key: cord-1002662-12fj1wwy
authors: Thurner, Lorenz; Fadle, Natalie; Bewarder, Moritz; Kos, Igor; Regitz, Evi; Thurner, Bernhard; Fischer, Yvan; Cetin, Onur; Rixecker, Torben; Hoffmann, Marie-Christin; Preuss, Klaus-Dieter; Schormann, Claudia; Neumann, Frank; Hartmann, Sylvia; Bock, Theresa; Kaddu-Mulindwa, Dominic; Bette, Birgit; Roemer, Klaus; Bittenbring, Joerg Thomas; Christofyllakis, Konstantinos; Bick, Angelika; Lesan, Vadim; Abdi, Zanir; Mang, Sebastian; Becker, André; Metz, Carlos; Seiler, Frederik; Lehmann, Johannes; Agne, Philipp; Adams, Thomas; Link, Andreas; Werner, Christian; Thiel-Bodenstaff, Angela; Reichert, Matthias; Danziger, Guy; Roth, Sophie; Papan, Cihan; Pilch, Jan; Pfuhl, Thorsten; Wuchter, Patrick; Herr, Christian; Lohse, Stefan; Schrezenmeier, Hubert; Boehm, Michael; Langer, Frank; Gäbelein, Gereon; Friesenhahn-Ochs, Bettina; Kessel, Christoph; Foell, Dirk; Bals, Robert; Lammert, Frank; Körper, Sixten; Rissland, Jürgen; Lensch, Christian; Stilgenbauer, Stephan; Becker, Sören L.; Smola, Sigrun; Krawczyk, Marcin; Lepper, Philipp M.
title: Autoantibodies against Progranulin and IL-1 receptor antagonist due to immunogenic posttranslational isoforms contribute to hyperinflammation in critically ill COVID-19
date: 2021-10-20
journal: bioRxiv
DOI: 10.1101/2021.04.23.441188
sha: fefe6dbbefb49cd6c7256aa65fa20660a8797fec
doc_id: 1002662
cord_uid: 12fj1wwy

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.

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 hightiter 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.

COVID-19 caused by SARS-CoV-2 shows a very wide spectrum of manifestations and severity ranging from completely asymptomatic infection, to mild cold symptoms or attenuation of the sense of taste and smell, to acute respiratory distress syndrome (ARDS), the latter often associated with thromboembolic complications (1) , (2) , (3) , (4) . Patients requiring intensive care treatment often present with a hyperinflammatory state, which has been compared with hemophagocytic lymphohistiocytosis (HLH) (5) . Accordingly, antiinflammatory or immunosuppressive drugs like dexamethasone or inhibitors of the IL-1ß-, IL-6-, JAK/STAT-, or the BCR-pathways have been studied in clinical trials (6) , (7) , (8) , (9) , (10) , (11) , (12) . Among these drugs, though, only dexamethasone and IL-6 pathway blockade have so far been identified to provide a significant clinical benefit, in terms of a reduced 28-day mortality in patients with severe COVID-19 (6) .

COVID-19 has been suggested to resemble hemophagocytic lymphohistiocytosis (HLH) (5), a condition associated with a hyperinflammatory dysregulation of the immune system.

Misdirected, or defective IFN I responses were reported as a key factor for inefficient immune responses in patients with COVID-19 (13) , (14) , (15) . In this context autoantibodies against IFN I were detected specifically in patients with severe courses of COVID-19 (16) .

Moreover, exuberant B-cell responses were regularly found in COVID-19 (17) , (18), along with a high prevalence of antiphospholipid- (19) , anti-Ro/La- (20) or anti-annexin-Vantibodies (21) . A role of proinflammatory antibodies has so far not yet been clearly established (22) .

Previously, we had identified neutralizing autoantibodies against progranulin (PGRN) in sera from patients with primary small vessel vasculitis (23) . Subsequently we found progranulin-Abs in various rheumatic and other autoimmune diseases, but only very rarely in healthy controls, elderly or obese subjects, patients with sepsis and patients with melanoma (26) .

PGRN, also called proepithelin, is a secreted precursor protein. Beside several other biological functions (27) , a major property of PGRN is its anti-inflammatory activity (28) , (29) , which is mediated by direct binding to TNFR1, TNFR2 and DR3 and thus antagonization of responses to TNF-α and TL1-a (30) , (31) . This has been demonstrated in vivo in several mouse models including collagen and collagen-antibody induced arthritis (30), OXA induced dermatitis (32) , and more relevant for COVID-19, also in LPS-induced lung injury/ARDS mouse models (33) , (34) , (35) . Both PGRN and TNF-α bind to cysteine-rich domain 2 and 3 of TNFR (36) . The proinflammatory effect of neutralizing PGRN-Abs was characterized in vitro by the analysis of TNF-α-induced cytotoxic effects by MTT-assays and by downmodulation of FoxP3 in CD4 + CD25 hi Tregs in inflammatory bowel diseases and rheumatic diseases (25) , (37) . With hyperinflammatory states often observed in patients with severe COVID-19, and in light of similarities between this viral condition, vasculitis and autoimmune diseases (4), the aim of the current study was to investigate the possible occurrence of antibodies directed against previously described anti-inflammatory antigens against as progranulin (23) or IL-10 (38) , but also against other hypothesis-driven selected secreted anti-inflammatory mediators like interleukin-1 receptor antagonist (IL-1-Ra).

The discovery cohort consisted of 30 patients with COVID-19 (25 male, 5 female, median age 60 years, 21 with critical disease requiring treatment in ICU and 9 with moderate to severe disease not in ICU, median plasma ferritin: 1547 ng/mL, median CRP 91.3 mg/L). The anti-PGRN ELISA demonstrated the presence of PGRN-autoantibodies in plasma of 11/30 patients (36.7%), of which 9 were treated in ICU and 10 were males (median ferritin: 2159.5 ng/ml, median CRP: 160.1 mg/l) ( Fig. 1 A) . In a control-cohort of non-COVID-19 ICU patients, only 1 of 28 patients (3.6%) had weakly detectable PGRN-Abs ( Fig. 1 B) . In autoantibody-positive patients with severe or critical COVID-19, the titers ranged from 1:1600 up to 1:3200 ( Fig. 1 C) . PGRN-Abs belonging to the IgM class were found in 10 of 11 patients with COVID-19, and in all of the 11 PGRN-Ab-positive patients IgG class autoantibodies were found, with IgG1 detectable in 8, IgG2 in 9, IgG3 in 6 and IgG4 in 8 patients ( Fig. 1 D) .

In a validation cohort of 64 patients (median age: 61 years, 53 male, 11 female) with critical COVID-19 requiring mechanical ventilation, 25 of 64 (39%) patients had PGRN-Abs (Supplementary Fig. 1 A) . The median age of PGRN-Ab-positive patients was 60 years. Very similar to the discovery cohort, the PGRN-Abs often belonged to both the IgM class and different IgG subclasses and had similar titers (Supplement Fig. 1 B and 1C) . In a cohort of 126 hospitalized patients with predominantly moderate COVID-19 (median age: 65.5 years, were not detected in 28 ICU patients without COVID-19 ( Fig. 1 G) . Within the discovery cohort titers of IL-1-Ra-Abs ranged between 1:800 to 1:1600 ( Fig. 1 H) . IL-1-Ra-Abs belonged to IgM and several IgG subclasses, except IgG4 ( Fig. 1 I) . The epitope region with the highest affinity was located C-terminal from amino acid 63 ( Supplementary Fig 2 A) . No visible difference in molecular weight of IL-1-Ra was observed in any of the IL-1-Ra-Abpositive patients with COVID-19 in conventional Western-blot. However, in the IEF, an additional, more negatively charged third band of IL-1-Ra appeared in samples from IL-1-Ra-Ab-positive patients. This isoform of IL-1-Ra was absent in controls without IL-1-Ra-Abs ( Fig. 2 C) . We then examined whether this additional isoform was related to a different phosphorylation state. Pretreatment with alkaline phosphatase before IEF led to the disappearance of both the normal second and the atypical additional third IL-1-Ra isoform 

Of 24 patients hospitalized mostly during the second and third SARS-CoV-2-wave in Germany, two to three longitudinal samples obtained at weekly intervals were available. In seven (#109, #110, #104, #115, Val 55, Val57, Val62) patients who were PGRN-and/or IL-1-Ra-Ab-negative at admission, seroconversion to PGRN-and/or IL-1-Ra-Ab-positivity could be observed. In these patients, the hyperphosphorylated isoforms were detected one to two weeks before the appearance of PGRN-and or IL-1-Ra-autoantibodies ( Figures 1 and 2) .

Enriched PGRN-Abs failed to show cross-reactivity in ELISA against recombinant HIStagged SARS-CoV-2 S1-, S2-, E-or M-proteins, nor against recombinant human FLAGtagged IL-1-Ra. In addition, antibodies against SARS-CoV-2 S1-, S2-, or M-proteins as well as against human IL-1-Ra could not be adsorbed by immobilized PGRN, but were detectable in the eluate of samples from patients with severe or critical COVID-19, excluding crossreactivity of PGRN-Abs ( Supplementary Fig 5) .

Using standard ELISA, we observed that PGRN levels were significantly decreased by more than 90% in the plasma of PGRN-Ab-positive patients with COVID-19 (median: 15.12 ng/mL; SEM 3.6 ng/mL), compared to (i) plasma of PGRN-Ab-negative patients with COVID-19 (median 161.23 ng/mL, SEM 48.14 ng/mL) (Mann-Whitney test: p < 0.0001) and

(ii) plasma of PGRN-Ab-negative patients from the ICU without COVID-19 (median 206.05 ng/mL; SEM 10.59 ng/mL) (Mann-Whitney test: p < 0.0001) (Fig. 3 A) . In plasma from a patient with IL-1-Ra-Abs and PGRN-Abs, but not in plasma from a patient seronegative for IL-1-Ra-and PGRN-Abs, immune complexes of both antigens were detectable Remarkably, one patient with critical COVID-19 and high titers of PGRN-Abs and IL-1-Ra-Abs required plasmapheresis due to exacerbating chronic inflammatory demyelinating polyneuropathy (CIDP). After 6 days of plasmapheresis, titers of IL-1-Ra and PGRN-Abs strongly decreased from 1:1600 to 1:100, the additional atypic isoform of IL-1-Ra was no longer detectable by IEF in plasma, and the IL-1-Ra plasma levels rose from 81.1 pg/ml to 2340.4 pg/ml (Supplementary Fig. 6 ).

Here we report the occurrence of autoantibodies to PGRN and IL-1-Ra in a considerable proportion (40% and 49.4%, resp.) of adult patients with critical COVID-19. Importantly, such autoantibodies were not, or barely detectable in COVID-19 patients with moderate, mild or asymptomatic disease, nor in non-COVID-19 ICU patients, SARS-CoV2-vaccinated or in healthy controls. Both IL-1-Ra and PGRN are known to exert anti-inflammatory activity, suggesting a causal link to the hyperinflammatory phenotype often observed in critically ill COVID-19 patients. It is noteworthy that PGRN-autoantibodies were first identified in plasma of 20-40% of patients suffering from primary small, middle and large vessel vasculitis (23) .

Critical forms of COVID-19 have been shown to be frequently associated with vasculitis-like characteristics (4), which may contribute to some of the complications of this disease.

IL-1-Ra is the physiologic inhibitor of IL-1α and IL-1β due to competition for IL-1R1. A proinflammatory imbalance between these proteins is found in gout crystallopathies, autoinflammatory or cryopyrin-associated periodic fever syndromes. Macrophage activation syndrome, which has many similarities to HLH, is a common What might have triggered the production of PGRN-and IL-1-Ra-Abs? A noteworthy observation in this context is that in contrast to rheumatologic diseases, where PGRN-Abs largely belong to the IgG1 class(23), in COVID-19 PGRN-Abs were distributed across the IgM and multiple IgG subclasses. Moreover, the PGRN-Ab titers were high, ranging from 1:1600 up to 1:3200 ( Fig. 1 C-D) . Similarly, IL-1-Ra-Abs were found at high titers and were also distributed across IgM and different IgG subclasses (Fig. 1 H-I) (37) . Moreover, we found that this second pSer81 isoform led to altered functions of PGRN, with a dramatic reduction in its affinity to TNFR1/2 and DR3 and consequently, a loss of PGRN's ability to antagonize the TNF-α and TL1A effects (37) . In autoimmune diseases PKCß1 was identified as the kinase and PP1 as the phosphatase relevant for phosphorylation and dephosphorylation of Ser81 PGRN, and inactivation of PP1 seemed to be responsible for the observed phosphorylation of PGRN at the Ser81 residue (37) .

Similarly, in our present work, we found that IL-1-Ra presented as an additional, hyperphosphorylated isoform. It was present exclusively in all patients with high titers of antibodies against IL-1-Ra. Ser97 was identified as a physiologically phosphorylated site and Thr111 as the atypically hyperphosphorylated amino acid of IL-1-Ra.

Interestingly, similar to the reported preceding appearance of pSer81 PGRN prior to PGRN-Abs in rheumatic diseases (37) Whatever the primary cause of the loss of self-tolerance to PGRN and IL-1-Ra may be, the present observations raise the question what potential clinical implications the autoantibodies might have. Notably, PGRN and IL-1-Ra plasma levels were substantially reduced in patients with PGRN-Abs and IL-1-Ra-Abs, respectively, as compared to seronegative patients (Fig 3   A ). This contrasts with the finding that patients with COVID-19 had elevated PGRN and IL-1-Ra plasma levels, as detected by multiplex assays (49)(50)(51)(52). Native Western blots could possibly provide an explanation for this discrepancy. Levels of free IL-1-Ra and PGRN were strongly decreased, and Ig-bound IL-1-Ra and PGRN represented the majority in antibody-positive patients (Fig. 3 B and 3 F) . Dilution series with rec. IL-1-Ra with or without addition of rec. or purified IL-1-Ra-Abs validated the commercial ELISA and showed, that it measures only free IL-1-Ra (supplementary Fig. 9 ).

Functional confirmation of the neutralizing effect of PGRN-Abs was obtained in a TNF-αinduced cytotoxicity assay, which showed that plasma from PGRN-Ab-positive patients was less effective than plasma from PGRN-Ab-negative patients in inhibiting the effect of TNFa in vitro (Fig. 3 C and 3 D) . The proinflammatory capacity of IL-1-Ra-Abs was confirmed with HEK IL-1 reporter cells, in which IL-1-Ra-Abs functionally neutralized both recombinant human IL-1-Ra and anakinra (Fig. 3 G and H) . It thus appears plausible that the autoantibodies detected in the present study have caused the observed massive reduction in the circulating levels of PGRN and IL-1-Ra. This notion is further supported by the observation of immunocomplexes of PGRN and IL-1-Ra. It is thus tempting to speculate that this reduction of two anti-inflammatory regulators might contribute to a proinflammatory milieu in a relevant subgroup of critically affected patients with COVID-19 and is associated with significantly higher morbidity and mortality. The resulting proinflammatory shift in the inflammatory balance due to PGRN-Abs and IL-1-Ra-Abs would represent a different mechanism compared to autoantibodies neutralizing type I IFN, which primarily weaken the antiviral immune response (16) . In addition to the in vitro data, PGRN-and IL-1-Ra-Abs were significantly more frequently observed in patients who required intensive care treatment.

Furthermore, the presence of IL-1-Ra-Abs or of IL-1-Ra-Abs in combination with PGRN-Abs was associated with an increased mortality from COVID-19 in hospitalized patients.

The current results raise several intriguing questions for future investigations. In particular, it will be interesting to clarify i) who is susceptible to develop hyperphosphorylated immunogenic isoforms of PGRN and IL-1-Ra and ii) what are the molecular underlying mechanisms for this formation of hyperphosphorylated isoforms in the context of COVID-19.

Another question is whether the presence of PRGN-Abs and IL-1-Ra-Abs (and the ensuing neutralization of PGRN and IL-1-Ra) in patients with critical COVID-19 represents a causal factor in the development of an autoimmune-like vasculitis (4).

A further potential implication of autoantibody-induced downregulation of two key inflammatory pathways is that targeted therapeutic approaches for this subgroup of patients might consist in targeted reinforcement of these impaired anti-inflammatory pathways.

A possible next step towards evaluating the potential of a more-targeted therapy might be to perform post-hoc analyses of plasma samples from prospective trials investigating agents modulating these pathways and related pathways like IL-6 and to look for correlations with baseline characteristics, response and outcome (6) , (9) , (8) , (7) , (53) , (11) , (54) . This would be of particular interest for the modulation of the IL-1 pathway, as anakinra, which is a recombinant fragment spanning from amino acids 26 to 177 of human IL-1-Ra, was in-vitro functionally neutralized by plasma of IL-1-Ra-Ab-positive patients. However, it is not possible to draw a simple conclusion about the clinical effect of anakinra in IL-1-Ra-Ab-positive patients.

Furthermore, the presence of IL-1-Ra-Abs has to be investigated in other autoimmune and autoinflammatory diseases.

Our study has several limitations. Unfortunately, no samples from prospective clinical trials or from different geographic regions and ethnic groups could be examined, and. Another weakness is that only selected known proinflammatory autoantibodies as PGRN-or IL-10-Abs or hypothesis-based possible candidate autoantibodies were investigated. This limited search, on the other hand, has led to a focused and detailed characterization.

In addition, the previously unknown hyperphosphorylated isoform of IL-1-Ra should be further characterized in future studies, similar to the reported distinct function of pSer81 PGRN in autoimmune diseases (37) . Future studies need to verify the frequency of these autoantibodies in critical COVID-19, to analyze the time course of their occurrence and their clinical relevance including the prognostic and predictive value and therapeutic options.

Finally, it should be addressed, whether these or other pathogenic autoantibodies play a role in multisystem inflammatory syndrome in children (MIS-C). individual whole blood cell lysates were utilized at a dilution of 1:100. ELISA was performed according to standard protocols. For the detection of the hyperphosphorylated pSer81 or the non-phosphorylated Ser81 PGRN isoform, phospho-Ser81-or non-phospho-Ser81 PGRNspecific Fabs, which had previously been selected by phage display screening (37) , were used at a concentration of 10 µg/ml. Following this, corresponding biotinylated antihuman Fab secondary antibodies and subsequently peroxidase-labeled streptavidin (Roche) were used.

Using QuickChange II Site-Directed Mutagenesis Kit (Stratagene) and IL1RN full-length FLAG-tagged DNA fragments, all candidate phosphorylation sites were mutagenized. In each case, the amino acids serine (S) and threonine (T) were replaced by alanine (A) and tyrosine (Y) was replaced by phenylalanine (F) disabling phosphorylation. This exchange was performed for IL1RN with S28A, S32A, S33A, T47A, Y49F, Y59F, S93A, S97A, T101A, T111A, S114A, S128A, S130A, T133A, T134A, S135A, S138A, T148A, S157A, T159A, T169A and Y172F. These FLAG-tagged mutants were cloned into pRTS and expressed in electroporated primary monocytes isolated by CD14 MACS (Pan Monocyte isolation kit, Miltenyi, Germany) from of a patient with hyperphosphorylated IL-1-Ra and IL-1-Ra-Abs and a control without hyperphosphorylated IL-1-Ra and negative for IL-1-Ra-Abs and subsequent cultivation with RPMI and 20% FCS with M-CSF at 10ng/ml and IL-4 at 1ng/ml.

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. After blocking with 1.5% (w/v) gelatin in Trisbuffered saline (TBS) and washing steps with TBS with Triton X-100, HIS-tagged pSer81specific recombinant Fabs (which had previously been selected by phage display screening (37)), were added at 10 µg/ml followed by washing steps with TBS with Triton X-100 and addition of lysates of PBMCs of patients with the pSer81 isoform. This was followed again by IL-1-Ra or PGRN were performed from 10,000 pg/ml, 1000 pg/ml, 100 pg/ml, 10 pg/ml and 0 pg/ml with or without the addition of 0.05µg rec. IL-1-Ra-Abs or rec. PGRN-Abs or from patient's plasma (Val16) purified IL-1-Ra-Abs or PGRN-Abs.

To assess the functional effects of PGRN-Abs in vitro, a nonradioactive viability assay (EZ4U Cell Proliferation Assay; Biomedica, Vienna, Austria) was performed. For this TNF-αinduced cytotoxicity indicator assay, we used the highly TNF-α-sensitive mouse fibrosarcoma WEHI-S cell line as target cells. In short, 4x10 4 WEHI-S cells were seeded into 200 μl of cell culture at 37°C and 5% CO2. To detect possible differences of TNF-a inhibiting activity in plasma between patients with or without PGRN-Abs, plasma of patients with COVID-19 with or without PGRN-Abs was added in dilutions from 1:8 to 1:512 to cultured WEHI-S cells, followed by administration of TNF-α (100 pg/ml). WEHI-S cells without addition of TNF-α and plasma, or solely with addition of TNF-α (100 pg/ml), were used as positive and negative controls, respectively. After 48 hours of incubation at 37°C, chromophore substrate was added to each well. This chromophore substrate is converted only by vital cells. The adsorption of the product was measured at an OD of 450 nm. Nonetheless the very short halflive of TNF-a compared to PGRN, to exclude any possible unknown interferences from other plasma components, the MTT assay was repeated with TNF-a (100 pg/ml) and recombinant PGRN (10ng/ml) with either recombinant PGRN-Abs, PGRN-Abs purified from plasma of patient Val16 at concentrations from 0µg/ml to 10 µg/ml or as a control SLP2-paraprotein purified from a patient with multiple myeloma at 10 µg/ml (56) .

For IL-1ß assay HEK-Blue™ IL-1β reporter cells (Invivogen, #hkb-il1bv2) were used, which react specifically to IL-1ß and IL-1α by induction of NF-κB/AP-1, leading to expression of a were screened by ELISA for reactivity against recombinant HIS-tagged SARS-CoV-2 S1and S2-proteins, N-protein and M-protein expressed in HEK293 cell (ABIN) and against reactivity against FLAG-tagged PGRN, precursor of IL-1-Ra isoform 1 and IL-1-Ra isoform 2. PGRN-Abs were purified from plasma patient #10 and #20 of the cohort with moderate and severe COVID-19 infection, respectively. Plasma from two healthy control, from a patient with rheumatologic disease with PGRN-Abs and without COVID-19, from a patient with rheumatologic disease without PGRN-Abs and without COVID-19 served as controls.

Comparrison of proportions was done with two-tailed Fisher exact test. IL-1 Ra and PGRN plasma levels were analyzed with D'Agostino and Pearson normality test and Shapiro-Wilk normality test. Plasma levels were then compared by nonparametric, two-tailed Mann-Whitney test. In the samples of patients with PRGN-Abs, an additional and more negatively charged PGRN isoform appeared in the gel, which was not detected in healthy controls and PGRN-Abnegative patients. B) ELISA for pSer81 PGRN isoform and for non-phosphorylated Ser81 PGRN isoform. C) WB and IEF of IL-1-Ra in IL-1-Ra-Ab-positive patients with moderate to severe COVID-19. An additional and differentially charged IL-1-Ra isoform appeared in patients of the discovery cohort, which was not detected in a healthy controls and IL-1-Ra-Ab-negative patients. D) IEF of IL-1-Ra isoforms from two IL-1-Ra-Ab-positive patients (#21 and #4) and a healthy control without IL-1-Ra-Abs before and after alkaline phosphatase treatment. Alkaline phosphatase treatment led to disappearance of the second phosphorylated 

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Progranulin deficiency exaggerates, whereas progranulin-derived Atsttrin attenuates, severity of dermatitis in mice

Prevention of lps-induced acute lung injury in mice by progranulin

Progranulin deficiency leads to severe inflammation, lung injury and cell death in a mouse model of endotoxic shock

Progranulin Improves Acute Lung Injury through Regulating the Differentiation of Regulatory T Cells and Interleukin-10 Immunomodulation to Promote Macrophage Polarization

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains

The molecular basis for development of proinflammatory autoantibodies to progranulin

Evidence for non-neutralizing autoantibodies against IL-10 signalling components in patients with inflammatory bowel disease

A frequent target of paraproteins in the sera of patients with multiple myeloma and MGUS

Association of a dominantly inherited hyperphosphorylated paraprotein target with sporadic and familial multiple myeloma and monoclonal gammopathy of undetermined significance: a case-control study

Gout-associated uric acid crystals activate the NALP3 inflammasome

A pilot study of IL-1 inhibition by anakinra in acute gout

Development and effect of antibodies to anakinra during treatment of severe CAPS: sub-analysis of a long-term safety and efficacy study

Neutralizing anti-IL-1 receptor antagonist autoantibodies induce inflammatory and fibrotic mediators in IgG4-related disease

Citrulline is an essential constituent of antigenic determinants recognized by rheumatoid arthritis-specific autoantibodies

T-cell epitope of the autoantigen myelin basic protein that induces encephalomyelitis

Hyper N-glycosylated SAMD14 and neurabin-I as driver CNS autoantigens of PCNSL

Serum protein profiling reveals a specific upregulation of the immunomodulatory protein progranulin in COVID-19

Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and RANTES with mild disease

Discrimination of COVID-19 from inflammation-induced cytokine storm syndromes by disease-related blood biomarkers

Distinct inflammatory profiles distinguish COVID-19 from influenza with limited contributions from cytokine storm

Blockage of interleukin-1β with canakinumab in patients with Covid-19

Interleukin-1 blockade with high-dose anakinra in patients with COVID-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study

Progranulin antibodies in autoimmune diseases

Autosomal-dominant inheritance of hyperphosphorylated paratarg-7

isoform, which occurs also in the healthy control, and of the third, atypical and hyperphosphorylated isoform

Only Ser97Ala and Thr111Ala led to the disappearance of the third band of IL-1-Ra. Regarding point-mutated, FLAG-tagged constructs of IL-1-Ra expressed in healthy control-derived monocytes (right) for every construct only two bands were seen, with the exception of Ser97Ala

This could be detected for IL-1-Ra-Abs and PGRN-Abs in patient #109, #110, Val62 and Val57. In patient #115 initially hyperphosphorylation of PRGN was observed while no hyperphosphorylation of IL-1-Ra was observed. One week later hyperphosphorylated IL-1-Ra appeared, followed by IL-1-Ra-Abs of IgM class one week thereafter. G) In patient #10, who presented initially with critical COVID-19 and was seropositive for PGRN-Abs, IL-1-Ra-Abs and both hyperphosphorylated isoforms, in a follow-up visit 10 month later neither these autoantibodies nor the hyperphosphorylated isoforms were detected anymore

A) PGRN plasma levels in patients with COVID-19 and ICU controls. PGRN plasma levels in controls from ICU without COVID-19 and without PGRN-Abs (median 206.05 ng/ml), and in patients with COVID-19 without PGRN-Abs (median 161.23 ng/ml) were higher compared to high-titer PGRN-Ab-positive patients with moderate to severe COVID-19 (median 15.21 ng/ml)

Western blots under non-reducing conditions and without SDS, showed beside converted, mature granulins at approximately 10kDa, reduced levels of free PGRN at approximately 80kDa in PGRN-Ab-positive (Val43, Val48, #109) compared to PGRN-Abnegative patients with COVID-19 (#97, #98) or with rheumatic disease

WEHI-S cells were incubated with TNF-α (or PBS) and plasma of PGRN-Ab-positive patients (COVID-19 #9 and #26) or matched PGRN-Abnegative patient (COVID #5), as indicated. The plasma of the patients #9 and #26 with PGRN-Abs resulted in a weaker inhibition of TNF-α-mediated cytotoxicity. The adsorbance of colored Formazan, as a marker for cell viability was detected at 450 nm. D) Effect of recombinant PGRN-Ab on inhibition of TNF-α-induced cytotoxicity. Purified PGRN-Ab from plasma of patient Val16 both at concentrations from 0µg/ml to 10µg/ml or purified SLP2-Ab at 10µg/ml as a control purified from a patient with multiple myeloma were added to WEHI-S cells incubated with TNF-α and recombinant PGRN. E) IL-1-Ra plasma levels in patients with COVID-19 and ICU controls. IL-1-Ra plasma levels were determined in patients with COVID-19 without IL-Ra-Abs (median 981.8 pg/mL) and in IL-1-Ra-Ab-positive patients with COVID-19 (median 214.4 pg/mL). Data are represented with median and interquartile range. F) Native Western-blot on band strength of IL-1

Effect of IL-1-Ra-Ab status on inhibition of IL-1ß. HEK IL-1 reporter cells were incubated with IL-1ß or TNF-α, rec. human IL-1-Ra, anti-IL-1-Ra-Ab, anti-SLP2-Ab as control, and 1:20 diluted plasma of IL-1-Ra-Abpositive patient (Val 16) or of a matched IL-1-Ra-Ab-negative patient

The diluted plasma of the patient Val 16 with IL-1-Ra-Abs resulted in a weaker inhibition of IL-1ß. The adsorbance of SEAP, as a marker for IL-1 pathway activation in HEK IL-1 reporter cell was detected at 650 nm. H) Effect of IL-1-Ra-Ab status on inhibition of IL-1ß

HEK IL-1 reporter cells were incubated with IL-1ß or TNF-α, anakinra, anti-IL-1-Ra-Ab, anti-SLP2-Ab as control, and plasma diluted 1:20 of IL-1-Ra-Ab-positive patient (Val 16) or a matched IL-1-Ra-Ab-negative patient