key: cord-0871088-e956y367
authors: Rodriguez-Perez, Ana I.; Labandeira, Carmen M.; Pedrosa, Maria A.; Valenzuela, Rita; Suarez-Quintanilla, Juan A.; Cortes-Ayaso, María; Mayán-Conesa, Placido; Labandeira-Garcia, Jose L.
title: Autoantibodies against ACE2 and angiotensin type-1 receptors increase severity of COVID-19
date: 2021-06-11
journal: J Autoimmun
DOI: 10.1016/j.jaut.2021.102683
sha: b106bf94009ec3f9a1352bf64dc6cc51b3bd4243
doc_id: 871088
cord_uid: e956y367

The renin-angiotensin system (RAS) plays a major role in COVID-19. Severity of several inflammation-related diseases has been associated with autoantibodies against RAS, particularly agonistic autoantibodies for angiotensin type-1 receptors (AA-AT1) and autoantibodies against ACE2 (AA-ACE2). Disease severity of COVID-19 patients was defined as mild, moderate or severe following the WHO Clinical Progression Scale and determined at medical discharge. Serum AA-AT1 and AA-ACE2 were measured in COVID-19 patients (n = 119) and non-infected controls (n = 23) using specific solid-phase, sandwich enzyme-linked immunosorbent assays. Serum LIGHT (TNFSF14; tumor necrosis factor ligand superfamily member 14) levels were measured with the corresponding assay kit. At diagnosis, AA-AT1 and AA-ACE2 levels were significantly higher in the COVID-19 group relative to controls, and we observed significant association between disease outcome and serum AA-AT1 and AA-ACE2 levels. Mild disease patients had significantly lower levels of AA-AT1 (p < 0.01) and AA-ACE2 (p < 0.001) than moderate and severe patients. No significant differences were detected between males and females. The increase in autoantibodies was not related to comorbidities potentially affecting COVID-19 severity. There was significant positive correlation between serum levels of AA-AT1 and LIGHT (TNFSF14; r(Pearson) = 0.70, p < 0.001). Both AA-AT1 (by agonistic stimulation of AT1 receptors) and AA-ACE2 (by reducing conversion of Angiotensin II into Angiotensin 1-7) may lead to increase in AT1 receptor activity, enhance proinflammatory responses and severity of COVID-19 outcome. Patients with high levels of autoantibodies require more cautious control after diagnosis. Additionally, the results encourage further studies on the possible protective treatment with AT1 receptor blockers in COVID-19.

1 Many recent studies have highlighted the major role of renin-angiotensin system (RAS) in 2 severity of COVID-19 [1] [2] [3] . Angiotensin converting enzyme 2 (ACE2) plays a key role in the 3 process and it is usually considered that ACE2 acts as a double-edged sword [4, 5] . First, ACE2 4 transforms components of the pro-inflammatory RAS axis such as angiotensin I (Ang I) and 5

particularly Ang II into components of the anti-inflammatory RAS axis such as Ang1-9 and 6

particularly Ang 1-7. Ang II acts on angiotensin type 1 (AT1) receptors and activates the 7 NADPH-oxidase complex producing superoxide and promoting cell pro-oxidative and pro-8 inflammatory responses [6, 7] , while Ang 1-7 acts on Mas and Mas-related receptors promoting 9 cell antioxidative and anti-inflammatory responses. Therefore, an increase in ACE2 activity is 10 essential to balance the RAS towards the anti-inflammatory response. Consistent with this, the 11 protective effects of ACE2 and its product Ang1-7 against experimental lung injuries have 12 previously been demonstrated [8] . Second, ACE2 is also the entry receptor for the virus [9] , and 13 upregulation of ACE2 expression may enhance cell infection. Third, both ACE2 functions 14 interact each other, since SARS-CoV-2 binding to ACE2 decreases the levels of ACE2 at the 15 cell surface [10, 11] , thus shifting the RAS balance towards the pro-inflammatory Ang II/AT1 16 axis, which leads to inflammation, fibrosis and progression of disease severity. 17

It is known that differences in immunological responses in different patients play a 18 major role in progression and severity of COVID-19. Progression and severity of several 19 1 women and 9 men) and 119 COVID-19 patients were enrolled in the study. The patient group 2 included, 31 mild, 68 moderate and 20 severe patients, considering the clinical course of the 3 disease at their medical discharge ( Table 1 ). The severe group included 16 patients who 4 required mechanical or non-invasive ventilation (scores 6-9) and 4 patients who died (score 10). 5

Mean age of patients was 56.5 years old (56.5±14.9 SD; range 20-93) and 48.3% were males. 6

The most relevant comorbidities in patient´s group included hypertension (33.8%), diabetes 7 (12.8%), dyslipidemia (38.5%), obesity (35.3%), heart disease (7.6%), lung disease (8.2%) and 8 renal disease (2.7%). Main clinical features are presented in Table 1 . No significant differences 9 in frequency of comorbidities were observed between control group and COVID-19 group. 10

Relationships between COVID-19 severity and different comorbidities were studied using chi-11 square test or Fisher's exact test (see Methods). A significant association (X 2 Pearson (2)=7.82; 12 p=0.020) was found between severity and diabetes; severity and obesity (X 2 Pearson (2)=13.92; 13 p=0.001) and severity and former smokers (X 2 Pearson (2)=6.56; p=0.038). 14 15

Serum anti-AT1 and anti-ACE2 autoantibodies. revealed significantly higher serum levels for AA-AT1 (p = 0.002) and for AA-ACE2 (p=0.013) 23 in COVID-19 group than in the control group. However, no significant differences were 24 detected between males and females for serum levels of AA-AT1 (p = 0.845) and AA-ACE2 (p 25 = 0.342) when sex differences were tested together with controls/patients, being the interaction 26 terms not significant for both levels of autoantibodies. (Figure 1A , B).

Then, we compared results from the three groups of patients with different severity. We 1 observed significant differences, demonstrating association between disease severity and serum 2 AA-AT1 and AA-ACE2 (Figure 2A The correlation coefficient between AA-AT1 and AA-ACE2 was statistically significant; 10 however, it is too low to consider that there is a relevant correlation between these variables and 11

suggests that other major factors contribute to the increase observed in serum in AA-ACE2 12 levels (see Discussion). Sex differences were studied together with severity ( Figure 2C , D; 13 Supplementary Table 1 ). However, no significant differences were detected in our patients 14 between males and females for serum levels of AA-AT1 (two-way ART-ANOVA coefficient, p 15 = 0.95) and AA-ACE2 (two-way ART-ANOVA coefficient, p = 0.14). 16 17

In order to know whether the increase in autoantibodies observed in our patients could be 20 related to comorbidities potentially affecting COVID-19 severity, serum levels of AA-AT1 and 21 only AA-ACE2 levels were significantly higher in patients with diabetes ( Figure 3A ). Moreover, we studied the possible relationship between COVID-19 25 severity and diabetes using chi-square test. A significant association (X 2 Pearson =7.82; p=0.020) 26 was found between severity and diabetes. Then, we analysed whether the relationship of disease 1 systemic sclerosis and primary Sjögren's syndrome [24] . The present results suggest that AA-2 AT1 contribute to COVID-19 severity, as AA-AT1 bind AT1 receptors and, together with 3

Angiotensin II, increase the activity of the RAS pro-inflammatory axis (summarized in Figure  4 4). It has been shown that Ang II enhances severity of several SARS experimental models, 5 which is reduced by AT1 blockers [8, 10] . In a recent study, we have observed that AT1 6 blockers (i. e. ARAII, ARBs, sartans) increased levels of transmembrane ACE2 in the lung 7 while decreased internalization of viral spike protein by several mechanisms [10] . The present 8 results suggest that patients with high levels of AA-AT1 are particularly suitable candidates to 9 treatment with AT1 receptor blockers, which have been considered beneficial for COVID- 19 10 outcome in several recent studies [25] [26] [27] . Increasing evidence indicates that treatments with 11 AT1 receptor blockers should not be discontinued in patients with COVID-19 or at risk of (TG2), has been revealed as a major mechanism for generation of AA-AT1. TG2 modifies and TG2-modified AT1 receptors act as neoantigens that promote AA-AT1 generation [16] and AT1 3 receptor sensitization [38] . Consistent with this, we observed a significant correlation between 4 AA-AT1 and LIGHT levels in COVID-19 patients. 5

A reduction in ACE2 activity at the cell surface (i. e. transmembrane ACE2) shifts the 6 RAS balance towards the pro-inflammatory axis, as ACE2 transforms Ang II into the anti- Our study has some limitations. A first limitation of the study is the relatively low 24 sample size. Severity groups were not balanced, as most of the patients belong to the moderate 25 group. Another limitation of this study is that we cannot exclude any possible influence of anti-26 COVID-19 medications in clinical outcomes, but most of the patients from the same severity group received similar treatments. The cross-sectional methodology cannot determine causality 1 but association, and we did not include changes in levels of autoantibodies with time. However, 2 we were particularly interested in the association between early (i. e. at diagnosis) levels of 3 autoantibodies and prediction of severity of the disease. Additional measurements during the 4 course of the disease would also be interesting. This may be possible in hospitalized patients. 5

However, patients of the mild group were sent home for quarantine, and sequential 6 measurements are practically impossible in this group. In a group of severe or moderate patients 7

with several autoantibody measurements, we did not observe significant changes between 8 samples during the disease course. Consistent with this, it was observed that the increase in AA-9 AT1 levels in preeclampsia usually declines 50% by a week after delivery [43] , and persists in 10 patients for more than 1 year postpartum [44] . In a more recent study, circulating AT1-AA were 11 

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