The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis
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Evaluation Summary:
This manuscript addresses a long-standing question regarding the highly variable cellular composition and functions as well as immune environments along the epididymis. Using multiple mouse models (bacterial infection and parabiosis between WT and Ccr2 KO) in conjunction with powerful scRNA-seq analyses, the authors provided solid evidence supporting the notion that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for sperm maturations and elimination of pathogens ascending the urogenital tract.
(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. The reviewers remained anonymous to the authors.)
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Abstract
The epididymis functions as transition zone for post-testicular sperm maturation and storage and faces contrasting immunological challenges, i.e. tolerance towards spermatozoa vs. reactivity against pathogens. Thus, normal organ function and integrity relies heavily on a tightly controlled immune balance. Previous studies described inflammation-associated tissue damage solely in the distal regions (corpus, cauda), but not in the proximal regions (initial segment, caput). To understand the observed region-specific immunity along the epididymal duct, we have used an acute bacterial epididymitis mouse model and analyzed the disease progression. Whole transcriptome analysis using RNAseq 10 days post infection showed a pro-inflammatory environment within the cauda, while the caput exhibited only minor transcriptional changes. High-dimensional flow cytometry analyses revealed drastic changes in the immune cell composition upon infection with uropathogenic Escherichia coli . A massive influx of neutrophils and monocytes was observed exclusively in distal regions and was associated with bacterial appearance and tissue alterations. In order to clarify the reasons for the region-specific differences in the intensity of immune responses, we investigated the heterogeneity of resident immune cell populations under physiological conditions by scRNASeq analysis of extravascular CD45+ cells. Twelve distinct immune cell subsets were identified, displaying substantial differences in distribution along the epididymis as further assessed by flow cytometry and immunofluorescence staining. Macrophages constituted the majority of resident immune cells and were further separated in distinct subgroups based on their transcriptional profile, tissue location and monocyte-dependence. Crucially, the proximal and distal regions showed striking differences in their immunological landscapes. These findings indicate that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for addressing the contrasting immunological challenges and thus, preserving tissue integrity and organ function.
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Author Response
Reviewer #1 (Public Review):
This study aimed to test the hypothesis that resident immune cells are strategically positioned along the epididymal duct to provide different immunological environments to prevent pathogens from ascending the urogenital tract. By using an epididymitis mouse model, the differential responses at different segments along the epididymis were examined at both histological and gene expression levels, and the data appeared to support their hypothesis. Furthermore, single-cell RNA-seq analyses identified the composition of resident immune cell types along the epididymal duct, and the parabiosis model further corroborated the major findings. Overall, the study was well conducted and the major conclusion seems well supported. The only caveat is the lack of elucidation on the direct or indirect …
Author Response
Reviewer #1 (Public Review):
This study aimed to test the hypothesis that resident immune cells are strategically positioned along the epididymal duct to provide different immunological environments to prevent pathogens from ascending the urogenital tract. By using an epididymitis mouse model, the differential responses at different segments along the epididymis were examined at both histological and gene expression levels, and the data appeared to support their hypothesis. Furthermore, single-cell RNA-seq analyses identified the composition of resident immune cell types along the epididymal duct, and the parabiosis model further corroborated the major findings. Overall, the study was well conducted and the major conclusion seems well supported. The only caveat is the lack of elucidation on the direct or indirect impact of the resident immune cells on sperm maturation.
We thank the reviewer for his/her feedback and the valuable comments.
We are aware of the fact that the current manuscript lacks further experimental evidence on the effects of immune cells on organ function, especially sperm maturation, and agree that this would constitute a relevant object to study. Although the assessment of the direct or indirect impact of particular immune cells on sperm maturation would require further intensive research, encompassing e.g. the consequences of targeted cell depletions (using several transgenic mouse models) with comprehensive follow-up analysis (i.e. by detecting anti-sperm antibodies, assessing the potential appearance of sperm-induced autoimmune reactions in vivo and conducting in vitro co-culture assays besides conducting sperm functional tests to evaluate capacitation and fertilization competencies). A study of this magnitude is outside of the scope of the present manuscript and would form a separate examination that alone would take more than a year to perform. Therefore, our intention was to submit this article as a ‘Tools and Resource’ article as it is providing a detailed overview of all immune cell types that are shaping the regional immunological landscape based on crucial information about their transcriptional profiles on single cell resolution. In our view the provided data are closing a gap in the current state of knowledge (particularly regarding the transcriptional identity and distribution of described immune cell populations) and will serve as a relevant common platform for current and future approaches.
Reviewer #2 (Public Review):
Pleuger et al. investigated the heterogeneity of resident immune cells in the murine epididymis. The response of immune cells in the different epididymal segments was characterized following acute bacterial infection by flow cytometry, and immunofluorescence microscopy. Single-cell RNA sequencing analysis and parabiosis experiments were performed to provide an atlas of resident immune cells and their etiology in the epididymis under steady-state conditions. The authors conclude that distinct immune cell phenotypes govern specific responses of the different epididymal segments during acute bacterial infection. Overall, the conclusions of this study are well supported by the data, but some specific aspects related to the region-specific phenotypes of resident immune cells need to be revisited.
- In order to conclude that there was an infiltration of neutrophils and monocytes following bacterial injection, the authors should provide flow cytometry quantification of the percentages of immune cell subsets relative to live cells, rather than relative to the CD45+ population.
Following the reviewer’s request, we have replaced the data previously shown in figure 2 by a completely new high-dimensional flow cytometry analysis including FltSNE visualization of CD45+ cell populations in different epididymal regions (IS, Caput, Corpus, Cauda) under different conditions (naive, sham, UPEC 10 days post infection). In addition, we have included bar diagrams displaying the percentage of all investigated immune cell subsets in relation to single live cells. The results displayed in the new figure are similar to previous shown data, but the overall figure layout and visualization method is clearer and more comprehensible. We thank the reviewer for the helpful comment.
- In general, all flow cytometry and immunofluorescence data should be presented and discussed with respect to previously published studies.
This is reflected in the discussion (line 564-575) and in addition by addressing similar points raised by the reviewers.
- A surprisingly low number of CX3CR1-EGFP cells was detected by immunofluorescence in the cauda. This is not in agreement with previous studies showing a similar % of CX3CR1-EGFP cells in the IS and cauda regions by immunofluorescence and flow cytometry. The authors need to discuss this discrepancy. Perhaps the different fixation procedures used in the current study compared to those used in previous studies could account for the loss of EGFP in epididymis cryo-sections. As such, cells that appear to be F4/80 positive but negative for EGFP by immunofluorescence might simply be due to the loss of cytoplasmic EGFP, while F4/80 immunogenicity remained intact.
Within our study, we have shown by combining scRNASeq, flow cytometry and immunostaining that distinct macrophage subgroups co-exist within the epididymis and that the diversity increases towards the cauda. Based on these data, we can assume that cells that appear to be F4/80 positive but negative for CX3CR1 (e.g. clusters 6-9 of the macrophage clustering show a very low level or even lack of Cx3cr1 expression) are distinct from CX3CR1+F4/80+ cells (e.g. clusters 1 and 2 of the macrophage subclustering, both showing a high expression of Cx3cr1). Therefore, our immunostaining (on Cx3cr1GFPCcr2RFP reporter mice) and flow cytometry data (on wild type C57BL/6J mice) in Figure 6 are in line with our transcriptomic data and strongly support the co-existence of both populations. We have seen the described gradient of macrophage numbers (decreasing from IS towards cauda) in all independently performed experiments (naive control group in infection experiments, steady-state characterization in wild type and transgenic mice). A previous study, however, demonstrated a constant CX3CR1+ cell ‘number’ throughout all epididymal regions (~5-6% in live cells, (Battistone et al., 2020)). Here, indeed we notice a discrepancy to our results that show a relatively high ‘number’ of CX3CR1+ cells in the initial segment of naive mice (20% in single live cells, new Figure 2G) that decreases towards the cauda (~5% in single live cells, data shown in the new Figure 2 of naive mice). [It needs to be mentioned that these numbers are slightly different to the percentage of CD45+ cells in single live cells shown in Figure 4 due to different settings in the flow cytometry (thresholding to exclude spermatozoa and debris)]. However, another study (Voisin et al., 2018) showed a comparable ratio of total macrophages within caput and cauda with a similar gradient throughout the epididymal regions (significantly lower ratio within the cauda compared to the caput). Although this study discriminated only between caput and cauda, these data are in line with our results.
Nevertheless, it needs to be noted that calculating the percentage of a population in single live cells is not representing an unbiased quantification approach as this calculation is highly dependent on previous gating (thresholding, aimed events, single cells as well as live cells; the latter is, in turn, dependent on the experimental procedures that may have an impact on the cell viability and antigen recognizability, see below). Rather, it provides important information about the population distribution among regions or conditions. For this reason, a comparison among studies as requested above is not expedient from our point of view. This as well as other studies are limited in the way that they lack an absolute quantification of immune cell populations as that would require e.g. a prior cell-counting or the relation of absolute cell numbers to mg of tissue as conducted in the parabiosis experiment shown in Figure 7 (that in turn is also limited for the epididymal regions due to the necessity of pooling tissue from several mice to obtain a sufficient cell number and thus, masking individual differences). Another alternative would be quantitative morphometric analysis of stained sections that has not been performed in the present study.
By comparing the protocol for the cell isolation and preparation of the single cell suspension between our study and previous reports (Battistone et al., 2020), it appears that different protocols have been applied that indeed could have a major impact. In this regard, the study of (Battistone et al., 2020) used a mixture of collagenase type I (0.5 mg/ml) and collagenase type II (0.5 mg/ml) and incubated tissue fragments for a short period (30 minutes) at 37°C. In contrast, in this study we have chopped the tissue fragments with scissors until no fragments were visible anymore then followed by enzymatic digestion (shaking at 37°C for 45 minutes with 1.5 mg/ml collagenase type IV and 60 U/ ml DNAse). Afterwards, we aspirated the digest 5-6 times through a 30G needle (to release pre-digested sticky cells from each other by shear forces) before passing through a 70 µm cell strainer. We have experienced that we can significantly increase the number of viable cells when using collagenase type IV for a longer time at the ideal concentration at 1.5 mg/ml (similar concentration and incubation duration with collagenase I resulted in a higher proportion of dead cells in the analysis). A longer incubation time increases the obtained cell numbers especially from the IS where the epithelial cells are densely connected to each other. In general, collagenase type IV has a lower tryptic activity than other collagenases and therefore, the usage of collagenase IV limits the damage on membrane proteins and receptors (an overview of the different collagenase types with respective references can be found at: https://www.worthington-biochem.com/products/collagenase/manual).
In summation, we agree with the reviewer that very likely methodological differences account for the mentioned discrepancy of our data to Battistone et al (2020) and raised this point in the revised discussion(ses line 559-564).
The statement "Intriguingly, our data revealed that distinct immunological landscapes exist within proximal (IS, caput) and distal regions (corpus, cauda), that are tailored to the respective needs of the microenvironments" implies that this is the first study that describes immune cell heterogeneity in the epididymis. Please rephrase this statement as previous studies have already shown the segment-specific heterogeneity of resident immune cells in this organ.
To address the reviewer's comment, we have rephrased the statement to “our data unraveled the transcriptional identity and tissue location of extravascular immune cells and further support the existence of distinct immunological environments along the epididymal duct that are tailored to the respective needs of the microenvironment” within the discussion section (line 555-558). Moreover, the previous investigations on epididymal immune cells were acknowledged and cited within the introduction (line 107-124) as well as in the discussion (line 549-554, line 564-575, line 580-584,). We hope that this satisfactorily addresses the reviewer’s critique.
The conclusion that macrophages constitute the major immune cell population of the murine epididymis is not supported by the data provided here. In fact, the authors found that macrophages account for only approximately 20% of CD45+ immune cells in the cauda. The authors should, therefore, modify their conclusion to state that macrophages constitute the major immune cell population in the IS. In fact, this conclusion would be more in line with previously published studies.
The reviewer is correct and we have changed the conclusion to “macrophages constitute the major immune cell population, especially within the IS” accordingly (see line 559-560).
The authors conclude that fewer intraepithelial CX3CR1-EGFP+ cells are present in the cauda, but they do not explain how they actually quantified these intraepithelial cells. A description of how these results were obtained is missing.
We agree with the reviewer that we did not quantify cells based on our immunostaining. All quantification approaches were obtained by flow cytometry on wild type mice with respective surface staining (acc. to previous selection of markers derived from scRNASeq, see Figure 6) and show only ratios, but no absolute numbers. An additional counting of the immunostained section would be required to ultimately determine whether these cells are quantitatively different in the cauda compared to the IS. The respective sentence, however, does not intend to compare the abundance of these cells among epididymal regions, rather it is stating that ‘the distal regions are populated by a more heterogeneous macrophage pool consisting of less intraepithelial CX3CR1+ macrophages, but higher abundance of interstitial pro-inflammatory monocyte-derived CCR2+MHC-II+, vasculature-associated TLF+ macrophages as well as CX3CR1-TLF-CCR2- macrophages’. This statement is pointing to the increasing macrophage heterogeneity towards the distal parts and is based on the clustering of the scRNASeq data, flow cytometry analysis and supported by the immunostaining that localized these populations in the epididymal compartments. For this reason flow cytometry and immunostaining are combined included in Figure 6 to display the ratio of identified macrophage subgroups to each other (Fig. 6B, bar diagram showing % of distinct subpopulations in total F4/80+ cells) with supportiving immunostaining using the same marker for localization.
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Evaluation Summary:
This manuscript addresses a long-standing question regarding the highly variable cellular composition and functions as well as immune environments along the epididymis. Using multiple mouse models (bacterial infection and parabiosis between WT and Ccr2 KO) in conjunction with powerful scRNA-seq analyses, the authors provided solid evidence supporting the notion that resident immune cells are strategically positioned along the epididymal duct, potentially providing different immunological environments required for sperm maturations and elimination of pathogens ascending the urogenital tract.
(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. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study aimed to test the hypothesis that resident immune cells are strategically positioned along the epididymal duct to provide different immunological environments to prevent pathogens from ascending the urogenital tract. By using an epididymitis mouse model, the differential responses at different segments along the epididymis were examined at both histological and gene expression levels, and the data appeared to support their hypothesis. Furthermore, single-cell RNA-seq analyses identified the composition of resident immune cell types along the epididymal duct, and the parabiosis model further corroborated the major findings. Overall, the study was well conducted and the major conclusion seems well supported. The only caveat is the lack of elucidation on the direct or indirect impact of the resident …
Reviewer #1 (Public Review):
This study aimed to test the hypothesis that resident immune cells are strategically positioned along the epididymal duct to provide different immunological environments to prevent pathogens from ascending the urogenital tract. By using an epididymitis mouse model, the differential responses at different segments along the epididymis were examined at both histological and gene expression levels, and the data appeared to support their hypothesis. Furthermore, single-cell RNA-seq analyses identified the composition of resident immune cell types along the epididymal duct, and the parabiosis model further corroborated the major findings. Overall, the study was well conducted and the major conclusion seems well supported. The only caveat is the lack of elucidation on the direct or indirect impact of the resident immune cells on sperm maturation.
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Reviewer #2 (Public Review):
Pleuger et al. investigated the heterogeneity of resident immune cells in the murine epididymis. The response of immune cells in the different epididymal segments was characterized following acute bacterial infection by flow cytometry, and immunofluorescence microscopy. Single-cell RNA sequencing analysis and parabiosis experiments were performed to provide an atlas of resident immune cells and their etiology in the epididymis under steady-state conditions. The authors conclude that distinct immune cell phenotypes govern specific responses of the different epididymal segments during acute bacterial infection. Overall, the conclusions of this study are well supported by the data, but some specific aspects related to the region-specific phenotypes of resident immune cells need to be revisited.
- In order to …
Reviewer #2 (Public Review):
Pleuger et al. investigated the heterogeneity of resident immune cells in the murine epididymis. The response of immune cells in the different epididymal segments was characterized following acute bacterial infection by flow cytometry, and immunofluorescence microscopy. Single-cell RNA sequencing analysis and parabiosis experiments were performed to provide an atlas of resident immune cells and their etiology in the epididymis under steady-state conditions. The authors conclude that distinct immune cell phenotypes govern specific responses of the different epididymal segments during acute bacterial infection. Overall, the conclusions of this study are well supported by the data, but some specific aspects related to the region-specific phenotypes of resident immune cells need to be revisited.
- In order to conclude that there was an infiltration of neutrophils and monocytes following bacterial injection, the authors should provide flow cytometry quantification of the percentages of immune cell subsets relative to live cells, rather than relative to the CD45+ population.
- In general, all flow cytometry and immunofluorescence data should be presented and discussed with respect to previously published studies.
- A surprisingly low number of CX3CR1-EGFP cells was detected by immunofluorescence in the cauda. This is not in agreement with previous studies showing a similar % of CX3CR1-EGFP cells in the IS and cauda regions by immunofluorescence and flow cytometry. The authors need to discuss this discrepancy. Perhaps the different fixation procedures used in the current study compared to those used in previous studies could account for the loss of EGFP in epididymis cryo-sections. As such, cells that appear to be F4/80 positive but negative for EGFP by immunofluorescence might simply be due to the loss of cytoplasmic EGFP, while F4/80 immunogenicity remained intact.
The statement "Intriguingly, our data revealed that distinct immunological landscapes exist within proximal (IS, caput) and distal regions (corpus, cauda), that are tailored to the respective needs of the microenvironments" implies that this is the first study that describes immune cell heterogeneity in the epididymis. Please rephrase this statement as previous studies have already shown the segment-specific heterogeneity of resident immune cells in this organ.
The conclusion that macrophages constitute the major immune cell population of the murine epididymis is not supported by the data provided here. In fact, the authors found that macrophages account for only approximately 20% of CD45+ immune cells in the cauda. The authors should, therefore, modify their conclusion to state that macrophages constitute the major immune cell population in the IS. In fact, this conclusion would be more in line with previously published studies.
The authors conclude that fewer intraepithelial CX3CR1-EGFP+ cells are present in the cauda, but they do not explain how they actually quantified these intraepithelial cells. A description of how these results were obtained is missing.
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