CXCR4high megakaryocytes regulate host-defense immunity against bacterial pathogens

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1. 

   eLife

   Aug 9, 2022

   Author Response

   Reviewer #1 (Public Review):

   The authors used single cell RNA-seq to assess the heterogeneity of megakaryocytes, thereby identifying a distinct CXCR4 high subpopulation that was also enriched in inflammatory genes and other chemokines or cytokines. They sort CXCR4 high cells and are able to investigate specific functional properties of this megakaryocyte population. This work complements prior studies which have suggested immune modulatory roles for certain megakaryocyte subsets such as the work of Pariser and colleagues (JCI 2021) on the antigen presentation capacity of lung megakaryocytes or the work by Liu et al (Advanced Science 2021) on immune surveillance gene expression in megakaryocytes (MKs).

   The strengths of the paper are:

   1. Analysis of scRNA-seq to identify MK subsets with validation

   1. The use of sorted …

   Author Response

   Reviewer #1 (Public Review):

   The authors used single cell RNA-seq to assess the heterogeneity of megakaryocytes, thereby identifying a distinct CXCR4 high subpopulation that was also enriched in inflammatory genes and other chemokines or cytokines. They sort CXCR4 high cells and are able to investigate specific functional properties of this megakaryocyte population. This work complements prior studies which have suggested immune modulatory roles for certain megakaryocyte subsets such as the work of Pariser and colleagues (JCI 2021) on the antigen presentation capacity of lung megakaryocytes or the work by Liu et al (Advanced Science 2021) on immune surveillance gene expression in megakaryocytes (MKs).

   The strengths of the paper are:

   1. Analysis of scRNA-seq to identify MK subsets with validation

   1. The use of sorted CXCR4 cells to interrogate the specific in vitro functions of this immune modulatory subset (using CXCR4 low MKs as a comparison) such as phagocytosis assays

   1. Elegant use of the PF4-Cre DTR model to ablate MKs while replenishing CXCR4 high cells as a means to assess functional effects of this subset in vivo which is a reasonable approach in the absence of a Cre that would specifically delete this subset.

   We appreciate the positive feedback from this reviewer.

   Potential weaknesses are:

   1. The unclear distinction between previously identified immune modulatory MK subsets such as the lung MKs which have antigen-processing capacity (Pariser et al) and the currently identified MK5 subset. The authors indicate that the MK5 subset has transcriptomic similarities to the previously described antigen-processing MK subset but this does not explain whether MK5 and/or CXCR4 high subset is indeed the primary. This is an important question because it would help address whether the immune modulatory roles are all concentrated in one MK subset or whether different MK subsets may play distinct roles in innate and adaptive immunity. For example, in Fig 3, there is a broad claim that MKs can modulate innate and adaptive immunity but it is not clear whether this claim is valid only for the specific MK5/CXCR4 subset.

   We totally agree with this argument. Our revised data showed that CXCR4high MKs, but not CXCR4low MKs, were able to phagocytose bacteria (Revised Fig 3F), process and present ovalbumin (OVA) antigens on their cell surface (Revised Fig 3G) to activate CD8+ OT-I T cells (Revised Fig 3H) and B3Z T cells (Revised Fig 3-S2), a T cell hybridoma which expresses TCR that specifically recognizes OVA. These revised data showed that CXCR4high MKs are an antigen processing and presentation subset in MKs.

   1. It would be helpful to understand whether the CXCR4 status of MKs can change over time. Are the CXCR4 high cells generated in infection (Fig 5) generated by the conversion of CXCR4 low cells (or non MK5 cells)? Or do CXCR4 high / MK5 cells differentiate from MK progenitors directly?

   Thanks for the suggested experiment. Our revised data showed that inflammatory treatment, including interferon γ, LPS, and L. monocytogenes could not increase CXCR4 expression in CXCR4low MKs (Revised Fig 4H and Fig 4-S4D). This experiment suggested that CXCR4high MKs might not be reprogramed from CXCR4low MKs. Furthermore, our HSPC tracing experiment showed that CXCR4high MKs were generated from HSPCs as efficiently as CXCR4low MKs during the acute inflammation-induced emergency megakaryopoiesis (Revised Fig 5E-G).

   Reviewer #2 (Public Review):

   Wang J. et al. examines bone marrow megakaryocyte (MK) heterogeneity, and the role that a specific subpopulation plays in the mouse immune response to Listeria monocytogenes infection. Using single cell RNA-sequencing (scRNAseq) the authors identified a bone marrow MK subpopulation, characterized by high CXCR4 expression. This subset referred to as MK-derived immune-stimulating cell (MDIC) population has immune-stimulatory properties and supports the migration and activation of innate immune cells potentially via TNFα and IL-6 secretion.

   In agreement with recent studies mapping in situ myelopoiesis which occurs near bone marrow sinusoidal vessels upon acute inflammatory stress with L. monocytogenes (Zhang J. et al Nature 2021), the authors observed a significant association of myeloid cells with perivascular CXCR4high MK but not with the more abundant CXCR4low MK subset. This study also revealed that MK in vivo deletion leads to a significant increase in the bacterial load in extramedullary hematopoietic organs accompanied by a reduction in the number of myeloid cells, although it is unclear if a similar MDIC population exists outside the bone marrow. Accordingly, it is unclear the effect of MK depletion in the context of L. monocytogenes infection in bone marrow myelopoiesis.

   Notably, in a rescue experiment, MDIC infusion was able to partially rescue the bacterial clearance defect in MK depleted and infected mice, further confirming the important role of MDICs in regulating bacterial immune responses.

   Using Pf4-cre reporter mice the authors further evaluated the capacity of bone marrow MDIC to enter circulation and migrate into organs upon bacterial infection potentially in response to an increase in CXCL12 expression in extramedullary organs. Finally, in agreement with recent studies (Haas S. et al Cell Stem Cell 2015), Wang et al. discovered that upon inflammatory stress, emergency hematopoietic stem cell-derived megakaryopoiesis is activated to restore platelets lost upon inflammation-induced thrombocytopenia but also to regulate immune response to bacterial infection.

   Overall, this study builds on recently published work regarding MK heterogeneity which technically is very challenging to investigate. Although it's suggested that MDIC greatly overlap with the recently described CD53+LSP1+ MK immune population (Sun S. et al Blood 2021), it is still unclear the extent to which these subsets overlap, accordingly, it's still unclear the relationship between bone marrow MDIC and previously described lung MK subsets, though to be enriched in immune function. Nevertheless, the authors performed a detailed characterization of bone marrow MDIC in homeostasis and in acute inflammatory stress, providing new evidence and mechanistic clues on the mechanisms by which MK subsets regulate immune function to bacterial infection.

   While this manuscript has many strengths, some of the author's conclusions and claims require further technical support and discussion. In particular:

   1. The potential mechanism via TNFα and IL-6 secretion is very interesting, however further data is necessary to support the author's claim. First, it's unclear if steady-state MDIC MK express TNFα and IL-6. If so, does this expression change upon infection?

   MDIC MKs (now referred to as CXCR4high MKs) expressed TNFα and IL-6 during the steady state, and maintained their expression levels upon L. monocytogenes infection (Revised Fig 2J).

   Second, mechanistically it would be important to evaluate or at least discuss how MDIC sense bacterial infection and respond by secreting TNFα and IL-6.

   Thanks for the suggestion. In this revision, we have included a brief discussion about previous studies that reported that MKs express multiple inflammation signals, which enable MKs to sense inflammation signals and express cytokines, as “MKs were reported to express multiple inflammation receptors, such as Fcγ receptors (Markovic et al., Br J Haematol 1995), Toll-like receptors (Beaulieu et al., Blood 2011; Ward et al., Thromb Haemost 2005), interleukin receptors (Navarro et al., J Thromb Haemost 1991; Yang et al., Br J Haematol 2000), and IFN receptors (Negrotto et al., J Thromb Haemost 2011), which might enable MKs to receive inflammation signals and express cytokines.” (Line 15-19, Page 13).

   Third, in Fig 2L and 2M it's missing a control for the effect of anti-TNFα and anti-IL-6 on phagocytes activity in the absence of MKs.

   Thanks for the suggested control. In this revision, we have confirmed the phagocytosis activity of immune cells by flow cytometry assays as suggested by this reviewer, in which we included the anti-TNFα and anti-IL-6 controls in the absence of MKs (Revised Fig 2M, N). Our revised data consistently showed that CXCR4high MKs enhanced the phagocytosis activity of neutrophils and macrophages through a TNFα and IL-6 dependent manner.

   Fourth, in Fig 2J and 2K it's unusual to evaluate TNFα and IL-6 levels by imaging.

   We agree with the argument. In this revision, we have further evaluated the expression of TNFα and IL-6 by flow cytometry, which consistently showed that CXCR4high MKs had higher expression levels of TNFα and IL-6 than CXCR4low MKs (Revised Fig 2J).

   1. The authors further explored the potential role of MKs in regulating adaptive immune function against bacterial infection, however these studies were very superficial and further studies are needed to substantiate this claim.

   We totally agree with this argument. In this revision, we have deleted the claim that MKs regulate adaptive immune function. Furthermore, Our revised data showed that CXCR4high MKs were able to phagocytose bacteria (Revised Fig 3F), and process and present ovalbumin (OVA) antigens on their cell surface (Revised Fig 3G) to activate CD8+ OT-I T cells (Revised Fig 3H) and B3Z T cells (Revised Fig 3-S2), a T cell hybridoma which expresses TCR that specifically recognizes OVA. These revised data suggested that CXCR4high MKs had antigen processing and antigen presentation capacity, which suggested that CXCR4high MKs might contribute to the regulation of adaptive immune function. We have included a brief discussion (Line 2-5, Page 14).

   1. Overall, the study relies heavily on subjective imaging quantification. The identification of CXCR4high and low MK subsets does not seem entirely objective and it is prone to inaccuracies due to the technical difficulty of bone imaging. The usage of other surface marker(s) for the MDIC subset would significantly improve the study. Accordingly, many of the experiments should be accompanied and/or replaced by flow cytometry analyses such as the phagocytosis experiments in Fig 2; quantification of MKs in Fig 4 H, I and N.

   We totally agree with this argument, and we have discussed that additional markers are warranted to further enrich CXCR4high MKs (Line 5-9 Page 14). Furthermore, we have further confirmed our imaging quantifications by flow cytometry, such as the bacterial phagocytosis ability of immune cells and CXCR4high MKs (Revised Fig 2M, N, Fig 2-S2A, B and Fig 3F) and the number of Tomato+ CXCR4high MKs in the liver, spleen, and lung (Revised Fig 4I, O and Fig 4-S4I).

   1. Regarding MK-deletion experiments, studies from the Passegue lab have shown that this will cause persistent bone marrow myeloid granulocyte/macrophage progenitor (GMP) formation during 5FU stress, most likely due to the reduction in the levels of PF4 and TGFb1 and the effect on hematopoietic stem cells. What happens to bone marrow myelopoiesis upon MK-deletion and bacterial infection? The authors describe a significant reduction in the liver and spleen but it's unclear the effect on the bone marrow. It would be helpful to discuss this point.

   Our revised results showed MK ablation increased the number of hematopoietic stem and progenitor cells and myelopoiesis in the bone marrow upon infection (Revised Fig 3-S1A-D). However, myeloid cells were reduced in the liver and spleen after MK ablation and bacterial infection (Revised Fig 3D-E). This further suggested the important role of CXCR4high MKs in promoting the migration and function of myeloid cells. We have included a brief discussion on this point (Line 10-14, Page 14).

   Reviewer #3 (Public Review):

   Overall this is an interesting study that adds significant knowledge to our understanding and characterization of Mks as immune cells. The identification of CXCR4hi Mks as immune regulatory cells is potentially important, particularly in the bacteria model used in this study.

   We appreciate the positive feedback of this reviewer.

   At this stage, the authors have however made a number of conclusions not yet supported by the data. In particularly differentiating the role of Mks versus the platelets they produce is not clear, so many conclusions about MDIC in immune responses need to be better supported and differentiated from platelet functions.

   We agree with this argument. We cannot exclude the role of platelets in immune responses. Our revised data showed that CXCR4high MKs produced fewer platelets (Revised Fig 1-S6D) but had more robust abilities in phagocytosis and antigen processing and presentation (Revised Fig 3F-H and Fig 3-S2), and stimulating innate immune cells by secreting cytokines (Revised Fig 2E-N and Fig 2-S2) than CXCR4low MKs. Furthermore, infusion with CXCR4high MKs, but not CXCR4low MKs, partially rescued the host-defense responses in MK ablated mice, which further supported the role of CXCR4high MKs in immune responses. However, the infusion rescue experiment with CXCR4high MKs did not fully rescue the host-defense responses in MK ablated mice (Revised Fig 3K-L). This is partially due to the reduced platelets in MK ablated mice as platelets are known for immune responses. We have discussed this possibility in the current version (Line 16-17, Page 9).

   Read the original source
2. 

   Version published to 10.7554/elife.78662 on eLife

   Jul 29, 2022
3. 

   eLife

   May 9, 2022

   Evaluation Summary:

   The manuscript studies the heterogeneity of megakaryocytes using single cell RNA-seq and identifies a subpopulation of CXCR4-high megakaryocytes with immune modulatory roles. The authors also perform functional studies which show that this subpopulation of megakaryocytes promotes bacterial phagocytosis by macrophages and neutrophils. This work would be of significant interest to researchers in the fields of immunology and host defense as well as researchers studying hematopoiesis and megakaryocyte biology because it provides new perspectives on megakaryocyte heterogeneity as well as the role of megakaryocytes in host defense and immune function.

   (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 …

   Evaluation Summary:

   The manuscript studies the heterogeneity of megakaryocytes using single cell RNA-seq and identifies a subpopulation of CXCR4-high megakaryocytes with immune modulatory roles. The authors also perform functional studies which show that this subpopulation of megakaryocytes promotes bacterial phagocytosis by macrophages and neutrophils. This work would be of significant interest to researchers in the fields of immunology and host defense as well as researchers studying hematopoiesis and megakaryocyte biology because it provides new perspectives on megakaryocyte heterogeneity as well as the role of megakaryocytes in host defense and immune function.

   (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 agreed to share their name with the authors.)

   Read the original source
4. 

   eLife

   May 9, 2022

   Reviewer #1 (Public Review):

   The authors used single cell RNA-seq to assess the heterogeneity of megakaryocytes, thereby identifying a distinct CXCR4 high subpopulation that was also enriched in inflammatory genes and other chemokines or cytokines. They sort CXCR4 high cells and are able to investigate specific functional properties of this megakaryocyte population. This work complements prior studies which have suggested immune modulatory roles for certain megakaryocyte subsets such as the work of Pariser and colleagues (JCI 2021) on the antigen presentation capacity of lung megakaryocytes or the work by Liu et al (Advanced Science 2021) on immune surveillance gene expression in megakaryocytes (MKs).

   The strengths of the paper are:

   1. Analysis of scRNA-seq to identify MK subsets with validation

   2. The use of sorted CXCR4 cells to …

   Reviewer #1 (Public Review):

   The authors used single cell RNA-seq to assess the heterogeneity of megakaryocytes, thereby identifying a distinct CXCR4 high subpopulation that was also enriched in inflammatory genes and other chemokines or cytokines. They sort CXCR4 high cells and are able to investigate specific functional properties of this megakaryocyte population. This work complements prior studies which have suggested immune modulatory roles for certain megakaryocyte subsets such as the work of Pariser and colleagues (JCI 2021) on the antigen presentation capacity of lung megakaryocytes or the work by Liu et al (Advanced Science 2021) on immune surveillance gene expression in megakaryocytes (MKs).

   The strengths of the paper are:

   1. Analysis of scRNA-seq to identify MK subsets with validation

   2. The use of sorted CXCR4 cells to interrogate the specific in vitro functions of this immune modulatory subset (using CXCR4 low MKs as a comparison) such as phagocytosis assays

   3. Elegant use of the PF4-Cre DTR model to ablate MKs while replenishing CXCR4 high cells as a means to assess functional effects of this subset in vivo which is a reasonable approach in the absence of a Cre that would specifically delete this subset.

   Potential weaknesses are:

   1. The unclear distinction between previously identified immune modulatory MK subsets such as the lung MKs which have antigen-processing capacity (Pariser et al) and the currently identified MK5 subset. The authors indicate that the MK5 subset has transcriptomic similarities to the previously described antigen-processing MK subset but this does not explain whether MK5 and/or CXCR4 high subset is indeed the primary antigen-processing subset in MKs. This is an important question because it would help address whether the immune modulatory roles are all concentrated in one MK subset or whether different MK subsets may play distinct roles in innate and adaptive immunity. For example, in Fig 3, there is a broad claim that MKs can modulate innate and adaptive immunity but it is not clear whether this claim is valid only for the specific MK5/CXCR4 subset.

   2. It would be helpful to understand whether the CXCR4 status of MKs can change over time. Are the CXCR4 high cells generated in infection (Fig 5) generated by the conversion of CXCR4 low cells (or non MK5 cells)? Or do CXCR4 high / MK5 cells differentiate from MK progenitors directly?

   Read the original source
5. 

   eLife

   May 9, 2022

   Reviewer #2 (Public Review):

   Wang J. et al. examines bone marrow megakaryocyte (MK) heterogeneity, and the role that a specific subpopulation plays in the mouse immune response to Listeria monocytogenes infection. Using single cell RNA-sequencing (scRNAseq) the authors identified a bone marrow MK subpopulation, characterized by high CXCR4 expression. This subset referred to as MK-derived immune-stimulating cell (MDIC) population has immune-stimulatory properties and supports the migration and activation of innate immune cells potentially via TNFα and IL-6 secretion.

   In agreement with recent studies mapping in situ myelopoiesis which occurs near bone marrow sinusoidal vessels upon acute inflammatory stress with L. monocytogenes (Zhang J. et al Nature 2021), the authors observed a significant association of myeloid cells with perivascular …

   Reviewer #2 (Public Review):

   Wang J. et al. examines bone marrow megakaryocyte (MK) heterogeneity, and the role that a specific subpopulation plays in the mouse immune response to Listeria monocytogenes infection. Using single cell RNA-sequencing (scRNAseq) the authors identified a bone marrow MK subpopulation, characterized by high CXCR4 expression. This subset referred to as MK-derived immune-stimulating cell (MDIC) population has immune-stimulatory properties and supports the migration and activation of innate immune cells potentially via TNFα and IL-6 secretion.

   In agreement with recent studies mapping in situ myelopoiesis which occurs near bone marrow sinusoidal vessels upon acute inflammatory stress with L. monocytogenes (Zhang J. et al Nature 2021), the authors observed a significant association of myeloid cells with perivascular CXCR4high MK but not with the more abundant CXCR4low MK subset. This study also revealed that MK in vivo deletion leads to a significant increase in the bacterial load in extramedullary hematopoietic organs accompanied by a reduction in the number of myeloid cells, although it is unclear if a similar MDIC population exists outside the bone marrow. Accordingly, it is unclear the effect of MK depletion in the context of L. monocytogenes infection in bone marrow myelopoiesis.

   Notably, in a rescue experiment, MDIC infusion was able to partially rescue the bacterial clearance defect in MK depleted and infected mice, further confirming the important role of MDICs in regulating bacterial immune responses.
   Using Pf4-cre reporter mice the authors further evaluated the capacity of bone marrow MDIC to enter circulation and migrate into organs upon bacterial infection potentially in response to an increase in CXCL12 expression in extramedullary organs. Finally, in agreement with recent studies (Haas S. et al Cell Stem Cell 2015), Wang et al. discovered that upon inflammatory stress, emergency hematopoietic stem cell-derived megakaryopoiesis is activated to restore platelets lost upon inflammation-induced thrombocytopenia but also to regulate immune response to bacterial infection.

   Overall, this study builds on recently published work regarding MK heterogeneity which technically is very challenging to investigate. Although it's suggested that MDIC greatly overlap with the recently described CD53+LSP1+ MK immune population (Sun S. et al Blood 2021), it is still unclear the extent to which these subsets overlap, accordingly, it's still unclear the relationship between bone marrow MDIC and previously described lung MK subsets, though to be enriched in immune function. Nevertheless, the authors performed a detailed characterization of bone marrow MDIC in homeostasis and in acute inflammatory stress, providing new evidence and mechanistic clues on the mechanisms by which MK subsets regulate immune function to bacterial infection.

   While this manuscript has many strengths, some of the author's conclusions and claims require further technical support and discussion. In particular:

   The potential mechanism via TNFα and IL-6 secretion is very interesting, however further data is necessary to support the author's claim. First, it's unclear if steady-state MDIC MK express TNFα and IL-6. If so, does this expression change upon infection? Second, mechanistically it would be important to evaluate or at least discuss how MDIC sense bacterial infection and respond by secreting TNFα and IL-6. Third, in Fig 2L and 2M it's missing a control for the effect of anti-TNFα and anti-IL-6 on phagocytes activity in the absence of MKs. Fourth, in Fig 2J and 2K it's unusual to evaluate TNFα and IL-6 levels by imaging.

   The authors further explored the potential role of MKs in regulating adaptive immune function against bacterial infection, however these studies were very superficial and further studies are needed to substantiate this claim.

   Overall, the study relies heavily on subjective imaging quantification. The identification of CXCR4high and low MK subsets does not seem entirely objective and it is prone to inaccuracies due to the technical difficulty of bone imaging. The usage of other surface marker(s) for the MDIC subset would significantly improve the study. Accordingly, many of the experiments should be accompanied and/or replaced by flow cytometry analyses such as the phagocytosis experiments in Fig 2; quantification of MKs in Fig 4 H, I and N.

   Regarding MK-deletion experiments, studies from the Passegue lab have shown that this will cause persistent bone marrow myeloid granulocyte/macrophage progenitor (GMP) formation during 5FU stress, most likely due to the reduction in the levels of PF4 and TGFb1 and the effect on hematopoietic stem cells. What happens to bone marrow myelopoiesis upon MK-deletion and bacterial infection? The authors describe a significant reduction in the liver and spleen but it's unclear the effect on the bone marrow. It would be helpful to discuss this point.

   Read the original source
6. 

   eLife

   May 9, 2022

   Reviewer #3 (Public Review):

   Overall this is an interesting study that adds significant knowledge to our understanding and characterization of Mks as immune cells. The identification of CXCR4hi Mks as immune regulatory cells is potentially important, particularly in the bacteria model used in this study.

   At this stage, the authors have however made a number of conclusions not yet supported by the data. In particularly differentiating the role of Mks versus the platelets they produce is not clear, so many conclusions about MDIC in immune responses need to be better supported and differentiated from platelet functions.

   Read the original source
7. 

   Version published to 10.1101/2021.06.09.447810v2 on bioRxiv

   Mar 26, 2022
8. 

   Version published to 10.1101/2021.06.09.447810v1 on bioRxiv

   Jun 10, 2021