Canonical Stimulation of the NLRP3 Inflammasome by Fungal Antigens Links Innate and Adaptive B-Lymphocyte Responses by Modulating IL-1β and IgM Production

The NLRP3 inflammasome is activated in response to different bacterial, viral, and fungal pathogens and serves as modulator of different pattern recognition receptors signaling pathways. One of the main functions of NLRP3 is to participate in IL-1β maturation which is important in the host defense against Pneumocystis and other fungal infections. However, dysregulation of NLRP3 and IL-1β secretion are also implicated in the pathophysiology of many auto-inflammatory disorders. Often time’s inflammatory flares are preceded by infectious illnesses questioning the role of infection in autoimmune exacerbations. However, we still do not fully understand the exact role that infection or even colonization plays as a trigger of inflammation. Herein, we investigated the role of NLRP3 in circulating B-lymphocytes following activation with two major microbial antigens (β-glucan and CpG). NLRP3 was determined essential in two independent B-lymphocytes processes: pro-inflammatory cytokine secretion and antibody regulation. Our results show that the β-glucan fungal cell wall carbohydrate stimulated B-lymphocytes to secrete IL-1β in a process partially mediated by Dectin-1 activation via SYK and the transcription factors NF-κB and AP-1. This IL-1β secretion was regulated by the NLRP3 inflammasome and was dependent on potassium efflux and Caspase-1. Interestingly, B-lymphocytes activated by unmethylated CpG motifs, found in bacterial and fungal DNA, failed to induce IL-1β. However, B-lymphocyte stimulation by CpG resulted in NLRP3 and Caspase-1 activation and the production and secretion of IgM antibodies. Furthermore, CpG-stimulated IgM secretion, unlike β-glucan-mediated IL-1β production, was mediated by the mammalian target of rapamycin (mTOR). Inhibition of NLRP3 and the mTOR pathway in CpG activated B-lymphocytes resulted in impaired IgM secretion suggesting their participation in antibody regulation. In conclusion, this study describes a differential response of NLRP3 to β-glucan and CpG antigens and identifies the NLRP3 inflammasome of human circulating B-lymphocytes as a modulator of the innate and adaptive immune systems.

The NLRP3 inflammasome is activated in response to different bacterial, viral, and fungal pathogens and serves as modulator of different pattern recognition receptors signaling pathways. One of the main functions of NLRP3 is to participate in IL-1β maturation which is important in the host defense against Pneumocystis and other fungal infections. However, dysregulation of NLRP3 and IL-1β secretion are also implicated in the pathophysiology of many auto-inflammatory disorders. Often time's inflammatory flares are preceded by infectious illnesses questioning the role of infection in autoimmune exacerbations. However, we still do not fully understand the exact role that infection or even colonization plays as a trigger of inflammation. Herein, we investigated the role of NLRP3 in circulating B-lymphocytes following activation with two major microbial antigens (β-glucan and CpG). NLRP3 was determined essential in two independent B-lymphocytes processes: pro-inflammatory cytokine secretion and antibody regulation. Our results show that the β-glucan fungal cell wall carbohydrate stimulated B-lymphocytes to secrete IL-1β in a process partially mediated by Dectin-1 activation via SYK and the transcription factors NF-κB and AP-1. This IL-1β secretion was regulated by the NLRP3 inflammasome and was dependent on potassium efflux and Caspase-1. Interestingly, B-lymphocytes activated by unmethylated CpG motifs, found in bacterial and fungal DNA, failed to induce IL-1β. However, B-lymphocyte stimulation by CpG resulted in NLRP3 and Caspase-1 activation and the production and secretion of IgM antibodies. Furthermore, CpG-stimulated IgM secretion, unlike β-glucanmediated IL-1β production, was mediated by the mammalian target of rapamycin (mTOR). Inhibition of NLRP3 and the mTOR pathway in CpG activated B-lymphocytes resulted in impaired IgM secretion suggesting their participation in antibody regulation.
In conclusion, this study describes a differential response of NLRP3 to β-glucan and CpG antigens and identifies the NLRP3 inflammasome of human circulating B-lymphocytes as a modulator of the innate and adaptive immune systems.
Keywords: β-glucan, B-lymphocytes, inflammasome, fungi, cpg, nlrP3, il-1β, igM NLRP3 Mediates IL-1β and IgM Frontiers in Immunology | www.frontiersin.org November 2017 | Volume 8 | Article 1504 inTrODUcTiOn The host immune system greatly determines the severity of fun gal diseases. In patients with an intact immune system, fungal infections are often clinically asymptomatic or manifest as a mild respiratory illness. In the immunocompromised host, however, fungal infections can disseminate and result in a lifethreatening event with high morbidity and mortality. Fungal diseases are on the rise, likely as a result of increasing use of immunosuppressive agents to treat malignancies and autoimmune diseases. Better understanding of fungal immunity will help with the develop ment of alternative antifungal therapeutic strategies that enhance specific aspects of host immunity. Blymphocytes, wellknown players of the adaptive immune response, react to fungal pathogens by generating antibodies and by releasing inflammatory cytokines (1,2). In the pres ence of T cells, Blymphocyte responses are characterized by isotype class switch and generation of memory and longlived plasma cells leading to the production of high affinity immu noglobulins (Igs) mostly of the IgG subtype. In the absence of T cells, Blymphocytes still generate Igs but these are of low affinity and mostly IgM. Tcell independent activation of Blymphocytes also results in the release of a variety of cytokines and chemokines which are mostly triggered by the activation of pattern recognition receptors (PRRs) such as tolllike recep tors (TLRs) and Clectin receptors (3). PRRs are expressed by most innate immune effector cells, including Blymphocytes and play a critical role in the detection of pathogens by recognizing conserved pathogenassociated molecular patterns like βglucan and CpG. βglucans are highly immunogenic carbohydrates found in the cell wall of many fungi including Aspergillus spp., Candida spp., and Pneumocystis while CpG are highly immuno reactive unmethylated motifs found in bacterial and fungal DNA (4,5). While each signal using specific PRRs, both have potent immunomodulatory properties and can activate Blymphocytes directly without the participation of T cells. Blymphocyte activation by βglucan and CpG results in the secretion of a specific profile of proinflammatory cytokines and chemokines important for the orchestration and activation of monocytes, macrophages, and neutrophils and therefore essential for host defense against fungal and other infections (6)(7)(8).
The NLRP3 inflammasome is generally triggered by infection or tissue damage and participates in the processing of mature and bioactive IL1β from its precursor and inactive form (proIL1β) (9,10). Since increased production of IL1β is known to be impor tant for the clearance of fungal infections and little is known about the contribution of Blymphocytes to the innate immune fungal defense, we sought to investigate the role of NLRP3 activation in Blymphocytes upon fungal βglucan stimulation and compare it with Blymphocyte responses to CpG.
The assembly of the inflammasome classically involves the recruitment of a Nodlike receptor (NLR), an adaptor protein (ASC) and a protease (procaspase1). Depending on the stimuli, the activation of NLRP3 can follow a canonical pathway that involves caspase1 activation or a noncanonical pathway that is independent of caspase1. In the particular case of βglucans, the data are contradictory and both pathways have been described (11)(12)(13). In Blymphocytes, however, the participation of NLRP3 in cytokine regulation and other processes has not been well characterized.
Herein, we describe a dual function of the NLRP3 inflamma some in activated peripheral human Blymphocytes as a modu lator of IL1β secretion as well as antibody production. While fungal βglucans andCpG were both able to elicit activation of the NLRP3 inflammasome, the mechanisms of activation were diver gent with βglucan inducing IL1β secretion and CpG an increase of IgM. Furthermore, CpG activation of NLRP3 was dependent on the activation of the mammalian target of rapamycin (mTOR) pathway while βglucan activation was mTORindependent.

MaTerials anD MeThODs reagents and antibodies
Endotoxinfree buffers and reagents were scrupulously used in all experiments. Curdlan, Zymosan, and Laminarin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Aspergillus fumigatus βglucan preparations were isolated as previously described (14). To ensure that all glucan preparations were free of endotoxin prior to use in culture, Curdlan, Zymosan, and A. fumigatus glu cans were vigorously washed 10 times with distilled physiological saline, incubated rotating overnight with polymyxin B (Sigma, St. Louis, MO, USA) at 4°C, then vigorously washed again with distilled physiological saline. The final preparations were assayed for endotoxin with the limulus amebocyte lysate method using Pyrosate Rapid Endotoxin Detection Kit (Associates of Cape Cod, East Falmouth, MA, USA) and found to consistently contain less than 0.25 EU/ml. Glucans were pulse sonicated 10 times using a Branson digital sonifier (VWR Scientific, Radnor, PA, USA) at 35% amplitude immediately before addition to the cultures. The Erk 1/2 inhibitor (PD98059), SYK inhibitors (Piceatannol and R406), and NFκB inhibitor (Bay117085) were all obtained from Calbiochem, Inc. (San Diego, CA, USA). AP1 inhibitor (SR 11302) was from R&D Systems, Inc. (Minneapolis, MN, USA). Caspase inhibitors (AcYVADCMK and AcYVADCHO) were purchased from Cayman Chemical (Ann Arbor, MI, USA), VX765 was from AdooQ Bioscience (Irvine, CA, USA), and Rapamycin was purchased from Selleck Chemicals (Houston, TX, USA). KCl and Adenosine 5′triphosphate (ATP) were from Sigma and oxidized ATP (oxATP) and MCC950 were from EMD Millipore (Billerica, MA, USA

leukocyte isolation and culture
All methods were carried out in accordance with relevant guide lines and regulations. Human Blymphocytes were isolated as previously described (7). Briefly, Blymphocytes were isolated from acid citrate dextrose anticoagulated blood obtained from deidentified healthy volunteer platelet donors in accordance with the current regulations by the AABB and the US Food and Drug Administration (15)  ]bis(4methoxy6nitro) benzenesulfonic acid hydrate (XTT) to a formazan dye through electron coupling in metabolically active mitochondria using the coupling reagent Nmethyldibenzopyrazine methyl sulfate. Only metabolically active cells are capable of mediating this reaction, which is detected by absorbance of the dye at 450-500 nm. Briefly, 50 µl of the XTT labeling mixture was added to the 100 µl of growth medium containing Blymphocytes and different concentrations of the inhibitors. The XTT labeling mixture was added in parallel samples 24 h after the addition of the various inhibitors. A set of blanks was also included that did not contain cells and was treated identically as the normal samples. In addition, a set of solvent controls was also included for each inhibitor. Absorbance was measured at 6 h after XTT addition. All treatments were performed in three replicates. Only inhibitor concentrations that elicited less than 20% net toxicity were used in these assays.

Preparation of cell lysates, electrophoresis, and immunoblotting
Total cellular proteins were obtained from Blymphocytes fol lowing the described culture conditions. Briefly, the cells were

statistical analyses
All data are presented as the mean ± SEM, from at least three independent experiments from different biological donors, unless otherwise specified. An experimental run included at least three different donors. The data were first analyzed using one way ANOVA, the differences between the individual groups were compared using multiple comparisons posttest unless otherwise indicated. Statistical analysis was performed using GraphPad Prism Version 5 (GraphPad Software, La Jolla, CA, USA).

il-1β is induced by Fungal β-glucans in a T-cell independent Manner
Blymphocytes respond to fungal βglucans and bacterial DNA in a Tcell independent manner by releasing cytokines and chemokines important for the acute inflammatory response (8,16). We have previously shown that βglucanstimulated peripheral human Blymphocytes are a source of IL8, IL6, and TNFα (7). Additionally, supernatant from βglucanstimulated Blymphocytes was able to significantly increase neutrophil chemotaxis suggesting the contribution of Blymphocytes in the acute inflammatory process (7). To further understand the role of Blymphocytes in fungal defense and since IL1β has been shown to participate in fungal clearance and neutrophil recruit ment (17,18), freshly isolated peripheral Blymphocytes from normal donors were stimulated with either βglucan (Curdlan) or CpG (CpG ODN 2006) and assessed for IL1β expression. RNA levels of IL1β were significantly elevated in βglucanstimulated cells but not in those stimulated with CpG ( Figure 1A). Protein levels of IL1β were next confirmed in the cell supernatants by ELISA. As expected, βglucanstimulated cells secreted significant amounts of active IL1β in a dose and time responsive manner (Figures 1B,C). The addition of βglucan to CpG did not enhance IL1β stimulation ( Figure 1D). Furthermore, IL1β secretion was not restricted to curdlan βglucans as other βglucan preparation such as zymosan and βglucan from Aspergillus were also potent inducers of IL1β (Figure 1E). Similarly, failure to induce IL1β secretion was not restricted to CpG ODN 2006, since two different CpG motifs present in A. fumigatus DNA (AF1 and AF2) (4) also failed to stimulate IL1β secretion ( Figure 1F). The differential response of Blymphocytes to βglucan and CpG was intriguing, though similarly observed by our group in the setting of IL8 and IgM (7). Our published studies demonstrated that βglucan induced IL8 secretion had no effect on IgM while CpG stimulated IgM secretion did not participate in IL8 secretion (7).

β-glucan and cpg induce activation of the nlrP3 inflammasome in human circulating B-lymphocytes
The NLRP3 inflammasome is crucial for the processing of biologically inactive IL1β (proIL1β) into the mature and bio logically active form (IL1β). It was therefore important for us to determine its role in βglucanmediated IL1β secretion. Protein expression of NLRP3, ASC, caspase1, and proIL1β as well as caspase1 activity were therefore assessed and found to increase significantly upon βglucan stimulation (Figures 2A,C). CpG stimulation did not induce proIL1β, a required step needed for mature IL1β production ( Figure 2B). However, despite the lack of increase in proIL1β, CpG also resulted in activation of the NLRP3 inflammasome (Figures 2B,C).
To further demonstrate that Caspase1 and NLRP3 were indeed important in βglucanmediated IL1β secretion and since primary human Blymphocytes are not suitable for lentivirus infection or transfection with other forms of interfering RNA, IL1β was measured in the cell supernatant of stimulated cells in the presence of different Caspase1 and NLRP3 inhibitors. As shown in Figures 2D-F, the use of the caspase1 inhibitors VX765 and YVADCMK, KCL, oxATP, and the specific NLRP3 inhibitor, MCC950 (19), resulted in significant decreases of IL1β secretion, confirming the participation of the canonical NLRP3 inflammasome pathway in βglucanmediated IL1β secretion. Absence of cell toxicity was confirmed for all the inhibitors as shown in Supplement S1 in Supplementary Material.
Since prior data suggested that in NLRP3 deficient mice IgM secretion and antibody production are impaired (8,(20)(21)(22), we hypothesized that NLRP3 may regulate IgM secretion. We and others have additionally shown that peripheral Blymphocytes secrete IgM in response to CpG (ODN 2006) (7, 23) (Supplement S2 in Supplementary Material). To better understand the role of NLRP3 activation in CpGstimulated Blymphocytes we further investigated if IgM was also triggered in response to AF1 and AF2. As shown in Figure 3A   in Supplementary Material). To ensure that this effect was specific to inhibition of the NLRP3 inflammasome, IL6, MMP7, and TNFα were also assessed in the presence of MCC950. As shown in Figure 3C, none of the other cytokines and metalloproteases were affected by the presence of the inhibitor further supporting the specific role of the NLRP3 inflammasome in IgM secretion. Additionally, IgM release was also impaired in the presence of KCL, oxATP, and caspase inhibitors (Figures 3D,E; Supplement S3A in Supplementary Material), all wellknown agents that affect the functioning of the NLRP3 inflammasome.

Dectin-1 and syk-Participate in nlrP3 activation by β-glucans While cpg signaling activation of nlrP3 regulates igM via mTOr
Since the inflammasome was activated by both βglucan and CpG and both are known to signal through very different PRRs, we next explored the specific signaling pathways that lead to IL1β and IgM secretion upon βglucan and CpG, respectively. βGlucans are known to signal predominantly through the Clectin receptor Dectin1 (6,7,24,25) while CpG is the natural ligand for TLR9 (4,26). Hence, we first explored the contribution of Dectin1 in βglucaninduced IL1β signaling. Blymphocytes were preincubated with either laminarin (a soluble βglucan known to bind to Dectin1 acting as a competitive inhibitor) or a specific Dectin1 blocking antibody prior to βglucan stimula tion. IL1β levels were significantly reduced in the presence of both, laminarin and the Dectin1 antibody, confirming the role of Dectin1 in βglucanmediated IL1β secretion (Figures 4A,B). βglucanmediated IL1β was also dependent on Syk and the transcription factors NFκB and AP1 (Figures 4C,D).
TLR9 is the main receptor for CpG motifs found in bacterial and fungal DNA (4,26) and it is known that stimulation by CpG also involves mTOR (4,6,(27)(28)(29). In contrast, βglucan stimula tion does not seem to require mTOR (6). Thus, to additionally understand the potential involvement of mTOR activation in IgM and IL1β regulation, phosphorylation of mTOR and other mTORrelated proteins were assessed after cells were stimulated for different period of time with CpG or βglucan. mTOR and other mTORrelated proteins (S6K, S6, and 4EBP1) tested were phosphorylated upon CpG but not after βglucan stimulation with the exception of 4EBP1 which seemed to be phosphorylated by both (Figure 5A; Supplement S3B in Supplementary Material). βglucan activation of 4EBP1 was inhibited in the presence of PD98059, a specific ERK1/2 inhibitor, and not by Rapamycin, a wellknown mTOR inhibitor, suggesting that 4EBP1 activation, in βglucanstimulated cells, was mTORindependent and required ERK1/2 (Supplement S4 in Supplementary Material), consistent with prior observations (30).
As our data suggested that NLRP3 regulates IgM via mTOR, we measured IgM levels after CpGstimulation in the presence  Figure 5C). Interestingly, Rapamycin inhibition of the NLRP3 inflammasome seemed to affect Caspase1 activation as shown in Figure 5D. Similar observa tions were made for MCC950, consistent with prior published observations (31).

Memory B-lymphocytes are the Main Producers of il-1β and igM
Peripheral Blymphocytes can be divided into naïve and memory cells. In order to further understand the subtype of Blymphocytes responsible for the secretion of IL1β and IgM, and since it is clearly established that naïve and memory cells produce different cytokine patterns upon stimulation (32,33). We isolated Blymphocytes by negative selection and then sorted them into CD20 + CD27 − (naïve) and CD20 + CD27 + (memory) cells. The two different subtypes of Blymphocytes were inde pendently stimulated with either βglucan or CpG. IL1β and IgM levels in the cell supernatant were then measured by ELISA.
As Figure 6A shows, we observed that IL1β was mostly secreted by memory cells whereas IgM production was greater from the sorted memory cells and significantly higher than naïve cells (p < 0.001), but was still seen at significant levels in the naïve cells upon CpG stimulation. Costimulation of the BCR receptor resulted in an increase in IgM secretion but it did not have a significant effect on IL1β secretion ( Figure 6B).

DiscUssiOn
In this study, we demonstrate that in human peripheral Blymphocytes the NLRP3 inflammasome is differentially acti vated by fungal βglucan and CpG antigens. Activation of NLRP3 by fungal βglucan resulted in the cleavage of proIL1β into its active form. While this is a wellknown function of the NLRP3 in other cell types such as macrophages and dendritic cells, its role in peripheral human Blymphocytes was not yet clearly understood. Furthermore, herein we have shown that IL1β secretion was mediated through the major βglucan receptor Dectin1 and required signaling through Syk, consistent with our prior observations of βglucan signaling in Blymphocytes and current knowledge in other immune cells (6,7,11,(34)(35)(36). IL1β regulation was mediated by the transcription factors NFκB and AP1 known to be necessary for the transcription of proIL1β (35,37). Once proIL1β is formed then subsequent activation of the NLRP3 inflammasome is necessary for the processing of the inactive proIL1β into the active or mature form. Activation of the inflammasome via the canonical and noncanonical pathways has been described in other cell types (13,35,38). For instance, in dendritic cells, Dectin1 participates not only in the activation of NFκB and synthesis of proIL1β by curdlanacti vated dendritic cells but also in the production of mature IL1β through a noncanonical inflammasome that recruits ASC and caspase8 to the complex CARDMaltBCL10 (38). Caspase8 has also been reported as an effector and regulator molecule of the canonical NLRP3 inflammasome that drives IL1β production (13). Herein, we demonstrate that stimulation of Blymphocytes with βglucan and CpG resulted in the activation of the canonical NLRP3 pathway and involved both ASC and caspase1 activa tion. Interestingly, while βglucanmediated IL1β secretion via Dectin1SykNFκB/AP1 pathways and canonical NLRP3 activation, CpG stimulation of Blymphocytes also resulted in the activation of the canonical NLRP3 inflammasome, but triggered the secretion of IgM and not IL1β. The lack of IL1β was likely due to the absence of proIL1β which limited the production of the active form since no substrate was available. Studies have shown that host immunity against disseminated candida, aspergillosis, and pneumocystis infection relies on IL1β to clearly mount an adequate immune response, particularly in early stages of disease (17,(39)(40)(41). Impaired IL1β secretion results in decreased recruitment of neutrophils, macrophages, and lymphocytes to the lungs, affecting the phagocytosis and killing of the fungi (17). IL1β also acts by activating the release of other inflammatory cytokines, like IL6 and TNFα as well as inducing Th17 polarization, all important mediators in antifungal defense (42). Furthermore, IL1β is also involved in the pathogenesis of many inflammatory diseases such as familial mediterranean fever, familial coldinduced autoinflammatory syndrome, steroid resistant asthma, and rheumatoid arthritis (43)(44)(45)(46). In most of these diseases, blocking IL1β by monoclonal antibodies or blocking the NLRP3 inflammasome provide symptomatic relief and are currently the standard clinical treatment strate gies. Understanding IL1β regulation in Blymphocytes offers a new way to potentially develop treatments that will continue to improve the clinical course of patients, not only during the acute infectious process, but potentially by ameliorating these chronic inflammatory conditions as well.
Over the last decade, microbiome studies have clearly dem onstrated that the lower respiratory tract is colonized by many organisms and is not sterile as initially assumed. It is very likely that the lung microbiome, similar to the intestinal microbiome, plays an important role in maintaining the wellfunctioning defense mechanisms of the respiratory mucosa (47). It is also highly probable that bacterial and fungal antigens, through activation of PRRs, help to keep the immune system in a basal activation state that ultimately results in the wellbeing of the host. However, if this balance is disrupted, and the microbiota loses diversity, the more prevalent microbial antigens through the same PRR would have no regulatory negative feedback resulting in an exaggerated inflammatory response. In the clini cal setting, single or paucity fungal colonization in the airway is not uncommon, particularly in immunosuppressed patients. While the clinical significance of this finding is unknown, in the majority of the cases it gets overlooked, as the thought is that it does not result in a clinically significant illness. While this may be true, our observations suggest that fungal antigens are potential triggers of IgM, IL1β, and other inflammatory cytokines (IL8, IL6, and TNFα) that can potentially act as chronic stimuli for Blymphocytes. Thus, it is important to recognize that fungal activated Blymphocytes, while they can contribute to host protection against fungal diseases in the right clinical setting, they can also result in a source of chronic inflammation.
Contrary to what we observed in βglucanstimulated Blymphocytes, our data also showed that CpG, while activating the NLRP3 inflammasome, did not result in IL1β secretion, but upregulated IgM secretion via the mTOR signaling pathway (see proposed mechanism in Figure 7). The role of NLRP3 in antibody production has been suggested in animal models (8); however it has not been investigated in human Blymphocytes. Herein, we showed that inhibition of NLRP3 decreased IgM production in a dosedependent manner while not affecting the secretion of other cytokines such as TNFα, IL6, and MMP7, suggesting that the production of IgM is seemingly controlled by the NLRP3 inflammasome.
The mTOR pathway is ubiquitously expressed in immune cells, including Blymphocytes (48,49). Upon activation it regulates important cell processes such as protein translation, cell growth and proliferation (50). mTOR functions as two signaling complexes, mTOR complex 1 (mTOR1) and mTOR complex 2 (mTOR2). Activation of mTOR1 results in phosphorylation of p70s6K (S6K), S6, and eukaryotic initiation factor 4Ebinding protein (4E BP1) which is important for the regulation of protein synthesis (50). Herein, we found phosphorylation of S6, S6K, and 4EBPE after CpG stimulation. Furthermore, the use of Rapamycin, a welldescribed mTOR1 inhibitor, decreased IgM secretion. The use of Rapamycin also affected NLRP3 and caspase1 activa tion, therefore impairing the function of NLRP3 inflammasome complex and suggesting that IgM regulation is not only mediated by NLRP3 but also by mTOR. Prior observations in macrophages also suggest that mTOR inhibition suppresses NLRP3 inflam masome activation in macrophages (31). In their study, mTOR regulated HK1dependent glycolysis was critical for NLRP3 acti vation. While further investigations are needed in Blymphocytes, studies to deeply understand the specific mechanism by which mTOR and NLRP3 are controlling antibody regulation in Blymphocytes studies have shown that mTOR participates in the regulation of high affinity antibodies (51). Furthermore, in ani mal models, impaired mTOR signaling in follicular and marginal Blymphocytes stimulated by LPS show a marked reduction of IgM and IgG (50,52). That mTOR is important for germinal center formation is known (48) however, little is known about the unique function of mTOR in circulating human Blymphocytes. Here, we have demonstrated the participation of mTOR in NLRP3 function and regulation of IgM. Interestingly, while βglucans are able to trigger different cytokines and chemokines, they fail to induce IgM in human peripheral Blymphocytes (7). These data differ from prior studies in murine models in which curdlan stimula tion seemed to induce IgM secretion (8). While the differences seen between the two experimental models could be explained by the differences in the experimental methodology and the lack of βglucan to induce proliferation in the human cells; herein, we have shown the inability of curdlan to trigger mTOR activation, a step found to be important for IgM secretion. Our observations are novel and raise awareness of the role of NLRP3 as regulator not only of IL1β and IL18 but also of IgM. Interestingly, despite NLRP3 activation, IL18 was not induced by βglucan or CpG in peripheral Blymphocytes (7).  Beyond their antibodyproducing role, Blymphocytes can affect the local immune environment through the release of cytokines and other proteins; however, how they modulate and contribute to the orchestration of the innate immune system is not clearly understood. Our group has recently shown that activated Blymphocytes participate in the recruitment of neu trophils by releasing IL8 and indirectly by contributing to the release of syndecan4 via MMP7 (6,7). Syndecan proteins are expressed on the cell membrane and can be shed into the extra cellular space by metalloproteases (53). Upon shedding they are free to bind to different cytokines participating in the regulation of cytokine influx and neutrophil recruitment (54)(55)(56). Herein, we further demonstrated that Blymphocytes also contribute to the pool of IL1β, a proinflammatory cytokine very important for antimicrobial host defense (42) and to the secretion of IgM antibodies. Naïve and memory Blymphocytes play different roles in the regulation of the immune response by releasing different cytokine profiles (2). Here, we investigated the specific Blymphocyte subtype responsible for IL1β and IgM secretion and contrary to what we have observed with MMP7, which was mostly released by naïve cells, memory cells were found to be the main source for both IL1β and IgM (6). While the exact mechanisms by which different Blymphocyte subtypes seem to preferentially secrete a specific cytokine milieu is not totally understood, these patterns are not fixed and can be influenced by the circumstances that surround their activation such as presence of Tlymphocytes and other inflammatory cells, by the integration of different signals from multiple receptors and by the proportion of the different Blymphocytes compartments (2,33,57). These are important observations with therapeutic implications as Blymphocyte reconstitution after Rituximab (Blymphocyte depleted agent) treatment favors naïve Blymphocytes (33). Increased numbers of naïve Blymphocytes, based on our observations, will result in a decrease of IL1β and IgM levels potentially contributing to a less inflammatory milieu. How these changes may affect antimi crobial and antiinflammatory host response need to be further investigated.
In summary, this study identified that the NLRP3 inflammasome is essential for two independent processes, proinflammatory cytokine secretion and antibody regulation. Whether NLRP3 activation resulted in cytokine or antibody regulation depended on the stimulating antigen with IL1β secretion being triggered by fungal βglucans and IgM by CpG. NLRP3 regulation was also differently regulated upon βglucan or CpG antigens and was mTORindependent when stimulated by βglucans but mTOR dependent upon CpG stimulation. The new role of the NLRP3 inflammasome and the mTOR pathway as regulators of IL1β and IgM in activated Blymphocytes studied here offers a potential new strategy to treat autoimmune disease in which IL1β and pathogenic IgM antibodies may play a role, particularly if trig gered by infectious agents. Further investigations are therefore needed to clarify the potential benefits of mTOR and NLRP3 inhibitors in these clinical settings.

aUThOr cOnTriBUTiOns
Concept and design; data acquisition; and data interpretation/ analysis: EC, MA, HD, and VV; manuscript drafting: EC, MA, and VV; manuscript review: all authors.
acKnOWleDgMenTs We thank Dr. Peikert and Dr. Limper and all the members Dr. Limper's laboratory and the Mayo Clinic Thoracic Diseases Research Unit for their many helpful discussions.

FUnDing
Funded by NIH grants K08 (HL112849) to EC and funds from the Annenberg Foundation.