Author Response:
Reviewer #1 (Public Review):
Summary:
The global decline of amphibians is primarily attributed to deadly disease outbreaks caused by the chytrid fungus, Batrachochytrium dendrobatidis (Bd). It is unclear whether and how skin-resident immune cells defend against Bd. Although it is well known that mammalian mast cells are crucial immune sentinels in the skin and play a pivotal role in the immune recognition of pathogens and orchestrating subsequent immune responses, the roles of amphibian mast cells during Bd infections are largely unknown. The current study developed a novel way to enrich X. laevis skin mast cells by injecting the skin with recombinant stem cell factor (SCF), a KIT ligand required for mast cell differentiation and survival. The investigators found an enrichment of skin mast cells provides X. laevis substantial protection against Bd and mitigates the inflammation-related skin damage resulting from Bd infection. Additionally, the augmentation of mast cells leads to increased mucin content within cutaneous mucus glands and shields frogs from the alterations to their skin microbiomes caused by Bd.
Strengths:
This study underscores the significance of amphibian skin-resident immune cells in defenses against Bd and introduces a novel approach to examining interactions between amphibian hosts and fungal pathogens.
Weaknesses:
The main weakness of the study is the lack of functional analysis of X. laevis mast cells. Upon activation, mast cells have the characteristic feature of degranulation to release histamine, serotonin, proteases, cytokines, and chemokines, etc. The study should determine whether X. laevis mast cells can be degranulated by two commonly used mast cell activators IgE and compound 48/80 for IgE-dependent and independent pathways. This can be easily done in vitro. It is also important to assess whether in vivo these mast cells are degranulated upon Bd infection using avidin staining to visualize vesicle releases from mast cells. Figure 3 only showed rSCF injection caused an increase in mast cells in naïve skin. They need to present whether Bd infection can induce mast cell increase and rSCF injection under Bd infection causes a mast cell increase in the skin. In addition, it is unclear how the enrichment of mast cells provides protection against Bd infection and alternations to skin microbiomes after infection. It is important to determine whether skin mast cells release any contents mentioned above.
We would like to thank the reviewer for taking the time to review our work and for providing us with valuable feedback.
Please note that amphibians do not possess the IgE antibody isotype1.
To our knowledge there have been no published studies using approaches for studying mammalian mast cell degranulation to examine amphibian mast cells. Notably, several studies suggest that amphibian mast cells lack histamine2, 3, 4, 5 and serotonin2, 6. While there are commercially available kits and reagents for examining mammalian mast cell granule content, most of these reagents may not cross-react with their amphibian counterparts. This is especially true of cytokines and chemokines, which diverged quickly with evolution and thus do not share substantial protein sequence identity across species as divergent as frogs and mammals. Respectfully, while following up on these findings is possible, it would involve considerable additional work to find reagents that would detect amphibian mast cell contents.
We would also like to respectfully point out that while mast cell degranulation is a feature most associated with mammalian mast cells, this is not the only means by which mammalian mast cells confer their immunological effects. While we agree that defining the biology of amphibian mast cell degranulation is important, we anticipate that since the anti-Bd protection conferred by enriching frog mast cells is seen after 21 days of enrichment, it is quite possible that degranulation may not be the central mechanism by which the mast cells are mediating this protection.
As noted in our manuscript, frog mast cells upregulate their expression of interleukin-4 (IL4), which is a hallmark cytokine associated with mammalian mast cells7. We are presently exploring the role of the frog IL4 in the observed mast cell anti-Bd protection. Should we generate meaningful findings in this regard, we will add them to the revised version of this manuscript.
We are also exploring the heparin content of frog mast cells and capacities of these cells to degranulate in vitro in response to compound 48/80. In addition, we are exploring in vivo mast cell degranulation via histology and avidin-staining. Should these studies generate significant findings, we will include them in the revised version of this manuscript.
Per the reviewer’s suggestion, in our revised manuscript we also plan to include data showing whether Bd infections affect skin mast cell numbers and how rSCF injection impacts skin mast cell numbers in the context of Bd infections.
In regard to how mast cells impact Bd infections and skin microbiomes, our data indicate that mast cells are augmenting skin integrity during Bd infections and promoting mucus production, as indicated by the findings presented in Figure 4A-C and Figure 5A-C, respectively. There are several mammalian mast cell products that elicit mucus production. In mammals, this mucus production is mediated by goblet cells while the molecular control of amphibian skin mucus gland content remains incompletely understood. Interleukin-13 (IL13) is the major cytokine associated with mammalian mucus production8, while to our knowledge this cytokine is either not encoded by amphibians or else has yet to be identified and annotated in these animals’ genomes. IL4 signaling also results in mucus production9 and we are presently exploring the possible contribution of the X. laevis IL4 to skin mucus gland filling. Any significant findings on this front will be included in the revised manuscript. Histamine release contributes to mast cell-mediated mucus production10, but as we outline above, several studies indicate that amphibian mast cells may lack histamine2, 3, 4, 5. Mammalian mast cell-produced lipid mediators also play a critical role in eliciting mucus secretion11 and our transcriptomic analysis indicates that frog mast cells express several enzymes associated with production of such mediators. We will highlight this observation in our revised manuscript.
We anticipate that X. laevis mast cells influence skin integrity, microbial composition and Bd susceptibility in a myriad of ways. Considering the substantial differences between amphibian and mammalian evolutionary histories and physiologies, we anticipate that many of the mechanisms by which X. laevis mast cells confer anti-Bd protection will prove to be specific to amphibians and some even unique to X. laevis. We are most interested in deciphering what these mechanisms are but foresee that they will not necessarily reflect what one would expect based on what we know about mammalian mast cells in the context of mammalian physiologies.
Reviewer #2 (Public Review):
Summary:
In this study, Hauser et al investigate the role of amphibian (Xenopus laevis) mast cells in cutaneous immune responses to the ecologically important pathogen Batrachochytrium dendrobatidis (Bd) using novel methods of in vitro differentiation of bone marrow-derived mast cells and in vivo expansion of skin mast cell populations. They find that bone marrow-derived myeloid precursors cultured in the presence of recombinant X. laevis Stem Cell Factor (rSCF) differentiate into cells that display hallmark characteristics of mast cells. They inject their novel (r)SCF reagent into the skin of X. laevis and find that this stimulates the expansion of cutaneous mast cell populations in vivo. They then apply this model of cutaneous mast cell expansion in the setting of Bd infection and find that mast cell expansion attenuates the skin burden of Bd zoospores and pathologic features including epithelial thickness and improves protective mucus production and transcriptional markers of barrier function. Utilizing their prior expertise with expanding neutrophil populations in X. laevis, the authors compare mast cell expansion using (r)SCF to neutrophil expansion using recombinant colony-stimulating factor 3 (rCSF3) and find that neutrophil expansion in Bd infection leads to greater burden of zoospores and worse skin pathology.
Strengths:
The authors report a novel method of expanding amphibian mast cells utilizing their custom-made rSCF reagent. They rigorously characterize expanded mast cells in vitro and in vivo using histologic, morphologic, transcriptional, and functional assays. This establishes solid footing with which to then study the role of rSCF-stimulated mast cell expansion in the Bd infection model. This appears to be the first demonstration of the exogenous use of rSCF in amphibians to expand mast cell populations and may set a foundation for future mechanistic studies of mast cells in the X. laevis model organism.
We thank the reviewer for recognizing the breadth and extent of the undertaking that culminated in this manuscript. Indeed, this manuscript would not have been possible without considerable reagent development and adaptation of techniques that had previously not been used for amphibian immunity research. In line with the reviewer’s sentiment, to our knowledge this is the first report of using molecular approaches to augment amphibian mast cells, which we hope will pave the way for new areas of research within the fields of comparative immunology and amphibian disease biology.
Weaknesses:
The conclusions regarding the role of mast cell expansion in controlling Bd infection would be stronger with a more rigorous evaluation of the model, as there are some key gaps and remaining questions regarding the data. For example:
1. Granulocyte expansion is carefully quantified in the initial time courses of rSCF and rCSF3 injections, but similar quantification is not provided in the disease models (Figures 3E, 4G, 5D-G). A key implication of the opposing effects of mast cell vs neutrophil expansion is that mast cells may suppress neutrophil recruitment or function. Alternatively, mast cells also express notable levels of csfr3 (Figure 2) and previous work from this group (Hauser et al, Facets 2020) showed rG-CSF-stimulated peritoneal granulocytes express mast cell markers including kit and tpsab1, raising the question of what effect rCSF3 might have on mast cell populations in the skin. Considering these points, it would be helpful if both mast cells and neutrophils were quantified histologically (based on Figure 1, they can be readily distinguished by SE or Giemsa stain) in the Bd infection models.
We thank the reviewer for this insightful suggestion. We are performing a further examination of skin granulocyte content during Bd infections and plan on including any significant findings in our revised manuscript.
We predict that rSCF administration results in the accumulation of mast cells that are polarized such that they ablate the inflammatory response elicited by Bd infection. Mammalian mast cells, including peritonea-resident mast cells, express csf3r12, 13. Although the X. laevis animal model does not permit nearly the degree of immune cell resolution afforded by mammalian animal models, we do know that the adult X. laevis peritonea contain heterogenous leukocyte populations. We anticipate that the high kit expression reported by Hauser et al., 2020 in the rCSF3-recruited peritoneal leukocytes reflects the presence of mast cells therein. As such and in acknowledgement of the reviewer’s suggestion, we also think that the cells recruited by rCSF3 into the skin may include not only neutrophils but also mast cells. Possibly, these mast cells have distinct polarization states from those enriched by rSCF. While the lack of antibodies against frog neutrophils or mast cells has limited our capacity to address this question, we will attempt to reexamine by histology the proportions of skin neutrophils and mast cells in the skins of frogs under the conditions described in our manuscript. Any new findings in this regard will be included in the revised version of this work.
2. Epithelial thickness and inflammation in Bd infection are reported to be reduced by rSCF treatment (Figure 3E, 5A-B) or increased by rCSF3 treatment (Figure 4G) but quantification of these critical readouts is not shown.
We thank the reviewer for this suggestion. We will score epithelial thickness under the distinct conditions described in our manuscript and present the quantified data in the revised paper.
3. Critical time points in the Bd model are incompletely characterized. Mast cell expansion decreases zoospore burden at 21 dpi, while there is no difference at 7 dpi (Figure 3E). Conversely, neutrophil expansion increases zoospore burden at 7 dpi, but no corresponding 21 dpi data is shown for comparison (Figure 4G). Microbiota analysis is performed at a third time point,10 dpi (Figure 5D-G), making it difficult to compare with the data from the 7 dpi and 21 dpi time points. Reporting consistent readouts at these three time points is important to draw solid conclusions about the relationship of mast cell expansion to Bd infection and shifts in microbiota.
Because there were no significant effects of mast cell enrichment at 7 days post Bd infection, we chose to look at the microbiome composition in a subsequent experiment at 10 days and 21 days post Bd infection, with 10 days being a bit more of a midway point between the initial exposure and day 21, when we see the effect on Bd loads. We will clarify this rationale in the revised manuscript.
The enrichment of neutrophils in frog skins resulted in prompt (12 hours post enrichment) skin thickening (in absence of Bd infection) and increased frog Bd susceptibility by 7 days of infection. Conversely, mast cell enrichment stabilized skin mucosal and symbiotic microbial environment, presumably accounting at least in part for the lack of further Bd growth on mast cell-enriched animals by 21 days of infection. Our question regarding the roles of inflammatory granulocytes/neutrophils during Bd infections was that of ‘how’ rather ‘when’ these cells affect Bd infections. Because the central focus of this work was mast cells and not other granulocyte subsets, when we saw that rCSF3-recruited granulocytes adversely affected Bd infections at 7 days post infection, we did not pursue the kinetics of these responses further. We plan to explore the roles of inflammatory mediators and disparate frog immune cell subsets during the course of Bd infections, but we feel that these future studies are more peripheral to the central thesis of the present manuscript regarding the roles of frog mast cells during Bd infections.
4. Although the effect of rSCF treatment on Bd zoospores is significant at 21 dpi (Figure 3E), bacterial microbiota changes at 21 dpi are not (Figure S3B-C). This discrepancy, how it relates to the bacterial microbiota changes at 10 dpi, and why 7, 10, and 21 dpi time points were chosen for these different readouts (Figure 5F-G), is not discussed.
Our results indicate that after 10 days of Bd infection, control Bd-challenged animals exhibited reduced microbial richness, while skin mast cell-enriched Bd-infected frogs were protected from this disruption of their microbiome. The amphibian microbiome serves as a major barrier to these fungal infections14, and we anticipate that Bd-mediated disruption of microbial richness and composition facilitates host skin colonization by this pathogen. Control and mast cell-enriched animals had similar skin Bd loads at 10 days post infection. However, by 21 days of Bd infection the mast cells-enriched animals maintained their Bd loads to levels observed at 10 days post infection, whereas the control animals had significantly greater Bd loads. Thus, we anticipate that frog mast cells are conferring the observed anti-Bd protection in part by preventing microbial disassembly and thus interfering with optimal Bd colonization and growth on frog skins. In other words, maintained microbial composition at 10 days of infection may be preventing additional Bd colonization/growth, as seen when comparing skins of control and mast cell-enriched frogs at 21 days post infection. By 21 days of infection, control animals rebounded from the Bd-mediated reduction in bacterial richness seen at 10 days. Considering that after 21 days of infection control animals also had significantly greater Bd loads than mast-cell enriched animals suggests that there may be a critical earlier window during which microbial composition is able to counteract _Bd_growth.
While the current draft of our manuscript has a paragraph to this effect (see below), we appreciate the reviewer conveying to us that our perspective on the relationship between skin mast cells and the kinetics of microbial composition and _Bd_loads could be better emphasized. We plan to revise our manuscript to include the above discussion points.
Bd infections caused major reductions in bacterial taxa richness, changes in composition and substantial increases in the relative abundance of Bd-inhibitory bacteria early in the infection. Similar changes to microbiome structure occur during experimental Bd infections of red-backed salamanders and mountain yellow-legged frogs15, 16. In turn, progressing _Bd_infections corresponded with a return to baseline levels of Bd-inhibitory bacteria abundance and rebounding microbial richness, albeit with dissimilar communities to those seen in control animals. These temporal changes indicate that amphibian microbiomes are dynamic, as are the effects of Bd infections on them. Indeed, Bd infections may have long-lasting impacts on amphibian microbiomes15. While Bd infections manifested in these considerable changes to frog skin microbiome structure, mast cell enrichment appeared to counteract these deleterious effects to their microbial composition. Presumably, the greater skin mucosal integrity and mucus production observed after mast cell enrichment served to stabilize the cutaneous environment during Bd infections, thereby ameliorating the Bd-mediated microbiome changes. While this work explored the changes in established antifungal flora, we anticipate the mast cell-mediated inhibition of Bd may be due to additional, yet unidentified bacterial or fungal taxa. Intriguingly, while mammalian skin mast cell functionality depends on microbiome elicited SCF production by keratinocytes17, our results indicate that frog skin mast cells in turn impact skin microbiome structure and likely their function. It will be interesting to further explore the interdependent nature of amphibian skin microbiomes and resident mast cells.
5. The time course of rSCF or rCSF3 treatments relative to Bd infection in the experiments is not clear. Were the treatments given 12 hours prior to the final analysis point to maximize the effect? For example, in Figure 3E, were rSCF injections given at 6.5 dpi and 20.5 dpi? Or were treatments administered on day 0 of the infection model? If the latter, how do the authors explain the effects at 7 dpi or 21 dpi given mast cell and neutrophil numbers return to baseline within 24 hours after rSCF or rCSF3 treatment, respectively?
Please find the schematic of the immune manipulation, Bd infection, and sample collection times below. We will include a figure like this in our revised manuscript.
The title of the manuscript may be mildly overstated. Although Bd infection can indeed be deadly, mortality was not a readout in this study, and it is not clear from the data reported that expanding skin mast cells would ultimately prevent progression to death in Bd infections.
We acknowledge this point. The revised manuscript will be titled: “Amphibian mast cells: barriers to chytrid fungus infections”.
Reviewer #3 (Public Review):
Summary:
Hauser et al. provide an exceptional study describing the role of resident mast cells in amphibian epidermis that produce anti-inflammatory cytokines that prevent Batrachochytrium dendrobatidis (Bd) infection from causing harmful inflammation, and also protect frogs from changes in skin microbiomes and loss of mucin in glands and loss of mucus integrity that otherwise cause changes to their skin microbiomes. Neutrophils, in contrast, were not protective against Bd infection. Beyond the beautiful cytology and transcriptional profiling, the authors utilized elegant cell enrichment experiments to enrich mast cells by recombinant stem cell factor, or to enrich neutrophils by recombinant colony-stimulating factor-3, and examined respective infection outcomes in Xenopus.
Strengths:
Through the use of recombinant IL4, the authors were able to test and eliminate the hypothesis that mast cell production of IL4 was the mechanism of host protection from Bd infection. Instead, impacts on the mucus glands and interaction with the skin microbiome are implicated as the protective mechanism. These results will press disease ecologists to examine the relative importance of this immune defense among species, the influence of mast cells on the skin microbiome and mucosal function, and open the potential for modulating mucosal defense.
We thank the reviewer for recognizing the significance and utility of the findings presented in our manuscript.
Weaknesses:
A reduction of bacterial diversity upon infection, as described at the end of the results section, may not always be an "adverse effect," particularly given that anti-Bd function of the microbiome increased. Some authors (see Letourneau et al. 2022 ISME, or Woodhams et al. 2023 DCI) consider these short-term alterations as encoding ecological memory, such that continued exposure to a pathogen would encounter an enriched microbial defense. Regardless, mast cell-initiated protection of the mucus layer may negate the need for this microbial memory defense.
We thank the reviewer their insightful comment. We will revise our discussion to include this possible interpretation.
While the description of the mast cell location in the epidermal skin layer in amphibians is novel, it is not known how representative these results are across species ranging in chytridiomycosis susceptibility. No management applications are provided such as methods to increase this defense without the use of recombinant stem cell factor, and more discussion is needed on how the mast cell component (abundance, distribution in the skin) of the epidermis develops or is regulated.
We appreciate the reviewer’s comment and would like to point out that the work presented in our manuscript was driven by comparative immunology questions more than by conservation biology.
We thank the reviewer for suggesting expanding our discussion to include potential management applications and potential mechanisms for regulating frog skin mast cells. While any content to these effects would be highly speculative, we agree that it may spark new interest and pave new avenues for research. To this end, our revised manuscript will include a paragraph to this effect.
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