Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo.

Besides monocyte (MO)-derived macrophages (MACs), self-renewing tissue-resident macrophages (trMACs) maintain the intracutaneous MAC pool in murine skin. Here, we have asked whether the same phenomenon occurs in human skin using organ-cultured, full-thickness skin detached from blood circulation and bone marrow. Skin stimulation ex vivo with the neuropeptide substance P (SP), mimicking neurogenic skin inflammation, significantly increased the number of CD68+MACs in the papillary dermis without altering intracutaneous MAC proliferation or apoptosis. Since intraluminal CD14+MOs were undetectable in the non-perfused dermal vasculature, new MACs must have differentiated from resident intracutaneous progenitor cells in human skin. Interestingly, CD68+MACs were often seen in direct cell-cell-contact with cells expressing both, the hematopoietic stem cell marker CD34 and SP receptor (neurokinin-1 receptor [NK1R]). These cell-cell contacts and CD34+cell proliferation were up-regulated in SP-treated skin samples. Collectively, our study provides the first evidence that resident MAC progenitors, from which mature MACs can rapidly differentiate within the tissue, do exist in normal adult human skin. That these NK1R+trMAC-progenitor cells quickly respond to a key stress-associated neuroinflammatory stimulus suggests that this may satisfy increased local MAC demand under conditions of wounding/stress.

Introduction

Macrophages (MACs) are mononuclear phagocytic leukocytes that play a key role in adaptive and innate immunity, and regulate tissue homeostasis [1-4]. While long believed to derive from circulating monocytes (MOs) [5-7], in most examined adult murine tissues, including skin, MACs are entirely or partially self-maintained from proliferating tissue-resident MACs (trMACs) of embryonal origin [8-11]. Moreover, during tissue inflammation, the contribution of MOs to the increase of MAC number is minimal and is due in large part to the proliferation of trMACs in murine tissues [10,12-14].

However, our current understanding of MAC ontogeny and differentiation in peripheral tissues largely relies on studies in mice and remains unclear whether these concepts are transferable to the human system, namely to human skin. Yet, the fact that patients with congenital monocytopenia still have skin MACs [15,16] supports the hypothesis that the pool of MACs in human skin is either self-maintained or generated by locally resident progenitor cells. Interestingly, it has already been demonstrated for human skin and upper airway mucosal mast cells, that they can mature from resident progenitor cells [17-19], and can be expanded in the absence of circulating progenitors, and bone marrow derived-stem cells.

Therefore, the current pilot study aimed to clarify whether, as in mice, the dermal MAC pool in adult human skin is self-maintained and can be expanded in the absence of hemoperfusion with circulating MOs and bone marrow derived-stem cells.

To address it, full-thickness hair-bearing human skin fragments were organ-cultured detached from blood circulation and bone marrow under serum-free conditions [20,21] and compared MAC number and activities in both a steady-state and pro-inflammatory conditions. For the latter, we simulated neurogenic inflammation through the administration of the prototypic stress-associated sensory neuropeptide, substance P (SP) [22], which acts primarily via neurokinin-1 receptor (NK1R) and Mas-related G Protein coupled receptor X2 (MRGPRX2) [23] and is a key mediator of neurogenic skin inflammation [22,24-26]. This design was also chosen because intracutaneous SP administration increases the number of intradermal MACs in several rodent models in vivo [24,25]. The number, proliferation and apoptosis of CD68+MACs [27,28] and of putative MAC precursors, namely of CD34+cells [29,30], was assessed in human dermis by quantitative (immuno-)histomorphometry [31]. Finally, preliminary mechanistic experiments were performed using the specific NK1R antagonist, aprepitant [32-34], in order to clarify how SP triggers the de novo generation of MAC in human skin.

Materials and methods

Human tissue collection and full-thickness skin organ culture

All experiments on human tissue were performed according to Helsinki guidelines. As a laboratory that specializes in hair research with special interest in the role of perifollicular macrophages in scalp skin, we purposely used healthy frontotemporal human hairy scalp skin samples from women undergoing cosmetic facelift surgery, obtained from collaborating plastic surgeons, after written patient consent and ethics committee approval from the University of Münster (n. 2015-602-f-S), which severely limited the amount of available human skin for organ culture. 4mm skin fragments were obtained from the skin samples upon arrival to the laboratory after overnight shipment, and organ cultured as previously described [20,35] with minor modifications.

To better conserve the viability of immunocytes, a mixture of William’s E and RPMI medium (1:1), which contains insulin, hydrocortisone and L-glutamine [20,21] was used.

After a 24h of equilibration period, skin punches were treated with 10−8, 10−10 M of SP or with a corresponding vehicle control (media only).

Alternatively, before and during SP stimulation, the selective NK1R antagonist, aprepitant [32-34] was administered at 10-7M, in order to prevent the effect of SP.

To test DNA synthesis, samples were treated for 24h with 10μM EdU (5-ethynyl-2'-deoxyuridine) [36].

To test if the endothelial cells can be activated by SP, skin biopsies were incubated either with 10−10 M of SP or with 2 different concentrations (0.5–50 ng/ml) of TNFα for 24h [21]. (for details, see S1 Text).

Immunohistochemistry/Immunofluorescence microscopy and quantitative (immuno-)histomorphometry

After acetone fixation, skin cryo-sections were incubated with the primary antibodies listed in S1 Table over night at 4°C, or 1h at 37°C, after appropriate pre-incubation with serum (S2 Table) and with the appropriate secondary antibody (S3 Table) or solutions provided by staining kits (for details, see S1 Text).

The number of single or double-positive cells were evaluated by quantitative (immuno-)histomorphometry [31] in the papillary dermis, in an area defined as 200μm from the basement membrane of the epidermis (S1 Fig), or in the whole skin section using Biozero-II Analyzer software (for details, see S1 Text).

Statistics

Data are expressed as number, percentage or fold change over vehicle or day 0 when vehicle was not determined. All data were analysed with GraphPad Prism 6 software (GraphPad Prism). Statistical significance was calculated by One-way ANOVA test for parametric data, or Kruskal-Wallis test for non-parametric data. Bonferroni’s test or Dunn’s test were used, respectively, as post hoc test. p<0.05 was regarded as significant.

Results and discussion

SP selectively increases the number of resident CD68+MACs in human papillary dermis ex vivo

After 24h of SP stimulation (10-10M and 10-8M [21]) ex vivo, the number of cells expressing CD68, a late endosomal glycoprotein which selectively demarcates human MACs [27,28], was significantly increased in human papillary dermis compared to control samples (Fig 1).

Fig 1

Effect of SP on CD68+cells.

Quantitative analysis and representative images of CD68+cells in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The number of positive cells was counted in the papillary dermis (200μm from the epidermis). N = 15–16 skin biopsies/group from 4 different donors. Fold change of Mean or Mean ± SEM. One-way ANOVA, post hoc test Bonferroni (#p<0.05). Scale bare: 50μm.

We detected a difference of CD68+cell number between samples treated with SP 10-10M and vehicle samples of 66.8 cell/mm2 (i.e. an increase of 34% compared to vehicle controls) (Fig 1). Given that the increase in the number of CD68+MACs consistently occurred within only 24 h of SP stimulation, and this in a non-blood-perfused tissue, this evidence constitutes a remarkable numeric enhancement.

Instead, the overall number of antigen-presenting cells in human skin, including MACs positive for MHC class II [37,38], remained essentially unchanged (S2A Fig).

This unexpected result rose the hypothesis that the increase number of CD68+MAC may be counteracted by the depletion of other MHCII+ cells, namely dendritic cells.

It is known, in fact, that under conditions of neurogenic inflammation, dermal dendritic cells fast respond and migrate to the lymph node [24,39]. Given that several subtypes of dendritic cells are present in human dermis and that it is still not entirely clear which is the best marker [40,41], we have opted for a double immunostaining protocol of MHCII with CD11c. Our results showed that the number of MHCII+CD11c+ dendritic cells is reduced by ca. 20% in SP treated samples as compared to vehicle samples (S2B Fig). This explains the essentially unaltered number of MHCII+cells after SP stimulation, since the decrease in CD11c+cells is counterbalanced by the observed increase in the number of (also MHCII+) CD68+MACs. Thus, intradermal MACs in human skin and/or their progenitor cells are highly responsive to SP stimulation, even in the absence of functional sensory skin innervation.

MOs are unlikely to significantly contribute to the de novo generation of CD68+MACs in human skin ex vivo

The newly formed dermal CD68+MACs could plausibly have derived from circulating MOs. However, when we have investigated the number of MOs trapped in the lumen of blood vessels by using double-immunostaining to visualize both the endothelium (CD31+cells) and CD14+MOs, hardly any CD14+MOs were found to be trapped in CD31+blood vessels (Fig 2A).

Fig 2

CD14+ MOs in human skin ex vivo.

(a) Representative images of CD14/CD31cells in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The CD14+cells were visualized in the entire dermis. N = 8 skin biopsies/group from 2 different donors (126 skin sections). Arrows indicate CD14+MOs trapped in the lumen of blood vessel cells (white arrows), and CD14+MOs localized close to blood vessels (violet arrows). (b) Quantitative analysis, and representative images of CD14/CD68 in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The percentage and number of double-positive cells was counted in the papillary dermis (200μm from the epidermis). N = 8 skin biopsies/group from 2 different donors. Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (# p<0.05). (c) Quantitative analysis, and representative images of P-selectin in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, SP or TNFα ex vivo. Staining intensity was evaluated in the entire dermis. N = 8 skin biopsies/group from 2 different donors. Fold change of Mean ± SEM, Kruskal-Wallis-Test, post hoc test Dunn, #p<0.05, ##p<0.01, ###p<0.001. Scale bare 50μm.

Furthermore, the number of MO-derived MACs (i.e. CD14+CD68+cells) [42], which represented ca. 75% of the total MAC population found in human skin ex vivo (Fig 2B), was unaffected by SP treatment (Fig 2B). Interestingly, Tamoutounour and colleagues, demonstrate that in mouse skin around 20% of CD68+MACs are Ly-6C- (i.e. the mouse analogue of CD14), indicating the existence of a pool of dermal MACs that is established prenatally and persists in adulthood, independently from circulating monocytes [10,42,43], showing that MACs are indeed “partially” self-maintained by proliferating tissue-resident MACs of embryonal origin.

Therefore, the fact that we also found around 25% of CD68+MACs in human skin to be negative for CD14 nicely correlates with mouse data and suggests that a substantial portion of human dermal MACs are also maintained independently from circulating monocytes.

In addition, H&E staining was also used to investigate whether blood was still trapped in capillaries after skin processing and organ culture, and to discriminate macrophage (large irregularly shaped cells) from monocytes (smaller and more rounded cells) [43,44] in the capillaries.

We could not find any MACs trapped in the blood vessels of any skin samples analysed (S3 Fig), confirming that the isolate CD14+cells found in capillaries were indeed CD14+ circulating MOs. We have also found very few histochemically stained red blood cells in skin samples at day 0 but not in vehicle or SP treated skin samples, i.e. organ cultured samples (S3 Fig). Therefore, while some blood remained trapped in the blood vessels after punches preparation, most of it was washed out during organ culture, further supporting our hypothesis that the newly generated MAC after SP stimulation did not derive from CD14+ circulating monocytes.

Moreover, MOs extravasation is strictly regulated via protein-protein interaction with endothelial cells [45]. In this process, several adhesion proteins are involved in the capturing and rolling phase, namely P-selectin [46,47]. P-selectin is a transmembrane lectin, whose expression is up-regulated by different cytokines, including TNFα [47].

To test if the endothelial cells are activated and overexpress P-selectin after SP stimulation, we treated the skin biopsies either with SP (10-10M) or with 2 different concentrations (0.5–50 ng/ml) of TNFα [21]. As reported, P-selectin was strongly up-regulated in CD31+cells in response to TNFα [47] (Fig 2C) indicating that endothelial cells can be activated ex vivo. Instead, SP did not promote P-selectin expression on them (Fig 2C).

Of interest, the fact that P-selectin expression is up-regulated in endothelial cells [45,46] of day 0 skin samples as compared to vehicle samples may well result from the pro-inflammatory environment triggered in the skin sample during the manipulation of the skin samples, from the trauma of surgical skin harvesting to the initiation of the culture (day 0) [48]. Thus, the expression of P-selectin is most likely restored to the baseline level during organ culture, as the skin gradually adjusts to its new ex vivo environment (Fig 2C).

This renders very unlikely that SP enhances the capacity of the extremely few CD14+MOs trapped in intracutaneous blood vessel ex vivo to extravasate in a P-selectin-dependent manner.

Therefore, together with the absence of functional blood flow in our assay system, the fact that SP did not activate the endothelial cells (Fig 2C) and the extremely rare presence of CD14+cells in human skin blood vessels (Fig 2A) virtually rules out extravasating MOs as a credible source of the substantial, SP-induced increase in the number of dermal CD68+cells.

The SP-induced MAC increase cannot be explained by suppression of MAC apoptosis or stimulation of MAC proliferation

Next, we checked whether the SP-induced MAC increase resulted from a protective effect of SP on dermal CD68+MACs apoptosis under ex vivo conditions. On the contrary, the percentage of apoptotic (TUNEL+ or active-caspase-3+) CD68+MACs was significantly up-regulated in SP-treated skin (Fig 3A), suggesting that the SP-induced increase in the intradermal MAC count was even higher than Fig 1 indicates. This also demonstrates that SP promotes human dermal MAC apoptosis within their physiological environment.

Fig 3

Proliferative and apoptotic MACs.

(a) Quantitative analysis and representative images of CD68/TUNEL or CD68/Caspase3 cells in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The percentage of double-positive cells was counted in the papillary dermis (200μm from the epidermis). N = 15–16 skin biopsies/group from 4 different donors (CD68/TUNEL) or N = 7–8 skin biopsies/group from 2 different donors (CD68/Caspase3). Mean ± SEM. One-way ANOVA, post hoc test Bonferroni (#p<0.05), Kruskal-Wallis-Test, post hoc test Dunn, (#p<0.05). Arrows indicate CD68+TUNEL+cells (green arrows), CD68+active-caspase3+ (yellow arrows). (b) Quantitative analysis and representative images of CD68/Ki-67 cells in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. Representative picture of the internal positive control for Ki-67+proliferative cells. The percentage of double-positive cells was counted in the entire dermis. N = 15–16 skin biopsies/group from 4 different donors. Mean ± SEM. Kruskal-Wallis-Test, post hoc test Dunn, ns. Arrow indicates CD68+Ki-67+cells (red). (c) Quantitative analysis, and representative images of MHCII+/Ki-67+cells in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The percentage of double-positive cells was counted in the papillary dermis (200μm from the epidermis). N = 11–12 skin biopsies/group from 3 different donors. Fold change of Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (#p<0.05, ##p<0.01). Arrow indicates MHCII+Ki-67+cells (pink). EP: epidermis; PD: papillary dermis. Scale bare: 50μm.

When the expression of proliferation-associated parameters (Ki-67, M-phase-specific phospho-histone 3 [PH3]), and DNA synthesis using EdU incorporation [49]) by CD68+MACs were evaluated, no significant differences were seen between vehicle- and SP-treated skin (Fig 3B; S4 Fig).

Moreover, MHCII is a molecule expressed in several mature cell populations in human skin in the dermis, namely antigen presenting cells (CD14+ and CD14- dendritic cells, and MACs), and non-antigen presenting cells [50]. We have already essentially excluded that CD14+cells and CD68+MACs, which also express MHCII, in the dermis as a credible source for the SP-induced increased in the number of dermal CD68+cells. Therefore, the only other MHCII+cell subtype we did not investigate that could give rise to MACs, are CD14- dendritic cells [51].

We have, therefore, determined the number of proliferative MHCII+cells in the papillary dermis. However, the percentage of proliferative MHCII+cells is significantly down-regulated either in vehicle or SP treated groups compared to freshly isolated skin (Fig 3C). The decrease in the proliferative MHCII+ cell number may be explained by the well-documented fact that MHCII+ dendritic cells, rather than macrophages [52], rapidly migrate out of the skin into the culture medium under tissue stress conditions [24,39]. This explain also the downregulation of MHCII+CD11c+ in S2B Fig.

This renders highly unlikely that any of the MHCII+cell populations in the dermis is a source of the increased number of CD68+cells.

Thus, the observed SP-induced increase in the number of CD68+MACs cannot be credibly explained by apoptosis-protection or the proliferation/self-renewal of CD68+MACs or other MHCII+cells.

Resident CD34+-progenitor cells in human dermis are the most likely source of intracutaneously generated CD68+MACs

These results strongly suggested that adult human dermis harbours immature, resident progenitors, from which mature CD68+MACs can rapidly be differentiated in loco, e.g. from previously deposited mesenchymal and/or hematopoietic stem cells (MSC, HSC). Immunohistology for classical MSC and/or HSC markers (c-Kit, CD34) [53,54] showed that almost all c-Kit+cells in the papillary dermis were CD68- and phenotypically represented mast cells [17,31], and that their number did not differ between vehicle- and SP-treated samples (Fig 4A). Instead, very rarely, CD68+CD34+cells could be seen in the papillary dermis of vehicle and SP-treated skin (Fig 4B), many of which expressed NK1R (S5A Fig) and were in direct physical contact with CD68+CD34-cells; the latter phenomenon notably increased after SP treatment (Fig 4C). Since endothelial cells can also express CD34 (S5B Fig) it is important to note that almost 40% of dermal CD34+cells were CD31- (S5B Fig), and thus likely represented CD34+HSCs (S5B Fig).

Fig 4

Identification of c-Kit+ cells and CD34+ progenitors in human skin.

(a) Quantitative analysis, and representative images of CD68/c-Kit in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The number of cells in close contact was counted in the papillary dermis (200μm from the epidermis). N = 15–16 skin biopsies/group from 4 different donors. Fold change of Mean ± SEM. Kruskal-Wallis-Test, ns, post hoc test Dunn, ns. (b) Representative pictures of CD34+ and CD68+ cells. The double positive cells were visualized in the papillary dermis (200μm from the epidermis). Yellow arrow indicates double positive CD68+CD34+ cell. (c) Quantitative analysis, and representative images of CD68/CD34. The number of cells in close contact was counted in the papillary dermis (200μm from the epidermis). N = 15–16 skin biopsies/group from 4 different donors. Fold change of Mean ± SEM. Kruskal-Wallis-Test, post hoc test Dunn (#p<0.05). Arrows indicate CD68+MACs in close contact with CD34+cells (white). Scale bare: 50μm.

SP promotes the proliferation of CD34+ dermal progenitor cells and their maturation into CD68+MACs

This in turn invited the hypothesis that the SP-induced increase in CD68+MACs (Fig 1) is primarily brought about by impacting on resident progenitor cells, from which these skin MACs differentiate. In fact, quantitative analysis of CD34/Ki-67 double-immunohistochemistry revealed that SP significantly upregulated the number of proliferating (i.e. Ki-67+) CD34+cells (Fig 5A), showing that SP can stimulate the proliferation of human dermal CD34+cells within their natural tissue niche.

Fig 5

Proliferative CD34+progenitor cells.

(a) Quantitative analysis, and representative images of CD34/Ki-67. The number of double positive cells was counted in the papillary dermis (200μm from the epidermis). N = 15–16 skin biopsies/group from 4 different donors. Mean ± SEM. Kruskal-Wallis Test, post hoc test Dunn (#p<0.5). Black arrows indicate CD34+Ki-67+cells. (b) Representative pictures of CD68+CD34+EdU+cells. The double positive cells (CD68+EdU+ or CD34+EdU+) were visualized in the papillary dermis. N = 12 skin biopsies/group from 3 different donors. Scale bare: 50μm.

Moreover EdU-incorporating CD34+cells were seen in close contact with EdU-incorporating CD68+cells (Fig 5B).

Direct activation of NK-1 receptor on CD34+MAC progenitors is involved in SP-induced MAC de novo generation ex vivo

Given that CD34+cells express NK1R (S5A Fig), and that the increase in MAC numbers occurs quite rapidly (within 24 hrs) after SP stimulation, our data support the hypothesis that SP induces MAC de novo generation via a direct effect on resident CD34+MAC progenitors in human skin. However, alternative indirect mechanisms may also be involved, such as mast cell activation and subsequent histamine release. In fact, SP does not activate mast cells only via NK1R, but also via Mas-related G Protein coupled receptor X2 (MRGPRX2), a receptor that is selectively expressed on mast cells and dorsal root ganglions [34,55]. Once activated by SP, mast cells secrete histamine, and several other cytokines, namely TNFα and IL-33, known to activate and promote MAC maturation [56-58].

Therefore, in this initial pilot study, we have begun to clarify whether SP-induced MAC de novo generation ex vivo results from a direct effect on the putative CD34+MAC progenitors, or indirect activation of mast cells by administering the selective NK1R antagonist, aprepitant [32-34], so that mast cell activation could be still possible via MRGPRX2.

These additional experiments confirm, once again, that SP stimulation increases significantly MAC number, CD34+cell proliferation, and enhanced the co-localization of CD68+cells with CD34 cells.

Most importantly, our new results show that the administration of aprepitant 2h before SP stimulation prevented 1) the up-regulation of CD68+cells, which number remained comparable to vehicle sample (Fig 6A), 2) the increase in CD34+cell proliferation (Fig 6B) and, 3) the stimulation of co-localizations between CD68+cells with CD34+cells, induced by SP administration (Fig 6C). Thus, our new results demonstrate that NK-1R-mediated signalling is exclusive involved in the SP-induced up-regulation of intradermal macrophage number ex vivo, and suggests a direct effect of SP on resident CD34+MAC progenitors in human skin: if mast cells were involved in the observed SP effects, they should have been activated by SP via MRGPRX2 [34,55] in the presence of aprepitant, which does not block MRGPRX2 signaling [34].

Fig 6

Aprepitant antagonizes the SP effect on CD34+cells and CD68+MAC.

(a) Quantitative analysis, and representative images of CD68 immunostainings in human skin fragments at day 0 or treated with vehicle, or SP, or aprepitant, or aprepitant and SP ex vivo. The number of single positive cells was counted in the papillary dermis. N = 11–12 skin biopsies/group from 3 donors. Fold change of Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (#p<0.05). Scale bare 50μm (b) Quantitative analysis, and representative images of CD34/Ki-67 immunostainings in human skin fragments at day 0 or treated with vehicle, or SP, or aprepitant, or aprepitant and SP ex vivo. The number of double positive cells was counted in the entire dermis. N = 11–12 skin biopsies/group from 3 donors. Fold change of Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (#p<0.05; ##p<0.01). Scale bare 10μm. (c) Quantitative analysis, and representative images of CD34/CD68 immunostainings in human skin fragments at day 0 or treated with vehicle, or SP, or aprepitant, or aprepitant and SP ex vivo. The number of cells in close contact was counted in the in the papillary dermis. N = 11–12 skin biopsies/group from 3 donors. Fold change of Mean ± SEM, Kruskal-Wallis Test, post hoc test Dunn (#p<0.5). Scale bare 10μm.

Conclusions

Although our immunohistomorphometry-based in situ findings should be systematically followed up by FACS analysis and single cell RNAseq so as to obtain further insights into how SP stimulation impacts on the tissue-resident immunocyte progenitor cells in human skin and their differentiation into CD68+ macrophages, these intriguing results suggest that CD68+MACs can indeed mature from resident intracutaneous CD31-CD34+mesenchymal stem cells in human skin, which lose CD34 expression upon differentiation. Together with the clinical observation that monocytopenia patient skin has normal MAC numbers and almost no proliferative CD68+cells, yet many CD34+cells [15], our ex vivo-data further support that human dermal CD68+trMACs arise from an expanding pool of CD34+MAC progenitors after SP stimulation.

Therefore, human skin can generate MACs de novo from pre-existing progenitors such as CD34+cells, at least under conditions of neurogenic inflammation, rather than from extravasated MOs. This designates dermal MACs as yet another key innate immunity protagonist besides mast cells [17,18], which can be expanded from resident progenitor cells present within human skin. This also raises the question whether the massive dermal MAC increase seen e.g. in lepromatous leprosy, leishmaniasis, granuloma annulare, and tattoo-associated granulomata [59,60] results not only from extravasating MOs, but also from the excessive local MAC maturation from resident, intracutaneous (CD34+)progenitor cells. If confirmed, this pathological intradermal MAC differentiation process in human skin would deserve to be targeted therapeutically.

Supporting material and methods.

(DOC)

Click here for additional data file.

Reference area.

Representative pictures of the defined reference areas in the dermis (200μm from the epidermis) used for our analysis. Scale bare 100 μm. D0: day 0.

(TIF)

Click here for additional data file.

Effect of SP on MHCII+cells.

(a) Quantitative analysis, and representative images of MHCII+cells in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The number of cells was counted in the papillary dermis (200μm from the epidermis). N = 11–12 skin biopsies/group from 3 different donors. Fold change of Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (#p<0.05).

(a) Quantitative analysis, and representative images of MHCII+CD11c+cells in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The number of double positive cells was counted in the papillary dermis (200μm from the epidermis). N = 11–12 skin biopsies/group from 3 different donors. Fold change of Mean ± SEM, One-way ANOVA, post hoc test Bonferroni (#p<0.05; ##p<0.01). Orange arrows indicate double positive cells.

Scale bare: 50μm.

(TIF)

Click here for additional data file.

H&E representation of skin capillaries.

Representative pictures showing skin capillaries and blood smear control. Erythrocytes (red arrows) were visualized in few capillaries only at day 0. Intraluminal MO (blue arrow) was detected in a single lumen at day 0. Perivascular MACs (green arrows) were identify at day 0 and Vehicle control. Blood smear control showing erythrocytes and circulating T-cells (black arrows).

(TIF)

Click here for additional data file.

Proliferative CD68+cells.

(a) Representative pictures of the internal positive control showing PH3+ cells in the epidermis. Very few PH3+CD68+MACs were visualized in D0 (day 0). This staining was qualitatively evaluated in 96 sections derived from 4 punches per conditions from 2 different donors. White arrow indicates a double positive CD68+PH3+cell (white).

(b) Representative pictures of the internal positive control showing EdU+ proliferative cells in the epidermis. Very few EdU+CD68+ MACs were visualized in vehicle and SP 10-10M treated human scalp skin. EdU+CD68+ cells were detected out of 32 sections derived from 4 punches per conditions from 1 donor. Pink arrow indicates a double positive CD68+EdU+ cells. EP: epidermis; PD: papillary dermis.

Scale bare 50μm.

(TIF)

Click here for additional data file.

Characterization of CD34+progenitor cells.

(a) Representative picture showing CD34/NK1R cells. Black Arrows indicate double positive CD34+NK1R+ cells.

(b) Quantitative analysis, and representative images of CD34/CD31 in immunofluorescence staining in human skin fragments at day 0 or treated with vehicle, or SP ex vivo. The percentage of double-positive cells was counted in the papillary dermis (200μm from the epidermis). N = 7–8 skin biopsies/group from 2 different donors. Mean ± Men. Green arrows indicate CD34+CD31-cells. Scale bare: 50μm.

(TIF)

Click here for additional data file.

Primary antibodies employed.

Antibodies used for immunofluorescence stainings are listed and described in detail.

(DOCX)

Click here for additional data file.

List of all the immunostainings.

Immunostaining performed and relevant details. Tris-buffered saline (TBS), phosphate buffered saline (PBS), 4’,6-diamidin-2’-phenylindoldihydrochlorid (DAPI).

(DOCX)

Click here for additional data file.

List of the secondary antibodies.

Fluorescein isothiocynate (FITC).

(DOCX)

Click here for additional data file.

11 Sep 2019

PONE-D-19-18895

Tissue-resident macrophages can be generated de novo in adult human skin during neurogenic inflammation from resident progenitor cells

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Reviewer #1: This manuscript considers the possibility that tissue macrophages can be generated in skin from resident progenitor cells when stimulated by substance P. This is potentially a very interesting manuscript that is novel. I have the following points:

1. As substance P appears to be the only neurogenic mediator used, then I suggest the title includes the name of this neuropeptide.

2. Substance P when added exogenously is able to activate mast cells, to release histamine/5-HT which could be the mechanism here. This should be investigated with histamine and 5-HT antagonists. Additionally an NK1 antagonist could be used. Also there are NK1 agonists. Thus there are simple pharmacological experiments which can be done to further this hypothesis and to give a much clearer indication that it is indeed NK1 receptor-dependent.

Reviewer #2: This manuscript describes a very interesting and intriguing mechanism of macrophage de novo generation from CD34+ mesenchymal stem cells in human skin. This observation may have important impacts in the understanding of several disease mechanisms linked with various skin pathologies.

The results presented are convincing, based on 4 different donors, however, some clarifications would be necessary.

1. Since the overall number of MHC class II cells remains unchanged after SP stimulation, but the number of CD68+ cells increased, it means some MHC class II cells were affected by SP and decreased in numbers. Which one? Dendritic cells?

2. It would be informative to clearly state the amount of increase in CD68+ cells upon SP stimulation, which seems to be rather small.

3. In the introduction, authors mentioned that MACs in skin are entirely or partially self-maintained from proliferating tissue-resident MACS, and not from MOs. However, they showed in figure 2 that 70 to 80% of MAC CD68+ cells were CD14+ positive, which means they are from MO origin. One would expect only a very limited proportion of CD14+CD68+ cells compared to CD14-CD68+ if MACs mostly originate from CD14- cells (MSCs or trMACs).

In figure 2, there was a mix-up between CD14-CD68+ and CD14+CD68-, please correct.

4. In figure 2a, how to discriminate between CD14+ MO and CD14+ MACs (as seen in Fig2b)?

Moreover, to confirm that blood was still trapped in capillaries (and has not been washed during skin processing), it would be useful to stain for red blood cells.

5. In figure 2c, how to explain the decrease of P-selectin between Day 0 and the test with the vehicle. My understanding is that the difference between Day 0 and vehicle is the 24h incubation period, more than a potential toxic effect of the vehicle. This means the tissue is deteriorating fast in this culture condition. Same comment about figure 3c. In this figure, how to explain that decreased proliferation is not observed for CD68+ cells (3b), but high for MHCII cells (3c)?

6. In the conclusion part, the mention that the findings were limited by unavailability of large skin samples is not convincing, since large amount of skin can be obtained from plastic surgeries such as abdominoplasties or breast surgeries. Indeed, a FACS analysis would have been highly informative.

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Reviewer #2: Yes: Francois Berthod

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3 Dec 2019

EDITOR COMMENTS

As you will recognize from the comments of the reviewers they raised some points of critique, especially regarding the role of histamine in your findings, characterization of cells and presentation of the manuscript.

Thank you. Below, we have attempted to address these items of critique along with the addition of substantial new data sets and improvements in the overall data presentation.

REVIEWER COMMENTS

Reviewer #1: This manuscript considers the possibility that tissue macrophages can be generated in skin from resident progenitor cells when stimulated by substance P. This is potentially a very interesting manuscript that is novel. I have the following points:

1. As substance P appears to be the only neurogenic mediator used, then I suggest the title includes the name of this neuropeptide.

Thanks a lot. Yes, this makes perfect sense. We have changed the title accordingly:

“Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo”.

2. Substance P when added exogenously is able to activate mast cells, to release histamine/5-HT which could be the mechanism here. This should be investigated with histamine and 5-HT antagonists. Additionally an NK1 antagonist could be used. Also there are NK1 agonists. Thus there are simple pharmacological experiments which can be done to further this hypothesis and to give a much clearer indication that it is indeed NK1 receptor-dependent.

We agree with the reviewer that mast cell activation, with subsequent histamine release, might be one of the mechanisms through which substance P could, in theory, promote macrophage de novo generation ex vivo. In fact, substance P does not activate mast cells only via the NK1R, but also via Mas-related G Protein coupled receptor X2 (MRGPRX2), a receptor that is selectively expressed on mast cells and dorsal root ganglions (1,2). Once activated by substance P, mast cells secrete histamine, and several other cytokines, namely TNFa and IL-33, known to activate and promote macrophage maturation (3–5). In addition, a plethora of other preformed and newly synthesized agents are released by mast cells upon degranulation. Therefore, histamine is only one of many other candidate mechanisms, if the observed macrophage effects were indeed indirectly mediated and dependent on mast cell degranulation. To dissect such indirect mechanisms is a very complex task and would require an extensive separate study.

However, our currently available data already tend to support the alternative explanation that substance P exerts direct effects on resident CD34+ macrophage progenitors in human skin, since we have shown that these cells also express NK1R (Fig S4a), and that the increase in macrophage numbers occurs quite rapidly (within 24 hrs). Incidentally, this corresponds well to our previous demonstration that the stimulation of resident human mast cell progenitors located in the connective tissue sheath of human scalp hair follicles ex vivo with the prototypic stress-associated neurohormone, CRH, suffices to induce substantial maturation of these progenitor cells into fully differentiated, functional and degranulating mast cells (6).

Following the reviewer’s excellent suggestion, we have performed 3 new skin organ culture experiments, in which we selectively antagonized only NK1R with aprepitant (7,8). As explained, this still leaves the possibility that mast cells could be activated by substance P via MRGPRX2 (1,2). These additional experiments confirm, once again, that substance P stimulation increases significantly macrophage number, CD34+ cell proliferation, and enhanced the co-localization of CD68+ cells with CD34+ cells.

Most importantly, our new results show that the administration of aprepitant 2h before substance P stimulation prevented 1) the up-regulation of CD68+ cells, which number remained comparable to vehicle sample, 2) the increase in CD34+ cell proliferation and, 3) the stimulation of co-localizations between CD68+ cells with CD34+ cells, induced by substance P administration (see revised Fig. 6a-c). Thus, our new results demonstrate that NK-1R-mediated signalling is exclusively involved in the substance P induced up-regulation of intradermal macrophage number ex vivo, and suggests a direct effect of substance P on resident CD34+ macrophage progenitors in human skin: if mast cells were involved in the observed substance P effects, they should have been activated by substance P via MRGPRX2 (1,2) in the presence of aprepitant, which does not block MRGPRX2 signalling .

We have added this data in the revised Results and Discussion session, page 14, and Fig 6a-c.

Reviewer #2: This manuscript describes a very interesting and intriguing mechanism of macrophage de novo generation from CD34+ mesenchymal stem cells in human skin. This observation may have important impacts in the understanding of several disease mechanisms linked with various skin pathologies. The results presented are convincing, based on 4 different donors, however, some clarifications would be necessary.

1. Since the overall number of MHC class II cells remains unchanged after SP stimulation, but the number of CD68+ cells increased, it means some MHC class II cells were affected by SP and decreased in numbers. Which one? Dendritic cells?

Thanks for raising this important point, which we had insufficiently addressed. Indeed, substance P acts also on other MHCII+ immunocytes which are, besides macrophages, namely monocytes (7,8), dendritic cells (7,9) and occasionally mast cells (10). It is known that, under conditions of neurogenic inflammation, dermal dendritic cells respond fast and migrate to the lymph node (11,12). Therefore, given that we did not see a decrease in the number of CD14+ monocytes (Fig 2b), just like the expert referee, we hypothesized that the MHC class II+ cell population decreasing after substance P stimulation are indeed dendritic cells. Given that several subtypes of dendritic cells are present in human dermis and that it is still not entirely clear which is the best marker to use (13,14), we have opted for a double immunostaining protocol of MHC class II with CD11c.

Our new results show that the number of MHCII+CD11c+ dendritic cells is reduced by ca. 20% in SP-treated skin samples compared to vehicle controls. This explains the essentially unaltered number of MHC class II+ cells after SP stimulation, since the decrease in CD11c+ cells is counterbalanced by the observed increase in the number of (also MHCII+) CD68+ macrophages. These new results are now presented and discussed on page 7, and shown in the new Fig S2b.

2. It would be informative to clearly state the amount of increase in CD68+ cells upon SP stimulation, which seems to be rather small.

Done as requested. We now state clearly in the revised Results and Discussion section (p.6) that the difference of CD68+ cell number between samples treated with Substance P 10-10M and vehicle samples is 66.8 cell/mm2 (i.e. an increase of 34% compared to vehicle control). Given that the increase in the number of CD68+ immunocytes consistently occurred within only 24 h of SP stimulation, and this in a non-blood-perfused tissue, this constitutes a remarkable numeric enhancement within a very short time window. This underscores the great potential of human dermis to regenerate macrophages from resident progenitors. We have now emphasized this in the revised manuscript (see Results and Discussion page 6, Fig 1)

3. In the introduction, authors mentioned that MACs in skin are entirely or partially self-maintained from proliferating tissue-resident MACS, and not from MOs. However, they showed in figure 2 that 70 to 80% of MAC CD68+ cells were CD14+ positive, which means they are from MO origin. One would expect only a very limited proportion of CD14+CD68+ cells compared to CD14-CD68+ if MACs mostly originate from CD14- cells (MSCs or trMACs).

Thanks for inviting us to clarify this. The sentence in the introduction “…including skin, MACs are entirely or partially self-maintained from proliferating tissue-resident MACs (trMACs) of embryonal origin” refers to murine tissues, including mouse skin. Indeed, the data of Tamoutounour et al. (2013) in mouse skin demonstrate that around 20% of CD68+ macrophage are Ly-6C- (i.e. the mouse analogue of CD14), indicating the existence of a pool of dermal macrophages that is established prenatally and persists in adulthood (7,8,15), showing that macrophages are indeed “partially” self-maintained by proliferating tissue-resident MACs of embryonal origin.

Therefore, the fact that we also found around 25% of CD68+ macrophage in human skin to be negative for CD14 is nicely in line with mouse data and suggests that a substantial portion of human dermal macrophages are also maintained independently from circulating monocytes. We have added this interesting and important discussion point in the revised Results and Discussion, see page 8.

In figure 2, there was a mix-up between CD14-CD68+ and CD14+CD68-, please correct.

Thank you very much for spotting this error, which we have corrected.

4. In figure 2a, how to discriminate between CD14+ MO and CD14+ MACs (as seen in Fig2b)?

Moreover, to confirm that blood was still trapped in capillaries (and has not been washed during skin processing), it would be useful to stain for red blood cells.

The aim of figure 2a is to identify monocytes trapped in the lumen of the blood vessels, that potentially could have extravasated and given rise to dermal macrophages. (Since macrophages are not circulating cells, the chance to find CD14+ macrophage trapped in blood vessels was obviously minimal.) However, we have followed the excellent suggestion of the reviewer and have used H&E to histochemical stain red blood cells and to discriminate macrophages (large irregularly shaped cells) from monocytes (smaller and more rounded cells) (16, 17) in the capillary lumen.

These additional analyses revealed that no MACs could be found to be trapped in the blood vessels of any of the skin samples analysed, confirming that the isolate, few intracapillary CD14+ cells that are found are indeed CD14+ circulating monocytes.

We have also found very few red blood cells in skin samples before the set-up of organ culture, but not in vehicle- or SP-treated skin samples, i.e. after organ culture. Therefore, while some blood remained trapped in the blood vessels after punches preparation, most of it was washed out during organ culture, further supporting our hypothesis that the newly generated MAC after SP stimulation did not derive from CD14+ circulating monocytes. These new data are now shown in the new Fig. S3 and are correspondingly addressed in the revised Results and Discussion section (p 8, fig S3).

5. In figure 2c, how to explain the decrease of P-selectin between Day 0 and the test with the vehicle. My understanding is that the difference between Day 0 and vehicle is the 24h incubation period, more than a potential toxic effect of the vehicle. This means the tissue is deteriorating fast in this culture condition. Same comment about figure 3c. In this figure, how to explain that decreased proliferation is not observed for CD68+ cells (3b), but high for MHCII cells (3c)?

Although organ-cultured human skin indeed represents a slowly degenerating assay system over time (17,18), it remains fully viable for several days, and most certainly during the short 24 h incubation period chosen here. If it were a fast-deteriorating system most other resident skin cells would have undergone massive apoptosis, which is clearly not the case here. Instead, as the reviewer pointed out, we did not detect a significant downregulation in the proliferation of CD68+ cells (fig 3b), CD34+ cells (fig 4c), or epidermal keratinocytes (fig 3b), and neither saw a significant up-regulation of apoptotic CD68+ cells in the vehicle group after 24 h compared to day 0. Therefore, the decrease in P-selectin expression, or proliferative MHC class II cell number between Day 0 and vehicle cannot be explained as consequence of rapid skin deterioration.

However, the fact that P-selectin expression is up-regulated in endothelial cells (19,20) of day 0 skin samples as compared to vehicle samples may well result from the pro-inflammatory environment triggered in the skin sample during the manipulation of the skin samples, from the trauma of surgical skin harvesting to the initiation of the culture (day 0) (21). Thus, the expression of P-selectin is most likely restored to the baseline level during organ culture, as the skin gradually adjusts to its new ex vivo environment. Instead, the decrease in the proliferative MHC class II+ cell number detected in vehicle samples compared to day 0 samples may be explained by the well-documented fact that dendritic cells, rather than macrophages (9), rapidly migrate out of the skin into the culture medium under tissue stress conditions (10,11). This has been briefly acknowledged in the Results and Discussion section (p 9 and p 11)

6. In the conclusion part, the mention that the findings were limited by unavailability of large skin samples is not convincing, since large amount of skin can be obtained from plastic surgeries such as abdominoplasties or breast surgeries. Indeed, a FACS analysis would have been highly informative.

While the reviewer is right about the availability of skin from the indicated sources, as a laboratory that specializes in hair research and has a special interest in the role of perifollicular macrophages in scalp skin (22–24), we purposely used small residual skin fragments derived from face-lift surgery, which severely limited the amount of available human skin for organ culture. This rationale is now explained more clearly in the revised Material and Methods section (p. 4). We fully agree with expert referee that FACS analysis would have very nicely complemented the current study. We now acknowledge in the conclusion that the pointers provided by the current, quantitative immunohistomorphometry-based study should be followed-up by complementing with FACS analyses (p 16).

References

1. Azimi E, Reddy VB, Shade K-TC, Anthony RM, Talbot S, Pereira PJS, et al. Dual action of neurokinin-1 antagonists on Mas-related GPCRs. JCI Insight. 2016 Oct 6;1(16):e89362.

2. Green DP, Limjunyawong N, Gour N, Pundir P, Dong X. A Mast-Cell-Specific Receptor Mediates Neurogenic Inflammation and Pain. Neuron. 2019 06;101(3):412-420.e3.

3. Varricchi G, Rossi FW, Galdiero MR, Granata F, Criscuolo G, Spadaro G, et al. Physiological Roles of Mast Cells: Collegium Internationale Allergologicum Update 2019. Int Arch Allergy Immunol. 2019;179(4):247–61.

4. Jackson-Jones LH, Rückerl D, Svedberg F, Duncan S, Maizels RM, Sutherland TE, et al. IL-33 delivery induces serous cavity macrophage proliferation independent of interleukin-4 receptor alpha. Eur J Immunol. 2016 Oct;46(10):2311–21.

5. Murray PJ, Allen JE, Biswas SK, Fisher EA, Gilroy DW, Goerdt S, et al. Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines. Immunity. 2014 Jul 17;41(1):14–20.

6. Ito N, Sugawara K, Bodó E, Takigawa M, van Beek N, Ito T, et al. Corticotropin-releasing hormone stimulates the in situ generation of mast cells from precursors in the human hair follicle mesenchyme. J Invest Dermatol. 2010;130(4):995–1004.

7. Haniffa M, Ginhoux F, Wang X-N, Bigley V, Abel M, Dimmick I, et al. Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation. J Exp Med. 2009 Feb 16;206(2):371–85.

8. McGovern N, Schlitzer A, Gunawan M, Jardine L, Shin A, Poyner E, et al. Human Dermal CD14+ Cells Are a Transient Population of Monocyte-Derived Macrophages. Immunity. 2014 Sep 18;41(3):465–77.

9. Toebak MJ, Gibbs S, Bruynzeel DP, Scheper RJ, Rustemeyer T. Dendritic cells: biology of the skin. Contact Dermatitis. 2009;60(1):2–20.

10. Kambayashi T, Allenspach EJ, Chang JT, Zou T, Shoag JE, Reiner SL, et al. Inducible MHC Class II Expression by Mast Cells Supports Effector and Regulatory T Cell Activation. J Immunol. 2009 Apr 15;182(8):4686–95.

11. Joachim RA, Handjiski B, Blois SM, Hagen E, Paus R, Arck PC. Stress-Induced Neurogenic Inflammation in Murine Skin Skews Dendritic Cells Towards Maturation and Migration: Key Role of Intercellular Adhesion Molecule-1/Leukocyte Function-Associated Antigen Interactions. Am J Pathol. 2008 Nov 1;173(5):1379–88.

12. Arck PC, Handjiski B, Peters EMJ, Peter AS, Hagen E, Fischer A, et al. Stress inhibits hair growth in mice by induction of premature catagen development and deleterious perifollicular inflammatory events via neuropeptide substance P-dependent pathways. Am J Pathol. 2003 Mar;162(3):803–14.

13. Zaba LC, Krueger JG, Lowes MA. Resident and “Inflammatory” Dendritic Cells in Human Skin. J Invest Dermatol. 2009 Feb 1;129(2):302–8.

14. Clark GJ, Silveira PA, Hogarth PM, Hart DNJ. The cell surface phenotype of human dendritic cells. Semin Cell Dev Biol. 2019 Feb 1;86:3–14.

15. Tamoutounour S, Guilliams M, Montanana Sanchis F, Liu H, Terhorst D, Malosse C, et al. Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin. Immunity. 2013 Nov 14;39(5):925–38.

16. Burke B, Lewis CE. The Macrophage. Second edition. Oxford ; New York: Oxford University Press; 2002. 680 p.

17. Lu Z, Hasse S, Bodo E, Rose C, Funk W, Paus R. Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions. Exp Dermatol. 2007 Jan;16(1):37–44.

18. Zhou L, Zhang X, Paus R, Lu Z. The renaissance of human skin organ culture: A critical reappraisal. Differ Res Biol Divers. 2018 Dec;104:22–35.

19. Gerhardt T, Ley K. Monocyte trafficking across the vessel wall. Cardiovasc Res. 2015 Aug 1;107(3):321–30.

20. Vestweber D. How leukocytes cross the vascular endothelium. Nat Rev Immunol. 2015 Nov;15(11):692–704.

21. Uchida Y, Gherardini J, Alam M, Keren A, Zhang H, Chéret J, et al. Human dermal Vδ1+T-cells recognize “stressed” HFs and may induce alopecia areata. J Dermatol Sci. 2017 May 1;86(2):e59.

22. Hardman JA, Muneeb F, Pople J, Bhogal R, Shahmalak A, Paus R. Human perifollicular macrophages undergo apoptosis, express Wnt ligands and switch their polarisation during catagen. J Invest Dermatol. 2019 Jun 21;

23. Christoph T, Müller-Röver S, Audring H, Tobin DJ, Hermes B, Cotsarelis G, et al. The human hair follicle immune system: cellular composition and immune privilege. Br J Dermatol. 2000;142(5):862–873.

24. Muneeb F, Hardman JA, Paus R. Hair growth control by innate immunocytes: Perifollicular macrophages revisited. Exp Dermatol. 2019 Apr;28(4):425–31.

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31 Dec 2019

Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo.

PONE-D-19-18895R1

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14 Jan 2020

PONE-D-19-18895R1

Tissue-resident macrophages can be generated de novo in adult human skin from resident progenitor cells during substance P-mediated neurogenic inflammation ex vivo.

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