IL-13 is a driver of COVID-19 severity

Abstract

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, in the lung, hyaluronan synthase 1 (Has1) was the most downregulated gene and hyaluronan accumulation was decreased. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator, and indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.

Figure 1.. Type 2 immune response in patients with severe COVID-19 disease.

(A-E) Cytokines were measured in plasma from 26 outpatients and 152 inpatients with COVID-19 infection at the University of Virginia Hospital using a 48-plex cytokine array. A) Heatmap of plasma cytokines, supplemental oxygen requirement and nasopharyngeal viral load, with rows ordered by patient status (outpatient (OP) vs inpatient (IP)) and columns by cytokine principal component 1 which included IL-13 (Table S2). B) Scatterplot comparing principal component 1 and 2 from Principal Component Analysis (PCA) of the plasma cytokines (orange inpatients and blue outpatients). C) Plasma IL-13 levels in COVID-19 patients who were or were not diagnosed with COVID-19 or D) did or did not require mechanical ventilation (Wilcox test). E) Proportion of COVID-19 patients requiring mechanical ventilation stratified by IL-13 plasma cytokine levels (Chi-square analysis). F) Kaplan-Meier survival analysis of the relationship between IL-13 level and mechanical ventilation. Comparison made to lowest IL-13 quantile (Logrank; Cox proportional hazards test adjusted for age, sex, and comorbidities). G) ROC curve with AUC plotted from: IL-13 alone (blue), IL-13 and IL-6 (red), or IL-13, IL-6, IL-8, and MIP-1b (black). G) IL-13 levels in 19 non-severe and 26 severe (requiring supplemental oxygen) COVID-19 patients from Virginia Commonwealth University Hospital (Wilcox test). * =p<0.05; ** =p< 0.005.

Figure 2.. Type 2 immune response in lungs of mice following infection with SARS-CoV-2.

10-week-old male mice (Tg K18-hACE2 2Prlmn) were infected with 5×10³ PFU of SARS-CoV-2 and lung tissue examined on day five post-infection by RNA-seq and immunohistochemistry (IHC). A) Type 2 gene expression in the lungs of infected vs uninfected mice (heat map of normalized values of manually curated list of type 2 immune pathway genes). B) Immunohistochemistry of the type 2 immunity proteins RELMa (RELMa) and Ym1 in the lungs of infected and uninfected mice (AW, airway). C) Quantification of IHC scoring for RELMa and Ym1 (mixed effect model). *=p<0.05; **=p<0.005

Figure 3.. neutralization protects from severe COVID-19 in K18-hACE2 mice.

IL-13 Mice were infected on day 0 with 5×10³ PFU of SARS-CoV-2 and administered 150 μg of anti-IL-13 or an IgG isotype control antibody intraperitoneally on days 0, 2, and 4. A) Clinical scores of illness severity on days 1–7 pi. Clinical scoring was measured by weight loss (0–5), posture and appearance of fur (piloerection) (0–2), activity (0–3) and eye closure (0–2). B) Weight loss on days 1–7 pi. C) Kaplan-Meier survival analysis in mice. D) Kaplan-Meier curve generated from data obtained from TriNetX: 1:1 matching based on 81 patients who had been prescribed Dupilumab independently of their COVID-19 diagnosis. E) Histopathology of lung tissue comparing infected mice with or without IL-13 neutralization to uninfected mice (PAS stain; inset arrow indicates PAS (+) goblet cell. F) Quantification of scoring for goblet cell metaplasia in the IgG isotype control and anti-IL-13 treatment groups. G) Immunohistochemistry of lung tissue stained for RELM-a (yellow) in parenchyma or airway, and DAPI (blue).H) Quantification of intensity of staining for RELMα following IL-13 neutralization (log transformed, mixed effect model). (N = 5 mice/group; A & B combined from three independently conducted experiments). *=p<0.05; **=p<0.005.

Figure 4.. Hyaluronan and COVID-19 disease.

Mice received i.p. injections of anti-IL-13 on days 0 and 2 pi, were euthanized on day 5 and lung tissue was split and placed either into trizol tissue reagent for RNA analysis or formaldehyde for paraffin embedding and immunohistochemistry. A) Gene expression in mouse lung of hyaluronan synthase (Has1) and the hyaluronan receptor Cd44 of infected mice with anti-IL-13, isotype control antibody and uninfected controls. B) Hyaluronan was measured in the plasma of COVID-19-negative controls and in patients with COVID-19 that did or did not require supplemental oxygen. Postmortem lung samples were obtained from fatal COVID-19 cases and control tissue from COVID-19 negative deaths. C) Quantification of hyaluronan deposition in fatal COVID-19 disease (N=11) and controls (N=8) (log transformed, mixed effect model) using hyaluronan-binding protein (HABP). D) Representative images of staining for hyaluronan (with HABP). E) Staining of hyaluronan in mouse lung (with HABP) and F) quantification of hyaluronan deposition in tissue following infection and neutralization of IL-13 (mixed effect model; combined 2 experiments). Mice were administered anti-CD44 or IgG2 isotype control on days 1, 2, 3 and 4 pi. G) Clinical scores and H) Kaplan-Meier survival curve for mice; combined two, independent experiments. *=p<0.05; **=p<0.005; ***=p<0.0005.