Gene expression profiles of Protein tyrosine phosphatase non-receptor 22, Tumor necrosis factor receptor-associated factor 1 and Interleukin-1 beta in patients with rheumatoid arthritis and healthy controls after severe acute respiratory syndrome-associated coronavirus-2 infection or vaccination
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Background. Recovery from coronavirus disease 2019 (COVID-19) probably leads to long-term symptoms, including immune system complications. Recent publications have reported that severe acute respiratory syndrome-associated coronavirus-2 (SARS-CoV-2) infection or, sometimes, vaccination against it may trigger autoimmune responses in vulnerable cohorts.
Purpose of the research. To investigate the differences in the expression of the Protein tyrosine phosphatase non-receptor type 22 (PTPN22), Tumor necrosis factor receptor-associated factor 1 (TRAF-1) and Interleukin-1 beta (IL-1β) genes in patients with rheumatoid arthritis (RA) and healthy controls following SARS-CoV-2 infection or vaccination.
Methods. Blood samples were collected from 61 patients diagnosed with RA post COVID-19 Rheumatoid arthritis patients after SARS-CoV-2 infection (RAI), and 40 controls (C) who had experienced at least one COVID-19 infection. RNA was extracted and used to prepare cDNA from each sample and was then used to analyse the expression of PTPN22, TRAF-1 and IL-1β using relative gene expression.
Results. The study covered all 61 patients; 17 had been vaccinated without prior COVID-19 infection Rheumatoid arthritis patients after SARS-CoV-2 vaccination (RAV), while 44 were diagnosed with RA after recovery from COVID-19 (RAI). A statistically significant decrease in the gene expression of TRAF-1 was observed in both RAI and (RAV) patients compared with the C group ( P =0.042). A consistently significant increase in gene expression of IL-1β was observed in both RAI and RAV samples compared with controls. However, the reduction in PTPN22 expression was not statistically significant.
Conclusion. In this study, TRAF-1 was significantly downregulated, but IL-1β was upregulated in patients with RA post either COVID-19 infection or vaccination, while PTPN22 showed a non-significant reduction. The study findings suggest that triggered immune response post SARS-CoV-2 exposure, through infection or vaccination, may influence molecular pathways involved in RA pathogenesis.
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The work presented is clear and the arguments well formed.
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Comments to Author
The work presents a relevant topic that can be further studied in future projects. The suggestions addressed made the article more understandable and provided a more insightful perspective, suggesting new avenues to explore regarding post-COVID-19 Rheumatoid Arthritis. After the adjustments, I was satisfied with the article and hope that this work does not stop here; I encourage the group to continue following the insights.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical …
Comments to Author
The work presents a relevant topic that can be further studied in future projects. The suggestions addressed made the article more understandable and provided a more insightful perspective, suggesting new avenues to explore regarding post-COVID-19 Rheumatoid Arthritis. After the adjustments, I was satisfied with the article and hope that this work does not stop here; I encourage the group to continue following the insights.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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The amendments made in the first round of review improved the paper, but there are some points that need to be addressed to clarify the the relevance of the findings further, as requested by reviewer 2. Please address these comments and resubmit your paper for further consideration. When resubmitting your work, please include a point by point response, as well as a tracked and a clean, corrected version of the manuscript. Best regards, Gustavo
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Comments to Author
Thank you for the revised manuscript but I do still several comments: 1.Abstract -The background of the abstract implied that COVID-19 induced RA and this did not reflect the scope and objective of this study as there were no appropriate pre-covid/unvaccinated/uninfected control arm. -Results: It was stated "A pronounced decrease in gene expression of TRAF-1 in both RAI and RAV patients compared to C group". Was this "pronounced decrease" statistically significant? (p
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image …
Comments to Author
Thank you for the revised manuscript but I do still several comments: 1.Abstract -The background of the abstract implied that COVID-19 induced RA and this did not reflect the scope and objective of this study as there were no appropriate pre-covid/unvaccinated/uninfected control arm. -Results: It was stated "A pronounced decrease in gene expression of TRAF-1 in both RAI and RAV patients compared to C group". Was this "pronounced decrease" statistically significant? (p
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
-
Reviewer #1: The manuscript presents a relevant topic, evaluating the gene expression of PTPN22, TRAF-1, and IL-1β in rheumatoid arthritis patients after infection or vaccination against SARS-CoV-2. The results are indicative of the post-COVID-19 immunological mechanisms that may lead to the development of rheumatoid arthritis. However, considering this is a cross-sectional study, some conclusions should be interpreted with caution, and some improvements could strengthen the manuscript. A general suggestion for the work: a control group of rheumatoid arthritis patients who did not contract COVID-19 or were not vaccinated could be added (although I understand that this group would be nearly impossible to obtain). Given the difficulty in finding such controls, RNAseq data from rheumatoid arthritis patients prior to COVID-19 could be …
Reviewer #1: The manuscript presents a relevant topic, evaluating the gene expression of PTPN22, TRAF-1, and IL-1β in rheumatoid arthritis patients after infection or vaccination against SARS-CoV-2. The results are indicative of the post-COVID-19 immunological mechanisms that may lead to the development of rheumatoid arthritis. However, considering this is a cross-sectional study, some conclusions should be interpreted with caution, and some improvements could strengthen the manuscript. A general suggestion for the work: a control group of rheumatoid arthritis patients who did not contract COVID-19 or were not vaccinated could be added (although I understand that this group would be nearly impossible to obtain). Given the difficulty in finding such controls, RNAseq data from rheumatoid arthritis patients prior to COVID-19 could be used, which would enrich the comparisons (although I also understand that this may not have been possible in this study). The absence of a control group of patients with pre-COVID-19 rheumatoid arthritis should be mentioned as a limitation. Including this group would have strengthened the conclusions, but I understand that it may not have been possible in the context of the study, so I leave this suggestion here for future studies. In the "Abstract" section, "Results" and "Conclusion," there is a contradiction with the observations of PTPN22 gene expression. "Results" states "The reduction in PPN22 expression was not statistically significant," and "Conclusion" states "The virus downregulated the relative expression of PTPN22." I believe this information should be aligned. Methodology - Add ethics approval Results In Figures 1, 2, and 3, statistical tests and p-values should be indicated in the graphs to facilitate interpretation of the results. The legend for Figure 2 is hidden behind the graph. In the "IL-1b Gene" section, there is a typo in "SYBER green," which I believe is "SYBR green." Discussion and References - Some statements in your discussion require specific references and citations in the text. For example: "In general, viral infections are known to be associated with the development of arthritis and RA that are diagnosed shortly after exposure to viruses." and "Some viruses have the capacity to enhance proliferation of human synovial cells and induce gene activation in synoviocytes, especially within synovial fibroblasts, which leads to the activation of pro-oncogenic genes and genes encoding pro-inflammatory cytokines." These and other statements, even if "basic," need to be evidence-based. I recommend citing the references appropriately after each statement. The reduced PTPN22 expression in RAI samples indicates that this gene may play an essential role in the development of RA. However, these results alone do not support the conclusion that PTPN22 plays a role in the development of RA after COVID-19. The conclusion regarding the role of PTPN22 should be reconsidered, as expression data alone do not directly support the functional involvement of PTPN22 and RA. The mouse results cited in the discussion, along with the PTPN22 gene expression results, may better support the hypothesis presented, since comparing gene expression between patients and controls alone is insufficient to conclude the findings. - In "(rs2488457 and rs2476601, a condition leading to an inflammatory response since T cell activation is not repressed by this gene, and decreased PTPN22 augments the development and symptoms of RA in a mouse model (18)," I believe the parenthesis needs to be closed. - Also in the discussion, a mechanism mediated by CD4+ T cells is suggested that could be leading to inflammation in the synovium. One suggestion for this or a future study could be to evaluate the expression of MHC class II molecules and interferon gamma, as this is a product of this activation of these lymphocytes when there is a reduction in PTPN22 expression, which is consistent with your results. The text also mentions the interaction of PTPN22 with Ncf1, which may contribute to the development of T cell-dependent autoimmunity. This reinforces the suggestion of evaluating the expression of molecular markers of T cells in patients, so as to further support the findings and also suggest the molecular mechanism that could be leading to the development of RA postoperatively. COVID-19. These points can be cited as future perspectives, not as a requirement of this study, as I understand that this is a cross-sectional study and these analyses may not be easy to conduct. - Add the citation of the Mirzaesmaeili and Abdul-Sater reference in the text. - TRAF-1 plays an essential role in cytokine production; it limits ASC ubiquitination, leading to a reduction in IL-1β and preventing NLRP3 inflammasome activation. Reducing NLRP3 inflammasome activation and inhibiting ASC oligomerization by TRAF-1 leads to a reduction in the production of pro-inflammatory cytokines involved in the innate immunity and inflammasome pathways. TRAF-1 expression in RAI patients was reduced compared to the control group, as well as to the RAV group, and this low expression is a profile already observed in patients with RA as referenced in [21] and [22] 36. These results lead us to believe that low TRAF-1 expression is not necessarily caused by SARS-CoV-2 infection, but rather may be a reflection of the expression profile of rheumatoid arthritis itself (as per references 21 and 36). This doubt could be resolved if gene expression in patients with pre-COVID-19 RA were monitored. Including a group with pre-COVID-19 RA would enrich the comparison, but I recognize that this may be beyond the scope or sample availability of this study. This is a suggestion for a future study. - Analysis of additional markers could enrich hypotheses about immunological mechanisms. For example, evaluating the expression of NF-kB, ERK, and STST1 could help support the suggested IL-1β-mediated mechanism. Conclusion The conclusion suggests that COVID-19 vaccines have a potential therapeutic benefit for rheumatoid arthritis. I recommend caution: based solely on gene expression results, this cannot be concluded or suggested, since clinical and symptomatic parameters were not evaluated. Furthermore, IL-1β is a cytokine involved in several diseases, cellular processes, and the innate immune response. The study presents relevant results, but some improvements are needed to improve the clarity of the results and interpretation of the scientific advances. Considering the limitations inherent to the cross-sectional design, the above suggestions aim to strengthen the manuscript and guide future studies. Reviewer #2: The methodology of this study won't be able to answer the primary objective. The primary objective was to ascertain whether SARS-CoV-2 infection and the vaccine against COVID-19 are involved in the development of rheumatoid arthritis. However, there was no control cohort which comprised of RA patients who developed RA before the onset of COVID / never had COVID infection/vaccine. Therefore, it is very difficult to ascertain how COVID-19 cause the development of RA as RA is a multifactorial disease as there was no comparisons with RA without COVID-19 infection. Suggest to revise the objective to : To study the differences of the gene expression in RA patients and healthy controls after COVID-19 infection. The title need to be revised as well to better reflect the new objective. In discussion, perhaps the authors can review the studies of gene expression in RA prior to COVID pandemic (historical cohort) to see whether is there indeed an change in the immunopathogenesis of RA after COVID-19.
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Comments to Author
The methodology of this study won't be able to answer the primary objective. The primary objective was to ascertain whether SARS-CoV-2 infection and the vaccine against COVID-19 are involved in the development of rheumatoid arthritis. However, there was no control cohort which comprised of RA patients who developed RA before the onset of COVID / never had COVID infection/vaccine. Therefore, it is very difficult to ascertain how COVID-19 cause the development of RA as RA is a multifactorial disease as there was no comparisons with RA without COVID-19 infection. Suggest to revise the objective to : To study the differences of the gene expression in RA patients and healthy controls after COVID-19 infection. The title need to be revised as well to better reflect the new objective. In discussion, perhaps …
Comments to Author
The methodology of this study won't be able to answer the primary objective. The primary objective was to ascertain whether SARS-CoV-2 infection and the vaccine against COVID-19 are involved in the development of rheumatoid arthritis. However, there was no control cohort which comprised of RA patients who developed RA before the onset of COVID / never had COVID infection/vaccine. Therefore, it is very difficult to ascertain how COVID-19 cause the development of RA as RA is a multifactorial disease as there was no comparisons with RA without COVID-19 infection. Suggest to revise the objective to : To study the differences of the gene expression in RA patients and healthy controls after COVID-19 infection. The title need to be revised as well to better reflect the new objective. In discussion, perhaps the authors can review the studies of gene expression in RA prior to COVID pandemic (historical cohort) to see whether is there indeed an change in the immunopathogenesis of RA after COVID-19.
Please rate the manuscript for methodological rigour
Poor
Please rate the quality of the presentation and structure of the manuscript
Poor
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-
Comments to Author
The manuscript presents a relevant topic, evaluating the gene expression of PTPN22, TRAF-1, and IL-1β in rheumatoid arthritis patients after infection or vaccination against SARS-CoV-2. The results are indicative of the post-COVID-19 immunological mechanisms that may lead to the development of rheumatoid arthritis. However, considering this is a cross-sectional study, some conclusions should be interpreted with caution, and some improvements could strengthen the manuscript. A general suggestion for the work: a control group of rheumatoid arthritis patients who did not contract COVID-19 or were not vaccinated could be added (although I understand that this group would be nearly impossible to obtain). Given the difficulty in finding such controls, RNAseq data from rheumatoid arthritis patients prior to …
Comments to Author
The manuscript presents a relevant topic, evaluating the gene expression of PTPN22, TRAF-1, and IL-1β in rheumatoid arthritis patients after infection or vaccination against SARS-CoV-2. The results are indicative of the post-COVID-19 immunological mechanisms that may lead to the development of rheumatoid arthritis. However, considering this is a cross-sectional study, some conclusions should be interpreted with caution, and some improvements could strengthen the manuscript. A general suggestion for the work: a control group of rheumatoid arthritis patients who did not contract COVID-19 or were not vaccinated could be added (although I understand that this group would be nearly impossible to obtain). Given the difficulty in finding such controls, RNAseq data from rheumatoid arthritis patients prior to COVID-19 could be used, which would enrich the comparisons (although I also understand that this may not have been possible in this study). The absence of a control group of patients with pre-COVID-19 rheumatoid arthritis should be mentioned as a limitation. Including this group would have strengthened the conclusions, but I understand that it may not have been possible in the context of the study, so I leave this suggestion here for future studies. In the "Abstract" section, "Results" and "Conclusion," there is a contradiction with the observations of PTPN22 gene expression. "Results" states "The reduction in PPN22 expression was not statistically significant," and "Conclusion" states "The virus downregulated the relative expression of PTPN22." I believe this information should be aligned. Methodology - Add ethics approval Results In Figures 1, 2, and 3, statistical tests and p-values should be indicated in the graphs to facilitate interpretation of the results. The legend for Figure 2 is hidden behind the graph. In the "IL-1b Gene" section, there is a typo in "SYBER green," which I believe is "SYBR green." Discussion and References - Some statements in your discussion require specific references and citations in the text. For example: "In general, viral infections are known to be associated with the development of arthritis and RA that are diagnosed shortly after exposure to viruses." and "Some viruses have the capacity to enhance proliferation of human synovial cells and induce gene activation in synoviocytes, especially within synovial fibroblasts, which leads to the activation of pro-oncogenic genes and genes encoding pro-inflammatory cytokines." These and other statements, even if "basic," need to be evidence-based. I recommend citing the references appropriately after each statement. The reduced PTPN22 expression in RAI samples indicates that this gene may play an essential role in the development of RA. However, these results alone do not support the conclusion that PTPN22 plays a role in the development of RA after COVID-19. The conclusion regarding the role of PTPN22 should be reconsidered, as expression data alone do not directly support the functional involvement of PTPN22 and RA. The mouse results cited in the discussion, along with the PTPN22 gene expression results, may better support the hypothesis presented, since comparing gene expression between patients and controls alone is insufficient to conclude the findings. - In "(rs2488457 and rs2476601, a condition leading to an inflammatory response since T cell activation is not repressed by this gene, and decreased PTPN22 augments the development and symptoms of RA in a mouse model (18)," I believe the parenthesis needs to be closed. - Also in the discussion, a mechanism mediated by CD4+ T cells is suggested that could be leading to inflammation in the synovium. One suggestion for this or a future study could be to evaluate the expression of MHC class II molecules and interferon gamma, as this is a product of this activation of these lymphocytes when there is a reduction in PTPN22 expression, which is consistent with your results. The text also mentions the interaction of PTPN22 with Ncf1, which may contribute to the development of T cell-dependent autoimmunity. This reinforces the suggestion of evaluating the expression of molecular markers of T cells in patients, so as to further support the findings and also suggest the molecular mechanism that could be leading to the development of RA postoperatively. COVID-19. These points can be cited as future perspectives, not as a requirement of this study, as I understand that this is a cross-sectional study and these analyses may not be easy to conduct. - Add the citation of the Mirzaesmaeili and Abdul-Sater reference in the text. - TRAF-1 plays an essential role in cytokine production; it limits ASC ubiquitination, leading to a reduction in IL-1β and preventing NLRP3 inflammasome activation. Reducing NLRP3 inflammasome activation and inhibiting ASC oligomerization by TRAF-1 leads to a reduction in the production of pro-inflammatory cytokines involved in the innate immunity and inflammasome pathways. TRAF-1 expression in RAI patients was reduced compared to the control group, as well as to the RAV group, and this low expression is a profile already observed in patients with RA as referenced in [21] and [22] 36. These results lead us to believe that low TRAF-1 expression is not necessarily caused by SARS-CoV-2 infection, but rather may be a reflection of the expression profile of rheumatoid arthritis itself (as per references 21 and 36). This doubt could be resolved if gene expression in patients with pre-COVID-19 RA were monitored. Including a group with pre-COVID-19 RA would enrich the comparison, but I recognize that this may be beyond the scope or sample availability of this study. This is a suggestion for a future study. - Analysis of additional markers could enrich hypotheses about immunological mechanisms. For example, evaluating the expression of NF-kB, ERK, and STST1 could help support the suggested IL-1β-mediated mechanism. Conclusion The conclusion suggests that COVID-19 vaccines have a potential therapeutic benefit for rheumatoid arthritis. I recommend caution: based solely on gene expression results, this cannot be concluded or suggested, since clinical and symptomatic parameters were not evaluated. Furthermore, IL-1β is a cytokine involved in several diseases, cellular processes, and the innate immune response. The study presents relevant results, but some improvements are needed to improve the clarity of the results and interpretation of the scientific advances. Considering the limitations inherent to the cross-sectional design, the above suggestions aim to strengthen the manuscript and guide future studies.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
-