Injury-induced pulmonary tuft cells are heterogenous, arise independent of key Type 2 cytokines, and are dispensable for dysplastic repair
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Evaluation Summary:
In this paper, the authors study distal tuft cells that are induced by influenza and bleomycinthe and find that Tuft cells originate from p63+ distal cells in virally-induced dysplastic regions of the lung as evidenced by lineage tracing. Interestingly, single cell sequencing reveals heterogeneity of tuft cells reminiscence of the murine tracheal tuft cells and supports a p63+ cell origin. They also found that the tuft cell induction is independent of the IL-25 and IL-4Ra pathway and since type 2 inflammation has been associated with tuft cell induction in the intestine, this suggests a different biology for the distal pulmonary tuft cells, although the inflammation-associated biology of the corresponding tracheal tuft cells has not been established. Somewhat surprisingly, tuft cell deficient mice do not develop abnormalities of alveolar regeneration following influenza and similarly, mucous metaplasia, which is associated with type 2 inflammation, was unchanged in the tuft cell deficient mice. Although the major findings of the study are negative, it provides important new information on these enigmatic cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors. This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2022.03.11.483948v1)
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Abstract
While the lung bears significant regenerative capacity, severe viral pneumonia can chronically impair lung function by triggering dysplastic remodeling. The connection between these enduring changes and chronic disease remains poorly understood. We recently described the emergence of tuft cells within Krt5 + dysplastic regions after influenza injury. Using bulk and single-cell transcriptomics, we characterized and delineated multiple distinct tuft cell populations that arise following influenza clearance. Distinct from intestinal tuft cells which rely on Type 2 immune signals for their expansion, neither IL-25 nor IL-4ra signaling are required to drive tuft cell development in dysplastic/injured lungs. In addition, tuft cell expansion occurred independently of type I or type III interferon signaling. Furthermore, tuft cells were also observed upon bleomycin injury, suggesting that their development may be a general response to severe lung injury. While intestinal tuft cells promote growth and differentiation of surrounding epithelial cells, in the lungs of tuft cell deficient mice, Krt5 + dysplasia still occurs, goblet cell production is unchanged, and there remains no appreciable contribution of Krt5 + cells into more regionally appropriate alveolar Type 2 cells. Together, these findings highlight unexpected differences in signals necessary for murine lung tuft cell amplification and establish a framework for future elucidation of tuft cell functions in pulmonary health and disease.
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Author Response
Reviewer #3 (Public Review):
The manuscript by Barr et al., investigates the molecular phenotype, regulation by type 2 immunity, and function, of ectopic tuft cells that appear in the lungs of mice recovering from infection by the mouse-adapted PR8 strain of influenza A virus. They use reporter mice and either bulk or single cell RNA sequencing to reveal the molecular heterogeneity among tuft cells present in lungs of mice 43 days after PR8 infection. Lineage tracing using a Krt5-CreER driver line was used to demonstrate the basal cell origin of ectopic tuft cells and mice harboring homozygous null alleles for either Pou2f3, Trpm5, IL4Ra or IL25, were evaluated to determine roles for tuft cells and type 2 immunity in regulation of dysplastic epithelial remodeling. Their data confirm that ectopic tuft cells are derived …
Author Response
Reviewer #3 (Public Review):
The manuscript by Barr et al., investigates the molecular phenotype, regulation by type 2 immunity, and function, of ectopic tuft cells that appear in the lungs of mice recovering from infection by the mouse-adapted PR8 strain of influenza A virus. They use reporter mice and either bulk or single cell RNA sequencing to reveal the molecular heterogeneity among tuft cells present in lungs of mice 43 days after PR8 infection. Lineage tracing using a Krt5-CreER driver line was used to demonstrate the basal cell origin of ectopic tuft cells and mice harboring homozygous null alleles for either Pou2f3, Trpm5, IL4Ra or IL25, were evaluated to determine roles for tuft cells and type 2 immunity in regulation of dysplastic epithelial remodeling. Their data confirm that ectopic tuft cells are derived from dysplastic Krt5-expressing cells that appear following PR8 infection, that pre-existing tuft cells play no role in basal cell dysplasia, and that ectopic tuft cells derived from dysplastic basal cells play no role in lung remodeling. Furthermore, they show that neither type 2 cytokines nor IL25, an upstream regulator of type 2 immune responses, play roles in regulating the pulmonary response to PR8 infection. Finally, they show that tuft cells are also induced in the lungs of bleomycin-injured mice and that the presence of tuft cells in alveolar regions of PR8-infected mice does not influence the inability of dysplastic basal cells to assume alveolar epithelial cell fates. The manuscript is well written and experiments were performed with rigorous experimental design and data of high quality. However, even though findings have potential importance and could be of interest, results seem preliminary and lack a strong rationale.
Major concerns:
- Studies of tuft cells in the gut and their response to type 2 immunity, which were the basis for this line of investigation into ectopic tuft cells in the PR8-infected lung, have shown that tuft cells are part of a feed-forward loop leading to tuft cell expansion and enhanced type 2 immune responses including increased abundance of goblet cells. Since ectopic pulmonary tuft cells are derived from dysplastic basal cells after PR8 infection, rather than the reverse, this is clearly not the case in lungs of PR8 infected mice. Furthermore, since tuft cells are derived from hyperplastic basal cells in lungs of PR8-infected mice, it would seem unlikely that they impact the extent of basal cell hyperplasia.
Ultimately the reviewer is correct in that the mechanisms at play in the post-flu lung promoting ectopic tuft cell expansion are clearly distinct from those in the small intestine. However, this was not a foregone conclusion, especially given that similar Type 2-dependent mechanisms clearly have a role in brush cell (now also termed tuft cell) expansion in the trachea. Regarding tuft cell influence on basal cell hyperplasia, we originally hypothesized that tuft cells differentiating from the migrating, proliferating basal cells may act in a feed-forward fashion to promote continued proliferation of the basal cells, akin to what happens upon tuft cell activation in the intestine. Nevertheless the Reviewer is correct in that our results show that basal cell hyperplasia is independent of tuft cell differentiation, and we feel this is valuable information for the field.
- Tuft cell expansion following parasitic infection of the gut and associated type 2 inflammation, and basal cell differentiation into tuft cells leading to their increased abundance following lung injury, are distinct processes and likely to be regulated through distinct mechanisms. As such, the rationale for investigating the roles of type 2 cytokines in the regulation of tuft cell appearance is rather weak. In the absence of data demonstrating how basal to tuft cell differentiation is regulated, this component of the study seems preliminary.
Amplification of tuft cells in the small intestine (Gerbe et al., 2016; Howitt et al., 2016; von Moltke et al., 2016) and upper airways (Ualiyeva et al., 2021, Bankova et al., 2018) are either totally dependent on or highly influenced by Type 2 cytokines, respectively. Accordingly, it was critical to examine whether a similar mechanism was at play in the lung after influenza injury, i.e. promoting tuft cell amplification downstream of Type 2 cytokines. While our findings demonstrate that post-flu tuft cells arise largely independent of Th2 signals, new findings in other tissues published after submission of the current manuscript do indeed demonstrate Th2 / ILC2-indepdent functions of tuft cells (O’Leary et al., DOI: 10.1126/sciimmunol.abj1080). Our findings support the existence of novel mechanisms regulating tuft cell differentiation, and as the Reviewer suggests, we hope to uncover these mechanisms in future work.
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Evaluation Summary:
In this paper, the authors study distal tuft cells that are induced by influenza and bleomycinthe and find that Tuft cells originate from p63+ distal cells in virally-induced dysplastic regions of the lung as evidenced by lineage tracing. Interestingly, single cell sequencing reveals heterogeneity of tuft cells reminiscence of the murine tracheal tuft cells and supports a p63+ cell origin. They also found that the tuft cell induction is independent of the IL-25 and IL-4Ra pathway and since type 2 inflammation has been associated with tuft cell induction in the intestine, this suggests a different biology for the distal pulmonary tuft cells, although the inflammation-associated biology of the corresponding tracheal tuft cells has not been established. Somewhat surprisingly, tuft cell deficient mice do not develop …
Evaluation Summary:
In this paper, the authors study distal tuft cells that are induced by influenza and bleomycinthe and find that Tuft cells originate from p63+ distal cells in virally-induced dysplastic regions of the lung as evidenced by lineage tracing. Interestingly, single cell sequencing reveals heterogeneity of tuft cells reminiscence of the murine tracheal tuft cells and supports a p63+ cell origin. They also found that the tuft cell induction is independent of the IL-25 and IL-4Ra pathway and since type 2 inflammation has been associated with tuft cell induction in the intestine, this suggests a different biology for the distal pulmonary tuft cells, although the inflammation-associated biology of the corresponding tracheal tuft cells has not been established. Somewhat surprisingly, tuft cell deficient mice do not develop abnormalities of alveolar regeneration following influenza and similarly, mucous metaplasia, which is associated with type 2 inflammation, was unchanged in the tuft cell deficient mice. Although the major findings of the study are negative, it provides important new information on these enigmatic cells.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors. This manuscript was co-submitted with: https://www.biorxiv.org/content/10.1101/2022.03.11.483948v1)
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Reviewer #3 (Public Review):
The manuscript by Barr et al., investigates the molecular phenotype, regulation by type 2 immunity, and function, of ectopic tuft cells that appear in the lungs of mice recovering from infection by the mouse-adapted PR8 strain of influenza A virus. They use reporter mice and either bulk or single cell RNA sequencing to reveal the molecular heterogeneity among tuft cells present in lungs of mice 43 days after PR8 infection. Lineage tracing using a Krt5-CreER driver line was used to demonstrate the basal cell origin of ectopic tuft cells and mice harboring homozygous null alleles for either Pou2f3, Trpm5, IL4Ra or IL25, were evaluated to determine roles for tuft cells and type 2 immunity in regulation of dysplastic epithelial remodeling. Their data confirm that ectopic tuft cells are derived from dysplastic …
Reviewer #3 (Public Review):
The manuscript by Barr et al., investigates the molecular phenotype, regulation by type 2 immunity, and function, of ectopic tuft cells that appear in the lungs of mice recovering from infection by the mouse-adapted PR8 strain of influenza A virus. They use reporter mice and either bulk or single cell RNA sequencing to reveal the molecular heterogeneity among tuft cells present in lungs of mice 43 days after PR8 infection. Lineage tracing using a Krt5-CreER driver line was used to demonstrate the basal cell origin of ectopic tuft cells and mice harboring homozygous null alleles for either Pou2f3, Trpm5, IL4Ra or IL25, were evaluated to determine roles for tuft cells and type 2 immunity in regulation of dysplastic epithelial remodeling. Their data confirm that ectopic tuft cells are derived from dysplastic Krt5-expressing cells that appear following PR8 infection, that pre-existing tuft cells play no role in basal cell dysplasia, and that ectopic tuft cells derived from dysplastic basal cells play no role in lung remodeling. Furthermore, they show that neither type 2 cytokines nor IL25, an upstream regulator of type 2 immune responses, play roles in regulating the pulmonary response to PR8 infection. Finally, they show that tuft cells are also induced in the lungs of bleomycin-injured mice and that the presence of tuft cells in alveolar regions of PR8-infected mice does not influence the inability of dysplastic basal cells to assume alveolar epithelial cell fates. The manuscript is well written and experiments were performed with rigorous experimental design and data of high quality. However, even though findings have potential importance and could be of interest, results seem preliminary and lack a strong rationale.
Major concerns:
1. Studies of tuft cells in the gut and their response to type 2 immunity, which were the basis for this line of investigation into ectopic tuft cells in the PR8-infected lung, have shown that tuft cells are part of a feed-forward loop leading to tuft cell expansion and enhanced type 2 immune responses including increased abundance of goblet cells. Since ectopic pulmonary tuft cells are derived from dysplastic basal cells after PR8 infection, rather than the reverse, this is clearly not the case in lungs of PR8 infected mice. Furthermore, since tuft cells are derived from hyperplastic basal cells in lungs of PR8-infected mice, it would seem unlikely that they impact the extent of basal cell hyperplasia.
2. Tuft cell expansion following parasitic infection of the gut and associated type 2 inflammation, and basal cell differentiation into tuft cells leading to their increased abundance following lung injury, are distinct processes and likely to be regulated through distinct mechanisms. As such, the rationale for investigating the roles of type 2 cytokines in the regulation of tuft cell appearance is rather weak. In the absence of data demonstrating how basal to tuft cell differentiation is regulated, this component of the study seems preliminary. -
Reviewer #2 (Public Review):
The authors study distal tuft cells that are induced by influenza and bleomycin. These cells are of unknown function. In this paper, the authors find that:
1. Tuft cells originate from p63+ distal cells in virally-induced dysplastic regions of the lung as evidenced by lineage tracing.
2. Single-cell sequencing reveals heterogeneity of tuft cells reminiscence of the murine tracheal tuft cells and supports a p63+ cell origin.
3. The tuft cell induction is independent of the IL-25 and IL-4Ra pathway. Since type 2 inflammation has been associated with tuft cell induction in the intestine, this suggests different biology for the distal pulmonary tuft cells, although the inflammation-associated biology of the corresponding tracheal tuft cells has not been established.
4. Tuft cell-deficient mice do not develop …Reviewer #2 (Public Review):
The authors study distal tuft cells that are induced by influenza and bleomycin. These cells are of unknown function. In this paper, the authors find that:
1. Tuft cells originate from p63+ distal cells in virally-induced dysplastic regions of the lung as evidenced by lineage tracing.
2. Single-cell sequencing reveals heterogeneity of tuft cells reminiscence of the murine tracheal tuft cells and supports a p63+ cell origin.
3. The tuft cell induction is independent of the IL-25 and IL-4Ra pathway. Since type 2 inflammation has been associated with tuft cell induction in the intestine, this suggests different biology for the distal pulmonary tuft cells, although the inflammation-associated biology of the corresponding tracheal tuft cells has not been established.
4. Tuft cell-deficient mice do not develop abnormalities of alveolar regeneration following influenza. Similarly, mucous metaplasia, which is associated with type 2 inflammation, was unchanged in the tuft cell-deficient mice.
5. Of note, the paper presents important negative results, but the function of tuft cells remains enigmatic. -
Reviewer #1 (Public Review):
In this manuscript, Barr and colleagues report some novel and surprising results in regards to the development and role of tuft cells during influenza-induced lung injury. The authors demonstrate beautifully how unlike in the intestine lung tuft cells do not require Il-25, Il-4Ra, or Trmp5 but do require Pou2f3. Interestingly, loss of tuft cells in Pou2f3 null mice did not affect basal cell or goblet cell differentiation in basal cell pods, raising the question as to what they are really doing there.
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