Autoantibodies against Progranulin and IL-1 receptor antagonist due to immunogenic posttranslational isoforms contribute to hyperinflammation in critically ill COVID-19
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Abstract
Hyperinflammation is frequently observed in patients with severe COVID-19. Inadequate and defective IFN type I responses against SARS-CoV-2, associated with autoantibodies in a proportion of patients, lead to severe courses of disease. In addition, hyperactive responses of the humoral immune system have been described.
In the current study we investigated a possible role of neutralizing autoantibodies against antiinflammatory mediators. Plasma from adult patients with severe and critical COVID-19 was screened by ELISA for antibodies against PGRN, IL-1-Ra, IL-10, IL-18BP, IL-22BP, IL-36-Ra, CD40, IFN-α2, IFN-γ, IFN-ω and serpinB1. Autoantibodies were characterized and the antigens were analyzed for immunogenic alterations.
In a discovery cohort with severe to critical COVID-19 high titers of PGRN-autoantibodies were detected in 11 of 30 (36.7%), and of IL-1-Ra-autoantibodies in 14 of 30 (46.7%) patients. In a validation cohort of 64 patients with critical COVID-19 high-titer PGRN-Abs were detected in 25 (39%) and IL-1-Ra-Abs in 32 of 64 patients (50%). PGRN-Abs and IL-1-Ra-Abs belonged to IgM and several IgG subclasses. In separate cohorts with non-critical COVID-19, PGRN-Abs and IL-1-Ra-Abs were detected in low frequency (i.e. in < 5% of patients) and at low titers. Neither PGRN-nor IL-1-Ra-Abs were found in 40 healthy controls vaccinated against SARS-CoV-2 or 188 unvaccinated healthy controls. PGRN-Abs were not cross-reactive against SARS-CoV-2 structural proteins nor against IL-1-Ra. Plasma levels of both free PGRN and free IL-1-Ra were significantly decreased in autoantibody-positive patients compared to Ab-negative and non-COVID-19 controls. In vitro PGRN-Abs from patients functionally reduced PGRN-dependent inhibition of TNF-α signaling, and IL-1-Ra-Abs from patients reduced IL-1-Ra- or anakinra-dependent inhibition of IL-1ß signaling. The pSer81 hyperphosphorylated PGRN isoform was exclusively detected in patients with high-titer PGRN-Abs; likewise, a hyperphosphorylated IL-1-Ra isoform was only found in patients with high-titer IL-1-Ra-Abs. Thr111 was identified as the hyperphophorylated amino acid of IL-1-Ra. In longitudinally collected samples hyperphosphorylated isoforms of both PGRN and IL-1-Ra emerged transiently, and preceded the appearance of autoantibodies. In hospitalized patients, the presence of IL-1-Ra-Abs or IL-1-Ra-Abs in combination with PGRN-Abs was associated with a higher morbidity and mortality.
To conclude, neutralizing autoantibodies to IL-1-Ra and PGRN occur in a significant portion of patients with critical COVID-19, with a concomitant decrease in circulating free PGRN and IL-1-Ra, indicative of a misdirected, proinflammatory autoimmune response. The break of self-tolerance is likely caused by atypical hyperphosphorylated isoforms of both antigens, whose appearances precede autoantibody induction. Our data suggest that these immunogenic secondary modifications are induced by the SARS-CoV-2-infection itself or the inflammatory environment evoked by the infection and predispose for a critical course of COVID-19.
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SciScore for 10.1101/2021.04.23.441188: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: This study was approved by the local Ethical Review Board (Bu 62/20) and conducted according to the Declaration of Helsinki. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources ELISA for autoantibodies against PGRN, IL-1-RA and IL-10: The ELISA for autoantibodies was performed as previously described. PGRNsuggested: NoneWhole blood lysates or lysates of PBMCs from PGRN- and/or IL-1-RA-antibody-positive patients and controls were analyzed by IEF for PGRN and SLP2 isoforms and plasma samples were analyzed for IL-1-RA isoforms. SLP2suggested: NoneWho… SciScore for 10.1101/2021.04.23.441188: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: This study was approved by the local Ethical Review Board (Bu 62/20) and conducted according to the Declaration of Helsinki. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources ELISA for autoantibodies against PGRN, IL-1-RA and IL-10: The ELISA for autoantibodies was performed as previously described. PGRNsuggested: NoneWhole blood lysates or lysates of PBMCs from PGRN- and/or IL-1-RA-antibody-positive patients and controls were analyzed by IEF for PGRN and SLP2 isoforms and plasma samples were analyzed for IL-1-RA isoforms. SLP2suggested: NoneWhole blood cell lysates or lysates of PBMCs from IL-1-RA-antibody positive patients were treated with alkaline phosphatase as previously described using FastAP thermo-sensitive alkaline phosphatase (Fermentas/VWR, Darmstadt, Germany). alkaline phosphatase ( Fermentas/VWR , Darmstadt , Germany).suggested: None(35) For ELISA for the pSer81 isoform of PGRN Nunc MaxiSorb plates were coated overnight at 4°C with rabbit antihuman PGRN antibodies directed against the C-terminus at a dilution of 1:2500 (v/v; LsBio, Seattle,WA, USA), followed by blocking with 1.5% (w/v) gelatin in TBS and washing steps with TBS-Tx [TBS, 0.1% (v/v) Tx100] antihuman PGRNsuggested: NoneTx100suggested: NoneFollowing this, corresponding biotinylated antihuman Fab secondary antibodies and subsequently peroxidase-labeled streptavidin (Roche) were used. antihuman Fab secondarysuggested: NoneELISA for antibodies against pSer81 PGRN isoform and their Ig class: To detect antibodies against pSer81 PGRN, Nunc MaxiSorp plates (eBioscience, Frankfurt, Germany) were precoated with murine anti-HIS mAb at a dilution of 1:2,500 (v/v; Sigma-Aldrich, Munich, Germany) at 4°C overnight. anti-HISsuggested: NonePGRN-antibody positive patient’s plasma (500 µl) was diluted 1:10 (v/v) in PBS and was incubated with the anti-FLAG matrix/FLAG-tagged PGRN complex and subsequently desorbed by elution with glycine buffer depleted. anti-FLAGsuggested: NoneExperimental Models: Cell Lines Sentences Resources For this purpose, 500 µl of lysate of HEK293 cells transfected with recombinant FLAG-tagged PGRN was incubated with 20 ml anti-FLAG matrix for 15 min at room temperature. HEK293suggested: NoneRecombinant DNA Sentences Resources Western blot, isoelectric focusing of PGRN and IL-1-RA, and ELISA for pSer81 or npSer81 PGRN: Western blotting and isoelectric focusing was performed as described (32). pSer81suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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