Lung Dysfunction and Systemic Inflammation: A Role for HO-1 and NLRP3 in a COVID-19 Murine Model

Lung Dysfunction and Systemic Inflammation: A Role for HO-1 and NLRP3 in a COVID-19 Murine Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Lung Dysfunction and Systemic Inflammation: A Role for HO-1 and NLRP3 in a COVID-19 Murine Model Sophia Kwon, Joanna Zhou, Jamie Antelo Rivero, Hailey Bernier, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8844647/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract RATIONALE. The COVID-19 (C19) pandemic caused significant mortality often due to lung injury and systemic inflammation, but there is significant heterogeneity in severity and the pathobiology is not well understood. We examined COVID-19-induced pulmonary and inflammatory sequelae using a murine noninfectious model to further define the models utility and to also understand the role of mediators such as heme oxgenase-1. METHODS. k18-hACE2 male mice oropharyngeally aspirated C19-spike or equal volume control. After 72 hours, we collected: pulmonary mechanics, bronchoalveolar lavage(BAL) and plasma, snap-froze right lung, and fixed/stained left lung for histologic injury assessment(Qupath). Cytokine elaboration in BAL and plasma was quantified(Luminex), and lung homogenates were probed for HO-1 and NLRP3 (Western). Statistical (SPSS and R) and pathways comparisons(Ingenuity Pathway Analysis) were made between control and C19. RESULTS Lung Mechanics. C19 exposure significantly reduced inspiratory capacity and static lung compliance;tissue elastance and airway hyperreactivity were increased. Histology: C19 exposure caused significant inflammation and thickened alveolar septae. Cytokines: C19 exposure led to inflammatory response in BAL and plasma with simultaneous activation of Type 1 and Type 2 pathways. Pathways. NLRP3 and HO-1 protein expression is significantly induced by C19. Regulator networks show involvement of multiple cell lines and lung damage. CONCLUSION: A noninfectious C19 murine model showed worsened lung parameters and increased inflammation. HO-1 and NLRP3 may be key mediators in the inflammatory process and induce both inflammatory and counter-regulatory effects. Further studies will focus on targeted therapeutic pathways that probe into the mechanistic relationship of HO-1 and NLRP3 in C19-related disease. Health sciences/Diseases Biological sciences/Immunology Health sciences/Medical research Health sciences/Pathogenesis COVID-19 Airway hyperreactivity lung injury noninfectious translational model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 BACKGROUND At the end of the public health emergency of the COVID-19 (C19) pandemic in May 2023 by the World Health Organization), there were over 766 million cases worldwide, with 1.13 million deaths in the United States alone. 1 C19 mortality is often attributed to inflammation, severe respiratory failure due to lung injury, catastrophic cascade of immune-mediated inflammation, and metabolic derangement. 2–11 However, the associated morbidity and mortality of C19 is quite heterogeneous. Therefore, further phenotyping and characterizing murine models of C19 induced disease may foster the identification of mechanistic pathways and biologically plausible therapeutic targets that may prevent future pandemics. Our laboratory has studied lung injury due to infectious and exposure-related causes such as particulate matter (PM). 12–18 Murine models exposed to live virus have demonstrated several physiologic sequelae of C19 infection, including acute lung injury, asthma, and hyperinflammatory response. 19–24 However, the high risks of infection and potential for mutation of a zoonotic disease requires rigorous, resource-intensive protocols and advanced BSL3 facilities to limit unintended infections. Moreover, the use of live virus models may restrict the ability to obtain more accurate lung function measurements, as invasive methods are often required instead of whole-body plethysmography. 19,20 Among the emerging virus-free methods of exposure for pre-clinical studies, spike protein has emerged as a key method of inducing acute lung injury in a murine model. 25,26 Spike protein offers the ability to easily modify experiments to account for different strains. Additionally, spike protein induces a multi-organ dysfunction in mouse models that include hyperinflammatory response, acute lung injury, ARDS, and long-term cognitive dysfunction. 25–27 Therefore, this work intends to utilize a non-infectious model to further validate its utility, expand phenotyping, characterize associated pathways and biologically plausible targets. One such target is Heme Oxygenase-1 (HO-1). HO-1 is the inducible isoform of HO and is vital in protecting the lung against oxidative damage. 28 HO-1 expressed in the lung is upregulated upon oxidant-induced lung injury. 28,29 It also has roles to downregulate the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in different models of murine lung inflammation. 30 In response to stress, HO-1 catalyzes the breakdown of heme into iron, carbon monoxide (CO), and biliverdin, which have potent antioxidant, anti-inflammatory, and anti-apoptotic functions. 31–33 However, recent studies have suggested potential worsened outcomes with HO-1, including murine models of pulmonary fibrosis that inhibit HO-1 show attenuated collagen deposition. 34 A pilot study of a PM-exposed cohort with global metabolomic serum profiling showed that HO-1-related metabolites including mesobilirubinogen and L-urobilinin were lower in those with airway hyperreactivity (AHR) and lung injury. 35 We have also shown preliminary work that HO-1 is attenuated in a murine model of high fat diet, and lower levels were associated with worse pulmonary function after particulate matter exposure. 36 How HO-1 and its by-products function in C19 is less clear. HO-1 was associated with poor COVID-19 progress and outcomes. 37,38 C19 increases free heme, driving cytokine storm, and may inhibit HO-1. In contrast, HO-1 genetic polymorphisms are linked to low baseline HO-1 levels and subsequent increased inflammation. 39 Amongst the by-products, free iron can be used for microbial replication processes, which may promote tissue injury and secondary infections. 40 Low-level CO has been used for its vasodilatory and anti-inflammatory properties in the treatment of tissue injury, lung disorders, and ARDS, but may also be a marker of inflammation and cytokine storm. 41 C19 has also been shown to bind to biliverdin and its by-product bilirubin, which helps it evade antibody immunity. 42,43 The mechanistic underpinnings of C19-associated disease and resultant mortality are not well understood, but furthering our understanding is key to developing targets for future interventions. 22,23,25 Proposed mechanisms of triggered inflammatory state include NLRP3, caspase-1, and caspase-8. 44,45 To address a critical gap in the current literature, we explore C19 spike protein exposure’s inflammatory effects on lung function, clinically relevant biomarkers, and perform functional pathway analysis, Fig. 1 . METHODS Murine Model of C19 Spike Exposure . Breeding pairs of heterozygous K18-hACE2 mice (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, Jackson Labs) had free access to standard chow/water and 12-hour light/dark cycles. All mice from each subsequent generation were genotyped by PCR as per vendor instructions and only males≥8 weeks old and ≥20g were used for subsequent experiments (Genotyping Core Laboratory, NYU). N = 30 male mice were utilized for pulmonary and biomarker assessment based on prior studies that showed pulmonary inflammation, and after preliminary studies using N = 5 female with C19 exposure had inconsistent results. 25 Inclusion/exclusion criteria were applied and provided in Supplemental Table 1 , and further explained in Supplemental Methods. All methods were performed in accordance with the relevant guidelines and regulations, including ARRIVE 2.0 guidelines (Animal Research: Reporting in Vivo Experiments; Essential 10) and the NYU IACUC (# 16–00447). 14,46–49 In addition, all mice were euthanized at the end of the experiments by terminal dose of ketamine/xylazine, followed by confirmatory bilateral thoracotomy and exsanguination in accordance to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals. 50 SARS-CoV-2 spike protein (SP; RayBiotech), 400 µg/kg in 2 mL/kg body weight or control buffer in 2 mL/kg body weight total (RayBiotech) was administered via oropharyngeal aspiration as previously described, Fig. 1 . 51–53 This dose has been previously shown to induce acute lung injury similar in appearance to cases of acute C19 in humans 72 hours after exposure in a non-infectious model using male K18-hACE2 mice. 25 Littermates were co-housed and exposed to either control buffer or SP on the same day to avoid batch bias. 72 hours after SP or control exposure, mice received intraperitoneal (ip) anesthesia (0.12 ml/10g bodyweight of a mixture of ketamine (100 mg/ml, Covetrus) and xylazine (10 mg/ ml, Troy Laboratories)), Fig. 1 . Lung Mechanics. N = 24 mice were tracheotomized with an 18g steel cannula for lung function and airway hyperreactivity measurements (methacholine challenge) (Flexivent; Scireq) as previously described. 14 N = 12 mice were excluded for improper Flexivent measurements, but were potentially used for biomarker collection. Sample Collection. Bronchoalveolar lavage (BAL) and plasma, via cardiac puncture, were collected immediately after lung function assessment. Lungs dedicated for histology were infused with 4% paraformaldehyde, fixed (at 25 cm of H 2 O pressure), mounted, electronically scanned, and assessed; (QuPath version 0.6.0). 54 Lungs for protein assessment and/or Evans blue assessment were snap frozen in liquid nitrogen. BAL and plasma were thawed once and assayed (Cytokine/Chemokine Panel 1; Millipore Sigma) for 33 analytes. Immunoblots. A portion of the right lung was lysed in NP-40 lysis buffer and probed for NLRP3 (Abcam AB 263899), MYD88 (Abcam AB2064), Caspase-1(Santa Cruz SC-392736), Caspase 8 (Santa Cuz SC-81656), RAGE (Santa Cruz SC-365154), and HO-1 (Abcam AB-189491) on a capillary based western blot (Wes, ProteinSimple). Each run was probed for beta-actin (Millipore Sigma, A2228) as a protein-loading control. Statistical Analysis. SPSS 28 (IBM, USA), R Studio (Ver 2025.05.0 + 496), R (Ver. 4.5.1), and Graphpad Prism 10.4.1 (Boston, MA, USA) were used for database management and statistics. Continuous data was compared by Student’s t-test or Mann-Whitney U as appropriate. Data from multiplex biomarker assays were first analyzed in semi-supervised hierarchical clustering, and then PCA for data visualization. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to assess upstream regulators, downstream effects, mechanistic and causal networks. 55 Additional details may be found in the Supplemental Methods . RESULTS C19 SP Aspiration induces Acute lung injury and Airway Hyperreactivity . We assessed the degree of C19-associated lung injury utilizing a comprehensive approach examining lung mechanics, proteinaceous leak, and histology. C19 mice (N = 5) had significantly lower inspiratory capacity (IC), static compliance (Cst), and increased tissue elastance (H) 72 hours after exposure compared to controls (N = 7), Fig. 2 A-C. Moreover, C19 exposure induced airway hyperreactivity with lower PC 200 , Fig. 2 D. Decreased Parameter A also affected hysteresis of the pressure-volume loop, compared to controls after 72 hours, Fig. 2 E-F. There was no significant difference in other spirometry metrics including baseline resistance, Supplemental Fig. 1 . C19 mice had an average of 25.50% (SD 14.91) macrophages on BAL cell count differential compared to controls 98.00% (1.79) macrophages, p = 0.01. C19 Induces multilobar inflammation . Mice with C19 exposure displayed more inflammatory changes on H&E stain compared to control mice. Representative images of pulmonary sections of buffer and C19-exposed mice are shown in Fig. 3 A-B respectively. Compared to controls, C19 mice had patchy areas of inflammation, thickened alveolar septae, and inflammatory cell accumulation in the interstitial, intra-alveolar, intrabronchial, and perivascular areas, Fig. 3 A ’-3B’ . There was also associated dense patches of inflammatory cells, collapse of the alveolar space, and focal atelectasis, Fig. 3 A ’’-3B’’ . A trained, blinded investigator (SK) annotated H&E stained sections of whole lung lobe (yellow) and areas of inflammation (green), Fig. 3 C. 54 Detected cells (red) were also identified using a threshold of 0.3 to detect alveolar and inflammatory cells, and compared in areas of inflammation to entire lung lobe, Fig. 3 C ’-C’’ . C19 (n = 3) had average of 57.78% of the lung lobe annotated as inflammation, compared to 0.37% in control mice (n = 3), p = 0.009, and 80.17% inflammatory cells compared to 0.89%, p = 0.002, Fig. 3 D. Ratio of lung to plasma EB was not different in C19 exposure (n = 7, mean 88.46 \(\pm\) SD 49.33) compared to control (n = 7, 63.69 \(\pm\) SD 41.17), p = 0.32. C19 SP exposure yielded a differential BAL and Plasma cytokine biomarker signature and inflammatory profile. In BAL , C19 exposure significantly increased proinflammatory cytokines (Fig. 4 A-D). Growth and differentiation associated factors G-CSF and LIF were increased after exposure to C19, while VEGF was decreased, Fig. 4 E-G. Chemokines of multiple cell lines including eosinophils and neutrophils, such as Eotaxin and KC, were also elevated after C19 exposure, Fig. 4 H-O. There was also activation of adaptive immune associated cytokines, with elevated IFN-γ, IL-4, and IL-9, but decreased IL-2, Fig. 4 P-S. Other assayed analytes including pro-inflammatory cytokine MIP-2 and growth factor GM-CSF, were not significantly different between control and C19 in BAL, Supplemental Fig. 2 . PCA using all measured analytes captured 78.5% of the total variance in 3 components, Fig. 4 T. The heat map shows that VEGF and IL-2 segregated with lower expression in C19 in cluster 1, vs generally higher expression of the other inflammatory analytes in C19 exposure in cluster 2–3, Fig. 4 U. In plasma , there was activation of both chemokines and the adaptive immune response-associated cytokines with IP-10, MIG and IL-13 significantly elevated in C19-exposed mice, Fig. 5 A-C. All other assayed analytes can be seen in Supplemental Fig. 3. PCA captured 68.7% of the variance in 3 components, Fig. 5 D. IL-13 and IP-10 segregated within Cluster 1, while MIG segregated to Cluster 2 in the heatmap, with both clusters associated with upregulation in C19-exposed mice, Fig. 5 E. BAL and plasma analytes were also plotted by fold-change and p-value in volcano plots, Supplemental Fig. 4A and B respectively . In BAL, 17/32 analytes significantly increased in fold-change(Eotaxin, MIP-1α, MIP-1β, RANTES, G-CSF, KC, IL-10, MIG, IFN-g, IL-12(p40), IL-4, IL-6, LIF, MCP-1, and TNF-α) and decreased in IL-2 and VEGF, Supplemental Fig. 4A . In plasma, MIG was identified as significantly upregulated (p < 0.05) Supplemental Fig. 4B . Multiple Inflammatory Pathways are Activated in C19 Exposure Transformed BAL and p-value data was input into IPA, and n = 14/192 significant canonical pathways that met threshold with p-value 2, Fig. 6 A. Multiple inflammatory pathways were activated including macrophage, T-cell response (pattern recognition receptors), and natural killer cells. The top significant identified canonical pathway, Macrophage Classical Activation Signaling Pathway is shown, Supplemental Fig. 5 . NRLP3 and HO-1 were significantly induced in the C19 spike protein exposures compared to control, Fig. 6 B. Caspase-1, Caspase-8, RAGE, and MYD-88 were not significantly different. Full Western images are provided in Supplemental Fig. 6–9 . Additionally, a regulators effects analysis was performed in IPA, and the analysis that involved HMOX1, the gene that encodes HO-1 with the highest consistency score, a measurement used to help rank the most highly connected and consistent networks from regulator to function, is presented, Fig. 6 C. Regulator networks showed recruitment of multiple cell lines including leukocytes, CD4 + T-lymphocytes, phagocytes, and macrophages, as well as damage of lung. Upstream regulators that were significantly activated or inhibited (|z-score|>2) are also displayed for BAL and plasma, Supplemental Fig. 10 . TreeMaps of disease and functions in BAL showed marked increased activity in multiple inflammatory pathway activation and cell recruitment pathways, Supplemental Fig. 11 . In plasma, the significance threshold was not met to fully develop a TreeMap. Full pathways analysis are available at: IPA_BAL and IPA Plasma. DISCUSSION C19 overwhelmed hospitals globally with high numbers of patients with multiorgan failure, often with cytokine release syndrome, and dysregulation of the immune system. 56 Future pandemics remain an ongoing threat, and it is critical to understand the mechanistic underpinnings of C19 to identify potential targets of future therapy. Our work contributes to the C19 literature by clarifying knowledge gaps validating a non-infectious murine body, quantifying lung function changes and end-organ dysfunction. Moreover, we have identified potential therapeutic targets by showing upregulation of both HO-1 and NRLP3. These targets are supported by our findings in IPA, showing the involvement of mediators central to HO-1 and NLRP3 activation. This noninfectious transitional model recapitulated many of the findings found using noninfectious models of COVID, and significantly overlaps many of the phenotypes seen in live-virus models. 25,57 Lung function decline after COVID infection was characterized by reduction in IC, compliance, volume, and hysteresis. 19 Our model differed by additionally showing airway hyperreactivity. Further, despite significant involvement of lungs after C19 exposure, there was no alveolar leak or an ARDS phenotype. Exposure to C19 induced a simultaneous elevation of several inflammatory and counterregulatory pathways in BAL, affecting Th1 (TNF- \(\alpha\) , IL-6, IFN- \(\gamma\) , IL-12(p40), IL-2, IP-10, MIG), Th2-associated response (IL-4, IL-9, Eotaxin), and Th17 associated mucosal response (IL-17, KC, G-CSF). This could indicate a severe, overactive inflammation. The suppression of IL-2 and VEGF could indicate T-cell exhaustion and failed tissue repair. 58,59 In plasma, although there were less significantly different inflammatory markers, there were similar patterns of mixed and often opposing immune signal from Th2 (IL-13), and Th1/interferon driven signaling (IP-10 and MIG) elevation after C19 exposure. There is similarity to cytokine release syndrome or cytokine storm seen in humans with C19, specifically in the elevation of IL-6, TNF-α, and IFN-γ. 60 IL-1, which was also seen as critical to the cytokine release in humans, was measured as different isoforms in the murine model. Of those, IL-1β trended higher in C19-exposure. The multiple and sometimes counter-regulatory pathway involvement may also be contributory to the pathogen-induced cytokine storm signaling pathway initially being identified as the top related canonical pathway in IPA, but it was not significant by z-score. We demonstrated that exposure to C19 spike protein can induce NRLP3 and HO-1, which may trigger downstream activation of inflammatory complexes. HO-1 is considered cytoprotective with antioxidant, anti-inflammatory, and antiviral properties reflective of activation of M2 macrophages. 37,61–63 However, its upregulation has been associated with poor outcomes in C19-infected patients, specifically worse hypoxia and mortality. 37,61,64 NLRP3 inflammasome activation in severe viral disease, such as in C19 and influenza, has also been thought to be at the center of immune-mediated dysregulation. 65–67 It plays both pro-inflammatory activation roles and anti-viral response, but excessive stimulation has been linked to cytokine storm and multi-organ failure. 67 In the lung, HO-1 activation is often seen to protect against the NLRP3 inflammasome via counterregulatory action in disease states such as cigarette-smoking induced COPD or LPS/sepsis-induced lung injury. 68–71 Simultaneous activation of HO-1 and NLRP3 has been seen in chronic inflammatory disease such as osteoarthritis. 72 The simultaneous activation of NLRP3 and HO-1 may indicate that there is activation of both inflammatory and counter-regulatory pathways, and point to possible chronicity with ongoing inflammation. This also concurs with the quantified cytokine profile of the murine model that showed upregulation of multiple immunoregulatory pathways. The involvement of HO-1 and NLRP3 is also evident from the pathway and regulator analysis. Macrophage classical activation signaling pathway was the most significant canonical pathway. Heme was also the most significant inhibited upstream regulator in BAL. This could be related to macrophage activation by heme breakdown in the HO-1 pathway. Heme is also an important NLRP3 activator, and its breakdown may be early counterregulatory mechanism action against NLRP3. 73 Further studies are needed to identify the complex mechanisms between HO-1 and NLRP3 activation in viral disease. This study has several limitations. Male mice were used exclusively because preliminary studies using female mice had inconsistent data. One possible reason for this may be because male mice weighed approximately 30% more than the female mice, and received a higher overall dose of SP. Another reason may be because female mice exhibit hormones that influence the pulmonary inflammation profile differently from male mice. Future studies could investigate the use of a fixed, higher dose of SP to observe if there are any sex-related effects. Our model differed in some aspects of lung dysfunction compared to both live and noninfectious models. There could be differences in live virus used compared to the noninfectious S1 spike protein. 19 Our model may also need a longer time interval to show increased resistance as the pulmonary disease progresses, or a higher dose to induce more profound pulmonary changes. We also could not recapitulate vascular permeability measures of alveolar leak through either Evans Blue or electric cell-substrate impedance measurements. 25,26 This could be due to small differences in technique of aspiration or injection of EB. It is also possible that the inflammatory profile would change over time, and that 72 hours may not represent the optimal time for peak inflammation. 57 Different doses and time intervals may have also shown different inflammatory profiles in not only the BAL, but also more peripherally in the plasma to represent more systemic inflammation. Moreover, it is possible that since this study did not use live virus, the inflammatory effects seen may not be generalizable to other strains of COVID or live virus studies. Nevertheless, this study represents an important step in studying C19-related inflammation using the isolated spike protein. The non-infectious model allowed us to measure the pulmonary changes that occur after exposure within a BSL-2 facility. There are also several aspects of the study that show moderate translatability of using a non-infectious model. Future Plans This work adds to the growing body of literature aimed at assessing reproducibility and translatability of noninfectious models of C19. Further, it guides future studies using noninfectious models of C19 to investigate inflammatory response and lung injury. Future plans include loss and gain of function studies to identify NLRP3 and HO-1 as potential targets to mitigate inflammatory response in C19. Additional time-course and dose studies may also be studied for future studies to help profile the inflammatory progression. Noninfectious aspiration models of additional environmentally relevant irritants also may lay the foundation for future experiments investigating possible synergistic inflammation from other concurrent exposures. Abbreviations AHR Airway Hyperreactivity ALI Acute Lung Injury ARDS Acute Respiratory Distress Syndrome C19 COVID-19 IACUC Institutional Animal Care and Use Committee IL Interleukin IP Interferon gamma inducible protein IRB Internal Review Board N Number NIH National Institutes of Health NYU New York University OAD Obstructive Airways Disease SP Spike protein TNF Tumor Necrosis Factor Abbreviations related to Flexivent Parameters Parameter A relates to hysteresis of PV loop Parameter K relates to hysteresis of PV loop Crs Compliance of the Respiratory System Cst Static compliance Ers Elastance FEV 0.1 Forced expiratory volume in 0.1 second G Tissue Damping H Tissue Elastance IC Inspiratory Capacity NPFE negative pressure-driven forced expiratory maneuver PC 200 Dose of methacholine that doubles baseline resistance Rrs Resistance of the Respiratory System Rn Newtonian Resistance Declarations Conflict of Inte rest Statement: The authors have no conflicts to declare. Ethics approval and consent to participate. This study was approved by the NYU IACUC # 16-00447 Consent for publication. Not applicable Availability of data and materials. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests. The authors declare that they have no competing interests. Funding. This work was supported by UL1TR001445 NCATS; KL2TR001446 (SK); Stony Wold-Herbert Fund (SK) and CDC/NIOSH U01- OH11855; -OH11300 and -OH01269 (AN). Authors’ contributions. AN was the primary investigator, had full access to all the data in the study and takes responsibility for the integrity and the accuracy of the data analysis. SK and AN participated in study conception and design; SK, AN, HB, JAR and JZ were responsible for data collection; SK and AN were responsible for data validation; SK, JZ, HB, JAR, GG and AN participated in data analysis; SK, JZ, HB, SP and AN undertook the statistical analysis. 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Ribatti, D. Immunosuppressive effects of vascular endothelial growth factor. Oncol Lett 24 , 369 (2022). Chen, P. , et al. Potential Pathophysiological Mechanisms Underlying Multiple Organ Dysfunction in Cytokine Release Syndrome. Mediators Inflamm 2022 , 7137900 (2022). Hara, Y. , et al. Heme oxygenase-1 as an important predictor of the severity of COVID-19. PLoS One 17 , e0273500 (2022). Singh, D., Wasan, H. & Reeta, K.H. Heme oxygenase-1 modulation: A potential therapeutic target for COVID-19 and associated complications. Free Radic Biol Med 161 , 263-271 (2020). Batra, N., De Souza, C., Batra, J., Raetz, A.G. & Yu, A.M. The HMOX1 Pathway as a Promising Target for the Treatment and Prevention of SARS-CoV-2 of 2019 (COVID-19). Int J Mol Sci 21 (2020). Su, W.L. , et al. Desaturation and heme elevation during COVID-19 infection: A potential prognostic factor of heme oxygenase-1. J Microbiol Immunol Infect 54 , 113-116 (2021). Yin, M., Marrone, L., Peace, C.G. & O'Neill, L.A.J. NLRP3, the inflammasome and COVID-19 infection. QJM 116 , 502-507 (2023). van den Berg, D.F. & Te Velde, A.A. Severe COVID-19: NLRP3 Inflammasome Dysregulated. Front Immunol 11 , 1580 (2020). Zhao, N., Di, B. & Xu, L.L. The NLRP3 inflammasome and COVID-19: Activation, pathogenesis and therapeutic strategies. Cytokine Growth Factor Rev 61 , 2-15 (2021). Zhang, Y., Wang, J., Wang, Y. & Lei, K. Nrf2/HO-1 signaling activation alleviates cigarette smoke-induced inflammation in chronic obstructive pulmonary disease by suppressing NLRP3-mediated pyroptosis. J Cardiothorac Surg 19 , 58 (2024). Luo, M., Hong, X.Q., Zhu, H., Li, G. & Tang, L. The HO-1 Signal Prevents HMGB1-Mediated Activation of NLRP3 Inflammasomes in Lipopolysaccharide-Induced Acute Lung Injury In Vitro. J Surg Res 247 , 335-343 (2020). Shutong, L. , et al. HO-1/autophagic flux axis alleviated sepsis-induced acute lung injury via inhibiting NLRP3 inflammasome. Cell Signal 100 , 110473 (2022). Huang, Y., Wang, A., Jin, S., Liu, F. & Xu, F. Activation of the NLRP3 inflammasome by HMGB1 through inhibition of the Nrf2/HO-1 pathway promotes bleomycin-induced pulmonary fibrosis after acute lung injury in rats. Allergol Immunopathol (Madr) 51 , 56-67 (2023). Chen, Z. , et al. Inhibition of Nrf2/HO-1 signaling leads to increased activation of the NLRP3 inflammasome in osteoarthritis. Arthritis Res Ther 21 , 300 (2019). Salgar, S., Bolivar, B.E., Flanagan, J.M., Anum, S.J. & Bouchier-Hayes, L. The NLRP3 inflammasome fires up heme-induced inflammation in hemolytic conditions. Transl Res 252 , 34-44 (2023). Additional Declarations No competing interests reported. Supplementary Files 03Bsupplementalfig012725.pdf 03ASupplementalMethods.docx SupplementalTable1.docx SupplementalFiguresLegends.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 08 Apr, 2026 Reviews received at journal 01 Apr, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers invited by journal 12 Mar, 2026 Editor assigned by journal 12 Mar, 2026 Editor invited by journal 03 Mar, 2026 Submission checks completed at journal 27 Feb, 2026 First submitted to journal 27 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8844647","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":607153692,"identity":"a9122941-ffe8-4e64-842d-2add9063eab3","order_by":0,"name":"Sophia Kwon","email":"","orcid":"","institution":"New York University","correspondingAuthor":false,"prefix":"","firstName":"Sophia","middleName":"","lastName":"Kwon","suffix":""},{"id":607153694,"identity":"1e7c52d0-71b7-43a7-b0d0-b7bcdb0a2810","order_by":1,"name":"Joanna Zhou","email":"","orcid":"","institution":"New York 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19:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8844647/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8844647/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104835556,"identity":"cb295b35-9d1d-4864-a774-5a6ee16fafa6","added_by":"auto","created_at":"2026-03-17 17:45:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":401120,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of Murine C19 SP Non-Infectious Model Timeline and Analysis Pipeline\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"01Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/30df1287bd44cbb53d811cc3.png"},{"id":104806392,"identity":"d54b70b3-8f5f-4a44-b501-1e44a943690d","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":109998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLung Mechanics Assessment in C19 Spike Exposed Mice.\u003c/strong\u003e \u003cstrong\u003eA. \u003c/strong\u003eInspiratory capacity (IC) \u003cstrong\u003eB.\u003c/strong\u003e Static compliance (Cst) \u003cstrong\u003eC.\u003c/strong\u003e Tissue Elastance (H) \u003cstrong\u003eD.\u003c/strong\u003e PC\u003csub\u003e200 \u003c/sub\u003eshowing more hyperreactivity after C19 exposure \u003cstrong\u003eE. \u003c/strong\u003eParameter A relates to hysteresis of PV loop \u003cstrong\u003eD.\u003c/strong\u003e Pooled average Pressure Volume Curve of Control and C19, with significant differences in pressure at all points except baseline, N5 for each group.\u003c/p\u003e","description":"","filename":"01Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/537e277e0cf9957ab7321c32.png"},{"id":104806400,"identity":"42f8887f-5f16-4b06-9571-e0e234980c6e","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":803670,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistology and Measures of Lung Injury Post C19 Exposure. \u003c/strong\u003eRepresentative histologic section of lungs of\u003cstrong\u003e \u003c/strong\u003ewhole lung lobe with area of terminal airway at 1x further examined indicated by a circle for \u003cstrong\u003eA.\u003c/strong\u003e Control and \u003cstrong\u003eB.\u003c/strong\u003e C19 exposure. \u003cstrong\u003eA’. \u003c/strong\u003eTerminal airway of representative control at 10x and \u003cstrong\u003eA’’. \u003c/strong\u003e40x with clear interstitial space (1), intra-alveolar space (2), peri/intrabronchial space (3), and perivascular area (4)\u003cstrong\u003e, \u003c/strong\u003ecompared to \u003cstrong\u003eB’.\u003c/strong\u003e C19 exposed mice with evidence of focal inflammation visible at 10x and \u003cstrong\u003eB’’. \u003c/strong\u003e40x. \u003cstrong\u003eC. \u003c/strong\u003eRepresentative image of lung section analyzed on Qupath with entire lung highlighted in yellow and compared to areas of inflammation highlighted in green. \u003cstrong\u003eC’. \u003c/strong\u003eTerminal airway at 10x and \u003cstrong\u003eC’’\u003c/strong\u003e. 40x with each identified cell and its nucleus outlined in red. Cells found in tissue and sequestered in the bronchoalveolar space are included in the final cell count. \u003cstrong\u003eD.\u003c/strong\u003e Table of histology parameters quantified by morphometry using Qupath of N=3 control vs N=3 C19 mice.\u003c/p\u003e","description":"","filename":"01Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/4904b3f1c82a6a67e5320501.png"},{"id":104806396,"identity":"12b56a90-34f8-453b-b4ed-5387a877b737","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":210380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBAL Biomarker Assessment. \u003c/strong\u003eC19 exposed mice had significantly higher expression of pro-inflammatory associated cytokines \u003cstrong\u003eA.\u003c/strong\u003e IL-6, \u003cstrong\u003eB. IL-12(p40) C. IL-17, and D. TNF-a. \u003c/strong\u003eGrowth and Differentiation factors E. GCSF, and F. LIF, were increased after exposure, while G. VEGF decreased. Chemokines H. Eotaxin, I. MCP-1, J. KC, K. IP-10, L. MIG, M. MIP-1a, N. MIP-1b, O. RANTES were also increased. P. IFN-g was also increased, while Q. IL-2 was decreased. R. IL-4 and S. IL-9 increased after C19 exposure. \u003cstrong\u003eT\u003c/strong\u003e. \u003cstrong\u003ePCA\u003c/strong\u003e captured 78.5% of the total variance. \u003cstrong\u003eU. BAL Biomarker Heatmap \u003c/strong\u003eshows 3 main clusters showing different patterns of upregulation of many cytokines / chemokines in cluster 2 and 3, and downregulation of IL-2 and VEGF. IL-3, IL-7, IL-13, and LIX are excluded from the heatmap because all values were at minimum detectable concentration, N \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;5 for each group.\u003c/p\u003e","description":"","filename":"01Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/23df4efd2b0d52e6ed38692e.png"},{"id":104808697,"identity":"39a84a86-f598-4134-ba9e-7d066120882e","added_by":"auto","created_at":"2026-03-17 12:39:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":118836,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma Biomarker Assessment. \u003c/strong\u003eC19 exposed mice had significantly higher expression of \u003cstrong\u003eA.\u003c/strong\u003e IP-10 \u003cstrong\u003eB.\u003c/strong\u003e MIG and \u003cstrong\u003eC.\u003c/strong\u003e IL-13 \u003cstrong\u003eD. PCA \u003c/strong\u003e68.7% of the variance was captured in 3 components. \u003cstrong\u003eE. Heatmap and Clustering \u003c/strong\u003eshows 3 clusters, with upregulation in C19 in cluster 1, equivalence in cluster 2, and downregulation in cluster 3.\u003c/p\u003e","description":"","filename":"01Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/74c5e2c184a71fe2225502bf.png"},{"id":104806398,"identity":"a645964e-6021-40c6-ad1f-cdcc158e0521","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":528854,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathways Assessment of the C19 affected Lung. A. Significant BAL Canonical Pathways \u003c/strong\u003e(p-value threshold 0.05).\u003cstrong\u003e \u003c/strong\u003eBars show top 14 activated (orange) and inhibited (blue) significant canonical pathways ranked by -log(p-value), with z-score \u0026gt;2 in white. \u0026nbsp;Line (black) shows ratio of analytes in pathway\u003cstrong\u003e B. Immunoblots. \u003c/strong\u003eControl (Lanes 1-3) compared to C19 exposed (Lanes 4-6), probed for NLRP3, Caspase-1, HO-1, RAGE, MYD-88, Caspase-8, and b-actin housekeeping gene. Blots have been cropped for clarity and conciseness. Contrast has been optimized for visibility and has been equally applied for controls and C19 exposure for each target protein. \u0026nbsp;\u003cstrong\u003eC. Regulator Effects Analysis \u003c/strong\u003ewith Highest Consistency Score\u003c/p\u003e","description":"","filename":"01Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/e495e88376ae917e08cb81be.png"},{"id":104836095,"identity":"e160a0b5-c0a8-4209-9a72-3eb5eba38037","added_by":"auto","created_at":"2026-03-17 17:51:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3249450,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/d6def484-0226-449f-a745-e9ea567005b3.pdf"},{"id":104808700,"identity":"894ec5bc-b3ea-48de-8416-bf11e9219087","added_by":"auto","created_at":"2026-03-17 12:39:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1537320,"visible":true,"origin":"","legend":"","description":"","filename":"03Bsupplementalfig012725.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/aa9f64f11eaea96e7f287cc8.pdf"},{"id":104806394,"identity":"ceb13979-ce51-4ae4-9614-49ed31135558","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":37997,"visible":true,"origin":"","legend":"","description":"","filename":"03ASupplementalMethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/1bb43f5c3d777c6b718e3042.docx"},{"id":104808632,"identity":"73edd171-88cd-43e5-8742-67fd15ff34ac","added_by":"auto","created_at":"2026-03-17 12:39:08","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":369043,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/3f8d2db587e6df84df0600ec.docx"},{"id":104806393,"identity":"2e207970-06d4-4791-817c-1b85afd164f4","added_by":"auto","created_at":"2026-03-17 11:49:07","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":15869,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFiguresLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-8844647/v1/0f3c0e9f036c30cba66731bf.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lung Dysfunction and Systemic Inflammation: A Role for HO-1 and NLRP3 in a COVID-19 Murine Model","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eAt the end of the public health emergency of the COVID-19 (C19) pandemic in May 2023 by the World Health Organization), there were over 766\u0026nbsp;million cases worldwide, with 1.13\u0026nbsp;million deaths in the United States alone.\u003csup\u003e1\u003c/sup\u003e C19 mortality is often attributed to inflammation, severe respiratory failure due to lung injury, catastrophic cascade of immune-mediated inflammation, and metabolic derangement.\u003csup\u003e2\u0026ndash;11\u003c/sup\u003e However, the associated morbidity and mortality of C19 is quite heterogeneous. Therefore, further phenotyping and characterizing murine models of C19 induced disease may foster the identification of mechanistic pathways and biologically plausible therapeutic targets that may prevent future pandemics.\u003c/p\u003e \u003cp\u003eOur laboratory has studied lung injury due to infectious and exposure-related causes such as particulate matter (PM).\u003csup\u003e12\u0026ndash;18\u003c/sup\u003e Murine models exposed to live virus have demonstrated several physiologic sequelae of C19 infection, including acute lung injury, asthma, and hyperinflammatory response.\u003csup\u003e19\u0026ndash;24\u003c/sup\u003e However, the high risks of infection and potential for mutation of a zoonotic disease requires rigorous, resource-intensive protocols and advanced BSL3 facilities to limit unintended infections. Moreover, the use of live virus models may restrict the ability to obtain more accurate lung function measurements, as invasive methods are often required instead of whole-body plethysmography.\u003csup\u003e19,20\u003c/sup\u003e Among the emerging virus-free methods of exposure for pre-clinical studies, spike protein has emerged as a key method of inducing acute lung injury in a murine model.\u003csup\u003e25,26\u003c/sup\u003e Spike protein offers the ability to easily modify experiments to account for different strains. Additionally, spike protein induces a multi-organ dysfunction in mouse models that include hyperinflammatory response, acute lung injury, ARDS, and long-term cognitive dysfunction.\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e Therefore, this work intends to utilize a non-infectious model to further validate its utility, expand phenotyping, characterize associated pathways and biologically plausible targets.\u003c/p\u003e \u003cp\u003eOne such target is Heme Oxygenase-1 (HO-1). HO-1 is the inducible isoform of HO and is vital in protecting the lung against oxidative damage.\u003csup\u003e28\u003c/sup\u003e HO-1 expressed in the lung is upregulated upon oxidant-induced lung injury.\u003csup\u003e28,29\u003c/sup\u003e It also has roles to downregulate the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in different models of murine lung inflammation.\u003csup\u003e30\u003c/sup\u003e In response to stress, HO-1 catalyzes the breakdown of heme into iron, carbon monoxide (CO), and biliverdin, which have potent antioxidant, anti-inflammatory, and anti-apoptotic functions.\u003csup\u003e31\u0026ndash;33\u003c/sup\u003e However, recent studies have suggested potential worsened outcomes with HO-1, including murine models of pulmonary fibrosis that inhibit HO-1 show attenuated collagen deposition.\u003csup\u003e34\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA pilot study of a PM-exposed cohort with global metabolomic serum profiling showed that HO-1-related metabolites including mesobilirubinogen and L-urobilinin were lower in those with airway hyperreactivity (AHR) and lung injury.\u003csup\u003e35\u003c/sup\u003e We have also shown preliminary work that HO-1 is attenuated in a murine model of high fat diet, and lower levels were associated with worse pulmonary function after particulate matter exposure.\u003csup\u003e36\u003c/sup\u003e How HO-1 and its by-products function in C19 is less clear. HO-1 was associated with poor COVID-19 progress and outcomes.\u003csup\u003e37,38\u003c/sup\u003e C19 increases free heme, driving cytokine storm, and may inhibit HO-1. In contrast, HO-1 genetic polymorphisms are linked to low baseline HO-1 levels and subsequent increased inflammation.\u003csup\u003e39\u003c/sup\u003e Amongst the by-products, free iron can be used for microbial replication processes, which may promote tissue injury and secondary infections.\u003csup\u003e40\u003c/sup\u003e Low-level CO has been used for its vasodilatory and anti-inflammatory properties in the treatment of tissue injury, lung disorders, and ARDS, but may also be a marker of inflammation and cytokine storm.\u003csup\u003e41\u003c/sup\u003e C19 has also been shown to bind to biliverdin and its by-product bilirubin, which helps it evade antibody immunity.\u003csup\u003e42,43\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe mechanistic underpinnings of C19-associated disease and resultant mortality are not well understood, but furthering our understanding is key to developing targets for future interventions.\u003csup\u003e22,23,25\u003c/sup\u003e Proposed mechanisms of triggered inflammatory state include NLRP3, caspase-1, and caspase-8.\u003csup\u003e44,45\u003c/sup\u003e To address a critical gap in the current literature, we explore C19 spike protein exposure\u0026rsquo;s inflammatory effects on lung function, clinically relevant biomarkers, and perform functional pathway analysis, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cb\u003eMurine Model of C19 Spike Exposure\u003c/b\u003e. Breeding pairs of heterozygous \u003cem\u003eK18-hACE2\u003c/em\u003e mice (Strain B6.Cg-Tg(K18-ACE2)2Prlmn/J, Jackson Labs) had free access to standard chow/water and 12-hour light/dark cycles. All mice from each subsequent generation were genotyped by PCR as per vendor instructions and only males\u0026ge;8 weeks old and \u0026ge;20g were used for subsequent experiments (Genotyping Core Laboratory, NYU). N\u0026thinsp;=\u0026thinsp;30 male mice were utilized for pulmonary and biomarker assessment based on prior studies that showed pulmonary inflammation, and after preliminary studies using N\u0026thinsp;=\u0026thinsp;5 female with C19 exposure had inconsistent results.\u003csup\u003e25\u003c/sup\u003e Inclusion/exclusion criteria were applied and provided in \u003cb\u003eSupplemental Table\u0026nbsp;1\u003c/b\u003e, and further explained in \u003cb\u003eSupplemental Methods.\u003c/b\u003e All methods were performed in accordance with the relevant guidelines and regulations, including ARRIVE 2.0 guidelines (Animal Research: Reporting in Vivo Experiments; Essential 10) and the NYU IACUC (# 16\u0026ndash;00447).\u003csup\u003e14,46\u0026ndash;49\u003c/sup\u003e In addition, all mice were euthanized at the end of the experiments by terminal dose of ketamine/xylazine, followed by confirmatory bilateral thoracotomy and exsanguination in accordance to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.\u003csup\u003e50\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSARS-CoV-2 spike protein (SP; RayBiotech), 400 \u0026micro;g/kg in 2 mL/kg body weight or control buffer in 2 mL/kg body weight total (RayBiotech) was administered via oropharyngeal aspiration as previously described, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003csup\u003e51\u0026ndash;53\u003c/sup\u003e This dose has been previously shown to induce acute lung injury similar in appearance to cases of acute C19 in humans 72 hours after exposure in a non-infectious model using male \u003cem\u003eK18-hACE2\u003c/em\u003e mice.\u003csup\u003e25\u003c/sup\u003e Littermates were co-housed and exposed to either control buffer or SP on the same day to avoid batch bias. 72 hours after SP or control exposure, mice received intraperitoneal (ip) anesthesia (0.12 ml/10g bodyweight of a mixture of ketamine (100 mg/ml, Covetrus) and xylazine (10 mg/ ml, Troy Laboratories)), Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLung Mechanics.\u003c/b\u003e N\u0026thinsp;=\u0026thinsp;24 mice were tracheotomized with an 18g steel cannula for lung function and airway hyperreactivity measurements (methacholine challenge) (Flexivent; Scireq) as previously described.\u003csup\u003e14\u003c/sup\u003e N\u0026thinsp;=\u0026thinsp;12 mice were excluded for improper Flexivent measurements, but were potentially used for biomarker collection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSample Collection.\u003c/b\u003e Bronchoalveolar lavage (BAL) and plasma, via cardiac puncture, were collected immediately after lung function assessment. Lungs dedicated for histology were infused with 4% paraformaldehyde, fixed (at 25 cm of H\u003csub\u003e2\u003c/sub\u003eO pressure), mounted, electronically scanned, and assessed; (QuPath version 0.6.0).\u003csup\u003e54\u003c/sup\u003e Lungs for protein assessment and/or Evans blue assessment were snap frozen in liquid nitrogen. BAL and plasma were thawed once and assayed (Cytokine/Chemokine Panel 1; Millipore Sigma) for 33 analytes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImmunoblots.\u003c/b\u003e A portion of the right lung was lysed in NP-40 lysis buffer and probed for NLRP3 (Abcam AB 263899), MYD88 (Abcam AB2064), Caspase-1(Santa Cruz SC-392736), Caspase 8 (Santa Cuz SC-81656), RAGE (Santa Cruz SC-365154), and HO-1 (Abcam AB-189491) on a capillary based western blot (Wes, ProteinSimple). Each run was probed for beta-actin (Millipore Sigma, A2228) as a protein-loading control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis.\u003c/b\u003e SPSS 28 (IBM, USA), R Studio (Ver 2025.05.0\u0026thinsp;+\u0026thinsp;496), R (Ver. 4.5.1), and Graphpad Prism 10.4.1 (Boston, MA, USA) were used for database management and statistics. Continuous data was compared by Student\u0026rsquo;s t-test or Mann-Whitney U as appropriate. Data from multiplex biomarker assays were first analyzed in semi-supervised hierarchical clustering, and then PCA for data visualization. \u003cb\u003eIngenuity Pathway Analysis (IPA) (Qiagen)\u003c/b\u003e was used to assess upstream regulators, downstream effects, mechanistic and causal networks.\u003csup\u003e55\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eAdditional details may be found in the Supplemental Methods\u003c/em\u003e.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eC19 SP Aspiration induces Acute lung injury and Airway Hyperreactivity\u003c/b\u003e. We assessed the degree of C19-associated lung injury utilizing a comprehensive approach examining lung mechanics, proteinaceous leak, and histology. C19 mice (N\u0026thinsp;=\u0026thinsp;5) had significantly lower inspiratory capacity (IC), static compliance (Cst), and increased tissue elastance (H) 72 hours after exposure compared to controls (N\u0026thinsp;=\u0026thinsp;7), Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C. Moreover, C19 exposure induced airway hyperreactivity with lower PC\u003csub\u003e200\u003c/sub\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD. Decreased Parameter A also affected hysteresis of the pressure-volume loop, compared to controls after 72 hours, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F. There was no significant difference in other spirometry metrics including baseline resistance, \u003cb\u003eSupplemental Fig.\u0026nbsp;1\u003c/b\u003e. C19 mice had an average of 25.50% (SD 14.91) macrophages on BAL cell count differential compared to controls 98.00% (1.79) macrophages, p\u0026thinsp;=\u0026thinsp;0.01.\u003c/p\u003e \u003cp\u003e \u003cb\u003eC19 Induces multilobar inflammation\u003c/b\u003e. Mice with C19 exposure displayed more inflammatory changes on H\u0026amp;E stain compared to control mice. Representative images of pulmonary sections of buffer and C19-exposed mice are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B respectively. Compared to controls, C19 mice had patchy areas of inflammation, thickened alveolar septae, and inflammatory cell accumulation in the interstitial, intra-alveolar, intrabronchial, and perivascular areas, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e\u0026rsquo;-3B\u0026rsquo;\u003c/b\u003e. There was also associated dense patches of inflammatory cells, collapse of the alveolar space, and focal atelectasis, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA\u003cb\u003e\u0026rsquo;\u0026rsquo;-3B\u0026rsquo;\u0026rsquo;\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eA trained, blinded investigator (SK) annotated H\u0026amp;E stained sections of whole lung lobe (yellow) and areas of inflammation (green), Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC.\u003csup\u003e54\u003c/sup\u003e Detected cells (red) were also identified using a threshold of 0.3 to detect alveolar and inflammatory cells, and compared in areas of inflammation to entire lung lobe, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u003cb\u003e\u0026rsquo;-C\u0026rsquo;\u0026rsquo;\u003c/b\u003e. C19 (n\u0026thinsp;=\u0026thinsp;3) had average of 57.78% of the lung lobe annotated as inflammation, compared to 0.37% in control mice (n\u0026thinsp;=\u0026thinsp;3), p\u0026thinsp;=\u0026thinsp;0.009, and 80.17% inflammatory cells compared to 0.89%, p\u0026thinsp;=\u0026thinsp;0.002, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD. Ratio of lung to plasma EB was not different in C19 exposure (n\u0026thinsp;=\u0026thinsp;7, mean 88.46\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003eSD 49.33) compared to control (n\u0026thinsp;=\u0026thinsp;7, 63.69\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\pm\\)\u003c/span\u003e\u003c/span\u003eSD 41.17), p\u0026thinsp;=\u0026thinsp;0.32.\u003c/p\u003e \u003cp\u003e \u003cb\u003eC19 SP exposure yielded a differential BAL and Plasma cytokine biomarker signature and inflammatory profile.\u003c/b\u003e In \u003cb\u003eBAL\u003c/b\u003e, C19 exposure significantly increased proinflammatory cytokines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-D). Growth and differentiation associated factors G-CSF and LIF were increased after exposure to C19, while VEGF was decreased, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G. Chemokines of multiple cell lines including eosinophils and neutrophils, such as Eotaxin and KC, were also elevated after C19 exposure, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-O. There was also activation of adaptive immune associated cytokines, with elevated IFN-γ, IL-4, and IL-9, but decreased IL-2, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eP-S. Other assayed analytes including pro-inflammatory cytokine MIP-2 and growth factor GM-CSF, were not significantly different between control and C19 in BAL, \u003cb\u003eSupplemental Fig.\u0026nbsp;2\u003c/b\u003e. \u003cem\u003ePCA\u003c/em\u003e using all measured analytes captured 78.5% of the total variance in 3 components, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eT. The heat map shows that VEGF and IL-2 segregated with lower expression in C19 in cluster 1, vs generally higher expression of the other inflammatory analytes in C19 exposure in cluster 2\u0026ndash;3, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eU.\u003c/p\u003e \u003cp\u003eIn \u003cb\u003eplasma\u003c/b\u003e, there was activation of both chemokines and the adaptive immune response-associated cytokines with IP-10, MIG and IL-13 significantly elevated in C19-exposed mice, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C. All other assayed analytes can be seen in \u003cb\u003eSupplemental Fig.\u0026nbsp;3.\u003c/b\u003e PCA captured 68.7% of the variance in 3 components, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD. IL-13 and IP-10 segregated within Cluster 1, while MIG segregated to Cluster 2 in the heatmap, with both clusters associated with upregulation in C19-exposed mice, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eE. BAL and plasma analytes were also plotted by fold-change and p-value in volcano plots, \u003cb\u003eSupplemental Fig.\u0026nbsp;4A and B respectively\u003c/b\u003e. In BAL, 17/32 analytes significantly increased in fold-change(Eotaxin, MIP-1α, MIP-1β, RANTES, G-CSF, KC, IL-10, MIG, IFN-g, IL-12(p40), IL-4, IL-6, LIF, MCP-1, and TNF-α) and decreased in IL-2 and VEGF, \u003cb\u003eSupplemental Fig.\u0026nbsp;4A\u003c/b\u003e. In plasma, MIG was identified as significantly upregulated (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003eSupplemental Fig.\u0026nbsp;4B\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMultiple Inflammatory Pathways are Activated in C19 Exposure\u003c/strong\u003e \u003cp\u003eTransformed BAL and p-value data was input into IPA, and n\u0026thinsp;=\u0026thinsp;14/192 significant canonical pathways that met threshold with p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 also had |z-score|\u0026gt;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eA. Multiple inflammatory pathways were activated including macrophage, T-cell response (pattern recognition receptors), and natural killer cells. The top significant identified canonical pathway, Macrophage Classical Activation Signaling Pathway is shown, \u003cb\u003eSupplemental Fig.\u0026nbsp;5\u003c/b\u003e.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eNRLP3 and HO-1 were significantly induced in the C19 spike protein exposures compared to control, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB. Caspase-1, Caspase-8, RAGE, and MYD-88 were not significantly different. Full Western images are provided in \u003cb\u003eSupplemental Fig.\u0026nbsp;6\u0026ndash;9\u003c/b\u003e. Additionally, a regulators effects analysis was performed in IPA, and the analysis that involved HMOX1, the gene that encodes HO-1 with the highest consistency score, a measurement used to help rank the most highly connected and consistent networks from regulator to function, is presented, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eC. Regulator networks showed recruitment of multiple cell lines including leukocytes, CD4\u0026thinsp;+\u0026thinsp;T-lymphocytes, phagocytes, and macrophages, as well as damage of lung.\u003c/p\u003e \u003cp\u003eUpstream regulators that were significantly activated or inhibited (|z-score|\u0026gt;2) are also displayed for BAL and plasma, \u003cb\u003eSupplemental Fig.\u0026nbsp;10\u003c/b\u003e. TreeMaps of disease and functions in BAL showed marked increased activity in multiple inflammatory pathway activation and cell recruitment pathways, \u003cb\u003eSupplemental Fig.\u0026nbsp;11\u003c/b\u003e. In plasma, the significance threshold was not met to fully develop a TreeMap.\u003c/p\u003e \u003cp\u003eFull pathways analysis are available at: IPA_BAL and IPA Plasma.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eC19 overwhelmed hospitals globally with high numbers of patients with multiorgan failure, often with cytokine release syndrome, and dysregulation of the immune system.\u003csup\u003e56\u003c/sup\u003e Future pandemics remain an ongoing threat, and it is critical to understand the mechanistic underpinnings of C19 to identify potential targets of future therapy. Our work contributes to the C19 literature by clarifying knowledge gaps validating a non-infectious murine body, quantifying lung function changes and end-organ dysfunction. Moreover, we have identified potential therapeutic targets by showing upregulation of both HO-1 and NRLP3. These targets are supported by our findings in IPA, showing the involvement of mediators central to HO-1 and NLRP3 activation.\u003c/p\u003e \u003cp\u003eThis noninfectious transitional model recapitulated many of the findings found using noninfectious models of COVID, and significantly overlaps many of the phenotypes seen in live-virus models.\u003csup\u003e25,57\u003c/sup\u003e Lung function decline after COVID infection was characterized by reduction in IC, compliance, volume, and hysteresis.\u003csup\u003e19\u003c/sup\u003e Our model differed by additionally showing airway hyperreactivity. Further, despite significant involvement of lungs after C19 exposure, there was no alveolar leak or an ARDS phenotype.\u003c/p\u003e \u003cp\u003eExposure to C19 induced a simultaneous elevation of several inflammatory and counterregulatory pathways in BAL, affecting Th1 (TNF-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\alpha\\)\u003c/span\u003e\u003c/span\u003e, IL-6, IFN-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\gamma\\)\u003c/span\u003e\u003c/span\u003e, IL-12(p40), IL-2, IP-10, MIG), Th2-associated response (IL-4, IL-9, Eotaxin), and Th17 associated mucosal response (IL-17, KC, G-CSF). This could indicate a severe, overactive inflammation. The suppression of IL-2 and VEGF could indicate T-cell exhaustion and failed tissue repair.\u003csup\u003e58,59\u003c/sup\u003e In plasma, although there were less significantly different inflammatory markers, there were similar patterns of mixed and often opposing immune signal from Th2 (IL-13), and Th1/interferon driven signaling (IP-10 and MIG) elevation after C19 exposure. There is similarity to cytokine release syndrome or cytokine storm seen in humans with C19, specifically in the elevation of IL-6, TNF-α, and IFN-γ.\u003csup\u003e60\u003c/sup\u003e IL-1, which was also seen as critical to the cytokine release in humans, was measured as different isoforms in the murine model. Of those, IL-1β trended higher in C19-exposure. The multiple and sometimes counter-regulatory pathway involvement may also be contributory to the pathogen-induced cytokine storm signaling pathway initially being identified as the top related canonical pathway in IPA, but it was not significant by z-score.\u003c/p\u003e \u003cp\u003eWe demonstrated that exposure to C19 spike protein can induce NRLP3 and HO-1, which may trigger downstream activation of inflammatory complexes. HO-1 is considered cytoprotective with antioxidant, anti-inflammatory, and antiviral properties reflective of activation of M2 macrophages.\u003csup\u003e37,61\u0026ndash;63\u003c/sup\u003e However, its upregulation has been associated with poor outcomes in C19-infected patients, specifically worse hypoxia and mortality.\u003csup\u003e37,61,64\u003c/sup\u003e NLRP3 inflammasome activation in severe viral disease, such as in C19 and influenza, has also been thought to be at the center of immune-mediated dysregulation.\u003csup\u003e65\u0026ndash;67\u003c/sup\u003e It plays both pro-inflammatory activation roles and anti-viral response, but excessive stimulation has been linked to cytokine storm and multi-organ failure.\u003csup\u003e67\u003c/sup\u003e In the lung, HO-1 activation is often seen to protect against the NLRP3 inflammasome via counterregulatory action in disease states such as cigarette-smoking induced COPD or LPS/sepsis-induced lung injury.\u003csup\u003e68\u0026ndash;71\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eSimultaneous activation of HO-1 and NLRP3 has been seen in chronic inflammatory disease such as osteoarthritis.\u003csup\u003e72\u003c/sup\u003e The simultaneous activation of NLRP3 and HO-1 may indicate that there is activation of both inflammatory and counter-regulatory pathways, and point to possible chronicity with ongoing inflammation. This also concurs with the quantified cytokine profile of the murine model that showed upregulation of multiple immunoregulatory pathways.\u003c/p\u003e \u003cp\u003eThe involvement of HO-1 and NLRP3 is also evident from the pathway and regulator analysis. Macrophage classical activation signaling pathway was the most significant canonical pathway. Heme was also the most significant inhibited upstream regulator in BAL. This could be related to macrophage activation by heme breakdown in the HO-1 pathway. Heme is also an important NLRP3 activator, and its breakdown may be early counterregulatory mechanism action against NLRP3.\u003csup\u003e73\u003c/sup\u003e Further studies are needed to identify the complex mechanisms between HO-1 and NLRP3 activation in viral disease.\u003c/p\u003e \u003cp\u003eThis study has several limitations. Male mice were used exclusively because preliminary studies using female mice had inconsistent data. One possible reason for this may be because male mice weighed approximately 30% more than the female mice, and received a higher overall dose of SP. Another reason may be because female mice exhibit hormones that influence the pulmonary inflammation profile differently from male mice. Future studies could investigate the use of a fixed, higher dose of SP to observe if there are any sex-related effects.\u003c/p\u003e \u003cp\u003eOur model differed in some aspects of lung dysfunction compared to both live and noninfectious models. There could be differences in live virus used compared to the noninfectious S1 spike protein.\u003csup\u003e19\u003c/sup\u003e Our model may also need a longer time interval to show increased resistance as the pulmonary disease progresses, or a higher dose to induce more profound pulmonary changes. We also could not recapitulate vascular permeability measures of alveolar leak through either Evans Blue or electric cell-substrate impedance measurements.\u003csup\u003e25,26\u003c/sup\u003e This could be due to small differences in technique of aspiration or injection of EB.\u003c/p\u003e \u003cp\u003eIt is also possible that the inflammatory profile would change over time, and that 72 hours may not represent the optimal time for peak inflammation.\u003csup\u003e57\u003c/sup\u003e Different doses and time intervals may have also shown different inflammatory profiles in not only the BAL, but also more peripherally in the plasma to represent more systemic inflammation. Moreover, it is possible that since this study did not use live virus, the inflammatory effects seen may not be generalizable to other strains of COVID or live virus studies. Nevertheless, this study represents an important step in studying C19-related inflammation using the isolated spike protein. The non-infectious model allowed us to measure the pulmonary changes that occur after exposure within a BSL-2 facility. There are also several aspects of the study that show moderate translatability of using a non-infectious model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFuture Plans\u003c/b\u003e This work adds to the growing body of literature aimed at assessing reproducibility and translatability of noninfectious models of C19. Further, it guides future studies using noninfectious models of C19 to investigate inflammatory response and lung injury. Future plans include loss and gain of function studies to identify NLRP3 and HO-1 as potential targets to mitigate inflammatory response in C19. Additional time-course and dose studies may also be studied for future studies to help profile the inflammatory progression. Noninfectious aspiration models of additional environmentally relevant irritants also may lay the foundation for future experiments investigating possible synergistic inflammation from other concurrent exposures.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eAHR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u0026nbsp;\u003c/strong\u003eAirway Hyperreactivity\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eALI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAcute Lung Injury\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eARDS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAcute Respiratory Distress Syndrome\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC19\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eCOVID-19\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIACUC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eInstitutional Animal Care and Use Committee\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIL\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eInterleukin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eInterferon gamma inducible protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eInternal Review Board\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eN\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eNumber\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNIH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNational Institutes of Health\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNYU\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNew York University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOAD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eObstructive Airways Disease\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eSpike protein\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTNF \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eTumor Necrosis Factor\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbbreviations related to Flexivent Parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParameter A\u0026nbsp;\u0026nbsp;\u003c/strong\u003erelates to hysteresis of PV loop\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParameter K\u0026nbsp;\u0026nbsp;\u003c/strong\u003erelates to hysteresis of PV loop\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCrs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eCompliance of the Respiratory System\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCst\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eStatic compliance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eErs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eElastance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFEV\u003csub\u003e0.1\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eForced expiratory volume in 0.1 second\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eTissue Damping\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eTissue Elastance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eInspiratory Capacity\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNPFE\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;negative pressure-driven forced expiratory maneuver\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePC\u003csub\u003e200\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eDose of methacholine that doubles baseline resistance\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRrs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eResistance of the Respiratory System\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRn\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eNewtonian Resistance\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Inte\u003c/strong\u003e\u003cstrong\u003erest Statement:\u003c/strong\u003e\u0026nbsp; The authors have no conflicts to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003eThis study was approved by the NYU IACUC # 16-00447\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials.\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis work was supported by UL1TR001445 NCATS; KL2TR001446 (SK); Stony Wold-Herbert Fund (SK) and CDC/NIOSH U01- OH11855; -OH11300 and -OH01269 (AN).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions.\u003c/strong\u003e AN was the primary investigator, had full access to all the data in the study and takes responsibility for the integrity and the accuracy of the data analysis. SK and AN participated in study conception and design; SK, AN, HB, JAR and JZ were responsible for data collection;\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eSK and AN were responsible for data validation; SK, JZ, HB, JAR, GG and AN participated in data analysis; SK, JZ, HB, SP and AN undertook the statistical analysis. All authors participated in data interpretation, writing and revision of the report and approval of the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u003c/strong\u003e\u0026nbsp; Work was produced in collaboration with the Experimental Pathology Research Laboratory at the New York University Langone Medical Center which is partially funded by the NYUCI Center Support Grant, \u0026ldquo;NIH/NCI 5 P30CA16087\u0026rdquo;.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. Cumulative Confirmed Deaths. in \u003cem\u003eWHO COVID-19 Dashboard - Daily cases and deaths\u003c/em\u003e (ed. 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The NLRP3 inflammasome fires up heme-induced inflammation in hemolytic conditions. \u003cem\u003eTransl Res\u003c/em\u003e \u003cstrong\u003e252\u003c/strong\u003e, 34-44 (2023).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"COVID-19, Airway hyperreactivity, lung injury, noninfectious translational model","lastPublishedDoi":"10.21203/rs.3.rs-8844647/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8844647/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eRATIONALE. \u003c/strong\u003eThe COVID-19 (C19) pandemic caused significant mortality often due to lung injury and systemic inflammation, but there is significant heterogeneity in severity and the pathobiology is not well understood. We examined COVID-19-induced pulmonary and inflammatory sequelae using a murine noninfectious model to further define the models utility and to also understand the role of mediators such as heme oxgenase-1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS. \u003c/strong\u003ek18-hACE2 male mice oropharyngeally aspirated C19-spike or equal volume control. After 72 hours, we collected: pulmonary mechanics, bronchoalveolar lavage(BAL) and plasma, snap-froze right lung, and fixed/stained left lung for histologic injury assessment(Qupath). Cytokine elaboration in BAL and plasma was quantified(Luminex), and lung homogenates were probed for HO-1 and NLRP3 (Western). Statistical (SPSS and R) and pathways comparisons(Ingenuity Pathway Analysis) were made between control and C19.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS Lung Mechanics.\u003c/strong\u003eC19 exposure significantly reduced inspiratory capacity and static lung compliance;tissue elastance and airway hyperreactivity were increased. \u003cstrong\u003eHistology:\u003c/strong\u003e C19 exposure caused significant inflammation and thickened alveolar septae. \u003cstrong\u003eCytokines:\u003c/strong\u003e C19 exposure led to inflammatory response in BAL and plasma with simultaneous activation of Type 1 and Type 2 pathways. \u003cstrong\u003ePathways.\u003c/strong\u003e NLRP3 and HO-1 protein expression is significantly induced by C19. Regulator networks show involvement of multiple cell lines and lung damage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONCLUSION: \u003c/strong\u003eA noninfectious C19 murine model showed worsened lung parameters and increased inflammation. HO-1 and NLRP3 may be key mediators in the inflammatory process and induce both inflammatory and counter-regulatory effects. Further studies will focus on targeted therapeutic pathways that probe into the mechanistic relationship of HO-1 and NLRP3 in C19-related disease.\u003c/p\u003e","manuscriptTitle":"Lung Dysfunction and Systemic Inflammation: A Role for HO-1 and NLRP3 in a COVID-19 Murine Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-17 11:49:02","doi":"10.21203/rs.3.rs-8844647/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T06:31:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T00:46:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T02:02:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"231771649594131841709912446076284748490","date":"2026-03-18T21:50:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27595568809744662244821045286656604766","date":"2026-03-18T19:12:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"141552087094035701063609468019416651797","date":"2026-03-12T15:08:48+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-12T10:27:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-12T07:17:42+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-03T14:38:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-27T21:29:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-02-27T15:02:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a53dbf06-112f-48f1-bd46-3341fc977c0f","owner":[],"postedDate":"March 17th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64606243,"name":"Health sciences/Diseases"},{"id":64606244,"name":"Biological sciences/Immunology"},{"id":64606245,"name":"Health sciences/Medical research"},{"id":64606246,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2026-04-24T06:39:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-17 11:49:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8844647","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8844647","identity":"rs-8844647","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}