Systemic inflammation recruits fast-acting anti-inflammatory innate myeloid progenitors from BM into lymphatics
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Evaluation Summary:
The authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. The cells appear in the lymphatics earlier than in peripheral blood. This type of trafficking is triggered by LPS administration and is anti-inflammatory.
(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 #2 agreed to share their name with the authors.)
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Abstract
Innate immune cellular effectors are actively consumed during systemic inflammation but the systemic traffic and the mechanisms that support their replenishment remain unknown. Here we demonstrate that acute systemic inflammation induces the emergent activation of a previously unrecognized system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors, but not other progenitors or stem cells, from bone marrow (BM) to lymphatic capillaries. The progenitor traffic to the systemic lymphatic circulation is mediated by Ccl19/Ccr7 and is NFκB independent, Traf6/IκB-kinase/SNAP23 activation which is responsible for the secretion of pre-stored Ccl19 by a subpopulation of CD205 + /CD172a + conventional dendritic cells type 2 (cDC2) and upregulation of BM myeloid progenitor Ccr7 signaling. The consequence of this progenitor traffic is anti-inflammatory with promotion of early survival and initiation of replenishment of lymph node cDC.
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Response to Reviewer #2 (Public Review):
The main strengths of this manuscript include: (1) the intriguing and novel observation of lymphatic migration early during inflammation; (2) the various techniques used to address the questions, including imaging and flow cyotmetric analysis, as well as functional assays; and (3) the thorough mechanistic model they have built through their investigation of signaling molecules and the chemokine-receptor interactions necessary for dendritic cell replenishment. Using the Lyve-1 mouse, they were able to identify vessels in the bone, suggesting a specific route for migration. They were also able to determine that the Lin- progenitors were in close proximity to these vessels upon LPS challenge and differentiated into dendritic cells. The ability of myeloid cells to rapidly release preformed CCL19 was …
Response to Reviewer #2 (Public Review):
The main strengths of this manuscript include: (1) the intriguing and novel observation of lymphatic migration early during inflammation; (2) the various techniques used to address the questions, including imaging and flow cyotmetric analysis, as well as functional assays; and (3) the thorough mechanistic model they have built through their investigation of signaling molecules and the chemokine-receptor interactions necessary for dendritic cell replenishment. Using the Lyve-1 mouse, they were able to identify vessels in the bone, suggesting a specific route for migration. They were also able to determine that the Lin- progenitors were in close proximity to these vessels upon LPS challenge and differentiated into dendritic cells. The ability of myeloid cells to rapidly release preformed CCL19 was also dependent on TRAF6, thus suggesting that mature cells in the lymph nodes initiate recruitment of CCR7+ myeloid progenitors, highlighting a novel circuitry of regeneration.
This study is very comprehensive…
We want to thank the reviewer #2 for his/her kind words and appreciation of the quality of our manuscript.
…though there are several questions remaining:
- the conclusion regarding the physiological role of this early response in survival is not well supported by the data;
We agree with the reviewer’s comment. We have modified the title and removed the survival analyses of Ccr7-deficient mice after lethal or sublethal doses of Ccr7. We have also re-phrased our statements in the Abstract, Introduction, Results and Discussion sections in this regard.
- the link with observations in humans is not robust;
The murine model was developed aiming to determine how myeloid progenitors (which were found to be enriched in the side population cells of human inflammatory lymphadenopathies) were reaching the tissue. The use of high-dose LPS intended to define short-term mechanisms of migration as we were aware that chronic administration of LPS results in multiple effects in the innate and adaptive immune systems that would hamper a fair interpretation of mechanisms of migration. We have re-phrased the manuscript to include an explanation of the rationale of our murine experiments subsequent to our human clinical trial observations.
- a number of questions regarding progenitor survival and proliferation remain. First, studies revealing enhanced mortality when CCR7 is blocked or when CCL19 production is lacking may be due to impacts on a variety of other cell types, most notably T regulatory cells.
We have removed the survival data on Ccr7-deficient mice treated with lethal or sublethal doses of LPS, and edited the manuscript as suggested in Abstract, Introduction, Results and Discussion sections. We have circumscribed our analysis to define the effect of anti-Ccr7 on preventing the migration of myeloid progenitors.
The reason these mice die faster was not carefully investigated and is unclear. While the authors conclude it is due to reduced anti-inflammatory dendritic cells, they provide very little data to support this.
We have modified the title of the manuscript, softened our conclusions and removed any statement suggesting that the effect of Traf6 deficiency depends on dendritic cells.
Second, data presented in the manuscript highlighting the presence of side population cells in human lymph nodes under specific conditions is consistent with the observations in the mouse model.
We appreciate the comment by the reviewer. The SP cells found in human LN corresponded to myeloid progenitors as identified in methylcellulose cultures of sorted SP cells. This myeloid progenitor population is the same as the one we identified in the mouse model.
However, the authors do not investigate functional potential in detail and do not account for abundance of mature cells in these lymph nodes (particularly the lymphoma patients, that may result in decreased frequency of HSPCs).
We did not quantify the absolute number of SP cells in LN since this was essentially impossible to determine given the nature of the experimental design in the clinical trial. The size and origin of these LNs was diverse as presented in Figures 1 and S1 and, unlike in the mouse model where complete regional LN chains were analyzed, the LNs sampled in the human trial did not include enough LN material to make the proposed quantification relevant.
Finally, though the findings are very interesting and the studies are robust, one potential concern is that TRAF6 is downstream of a variety of innate signaling pathways and, in general, the dysfunction of myeloid cells may be profound and beyond the conclusion of directing migration, as TRAF6-dependent proliferation may also contribute to the observations made in vivo.
We agree with the reviewer. We have softened our statements and made them descriptive. Myeloid cell Traf6 signaling is crucial for anti-inflammatory effects and seems to be crucial to induce mature myeloid-cell dependent migration of myeloid progenitors. Whether these two processes coincide within the same cell type or depend on different myeloid cell types was not completely unveiled. We have revised the Discussion section and clarified this point.
Overall, this is a compelling story and reveals a novel migratory pathway that may operate in a variety of settings to replenish immune cells to maintain homeostasis, and how this trafficking is impacted in different immune/inflammatory and diseased states warrants more investigation.
We want to thank the reviewer for their kind words and appreciation of the quality of our manuscript. We have softened the statements and conclusions linking the myeloid progenitor migration with the increased mortality observed in LysM-Traf6 deficient mice.
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Response to Reviewer #1 (Public Review):
[...] The studies are well performed and the data supports the conclusions. The role of this signaling axis in the recruitment of GMPs/MDPs has not been investigated in this detail.
We want to thank the reviewer for their kind words and appreciation of the quality of our manuscript.
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Reviewer #2 (Public Review):
This manuscript addresses how myeloid cells are rapidly regenerated during periods of consumptive stress, such as that what occurs during infection. The authors defined a novel migration pattern activated upon inflammation wherein bone marrow-derived myeloid progenitors rapidly seed lymph nodes to produce dendritic cells. Using an in vivo model (injection of LPS) they demonstrated systemic inflammation was necessary for triggering this migratory pathway. A key observation was that prior to detection in the blood, myeloid progenitors were detected in the lymphatics, including the thoracic duct and lymph nodes. Using a combination of imaging strategies, in vitro assays, and transplantation assays the specific myeloid differentiation of these progenitors was revealed: progenitors in lymphatics did not have stem …
Reviewer #2 (Public Review):
This manuscript addresses how myeloid cells are rapidly regenerated during periods of consumptive stress, such as that what occurs during infection. The authors defined a novel migration pattern activated upon inflammation wherein bone marrow-derived myeloid progenitors rapidly seed lymph nodes to produce dendritic cells. Using an in vivo model (injection of LPS) they demonstrated systemic inflammation was necessary for triggering this migratory pathway. A key observation was that prior to detection in the blood, myeloid progenitors were detected in the lymphatics, including the thoracic duct and lymph nodes. Using a combination of imaging strategies, in vitro assays, and transplantation assays the specific myeloid differentiation of these progenitors was revealed: progenitors in lymphatics did not have stem cell function but maintained potential to generate dendritic cells. Using adoptive transfer experiments they determined that labeled progenitors did not home to the bone marrow after LPS. Moreover, prior to their detection in the lymph nodes, these progenitors were found in close proximity to lymphatic endothelial cells in the bone, as determined with intra vital imaging of Lyve-1-GFP mice. They also observed the existence of Lyve-1+ vessels in the bone of LPS treated mice, rarely observed in controls. Therefore, it was concluded that myeloid progenitors are released from the bone marrow and enter the lymphatics very rapidly upon LPS challenge via a network of lymphatic vessels in the bone.
To determine mechanisms that were required for this migratory pathway, they first focused on the signaling molecule TRAF6, a key signaling protein downstream of TLR signaling. Using Mx1-Cre inducible TRAF6 deficiency they observed reduce mobilization of progenitors and found a cell-autonomous defect in migration towards LPS-stimulated cells in vitro. These chemotactic assays were used to identify the specific role of myeloid cells in driving migration of progenitors. The authors ruled out the role of NF-kB signaling via over-expressing the degradation-resistant mutant of IkBa, but revealed that protein-trafficking was necessary for progenitor mobilization. Analysis of chemokines and potential factors that could drive this trafficking pattern identified the chemokine CCL19 and its receptor CCR7 in migration. In vivo targeting of this pathway via antibody blockade experiments demonstrated that CCL19 and CCR7 were required for the myeloid progenitor mobilization, and, furthermore, that the mature myeloid (CD11b+CD11c+) cells in the LNs were sources of CCL19.
The main strengths of this manuscript include: (1) the intriguing and novel observation of lymphatic migration early during inflammation; (2) the various techniques used to address the questions, including imaging and flow cyotmetric analysis, as well as functional assays; and (3) the thorough mechanistic model they have built through their investigation of signaling molecules and the chemokine-receptor interactions necessary for dendritic cell replenishment. Using the Lyve-1 mouse, they were able to identify vessels in the bone, suggesting a specific route for migration. They were also able to determine that the Lin- progenitors were in close proximity to these vessels upon LPS challenge and differentiated into dendritic cells. The ability of myeloid cells to rapidly release preformed CCL19 was also dependent on TRAF6, thus suggesting that mature cells in the lymph nodes initiate recruitment of CCR7+ myeloid progenitors, highlighting a novel circuitry of regeneration.
This study is very comprehensive, though there are several questions remaining: (1) the conclusion regarding the physiological role of this early response in survival is not well supported by the data; (2) the link with observations in humans is not robust; (3) a number of questions regarding progenitor survival and proliferation remain. First, studies revealing enhanced mortality when CCR7 is blocked or when CCL19 production is lacking may be due to impacts on a variety of other cell types, most notably T regulatory cells. The reason these mice die faster was not carefully investigated and is unclear. While the authors conclude it is due to reduced anti-inflammatory dendritic cells, they provide very little data to support this. Second, data presented in the manuscript highlighting the presence of side population cells in human lymph nodes under specific conditions is consistent with the observations in the mouse model. However, the authors do not investigate functional potential in detail and do not account for abundance of mature cells in these lymph nodes (particularly the lymphoma patients, that may result in decreased frequency of HSPCs). Finally, though the findings are very interesting and the studies are robust, one potential concern is that TRAF6 is downstream of a variety of innate signaling pathways and, in general, the dysfunction of myeloid cells may be profound and beyond the conclusion of directing migration, as TRAF6-dependent proliferation may also contribute to the observations made in vivo.
Overall, this is a compelling story and reveals a novel migratory pathway that may operate in a variety of settings to replenish immune cells to maintain homeostasis, and how this trafficking is impacted in different immune/inflammatory and diseased states warrants more investigation.
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Reviewer #1 (Public Review):
In this manuscript the authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. This traffic is mediated by Ccl19/Cccr7 and is NfkB independent but Traf activation dependent. This type of trafficking is anti-inflammatory with promotion of early survival.
Specifically, authors work shows the traffic of DC-biased myeloid progenitors through direct transit from BM to bone lymphatic capillaries. This type of trafficking is highly activated in endotoxic inflammation. Giving LPS to mice results in massive mobilization of myeloid progenitors from the BM to lymph and retention in LN takes place. This happens rapidly and …
Reviewer #1 (Public Review):
In this manuscript the authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. This traffic is mediated by Ccl19/Cccr7 and is NfkB independent but Traf activation dependent. This type of trafficking is anti-inflammatory with promotion of early survival.
Specifically, authors work shows the traffic of DC-biased myeloid progenitors through direct transit from BM to bone lymphatic capillaries. This type of trafficking is highly activated in endotoxic inflammation. Giving LPS to mice results in massive mobilization of myeloid progenitors from the BM to lymph and retention in LN takes place. This happens rapidly and before the appearance of these cells in PB. This type pf LPS challenge induces Ccr7 expression on GMPs as well as secretion of CcL9 in the LN. Importantly, loss of CcL9 or neutralizing Ccr7 inhibits GMP/MDP migration to the LN and inflammation induce mortality.
The studies are well performed and the data supports the conclusions. The role of this signaling axis in the recruitment of GMPs/MDPs has not been investigated in this detail.
-
Evaluation Summary:
The authors demonstrate that acute systemic inflammation induces a new system of rapid migration of granulocyte-macrophage progenitors and committed macrophage-dendritic progenitors but not other progenitors or stem cells from BM to lymphatic capillaries. The cells appear in the lymphatics earlier than in peripheral blood. This type of trafficking is triggered by LPS administration and is anti-inflammatory.
(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 #2 agreed to share their name with the authors.)
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