Neuron-associated macrophage proliferation in the sensory ganglia is associated with peripheral nerve injury-induced neuropathic pain involving CX3CR1 signaling

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    Evaluation Summary:

    Guimaraes et al address the origin of the macrophage increase in sensory ganglia after peripheral nerve injury. The authors show that there is no major influx by blood-derived monocytes into ganglia after injury and that resident macrophages proliferate, which is dependent on CX3CR1 signaling. Overall the work is clear and sound and should be of interest to immunologists and neurobiologists.

    (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 and Reviewer #3 agreed to share their names with the authors.)

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Abstract

Resident macrophages are distributed across all tissues and are highly heterogeneous due to adaptation to different tissue-specific environments. The resident macrophages of the sensory ganglia (sensory neuron-associated macrophages, sNAMs) are in close contact with the cell body of primary sensory neurons and might play physiological and pathophysiological roles. After peripheral nerve injury, there is an increase in the population of macrophages in the sensory ganglia, which have been implicated in different conditions, including neuropathic pain development. However, it is still under debate whether macrophage accumulation in the sensory ganglia after peripheral nerve injury is due to the local proliferation of resident macrophages or a result of blood monocyte infiltration. Here, we confirmed that the number of macrophages increased in the sensory ganglia after the spared nerve injury (SNI) model in mice. Using different approaches, we found that the increase in the number of macrophages in the sensory ganglia after SNI is a consequence of the proliferation of resident CX3CR1 + macrophages, which participate in the development of neuropathic pain, but not due to infiltration of peripheral blood monocytes. These proliferating macrophages are the source of pro-inflammatory cytokines such as TNF and IL-1b. In addition, we found that CX3CR1 signaling is involved in the sNAMs proliferation and neuropathic pain development after peripheral nerve injury. In summary, these results indicated that peripheral nerve injury leads to sNAMs proliferation in the sensory ganglia in a CX3CR1-dependent manner accounting for neuropathic pain development. In conclusion, sNAMs proliferation could be modulated to change pathophysiological conditions such as chronic neuropathic pain.

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  1. Author Response

    Reviewer #1 (Public Review):

    The authors address the origin of the macrophage increase in sensory ganglia after peripheral nerve injury, showing that there is no major influx by blood-derived monocytes into ganglia after injury and that resident macrophages proliferate, which is dependent on CX3CR1 signaling.

    • Interesting and relevant question, mainly addressed with adequate experimental approaches.
    • Most conclusions are supported by the data, however, some important controls and experiments are missing.
    • The authors should demarcate their results from the study of Iwai et al, 2021 which addresses similar questions.

    Thank you for the positive comments, we hope that our point-by-point responses below and the important changes/inclusions in the MS satisfactorily addressed your concerns. We agree that some important controls were missing, and we have included additional data in the revised manuscript. Regarding the Iwai et al. paper, it is in line with our hypothesis. In fact, they suggest that in trigeminal ganglia (TG), resident macrophages proliferate after peripheral injury, although they detected few blood monocytes infiltrating the TG. Our paper, besides to confirm Iwai et al. results, by using different and complementary approaches are more specific compared to BM transfer in irradiated mice, we also advanced in terms of the mechanisms that these cells proliferate (CX3CR1 signalling) and the impact of these proliferation for neuropathic pain development. We discussed these points in the new version of the MS. Please see page 4 lines 88-93.

    Reviewer #2 (Public Review):

    The investigators looked at mφs in lumbar DRG after a spared nerve injury in which two of the three branches of the sciatic nerve are transected and the third left intact. This is a classical preparation for studying neuropathic pain. This paper demonstrates that the increase of mφs is an increase in the number of CX3CR1+ (resident) mφs and not CCR2+ (infiltrating mφs) by using CX3CR1 and CCR2 individual reporter mice. Using a CX3CR1 conditional knockout (KO) mouse, they found that this receptor must be present on the mφs for the increase in number to occur. Next, they did a parabiosis experiment with GFP+ mice and found that neither of these mφ subtypes infiltrated into the DRG. To examine proliferation, they injected animals with Ki67 and found this label, which is an indication of proliferation, was present in the CX3CR1+ mφs (but not the CCR2+ mφs). Finally, they identified the CX3CR1 mφs to be the cells that express TNFα and IL-1β but not IL-6.

    An experiment that would be useful would be to determine if there is an increase or a decrease in the availability to mφs of the ligand CXC3L1 after the spared nerve injury. The authors state from the work of others that membrane-bound CX3CL1 is constitutively expressed and that it is decreased after nerve injury. They hypothesize that this indicates a release of the chemokine, but such a decrease could also indicate a decrease in expression. A few sentences on what is known in other systems on the importance and mode of action of membrane-bound and non-membrane-bound CX3CL1 would be useful.

    Thanks to the reviewer for a great summary of our manuscript. We have now performed a time course of Cx3cl1 expression in the DRG after the spared nerve injury and it was included in figure 7A. We also apologise for the lack of information regarding the importance and mode of action of membrane-bound and non-membrane-bound CX3CL1, which is now included in the discussion section (Page 16).

    The main weakness of the manuscript is that many highly relevant previous findings, in some cases reporting nearly identical experiments sometimes with the same and sometimes with somewhat different results, are not mentioned. Kalinski et al. (which is cited but not in this context) reported a very similar parabiosis experiment. While they did not identify subtypes of mφs, they found an increase in infiltration of mφs, which was small (though statistically significant) compared to the larger increase that occurred in the distal nerve. In 2013 and 2018, Niemi et al. and Lindborg et al (J Neurosci and J

    Neuroinflammation respectively) reported that mφs in the DRG are somewhat decreased in a CCR2 KO mouse, suggesting again that there is some infiltration of mφs into the DRG after axotomy. They also showed that the mφ chemokine CCL2 increases in the DRG after sciatic nerve injury. With regard to proliferation, Yu et al. in 2020 (which again is cited but not in this context) also used a spared nerve paradigm stained DRGs for CX3CR1+ mφs and found an increase. They then stained DRG sections for Ki67 and demonstrated proliferation in this population. An earlier reference by Krishnan et al in 2018 published in J Neuropathol Exp Neurol is entitled "An Intimate Role for Adult Dorsal Root Ganglia Resident Cycling Cells in the Generation of Local Macrophages and Satellite Glial Cells". With regard to cytokine expression, in 1995, Murphy et al published a paper in J Neurosci demonstrating induction of interleukin-6 in axotomized sensory neurons.

    Thank you for the comment. These papers, you have indicated, are the main reason we have idealised our MS. The controversy regarding the possible infiltration of peripheral blood monocytes for the increase in the number of macrophages in the sensory ganglia after peripheral nerve injury. Furthermore, some of these papers you also indicated, came out during the execution of this manuscript, and they also brought controversies or did not explore some points. Therefore, we believe that our work by using different and complementary approaches strongly support the hypothesis that after peripheral nerve injury, peripheral blood monocytes did not infiltrate the DRGs significantly, but that the increase in the macrophages population is due to the proliferation of resident macrophages. Furthermore, we provided novel mechanistic evidence of the role of CX3CR1 signalling for the proliferation of these cells (figures 7 and S6). In addition, our new experiments suggested by the referees and editor suggest that CX3CR1-dependent proliferation of DRG macrophages is involved in the development of neuropathic pain (Figures 6D and 7E). We will make these points clear in the new version of the MS. Please see pages 11, 12, 14 and 17 (discussion and introduction section).

    Reviewer #3 (Public Review):

    This paper addresses the mechanism underlying a well-documented finding whereby the numbers of resident macrophages increase in dorsal root ganglia following peripheral nerve injury. It delineates the relative contribution of monocyte recruitment via circulation and local proliferation. The paper is clearly structured and written, and the data overall support the main conclusion that the increase in nerve-associated macrophages is primarily driven by proliferation, not monocyte recruitment. Its main weakness is that the question that is being asked is rather restricted, so the additional insight gained for the field will be incremental. It would be particularly interesting in the future to address whether the existence of a protective barrier indeed is the reason peripheral cells are not recruited to the nerve injury lesion and to assess e.g. whether forced breaching of this barrier results in monocyte influx and altered injury response.

    We appreciate your comments and suggestions. In the new version of the MS, we are presenting a series of novel experiments that confirm and support our initial hypothesis. Furthermore, novel experiments also explore the importance of the phenomenon we have explored in the context of neuropathic pain development. Regarding your suggestion about the next steps, we are working now in an attempt to understand why these cells are not able to infiltrate the DRGs after injury. Interestingly, one paper that came out during the revision of this work, showed that CD8+ T cells that are not able to infiltrate the DRGs after nerve injury in adult mice, start to infiltrate the DRGs of old mice (Zhou et al. 2022), indicating that ageing process may promote changes in this protective barrier. In addition, we have published a recent paper indicating that immune cells infiltrate the dorsal root leptomeninges after SNI (Maganin et al. 2022). We included these references and discussed these points in the new version of our MS. Please see page 15 lines 366 and 370.

    References:

    Zhou, L., G. Kong, I. Palmisano, M. T. Cencioni, M. Danzi, F. De Virgiliis, J. S. Chadwick, G. Crawford, Z. Yu, F. De Winter, V. Lemmon, J. Bixby, R. Puttagunta, J. Verhaagen, C. Pospori, C. Lo Celso, J. Strid, M. Botto, and S. Di Giovanni. 2022. "Reversible CD8 T cell-neuron cross-talk causes aging-dependent neuronal regenerative decline." Science 376 (6594): eabd5926. https://doi.org/10.1126/science.abd5926.

    Maganin, A. G., G. R. Souza, M. D. Fonseca, A. H. Lopes, R. M. Guimarães, A. Dagostin, N. T. Cecilio, A. S. Mendes, W. A. Gonçalves, C. E. Silva, F. I. Fernandes Gomes, L. M. Mauriz Marques, R. L. Silva, L. M. Arruda, D. A. Santana, H. Lemos, L. Huang, M. Davoli-Ferreira, D. Santana-Coelho, M. B. Sant'Anna, R. Kusuda, J. Talbot, G. Pacholczyk, G. A. Buqui, N. P. Lopes, J. C. Alves-Filho, R. M. Leão, J. C. O'Connor, F. Q. Cunha, A. Mellor, and T. M. Cunha. 2022. "Meningeal dendritic cells drive neuropathic pain through elevation of the kynurenine metabolic pathway in mice." J Clin Invest 132 (23). https://doi.org/10.1172/JCI153805.

  2. Evaluation Summary:

    Guimaraes et al address the origin of the macrophage increase in sensory ganglia after peripheral nerve injury. The authors show that there is no major influx by blood-derived monocytes into ganglia after injury and that resident macrophages proliferate, which is dependent on CX3CR1 signaling. Overall the work is clear and sound and should be of interest to immunologists and neurobiologists.

    (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 and Reviewer #3 agreed to share their names with the authors.)

  3. Reviewer #1 (Public Review):

    The authors address the origin of the macrophage increase in sensory ganglia after peripheral nerve injury, showing that there is no major influx by blood-derived monocytes into ganglia after injury and that resident macrophages proliferate, which is dependent on CX3CR1 signaling.

    - Interesting and relevant question, mainly addressed with adequate experimental approaches.

    - Most conclusions are supported by the data, however, some important controls and experiments are missing.

    - The authors should demarcate their results from the study of Iwai et al, 2021 which addresses similar questions.

  4. Reviewer #2 (Public Review):

    The investigators looked at mφs in lumbar DRG after a spared nerve injury in which two of the three branches of the sciatic nerve are transected and the third left intact. This is a classical preparation for studying neuropathic pain. This paper demonstrates that the increase of mφs is an increase in the number of CX3CR1+ (resident) mφs and not CCR2+ (infiltrating mφs) by using CX3CR1 and CCR2 individual reporter mice. Using a CX3CR1 conditional knockout (KO) mouse, they found that this receptor must be present on the mφs for the increase in number to occur. Next, they did a parabiosis experiment with GFP+ mice and found that neither of these mφ subtypes infiltrated into the DRG. To examine proliferation, they injected animals with Ki67 and found this label, which is an indication of proliferation, was present in the CX3CR1+ mφs (but not the CCR2+ mφs). Finally, they identified the CX3CR1 mφs to be the cells that express TNFα and IL-1b but not IL-6.

    An experiment that would be useful would be to determine if there is an increase or a decrease in the availability to mφs of the ligand CXC3L1 after the spared nerve injury. The authors state from the work of others that membrane-bound CX3CL1 is constitutively expressed and that it is decreased after nerve injury. They hypothesize that this indicates a release of the chemokine, but such a decrease could also indicate a decrease in expression. A few sentences on what is known in other systems on the importance and mode of action of membrane-bound and non-membrane-bound CX3CL1 would be useful.

    The main weakness of the manuscript is that many highly relevant previous findings, in some cases reporting nearly identical experiments sometimes with the same and sometimes with somewhat different results, are not mentioned. Kalinski et al. (which is cited but not in this context) reported a very similar parabiosis experiment. While they did not identify subtypes of mφs, they found an increase in infiltration of mφs, which was small (though statistically significant) compared to the larger increase that occurred in the distal nerve. In 2013 and 2018, Niemi et al. and Lindborg et al (J Neurosci and J Neuroinflammation respectively) reported that mφs in the DRG are somewhat decreased in a CCR2 KO mouse, suggesting again that there is some infiltration of mφs into the DRG after axotomy. They also showed that the mφ chemokine CCL2 increases in the DRG after sciatic nerve injury. With regard to proliferation, Yu et al. in 2020 (which again is cited but not in this context) also used a spared nerve paradigm stained DRGs for CX3CR1+ mφs and found an increase. They then stained DRG sections for Ki67 and demonstrated proliferation in this population. An earlier reference by Krishnan et al in 2018 published in J Neuropathol Exp Neurol is entitled "An Intimate Role for Adult Dorsal Root Ganglia Resident Cycling Cells in the Generation of Local Macrophages and Satellite Glial Cells". With regard to cytokine expression, in 1995, Murphy et al published a paper in J Neurosci demonstrating induction of interleukin-6 in axotomized sensory neurons.

  5. Reviewer #3 (Public Review):

    This paper addresses the mechanism underlying a well-documented finding whereby the numbers of resident macrophages increase in dorsal root ganglia following peripheral nerve injury. It delineates the relative contribution of monocyte recruitment via circulation and local proliferation. The paper is clearly structured and written, and the data overall support the main conclusion that the increase in nerve-associated macrophages is primarily driven by proliferation, not monocyte recruitment. Its main weakness is that the question that is being asked is rather restricted, so the additional insight gained for the field will be incremental. It would be particularly interesting in the future to address whether the existence of a protective barrier indeed is the reason peripheral cells are not recruited to the nerve injury lesion and to assess e.g. whether forced breaching of this barrier results in monocyte influx and altered injury response.