked to loss of mitochondrial membrane potential and disrupted mitochondrial network dynamics [23]. In healthy cells, mitochondrial morphology depends on a dynamic equilibrium between fission and fusion events. Mitochondrial fusion is coordinated by mitofusins (MFN1, MFN2), whereas dynamin-related protein 1 (DRP1) is defined as the major mediator of mitochondrial fission [24]. In addition to dynamic regulation of mitochondrial morphology, MFN2 also modulates ER-mitochondria tethering [25]. Induction of stress conditions has been closely associated with a dynamic remodeling of mitochondrial morphology [26,27]. Under acute ER stress conditions, a protective mechanism mediated by enhanced mitochondrial fusion can be stimulated to prevent pathologic mitochondrial fragmentation and promote mitochondrial metabolism, which is dependent on membrane potential [27]. Under severe stress conditions, there is an imbalance in the highly conserved dynamic processes towards mitochondrial fission, which is linked to the loss of mitochondrial membrane potential that acts as a signal to facilitate fragmentation and degradation of damaged mitochondria by mitophagy [26,28]. Recent studies suggest that perturbations in mitochondrial fission contribute to NLRP3 inflammasome-mediated inflammation [29,30].

This study aimed to investigate whether SERCA inhibition, and subsequent induction of ER stress, activates the NLRP3 inflammasome leading to sterile inflammation in human monocytes. Furthermore, the relationship between mitochondrial dysfunction and NLRP3 inflammasome activation under these conditions was also evaluated in human THP-1 monocytes.

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3. Results

3.1. SERCA Inhibition Induces ER Stress in Human Monocytes

First, induction of ER stress was evaluated in human monocytes treated with TG, a classic SERCA inhibitor. For that, activation of the ER stress-induced UPR signaling pathway was assessed in THP-1 cells treated with 5 or 10 μM TG in a time-dependent manner (1–24 h) by measuring the protein levels of UPR markers namely ATF4, XBP1s, GRP78, and CHOP. ATF4 protein levels were found significantly increased at all time points in TG-treated THP-1 monocytes when compared with controls cells (Figure 1A). A time-dependent increase in XBP1s levels was observed in TG-treated cells, which reached statistical significance at 24 h (Figure 1B). The levels of CHOP and GRP78 showed a tendency to increase, namely at 8 and 24 h of TG exposure, respectively, although it was not statistically significant (Figure 1C,D). The results showed that SERCA inhibition triggered by TG induces ER stress in human monocytes, as demonstrated by the activation of the UPR pathway.

3.2. SERCA Inhibition Is Not Able to Upregulate ER Stress Response Strategies

Given that Sigma-1R plays a key role in cellular stress signaling, namely under ER stress conditions, its protein levels were evaluated by WB in THP-1 monocytes treated with 5 or 10 μM TG in a time-dependent manner (1–24 h). No differences were observed in Sigma-1R levels between controls and TG-treated cells, indicating that ER stress induced by SERCA inhibition does not upregulate Sigma-1R in order to mitigate ER stress (Figure 2).

3.3. SERCA Inhibition Fails to Promote IL-1β Secretion in Human Monocytes

Afterwards, the role of SERCA inhibition on NLRP3 inflammasome activation was investigated by measuring the levels of IL-1β secreted by control and TG-treated THP-1 monocytes. In cells treated with LPS, the secretion of IL-1β increased by about 400-fold in comparison with control cells. The canonical NLRP3 activators LPS plus ATP augmented IL-1β secretion by about 200-fold in comparison with LPS-treated monocytes (Figure 3). No increment in IL-1β levels was detected in supernatants from THP-1 cells treated with LPS plus vehicle or treated with LPS plus TG, suggesting that TG is not able to activate the NLRP3 inflammasome that promotes IL-1β release to the extracellular space.

After demonstrating that TG-induced SERCA inhibition is not able to activate NLRP3 inflammasome in the THP-1 cell line of human monocytes, its effect was also investigated in primary monocytes isolated from peripheral blood (Figure 4). Differences were not observed when comparing the IL-1β levels secreted by control and TG-treated primary monocytes (Figure 4A). Given that NLRP3 inflammasome activation is classically described as a two-signal model, its activity was further assessed in LPS-primed cells. Once again, the secreted levels of IL-1β were similar between monocytes incubated with LPS alone or with LPS plus 10 μM TG (Figure 4B).

These findings indicate that TG-induced SERCA inhibition does not work as an activator of the NLRP3 inflammasome in primary monocytes as well, supporting that this effect is specific of this type of cell from the innate immune system.

3.4. NLRP3 Inflammasome-Associated Triggers Are Not Promoted by SERCA Inhibition

Once it was demonstrated that SERCA inhibition does not activate the NLRP3 inflammasome in human monocytes, some key mitochondrial events that have been described as triggers of inflammasome activation were evaluated, including ROS production, membrane depolarization, and fission. The alterations induced by TG on mitochondrial membrane potential were assessed with the TMRE fluorescent probe using FCCP as a positive control (Figure 5A). In cells treated with this uncoupler of the electron transport chain FCCP, TMRE fluorescence was approximately 50% of that displayed by control cells. On the other hand, no differences were observed in TMRE fluorescence between control and TG-treated cells at 4 and 8 h, suggesting that SERCA does not induce loss of mitochondrial membrane potential in THP-1 monocytes.

The mitochondrial ROS production in human monocytes was assessed through the MitoSOX probe (Figure 5B). Both control and TG-treated THP-1 cells exhibited similar levels of MitoSOX fluorescence, showing that SERCA inhibition induced by TG does not stimulate the accumulation of oxidant species within mitochondria.

Given that DRP1-mediated mitochondrial fission can trigger NLRP3 inflammasome activation, the levels of DPR1 phosphorylated at Ser616 were evaluated by WB in the absence or presence of SERCA inhibition in human monocytes. No relevant differences were observed in p-DPR1 levels between control and TG-treated THP-1 cells from 1–24 h of TG exposure (Figure 5C).

Taken together, these findings suggest that molecular triggers of NLRP3 inflammasome activation, such as mitochondrial ROS, mitochondrial membrane loss, and fission are not affected by SERCA inhibition in human monocytes.

3.5. SERCA Inhibition Increases Mitochondrial Fusion and Ca²⁺ Content

Then, the effect of SERCA inhibition on mitochondrial fusion was analyzed in human THP-1 monocytes. For that purpose, protein levels of Mitofusin 2, a protein mediator of mitochondria fusion were assessed by WB in controls and in TG-treated cells (Figure 6A). A significant upregulation of Mitofusin 2 was detected upon 8 and 24 h of exposure to TG when compared to control conditions, suggesting that TG promotes mitochondrial fusion. These findings were further supported by results obtained by TEM, showing that the presence of fused mitochondria is increased in monocytes treated with 10 µM TG when compared to untreated cells (Figure 6B).

Mitochondrial Ca²⁺ content was also evaluated under SERCA inhibition conditions in human monocytes, using the Rhod-2AM fluorescent probe (Figure 6C). When compared with control cells, a significant augment in Rhod-2AM fluorescence was found in THP-1 monocytes treated with TG, particularly after 4 and 8 h incubation, suggesting that SERCA inhibition in these innate immune cells is followed by a rise in mitochondrial Ca²⁺ content. Moreover, control monocytes were incubated with the selective inhibitor of the MCU, RU 360, in order to ensure that Rhod-2 fluorescence is specifically associated with mitochondria Ca²⁺ and is not result of probe mislocalization. A significant reduction in Rhod-2 fluorescence was observed in cells treated with RU 360 in comparison with untreated cells, demonstrating that mitochondria Ca²⁺ is the main source of the observed Rhod-2 fluorescence.

Finally, susceptibility of human monocytes towards SERCA inhibition was assessed by the resazurin assay in cells treated with 5 or 10 μM TG for 24 h. Both TG concentrations were shown to decrease cell viability by approximately 20% (Figure 6D).

4. Discussion

Activation of the NLRP3 inflammasome has been implicated in a wide range of diseases. However, the underlying mechanisms remain largely uncertain [31,32]. In order to clarify the signaling pathways that regulate the NLRP3 inflammasome in human monocytes, the role of SERCA inhibition induced by exposure to TG was evaluated in these cells from the innate immune system. For that purpose, both human THP-1 monocytic cell line and primary monocytes were used. The low yield of monocytes isolated from peripheral blood strongly impacted the number of parameters analyzed on these cells. To overcome this limitation, THP-1 human monocytes were used as an in vitro model, allowing us to further explore NLRP3 inflammasome activation.

First, it was demonstrated that inhibition of SERCA activates the ER stress-induced UPR in THP-1 monocytes. More specifically, TG activated both the PERK and IRE1α branches of the ER UPR, as shown by the upregulation of ATF4 and XBP1s, respectively, and ER stress was found to be an early event. The PERK and IRE1α UPR branches were the particular focus of this study, given their crucial role in triggering inflammatory responses during ER stress. Under stressful conditions, components of the PERK and IRE1α UPR arms interact with inflammatory signaling cascades, which results in the upregulation of inflammatory genes and subsequent induction of inflammatory responses. Activated IRE1 interacts with IKK and JNK, leading to activation of the inflammatory mediators NF-κB and AP-1. When activated by phosphorylation, eIF2α, which is a signaling mediator at the PERK UPR branch, is also able to activate NF-κB by inhibiting the translation of IκBα [33].

UPR activation in TG-treated cells was further supported by the tendency of increase in other ER stress markers such as GRP78 and CHOP. These data are in accordance with studies that have emerged over the years reporting consistently the activation of the UPR in TG-treated mammalian cells [8,34,35]. ATF4 transcription factor regulates the expression of numerous genes, including the pro-apoptotic factor CHOP. These mediators of the PERK UPR arm play an important role in committing cells to apoptosis under chronic ER stress conditions [35]. Our findings suggest that human monocytes are able to trigger strategies to cope with chronic ER stress upon SERCA inhibition, as indicated by the 17–25% decrease in viability observed in cells treated with 5 or 10 μM TG for 24 h, respectively. Based on ISO 10993-5:2009, “Biological evaluation of medical devices-Part5: Tests for in vitro cytotoxicity”, a reduction in cell viability below 30% is not considered a cytotoxic effect. Therefore, under the present experimental conditions, TG is non-cytotoxic.

Concerning the molecular mechanisms that could be involved in the adaptive response triggered by innate immune cells under stressful conditions, the focus was on Sigma-1R-mediated stress response. Structurally, Sigma-1R is an ER membrane protein that contains an N-terminus that faces the cytosol and a C-terminus that faces the ER lumen [36]. Its major action site is the ER, where it can bind specific signaling molecules, such as the ER chaperone GRP78/BiP [16,17,37]. Functionally, in its dormant state, Sigma-1R forms a complex with GRP78/BiP. Under ER stress conditions, Sigma-1R dissociates from GRP78 and regulates the chaperone activity at the ER by stabilizing the IRE1 and IP₃Rs [16,38,39]. Mori and colleagues showed that Sigma-1R is able to associate with activated IRE1, which upregulates ER chaperones [38,39]. Regarding its action on IP₃Rs, Hayashi and Su found that Sigma-1R-IP₃R complexes prolong ER-to-mitochondria Ca²⁺ flux under chronic ER stress conditions. Moreover, it was observed that ER stress induced either by TG or tunicamycin, specific ER stressors, promotes the transient expression of Sigma-1R, which was not detected after 3 h. According to these authors, the Sigma-1R upregulation represents a cellular response to stress, and its inhibition 3 h upon ER stress induction can be explained by a decline in the stability of Sigma-1R protein and/or mRNA [16]. Our data suggest that SERCA inhibition does not activate Sigma-1R-mediated stress response in THP-1 monocytes.

TG is a specific blocker of the SERCA pump, which is crucial for reallocate released Ca²⁺ back into the ER and maintain Ca²⁺ homeostasis. Since Ca²⁺ can activate physiological and pathological signaling pathways, a tight regulation of intracellular Ca²⁺ concentrations is crucial to maintain proper Ca²⁺ dynamics and thus preserve normal cell function [4,5]. Due to its mechanism of action, TG administration has been intimately associated with a rapid depletion of ER Ca²⁺ stores and, subsequently, with an increase in intracellular Ca²⁺ levels [2,8]. Our findings showing enhanced levels of the fusion protein Mfn2 in TG-treated THP-1 cells, together with TEM representative images that show an increase in fused mitochondria in these cells, suggest that mitochondria fusion might be an adaptive response triggered by human monocytes to cope with stress induced by SERCA inhibition. The imbalance of mitochondrial dynamics towards mitochondrial fusion can be responsible for enhanced mitochondrial Ca²⁺ uptake observed in TG-treated THP-1 cells, which is required to activate enzymes involved in the TCA cycle and ATP production [40,41]. This hypothesis is supported by a recent study developed by Lebeau and collaborators that shows that TG-induced ER stress remodels mitochondrial morphology to prevent mitochondrial fragmentation and thus promote mitochondrial metabolism in response to ER stress. Moreover, it was demonstrated that the mechanism that protects mitochondria during ER stress is orchestrated through the PERK branch of the ER UPR. Upon activation, PERK selectively activates eIF2α by phosphorylation, which, in turn, promotes protective stress-induced mitochondrial hyperfusion [27]. This protective mechanism promotes elongation of mitochondria and avoids fragmentation that triggers mitophagy, and it was previously described as a cellular response to other types of stress, such as starvation, ribosome inhibition, and UV irradiation [42,43,44]. According to Sabouny et al., mitochondria hyperfusion mediated by the Keap1–Nrf2 pathway in response to stress involves the degradation of the mitochondrial fission protein DRP1 [44]. The enhanced mitochondrial fusion as a protective response to ER stress was further strengthened by data showing that mitochondrial morphology regulates mitochondrial Ca²⁺ uptake in order to preserve intracellular Ca²⁺ homeostasis [45]. These authors observed that the increment in mitochondrial fusion, which was achieved through the expression of an abnormal form of the fission protein DRP1, increases both mitochondrial Ca²⁺ uptake and Ca²⁺ retention capacity in C2C12 cells. Interestingly, they also reported a significant reduction in Ca²⁺ uptake and retention within mitochondria in cells expressing a fission phenotype that was induced by knockdown of the gene encoding the fusion protein Mfn2 [45]. Additionally, we observed that modulation of mitochondrial dynamics towards increased fusion is independent of alterations in mitochondrial membrane potential. This finding is also corroborated by Kowaltowski and coworkers, showing that the increase in mitochondria fusion in cells expressing abnormal DRP1 is independent of mitochondrial depolarization [45]. The preservation of mitochondrial membrane potential after TG treatment in THP-1 monocytes could be explained by the activity of Ca²⁺ extrusion systems that avoid mitochondrial Ca²⁺ overload [46].

ER stress affects mitochondrial function, and, depending on the level of stress, mitochondrial dysfunction can be promoted. Under chronic/severe ER stress, the extensive mitochondria Ca²⁺ influx is followed by mitochondrial fragmentation, increased ROS production, and loss of membrane potential, which can act as a signal to programmed cell death [47]. Together, the findings herein presented, showing absence of increased mitochondrial fission, membrane depolarization, or accumulation of ROS, indicate that SERCA inhibition in human monocytes does not promote mitochondrial damage, supporting that these innate immune cells are able to cope with stress. Furthermore, the above-mentioned mitochondrial alterations have been implicated in NLRP3-mediated inflammation [23]. Accumulation of mitochondrial ROS and disrupted mitochondrial membrane potential are classical activators of the NLRP3 inflammasome [20,21,22,23]. Although the role of mitochondrial dynamics in NLRP3 inflammasome activation is less explored, it has been proposed that fission of the mitochondrial network also plays a role [29]; however, the mechanisms involved are still controversial. On the one hand, Li and coworkers found that inhibition of DRP1-mediated mitochondrial fission blocks NOX2 oxidase signaling, preventing NLRP3 inflammasome activation in palmitate-treated endothelial cells [48]. NOX2 is a NADPH oxidase responsible for the production of ROS in many cells [49]. On the other hand, Park and colleagues demonstrated that activation of the NLRP3 inflammasome in response to LPS plus ATP is regulated by DRP1 in mouse bone marrow-derived macrophages. By silencing DRP1, these authors provided evidence that defective mitochondrial fission activates the NLRP3-dependent caspase-1 and subsequent IL-1β secretion [29]. Although there were no changes in relevant mitochondrial triggers of NLRP3 inflammasome activation upon SERCA inhibition in THP-1 human monocytes, secreted IL-1β levels were measured as a readout of NLRP3 inflammasome activation by TG-treated THP-1 cells. Under these conditions, extracellular IL-1β secretion was not promoted, and therefore, it can be concluded that NLRP3 inflammasome is not activated upon SERCA inhibition in THP-1 human monocytes. These findings obtained in THP-1 monocytes are supported by a previous study showing that IL-1β secretion in response to TG treatment is not detected in PMA-differentiated THP-1 macrophages [19]. Accordingly, the impact of SERCA inhibition on NLRP3 inflammasome was also evaluated in primary monocytes isolated from peripheral blood of healthy subjects exposed to TG, in the presence or absence of LPS priming. In both cases, extracellular IL-1β levels were not increased, supporting the view that absence of NLRP3 inflammasome activation is a specific early stress response of human monocytes triggered upon SERCA inhibition. Menu and co-workers have demonstrated that TG induces NLRP3 activation in murine LPS-primed bone marrow-derived macrophages [19]. Although both monocytes and macrophages are key components of the innate immune system, the differential response to stress induced by SERCA inhibition suggests that NLRP3 activation in the immune system occurs in a cell type-specific manner. The difference in the response between macrophages and monocytes may be explained by its functions on the innate immune system. As described in the literature, macrophages are the main innate immune cells involved in acute inflammatory responses, which are not so exacerbated in their precursors, the monocytes [50].

5. Conclusions

This work provides novel evidence that NLRP3 inflammasome is not activated by SERCA inhibition in human monocytes, either in primary monocytes or in the THP-1 monocytic cell line. Our findings from THP-1 cells showing increased mitochondrial fusion under these stressful conditions suggest that human monocytes trigger adaptive strategies through remodeling of mitochondrial morphology towards fusion in order to preserve metabolism and energy production. This mechanism, called mitochondrial hyperfusion, prevents mitochondrial dysfunction in response to ER stress caused by SERCA inhibition, namely ROS accumulation, membrane depolarization, and fission, thus avoiding NLRP3 inflammasome activation and, subsequently, the release of the pro-inflammatory cytokine IL-1β. Therefore, human monocytes can counteract early ER stress due to SERCA inhibition by modulating mitochondrial morphology and function in order to preserve energy production and cell survival and to prevent sterile inflammation induced by NLRP3 inflammasome activation.