Inflammation is a defence response to tissue damage that requires tight regulation in order to prevent impaired healing. Tissue-resident macrophages have a key role in tissue repair1, but the precise molecular mechanisms that regulate the balance between inflammatory and pro-repair macrophage responses during healing remain poorly understood. Here we demonstrate a major role for sensory neurons in promoting the tissue-repair function of macrophages. In a sunburn-like model of skin damage in mice, the conditional ablation of sensory neurons expressing the Gαi-interacting protein (GINIP) results in defective tissue regeneration and in dermal fibrosis. Elucidation of the underlying molecular mechanisms revealed a crucial role for the neuropeptide TAFA4, which is produced in the skin by C-low threshold mechanoreceptors—a subset of GINIP+ neurons. TAFA4 modulates the inflammatory profile of macrophages directly in vitro. In vivo studies in Tafa4-deficient mice revealed that TAFA4 promotes the production of IL-10 by dermal macrophages after UV-induced skin damage. This TAFA4–IL-10 axis also ensures the survival and maintenance of IL-10+TIM4+ dermal macrophages, reducing skin inflammation and promoting tissue regeneration. These results reveal a neuroimmune regulatory pathway driven by the neuropeptide TAFA4 that promotes the anti-inflammatory functions of macrophages and prevents fibrosis after tissue damage, and could lead to new therapeutic perspectives for inflammatory diseases.
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All data supporting the findings of this study are found within the manuscript and its Supplementary Information, and are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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We thank V. Feuillet for reading the manuscript and for comments and discussions, B. Escaliere for statistical insights, J. Galluso for mouse breeding and genotyping, and M. Lucattelli and L. Zitvogel for providing Fpr1-knockout bone marrow cells. We thank S. Memet and J. P. Gorvel for providing the IL-10GFP/GFP mice and S. Sarrazin and M. Sieweke for providing the CX3CR1CreERT2:R26-YFP mice. We thank the Centre d’Immunologie de Marseille-Luminy (CIML) mouse house and core cytometry, imaging and histology facilities. The S.U. laboratory received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation program, under grant agreement no. 648768; from the Agence Nationnale de la Recherche (no. ANR-14-CE14-0009-01); and from the Fondation pour la Recherche Médicale (no. ECO201906009090). This work was also supported by institutional grants from INSERM, CNRS, Aix-Marseille University and Marseille-Immunopole to the CIML.
Inserm Transfert have filed a provisional international patent application (WO2020/064907) on the clinical use of TAFA4 for treating inflammatory diseases, which lists S.U., G.H., G.D. and A.M. as authors. A.M. is the founder of Tafalgie Thérapeutics, which exploits a patent on the clinical use of TAFA4 (DI 06104-01). All other authors declare no competing interests.
Peer review information Nature thanks Isaac Chiu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 UV overexposure triggers a sequence of skin inflammation and repair over 35 days and GINIP+ neuron activation.
a, Heat map representing the expression kinetics of genes involved in skin inflammation and repair over time (days) post-irradiation. Ears from wild-type mice were collected before UV irradiation (day (D)0) or at D3, D7, D14 and D35 post-irradiation and total extracted RNA was analysed by Fluidigm. Genes encoding proinflammatory cytokines and chemokines were expressed at D3 and D7 delineating the inflammatory phase (red cluster). Pro-repair genes were upregulated at D14 and D35 (green cluster) delineating the resolution/remodelling phases. The gene Fam19a4 (also known as Tafa4) that encodes TAFA4 is highlighted in yellow (n = 4–8 mice per group). b, Sensory neurons from C2/C3 DRGs innervate the ear skin. The fluorescent tracer DiI was injected intradermally in the right ears of wild-type mice and the left ears were injected with PBS (n = 3 independent DRGs per group). c, The trigeminal ganglia (TG) and cervical DRG (C2 to C5) were collected 48 h post-injection and analysed by fluorescent microscopy. d, Quantification of DiI+ neurons per DRG fields of view; PBS-injected control side (blue) or DiI-injected side (red). e, Additional representative confocal images (related to Fig. 1c) of C2 DRGs labelled for GINIP (red), CGRP (blue) and ATF3 (green), from unexposed (left) and UV-exposed (right) mice at D3 post-irradiation. f, Quantification of total GINIP+ and CGRP+ neurons per DRGs (left) and total CGRP+ATF3+ and GINIP+ATF3+ DRG neurons (right) from unexposed (blue) and UV-exposed (red) mice at D3 post-irradiation (n = 8 mice per group). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 2 Conditional ablation of GINIP+ neurons in GINIP–DTR mice and skin histopathological analysis after UV exposure.
a, Representative confocal images for DAPI (blue) and GINIP (green) staining of C3 DRGs from control DTR-control (top) and GINIP-DTR (bottom) mice, 10 weeks after diphtheria toxin (DT)-treatment. b, Absolute number of GINIP+, CGRP+, TAFA4+, TH+ and IB4+ DRG neurons were quantified in DTR-control (blue) and GINIP-DTR (red) mice (n = 4–6 independent DRG per group). c, Representative confocal images for Beta3-tubulin (blue) and GINIP (green) staining of mouse ear skin sections as in a. d, Scratching episodes were monitored for 30 min at each time point post-irradiation indicated (n = 6–10 mice per group). e, Representative H&E staining of ears from DTR (left) and GINIP-DTR (right) mice at D35 post-irradiation. f–i, Histopathological analysis for leukocyte infiltration (f), epidermal thickness (g), fibrosis (h) and fibrosis extension (i) (n = 5 mice per group); related to Fig. 1g–j. Criteria used for the histopathological scoring are described in the Methods. j, Representative Masson trichrome (top) and H&E staining (bottom) of back skin from DTR-control (left) and GINIP-DTR (right) mice at D35 post-irradiation. All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
a, Flow cytometry gating strategy using CD11b, CD11c, CD64, MHC-II, CD103, c-Kit, Ly6G and CD24 marker expression. Dendritic cells (DC; CD11c+MHC-II+), mast cells (MC; CD11b−Kit+), lymphoid cells (Lymϕ; CD11b−CD103−/+), granulocytes (CD11b+CD24+, Ly6G+) and the monocyte/macrophage compartment (Mo/Mϕ; CD11c−MHC-II−CD11b+CD24−Ly6G−CD64int/+) are shown. b, Absolute numbers per mg of skin for dendritic cells, MC, Lymϕ, Gr and monocyte/macrophage populations at D14 post-irradiation in DTR-control (blue) or GINIP-DTR (red) mice (n = 8 mice per group). c, Gating strategy for skin dendritic cell subsets (CD11c+MHC-II+) and monitoring of Langerhans cells (LC; EpCAM+Langerin+) (n = 5–8 mice per group). d, FACS plot for Langerhans cells (left) and quantification (right) in DTR-control and GINIP-DTR mice, unexposed (no UV) or at D14 post-irradiation (UV) (n = 5–10 mice per group). e, Percentage of Langerhans cells among dendritic cells over time post-UV exposure in wild-type and Tafa4-knockout mice. f, Representative flow cytometry analysis of skin immune cells and DRG neurons for the expression of DTR in GINIP–DTR mice. g, Confocal analysis of whole mount ear skin from Nav1.8-RFP mice stained for GINIP+ neurons and CD206+ dermal resident macrophages. Hair follicles (HF) are highlighted within white dashed squares. In the skin, Nav1.8 expression (RFP) is restricted to sensory neuron axonal extensions. In DRG (f), DTR expression is restricted to neuronal cellular bodies in DRGs. These markers were not detected in immune cells. All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 4 Flow cytometry and t-SNE analysis of skin monocyte/macrophage subsets in GINIP-DTR mice.
a, Classical flow cytometry gating strategy for monocyte and macrophage subsets in the skin. b, t-SNE analysis including CCR2, Ly6C, MHC-II, CD64, CD206 and TIM4 markers was performed to cluster monocyte/macrophage populations at day 14 post-irradiation in the skin of GINIP-DTR and DTR-control mice (see also Methods for t-SNE analysis). c, Representative plots for CCR2−/+CD206− monocyte subsets on D3, D7 and D14 post-irradiation in the skin of control (DTR) or GINIP-DTR mice. d, Absolute numbers of Ly6C+ monocytes, intermediate monocytes, patrolling monocytes and monocyte-derived dendritic cell subsets per mg of ear skin (n = 9–13 mice per group) as in c. e, Representative FACS plots for CD206+ dermal resident macrophages as in c. f, Absolute numbers of DN and MHC-II+ macrophage subsets per mg of ear skin (n = 9–13 mice per group) as in c. g, Representative FACS plots for CD206+ dermal resident macrophage subsets at D14 post-irradiation in back skin from control (DTR) or GINIP-DTR mice. h, Absolute numbers of macrophage subsets per mg of back skin (n = 12 mice per group) from DTR-control (grey) or GINIP-DTR (coloured) mice, at D14 post-irradiation. All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
a, Representative confocal images of whole mount ear skin from wild-type mice stained with an anti-Beta3-tubulin (blue) and anti-GINIP (green) antibodies. GINIP+ lanceolate barrel structures innervating hair follicles (HF) are highlighted within dashed squares. b, Confocal analysis as in a with an additional anti-TH antibody staining (red) shows GINIP+TH+ C-LTMR axon terminals reaching the hair follicle. c, Schematically, GINIP+ neurons consist of two subsets of sensory neurons projecting in the inter-follicular regions of the epidermis as free nerve endings (IB4+) and hair follicles as C-LTMRs (TAFA4+). d, Confocal images of DRG sections from control wild-type (left) or Tafa4-knockout (right) mice after immunofluorescent (IF) staining with an anti-GINIP antibody (green) and an anti-TAFA4 antibody (1D8; red) and DAPI (blue). e, Additional confocal images of C3 DRGs (IF) stained with IB4 (blue), anti-TAFA4 (1D8, red) and anti-ATF3 (green) antibodies in wild-type mice (related to Fig. 3a), unexposed (left) or at D3 post-irradiation (right). f, Absolute number of TAFA4+ and IB4+ neurons (left; n = 6–12 mice per group) and absolute number of TAFA4+ATF3+ and IB4+ATF3+ neurons per DRG (right) from unexposed (blue) and exposed D3 post-irradiation (red) wild-type mice, (n = 8 mice per group). g, DAPI staining (blue) in situ hybridization (ISH) for Tafa4 mRNA (red) in DRG as in d. h, Quantification of TAFA4 levels (determined by ELISA) in DRGs from wild-type mice unexposed (D0) to D14 post-irradiation (n = 3 mice per time point; n.d., not determined). i, Tafa4 mRNA expression (RT–qPCR) in peripheral tissues and DRGs (n = 3 mice per time point; n.d., not detected). j, Quantification of Tafa4 mRNA expression in peripheral blood mononuclear cells (PBMCs), bone marrow, BMDMs and sorted CD206+ dermal macrophages compared to DRGs (n = 3 independent samples; n.d., not detected). k, Tafa4 mRNA expression in C3 DRGs from D0 to D35 post-irradiation (n = 3 mice per time point). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test except h, Kruskal–Wallis. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 6 TAFA4 regulates skin inflammation after UV exposure but does not affect IL-10 production in Treg cells and mast cells.
a, Changes in ear skin thickness over time post-irradiation in wild-type control (blue) and Tafa4-knockout (red) mice (n = 9 mice per group). b, c, Representative H&E images of ears from wild-type (left) or Tafa4-knockout (right) mice at D35 post-irradiation and histopathological scoring for leukocyte infiltration, epidermal thickness, fibrosis, fibrosis extension (b) and cumulative fibrosis score and total pathological scores (c) (n = 12 mice per group). See detailed scoring in Methods. d, IL-6, IL-1β and chemokines CCL2, CXCL1 and CCL4 levels (measured by CBA) and Il10 mRNA level, in ear skin from wild-type (blue) or Tafa4-knockout (red) mice from D0 to D35 post-irradiation (n = 3–11 mice per group and time point). e, f, FACS analysis and detection of IL-10+ immune cells from control (wild-type; grey) and IL-10GFP/WT (red) mice using an anti-GFP antibody (e) and using an anti-IL-10 antibody (f), in skin dendritic cell subsets, Lymϕ cells, monocytes, mast cells (MC) and macrophage subsets in IL-10GFP/GFP mice (grey) and wild-type mice (blue). g, h, Gating strategy for Treg cells (CD4+TCRβ+FoxP3+) (g) and absolute number per mg of ear skin before (D0) or post-irradiation, in DTR-control (blue) or GINIP-DTR (red) mice (h) (n = 9–18 mice per group and per time point). i, Representative FACS plots for IL-10 expression in Treg cells from wild-type (left) or Tafa4-knockout (right) mice before (blue) or post-irradiation (red). j, Gating strategy (left) and absolute number (right) of mast cells (MC) per mg of skin from DTR-control (blue) or GINIP-DTR (red) mice, showing MC depletion (D3) and repopulation (D7) after UV exposure (n = 8–9 mice per group and time point). k, Representative FACS plots for IL-10 expression in TIM4+ macrophages (blue) and MC (yellow) before (no UV, top) and at D10 post-irradiation (bottom) in wild-type mice. l, m, Representative FACS plot for IL-10 expression in MC from wild-type (blue) or Tafa4-knockout (red) mice at D10 post-irradiation (l) and IL-10 median fluorescence intensity (MFI) analysis (n = 6–10 mice per group) (m). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test except a, two-way ANOVA. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 7 TAFA4 promotes IL-10 production by dermal resident macrophage subsets in vivo and directly upregulates Il10 in macrophages in vitro.
a–c, Representative FACS plots (top) for intracellular IL-10 expression in DN macrophage (a), MHC-II+ macrophage (b) and TIM4+ macrophage (c) subsets from wild-type (blue) and Tafa4-knockout (red) mice at D10 post-irradiation, and IL-10 MFI quantifications (bottom) in the respective macrophage subsets at D3, D7 and D10 post-irradiation (n = 6–9 mice per group and time point). d, BMDMs, derived from bone marrow of wild-type or Fpr1-knockout mice, were challenged in a migration assay (transwell) using medium alone (CTL−), MCP-1 (CTL+) or increasing concentrations of TAFA4. Absolute number of macrophages in the bottom wells were analysed by FACS (n = 3 independent samples per group). e, IL6 mRNA level in thioglycollate-elicited macrophages in the presence (green) or absence (blue) of TAFA4 (related to Fig. 3i) (n = 6–8 independent sample per group). f, BMDMs as in d were activated in vitro by LPS alone (blue) or in the presence of TAFA4 (green). Gene expression was analysed by RT–qPCR for Tnf, Il6 and Il10 and compared to non-activated BMDM (CTL) (n = 4–5 independent samples). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 8 Fate-mapping and shield-irradiated bone-marrow chimeras revealed that TAFA4 regulates the dynamic between embryo-derived and monocyte-derived macrophages after UV exposure.
a, Strategy for embryonic progenitor fate mapping using the CX3CR1CreERT2:R26-YFP mice. Tamoxifen (Tam) was injected in pregnant females at E16.5 and offspring were analysed at 6 weeks of age. b, Percentages of YFP+ cells in the indicated cell types (n = 5 mice). LCs, Langerhans cells. c, Experimental scheme for monocyte tracing using CD45.1+ bone-marrow shield-irradiated chimeras. d, e, Percentage of CD45.1 chimerism within the indicated monocyte/macrophage subsets in 2-month-old chimeric mice in the steady state (n = 7 mice per group) (d) or after an additional 4 months following UV irradiation (purple) or no UV irradiation (grey) (n = 5 mice per group) (e). f, Gating strategy for monocyte/macrophage analysis in wild-type CD45.1 bone-marrow chimera before UV exposure (top) and then at D7 post-irradiation in wild-type (middle) and in Tafa4-knockout (bottom) recipient chimeras. g, Relative expression of the markers CD45.1, Ly6C, CD64, CD206, MHC-II and TIM4 used for t-SNE analysis of skin monocyte/macrophage subsets from wild-type or Tafa4-knockout recipient chimeras (related to Fig. 4b). h, Absolute numbers (n = 9 mice per group) per mg of ear skin (left) and CD45.1 chimerism levels (right) (n = 7 mice per group) for DN macrophages and MHC-II+ macrophages in wild-type (blue) and Tafa4-knockout (red) bone-marrow-chimeric mice on D7 post-irradiation. i, Representative annexin-V staining of DN macrophages (left), MHC-II+ macrophages (middle) and TIM4+ macrophages (right) at D3 post-irradiation from DTR (blue) or GINIP-DTR (red) mice. j, MFI for annexin-V in macrophage subsets as in i (n = 4 mice per group). k, Representative annexin-V as in i at D2 post-irradiation for wild-type (blue) or Tafa4-knockout (red) mice. l, MFI for annexin-V in macrophage subsets as in j (n = 5–6 mice per group). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 9 IL-10 defect and dermal macrophage depletion recapitulate the Tafa4-knockout mouse phenotype after UV exposure.
a, Experimental scheme for in vivo neutralization of IL-10 in wild-type mice. b, Ear thickness, at D14 post-irradiation, of wild-type (blue) or Tafa4-knockout (red) mice treated with an IgG2a isotype control (iso) and of wild-type mice treated with anti-IL-10 blocking antibodies (purple) (n = 10 mice per group). c, Absolute numbers of inflammatory macrophages (left) and TIM4+ macrophages (right) in the ear skin of the indicated 3 groups of mice (as in b) (n = 6–9 mice per group). d, Representative FACS plots of monocyte/macrophage subsets at D0 and D3 post-irradiation in IL-10WT/GFP and IL-10GFP/GFP (Il10-knockout) mice. e, Absolute number of TIM4+ macrophages (left) and inflammatory macrophages (right) per mg of skin at D7 post-irradiation in IL-10WT/GFP (grey) and IL-10GFP/GFP (coloured) (n = 8–10 mice per group). f, Ear thickness before (no UV) and at D14 post-irradiation (UV) in IL-10WT/GFP (blue) and IL-10GFP/GFP (purple) mice (n = 9–12 mice per group). g, Experimental scheme for dermal resident macrophage depletion: two i.p. injections, at 3 days (D – 3) and 2 days (D – 2) before D0, of the CSF-1R blocking antibody AFS98, are effective to deplete DN, TIM4+, MHC-II+ and Langerhans cells in wild-type mice. h, Absolute number of macrophages per mg of ear skin before (CTL) and after AFS98 injection (coloured) (n = 13 mice per group). i, IL-10 level detected by CBA in wild-type (blue) and Tafa4-knockout (red) mice after isotype control or AFS98 antibody injection (n = 4–5 mice per group). j, Absolute number of myeloid cell subsets per mg of ear skin after isotype control or AFS98 antibody injection (coloured) (n = 11 mice per group). k, Left, representative FACS plots of monocyte/macrophage subsets over the time course of macrophage repopulation (D0, D2 and D4) after AFS98 injection and complete macrophage depletion (D0) in wild-type mice; right, representative FACS plots at D6 after AFS98 injection in wild-type (blue) and Tafa4-knockout (red) mice showing the acquisition of TIM4 by monocyte-derived macrophages (see bottom panels). l, Representative FACS plots of monocyte/macrophage subsets at D0 and D3 post-UV exposure in wild-type and Tafa4-knockout mice after macrophage depletion with the blocking anti-CSF-1R antibody (AFS98). m, Absolute number of TIM4+ macrophages (left) and inflammatory macrophages (right) per mg of skin at D7 post-irradiation in wild-type (grey) and Tafa4-knockout (coloured) mice treated with an IgG2a isotype control (Iso+UV), or treated with anti-CSF-1R antibody (AFS+UV) (n = 8–9 mice per group). n, Ear thickness in unexposed mice (no UV), and at D14 post-irradiation in wild-type (blue) and Tafa4-knockout (red) mice treated as in m (n = 10 mice per group). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
Extended Data Fig. 10 Adoptive transfer of TIM4+ macrophages is sufficient to reduce tissue damage in GINIP-DTR mice.
a, Experimental scheme for intradermal adoptive transfer of CD45.1+TIM4+ macrophages. b, Gating strategy for sorting CD45.1+ inflammatory macrophages (pink) and TIM4+ macrophages (cyan). c, Ear thickness for unexposed (no UV) or UV-exposed mice at D7 post-irradiation, in DTR (blue), GINIP–DTR (red) mice and GINIP-DTR mice injected with inflammatory macrophages (pink) or with TIM4+ macrophages (cyan) (n = 7–10 mice per group). d, Representative images of ears at D7 post-irradiation from each group as in c. e, Experimental scheme for TAFA4 rescue in Tafa4-knockout mice. f, Representative FACS plot for intracellular staining of IL-10 in TIM4+ macrophages at D14 post-irradiation, in wild-type mice treated with saline (blue) or Tafa4-knockout mice treated with saline (red) or Tafa4-knockout mice treated with TAFA4 (green). g, Model for TAFA4 functions in vivo after UV exposure: (1) role of TAFA4 during the inflammatory phase. Skin overexposure to UV induces the release of the neuropeptide TAFA4 by C-LTMRs. TAFA4 promotes the production of IL-10 by embryonic-derived dermal resident TIM4+ macrophages. The production of IL-10 is essential for their survival and protect the skin from over-inflammation. Tissue lesions also induce the recruitment of monocytes in the skin, where they differentiate into TNF+ inflammatory macrophages. In the absence of TAFA4 production, the number of TIM4+ macrophages and IL-10 levels are reduced, promoting the expansion of inflammatory macrophages. (2) Role of TAFA4 during the resolution phase. The TAFA4–IL-10 axis is still active, promoting the maintenance of both embryonic- and monocyte-derived IL-10+ TIM4+ macrophages, which are required for tissue repair. In the absence of TAFA4 production, IL-10 production by the three subsets of CD206+ dermal macrophages, is compromised, leading to persistent inflammation and fibrotic scars. h, Repeated measure over time of ear thickness changes (μm) of wild-type (blue) and Tafa4-knockout (red) mice treated daily with imiquimod (IMQ) during six consecutive days and followed up to 15 days (n = 5 mice per group). All data are representative of at least two independent experiments and presented as mean ± s.e.m. P values determined by one-way ANOVA with Tukey’s multiple comparisons test. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.
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Hoeffel, G., Debroas, G., Roger, A. et al. Sensory neuron-derived TAFA4 promotes macrophage tissue repair functions. Nature 594, 94–99 (2021). https://doi.org/10.1038/s41586-021-03563-7