Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

A monocyte–leptin–angiogenesis pathway critical for repair post-infection

Abstract

During infection, inflammatory monocytes are thought to be key for bacterial eradication, but this is hard to reconcile with the large numbers of neutrophils that are recruited for each monocyte that migrates to the afflicted tissue, and the much more robust microbicidal functions of the neutrophils. However, unlike neutrophils, monocytes have the capacity to convert to situationally specific macrophages that may have critical functions beyond infection control1,2. Here, using a foreign body coated with Staphylococcus aureus and imaging over time from cutaneous infection to wound resolution, we show that monocytes and neutrophils are recruited in similar numbers with low-dose infection but not with high-dose infection, and form a localization pattern in which monocytes surround the infection site, whereas neutrophils infiltrate it. Monocytes did not contribute to bacterial clearance but converted to macrophages that persisted for weeks after infection, regulating hypodermal adipocyte expansion and production of the adipokine hormone leptin. In infected monocyte-deficient mice there was increased persistent hypodermis thickening and an elevated leptin level, which drove overgrowth of dysfunctional blood vasculature and delayed healing, with a thickened scar. Ghrelin, which opposes leptin function3, was produced locally by monocytes, and reduced vascular overgrowth and improved healing post-infection. In sum, we find that monocytes function as a cellular rheostat by regulating leptin levels and revascularization during wound repair.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Monocytes are recruited to low-dose S. aureus skin infection.
Fig. 2: Monocytes do not contribute to S. aureus clearance but are critical for tissue repair.
Fig. 3: Hypodermal expansion and elevated leptin in CCR2-deficient mice post-infection.
Fig. 4: Leptin drives dysregulated angiogenesis and delayed healing post-infection.

Data availability

The single-cell RNA-sequencing dataset analysed in this study is available in the Gene Expression Omnibus under accession code GSM2910020.

References

  1. Shi, C. & Pamer, E. G. Monocyte recruitment during infection and inflammation. Nat. Rev. Immunol. 11, 762–774 (2011).

    CAS  Article  Google Scholar 

  2. Guilliams, M., Mildner, A. & Yona, S. Developmental and functional heterogeneity of monocytes. Immunity 49, 595–613 (2018).

    CAS  Article  Google Scholar 

  3. Dixit, V. D. et al. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J. Clin. Invest. 114, 57–66 (2004).

    CAS  Article  Google Scholar 

  4. Tsou, C. L. et al. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J. Clin. Invest. 117, 902–909 (2007).

    CAS  Article  Google Scholar 

  5. Cho, J. S. et al. Neutrophil-derived IL-1β is sufficient for abscess formation in immunity against Staphylococcus aureus in mice. PLoS Pathog. 8, e1003047 (2012).

    CAS  Article  Google Scholar 

  6. Feuerstein, R., Seidl, M., Prinz, M. & Henneke, P. MyD88 in macrophages is critical for abscess resolution in staphylococcal skin infection. J. Immunol. 194, 2735–2745 (2015).

    CAS  Article  Google Scholar 

  7. Brandt, S. L. et al. Macrophage-derived LTB4 promotes abscess formation and clearance of Staphylococcus aureus skin infection in mice. PLoS Pathog. 14, e1007244 (2018).

    Article  Google Scholar 

  8. Thammavongsa, V., Missiakas, D. M. & Schneewind, O. Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science 342, 863–866 (2013).

    ADS  CAS  Article  Google Scholar 

  9. Willenborg, S. et al. CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair. Blood 120, 613–625 (2012).

    CAS  Article  Google Scholar 

  10. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284, 1318–1322 (1999).

    ADS  CAS  Article  Google Scholar 

  11. Mack, M. et al. Expression and characterization of the chemokine receptors CCR2 and CCR5 in mice. J Immunol 166, 4697–4704 (2001).

    CAS  Article  Google Scholar 

  12. Croxford, A. L. et al. The cytokine GM-CSF drives the inflammatory signature of CCR2+ monocytes and licenses autoimmunity. Immunity 43, 502–514 (2015).

    CAS  Article  Google Scholar 

  13. Carneiro, M. B. et al. Th1-Th2 cross-regulation controls early Leishmania infection in the skin by modulating the size of the permissive monocytic host cell reservoir. Cell Host Microbe 27, 752–768.e757 (2020).

    CAS  Article  Google Scholar 

  14. Tamoutounour, S. et al. Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin. Immunity 39, 925–938 (2013).

    CAS  Article  Google Scholar 

  15. Baranska, A. et al. Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal. J. Exp. Med. 215, 1115–1133 (2018).

    CAS  Article  Google Scholar 

  16. Sierra-Honigmann, M. R. O. et al. Biological Action of Leptin as an Angiogenic Factor. Science 281, 1683–1686 (1998).

    ADS  CAS  Article  Google Scholar 

  17. Bouloumié, A., Drexler, H. C., Lafontan, M. & Busse, R. Leptin, the product of Ob gene, promotes angiogenesis. Circ. Res. 83, 1059–1066 (1998).

    Article  Google Scholar 

  18. Abbasi, S. et al. Distinct regulatory programs control the latent regenerative potential of dermal fibroblasts during wound healing. Cell Stem Cell 27, 396–412 e396 (2020).

    CAS  Article  Google Scholar 

  19. Klok, M. D., Jakobsdottir, S. & Drent, M. L. The role of leptin and ghrelin in the regulation of food intake and body weight in humans: a review. Obes. Rev. 8, 21–34 (2007).

    CAS  Article  Google Scholar 

  20. Shook, B. A. et al. Dermal adipocyte lipolysis and myofibroblast conversion are required for efficient skin repair. Cell Stem Cell 26, 880–895 e886 (2020).

    CAS  Article  Google Scholar 

  21. Zhang, L. J. et al. Dermal adipocytes protect against invasive Staphylococcus aureus skin infection. Science 347, 67–71 (2015).

    ADS  CAS  Article  Google Scholar 

  22. Dokoshi, T. et al. Hyaluronidase inhibits reactive adipogenesis and inflammation of colon and skin. JCI Insight 3, e123072 (2018).

    Article  Google Scholar 

  23. Seim, I., Crisp, G., Shah, E. T., Jeffery, P. L. & Chopin, L. K. Abundant ghrelin gene expression by monocytes: putative implications for fat accumulation and obesity. Obes. Med. https://doi.org/10.1016/j.obmed.2016.12.001 (2017).

  24. Frank, S., Stallmeyer, B., Kämpfer, H., Kolb, N. & Pfeilschifter, J. Leptin enhances wound re-epithelialization and constitutes a direct function of leptin in skin repair. J. Clin. Invest. 106, 501–509 (2000).

    CAS  Article  Google Scholar 

  25. Wynn, T. A. & Vannella, K. M. Macrophages in tissue repair, regeneration, and fibrosis. Immunity 44, 450–462 (2016).

    CAS  Article  Google Scholar 

  26. Gonzalez-Perez, R. R., Lanier, V. & Newman, G. Leptin’s pro-angiogenic signature in breast cancer. Cancers 5, 1140–1162 (2013).

    CAS  Article  Google Scholar 

  27. Dal-Secco, D. et al. A dynamic spectrum of monocytes arising from the in situ reprogramming of CCR2+ monocytes at a site of sterile injury. J. Exp. Med. 212, 447–456 (2015).

    CAS  Article  Google Scholar 

  28. Baba, T. et al. Genome and virulence determinants of high virulence community-acquired MRSA. Lancet 359, 1819–1827 (2002).

    CAS  Article  Google Scholar 

  29. Wang, R. et al. Identification of novel cytolytic peptides as key virulence determinants for community-associated MRSA. Nat. Med. 13, 1510–1514 (2007).

    CAS  Article  Google Scholar 

  30. Pang, Y. Y. et al. agr-Dependent interactions of Staphylococcus aureus USA300 with human polymorphonuclear neutrophils. J. Innate Immun. 2, 546–559 (2010).

    CAS  Article  Google Scholar 

  31. Harding, M. & Kubes, P. Innate immunity in the vasculature: Interactions with pathogenic bacteria. Curr. Opin. Microbiol. 15, 85–91 (2012).

    CAS  Article  Google Scholar 

  32. McDonald, B. et al. Intravascular danger signals guide neutrophils to sites of sterile inflammation. Science 330, 362–366 (2010).

    ADS  CAS  Article  Google Scholar 

  33. Yipp, B. G. et al. Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo. Nat. Med. 18, 1386–1393 (2012).

    CAS  Article  Google Scholar 

  34. Zwick, R. K. et al. Adipocyte hypertrophy and lipid dynamics underlie mammary gland remodeling after lactation. Nat. Commun. 9, 3592 (2018).

    ADS  Article  Google Scholar 

  35. Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902.e1821 (2019).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank T. Nussbaumer for mice husbandry and genotyping; R. Gamutin for monitoring infected mice in the University of Calgary biohazard animal facility; P. Colarusso, A. Chojnacki and L. Swift at the University of Calgary Live Cell Imaging Facility for microscope usage and image analysis support; J. Zindel for providing the R scripts for image analysis; W. Y. Lee for microscope assistance and maintenance and assistance with experiments; H. Kuipers for use of the Cytek Aurora, which was funded through the Canadian Foundation for Innovation; P. Mukherjee and the Microscopy and Imaging Facility at the University of Calgary for scanning electron microscopy assistance; Y. Ou and S. Liu and the Alberta Precision Research Laboratories for assistance with histology; B. G. J. Surewaard for providing the S. aureus MW2 strain; and T. Kieffer for providing the Leprfl/fl mice. R.M.K. was supported by the Alberta Graduate Excellence Scholarship and the University of Calgary Doctoral Scholarship. H.B.S. and S.S. were supported by CIHR Vanier scholarships. This work was supported by foundation grants from the Canadian Institute of Health Research (FDN148380 to K.A.S. and FDN143248 to P.K.).

Author information

Authors and Affiliations

Authors

Contributions

R.M.K., J.F.D. and P.K. designed the experiments. R.M.K., H.B.S., R.S., W.Y.L., E.L., and C.M.K. performed the experiments. Specifically, R.M.K. performed all experiments, with the following exceptions: H.B.S. assisted with operating the confocal microscope (Extended Data Fig. 2i,j) and performed the whole-mount immunofluorescence (Extended Data Fig. 7g), R.S. assisted with mouse treatments and the monocyte sorting for adoptive transfer experiments (Fig. 2l–o), W.Y.L. performed bone marrow transfers (Fig. 4l–p) and assisted with mouse treatments; E.L. performed immunofluorescence and imaging of BODIPY-stained skin sections (Extended Data Fig. 7e) and C.M.K. performed immunofluorescence staining and imaging for Extended Data Fig. 7h. R.M.K. analysed all data except for the scRNA-seq dataset, which was analysed by S.S.. J.Y.N. and Y.S. provided ghrelin-deficient bones for bone marrow transfer. B.G.J.S. contributed technical and experimental support for the S. aureus infection model and provided critical review of the paper. K.A.S. provided expertise in immunofluorescence staining and critical review of the paper. M.M. provided the MC-21 monocyte-depleting antibody. J.B. supervised the single-cell sequencing analysis. R.M.K., J.F.D. and P.K. wrote the manuscript with input from all co-authors. All authors read and approved the manuscript for submission. J.F.D. and P.K. supervised the study.

Corresponding authors

Correspondence to Justin F. Deniset or Paul Kubes.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature thanks Vishwa Dixit, Miriam Merad, Victor Torres and the other, anonymous, reviewer for their contribution to the peer review of this work. Peer review reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of the S. aureus bead infection in vivo.

a–c, Literature review of infectious dose used to study S. aureus skin infections, arranged by low-dose to very high dose (a), absolute value for infectious dose for each type of infection (b), and proportion of papers that used planktonic vs foreign body-associated S. aureus (c). d, e, C57 mice were infected with low-dose S. aureus bead or 500 CFU in 50 μL saline (planktonic) and CFUs were measured over time indicated. d, Percentage of mice with an active infection with detectable CFUs. n = 10 mice (for 24 h, 72 h, 7 d) and n= 5 (for 21 d) mice per group from 2 independent experiments; two-sided Chi square test at individual time points; P= 0.0603 (24 h), P= 0.1213 (72 h), P= 0.0028 (7 d). e, Quantification of skin CFUs post-infection. n = 10 mice (for 24 h, 72 h, 7 d) and n= 5 (for 21 d) mice per group from 2 independent experiments; unpaired two-sided Student t-test at individual time points (24 h), unpaired two-sided Mann Whitney U-test between individual time points (72 h, 7 d); data are median; P = 0.6470 (24 h), P = 0.0178 (72 h), P = 0.0082 (7 d). f, C57 mice were infected with low-dose S. aureus bead or 500 CFU in 50 μL saline (planktonic) and skin infections were processed for scanning electron microscopy at 24 h. Representative image of bead and planktonic infection. Image representative from n= 3 mice per group from 2 independent experiments. g, C57 mice were infected with low-dose S. aureus bead or 500 CFU in 50 μL saline (planktonic) and CFUs were measured over time indicated. Quantification of CFUs in skin-draining lymph node, kidney, liver, spleen, and blood. n = 10 mice (for 24 h, 72 h, 7 d) and n= 5 (for 21 d) mice per group from 2 independent experiments; unpaired two-sided Mann Whitney U-test between individual time points; data are median; LN 72h: P = 0.0251, Kidney 72 h: P = 0.0325, all other groups P> 0.05. h, Representative image of naïve skin in wildtype mice, representative of n = 3 mice from 2 independent experiments. Scale bar, 100 μm. i, Representative image of a 24 h infection in wildtype mice infected with GFP-expressing MW2 S. aureus bead, representative of n =  3 mice from 2 independent experiments. Infection site is highlighted with a dashed line. Scale bar, 100 μm. j, k, C57 mice were infected with GFP-expressing S. aureus bead and flow cytometry was performed at 24 h. j, Gating strategy to identify immune cells positive for GFP+ S. aureus. k, Quantification of S. aureus GFP+ immune cells shown as frequency of CD45+ cells. n= 6 mice per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P< 0.0001. l, m, Cx3cr1GFP/+Catchupivm-red mice were infected with low-dose S. aureus bead and monocyte and neutrophil behaviour was imaged in vivo at 24 h post-infection. l, Representative spider plot showing track displacement length of CX3CR1gfp+ monocytes and tdTomato+ neutrophils at the site of infection over a 20-minute time-lapse video. Plots are representative of n =  4 mice per group from 2 independent experiments. m, Quantification of the average velocity of neutrophils and CX3CR1gfp+ cells. n= 4 mice per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0135.

Extended Data Fig. 2 Assessment of immune cell recruitment to S. aureus skin infection at 24 h post-infection.

a–f, Cx3cr1GFP/+Ccr2RFP/+ mice were infected with low (500 CFU/bead) or high (107 CFU/bead) dose S. aureus bead and flow cytometry was performed at 24 h. a, Gating strategy to identify immune cell populations. b, Representative flow cytometry plots and quantification of CD11b+ Ly6G+ neutrophils and CD11b+ Ly6G non-neutrophils at low and high dose bead. Quantification shown as frequency of Live, CD45+ cells. n= 5 mice (high dose) and n= 6 mice (low-dose) from 2 independent experiments; mixed-effects analysis with Šídák’s multiple comparisons test; data are mean ± s.e.m.; P= 0.2635 (low-dose CD11b+ LY6G+ vs low-dose CD11b+ LY6G), P< 0.0001 (high dose CD11b+ LY6G+ vs high dose CD11b+ LY6G), P = 0.0008 (CD11b+ LY6G+ low-dose vs CD11b+ LY6G+ high dose), P = 0.0455 (CD11b+ LY6G low-dose vs CD11b+ LY6G high dose). c, Representative histogram of CX3CR1gfp expression in CD11b+ LY6G+ neutrophils, CD11b+ LY6G non-neutrophils, and non-neutrophils from GFP FMO control. d, Representative flow cytometry plot showing expression of LY6C and CD64 gated on single cells/live/CD45+/CD11b+ LY6G/CX3CR1gfp+ cells, showing populations of GFP+ LY6Chi CD64low/+ monocytes and GFP+ LY6Clow CD64+ macrophages recruited to low-dose S. aureus bead. Quantification shown as frequency of CX3CR1gfp+ cells. n= 4 mice per group, data representative from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P< 0.0001. e, Representative flow cytometry plots showing expression of CX3CR1gfp+ cells gated on single cells/live/CD45+ cells. Quantification of total CX3CR1gfp+ cells shown as frequency of live, CD45+ cells. n= 4 mice per group, data representative from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0015. f, Quantification of GFP+ LY6Chi CD64low/+ monocytes shown as frequency of live/CD45+/CX3CR1gfp+ cells in high and low-dose S. aureus bead. unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.6772. g, h, C57 mice were infected with S. aureus bead, heat-killed S. aureus bead or sterile bead and flow cytometry was performed at 24 h. g, Experiment timeline. h, Quantification of total numbers of CX3CR1+ myeloid cells gated from single cells/live/CD45+/CD11b+/LY6G. n= 6 mice (S. aureus, sterile bead) and n= 7 mice (heat-killed S. aureus) from 2 independent experiments; one-way ANOVA with Tukey’s multiple comparisons test; data are mean ± s.e.m.; P < 0.0001. i, j, Cx3cr1GFP/+Ccr2RFP/+ mice were infected with S. aureus bead and imaged with spinning disk confocal intravital microscopy at 24 h, 72 h, and 7 d post-infection. i, Representative images showing monocyte heterogeneity during infection, representative of n = 4 mice (24 h) and 3 mice (72 h, 7 d) per group from 4 independent experiments. j, Quantification of the average intensity of GFP and RFP expression in monocytes. Each dot represents the average of 3 FOV from one mouse. n = 4 mice (24 h) and 3 mice (72 h, 7 d) per group from 4 independent experiments; mixed effects analysis with Šidák’s multiple comparisons test; data are mean ± s.e.m.; **P < 0.01, ***P < 0.001, ****P < 0.0001. k, l, Cx3cr1GFP/+Ccr2RFP/+ and Cx3cr1GFP/+Ccr2RFP/RFP mice were infected with S. aureus bead and flow cytometry was run at 24 h and 72 h. k, Gating strategy used to identify CX3CR1gfp+ monocyte populations. l, Quantification of total numbers of CX3CR1gfp+ LY6Chi CD64low/+ and CX3CR1gfp+ LY6Clow CD64hi monocytes/macrophages. n = 6 mice (24 h) and 3 mice (72 h) per group from 2 independent experiments; two-way ANOVA with Šidák’s multiple comparison; data are mean ± s.e.m.; For CX3CR1gfp+ LY6Chi CD64low/+: P= 0.0027 (Cx3cr1GFP/+Ccr2RFP/+ 24h vs Cx3cr1GFP/+Ccr2RFP/+ 72h), P = 0.9980 (Cx3cr1GFP/+Ccr2RFP/RFP 24 h vs Cx3cr1GFP/+Ccr2RFP/RFP 72h), P= 0.0004 (Cx3cr1GFP/+Ccr2RFP/+ 24 h vs Cx3cr1GFP/+Ccr2RFP/RFP 24 h), P= 0.9390 (Cx3cr1GFP/+Ccr2RFP/+ 72 h vs Cx3cr1GFP/+Ccr2RFP/RFP 72 h); For CX3CR1gfp+ Ly6Clow CD64hi: P= 0.0138 (Cx3cr1GFP/+Ccr2RFP/+ 24 h vs Cx3cr1GFP/+Ccr2RFP/+ 72 h), P = 0.9923 (Cx3cr1GFP/+Ccr2RFP/RFP 24 h vs Cx3cr1GFP/+Ccr2RFP/RFP 72 h), P= 0.3943 (Cx3cr1GFP/+Ccr2RFP/+ 24 h vs Cx3cr1GFP/+Ccr2RFP/RFP 24 h), P= 0.0042 (Cx3cr1GFP/+Ccr2RFP/RFP 72 h vs Cx3cr1GFP/+Ccr2RFP/RFP 72 h) .

Extended Data Fig. 3 Characterization of monocyte/macrophage populations at the S. aureus skin infection.

a–c, CCR2-creERT2;zsGreen+/− mice were treated with tamoxifen, infected with low-dose S. aureus bead, and imaged or processed for flow cytometry analysis at indicated time points. a, Experiment timeline for fate mapping with CCR2-creERT2;zsGreen+/− mice. b, Representative image of zsGreen+ cells recruited to the S. aureus skin infection at 24 h and 14 d post-infection. Infection site is highlighted with a dashed line. Image representative of n = 3 mice per group from 2 independent experiments. Scale bar, 100 μm. c, Quantification of zsGreen+ LY6Chi CD64low/+ monocytes and zsGreen+ LY6Clow CD64+ macrophages shown as frequency of zsGreen+ cells. n = 3 mice per group from 2 independent experiments, one-way ANOVA with Tukey’s multiple comparison test; data are mean ± s.e.m.; For zsGreen+ LY6Chi CD64low/+, P= 0.1570 (24 h vs 72 h), P= 0.1965 (24 h vs 14 d), P= 0.9829 (72 h vs 14 d); For zsGreen+ LY6Clow CD64+: P= 0.6387 (24 h vs 72 h), P= 0.2125 (24 h vs 14 d), P= 0.6115 (72 h vs 14 d). d–g, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with S. aureus bead and skin infections were processed for spectral flow cytometry analysis of monocyte/macrophage populations at indicated time points. d, Gating strategy used to identify immune cell populations isolated from skin infections. e, Pie chart representing the proportion of monocyte subsets in Ccr2RFP/+ mice shown as frequency of LY6G CD11b+ myeloid cells. f, Quantification of total numbers of P1-P5 monocyte/macrophage subsets in Ccr2RFP/+ and Ccr2RFP/RFP mice. n = 4 mice (Ccr2RFP/+: naïve, 72 h, 14 d; Ccr2RFP/RFP:  14 d) and 3 mice (Ccr2RFP/+: 24 h; Ccr2RFP/RFP: naïve, 24 h, 72 h) per group from 3 independent experiments; mixed effects analysis with Šidák’s multiple comparisons test; data are mean ± s.e.m.; For P1: P= 0.0018 (Ccr2RFP/+ 24 h vs Ccr2RFP/RFP 24 h); For P2: P= 0.0240 (Ccr2RFP/+ 24 h vs Ccr2RFP/RFP 24 h); P= 0.0017 (Ccr2RFP/+ 72 h vs Ccr2RFP/RFP 72 h); For P3: P= 0.0010 (Ccr2RFP/+ 72 h vs Ccr2RFP/RFP 72 h); For P5: P= 0.0486 (Ccr2RFP/+ 72 h vs Ccr2RFP/RFP 72 h); All other groups P> 0.05. g, Quantification of total numbers immune cell populations in Ccr2RFP/+ and Ccr2RFP/RFP mice. For quantification of neutrophils, CD11b+ dendritic cells, CD11b- non-myeloid cells: n= 4 mice (Ccr2RFP/+: naïve), 3 mice (Ccr2RFP/RFP: naïve), 9 mice (Ccr2RFP/+: 24 h), 8 mice (Ccr2RFP/RFP: 24 h), 7 mice (Ccr2RFP/+: 72 h), 6 mice (Ccr2RFP/RFP: 72 h), 9 mice (Ccr2RFP/+: 14 d) and 11 mice (Ccr2RFP/RFP: 14 d) per group from 5 independent experiments. For quantification of CD4+ T cells, CD8+ T cells, λδ T cells, B cells, NK cells and Lin cells: n = 4 mice (Ccr2RFP/+: naïve, 72 h, 14 d; Ccr2RFP/RFP: 14 d) and 3 mice (Ccr2RFP/+: 24 h; Ccr2RFP/RFP: naïve, 24 h, 72 h) per group from 3 independent experiments. mixed effects analysis with Šidák’s multiple comparisons test; data are mean ± s.e.m.; P> 0.05 (for all groups at all time points for Ccr2RFP/+ vs Ccr2RFP/RFP).

Extended Data Fig. 4 Neutrophil depletion results in significant CFU burden in skin at 72 h post-infection.

a–c, C57/BL6 mice were treated with neutrophil depleting antibody (LY6G 1A8) or isotype control antibody (2A3) and infected with low-dose S. aureus bead. a, Quantification of bacterial CFUs from skin at 24 and 72 h. n = 5 mice per time point from 2 independent experiments; two-way ANOVA with Šídák’s multiple comparisons test; data are mean ± s.e.m.; P = 0.9335 (24 h 2A3 vs 24 h 1A8), P= 0.0038 (72 h 2A3 vs 72 h 1A8), P = 0.6611 (24 h 2A3 vs 72 h 2A3), P= 0.0076 (24 h 1A8 vs 72 h 1A8). b, Quantification of abscess size at 24 and 72 h. n = 5 mice per time point from 2 independent experiments; two-way ANOVA with Šídák’s multiple comparisons test; data are mean ± s.e.m.; P = 0.9831 (24 h 2A3 vs 24 h 1A8), P= 0.0062 (72 h 2A3 vs 72 h 1A8), P = 0.8751 (24 h 2A3 vs 72 h 2A3), P= 0.0043 (24 h 1A8 vs 72 h 1A8). c, Representative image of a 72 h infection in isotype or 1A8-treated mouse.

Extended Data Fig. 5 A tattoo identifies the infection region over 30 days.

a–d, Mice were tattooed with green tattoo ink prior to infection with low-dose S. aureus bead. a, Representative photo of a mouse with tattoo prior to infection with S. aureus bead (left) and immediately after injection of the S. aureus bead (right). b, Diagram showing placement of tattoo around S. aureus infection/wound. c, Representative intravital image of autofluorescent tattoo ink in skin. Scale bar, 100 μm. d, Representative photos of the tattoo points visible in mouse skin at 7 d, 14 d and 30 d post-infection. e–g, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with low-dose S. aureus bead and imaged at 14 days post-infection. e, Quantification of wound size. n = 8 for Ccr2RFP/+ and n = 13 for Ccr2RFP/RFP mice from 6 independent experiments. f, Representative photos of a wound from Ccr2RFP/+ and Ccr2RFP/RFP mice at 14 days post-infection. Black arrow points to wound. g, Representative intravital images showing 3D projection of vasculature in remote regions of skin at 14 days post-infection in Ccr2RFP/+ (top) and Ccr2RFP/RFP (bottom) mice. Scale bar, 200 μm. Images representative of n = 7 for Ccr2RFP/+ and n = 12 for Ccr2RFP/RFP mice from 6 independent experiments. h, Representative photos of a healed infection from a Ccr2RFP/+ mouse and a wound present from a Ccr2RFP/RFP mouse at 30 days post-infection. Green arrows point to tattoo markings in skin. i–k, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with low-dose S. aureus bead and imaged at 90 days post-infection. Quantification of wound presence (i), vasculature (j), and wound size (k) at 90 days post-infection in Ccr2RFP/+ and Ccr2RFP/RFP mice. For j–k, n = 12 mice (Ccr2RFP/+) and n = 13 mice (Ccr2RFP/RFP) from 2 independent experiments; data are mean ± s.e.m.

Extended Data Fig. 6 Measurement of cytokines by ELISA.

a–e, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with S. aureus bead and skin homogenates were analyzed for multiplex ELISA at indicated time points. a, b, Multiplex ELISA of matrix metalloproteinases MMP-2, MMP-3, MMP8, proMMP-9 and MMP-12 (a), growth factors and repair mediators Angiopoietin-2, EGF, FGF-2, HGF, PLGF-2, SDF-1 and VEGF-A (b) from skin homogenates of Ccr2RFP/+ and Ccr2RFP/RFP mice at 7- and 14-days post-infection. For a–b, n = 4 mice per group from 2 independent experiments; data are mean ± s.e.m.; two-way ANOVA with Šídák’s multiple comparisons test; P> 0.05 (Ccr2RFP/+ vs Ccr2RFP/RFP at all time points for all mediators). c, Measurement of Leptin from naïve skin of Ccr2RFP/+ and Ccr2RFP/RFP mice. n = 4 per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P = 0.1769. d, Measurement of pro- and anti-inflammatory cytokines from skin homogenates of Ccr2RFP/+ and Ccr2RFP/RFP mice at 7- and 14-days post-infection. n = 6 mice (Ccr2RFP/+, 7 d and 14 d), n = 3 mice (Ccr2RFP/RFP 7 d), and n= 5 mice (Ccr2RFP/RFP 14 d) from 2 independent experiments; Mixed effects model with Šídák’s multiple comparisons test; data are mean ± s.e.m.; P> 0.05 (Ccr2RFP/+ vs Ccr2RFP/RFP at all time points for all mediators).

Extended Data Fig. 7 Dynamic hypodermal expansion and resolution during S. aureus skin infection in wildtype mice.

a, b, C57 mice were infected with low-dose S. aureus bead and skin infections were processed for H&E staining at indicated time points. a, Representative H&E staining in skin infections. Infection and hypodermis are identified with a label. Scale bar, 100 μm. b, Quantification of hypodermis thickness in C57 mice after infection with S. aureus bead. n = 7-8 measurements for each mouse, 3 mice per group, 2 independent experiments; one-way ANOVA with Tukey’s multiple comparisons test; data are mean ± s.e.m.; P< 0.0001 (for 24 h vs. naïve, 24 h vs 72 h, 24 h vs 7 d, 24 h vs 14 d); P> 0.05 (all other groups). c, d, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with low-dose S. aureus bead and skin infections were processed for H&E staining at indicated time points. c, Representative H&E images of hypodermal adipocyte size in Ccr2RFP/+ and Ccr2RFP/RFP mice at 72 h post-infection. Scale bar, 30 μm. d, Quantification of hypodermis thickness in naïve C57 mice versus remote regions of the skin of Ccr2RFP/+ and Ccr2RFP/RFP mice at 24 h post-infection. n= 16 measurements from 4 mice per group from 2 independent experiments; one-way ANOVA with Tukey’s multiple comparisons test; data are mean ± s.e.m.; P> 0.05 (all groups). e, f, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with low-dose S. aureus bead and skin infections were processed for BODIPY staining by immunofluorescence. e, Representative confocal immunofluorescence of skin sections showing BODIPY+ adipocytes and lipid droplets in Ccr2RFP/+ and Ccr2RFP/RFP mice at 14 days post-infection with S. aureus bead. Scale bar, 300 μm. f, Quantification of total BODIPY+ signal in the wounds of Ccr2RFP/+ and Ccr2RFP/RFP mice at 14 days post-infection. n = 6 mice (Ccr2RFP/+: 7 d, 14 d), 5 mice (Ccr2RFP/RFP: 7 d) and 4 mice (Ccr2RFP/RFP: 14 d) per group from 2 independent experiments; data are mean ± s.e.m.; P= 0.9688 (Ccr2RFP/+ 7 d vs Ccr2RFP/+ 14 d), P= 0.90357 (Ccr2RFP/RFP 7 d vs Ccr2RFP/RFP 14 d), P= 0.9097 (Ccr2RFP/+ 7 d vs Ccr2RFP/RFP 7 d), P= 0.0102 (Ccr2RFP/+ 14 d vs Ccr2RFP/RFP 14 d). g, Ccr2RFP/+ and Ccr2RFP/RFP mice were infected with low-dose S. aureus bead and skin infections were processed at 14 d for whole mount immunofluorescent staining for leptin and LipidTOX. Representative whole mount images of 14-day wounds from Ccr2RFP/+ and Ccr2RFP/RFP mice. Scale bars, 500 μm (left) and 50 μm (inset). Image representative of n= 3 mice per group from 2 independent experiments. h, Representative immunofluorescence staining for perilipin-1 and leptin in Ccr2RFP/RFP skin at 14 days post-infection. Scale bars, 50 μm. Image representative of n= 3 mice per group from 2 independent experiments.

Extended Data Fig. 8 Leptin stimulates angiogenesis following S. aureus skin infection.

a–d, Ccr2RFP/+ mice were infected with low-dose S. aureus bead, treated with vehicle or SMLA, and imaged at 14 days post-infection. a, Experiment timeline showing SMLA treatment to Ccr2RFP/+ mice. b, Quantification of wound presence. c, Quantification of vasculature volume. d, Quantification of wound size at 14 d. For (b-d), n = 8 (vehicle) and n= 7 (SMLA) per group from 2 independent experiments; For (c-d), data are mean ± s.e.m. e–i, Ccr2RFP/+ mice were infected with low-dose S. aureus bead, treated with vehicle or leptin, and imaged at 14 days post-infection. e, Experiment timeline showing leptin treatment to Ccr2RFP/+ mice. f, Representative images showing effect of leptin treatment on blood vasculature in Ccr2RFP/+ mice at 14 d post-infection. Top: 2D stitch of wound, bottom: 3D projection of vasculature. Scale bars, 300 μm (top) and 100 μm (bottom). Images representative of n = 8 mice (vehicle) and 9 mice (leptin) per group from 2 independent experiments. g, Quantification of blood vasculature. n = 4 per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0310. h, Quantification of wound presence. n = 8 mice (vehicle) and 9 mice (leptin) per group from 2 independent experiments. i, Quantification of wound size. n = 4 per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0571. j–m, Ccr2RFP/RFP mice were infected with low-dose S. aureus bead, treated with vehicle or leptin, and imaged at 14 days post-infection. j, Experiment timeline for leptin administration to Ccr2RFP/RFP mice infected with S. aureus bead. k, Quantification of wound presence. l, Quantification of vasculature volume. Data are mean ± s.e.m. m, Quantification of wound size. Data are mean ± s.e.m. For (k–m), n= 6 mice (vehicle) and n= 4 mice (leptin) from 2 independent experiments.

Extended Data Fig. 9 Leptin-LepR signaling drives vasculature formation during S. aureus skin infection.

a, Uniform Manifold Approximation and Projection (UMAP) of single cells isolated from murine back skin. b–e, UMAP showing expression of Pecam1 (b), Lyve1 (c), Flt1 (d) and LepR (e). f, Violin plot showing Lepr expression. g–k, Tamoxifen-treated LepRfl/flCdh5(PAC)-CreERT2 and LepRfl/fl control mice were treated with MC-21, infected with S. aureus bead, and imaged at 14 d post-infection. g, Experiment timeline for tamoxifen treatment, MC-21 administration, infection, and imaging. h, Representative images at 14 d post-infection. Top: 2D stitch of entire wound, bottom: 3D projection of vasculature. Scale bars, 200 μm. Images representative of n = 7 per group from 2 independent experiments. i, Quantification of wound presence. n = 7 per group from 2 independent experiments. j, Quantification of vasculature volume. n = 7 per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0180. k, Quantification of wound size. n = 7 per group from 2 independent experiments; unpaired two-sided Student t-test; data are mean ± s.e.m.; P= 0.0425. l, Quantification of wound size at 14 d post-infection in Ccr2RFP/RFP mice after vehicle or ghrelin treatment. n= 6 (vehicle) and n= 9 (ghrelin) from 2 independent experiments. Data are mean ± s.e.m. m–p, Ccr2RFP/+ mice were infected with low-dose S. aureus bead, treated with vehicle or ghrelin, and imaged at 14 days post-infection. m, Experiment timeline showing ghrelin treatment to Ccr2RFP/+ mice. n, Quantification of wound presence. n = 10 per group from 2 independent experiments; two-sided Chi-squared test; P= 0.0191. o, Quantification of vasculature volume. n= 9 mice (vehicle) and n= 4 mice (ghrelin) from 2 independent experiments; data are mean ± s.e.m. p, Quantification of wound size. n = 10 per group from 2 independent experiments; data are mean ± s.e.m.

Extended Data Fig. 10 Model for the role of monocytes during S. aureus skin infection.

Monocytes are recruited in a CCR2-dependent manner to a low-dose S. aureus skin infection to regulate tissue by limiting leptin-mediated angiogenesis through local production of ghrelin. In CCR2-deficient mice, or after anti-CCR2 monocyte depletion, there is no monocyte recruitment to the skin infection, which results in pathologic angiogenesis driven by leptin and delayed healing. Monocytes function as a cellular rheostat by regulating leptin levels and revascularization during tissue repair post-infection.

Supplementary information

Supplementary Table 1

Antibodies used in the study.

Reporting Summary

Peer Review File

Supplementary Video 1

Neutrophil and monocyte behaviour at the S. aureus infection site. Cx3CR1GFP/+ CatchupIVM-red mice were infected with S. aureus bead were imaged at the infection site to capture immune cell behaviour.

Supplementary Video 2

Imaging blood vasculature at 14 days post infection. Cx3cr1GFP/+Ccr2RFP/+ and Cx3cr1GFP/+Ccr2RFP/RFP mice infected with S. aureus bead were imaged at 14 days post infection. A fluorescent vascular dye was injected i.v. to visualize the blood vessels inside the wound.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kratofil, R.M., Shim, H.B., Shim, R. et al. A monocyte–leptin–angiogenesis pathway critical for repair post-infection. Nature 609, 166–173 (2022). https://doi.org/10.1038/s41586-022-05044-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-022-05044-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing