key: cord-0289088-fzssgy8k
authors: Pfeifer, J.; Thurner, B.; Kessel, C.; Fadle, N.; Regitz, E.; Hoffmann, M.-C.; Kos, I.; Preuss, K.-D.; Fischer, Y.; Roemer, K.; Lohse, S.; Heyne, K.; Detemple, M.-C.; Fedlmeier, M.; Juenger, H.; Sauer, H.; Meyer, S.; Rohrer, T.; Wittkowski, H.; Masjosthusmann, K.; Becker, S. L.; Smola, S.; Bewarder, M.; Boehm, M.; Anton, J.; Pino-Ramirez, R. M.; Abdul-Khaliq, H.; Foell, D.; Thurner, L.
title: Autoantibodies against IL-1-receptor-antagonist in multisystem inflammatory syndrome in children
date: 2021-09-12
journal: nan
DOI: 10.1101/2021.09.08.21263027
sha: 99b75e17f9288859e5d86b64e4f8a71f97eca8cb
doc_id: 289088
cord_uid: fzssgy8k

Multisystem inflammatory syndrome in children (MIS-C or PIMS) is a rare but serious complication after an infection with SARS-CoV-2. A possible involvement of pathogenetically relevant autoantibodies has been discussed. Recently neutralizing autoantibodies against anti-inflammatory receptor antagonists progranulin (PGRN) and IL-1-receptor antagonist (IL-1-Ra) were discovered in adult patients with critical COVID-19. Plasma of an index case with severe PIMS/MIS-C was analyzed for autoantibodies against IL-1-Ra and PGRN. The study was extended by a case series of 12 additional patients. In addition to ELISA for of antibodies, IL-1-Ra plasma levels were determined and IL-1-Ra was analyzed by Western-blot and isoelectric focusing. Functional activity of the autoantibodies was examined in vitro with IL-1beta reporter assays. Antibodies against IL-1-Ra could be detected in 10 of 13 (76.9%) patients with PIMS/MIS-C, but not in controls. In contrast to critical COVID-19 in adults, no IL-1-Ra antibodies of the IgM class were detected in PIMS/MIS-C. IL-1-Ra-antibodies exclusively belonged to IgG1. No antibodies directed against PGRN were detected. Western blots and ELISA showed a concomitant reduction of free IL-1-Ra plasma levels in the presence of IL-1-Ra-antibodies. The antibodies inhibited IL-1-Ra function in IL-1beta reporter cell assays. Notably, an additional, hyperphosphorylated, transiently occurring atypical isoform of IL-1-Ra was observed in all IL-1-Ra autoantibody-positive patients. To conclude, IL-1-Ra autoantibodies were observed in high frequency in children with PIMS/MIS-C. They may represent a diagnostic and pathophysiologically relevant marker for PIMS/MIS-C. Their generation is likely to be triggered by an atypical, hyperphosphorylated isoform of IL-1-Ra.

SARS-CoV-2 infection typically takes a mild or asymptomatic course in children. Pediatric inflammatory multisystem syndrome (PIMS) or multisystem inflammatory syndrome in children (MIS-C) is a rare but serious complication that usually occurs after an interval of 2 to 6 weeks after infection with SARS-CoV-2 (1)(2)(3). All affected children have persistent fever, other clinical features may vary. Acute abdominal pain, diarrhea or vomiting, muscle pain, headache and fatigue are frequently. Bilateral conjunctival injection (hyperemia) and exanthema, swollen hands and feet and a "strawberry tongue" resembling Kawasaki´s disease have been described in a high percentage of patients with PIMS/MIS-C. Affection of the cardiovascular system with myocardial dysfunction, arterial hypotension or systemic shock all have been regularly observed. Myocarditis, myocarditis, pericarditis and involvement of the valves and coronary arteries (dilatation or aneursyma) may develop. Laboratory studies regularly show intensive inflammation with elevated serum levels of C-reactive protein, procalcitonin, ferritin, cardiac involvement with elevated troponin and N-terminal pro-B-type natriuretic peptide, as well as hyponatremia, markers of coagulopathy (elevated D-dimers, prolonged prothrombin time and partial thromboplastin time) and hematologic abnormalities (anemia, lymphocytopenia and thrombocytopenia or thrombocytosis). Most affected children will need intensive care due to multiorgan failure and shock (4)(5)(6)(7)(8) (9) .

Little is known about the pathogenesis of PIMS/MIS-C. It has been suggested that the formation of pathogenetically relevant autoantibodies contributes to a hyperinflammatory state (10) . This is based on the observations that i) there is a interval between the infection with SARS-CoV-2 and the manifestation of the disease in the majority of cases and ii) the patients typically respond to intravenous immunoglobulins (IVIGs) and glucocorticoids.

Besides stabilization with fluids, vasoactive or inotropic drugs, oxygen, acetylsalicylic acid in cases with cardiac involvement and thrombocytosis, anticoagulation in severe impairment of the left ventricle, the administration of IVIGs and steroids are presently standard-of-care. The American college of Rheumatology recommends high dose anakinra for IVIG-refractory PIMS/MIS-C, a recombinant modified human IL-1-Ra (11)(12) (13) .

Recently, in adult patients with severe and critically ill with COVID-19, we discovered neutralizing autoantibodies against progranulin (PGRN), an anti-inflammatory ligand of the pro-inflammatory receptors TNFR1/TNFR2 and DR3, and a direct receptor antagonist of TNF-α as well as TL1a. In these patients we also discovered autoantibodies against interleukin-1 receptor antagonist (IL-1-Ra), the ligand of the pro-inflammatory IL-1 receptor and antagonist of IL-1α and Il-1β.

In addition, we found evidence that the autoantibodies probably have been evoked by a preceding and transient hyperphosphorylation of the antigens. The antibodies belonged to a broad range of Ig classes including IgM and several IgG subclasses, indicating recent immune responses (14) . PGRN-antibodies (Abs) and the immunogenic pSer81 PGRN isoform have been initially described in systemic primary vasculitides (15) (16) . As critical COVID-19 can partially resemble hyperinflammatory syndromes and has similarities to hemophagocytic lymphohistiocytosis (HLH), autoinflammatory syndromes (17), Kawasaki disease (9) , and vasculitides (18) we performed a targeted screen for these autoantibodies and posttranslationally altered isoforms of the corresponding autoantigens, in a young girl affected with PIMS/MIS-C.

A formerly healthy girl, with preexisting mild allergic rhinitis due to pollen allergy, in the age group between 6 and 10 years was admitted with a history of persisting high fever for 5 days, abdominal pain, vomiting, diarrhea, slight bilateral (non-purulent) conjunctivitis and exanthema. The general condition was poor. Six weeks before admission she had a PCRconfirmed mild infection with Sars-CoV-2 detected due to another positive case in the family.

On admission, echocardiography showed a reduced left ventricular shortening fraction (25%), mitral regurgitation, and progressive dilatation of the right coronary artery occurred (maximal ostial diameter 6 mm, Z-score +8,8), interestingly with a congenitally single coronary ostium.

Bilateral pleural effusions and ascites were present, as well as tachycardia, tachypnoea and arterial hypotension. Laboratory results on admission showed elevated C-reactive protein of 177 mg/l, elevated NT-pro-BNP of 2,069 pg/ml (in the course max. 52,000 pg/ml), hyponatremia of 129 mmol/l and hypokalemia of 3.0 mml/l. Antibodies against Sars-CoV-2 were present.

The diagnosis of PIMS/MIS-C was suspected. Differential diagnoses were Kawasaki disease, macrophage activation syndrome, bacterial sepsis, toxic shock syndrome and other inflammatory or infectious diseases. Treatment with volume resuscitation, high-dose acetylsalicylic acid (30 mg/kg/d), IVIG (2 g/kg) and antibiotic treatment with piperacillin / tazobactam and metronidazole was initiated. Due to development of shock symptoms she was transferred to pediatric ICU requiring inotropic support (epinephrine, milrinone, levosimendan). The therapy included non-invasive ventilation (max. FiO2 0.5), bilateral pleural and ascites drainages, fluid and electrolyte management, buffering, erythrocyte transfusion, substitution of antithrombin-III, albumin and fresh-frozen-plasma. After All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  exclusion of a bacterial or viral infection, pulse treatment with intravenous methylprednisolone 30 mg/kg/d for 5 days, followed by prednisolone 2 mg/kg/d (with gradual tapering) was commenced on the second day. A crucial improvement was seen only after the onset of glucocorticoid treatment. 72 h after defervescence the dose of acetylic salicylic acid was reduced to 100 mg per day. The patient underwent a therapy with enalaprile, metoprolole and decongestion with diuretics. On day 16 the girl could be discharged. In follow-up, echocardiographic findings and blood parameters were normalized (restitutio ad integrum). Table 1 :

Plasma samples of the index case and of a case series of further 12 children with PIMS/MIS-C were analyzed. All patients survived PIMS/MIS-C. Table 1 :

In the index case IL-1-Ra-Abs, but no PGRN-Abs were detected. The titers of IL-1-Ra-Abs at presentation were 1:800 (Fig. 1) . IL-1-Ra-Abs belonged to IgG subclass 1 (Fig. 1) , no IL-1-Ra-Abs of IgM class were observed. Three months later the titer had fallen off to 1:400 and no IL-1-Ra-Abs could be detected seven month later.

In the series of further 12 patients with PIMS/MIS-C, in 9 patients IL-1-Ra-Abs were detected with titers ranging between 1:200 to 1:800. All IL-1-Ra-Abs belonged exclusively to IgG1 in all patients. Autoantibodies against progranulin were not detectable in any of these patients with PIMS/MIS-C. In contrast, IL-1-Ra-Abs were not detectable in 6 patients with Kawasaki's disease. Moreover, IL-1-Ra-Abs were not found in plasma or serum samples of 33 children of the control group.

IEF performed on total protein from plasma of the index case at the time of presentation, revealed the presence of an additional, more negatively charged third band of IL-1-Ra.

Pretreatment with alkaline phosphatase before IEF led to the disappearance of both the normally occurring second and the atypical additional third isoform IL-1-Ra isoform, indicating a hyperphosphorylation (Fig. 1) . In the further course, the hyperphosphorylated third signal of IL-1-Ra was no longer detectable at 3 or 7 months after the onset of the inflammatory multisystem syndrome in plasma of the index-patient.

The hyperphosphorylated IL-1-Ra isoform was observed in all 10 patients with IL-1-Ra-Abs but not in any of the controls (Fig. 2 ).

Using standard ELISA, we found that the plasma levels of free IL-1-Ra were strongly reduced Native Western blot of total plasma protein produced a significantly weakened Il-1-Ra signal.

Notably, though, an additional signal representing IgG-bound IL-1-Ra was found during acute MIS-C that was absent 7 month later, when the patient was seronegative for IL-1-Ra-Abs Remarkably, in a patient PIMS-6 of the case series of whom a follow-up sample was available, the initial plasma sample was positive for IL-1-Ra-Abs and the hyperphosphorylated isoform of IL-1-Ra and the initial IL-1-Ra plasma level detected by ELISA was 323.49 pg/ml, whereas in the follow-up sample 5 weeks later lacked both IL-1-Ra-Abs and the hyperphosphorylated IL-1-Ra isoform ( Fig 2F) and the IL-1-Ra plasma level had increased to 1642 pg/ml. Table 3 :

Next, to investigate possible functional effects of the IL-1-Ra-Abs an in vitro IL-1ß assay was applied. Addition of plasma from patients with acute inflammatory syndrome and seropositive for IL-1-Ra-Abs significantly weakened the antagonism by recombinant IL-1-Ra, resulting in a stronger stimulatory effect of IL-1ß. The attenuation was comparable to the one seen upon All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  the addition of a recombinant anti-human IL-1-Ra-antibody and to the addition of plasma from an adult patient with severe COVID-19 that had tested positive for IL-1-Ra-Abs. In contrast, though, addition of plasma from the same patient, at the same dilution, but 7 months after PIMS/MIS-C and lacking IL-1-Ra-Abs, did not weaken the effect of rec. IL-1-Ra (Fig. 3 B).

Here we report on the high prevalence of neutralizing autoantibodies against the antiinflammatory molecule IL-1-Ra in a case-series of patients with PIMS/MIS-C.

Autoantibodies against IL-1-Ra were just recently described by our group in adults with critical COVID-19 (14) ; their occurrence was also reported in IgG4 associated diseases (21) .

In adults with critical COVID-19, IL-1-Ra-Abs were detected at a frequency of about 50%, along with autoantibodies against PGRN (in about 40% of patients). Moreover, in adults with critical COVID-19 we also found hyperphosphorylated isoforms of both IL-1-Ra and PGRN.

All IL-1-Ra-and PGRN-Abs in adult COVID-19 patients belonged to IgM and different IgG subclasses.

In contrast to the findings in adult patients with critical COVID-19, no antibodies directed against PGRN were found in children with PIMS/MIS-C. IL-1-Ra-Abs were found at an even higher percentage in these children (10 of 13; 76,9%), compared to the adults with critical COVID-19. Notably, the plasma levels of free IL-1-Ra were strongly reduced in all patients with autoantibodies and simultaneously antibody-bound IL-1-Ra was detected. IL-1-Ra-Abs in PIMS/MIS-C belonged exclusively to IgG1 subclass, no antibodies of IgM class as in adults with critical COVID-19 were detected (14) . The titers of IL-1-Ra-abs were lower To further shed light on this issue, in-depth analysis of biobanked serum and plasma samples All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  from children with complication-free courses after SARS-CoV-2 infection might be a next step. It should also prove useful to employ stimulation assays with various cytokines in order to trigger the hyperphosphorylated isoform of IL-1-Ra in established cell lines, primary cells of adults, and primary cells of children who i) had developed PIMS/MIS-C and ii) had a complication-free course after SARS-CoV-2 infection. Animal models have shown that low IL-1-Ra levels, either due to neutralization after immunization or due to deficiency, induce or exacerbate inflammatory syndromes such as colitis, arthropathy, vasculitis or psoriasis-like manifestations (22)(23) preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  or had positive PCR, with the exception of one patient who had only reported contact to SARS-CoV2.

In addition, 6 children with Kawasaki disease (age: min 1.4 years, max 7.5 years) and samples of 33 children with suspected growth retardation (age: min 3 years, max 15.3 years, median age 10.6 years) were included.

The ELISA for autoantibodies was performed as previously described (19) . In short, the antigens were obtained using the coding sequences of the GRN gene encoding PGRN, isoform 

Isoelectric focusing (IEF) and Western blotting (including native Western blotting with nonreducing sample pretreatment and gradient gels without SDS) was performed. Plasma samples were analyzed for IL-1-Ra isoforms. Plasma from IL-1-Ra-Ab-positive patient was treated with alkaline phosphatase as previously described using FastAP thermo-sensitive alkaline All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  phosphatase (Fermentas/VWR, Darmstadt, Germany) (20) .

IL-1-Ra plasma levels were determined with a commercially available ELISA kit (Invitrogen/ThermoFisher #BMS2080) according to the manufacturer's instructions.

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 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. showing IgG-and IgM-bound IL-1-Ra. Native Western-blots of plasma from patients with Kawasaki disease J), or of healthy controls K) did neither show reduced bands of free IL-1-Ra nor immunocomplexes of IL-1-Ra. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;  represented with median and interquartile range. **P≤0.01; ***P All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ; 

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263027 doi: medRxiv preprint

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ; https://doi.org/10.1101/2021.09.08.21263027 doi: medRxiv preprint

All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 12, 2021. ;

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