Ethics statement

Mice were maintained according to National Institutes of Health guidelines for the use of experimental animals with the approval of Vanderbilt University Medical Center (protocol #M1600215) and Indiana University (#10500) Committees for the Use and Care of Animals. Experiments were performed following the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and the US Animal Welfare Act.

Animals

C57BL/6J breeding pairs were initially obtained from the Jackson Laboratory and maintained by breeding at Vanderbilt University Medical Center (VUMC), Nashville, TN, USA. MMDTR or Csfr1^{LsL-DTR-mCherry}_LysM^Cre mice were generated by breeding the Csf1r-HBEGF/mCherry) 1Mnz/J plus LysM^cre/cre mice as previously reported [37] and allowed for the detection of monocytes/macrophages by mCherry fluorescence. EGFP-LysM mice were donated by Dr. Nadia Carlesso (City of Hope, Duarte, CA, USA). Catchup^IVM-red mice [38] were donated by Dr. Matthias Gunzer (Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg-Essen, Essen, Germany). Wild-type BALB/c and IFNAR -/- mice were a gift from Dr. Stokes Peebles (Vanderbilt University Medical Center- VUMC). C57BL/6 Socs1^fl mice were obtained from Warren Alexander (Walter and Eliza Hall Institute, Parkville, Victoria, Australia) [39], and this strain was crossed with LysM^cre/cre mice (Jackson Laboratory) to create SOCS1^Δmyel mice. C57BL/6 Socs1^fl littermates were used as controls.

Induction of hyperglycemia

For streptozotocin (STZ)-induced hyperglycemia, 6- to 8-week-old male C57BL/6J mice were treated by i.p. injection with 40 mg/kg of STZ (Adipogen) dissolved in 0.1 M sodium citrate buffer once daily for 5 consecutive days [40]. Euglycemic control mice received on the vehicle control, citrate buffer. Mice were considered hyperglycemic when blood glucose levels were >250 mg/dl. Mice were treated with STZ to induce hyperglycemia 30 days before MRSA skin infection.

S. aureus strains

The MRSA USA300 LAC strain was a gift from Bethany Moore (University of Michigan, Ann Arbor, Michigan, USA [41]. The bioluminescent USA300 (NRS384 lux) strain was a gift from Roger Plaut (Food and Drug Administration, Silver Spring, Maryland, USA [42]). The methicillin-susceptible S. aureus (MSSA) Newman strain was obtained from Eric Skaar (Vanderbilt University Medical Center, Nashville, TN). The GFP-expressing USA300 LAC strain was a gift from William Nauseef (University of Iowa, Iowa City, Iowa) [43]. MRSA stocks were stored at –80°C. MRSA was cultured as previously described [44].

MRSA skin infection and treatments

The murine skin infection model was adapted from a previous study [44]. Male mice between 6 and 12 weeks of age were used for MRSA skin infection. Mice were infected with approximately 3 × 10⁶ MRSA, and biopsies and sample collection were taken at various times, ranging from 6 hours to 9 days after infection, as previously described [26,44]. Lesion size was measured daily via caliper, and the affected area was calculated using the standard equation for the area (length x width) [45]. The iKIR (DTHFRTFRSHSDYRR) and scrambled-KIR peptide control (DTHFARTFARSHSDYRRI) were obtained from GenScript [28]. A lipophilic palmitoyl group was added to the N-terminus of both sequences to facilitate cell penetration [31]. Mice were treated intraperitoneally with 50 μg of either the iKIR peptide or the scrambled peptide control [28] 1 hour prior to infection and once-daily following infection. Mice were injected intraperitoneally with 40mg/kg of the anti-IFNGR or IFNAR antibody 3 hours before infection, followed by infection for 24 hours.

Skin biopsy collection and determination of in vivo bacterial load

8 mm punch biopsies were collected from naive and infected skin at different time points post-infection and used to determine bacterial counts, histological analysis, cytokine production, mRNA expression, and flow cytometry analysis [26]. For bacterial counts, skin biopsy samples were collected, weighed, processed, and homogenized in tryptic soy broth (TSB) media. Serial dilutions were plated on tryptic soy agar. Colony-forming units (CFUs) were counted after incubation overnight at 37°C and corrected for tissue weight. Results are presented as CFU/g tissue.

Histopathology analysis

For histological analysis, 8 μm skin sections were stained with Hematoxylin and eosin or gram staining to visualize bacteria in the infected skin, as we have previously shown [26]. Images of tissue sections were visualized and acquired using the Nikon Eclipse Ci and Nikon Ds-Qi2 (Nikon, Tokyo, Japan).

In vivo bioluminescence imaging (BLI) and analysis with IVIS

An IVIS Spectrum/CT (Perkin Elmer) in vivo optical instrument was used to image bacterial bioluminescence and phagocyte fluorescence in the mice. Bioluminescence and fluorescent imaging and analysis were performed as previously described [46].

Skin single cell isolation and staining for flow cytometry

Skin biopsy specimens were collected and minced before digestion in 1mL of DMEM with 1 mg/ mL collagenase D (Roche Diagnostics) for 3 hours at 37°C. Single-cell suspensions were treated with CD16/32 Fc blocking antibodies (Biolegend; catalog 101310; clone 93) to prevent non-specific antibody binding and stained with the fluorescent-labeled antibodies for 20 minutes followed by fixation using 1% paraformaldehyde. The following antibodies were utilized: F4/80-FITC (Biolegend; catalog 123107; clone BM8), Ly6G-PerCP/Cy5.5 (Biolegend; catalog 127616; clone 1A8) Ly6C-AF647 (Biolegend; catalog 128010; clone HK14), CD11b-PE/Cy7 (Biolegend; catalog 101216; clone M1/70). Analyses were completed using FlowJo software (FlowJo, Ashland, OR).

Detection of cytokines and chemokines

Biopsy samples were collected, weighed, and homogenized in TNE cell lysis buffer containing phosphatase and protease inhibitors and centrifuged to remove cellular debris. Skin biopsy homogenates were then analyzed using the pro-inflammatory-focused 18-plex Discovery Assay from Eve Technologies (Eve Technologies, Calgary, AB) to detect cytokines and chemokines. Levels of IFNγ, IFNα, and IFNβ were measured the time points indicated in the legends by ELISA, (IFNγ -Invitrogen #88–7314) (IFNα-Biolegend #439407) (IFNβ-PBL #42120–1). Protein concentration was corrected for tissue weight.

Detection of reactive oxygen and nitrogen species

In vivo and in vitro nitrate/nitrite levels were measured using a modified Griess assay (Sigma Aldrich #G4410) following manufacturers’ protocol. In vivo hydrogen peroxide (H₂O₂) production was measured with the Amplex Red Assay (ThermoFisher #A2218) as we have previously shown [47].

Immunoblotting

Western blots were performed as previously described [29]. Protein samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with commercially available primary antibodies against SOCS-1, total-STAT-1, phosphorylated STAT-1 (Y701), total- and phosphorylated STAT-3 (S727) (all at 1:1000; Cell signaling), or β-actin (1:10,000; Invitrogen). Membranes were then washed and incubated with appropriate fluorophore-conjugated secondary antibodies (1:10,000, anti-rabbit IgG, IRDye 800CW antibody, #926–32211, Licor). Relative band intensities were quantified using ImageJ software (NIH), as previously described [29].

RNA isolation and quantitative real-time PCR

Skin biopsies samples were collected, and total RNA was isolated using lysis buffer (Buffer RLT; QIAGEN) following the manufacturer’s protocol. The RT² First Strand Kit reverse transcription system (QIAGEN) was used for cDNA synthesis, and quantitative PCR (qPCR) was performed on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories). Relative gene expression was calculated using the comparative threshold cycle (C_t) and expressed relative to control or WT groups beta actin (ΔΔCt method). Primers for β-actin and Socs1 were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

Nanostring and gene enrichment analysis

Global gene expression in infected skin biopsies from wild-type hyperglycemic and euglycemic mice was assessed by the NanoString nCounter gene expression system (Nanostring, Seattle, WA). Mouse Myeloid Innate Immunity panel for 770 genes in 19 different pathways was used for the analysis. Designed CodeSet underwent extensive quality control to avoid cross-hybridization to non-target molecules in samples. RNA was extracted from the infected skin using Trizol. The purity and concentration of the RNA were confirmed spectrophotometrically with a Nanodrop (Thermo Scientific, Waltham, MA). 100 ng of RNA (20ng/μl) was hybridized with probe CodeSet before running samples on NanoString. nSolver 3.0 software used to assess the quality of the run, followed by the deletion of the low-quality sample from further analysis. Two-sided hypergeometric statistical analysis was performed with the Kappa Score threshold setting of 0.3. Enrichment depletion was calculated based on Benjamini-Hochberg Correlated p-values of <0.05.

Bone marrow-derived macrophage generation

Bone marrow cells were flushed from both tibias and femurs of mice with ice-cold PBS. Cells were centrifuged, and red blood cells were lysed using ACK buffer. Cells were adjusted to 1x10⁶ cells/mL in DMEM with FBS (5%), M-CSF (20ng/mL), and GM-CSF (10ng/mL) and maintained at 37°C with 5% CO_2, and the cell culture media containing M-CSF and GM-CSF was changed every 3 days until day 7 when the cell was fully differentiated.

Phagocytosis and killing assays

Bacterial phagocytosis and killing were performed as previously shown [26,28,29,47]. Briefly, BMDMs (2 × 10⁵/well) were plated into two individual 96-well plates with opaque walls and clear bottoms. Cells were pretreated with 10 μM iKIR or scrambled (SCR) KIR peptide for 1 hour before the addition of GFP-MRSA at a multiplicity of infection of 50:1 [26]. Infected cells were incubated 1 hour to allow phagocytosis, and both plates were washed with warm PBS, and GFP fluorescence was measured on the first plate. The second plate was then maintained in PBS with SCR KIR or PBS with iKIR peptide and was incubated for another 2 hours for killing assays. To determine the role of NO in iKIR-mediated microbial killing, cells were treated with 50 μM of the iNOS inhibitor 1400W dihydrochloride (Tocris). To measure the intensity of intracellular GFP fluorescence, extracellular fluorescence was quenched with 500 μg/mL trypan blue, and the GFP fluorescence was quantified using a fluorimeter plate reader. Trypan blue served as a blank. A reduction in GFP fluorescence in the killing plate relative to the phagocytosis plate indicated bacterial killing [26,48].

Statistical analysis

Results are shown as a mean +/- SEM and were analyzed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA). For comparisons between two experimental groups, a Mann-Whitney test was used, and for comparisons among three or more experimental groups, one-way ANOVA followed by Bonferroni multiple comparison test was used. P < 0.05 was considered significant.

SOCS-1 impairs skin host defense

To explore the role of SOCS-1 in S. aureus skin infection, we initially examined Socs1 mRNA expression in the MRSA-infected skin of C57BL6/J mice. We observed a gradual increase in skin Socs1 mRNA expression and protein levels at days 1 and 3 post-infection (Figs 1A and S1A). Next, we investigated the consequences of SOCS-1 inhibition in skin host defense using pharmacologic and genetic approaches. SOCS-1 binds to JAK receptors via its SH2 domain and a short motif upstream of SH2, known as the KIR (kinase inhibitory region) that directly inhibits the kinase activity of JAK tyrosine kinases. The KIR of SOCS-1 occupies the substrate binding groove of JAK and prevents subsequent phosphorylation. Previously, we have utilized a SOCS-1 KIR blocking peptide termed iKIR that enhanced STAT-1 and STAT-3 phosphorylation in macrophages to promote cytokine storm during sepsis [28]. Here, we examined if this peptide could influence skin host defense. During infection with bioluminescent MRSA, wild-type mice were treated 1 hour before infection with the iKIR peptide or scrambled (SCR) KIR peptide control [28,31,49] followed by daily treatments post-infection. Mice treated with iKIR showed decreased infection area and bacterial loads when compared to infected mice treated with the scrambled control KIR (Fig 1B and 1C), as evidenced by decreased bacterial bioluminescence, as well as decreased CFUs (Fig 1D). The beneficial effect of iKIR treatment is not restricted to MRSA as we also observed that iKIR treatment decreased bacterial load during infection with the MSSA Newman strain (S1B Fig). Interestingly, iKIR treatment decreased bacterial burden in the skin of mice as early as six hours post-infection that persisted over the course of a nine-day infection (Fig 1D) but did not appear to directly impact bacterial viability or growth during axenic cultures (S1C Fig). Lower bioluminescent signals also correlated with a more compact abscess in iKIR-treated mice (Fig 1E).

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Fig 1. Inhibition of SOCS-1 actions improves subcutaneous skin infection outcome.

A) mRNA expression of Socs1 in the skin of mice infected subcutaneously with MRSA at day 1 and day 3 post-infection as determined by qPCR. B) Bioluminescent infection area in mice treated with SCR or SOCS-1 iKIR peptide using the in vivo animal imaging (IVIS Spectrum) detection system. C) Right–Total flux (photons/sec) of bioluminescent MRSA detected in mice treated as in B using IVIS Spectrum. Left–Representative images of bioluminescent MRSA in the skin of mice treated as in B using planar bioluminescent imaging. D) Bacterial load determined by CFU measured in the skin biopsy homogenates from mice treated as in B determined at the indicated time points after infection. E) Representative H&E stains from mice treated as in B and shown at 10X magnification F) Infection area measured every other day for 9 days post-infection in SOCS1^fl and SOCS1^Δmyel mice. G) Bacterial load determined by CFU measured in the skin biopsy homogenates in SOCS1^fl and SOCS1^Δmyel mice at day 9 post-infection. Data represent the mean ± SEM from 3–9 mice from 2–3 independent experiments. *p < 0.05 vs. SCR KIR.

https://doi.org/10.1371/journal.ppat.1009387.g001

SOCS-1 regulates both the adaptive and innate immune response [30]. As neutrophils and macrophages are required to control MRSA skin infection [5,6], we further studied the role of myeloid-specific SOCS-1 actions during skin infection. Infection in Socs1^Δmyel mice resulted in smaller lesion size over time and decreased bacterial burden in the skin at day 9 post-infection (Fig 1F and 1G). Together, these data suggest that phagocyte SOCS-1 expression negatively impacts MRSA skin infection outcomes.

SOCS-1 inhibition increases bacterial phagocytosis and killing in macrophages

Due to the impact of SOCS-1 inhibition on bacterial clearance in the skin, we hypothesized that SOCS-1 might inhibit macrophage antimicrobial effector functions, such as phagocytosis and bacterial killing. We next examined Gram stains in the infected skin sections of wild-type mice treated with iKIR or scrambled KIR to determine whether differences in bacterial ingestion, niche location, and burden were evident between iKIR-treated or scrambled KIR-treated animals. MRSA was found mostly within cells in iKIR-treated mice, while a higher abundance of extracellular bacteria was observed in the skin of scrambled KIR-treated animals at day 1 post-infection (Fig 2A). When bone marrow derived macrophages (BMDMs) from both Socs1^Δmyel mice and wild-type mice treated with the iKIR peptide were challenged with GFP-MRSA, we observed enhanced bacterial phagocytosis when compared to Socs1^fl BMDMs, or SCR KIR treated cells (Fig 2B). Importantly, SOCS-1 inhibition enhanced bacterial killing in BMDMs (Fig 2C). These data suggest that SOCS-1 is a negative regulator of macrophage antimicrobial effector functions that might be involved in MRSA skin infection control.

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Fig 2. SOCS-1 inhibition enhances antimicrobial effector functions in BMDMs.

A) Gram staining of skin biopsies collected at day 1 post-MRSA skin infection from iKIR and scrambled KIR treated mice. Gram staining to label gram-positive bacteria is shown in purple/brown. Magnifications are as shown. Black arrows indicate extracellular MRSA clusters. Images are representative of 3–5 mice per group. B) Phagocytosis of GFP tagged MRSA by BMDM’s from SOCS1^fl and SOCS1^Δmyel mice or BMDM’s from WT mice treated with the SCR KIR or iKIR peptide. C) Determination of Bacterial killing of GFP tagged MRSA by BMDMs from B as described in Phagocytosis and Killing assays—Methods. D) mRNA expression of Nos2 in the skin of infected mice treated with either SCR KIR or iKIR peptide at day 1 post-infection as determined via qPCR. E) Determination of bacterial killing of GFP-tagged MRSA as in C with BMDMs from WT mice treated with either SCR KIR, iKIR, or iKIR+ an iNOS inhibitor (1400W dihydrochloride). F) Measurement of nitric oxide in the supernatant of BMDMs from E. G) H₂O₂ levels in the skin of mice treated with either SCR KIR or iKIR at day 1 post infection as determined by Amplex Red assay. Data represent the mean ± SEM from 3–6 mice from 2–3 independent experiments. *p < 0.05 vs. SOCS1^fl or SCR KIR treated mice. #p < .05 vs. iKIR treated mice.

https://doi.org/10.1371/journal.ppat.1009387.g002

S. aureus is sensitive to killing via nitric oxide (NO) [5,9,50]. To identify if SOCS-1 targets NO production in macrophages, we initially determined Nos2 expression (the gene that encodes the inducible nitric oxide synthase protein) (iNOS) in iKIR treated and infected mice. We found significantly higher Nos2 expression in the skin of iKIR-treated animals than mice treated with the scrambled peptide control at day 1 post-infection (Fig 2D). As increased NO release could lead to more efficient bacterial killing in vivo, we determined whether increased iNOS/NO in iKIR treated mice accounts for improved microbial killing. BMDMs were treated with iKIR and an iNOS inhibitor (1400W dihydrochloride), followed by bacterial killing determination. iNOS inhibition ablated the beneficial effects of SOCS-1 inhibition on bacterial killing (Fig 2E). Notably, SOCS-1 inhibition increased NO release in MRSA-challenged BMDMs, and the treatment with the iNOS inhibitor reversed this trend (Fig 2F). Furthermore, when we examined whether SOCS-1 could regulate the production of the reactive oxygen species (ROS) known to be involved in S. aureus killing, we did not detect differences in skin H₂O₂ production between scrambled KIR and iKIR-treated and infected mice (Fig 2G). Together these data demonstrate a role for SOCS-1 in regulating antimicrobial programs in macrophages dependent on NO release.

SOCS-1 inhibits STAT-1 dependent production of pro-inflammatory cytokines and neutrophil recruitment during skin infection

Next, we examined the impact of SOCS-1 inhibition on the inflammatory milieu during skin infection. Initially, we determined whether SOCS-1 inhibition influenced STAT-1 activation in vivo. We found that while S. aureus infection induces STAT-1 phosphorylation (Y701), iKIR treatment further enhanced STAT-1 phosphorylation in the infected skin compared to mice treated with the scrambled KIR control (Fig 3A). Importantly, iKIR did not change SOCS-1 expression or impact STAT-3 phosphorylation (S727) during MRSA infection (Fig 3A). We also confirmed that skin infection in Socs1^Δmyel mice increased pSTAT-1 abundance compared to infected wildtype control animals. These data suggest that the SOCS-1/STAT-1 signaling axis is a component of skin host defense.

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Fig 3. SOCS-1 inhibition increases pro-inflammatory cytokines and immune cell recruitment during skin infection.

A) Representative Western blots for SOCS-1, pSTAT-1 (Y701), tSTAT-1, pSTAT-3 (S727), tSTAT-3, and Actin from biopsies collected at day 3 post-infection in SCR KIR and iKIR treated animals. The numbers represent mean densitometry analysis of the bands ± SEM (n = four to five mice/group). B) Heat-map of proteins involved in the inflammatory immune response and its resolution in mice treated with either iKIR or SCR KIR at day 1 post-infection as measured using bead array multiplex (Eve Technologies). Proteins are listed on the left-hand y-axis, grouped alphabetically in clades. Red indicates higher abundance, whereas green represents lower abundance. Each column for each condition represents a technical replicate (n = 4-5/group). C) Total flux (photons/sec) of EGFP detected in EGFP-LysM mice at day 3 post infection treated with either SCR or iKIR. Representative pictures are shown below. D) Total flux of mCherry signal detected in MMDTR mice at day 3 post infection treated with either SCR or iKIR, representative pictures are shown below. E) Total flux of tdTomato signal detected in Catchup^IVM-red mice at day 3 post infection treated with either SCR or iKIR with representative picture shown below. F) Total number of tdTomato+ cells in biopsies collected from mice in E. Data represent the mean ± SEM from 3–5 mice from 2–3 independent experiments. *p < 0.05 vs. SCR KIR treated mice.

https://doi.org/10.1371/journal.ppat.1009387.g003

We then determined whether iKIR enhances the production of 17 cytokines and chemokines known to modulate the skin inflammatory response and host defense [5]. Our data show that iKIR enhances specifically the production of pro-inflammatory cytokines known to drive proper abscess formation and improved infection outcome (IL-1β) [3], neutrophil recruitment (CXCL1 and CXCL2) [51], but not monocyte recruitment (CCL2) [52] in response to MRSA skin infection (Figs 3B and S2A). These data suggest that SOCS-1 controls a specific group of cytokines and chemokines known to influence neutrophil migration and functions to prevent an efficient host immune response to MRSA skin infection.

Since IL-1β, CXCL1, and CXCL2 drive phagocyte recruitment during skin infection [52] and iKIR enhances the abundance of these mediators at days 1 and 3 after infection (Figs 3B and S2B), we aimed to investigate whether iKIR treatment increases neutrophil and/or monocyte-derived macrophage migration to the site of infection. To examine specific phagocyte recruitment, we detected the accumulation of specific phagocytes using different transgenic mice that express fluorescent proteins specifically in myeloid cells, monocytes/macrophages, and neutrophils using IVIS optical imaging [37,38,53]. We first infected and treated EGFP-LysM mice (mainly neutrophils as well as monocytes and macrophages express EGFP) [53]. We detected a non-significant increase in GFP signal in the skin of iKIR-treated mice when compared to infected animals treated with the scrambled control (Fig 3C). To examine whether iKIR influences monocyte and macrophage recruitment, we infected and treated MMDTR mice (CSFR1+ cells express the mCherry fluorescent protein) and also found no differences between SCR KIR and iKIR treated animals (Fig 3D). To determine if iKIR specifically influences neutrophil recruitment, we infected and treated Catchup^IVM-red mice in which Ly6G⁺ cells express the tdTomato fluorophore. We found iKIR treatment greatly increased the tdTomato signal during infection (Fig 3E). We confirmed these findings by flow cytometry analysis where we did not detect differences in total macrophage (CD11b+ F4/80+) numbers but increased neutrophil abundance (CD11b+ tdTomato+) when compared to SCR KIR treated mice (Fig 3F). These data suggest that SOCS-1 is an endogenous inhibitor of neutrophil recruitment by hampering the secretion of chemoattractants such as CXCL1 an IL-1β that are involved in skin host defense [7,53].

SOCS-1/type I interferon axis mediates skin host defense

SOCS-1 is part of a negative feedback loop between type I and type II IFN production and STAT-1 activation [30]. Whether SOCS-1 regulates the production of type I and type II IFNs during skin infection remains to be determined. We detected higher IFNγ, IFNα, and IFNβ levels in skin biopsies from iKIR-treated and infected mice than scrambled KIR-treated mice at day 3 post-infection (Fig 4A). IFNβ was also significantly increased in the supernatant of iKIR BMDMs co-cultured with MRSA (S3A Fig). Due to the prominent role of IFNγ in enhancing phagocyte antimicrobial effector functions [14], we first blocked IFNγ actions in iKIR-treated and infected mice using an anti-interferon gamma receptor (IFNGR) antibody or IgG control antibody before infection and treatment with either iKIR or scrambled KIR peptide. Mice treated with the anti-IFNGR antibody plus scrambled KIR showed higher bacterial burden than mice treated with the control antibody as early as day 1 post-infection (Fig 4B). However, while iKIR plus IgG control decreased bacterial load, the treatment of mice with anti-IFNGR antibody did not prevent iKIR effects on bacterial clearance, demonstrating that IFNγ is not solely involved in the inhibitory effects of SOCS-1 during MRSA skin infection (Fig 4B).

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Fig 4. Inhibition of type I interferon signaling removes the benefit of SOCS-1 inhibition.

A) Interferon levels in the skin at day 1 post-infection in SCR KIR and iKIR treated animals as measured via ELISA. B) Bacterial load as determined via CFU in skin biopsy homogenates of SCR KIR and iKIR treated mice treated with either IFNGR antibody or IgG control. C) Bioluminescent infection area in the skin of SCR KIR and iKIR treated animals treated with or without an IFNAR blocking antibody at day 1 post-infection. D) Bacterial load determined by CFU measured in the skin biopsy homogenates from mice treated as in C at day 1 post-infection. E) Representative images of bioluminescent MRSA in the skin of mice treated as in C using planar bioluminescent imaging with average flux (photons/sec) below F) Nitrite/Nitrate as measured via Griess assay from biopsies collected from mice treated as in C at day 1 post-infection. G) Surface lesion size as measured via caliper at day 3 post-infection in BALB/c or BALB/c IFNAR -/- mice treated with either iKIR or SCR KIR. H) Bacterial load determined by CFU measured in the skin biopsy homogenates from mice treated as in G at day 3 post-infection. I) Representative images of bioluminescent MRSA in the skin of mice treated as in G using planar bioluminescent imaging with average flux (photons/sec) below. J) Nitrite/Nitrate as measured via Griess assay from biopsies collected from mice treated as in I at day 3 post-infection. Data represent the mean ± SEM from 3–9 mice from 2–3 independent experiments. *p < 0.05 vs. SCR KIR+αIGG or SCR treated WT mice. #p<0.05 vs. iKIR+ αIGG or iKIR treated WT mice.

https://doi.org/10.1371/journal.ppat.1009387.g004

Next, we determined whether iKIR actions are dependent on IFNα/β using both an IFNAR blocking antibody and IFNAR^-/- mice. While the pretreatment of mice with an anti-IFNAR antibody and scrambled KIR increased bacterial skin load, the blocking antibody also impaired iKIR-mediated decreases in lesion size (Fig 4C) and bacterial burden (Fig 4D and 4E). Treatment with the IFNAR antibody also decreased in vitro bacterial killing of iKIR treated BMDM (S3B Fig). We then confirmed that type I interferons were a potential mediator of improved skin host defense during SOCS-1 inhibition in a genetic model. Wild-type and IFNAR^-/- mice were treated with either iKIR or scrambled KIR peptide, followed by MRSA skin infection. Our data showed that IFNAR^-/- mice had increased lesion size at day 3 post-infection (Fig 4G) and bacterial load compared to WT mice (Fig 4H and 4I). Furthermore, IFNAR^-/- mice were refractory to iKIR treatment (Fig 4G, 4H and 4I). Next, we aimed to link SOCS-1/type I IFN and NO in in vivo MRSA skin infection. Our data showed that SOCS-1 inhibition enhances NO in vivo and blocking both SOCS-1 and IFNAR actions restored NO levels to the levels observed in scrambled KIR-treated mice (Fig 4F and 4J). Together, these data show a previously unknown operative axis of SOCS-1 and type I IFNs mediating NO-mediated microbial killing during bacterial skin infection.

SOCS-1 drives impaired skin host defense in hyperglycemic mice

Patients with hyperglycemia are more susceptible to MRSA skin infections than euglycemic individuals [44,54]. Therefore, we hypothesized that hyperglycemia enhances SOCS-1 expression and that blocking SOCS-1 actions might be a potential therapeutic approach to treat skin infections under hyperglycemic conditions. Initially, we determined Socs1 mRNA expression over time in the skin of infected hyperglycemic and euglycemic mice. Our data show that Socs1 expression was significantly upregulated in the skin of hyperglycemic animals at both day 1 and day 3 post-infection when compared to euglycemic animals (Fig 5A). Enhanced SOCS-1 expression correlated with decreased pSTAT-1 in skin biopsies of infected hyperglycemic mice (Fig 5B and 5C). Since SOCS-1 expression is increased in the skin of infected hyperglycemic mice, we asked whether the infection in hyperglycemic mice is characterized by an overall decrease in genes involved in IFN responses. Interestingly, our data show an overall downregulation of the IFN gene signature at day 1 post-infection in hyperglycemic mice (Fig 5D and 5E). When we examined specific IFN-related genes, we observed inhibition of Ifna1, Ifnb1, Ifng, and Stat1 in the infected skin of hyperglycemic mice. Importantly, we observed increased Socs1 (confirming the results in Fig 4A and 4B), Socs3, Irf2, Irf3, and Irf7 (Fig 5E) in infected hyperglycemic mice. We then determined whether myeloid-specific SOCS-1 expression is involved in poor skin host defense in hyperglycemic mice. Our data show that hyperglycemic Socs1^Δmyel mice show decreased lesion size (Fig 5F) and bacterial load (Fig 5G) when compared to hyperglycemic Socs1^fl mice on day 9 post-infection. Next, we sought to investigate if iKIR could restore skin host defense and represent a potential therapeutic approach to treating antibiotic-resistant skin infections. Daily treatment of the hyperglycemic mice with iKIR significantly reduced lesions size and bacterial burden (Fig 5H and 5I). Together these data suggest that enhanced Socs1 expression during hyperglycemia is detrimental to skin host defense and that iKIR treatment could represent host-directed immunotherapy to treat these antibiotic-resistant pathogens in the skin.

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Fig 5. SOCS-1 impairs skin host defense in hyperglycemic mice.

A) Expression of Socs1 mRNA at day 1 and day 3 post-infection in biopsies collected from STZ induced hyperglycemic and control mice B) Representative Western blot for SOCS-1, pSTAT-1, and tSTAT-1 from biopsies collected at day 3 post-infection in STZ-induced hyperglycemic and control mice C) Densitometry quantification of separate Western Blots as shown in B. D) IFN signaling score determined using NanoSolver in mRNA isolated from infected skin of iKIR and scrambled KIR-treated mice. E) Heatmap of genes analyzed via NanoString from mRNA collected in D. F) Surface lesions size in STZ induced hyperglycemic and euglycemic SOCS1^fl and SOCS1^Δmyel mice over a 9 day subcutaneous MRSA infection. G) Bacterial load determined by CFU measured in the skin biopsy homogenates from mice treated as in F at day 9 post-infection. H) Surface lesions size in STZ induced hyperglycemic and euglycemic mice treated with either SCR KIR or iKIR over the course of a 9 day subcutaneous MRSA infection. I) Bacterial load determined by CFU measured in the skin biopsy homogenates from mice treated as in H at day 9 post-infection. Data represent the mean ± SEM from 3–6 mice from 2–3 independent experiments. *p < 0.05 vs. Vehicle controls, SOCS1^fl or SCR KIR treated mice.

https://doi.org/10.1371/journal.ppat.1009387.g005