Interplay between Multisystem Inflammatory Syndrome in Children, Interleukin 6, Microbiome, and Gut Barrier Integrity

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, I read with interest your manuscript reviewing the interplay between MIS-C, IL-6, microbiome and gut barrier integrity. Basically, you propose an interesting model, where the gut microbiome alteration and SARSCoV2 persistence in the gut  may enhance zonulin release and therefore reduce the intestinal barrier, leading to increase spike protein circulation. This model is coherent with the proposed superantigen activity acted by SARSCoV2 spike protein and, in general, the “superantigen model”, that you didn't review appropriately  (see for example Sacco et al. Nat Med. 2022 May;28(5):1050-1062 and Noval Rivas et al, Front. Immunol. (2022) 13:941009)). Beside the proposed model I believe the manuscript report a lot of information that is not relevant to the discussion, for example the introduction, on why COVID 19 is less severe in children, paragraph 2, some parts of paragraph 4.1 from line 224 to line 242, lines 344-365 page 8, lines 375-381 of page 8 etc

You propose that high zonulin levels […] are due to excessive concentrations of IL-6 […] but this is disputable: high IL-6 levels are secondary to the “cytokine storm” during MIS-C, therefore it is secondary, and not the cause of, increased zonulin levels (that triggers MIS-C) in your pathogenic model.

One crucial point is that MIS-C is very different from COVID19 and adult long-COVID, therefore any comparison between therapeutic responses in those clinical entities (and there are a few in your manuscript, for example, lines 399-411 on page 9) are not appropriate

Conclusions are not coherent with the main body of the review

 

You propose that prophylaxis with probiotics may reduce the incidence of MIS-C. I think this is just a hypothesis, but the main problem would be how to identify subjects who could benefit from it, as many children developing MIS-C do not have a positive history of symptomatic SARSCoV2 infection 

Comments on the Quality of English Language

Wording is accurate, though I suggest to carefully review the manuscript as the concepts are not always straigthforward and the manuscript is not fluid

Author Response

Dear authors, I read with interest your manuscript reviewing the interplay between MIS-C, IL-6, microbiome and gut barrier integrity. Basically, you propose an interesting model, where the gut microbiome alteration and SARSCoV2 persistence in the gut may enhance zonulin release and therefore reduce the intestinal barrier, leading to increase spike protein circulation. This model is coherent with the proposed superantigen activity acted by SARSCoV2 spike protein and, in general, the “superantigen model”, that you didn't review appropriately (see for example Sacco et al. Nat Med. 2022 May;28(5):1050-1062 and Noval Rivas et al, Front. Immunol. (2022) 13:941009

 

R: We thank the reviewer for this important suggestion. We have added the following sub-header in lines 331-389. The works from Sacco et al. and Noval Rivas were cited and described. The work from Sacco et al was cited in lines 104, 207, 336, 400, and 554. The work from Noval Rivas was cited in line 343.

 

 

4.3. The superantigen hypothesis of SARS-CoV-2 spike protein

 

          The proposed superantigen activity of the SARS-CoV-2 spike protein has been a topic of investigation, particularly in relation to MIS-C. On one hand, the study by Sacco et al. [19] identified distinct immunopathologic signatures in pediatric COVID-19 and MIS-C and pointed out that MIS-C is characterized by prominent type II IFN and NF-κB-dependent  signatures, matrisome activation, and increased levels of circulating spike protein. Only two of the 15 MIS-C patients showed a positive PCR on a nasopharyngeal swab within 7 days of the hospitalization, indicating that elevated spike protein levels were not caused by a prolonged respiratory tract infection, even though they did not look into the presence of SARS-CoV-2 mRNA in stool samples [19]. The study by Noval Rivas [96] suggested that “continuous and prolonged exposure to superantigen-like and neurotoxin-like viral motifs of the SARS-CoV-2 spike may promote autoimmunity leading to the development of post-acute COVID-19 syndromes, including MIS-C and long COVID, as well as neurological complications resulting from SARS-CoV-2 infection”. On the other hand, some studies also highlight that the SARS-CoV-2 spike protein does not exhibit superantigen-like activity [97][98]. In experimental setups, the spike protein was compared to staphylococcal enterotoxin B (SEB), a well-known superantigen. The study showed that while SEB induced a significant production of pro-inflammatory cytokines, the SARS-CoV-2 spike did not trigger a similar response in T cells, indicating that it lacks the intrinsic ability to function as a superantigen [97].

Other study examined the T cell response to SARS-CoV-2 peptide pools in children in the subacute phase of MIS-C in order to corroborate this observation. Despite their abundance, Vβ21.3-bearing CD4+ and CD8+ T cells did not belong to the SARS-CoV-2-specific T cell population. According to this research, most children's T cell repertoire grew in response to an antigen unrelated to SARS-CoV-2 [99]. Although the antigen that triggers MIS-C is unknown, it might come from the associated leaky gut [33].          

Burns [98] wrote: " If spike peptides are not superantigens, then what is the role of the spike protein in MIS-C pathogenesis?” Several groups reported the persistence of the spike antigen in the circulation of patients with acute MIS-C. However, Sigal et al. [100] used a sensitive electro-chemiluminescent immunoassay in patients with MIS-C and showed no persistence of spike antigen in the plasma. Thus, these results do not support the hypothesis that circulating spike protein in patients with MIS-C is involved in disease pathogenesis."

    In 2022, reports from different countries reported a decrease in the prevalence of MIS-C in association with Omicron variant waves of SARS-CoV-2 infection. This questioned again the spike superantigen theory significance in the pathophysiology of MIS-C. A research conducted in 12 Israeli hospitals over a 16-week period during each of the three pandemic waves (Alpha, Delta, and Omicron), found that during the Omicron wave, there were fewer admissions to critical care and less severe cardiac outcomes. According to national data, there were 54.5 MIS-C incidents per 100,000 people under the age of 18 during Alpha, 49.2 during Delta, and 3.8 during Omicron. The reduced frequency of MIS-C during the Omicron wave may have been caused by vaccination, prior SARS-COV-2 infection acting as a protective factor, or changes in the spike protein that resulted in diminished pathogenesis [101]. A study found that at the junction between the S1 and S2 subunits, the SARS-CoV-2 spike component S1 includes an insertion of four amino acids, P681RRA684 (PRRA), next to the cleavage site R685↑S686 [102]. Only SARS-CoV-2 and the β-coronaviruses in the SARS-like subfamily possess the polybasic segment PRRA [102]. The PRRA insert was discovered to be a component of the E661–R685 motif, a group of 25 amino acids with sequence and structural similarities to a portion of the SAg Staphylococcal enterotoxin B (SEB) [103].

It's crucial to keep in mind that the furin-like cleavage site R685↑S686 is located next to this SAg-like motif [102]. Because the furin cleavage site contains the SAg-like motif, it is crucial for the acidic furin epitope to recognize it. In fact, viral mutations that eliminated the furin cleavage site have been shown to significantly decrease SARS-CoV-2-induced pathogenesis in mice and hamsters (47, 48).

        

 

Reviewer: Beside the proposed model I believe the manuscript report a lot of information that is not relevant to the discussion, for example the introduction, on why COVID 19 is less severe in children, paragraph 2, some parts of paragraph 4.1 from line 224 to line 242, lines 344-365 page 8, lines 375-381 of page 8 etc

 

R: We thank reviewer for these important suggestions. We have deleted unnecessary data and added more information specifically for MIS-C. New data was highlighted in yellow in lines 61-81. Regarding the other paragraphs, we also deleted reductant information.

 

Reviewer: You propose that high zonulin levels […] are due to excessive concentrations of IL-6 […] but this is disputable: high IL-6 levels are secondary to the “cytokine storm” during MIS-C, therefore it is secondary, and not the cause of, increased zonulin levels (that triggers MIS-C) in your pathogenic model.

R: We thank reviewer for this observation. We have expanded the information showing that IL-6 is also an essential cytokine for initial viral replication. We have added this text in lines 397-401:

It has been demonstrated that the SARS-CoV-2 spike and nucleocapsid proteins alone can cause monocytes and macrophages to produce IL-6. Of interest, children with MIS-C exhibit hyper-phagocytosis and enhanced monocyte recruitment [106]. In certain COVID-19 patients, this IL-6 overexpression may be the catalyst that starts the dysregulated immune response [107] [108].

 

And in lines 422-428 we wrote:

In the context of ulcerative colitis (UC), a study found that at physiological levels, IL-6 controls epithelial barrier function by modulating the expression of TJs-related proteins. In contrast, IL-6 levels in the plasma of UC patients were elevated and increased as the disease worsened. Overproduction of IL-6 has also been shown to damage the intestinal epithelial cell barrier and control barrier function by increasing zonulin release. Conversely, when an anti-IL-6 antibody was added, the amount of zonulin was lower than in the control group [126].

 

 

 

Reviewer: One crucial point is that MIS-C is very different from COVID19 and adult long-COVID, therefore any comparison between therapeutic responses in those clinical entities (and there are a few in your manuscript, for example, lines 399-411 on page 9) are not appropriate

R: We appreciate the reviewer for this important comment. Although MIS-C and COVID-19 are different, they share common pathophysiological mechanisms, such as hyper-inflammation, cytokine storms, and immune dysregulation. The presence of elevated levels of pro-inflammatory cytokines, including IL-6 TNF- α, and Il-1β has been observed in both conditions. These overlaps justify the comparison of therapeutic responses, as insights gained from one condition could suggest treatment strategies for the other. For example, larazotide, a zonulin antagonist was used by Yonker et al to treat children with MIS-C, with excellent results.

https://www.jci.org/articles/view/149633

Larazotide could be investigated for its potential to decrease zonulin levels in adults. On the other side, Brogna et al demonstrated that early antibiotic treatment appears to be essential for halting the progression of disease, controlling toxin release from infected bacteria, and preventing viral replication in the GM.

https://onlinelibrary.wiley.com/doi/full/10.1002/jmv.29507

 

Reviewer: Conclusions are not coherent with the main body of the review

R: Our special thanks for this important observation. We have completely re-organized the conclusions, covering most important aspects of this work.

 

Reviewer: You propose that prophylaxis with probiotics may reduce the incidence of MIS-C. I think this is just a hypothesis, but the main problem would be how to identify subjects who could benefit from it, as many children developing MIS-C do not have a positive history of symptomatic SARSCoV2 infection 

 

R: Respectfully, we did not propose prophylaxis with antibiotics.

 In lines 453-459 we wrote:

“Furthermore, it is noteworthy that a considerable proportion of patients who were administered antibiotics within the initial three days and throughout the full seven days of the acute phase did not experience long COVID. One of the main contributing factors to the development of the disease appears to be the bacteriophage behavior of SARS-CoV-2 during the acute and post-COVID-19 phases. Early antibiotic treatment appears to be essential for halting the progression of disease, controlling toxin release from infected bacteria, and preventing viral replication in the GM [133].”

 

From a clinical point of view, it may seem incorrect to administer antibiotics instead of an antiviral, however, Brogna et al demonstrated that this virus infects commensal bacteria in the intestine, so the timely use of antibiotics could inhibit the excessive multiplication of the virus inside the bacteria.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Your review article on interplay between multisystem inflammatory syndrome in children, interleukin 6, microbiome, and gut barrier integrity is undeniably a contribution to the society of scientists and also clinicians, well-organized and easy-to-understand material.

However, I have some objections and suggestions:

1.       Please use the WHO-recommended abbreviation - long COVID - instead of Long COVID-19 or long-COVID-19  throughout the text;

2.       Line 124. Please edit the title of the second section – Long COVID-19 and (MIS-C) are different – by writing Long COVID correctly and removing the brackets;

3.       I recommend moving the description of zonulin from lines 260 and 261 higher, where the emphasis is placed on zonulin for the first time (around line 251);

4.       Please correct 4.2. heading from MISC to MIS-C;

5.       Line 487 and 488 should be reviewed and the sentence corrected: Of the 147 patients, 78 (53.1%) in the probiotic group experienced complete remission, while 41 (28.1%) in the placebo group did.

6.       Please critically evaluate the used references, it is possible that some of the references that are older than 10 years are not all necessary.

Author Response

Your review article on interplay between multisystem inflammatory syndrome in children, interleukin 6, microbiome, and gut barrier integrity is undeniably a contribution to the society of scientists and also clinicians, well-organized and easy-to-understand material.

However, I have some objections and suggestions:

1. Please use the WHO-recommended abbreviation - long COVID - instead of Long COVID-19 or long-COVID-19 throughout the text;

 

R: Thank you for this important clarification. We have corrected them.

 

2.  Line 124. Please edit the title of the second section – Long COVID-19 and (MIS-C) are different – by writing Long COVID correctly and removing the brackets;

 

R: We have corrected that mistake. Thank you.

 

3.  I recommend moving the description of zonulin from lines 260 and 261 higher, where the emphasis is placed on zonulin for the first time (around line 251);

 

R: Thank you very much for this recommendation. We have relocated that paragraph.

 

 

4.  Please correct 4.2. heading from MISC to MIS-C;

 

R: Corrected. Thank you.

 

 

5.  Line 487 and 488 should be reviewed and the sentence corrected: Of the 147 patients, 78 (53.1%) in the probiotic group experienced complete remission, while 41 (28.1%) in the placebo group did.

 

R: Corrected, thank you.

 

 

6.  Please critically evaluate the used references, it is possible that some of the references that are older than 10 years are not all necessary.

 

R: We thank the reviewer for this suggestion. However, we have included old references to give credit to the original authors, since it is very common nowadays to cite review works instead of the original works.

Reviewer 3 Report

Comments and Suggestions for Authors

Zari and colleagues provide a comprehensive overview of the current knowledge regarding the role of the gut barrier and microbiome in steering the multisystem inflammatory syndrome during SARS-CoV-2 infection, particularly in children. They specifically analyze the link between dysbiosis and MIS-C, highlighting the role of SARS-CoV-2 and subsequent IL-6 production in altering the gut barrier. Finally, they focus on current therapeutic possibilities under study.

However, considering the topic, it is surprising that certain basic knowledge notions are not mentioned. The entire manuscript focuses on the leakage of the gut barrier, yet the authors do not consider a fundamental aspect: a large portion of the blood draining from the gut does not directly reach systemic circulation; instead, it passes through the liver via the hepatic portal vein. Consequently, it has been well known for decades that the liver is responsible for immune surveillance of antigens originating from the gut. (PMID: 38658756). This concept is not directly linked to COVID infection but should at least be mentioned in the introduction or discussion.

It is also crucial to mention, especially from a therapeutic perspective, the phenomenon of COVID rebound observed in patients treated with antiviral drugs during their initial infection (PMID: 36069968). Specifically, several reports have shown that antiviral treatment during SARS-CoV-2 infection (e.g., nirmatrelvir) reduces the risk of progression to severe COVID-19 but attenuates the development of SARS-CoV-2-specific antibody and T cell responses (PMID: 36946379). In some patients, this results in a rebound of viral load and a rapid relapse of COVID-19 symptoms shortly after completing early and effective nirmatrelvir treatment. Moreover, this mechanism could impair the development of effective long-term immunity. This circumstance needs to be mentioned when suggesting possible therapeutic strategies that impact the immune response.

Author Response

Zari and colleagues provide a comprehensive overview of the current knowledge regarding the role of the gut barrier and microbiome in steering the multisystem inflammatory syndrome during SARS-CoV-2 infection, particularly in children. They specifically analyze the link between dysbiosis and MIS-C, highlighting the role of SARS-CoV-2 and subsequent IL-6 production in altering the gut barrier. Finally, they focus on current therapeutic possibilities under study.

However, considering the topic, it is surprising that certain basic knowledge notions are not mentioned. The entire manuscript focuses on the leakage of the gut barrier, yet the authors do not consider a fundamental aspect: a large portion of the blood draining from the gut does not directly reach systemic circulation; instead, it passes through the liver via the hepatic portal vein. Consequently, it has been well known for decades that the liver is responsible for immune surveillance of antigens originating from the gut. (PMID: 38658756). This concept is not directly linked to COVID infection but should at least be mentioned in the introduction or discussion.

R: We thank reviewer # 3 for this important clarification. He (she) correctly pointed out that “a large portion of the blood draining from the gut does not directly reach systemic circulation; instead, it passes through the liver via the hepatic portal vein. Consequently, it has been well known for decades that the liver is responsible for immune surveillance of antigens originating from the gut. (PMID: 38658756).”.  We have updated this important information as follows in lines 268-287:

It is known that a significant amount of blood that exits the gastrointestinal tract goes via the hepatic portal vein in the liver before entering the systemic circulation. As a result, it has long been understood that the liver is in control of immune surveillance against antigens coming from the gut [94]. It is important to mention that SARS-CoV-2 infection has been shown to impair liver immunity through several mechanisms, leading to liver injury and dysfunction. Hepatobiliary epithelial cells cholangiocytes in particular express the angiotensin-converting enzyme 2 (ACE2) receptor, which allows SARS-CoV-2 to infect these cells [95]. This direct infection can aggravate liver failure as the virus has been found in COVID-19 patients' liver tissues, suggesting that this organ may be a target for viral replication [96] [97]. A cytokine storm may result from the exacerbated immunological response that the infection sets off, which is defined by the release of pro-inflammatory cytokines. The liver's capacity to develop a successful immune response may be hampered by this systemic inflammation, aggravating liver dysfunction and damage in adults [98] and in children [99]. According to a 2020 study, younger children—those under three years old—were more likely than older children to develop liver damage from COVID-19. The likely cause was early-life liver immaturity [100]. Upon admission, children frequently had higher liver enzyme levels than adults, who typically had an increase in enzyme levels no earlier than the second week of hospitalization [101]. Similar findings were reported in other investigation, with MIS-C showing these findings more frequently than in COVID-19 [99].

 

 

It is also crucial to mention, especially from a therapeutic perspective, the phenomenon of COVID rebound observed in patients treated with antiviral drugs during their initial infection (PMID: 36069968). Specifically, several reports have shown that antiviral treatment during SARS-CoV-2 infection (e.g., nirmatrelvir) reduces the risk of progression to severe COVID-19 but attenuates the development of SARS-CoV-2-specific antibody and T cell responses (PMID: 36946379). In some patients, this results in a rebound of viral load and a rapid relapse of COVID-19 symptoms shortly after completing early and effective nirmatrelvir treatment. Moreover, this mechanism could impair the development of effective long-term immunity. This circumstance needs to be mentioned when suggesting possible therapeutic strategies that impact the immune response.

R: We thank reviewer for this important observation. We have added this discussion in lines 472-480.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Dear Authors thank you very much for addressing all my objections to your work. I still have minor clues on a couple of conclusions you make but I believe their discussion would not add much value to your paper, so I will refrained to express them , in general your manuscript is well built and the model pretty well described

Comments on the Quality of English Language

A quick review should be done as there are some minor grammatical errors and mistyping