Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation, however, neutrophils are necessary for optimal viral control. In this study, we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12−/− mice are protected from lethality during IAV infection and show decreased vascular permeability, fewer pulmonary neutrophils, and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12−/− neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12−/− dendritic cells was not due to a difference in CXCL1 protein stability, but instead to a decrease in Cxcl1 mRNA stability. Together, these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses.
Severe disease during influenza A virus (IAV) infection is associated with excessive cytokine production and an exaggerated innate immune response, leading to substantial tissue damage and impaired respiratory function (1–3). The heightened morbidity and mortality among otherwise healthy adults infected with the 2009 pandemic H1N1 strain emphasized the threat posed by emerging strains to which there is little or no pre-existing immunity in the population.
The presence of elevated neutrophil chemoattractants and massive neutrophil infiltration of the lungs in lethal cases of IAV suggested a detrimental role for neutrophils during IAV infection (1, 4, 5). Subsequent studies have revealed a much more nuanced relationship between neutrophils and the IAV-infected lung. Neutrophils are among the first immune cells to arrive in the lungs, where they contribute to viral clearance through phagocytosis of viral particles and IAV-infected apoptotic cells (6). Neutrophil depletion in mice during IAV infection results in increased viral titers and mortality (7, 8), however, mice deficient in key neutrophil effector molecules such as myeloperoxidase or chemoattractants such as CXCL2 have less severe disease (9, 10). A recent study provided an excellent framework for understanding these dissonant results, finding that enhancement of inflammatory signaling networks driven largely by neutrophils is an early predictor of lethality (7, 11). Key effector molecules downstream of activation of these networks include neutrophil chemoattractants (CXCL1, 2, and 5) and their receptor (CXCR2). Partial depletion of neutrophils reduced mortality during lethal infection, supporting the notion that control of early inflammatory responses is key to survival in high-dose infections (11).
Nlrp12 is a member of the nucleotide binding domain and leucine rich repeat containing receptor family, which has been implicated in regulation of proinflammatory signaling in the context of bacterial infections, tumorigenesis, and autoimmunity (12–14). We and others have recently shown that Nlrp12-deficient cells produce decreased CXCL1, a potent neutrophil chemoattractant, during bacterial infections (13, 15). Given the complex role of neutrophils during IAV infection, we sought to determine whether Nlrp12 influenced host susceptibility to IAV infection. We report that Nlrp12−/− mice have significantly improved survival following IAV infection in comparison with wild-type (WT) mice. Nlrp12−/− mice maintained their ability to control infection and have decreased CXCL1-driven pulmonary vascular permeability and pulmonary neutrophil recruitment.
Materials and Methods
The generation of Nlrp12−/− mice has been described elsewhere (16). Mice were backcrossed to C57BL/6N or BALB/cJ mice for at least nine generations and maintained in a specific pathogen-free facility. C57BL/6N mice were purchased from the Charles River Laboratories and used as WT controls unless otherwise stated; BALB/cJ and C57BL/6J mice were purchased from the Jackson Laboratory. Both male and female mice 6–10 wk of age were used, however, mice were sex, age, and weight matched for individual experiments. The Institutional Animal Care and Use Committee at the University of Iowa approved all protocols used in this study.
Virus and in vivo infection
Mouse-adapted IAV strain A/PR/8/34 was propagated as previously described (17). Mice were anesthetized with ketamine and xylazine and infected intranasally with virus diluted in 50 μl sterile DMEM. Weight was monitored daily and mice were euthanized upon losing 30% of their starting weight. For CXCL1 blocking experiments, mice were administered 5 μg anti-CXCL1 (MAB453, clone 48415; R&D Systems) or isotype control Ab i.p. immediately prior to infection, and 24 and 72 h postinfection.
Lung titers, sectioning, and histology
To measure virus titers, lungs were homogenized using a Tissue-Tearor (Biospec), then homogenates were clarified by centrifugation and immediately frozen at −80°C. A standard plaque assay on Madin-Darby canine kidney cells was subsequently used to quantify infectious virus. For histology, lungs were fixed in 10% neutral buffered formalin, embedded, cut at 5 μm, and stained with H&E. A single H&E-stained slide from each of five separate animals per group was evaluated by a board-certified veterinary pathologist in a blinded fashion, and both necrosis and pulmonary inflammation were assessed using semiquantitative scoring. Necrosis was defined by cellular necrosis of the bronchiolar epithelial cells and/or the pneumocytes, which was accompanied by accumulation of necrotic cellular debris in these regions. The entire slide was evaluated and included both the right and left lung lobes. Scores were 0 = no necrosis; 1 = rare (1–3) scattered areas of necrosis throughout the lung; 2 = multifocal (3–9) scattered areas of necrosis throughout the lung; 3 = numerous (>10) areas of necrosis throughout the lung. Inflammation was defined by the accumulation of inflammatory cells, primarily viable and degenerate neutrophils, within airways and/or alveolar spaces. Scores were 0 = no inflammation; 1 = 1–10% of the pulmonary parenchyma contains inflammatory cell infiltrates; 2 = 10–50% of the pulmonary parenchyma contains inflammatory cell infiltrates; 3 = >50% the pulmonary parenchyma contains inflammatory cell infiltrates.
Cytokines and chemokines were quantified in cell culture supernatants and lung homogenate supernatants using DuoSet mouse ELISA kits from R&D Systems: CXCL1, CXCL2, CXCL5, IL-1β, CCL2, and CCL5; or ReadySetGo! mouse ELISA kits from eBioscience: IL-6, TNF-α, IL-10, IL-1α, IL-12p40, and IFN-γ following the manufacturer’s instructions.
For some experiments, intravascular staining for CD45.2 was performed as described (18) prior to tissue harvest. Single-cell suspensions were prepared by pressing tissues through wire mesh screens. Live cells were enumerated by Trypan blue exclusion. Next, 1 × 106 cells per well were blocked with 2% rat serum and anti-mouse CD16/32 (clone 2.4G2; Tonbo Biosciences) in FACS buffer for 30 min at 4°C. Following blocking, cells were stained in FACS buffer with fluorochrome-conjugated Abs in the dark for 30 min at 4°C. Cells were then fixed in FACS Lysis Buffer (BD) per the manufacturer’s instructions and resuspended in PBS. The following fluorochrome-conjugated Abs were used: CD4 (clone GK1.5), CD8a (53-6.7), CD11a (M17/4), CD11b (M1/70), CD45.2 (104), CD49d (R1-2), CD64 (X54-5/7.1FC), CD90.2 (30-H12), Ly6G (1A8), Ly6C (HK1.4), and I-A/I-E (M5/114.15.2) from BioLegend; CD4 (RM4-5) from eBioscience; and CD11c (HL3) from BD Biosciences. Data were acquired on a BD LSR II and analyzed with FlowJo software (FlowJo). For detection of intracellular CXCL1, mice were treated with 500 μg monensin (Sigma-Aldrich) i.p. 5 h prior to harvest to inhibit cytokine/chemokine secretion. Tissue processing and surface staining was performed as above, but 0.1 mg/ml monensin and 5 μg/ml brefeldin A (Sigma-Aldrich) was present in all solutions until fixation. Cells were fixed, permeabilized, and stained using a Transcription Factor Staining Kit (eBioscience) according to the manufacturer’s instructions. CXCL1 was detected by biotinylated anti-CXCL1 (BAF453; R&D Systems) and APC-labeled streptavidin (eBioscience). Live cells were sorted for gene expression analysis on a BD FACS Aria II. Hoechst dye was added to cells prior to sorting to identify live cells, which were collected and processed for gene expression analysis (as below) after sorting.
We assessed microvascular permeability by extravasation of Evans blue dye as previously described (19). Briefly, mice were injected i.v. with 20 mg/kg Evans blue dye. Then 1 h later, lungs were perfused with PBS, weighed, and homogenized. Evans blue dye in lung homogenates was extracted with formamide (Sigma-Aldrich) at 60°C for 18 h. Absorbance of lung homogenate supernatants at 620 and 740 nm was measured to calculate micrograms Evans blue dye per gram of lung tissue.
In vitro infection and stimulation of bone marrow–derived cells
Bone marrow–derived dendritic cells (BMDCs) and bone marrow–derived macrophages (BMMs) were generated as described (13, 20). Bone marrow neutrophils were purified by negative selection with an EasySep Mouse Neutrophil Enrichment Kit (Stemcell Technologies), following the manufacturer’s instructions. On day 6 of culture, 1 × 106 BMDCs were infected with IAV (multiplicity of infection = 5) in serum-free infection medium for 60 min, then washed twice with PBS and incubated in complete medium until harvest. Alternatively, BMDCs were treated with 1 μg/ml R848 (InvivoGen) in complete medium until harvest. For transcript stability experiments, 10 μg/ml actinomycin D (ActD) (Sigma-Aldrich) was added to BMDCs, BMMs, or neutrophils after 3 h of 1 μg/ml R848 stimulation. For protein stability experiments, 10 μg/ml cycloheximide (Sigma-Aldrich) was added to BMDCs after 6 h of R848 stimulation. To assess neutrophil death, freshly isolated neutrophils were incubated with 30 ng/ml PMA (Sigma-Aldrich), 4 ng/ml CXCL1 (#250-11; PeproTech) or in complete medium until harvest. Incubation with IAV was performed as for BMDCs (see above).
Gene expression analysis
RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) per the manufacturer’s instructions. cDNA was synthesized using Superscript III First Strand Synthesis SuperMix (Invitrogen). Quantitative PCR was performed using cDNA, primer pairs for Cxcl1, Il6, and Actb (PPM03058C, PPM03015A, and PPM02945B; Qiagen), and PerfeCta SYBR Green Fastmix (Quanta Biosciences). Reactions were run in a QuantStudio 6 Flex (Applied Biosystems) with a standard cycling protocol (90°C 2 min; 40 cycles of 95°C 15 s, 60°C 1 min). Quantitative PCR data were analyzed using the 2−ΔΔCt method where ΔΔ cycle threshold (Ct) = [(Ct gene of interest-Ct housekeeping) stimulated − (Ct gene of interest-Ct housekeeping) unstimulated]. Actb was chosen as a housekeeping gene.
Lysates were prepared in radioimmunoprecipitation assay buffer (Cell Signaling Technology) with 1 mM PMSF per the manufacturer’s instructions. Proteins were separated on a NuPAGE gel (Invitrogen) and transferred to a polyvinylidene difluoride membrane using the XCell II blotting system (Invitrogen). Membranes were blocked with 5% nonfat milk and incubated with primary Ab overnight at 4°C. Primary Abs were purchased from Cell Signaling Technologies: IRAK1 (#4504), phospho-IkBa (#2859), IkBa (#4814), phospho-p38 MAPK (#4511), p38 MAPK (#8690), phospho-p44/42 MAPK (ERK1/2, #4370), p44/p42 MAPK (ERK1/2, #4695), NFkB2 p100/p52 (#4882), phospho–NF-κB p65 (#3033), NF-κB p65 (#8242) phospho-TBK1 (#5483), TBK1 (#3504). CXCL1 (PA1-29220; Thermo Fisher Scientific), GAPDH (CB1001), and total actin Ab (MAB1501) were purchased from EMD Millipore. HuR (sc-5261), IRAK1 (sc-5288), phospho-MAPKAPK2 (sc-293139), MAPKAPK2 (sc-393609), and tristetraprolin (TTP) (sc-374305) were purchased from Santa Cruz Biotechnology. TRAF2 (592) was purchased from Medical & Biological Laboratories. Following washing, membranes were incubated with HRP-tagged anti-mouse IgG (1706516; Bio-Rad) or anti-rabbit IgG (NA934; GE Healthcare) and developed using SuperSignal West Pico or Femto substrate (Thermo Fisher Scientific).
Data were graphed and the indicated statistical tests performed using GraphPad Prism software.
Nlrp12 deficiency improves survival following IAV infection
To determine whether Nlrp12 is important for protection during a high-dose IAV infection, we infected WT C57BL/6N and Nlrp12−/− mice with a 4LD50 inoculum of IAV, and monitored survival and weight loss as a measure of morbidity. Strikingly, 67% of Nlrp12−/− mice survived the infection, compared with 0% survival in the WT group at day 14 postinfection (Fig. 1A). Apart from a failure of WT mice to regain weight after day 10, no significant differences in weight loss were apparent (Fig. 1A). Similar results were observed in Nlrp12−/− mice backcrossed onto a BALB/c background (Supplemental Fig. 1A).
T cell responses are critical for protection during a primary IAV infection, and augmentation of the CD8+ T cell response during high-dose infections improves survival (17). A recent study suggested that Nlrp12−/− CD4+ and CD8+ T cells exhibit increased expansion and proinflammatory cytokine production upon stimulation (12), prompting us to investigate the possibility that Nlrp12−/− mice had enhanced T cell responses during high-dose IAV infection. Interestingly, we found no difference in the magnitude of the IAV-specific CD8+ or CD4+ T cell responses (Fig. 1B, 1C, Supplemental Fig. 1B, 1C) (21). Consistent with the importance of IAV-specific CD8+ T cells for viral clearance, we observed no difference in virus titers in the lungs of WT and Nlrp12−/− mice at day 3 or 7 postinfection (Fig. 1D). Histopathologic analysis indicated more pathology overall in the lungs of WT mice than Nlrp12−/− mice at day 5 postinfection, with increased hemorrhage and necrotic debris in airways and alveoli (Fig. 1E, 1F).
Nlrp12 modulates CXCL1 during IAV infection
Unrestrained proinflammatory cytokine production is a hallmark of severe disease during IAV infections in humans and mice (2). Although IL-1β, IL-6, and TNF-α are closely associated with disease severity and weight loss, we saw no significant differences in the amount of these cytokines at early or late time points in the lungs of WT and Nlrp12-deficient mice (Fig. 2A–C). In contrast, neutrophil chemoattractant CXCL1 was dramatically reduced in Nlrp12−/− mice compared with WT mice at day 3 but not day 7 postinfection (Fig. 2D). Neutrophil chemoattractants CXCL2 and CXCL5 were not different between the two groups, nor were a number of other cytokines and chemokines involved in inflammation and recruitment of innate and adaptive immune cells (Fig. 2E, 2F, Supplemental Fig. 1D–I). Therefore, Nlrp12−/− mice have a specific reduction in pulmonary CXCL1 during the early phase of IAV infection.
Nlrp12−/− mice have decreased pulmonary neutrophils
Neutrophils contribute to both viral clearance and immunopathology during IAV infection (7). They are recruited to the lungs early and peak 3–5 d after lethal PR8 infection (8). Following IAV infection, we assessed inflammatory cell infiltration into the lung by flow cytometry (Fig. 3, Supplemental Fig. 2). At day 3 postinfection, we found that pulmonary neutrophils were significantly decreased, both in absolute number and percentage, in the lungs of Nlrp12−/− mice compared with WT (Fig. 3, Supplemental Fig. 2B). Intravascular staining revealed no difference in the percentage of neutrophils present in the lung vasculature of Nlrp12−/− mice compared with WT (Supplemental Fig. 2B). This difference in pulmonary neutrophils was still present at days 5 and 7 postinfection, but we observed no significant differences in the number of macrophages, monocytes, or DCs at any of the time points measured (Fig. 3, Supplemental Fig. 2B). The significant increases in the frequency of DCs and macrophages at days 5 and 7 postinfection are due to the decreased total number of hematopoietic cells, and do not likely represent compensatory recruitment of these cells to the lungs (Supplemental Fig. 2B). Previous studies have shown that IAV accelerates apoptosis in human neutrophils (22). We were interested to determine whether a difference in survival contributes to the decrease in neutrophils present in the IAV-infected lungs of Nlrp12−/− mice. We quantified neutrophil death following incubation with IAV, CXCL1, or PMA and found no increase in death among Nlrp12−/− neutrophils compared with WT neutrophils (Supplemental Fig. 2C). Therefore, increased death of neutrophils in the lungs of Nlrp12−/− mice is not likely to be driving the decrease in those cells in the lungs following IAV infection. We further quantified pulmonary neutrophil recruitment in littermate Nlrp12+/+, Nlrp12+/−, and Nlrp12−/− mice following infection with IAV and found that both Nlrp12+/− and Nlrp12−/− mice had significantly fewer pulmonary neutrophils than Nlrp12+/+ mice (Fig. 4A, 4B), suggesting haploinsufficiency. We also compared neutrophil numbers in the lungs of IAV-infected C57BL/6N and C57BL/6J sub-strains as C57BL/6J mice carry a missense mutation in Nlrp12 (13, 23). Consistent with our findings in the context of bacterial infection, C57BL/6J mice had significantly fewer infiltrating pulmonary neutrophils compared with C57BL/6N mice 3 d after IAV infection (Fig. 4C, 4D). Together, these results strongly support a role for Nlrp12 in modulating pulmonary neutrophil accumulation during IAV infection.
Nlrp12−/− mice have decreased vascular permeability during IAV infection
Pulmonary edema due to diminished barrier integrity in IAV-infected lungs is a serious consequence of both the viral infection and the resultant immune response, and is associated with increased mortality (24). Neutrophil-derived reactive oxygen species and cytokines have been implicated in epithelial-endothelial barrier damage during IAV infections (25). The observed decrease in pulmonary neutrophils in Nlrp12−/− mice led us to ask whether there would also be less pulmonary vascular permeability in Nlrp12−/− mice compared with WT mice. Utilizing Evans blue dye extravasation as an indicator of vascular permeability we found that at day 5 postinfection Nlrp12−/− mice had significantly decreased vascular permeability compared with WT mice (Fig. 5A).
Binding of CXCL1 to CXCR2 on endothelial cells increases microvascular permeability in the lungs (26). When a CXCL1-blocking Ab was administered to WT mice during IAV infection, vascular permeability and pulmonary neutrophils, but not DCs or macrophages, were decreased at day 5 postinfection (Fig. 5B–E) similar to our findings with Nlrp12−/− mice. Therefore, Nlrp12-dependent modulation of CXCL1 levels plays a role in vascular permeability during IAV infection.
Cellular source of CXCL1 in the lungs during IAV infection
We next determined the cellular source of CXCL1 in IAV-infected lungs. We treated IAV-infected mice with monensin to prevent cells from secreting CXCL1, then assessed intracellular CXCL1 by flow cytometry. We found that a higher percentage of CD45.2+ hematopoietic cells than CD45.2− nonhematopoietic cells were CXCL1+, and there was no significant difference in the proportion of CXCL1+ nonhematopoietic cells between WT and Nlrp12−/− mice (Fig. 6A, 6B). Among immune cell subsets, the frequency of CXCL1+ neutrophils and DCs was significantly decreased in Nlrp12−/− mice, but there was no difference in the proportion of CXCL1+ macrophages at day 2 or 3 postinfection (Fig. 6C). To determine whether the decrease in CXCL1 was due to a change in Cxcl1 transcription, we sorted the same cell populations from IAV-infected lungs and measured Cxcl1 transcript levels. We found decreased Cxcl1 message in DCs and neutrophils at day 2 postinfection, and decreased Cxcl1 message in DCs, neutrophils, and macrophages at day 3 postinfection (Fig. 6D).
Nlrp12 is highly expressed in neutrophils, moderately expressed in BMDCs, and only modestly expressed in BMMs (16). We therefore determined the relative expression of Nlrp12 in CXCL1-producing subsets of cells in the IAV-infected lung. Consistent with published data, we found that neutrophils had the greatest expression of Nlrp12 at days 2 and 3 postinfection, over 10× more than in DCs and 100× more than in macrophages (Fig. 6E). Together, these data demonstrate that both Nlrp12 and Cxcl1 are highly expressed in hematopoietic cells.
Decreased Cxcl1 mRNA stability in Nlrp12−/− BMDCs
Induction of Cxcl1 can occur downstream of NF-κB activation, and previous studies have implicated Nlrp12 in the regulation of NF-κB activation (14, 27, 28). We therefore infected BMDCs with IAV and performed immunoblotting to assess activation of NF-κB and MAPK family members. Interestingly, we found no pronounced differences in activation of NF-κB or MAPK family members, despite decreased CXCL1 production by IAV-infected Nlrp12−/− BMDCs (Supplemental Fig. 3A–C). These data indicated that although the decrease in CXCL1 production by DCs in IAV-infected lungs is recapitulated in IAV-infected Nlrp12−/− BMDCs, this is unlikely due to alterations in signaling pathways that ultimately converge on Cxcl1 transcription.
IAV does not robustly infect BMDCs (29), thus it was possible that suboptimal or asynchronous stimulation of virus-sensing pattern recognition receptors (PRRs) was obscuring signaling differences. To circumvent this, we treated BMDCs with the TLR7 agonist R848 and again observed decreased CXCL1, but not IL-6, protein, and mRNA in Nlrp12−/− compared with WT BMDCs (Fig. 7A–D). Similar to results from IAV infection, we again did not see any differences in activation of NF-κB or MAPK family members between WT and Nlrp12−/− BMDCs following challenge with R848, nor did we see any difference in CXCL1 protein stability (Supplemental Fig. 3D–F).
Given a lack of evidence for alterations in signaling pathways that culminate in transcription of proinflammatory mediators including CXCL1, we next investigated the possibility that Cxcl1 mRNA stability was different in Nlrp12−/− BMDCs compared with WT BMDCs. We measured Cxcl1 and Il6 transcript decay by quantitative RT-PCR (qRT-PCR) in R848-treated BMDCs following transcription blockade with ActD. Interestingly, we found an increased rate of Cxcl1 transcript decay in Nlrp12−/− BMDCs compared with WT BMDCs, whereas the rate of Il6 transcript decay was unchanged (Fig. 7E, 7F). The half-life of Cxcl1 transcript was ∼30 min longer in WT BMDCs than in Nlrp12−/− BMDCs, but no difference in Cxcl1 transcript stability was observed in BMMs or neutrophils (Supplemental Fig. 4A, 4B). Together, these data suggest that Nlrp12 plays a role in Cxcl1 transcript stability specifically in BMDCs.
Stability of transcripts including Cxcl1, Il6, and Tnf can be modulated through conserved AU-rich elements (AREs) in their 3′ UTRs (30, 31). ARE binding proteins mediate the regulatory function of AREs by stabilizing or destabilizing specific mRNAs in response to upstream signaling, commonly from PRRs or cytokine receptors. Cxcl1 mRNA is present at very low levels in resting myeloid cells, reflecting that the continuous transcription of Cxcl1 is followed by the binding of these transcripts by the ARE-binding protein TTP, which leads to transcript degradation. Upon stimulation of macrophages with LPS, Cxcl1 transcription increases and the mRNA is stabilized in part via p38 MAPK and MAPKAPK2-mediated phosphorylation of TTP, which affects its cellular localization and ability to recruit degradation machinery to the mRNA (30). Under different stimulatory conditions, stabilizing (HuR) and destabilizing factors (SF2 via TRAF2/5 and Act1, KSRP) affect the abundance of Cxcl1 mRNA (32). We thus asked if the regulation of Cxcl1 mRNA by Nlrp12 involved one of these pathways. WT and Nlrp12−/− BMDCs were stimulated with R848 followed by immunoblotting for ARE-binding and associated proteins. We found no differences in the abundance or activation of any of these proteins, nor did we see a difference in the activation of translation initiation factor eIF4E (Supplemental Fig. 4C). Together, these data indicate that the mechanism by which Nlrp12 affects Cxcl1 transcript stability in BMDCs is likely through pathways distinct from those that have been described downstream of either LPS or cytokine stimulation.
This work demonstrates that Nlrp12 contributes to disease severity during IAV infection through its effect on CXCL1 production, neutrophil recruitment to the lungs, and vascular permeability. Our finding that Nlrp12−/− mice have decreased—but not ablated—pulmonary neutrophils and improved survival is important because it supports a protective effect of neutrophils during IAV infection that is dependent on the magnitude of the neutrophilic response. We report a 25–40% decrease in the number of pulmonary neutrophils during infection, and show that this curtails enough of the immunopathology driven by excessive innate inflammatory responses without negatively affecting viral clearance or CD8+ T cell responses, to result in improved survival. We also show that treatment with CXCL1-blocking Ab results in decreased neutrophil recruitment and decreased vascular permeability, consistent with the notion that downmodulation of early inflammatory mediators decreases disease severity.
The specific decrease in CXCL1 has broad implications for the overall course of disease due to direct and indirect effects on barrier function in the IAV-infected lung. The main complication of IAV infection is viral pneumonia, which can lead to acute respiratory distress syndrome, respiratory failure, and death (24). Respiratory failure is precipitated by damage to the epithelial-endothelial barrier in the alveolus, which allows accumulation of proteinaceous fluid and immune cells that dramatically impair efficient gas exchange. CXCL1 can affect the barrier in at least two ways: first, by binding to CXCR2 on endothelial cells, causing retraction of the endothelial cells to increase microvascular permeability (26); second, by attracting CXCR2-expressing neutrophils to the site of infection, where they have the capacity to directly damage the epithelial cells in the lungs in the course of fighting infection (11). Thus, the decrease in CXCL1 observed in Nlrp12−/− animals may be protective to both sides of this critical barrier in the lung, ultimately lessening disease severity.
An important avenue of study will be to determine the mechanism by which Nlrp12 deficiency affects CXCL1 production. A recent study reported that CXCL1, CXCL5, IL-17A, and neutrophil recruitment were decreased in Nlrp12−/− mice during pulmonary Klebsiella pneumoniae infection (15). Signaling through IL-17R leads to stabilization of Cxcl1 and Cxcl5 mRNA (32), which may explain the decrease observed during K. pneumoniae infection. However, IL-17A was not detected in the lungs of WT or Nlrp12−/− mice during IAV infection, thus it is unlikely to be driving the observed decrease in Cxcl1 transcript stability. It is particularly intriguing that we observe a defect in CXCL1, and not other neutrophil chemoattractants or proinflammatory mediators with similar patterns of transcriptional regulation. It is well established that modulation of neutrophil chemoattractant transcript stability is an important determinant of both the duration and magnitude of their expression, and that ARE-mediated instability is a key regulatory mechanism. Despite this generality, it is also clear that the precise regulation of the production of the chemokine reflects not only which gene is being regulated, but also varies according to the cell type in which the gene is being expressed as well as by the specific conditions under which that cell is being stimulated. Differential regulation of TNF-α is illustrative of this mechanistic heterogeneity: Tnf mRNA is stabilized following LPS stimulation of macrophages, resulting in increased expression, but following stimulation of macrophages through TLR7 or TLR9, upregulation is not through modification of transcript stability but rather occurs at the level of translation (33). Given this highly variant regulation and the fact that most studies of Cxcl1 stability in myeloid cells have been performed in LPS-treated macrophages, it is not unexpected that a separate pathway would act to specifically modify Cxcl1 transcript stability in BMDCs in response to R848 stimulation.
The importance of host genetic background to outcome during IAV infection has been demonstrated in a number of studies (34). Large-scale efforts such as the collaborative cross that seeks to mimic the genetic diversity found in the human population are validating the importance of specific genes and discovering new correlates of protection (35). To our knowledge, there has not been a direct comparison of C57BL/6N and C57BL/6J substrains during IAV infection. The data presented in this study indicate that there is a significant reduction in the number of pulmonary neutrophils in the lungs of C57BL/6J mice compared with C57BL/6N mice following IAV infection. In the context of these data and our previous work, we believe this difference in neutrophil recruitment is due to the missense mutation in Nlrp12 in C57BL/6J mice (13, 23), which phenocopies the neutrophil recruitment defect in Nlrp12−/− mice. This substantial phenotypic difference between substrains highlights the importance of ensuring that findings in knockout mice are confirmed using substrain-matched controls or littermates when possible.
In conclusion, the experiments described in this study show that Nlrp12−/− mice are protected from lethality during IAV infection, exhibiting less pulmonary microvascular permeability, CXCL1, and neutrophil recruitment. Modulation of the innate immune response to IAV is being increasingly appreciated as a key early intervention with durable protective effects. Our findings add Nlrp12 to the intracellular PRRs that contribute to early inflammatory responses during IAV infection.
We are grateful to members of the Inflammation Program, Eric Elliott, Bruce Hostager, Nurbek Mambetsariev, Alexis Miller, Diogo Valadares, and Alicia Wallis for valuable discussions and technical assistance. We thank Tamara Mirzapoiazova for generosity with microvascular permeability-related protocols and the University of Iowa Flow Cytometry Facility, a Carver College of Medicine/Holden Comprehensive Cancer Center core research facility, as well as the University of Iowa Comparative Pathology Laboratory, for expertise.
This work was supported by National Institutes of Health grants R01 AI118719 (to F.S.S.), R01 AI104706 (to S.L.C.), and an American Lung Association/American Academy of Allergy, Asthma & Immunology Foundation grant (to S.L.C.). E.E.H. was supported by National Institutes of Health Grant T32 AI007485 (to G.A.B.). This article is based upon work supported in part by facilities and equipment provided by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development.
The online version of this article contains supplemental material.
The authors have no financial conflicts of interest.