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Does methotrexate influence COVID-19 infection? Case series and mechanistic data

Does methotrexate influence COVID-19 infection? Case series and mechanistic data Background: To investigate whether methotrexate treatment may affect the susceptibility to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: Clinical assessment of symptoms, SARS-CoV-2 RNA, and anti-SARS-CoV-2 IgG in an initial case series of four families and confirmatory case series of seven families, within which one family member developed coronavirus disease 19 (COVID-19) and exposed another family member receiving methotrexate treatment; experimental part with methotrexate treatment of mice and organoids followed by the assessment of mRNA and protein expression of the SARS-CoV-2 receptor angiotensin-converting enzyme (ACE)-2. Results: In the initial case series, three of four women on a joint ski trip developed COVID-19, while the fourth woman, under treatment with methotrexate, remained virus-free. Two of the three diseased women infected their husbands, while the third husband treated with methotrexate remained virus-free. In addition, 7 other families were identified in a follow-up case series, in which one member developed COVID-19, while the other, receiving methotrexate, remained healthy. Experimentally, when mice were treated with methotrexate, ACE2 expression significantly decreased in the lung, in the intestinal epithelium, and in intestinal organoids. Conclusion: These clinical and experimental data indicate that methotrexate has certain protective effects on SARS- CoV-2 infection via downregulating ACE2. Keywords: Methotrexate, Coronavirus disease 19, Infection Background affected organs and the development of coronavirus dis- Severe acute respiratory syndrome coronavirus 2 (SARS- ease 19 (COVID-19 [2]. CoV-2) enters epithelial and other cells through binding The level of ACE expression by lung epithelia may in- to angiotensin-converting enzyme 2 (ACE2) expressed fluence the susceptibility to SARS-CoV-2 infection. For on various epithelial cells including those of the lungs instance, low ACE2 expression in young mammals can and the gut [1]. SARS-CoV-2 entry into human cells potentially explain the overall low susceptibility of chil- triggers cell damage associated with a robust and some- dren to COVID-19 and other zoonotic coronaviruses times overshooting inflammatory responses in the [3]. Furthermore, smoking as well as high salt diet have shown to increase ACE2 expression, which may explain the higher risk for COVID-19 in smokers and those with * Correspondence: georg.schett@uk-erlangen.de hypertension, respectively [4, 5]. In addition, administra- Department of Internal Medicine 3, Friedrich-Alexander University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany tion of recombinant soluble ACE2 saturates cellular sur- Deutsches Zentrum fuer Immuntherapie (DZI), Friedrich-Alexander face ACE2 outcompetes viral binding sites and hence is University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 2 of 6 effective in preventing SARS-CoV-2 infection of human Germany) using the EUROIMMUN Analyzer I platform tissue organoids [6]. according to the manufacturer’s protocol and as de- To date, little is known on whether anti-inflammatory scribed previously [7]. Optical density was determined at drugs used for the treatment of immune-mediated 450 nm with reference wavelength at 630 nm. A cut-off chronic inflammatory diseases (IMIDs), including arth- of ≥0.8 (OD450nm) was considered as positive. Assays ritis, could influence ACE2 expression and affect suscep- were performed in line with the guidelines of the Ger- tibility to SARS-CoV-2 infection. Herein, we report on man Medical Association (RiliBAK) with stipulated in- case series of families, in which one family member de- ternal and external quality controls. veloped COVID-19, while the other family member, tak- ing methotrexate (MTX), remained virus-free and Mice healthy despite substantial viral exposure. Female, C57Bl/6 N mice were maintained under specific pathogen-free conditions at the Präklinisches Experi- Methods mentelles Tierzentrum (PETZ) Erlangen, Germany, and Ethical approval approved by the local ethics authorities of the Regierung Ethical approval (#157_20 B) to conduct this analysis of Unterfranken (#55.2-2532.1-59/14). Nine-week-old was granted by the institutional review board (IRB) of mice were acclimated for 1 week, followed by a 3-week the University Clinic of Erlangen. Written informed con- co-housing period before i.p. treatment with 2 mg/kg sent was obtained from the study participants. mouse methotrexate (n = 6) or vehicle (PBS; n =6) started for three consecutive days before analysis. Two Participants independent experiments were performed. Data were obtained from [1] an initial patient group of 4 women performing a ski holiday at Ischgl (Austria) and Organoids their respective husbands and 7 confirmatory cases from Intestinal organoids were generated as previously de- Italy [2], Brazil [4] and Germany [1] comprising families scribed [8]. In short, the small intestine of wild-type with one member infected with COVID-19 and the C57Bl/6 J was removed, longitudinally opened, and villi other member taking methotrexate staying healthy. In were scraped out. Isolated intestinal crypts were resus- the initial patient group, methotrexate was used for pended in 25 μl/well growth factor reduced Matrigel treatment of psoriasis [1] and rheumatoid arthritis [1]. (Corning, NY, USA). Organoids were cultured in the in- In the confirmatory cases, methotrexate was used for cubator for at least 7 days before any experiments were treatment of rheumatoid arthritis [4] and psoriatic arth- performed. Medium was changed every 2 to 3 days and ritis/spondyloarthritis [3]. Doses of methotrexate were organoids were split once per week. Organoids were between 10 and 20 mg/week. stimulated with 0.25, 0.5, or 1 mM methotrexate for 6 h before analysis. Symptoms Participants were asked for their clinical symptoms Western blot (cough, rhinitis, throat pain, fever, headache, fatigue, Organoids were washed two times with PBS and then musculoskeletal pain, anosmia, shortness of breath, and subsequently lysed with RIPA buffer. Protein extract diarrhea) according to a questionnaire that was used for were separated on a 4–12% SDS-polyacrylamide gel, a field study on anti-SARS-CoV-2 IgG [7]. The observa- transferred to a nitrocellulose membrane, and stained tion period in the initial and confirmatory cases was with antibodies against murine ACE2 (R&D systems). 1 month. An antibody against murine β-actin (Abcam) was used as loading control. Densitometry analysis was performed SARS-CoV-2 RNA test using ImageJ. RNA quantification was down from mucosal swabs using Cobas SARS-CoV-2 real-time RT-PCR Test (Roche, Ba- sel, CH). Histology Lung and intestinal tissue were fixed in 4% formalin. Anti-SARS-CoV-2 antibody testing After deparaffinization, rehydration, and blocking, paraf- Serum samples were taken between March 18 and April fin sections were stained using a primary antibody 30 for anti-SARS-CoV-2 IgG tests. Immunoglobulin G against murine ACE2 (R&D systems). Slides have been (IgG) antibodies against the S1 domain of the spike pro- developed using a DAB staining solution kit after incu- tein of SARS-CoV-2 were tested by the recent CE ver- bation with a HRP-conjugated anti-goat secondary anti- sion (April 2020) of the commercial enzyme-linked body. Quantification of stained area per slide was immunosorbent assay from Euroimmun (Lübeck, performed using ImageJ. Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 3 of 6 Statistical analysis (Fig. 1a). Anosmia was reported by all of them. The Data are expressed as mean ± s.d. Analysis was per- fourth woman taking methotrexate developed no symp- formed using Student’s t test, single comparison, or ana- toms. Five days after the start of symptoms, RNA tests lysis of variance (ANOVA) test for multiple comparisons for SARS-CoV-2 were taken from mucosal swabs and (two-way ANOVA followed by Tukey’s or Bonferroni’s the three women with symptoms were all tested positive. multiple comparisons test). All experiments were con- The woman taking methotrexate showed a negative ducted at least two times. P values of 0.05 were consid- SARS-CoV-2 RNA test. Three weeks later, serums of all ered significant and are shown as p < 0.05 (*), p < 0.01 four women were tested for anti-SARS-CoV-2 IgG (**), or p < 0.001 (***). Graph generation and statistical (Euroimmun ELISA). The three symptomatic women, analyses were performed using the Prism version 8 soft- who had previously tested positive for SARS-CoV-2 ware (GraphPad, La Jolla, CA). RNA, were also highly positive for anti- SARS-CoV-2 IgG, while the one on methotrexate remained negative. Results The three infected women recovered within 2 weeks Initial case series without complications. We were first contacted by a group of four Caucasian All women lived with their husbands in the respective women, aged between 53 and 62 years, who lived in dif- households. No other family members lived in these ferent households in Germany and had traveled for ski households. Two of the three husbands of the infected holidays to Ischgl (Tyrol, Austria) from Feb 29 to March women developed anosmia, fatigue, headache, and cough 4, 2020. They shared the car for going there (approxi- within 7 days after return of their wives. The third hus- mately 4-h ride) and stayed in the same room the entir- band, living with one of the infected women, did not de- ety of their holiday. They regularly visited ski huts and velop any symptoms. He was on stable treatment with restaurants for lunch and dinner. All of them were in methotrexate (20 mg/week) for psoriasis. Mucosal swab good physical condition and none had concomitant dis- was done, in which that taken from one of the two in- eases with the only exception that one of the four was fected husbands confirmed for COVID-19 RNA. More- on stable methotrexate treatment (15 mg/week) for over, all four husbands (including the one of the healthy rheumatoid arthritis. Her arthritis was in remission. woman) were tested for anti-SARS-CoV-2 IgG. The two All four remained healthy during the holidays, but 2 symptomatic husbands were highly positive for anti- days after their return, three developed infectious symp- SARS-CoV-2 IgG, while the asymptomatic husband on toms including fever, cough, headache, and fatigue methotrexate as well as the husband from the non- Fig. 1 MTX inhibits SARS-CoV-2 infection in highly exposed individuals. a Initial patient group of four women and their respective husbands. b Confirmatory cases. Symptoms related to COVID-19 and results of mucosal swabs for SARS-CoV-2 RNA and SARS-CoV-2 IgG antibody serum testing are shown. Red squares: positive or present; white squares: negative or absent; gray squares: not tested. Methotrexate use indicated by blue squares Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 4 of 6 infected woman remained IgG negative. Both husbands expression of ACE2, the receptor of the SARS-CoV-2 with COVID-19 recovered from the infection without [1]. We therefore treated mice with MTX for 3 days and complications. then tested the RNA expression of ACE2 in the lungs and in the intestine by RT-PCR. In addition, we tested Confirmatory cases ACE2 protein expression by immunohistochemistry in Based on these observations we searched for additional both organs. MTX treatment led to a significant down- families, in which one of the family members developed regulation of ACE2 mRNA (Fig. 2a) and protein expres- RNA- confirmed COVID-19, while the other family sion (Fig. 2b and c) in the lungs and in the intestine. member taking MTX remained healthy. We identified 7 Furthermore, when intestinal organoids were treated additional couples or parents/children, in whom the per- with methotrexate at concentration reflecting human son taking methotrexate remained healthy and SARS- plasma levels in rheumatoid arthritis [9], ACE2 protein CoV-2 RNA negative despite living in the same house- expression was down regulated (Fig. 2d). hold and in close contact with a COVID-19 case. Four of the seven cases received MTX for rheumatoid arth- Discussion ritis, while the other three individuals had psoriatic arth- MTX is a widely and long-used drug to effectively treat ritis or peripheral spondyloarthritis. Details of the IMIDs, in particular rheumatoid and psoriatic arthritis disease symptoms are shown in Fig. 1b. [10]. While doses of MTX used for the treatment of arthritis have potent ant-inflammatory properties, they Mechanistic studies are not generally immune suppressive and have not been As these observations suggested that MTX treatment is shown to increase the risk of viral infections. MTX in- associated with at least some level of protection from duces the generation of adenosine, which has anti- COVID-19, we hypothesized that MTX may affect inflammatory properties and likely mediated the Fig. 2 MTX down-regulates ACE2 expression. a Real-time PCR showing quantification of mRNA for ACE2 in the various segments off the intestine and the lung in mice treated with methotrexate (MTX; N = 3 per group per experiment; data summarized from two independent experiments). b Representative immune histochemistry staining for ACE2 (brown color, arrowheads) in the intestine and the lung of mice treated with methotrexate or vehicle for 3 days. Isotype control antibody staining is also shown. c Quantification of ACE2 expression in immune histochemistry (N = 3 per group per experiment; data summarized from two independent experiments). d Representative immunoblot for ACE2 and actin (control) in intestinal organoids treated with two doses of methotrexate including densitometric quantification Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 5 of 6 inhibitory effect of MTX on arthritis [11, 12]. It is inter- SARS-CoV-2 infection via downregulation of the expres- esting that adenosine inhibits lung inflammation in the sion of the viral receptor ACE2. However, to better de- context of LPS challenge and also inhibits NETosis and fine the inhibitory effect of methotrexate on the thrombosis [13, 14], which are hallmarks of COVID-19 development of COVID-19, larger data sets including [15]. Previous data have also suggested a link between registry data and population-based data in high preva- ACE2 expression and adenosine metabolism but have lence areas are needed. not described the regulation of ACE2 by MTX [16]. Abbreviations Our data show that MTX-treated patients can remain ACE2: Angiotensin-converting enzyme 2; COVID-19: Coronavirus disease 19; healthy despite long and close contact to SARS-CoV-2- IMIDs: Immune-mediated chronic inflammatory diseases; MTX: Methotrexate; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2 infected individuals, which is remarkable. Since MTX mitigates but not abrogates the expression of ACE2, the Acknowledgements protective effect of MTX is definitely not complete and Nothing to acknowledge. may be overridden by other factors such as high viral Authors’ contributions load or the aforementioned inducers of ACE2 expres- MMZ, FS, and KD did the experimental work. GB, RC, CF, and BM recruited sion. Cohorts investigating IMID patients with COVID- the patients. GS wrote the manuscript. The authors read and approved the 19 have not shown a difference in the prevalence of final manuscript. MTX between mild (non-hospitalized) and severe (hos- Funding pitalized) patients [17–19]. These observations suggest This work was supported by the German Research Council (DFG) with that MTX may not inhibit the hyper-inflammatory re- projects CRC1181 (project INST 90/925-1; to GS), FOR 2886 (project SCHE 1583/15-1, to GS), and SCHE 1583/14-1 (to GS); the Bundesministerium für sponse in the patients infected by SARS-CoV-2. In con- Bildung und Forschung (BMBF) with project Mascara (to GS); the IMI-funded trast, our data support the role of MTX in viral entry project RTCure (115142) (to GS); the ERC grant 4D NanoScope from the Euro- through inhibition of ACE2 expression, which is differ- pean Union (to GS); and the Schreiber Stiftung (to GS). ent from the regulation of SARS-CoV-2-induced host re- Availability of data and materials sponse. The observation that the MTX-treated patients The datasets used and/or analyzed during the current study are available of the two case series remained RNA-negative under- from the corresponding author on reasonable request. lines this concept. Declarations A limitation of this study is that we cannot conclude from the case series and from the experimental data that Ethics approval and consent to participate methotrexate indeed lowers the risk for COVID-19. The All patients provided written informed consent and the institutional review board (IRB)/ethics committee (Ethik-Kommission der Friedrich-Alexander- study has not been designed to answer this question, Universität Erlangen-Nürnberg) approved the study. which can only be addressed in larger prospective co- horts. Furthermore, murine data have certain limitations Consent for publication Not applicable. since mouse ACE2 bind less efficiently than human ACE2 to SARS viruses [20]. Nonetheless, the data open Competing interests doors to study such concept and to consider that anti- The authors declare no competing interests. inflammatory drugs may not only influence the course Author details of SARS-CoV-2 infection via modulation of inflamma- Department of Internal Medicine 3, Friedrich-Alexander University (FAU) tory responses but also susceptibility to the infection via Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany. Deutsches Zentrum fuer Immuntherapie (DZI), Friedrich-Alexander influencing virus entry. In addition, our data extend the University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, concept that anti-inflammatory drugs used to treat dis- Erlangen, Germany. Department of Rheumatology and Clinical Immunology, eases like rheumatoid arthritis are not broadly immune Charité, Berlin, Germany. Department of Clinical Sciences and Community Health, Research Center for Adult and Pediatric Rheumatic Diseases, suppressive and may dampen rather than increase University of Milan, G. Pini Hospital, Milan, Italy. Cobra Clinic of COVID-19 risk. In fact, several inflammatory pathways Rheumatology and Research Center, São Paulo, Brazil. Department of of the host that are induced by SARS-CoV-2 are shared Rheumatology, University of Sao Paulo, São Paulo, Brazil. by IMIDs such as arthritis [21]. In support, most studies Received: 7 October 2020 Accepted: 22 February 2021 so far showed that IMIDs treated with inhibitors of pro- inflammatory cytokines have no evidence for a more se- vere course of Covid-19 and with some treatments, e.g., References 1. Hoffmann M, Kleine-Weber H, Schroeder S, Krueger N, Herrler T, Erichsen S, TNF inhibitors, an even milder case of COVID-19 has et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked been observed [16–18]. by a clinically proven protease inhibitor. Cell. 2020;181:271–80. 2. Huang C, Wang J, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020;395: Conclusion 497–506. Taken together these clinical and functional data indi- 3. Chen K, Bi J, Su Y, Chappell MC, Rose JC. Sex-specific changes in renal cate that MTX may have a certain protective effect on angiotensin-converting enzyme and angiotensin-converting enzyme 2 gene Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 6 of 6 expression and enzyme activity at birth and over the first year of life. Reprod Sci. 2016;23:200–10. 4. Bernardi S, Toffoli B, Zennaro C, Tikellis C, Monticone S, Losurdo P, et al. High-salt diet increases glomerular ACE/ACE2 ratio leading to oxidative stress and kidney damage. Nephrol Dial Transplant. 2012;27:1793–800. 5. Liu Y, Zhao L, Zhang Q, Zhang L, Ren G. Effect of long-term smoking on expression of serum ACE and ACE2 as well as its significance. J Taishan Med Coll. 2019;40:258–60. 6. Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, et al. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell. 2020. https://doi.org/10.1016/j.cell.2 020.04.004. 7. Simon D, Tascilar K, Kroenke G, Kleyer A, Zaiss MM, Heppt F, et al. Patients with immune-mediated inflammatory diseases receiving cytokine inhibitors have low prevalence of SARS-CoV-2 seroconversion. Nat Commun. 2020;11: 8. Tajik N, Frech M, Schulz O, Schälter F, Lucas S, Azizov V, et al. Targeting zonulin and intestinal epithelial tight barrier function to prevent onset of arthritis. Nat Commun. 2020;11:1995. 9. Leeb B, et al. Methotrexate serum-level determinations during low-dose therapy of rheumatoid and psoriatic arthritis. Int J Clin Pharmacol Res. 1989; 9:209–15. 10. Weinblatt ME. Methotrexate: who would have predicted its importance in rheumatoid arthritis? Arthritis Res Ther. 2018;20:103. 11. Cronstein BN, Aune TM. Methotrexate and its mechanisms of action in inflammatory arthritis. Nat Rev Rheumatol. 2020;16:145–54. 12. Kremer J. Towards a better understanding of methotrexate. Arthritis Rheumatol. 2004;50:1370–82. 13. Friebe D, Yang T, Schmidt T, Borg N, Steckel B, Ding Z, Schrader J. Purinergic signaling on leukocytes infiltrating the LPS-injured lung. PLoS One. 2014;9:e95382. 14. Ali RA, Gandhi AA, Meng H, Yalavarthi S, Vreede AP, Estes SK, et al. Adenosine receptor agonism protects against NETosis and thrombosis in antiphospholipid syndrome. Nat Commun. 2019;10:1916. 15. Leppkes M, Knopf J, Naschberger E, Lindemann A, Singh J, Herrmann I, et al. Vascular occlusion y neutrophil extracellular taps in COVID-19. EBioMedicine. 2020;58:102925. 16. Patel VB, Zhong JC, Grant MB, Oudit GY. Role of the ACE2/angiotensin 1-7 Axis of the renin-angiotensin system in heart failure. Circ Res. 2016;118: 1313–26. 17. Haberman R, Axelrad J, Chen A, Castillo R, Yan D, Izmirly P, et al. Covid-19 in immune-mediated inflammatory diseases — case series from New York. N Engl J Med. 2020;58:102925. 18. Haberman RH, Castillo R, Chen A, Yan D, Ramirez D, Sekar V, et al. COVID-19 in patients with inflammatory arthritis: a prospective study on the effects of comorbidities and DMARDs on clinical outcomes. Arthritis Rheumatol. 2020. 19. Gianfrancesco M, Hyrich K, Al-Adely S, Carmona L, Danila MI, Gossec L, et al. Characteristics associated with hospitalization for COVID-19 in people with rheumatic disease: data from the COVID-19 Global Rheumatology Alliance physician-reported registry. Ann Rheum Dis. 2020. 20. Li W, Greenough TC, Moore MJ, Vasilieva N, Somasundaran M, Sullivan JL, et al. Efficient replication of severe acute respiratory syndrome coronavirus in mouse cells is limited by murine angiotensin converting enzyme 2. J Virol. 2004;78:11429–33. 21. Schett G, Sticherling M, Neurath MF. COVID-19: risk for cytokine targeting in chronic inflammatory diseases? Nat Rev Immunol. 2020;20:271–2. Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Arthritis Research & Therapy Springer Journals

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Abstract

Background: To investigate whether methotrexate treatment may affect the susceptibility to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: Clinical assessment of symptoms, SARS-CoV-2 RNA, and anti-SARS-CoV-2 IgG in an initial case series of four families and confirmatory case series of seven families, within which one family member developed coronavirus disease 19 (COVID-19) and exposed another family member receiving methotrexate treatment; experimental part with methotrexate treatment of mice and organoids followed by the assessment of mRNA and protein expression of the SARS-CoV-2 receptor angiotensin-converting enzyme (ACE)-2. Results: In the initial case series, three of four women on a joint ski trip developed COVID-19, while the fourth woman, under treatment with methotrexate, remained virus-free. Two of the three diseased women infected their husbands, while the third husband treated with methotrexate remained virus-free. In addition, 7 other families were identified in a follow-up case series, in which one member developed COVID-19, while the other, receiving methotrexate, remained healthy. Experimentally, when mice were treated with methotrexate, ACE2 expression significantly decreased in the lung, in the intestinal epithelium, and in intestinal organoids. Conclusion: These clinical and experimental data indicate that methotrexate has certain protective effects on SARS- CoV-2 infection via downregulating ACE2. Keywords: Methotrexate, Coronavirus disease 19, Infection Background affected organs and the development of coronavirus dis- Severe acute respiratory syndrome coronavirus 2 (SARS- ease 19 (COVID-19 [2]. CoV-2) enters epithelial and other cells through binding The level of ACE expression by lung epithelia may in- to angiotensin-converting enzyme 2 (ACE2) expressed fluence the susceptibility to SARS-CoV-2 infection. For on various epithelial cells including those of the lungs instance, low ACE2 expression in young mammals can and the gut [1]. SARS-CoV-2 entry into human cells potentially explain the overall low susceptibility of chil- triggers cell damage associated with a robust and some- dren to COVID-19 and other zoonotic coronaviruses times overshooting inflammatory responses in the [3]. Furthermore, smoking as well as high salt diet have shown to increase ACE2 expression, which may explain the higher risk for COVID-19 in smokers and those with * Correspondence: georg.schett@uk-erlangen.de hypertension, respectively [4, 5]. In addition, administra- Department of Internal Medicine 3, Friedrich-Alexander University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany tion of recombinant soluble ACE2 saturates cellular sur- Deutsches Zentrum fuer Immuntherapie (DZI), Friedrich-Alexander face ACE2 outcompetes viral binding sites and hence is University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 2 of 6 effective in preventing SARS-CoV-2 infection of human Germany) using the EUROIMMUN Analyzer I platform tissue organoids [6]. according to the manufacturer’s protocol and as de- To date, little is known on whether anti-inflammatory scribed previously [7]. Optical density was determined at drugs used for the treatment of immune-mediated 450 nm with reference wavelength at 630 nm. A cut-off chronic inflammatory diseases (IMIDs), including arth- of ≥0.8 (OD450nm) was considered as positive. Assays ritis, could influence ACE2 expression and affect suscep- were performed in line with the guidelines of the Ger- tibility to SARS-CoV-2 infection. Herein, we report on man Medical Association (RiliBAK) with stipulated in- case series of families, in which one family member de- ternal and external quality controls. veloped COVID-19, while the other family member, tak- ing methotrexate (MTX), remained virus-free and Mice healthy despite substantial viral exposure. Female, C57Bl/6 N mice were maintained under specific pathogen-free conditions at the Präklinisches Experi- Methods mentelles Tierzentrum (PETZ) Erlangen, Germany, and Ethical approval approved by the local ethics authorities of the Regierung Ethical approval (#157_20 B) to conduct this analysis of Unterfranken (#55.2-2532.1-59/14). Nine-week-old was granted by the institutional review board (IRB) of mice were acclimated for 1 week, followed by a 3-week the University Clinic of Erlangen. Written informed con- co-housing period before i.p. treatment with 2 mg/kg sent was obtained from the study participants. mouse methotrexate (n = 6) or vehicle (PBS; n =6) started for three consecutive days before analysis. Two Participants independent experiments were performed. Data were obtained from [1] an initial patient group of 4 women performing a ski holiday at Ischgl (Austria) and Organoids their respective husbands and 7 confirmatory cases from Intestinal organoids were generated as previously de- Italy [2], Brazil [4] and Germany [1] comprising families scribed [8]. In short, the small intestine of wild-type with one member infected with COVID-19 and the C57Bl/6 J was removed, longitudinally opened, and villi other member taking methotrexate staying healthy. In were scraped out. Isolated intestinal crypts were resus- the initial patient group, methotrexate was used for pended in 25 μl/well growth factor reduced Matrigel treatment of psoriasis [1] and rheumatoid arthritis [1]. (Corning, NY, USA). Organoids were cultured in the in- In the confirmatory cases, methotrexate was used for cubator for at least 7 days before any experiments were treatment of rheumatoid arthritis [4] and psoriatic arth- performed. Medium was changed every 2 to 3 days and ritis/spondyloarthritis [3]. Doses of methotrexate were organoids were split once per week. Organoids were between 10 and 20 mg/week. stimulated with 0.25, 0.5, or 1 mM methotrexate for 6 h before analysis. Symptoms Participants were asked for their clinical symptoms Western blot (cough, rhinitis, throat pain, fever, headache, fatigue, Organoids were washed two times with PBS and then musculoskeletal pain, anosmia, shortness of breath, and subsequently lysed with RIPA buffer. Protein extract diarrhea) according to a questionnaire that was used for were separated on a 4–12% SDS-polyacrylamide gel, a field study on anti-SARS-CoV-2 IgG [7]. The observa- transferred to a nitrocellulose membrane, and stained tion period in the initial and confirmatory cases was with antibodies against murine ACE2 (R&D systems). 1 month. An antibody against murine β-actin (Abcam) was used as loading control. Densitometry analysis was performed SARS-CoV-2 RNA test using ImageJ. RNA quantification was down from mucosal swabs using Cobas SARS-CoV-2 real-time RT-PCR Test (Roche, Ba- sel, CH). Histology Lung and intestinal tissue were fixed in 4% formalin. Anti-SARS-CoV-2 antibody testing After deparaffinization, rehydration, and blocking, paraf- Serum samples were taken between March 18 and April fin sections were stained using a primary antibody 30 for anti-SARS-CoV-2 IgG tests. Immunoglobulin G against murine ACE2 (R&D systems). Slides have been (IgG) antibodies against the S1 domain of the spike pro- developed using a DAB staining solution kit after incu- tein of SARS-CoV-2 were tested by the recent CE ver- bation with a HRP-conjugated anti-goat secondary anti- sion (April 2020) of the commercial enzyme-linked body. Quantification of stained area per slide was immunosorbent assay from Euroimmun (Lübeck, performed using ImageJ. Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 3 of 6 Statistical analysis (Fig. 1a). Anosmia was reported by all of them. The Data are expressed as mean ± s.d. Analysis was per- fourth woman taking methotrexate developed no symp- formed using Student’s t test, single comparison, or ana- toms. Five days after the start of symptoms, RNA tests lysis of variance (ANOVA) test for multiple comparisons for SARS-CoV-2 were taken from mucosal swabs and (two-way ANOVA followed by Tukey’s or Bonferroni’s the three women with symptoms were all tested positive. multiple comparisons test). All experiments were con- The woman taking methotrexate showed a negative ducted at least two times. P values of 0.05 were consid- SARS-CoV-2 RNA test. Three weeks later, serums of all ered significant and are shown as p < 0.05 (*), p < 0.01 four women were tested for anti-SARS-CoV-2 IgG (**), or p < 0.001 (***). Graph generation and statistical (Euroimmun ELISA). The three symptomatic women, analyses were performed using the Prism version 8 soft- who had previously tested positive for SARS-CoV-2 ware (GraphPad, La Jolla, CA). RNA, were also highly positive for anti- SARS-CoV-2 IgG, while the one on methotrexate remained negative. Results The three infected women recovered within 2 weeks Initial case series without complications. We were first contacted by a group of four Caucasian All women lived with their husbands in the respective women, aged between 53 and 62 years, who lived in dif- households. No other family members lived in these ferent households in Germany and had traveled for ski households. Two of the three husbands of the infected holidays to Ischgl (Tyrol, Austria) from Feb 29 to March women developed anosmia, fatigue, headache, and cough 4, 2020. They shared the car for going there (approxi- within 7 days after return of their wives. The third hus- mately 4-h ride) and stayed in the same room the entir- band, living with one of the infected women, did not de- ety of their holiday. They regularly visited ski huts and velop any symptoms. He was on stable treatment with restaurants for lunch and dinner. All of them were in methotrexate (20 mg/week) for psoriasis. Mucosal swab good physical condition and none had concomitant dis- was done, in which that taken from one of the two in- eases with the only exception that one of the four was fected husbands confirmed for COVID-19 RNA. More- on stable methotrexate treatment (15 mg/week) for over, all four husbands (including the one of the healthy rheumatoid arthritis. Her arthritis was in remission. woman) were tested for anti-SARS-CoV-2 IgG. The two All four remained healthy during the holidays, but 2 symptomatic husbands were highly positive for anti- days after their return, three developed infectious symp- SARS-CoV-2 IgG, while the asymptomatic husband on toms including fever, cough, headache, and fatigue methotrexate as well as the husband from the non- Fig. 1 MTX inhibits SARS-CoV-2 infection in highly exposed individuals. a Initial patient group of four women and their respective husbands. b Confirmatory cases. Symptoms related to COVID-19 and results of mucosal swabs for SARS-CoV-2 RNA and SARS-CoV-2 IgG antibody serum testing are shown. Red squares: positive or present; white squares: negative or absent; gray squares: not tested. Methotrexate use indicated by blue squares Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 4 of 6 infected woman remained IgG negative. Both husbands expression of ACE2, the receptor of the SARS-CoV-2 with COVID-19 recovered from the infection without [1]. We therefore treated mice with MTX for 3 days and complications. then tested the RNA expression of ACE2 in the lungs and in the intestine by RT-PCR. In addition, we tested Confirmatory cases ACE2 protein expression by immunohistochemistry in Based on these observations we searched for additional both organs. MTX treatment led to a significant down- families, in which one of the family members developed regulation of ACE2 mRNA (Fig. 2a) and protein expres- RNA- confirmed COVID-19, while the other family sion (Fig. 2b and c) in the lungs and in the intestine. member taking MTX remained healthy. We identified 7 Furthermore, when intestinal organoids were treated additional couples or parents/children, in whom the per- with methotrexate at concentration reflecting human son taking methotrexate remained healthy and SARS- plasma levels in rheumatoid arthritis [9], ACE2 protein CoV-2 RNA negative despite living in the same house- expression was down regulated (Fig. 2d). hold and in close contact with a COVID-19 case. Four of the seven cases received MTX for rheumatoid arth- Discussion ritis, while the other three individuals had psoriatic arth- MTX is a widely and long-used drug to effectively treat ritis or peripheral spondyloarthritis. Details of the IMIDs, in particular rheumatoid and psoriatic arthritis disease symptoms are shown in Fig. 1b. [10]. While doses of MTX used for the treatment of arthritis have potent ant-inflammatory properties, they Mechanistic studies are not generally immune suppressive and have not been As these observations suggested that MTX treatment is shown to increase the risk of viral infections. MTX in- associated with at least some level of protection from duces the generation of adenosine, which has anti- COVID-19, we hypothesized that MTX may affect inflammatory properties and likely mediated the Fig. 2 MTX down-regulates ACE2 expression. a Real-time PCR showing quantification of mRNA for ACE2 in the various segments off the intestine and the lung in mice treated with methotrexate (MTX; N = 3 per group per experiment; data summarized from two independent experiments). b Representative immune histochemistry staining for ACE2 (brown color, arrowheads) in the intestine and the lung of mice treated with methotrexate or vehicle for 3 days. Isotype control antibody staining is also shown. c Quantification of ACE2 expression in immune histochemistry (N = 3 per group per experiment; data summarized from two independent experiments). d Representative immunoblot for ACE2 and actin (control) in intestinal organoids treated with two doses of methotrexate including densitometric quantification Schälter et al. Arthritis Research & Therapy (2021) 23:166 Page 5 of 6 inhibitory effect of MTX on arthritis [11, 12]. It is inter- SARS-CoV-2 infection via downregulation of the expres- esting that adenosine inhibits lung inflammation in the sion of the viral receptor ACE2. However, to better de- context of LPS challenge and also inhibits NETosis and fine the inhibitory effect of methotrexate on the thrombosis [13, 14], which are hallmarks of COVID-19 development of COVID-19, larger data sets including [15]. Previous data have also suggested a link between registry data and population-based data in high preva- ACE2 expression and adenosine metabolism but have lence areas are needed. not described the regulation of ACE2 by MTX [16]. Abbreviations Our data show that MTX-treated patients can remain ACE2: Angiotensin-converting enzyme 2; COVID-19: Coronavirus disease 19; healthy despite long and close contact to SARS-CoV-2- IMIDs: Immune-mediated chronic inflammatory diseases; MTX: Methotrexate; SARS-CoV-2: Severe acute respiratory syndrome coronavirus 2 infected individuals, which is remarkable. Since MTX mitigates but not abrogates the expression of ACE2, the Acknowledgements protective effect of MTX is definitely not complete and Nothing to acknowledge. may be overridden by other factors such as high viral Authors’ contributions load or the aforementioned inducers of ACE2 expres- MMZ, FS, and KD did the experimental work. GB, RC, CF, and BM recruited sion. Cohorts investigating IMID patients with COVID- the patients. GS wrote the manuscript. The authors read and approved the 19 have not shown a difference in the prevalence of final manuscript. MTX between mild (non-hospitalized) and severe (hos- Funding pitalized) patients [17–19]. These observations suggest This work was supported by the German Research Council (DFG) with that MTX may not inhibit the hyper-inflammatory re- projects CRC1181 (project INST 90/925-1; to GS), FOR 2886 (project SCHE 1583/15-1, to GS), and SCHE 1583/14-1 (to GS); the Bundesministerium für sponse in the patients infected by SARS-CoV-2. In con- Bildung und Forschung (BMBF) with project Mascara (to GS); the IMI-funded trast, our data support the role of MTX in viral entry project RTCure (115142) (to GS); the ERC grant 4D NanoScope from the Euro- through inhibition of ACE2 expression, which is differ- pean Union (to GS); and the Schreiber Stiftung (to GS). ent from the regulation of SARS-CoV-2-induced host re- Availability of data and materials sponse. The observation that the MTX-treated patients The datasets used and/or analyzed during the current study are available of the two case series remained RNA-negative under- from the corresponding author on reasonable request. lines this concept. Declarations A limitation of this study is that we cannot conclude from the case series and from the experimental data that Ethics approval and consent to participate methotrexate indeed lowers the risk for COVID-19. The All patients provided written informed consent and the institutional review board (IRB)/ethics committee (Ethik-Kommission der Friedrich-Alexander- study has not been designed to answer this question, Universität Erlangen-Nürnberg) approved the study. which can only be addressed in larger prospective co- horts. Furthermore, murine data have certain limitations Consent for publication Not applicable. since mouse ACE2 bind less efficiently than human ACE2 to SARS viruses [20]. Nonetheless, the data open Competing interests doors to study such concept and to consider that anti- The authors declare no competing interests. inflammatory drugs may not only influence the course Author details of SARS-CoV-2 infection via modulation of inflamma- Department of Internal Medicine 3, Friedrich-Alexander University (FAU) tory responses but also susceptibility to the infection via Erlangen-Nürnberg and Universitätsklinikum Erlangen, Erlangen, Germany. Deutsches Zentrum fuer Immuntherapie (DZI), Friedrich-Alexander influencing virus entry. In addition, our data extend the University (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen, concept that anti-inflammatory drugs used to treat dis- Erlangen, Germany. Department of Rheumatology and Clinical Immunology, eases like rheumatoid arthritis are not broadly immune Charité, Berlin, Germany. Department of Clinical Sciences and Community Health, Research Center for Adult and Pediatric Rheumatic Diseases, suppressive and may dampen rather than increase University of Milan, G. Pini Hospital, Milan, Italy. Cobra Clinic of COVID-19 risk. In fact, several inflammatory pathways Rheumatology and Research Center, São Paulo, Brazil. Department of of the host that are induced by SARS-CoV-2 are shared Rheumatology, University of Sao Paulo, São Paulo, Brazil. by IMIDs such as arthritis [21]. 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Published: Jun 10, 2021

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