key: cord-0761154-u07jsjmf
authors: Pascolini, Simona; Vannini, Antonio; Deleonardi, Gaia; Ciordinik, Michele; Sensoli, Annamaria; Carletti, Ilaria; Veronesi, Lorenza; Ricci, Chiara; Pronesti, Alessia; Mazzanti, Laura; Grondona, Ana; Silvestri, Tania; Zanuso, Stefano; Mazzolini, Marcello; Lalanne, Claudine; Quarneti, Chiara; Fusconi, Marco; Giostra, Fabrizio; Granito, Alessandro; Muratori, Luigi; Lenzi, Marco; Muratori, Paolo
title: COVID‐19 and immunological dysregulation: can autoantibodies be useful?
date: 2020-09-29
journal: Clin Transl Sci
DOI: 10.1111/cts.12908
sha: fdee8c78c51eabe7059c8f046a107c16a8554747
doc_id: 761154
cord_uid: u07jsjmf

Coronavirus disease 2019 (COVID‐19) is often associated with interstitial pneumonia. However, there is insufficient knowledge on the presence of autoimmune serological markers in patients with COVID‐19. We analyzed the presence and role of autoantibodies in patients with COVID‐19‐associated pneumonia. We prospectively studied 33 consecutive patients with COVID‐19, 31 (94%) of whom had interstitial pneumonia, and 25 age‐ and sex‐matched patients with fever and/or pneumonia with etiologies other than COVID‐19 as the pathological control group. All patients were tested for the presence of antinuclear antibodies (ANAs), anti‐antiphospholipid antibodies (APLs), and anti‐cytoplasmic neutrophil antibodies (ANCAs). Clinical, biochemical, and radiological parameters were also collected. Fifteen of 33 (45%) patients tested positive for at least one autoantibody, including 11 who tested positive for ANAs (33%), 8 who tested positive for anti‐cardiolipin antibodies (IgG and/or IgM) (24%), and 3 who tested positive for anti‐β2‐glycoprotein antibodies (IgG and/or IgM) (9%). ANCA reactivity was not detected in any patient. Patients that tested positive for autoantibodies had a significantly more severe prognosis than other patients did: 6 of 15 (40%) patients with autoantibodies died due to COVID‐19 complications during hospitalization, whereas only 1 of 18 (5.5%) patients who did not have autoantibodies died (p = 0.03). Patients with poor prognosis (death due to COVID‐19 complications) had a significantly higher respiratory rate at admission (23 breaths per minute vs. 17 breaths per minute; p = 0.03) and a higher frequency of autoantibodies (86% vs. 27%; p = 0.008). In conclusion, autoantibodies are frequently detected in patients with COVID‐19 possibly reflecting a pathogenetic role of immune dysregulation. However, given the small number of patients, the association of autoantibodies with an unfavorable prognosis requires further multicenter studies.

With almost 20 million cases and more than 700,000 fatalities to date, 1 Health Organization reports a worldwide mortality of 3.7%. 1 However, up to 29% of critically ill patients die in the hospital, and 50% of patients who receive invasive respiratory support are likely to die during treatment. 6 The predominant cause of death is severe lung failure due to bilateral interstitial pneumonia; acute and organizing diffuse alveolar damage and SARS-CoV-2 persistence in the respiratory tract are the predominant histopathologic findings of postmortem examinations and the leading causes of death. 7 Multiorgan involvement is also observed.

Because of the rapid spread of SARS-CoV-2, physicians have not always been able to follow evidence-based medicine and standardized protocols. Further, the necessity of treating patients with a high risk of mortality has led many practitioners to make common sense-driven treatment decisions. Based on experience from the 2003 SARS and 2012 MERS epidemics and the work of some centers, it was possible to establish some cardinal points on the pathogenesis and treatment of COVID-19:

1) The most common symptoms of COVID-19 are fever, fatigue, and respiratory symptoms, including cough, sore throat, and shortness of breath. Diarrhea and gastrointestinal symptoms are also reported, with 50% of patients having positive results of reverse transcriptase polymerase chain reaction (RT-PCR) tests in feces samples. 8 2) Anywhere from 1.6% to 56.5% of patients may be asymptomatic. [9] [10] [11] [12] [13] Combined with the long incubation period (from 2 to 14 days), this may account for the high level of contagion and global spread.

3) COVID-19 pneumonia is associated with lung damage, and ARDS and robust interferon immunosuppression with lymphopenia are part of the virally induced

This article is protected by copyright. All rights reserved immunosuppression. 8 This loss of "front-line" antiviral defenses may activate a "second wave" of more tissue-aggressive immunity, including exaggerated interleukin-6 (IL-6) production with a secondary cytokine storm and tissue damage. 14 This cytokine storm may play a major role in the pathogenesis of the second phase of COVID-19, initiating viral sepsis and inflammation-induced lung injury that leads to other complications, including ARDS, organ failure, and death. 

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This study was approved by the local ethics committee. Oral or written consent was obtained from the patients.

From March 30, 2020 to May 10, 2020, we prospectively enrolled 47 consecutive patients referred to our hospital for suspected SARS-CoV-2 infection. All patients were evaluated at admission by clinical and respiratory parameters (blood pressure, heart rate, respiratory rate, and arterial blood gas). Complete laboratory panels were obtained from all patients (white blood cells, hemoglobin, platelets, transaminases, bilirubin, urea, creatinine, D-dimer, international normalized ratio, partial thromboplastin time, immunoglobulins, lactate dehydrogenase, IL-6, RCP, and ferritin), and every patient underwent high-resolution lung computed tomography. Fourteen patients were excluded from the study due to repeated negativity of SARS-CoV-2 RT-PCR results, and 33 patients were included for analysis.

Clinical, laboratory, and radiological characteristics and treatment and outcome data were inserted daily in a dedicated database from the electronic medical records.

We also included 25 age-and sex-matched patients with fever and/or pneumonia with etiologies other than COVID-19 as the pathological control group.

Throat and nose swab specimens were collected for the extraction of SARS-CoV-2 RNA 

The clinical and laboratory parameters at baseline are shown in Table 1 . Sixteen (48.4%) of the 33 patients were women, and 17 (51.5%) were men. The median age of the

This article is protected by copyright. All rights reserved patients was 70 years. Twenty-six (78.7%) of 33 patients were admitted to the hospital and discharged after recovery, and 7 (21.2%) died during hospitalization.

Fifteen of 33 patients (45.4%) had at least one autoimmune reactivity: 11 (33.3%) had ANA reactivity, 8 (24.2%) had anti-cardiolipin antibody (IgG and/or IgM) reactivity, and 3 (9.1%) had anti-β2-glycoprotein I antibody (IgG and/or IgM) reactivity. ANCA reactivity was not detected in any patient. Of the ANA-positive cases, 4 (36.3%) had nucleolar staining, 4 (36.3%) had speckled staining, 2 (18.1%) had indeterminate staining, and 1 (9.1%) had homogenous staining ( Table 2 ). The median titer of ANA reactivities was 1:640 (range 1:160-1:5120). Of the APL-positive cases, we observed anti-cardiolipin IgG positivity in 5 cases, anti-cardiolipin IgM positivity in 6 cases, anti-β2-glycoprotein IgG positivity in 2 cases, and anti-β2-glycoprotein IgM positivity in 2 cases, with some overlap (Table 5) .

There were three patients with seropositivity in the control group. Two patients tested positive for ANAs with a speckled pattern (titer 1:80), and one patient was positive for anti-cardiolipin IgG (34 GPL/mL). The frequency of autoantibody positivity in the COVID-19 group was significantly higher than that in the control group (45% vs. 12%; p = 0.03).

The autoantibody-positive subgroup had higher lactate values than the autoantibodynegative subgroup (1 mmol/L vs. 0.86 mmol/L; p = 0.03). Additionally, the autoantibodypositive subgroup had a significantly more severe prognosis; 6 of 15 patients in this subgroup (40%) died due to COVID-19 complications, whereas only 1 of 18 (5.5%) patients in the autoantibody-negative subgroup died (p = 0.03; Table 3 ).

Nine of 33 (27.2%) patients enrolled in the study were admitted to the ICU, two of whom died in the ICU. Both of the patients who died were seropositive for ANAs.

We ultimately divided our patients into those with a good prognosis and those with a poor prognosis (death due to COVID-19 complications). The subgroup with a poor prognosis had a significantly higher prevalence of autoantibodies than the subgroup with a good prognosis (86% vs. 27%; p = 0.008). Furthermore, patients with a poor prognosis had a significantly higher respiratory rate (23 breaths per minute vs. 16 breaths per minute; p =

This article is protected by copyright. All rights reserved 0.03) upon admission and higher IL-6 and serum C-reactive protein levels, although these differences were not significant ( Table 4 ).

The raw data of each patient (both study and control populations) are reported in the supplementary excel file.

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In our single-center study, 45.4% (15 of 33) of patients with COVID-19 had reactivity to at least one autoantibody. Specifically, 33.3% (11 patients) of the patients had ANA reactivity, characterized in one patient by anti-histone antibodies on specific ENAs. A recent study from China 22 including 21 patients documented a slightly higher prevalence of ANA positivity (50% vs. 33%). However, we examined ANA positivity by indirect immunofluorescence, whereas that study examined reactivity by ANA immunoblot.

Furthermore, the studies enrolled two different subsets of COVID-19 patients. In the Chinese study, all patients were in severe (38.1%) or critical (61.9%) condition and were in the ICU of the Huangshi Central Hospital. Conversely, we selected patients admitted from the Emergency Department to a General Medicine Unit or to a Subintensive Care Unit. Nine patients (27.2%) were later admitted to the ICU, only four of whom died. We only observed 7 deaths (21.2%) in this study; this is in line with the overall in-hospital mortality rates from larger studies (24% 23 and 28% 17 ). Furthermore, the aforementioned Chinese study 22 described a subgroup of patients with anti-ENA positivity (specifically anti-SSA Ro52 and anti-SSA Ro60), which we did not observe in our study. This discrepancy among reactivities could be attributable to the low numbers of patients enrolled in the two studies and to the different genetic backgrounds of the populations, which could lead to different antibody phenotypes. It is of interest that the prevalence of autoantibodies was higher in critical cases of COVID-19 than in less severe cases in both of these studies.

The nucleolar pattern of ANAs is often associated with the interstitial pneumonia that characterizes the clinical course of systemic sclerosis. 24 Samples from four of our patients showed nucleolar staining of ANAs, and these patients also had a radiological definitive diagnosis of interstitial pneumonia, but none had reactivity against 0 Scl-7.

Such reactivity usually represents the target antigen in diffuse systemic sclerosis.

We observed an overall frequency of APLs of more than 21% (7 cases), which is at least two-fold more than that reported by Harzallah and colleagues in a series of 56 patients with COVID-19. 14 Furthermore, the presence of APLs in this study was significantly associated with poor prognosis. Unlike Tang et al, 16 who identified coagulopathy as an independent negative prognostic factor, we did not observe any difference in these parameters among patients with poor prognosis and those with good prognosis. This

This article is protected by copyright. All rights reserved difference could be due, at least in part, to the different selection criteria and is also likely affected by the lower number of cases in our series (183 vs. 33). However, we did observe a significant association between autoreactivities and poor prognosis in our series; all but one of our severely ill patients had at least one autoreactivity ( Table 4 ).

The majority of post-infectious APLs differ immunochemically from those seen in patients with autoimmune disease. 25 It is of particular interest that none of our patients had ANCA reactivity. ANCAs are reactive against multiple antigens in the cytoplasmic and perinuclear regions in neutrophils and monocytes. The most prevalent ANCAs in ANCA-associated vasculitis target MPO and PR3 and are strongly associated with small vessel vasculitis. 28 It is believed that, in genetically predisposed patients, the first neutrophil immune response may lead to antigen presentation and chronic immune dysregulation in small vessel vasculitis. 28 However, the immunopathology of vasculitis is still unclear. Several models have suggested that T-lymphocytes may play a leading role in the loss of tolerance and development of autoimmunity. Additionally, some studies have evaluated the cytokine profile in these patients, showing that the cytokines IL-6, IL-10, TNF-α, IFN-γ, and IL-17A are increased in patients with AAV infection. 29, 30 The lack of expression of these markers in our study may be because of the small number of cases. Additionally, our serological tests may have been performed too early to detect antigen presentation and the subsequent rise in autoreactivity titers. By contrast, it is possible that the initial viral immunosuppression may decrease the first neutrophil response and inhibit ANCA production.

Our study has some limitations. It would be inappropriate and risky to attribute clinical significance to the high frequency of autoreactivities among patients with a poor 

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Accepted Article This article is protected by copyright. All rights reserved

A-a O2 gradient (mmHg) median (range)

SBP (mmHg) median (range)

DBP (mmHg) median (range)

HR (bpm) median (range)

We thank all patients and their families involved in the study. We thank all health-care workers involved in the diagnosis and treatment of patients. 

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This article is protected by copyright. All rights reserved INR ratio median (range) 1. Abbreviations: ACL: anti-cardiolipin antibody, Ab2GP: anti-β2-glycoprotein antibody, IgG:, IgM: . Positivity > 40 GPL or MGL/mL for ACL and > 40 Ui/mL for Ab2GP.

Accepted Article