Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19

Abstract

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Keywords: COVID-19; autoimmunity; immunoglobulin A; pulmonary surfactant; pulmonary-associated surfactant protein.

Figure 1.

Surfactant protein B (SP-B) IgA are found in BAL fluid (BALF) of patients with severe COVID-19. (A) The top 30 IgA-bound proteins in BALF of patients with severe COVID-19 (n = 18) are presented in the left heatmap (red) and grouped by their respective physiological compartments. Each column represents a patient. The right heatmap (blue) shows results of IgA:protein precipitates of BALF from patients without COVID-19. Protein concentrations were normalized to the IgA heavy chain signals, and log₂ transformed. (B) Schematic workflow of the experimental setup for the identification of IgA-bound antigens. BALF was processed with peptide M pulldown columns, and IgA-bound proteins were measured and identified via liquid chromatography–tandem mass spectrometry. (C) The quantitative comparison revealed a significantly higher concentration of IgA:SP-B complexes in the severe COVID-19 versus non-COVID-19 BALF (*P = 0.03, Mann-Whitney test). Alpha-ETF = electron transfer flavoprotein subunit alpha; EXOC3L = exocyst complex component 3-like protein; glycopr. = glycoprotein; immunoprec. = immunoprecipitate; LC-MS/MS = liquid chromatography with tandem mass spectrometry; norm. = normalized; pr. = protein; sub. = subunit.

Figure 2.

Lungs of patients with severe coronavirus disease (COVID-19) harbor colocalized surfactant protein B (SP-B) and IgA. (A) Schematic of the staining strategy used to detect colocalization (merge = yellow) of IgA (fluorescein isothiocyanate [FITC] = green) with SP-B (Alexa 594 = red) in lung tissue from fatal COVID-19, influenza, or patients with cancer, by immunofluorescence. (B) Double immunofluorescence staining for SP-B and IgA in formalin-fixed paraffin-embedded sections from lung tissue of patients with COVID-19 (n = 4), (C) in biopsy specimens from patients with influenza (n = 2), and (D) in uninfected patients with metastatic melanoma (n = 3). Consistently, colocalization of SP-B with IgA was only observed in samples from deceased patients who had COVID-19 and not in the lungs of the other two groups. Scale bars, 100 μm.

Figure 3.

Lungs of patients with severe coronavirus disease (COVID-19) harbor colocalized surfactant protein C (SP-C) and IgA. (A) Double immunofluorescence staining for SP-C (Alexa 594 = red) and IgA (fluorescein isothiocyanate [FITC] = green) in formalin-fixed paraffin-embedded sections from lung tissue of patients with COVID-19 (n = 4), (B) in biopsy specimens from patients with influenza (n = 2), and (C) in uninfected patients with metastatic melanoma (n = 3). Colocalization of SP-C with IgA was consistently seen only in samples from deceased patients who had COVID-19 and not in the lungs of the other two groups. Scale bars, 100 μm.

Figure 4.

IgA targeting surfactant proteins B and C (SP-B and -C) are elevated in blood samples from patients with severe coronavirus disease (COVID-19). (A) Schematic representation of the IgA enzyme-linked immunosorbent assay (ELISA) experiment using recombinant proteins as coating and diluted plasma from patients as the antibody source. (B) SP-B (left), SP-C (center), or poractant alfa (right) ELISAs revealed significantly more IgA against the target proteins in plasma from patients with severe COVID-19 (n = 77) compared with those with mild COVID-19 (n = 12) or healthy control subjects (n = 12). (C) Receiver operating characteristics analysis demonstrated that the presence of IgA against either SP-B (left), SP-C (center), or poractant alfa (right) was significantly associated with the requirement of the patient for oxygen supplementation. (D) Comparison of the surfactant-specific IgA concentrations in serum from patients with COVID in the validation cohort (n = 60), patients with bacterial pneumonia and without severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (n = 30), and noninfected control patients without pneumonia (n = 10). ELISAs consistently detected significantly higher concentrations of IgA against SP-B (left), SP-C (center), and poractant alfa (right) in patients with severe COVID-19 compared with patients without COVID-19. All data are represented as mean ± 95% confidence interval. For multiple comparisons, the Kruskal-Wallis test with Dunn’s correction was used. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. NS = not significant; TMB = substrate for horseradish peroxidase.

Figure 5.

Plasma and isolated IgA from patients with severe coronavirus disease (COVID-19) inhibit the function of pulmonary surfactant. (A) Incubation of poractant alfa (40 mg/ml) with plasma from patients with severe COVID-19 resulted in a significant increase in surface tension, whereas plasma from patients with mild COVID-19 and patients in the noninfected healthy control did not experience notable changes. (B) Purified IgA (0.2 mg/ml) from plasma of patients with severe COVID-19 (n = 5) caused a significant increase of surface tension compared with PBS control subjects (n = 3). (C) Graphical summary of our results showing that auto-IgA occurs in the alveoli and blood of severely diseased patients with COVID-19, binds to surfactant proteins, and causes impairment of pulmonary surfactant function. All data are represented as mean ± SD. For single comparisons, a Mann-Whitney test was used, and for multiple comparisons, a Kruskal-Wallis test with Dunn's correction was applied. *P < 0.05 and **P < 0.01. NS = not significant; PBS = phosphate-buffered saline.