Nociceptor neuroimmune interactomes reveal cell type- and injury-specific inflammatory pain pathways

Aakanksha Jain, Benjamin M. Gyori, Sara Hakim, Samuel Bunga, Daniel G Taub, Mari Carmen Ruiz-Cantero, Candace Tong-Li, Nicholas Andrews, Peter K Sorger, Clifford J Woolf

Preprint posted on 3 February 2023 https://www.biorxiv.org/content/10.1101/2023.02.01.526526v1

Discovering the hidden connections between neurons and immune cells in pain perception

Selected by Patrick Penndorf

Categories: biochemistry, bioinformatics, cell biology, genetics, genomics, immunology, molecular biology, neuroscience

Introduction

Background
Nociception describes the sensing and processing of noxious stimuli¹. In other words, these are the processes that are necessary for our perception of pain. As for the other senses, there specific neurons responsible to detect certain signals, in the case of pain, they are called nociceptors. The anatomical structure of these neurons is somewhat special. In fact, their cell bodies are located far away from their innervation target. They can be found to accumulate in so-called dorsal root ganglia (DRG). These hubs of neurons and glia are located next to the spine. Remarkably, their axons both sense the stimuli and forward these signals to the brain^1-4.

When it comes to sensing pain, it has been previously shown that the immune and nervous system work together. For example, factors produced by the immune system can sensitize nociceptive to respond more sensitively to painful stimuli^5-7. Conversely, merely activating nociceptive neurons can lead to immune cell influx⁸. By the same token, leukocytes can be activated by neuropeptides, such as Substance P, that are released from nociceptive neurons⁹. Neuroimmune interactions seem to be important in wound healing¹⁰. Despite the availability of various skin damage models, surprisingly few studies have focused on neuroimmune interactions. In this preprint, Jain et al. use single cell sequencing approaches to unravel interactions between neurons and immune cells in different skin damage models.

Key Findings

Immune cell recruitment differs among skin damage models

The authors of this preprint set out to investigate neuroimmune interactions in models of mechanical-, burn- and inflammatory-associated pain. Therefore, they inflicted tissue damage in the skin of mice by incision, UV-light or zymosan injection, respectively. As expected, these treatments led to a higher sensitivity of animals to heat. Using single cell sequencing they analyzed leukocyte populations present at the time of strongest hypersensitivity to pain in each condition.

The authors could observe a strong increase in neutrophils in the zymosan model. In the skin incision condition, recruited macrophages dominated over neutrophil influx. These changes were seemingly accompanied by a contraction of regulatory skin macrophages. However, UV exposure triggered only modest changes regarding immigrating cell populations. Contralateral skin (unaffected skin anatomically opposite of the treated area) did not show differences across the different conditions.

Transcriptomic changes in associated nociceptive neurons

Using a dataset from a previous publication, Jain and colleagues found that various types of DRG neurons differentially express immune-regulating ligands and receptors. This suggests a potential role for a neuron subtype-specific immune cell regulation. Interestingly, the authors also found receptors on neurons that were hitherto assumed to be immune specific.

Creating an interactome database for cellular interactions

The authors then used various databases and interactomes to identify ligands, receptors, enzymes and ion channels that might underlie potential neuroimmune interactions. For all their protein datasets they computed potential interactions. Excitingly, an online tool to browse these interactions – using a literature-based text-mining mechanism – is available and can continuously be updated. The authors used this tool to identify statistically significant interactions for each damage model. Although immune-immune and neuron-neuron interactions could be assessed, the investigators focused on immune-neuron interactions to identify pathways relevant for immune-related pain signaling. Each skin damage model was compared to the pooled data from the contralateral skin which acted as the reference control. Comparing the different models, the number of interactions for each immune cell population differed. However, the highest absolute count of interactions were found to involve different macrophage populations.

Identifying interactions

Many previously identified interactions in neuroimmune crosstalk were also found by Jain and colleagues. Additionally, they were able to identify previously unknown interactions. The frequency of a given interaction varied across conditions. Some interactions were found to be unique to a specific skin damage model. Importantly, the tool is also able to predict between which neurons and immune cells a specific interaction might occur.

Unraveling a novel pathway for pain desensitization

Having detected an increased level of the thrombospondin encoded by the Thbs1 gene in leukocytes (dominating in neutrophils) and observing strong expression of its respective receptor in mouse DRG neurons, the authors hypothesized an important role in pain signaling. Of note, this interaction was present in all skin damage models. Interestingly this ligand, TSP-1, is originally known for a variety of other functions related to synaptogenesis¹¹, tissue repair¹² and coagulation^13,14.Using a capsaicin-induced peripheral sensitization assay, the investigators could however prove that addition of TSP-1 can antagonize PGE2-induced hypersensitivity which leads to higher reactivity to capsaicin.

Why I chose this preprint

Interdisciplinary research has shown how closely connected the “systems” in our bodies are. Advancing our knowledge by identifying new regulatory mechanisms between various cell types will pave the way for a better understanding of how an organism is functioning and how signals are integrated. Given the various processes underlying neuroimmune interactions, a compendium of these interactions, as established in this preprint, is an essential step forward. Their freely accessible online resource which contains those interactions can dramatically facilitate the search for targets and mechanisms for other researchers.

Open questions and future directions

Since one will be able to access the dataset the authors used, not many questions will have to be left unanswered. In other studies, it would nevertheless be interesting to see to what extent interactions and expression levels differ when the amount of tissue damage is varied in a single skin damage model. However, more importantly, assessing the differences in these interactions between sterile and infection-associated inflammation could pave the way to further differentiate neuroimmune responses. Although this study did not identify changes in neuronal transcription among the three skin damage models, characterizing the function of immune-typical receptors on neurons will offer new insights. Finally, this study identified interactions involving metabolites. Future studies analyzing these interactions more closely might enable us to understand the connection between a certain metabolic state of a cell (or even a tissue) and the resulting immune responses better. It goes without saying that these insights might then also open up new hypotheses about disease-associated pain sensation which will have important clinical implications.

Questions for the authors

Did you see a differential role of metabolites associated with glycolysis versus oxidative phosphorylation related to specific interactions?
In terms of clinical implications, how might a network of interactions be helpful for the development of new therapeutic targets for inflammatory pain?

References

1. Dubin AE, Patapoutian A. Nociceptors: the sensors of the pain pathway. J Clin Invest. 2010 Nov;120(11):3760-72. doi: 10.1172/JCI42843. Epub 2010 Nov 1. PMID: 21041958; PMCID: PMC2964977.

2. Crawford LK, Caterina MJ. Functional Anatomy of the Sensory Nervous System: Updates From the Neuroscience Bench. Toxicol Pathol. 2020 Jan;48(1):174-189. doi: 10.1177/0192623319869011. Epub 2019 Sep 25. PMID: 31554486.

3. Todd AJ. Neuronal circuitry for pain processing in the dorsal horn. Nat Rev Neurosci. 2010 Dec;11(12):823-36. doi: 10.1038/nrn2947. Epub 2010 Nov 11. PMID: 21068766; PMCID: PMC3277941.

4. Meltzer S, Santiago C, Sharma N, Ginty DD. The cellular and molecular basis of somatosensory neuron development. Neuron. 2021 Dec 1;109(23):3736-3757. doi: 10.1016/j.neuron.2021.09.004. Epub 2021 Sep 29. PMID: 34592169; PMCID: PMC8639614.

5. Shutov LP, Warwick CA, Shi X, Gnanasekaran A, Shepherd AJ, Mohapatra DP, Woodruff TM, Clark JD, Usachev YM. The Complement System Component C5a Produces Thermal Hyperalgesia via Macrophage-to-Nociceptor Signaling That Requires NGF and TRPV1. J Neurosci. 2016 May 4;36(18):5055-70. doi: 10.1523/JNEUROSCI.3249-15.2016. PMID: 27147658; PMCID: PMC4854968.

6. Grace PM, Tawfik VL, Svensson CI, Burton MD, Loggia ML, Hutchinson MR. The Neuroimmunology of Chronic Pain: From Rodents to Humans. J Neurosci. 2021 Feb 3;41(5):855-865. doi: 10.1523/JNEUROSCI.1650-20.2020. Epub 2020 Nov 25. PMID: 33239404; PMCID: PMC7880288.

7. Lowy DB, Makker PGS, Moalem-Taylor G. Cutaneous Neuroimmune Interactions in Peripheral Neuropathic Pain States. Front Immunol. 2021 Apr 12;12:660203. doi: 10.3389/fimmu.2021.660203. PMID: 33912189; PMCID: PMC8071857.

8. Michoud F, Seehus C, Schönle P, Brun N, Taub D, Zhang Z, Jain A, Furfaro I, Akouissi O, Moon R, Meier P, Galan K, Doyle B, Tetreault M, Talbot S, Browne LE, Huang Q, Woolf CJ, Lacour SP. Epineural optogenetic activation of nociceptors initiates and amplifies inflammation. Nat Biotechnol. 2021 Feb;39(2):179-185. doi: 10.1038/s41587-020-0673-2. Epub 2020 Sep 21. PMID: 32958958; PMCID: PMC7878280.

9. Mashaghi A, Marmalidou A, Tehrani M, Grace PM, Pothoulakis C, Dana R. Neuropeptide substance P and the immune response. Cell Mol Life Sci. 2016 Nov;73(22):4249-4264. doi: 10.1007/s00018-016-2293-z. Epub 2016 Jun 17. PMID: 27314883; PMCID: PMC5056132.

10. Hoeffel G, Debroas G, Roger A, Rossignol R, Gouilly J, Laprie C, Chasson L, Barbon PV, Balsamo A, Reynders A, Moqrich A, Ugolini S. Sensory neuron-derived TAFA4 promotes macrophage tissue repair functions. Nature. 2021 Jun;594(7861):94-99. doi: 10.1038/s41586-021-03563-7. Epub 2021 May 19. PMID: 34012116.

11. Wang B, Guo W, Huang Y. Thrombospondins and synaptogenesis. Neural Regen Res. 2012 Aug 5;7(22):1737-43. doi: 10.3969/j.issn.1673-5374.2012.22.009. PMID: 25624796; PMCID: PMC4302456.

12. Kyriakides TR, Maclauchlan S. The role of thrombospondins in wound healing, ischemia, and the foreign body reaction. J Cell Commun Signal. 2009 Dec;3(3-4):215-25. doi: 10.1007/s12079-009-0077-z. Epub 2009 Oct 21. PMID: 19844806; PMCID: PMC2778594.

13. Isenberg JS, Romeo MJ, Yu C, Yu CK, Nghiem K, Monsale J, Rick ME, Wink DA, Frazier WA, Roberts DD. Thrombospondin-1 stimulates platelet aggregation by blocking the antithrombotic activity of nitric oxide/cGMP signaling. Blood. 2008 Jan 15;111(2):613-23. doi: 10.1182/blood-2007-06-098392. Epub 2007 Sep 21. PMID: 17890448; PMCID: PMC2200855.

14. Bonnefoy A, Daenens K, Feys HB, De Vos R, Vandervoort P, Vermylen J, Lawler J, Hoylaerts MF. Thrombospondin-1 controls vascular platelet recruitment and thrombus adherence in mice by protecting (sub)endothelial VWF from cleavage by ADAMTS13. Blood. 2006 Feb 1;107(3):955-64. doi: 10.1182/blood-2004-12-4856. Epub 2005 Oct 4. PMID: 16204318; PMCID: PMC1895898.

Tags: inflammation, interdisciplinary, mouse, neuroimmunology, pain, sequencing, text-mining

Posted on: 5 July 2023

doi: https://doi.org/10.1242/prelights.34994

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Author's response

Dr. Aakanksha Jain shared

1. This is a great question! While we considered various enzymes that produce metabolites, we did not look into the overall metabolic states of individual cells. We do find both inflammatory and reparative macrophages suggesting the dependence on both glycolysis and Oxphos pathways. We found the most number of interactions between inflammatory macs subset and neurons however, it will be interesting to see if the glycolytic pathway indeed underlies these interactions.

2. We found TSP-1 – CD47 interaction an attractive pathway to investigate further because TSP-1 has been shown to bind to the potential gabapentin receptor and CD47 blockade is currently being explored as a cancer immunotherapy. More broadly, we would like to use these predicted interactions to identify downstream signaling nodes that can be targeted such as kinases or transcriptional factors, by doing pathway analyses. We are also currently cross-referencing these data with the druggable target database to identify more clinically applicable axes.

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This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.

Nociceptor neuroimmune interactomes reveal cell type- and injury-specific inflammatory pain pathways

Introduction

Key Findings

Why I chose this preprint

Open questions and future directions

Questions for the authors

References

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