Temporal single-cell analysis reveals age-associated delay in immune resolution after respiratory viral infection
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eLife Assessment
This study presents valuable findings that could potentially allow a deeper understanding of the immunopathogenesis underlying influenza infection in aged mice. The results are based on solid evidence that define putative immune determinants underlying immunopathology in the aged lung. This study will be of interest to researchers pursuing aging research, as well as to immunologists.
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Abstract
Aging is a major risk factor for increased morbidity and mortality following acute respiratory virus infections. To elucidate the immune determinants underlying viral pathogenesis and delayed lung repair in the aged lung, a comprehensive time-course study was conducted. Single-cell RNA sequencing (scRNAseq) and high-dimensional flow cytometry were utilized to compare lungs from young and aged mice infected with influenza A virus (IAV). Aged hosts displayed diminished alveolar macrophage (AM) and dendritic cell (DC) but elevated monocyte-derived macrophage (MoM) and interstitial macrophage (IM) presence following infection. Additionally, enhanced accumulation of adaptive immune cells, including CD4 + tissue-resident helper (T RH ) cells, CD8 + tissue-resident memory (T RM ) cells, and a B cell subset resembling age-associated B cells, was observed in the memory phase. Pathway analysis revealed that elevated type I and II interferon (IFNα/γ) signaling, especially in MoM/IM subsets, distinguished the aged hosts from the young. Inhibition of IFNα/γ signaling after viral clearance improved long-term respiratory outcomes and reduced both IM and T RH populations in aged mice. These findings highlight the pivotal role of IFNα/γ signaling, likely within MoM/IM subsets, in driving the exuberant persistence of adaptive immune cells and chronic immunopathology in the aged lung following acute viral infection.
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eLife Assessment
This study presents valuable findings that could potentially allow a deeper understanding of the immunopathogenesis underlying influenza infection in aged mice. The results are based on solid evidence that define putative immune determinants underlying immunopathology in the aged lung. This study will be of interest to researchers pursuing aging research, as well as to immunologists.
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Reviewer #1 (Public review):
Summary:
In this report, Dr Jie Sun and colleagues employed high-resolution single-cell technologies (transcriptomic + cytometry) to build a temporal map of lung responses after IAV infection in young and old mice. They performed detailed analyses of several innate and adaptive immune compartments and described how age influences each of them. The data are robustly generated, and the analyses provide interesting observations that could be associated with disease severity in aged mice. Mechanistically, the authors provide evidence that IFNa/g signaling after viral clearance could mediate some long-term respiratory outcomes, possibly by acting on MoIMs.
Strengths:
(1) Comprehensive temporal profiling of lung responses.
(2) Combination of scRNA_seq and flow cytometry.
(3) Mechanistic part assessing the role of …
Reviewer #1 (Public review):
Summary:
In this report, Dr Jie Sun and colleagues employed high-resolution single-cell technologies (transcriptomic + cytometry) to build a temporal map of lung responses after IAV infection in young and old mice. They performed detailed analyses of several innate and adaptive immune compartments and described how age influences each of them. The data are robustly generated, and the analyses provide interesting observations that could be associated with disease severity in aged mice. Mechanistically, the authors provide evidence that IFNa/g signaling after viral clearance could mediate some long-term respiratory outcomes, possibly by acting on MoIMs.
Strengths:
(1) Comprehensive temporal profiling of lung responses.
(2) Combination of scRNA_seq and flow cytometry.
(3) Mechanistic part assessing the role of IFNa/g signaling.
Weaknesses:
(1) Descriptive nature of the study.
(2) Lack of quantification of lung lesions.
(3) Lung functional measurements were only assessed in aged mice (with or without treatment).
(4) No assessment of global and virus-specific humoral responses, which could be related to changes in B cells.
(5) Recently described "pro-repair" Ly6G+ macrophages after IAV infection (PMID: 39093958) are not considered here, and the gating strategy encompasses them in the neutrophil gate.
(6) The authors suggest that IMs in the aged lung may serve as a major contributor to the pathogenesis of long-term sequelae observed in aged hosts, but do not assess this possibility experimentally.
Addressing the weaknesses identified above would substantially strengthen the conclusions of the manuscript.
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Reviewer #2 (Public review):
Summary:
In this paper, the authors leverage single-cell approaches to delve deeper into the host responses and immune cells involved in immunopathogenesis of influenza virus infection in aged mice. The dynamics of gene expression and immune cell frequencies were also tracked at multiple time-points to examine the acute and chronic changes in young and aged mice after influenza virus infection. Their analyses demonstrated that the immune cell frequencies and gene signatures differed in young and aged mice, especially macrophages, T cells and B cells. Furthermore, interferon pathways were found to be differentially regulated in the young and aged mice, and blocking the interferon pathway with monoclonal antibodies led to improvement in lung respiratory functions and reduced inflammation.
Strengths:
A strength …
Reviewer #2 (Public review):
Summary:
In this paper, the authors leverage single-cell approaches to delve deeper into the host responses and immune cells involved in immunopathogenesis of influenza virus infection in aged mice. The dynamics of gene expression and immune cell frequencies were also tracked at multiple time-points to examine the acute and chronic changes in young and aged mice after influenza virus infection. Their analyses demonstrated that the immune cell frequencies and gene signatures differed in young and aged mice, especially macrophages, T cells and B cells. Furthermore, interferon pathways were found to be differentially regulated in the young and aged mice, and blocking the interferon pathway with monoclonal antibodies led to improvement in lung respiratory functions and reduced inflammation.
Strengths:
A strength of this study is that multiple time points are considered for analyses, allowing assessment of temporal changes in gene expression and immune cell frequencies after virus infection during the acute and chronic phases of the disease. The data presented could also serve as a potential resource for other researchers interested in understanding the host responses to the influenza virus, especially in aged mice. Another interesting finding was that blocking interferon signalling can reduce the chronic severe symptoms caused by the influenza virus in aged mice.
Weaknesses:
The manuscript could greatly benefit from more rigorous approaches, particularly in the statistical analyses and data visualisation. Moreover, the scientific rationale and logic for several parts of the manuscript can be improved. Finally, the authors did not adequately dissect whether the contribution of host responses was from virus infection or from bystander effects. Specifically, my major comments are as follows:
(1) While it is interesting to compare the difference in host responses between aged and young mice, the authors should also more deeply characterise the differences in phenotypic and infection kinetics between aged and young mice, so that the readers can better appreciate the effects of virus infection and host immune tolerance to viral infection. For instance, what are the differences in virus infection kinetics between the aged and young mice? Are the levels of infection different? Are the virus dynamics and kinetics different between aged and young mice? Besides lung tissue damage, are there also tissue damage or inflammatory responses beyond lung tissues that differ between aged and young mice?
(2) Figure 1B: Could the authors quantify the extent of tissue damage in aged and young adults? It is challenging to interpret the extent of tissue damage, especially across the different time points.
(3) Figure 1D: The authors claim that the senescence signatures are higher in aged mice, justifying that the pathway analyses are consistent with ageing signatures. However, it is also important to note that the senescence signatures were insignificant in aged mice after day 14. Is this expected?
(4) Figure 1E: The stacked bar charts are difficult to read. It is unclear if the cell type frequencies or proportions are significantly changed, especially as the authors are showing these changes with averaged values. Moreover, the authors should keep the colours of the bar charts consistent with the UMAP.
(5) Figure 1F-M: The charts show increased frequencies of innate and adaptive immune cells in aged mice. How about the young mice? Which type of cells are increased to allow these mice to be more tolerant to infection?
(6) Figure 2D and Figure S2C: Besides showing the dynamics of the different clusters, the authors should also display the statistics for individual mice. If the analyses have to be pooled for the single-cell analysis, the authors should declare the challenges and show the statistical comparisons for the flow cytometry.
(7) Figure 3E: The authors should not claim differences in somatic hypermutation based on gene expression. This will require BCR sequencing and evidence for clonal expansion to confirm that there are differences in somatic hypermutation. Moreover, the authors did not measure the quality and quantity of antibody responses between aged and young mice. The claims for the antibody responses are thus extrapolated, and the B cell identities cannot be identified without any functional or phenotypic readouts.
(8) Figure 4H. Why did the authors not perform the experiments for aged mice with a higher virus dose? Also, the spider plots do not display the variability between individual mice, making it challenging to interpret whether the changes were statistically different between the conditions.
(9) Figure 5A. Is the interferon pathway the only pathway that was significantly enriched in the aged mice? Is it the top pathway? The authors should also show the other pathways that were significantly enriched in aged mice. Did the authors also analyse whether the differences in interferon pathways were caused by infected cells or by bystander cells?
(10) Figure 5B: Based on the pathway analyses, the peak responses for interferon are at day 9 post-infection. However, the interferon treatment is performed on day 14, where differences were less apparent. Why did the authors choose to do the interferon treatment at day 14 instead?
(11) Figure 6: How about interferon-mediated cell-cell interactions? The authors should consider using established libraries such as Cell Chat to determine if there are any cell-cell communications that lead to differences in interferon responses and signaling.
(12) Throughout the whole manuscript, the authors kept emphasising that the aged mice displayed uncoordinated immune responses, yet, based on the pathway analyses and phenotypic characterisation, it appears that only interferon was mainly dysregulated. I would thus like to recommend that the authors adjust the tone of the manuscript to tailor it to the results obtained from their investigations.
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