NLRP3-Inflammasome Inhibition during Respiratory Virus Infection Abrogates Lung Immunopathology and Long-Term Airway Disease Development

Respiratory syncytial virus (RSV) infects most infants by two years of age. It can cause severe disease leading to an increased risk of developing asthma later in life. Previously, our group has shown that RSV infection in mice and infants promotes IL-1β production. Here, we characterized the role of NLRP3-Inflammasome activation during RSV infection in adult mice and neonates. We observed that the inhibition of NLRP3 activation using the small molecule inhibitor, MCC950, or in genetically modified NLRP3 knockout (Nlrp3−/−) mice during in vivo RSV infection led to decreased lung immunopathology along with a reduced expression of the mucus-associated genes and reduced production of innate cytokines (IL-1β, IL-33 and CCL2) linked to severe RSV disease while leading to significant increases in IFN-β. NLRP3-inflammasome inhibition or deletion diminished Th2 cytokines and inflammatory cell infiltration into the lungs. Furthermore, NLRP3 inhibition or deletion during early-life RSV infection led to reducing viral-exacerbated allergic response in a mouse model of RSV-induced allergy exacerbation. Here, we demonstrated the critical role of NLRP3-inflammasome activation in RSV immunopathology and the related long-term airway alteration. Moreover, these findings suggest the NLRP3-inflammasome as a potential therapeutic target to attenuate severe RSV disease and limit childhood asthma development.


Introduction
RSV infection is the leading cause of childhood hospitalization and increases the risk for developing childhood asthma and recurrent wheezing [1,2]. RSV can skew the immune response away from antiviral and towards a Th2-type response by inhibiting the production of type 1 interferon (IFN) [3]. Severe RSV disease has been linked with a strong Th2 immune response that led to lung pathologies, such as allergy and asthma, by promoting an inflammatory cellular environment [4][5][6].
An important source of Th2 cytokines are the innate lymphoid 2 cells (ILC2). Increased numbers of these cells have been observed during RSV infection and have been related with the development of lung inflammation and mucus production [7,8]. The ILC2 are activated directly by epithelium-associated cytokines IL-25, IL-33 and TSLP [9], which are strong drivers of Th2 immune responses [10]. It has also been shown that IL-1β is a critical activator of ILC2 since it can regulate the expression of epithelial cytokine receptors, promote proliferation and cytokine production [11]. Previously, our group has shown that Viruses 2021, 13, 692 2 of 16 RSV infection in mice and infants promotes IL-1β pathway activation, which leads to the induction of innate cytokines (CCL2, TSLP and IL-33) by the airway epithelium [12] and that the inhibition of the IL-1β pathway with IL-1 receptor antagonist during early-life RSV infection altered RSV immunopathology and diminished subsequent exacerbated allergic disease [13].
The current study characterized the direct role of NLRP3-inflammasome in the development of RSV immunopathology and the long-term alteration in the lung immune environment. Here, we demonstrate that by inhibiting NLRP3-Inflammasome activity by using a specific inhibitor (MCC950) or NLRP3 knockout mice, the RSV immunopathology is abolished as well as subsequent allergic exacerbation following early-life RSV. These studies further suggest the NLRP3-inflammasome as a possible therapeutic target to mitigate RSV immunopathology and its associated long-term airway alterations.

Mice Models
All experiments involving the use of animals were approved by the University of Michigan Care and Use of Animals Committee. Female and male (BALB/c, C57BL/6) mice of 6 to 8 weeks of age were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Nlrp3−/− mice on C57BL/6 background have been reported [22]. For adult infection mice model or for in house breeding. All mice were maintained under standard pathogen-free conditions.

Adult Mice Model
Female BALB/c mice 6-7 weeks old were intratracheally infected with 50 µL/mouse of RSV (2 × 10 5 pfu) [23] Line 19 antigenic subgroup A. The mice were treated intraperitoneally with MCC950 (Cayman Chemicals, Ann Arbor, MI, USA) (20 mg/kg/mouse), or with vehicle (EtOH final concentration 0.5%) every day during the time of infection. On Day 6 post-infection, we collected samples for flow cytometry and bronchoalveolar lavages (BAL) and on Day 8 post-infection, we collected samples for histology and RNA extraction.

Neonatal Model
Female and male neonatal mice from in house breeding colony, BALB/c, C57BL/6, or Nlrp3−/− mice and intranasally infected with 5 µL/mouse of RSV (1 × 10 5 pfu) Line 19 antigenic subgroup A at 7 days of age as described previously [24]. The mice were treated intraperitoneally with MCC950 (20 mg/kg/mouse) or with vehicle (EtOH final concentration 0.5%) every day during the time of infection. On Day 6 post-infection, we

RSV/CRA Model
Neonatal mouse model was performed, followed by CRA allergy model. Cockroach allergen (CRA) (HollisterStier Allergy, Spokane, WA, USA) was administered via intratracheal instillation into the lungs over 3 consecutive days, starting at 4-weeks post-infection, followed by 4 challenges 2 weeks later to elicit an allergic response as previously described [24]. As a control, we have naïve and CRA only treated groups. In brief, mice were sensitized intratracheally with 500 protein nitrogen units (pnu) of CRA, over 3 consecutive days. Next, mice were challenged at Days 14, 20, 22 and 23 with 500 pnu of CRA. Samples were harvested on Day 24.

Respiratory Syncytial Virus
Our laboratory uses antigenic subgroup A, Line 19 RSV, obtained initially from a sick infant at the University of Michigan Hospital System. This isolate has been shown to mimic human infection by eliciting airway mucus production in animal models [23].

Quantitative RT-PCR
Lung tissue was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) to extract RNA cDNA was synthesized using murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA, USA) and incubated at 37 • C for one hour followed by incubation at 95 • C for 5 min to stop the reaction. Real-time quantitative PCR (qPCR) was multiplexed using Taqman primers, with a FAM-conjugated probe and GAPDH with a VIC-conjugated probe (Applied Biosystems, Foster City, CA, USA) to measure Ifnb, Il4, Il5 and Il13 gene expression. Fold change was quantified using the 2 −∆∆ cycle threshold (CT) method. Custom primers were designed to measure RSV F gene, Muc5ac and Gob5 mRNA levels as described [13]. All reactions were run on a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Lung Histology
The left lung was perfused with 4% (v/v) formaldehyde for fixation and embedded in paraffin. Five-micrometer lung sections were stained with periodic acid-Schiff (P.A.S., Middleton, WI, USA) to detect mucus production and inflammatory infiltrates were observed with hematoxylin and eosin stain (H&E, Middleton, WI, USA). Photomicrographs were captured using a Zeiss Axio Imager Z1 and Axio Vision 4.8 software (Zeiss, Munich, Germany).

Enzyme-Linked Immunosorbent Assays
Murine IL-1β and IL-33 proteins were quantified from Bronchial alveolar lavage (BAL) samples taken from naïve or infected mice after euthanasia. For IL-1β and IL-33, R&D Duo set ELISA kit was used (R&D Systems, Minneapolis, MN, USA) per the manufacturer's instructions.

Protein Lung Extraction and Cytokine Quantification
Lung protein was extracted using a tissue cell lysis reagent T-PER (Thermo Fisher, Rockford, IL, USA), following the manufacturer's instructions. The total protein was quantified using Pierce BCA assay kit (Thermo Fisher, Rockford, IL, USA) and the protein levels of IL-4, IL-5, IL-13, IL-17 and CCL2/MCP-1 were measured with a Bio-Plex cytokine assay (Bio-Rad Laboratories, Hercules, California, USA).

Flow Cytometry
The lungs were removed to analyzed leukocytes population. Lung single cells were isolated by enzymatic digestion with 1 mg/mL collagenase A (Roche, Indianapolis, IN,

Statistical Analysis
Data were analyzed by Prism 8 (GraphPad Software, San Diego, CA, USA). Results are expressed as mean ± standard error. Statistical significance was measured by one-way or two-way ANOVA followed by post hoc Student's t-test as appropriate. A p ≤ 0.05 was considered significant

NLRP3-Inflammasome Inhibition Abrogates RSV Immunopathology in Adult Mice
It has been described that RSV infection upregulates IL-1β through the activation of NLRP3-inflammasome [12,13,18]. To characterize the role of NLRP3-inflammasome inhibition, we infected adult BALB/c mice with RSV Line 19 and treated them daily with either an NLRP3 inhibitor (MCC950) or vehicle (0.5% EtOH) ( Figure 1A). The histopathological findings reveal that inhibition of NLRP3-inflammasome during RSV infection using MCC950, a direct inhibitor of the NLRP3 protein [16], strongly reduced lung inflammatory infiltration and decreased mucus deposition in the airways, when compared with infected controls ( Figure 1B). MCC950 treatment correspondingly decreased mucus-related gene expression Gob5 and Muc5ac ( Figure 1C). Significantly decreased expression of mucus inducing Il13 ( Figure 1D) and the Th2 cytokine Il5 ( Figure 1E) with significant upregulation of antiviral Ifnb ( Figure 1F) were also observed. No significant difference was observed in the expression of RSV fusion gene in the lung of the animals infected and treated when compared with infected controls (data not shown). These data suggest that NLRP3-inflammasome inhibition during RSV infection abrogates viral lung immunopathology and promotes innate antiviral responses by increasing type 1 interferon.

RSV Immunopathology Is Dependent on NLRP3-Inflammasome Function
To characterize the role of NLRP3-inflammasome activation in the inflammatory immune response, we evaluated bronchoalveolar lavages (BAL) of adult mice infected with RSV under MCC950 treatment and control. First, we analyzed the production of IL-1β in the BAL of the adult mice and we observed that MCC950 treatment decreased levels of IL-1β in the sample when compared with control group (Figure 2A). Furthermore, NLRP3 inhibition diminished production of IL-33 in the BAL fluid compared with controls ( Figure 2B). Levels of IL-4, IL-13 and CCL2, were significantly decreased in total lung protein extracts ( Figure 2C,E), with no significant differences observed in IL-5 and IL-17 ( Figure 2C,E). Total leukocyte numbers were analyzed to characterize the inflammatory response and we observed significantly reduced total numbers of interstitial macrophages, CD11c+CD11b+ antigen-presenting cells (APC), neutrophils and type 2 innate lymphoid cells (ILC2) in the MCC950 treated mice compared with control RSV infection ( Figure 2F,I) with no difference in T cells ( Figure 2J). These data suggest that NLRP3-inflammasome activity has an important role during RSV immune response, which leads to Th2 immune response and severe disease.

NLRP3-Inflammasome Inhibition Modifies RSV Immunopathology in Neonate Mice
The severity of RSV infection and immunopathology has been linked with different factors. For example, the age at which a child is infected, as preterm babies and infants are more susceptible to developing severe RSV disease [25]. To examine the role of NLRP3inflammasome inhibition on RSV immunopathology, we intranasally infected neonate BALB/c mice at 7 days of age treated with MCC950 or vehicle ( Figure 3A). Examination of histopathology determined that the MCC950 treatment reduced mucus production and goblet cell metaplasia compared with vehicle-treated controls ( Figure 3B) as well as a correlative decrease in mucus gene expression of Gob5 ( Figure 3C). Importantly, a reduction of Th2 cytokine gene expression of Il4, Il5 and Il13 was observed in MCC950-treated mice ( Figure 3D-F). Examination of lung inflammation demonstrated decreased numbers of neutrophils, interstitial macrophages, ILC2 and CD8+ T cells in the RSV+MCC950treated group compared with controls ( Figure 4A,D). Thus, similar to adults, neonatal mice treated with MCC950 during RSV infection presented decreased RSV immunopathology, suggesting the NLRP3-inflammasome plays an important role in the RSV severity during early-life.

Decreased RSV Immunopathology in Nlrp3−/− Neonate Mice
To further investigate the role of NLRP3 inflammasome during neonatal RSV infection, we used Nlrp3−/− neonatal mice and as a control, we used age-matched C57BL/6 mice ( Figure 5A). The histopathological finding showed that Nlrp3−/− neonatal mice presented decreased inflammatory airway infiltration and reduced mucus deposition compared to C57BL/6 infected group ( Figure 5B). Significant downregulation of the expression of Gob5, Il4, Ccl2 and Ifnb was observed in Nlrp3−/− neonatal mice when compared with control mice (Figure 5C-F). Inflammatory lung infiltration was evaluated by flow cytometry and we observed that Nlrp3−/− mice presented significantly decreased numbers of interstitial macrophages, CD11c+CD11b+ APC, neutrophils and CD8+CD69+ T cells ( Figure 6A,D). These data demonstrated the critical role of NLRP3-inflammasome in the RSV immunopathology in neonatal mice.

Decreased RSV Immunopathology in Nlrp3−/− Neonate Mice
To further investigate the role of NLRP3 inflammasome during neonatal RSV infection, we used Nlrp3−/− neonatal mice and as a control, we used age-matched C57BL/6 mice ( Figure 5A). The histopathological finding showed that Nlrp3−/− neonatal mice presented decreased inflammatory airway infiltration and reduced mucus deposition compared to C57BL/6 infected group ( Figure 5B). Significant downregulation of the expression of Gob5, Il4, Ccl2 and Ifnb was observed in Nlrp3−/− neonatal mice when compared with control mice (Figure 5C-F). Inflammatory lung infiltration was evaluated by flow cytometry and we observed that Nlrp3−/− mice presented significantly decreased numbers of interstitial macrophages, CD11c+CD11b+ APC, neutrophils and CD8+CD69+ T cells ( Figure 6A,D). These data demonstrated the critical role of NLRP3-inflammasome in the RSV immunopathology in neonatal mice.

NLRP3-Inflammasome Inhibition during Early-Life RSV Infection Attenuated Secondary Allergic Exacerbation
Severe RSV infection is the most prominent cause of bronchiolitis and childhood hospitalization in infants under six months of age. This group of infants is the most susceptible to developing severe RSV disease and asthma later in life [1,2]. To evaluate whether the NLRP3-inflammasome during early-life impacts long term disease phenotypes, we used neonatal RSV infection followed by the cockroach allergic model ( Figure 7A). Neonatal RSV infected mice treated with MCC950 or vehicle during the infection were sensitized and challenged with cockroach allergen (CRA) at 5 weeks of age. As controls, naïve and CRA only treated age-matched animals were used. Lung histology showed that inhibition of NLRP3 during early life RSV infection (RSV+MCC950/CRA) did not have an exacerbated allergic phenotype compared to the control treated neonatal RSV infected mice (RSV+vehicle/CRA) ( Figure 7B). Lung cytokine gene expression of Il4 and ll-5 was significantly downregulated with a trend towards decreased Il13 in the RSV+MCC950/CRA compared with RSV+vehicle/CRA group ( Figure 7C,E). Inflammatory infiltration in the lung was evaluated and we observed significantly decreased numbers of APC, including CD11c+CD11b+, CD11c+CD103+ and interstitial macrophages in the RSV+MCC950/CRA when compared with RSV+vehicle/CRA ( Figure 7E,G). Altogether, these data indicate that NLRP3-inflammasome has a role during early-life RSV infection that alters the immune response to the secondary lung insult in the adult mice.

NLRP3-Inflammasome Inhibition during Early-Life RSV Infection Attenuated Secondary Allergic Exacerbation
Severe RSV infection is the most prominent cause of bronchiolitis and childhood hospitalization in infants under six months of age. This group of infants is the most susceptible to developing severe RSV disease and asthma later in life [1,2]. To evaluate whether the NLRP3-inflammasome during early-life impacts long term disease phenotypes, we used neonatal RSV infection followed by the cockroach allergic model ( Figure 7A). Neonatal RSV infected mice treated with MCC950 or vehicle during the infection were sensitized and challenged with cockroach allergen (CRA) at 5 weeks of age. As controls, naïve and CRA only treated age-matched animals were used. Lung histology showed that inhibition of NLRP3 during early life RSV infection (RSV+MCC950/CRA) did not have an exacerbated allergic phenotype compared to the control treated neonatal RSV infected mice (RSV+vehicle/CRA) ( Figure 7B). Lung cytokine gene expression of Il4 and ll-5 was significantly downregulated with a trend towards decreased Il13 in the RSV+MCC950/CRA compared with RSV+vehicle/CRA group ( Figure 7C,E). Inflammatory infiltration in the lung was evaluated and we observed significantly decreased numbers of APC, including CD11c+CD11b+, CD11c+CD103+ and interstitial macrophages in the RSV+MCC950/CRA when compared with RSV+vehicle/CRA ( Figure 7E,G). Altogether, these data indicate that NLRP3-inflammasome has a role during early-life RSV infection that alters the immune response to the secondary lung insult in the adult mice.

Discussion
The current studies demonstrate that inhibition of NLRP3-Inflammasome activity by a specific inhibitor (MCC950) or in NLRP3 knockout mice abrogated RSV immunopathology as well as the subsequent allergic airway exacerbation after early-life RSV infection. NLRP3inflammasome is a multiprotein complex, which plays a crucial role in innate immunity that elicits the release of proinflammatory cytokines like IL-1β and IL-18 [15,16]. Increased levels of IL-1β has been linked with the severity of disease in various respiratory virus infections, including RSV [12,13,26], rhinovirus [27][28][29] and SARS-CoV2 [30][31][32]. Since aberrant activation of NLRP3-inflammasome and IL-1 are associated with numerous chronic diseases, including cardiovascular, atherosclerosis and diabetes, the interest in developing or identifying potent and specific NRLP3 inhibitors has been of increased interest [16]. Targeting NLRP3 activation could offer a safer choice to prevent immunosuppressive side effects that can arise by targeting other downstream products of the NLRP3-inflammasome, such as IL-1, IL-18, or IL-33 [16]. Importantly, targeting NLRP3-activation resulted in decreased IL-1β, IL-33 and CCL2 production with significantly increased type 1 IFN-β, suggesting that targeting NLRP3 rebalanced the antiviral immune response decreasing detrimental proinflammatory pathways.
IL-1β and IL-33 belong to the IL-1 family of cytokines and receptors primarily associated with innate immunity [11,33,34]. It has been shown that IL-1β and IL-33 are critical activators of ILC2 that induce proliferation and cytokine production (e.g., IL-5 and IL-13) and also regulate the expression of epithelial cytokine receptors [11]. The inhibition of NLRP3-inflammasome during RSV infection in adult and neonate mice diminished the Th2 cytokine expression, likely related to the activation and proliferation of ILC2, which are a significant source of IL-13 in the lungs [9,35]. These data correlated with the decreased mucus production that has been associated with the production of IL-13 and the development of goblet cell hyperplasia [36]. IL-1β also impacts the airway epithelium immune homeostasis by increasing the production of IL-33 and CCL2 from airway epithelial cells [12,37]. Altogether, these data suggest that NLRP3-inflammasome inhibition impacts the Th2 immune response by modifying the activation and proliferation of ILC2 through the reduction of IL-1β and IL-33 in the airway.
Previously, our group has shown that RSV infection in mice and infants promotes IL-1β pathway activation, which leads to the induction of innate cytokines (CCL2, TSLP and IL-33) by the airway epithelium [12,13]. Our previous studies have indicated that primary cultures of alveolar epithelial cells type II from naïve mice had increased expression of epithelial cell-derived cytokines IL-33, TSLP and CCL2 after in vitro infection with RSV [12]. We have also shown that early-life RSV infection generated persistent expression of IL-33 in alveolar epithelial cells and TSLP within the bronchial airway epithelial cells [24].
Here, we observed decreased production and expression of pulmonary CCL2 where NLRP3-inflammasome was inhibited (MCC950) or genetically abrogated in Nlrp3−/− mice. CCL2 is a potent chemoattractant of myeloid and lymphoid cells and it has been suggested that this chemokine can impact leukocyte phenotype, polarization, effector molecule secretion, autophagy and survival, in a context-dependent manner [38]. Abnormal upregulation of CCL2 results in sustained and exacerbated cell recruitment and uncontrolled inflammation [38], as seen in rheumatoid arthritis, atherosclerosis, diabetes and COVID-19, among others [39][40][41][42][43][44]. When NLRP3 was blocked during RSV infection a downregulation of CCL2 expression and diminished total numbers of antigen-presenting cells (CD11c+CD11b+ and interstitial macrophages) was observed. Thus, NLRP3 activation provides a central and early role for regulating the immunopathology response during RSV infection that may have long term effects.
Clinical studies have supported a link between early-life severe RSV infection and airway disease development later in life, including asthma [1,2,6]. In this work, we observed that inhibition of the NLRP3 inflammasome during early-life RSV infection impacts the subsequent response to a secondary insult (CRA) later in life by decreasing inflammatory lung infiltration, including APC that led to decreased lung Th2 cytokine phenotype (Figure 8). Similar results were observed using interleukin 1 receptor antagonist (IL-1ra) [13], corroborating the importance of this pathway. Multiple early life events in the lung have been associated with NLRP3 activation, including viral and bacterial infections, Bronchopulmonary dysplasia and even septic events, which have been associated with increased susceptibility to later disease [13,[45][46][47]. Perhaps targeting NLRP3 during these early life events may provide a common therapeutic pathway for controlling long term sequelae that are often observed later in these children, including childhood asthma. flammatory lung infiltration, including APC that led to decreased lung Th2 cytokine phenotype ( Figure 8). Similar results were observed using interleukin 1 receptor antagonist (IL-1ra) [13], corroborating the importance of this pathway. Multiple early life events in the lung have been associated with NLRP3 activation, including viral and bacterial infections, Bronchopulmonary dysplasia and even septic events, which have been associated with increased susceptibility to later disease [13,[45][46][47]. Perhaps targeting NLRP3 during these early life events may provide a common therapeutic pathway for controlling long term sequelae that are often observed later in these children, including childhood asthma.

Conflicts of Interest:
The authors declare no conflict of interest.