NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function

Gram-positive bacteria, including Staphylococcus aureus are endemic in the U.S., which cause life-threatening necrotizing pneumonia. Neutrophils are known to be critical for clearance of S. aureus infection from the lungs and extrapulmonary organs. Therefore, we investigated whether the NLRP6 inflammasome regulates neutrophil-dependent host immunity during pulmonary S. aureus infection. Unlike their wild-type (WT) counterparts, NLRP6 knockout (KO) mice were protected against pulmonary S. aureus infection as evidenced by their higher survival rate and lower bacterial burden in the lungs and extrapulmonary organs. In addition, NLRP6 KO mice displayed increased neutrophil recruitment following infection, and when neutrophils were depleted the protective effect was lost. Furthermore, neutrophils from the KO mice demonstrated enhanced intracellular bacterial killing and increased NADPH oxidase-dependent ROS production. Intriguingly, we found higher NK cell-mediated IFN-γ production in KO mouse lungs, and treatment with IFN-γ was found to enhance the bactericidal ability of WT and KO neutrophils. The NLRP6 KO mice also displayed decreased pyroptosis and necroptosis in the lungs following infection. Blocking of pyroptosis and necroptosis in WT mice resulted in increased survival, reduced bacterial burden in the lungs, and attenuated cytokine production. Taken together, these novel findings show that NLRP6 serves as a negative regulator of neutrophil-mediated host defense during Gram-positive bacterial infection in the lungs through regulating both neutrophil influx and function. These results also suggest that blocking NLRP6 to augment neutrophil-associated bacterial clearance should be considered as a potential therapeutic intervention strategy for treatment of S. aureus pneumonia.

Introduction Acute pneumonia is a leading cause of childhood mortality (<5 years of age) accounting for the death of 920,136 children annually [1], and methicillin-resistant Staphylococcus aureus (MRSA) has been implicated in severe life-threatening infections, including necrotizing pneumonia and sepsis [2]. In addition, S. aureus infection is also one of the major causes of secondary pneumonia following influenza infection [3]. Furthermore, S. aureus has developed resistance to multiple antibiotics and effective treatment strategies against this bacterium are limited [2,4]. Therefore, S. aureus is a serious threat to human health and novel therapeutic strategies are needed.
The lung pathology induced by S. aureus is attributed to virulence factors, the intense inflammatory response, and evasion of host defense mechanisms, including neutrophil-mediated ROS-dependent bacterial killing [5,6]. Regarding innate immune responses, nucleotidebinding oligomerization domain-like receptor (NLR) pyrin domain-6 (NLRP6) is a recently identified NLR present in the cytosol of innate immune cells [7]. Co-transfection of plasmids containing NLRP6 and apoptosis-associated speck-like protein containing card (ASC) in 293T cells led to activation of NF-kB and in COS-7L cells to a synergistic increase in caspase-1 activation and IL-1β secretion [8]. Under homeostatic conditions Levy et al have demonstrated that NLRP6 co-localizes with ASC and caspase-1 to form a complex resulting in IL-18 secretion from intestinal epithelial cells, which is essential to prevent development of dysbiosis [9]. Together these results suggest that NLRP6 can co-localize with ASC for inflammasome formation and activation. However, the mechanisms of NLRP6 inflammasome activation and its role in host defense specifically during pulmonary microbial infection has not been explored. Moreover, it remains unknown whether the NLRP6 inflammasome is activated by microbial infections and whether NLRP6 co-localizes with ASC and caspase-1 during such infections to induce pyroptosis. In a mouse model of systemic infection, Anand et al [7] found that the NLRP6 negatively regulates host defense against Listeria and Salmonella infections as NLRP6 KO mice showed higher survival, decreased bacterial burden, and attenuated pathology compared to WT mice. In contrast, a study reported by Wlodarska et al [10] revealed a positive regulatory role of the NLRP6 inflammasome on immune function during enteric infection with Citrobacter rodentium. In this investigation, NLRP6 KO mice were shown to have an increased C. rodentium burden in the intestine, which correlated with extensive mucosal damage in the KO mice compared with WT controls. However, these results cannot be extrapolated to other bacterial infections in different organs, such as the lung, because Gram-positive bacteria have unique virulence factors and immune evasion strategies and the route of administration of bacteria also dictates host responses. Thus, the role of the NLRP6 inflammasome in pulmonary immunity against Gram-positive infections remains unclear. To this end, we have used NLRP6 KO mice to demonstrate how S. aureus exploits the NLRP6 inflammasome to dampen neutrophil function and enhance pyroptosis and necroptosis to increase mortality during acute bacterial pneumonia. Our results show NLRP6 as a potential therapeutic target for treatment of S. aureus-infected pneumonic patients.

NLRP6 expression is increased in the lungs of pneumonic patients and S. aureus-infected mice
To determine whether NLRP6 is upregulated in the lungs of pneumonic patients, we stained human pneumonic and normal lung tissue sections with anti-NLRP6 antibody and found that NLPR6 was upregulated in key innate immune cells in the lungs, such as neutrophils (lipocalin-2 + ), macrophages (CD68 + ), and epithelial cells (CD326 + ) ( Fig 1A). Next, we determined if NLRP6 expression is upregulated in S. aureus-induced pneumonia in mice. In line with the results seen in human pneumonic lung sections, NLRP6 was upregulated in neutrophils (Ly6G + ), macrophages (F4/80 + ), and epithelial cells (CD 326 + ) of mouse lungs following S. aureus infection (Fig 1B). Consistent with the immunofluorescence results, the expression of NLRP6 was also increased in lung lysates of human pneumonic patients, S. aureus-infected human cell lines (THP-1 and HL-60), and mouse bone marrow-derived macrophages (BMDMs) (Fig 1C-1E).
To investigate whether NLRP6 triggers inflammasome activation during S. aureus infection, we infected BMDMs from WT and NLRP6 KO (KO) mice with S. aureus (MOI: 20) and measured the extent of caspase-1 activation at 8 hours post-infection. Both cleaved caspase-1 (p-20) in macrophage lysates and IL-1β levels in culture supernatants following infection were attenuated in the NLRP6 KO samples compared to the WT control (Fig 1F and 1G) indicating the activation of the NLRP6 inflammasome by S. aureus. Similarly, IL-18 was also reduced in NLRP6 KO BMDMs after S. aureus infection (S1A Fig). Since IL-1β was sharply reduced in the KO BMDMs, we determined if NLRP3 inflammasome is still intact in these BMDMs. IL-1β production in NLRP6 KO BMDMs after treatment with specific NLRP3 agonists (ATP and Nigericin) was comparable with that of WT BMDMs suggesting that NLRP3 is indeed intact in NLRP6 KO macrophages (S1B Fig). S. aureus has several virulence components, such as α-hemolysin (hla), clumping factor B, β toxin, phenol-soluble modulins, and panton-valentine leukocidins [5] that could potentially activate the NLRP6 inflammasome. Because hla has been reported to activate the NLRP3 inflammasome in human and mouse monocytic cells under similar conditions [11,12], we investigated whether hla can also activate the NLRP6 inflammasome in BMDMs and found this to be the case (Fig 1E and 1F). Consistent with the in vitro results, NLRP6 KO mice showed lower levels of IL-1β in bronchoalveolar lavage fluid (BALF) after infection with S. aureus, providing evidence of NLRP6 inflammasome activation in vivo ( Fig 1H). However, there was no difference observed in the levels of IL-18 between WT and the KO mice (S1C Fig). ASC is known to be an integral part of NLRP3 inflammasome signaling although it is dispensable for NLRC4 inflammasome activation [13]. We infected BMDMs with S. aureus and performed immunofluorescence assay to observe whether NLRP6 co-localizes with ASC and caspase-1. We found that NLRP6 co-localized with ASC and caspase-1 during S. aureus infection (Fig 1I and 1J   Lungs tissue sections from healthy controls and pneumonic patients were stained with antibodies against neutrophils (lipocalin-2 + ), macrophages (CD68 + ), and epithelial (CD326 + ) cells. NLRP6 is shown in red, neutrophils, macrophages, and epithelial cell markers are in green, and DAPI is blue. NLRP6-positive cells are indicated by white arrowheads. (B) Immunofluorescence microscopy was performed on lung sections from healthy controls and S. aureus-infected mice to assess expression of NLRP6. Neutrophils (Ly6G + ), macrophages (F4/80 + ), and epithelial cells (CD 326 + ) are stained green and NLRP6 is red. NLRP6-positive cells are indicated by white arrowheads. (C) NLRP6 expression was analyzed by western blot in lysates of human healthy control tissue, pneumonic lung tissue, and HL-60 (human neutrophil-like) cells infected with S. aureus (MOI10) for 4 hours. (D) THP-1 (human monocytic) cells were infected with S. aureus (MOI 20) or purified α-hemolysin (hla) for 8 hours and expression of NLRP6 was assessed by western blotting. (E, F) Bone marrow-derived macrophages (BMDMs) were infected with S. aureus (MOI: 20) or hla from S. aureus (50 μg/ml) for 8 hours (E). Expression of NLRP6 protein was analyzed by immunoblotting. (F) Caspase-1 processing by the NLRP6 inflammasome in response to S. aureus or hla in BMDMs. (G) IL-1β secretion by WT and NLRP6 KO BMDMs following S. aureus infection. (H) IL-1β secretion by WT and NLRP6 KO mice after S. aureus (5 X 10 7 CFU/mouse) infection (N = 6-8/group). (I) BMDMs from WT mice were infected with S. aureus (MOI: 50) and stained for NLRP6 (red) and ASC (green). The white arrowheads demonstrate co-localization of NRP6 and ASC. (J) BMDMs from WT mice were infected with S. aureus (MOI: 50) and stained for NLRP6 (red) and caspase-1 (green). The white arrowheads show co-localization of NRP6 and caspase-1. The graphs show the mean ± SEM of three independent experiments. The images shown are the representative of five different fields from three independent experiments. Magnification: 40X. SA: S. aureus, hla: α-hemolysin. Ã , p<0.05, ÃÃ , p<0.01, and ÃÃÃ p<0.001.

NLRP6 deficiency confers host protection during S. aureus-induced pneumonia
To assess the role of the NLRP6 inflammasome in pulmonary host defense against S. aureus, we infected WT, ASC KO, and NLRP6 KO mice intratracheally with a lethal dose of S. aureus (USA 300) (2X10 8 CFUs per mouse) and observed the survival patterns for 10 days. Although all WT mice died within 3 days, 70% of NLRP6 KO mice survived longer than 10-days postinfection (Fig 2A). Furthermore, ASC KO mice displayed a survival pattern similar to that of the NLRP6 KO mice (Fig 2A). To determine whether the difference in survival is due to differences in the bacterial burden in various organs, we measured the bacterial burden in the lung, BALF, and extra-pulmonary organs after infecting mice with a sub-lethal inoculum (5X10 7 CFU) of S. aureus. As compared to NLRP6 KO mice, WT mice had higher bacterial burdens in the lungs, BALF, and liver at both 12 and 24-hours post-infection (Fig 2B-2D). Accordingly, the total protein in the BALF, which is a measure of pulmonary leakage, was higher in WT mice compared to their NLPR6 KO counterparts (Fig 2E).
To determine whether the detrimental effects of NLRP6 are bacterial strain specific, we infected WT and KO mice with a methicillin-susceptible Staphylococcus aureus strain (MSSA, Newman strain) and measured the bacterial burden in lungs and BALF at 24-hours post-infection. Consistent with the results seen with the USA 300 strain, the bacterial burden of the methicillin-sensitive strain was also higher in the lungs and BALF of WT mice compared to that of KO mice (Fig 2F and 2G). NLRP6 inflammasome has been implicated in regulating intestinal microbiota [9,14]. To determine if observed difference in the phenotype between WT and NLRP6 KO mice is due to difference in gut microbiota, we co-housed WT and NLRP6 KO mice for 4 weeks and infected them with S. aureus. As observed with non-cohoused mice, co-housed NLRP6 KO mice had significantly less bacterial burden in the lungs and BALF as compared to co-housed WT mice (Fig 2H and 2I). These data suggest that NLRP6 regulates pulmonary S. aureus infection independent of microbiota composition.

Both hematopoietic and non-hematopoietic cells contribute to host susceptibility
Since we found upregulation of NLRP6 in both hematopoietic (neutrophils and macrophages) and non-hematopoietic cells (epithelial cells) (Fig 1B), we sought to determine if NLRP6 from each of these compartments is detrimental to bacterial clearance of S. aureus-induced pneumonia. Using bone marrow chimeric mice, we found that WT mice that received KO bone marrow (KO!WT) had lower bacterial burdens in the lungs and BALF than WT mice that received WT bone marrow (WT!WT) (Fig 2J and 2K). However, KO mice that received WT bone marrow (WT!KO) showed no increase in bacterial burden in the lungs and BALF compared to KO mice that received KO bone marrow (KO!KO) (Fig 2J and 2K). Together, these results suggest that NLRP6 derived from both hematopoietic and non-hematopoietic compartments is detrimental for bacterial clearance during S. aureus pneumonia.

Enhanced host protection in NLRP6 KO mice is dependent on neutrophils
Neutrophils have been shown to be essential for containing pulmonary Staphylococcal infections [15,16]. To determine if neutrophils confer augmented host protection in NLRP6 KO mice, we depleted neutrophils in the KO mice prior to infection with a lethal dose of S. aureus and observed survival. We found that depletion of neutrophils reversed the survival benefit observed in the NLRP6 KO mice suggesting that protection is neutrophil-dependent ( Fig 3A). Since neutrophils are essential for survival, we investigated if disruption of NLRP6 affects recruitment of neutrophils into alveolar spaces during S. aureus pneumonia. Compared to WT mice, KO mice had more leukocytes, including neutrophils, and macrophages recruited into alveolar spaces (Fig 3B-3D). Furthermore, to measure neutrophil accumulation in the lung parenchyma, we performed a myeloperoxidase (MPO) assay and found that KO mice had more MPO activity than WT mice ( Fig 3E).

Deletion of NLRP6 enhances bacterial killing of neutrophils through increased IFN-γ and ROS production
Since NLRP6 was found to be upregulated in neutrophils and macrophages in the lungs, we wanted to know whether deletion of NLRP6 affects the function of these cells. To this end, we compared the intracellular killing ability of bone marrow derived-neutrophils (BMDNs) and BMDMs isolated from both WT and NLRP6 KO mice following infection with S. aureus (MOI:10). Our results indicate that neutrophils, but not macrophages, from NLRP6 KO mice had improved killing ability compared to WT cells as depicted by the reduction in intracellular CFUs in KO neutrophils ( Fig 4A and S2A Fig). However, the rate of phagocytosis was similar in neutrophils from both groups (WT and KO) (S2B Fig). It is known that neutrophils use  NADPH oxidase-dependent reactive oxygen species (ROS) to kill S. aureus intracellularly [17][18][19]. To determine if differences in killing ability of WT and NLRP6 KO neutrophils is due to an alteration in ROS production, we compared ROS production by neutrophils from WT and KO mice after infection with S. aureus and found that KO neutrophils produced more ROS than WT neutrophils ( Fig 4B). To confirm these in vitro findings, we assessed the expression of NADPH oxidase components in lung homogenates from infected mice by western blotting. The expression of p47 phox , p67 phox , and gp91 phox was increased in the lungs from NLRP6 KO mice compared to those from WT mice, supporting the finding of higher ROS production by KO neutrophils (Fig 4C).
IFN-γ has been shown to activate phagocytic cells during intra-pulmonary S. aureus infection [20]. Therefore, we measured the levels of IFN-γ secreted in the BALF of WT and NLRP6 KO mice after infection with S. aureus. Interestingly, we found that IFN-γ production was higher in KO mice compared to that of WT mice ( Fig 4D). The production of IFN-γ requires activation of MAPK pathways [21]. Accordingly, we found higher MAPK activity in NLRP6 KO lungs than in WT lungs ( Fig 4E). IFN-γ has been shown to induce ROS production in human mast cells during Staphylococcal infection [22]. Based on this observation, we assessed if IFN-γ contributes to the enhanced bacterial killing by neutrophils through induction of ROS. To this end, BMDNs from WT and KO mice were pre-treated with IFN-γ and subsequently infected with S. aureus (MOI of 10) followed by assessment of ROS production. Treatment of neutrophils with IFN-γ increased NADPH oxidase activity and ROS production ( Fig  4F and 4G). The activation of phagocytes by IFN-γ involves activation of signal transducer and activator of transcription (STAT) proteins [23]. Supporting this observation, higher expression of phospho-STAT3 was found in the lungs of KO mice compared to WT (Fig 4E). Collectively, these results suggest that genetic ablation of NLRP6 increases bacterial killing by neutrophils through increased IFN-γ and ROS production, which are associated with higher NADPH oxidase activity.

IFN-γ mediates bacterial clearance in NLRP6 KO mice
Our in vivo experiments demonstrate that NLRP6 KO mice exhibit improved bacterial clearance (Fig 2B-2D), higher neutrophil accumulation, as well as enhanced IFN-γ production (Figs 3C and 4D). Moreover, our in vitro experiments using BMDNs demonstrate that IFN-γ enhance bacterial killing by neutrophils through increased ROS production ( Fig 4G). Therefore, we examined if blocking IFN-γ hinders bacterial clearance in the KO mice following infection with S. aureus. In this context, we administered anti-mouse IFN-γ antibody (100 μg/ mouse i.p.) to one group of the KO mice and a similar volume of isotype antibody to another group of mice 12 hours before infection with S. aureus. Blocking of IFN-γ in the KO mice increased the bacterial burden in the lungs and BALF suggesting that IFN-γ mediates bacterial clearance in the KO mice (Fig 5A and 5B).

Natural killer and CD4 + T cells are the primary sources of IFN-γ
We next sought to identify the cellular sources of IFN-γ during S. aureus infection. To this end, we performed flow cytometric analysis of lung cells from WT and KO mice following infection with S. aureus and found that NLRP6 KO mice had more IFN-γ-positive NK and CD4 + T cells (Fig 6A-6D). In this regard, Nguyen et al reported NK cells as the major source of IFN-γ during S. aureus infection [20]. We also found CD8 + T cells and γδT cells produce IFNγ, although there was no difference between WT and KO mice in the total number of IFN-γpositive CD8 or γδT cells in the lungs (S3A- S3D Fig). To determine whether increased IFN-γ production in the KO mice is due to higher MAPK activity in NK and CD4 T cells, we isolated these cells from the lungs of WT and KO mice and treated them with MAPK inhibitor prior to infection with S. aureus. However, blocking MAPK did not reduce IFN-γ secretion by NK and CD4 T cells ( S3E Fig and S3F Fig). Furthermore, we measured the number of NK and CD4 T cells in the lungs after infection through flow cytometry and found that the KO mice had more NK and CD4 T cells accumulated in the lungs compared to that of WT mice (Fig 6E). These results together suggest that increased IFN-γ observed in the KO mice is due to enhanced numbers of NK and CD4 T cells recruited during infection.

Deletion of NLRP6 decreases S. aureus-induced pyroptosis and necroptosis
Since we found decreased neutrophil recruitment and increased protein leakage in the lungs of WT mice compared to KO mice (Figs 3C and 2E), we hypothesized that cell death in the lungs may be responsible for these results during S. aureus infection. In this regard, LDH, IL-1α, and HMGB-1 are intracellular molecules released exclusively after cell death and are thus termed alarmins [24,25]. Therefore, we measured the levels of these alarmins in an in vivo setting and found their expression to be increased in WT mice compared to NLRP6 KO mice. This suggests that NLRP6 enhances inflammatory modes of cell death during S. aureus-induced pneumonia (Fig 7A-7C). We also measured the extent of cell death in BMDNs following infection with S. aureus. Neutrophils from WT mice exhibited increased cell death, as seen by increased cytotoxicity (LDH release), compared to that of KO neutrophils ( Fig 7D). Next, to determine the nature of cell death, BMDNs from WT and KO mice were pre-treated with either Ac-YVAD-CMK (Caspase-1 inhibitor) or Necrostatin-1s (Nec-1s, necroptosis inhibitor) and infected with S. aureus. Pre-treatment of neutrophils with Ac-YVAD-CMK or Nec-1s reduced the cell death in WT neutrophils suggesting that the nature of cell death is both pyroptosis and necroptosis ( Fig 7D). In this regard, it is reported that caspase-1 and gasdermin-D mediate pyroptosis during bacterial infection [26][27][28]. To validate that NLRP6 induces pyroptosis, we assessed the expression of caspase-1 and gasdermin-D in BMDMs from WT and NLRP6 KO mice after infection with S. aureus (MOI of 20) for 8 hours. Both cleaved caspase-1 (Fig 1F)  and gasdermin-D (Fig 7E) expression were higher in WT mice compared to KO mice. Also, we performed immunofluorescence microscopy on BMDMs to quantify caspase-1 and gasdermin-D expression and found increased caspase-1 and gasdermin-D-positive cells in BMDMs from WT mice compared to NLRP6 KO mice (Fig 7F and 7G; S4A and S4B Fig).
It has been reported that S. aureus induces pathology in the lungs by a distinct cell death mechanism known as necroptosis [29]. Receptor-interacting-serine-threonine kinase-1 (RIP1), RIP3, and mixed lineage kinase-domain like protein (MLKL) are the core protein kinases that initiate and execute necroptosis [29][30][31]. Recently, caspase-8 has been shown to negatively regulate necroptosis during Salmonella infection in an enteritis model [32]. Thus, to explore whether NLRP6 can enhance necroptosis in the lungs during S. aureus infection, we assessed the expression of phospho-MLKL RIP1, RIP-3, and caspase-8 in lung homogenates obtained from S. aureus-infected WT and NLRP6 KO mice through western blotting. Interestingly, both phospho-MLKL and RIP-3 were higher in the lungs of WT mice compared to NLRP6 KO mice (Fig 7H). To further confirm this finding at the cellular level, we infected Role of NLRP6 in Gram-positive Pneumonia both WT and NLRP6 KO BMDMs with S. aureus for 8 hours and quantified necroptosis using immunofluorescence microscopy. The immunofluorescence assay also revealed more phospho-MLKL and RIP-3-positive cells in WT macrophages compared to NLRP6 KO macrophages after S. aureus infection, confirming that NLRP6 increases necroptosis (Fig 7I and S4C  Fig).
Since S. aureus has been shown to induce necroptosis in human cells, such as THP-1 cell lines [29], we examined if activation of the necroptosis pathway occurs in lungs of human patients during pneumonia. Immunofluorescence microscopy conducted on lung sections Role of NLRP6 in Gram-positive Pneumonia obtained from pneumonia patients displayed more necroptosis, as evidenced by increased phospho-MLKL and RIP-3 expression, compared to that of healthy control lungs (Fig 7J).

Blocking pyroptosis and necroptosis in WT mice augments host defense
We found that NLRP6 enhances both pyroptosis and necroptosis pathways during pulmonary S. aureus infection (Fig 7A-7I). Because pyroptosis has been found to be beneficial for bacterial clearance during intracellular bacterial infections [28], we examined whether pyroptosis is advantageous during S. aureus infection. Blockade of pyroptosis in mice by intra-peritoneal administration of Ac-YVAD-CMK (caspase-1 inhibitor) 12 hours prior to infection with S. aureus resulted in reduced pulmonary leakage, LDH release, and bacterial burden in lungs and BALF (Fig 8A-8D). In addition, blockade of pyroptosis suppressed cytokine secretion and enhanced survival of WT mice (Fig 8E-8H), confirming that pyroptosis is detrimental during S. aureus infection. It should be noted that the bacterial burden in WT mice receiving Ac-YVAD-CMK was still high when compared with NLRP6 KO mice (Fig 8C), suggesting that an NLRP6-dependent, but caspase-1-independent, mechanism also exists to increase susceptibility to S. aureus infection.
Next, to confirm that NLRP6-mediated necroptosis is detrimental for host defense, we blocked necroptosis using an MLKL inhibitor (GW806742X) 12 hours before infection with S. aureus. There was a decrease in total protein and LDH release in the BALF of WT mice treated with GW806742X, which was comparable to that seen in NRLP6 KO mice (Fig 9A and 9B). Moreover, blocking necroptosis reduced the bacterial burden in lungs and BALF and ameliorated the inflammatory cytokine levels in WT mice (Fig 9C-9G). Also, leukocyte recruitment (neutrophils and macrophages) and survival were also increased in WT mice treated with necroptosis inhibitor (Fig 9H-9K). Inhibition of necroptosis using a RIP-1 inhibitor (Nec-1s) also reduced total protein leakage and LDH release in the BALF (S5 Fig). Collectively, these results reveal that NLRP6-mediated pyroptosis and necroptosis are detrimental to bacterial clearance and host survival during pulmonary S. aureus infection.

Discussion
Lung diseases induced by Gram-positive pathogens are an important cause of morbidity and mortality in both immunocompetent and immunocompromised individuals [13,33]. Although antibiotics decrease the mortality rates of bacterial pneumonia, the efficacy is somewhat limited due to the substantial number of immunocompromised individuals, growing number of elderly patients, and the rise of multi-antibiotic resistant bacterial strains. Thus, alternative therapeutic approaches, including the manipulation of host signaling events, are needed. However, detailed understanding of the host innate immune response is critical for the design of potential therapeutic interventions. Because the lung is continuously exposed to pathogens and their virulence factors, this organ possesses a multifaceted host defense system. Moreover, a successful immune response in the lung is critical for efficient clearance of microbial pathogens and therefore, the innate immune system possess germline-encoded patternrecognition receptors.
The NOD-like receptors (NLRs), including inflammasomes, are specialized cytosolic pattern-recognition receptors/sensors necessary for clearance of invading cytosolic pathogens. Under normal homeostatic conditions, the NLRP6 inflammasome is highly expressed in intestinal epithelial cells [9,14,34,35] where it co-localizes with ASC and caspase-1 [9]. It is also expressed in immune cells including neutrophils, T-cells, macrophages, and dendritic cells [7]. Despite high expression of NLRP6 in the lower respiratory tract, the role of NLRP6 in lung inflammation has not previously been explored. In the current study, we demonstrate that NLRP6 is upregulated in neutrophils, macrophages, and epithelial cells in the lungs of human pneumonia patients. Furthermore, NLRP6 is upregulated in myeloid and non-myeloid cells in the lungs and co-localizes with ASC in a mouse model of pulmonary S. aureus infection. We also found that the important virulence factor, α-hemolysin, can activate the NLRP6 inflammasome.
The immune response to S. aureus is manifested by vascular leakage, neutrophil recruitment into the alveolar space, and upregulation of cytokines and chemokines [5,6]. The current study demonstrates that the NLRP6 inflammasome increases susceptibility to S. aureusinduced lung infection. Delving into the mechanisms underlying this, we found that NLRP6 dampens NK cell-mediated IFN-γ secretion thereby hindering ROS-dependent bacterial Role of NLRP6 in Gram-positive Pneumonia clearance by neutrophils. Moreover, our study identifies NK cells and CD4-T cells as the primary source of IFN-γ during acute pulmonary S. aureus infection. In agreement with these findings, studies of Listeria and Salmonella infections [7] have also shown that NLRP6 signaling is detrimental to host defense. Nonetheless, in a non-infectious model, the NLRP6 inflammasome was found to be important for epithelial self-renewal, proliferation, and mucus secretion, which were essential for protection against chemical-induced colitis [9,34].
Studies from different groups have shown that NLRP6 inflammasome regulates gut microbiota composition [7,9,14]. NLRP6 KO mice were found to have different microbiota configuration which make them more susceptible to chemical-induced colitis compared to the WT mice [14]. In contrast, recent studies have demonstrated that NLRP6 and ASC-related inflammasome do not regulate gut microbiota composition [36,37]. Although it remains debatable whether the NLRP6 inflammasome influence gut microbiome, the reported difference in microflora composition in the KO mice can be nullified by co-housing the mice together with WT for 4 weeks [7]. In addition to colitis, microbiota have been shown to influence various disease conditions such as rheumatoid arthritis [38], diabetes [39], inflammatory bowel disease [40], and colorectal cancer [41]. In our study, however, co-housing of WT with NLRP6 KO mice did not change the resistant phenotype of the KO mice against S. aureus. In this context, similar report has been demonstrated by Anand et al, showing that co-housing does not alter NLRP6 mediated immune mechanism during Salmonella and Listeria infection [7].
It is widely accepted that hematopoietic and non-hematopoietic (stromal) cells in the lung produce numerous proinflammatory mediators, including cytokines and chemokines. Although hematopoietic cells secrete chemokines or neutrophil chemo-attractants, including CXCL1/KC and CXCL2/MIP-2, the stromal cells (alveolar epithelial cells) secrete other neutrophil chemo-attractants, such as CXCL5/LIX and CXCL15/lungkine [42]. Our observations indicate that NLRP6 from both cell types contributes to the enhanced susceptibility to S. aureus-induced pneumonia. These conclusions are consistent with previous studies of the role of hematopoietic and non-hematopoietic cells in the context of bacterial infections in the lungs. In this context, NLRP6 in both hematopoietic and non-hematopoietic cells increases susceptibility to Listeria and Salmonella infections [7]. Similarly, CXCL1/KC secreted by both hematopoietic and stromal cells was found to be crucial for bacterial elimination and neutrophil accumulation in the lungs following Klebsiella pneumoniae infection [43]. Nevertheless, it is clear from this investigation that neutrophil accumulation and function are critical for host protection against S. aureus.
Pyroptosis and necroptosis are two distinct inflammatory modes of cell death. While pyroptosis is mediated by caspase-1 [26][27][28] and executed by gasdermin-D [26,27], necroptosis is regulated by RIP1, RIP3, and MLKL (caspase-1 independent) [29,30]. Pyroptosis has been shown to play an essential role in limiting several intracellular bacterial infections such Salmonella typhimurium, Legionella pneumophila, and Burkholderia thailandensis [28]. However, extensive caspase-1 activation and subsequent pyroptosis have also been associated with the severity of several diseases such as myocardial infarction [44], inflammatory bowel disease [45], and endotoxic shock [46]. Pertaining to these observations, we report that during S. aureus infection, NLRP6-mediated pyroptosis is detrimental for host survival. Furthermore, blocking pyroptosis reduced the hyper-inflammatory milieu and subsequently increased survival suggesting that pyroptosis triggers exaggerated inflammation during S. aureus infection. This difference in the role of pyroptosis can be attributed to differences in pathogenic properties and life styles of bacterial pathogens. While S. aureus is predominantly an extracellular pathogen, studies have shown that it can also survive intracellularly [47] and can resist anaerobic conditions [48]. Necroptosis induced by S. aureus is responsible for pathology in the lung [29]; however, its relationship with inflammasomes was previously unknown. Although ASC and NLRP3 have been linked with pore-forming toxin-induced necroptosis [31], the precise role of inflammasomes in the induction of necroptosis is not clear. In this study, we used both in vivo and in vitro experiments to show that NLRP6 mediates necroptosis of immune cells during acute pulmonary S. aureus infection. Moreover, S. aureus exploits NLRP6 to drive necroptosis, which is accompanied by an intense inflammatory response and loss of macrophages and neutrophils. It is possible that reduced cell death in NLPR6 KO mice attributed to higher leukocyte accumulation in the lungs of these mice. Since TNF-α has been shown to induce necroptosis [49,50], the reduction of TNF-α found in the NLRP6 KO mice suggests that NLRP6 can trigger necroptosis via the TNF-α pathway. However, more comprehensive future studies will be needed to identify the detailed molecular mechanisms underlying NLRP6-mediated necroptosis. Future studies are also needed to determine whether other toxins or virulence factors produced by S. aureus can also activate the NLRP6 inflammasome.
In conclusion, the present study reveals the detrimental role of NLRP6 during S. aureus pneumonia (S6 Fig). Furthermore, NLRP6 in both hematopoietic and resident lung compartments contributes to S. aureus-induced lung inflammation. Not only does NLRP6 subdue neutrophil function by dampening IFN-γ and ROS production, it also triggers pyroptosis and necroptosis in the lungs that may lead to hyper-inflammation, loss of neutrophils, and mortality. However, future studies are essential to determine whether NLRP6 interacts with other inflammasomes such as NLRP3 and/or NLRC4 to induce pyroptosis and necroptosis during S. aureus infection. Comprehensive studies using specific double-or triple-KO mice would be useful to delineate these interactions in a conclusive manner. Furthermore, extending upon our findings, we propose that functional single nucleotide polymorphisms in human NLRP6 may have effects on host defense mechanisms against gram-positive microbes.

Ethics statement
Mouse experiments were conducted in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University (protocol number 16-072). All animal experiments were performed in a manner to ensure minimal pain and distress. Nlrp6 -/-, Asc -/-, and Caspase-1/11 -/were obtained from the Millennium Pharmaceuticals (Cambridge, MA) whereas C57Bl/6 mice were obtained from Taconic (Rensselaer, NY) and Jackson (Bar Harbor, ME) Laboratories. THP-1 (human monocytic) cells and HL-60 (human neutrophil-like) cells were purchased from ATCC (Manassas, VA). Lysates of human healthy control tissue and pneumonic lung tissue were obtained from Novus Biologicals, CO.

Immunofluorescence microscopy
Immunofluorescence microscopy of lung sections was performed as described previously [51]. Human lung sections from lungs without evidence of infection or injury (control) or from patients who died due to ALI/ARDs (pneumonic) were used from BioChain Institute Inc. (Newark, CA). Mouse lung sections were from saline-challenged or S. aureus-infected mice. Lung sections were incubated with anti-NLRP6 (Abgent, CA) and antibodies for surface markers including anti-lipocalin Ab for neutrophils (R&D Systems, MN), or anti-CD68 Ab for macrophages (BioLegend, CA), and anti-CD326 Ab for alveolar epithelial cells (BioLegend, CA). For mouse lung sections, we used antibodies for surface markers including anti-Ly6G for PMNs (BioLegend, CA), anti-F4/80 for macrophages (BioLegend, CA), and anti-CD326 for alveolar epithelial cells along with anti-NLRP6 Ab (Sigma, MO). Appropriate Alexa-conjugated secondary antibodies (Invitrogen, CA) were used. For detection of necroptosis and pyroptosis through an immunofluorescence assay, antibodies against mouse NLRP6 and ASC (Sigma, MO), p-MLKL, (Abcam, MA), RIP3 (Cell signaling, MA), caspase-1 (Adipogen, CA), and gasdermin-D (Santa cruz, CA) were used. Excess antibodies were washed off, and the cells were labeled with secondary antibodies, such as mouse IgG/IgM (H+L) Alexa fluor 488, 568 (Invitrogen, CA). Images were obtained using an Axiocam digital camera (Zeiss, NY) connected to a Zeiss Axioskop 2 Plus microscope.

Immunoblotting and cytokine measurement
BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0.1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47 phox, P67 phox, gp91 phox , phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 4 0 C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer's protocol (eBioscience, CA).

Animals
Eight to twelve-week-old WT mice (C57BL/6 background) were used. Equal age-and gendermatched NLRP6 KO, ASC KO, and Caspase-1/11 DKO mice on the C57BL/6 background were used throughout the experiments. Mice were kept on a 12:12 hour light/dark cycle under specific pathogen-free condition with free access to food and water. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Louisiana State University.

Pneumonia model
To induce pneumonia, mice were anesthetized using ketamine (100 mg/kg) and xylazine (5 mg/kg) prior to intratracheal inoculation of S. aureus (USA 300 strain). A small midline incision was made on the ventral aspect of the neck and excess fat was separated to expose the trachea. Fifty microliters of bacterial suspension containing 5X10 7 CFU of log phase S. aureus in isotonic saline was injected into the lungs by piercing trachea using a 28.5-gauge needle. At 12-and 24-hours post-infection, mice were euthanized to collect BALF, lungs, and liver for quantification of bacterial burden and leukocyte recruitment. BALF and homogenized organs were serially diluted and plated onto Tryptic soy agar (TSA) plates for bacterial enumeration.
For survival experiments, we used 2 X 10 8 CFUs/mouse of S. aureus and observed survival for 12 days post-infection.

BALF collection
BALF was collected as described in our previous publication [51]. Briefly, after specific time points, mice were humanly euthanized, and the trachea was exposed. Using a 20-gauge catheter, 0.8 ml of PBS (heparin and dextrose added) was instilled inside the lungs and collected in a clean tube. The process was repeated a total of four times so that a minimum of 2.8-3 ml of BALF was collected from each mouse. Total leukocyte count was performed in a hemocytometer using 10 μl of BALF and the differential count was done under light microscopy using cytospin slides stained with DiffQuik reagent. The remaining cell-free BALF was preserved at -80 o C for cytokine analysis.

Co-housing experiments
Co-housing experiments were performed as described by Anand et al [7]. In brief, age and sex matched WT and NLRP6 KO mice were co-housed together in 1:1 ratio for 4 weeks. After cohousing, WT and the KO mice were infected with S. aureus (i.t.) and euthanized 24 hours post-infection to measure the bacterial burden.

Bone marrow chimeras
Bone marrow chimeras were generated as described previously [51,52]. In brief, the recipient mice were lethally irradiated with a 1000-rad inoculum from a cesium source. Bone marrow cells collected from healthy donor mice were injected into recipient mice via tail vein (8 million cells per mouse). The chimeric mice were kept under 0.2% neomycin sulfate treatment for 15 days after transplantation. After 8 weeks of transplantation, the chimeric mice were infected with 5X 10 7 CFU of S. aureus. The mice were euthanized at 24-hour post-infection to estimate cellular recruitment and bacterial burden in the lungs.

MPO assay
For MPO activity assay, lungs obtained from WT and KO mice after infection were homogenized. The supernatants obtained after centrifugation were mixed with 50 mM potassium phosphate buffer (with 0.5% hexadecyltrimethylammonium bromide) in 1:5 ratio and then centrifuged again. The supernatants were transferred to a 96-well plate. Absorbance was measured using a spectrophotometer at 460 nm after adding hydrogen peroxide/O-dianisidine hydrochloride buffer.

Neutrophil killing assay
The intracellular killing assay was performed as described [54] with slight modification. Briefly, bone marrow neutrophils from NLRP6 KO and WT mice were isolated, infected with S. aureus (MOI: 10), and treated with gentamicin for 30 minutes at designated time points post-infection (30 min, 60 min, 90 min, and 120 min) to kill extracellular bacteria. Cells were washed several times with sterile PBS to remove excess gentamicin and were then lysed with 0.1% triton X to release intracellular bacteria. The lysate was serially diluted with PBS, plated onto the TSA, and incubated at 37 0 C overnight for bacterial load estimation.

ROS detection
Total neutrophils, isolated from bone marrow of WT and NLRP6 KO mice using a magnetic negative selection cell isolation kit (STEMCELL Technologies, Vancouver, Canada), were infected with S. aureus (MOI 10). Total intracellular ROS production was quantified using a Fluorometric kit (AA Bioquest, CA). The effect of IFN-γ on ROS production was determined by pretreating neutrophils with either 20 ng/ml of recombinant mouse IFN-γ (R&D Systems, MN) or an equal volume of PBS for 30 minutes before infection with S. aureus. The total ROS production was quantified after 30 minutes of infection using a spectrophotometer.

Measurement of cell death
BMDNs were isolated from WT and NLRP6 KO mice and pretreated with Nec-1s (300 μM) or Ac-YVAD-CMK (100 μg/ml) or DMSO for 30 minutes before infection with S. aureus (MOI: 20). The percentage of cytotoxicity in BMDNs and LDH release into the alveolar space after S. aureus infection were measured using the Cytotox-ONE TM homogenous membrane integrity assay kit (Promega, WI). HMGB-1 was measured using a commercially available kit from Chondrex Inc, WA.

Statistics
Data are represented as mean ± SEM. The Mann-Whitney test was used to compare the bacterial burden between two groups. Student's t-test was used whenever the data were parametric in nature. We used one-way ANOVA followed by Bonferroni's multiple comparison test wherever more than two groups were involved. All statistical analyses were performed using GraphPad Prism 7 software. The survival curve was analyzed using Log-rank (Mantel Cox) test. All experiments were performed thrice. Significant differences are indicated by Ã (p<0.05), ÃÃ (p<0.01), ÃÃÃ (p<0.001), and ÃÃÃÃ (p<0.0001). inflammasome (1). The activated NLRP6 inflammasome dampens IFN-γ production by NKcells (2). Since IFN-γ is important for NADPH oxidase activity, reduction of IFN-γ secretion leads to reduced activity of NADPH oxidase (3) and ultimately reduced ROS production (4) ultimately compromising the function of neutrophils (5). Reduced ROS production by neutrophils leads to reduced bacterial clearance (6). Upon activation, NLRP6 increases the activity of caspase-1 and gasdermin-D (7) thereby triggering pyroptosis (8). Similarly, NLRP6 triggers necroptosis by upregulating RIP-3 and p-MLKL in the lungs (9&10). Both of these inflammatory cell death mechanisms (11) lead to exaggerated inflammation and loss of immune cells (12). Ultimately, there will be less bacterial clearance (5) and increased mortality (13).