# Sensory neurons shape local macrophage identity via TGF-β signaling

Julia Kolter, Clarissa-Laura Döring, Samah Sarout, Sebastian Baasch, Lloyd Steele, Reem Alsumati, Gabriel V Lucena Silva, Conceição E Aníbal Silva, Isadora Marques Paiva, Zohreh Mansoori Moghadam, et al.

► To cite this version:

Julia Kolter, Clarissa-Laura Döring, Samah Sarout, Sebastian Baasch, Lloyd Steele, et al. Sensory neurons shape local macrophage identity via TGF-β signaling. Immunity, 2025, 58 (10), pp.2556-2573.e8. 10.1016/j.immuni.2025.08.004. hal-05377114

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# Immunity

## Sensory neurons shape local macrophage identity via TGF-β signaling

### Graphical abstract

<figure><img src="image_2.png" /></figure>

### Authors

Julia Kolter, Clarissa-Laura Döring, Samah Sarout, ..., Thiago M. Cunha, Vincent Flacher, Philipp Henneke

### Correspondence

julia.kolter@uniklinik-freiburg.de

### In brief

Specialized macrophages are known to persist within tissue niches, but the molecular mechanisms guiding their sustained functional adaptation remain unclear. Kolter and Döring et al. show that local interactions between sensory nerves and macrophages activate TGF-β, which imprints and maintains nerve-associated macrophages that contribute to regeneration after tissue injury.

### Highlights

* Embryonic and monocyte-derived macrophages colonize sensory nerves during sprouting

* Adaptation is driven by localized activation of TGF-β upon nerve contact

* TGF-β signaling ensures long-term maintenance of macrophages on axons

* This conserved neuro-immune interaction is essential for nerve regeneration

<figure><img src="image_3.png" /></figure>
## Article

# Sensory neurons shape local macrophage identity via TGF-β signaling

Julia Kolter,¹,¹⁶,¹⁷,* Clarissa-Laura Döring,¹,²,¹⁶ Samah Sarout,¹ Sebastian Baasch,¹,³ Lloyd Steele,⁴ Reem Alsumati,¹

Gabriel V. Lucena Silva,⁵ Conceição E. Aníbal Silva,¹,⁵ Isadora Marques Paiva,⁵ Zohreh Mansoori Moghadam,¹

Vitka Gres,¹ Florens Lohrmann,¹ Philipp Aktories,⁶ Theresa Buchegger,⁷ Mitchell Bijnen,⁸ Layal Doumard,⁹

Benjamin Voisin,⁹,¹⁰ Christelle Le Foll,¹¹ Nico Lachmann,⁷,¹² Melanie Greter,⁸ Katrin Kierdorf,⁶,¹³ Muzlifah Haniffa,⁴,¹⁴

Thiago M. Cunha,⁵ Vincent Flacher,⁹ and Philipp Henneke¹,¹³,¹⁵

¹Institute for Infection Control and Prevention, Medical Center and Faculty of Medicine, University of Freiburg, Freiburg, Germany

²Faculty of Biology, University of Freiburg, Freiburg, Germany

³Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Freiburg, Germany

⁴Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK

⁵Center for Research in Inflammatory Diseases (CRID), Department of Pharmacology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil

⁶Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany

⁷Department of Pediatric Pneumology, Allergology and Neonatology and Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany

⁸Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland

⁹CNRS I2CT/UPR3572 Immunology, Immunopathology and Therapeutic Chemistry, Strasbourg Drug Discovery and Development Institute (IMS), Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France

¹⁰University of Strasbourg, CNRS, UPR3572 Strasbourg, France

¹¹Institute of Veterinary Physiology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland

¹²Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Hannover, Germany

¹³Centre for Integrative Biological Signalling Studies (CIBSS), University of Freiburg, Freiburg, Germany

¹⁴Biosciences Institute, Newcastle University, Department of Dermatology and NIHR Newcastle Biomedical Research Centre, Newcastle upon Tyne, UK

¹⁵Center for Chronic Immunodeficiency (CCI), Medical Center and Faculty of Medicine, University of Freiburg, Freiburg, Germany

¹⁶These authors contributed equally

¹⁷Lead contact

*Correspondence: julia.kolter@uniklinik-freiburg.de

https://doi.org/10.1016/j.immuni.2025.08.004

## SUMMARY

Resident macrophages play integral roles in maintaining tissue homeostasis and function. In the skin, prenately seeded, specialized macrophages patrol sensory nerves and contribute to their regeneration after injury. However, mechanisms underlying the long-lasting postnatal commitment of these nerve-associated macrophages remain largely elusive. Here, we found that traumatic injury induced recruitment of myeloid progenitor cells to the sprouting axons of sensory nerves, where they gradually acquired a nerve-associated macrophage-like profile. This identity change was driven and maintained by the immediate microenvironment, particularly by transforming growth factor-β (TGF-β), which was locally activated by the physical interaction with nerves and integrin-mediated cleavage, and by the neuropeptide calcitonin gene-related peptide (CGRP). Moreover, TGF-β-mediated macrophage specification was essential for proper nerve regeneration following injury. Overall, we identify TGF-β as a conserved, spatially regulated mediator governing local imprinting and long-term specialization of macrophages within the subtissular neuronal niche, offering insights into precise bidirectional neuro-immune crosstalk underlying skin homeostasis.

## INTRODUCTION

The skin acts as a protective barrier to the environment, with macrophages representing the most abundant resident immune cell type. Macrophages play important roles in tissue homeostasis and barrier defense. They adapt to various microenvironments and consequently display substantial variations in

immunophenotype, morphology, transcriptome, and function. Generally, tissue-specific cues, like metabolites, growth factors, and microbial products, contribute to distinct transcriptional programs.¹ However, factors directing precise diversification of macrophages in a structurally complex tissue like the skin are poorly defined. By contrast, some insights have been gained from macrophages in less complex environments. For instance,
<figure><img src="image_4.png" /><figcaption>Figure 1. sNAMs recruited upon injury derive from bone marrow progenitors</figcaption></figure>

(A) TdTomo labeling in ear punches of Cx3cr1<sup>creERT2/gfp</sup> R26<sup>tdT/tdT</sup> mice, induced prior to injury. Dashed lines indicate regeneration rings at the injury center (n = 4). Scale bar, 100 µm, zoom-ins 20 µm.

(B) TdTomo expression in CD64⁺ dermal macrophages in Mrc1<sup>creERT2/creERT2</sup> or Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> 10 days post injury (dpi), measured via flow cytometry (n = 5).

(C) TdTomo expression in the regeneration rings (dashed lines) of Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup> mice. Scale bar, 500 µm, zoom-in 50 µm.

(D) Quantification of tdTomato⁺ F4/80⁺ cells in regeneration rings of Figure 1C.

(E) TdTomo expression in macrophages in ear punches of Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> mice. Scale bar, 50 µm, zoom-in 40 µm. Arrows point to exemplary tdTomato⁺ sNAM.

(F) Quantification of tdTomato⁺ F4/80⁺ cells in regeneration rings of Figure 1E.

(G) Ear-shielded, irradiated CD45.1 mice transplanted with Cx3cr1<sup>gfp/+</sup> and vice versa, punched 8 weeks post-transfer, and analyzed 14 days later.

(H) Quantification of CX₃CR1<sup>high</sup> macrophages in chimeric mice 14 days post injury (n = 3–4) (two-tailed unpaired t test).
exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-β in the alveolar space activates the master transcription factor peroxisome proliferator-activated receptor (PPAR)-γ in alveolar macrophages, driving signature gene expression and differentiation early in life.²,³ Moreover, tissue complexity requires the acquisition of distinct functions by macrophages residing in diverse anatomical niches of a single organ, e.g., the red pulp and marginal zones of the spleen. At an even higher resolution, macrophages associate with specific tissue substructures, such as blood vessels and peripheral nerves.⁴,⁵ We have previously reported on a small subset of dermal macrophages associated with sensory nerves, which patrol axons and contributes to nerve repair upon injury.⁶ These sensory nerve-associated macrophages (sNAMs) exhibit a distinct transcriptional profile and immunophenotype, characterized by high fractalkine receptor (CX₃CR1) and low mannose receptor (CD206) expression. sNAMs are of embryonic origin and capable of self-maintenance, whereas the majority of interstitial dermal macrophages is rapidly replaced by circulating monocytes.⁶,⁷ These findings support the generally accepted model of macrophages receiving tissue-specific contributions from embryonic and adult hematopoiesis, ranging from exclusively yolk-sac-derived microglia in the adult central nervous system (CNS) to mostly monocyte-derived macrophages in the colon.¹,⁸⁻¹⁰ Nevertheless, even in the gut, self-renewing macrophages are found close to enteric neurons and the submucosal vasculature,¹¹ suggesting that their longevity is instructed by tissue niche signals. Unlike the CNS, macrophages of the peripheral nervous system (PNS) remain incompletely understood.¹² Macrophages within the dorsal root ganglia (DRGs) and larger nerves, e.g., the sciatic nerves, are of mixed origin and exhibit transcriptional signatures resembling activated microglia.¹³ Furthermore, larger PNS nerves contain distinct populations of epineurial and endoneurial macrophages.¹⁴ In the intestine, muscularis macrophages in the myenteric plexus coordinate gastrointestinal mobility and support neuronal survival and maturation.¹¹,¹⁵,¹⁶ However, in the gut and most tissues, nerve-associated and interstitial macrophages (IMs) are difficult to distinguish due to low abundance, overlapping marker expression, and lack of physical separation. As a result, discriminating traits in their origin and function are understudied, as are the molecular determinants provided by different peripheral nerves.

Here, we combined *in vitro* and *in vivo* approaches to identify the molecular signals and their sources mediating the specific interaction between macrophages and sensory nerves in the dermis. We found that macrophages are rapidly recruited to sprouting axons during perinatal development and after injury, which is accompanied by profound transcriptional and phenotypic changes. Transcriptional profiling revealed a critical role for TGF-β in sNAM differentiation, complemented by neuron-

derived calcitonin gene-related peptide (CGRP). TGF-β was locally activated in the neuronal microenvironment by integrin β5-expressing sNAMs, contributing to their transcriptional identity and self-maintenance. Moreover, TGF-β signaling was essential for the differentiation of recruited sNAM-like macrophages after injury. Consequently, disruption of TGF-β-mediated crosstalk between neurons and macrophages impaired peripheral nerve regeneration and wound healing after physical trauma, underscoring the importance of specialized immune cells in reinstating tissue homeostasis.

## RESULTS

### sNAMs recruited upon injury derive from bone marrow progenitors

At the site of injury, sensory nerves sprout and form a dense axon network, the so-called regeneration ring.⁶,¹⁷ Whereas bona fide sNAMs are of prenatal origin and self-maintain in homeostasis, previous data indicated that macrophages colonizing the sprouting nerves in the regeneration ring are not derived from resident sNAMs, despite their similar immunophenotype.⁶ Since these “sNAM-like cells” also appeared in monocyte-deficient mice, we previously concluded that they may be derived from IMs. Here, we aimed to explore their adaptation process to neurons with comprehensive fate mapping approaches. Since sNAMs are the only dermal macrophages with high CX₃CR1 expression in the adult skin, we induced Cx3cr1<sup>creERT2/gfp</sup> R26<sup>tdT/tdT</sup> mice with tamoxifen (TAM) before injury. Macrophages associated with β3-tubulin<sup>+</sup> nerves outside of the injured area were reliably labeled with GFP and tdTomato, whereas the majority of sNAM-like GFP<sup>+</sup> cells associated with sprouted axons in the regeneration ring lacked tdTomato expression, indicating a non-sNAM origin (Figures 1A, S1A, and S1C). In line with their CX₃CR1 upregulation upon recruitment, macrophages in the regeneration ring were tdTomato-labeled if TAM was administered after injury (Figures S1B and S1C). Next, we assessed whether these recently recruited cells derived from differentiated IMs, which, in contrast to sNAMs, strongly express CD206.⁶ We employed the recently developed Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup> mice, in which >90% of CD206-expressing dermal macrophages were labeled with tdTomato after TAM induction,¹⁸,¹⁹ whereas blood monocytes remained tdTomato-negative (Figures S1D and S1E). Secondly, we used Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> mice, in which cells derived from hematopoietic stem cells (HSCs) expressed tdTomato<sup>20</sup> (Figures S1D and S1F). Upon punch injury, most IMs remained labeled in the Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup> mice, whereas recruited sNAMs in the regeneration ring were tdTomato-negative (Figures 1B-1D). Instead, they showed strong labeling in Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> mice (Figures 1B, 1E, and 1F). Of note, endothelial cells also expressed tdTomato in Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> mice,

<footnote>(I) CX₃CR1<sup>high</sup> macrophages in ear punches of mice in which either donor (upper panel) or host (lower panel) cells were transgenic for Cx3cr1<sup>gfp/+</sup> (n = 3-4). Regeneration rings are shown to the right of the dashed lines. Scale bar, 50 µm.</footnote>

<footnote>(J) Macrophages in the regeneration ring of Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup> mice, induced with TAM in parallel to punch injury and analyzed 14 days later (n = 6). Scale bar, 50 µm.</footnote>

<footnote>(K) Quantification of tdTomato<sup>+</sup>F4/80<sup>+</sup> cells in regeneration rings of Figure 1J. Data are mean ± SEM. **p < 0.01, ***p < 0.0001 (one-way ANOVA, Tukey's post hoc test unless stated otherwise). See also Figure S1.</footnote>
hampering immunofluorescence analyses (Figures S1G and S1H). To complement this approach, we performed bone marrow transplantation in ear-shielded mice 8 weeks before injury (Figures 1G and S1–S1K). Cd45.1 mice transplanted with Cx3cr1<sup>gfp/+</sup> bone marrow showed robust recruitment of GFP<sup>+</sup> CX<sub>3</sub>CR1<sup>high</sup> cells into the regeneration ring, while the recruited cells in Cx3cr1<sup>gfp/+</sup> mice transplanted with Cd45.1 bone marrow did not express GFP (Figures 1G–1I, S1L, and S1M). These results again argued that sNAMs recruited after injury derived from HSCs and not from resident dermal IMs. However, tissue-resident IMs may compensate for the lack of infiltrating monocytes by adopting an sNAM phenotype, as indicated by the normal abundance of sNAMs in monocyte-deficient mice after injury.⁶ When inducing *Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup>* mice simultaneously with the punch injury, the majority of sNAMs within the regeneration ring were labeled 14 days post injury, while monocytes did not express tdTomato at any stage (Figures 1J, 1K, and S1N). This finding supported our hypothesis that the acquisition of the distinct sNAM phenotype is a stepwise process, where monocytes infiltrate the dermis and upregulate CD206 before completing their adaptation into sNAMs. Moreover, phenotypically mature IMs remained only transiently plastic for further transformation into sNAMs.

Monocytes recruited to peripheral nerves rapidly
undergo transcriptional adaptation

To distinguish recruited from resident sNAMs after tissue extrac-
tion, we induced Cx3cr1<sup>creER/gfp</sup> R26<sup>tdT/tdT</sup> mice prior to injury
(Figures 1A and 2A). IMs did not express CX<sub>3</sub>CR1 under any of
these conditions and thus was not labeled. We then sorted
tdTomato<sup>+</sup> GFP<sup>+</sup> bona fide sNAMs and double negative IMs
before and 10 days post injury for RNA sequencing (RNA-seq).
TdTomato<sup>neg</sup> GFP<sup>+</sup> cells, representing recently recruited
sNAMs, were only detected after injury (Figure S2A). sNAMs ex-
hibited a characteristic core transcriptional signature, including
genes such as Axl, Bcl2a1, and Cd9, as observed before<sup>6</sup>, that
persisted after injury (Figures 2B, 2C, and S2B). One of the high-
est upregulated genes compared with IMs was Ramp3, the re-
ceptor for the neuropeptide CGRP (Figure S2B). Upon injury,
sNAMs displayed 105 significantly upregulated genes, mainly
associated with proliferation and DNA repair (Figures 2D and
S2C). Notably, recruited TdTomato<sup>neg</sup> sNAMs displayed a tran-
scriptional profile between resident sNAMs and IMs, in line
with their transitional state in the tissue 10 days post injury
(Figures 2B and S2D). Importantly, sNAM signature genes such
as Itgav, Bcl2a1, and Cd72 were also increased in recruited
sNAMs (Figure 2C). By contrast, genes that were upregulated
in resident sNAMs were associated with tissue residency, e.g.,
the phosphatidylserine receptor gene Timd4 (Figure 2D). In re-
cruited sNAMs, on the other hand, genes associated with cell
migration and neurogenesis were enriched (Figure 2E). Signature
genes, such as Axl, F11r, and Ramp1/3, were upregulated in
both sNAM populations after injury, whereas hallmark genes of
IMs, such as Mrc1, were consistently downregulated
(Figures 2F, 2G, and S2E). Altogether, these data suggest that
bone marrow-derived macrophages can converge with embry-
onic, tissue-adapted sNAMs with respect to immunophenotype
and transcriptional profile upon availability of newly formed,
neuron-associated dermal niches.

Sensory neurons drive macrophage adaptation

To further dissect the macrophage-sensory neuron interaction,
we cocultured macrophage colony-stimulating factor (M-CSF)-
induced bone-marrow-derived macrophages (BMDMs) from
Cx3cr1 gfp/+ mice with DRG-derived sensory neurons (Figure
3A). DRGs contain the cell bodies of dermal sensory neurons,
including diverse subtypes, expressing neuropeptides such as
CGRP and substance P upon differentiation (Figures S3A and
S3B). We found that macrophages preferentially localized to
neuronal somata and outgrowing neurites, which was accompa-
nied by distinct morphological changes, i.e., elongation of the
cell body and physical alignment along axonal processes
(Figure 3B). Moreover, macrophages showed patrolling
behavior along the neurites in live cell imaging (Figure 3C;
Video S1) and strongly increased CX<sub>3</sub>CR1 expression, reminiscent
of sNAMs in vivo (Figures 3B, 3D, 3E, S3C, S3D, and
S3E). However, in contrast to sNAMs in the tissue, CD206 was
not downregulated in cocultured macrophages (Figures 3D
and 3E). F4/80 was significantly decreased, whereas the macro-
phage markers CD11b, CD45, and CD64 were not altered
(Figures 3E, S3F, and S3G). Sorted macrophages from cocul-
tures with sensory neurons showed a substantially altered tran-
scriptome with 5,393 differentially expressed genes compared
with monocultured macrophages (Figures 3F and S3H). Gene
Ontology (GO) term enrichment analysis revealed upregulated
gene sets associated with nerve development and differentia-
tion (Figure 3G). Furthermore, cocultured macrophages shared
a common core transcriptional signature with bona fide sNAMs
extracted from the skin, including Axl, F11r, and Pmepa1
(Figures 3F, 3H, and 3I). Notably, upregulation of these sNAM-
associated genes was dependent on the viability of the neurons,
as dead DRG cells did not induce similar transcriptional changes
(Figure S3I). Overall, these results demonstrate that sensory
neurons induced profound transcriptional and phenotypic
changes in macrophages with substantial similarities in vivo
and ex vivo.

Sensory neurons imprint macrophages via TGF-β
signaling

Next, we investigated signaling pathways activated in macro-
phages by DRG neurons *in vitro*. “Response to TGF-β” and
“TGF-β receptor signaling pathway” were among the most
prominently enriched GO terms (Figures 4A and 4B). Tgfbr1
and genes downstream of TGF-β signaling, including Smad3
and Smad7, were upregulated along with genes under the regu-
latory influence of TGF-β, such as Gcnt2, Pmepa1, and Runx3
(Figures 4C and 4D). TGF-β has pleiotropic effects on different
cell types, and diverging effects have been reported in macro-
phages, ranging from dampening inflammation to induction of
migration.<sup>21,22</sup> TGF-β also promotes differentiation of alveolar
macrophages<sup>3</sup> and embryonic microglia,<sup>23</sup> whereas the influ-
ence on skin macrophages remains largely unclear. Thus, we
first analyzed the transcriptional profiles of BMDM treated
with recombinant, activated TGF-β *in vitro* (Figures S4A–S4C).
TGF-β-treated macrophages showed marked transcriptional
similarities to macrophages cocultured with neurons, including
upregulation of sNAM signature genes, such as Axl, F11r, and
Fcrls (Figures 4E and S4C). Moreover, TGF-β induced a strong
concentration-dependent upregulation of CX<sub>3</sub>CR1 and a
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<figure><img src="image_11.png" /><figcaption>**Figure 2. Rapid transcriptional adaptation of monocytes recruited to peripheral nerves**</figcaption></figure>

(A) Experimental setup to sort sNAMs and interstitial macrophages (IMs) from steady state and 10 days post injury (inj) for RNA-seq. Littermate mice were punched in the left ear (inj) 21 days after TAM, and the right ear was left intact (steady state). Macrophages were sorted from both ears 10 days later and processed for RNA-seq. RecNAM: recruited sNAMs.

(B) Principal-component analysis (PCA) of sorted macrophage subsets.

(C) Heatmap depicting the top 50 upregulated "sNAM core" genes.⁸ Values were Z score normalized for each gene across samples.

(D) Volcano plots of differentially expressed genes (DEGs) in sNAMs upon injury. Yellow/green dots represent a log2-transformed fold change > 1 and *p* ≤ 0.05 (*padj* = two-tailed Benjamini-Hochberg test *p* value).

(E) GO biological pathways in recNAM in comparison to sNAMs (>1.5-fold change, FDR <0.01).

(F) Normalized counts of selected genes similarly expressed in sNAM subsets.

(G) Normalized counts of *Mrc1* and *Ramp3* in sorted macrophage subsets.

Data are mean ± SEM. \* *p* < 0.1, \*\* *p* < 0.01, \*\*\* *p* < 0.0001 (repeated measures [RMs] one-way ANOVA, Tukey's post hoc test).

See also Figure S2.

concomitant downregulation of F4/80 and CD206 (Figures 4F, S4D, and S4E). Thus, TGF-β caused phenotypic changes in macrophages, largely resembling those in cocultures. In line with this, we found increased phosphorylated SMAD2 and 3 in cocultured macrophages, indicating activation of the canonical TGF-β signaling pathway (Figure 4G).

ELISAs revealed TGF-β1 secretion by both macrophages and DRG neurons alone, further increased in cocultures (Figure S4F). We confirmed the expression in both cell types on the gene

expression (Figure S4G) and protein level (Figure 4H). Genetic deletion of *Tgfb1* in macrophages did not reduce CX₃CR1 surface expression in cocultures (Figure 4I), suggesting a minor role of autocrine TGF-β1 in this context. However, when we treated DRG cells with *Tgfb1*-specific small interfering RNAs (siRNAs) prior to coculturing, CX₃CR1 expression in macrophages was significantly decreased (Figures 4J, 4K, and S4H). To further explore the impact of neuron-derived TGF-β1, we next generated Nav1.8cre/+ *Tgfb1fl/fl* mice. Na<sub>v</sub>1.8 is expressed
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<figure><img src="image_19.png" /><figcaption>**Figure 3. Sensory neurons drive macrophage adaptation**</figcaption></figure>

(A) Coculture timeline of macrophages (Macs) and DRG neurons.

(B) Immunofluorescence (IF) of Macs associated with axons (left) or neuron somata (bottom right) after 5 days. Zoom: 3D reconstruction along neurites. Scale bars, 20 µm; zoom, 3 µm.

(C) Live cell imaging showing Macs pre-labeled with CellTracker Orange (CTO) moving along neurites (arrows) at 3 days. Time (t) in minutes. Scale bar, 20 µm.

(D) Mac surface markers after 5 days in mono- and coculture.

(E) Surface marker expression of Macs as mean fluorescence intensity (MFI) (n = 13).

(F) Volcano plot of differentially expressed genes of Macs in mono- and coculture. Gray/green dots: log₂-fold change >1. padj = two-tailed Benjamini-Hochberg test *p* value.

(G) GO terms in Macs in coculture, including “nerve” and “neuron.”

(H) Heatmap of top 100 DEG upregulated in Macs in co- vs. monoculture compared with sNAMs and IMs *in vivo* (Figure 2).

(I) Normalized counts of sNAM signature genes in mono- vs. co-cultured Macs.

Data are mean ± SEM. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001; ns, not significant (two-tailed paired t test).

See also Figure S3.
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<figure><img src="image_33.png" /><figcaption>Figure 4. Sensory neurons imprint macrophages via TGF-β signaling</figcaption></figure>

(A) GO slim biological processes enriched in cocultured Macs with highest fold enrichment (base mean >100).

(B) Fast gene set enrichment analysis (fgsea) of ranked DEG (co- vs. mono-cultures). Positive enrichment indicates genes of the "TGF_BETA_SIGNAL_PATHWAY" enriched in coculture.

(C) Normalized counts of TGF-β-related genes (n = 3). Two-tailed paired t test.

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by many DRG nociceptors, and Nav1.8<sup>cre</sup> mice are widely used to target neuro-immune interactions in skin and other peripheral tissues.<sup>24–26</sup> Conditional deletion of Tgfb1 in Na<sub>v</sub>1.8<sup>+</sup> nociceptors alone did not replicate the effect observed with siRNA-mediated silencing (Figure 4L). Transcriptional analysis of sorted DRG neurons (encompassing both Nav1.8<sup>+</sup> and Nav1.8<sup>-</sup> populations) extracted from Nav1.8<sup>cre/+</sup> Tgfb1<sup>fl/fl</sup> mice revealed only a slight, non-significant reduction in overall Tgfb1 expression after treatment (Figure S4I), suggesting that other DRG neuron subtypes contributed to TGF-β1 production or compensated for the loss of TGF-β1 in Nav1.8<sup>+</sup> neurons. Supporting this, the broader siRNA-mediated approach resulted in a marked five-fold reduction in total Tgfb1 expression in DRG cultures (Figure S4H).

Altogether, these results pointed to a role for TGF-β in imprinting macrophages within the neuronal microenvironment. In line with this, a selective inhibitor of the TGFBR1 kinase (SB-525334), or the conditional genetic deletion of Tgfb2 in macrophages, prevented CX<sub>3</sub>CR1 upregulation during coculture with DRG neurons (Figures 4M, 4N, and S4J–S4M). Furthermore, sNAM signature genes F11r, Fcrls, and Pmepa1 were downregulated (Figure S4N). Of note, inhibition of TGF-β signaling led to a further increased expression of CD206 in macrophages in mono- and cocultures (Figures 4M, 4N, S4K, and S4M). On the other hand, macrophages treated with activated TGF-β alone lost CD206 expression, reminiscent of sNAMs *in vivo* (Figure 4F lower panel, S4D). These observations suggested that additional factors in the cocultures modulated the macrophage phenotype, including CD206. Thus, we next analyzed the neuropeptide CGRP, which is secreted by DRG neurons. When comparing the transcriptomes of cocultured BMDM to transcriptomes of CGRP-treated BMDM,<sup>24</sup> we found a strong enrichment of CGRP-induced genes, including Thrombospondin (Thbs1) and a CGRP receptor (Ramp3) (Figures S4O and S4P).<sup>24</sup> Moreover, CGRP induced CD206 expression in macrophages in a concentration-dependent fashion, while CX<sub>3</sub>CR1 expression remained stable (Figure S4Q). When macrophages were simultaneously exposed to CGRP and TGF-β, CD206 remained upregulated, similar to the phenotype in cocultured macrophages (Figure S4R). Finally, RAMP1- and 3-deficient macrophages showed downregulation of Thbs1, Tgfb1<sup>r</sup>, and Pmepa1 in cocultures (Figure 4O), indicating that

CGRP secreted by neurons was indeed sensed by macrophages and impacted their immunophenotype.

Collectively, we have identified TGF-β signaling as a key driver of the transcriptional and immunophenotypic adaptation of macrophages to sensory neurons with additional modulation by the neuropeptide CGRP.

## TGF-β drives sNAM differentiation and self-maintenance

We next examined if sNAMs exhibited a TGF-β-driven transcriptional signature *in situ*. Indeed, sNAMs showed high expression of Tgfb1<sup>r</sup> and the TGF-β target genes Gcnt2, Pmepa1<sup>r</sup>, and Zeb1<sup>r</sup> in steady state and after axonal injury (Figure 5A). Moreover, genes driving the leading edge of the gene set enrichment analysis for TGF-β in cocultured macrophages *in vitro* (Figure 4B) were also upregulated in sNAMs in adult skin (Figure 5B). Additionally, in early postnatal development (P14), when most dermal macrophages still express CX<sub>3</sub>CR1,<sup>6</sup> all dermal macrophages showed increased expression of TGF-β-related genes (Figure 5B). This indicated that TGF-β may globally steer early-life development of dermal macrophages, whereas in adults this signature was exclusively maintained in sNAMs derived from prenatal sources, potentially due to microenvironmental cues. Notably, whereas Ramp1<sup>r</sup> and Ramp3<sup>r</sup> were significantly upregulated in sNAMs (Figures 2F and 2G), we did not find consistent induction of known CGRP-responsive genes, including Thbs1<sup>r</sup>, compared with IMs (Figures S5A and S5B), suggesting that sNAMs residing on nerve axons may not be continuously exposed to high amounts of CGRP in homeostasis. By contrast, we found a pronounced CGRP signature in sNAMs following injury, in particular in recruited sNAMs (Figures S5A and S5B), potentially reflecting increased CGRP release from nerve endings at sites of skin damage.<sup>24</sup>

We next analyzed if DRG neurons produced TGF-β *in vivo*. In publicly available single-cell RNA-seq (scRNA-seq) datasets,<sup>27</sup> we indeed found expression of Tgfb1<sup>r</sup> and Tgfb2<sup>r</sup> in CGRP<sup>+</sup> neurons and Aδ- and Aβ-low threshold mechanoreceptors (Figures 5C and S5C). We next employed Cx3cr1<sup>creERT2/+</sup> R26<sup>rfp/ylfp</sup> mice to specifically target sNAMs in the skin. Only sNAMs expressed YFP after TAM induction, in contrast to CD206<sup>+</sup> IMs and monocytes (Figures S5D and S5E). Sorted YFP<sup>+</sup> macrophages expressed signature genes such as Fcrls,

(D) Heatmap of top 25 TGF-β-related genes, upregulated in cocultured Macs.

(E) Heatmap of top 100 DEGs in Macs treated with 0.02 ng/mL recombinant TGF-β compared with Macs in mono- or co-cultures.

(F) MFI of CX<sub>3</sub>CR1-GFP and CD206 in Macs cultured 5 days with recombinant TGF-β (n = 7).

(G) Anti-phospho-SMAD2/3 (pSMAD2/3) staining in Macs cultured alone, with DRG neurons, or 2 ng/mL TGF-β for 3 days (n = 7–9).

(H) TGF-β1 staining of DRGs and Macs cultured alone or together for 5 days. Scale bar, 20 µm.

(I) Relative MFI for CX<sub>3</sub>CR1 of cocultured Macs, induced with OH-TAM during differentiation, from Cx3xr1<sup>CreERT2/+</sup>Tgfb1<sup>fl/fl</sup> R26<sup>rfp/ylfp</sup> or Cx3cr1<sup>CreERT2/+</sup>R26<sup>rfp/ylfp</sup> mice after 5 days. MFI normalized per experiment (n = 4–5). Two-tailed unpaired t test.

(J) Experimental design of DRG-Mac cocultures with Tgfb1<sup>r</sup> siRNA.

(K) Fold change of CX<sub>3</sub>CR1-GFP MFI in Macs cocultured with DRGs previously treated with non-targeting or Tgfb1<sup>r</sup> siRNA (n = 4). Two-tailed paired t test.

(L) CX<sub>3</sub>CR1-GFP MFI in Macs cocultured for 5 days with DRG cells from Nav1.8<sup>+/+</sup>Tgfb1<sup>fl/fl</sup> (Cre<sup>-</sup>) or Nav1.8<sup>Cre/+</sup>Tgfb1<sup>fl/fl</sup> mice (Cre<sup>+</sup>) (n = 6). Two-tailed paired t test.

(M) MFI of CX<sub>3</sub>CR1 and CD206 in Macs in mono- and co-cultures treated with TGFBR1 inhibitor SB525334 (SB) or DMSO for 5 days (n = 3–5). Mixed-effect analysis, Sidák's test.

(N) CD206 and β3-tubulin staining of cocultures with Cx3cr1<sup>gfp/+</sup> Macs ± SB525334 (SB). Scale bar, 50 µm.

(O) sNAM signature gene expression relative to *Hprt* in wt and *Ramp1/3*-deficient Macs cocultured with wt DRG cells for 5 days (n = 3). Two-tailed unpaired t test. Data are mean ± SEM. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001; ns, not significant (RM one-way ANOVA, Tukey's post hoc test unless stated otherwise). See also Figure S4.
<figure><img src="image_34.png" /><figcaption>Figure 5. TGF-β drives sNAM differentiation and self-maintenance</figcaption></figure>

(A) TGF-β-related genes in RNA-seq of sorted dermal macrophages.

(B) Heatmap depicting genes driving the leading edge of GSEA for TGF-β signaling in Figure 4B, in IMs and sNAMs in the dermis of P14 and adult mice (microarray data, $^6$).

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Axl, and Ramp1 and 3 (Figure S5F), and TGF-β-related genes were strongly upregulated compared with YFP⁻ macrophages (Figure S5G). Next, we crossed these mice with conditional *Tgfbr2 flox/flox* mice to selectively disrupt TGF-β signaling in sNAMs. *Tgfbr2* deletion in YFP⁺ macrophages resulted in their depletion from dermal nerves within 4 weeks post-TAM administration (Figures 5D, 5E, and S5H–S5J). Whole-mount imaging revealed a marked increase of CD206 surface expression in the rare YFP⁺ macrophages associated with sensory nerves in the absence of *Tgfbr2* (Figures 5D and 5F), indicating that TGF-β signaling indeed suppressed CD206 in sNAMs. Additionally, remaining YFP⁺ cells sorted from the skin of *Cx3cr1 creERT2/+ Tgfbr2 flox/flox* mice exhibited reduced expression of *Cx3cr1* and *Axl* (Figure 5G). Collectively, these analyses suggested that sNAMs continuously depend on homeostatic TGF-β signaling for their self-maintenance and transcriptional identity.

Next, we explored whether the specific role of TGF-β in
creating a local macrophage niche was conserved from mice
to humans. Indeed, various neuronal subtypes in human DRG
expressed *TGFB1* in a published spatial transcriptomic human
dataset (Figure S5K).²⁸ We then reprocessed and integrated
scRNA-seq data from 32 datasets and 251 samples of human
adult skin²⁹ and filtered for myeloid cells from healthy individuals,
yielding 82,751 cells post-quality control (Figures S5L and S5M).
Myeloid cells were annotated into monocytes, neutrophils, DCs,
and macrophages based on annotations and signature genes
from previous publications³⁰,³¹ (Figures S5M–S5O). We next
searched for cells expressing an sNAM signature and identified
a distinct population of macrophages enriched for these genes,
including *CX3CR1*, *AXL*, *F11R*, *TGFBR1*, and *CD9* (Figures 5H,
S5M, and S5O). Similar to mouse skin, these macrophages re-
constituted a minority of total dermal macrophages, ranging
from 0.47% to 16.67% (5.17% ± 6.82%), with higher abundance
in facial and scalp datasets. Moreover, the cluster robustly ex-
pressed TGF-β-regulated genes, such as *MMP14*, *SMAD2*,
and *SMAD7* (Figure 5I). Subsequently, we differentiated human
skin fibroblast-derived induced pluripotent stem cells (iPSCs)
to sensory neurons, which formed dense neurite networks
*in vitro* and secreted neuropeptides, such as CGRP and sub-

stance P (Figure S6A).³² iPSC-derived sensory neurons were co-cultured with human iPSC-derived macrophages (iMacs), which have been demonstrated to differentiate into tissue macrophages when exposed to organ-specific cues³³,³⁴ (Figure 5J). Indeed, we found elongated iMacs in close proximity to sensory neurons, patrolling along neurites and often accumulating in ganglia-like structures (Figures 5K and 5M; Video S2). In cocultures, these ganglia-like structures showed strong TGF-β1 expression, which was not observed when neurons were cultured alone (Figures 5L and S6B). In line with our mouse data, we observed a significant upregulation of CX₃CR1 in cocultures and in iMacs treated with TGF-β (Figures 5N, S6C, and S6D). Other surface markers, including CD206, CD64, CD11b, and CD14, remained unchanged (Figures 5N and S6C). Inhibition of TGF-β signaling decreased CX₃CR1 expression in cocultured iMacs (Figures 5O and S6E). Next, we investigated how macrophages impacted the differentiation and growth of neurons in this model by differentiating iPSC-derived neuron precursor cells (NPCs) with iMacs or alone (Figure S6F). We observed a pronounced increase in nerve density and formation of nerve bundle-like structures when macrophages were present (Figures 5P and 5Q). Collectively, these findings suggest an evolutionarily conserved communication network from mice to humans, in which sensory neurons regulate macrophage behavior through TGF-β signaling, while macrophages, in turn, support axon growth.

Local activation of TGF-β by macrophages requires
physical association with sensory neurons

TGF-β is involved in the development and/or survival of alveolar macrophages, epidermal Langerhans cells (LCs), and embryonic microglia,³,²³,³⁵ yet is thought to be dispensable for the self-maintenance of adult microglia and other tissue macrophages, including those in the dermis.³,³⁶,³⁷ Microglia and colonic macrophages, both targeted in our inducible system due to their high CX₃CR1 expression, were indeed not quantitatively affected by the loss of TGF-β signaling (Figures 6A, S6G, and S6H). Furthermore, unlike sNAMs, genetic ablation of *Tgfbr2* did not affect CX₃CR1 and CD206 expression in these cells (Figures S6I and

(C) *Tgfbi*−3 expression in neuronal subtypes of DRGs in GEO: GSE19088,²⁷ LTMR: low-threshold mechanoreceptor, SST: somatostatin+ pruriceptors.

(D) *Cx3cr1*<sup>creERT2/+</sup> *R26*<sup>fp/ylfp</sup> *Tgfbr2*<sup>flox/flox</sup> and *Cx3cr1*<sup>creERT2/+</sup> *R26*<sup>fp/flox/flox</sup> mice were induced with TAM 4 weeks prior to analysis. Images depict CD206 expression, and arrows point to sNAMs. Scale bar, 50 µm, zoom-in, 20 µm.

(E) Quantification of YFP⁺ macrophages normalized to recombination efficiency in ear skin of mice described in (D) (n = 15). Two-tailed unpaired t test.

(F) 3D reconstruction of YFP⁺ sNAMs and quantification of CD206 expression. Mean intensity was quantified in individual sNAMs in 3–6 field of view (FOV) per sample (n = 4). Scale bar, 10 µm. Two-tailed unpaired t test.

(G) Relative gene expression of depicted genes in YFP⁺ and YFP⁻ macrophages sorted from mice in (D) (n = 6–7). Two-way ANOVA, Tukey's post hoc test.

(H) Expression of core sNAM gene signature in annotated myeloid cell clusters in human skin scRNA-seq datasets.

(I) Expression of TGF-β-associated genes in clusters of Figure 5H.

(J) Timeline of human coculture with iPSC-derived sensory neurons (SN) and iPSC-derived macrophages (iMacs). SN were differentiated from neuron precursor cells (NPCs).

(K) Confocal images of cocultured iMacs after 5 days. White boxes (1–3): elongated iMacs at ganglia-like structures of SNs. Scale bar, 50 µm; zoom-in, 20 µm.

(L) Ganglia-like structures in coculture expressing TGF-β. Scale bar, 50 µm.

(M) Live cell imaging at 3 days showing iMacs (pre-labeled with CTO) migrating along neurites (red arrowhead) and clustering at ganglia-like structures (white arrowhead). SN were labeled with CellTracker Green. Time (t) in minutes. Scale bars, 50 µm.

(N) MFI of *CX₃CR1* and *CD206* in *iMacs* (*CD14⁺* *CD11b⁺*) after 10 days (*n* = 6). Two-tailed unpaired t test.

(O) MFI of *CX₃CR1* and *CD206* in *iMacs* (*CD14⁺* *CD11b⁺*) in coculture ± *TGFBR1* inhibitor SB525334 (SB) after 5 days (*n* = 10). Two-tailed unpaired t test.

(P) Neurofilament M (NFM) staining of SN alone or differentiated with iMacs in coculture. Scale bar, 100 µm.

(Q) Quantification of NFM⁺ area. Three independent experiments, two-tailed unpaired t test.

Data are mean ± SEM. \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001, \*\*\*\*p < 0.0001; ns, not significant (one-way ANOVA, Tukey's post hoc test unless stated otherwise).
See also Figures S5 and S6.
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<figure><img src="image_46.png" /><figcaption>Figure 6. Local activation of TGF-β by macrophages requires physical association with sensory neurons</figcaption></figure>

(A) Absolute counts of YFP+ microglia (CD45lo CD11bhi CD64hi/Tmem119hi) and colonic macrophages (CD45hi CD11bhi CD64hi Linneg) 4 weeks post-TAM. Two-tailed unpaired t test.

(B) MFI of CX3CR1 in Macs cultured with M-CSF, GM-CSF, or both for 5 days (n = 6).

(C) *Csf1* and *Il34* expression in DRG neurons, cultured alone or with Macs for 3 days (n = 4).

(D) M-CSF concentration after 24 h of culture ± 20 ng/mL M-CSF (n = 4).

(E) M-CSF staining of cocultures after 5 days. Scale bar, 20 µm.

(F) Experimental setup for conditioned (con.) medium and transwell assays.

(G) MFI of CX3CR1 and CD206 in Macs cocultured, treated with con. medium from cocultures, or cultured in transwells with DRGs after 5 days (n = 8).

(H) Heatmap of DEGs for metalloproteases in mono- and cocultures.

(I) Heatmap of DEGs for integrins.

(J) Normalized counts of *ltgb5* in mono- or cocultures. Two-tailed paired t test.

(K) Normalized counts of *ltgb5* in dermal macrophages of RNA-seq data from Figure 2.

(L) Mac surface markers in cocultures treated with αvβ5 inhibitor cilengitide (Cil) after 5 days, measured by flow cytometry (n = 8). Two-tailed paired t test.
Data are mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant (RM one-way ANOVA, Tukey's post hoc test unless stated otherwise).
See also Figure S6.

S6J). However, YFP+ microglia showed decreased *Fcrls* expression (Figure S6J), consistent with the established role of TGF-β in maintaining microglia homeostasis.²³,³⁶ Alveolar macrophages and LCs were not targeted in our system due to their lack of CX₃CR1 and, accordingly, Cre expression.³⁸ However, since

CX₃CR1 was one of the highest expressed genes in macrophages treated with TGF-β (Figures 4E and 4F), we wondered why alveolar macrophages, known to be exposed to TGF-β *in vivo*, generally do not express CX₃CR1 in adult tissues. Notably, TGF-β upregulates the master transcriptional regulator
<figure><img src="image_47.png" /><figcaption>Figure 7. TGF-β signaling in macrophages impacts neuronal growth</figcaption></figure>

(A) Experimental setup to deplete *Tgfbr2* in sNAMs in punch injury.

(B) Quantification of YFP+ macrophages in ear skin normalized to recombination efficiency. Two-tailed unpaired t test, *n* = 6–8 in 4 independent experiments.

(C) YFP+ macrophages recruited to injured nerve endings in mice described in (A). Scale bar, 50 µm.

(D) CD206 mean intensity in YFP+ macrophages at the injury site. Two-tailed unpaired t test. *n* = 5 FOV in 4 mice in 2 independent experiments.

(E) Experimental setup to deplete *Tgfbr2* both in resident and recruited sNAMs in punch injury. *n* = 7–10 in 2–3 independent experiments.

(F) Ly6C+ macrophages in the skin at 9 and 19 dpi.

(G) YFP+ macrophages at 9 and 19 dpi.

(H) Relative gene expression of sNAM signature genes in sorted YFP+ macrophages at 19 dpi. Two-tailed unpaired t test, *n* = 3–6.

(I) Injured nerves at punch site, 19 dpi. Dashed lines indicate regeneration rings. Scale bar, 50 µm.

(J) F4/80 and YFP expression in macrophages associated with regeneration rings (dashed lines). Scale bar, 50 µm.

(K) YFP+ macrophages associated with nerves at the injury site, quantified by microscopy in 6 FOVs per sample. *n* = 5. Two-tailed unpaired t test.

(L) Regeneration rings at 19 dpi. Scale bar, 500 µm.

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PPAR-γ in alveolar macrophages,³ yet PPAR-γ was downregulated in BMDM cocultured with neurons or treated with activated TGF-β (Figure S6K). In the lungs, epithelial cell-derived GM-CSF contributes to PPAR-γ induction. In line with this notion, BMDM differentiated with TGF-β plus GM-CSF instead of M-CSF completely lacked CX₃CR1 expression (Figure S6L). In addition, CX₃CR1 expression was abrogated in fully differentiated macrophages when GM-CSF was added to the medium, even in the presence of TGF-β (Figures 6B and S6M). Moreover, GM-CSF led to downregulation of CX₃CR1 in macrophages cultured with DRG neurons (Figures 6B and S6N), indicating that the immunophenotypic properties of TGF-β were indeed modulated by further signals, such as growth factors. Of note, when M-CSF-derived BMDM were cultured without M-CSF after initial differentiation, they displayed high rates of cell death, while BMDM cocultured with DRG neurons survived for at least 5 days without morphological alterations (Figure S6O). Accordingly, we found DRG neurons to express Csf1 and Il34 (Figures 6C and S6P). Multiplex cytokine arrays and histology confirmed that DRG neurons produced M-CSF, resulting in elevated concentrations in cocultures even without exogenous supplementation (Figures 6D and 6E). Conversely, exogenous M-CSF was undetectable in monocultures, likely due to rapid uptake by macrophages. Thus, neurons provide multiple soluble factors that may contribute to maintaining macrophages within their niche.

It seems worth noting that the TGF-β-dependent sNAMs represent a minor macrophage population in the dermis (2%-3% of dermal macrophages), and that the majority of dermal macrophages lack CX₃CR1 yet highly express CD206. This implies that TGF-β must act with high spatial precision, likely restricted to the neuronal niche, in order to mediate distinct effects on sNAMs and immediately adjacent IMs. When we cultured BMDM either with conditioned medium from cocultures or separated from DRG neurons in a transwell system by a membrane with 0.4 µm pores, we found that the physical contact with neurons was required to induce the sNAM-like phenotype in macrophages (Figures 6F, 6G, and S6Q). Notably, TGF-β is secreted in a biologically inactive complex with the disulfide-linked latency-associated peptide (LAP), which is activated by enzymatic cleavage, e.g., by certain matrix metalloproteases, or by mechanical force, i.e., by binding of integrins to the LAP domain.²² Consistent with this, BMDM cocultured with neurons upregulated genes encoding metalloproteases and integrins (Figures 6H and 6I), for instance, Mmp14, which is known to activate TGF-β (Figures 6H, S6R, and S6S). The gene encoding the integrin subunit β5 (Itgb5) was highly expressed, both in vitro and in vivo (Figures 6I, 6J, and 6K). This subunit forms a cell surface heterodimer with the integrin αv subunit, together mediating binding of latent TGF-β.³⁹ Treatment of the cocultures with a peptide inhibitor of integrin αvβ5 (Cilengitide) significantly decreased CX₃CR1 and simultaneously increased CD206 and F4/80 expression (Figures 6L and S6T), suggesting that integrin αvβ5 mediated local TGF-β activation when macrophages were

physically associated with neurons. Thus, TGF-β acts in a locally restricted fashion on macrophages, thereby modulating macrophages directly recruited to neurons, while sparing more distant cells. Moreover, the concerted action with tissue-specific factors such as GM-CSF induces a context-specific response of macrophages in different tissues.

## TGF-β signaling in macrophages impacts neuronal growth

Since macrophages support axonal outgrowth and repair after injury,⁶,⁴⁰ we next assessed the effect of TGF-β signaling in sNAMs on the sprouting of nerves after skin injury in TAM-treated Cx3cr1<sup>creERT2/+</sup> R26<sup>Yfp/Yfp</sup> Tgfbr2<sup>flox/flox</sup> and control mice without the floxed gene (Figure 7A). Under these conditions, resident sNAMs proliferate on injured axons immediately adjacent to the injury site and phagocytose myelin.⁶ In Cx3cr1<sup>creERT2/+</sup> Tgfbr2<sup>flox/flox</sup> mice, we found YFP<sup>+</sup> nerve-associated macrophages to be reduced, indicating that TGF-β signaling was required for their self-maintenance and accumulation under injury (Figures 7B and S7A). Furthermore, remaining YFP<sup>+</sup> macrophages at axonal injury sites expressed CD206 (Figures 7C and 7D). Nevertheless, sprouting of injured axons was only marginally affected (Figure S7B). We hypothesized that recruited sNAMs functionally compensated for the loss of prior resident sNAMs since these cells—which only upregulate CX₃CR1 upon associating with nerves after injury—were not targeted by this approach. We thus extended targeting to incoming macrophages at the lesion site by continuously administering TAM, thereby ensuring deletion in CX₃CR1-expressing cells throughout the injury response (Figure 7E). Following this treatment, both prior resident macrophages and those recently recruited into the sensory nerve niche after injury displayed robust YFP expression (Figure S7C), whereas dermal IMs and inflammatory monocytes remained YFP<sup>neg</sup> (data not shown, Figure S7D). Nine days post injury, at early stages of nerve sprouting, fewer Ly6C<sup>hi</sup> macrophages and neutrophils were observed at the site of injury in Cx3cr1<sup>creERT2/+</sup> Tgfbr2<sup>flox/flox</sup> mice, although neutrophils and monocytes were slightly elevated in the peripheral blood of the latter (Figures 7F and S7E–S7G). Nineteen days post injury, Ly6C<sup>hi</sup> macrophages decreased in control mice, which was paralleled by an increase of YFP<sup>+</sup> macrophages (Figures 7F and 7G). Cx3cr1<sup>creERT2/+</sup> Tgfbr2<sup>flox/flox</sup> mice displayed more Ly6C<sup>+</sup> macrophages and strongly decreased numbers of YFP<sup>+</sup> macrophages at this time point (Figures 7G and S7H–S7J), indicating a failure of monocytes to differentiate into sNAMs in the absence of TGF-β signaling. In line with this, remaining YFP<sup>+</sup> macrophages at the injury site in Cx3cr1<sup>creERT2/+</sup> Tgfbr2<sup>flox/flox</sup> mice were frequently located outside of the regeneration ring, i.e., did not colocalize with resprouting nerves, and failed to acquire the typical sNAM morphology (Figures 7I–7K and S7K). In addition, marker genes of sNAM identity were downregulated, whereas surface protein expression of CD206 was increased (Figures 7H, S7L, and S7M).

(M) β3-tubulin<sup>+</sup> area at injury sites at 9 and 19 dpi. Two-way ANOVA, Šidák's post hoc test. *n* = 14–20 ears at 9 dpi, *n* = 13–14 ears at 19 dpi, and *n* = 6 for Cre-negative mice in 2–3 independent experiments each.

(N) Wound area at 19 dpi. One-way ANOVA, Tukey's post hoc test. *n* = 13–4 ears, *n* = 6 for Cre-negative mice in 2 independent experiments. Data are mean ± SEM. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001; ns, not significant (two-way ANOVA, Tukey's post hoc test unless stated otherwise). See also Figure S7.
Control mice exhibited large hyperinnervated areas at injury sites, i.e., fully closed circular regeneration rings, which were densely colonized by YFP⁺ macrophages (Figures 7J and 7L). By contrast, nerve sprouting was impaired at both time points when TGF-β receptor signaling was disrupted in CX₃CR1⁺ macrophages (Figures 7L, 7M, and S7N). Moreover, the lesion areas were enlarged in Cx3cr1<sup>creERT2/+</sup> Tgfb1<sup>fl/fl</sup> mice in comparison to control mice after 19 days (Figure 7N). Notably, conditional deletion of TGF-β1 (Tgfb1) specifically in either sNAMs or Nav1.8⁺ nociceptors alone did not impair neuronal growth (Figures S7O and S7P), suggesting that TGF-β1 was produced by multiple neuronal sources in the regeneration ring. This finding was consistent with our *in vitro* experiments (Figures 4I, 4L, and S4I) and supported by broad expression of Tgfb1 across different DRG neuron subsets (Figures 5C and S5C).

Collectively, these data indicate that the local sensing of TGF-β by macrophages is required for their spatially confined adaptation to the sensory nerve niche, and this functional commitment of specialized macrophages is a prerequisite for tissue reinnervation upon injury.

DISCUSSION

The immune system and the nervous system interact and communicate to synchronize and maintain tissue integrity and to avoid potentially pathogenic dysregulation. Here, we uncovered that macrophages were recruited to sensory nerves upon niche availability emerging after nerve injury. The physical interaction with sensory nerves drove a transcriptional program and long-lasting differentiation to sNAMs, which involved TGF-β as a molecular link propagating bi-cellular communication, ultimately enabling nerve regeneration.

Neuro-immune interactions have recently received increasing attention.<sup>17,24,26,41,42</sup> In various tissues, macrophages have been found to associate with peripheral nerves.<sup>11,43,44</sup> Their phenotype and function strongly depend on tissue and nerve type, e.g., macrophages in the enteric nervous system express β2-adrenergic receptors and mediate neuronal protection in enteric infection.<sup>45,46</sup> Thus, macrophages adapt to their immediate microenvironment and contribute to its homeostasis. By combining selective gene targeting with cocultures, we have uncovered how macrophages are guided and maintained in resting and activated neuronal niches: TGF-β signaling, in combination with direct cell-cell contact, drove the functional intratissular macrophage differentiation, which supported neuronal regeneration. These findings align with recent observations by Viola et al., who found that TGF-β imprints intestinal muscularis macrophages, which then regulate enteric neuron density.<sup>16</sup> However, nerve-associated macrophages could not be specifically targeted in this study due to the lack of specific markers. Collectively, our results and those of Viola et al. suggest that TGF-β signaling is a conserved mechanism supporting macrophages in close contact with peripheral nerves.

TGF-β is particularly interesting in this context due to its secretion in an inactive form that requires local activation. The latent complex is bound to cell surfaces via milieu molecules, such as glycoprotein A repetitions predominant protein (GARP or LRRC32) or LRRC33, or to the extracellular matrix via latent TGF-β binding protein (LTBP).<sup>22,47</sup> In particular, interaction with cognate integrins in combination with cytoskeleton-associated

traction force enables a localized release. Thus, TGF-β action
can be extremely spatially confined, relying on activation by tightly
controlled integrin expression on specific cell subsets and not on
the rather ubiquitous presence of diffusible TGF-β. In the CNS, it
was shown that integrin-bearing astrocytes and microglia ex-
pressing milieu molecules cooperatively activate TGF-β with little
spread of TGF-β activity to neighboring microglia.⁴⁷ This model
provides a compelling mechanistic explanation for the localized
imprinting of macrophages within the dermis, where sNAMs and
phenotypically distinct interstitial and perivascular macrophages
coexist site by site. Whereas ablation of TGF-β signaling in sNAMs
impaired macrophage adaptation and nerve sprouting, the ge-
netic deletion of *Tgfb1* in either sNAMs or Nav1.8⁺ nociceptors
was not sufficient to induce the same phenotype. Yet, impaired
sNAM differentiation *in vitro* upon targeting *Tgfb1* in total DRG
neurons by siRNA supports a model where neuron-derived
TGF-β1 is pivotal in macrophage transformation. However, given
the expression of TGF-β in a variety of tissue cells, including mac-
rophages, we propose that its localized activation in the neuronal
microenvironment is particularly critical for the precision and
spatial confinement of the adaptation process.

Notably, TGF-β has been considered to be unique in its role for alveolar macrophages and LCs, but not for other tissue macrophages, including those in the dermis.<sup>3,48</sup> Nonetheless, specific tools like inducible *in vivo* signal disruption enabled us to uncover the relevance of TGF-β for the local imprinting of a small, yet highly dedicated, dermal macrophage subset. Tissue heterogeneity thus needs to be considered when studying resident macrophages. As peripheral nerves require protection from external stressors and inflammation, TGF-β might be an important local regulator for immune activation of incoming and resident macrophages. In combination with further niche-specific cues, TGF-β may then shape the identity of specific macrophage subsets, leading to context-dependent transcriptional programs. Underlying molecular mechanisms may be conserved across sites and modified, for instance, by growth factors, which promote macrophage maintenance and orchestrate differential gene networks in combination with TGF-β.

The PNS is essential for tissue health and has, in contrast to the CNS, the ability to regenerate. After injury, the proximal ends of lesioned axons form growth cones with a limited potential to sprout. The cues driving regeneration are incompletely understood; however, peripheral immune cells are involved.<sup>49</sup> In the ear punch model, next to sNAMs, tissue-resident T-cells promote nerve regeneration upon colonization by *Staphylococcus aureus* via IL-17 signaling.<sup>17</sup> Recently, wound innervation was shown to mediate tissue healing via CGRP-mediated immune cell coordination.<sup>24</sup> Corroborating this, we observed enlarged lesion sites and impaired nerve sprouting when TGF-β signaling was disrupted in sNAMs. Notably, the CGRP gene signature was most pronounced in sNAMs and not IMs following injury, suggesting that sNAMs are central in the regenerative effects observed by us and others.<sup>24</sup> Consequently, macrophage imprinting via TGF-β represents a critical step, initiating the neuro-immune regenerative axis and promoting wound repair.

In summary, we found that sensory neurons facilitate
the recruitment and adaptation of dedicated tissue macrophages
to their axons, thus forming a specific subniche. Macrophage dif-
ferentiation and maintenance in this microenvironment depended
on spatially restricted activation of TGF-β and CGRP, enabling precise neuro-immune communication. Monocyte-derived macrophages rapidly differentiated into sNAMs when additional axons sprouted, suggesting that limited recruitment of monocytes in homeostasis was due to the presence of self-maintaining resident macrophages in the niche rather than a lack of plasticity. Finally, TGF-β-induced conversion of macrophages to sNAMs was required for neurons to properly regenerate after cutaneous injury, contributing to tissue repair. Overall, a better understanding of the complex immune-mediated and potentially druggable functions of specialized tissue macrophages holds promise for the therapy of peripheral nerve injuries and neuropathies, as well as chronic skin diseases.

## Limitations of the study

The profound axon sprouting of neurons upon injury enabled us to investigate macrophage adaptation in real time in the adult skin. We hypothesized that similar mechanisms may occur in skin innervation during fetal development. As skin innervation starts at E13.5, interference with molecular pathways or cell replacement must be performed during pregnancy, which represents a technical challenge but offers promising prospects for future studies. Furthermore, it has yet to be established whether TGF-β signaling is required for human macrophage-mediated sensory neurons. While we found that integrin β5 contributes to TGF-β activation in vitro, we cannot exclude additional local cleavage of TGF-β by proteases produced by sensory neurons or associated cells, e.g., Schwann cells, as shown recently in the CNS, where integrin β8 expressed by radial glia activated autocrine TGF-β1 from microglia during development.⁵⁰ Finally, since TGF-β1 has been reported to exert neurotrophic effects,⁵¹ its local activation by sNAMs may contribute to neuronal outgrowth under injury, yet this hypothesis warrants further investigations.

## RESOURCE AVAILABILITY

### Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Julia Kolter (julia.kolter@uniklinik-freiburg.de).

### Materials availability

This study did not generate new, unique reagents.

### Data and code availability

RNA-seq data have been deposited at the NCBI GEO database as GEO: GSE250457 and are publicly available as of the date of publication. This paper does not report original code. Any additional information to reanalyze the data reported in this paper is available from the lead contact upon request.

## ACKNOWLEDGMENTS

We are indebted to Anita Imm and the Lighthouse facility (Freiburg) for their assistance with cell sorting and confocal microscopy, to Marco Prinz (Freiburg) and Steffen Jung (Israel) for provision of mice, and to the Center for Experimental Models and Transgenic Service (Freiburg) for excellent technical support. This work was supported by the Hans A. Krebs Medical Scientist Programme (Faculty of Medicine, Freiburg) (J.K. and S. B.); the German Research Foundation (DFG) under TRR359 (#491676693, J.K., P.H., and K.K.), TRR167 (#259373024, P.H. and K.K.), CRC1160 (#256073931, P.H. and K.K.), CRC1479 (#441891347, K.K.), (HE3127/9, HE3127/12, and HE3127/16, P.H.), and (LA3680/9-1 and LA3680/10-1, N.L.); under Germany's Excellence Strategy (CIBSS-EXC-2189, #390939984, P.H. and K.K.); the

Agence Nationale de la Recherche (ANR) under ANR/ERAPerMed BATMAN (ANR-18-PERM-0001) (V.F. and L.D.), ANR AUTOMATE (ANR-20-CE15-0018-01) and ANR LabCom INCREASE (ANR-22-LCV2-0009-01) (V.F.), a Marie Slodowska-Curie Individual Fellowship (H2020-MSCA-IF-2019 896095 ViriVITES, B.V.), the Swiss National Science Foundation (#207960, C.L.F.), the European Research Council under the European Union's (EU) Horizon 2020 research and innovation program (#852178, N.L.); the EU (#101100859 and #101158172, N.L.); and the German Center of Lung Research (N.L.). Experimental schemes were created with Biorender.com.

## AUTHOR CONTRIBUTIONS

J.K. and C.D. conceptualized the study, designed experiments, and analyzed the data. J.K., C.D., S.S., S.B., R.A., Z.M.M., V.G., F.L., and P.A. performed experiments; L.S. and M.H. provided and analyzed human skin scRNA-seq data; G.V.L.S. and C.E.A.S. analyzed published scRNA-seq datasets; T.B. and N.L. generated iPSC-derived macrophages; B.V., L.D., and V.F. generated iPSC-derived neuron precursor cells; I.M.P. and T.C. provided intellectual input; K.K., M.B., C.L.F., and M.G. provided mice and critical revision; J.K., C.D., and P.H. wrote the manuscript; J.K. and P.H. supervised the project and acquired funding.

## DECLARATION OF INTERESTS

N.L. has filed a patent application on human iPSC-derived macrophage generation (IP PCT/EP2018/061574).

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* EXPERIMENTAL MODEL AND SUBJECT DETAILS

* Mice

* METHOD DETAILS

* Animal models and *in vivo* interventions

* Tissue preparation

* Cell cultures

* iPSC-derived cells

* Cocultures

* *In vitro* treatments

* Flow cytometry and cell sorting

* Antibodies used for flow cytometry

* ELISA

* RNA extraction and qRT-PCR

* RNAseq

* Single cell RNAseq analysis

* Histology

* Live-cell imaging

* Oligonucleotide sequences for qRT-PCR

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.immuni.2025.08.004.

Received: December 15, 2023

Revised: August 1, 2025

Accepted: August 6, 2025

Published: September 5, 2025

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57. Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550. https://doi.org/10.1186/s13059-014-0550-8.

58. Thomas, P.D., Ebert, D., Muruganujan, A., Mushayahama, T., Albou, L.P., and Mi, H. (2022). PANTHER: Making genome-scale phylogenetics accessible to all. Protein Sci. 31, 8–22. https://doi.org/10.1002/pro.4218.

59. Afgan, E., Nekrutenko, A., Grüning, B.A., Blankenberg, D., Goecks, J., Schatz, M.C., Ostrovsky, A.E., Mahmoud, A., Lonie, A.J., and Syme, A. (2022). The Galaxy platform for accessible, reproducible and collaborative
biomedical analyses: 2022 update. Nucleic Acids Res. 50, W345–W351.
https://doi.org/10.1093/nar/gkac247.

60. Hao, Y., Hao, S., Andersen-Nissen, E., Mauck, W.M., 3rd, Zheng, S., Butler, A., Lee, M.J., Wilk, A.J., Darby, C., Zager, M., et al. (2021). Integrated analysis of multimodal single-cell data. Cell 184, 3573–3587. e29. https://doi.org/10.1016/j.cell.2021.04.048.

61. Lopez, R., Regier, J., Cole, M.B., Jordan, M.I., and Yosef, N. (2018). Deep generative modeling for single-cell transcriptomics. Nat. Methods 15, 1053–1058. https://doi.org/10.1038/s41592-018-0229-2.

62. Wolf, F.A., Angerer, P., and Theis, F.J. (2018). SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15. https://doi.org/10.1186/s13059-017-1382-0.
## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3">Antibodies</td></tr><tr><td>Anti-Beta-III-tubulin (clone TuJ-1)</td><td>R&amp;D</td><td>Cat# NL1195V; RRID: AB_357520</td></tr><tr><td>Anti-CD11b (clone M1/70)</td><td>Invitrogen</td><td>Cat# MA1-1008; RRID: AB_11153179</td></tr><tr><td>Anti-CD11c (clone HL3)</td><td>BD Biosciences</td><td>Cat# 550261; RRID: AB_398460</td></tr><tr><td>Anti-CD16/32 (clone 93)</td><td>Invitrogen</td><td>Cat# 14-0161-82; RRID: AB_467133</td></tr><tr><td>Anti-CD206 (clone C068C2)</td><td>Biolegend</td><td>Cat# 141708; RRID: AB_10900231</td></tr><tr><td>Anti-CD3e (clone 145-2C11)</td><td>Invitrogen</td><td>Cat# A14714; RRID: AB_2534230</td></tr><tr><td>Anti-CD45 (clone 30-F11)</td><td>Invitrogen</td><td>Cat# MCD4528; RRID: AB_10373710</td></tr><tr><td>Anti-CD45.1 (clone A20)</td><td>Invitrogen</td><td>Cat# 12-0453-82; RRID: AB_465675</td></tr><tr><td>Anti-CD45.2 (clone 104)</td><td>Biolegend</td><td>Cat# 109806; RRID: AB_313443</td></tr><tr><td>Anti-CD64 (clone X54-5/7.1)</td><td>Biolegend</td><td>Cat# 139308; RRID: AB_2561963</td></tr><tr><td>Anti-CGRP (clone 4901)</td><td>Santa Cruz</td><td>Cat# sc-57053; RRID: AB_2259462</td></tr><tr><td>Anti-CSF1 (polyclonal)</td><td>Bioss</td><td>Cat# BS-4910R; RRID: AB_2832205</td></tr><tr><td>Anti-CX3CR1 (clone SA011F11)</td><td>Biolegend</td><td>Cat# 149008; RRID: AB_2564492</td></tr><tr><td>Anti-F4/80 (clone Cl:A3-1)</td><td>Invitrogen</td><td>Cat# MF48000; RRID: AB_10376289</td></tr><tr><td>Anti-GFP (polyclonal)</td><td>Rockland</td><td>Cat# 600-101-215; RRID: AB_218182</td></tr><tr><td>Anti-Goat anti-rabbit</td><td>Invitrogen</td><td>Cat# A-11035; RRID: AB_2534093</td></tr><tr><td>Anti-LAP TGF-β1 (clone TW7-16B4)</td><td>Biolegend</td><td>Cat# 141404; RRID: AB_10730610</td></tr><tr><td>Anti-Ly6-C (clone HK1.4)</td><td>Biolegend</td><td>Cat# 128036; RRID: AB_2562353</td></tr><tr><td>Anti-Ly6-G (clone 1A8)</td><td>Biolegend</td><td>Cat# 127606; RRID: AB_1236494</td></tr><tr><td>Anti-MHCII (clone AF6-120.1)</td><td>Invitrogen</td><td>Cat# 48-5320-82; RRID: AB_10669941</td></tr><tr><td>Anti-Neurofilament M (Poly28410)</td><td>Biolegend</td><td>Cat# 841001; RRID: AB_2565457</td></tr><tr><td>Anti-NK1.1 (clone PK136)</td><td>Biolegend</td><td>Cat# 156504; RRID: AB_2783135</td></tr><tr><td>Anti-pSMAD2/3 (clone O72-670)</td><td>BD Biosciences</td><td>Cat# 562586; RRID: AB_11151915</td></tr><tr><td>Anti-Siglec-F (clone S17007L)</td><td>Biolegend</td><td>Cat# 155508; RRID: AB_2750237</td></tr><tr><td>Anti-Substance P (clone NC1/34HL)</td><td>Santa Cruz</td><td>Cat# sc-21715; RRID: AB_628299</td></tr><tr><td>Anti-Tmem119 (clone V3RT1GOsz)</td><td>Invitrogen</td><td>Cat# 14-6119-82; RRID: AB_2848273</td></tr><tr><td>Human anti-CD11b (clone ICRF44)</td><td>Invitrogen</td><td>Cat# 25-0118-42; RRID: AB_1582272</td></tr><tr><td>Human anti-CD14 (clone M5E2)</td><td>BD Biosciences</td><td>Cat# 558121; RRID: AB_397041</td></tr><tr><td>Human anti-CD206 (clone 19.2)</td><td>Invitrogen</td><td>Cat# 53-2069-42; RRID: AB_2574416</td></tr><tr><td>Human anti-CD64 (clone 10.1)</td><td>Biolegend</td><td>Cat# 983202; RRID: AB_2650652</td></tr><tr><td>Human anti-CX3CR1 (clone 2A9-1)</td><td>Biolegend</td><td>Cat# 341610; RRID: AB_2087424</td></tr><tr><td colspan="3">Biological samples</td></tr><tr><td>Fetal bovine serum</td><td>Corning</td><td>Cat# 35-079-CV</td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>Cat# 16050130</td></tr><tr><td>Normal Goat Serum</td><td>abcam</td><td>Cat# ab138478</td></tr><tr><td colspan="3">Chemicals, peptides, and recombinant proteins</td></tr><tr><td>Ascorbic acid</td><td>Sigma Aldrich</td><td>Cat# A1300000</td></tr><tr><td>Ascorbic acid 2-phosphate</td><td>Sigma Aldrich</td><td>Cat# 49752</td></tr><tr><td>B-27</td><td>Gibco</td><td>Cat# 12587010</td></tr><tr><td>Bovine serum albumine</td><td>Sigma-Aldrich</td><td>Cat# A7906</td></tr><tr><td>CGRP, recombinant</td><td>R&amp;D Systems</td><td>Cat# 1161/100U</td></tr><tr><td>CHIR99021</td><td>Sigma Aldrich</td><td>Cat# SML1046</td></tr><tr><td>Cilengitide</td><td>Tocris</td><td>Cat# 5870/10</td></tr><tr><td>Ciprofloxacin</td><td>Fresenius Kabi</td><td>n.a.</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Collagenase II</td><td>Worthington</td><td>Cat# LS004176</td></tr><tr><td>Collagenase IV</td><td>Worthington</td><td>Cat# LS004188</td></tr><tr><td>Corn oil</td><td>Sigma-Aldrich</td><td>Cat# C8267</td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific</td><td>Cat# D9542</td></tr><tr><td>DAPT</td><td>Selleckchem</td><td>Cat# S2215</td></tr><tr><td>Dispase</td><td>StemCell Technologies, Inc.</td><td>Cat# 07913</td></tr><tr><td>DMEM</td><td>Gibco</td><td>Cat# 61965026</td></tr><tr><td>DMEM F12</td><td>Gibco</td><td>Cat# 10565018</td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>Cat# D8418</td></tr><tr><td>Dnase I</td><td>Roche</td><td>Cat# 04716728001</td></tr><tr><td>DPBS</td><td>Thermo Fisher Scientific</td><td>Cat# 14190094</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>Cat# E5134</td></tr><tr><td>Fixable Viability Dye</td><td>Invitrogen</td><td>Cat# 65-0863-14</td></tr><tr><td>Gentamycin</td><td>Sigma-Aldrich</td><td>Cat# G1397</td></tr><tr><td>GM-CSF</td><td>Peprotech</td><td>Cat# 315-03</td></tr><tr><td>hBDNF</td><td>Peprotech</td><td>Cat# 450-02</td></tr><tr><td>hbFGF</td><td>Peprotech</td><td>Cat# 100-18B</td></tr><tr><td>hBMP4</td><td>Peprotech</td><td>Cat# 120-05</td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific</td><td>Cat# 14175053</td></tr><tr><td>hGDNF</td><td>Peprotech</td><td>Cat# 450-10</td></tr><tr><td>hIL-3</td><td>Peprotech</td><td>Cat# 200-03</td></tr><tr><td>hM-CSF</td><td>Peprotech</td><td>Cat# 300-25</td></tr><tr><td>hNGF</td><td>Peprotech</td><td>Cat# 450-01</td></tr><tr><td>hSCF</td><td>Peprotech</td><td>Cat# 300-07</td></tr><tr><td>hVEGF</td><td>Peprotech</td><td>Cat# 100-20A</td></tr><tr><td>Hydroxytamoxifen</td><td>Sigma-Aldrich</td><td>Cat# T7904</td></tr><tr><td>insulin</td><td>Sigma Aldrich</td><td>Cat# I3536</td></tr><tr><td>L-alanyl-L-glutamine</td><td>Corning</td><td>Cat# 25-015-Cl</td></tr><tr><td>LDN193189</td><td>Sigma Aldrich</td><td>Cat# TA9491598594</td></tr><tr><td>Lipofectamine RNAiMAX</td><td>ThermoFisher</td><td>Cat# 13778030</td></tr><tr><td>M-CSF, murine</td><td>Peprotech</td><td>Cat# 315-02</td></tr><tr><td>N-2 Supplement (S100x)</td><td>Gibco</td><td>Cat# 17502048</td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>Cat# S5761</td></tr><tr><td>Neurobasal A</td><td>Gibco</td><td>Cat# 10888022</td></tr><tr><td>Opti-MEM</td><td>Gibco</td><td>Cat# 51985034</td></tr><tr><td>Paraformaldehyd</td><td>Electron Microscopy Sciences</td><td>Cat# 15700</td></tr><tr><td>Pen/Strep</td><td>PAN Biotech</td><td>Cat# P06-07100</td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>Cat# P1644</td></tr><tr><td>Poly-D-Lysin</td><td>Sigma-Aldrich</td><td>Cat# P7280</td></tr><tr><td>ProLong Diamond Antifade Mountant</td><td>Thermo Fisher Scientific</td><td>Cat# P36965</td></tr><tr><td>RBC lysis Buffer</td><td>Invitrogen</td><td>Cat# 00-4333-57</td></tr><tr><td>SB-525334</td><td>Cayman chemicals</td><td>Cat# 16281</td></tr><tr><td>SB-431542</td><td>Sigma Aldrich</td><td>Cat# S4317</td></tr><tr><td>Sodium selenite</td><td>Sigma Aldrich</td><td>Cat# S9133</td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>Cat# T5648</td></tr><tr><td>TGF-β1</td><td>Peprotech</td><td>Cat# 100-21</td></tr><tr><td>Trace element A</td><td>Corning</td><td>Cat# 25-021-Cl</td></tr><tr><td>Trace element B</td><td>Corning</td><td>Cat# 25-022-Cl</td></tr><tr><td>Trace element C</td><td>Corning</td><td>Cat# 25-023-Cl</td></tr></tbody></table>

(Continued on next page)
Continued

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>Transferrin</td><td>Sigma Aldrich</td><td>Cat# T8158</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>Cat# 93418</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>Cat# P2287</td></tr><tr><td>Y-27632</td><td>Tocris</td><td>Cat# 1254</td></tr></tbody></table>

Critical commercial assays

<table><tr><td>ABsolute qPCR SYBR Green</td><td>Thermo Fisher Scientific</td><td>Cat# AB1159A</td></tr><tr><td>BD Cytofix™ Fixation Buffer</td><td>BD Biosciences</td><td>Cat# 554655</td></tr><tr><td>BD Phosflow™ Perm Buffer III</td><td>BD Biosciences</td><td>Cat# 558050</td></tr><tr><td>LEGENDplex-kit</td><td>Biolegend</td><td>Cat# 740677</td></tr><tr><td>RNAeasy Micro Kit</td><td>QIAGEN</td><td>Cat# 74004</td></tr><tr><td>Superscript™ IV VILO™ Master Mix</td><td>Thermo Fisher Scientific</td><td>Cat# 11756050</td></tr><tr><td>TGF-β1 ELISA</td><td>R&amp;D Systems</td><td>Cat# DY1679</td></tr><tr><td>Trifecta siRNA Kit, Tgfb1</td><td>Trifecta</td><td>mm.Ri.Tgfb1.13</td></tr></table>

Deposited data

<table><tr><td>Bulk RNAseq of dermal macrophages in homeostasis and after injury</td><td>This paper</td><td>GEO: GSE250457</td></tr><tr><td>Bulk RNAseq of co-cultured BMDMs</td><td>This paper</td><td>GEO: GSE250457</td></tr><tr><td>scRNAseq of murine DRG</td><td>Sharma et al.<sup>27</sup></td><td>GEO: GSE19088</td></tr><tr><td>scRNAseq of human DRG</td><td>Tavares-Ferreira et al.<sup>28</sup></td><td>N/A</td></tr><tr><td>scRNAseq of human skin donors</td><td>Steele et al.<sup>29</sup></td><td>N/A</td></tr><tr><td>Bulk RNAseq of CGRP-treated BMDM</td><td>Lu et al.<sup>24</sup></td><td>GEO: GSE255049</td></tr><tr><td>Microarray data of mouse dermal macrophages</td><td>Kolter et al.<sup>6</sup></td><td>GEO: GSE114526</td></tr></table>

Experimental models: Cell lines

<table><tr><td>iPSC cell line LVPC118</td><td>Jung et al.<sup>52</sup></td><td>N/A</td></tr></table>

Experimental models: Organisms/strains

<table><tr><td>Mouse: C57BL/6J</td><td>The Jackson Laboratory</td><td>JAX: 000664</td></tr><tr><td>Mouse: B6(Cg)-Cxcr4tm1.1(cre/ERT2)Stum/J</td><td>The Jackson Laboratory</td><td>JAX: 40559</td></tr><tr><td>Mouse: B6.129(Cg)-Scn10atm2(cre)Jwo/TjpJ</td><td>The Jackson Laboratory</td><td>JAX: 036564</td></tr><tr><td>Mouse: B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2)Jung/J</td><td>Steffen Jung (Israel)</td><td>Yona et al.<sup>38</sup></td></tr><tr><td>Mouse: B6.129P2(Cg)-Cx3cr1tm1Litt/J</td><td>Steffen Jung (Israel)</td><td>Yona et al.<sup>38</sup></td></tr><tr><td>Mouse: B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J</td><td>The Jackson Laboratory</td><td>JAX: 006148</td></tr><tr><td>Mouse: B6.Cg-Gt(ROSA)26Sortm9(CAGtdTomato) Hze/J</td><td>The Jackson Laboratory</td><td>JAX: 007909</td></tr><tr><td>Mouse: B6;129-Tgfbr2tm1Karl/J</td><td>The Jackson Laboratory</td><td>JAX:012603</td></tr><tr><td>Mouse: C57BL/6J-Tgfb1em2Lutzy/Mmjax</td><td>The Jackson Laboratory</td><td>JAX:065809</td></tr><tr><td>Mouse: Mrc1-cre/ERT2 x B6.Cg-Gt(ROSA)26Sortm9 (CAG-tdTomato)Hze/J</td><td>Marco Prinz (Freiburg)</td><td>Masuda et al.<sup>18</sup></td></tr></table>

Oligonucleotides

<table><tr><td>Primers for qRT-PCR, see methods</td><td>This paper</td><td>N/A</td></tr></table>

Software and algorithms

<table><tr><td>Fiji 1.47v</td><td>ImageJ</td><td>https://fiji.sc/</td></tr><tr><td>Imaris v10.1.1</td><td>Bitplane</td><td>https://imaris.oxinst.com</td></tr><tr><td>Kaluza v2.1</td><td>Beckman Coulter</td><td>https://www.beckman.de/flow-cytometry/software/kaluza</td></tr><tr><td>Panther v18.0</td><td>Paul Thomas</td><td>http://www.pantherdb.org/</td></tr><tr><td>Prism v9.0</td><td>GraphPad Software</td><td>https://www.graphpad.com/scientific-software/prism/</td></tr><tr><td>R v4.3.1</td><td>The R foundation</td><td>https://www.r-project.org/</td></tr><tr><td>ZEN 3.0</td><td>Zeiss</td><td>https://www.zeiss.de/mikroskopie/produkte/mikroskopsoftware/zen.html</td></tr></table>
# EXPERIMENTAL MODEL AND SUBJECT DETAILS

## Mice

All mice were on C57BL/6 genetic background. C57BL/6J and C57BL/6N mice were purchased from Jackson Laboratories (USA) or Charles River Laboratory (Germany). Cx3cr1<sup>gfp/+</sup> mice were a kind gift of Steffen Jung (Weizmann Institute, Rehovot, Israel). Cx3cr1<sup>creERT2</sup> mice were described in.³⁸ For fate mapping analysis, mice were crossed to either floxed Rosa26-stop-EYFP (#006148; R26<sup>fp/yfp</sup>) or Rosa26-stop-tdTomato (#007914; R26<sup>tdT/tdT</sup>) mice purchased from Jackson Laboratory and Cx3cr1<sup>gfp/+</sup> mice in second generation to obtain of Cx3cr1<sup>creERT2/gfp</sup> R26-tomato mice. Mrc1<sup>creERT2/creERT2</sup> R26<sup>tdT/tdT</sup> and Cxcr4<sup>creERT2/+</sup> R26<sup>tdT/tdT</sup> mice were obtained from Marco Prinz (University of Freiburg, Germany). Mrc1<sup>creERT2/creERT2</sup> and Cx3cr1<sup>creERT2</sup> R26<sup>fp/yfp</sup> mice were bred to Tgfb2<sup>flox/flox</sup> mice provided by Katrin Kierdorf (University of Freiburg, Germany). Cx3cr1<sup>creERT2/+</sup> Tgfb1<sup>flox/flox</sup> mice were obtained from Melanie Greter (ETH Zurich, Switzerland). Tgfb1<sup>flox/flox</sup> mice were bred to Nav1.8<sup>cre</sup> mice (#006148, Jackson Laboratory). Ramp1/3<sup>-/-</sup> mice were provided by Christelle Le Foll (University of Zurich, Switzerland). Cd45.1 (#002014) and beta-actin-EGFP (#006567) mice were purchased from Jackson Laboratory. Mice were bred under specific pathogen-free conditions in the animal facilities of the University of Freiburg and housed in groups of up to five mice. Mice were kept in 12h light/dark cycles and food and water was provided ad libitum. Adult mice were between 6 and 20 wk of age if not mentioned otherwise. Both male and female mice were used for experiments, for transplantations mice were sex-matched. For RNAseq, 10 weeks old female mice were used. Littermates were randomly assigned to experimental groups. All animal experiments were approved by the Federal Ministry for Nature, Environment and Consumer's protection of the state of Baden-Wuerttemberg.

# METHOD DETAILS

## Animal models and *in vivo* interventions

Cre recombinase was induced as previously described.⁶ Briefly, 4 mg TAM were dissolved in 200 µl corn oil (both Sigma) and injected subcutaneously at 4 injections sites in mice under isoflurane narcosis. The treatment was repeated after 48 h. For bone marrow transplantations, mice were anesthetized with Ketamine and Xylazin. One ear of each recipient mouse was shielded with a customized lead device and lethally irradiated with 9 Gy. Subsequently, mice received 1x10⁷ donor-derived bone marrow cells i.v. For ear punches, mice were punched in the center of the ear pinnae using a sterile stainless steel 2-mm hole punch (Type 292-2, Zoonlab, Germany).

## Tissue preparation

Dermal macrophages were isolated as described recently.⁵³ Briefly, mouse ears (dermis and epidermis) were minced into small pieces and subjected to enzymatic digestion by Dispase (0.25 U/ml; STEMCELL Technologies), Collagenase II (1 mg/ml; Worthington), and DNase I (0.04 mg/ml; Roche) in HBSS with 5% FCS at 1400rpm at 37°C for 2h. For colon macrophages, the colon was opened longitudinally and rinsed. Epithelial cells were dissociated by shaking twice for 15 min at 37°C in 2 mM EDTA and 10 mM HEPES in HBSS. Remaining tissue was washed, minced, and digested three times for 15 min at 37°C with 0.3 mg/ml Collagenase IV (Worthington), 5 U/ml Dispase (Corning), and 0.5 mg/ml DNase I (Roche) in HBSS supplemented with 2% FCS. After digestion, the samples were filtered with a 70µm cell strainer.

Peripheral blood was withdrawn from the retro-orbital sinus, and red blood cells were lysed with RBC lysis buffer (eBioscience). For microglia isolation, mice were transcardially perfused with ice-cold PBS. Brains were removed, homogenized using a Dounce homogenizer (Carl Roth) and the cell suspension was filtered through a 100 µm cell strainer. Myelin was removed by resuspension in 37 % Percoll (Sigma Aldrich) and centrifugation for 20 min at 300g (RT), with acceleration set to minimum and break disabled. To harvest neurons, DRG from all spine sections were collected. In the first step of digestion, DRG were incubated for 60 min in 10 mg/ml collagenase II dissolved in sterile Hanks Balanced Salt Solution (HBSS). In the second step, DRG were digested with 0.25 % Trypsin (Gibco) for 20 min. Subsequently, DRG were triturated by repeated pipetting in DMEM F12 (Gibco) medium and filtered through a 70 µm strainer and centrifuged for 5 min at 200 g.

## Cell cultures

For BMDM culture, bone marrow from femur and tibia was extracted and plated in DMEM medium, containing 10 % fetal bovine serum (FBS), 20 ng/mL M-CSF (Peprotech) and 0.05 % Ciprofloxacin (Frisenius Kabi). After five days of differentiation, BMDM were harvested. For DRG cultures, pelleted DRG cells were seeded in DMEM F12 and Neurobasal (Gibco) medium (1:1) containing 10% horse serum (Gibco), 10 ng/ml human NGF (Peprotech) and 0.05 % Ciprofloxacin in poly-D-lysine (Sigma Aldrich)-coated wells. The medium was changed every third day. Cells were treated for 10 min at 37°C with accutase solution (Sigma Aldrich) for harvesting. Cell cultures were visually inspected by light microscopy to verify cell density and purity before each experiment.

## iPSC-derived cells

### Human iPSC-derived macrophages

Human iPSC-derived macrophages were generated as described previously.⁵⁴ iPSC cells were cultured on Geltrex™-coated tissue culture flasks using E8 medium (containing DMEM/F-12 (GIBCO, Life Technologies), 64 mg/L ascorbic acid 2-phosphate, 14 µg/L sodium selenite, 543 mg/L, NaHCO₃, 20 mg/L insulin and 10.7 mg/L human recombinant transferrin (all from Sigma-Aldrich).
supplemented with 100 ng/mL hbFGF and 2 ng/mL hTGFβ (both from Peprotech) under standard humidified conditions at 37 °C and 5 % CO₂. The cells were split twice a week using Accutase™ (Stemcell Technologies) and under the addition of 10 µM Y-27632 (Tocris, Bristol, UK). A complete medium change using Y-27632-free medium was performed 48 h after splitting. In order to start mesoderm priming, 5 x 10⁵ iPS cells were seeded in 3 mL of mesoderm priming I medium (E8 medium supplemented with 10 µM Y-27632, 50 ng/mL hVEGF and hBMP4 and 20 ng/mL hSCF (all from Peprotech) using CELLSTAR 6-well plates placed on an orbital shaker at 70 rpm, leading to the formation of embryoid bodies (EBs). On day 2, the medium was changed to E6 medium (containing only hVEGF, hBMP4 and hSCF). On day 4 after mesoderm priming I, supernatant was discarded, and 3 mL of mesoderm priming II medium (E6 medium supplemented with 50 ng/mL hVEGF and hBMP4, 20 ng/mL hSCF and 25 ng/mL hIL-3) was added and the shaking speed was increased to 85 rpm. Mesoderm priming medium II was refreshed at day 7 before macrophage differentiation (hematopoietic differentiation) was started at day 10. For this, the medium was removed and the EBs were transferred to a 6-well tissue culture plate using 2 mL of differentiation medium (X-VIVO containing 1% (v/v) P/S and L-Glu, 0.1% (v/v) β-Mercaptoethanol supplemented with 25 ng/mL hIL-3 and 50 ng/mL hM-CSF. Macrophage production started from day 4 onwards, and macrophages could be continuously harvested 1-2x per week.

## Human iPSC-derived sensory neurons

iPSC cells (line LVPC118) were obtained from Dr. S. Viville, Strasbourg⁵² and differentiated as previously described.³² Briefly, LVPC118 iPSC were seeded (day 0) into cell culture flasks coated with Geltrex (Thermofisher) at a density of 90,000 cells/cm². Until day 10, LVPC118 were cultured in Basal medium, consisting of 1:1 (vol:vol) DMEM-F12 (Lonza) : NeuroBasal medium supplemented with 2% B-27, 1% N2, 1% MEM non-essential amino-acid (Thermofisher), 1% L-alanyl-L-glutamine, 0.1% trace element A, 0.1% trace element B, 0.1% trace element C (Corning), 50 µg/mL gentamycin (Lonza), 5 µM Y-27632, 0.01 mM β-mercaptoethanol (Thermofisher) and 50 µg/mL ascorbic acid (Sigma). From day 0 to day 4, Basal medium was supplemented with 40 µM SB431542, 0.2 µM LDN193189 and 3 µM CHIR99021 (Sigma). From day 5 to day 6, Basal medium was supplemented 3 µM CHIR99021. From day 9 to day 10, Basal medium was supplemented with 10 µM DAPT (Selleckchem). At day 11, neuron precursor cells (NPCs) were harvested using Accutase and cryopreserved for further use. NPCs were thawed and seeded in poly-D-lysin precoated wells with DMEM F12 and Neurobasal medium (1:1) containing N2 (Gibco, 100x), B27 (Gibco, 50x), 10 µM DAPT (Selleckchem), 50 µg/ml ascorbic acid (Sigma), 5 µM Y-27632 dihydrochloride (Tocris), 10 ng/ml human GDNF (Peprotech), 20 ng/ml human BDNF (Peprotech) and 0.5 % Streptomycin and Penicillin (Pan Biotech). Medium was changed the next day. From the third day on, DAPT was replaced by 10 ng/ml human NGF and the medium was changed every other day.

## Cocultures

For mouse cell cultures, BMDM were added at a density of 25,000 cells/cm² to DRG cultures (10,000 cells/cm²) and cocultured for 5 days. As a control, BMDM in monocultures were cultured in the same medium. In all approaches, 20 ng/ml M-CSF was added to the cultures. Medium was changed every second day. For experiments with conditioned medium, medium was taken from cocultures after two and four days. Medium was centrifuged and the supernatant was mixed 1:1 with fresh media for DRG cells, 20 ng/ml M-CSF and 10 ng/ml NGF. Conditioned medium was then added to BMDM on day two and four of monoculture. For transwell approaches, 50,000 BMDM were seeded into 24-well cell culture inserts with a PET membrane and 0.4 µm pore size (Carl Roth) were used which were placed on wells containing DRG cells. DRG medium with 20 ng/ml M-CSF and 10 ng/ml NGF was added and changed every second day.

For human cocultures, iMacs were added at a density of 25,000 cells/cm² to iPSC-derived sensory neurons (100,000 cells/cm²) at day 2 or 7 of differentiation and cocultured for 5, 10 or 12 days. For cocultures, medium for sensory neurons containing NGF was used and Y-27632 dihydrochloride was replaced with 50 ng/ml human M-CSF. For control, iMacs were cultured alone in the same medium. Medium was changed every second day. For BMDM cultures with heat-killed neurons, DRG cells were incubated for 20 min at 65°C and subsequently cooled on ice prior to adding them to BMDM.

## In vitro treatments

BMDM as well as human iMacs were treated with different concentrations (0.2 - 5ng) of recombinant human TGF-β1 (Peprotech), reconstituted according to manufacturer's instructions. Citric acid diluted in PBS and 0.1% BSA was used as the vehicle control. For the inhibition of TGF-β signaling, 1 µM of TGF-β RI Kinase Inhibitor VIII SB-525334 (Cayman chemicals) was used and DMSO as vehicle control. To inhibit integrin αVβ5, 5 µM Cilengitide (Tocris) was added to the cultures. For all treatments, cells were cultured with medium used for neuron cultures with additional 20 ng/ml murine M-CSF for mouse and 50 ng/ml human M-CSF for human macrophages. To determine M-CSF production by neurons, BMDM were initially differentiated with M-CSF, then M-CSF was removed after reseeding in mono- or cocultures. For tamoxifen inductions, 2mM 4-Hydroxytamoxifen (OH-TAM) in ethanol or ethanol alone was added to the culture, and medium was exchanged after 3 days and cells were harvested on day five. For the knockdown of TGF-β1 in DRG cells, a RNAi kit containing three DsiRNAs targeting *Tgfb1* was purchased from IDT (TriFECTa kit, Identifier: mm. Ri.Tgfb1.13). After 4 days of differentiation, DRG cells were transfected in 100 µl of Opti-MEM (Gibco) containing 1 µl of Lipofectamine RNAiMAX (ThermoFisher) and 50 nM of either *Tgfb1* silencing RNA or non-targeting siRNA for 6 h. Following transfection, the cells were washed and cultured in DRG medium for an additional 2 days before the addition of BMDM.
Flow cytometry and cell sorting

To block Fcγι/III receptors, cells were incubated with anti-CD16/32 antibody (eBioscience) for 5min at 4°C in FACS buffer (PBS, 2 %FCS, 2 mM EDTA). Subsequently, the respective antibodies were added to a mastermix in FACS buffer for surface staining and incubation for 30 min at 4°C. To control for cell viability, DAPI or fixable viability dye™ (eBioscience) were used according to manufacturer's instructions. In cell cultures, dead cells were additionally excluded based on their FSC/SSC properties. For pSMAD2/3 staining, cells were fixed with Cytofix Fixation Buffer (BD), permeabilized with Phosflow Perm Buffer III (BD), and subsequently stained for surface markers and pSMAD2/3. Fluorescence-minus-one (FMO) controls were used to set gates for antibody stainings; wild-type or Cre-negative cells were used to set gates for fluorescence reporters. For analysis, a 3-laser flow cytometer (Gallios™, Beckman Coulter) was used and data were processed with the Kaluza software (v2.1, Beckman Coulter) or FlowJo (v10, LLC). Dermal cells and cells from cocultures were purified by cell sorting with a MoFlo Astrios AQ or a CytoFlex SRT (Beckman Coulter). Cell purity was assessed by regular backsorting of samples, assuring >95% purity of sorted populations. Total cell numbers were quantified using Precision Count Beads (BioLegend) according to the manufacturer's instructions.

Antibodies used for flow cytometry

The following anti-mouse antibodies were used for flow cytometry stainings: CD3ε (145-2C11, BioLegend), CD11b (M1/70, Invitrogen), CD11c (HL3, BD), CD16/32 (93, Biolegend), CD45 (30-F11, Invitrogen), CD45.1 (A20, Invitrogen), CD45.2 (104, BioLegend), CD64 (X54-5/7.1, BioLegend), CD206 (C068C2, BioLegend), β3-tubulin (TuJ-1, R&D), CX<sub>3</sub>CR1 (SA011F11, BioLegend), F4/80 (CI: A3-1, Thermo Fisher Scientific), Ly6C (HK1.4, BioLegend), Ly6G (1A8, BioLegend), MHC II (AF6-120.1, Invitrogen), NK1.1 (PK136, BioLegend), Siglec-F (S17007L, BioLegend), LAP (TGF-β1) (TW7-16B4, BioLegend), Tmem119 (V3RT1GOsz, Invitrogen) and pSMAD2/3 (O72-670, BD Bioscience). The following anti-human antibodies were used for staining: CD11b (ICRF44, Invitrogen), CD14 (M5E2, BD Pharmingen), CD64 (10.1, BioLegend), CD206 (19.2, Invitrogen), CX<sub>3</sub>CR1 (2A9-1, BioLegend), and Neurofilament M (Poly28410, BioLegend).

ELISA

For ELISA, cells were cultured in DRG medium without serum in 48-well plates. Medium was changed on the second day of culture and collected on day five for analysis. TGF-β1 ELISA (R&D Systems, DY1679) was performed according to manufacturer's instructions. To measure CSF1, supernatants from BMDM and DRG cells—cultured either alone or in co-culture, with or without additional M-CSF—were collected and analyzed using a bead-based immunoassay LEGENDplex-kit (740677, BioLegend), according to the “LEGENDplex™ Multi-Analyte Flow Assay Kit” protocol. Cytokines were measured with a BD LSR Fortessa SORP flow cytometer and data was analyzed with the Legendplex Qognit platform.

RNA extraction and qRT-PCR

For RNA extraction, cell populations were sorted in RLT buffer containing 1% β-mercaptoethanol. RNA was extracted with the ExtractME® total RNA micro spin kit or RNeasy Micro Kit Plus (Qiagen) according to manufacturer's instructions. cDNA synthesis was performed by using SuperScript™ IV VILO mix (Thermo Fisher). For qRT-PCR, ABsolute qPCR SYBR Green was used (Thermo Fisher Scientific) and samples were analyzed with a LightCycler 480 (Roche). Gene expression was compared to Gadph if not indicated otherwise

RNAseq

Library preparation and RNAseq for dermal macrophages were performed at the Genomics Core Facility “KFB - Center of Excellence for Fluorescent Bioanalytics” (University of Regensburg, Regensburg, Germany; www.kfb-regensburg.de). The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first strand cDNA from 250 pg total-RNA. Double-stranded cDNA was amplified by LD PCR (13 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). 150 pg of input cDNA were tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step 1:5 dilutions of index 1 (i7) and index 2 (i5) primers were used. The libraries were quantified using the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on a NextSeq 500 instrument controlled by the NextSeq Control Software (NCS) v2.2.0, using two 75 Cycles High Output Kits with the dual index, single-read (SR) run parameters. Image analysis and base calling were done by the Real Time Analysis Software (RTA) v2.4.11. The resulting.bcl files were converted into.fastq files with the bcl2fastq v2.18 software. Trimmed (Cutadapt) raw reads were aligned to the mm10 murine reference genome with STAR (Version 2.7.2b)⁵⁵ and quantified with feature counts.⁵⁶

RNA-sequencing and raw data processing for cell culture samples was performed by CeGaT (Tübingen, Germany). Libraries were prepared using 10 ng RNA and the SMART-Seq Stranded Kit (Takara). Sequencing was performed with 2x 100 bp read length and 50 million clusters/probe on NovaSeq 6000 (Illumina). Demultiplexing of the sequencing reads was performed with Illumina bcl2fastq (2.20). Trimmed raw reads were aligned to mm10 using STAR (version 2.7.3). Differential expression analysis between groups was performed with DESeq2 (version 1.24.0)⁵⁷ in R (version 3.6.1) (R Core Team 2015). Data were analyzed with the RStudio software to perform principal component analysis between the samples and to visualize expression patterns in volcano plots. Heatmaps were generated with Morpheus (https://software.broadinstitute.org/morpheus/). Gene ontology enrichment analysis on upregulated DEGs
was performed with Panther 18.0.⁵⁸ The fgsea package on the Galaxy Freiburg platform⁵⁹ was used for gene set enrichment analysis (GSEA). At indicated conditions, genes were preranked by log₂ fold change and enrichment in the “TGF_BETA_SIGNALING_PATH-WAY” was determined using a gene set merged from curated canonical pathway gene sets (M2, Molecular Signatures Database, https://www.gsea-msigdb.org/), including TGF-β related gene sets of Reactome, BioCarta and WikiPathways databases.

## Single cell RNAseq analysis

### Mouse and human DRG

Public data from mouse DRG in healthy conditions²⁷ and human DRG²⁸ were reanalyzed. The datasets were downloaded and the RDS file was imported into R (R Core Team, 2023) environment version v4.3.1 and Seurat v4.3.0.⁶⁰ For mouse DRG data, the pre-processing step was performed by filtering genes expressed in at least 300 cells and outlier cells based on three metrics (nCountRNA > 50,000 and nFeature_RNA > 6,500 and mitochondrial percentage expression > 5) were filtered out. The percentage of mitochondrial genes was regressed out using the SCTransform function and Principal Component Analysis (PCA) were performed using the top 45 PCs for dimension reduction. For pre-processing of human DRG data, cells based on three metrics (nCountRNA > 30,000 and nFeature_RNA > 6,000 and mitochondrial percentage expression > 10) were filtered out. The percentage of mitochondrial genes was regressed out in the SCTransform function and Principal Component Analysis (PCA) & dimension reduction was performed using the top 50 PCs. In both datasets, t-distributed stochastic neighbor embedding (t-SNE) was used for dimension reduction and clusters were identified using the author's annotations. Visualization of genes illustrating expression levels was performed using R/Seurat commands (DimPlot, FeaturePlot, and DotPlot) with ggplot2 v3.4.2 and scCustomize v1.1.1 R packages.

### Human skin

To establish a dataset of human skin macrophages, we used re-processed raw scRNAseq data as previously described.²⁹ This included publicly available data generated from fresh skin biopsies using the 10x Genomics Chromium platform. We selected myeloid cells specifically and reintegrated these cells using scVI.⁶¹ We selected 6000 highly variable genes (HVGs) as features for the integration. Batch-aware HVG selection was performed by setting the batch key to sample ID. For feature (HVG) selection, we did not include mitochondrial genes, cell cycle genes, from https://github.com/haniffalab/FCA_Whole Embryo/blob/main/resources/pan_fetal_cc genes.csv); hypoxic genes, and ribosomal genes. The following hyperparameters were used for the scVI model: number of layers: 2; number of latent dimensions: 30; gene likelihood: zero-inflated negative binomial distribution, and dispersion: gene-batch. An early stopping patience of 5 epochs was used. The batch key was “sampleID” and no other covariates were passed to the model for correction. We included cells from healthy and phenotypically normal (non-lesional) skin only. Using the output embeddings from scVI, we performed kNN graph construction (k=20). We performed unsupervised clustering of the kNN using a graph community detection algorithm (Leiden). For each cluster, we calculated differentially expressed genes using scanpy.tl. rank genes_groups, which we used to label clusters. We visualized the clusters in two dimensions using UMAP. For gene expression dot plots, we normalized values using the shifted logarithm with a scaling factor of 10,000. The functions were implemented using scanpy.⁶²

### Histology

For ear skin whole mounts, mouse ears were split into dorsal and ventral parts, fixed overnight at 4°C in 1% paraformaldehyde (Electron Microscopy Sciences) and stained with primary and secondary antibodies diluted in washing buffer consisting of 1x PBS, 1% BSA and 0.025% (v/v) Triton X-100 (Sigma). For imaging of cell cultures, cells were fixed for 15 min with 4% Paraformaldehyde and stained with primary and secondary antibodies diluted in staining buffer containing 1x PBS, 10% normal goat serum (abcam), 0.05% Tween20 (Sigma-Aldrich).

For the detection of basement membranes, nerves and macrophages, the following antibodies were used: anti-collagen IV (ab19808, abcam), anti-beta-III tubulin Northern Lights TM NL-637 (TuJ-1, R&D Systems), anti-F4/80 PE (BM8, Invitrogen), anti-F4/80 BV421 (BM8, Invitrogen), anti-CD206 PE (19.2, Invitrogen). Secondary antibodies included goat anti-rabbit AF633, AF546 and AF488 (Invitrogen). Yellow fluorescent protein (YFP) was enhanced with anti-GFP DyLight488 antibody (600-141-215, Rockland). For cultured cells, the following antibodies were used: anti-CGRP AF647 (4901, Santa Cruz), anti-CGRP (4901, Santa Cruz) anti-Substance P (NC1/34HL, Santa Cruz), anti-CSF1 (BS-4910R, ThermoFisher), anti-TGF-β1 PE (TW7-16B4, BioLegend), and Neurofilament M (Poly28410, BioLegend). Confocal microscopy on skin whole mounts was performed with a LSM 880 or LSM980 confocal microscope equipped with a 20×/0.8 NA Plan-Apochromat objective (Carl Zeiss Microimaging) using 1-µm optical slices. Images are displayed as maximum-intensity projections of 10-200µm thick z-stacks. For three-dimensional reconstructions, 20-50µm volumes were recorded in 0.5-1µm sections (2048x2048 pixel resolution) and analyzed with the IMARIS software (Bitplane, Switzerland). For evaluation of axon sprouting, 3mm x 3mm composite tiled images were recorded around the center of the ear punch with 140µm Z-stacks. Mean intensity of CD206 was measured in ZEN 3.0 (Zeiss). For quantifications, a minimum of 3 fields of view (FOV) per sample were quantified in Image J (1.47v, NIH, USA) to calculate the depicted mean values. Cell numbers were calculated with the ImageJ plugin Cell Counter. For nerve density evaluation in the human culture, three tiled images (850 µm × 850 µm each with 12.42 µm Z-stacks) per well were taken at random positions. Samples with disintegrated structures after sample preparation were excluded from further analysis. Tiles and Z-stacks were merged and the areas of β3-tubulin positive staining in mice and NFM positive staining in human were evaluated using Image J (1.47v, NIH, USA). Images were taken with the same parameters on the same experimental day for each independent experiment and the color threshold was set according to controls of each group and applied to all other samples to ensure comparability.
Live-cell imaging

For time-lapse live cell imaging, cells were labelled using cell tracker orange CMRA or cell tracker green (both ThermoFisher Scientific). Macrophages and neurons were stained separately before the start of the coculture for 30 min in DMEM/F12 medium at 37°C and washed with medium. Cocultures were seeded in 8-well µ-slides (ibidi, Germany). Time-lapse imaging was performed with a LSM 710 or CD7 LSM 900 microscope equipped with a 20x/0.8 NA Plan-Apochromat objective (Carl Zeiss Microimaging) and data were processed in ZEN (Zeiss, Germany).

Oligonucleotide sequences for qRT-PCR

Mouse Oligonucleotides for qRT-PCR included the following sequences (5'-3'):

- Gapdh (for: ACTCCACTCACGGCAAATTC, rev: TCTCCATGGTGGTGAAGACA)

- Il1b (for: TTCAGGCAGGCAGTATCACTC, rev: GAAGGTCCACGGGAAAGACAC)

- Axl (for: TGAGCCAACCGTGGAAAGAG, rev: AGGCCACCTTATGCCGATCTA)

- Plxna4 (for: GCAAGGAAGCTTTTGCCGAG, rev: TGAGTCCTTTCTCCACACGC)

- Mrc1 (for: GGCTGATTACGAGCAGTGGA, rev: ATGCCAGGGTCACCTTTCAG)

- Cx3cr1 (for: AGGACACAGCCAGACAAG, rev: TCAGGGGAGAAAGCAAG)

- Ramp3 (for: GTGAGTGTGCCCAGGTATGC, rev: CGACAGGTTGCACCACTTC)

- Ramp1 (for: CCATCTCTTCATGGTCACTGC, rev: AGCGTCTTCCCAATAGTCTCC)

- Fcrls (for: CTTCTGGTCTTCGCTCCTGTC, rev: ATGGTGTAGCTTGAAGCACTG)

- Pmepa1 (for: TGGAGTTCGTGCAAATCGTG, rev: TCCGAGGACAGTCCATCGTC)

- F11r (for: GTGCTTGTACCTCCATCCAAGCCG, rev: GAGGGTGGGGAACCATCATGCTC)

- Mmp14 (for: AGGCTGATTTGGCAACCATGA, rev: CCCACCTTAGGGGTGAATTCTG)

- Itgb5 (for: GCTGCTGTCTGCAAGCACAA, rev: AAGCAAGGCAAGCGATGGA)

- Zeb1 (for: TGAGCACACAGGTAAGAGGCC, rev: GGCTTTTCCCCAGAGTGCA)

- Gcnt2 (for: TCAGCCTATCTCTCATTGCTGC, rev: CGCTGTCCGCTGTATAAAAAACT)

- Smad7 (for: AAGTGTTTCAGGTGGCCGGATCTCAG, rev: ACAGCATCTGGACAGCCTGCAGTTG)

- Tgfb1 (for: TGACGTCACTGGAGTTGTACGG, rev: GGTTCATGTCATGGATGGTGC)

- Csf1 (for: TGCTAAGTGCTCTAGCCGAG, rev: CCCCCAACAGTCAGCAAGAC)

- Il-34 (for: CTTTGGGAAACGAGAATTTGGAGA, rev: GCAATCCTGTAGTTGATGGGGAAG)

- Hprt (for:TCAGTCAACGGGGGACATAAA, rev: GGGGCTGTACTGCTTAACCAG)

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed with Prism 9.0 (GraphPad). For comparison of two groups, unpaired two-tailed Student's t-tests were applied unless stated otherwise. For multiple comparisons, one-way or two-way ANOVAs were performed, followed by multiple comparison tests as stated in the legends. For paired data (i.e., measurement from the same mouse or donor cells measured across different conditions), statistical analyses were conducted using repeated-measures (RM) one-way or two-way ANOVA. For datasets with missing values, mixed-effect models were applied. Differences were considered statistically significant if p-values were ≤ 0.05. (ns, not significant; \*p<0.05; \*\*p<0.01; \*\*\*p<0.001, \*\*\*\*<0.0001). Data are represented as mean±SEM if not stated otherwise. Sample sizes and experimental repeats are indicated in the figure legends.