Enhanced Airway Epithelial Response to SARS-CoV-2 Infection in Children is Critically Tuned by the Cross-Talk Between Immune and Epithelial Cells

  1. Vladimir G. Magalhães
  2. Sören Lukassen
  3. Maike Drechsler
  4. Jennifer Loske
  5. Sandy S. Burkart
  6. Sandra Wüst
  7. Eva-Maria Jacobsen
  8. Jobst Röhmel
  9. Marcus A. Mall
  10. Klaus-Michael Debatin
  11. Roland Eils
  12. Stella Autenrieth
  13. Aleš Janda
  14. Irina Lehmann
  15. Marco Binder

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Abstract

To cope with novel virus infections to which no prior adaptive immunity exists, the body strongly relies on the innate immune system. In such cases, including infections with SARS-CoV-2, children tend to fair better than adults. In the context of COVID-19, it became evident that a rapid interferon response at the site of primary infection is key for successful control of the virus and prevention of severe disease. The airway epithelium of children was shown to exhibit a primed state already at homeostasis and to respond particularly well to SARS-CoV-2 infection. However, the underlying mechanism for this priming remained elusive. Here we show that interactions between airway mucosal immune cells and epithelial cells are stronger in children, and via cytokine-mediated signaling lead to IRF-1-dependent upregulation of the viral sensors RIG-I and MDA5. Based on a cellular in vitro model we show that stimulated human peripheral blood mononuclear cells (PBMC) can induce a robust interferon-beta response towards SARS-CoV-2 in a lung epithelial cell line otherwise unresponsive to this virus. This is mediated by type I interferon, interferon-gamma and TNF, and requires induction of both, RIG-I and MDA5. In single cell-analysis of nasal swab samples the same cytokines are found to be elevated in mucosal immune cells of children, correlating with elevated epithelial expression of viral sensors. In vitro analysis of PBMC derived from healthy adolescents and adults confirm that immune cells of younger individuals show increased cytokine production and potential to prime epithelial cells. In co-culture with SARS-CoV-2-infected A549 cells, PBMC from adolescents significantly enhance the antiviral response. Taken together, our study suggests that higher numbers and a more vigorous activity of innate immune cells in the airway mucosa of children tune the set-point of the epithelial antiviral system. This likely is a major contributor to the robust immune response to SARS-CoV-2 in children. Our findings shed light on the molecular underpinnings of the stunning resilience of children towards severe COVID-19, and may propose a novel concept for immunoprophylactic treatments.

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    The authors do not wish to provide a response at this time.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary: This study by Magalhaes et al sheds light on the molecular underpinnings of the relative resistance of children to severe COVID-19. The authors found that priming of epithelial cells by resident immune cells to express tonic levels PRR receptors MDA-5 and RIG-I predisposes the epithelial cells for a faster and more robust onset of IFN-beta production upon SARS-CoV-2 infection. The study uses a combination of in vitro and ex vivo models, as well as mining of scRNA-Seq datasets from clinical specimens.

    Major comments: The claims and conclusions are supported by the data and therefore no new experiments are needed.

    Optional

    1. The use of primary cells (i.e. human airway epithelial cultures cross talking to immune cells) would make this study more compelling, although I assume that the major findings would be recapitulated in such models.
    2. It is not clear how the use of Yersinia enterocolitica to trigger activation of PBMC is relevant to this story. Using different (commensal) pathogens to achieve PBMC activation may yield different and more physiologically relevant results.
    3. The manuscripts would greatly benefit from improved structure and focus, particularly in the Abstract, Introduction and Results sections. The text is very dense, and makes it difficult for the reader to follow the flow and to distinguish important from less important information. Particularly, the introduction starts very broadly introducing COVID-19, which I think we are by now all familiar with. Directly starting with the burning question why kids get less sick with SARS-CoV-2 would capture the readers' attention better. Figure 1 a is beautiful for a review but much too dense to help the reader as a graphical abstract. In the results section, for each experiment, leading with clearly stating the rationale of the specific question, the gap in knowledge and why the gap is there, then followed by the results, then summarizing the impact of said results, would make this a much more enjoyable read and help the reader evaluate the novelty and impact better, particularly for Figures 1, 2, and 3 (but also all others). The interaction wheel graphs (Figure 4. are amazing, but are not properly explained in the text (do I read this right that in adults, all the crosstalk is basically performed by proliferating T-cells?). In all, these scientific writing issues sell an otherwise beautiful story short.

    Referees cross-commenting

    I agree with reviewers 1 and 2 that the use of primary cells would significantly elevate the story. However, I think this should be "optional", as I do not think it would change the findings.

    Significance

    General assessment:

    The main strength of the study are its topic and clearly relevant question: why do kids rarely get severe COVID-19? The main novelty is the answer to this question, that immune cell-epithelial crosstalk in children elevates the tonic expression of MDA5 and RIG-I via the IRF1 axis, leading to faster onset of IFN production and signaling upon SARS-CoV-2 challenge, which ultimately mounts an antiviral response detrimental to robust SARS-CoV-2 replication. The study uses an innovative combination of in vivo and ex vivo experiments and analysis of clinical specimens.

    The significant advance of this study to the field is clear to this reviewer, although it could be much better stated in the manuscript, as described at length above. The study is of great interest to the field of immunology and virology, and also has clinical and translational impact with respect to risk assessment for severe COVID-19 per age group, as well as epidemiological considerations for infection control.

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    Referee #2

    Evidence, reproducibility and clarity

    In the manuscript entitled "Enhanced airway epithelial response to SARS-CoV-2 infection in children is critically tuned by the crosstalk between immune and epithelial cells" Gonçalves Magalhães et al address the molecular interactions that result in greater antiviral responses to coronavirus infections in children versus adults. The authors have re-analyzed previously reported scRNA data from the nasal passages of healthy donors and found heightened pro-inflammatory responses in the nasal passages of children relative to the signatures in adults. Thus, the authors posit that this could result in priming of epithelial cells subsequently boosting antiviral responses to SARS-CoV-2 infection in a RIG-I and MDA5 dependent manner. Beyond the expected antiviral protection conferred by type I IFNs, the authors demonstrate type II IFN and TNF can also promote antiviral priming. Although epithelial and immune cell crosstalk has been previously demonstrated, this study proposes an age-dependent differences in the magnitude of this crosstalk. Indeed, bacterial stimulation of PBMCs derived from children resulted in greater cytokine [production and A549 priming. The article was well written, the data presented is compelling, and appropriately controlled. Importantly, the authors appropriately acknowledge the study limitations. The mechanisms that govern the increased inflammatory responsiveness of tissues derived from children remain to be addressed.

    Major comments:

    • Although the authors acknowledge this limitation, do primary epithelial cells respond to priming? Is there an age difference in viral detection and/or the response to priming?
    • Optional: Does the deletion of IRF3 phenocopy MAVS deficiency in the context of type I IFN priming and blockade of IFN replication (Fig 2D)? Does priming induced increases in IRF1 and IRF7 and is this sufficient to overcome the loss of IRF3 (PMID: 25520509)?

    Minor comments:

    • Are the differences observed in MDA5 and RIG-I expression after PBMC stimulation across A549 IFNR KO cells significant?
    • Figure 2C could be strengthened by the addition of total IRF3 and STAT2 immunoblot.
    • Figure 3C, include tSTAT2 control.
    • There is one typo on line 610. Change OSA2 to OAS2.
    • Please expand the discussion to include relevant work on IFN and TNF-mediated antiviral priming (PMID: 16537619) and epithelial-immune cell crosstalk (PMID: 36563691).

    Significance

    Advance: This study expands upon previous work by the authors that described that children have higher expression of RLRs and in epithelial and immune cells relative to adults. The current work, provides an incremental but important advance to the previous study by demonstrating that PBMC-mediated priming of epithelial cells (A549). However, it remains to be addressed whether epithelial cells from children have increased capacity to detect SARS-CoV-2 infection or respond to priming.

    Audience: This work is of interest to pediatric clinicians, virologists, immunologists, cell biologists, bioinformaticians.

    Reviewer expertise: viral innate immunity; IFN regulation

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    Referee #1

    Evidence, reproducibility and clarity

    In the manuscript entitled "Enhanced Airway Epithelial Response to SARS-CoV-2 Infection in Children is Critically Tuned by the Cross-Talk Between Immune and Epithelial Cells", the authors report a mechanism how airway epithelium of children is in a primed state to more efficiently defense SARS-CoV-2 infection. They interestingly found that the expression level of MDA5 and RIG-I are essential for this primed stage. In details, they discovered that enhanced immune-epithelial cell interactions through cytokines play a major role in the upregulation of these two RNA sensors, where IRF1 may be the most important regulator. They fully used their single cell sequencing data and established a relative convincing in vitro model to validate their hypothesis. This story is quite attractive to me since they explained the behind mechanisms for a clinical phenomenon. However, there are still some issues need to be addressed.

    Major comments:

    1. A549 is a cancer cell line and cannot support SARS-CoV-2 replication. The authors reply on it too much. Some important experiments need to be performed in primary airway epithelial cells.
    2. MDA5 and RIG-I protein levels need to be detected in some important experiments such as Figure 4D, Figure 5A, Figure 5B, Figure 6B.

    Minor comments:

    1. In figure 1B, the authors claimed that "As expected, pre-treatment alone did not trigger notable IFN-β transcription". But in my mind, pretreatment elevated IFN- β mRNA at least 5 folds compared to mock group (from 10-2.5 to 10-1.8). The authors need to make the statement more accurate.
    2. In figure 2A, the authors used IFNARKO cells. This should be mentioned in the manuscript.
    3. The statement of Figure 2C should be more accurate, "This was also confirmed at the downstream level of STAT2 activation, at which phosphorylation was still observed upon infection of RIG-I or MDA5 single KOs with SARS-CoV- 2, but was fully diminished only in the double KO cells (Fig 2C)." IRF3 phosphorylation is not downstream of STAT2. More downstream event can be shown as STAT1/STAT2/IRF9 nuclear translocation or ISGs transcription. Also, total STAT2 levels should be shown here.
    4. It is quite surprising that none of IRNAR, RIG-I, MDA5 affected SARS-CoV-2 replication in untreated cells.
    5. If we compare the mRNA induction of MDA5 and RIG-I between Figure 4D and Figure 6B, they are not in the same amplitude. There are 10 folds induction in Figure 4D while 100-300 folds in Figure 6B.

    Significance

    This is an interesting study with clear data. They fully used their single cell sequencing data and established a relative convincing in vitro model to validate their hypothesis. They uncovered an important mechanism why airway epithelium of children is in a primed state and more resistant to SARS-CoV-2 infection.

    The limitation here is that they do not have in vivo model to support their conclusions.

    Nevertheless, this manuscript is still able to answer a clinical question. Why children are much more resistant to SARS-CoV-2 infections compared to adults? It has good clinical significance. I believe that a broad audience will be interested in this story.

    I study virus and cancer induced metabolic reprogramming, virus and host interaction and innate immune regulation, and mass spectrometry (metabolomics and proteomics).

  5. Version published to 10.1101/2023.05.17.541103v1 on bioRxiv