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Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins

Nature Microbiology volume 7, pages 62–72 (2022)Cite this article

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Abstract

Swift recruitment of phagocytic leucocytes is critical in preventing infection when bacteria breach through the protective layers of the skin. According to canonical models, this occurs via an indirect process that is initiated by contact of bacteria with resident skin cells and which is independent of the pathogenic potential of the invader. Here we describe a more rapid mechanism of leucocyte recruitment to the site of intrusion of the important skin pathogen Staphylococcus aureus that is based on direct recognition of specific bacterial toxins, the phenol-soluble modulins (PSMs), by circulating leucocytes. We used a combination of intravital imaging, ear infection and skin abscess models, and in vitro gene expression studies to demonstrate that this early recruitment was dependent on the transcription factor EGR1 and contributed to the prevention of infection. Our findings refine the classical notion of the non-specific and resident cell-dependent character of the innate immune response to bacterial infection by demonstrating a pathogen-specific high-alert mechanism involving direct recruitment of immune effector cells by secreted bacterial products.

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Fig. 1: S. aureus PSM toxins are critical to the early leucocyte influx to the site of skin infection: experiments with bacteria.
Fig. 2: Genome-wide gene expression experiments reveal EGR1 as the main mediator of the early human neutrophil response to S. aureus.
Fig. 3: PSMs trigger EGR1 transcription via the MEK/ERK pathway.
Fig. 4: Leucocyte attraction via the FPR2–EGR1 pathway is direct and independent of resident skin cells.
Fig. 5: EGR1 signalling has a significant impact on S. aureus skin infection.

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Data availability

Microarray data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE103779. All other data generated or analysed during this study are included in this paper or in the Supplementary Information. Source data are provided with this paper.

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Acknowledgements

This study was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID) and the National Cancer Institute (NCI), US National Institutes of Health (NIH), project numbers ZIA AI000904 (M.O.), ZIA AI001079 (F.R.D.), ZIA AI001171 (D.L.B.), ZIA BC010725 (J.M.W.), and by federal funds from the NCI (contract no. HSN261200800001E, to J.M.W.). F.M.F.A. received a scholarship from Shaqra University, Al Quwaiiyah, Saudi Arabia. We thank K. v. Kessel, University of Utrecht, for supplying FLIPr.

Author information

Author notes

  1. Thuan H. Nguyen

    Present address: University of Maryland School of Medicine, Baltimore, MD, USA

  2. Kevin M. Rigby

    Present address: miRagen Therapeutics, Inc., Boulder, CO, USA

  3. Saba Firdous

    Present address: Chlamydia Pathogenesis Section, NIAID, Bethesda, MD, USA

  4. Janice Chiou

    Present address: Graduate School in Biomedical Science, Cedars Sinai Medical Center, Los Angeles, CA, USA

  5. Matthew D. Park

    Present address: Icahn School of Medicine at Mount Sinai, New York, NY, USA

  6. Rachelle L. Hunt

    Present address: Microbial Pathogenesis Department, Yale University, New Haven, CT, USA

  7. Fawaz M. F. Almufarriji

    Present address: School of Molecular and Cell Biology, University of Leeds, Leeds, UK

  8. Vee Y. Tan

    Present address: Tuberculosis Research Section, NIAID, Bethesda, MD, USA

  9. Joshua W. McCausland

    Present address: Johns Hopkins University, Baltimore, MD, USA

  10. Emilie L. Fisher

    Present address: Vanderbilt University, Nashville, TN, USA

  11. Anthony J. Yeh

    Present address: William Carey University College of Osteopathic Medicine, Hattiesburg, MS, USA

  12. Justin S. Bae

    Present address: Harvard University, Cambridge, MA, USA

  13. These authors contributed equally: Thuan H. Nguyen, Gordon Y. C. Cheung.

Authors and Affiliations

  1. Pathogen Molecular Genetics Section, Laboratory of Bacteriology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Thuan H. Nguyen, Gordon Y. C. Cheung, Kevin M. Rigby, Amer E. Villaruz, Ryan Liu, Pipat Piewngam, Saba Firdous, Janice Chiou, Matthew D. Park, Rachelle L. Hunt, Fawaz M. F. Almufarriji, Vee Y. Tan, Titus K. Asiamah, Joshua W. McCausland, Emilie L. Fisher, Anthony J. Yeh, Justin S. Bae & Michael Otto

  2. Pathogen–Host Cell Biology Section, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Kevin M. Rigby, Adeline R. Porter, Scott D. Kobayashi & Frank R. DeLeo

  3. Biological Imaging Section, Research Technologies Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Olena Kamenyeva & Juraj Kabat

  4. Genomics Unit, Research Technology Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA

    Daniel E. Sturdevant

  5. Laboratory of Cancer and Immunometabolism, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA

    Ji Ming Wang

  6. T-Lymphocyte Biology Unit, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

    Daniel L. Barber

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  1. Thuan H. Nguyen
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Contributions

M.O. conceptualized the study. D.L.B. and T.H.N. assisted in planning the leucocyte influx experiments, and F.R.D., the microarray experiments. M.O. and F.R.D. supervised experiments. G.Y.C.C. and T.H.N. designed and set up, and G.Y.C.C., T.H.N., R.L., J.S.B., P.P., S.F., J.C., M.D.P., R.L.H., F.M.F.A., V.Y.T., T.K.A., J.W.M., E.L.F. and A.J.Y. performed the leucocyte influx and intravital imaging experiments. O.K. performed confocal microscopy. J.K., O.K., G.Y.C.C. and T.H.N. analysed imaging data. K.M.R., A.R.P. and S.D.K. performed neutrophil array and corresponding control experiments. D.E.S. analysed array data. T.H.N. and A.E.V. performed RT-qPCR, and S.F. and T.H.N., pathway analysis. J.M.W. supplied FPR2−/− mouse breeding pairs. M.O. wrote the paper.

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Correspondence to Michael Otto.

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Peer review information Nature Microbiology thanks Mia Phillipson and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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

Extended data

Extended Data Fig. 1 Bacterial numbers and psm gene expression during early ear infection.

a, CFU in ears infected with wild-type LAC or Δpsm S. aureus, and with PBS as control to analyze for contaminating skin bacteria. n = 3/group. b, Expression of the psmα locus by S. aureus LAC bacteria during early ear and back skin infection. n = 4/group and time point. a,b, Error bars show the mean ± SD.

Source data

Extended Data Fig. 2 S. aureus PSM toxins are critical to the early leukocyte influx to the site of skin infection – Experiments with pure peptides.

a, Neutrophil influx measured after injection of 3 μl of a 1 μM solution of PSMα3 peptide in water over 6 h p. i. (n = 3), and influx of total leukocytes, neutrophils, and non-neutrophil leukocytes 5 h p. i. after injection of 3 μl of a 1 μM solution of PSMα3 or PSMα3K12A, or 3 μl water as control. n = 7/group (peptides); n = 4/group (water control). Statistical analysis is by 1-way ANOVA with Dunnett’s post-test versus the data obtained at 1 h p. i. (left panel) or Tukey’s post-tests (other panels). Error bars show the mean ± SD. b, Selected representative images. Green, leukocytes; red, blood vessels.

Source data

Extended Data Fig. 3 Establishment of sublytic and pro-inflammatory culture filtrate dilutions or pure PSMs and impact of FLIPr.

a, Test of S. aureus culture filtrate dilutions and PSMα3 and δ-toxin concentrations on cytolytic activity toward human neutrophils by release of LDH. n = 3/group. Error bars show the mean ± SD. b-d, Test by flow cytometry of the impact of FLIPr (green line) on blocking of induced pro-inflammatory effect in human neutrophils (expression of CD11b) by different concentrations of PSMα3 or δ-toxin. FLIPr was tested at concentrations between 0.5 and 10 μg/ml. Shown are the 0.5 μg/ml results for PSMα3 and the 1.0 μg/ml results for δ-toxin. Tests were performed ranging from n = 1 to n = 6 at concentrations close to the range of interest. A gate was set during data analysis to exclude dead cells and debris (as indicated in panel d). Error bars show the mean ± SD.

Source data

Extended Data Fig. 4 Verification of differential EGR1 expression in selected comparisons by RT-qPCR.

a, Comparison of the impact of diluted culture filtrates of S. aureus wild-type (WT) versus Δpsm and Δpsmα mutants on EGR1 expression in human neutrophils. Statistical analysis is by 1-way ANOVA with Tukey’s post-test. n = 14. b, Impact of FLIPr on stimulation by S. aureus WT culture filtrate. n = 5. c, Impact of FLIPr on stimulation by PSMα3. n = 3. b,c, Statistical analysis is by two-tailed unpaired Student’s t-test. a-c, Error bars show the mean ± SD.

Source data

Extended Data Fig. 5 Determination of leukocyte infiltration into spleens for normalization in the adoptive transfer experiment.

a-c, Exemplary confocal pictures of spleens. Leukocytes were labeled with different dyes and visualized as follows: leukocytes from wild-type mice in magenta, from FPR2-/- mice in green, and from EGR1-/- mice in blue. Note that computation and analysis was performed in a 3D manner; the pictures only show 2D slices. The exemplary pictures shown here are from the same mice as those in Extended Data Fig. 6.

Extended Data Fig. 6 Leukocyte attraction via the FPR2-EGR1 pathway is direct and independent of resident skin cells – exemplary confocal microscopy pictures.

a, The pictures show the unprocessed images and the two steps of processing: masking of hairs and ROI determination (see methods). Leukocytes are labeled with different dyes: leukocytes from wild-type mice in magenta, from FPR2-/- mice in green, and from EGR1-/- mice in blue. Dye-labeled bacteria are in cyan. Central, non-shaded areas represent the analyzed, computed regions of interest (ROIs). Note that computation and analysis was performed in a 3D manner; the pictures only show 2D slices. b, Processed WT mouse picture in higher magnification centered on the injected bacteria. The exemplary pictures shown here are from the same mice as those in Extended Data Fig. 5 showing spleen controls.

Extended Data Fig. 7 Leukocyte attraction via the FPR2-EGR1 pathway is direct and independent of resident skin cells – FPR2-/- and EGR1-/- comparisons.

Shown are the comparisons with FPR2-/- and EGR1-/- recipient mice versus FPR2-/- and EGR1-/- donor leukocyte comparisons, in analogy to the comparisons with wild-type recipient mice versus wild-type donor leukocytes shown in Fig. 4c,d,f,g. Statistical analysis is by repeated measures ANOVA with Dunnett’s post-test versus WT (wild-type). Error bars show the mean ± SD. n = 4-6. For the comparisons with mixed group numbers, a mixed model (rather than ANOVA) was automatically employed by Prism for data analysis.

Source data

Extended Data Fig. 8 Activation of mouse neutrophils by fMLP.

Neutrophil activation was assessed by determination of Ca2+ flux. 1 × 105 neutrophils isolated from wild-type, EGR1-/-, and FPR2-/- mice were stimulated with fMLP in DMSO at the indicated concentrations. DMSO controls are shown. Error bars show the mean ± SD. n = 8/group.

Source data

Supplementary information

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Numerical source data and associated statistical analysis.

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Numerical source data and associated statistical analysis.

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Numerical source data and associated statistical analysis.

Source Data Extended Data Fig. 8

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Nguyen, T.H., Cheung, G.Y.C., Rigby, K.M. et al. Rapid pathogen-specific recruitment of immune effector cells in the skin by secreted toxins. Nat Microbiol 7, 62–72 (2022). https://doi.org/10.1038/s41564-021-01012-9

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