Keywords: Innate immunity, dermal white adipose tissue, dermal fibroblasts, adipocyte progenitors, adipocytes,Staphylococcus aureus, infection, cathelicidin, antimicrobial peptides, Transforming growth factor beta

Dermal PDGFRA⁺THY1^hi adipocyte progenitors and their antimicrobial response are lost with advancing age

To establish the relative contribution from cells of the adipocyte lineage to express cathelicidin, mice were generated to permit conditional deletion ofCamp by insertingLoxP sites flanking exons 2~4 (Fig. 1A). TheseCamp^flox/flox mice were crossed withPdgfra-cre orAdipoq-cre mice to deleteCamp in dFB that expressPdgfra (Camp^f/fPdgfra-cre) or cells in the later pAD stage that expressAdipoq (Camp^f/fAdipoq-cre). Flow cytometry and RTqPCR validated thatPdgfra orcre expression was highly enriched on THY1⁺CD45⁻ dermal fibroblasts (dFB) but not in THY1⁻CD45⁺ myeloid immune cells or spleen tissue (Fig. S1A–B). UponS. aureus infection,Camp expression was significantly suppressed in theCamp^f/fPdgfra-cre orCamp^f/fAdipoq-cre skin, but not in the spleen or blood cells (Fig. 1B andS1C–E).Camp^f/fPdgfra-cre andCamp^f/fAdipoq-cre mice became significantly more susceptible toS. aureus infection compared to WT littermate controls (Fig. 1C andFig. S1F–H). These results reinforced the conclusion that cathelicidin derived from cells of the adipocyte lineage plays a role in defense againstS. aureus infection.

In general, innate immune defense is maximal in late fetal and early postnatal life and declines with aging (Futata et al., 2012;Georgountzou and Papadopoulos, 2017;Shaw et al., 2013). An age-dependent decrease of the ability of skin to clear bacteria was seen by increased bacterial CFU in the infection edge area with advancing age (Fig. 1D andS1I). To understand the contribution of adipogenesis to this loss of innate immune defense, we next examined the expression of cathelicidin in the cells of the adipocyte lineage during aging. A progressive loss of the capacity ofS. aureus to trigger reactive adipogenesis was observed with advancing age, as measured by lesser staining of cathelicidin (Fig. 1E) and lower expression ofPref1 andCamp mRNA (Fig. S1J andFig. 1F ) in dWAT at sites of infection. Loss of dermal reactive adipogenesis in response toS. aureus correlated with an age-dependent increase in the mRNA expression ofSpp1, a pro-fibrotic marker (Fig. 1F).

Next, we established a flow cytometry method to define cell populations within dFB that are activated uponS. aureus infection. Adipogenic dFB were gated as CD31⁻;CD45⁻;PDGFRA⁺;THY1^hi cells (Chia et al., 2016;Rivera-Gonzalez et al., 2016).S. aureus infection triggered an increase in surface expression of PDGFRA and Sca1 in THY1^hi;PDGFRA⁺ cells but not in THY1^lo;PDGFRA⁺ cells (Fig. 1G–1H andFig. S1K). This suggested that reactive adipogenesis was restricted to THY1^hi dFB. To determine whether age-related loss of reactive adipogenesis was associated with the loss of THY1^hi dFB, THY1 expression was analyzed in PDGFRA⁺ dFB in the late embryo and in mice up to 2 years of age. Fibroblasts from eWAT were also analyzed because eWAT has been previously reported to have age-associated dysfunction in adipogenesis and increased production of pro-inflammatory cytokines and fibrotic features (Kirkland et al., 1993;Sun et al., 2013). Flow cytometry analyses revealed that PDGFRA⁺ fibroblasts in eWAT progressively lost THY1 expression with advancing age (from 3 weeks, 2 months, 1 year to 2 years), and by 1~2 years of age PDGFRA⁺ fibroblasts in eWAT were mostly THY1^lo (Fig. 1I–K). In the skin, E14 embryonic PDGFRA⁺ dFB were mostly THY1^lo (>97%), and became mostly THY1^hi (>90%) by postnatal day 1 (P1). Similar to eWAT, THY1 expression became then progressively lost in dermal PDGFRA⁺ fibroblasts during postnatal development and aging (Fig. 1I–K). Thus, age-related loss of THY1 expression is a common feature of PDGFRA⁺ fibroblasts in both eWAT and skin.

Next, age-related changes in PDGFRA⁺ dFB were evaluated in parallel with changes in myeloid or lymphoid-derived immune cells, including CD11B⁺F4/80⁺ macrophage, CD11B⁺Ly6G^hi neutrophils, CD11C⁺F4/80⁻ dendritic cells (DC), CD45⁺ T cell receptor(TCR)γδ⁺ T cells and CD45⁺TCRαβ⁺ T cells (Fig. 1L andS1M–N). PDGFRA⁺THY1^hi dFB represented the major resident innate cell type (~75% of total dermal cells) in neonatal (P1) skin. During postnatal development (3 weeks~2 months), the decline in THY1^hi dFB was accompanied by an increase in myeloid-derive immune cell populations. Upon aging (1~2 years), the THY1^hi dFB population continued to decline, as did macrophages and DCs, whereas CD45⁺TCRαβ⁺ cells increased both in the skin and in eWAT (Fig. 1L andS1M–N)..

Next we characterized if age-related changes of adipogenic dFB correlated with changes in dermal fibrogenesis and adipogenesis. As shown inFig. 2A, while dWAT and the fibrogenic features (collagen staining and dermal hydroproline content) of dermis were not developed in E14 skin, neonatal skin featured large dWAT with immature adipocytes and numerous small lipid droplets (Fig. 2A–2B andS2A). Dermal adipocytes matured (indicated by large lipid droplets) by 3 weeks of age and were then gradually lost during adulthood and aging (2 months through 2 years) (Fig. 2A). Loss of immature dermal fat with advancing age was also supported by early peak at P1 in the mRNA expression ofPref1 andCamp, genes associated with early adipogenesis, followed by their progressive decline from postnatal development through aging (Fig. 2C–D). Immunostaining for cathelicidin confirmed that it was abundantly expressed by P1 immature dermal AD whereas its expression was decreased in mature 3 week AD and became undetectable in the 1 year aged AD (Fig. S2B). Deletion ofCamp in Pdgfra⁺ dFB (Camp^f/fPdgfra-cre) or Adipoq⁺ AD (Camp^f/fAdipoq-cre) led to >95% and ~75% reduction inCamp mRNA expression in neonatal skin, respectively (Fig. 2E), confirming that differentiating immature AD are the major cellular source of cathelicidin in neonatal skin. The expression of mature adipocyte markers, includingAdipoq andFabp4, peaked at 3 weeks ~ 2 months, then decreased progressively from 2 months~2 years (Fig. 2C andS2C), supporting our histological observation that mature AD were lost with age. Age-dependent loss of immature dermal fat coincided with an age-dependent gain in dermal fibrotic features, especially in the dWAT layer, as shown by collagen staining, hydroxyproline measurement andCol1a1 mRNA expression (Fig. 2A–2C andS2D). Elastic fibers in dFB also developed postnatally, and they became less dense in the lower dermis with advancing age (Fig. S2A). By 2 years of age the dermis appeared thinner andCol1a1 mRNA expression was low (Fig. 2A andS2D), the dWAT layer became devoid of AD, the dWAT showed extensive collagen staining (Fig. 2A andS2A). Together, these results show that age-dependent loss of adipogenic dFB is associated with a progressive loss in immature dermal fat and gain of fibrotic features.

To define adipogenic potential of the cells associated with the age related changes observed in mouse skin, dFB were isolated from skin at various ages and were differentiated into adipocytes in vitro. While uncommitted embryonic E14 dFB were only partially adipogenic, neonatal P1 dFB were highly adipogenic and this adipogenic potential gradually declined during postnatal development (3 weeks~2 months) and was lost in aged 1 year dFB (Fig. 2F). Time course analysis of differentiating neonatal dFB showed that immature AD with small lipid droplets were formed as early as 2 days post differentiation (p.d.), and by day 4~7p.d. >90% of cells became mature AD with large lipid droplets (Fig. 2G). Gene expression analyses revealed four distinct expression kinetics: (1)Col1a1 andThy1 mRNA expression was highest in undifferentiated cells and gradually decreased as cells converted toward AD; (2)Pdgfra,Sca1,Pref1 orPparg1 mRNA expression was detected prior to differentiation and were further elevated on day 1~2p.d. followed by a robust decrease at day 4p.d.; (3)Camp expression was induced as early as day 1 p.d. and peaked around day 2p.d.; (4)Pparg2,Adipoq orFabp4 mRNA expression was induced later around day 2~4p.d. and continued to increase as adipocytes matured (Fig. 2H–I).Camp^f/fPdgfra-cre dFB orCamp^f/fAdipoq-cre dFB showed loss ofCamp expression and secretion through differentiationin vitro (Fig. S2E–G), confirming that these adipose precursor cells are the major cellular source for cathelicidin. The ability of differentiating neonatal dFB to expressCamp andPparg2 mRNA (Fig. 2J andS2H) and to secrete cathelicidin protein into conditioned medium (Fig. S2I–J) was progressively lost in dFB isolated from 3 weeks, 2 months and 1 year skin. In contrast, adult dFB produced higherCol1a1 mRNA expression during differentiation (Fig. 2J), suggesting that decreased adipogenic function is accompanied by increased fibrogenic function in adult dFB. Functionally, the supernatant from differentiating P1 dFB potently inhibited the growth ofS. aureus (SA113 and USA300) (Fig. 2K), whereas the age-related decline in antimicrobial protein expression correlated with decreased capacity of differentiating dFB to inhibit the growth ofS. aureus (Fig. 2L–M). Therefore, taken together, these data show a loss of immature dermal fat and the adipogenic-antimicrobial function of dFB occurs with age.

Activation of the TGFβ pathway is associated with the loss of antimicrobial function of dFB

To better understand the change in antimicrobial and adipogenic function of dFB, we profiled the transcriptomes of primary dFB isolated at different ages by RNA-sequencing (RNA-seq). Because fibroblast populations become heterogeneous during postnatal development (Driskell et al., 2013) (Fig. S3A), Sca1⁺ dFB were sorted from all postnatal groups to focus on the dFB population committed to the adipose lineage. Sca1 labeling purity was confirmed by flow cytometry (Figure S3B), and sorted Sca1⁻ dFB were confirmed to be unable to differentiate into AD compared to Sca1⁺ dFB (Fig. S3C). Because dFB lost their adipogenic function by 2 months of age (Fig. 2E), we chose 2 months as the most advanced age group for RNA-seq analyses.

Principle component analysis revealed a distinction in the transcriptomes of dFB isolated from different age groups. Embryonic E14 dFB and 2 month Sca1⁺ dFB samples were clearly separated from P1 Sca1⁺ dFB (Fig. 3A). Time course analyses identified seven gene clusters with multiple Gene Ontologies (GOs) undergoing distinct temporal changes (Fig. 3B andS3D–I). To identify top putative regulators that drive these age-associated changes, we performed Ingenuity Pathway Analysis (IPA) and identified TGFβ signaling as the top activated signaling pathway in 2 month Sca1⁺ dFB compared to P1 Sca1⁺ dFB (Fig. 3C). IPA also predicted that TGFβ signaling activities were significantly altered in dFB from other age groups compared to P1 Sca1⁺ dFB (Fig. S3J–K). Consistently, RNA-seq and RTqPCR analyses (Fig. 3D–G) revealed that the expression of the key genes in the TGFBR pathway, includingTgfbr1 or Tgfbr2, Tgfb1 or Tgfb2 andInhba, was elevated in 2 months compared to P1 Sca1+ dFB. Several TGFβ pro-fibrotic genes downstream of TGFβ includingActa2 (gene coding for alpha smooth muscle actin-SMA),Ctgf,Spp1, Pai1, Mmp13, Saa3 and Il6 were strongly elevated in 2 month cells compared to P1 cells. In contrast, the expression of several pro-adipogenic genes includingPref1,Pparg, Sca1, Lpl (Gregoire et al., 1998) andDcn (a natural inhibitor of TGFβ signaling (Border et al., 1992)) was suppressed in 2 month cells compared to P1 cells. Similar to 2 month cells, embryonic E14 fibroblasts expressed and secreted significantly higher amounts ofTgfb2 transcript and TGFβ2 protein, respectively (Fig. 3E–F). The expression of proadipogenic genes, includingPparg,Sca1 andDcn was suppressed in E14 compared to P1 cells, but profibrotic genes includingActa2 andSpp1 were only moderately elevated in E14 cells, suggesting that pro-fibrotic effect of TGFβ is preferentially suppressed in embryonic cells. Together, these results indicated that TGFβ could play a role in the age-related pro-adipogenic to pro-fibrotic switch of dFB.

TGFβ2 drives loss of adipogenic-antimicrobial function in neonatal dFB

To determine if the presence of TGFβ ligands were functionally relevant, neonatal dFB were treated with recombinant mouse TGFβ2. TGFβ2, as low as 0.1 ng/mL, potently suppressed the adipogenic function of P1 cells, and at higher doses (3~10 ng/mL) it not only completely abolished adipogenic function but also triggered a fibrotic phenotype, characterized by extensive cell striation and spindle-shaped morphology (Fig. 4A). In parallel, the induction of adipocyte marker genes, secretion of cathelicidin and FABP4 proteins, as well as suppression ofCol1a1 mRNA upon adipocyte differentiation was inhibited by TGFβ2 (Fig. 4B–C). In undifferentiated P1 dFB, TGFβ2 triggered a dose-dependent decrease in the expression of pro-adipogenic genes (Sca1, Pref1, Pparg1 and Dcn) and an increase in the expression of pro-fibrotic and inflammatory genes (Spp1, Acta2, Mmp13, Pai1, Ctgf andIl6) (Fig. 4D–E). Similar results were also seen in neonatal dFB treated with TGFβ1, except that TGFβ1 was 3~10 times less potent than TGFβ2 (Fig. S4A–B).

To characterize the dynamic interplay between pro-fibrotic and anti-adipogenic functions of TGFβ, Sca1 and SMA expression was evaluated in PDGFRA⁺THY1⁺ dFB by flow cytometry (Fig. S4C). Cultured neonatal PDGFRA⁺THY1⁺ dFB exhibited high Sca1 and low SMA mRNA expression. Treatment with TGFβ2 or TGFβ1 for 2 days led to a rapid dose-dependent decrease in Sca1 and increase in SMA expression (Fig. 4F–G andFig. S4D). Flow cytometry analyses of cultured dFB from various ages revealed that while neonatal PDGFRA⁺THY1⁺ dFB were mostly Sca1^hiSMA^lo, dFB gradually lost Sca1 and gained SMA expression with age, and by 1 year most cells were Sca1^loSMA^hi (Fig. 4H–I). Cultured 2 month or 1 year dFB strongly resembled neonatal dFB treated for 2 days with TGFβ2 (Fig. 4F). The antimicrobial activity of differentiating neonatal dFB againstS. aureus was completely suppressed with addition of TGFβ2 (Fig. 4J–K andS4E). These results demonstrate that TGFβ2 is a potent suppressor of dFB’s capacity to differentiate into AD and provide antimicrobial activity.

TGFβ exerts anti-adipogenic effects on dFB by activating the TGFBR-SMAD2,3 pathway

To understand the signaling pathway mediating the effect of TGFβ on dFB, downstream signaling molecules of the TGFβ pathway were evaluated. Phosphoblotting analyses of neonatal dFB treated with TGFβ2 showed that TGFβ2 induced a transient increase of SMAD2,3 and AKT phosphorylation (Fig. 5A). To determine the role of SMAD3 and AKT in the effects of TGFβ, small pharmacological inhibitors specific to SMAD3 (SIS3) (Jinnin et al., 2006), AKT (Wortmannin) (Zhang et al., 2016) or TGFBR (SB431542-SB) (Inman et al., 2002;Mordasky Markell et al., 2010) were applied. Pretreatment with either SMAD3 or TGFBR inhibitors, but not an AKT inhibitor, rescued TGFβ-driven suppression ofPparg1 and induction ofCtgf mRNA expression (Fig. 5B), and restored the adipogenic function and the ability of neonatal dFB to produce and secrete cathelicidin (Fig. 5C–D andFig. S5A). These results demonstrate that TGFβ exerts its pro-fibrotic and anti-adipogenic effects on dFB via the TGFBR-SMAD2,3 pathway.

Inhibition of TGFBR function boosts dFB adipogenic potential and enhances resistance to skin infection

To test the hypothesis that TGFβ2 is responsible for the age-dependent loss of adipogenic capacity of dFB, primary dFB isolated from mice of various ages were treated in culture for 3 days with a TGFBR inhibitor (SB). Inhibition of TGFBR reversed the loss of Sca1 and gain of SMA observed in PDGFRA⁺THY1⁺ dFB during aging (Fig. 5E–G). The age-associated suppression of pro-adipogenicPparg1 and increase of the pro-fibroticSpp1 were also reversed by SB treatment (Fig. 5H–I). Inhibition of TGFBR enabled embryonic and adult dFB to differentiate into mature adipocytes (Fig. 5J) and express cathelicidin (Fig. 5K).Col1a1 expression was also suppressed upon TGFBR inhibition (Fig. S5B). Furthermore, the antimicrobial potential of the conditioned medium from differentiating embryonic dFB was enhanced when TGFBR was inhibited (Fig. 5L).

TGFβ signaling is active in the dermis and promotes loss of antimicrobial defense of mice

To investigate the role of the TGFβ pathway in dFBin vivo, activation of phosph-SMAD2,3 and the abundance of TGFβ was examined in mice of various ages. Increased pSMAD2,3 was seen in adult dermis (3 weeks~2 years) compared to neonatal dermis (Fig. 6A). P1 and 3 week skin showed random pSMAD2,3 distribution through the dermis while 2 month~2 year skin showed prominent pSMAD2,3 staining in the dWAT layer (Fig. 6A). This suggested that pSMAD2,3 signal may be preferentially turned on only in the committed pAd in dWAT during adulthood. Circulating TGFβ2 concentrations in serum were higher in neonatal (P1~P7) and young adult mice (3 weeks) but then declined progressively with advancing age (2 months~2 years) (Fig. 6B). In contrast, circulating TGFβ1 concentrations were lower in neonates and became elevated from adulthood through aging (Fig. 6C). To determine whether TGFβ may promote dermal fat loss in adulthood, adult mice were treated intradermally with TGFBR inhibitor (SB) for 5 days (Fig. 6D). SB-treated skin showed notable expansion of the dWAT layer (Fig. 6D) and higher expression ofCamp andAdipoq, and lower expression of pro-fibrotic genes (Col1a1 andCtgf) (Fig. 6E).

To determine if these observations could translate into reduced susceptibility to infectionin vivo, we administrated SB after infecting 1 year adult mouse skin withS. aureus strain USA300 (Fig. 6F). Treating the infected mice with SB improved resistance toS. aureus skin infection as shown by a decrease in lesion size and bacterial CFU at both the infection edge and in the central area of the lesion (Fig.6F–G). RTqPCR analyses revealed that SB administration not only increased the expression of pAd or AD-related genes, includingPdgfra andCamp, but also suppressed expression of the pro-fibrotic geneSpp1 at the infection site (Fig. 6H andS5C). Infection triggered adipocyte hyperplasia and cathelicidin protein production from dWAT that was enhanced in SB-treated mice (Fig. 6I). In addition, to inhibit TGFBR specifically in dFB, we generated conditional heterozygous deletion ofTgfbr2 by crossingTgfbr2^flox/flox mice toPdgfra-cre (Tgfbr2^flox/+Pdgfra-cre) (Fig. 6J). No homozygousTgfbr2^flox/floxPdgfra-cre offspring were generated despite extensive breeding, and thus experiments were only conducted with heterozygous offspring. dFB isolated fromTgfbr2^flox/+Pdgfra-cre mice expressed ~50% lessTgfbr2 mRNA compared to WT dFB (Fig. S5D).Tgfbr2^flox/+Pdgfra-cre dFB were less responsive to exogenous TGFβ2 treatment as shown by an impaired induction of profibrotic genes includingCtgf,Acta2 andSpp1 (Fig. 6K–L). Importantly, when mice were subjected toS. aureus infection, the fibrotic response in the infected skin was significantly reduced in compared to WT control (Fig. 6M). Furthermore, more lipid droplets and cathelicidin protein were detected in the infectedTgfbr2^flox/+Pdgfra-cre dermis compared to infected WT controls (Fig. 6N). Overall bacterial abundance was lower, but not statistically significant, in the infected skin ofTgfbr2^flox/+Pdgfra-cre mice compared to WT controls (Fig. S5E–G), suggesting that heterozygous deletion ofTgfbr2 in Pdgfra⁺ dFB may not be sufficient to drive a clinical phenotype. These changes demonstrate that TGFβ critically drives age-dependent loss of the innate antimicrobial function of the skin.

TGFβ promotes loss of adipogenic and antimicrobial defense function of primary human dermal fibroblasts

To determine if our observations in mice may also be relevant to humans, we evaluated dWAT in human skin samples (Caucasian; non-obese) from neonates (gestational week, GW 29~38), young adults (18 ~25 years), mid-age adults (50~65 years) and elderly donors (>75 years). Neonatal skin, either preterm (GW 29~32) or full term (GW 38~40), contained large volume of dWAT and thin a dermis, while in adulthood dWAT volume became gradually reduced and was eventually lost in mid-age and elderly skin samples (Fig. 7A andS6A). A similar trend of dWAT loss during adulthood was also observed in human skin biopsies (Chinese; non-obese) collected from young children (2~6 years), young adult (18~25 years) and mid-age adult (50~65 years) (Fig. S6B). Similar to mouse skin, the mRNA expression ofPREF1 or human cathelicidinCAMP was significantly higher in human neonatal skin compared to mid-age or elderly skin samples (Fig. 7B andS6C). Immunostaining of human skin sections for the human cathelicidin peptide LL-37 showed that it was abundantly expressed by neonatal dermal AD but not in the larger AD in aged skin (Fig. S6D). dWAT loss in adulthood coincided with an increase of dermal thickness, fibrogenesis as measured by collagen staining as well asCOL1A1 mRNA upregulation (Fig. 7A,S6A andS6E). In elderly human skin, similar to 2 year mouse skin, a decrease in dermal thickness, collagen content andCOL1A1 mRNA expression was observed (Fig. 7A,S6A andS6E), suggesting that prolong activation of dFB may eventually lead to loss of cells’ profibrogenic function in elderly skin.

To determine if TGFβ can inhibit adipogenic function of human neonatal dFB, primary neonatal dFB isolated from 5 donors were differentiated into adipocytes with or without TGFBR inhibitor or recombinant TGFβ2. Neonatal dFB from three donors had high adipogenic capacity and highly expressedCAMP andFABP4 mRNA, both of which could be completely inhibited with TGFβ2 treatment (Fig. S7A andFig. 7C). In contrast, dFB isolated from the other two neonatal donors had low basal adipogenic capacity but became highly adipogenic and expressed highCAMP andFABP4 mRNA when TGFBR inhibitor (SB) was added (Fig. S7A andFig. 7C). Adipogenic capacity positively correlated withPPARG mRNA expression in undifferentiated cells prior to or after SB treatment (Fig. 7D).

To define the role of TGFβ in controlling the pro-adipogenic and pro-fibrotic functions of human dFB, we next focused on the neonatal dFB with low basal adipogenic capacity. Under basal conditions these cells had a notable fibrotic morphology, characterized by stratification and clustering of cells into aggregates (Fig. 7E), and this phenotype was further exacerbated upon treatment with TGFβ2, but was potently diminished upon treatment with SB (Fig. 7E). Flow cytometry analysis for SMA protein expression revealed that untreated cells were composed of mixed populations with low, medium and high expression of SMA, and TGFBR inhibitor treatment shifted these cell populations to mostly SMA^lo state, whereas TGFβ2 treatment shifted them toward mostly SMA^hi state (Fig. 7F). These cells expressed low basalPPARG mRNA and highACTA2 andCTGF (fibrotic gene) mRNA, and recombinant TGFβ2 further decreasedPPARG and increasedACTA2 andCTGF expression, whereas SB treatment strongly increasedPPARG and almost completely suppressedCTGF expression (Fig. S7B–C). Thus, by default, these cells were weakly adipogenic, and TGFBR inhibitor treatment restored their adipogenic function and the ability to expressCAMP (Fig. 7E andS7C). These results demonstrate that endogenous TGFβ pathway is constitutively active in neonatal human dFB with low basal adipogenic capacity, driving them to shift from a pro-adipogenic to a pro-fibrotic phenotype. Furthermore, antimicrobial assay using conditioned medium collected from undifferentiated and differentiated human neonatal dFB treated with SB demonstrated that only differentiating adipocytes treated with TGFBR inhibitor significantly suppressed growth of twoS. aureus strains, including SA113 and USA300 (Fig. 7G). In addition, inhibition of TGFBR in primary adult dFB isolated from four mid-age adult donors also showed a significant decrease of mRNA expression forCTGF andCOL1A1 and an increase inPPARG (Fig. 7H), suggesting that TGFβ-mediated pro-adipogenic to pro-fibrotic switch also occurs in human adult cells. Together, these results are consistent with observations in mice and demonstrate that TGFβ drives loss of the adipogeni-cantimicrobial function of primary human dFB.

Animals and animal care

All animal experiments were approved by the University of California, San Diego (UCSD), Institutional Animal Care and Use committee. C57BL/6 wildtype mice were originally purchased from Jackson laboratory, and were then breeded and maintained in animal facility of UCSD. For aging mouse model, dorsal skin biopsies were collected from C57BL/6 wildtype male mice with various ages (E14, P1, 3W, 2 months and 1 year old).Pdgfra-cre mice (Stock No: 013148),Adipoq-cre mice (Stock No: 028020) andTgfbr2^flox/flox mice (Stock No: 012603) were originally purchased from Jackson laboratory then breeded and maintained in animal facility of UCSD.Camp^flox/flox mice, in which exon 2 and exon 4 of Camp gene were flanked by LoxP sites, were generated by Ozgene (Bentley DC, WA, Australia) Fibroblasts or adipocyte specific deletion ofCamp were generated by breedingCamp^flox/flox mice withPdgfra-cre mice orAdipoq-cre mice respectively.Tgfbr2 specific deletion in fibroblasts was achieved by breedingTgfbr2^flox/flox mice withPdgfra-cre mice. Probably due to early embryonic lethality, we were not able to get the homozygous deletion ofTgfbr2 in fibroblasts (Tgfbr2^flox/flox;Pdgfra-cre) over more than one year of breeding, so the heterozygous deletion ofTgfbr2 in fibroblasts (Tgfbr2^flox/+;Pdgfra-cre) was used instead as theTgfbr2 heterozygous deletion model to study the role of TGFBR pathway in the infection triggered dermal adipogenesis-antimicrobial response. Dorsal skin biopsies around the same area were collected from mice with various ages for analysis to control the consistency of the tissue site collected over the whole lifetime of the mouse

Mouse model of S. aureus skin infection

Skin infection experiments were done as described before (Zhang et al., 2015).Staphylococcus aureus strain USA300 (MRSA) was used for in vivo infection. In brief, the backs of sex-matched and age-matched adult wildtype or indicated strain of mutant mice were shaved and hair removed by chemical depilation (Nair) then injected subcutaneously with 100 μL of a mid-logarithmic growth phase of S. aureus (1× 10⁷ CFU of bacteria) in PBS. Mice were sacrificed after day 3 and skin biopsy covering the infection area was harvested. Skin biopsies were homogenized in 1 mL Trizol (for RNA) or PBS (for CFU counting) with 2 mm zirconia beads in a mini-bead beater 16 (Biospect, Bartlesville, OK). To count CFU, homogenized skin samples were serially diluted, plated onto Tryptic Soy Agar, and enumerated after 18 hrs to quantify the CFU per gram of tissue. For administration of TGFBR inhibitor, SB431542 (0.125 mg in 100uL of 2% DMSO + 30% PEG300 + PBS) or vehicle control was injected intradermally within the infection site at 24 hrs and 48 hrs post infection and skin biopsies were collected at day 3 post infection for RNA extraction or CFU count as described above. For all animal studies, animals were randomly selected without formal pre-randomization and quantitative measurements were done without the opportunity for bias.

Chemicals, antibodies and dyes

IBMX (3-Isobutyl-1-methylxanthine), dexamethasone, indomethacin, and recombinant human insulin were purchased from Sigma-Aldaich (St. Louis, MO). SB431542 and SIS3 were purchased from Selleckchem (Houston, TX). Wortmannin was purchased from EMD Millipore (Billerica, MA). Rabbit anti-CRAMP and rabbit anti-LL-37 antibodies were made from our lab as described previously (Zhang et al., 2015;Zhang et al., 2016); goat anti-COLIV, mouse anti-FABP4 and rabbit anti-SMAD2,3 antibodies are from Abcam (Cambridge, MA); Lipid dye bodipy is from Molecular Probes (Eugene, OR). Rabbit anti-Phospho-SMAD2, 3, rabbit anti-phospho-AKT antibody and mouse anti-AKT antibody were purchased from Cell Signaling (Danvers, MA). Recombinant mouse TGFβ1 and TGFβ2 were purchased from R&D Systems (Minneapolis, MN).

Human skin sample collection

Fresh adult human (Caucasian) full thickness skin biopsies, from the back of healthy and non-obese male donors between 18~90 years of age, were collected by the Dermatology clinics, University of California San Diego. Fresh adult human (Chinese) full thickness skin biopsies were collected by Hospital for skin disease, Institute of Dermatology, Chinese Academy of Medical Science and Peking Union Medical College. All sample acquisitions, were approved and regulated by the University of California San Diego Institutional Review Board (reference number 140144) or by Institute of Dermatology, Chinese Academy of Medical Science Medical Ethics Committee (reference number 2012003). The informed consent was obtained from all subjects prior to skin biopsies. Fresh neonatal full thickness skin samples, from the back of neonatal donors, were obtained from the international institute for advancement of Medicine (IIAM; Exton, PA). The informed consent was obtained from parent of neonatal donor prior to skin collection. All human biopsies were taken from the back skin, where it is relatively protected from sun exposure compared to other regions such as face and neck of the body. Upon collection, these samples were directly fixed with PFA then proceed for either paraffin embedding or OCT embedding for histological or immunofluorescent analyses.

Histology, collagen trichrome staining and immunohistochemistry (IHC)

Paraffin embedding and sectioning was performed by the Moores cancer center histology core at UCSD. For OCT embedding, tissue biopsies were first fixed with 4% PFA for 2 hours then cryoprotected by sucrose for 48 hours prior to being embedded in OCT compound and stored in −80 ⁰C. Histological analysis was performed using either paraffin sections or frozen sections by Hematoxylin-Eosin (H&E) staining. Collagen was stained by the Richard-Allan Scientific Gomori Trichrome green collagen staining kit (ThermoFisher Scientific, Waltham, MA, USA). Elastic staining (Thermo Fisher Scientific, Waltham, MA, USA) and hydroxyproline assay (BioVision) were performed on human or mouse tissues according to manufacturer’s instructions. For IHC, fixed and permeabilized tissue sections were blocked with Image-iT FX reagent (invitrogen) before incubating with primary antibodies followed by appropriate 488- or Cy3-coupled secondary antibodies. Nuclei were counter-stained with DAPI. All images were taken with an Olympus BX41 microscope (widefield) or Zeiss LSM510 confocal microscope as indicated.

Primary dermal fibroblast isolation and culture

For neonatal mouse dorsal skin, epidermis was first removed from the dermis by overnight dispase digestion as described before (Li et al., 2017), and for adult skin hair was first removed by clipper then cut into 5mm wide strips prior to dispase digestion. The dermis was then cut into small pieces (~1 mm) by scalpel then digested with 2.5 mg/mL Collagenase D and 30 ng/mL DNAse1 for 2 hours at 37 ⁰C with constant rocking to release dermal fibroblasts. Cells mixture was then filtered through 30 μm filter into single cell suspension then treated with red blood cell lysis buffer. Isolated dermal fibroblasts were cultured in growth medium (DMEM supplemented with 10% FBS, glutamax and antibiotics-antimicotics) in a humidified incubator at 5% CO2 and 37°C under sterile conditions. Fresh medium was replenished daily to remove debris or dead cells. Primary cells were then trypsinized within 3 days and replated at 5 × 10⁴/mL for in vitro assays, and only passage 1 cells were used for experiment. To induce adipocyte differentiation, two day post-confluent dFB were switched to adipocyte differentiation medium containing 2 μM Dexamethasone, 250 μM IBMX, 200 μM Indomethacin and 10 μg/mL recombinant human insulin. Fresh differentiation medium was changed at day 2, 4, and 7 during the differentiation time course.

Flow cytometry and analysis (FACS)

FACS analysis of primary dermal fibroblasts was modified from previously reported method for adipogenic fibroblasts (Chia et al., 2016;Rivera-Gonzalez et al., 2016). Briefly, freshly isolated dFB from mouse skin or primary cultured of dFB first were stained with zombie violet viability dye (BioLegend, 423114) to stain dead cells. Cells were then blocked with anti-mouse CD16/32 (eBioscience, 14016185), followed by staining with an antibody cocktails containing PECy7-CD45 (BioLegend, 147704), PerCP-Cy5.5-CD31 (BioLegend, 102522), PE-THY1 (BioLegend, 105308), APC-PDGFRA (CD140a) (eBioscience, 17140181) and BV605-Sca1 (BioLegend, 108133). Stained cells were then fixed and permeabilized using the intracellular fixation and permeabilization buffer set (eBioscience), then intracellular SMA was stained by AF488-SMA (eBioscience, 53976082). For FACS staining of T cell populations, cells were stained with an antibody cocktail including APC-CD4 (Biolegend, 100516), BV510-CD8a (Biolegend, 100752), APC-Cy7-TCRβ (Biolegend, 12–5711-82), PE-TCRδγ (eBioscience, 12–5711-82), and PerCP-Cy5.5-CD45.2 (Biolegend, 109828). For FACS staining of other myeloid immune cell panel, cells were stained with an antibody cocktail including PECy7-CD11B (BioLegend, 101216), FITC-Ly6G (eBioscience, 11593182), PE-F4/80 (eBioscience,12480182), APC-CD11C (BioLegend, 117310), AF700-MHCII and (eBioscience, 56532182) FACS analysis for protein expression of each cell marker was performed by the BD FACSCanto RUO machine and analyzed by FlowJo V10 software. Dead cells stained positive with zombie violet dye were excluded from the analyses.

Reverse transcription-quantitative PCR (RTqPCR) analyses

Total cellular RNA was extracted using the PureLink RNA isolation kit with RNase-free DNase1 digestion to remove genomic DNA contamination (Qiagen) and 500 ng of RNA was reverse transcribed to cDNA using iScript cDNA synthesis kit (Bio-rad). Quantitative, real-time PCR was performed on the CFX96 real time system (Bio-rad) using SYBR Green Mix (Bimake, Houston, Tx). All of the primers used with SYBR green were designed to span at least one exon to minimize the possibility of nonspecific amplification from the genomic DNA. The expression ofTbp gene (TATA-Box Binding Protein) was used as a house keeping gene to normalize data for the expression of mouse genes, andHPRT was used as a house keeping gene for the expression of human genes. Specific primer sequences are shown insupplementary Table S1 (mouse genes) andTable S2 (human genes).

Immunoblotting analyses

10ul of cell medium was separated on a 10–20% Tris-Tricine precast gel (Biorad), transferred to PVDF membrane (Biorad), followed by immunoblotting using indicated primary antibodies followed by fluorescent secondary antibodies (LICOR) and imaging using fluorescent Odyssey System (LICOR).

TGFβ1 and TGFβ2 ELISA

Mouse TGFβ1 or TGFβ2 ELISA Kit was purchased from R&D Systems (Minneapolis, MN) and TGFβ1 orTGFβ2 concentrations in conditioned medium from primary mouse dermal fibroblasts or mouse serum were determined according to manufacturer’s instruction.

In vitro bacterial killing assay

Antibiotic free and phenol-red free conditioned medium was collected from differentiating dermal fibroblasts as described before (Zhang et al., 2015). The conditioned medium (100 μL) was then mixed with 10⁵/ml CFU (colony forming unit) of indicated bacterial strain in 96 well tissue culture microtiter plates and incubated at 37°C for 10 ~48 hrs prior to plating for CFU counting. The optical density at 595 nm (OD595) was also measured every 2~4 hours by a spectrometer.

Sample preparation for RNA-seq

Primary dermal fibroblasts, isolated from dorsal skin of mouse at E14, E17, P1, 3 weeks and 2 months of age, were harvested at the first passage. To enrich fibroblasts that have committed to adipocyte lineage, Sca1+ dFB were purified from P1, 3 week and 2 month dFB using the anti-Sca1 microbeads kit and MACS columns and separators (Miltenyi Biotec, San Diego, CA). Purity of cells from each age group was greater than 90% as confirmed by FACS analyses. Total cellular RNA was extracted using the PureLink RNA isolation kit with DNase1 digestion to remove genomic DNA contamination (Life Technologies) and 10 ng total RNA (n=3 for each age group) was used for SMART-seq2.

SMART-seq2

SMART-seq2 was performed as previously described with minor modifications (Picelli et al., 2013; Picelli et al., 2014). Briefly, 10 ng total RNA (RIN >9.6) was reversed transcribed using Super Script II (Invitrogen). cDNA was pre-amplified for 10 cycles using high fidelity KAPA HiFi HotStart Ready Mix (Kapa Biosystems). Pre-amplified cDNAs were cleaned with AMPure XP beads (Beckman Coulter) at a 1:1 ratio and eluted with elution buffer (Qiagen). Tn5-mediated tagmentation was performed on 18 ng total cDNA using the Nextera DNA Sample Preparation Kit (Illumina) at 55°C for 5 minutes and deactivated with PM buffer (Qiagen). Adapter-ligated fragments were amplified for 8 continuous cycles using universal Ad1 and unique Ad2.xx barcodes (IDT). Amplified PCR fragments were purified with AMPure XP beads as suggested (Beckman Coulter). Size selected libraries were quantified using Qubit (Thermo), loaded on a High-Sensitivity DNA chip for quality control (Agilent), and quantified using KAPA for Illumina Sequencing Platforms (Illumina). Libraries were multiplexed and sequenced as paired-end, 43 bps on a NextSeq 500 Illumina Sequencing Platform (Illumina) (Cluster density = 252K/mm2, Clusters PF = 78.8%, Q30 = 90.7%).

Transcript alignment, quantification and filtering

Paired-end reads were aligned to the mouse genome (mm10/gencode.vM4) with bowtie (version 1.0.0) and quantified using the RNA-seq by Expectation-Maximization algorithm (RSEM) (version 1.2.12) with the following standard parameters:rsem-calculate-expression -p $CORES --paired-end (Li and Dewey, 2011). Samples displaying >12,000,000 aligned reads and >77% alignment were considered for downstream analyses. Samples were filtered and only protein-coding genes and lncRNAs expressed at minimum 1 TPM in at least one sample in all biological replicates were considered for downstream analyses.

SMART-seq2 and IPA analyses

Differential expression dynamics across E14, E17, P1-Sca1⁺, 3W-Sca1⁺, 2M-Sca1⁺ samples was identified using the single-time series, two-step regression model algorithm Next maSigPro using with the following parameters under the T.Fit, get.siggenes and see.genes functions:alfa = 0.01, vars=”all”, rsq = 0.7, k=7 (Nueda et al., 2014). Principle component analyses were performed using the R ggbiplot package. Pathway and upstream regulator analyses were performed by Ingenuity pathway analysis (IPA) from Qiagen.

Statistics

Experiments were repeated at least 3 times with similar results. Statistical significance was determined using Student’s unpaired two-tailed t-test, or one way ANOVA multiple comparison test as indicated in the legend (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

KEY RESOURCES TABLE.

Highlights.

1. Neonatal skin is enriched with immature fat and adipogenic dermal fibroblasts

2. Dermal immature fat and the adipogenic-antimicrobial dFB are lost with age

3. TGFβ pathway promotes the age-related adipogenic to fibrotic switch of dFB

4. Inhibition of TGFBR restores antimicrobial function of dFB in adult mice

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Supplementary Materials