key: cord-0696861-2d4zypdw authors: Gaurav, Rohit; Anderson, Daniel R.; Radio, Stanley J.; Bailey, Kristina L.; England, Bryant R.; Mikuls, Ted R.; Thiele, Geoffrey M.; Strah, Heather M.; Romberger, Debra J.; Wyatt, Todd A.; Dickinson, John D.; Duryee, Michael J.; Katafiasz, Dawn M.; Nelson, Amy J.; Poole, Jill A. title: IL-33 Depletion in COVID-19 Lungs date: 2021-07-07 journal: Chest DOI: 10.1016/j.chest.2021.06.058 sha: 560cbf73c93a98a92dab6c6fa9f9c56cf577124b doc_id: 696861 cord_uid: 2d4zypdw nan Interleukin-33 (IL-33) is an alarmin that plays an integral role in lung homeostasis through its actions in wound repair, fibrosis, and remodeling processes (1) . Stored in the nucleus, IL-33 is released to the cytoplasm and extracellular fluids following insult or damage induced by various infectious, noxious, or environmental agents (2) . In addition to its role in allergic asthma (3, 4) , studies have demonstrated elevated IL-33 in chronic obstructive pulmonary disease (COPD) plasma (5) , COPD airways (1) and idiopathic pulmonary fibrosis (IPF) lung tissues (6) , but a comparative analysis of lung IL-33 expression in the setting of infectious sequalae are lacking. Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes an inflammatory cascade resulting in reduced diffusion capacity, hypoxia, and death (7, 8) . The REACT COVID investigators screened serum from 100 COVID-19 subjects for cytokines (i.e. IL-6, TNF, IL-8, IL-1ß, GM-CSF, IL-33, IFN-ɣ, IL-10) and they found that increased serum IL-33 levels (as well as TNF) were independently predictive of poor outcomes with SARS-CoV-2 in patients age <70 years (adjusted odds ratio for IL-33: 11.14; 95% confidence intervals: 1.01-123.72) (9) . The objective of this study was to characterize IL-33 expression in the lungs of patients with fulminant COVID-19, comparing this expression with that observed in other inflammatory lung diseases. The integrated densities (the product of area and mean gray value) of each protein were measured as single color on black background with color threshold by Image J (version: 2.1.0/1.53c). Statistical analysis was conducted averaged densities of each patient with Prism 9 (version: 9.0.0) using Mann-Whitney test versus control group with p<0.05 accepted as statistically significant. Tissue IL-33 expression was significantly increased in IPF (6.57-fold, p=0.0012) and COPD (3.91-fold, p=0.0012) compared to controls, whereas COVID-19 patients had low to negligible IL-33 expression that was significantly reduced as compared to controls (0.03-fold; p=0.0003) ( Figure 1A -C). Co-staining with prosurfactant protein C (proSP-C) was used to assess type II alveolar epithelial cells (AEC2) and vimentin was used to assess mesenchymal cells (i.e., fibroblasts, smooth muscle cells, and endothelial cells) and macrophages ( Figure 1A between COPD and controls ( Figure 1D ). As compared to controls, AEC2 numbers were decreased in COVID-19 (0.01-fold, p=0.0003) and COPD (0.43-fold, p=0.0047) lungs with no difference between IPF and controls ( Figure 1E ). In post-COVID fibrosis, IL-33, vimentin, and AEC2 were all increased to levels at or above that demonstrated in COPD and IPF ( Figure 1A-E) . Lung tissue IL-33 was expressed in basal epithelial cells, AEC2, endothelial cells, fibroblasts, macrophages, and other progenitor cells with markedly increased expression in COPD and IPF. However, IL-33 was nearly entirely depleted from the lung tissue of COVID subjects with negligible availability or reserve in the nucleus of any lung cell. Some (5 of 8) COVID-19 lungs demonstrated extracellular or cytoplasmic IL-33 expression, but at extremely low levels to suggest release and depletion. These findings also corresponded to a near absence of proSP-C + AEC2 in the COVID-19 lungs to potentially suggest cellular death and/or lack of progenitor epithelial cells to aid in lung repair and recovery processes. There was wider patient variability in vimentin expression with COVID-19, which could reflect variation in time course of COVID infection (timing unknown). Similar to COVID-19 (9), serum IL-33 levels are also increased with influenza and lung IL-33 increases in healthy controls infected with influenza (10). However, there remains a gap of knowledge as to whether other fulminant infectious respiratory diseases are also associated with IL-33 exhaustion. Corticosteroids are commonly utilized in COVID-19 and can downregulate several cytokines (11) , but IL-33 has been recognized to be non-responsive to glucocorticoid therapy (3, 4) . Moreover, all bio-banked lungs received glucocorticoids in the standard optimization procedure prior to harvest. It remains possible that circulating IL-33-producing cells are also important in the response to SARS-CoV-2 infection; however, the striking depletion of lung tissue IL-33 suggests J o u r n a l P r e -p r o o f the importance of a lung compartment specific source of IL-33. It is also noted that IL-33 is cleaved to a number of inflammatory products that potentially were not detected; however, the antibody utilized recognizes mature and cleaved forms through recognition of the Ser112-Thr270 amino acid sequence of IL-33. Nonetheless, these studies underscore the complexity of IL-33 in lung disease as lung IL-33 expression was strikingly increased in chronic lung disease whereby serum IL-33 levels can also be variably elevated (5, 6) . IL-33 is a key mediator in danger signaling, but also in wound repair and lung recovery processes that can be marked by dysregulated fibrosis. Investigating IL-33 levels in the lung of COVID-19 survivors would also provide insight into restoration of IL-33 in normal homeostasis. Indeed, IL-33 and AEC2 expression was increased in post-COVID fibrosis lung to support these future studies. Whether replenishment of IL-33 by lung progenitor cells as observed in chronic disease states would be beneficial or harmful in the setting of overwhelming infection is not known. In contrast, blocking viral-mediated IL-33 release early in the infectious process could be explored. Considering an integral role of IL-33 in Th2 diseases, future studies could also utilize asthmatic lung samples in comparison studies. A limitation of this study is that IL-33 protein expression was assessed by immunohistochemistry due to availability, and IL-33 investigations in various compartments including lavage fluid, tissue, serum at both protein and gene expression level to fully inform the role of IL-33 in SARS-CoV-2 are warranted. In conclusion, these studies strengthen the relationship of IL-33 in COVID-19 to suggest that additional and longitudinal assessments are warranted to understand the mechanisms and timing of lung IL-33 expression and regulation for promoting damage or driving wound repair processes to inform potential interventional strategies. Poole takes responsibility for (is the guarantor of) the content of the manuscript, including the data and analysis DRA: Conception of the work; acquisition of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. SJR: Conception of the work; acquisition, analysis, & interpretation of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. KLB: Design of the work; acquisition & interpretation of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. BRE: Conception of the work; analysis & interpretation of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. TRM: Conception of the work; analysis & interpretation of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. GMT: Conception of the work; analysis & interpretation of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. HMS: Conception of the work; acquisition of data for the work; critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. DJR: Conception of the work; analysis of data for the work critical analysis and revision of the draft; approved the manuscript; and is accountable for all aspects of the work. JDD: Conception of the work; acquisition of data for the work Long-term IL-33-producing epithelial progenitor cells in chronic obstructive lung disease The R213G polymorphism in SOD3 protects against allergic airway inflammation IL-33 promotes airway remodeling in pediatric patients with severe steroid-resistant asthma Alternative splicing of interleukin-33 and type 2 inflammation in asthma Factors associated with plasma IL-33 levels in patients with chronic obstructive pulmonary disease Full-length IL-33 promotes inflammation but not Th2 response in vivo in an ST2-independent fashion COVID-19 Does Not Lead to a "Typical" Acute Respiratory Distress Syndrome Uncontrolled Innate and Impaired Adaptive Immune Responses in Patients with COVID-19 ARDS Inflammatory phenotyping predicts clinical outcome in COVID-19 Handicapped retroviral vectors efficiently transduce foreign genes into hematopoietic stem cells How corticosteroids control inflammation: Quintiles Prize Lecture The authors wish to thank Lisa Chudomelka for assistance with manuscript preparation/submission.