Tumor Necrosis Factor-α Is Required for Mast Cell-Mediated Host Immunity Against Cutaneous Staphylococcus aureus Infection

Tumor Necrosis Factor-α Is Required for Mast Cell-Mediated Host Immunity Against Cutaneous... Abstract Background Mast cells (MCs) play a key role in immune process response to invading pathogens. Methods This study assessed the involvement of MCs in controlling Staphylococcus aureus infection in a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice. Results KitW-sh/W-sh mice developed significantly larger skin lesions after the cutaneous S. aureus challenge, when compared to wild-type (WT) mice, while MC dysfunction reduced the inflammation response to S. aureus. The levels of tumor necrosis factor (TNF)-α in skin tissues were significantly decreased in KitW-sh/W-sh mice upon infection. Moreover, the exogenous administration of MCs or recombinant TNF-α effectively restored the immune response against S. aureus in KitW-sh/W-sh mice via the recruitment of neutrophils to the infected site. These results indicate that the effects of MC deficiency are largely attributed to the decrease in production of TNF-α in cutaneous S. aureus infection. In addition, S. aureus-induced MC activation was dependent on the c-kit receptor-activated phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway, which was confirmed by treatment with Masitinib (a c-kit receptor inhibitor), Wortmannin (a PI3K inhibitor), and pyrrolidine dithiocarbamate (a NF-κB inhibitor), respectively. Conclusions The present study identifies the critical role of MCs in the host defense against S. aureus infection. inflammation response, mast cells, Staphylococcus aureus, TNF-α Mast cells (MCs) are a minority population of cells in the blood stream but prominently occur in the skin, lungs, digestive tract, and the nose [1, 2]. Functionally, MCs play an important role in regulation of angiogenesis, immune tolerance, and defense against pathogens, but the best-known role of MCs is in allergy reaction due to their granule-containing histamine and heparin [3, 4]. Further research on MCs and their functions in infectious diseases could help in better understanding the inmate immunity and host defense against pathogens, which is of clinical significance. Staphylococcus aureus is one of major pathogens in the skin and soft-tissue infections, which lead to abscess, cellulitis, folliculitis, and impetigo, resulting in a significant public health problem [5, 6]. Methicillin-resistant S. aureus could lead to complicated and difficult treatment of skin infection [5]. Especially in the USA 300 strain, the difficulty of treatment depended mainly on virulence other than antibiotic resistance [7]. A couple of virulent factors, including delta-hemolysin (Hld), phenol soluble modulins (PSMs), alpha-hemolysin (Hla), and Panton-Valentine leucocidin (PVL), were involved in the pathogenesis of S. aureus infection [8–11]. Human skin is composed of the epidermis and dermis, providing physical protection against S. aureus infection. A variety of immune cells are present in skin tissues [12, 13]. MCs in the skin function as sentinels in the host immunity and are activated by invading pathogens or inflammatory mediators. MC-derived cytokines participate in the inflammatory process, such as the recruitment of neutrophils and other immune cells into the infected site, resulting in pathogen elimination. MCs per se also have been reported to produce and secrete antimicrobial peptides [14, 15]. Indeed, MCs have been shown to play crucial roles in the clearance of group A Streptococcus, Pseudomonas aeruginosa, and vaccinia virus and improve the outcome of skin infectious diseases [16–18]. KitW-sh/W-sh mice with the KitW-sh inversion mutation, which were generated by Grimbaldeston et al [19, 20], have been shown to have MC deficiency in all tissues, but remained intact for major classes of other differentiated hematopoietic and lymphoid cells. In the present study, the cutaneous infection model of MC-deficient mice (KitW-sh/W-sh) was used to dissect the function of MCs in controlling S. aureus infection. MATERIALS AND METHODS Animals Mast cell-deficient mice (KitW-sh/W-sh) with C57BL/6 background were kindly provided by Dr. Guo-Ping Shi of Harvard Medical School [21], whereas age- and gender-matched wild-type (WT) C57BL/6 mice were used as controls. All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University. Bacterial Culture The S. aureus strain was a clinical isolate (multilocus sequence type ST15 and agr type II) with Hld+, PSMα+, Hla+, and PVL−. Staphylococcus aureus was grown in tryptic soy broth (TSB) at 37°C with 5% CO2, with shaking (200rpm) to the mid-logarithmic phase. Then, the bacteria were resuspended in phosphate-buffered saline (PBS). The concentrations were determined by measuring the absorbance at 600 nm and verified using colony-forming units (CFUs) assay on TSB agar [22]. Cell Culture An MC line P815 was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4.5 mg/mL glucose, in a humidified incubator with 5% CO2 at 37°C. Bone marrow MCs (BMMCs) were generated from the femoral bone marrow cells of mice and maintained with the presence of 10% pokeweed mitogen-stimulated spleen-conditioned medium, as previously described [23]. After 4 weeks of culture, more than 99% of cells developed into MCs. BMMC and P815 cells (1 × 106/well) were treated with Masitinib (10 μM; Selleck) for 30 minutes before S. aureus stimulation for 4 h and 8 h (Figure 5 and 6), while P815 cells were treated with Masitinib for 30 minutes before S. aureus infection for 15, 30, and 60 minutes (Figure 7A and B [WB]). P815 cells (1 × 106/well) were pretreated with Wortmannin (10 μM; Abmole) or pyrrolidine dithiocarbamate ([PDTC] 25 μM; Abmole) for 30 minutes before 4 h exposure to S. aureus (Figure 7C and D). A Mouse Model of Staphylococcus aureus Skin Infection Mice, with 6–8 weeks old, on a C57BL/6 genetic background were used in S. aureus infection experiments. The mouse skin on the posterior back was shaved and intradermally injected with 100 µL S. aureus at 3 × 107 CFUs, while sham mice were given 100 µL PBS injections. For recombinant tumor necrosis factor (rTNF)-α treatment experiments, 200 ng of mouse rTNF-α (Peprotech EC Ltd.) in 100 µL normal saline was intradermally injected along with 100 µL S. aureus (3 × 107 CFUs) into WT or KitW-sh/W-sh mice. The size of the lesion was monitored and measured using Image J software (National Institutes of Heath). Mice were euthanized and skin specimens were collected for histology at indicated time points after infection. Some skin specimens were weighted and homogenized in 1 mL PBS using a homogenizer (Jingxin) for assessment of bacterial load and cytokine levels. Reconstitution of Mast Cells in KitW-sh/W-sh Mice For the reconstitution of MCs in knockout mice, BMMCs (106 cells in 200 µL DMEM) were injected into the shaved back skin of KitW-sh/W-sh mice at 4 weeks old. Then, these mice were cutaneously infected with S. aureus at 4 weeks after BMMC injection. Histological Analysis Mouse skin specimens were fixed in 10% paraformaldehyde and embedded in paraffin. Four micrometer-thick tissue sections were stained with hematoxylin-eosin (H&E). The stained sections were reviewed for morphology and subjected to morphometric analysis under a photomicroscope (Leica). To evaluate the infiltration of neutrophils into the skin tissue, 6 randomly selected fields (magnification, ×50) were examined, and the average value of cells/mm2 was determined for each skin sample. Immunohistochemistry Four micrometer sections of paraffin-embedded skin tissues were blocked with 0.5% bovine serum albumin (BSA) for 30 minutes and incubated with anti-myeloperoxidase (MPO) antibody (Servicebio). Subsequently, a secondary antibody (Servicebio) was added and incubated at 37°C for 60 minutes. The stained sections were reviewed for morphology and subjected to morphometric analysis under a photomicroscope (Leica). Immunofluorescent Staining The paraffin-wax sections of murine skin tissue were placed on glass slides. The nonspecific binding of antibodies was blocked by incubation with 1% BSA for 1 hour before incubation with primary antibodies. Alexa Fluor 488 anti-mouse CD117 (c-Kit) antibody (1: 50; BioLegend) and phycoerythrin antimouse TNF-α antibody (1: 50; BioLegend) were mixed and used for double staining. The sections were incubated with primary antibodies overnight at 4°C, followed by incubation at 37°C for 30 minutes. Then, 4’,6-diamidino-2-phenylindole was added for nuclei detection before viewing by confocal microscopy (Olympus). Quantitative Reverse-Transcription Polymerase Chain Reaction Ribonucleic acid was isolated from the skin specimens or MCs using TRIzol reagent (Invitrogen) and reversely transcribed into complementary deoxyribonucleic acid (cDNA) using the reverse-transcriptase cDNA synthesis system (Applied Biosystems). Quantitative polymerase chain reaction was performed using a Sequence Detection Software (Bio-Rad). The primer sequences of target genes are listed in Supplementary Table 1. Enzyme-Linked Immunosorbent Assay The levels of interleukin (IL)-β, IL-6, TNF-α, and cathelicidin-related antimicrobial peptide (CRAMP) in the skin homogenates and culture supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems Inc.). Western Blotting Western blotting was performed as previously described [24]. Antibodies were used against the following proteins: PI3K, p-PI3K, P65 NF-κB, p-P65 NF-κB, and β-actin (Cell Signaling Technology); AKT and p-AKT (Abcam). The protein signal was developed using the enhanced chemiluminescence reagent (Lianke) and detected using a digital image system (Proteinsimple). The signal intensity was further quantified using Image J software (National Institutes of Health). Antimicrobial Assay To assess the direct antibacterial effect of the conditional medium (CM) derived from the MC culture, 1 × 106 of cells were infected with S. aureus (MOI, 20) and cultured for 4 hours. The cell culture medium was passed through a 0.2-μm pore-sized filter. One hundred CFUs of S. aureus (10 μL) were incubated with the CM (90 μL) for 0.5 hours, 1 hour, 3 hours, and 16 hours, respectively, at 37°C before CFU counting. Statistical Analysis All results were expressed as mean ± standard error, unless otherwise stated. The statistical significance of the difference between 2 or multiple groups was analyzed, using Student’s t test or one-way analysis of variance as appropriate. Colony-forming units in the tissue homogenates were compared using the Mann-Whitney U test. A P value <.05 was considered statistically significant. RESULTS KitW-sh/W-sh Mice Develop Larger Skin Lesions After Cutaneous Staphylococcus aureus Infection Compared With Wild-Type Mice To investigate whether MCs contribute to host defense against skin S. aureus infection, WT and KitW-sh/W-sh mice were intradermally inoculated with S. aureus at 3 × 107 CFUs per mouse. The sizes of the skin lesions were recorded over time (Figure 1A and B). It was found that KitW-sh/W-sh mice developed larger skin lesions than WT animals and the maximum size reached up to 85.80 ± 8.11 mm2 at day 3. The difference in skin lesions between both groups appeared to be indistinguishable at day 14. Moreover, bacterial load in the skin tissue of KitW-sh/W-sh mice was 10-fold higher than those in WT mice 3 days after infection (Figure 1C). Although there was no difference in skin neutrophil count between uninfected KitW-sh/W-sh and WT mice, a remarkable reduction in neutrophil level was observed in the skin tissues of KitW-sh/W-sh mice through H&E and MPO staining 72 hours after intradermal infection, compared with S. aureus-stimulated WT mice (Figure 1D and E). Figure 1. View largeDownload slide KitWsh/Wsh mice display susceptibility to Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 1, 3, 5, 7, and 14 after infection. (B) Lesion area (mm2). (C) Bacterial load at day 3 postinfection. Individual values and median were shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections at day 3 postinfection. *, P < .05 and **, P < .01. Abbreviation: PBS, phosphate-buffered saline. Figure 1. View largeDownload slide KitWsh/Wsh mice display susceptibility to Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 1, 3, 5, 7, and 14 after infection. (B) Lesion area (mm2). (C) Bacterial load at day 3 postinfection. Individual values and median were shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections at day 3 postinfection. *, P < .05 and **, P < .01. Abbreviation: PBS, phosphate-buffered saline. Mast Cell Deficiency Reduces Inflammation Response to Staphylococcus aureus Infection Next, the effect of MC deficiency on inflammation response to S. aureus infection was evaluated. The levels of inflammation cytokines, including IL-1β, IL-6, IL-17A, keratinocyte-derived cytokine, macrophage inflammatory protein-2, TNF-α, and antibacterial peptides such as CRAMP and regenerating islet-derived IIIγ were detected (Figure 2A and B). The data revealed that the expression of TNF-α, IL-6, IL-17A, and CRAMP was significantly lower in skin specimens obtained from KitW-sh/W-sh mice than those from WT mice 3 days after infection. To determine the main cell source of TNF-α in the infected skin, tissue sections were co-immunostained for the expression of c-kit (the marker for MCs) and TNF-α. The confocal imagines clearly revealed that S. aureus infection was able to robustly activate TNF-α expression in MCs of WT mice (Figure 2C). Figure 2. View largeDownload slide KitW-sh/W-sh mice exhibit an impaired inflammation response upon skin Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5 per group) were intradermally injected with S. aureus. (A) Transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP-2), tumor necrosis factor (TNF)-α, cathelicidin-related antimicrobial peptide (CRAMP), and regenerating islet-derived IIIγ (RegIIIγ)were detected using quantitative reverse-transcription polymerase chain reaction at day 3 after infection. (B) Protein levels of IL-1β, IL-6, TNF-α, and CRAMP were analyzed using enzyme-linked immunosorbent assay at day 3 after infection. (C) Enhanced production of TNF-α was observed in c-kit+ skin mast cells of WT mice 6 hours postinfection. Magnification, ×180; scale bar, 20 μm. *, P < .05 and **, P < .01. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 2. View largeDownload slide KitW-sh/W-sh mice exhibit an impaired inflammation response upon skin Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5 per group) were intradermally injected with S. aureus. (A) Transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP-2), tumor necrosis factor (TNF)-α, cathelicidin-related antimicrobial peptide (CRAMP), and regenerating islet-derived IIIγ (RegIIIγ)were detected using quantitative reverse-transcription polymerase chain reaction at day 3 after infection. (B) Protein levels of IL-1β, IL-6, TNF-α, and CRAMP were analyzed using enzyme-linked immunosorbent assay at day 3 after infection. (C) Enhanced production of TNF-α was observed in c-kit+ skin mast cells of WT mice 6 hours postinfection. Magnification, ×180; scale bar, 20 μm. *, P < .05 and **, P < .01. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Adoptive Transfer of Bone Marrow Mast Cells Reconstitutes Host Defense Against Staphylococcus aureus Infection in KitW-sh/W-sh Mice To determine the susceptibility of KitW-sh/W-sh mice to S. aureus, which was mainly attributed to MC deficiency, we performed an adoptive MC transfer experiment. The results showed that KitW-sh/W-sh mice treated with WT-derived BMMCs had significantly smaller lesions than MC-deficiency mice at day 3 after the infection (Figure 3A and B). Correspondingly, MC-reconstituted KitW-sh/W-sh mice exhibited markedly improved bacterial clearance to S. aureus (Figure 3C). The impaired recruitment of neutrophils in KitW-sh/W-sh mice was largely reversed after the adoptive injection of BMMCs upon infection (Figure 3D and E). Simultaneously, the levels of TNF-α and CRAMP in MC-reconstituted KitW-sh/W-sh mice significantly increased after infection, compared with non-MC-treated animals (Figure 3F). Figure 3. View largeDownload slide Bone marrow mast cell (BMMC) transfer reconstitutes host resistance to Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT), KitW-sh/W-sh, and reconstituted KitW-sh/W-sh mice (n = 6 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 3 after infection. (B) Lesion area (mm2) at day 3 postinfection. (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections 3 days after infection. (F) Enzyme-linked immunosorbent assay of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and cathelicidin-related antimicrobial peptide (CRAMP) 3 days after infection. *, P < .05 and **, P < .01. Figure 3. View largeDownload slide Bone marrow mast cell (BMMC) transfer reconstitutes host resistance to Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT), KitW-sh/W-sh, and reconstituted KitW-sh/W-sh mice (n = 6 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 3 after infection. (B) Lesion area (mm2) at day 3 postinfection. (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections 3 days after infection. (F) Enzyme-linked immunosorbent assay of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and cathelicidin-related antimicrobial peptide (CRAMP) 3 days after infection. *, P < .05 and **, P < .01. Exogenous Recombinant Tumor Necrosis Factor-α Administration Restores Immune Response Against Staphylococcus aureus Infection in KitW-sh/W-sh Mice Neutrophils are essential for the effective clearance of S. aureus in lesions. Given that MC-derived TNF-α expression after infection could significantly enhance Th17 cell-dependent neutrophil-rich inflammatory response [25, 26], we explored whether the administration of rTNF-α rescued KitW-sh/W-sh mice with skin S. aureus infection. In KitW-sh/W-sh mice, it was found that treatment with 200 ng of rTNF-α resulted in a significant reduction in lesion size 3 days after infection, when compared with vehicle treatment (Figure 4A and B). KitW-sh/W-sh mice also exhibited a reduction in bacterial load after the administration of rTNF-α, along with more abundant neutrophil infiltration (Figure 4C–E), and an increase in the expression of IL-17A and CRAMP (Figure 4F). These results demonstrate that TNF-α participates in MC-mediated skin immune response to S. aureus infection. Figure 4. View largeDownload slide Recombinant tumor necrosis factor (rTNF)-α restores host defense against cutaneous Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 mice per group) were intradermally injected with 100 µL mouse rTNF-α (200 ng) or normal saline (NS) along with 100 µL S. aureus (3 × 107 colony-forming units [CFU]). (A) Representative photographs of lesions at day 1, 3, 5, and 7 after infection. (B) Lesion area (mm2). (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E) and myeloperoxidase (MPO) staining of skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained slides 3 days after infection. (F) Transcriptional levels of interleukin (IL)-17A and cathelicidin-related antimicrobial peptide (CRAMP) were detected using quantitative reverse-transcription polymerase chain reaction 3 days after infection.*, P < .05 and **, P < .01. Abbreviation: mRNA, messenger ribonucleic acid. Figure 4. View largeDownload slide Recombinant tumor necrosis factor (rTNF)-α restores host defense against cutaneous Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 mice per group) were intradermally injected with 100 µL mouse rTNF-α (200 ng) or normal saline (NS) along with 100 µL S. aureus (3 × 107 colony-forming units [CFU]). (A) Representative photographs of lesions at day 1, 3, 5, and 7 after infection. (B) Lesion area (mm2). (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E) and myeloperoxidase (MPO) staining of skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained slides 3 days after infection. (F) Transcriptional levels of interleukin (IL)-17A and cathelicidin-related antimicrobial peptide (CRAMP) were detected using quantitative reverse-transcription polymerase chain reaction 3 days after infection.*, P < .05 and **, P < .01. Abbreviation: mRNA, messenger ribonucleic acid. Staphylococcus aureus-Induced Mast Cell Activation Is Dependent on the Expression of c-Kit Receptor Previous studies have showed the importance of c-kit receptor in mediating MC activity [27, 28]. To assess the role of c-kit receptor in controlling S. aureus infection, the expression of kinds of inflammatory mediators in BMMCs was investigated (Figure 5A). It was found that Masitinib, the c-kit receptor inhibitor, could markedly reduce the levels of IL-6 and TNF-α from MCs after infection. Moreover, the data were consistent with those obtained from immortalized P815 MCs after incubation with S. aureus (Figure 6A), confirming that c-kit receptor activation is involved in the functional regulation of MCs against bacterial infection. Di Nardo et al [16] demonstrated that activated murine MCs produced peptides such as CRAMP (the murine homolog of human LL-37), and they showed potent antimicrobial activity. The current data also revealed that the levels of CRAMP in MCs (BMMCs and P815) were significantly increased after exposure to S. aureus. Furthermore, Masitinib remarkably reduced the bacteria-induced expression of CRAMP, along with the attenuated antimicrobial potency of P815 cells against S. aureus (Figure 5B and Figure 6B and C). These results reveal that c-kit-related signaling participates in the antibacterial immune response of MCs. Figure 5. View largeDownload slide Staphylococcus aureus induces bone marrow mast cell (BMMC) activation dependent of C-kit receptor. Bone marrow mast cells pretreated or not pretreated with Masitinib for 30 minutes were incubated with S. aureus. (A) The transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α in BMMCs were analyzed using quantitative reverse-transcription polymerase chain reaction at 4 hours and 8 hours after infection. (B) The transcriptional levels of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) 4 hours and 8 hours after infection *, P < .05 and **, P < .01. Abbreviations: mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 5. View largeDownload slide Staphylococcus aureus induces bone marrow mast cell (BMMC) activation dependent of C-kit receptor. Bone marrow mast cells pretreated or not pretreated with Masitinib for 30 minutes were incubated with S. aureus. (A) The transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α in BMMCs were analyzed using quantitative reverse-transcription polymerase chain reaction at 4 hours and 8 hours after infection. (B) The transcriptional levels of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) 4 hours and 8 hours after infection *, P < .05 and **, P < .01. Abbreviations: mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 6. View largeDownload slide Staphylococcus aureus induces P815 cell activation via the c-kit receptor. P815 cells were pretreated or not pretreated with Masitinib for 30 minutes before S. aureus stimulation. (A) The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) detection of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed in P815 cells at 4 hours and 8 hours after bacterial incubation. (B) The qRT-PCR detection of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) mRNA 4 hours and 8 hours after infection. (C) The antimicrobial activity of the conditional medium from P815 cells after S. aureus infection. *, P < .05 and **, P < .01; ∆P < .05 vs phosphate-buffered saline (PBS) group; #P < .05 vs S. aureus + Masitinib group. Abbreviation: CFU, colony-forming units. Figure 6. View largeDownload slide Staphylococcus aureus induces P815 cell activation via the c-kit receptor. P815 cells were pretreated or not pretreated with Masitinib for 30 minutes before S. aureus stimulation. (A) The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) detection of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed in P815 cells at 4 hours and 8 hours after bacterial incubation. (B) The qRT-PCR detection of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) mRNA 4 hours and 8 hours after infection. (C) The antimicrobial activity of the conditional medium from P815 cells after S. aureus infection. *, P < .05 and **, P < .01; ∆P < .05 vs phosphate-buffered saline (PBS) group; #P < .05 vs S. aureus + Masitinib group. Abbreviation: CFU, colony-forming units. C-kit/PI3K/AKT/p65-NF-κB Signaling Mediates Antimicrobial Activity and Inflammation Response of Mast Cells to Staphylococcus aureus Infection Finally, the underlying molecular mechanisms by which MCs produced TNF-α and CRAMP after S. aureus infection were investigated. The present data in Figure 7A and B revealed that S. aureus profoundly induced the phosphorylation of PI3K, AKT, and P65-NF-κB in MCs, which was remarkably attenuated by pretreatment with the c-kit receptor inhibitor Masitinib. However, Masitinib alone had no effect on the phosphorylation of these molecules in non-infected cells (Supplementary Figure 1). The S. aureus-induced production of TNF-α and CRAMP was significantly reduced by pretreatment with the specific PI3K inhibitor Wortmannin or the specific NF-κB inhibitor PDTC (Figure 7C and D). Therefore, S. aureus-induced MC activation was dependent on c-kit receptor-activated PI3K/AKT/P65-NF-κB signaling. Figure 7. View largeDownload slide The c-kit/phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway mediates immune response in mast cells upon Staphylococcus aureus challenge. (A and B) Mast cells P815 were pretreated or not pretreated with Masitinib for 30 minutes. The phosphorylation of PI3K, AKT, and P65-NF-ĸB was detected by Western blotting at 15, 30, and 60 minutes postinfection. (C) Mast cells P815 were incubated with dimethyl sulfoxide (DMSO), Wortmannin, and pyrrolidine dithiocarbamate (PDTC), respectively, for 30 minutes before 4 hours exposure to S. aureus. The quantitative reverse-transcription polymerase chain reaction analysis of cathelicidin-related antimicrobial peptide (CRAMP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed. (D) The enzyme-linked immunosorbent assay of CRAMP, IL-6, and TNF-α was performed at 4 hours after infection. *, P < .05 and **, P < .01. Figure 7. View largeDownload slide The c-kit/phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway mediates immune response in mast cells upon Staphylococcus aureus challenge. (A and B) Mast cells P815 were pretreated or not pretreated with Masitinib for 30 minutes. The phosphorylation of PI3K, AKT, and P65-NF-ĸB was detected by Western blotting at 15, 30, and 60 minutes postinfection. (C) Mast cells P815 were incubated with dimethyl sulfoxide (DMSO), Wortmannin, and pyrrolidine dithiocarbamate (PDTC), respectively, for 30 minutes before 4 hours exposure to S. aureus. The quantitative reverse-transcription polymerase chain reaction analysis of cathelicidin-related antimicrobial peptide (CRAMP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed. (D) The enzyme-linked immunosorbent assay of CRAMP, IL-6, and TNF-α was performed at 4 hours after infection. *, P < .05 and **, P < .01. DISCUSSION Mast cells have been shown to contribute to innate immune response against pathogens in addition to the role in allergic responses [16, 17]. However, the roles of MCs in protecting the skin against bacterial infection remain to be defined. This study utilized a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice to assess the effects of MCs on S. aureus infection. The present data revealed that MCs play an essential role in the host defense against cutaneous S. aureus infection. In particular, upon S. aureus infection, MCs released TNF-α to mediate neutrophil recruitment to the infection site, resulting in the promotion of bacterial clearance. Furthermore, MC-derived TNF-α was regulated by the activation of the c-kit-mediated PI3K/AKT/p65-NF-κB signaling pathway. During skin infection, a variety of immune cells and cytokines could coordinate and orchestra host skin immunity [29, 30]. Previous studies have shown that the activation of MCs is evolved in host defense against bacteria. Siebenhaar et al [17] found that MCs are important for the clearance for P. aeruginosa through the upregulation of endothelin-1 and the accumulation of neutrophil infiltrations in P. aeruginosa- infected skin. Di Nardo et al [16] demonstrated that MCs were able to trigger the anti-group A Streptococcus immunity in the skin, in part, by releasing CRAMP. The antimicrobial peptide CRAMP released from skin MCs was shown to inactivate vaccinia virus infectivity [18]. Furthermore, MCs have been shown to play an essential role in defending against Chlamydia pneumoniae lung infection by recruiting neutrophils and lymphocytes into the airspace [31]. However, peritoneal MCs have no effect on S. aureus growth after intraperitoneal infection, although they could be activated in vitro [32]. Lê et al [33] found that the activation of MCs induced by P. aeruginosa contributes to the increase in alveolar-capillary permeability. Therefore, the precise role of MCs in the host immunity could be dependent on the types of pathogens and sites of infection. In the current study, using KitW-sh/W-sh mice, we demonstrated that MC-deficient mice were significantly more susceptible to S. aureus skin infection than WT mice, along with larger skin lesions and higher bacterial loads. To rule out the broader effect from loss of c-Kit in KitW-sh/W-sh mice, an adoptive transfer of BMMCs into the skin of KitW-sh/W-sh mice before S. aureus infection was further performed. Compared with controls, the knockout mice that received WT-derived BMMCs had significantly smaller lesions and markedly decreased bacterial burden 3 days after the infection. Consistently, the impaired recruitment of neutrophils and suppressed inflammatory response were largely reconstituted in KitW-sh/W-sh mice with adoptive BMMC administration. Therefore, the present study revealed that MC deficiency definitely leads to impaired host defense against skin S. aureus infection. However, it should be noted that the Kit-independent MC-deficient Mcpt5-Cre+ × R-DTA mice would be an additional ideal mouse model for the verification of the role of MCs in diverse models of diseases [32, 34, 35]. Instant and appropriate inflammatory response is essential for cutaneous immunity against pathogens. Tumor necrosis factor-α is an early response cytokine that facilitates neutrophil infiltration into the lesion site and clears pathogens [36, 37]. The bacterial load was significantly higher in the S. aureus-induced experimental brain abscess in TNF-α−/− mice, when compared with WT mice [38]. Mast cell-derived TNF enhanced Th17 cell-dependent inflammatory response with rich neutrophil infiltration in the airway [25], and IL-17 from resident epidermal γδT cells promoted neutrophil recruitment into the site of skin S. aureus infection [26]. The blockade of TNF-α by infliximab was able to reduce the expression of inflammation cytokines including IL-17 [39]. The present study revealed that TNF-α was rapidly produced after S. aureus infection, and involved in neutrophil recruitment into the skin lesion. This finding was further supported by the fact that the administration of rTNF-α restored host resistance to S. aureus in MC-deficient mice. Previous studies have supported that CRAMP produced by MCs contributed to host defense against pathogens in skin lesions infected by group A Streptococcus and vaccinia virus [16, 18]. Our data revealed that upregulated expression of IL-17A and CRAMP occurred in skin lesions injected with rTNF-α, which enhanced host antibacterial immunity. Taken together, these results demonstrate that MC-facilitated inflammatory response, which is mainly mediated by TNF-α, would be beneficial to the ultimate outcome of skin S. aureus infection. Staphylococcus aureus can cause serious skin infections due to the expression of diverse toxins such as PVL, Hla, Hld, and other cytolytic toxins, which are able to initiate the innate immune system. Previous studies have shown that PVL and Hla contribute to the pathology of tissues through the activation of macrophages and recruitment of excessive neutrophils [5, 9]. Furthermore, the host immune system, as a target for invading pathogens, recognizes them through pattern recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [10, 22]. Thus, understanding the host response to bacteria and their elements would be helpful for developing novel therapeutic strategies. TLRs sense pathogen-associated molecular patterns from pathogens and primarily mediate signaling via its interaction with adaptor proteins MyD88 to activate the NF-κB pathways [22, 40, 41]. NOD2 stimulation also leads to direct activation of NF-κB and increases expression of its target genes. During skin infection, S. aureus-induced IL-6 response depends on the activation of NOD2 [10]. Blocking TLR or NOD with specific antibodies abolished peptidoglycan-induced activation of MCs [42]. In addition to TLRs and NOD, other receptors are also involved in response to pathogens. FimH receptor CD48 has been found to be able to detect the presence of Escherichia coli and S. aureus [43, 44]. CD48 triggered the TNF-α release in MCs after E. coli infection through FimH fimbrial adhesion [43]. Similarly, infection of human cord blood-derived MCs with S. aureus induced CD48 activation and TNF-α release [44]. The c-kit has recently been shown to be essential in the mediation of MC maturation and survival. Stem cell factor stimulation led to secondary signaling events, including the phosphorylation of PI3K, AKT, and p65-NF-κB proteins in cells [45, 46]. Our previous studies have defined that the activation of AKT and NF-κB by S. aureus was a critical molecular mechanism for maintaining the balance between host antibacterial immunity and tissue injury [22, 24]. Oviedo-Boyso et al [47] reported that PI3K-AKT-P65 pathway was also important for the internalization of S. aureus in endothelial cells. Our current data showed that, upon S. aureus infection, skin MCs were able to produce and secrete TNF-α and CRAMP through the activation of the c-kit-dependent PI3K-AKT-p65-NF-κB signaling pathway. CONCLUSIONS In conclusion, we have demonstrated that MCs facilitate neutrophil recruitment to the infection site and prompt cutaneous host defense against S. aureus. The c-kit-mediated TNF-α production of MCs initiates this critical early innate immune response. The present study provides a novel therapeutic strategy to augment and regulate the function of MCs to combat S. aureus infection. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by grants from the National Natural Science Foundation of China (81770008, 81570005, and 81370176) and Major Science and Technology Special Project of Zhejiang Province (2014C03033). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. References 1. Gibson S , Miller HR . Mast cell subsets in the rat distinguished immunohistochemically by their content of serine proteinases . Immunology 1986 ; 58 : 101 – 4 . Google Scholar PubMed 2. Caughey GH . Mast cell proteases as protective and inflammatory mediators . Adv Exp Med Biol 2011 ; 716 : 212 – 34 . Google Scholar CrossRef Search ADS PubMed 3. da Silva EZ , Jamur MC , Oliver C . Mast cell function: a new vision of an old cell . J Histochem Cytochem 2014 ; 62 : 698 – 738 . Google Scholar CrossRef Search ADS PubMed 4. Caughey GH . Mast cell proteases as pharmacological targets . 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Iwaki S , Tkaczyk C , Satterthwaite AB et al. Btk plays a crucial role in the amplification of Fc epsilonRI-mediated mast cell activation by kit . J Biol Chem 2005 ; 280 : 40261 – 70 . Google Scholar CrossRef Search ADS PubMed 47. Oviedo-Boyso J , Cortés-Vieyra R , Huante-Mendoza A et al. The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells . Infect Immun 2011 ; 79 : 4569 – 77 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Infectious Diseases Oxford University Press

Tumor Necrosis Factor-α Is Required for Mast Cell-Mediated Host Immunity Against Cutaneous Staphylococcus aureus Infection

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© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
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0022-1899
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Abstract

Abstract Background Mast cells (MCs) play a key role in immune process response to invading pathogens. Methods This study assessed the involvement of MCs in controlling Staphylococcus aureus infection in a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice. Results KitW-sh/W-sh mice developed significantly larger skin lesions after the cutaneous S. aureus challenge, when compared to wild-type (WT) mice, while MC dysfunction reduced the inflammation response to S. aureus. The levels of tumor necrosis factor (TNF)-α in skin tissues were significantly decreased in KitW-sh/W-sh mice upon infection. Moreover, the exogenous administration of MCs or recombinant TNF-α effectively restored the immune response against S. aureus in KitW-sh/W-sh mice via the recruitment of neutrophils to the infected site. These results indicate that the effects of MC deficiency are largely attributed to the decrease in production of TNF-α in cutaneous S. aureus infection. In addition, S. aureus-induced MC activation was dependent on the c-kit receptor-activated phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway, which was confirmed by treatment with Masitinib (a c-kit receptor inhibitor), Wortmannin (a PI3K inhibitor), and pyrrolidine dithiocarbamate (a NF-κB inhibitor), respectively. Conclusions The present study identifies the critical role of MCs in the host defense against S. aureus infection. inflammation response, mast cells, Staphylococcus aureus, TNF-α Mast cells (MCs) are a minority population of cells in the blood stream but prominently occur in the skin, lungs, digestive tract, and the nose [1, 2]. Functionally, MCs play an important role in regulation of angiogenesis, immune tolerance, and defense against pathogens, but the best-known role of MCs is in allergy reaction due to their granule-containing histamine and heparin [3, 4]. Further research on MCs and their functions in infectious diseases could help in better understanding the inmate immunity and host defense against pathogens, which is of clinical significance. Staphylococcus aureus is one of major pathogens in the skin and soft-tissue infections, which lead to abscess, cellulitis, folliculitis, and impetigo, resulting in a significant public health problem [5, 6]. Methicillin-resistant S. aureus could lead to complicated and difficult treatment of skin infection [5]. Especially in the USA 300 strain, the difficulty of treatment depended mainly on virulence other than antibiotic resistance [7]. A couple of virulent factors, including delta-hemolysin (Hld), phenol soluble modulins (PSMs), alpha-hemolysin (Hla), and Panton-Valentine leucocidin (PVL), were involved in the pathogenesis of S. aureus infection [8–11]. Human skin is composed of the epidermis and dermis, providing physical protection against S. aureus infection. A variety of immune cells are present in skin tissues [12, 13]. MCs in the skin function as sentinels in the host immunity and are activated by invading pathogens or inflammatory mediators. MC-derived cytokines participate in the inflammatory process, such as the recruitment of neutrophils and other immune cells into the infected site, resulting in pathogen elimination. MCs per se also have been reported to produce and secrete antimicrobial peptides [14, 15]. Indeed, MCs have been shown to play crucial roles in the clearance of group A Streptococcus, Pseudomonas aeruginosa, and vaccinia virus and improve the outcome of skin infectious diseases [16–18]. KitW-sh/W-sh mice with the KitW-sh inversion mutation, which were generated by Grimbaldeston et al [19, 20], have been shown to have MC deficiency in all tissues, but remained intact for major classes of other differentiated hematopoietic and lymphoid cells. In the present study, the cutaneous infection model of MC-deficient mice (KitW-sh/W-sh) was used to dissect the function of MCs in controlling S. aureus infection. MATERIALS AND METHODS Animals Mast cell-deficient mice (KitW-sh/W-sh) with C57BL/6 background were kindly provided by Dr. Guo-Ping Shi of Harvard Medical School [21], whereas age- and gender-matched wild-type (WT) C57BL/6 mice were used as controls. All animal experiments were approved by the Animal Care and Use Committee of Zhejiang University. Bacterial Culture The S. aureus strain was a clinical isolate (multilocus sequence type ST15 and agr type II) with Hld+, PSMα+, Hla+, and PVL−. Staphylococcus aureus was grown in tryptic soy broth (TSB) at 37°C with 5% CO2, with shaking (200rpm) to the mid-logarithmic phase. Then, the bacteria were resuspended in phosphate-buffered saline (PBS). The concentrations were determined by measuring the absorbance at 600 nm and verified using colony-forming units (CFUs) assay on TSB agar [22]. Cell Culture An MC line P815 was obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 4.5 mg/mL glucose, in a humidified incubator with 5% CO2 at 37°C. Bone marrow MCs (BMMCs) were generated from the femoral bone marrow cells of mice and maintained with the presence of 10% pokeweed mitogen-stimulated spleen-conditioned medium, as previously described [23]. After 4 weeks of culture, more than 99% of cells developed into MCs. BMMC and P815 cells (1 × 106/well) were treated with Masitinib (10 μM; Selleck) for 30 minutes before S. aureus stimulation for 4 h and 8 h (Figure 5 and 6), while P815 cells were treated with Masitinib for 30 minutes before S. aureus infection for 15, 30, and 60 minutes (Figure 7A and B [WB]). P815 cells (1 × 106/well) were pretreated with Wortmannin (10 μM; Abmole) or pyrrolidine dithiocarbamate ([PDTC] 25 μM; Abmole) for 30 minutes before 4 h exposure to S. aureus (Figure 7C and D). A Mouse Model of Staphylococcus aureus Skin Infection Mice, with 6–8 weeks old, on a C57BL/6 genetic background were used in S. aureus infection experiments. The mouse skin on the posterior back was shaved and intradermally injected with 100 µL S. aureus at 3 × 107 CFUs, while sham mice were given 100 µL PBS injections. For recombinant tumor necrosis factor (rTNF)-α treatment experiments, 200 ng of mouse rTNF-α (Peprotech EC Ltd.) in 100 µL normal saline was intradermally injected along with 100 µL S. aureus (3 × 107 CFUs) into WT or KitW-sh/W-sh mice. The size of the lesion was monitored and measured using Image J software (National Institutes of Heath). Mice were euthanized and skin specimens were collected for histology at indicated time points after infection. Some skin specimens were weighted and homogenized in 1 mL PBS using a homogenizer (Jingxin) for assessment of bacterial load and cytokine levels. Reconstitution of Mast Cells in KitW-sh/W-sh Mice For the reconstitution of MCs in knockout mice, BMMCs (106 cells in 200 µL DMEM) were injected into the shaved back skin of KitW-sh/W-sh mice at 4 weeks old. Then, these mice were cutaneously infected with S. aureus at 4 weeks after BMMC injection. Histological Analysis Mouse skin specimens were fixed in 10% paraformaldehyde and embedded in paraffin. Four micrometer-thick tissue sections were stained with hematoxylin-eosin (H&E). The stained sections were reviewed for morphology and subjected to morphometric analysis under a photomicroscope (Leica). To evaluate the infiltration of neutrophils into the skin tissue, 6 randomly selected fields (magnification, ×50) were examined, and the average value of cells/mm2 was determined for each skin sample. Immunohistochemistry Four micrometer sections of paraffin-embedded skin tissues were blocked with 0.5% bovine serum albumin (BSA) for 30 minutes and incubated with anti-myeloperoxidase (MPO) antibody (Servicebio). Subsequently, a secondary antibody (Servicebio) was added and incubated at 37°C for 60 minutes. The stained sections were reviewed for morphology and subjected to morphometric analysis under a photomicroscope (Leica). Immunofluorescent Staining The paraffin-wax sections of murine skin tissue were placed on glass slides. The nonspecific binding of antibodies was blocked by incubation with 1% BSA for 1 hour before incubation with primary antibodies. Alexa Fluor 488 anti-mouse CD117 (c-Kit) antibody (1: 50; BioLegend) and phycoerythrin antimouse TNF-α antibody (1: 50; BioLegend) were mixed and used for double staining. The sections were incubated with primary antibodies overnight at 4°C, followed by incubation at 37°C for 30 minutes. Then, 4’,6-diamidino-2-phenylindole was added for nuclei detection before viewing by confocal microscopy (Olympus). Quantitative Reverse-Transcription Polymerase Chain Reaction Ribonucleic acid was isolated from the skin specimens or MCs using TRIzol reagent (Invitrogen) and reversely transcribed into complementary deoxyribonucleic acid (cDNA) using the reverse-transcriptase cDNA synthesis system (Applied Biosystems). Quantitative polymerase chain reaction was performed using a Sequence Detection Software (Bio-Rad). The primer sequences of target genes are listed in Supplementary Table 1. Enzyme-Linked Immunosorbent Assay The levels of interleukin (IL)-β, IL-6, TNF-α, and cathelicidin-related antimicrobial peptide (CRAMP) in the skin homogenates and culture supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems Inc.). Western Blotting Western blotting was performed as previously described [24]. Antibodies were used against the following proteins: PI3K, p-PI3K, P65 NF-κB, p-P65 NF-κB, and β-actin (Cell Signaling Technology); AKT and p-AKT (Abcam). The protein signal was developed using the enhanced chemiluminescence reagent (Lianke) and detected using a digital image system (Proteinsimple). The signal intensity was further quantified using Image J software (National Institutes of Health). Antimicrobial Assay To assess the direct antibacterial effect of the conditional medium (CM) derived from the MC culture, 1 × 106 of cells were infected with S. aureus (MOI, 20) and cultured for 4 hours. The cell culture medium was passed through a 0.2-μm pore-sized filter. One hundred CFUs of S. aureus (10 μL) were incubated with the CM (90 μL) for 0.5 hours, 1 hour, 3 hours, and 16 hours, respectively, at 37°C before CFU counting. Statistical Analysis All results were expressed as mean ± standard error, unless otherwise stated. The statistical significance of the difference between 2 or multiple groups was analyzed, using Student’s t test or one-way analysis of variance as appropriate. Colony-forming units in the tissue homogenates were compared using the Mann-Whitney U test. A P value <.05 was considered statistically significant. RESULTS KitW-sh/W-sh Mice Develop Larger Skin Lesions After Cutaneous Staphylococcus aureus Infection Compared With Wild-Type Mice To investigate whether MCs contribute to host defense against skin S. aureus infection, WT and KitW-sh/W-sh mice were intradermally inoculated with S. aureus at 3 × 107 CFUs per mouse. The sizes of the skin lesions were recorded over time (Figure 1A and B). It was found that KitW-sh/W-sh mice developed larger skin lesions than WT animals and the maximum size reached up to 85.80 ± 8.11 mm2 at day 3. The difference in skin lesions between both groups appeared to be indistinguishable at day 14. Moreover, bacterial load in the skin tissue of KitW-sh/W-sh mice was 10-fold higher than those in WT mice 3 days after infection (Figure 1C). Although there was no difference in skin neutrophil count between uninfected KitW-sh/W-sh and WT mice, a remarkable reduction in neutrophil level was observed in the skin tissues of KitW-sh/W-sh mice through H&E and MPO staining 72 hours after intradermal infection, compared with S. aureus-stimulated WT mice (Figure 1D and E). Figure 1. View largeDownload slide KitWsh/Wsh mice display susceptibility to Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 1, 3, 5, 7, and 14 after infection. (B) Lesion area (mm2). (C) Bacterial load at day 3 postinfection. Individual values and median were shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections at day 3 postinfection. *, P < .05 and **, P < .01. Abbreviation: PBS, phosphate-buffered saline. Figure 1. View largeDownload slide KitWsh/Wsh mice display susceptibility to Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 1, 3, 5, 7, and 14 after infection. (B) Lesion area (mm2). (C) Bacterial load at day 3 postinfection. Individual values and median were shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections at day 3 postinfection. *, P < .05 and **, P < .01. Abbreviation: PBS, phosphate-buffered saline. Mast Cell Deficiency Reduces Inflammation Response to Staphylococcus aureus Infection Next, the effect of MC deficiency on inflammation response to S. aureus infection was evaluated. The levels of inflammation cytokines, including IL-1β, IL-6, IL-17A, keratinocyte-derived cytokine, macrophage inflammatory protein-2, TNF-α, and antibacterial peptides such as CRAMP and regenerating islet-derived IIIγ were detected (Figure 2A and B). The data revealed that the expression of TNF-α, IL-6, IL-17A, and CRAMP was significantly lower in skin specimens obtained from KitW-sh/W-sh mice than those from WT mice 3 days after infection. To determine the main cell source of TNF-α in the infected skin, tissue sections were co-immunostained for the expression of c-kit (the marker for MCs) and TNF-α. The confocal imagines clearly revealed that S. aureus infection was able to robustly activate TNF-α expression in MCs of WT mice (Figure 2C). Figure 2. View largeDownload slide KitW-sh/W-sh mice exhibit an impaired inflammation response upon skin Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5 per group) were intradermally injected with S. aureus. (A) Transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP-2), tumor necrosis factor (TNF)-α, cathelicidin-related antimicrobial peptide (CRAMP), and regenerating islet-derived IIIγ (RegIIIγ)were detected using quantitative reverse-transcription polymerase chain reaction at day 3 after infection. (B) Protein levels of IL-1β, IL-6, TNF-α, and CRAMP were analyzed using enzyme-linked immunosorbent assay at day 3 after infection. (C) Enhanced production of TNF-α was observed in c-kit+ skin mast cells of WT mice 6 hours postinfection. Magnification, ×180; scale bar, 20 μm. *, P < .05 and **, P < .01. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 2. View largeDownload slide KitW-sh/W-sh mice exhibit an impaired inflammation response upon skin Staphylococcus aureus infection. Wild-type (WT) and KitW-sh/W-sh mice (n = 5 per group) were intradermally injected with S. aureus. (A) Transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP-2), tumor necrosis factor (TNF)-α, cathelicidin-related antimicrobial peptide (CRAMP), and regenerating islet-derived IIIγ (RegIIIγ)were detected using quantitative reverse-transcription polymerase chain reaction at day 3 after infection. (B) Protein levels of IL-1β, IL-6, TNF-α, and CRAMP were analyzed using enzyme-linked immunosorbent assay at day 3 after infection. (C) Enhanced production of TNF-α was observed in c-kit+ skin mast cells of WT mice 6 hours postinfection. Magnification, ×180; scale bar, 20 μm. *, P < .05 and **, P < .01. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Adoptive Transfer of Bone Marrow Mast Cells Reconstitutes Host Defense Against Staphylococcus aureus Infection in KitW-sh/W-sh Mice To determine the susceptibility of KitW-sh/W-sh mice to S. aureus, which was mainly attributed to MC deficiency, we performed an adoptive MC transfer experiment. The results showed that KitW-sh/W-sh mice treated with WT-derived BMMCs had significantly smaller lesions than MC-deficiency mice at day 3 after the infection (Figure 3A and B). Correspondingly, MC-reconstituted KitW-sh/W-sh mice exhibited markedly improved bacterial clearance to S. aureus (Figure 3C). The impaired recruitment of neutrophils in KitW-sh/W-sh mice was largely reversed after the adoptive injection of BMMCs upon infection (Figure 3D and E). Simultaneously, the levels of TNF-α and CRAMP in MC-reconstituted KitW-sh/W-sh mice significantly increased after infection, compared with non-MC-treated animals (Figure 3F). Figure 3. View largeDownload slide Bone marrow mast cell (BMMC) transfer reconstitutes host resistance to Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT), KitW-sh/W-sh, and reconstituted KitW-sh/W-sh mice (n = 6 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 3 after infection. (B) Lesion area (mm2) at day 3 postinfection. (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections 3 days after infection. (F) Enzyme-linked immunosorbent assay of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and cathelicidin-related antimicrobial peptide (CRAMP) 3 days after infection. *, P < .05 and **, P < .01. Figure 3. View largeDownload slide Bone marrow mast cell (BMMC) transfer reconstitutes host resistance to Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT), KitW-sh/W-sh, and reconstituted KitW-sh/W-sh mice (n = 6 per group) were intradermally injected with S. aureus. (A) Representative photographs of lesions at day 3 after infection. (B) Lesion area (mm2) at day 3 postinfection. (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E)- and myeloperoxidase (MPO)-stained skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained tissue sections 3 days after infection. (F) Enzyme-linked immunosorbent assay of interleukin (IL)-6, tumor necrosis factor (TNF)-α, and cathelicidin-related antimicrobial peptide (CRAMP) 3 days after infection. *, P < .05 and **, P < .01. Exogenous Recombinant Tumor Necrosis Factor-α Administration Restores Immune Response Against Staphylococcus aureus Infection in KitW-sh/W-sh Mice Neutrophils are essential for the effective clearance of S. aureus in lesions. Given that MC-derived TNF-α expression after infection could significantly enhance Th17 cell-dependent neutrophil-rich inflammatory response [25, 26], we explored whether the administration of rTNF-α rescued KitW-sh/W-sh mice with skin S. aureus infection. In KitW-sh/W-sh mice, it was found that treatment with 200 ng of rTNF-α resulted in a significant reduction in lesion size 3 days after infection, when compared with vehicle treatment (Figure 4A and B). KitW-sh/W-sh mice also exhibited a reduction in bacterial load after the administration of rTNF-α, along with more abundant neutrophil infiltration (Figure 4C–E), and an increase in the expression of IL-17A and CRAMP (Figure 4F). These results demonstrate that TNF-α participates in MC-mediated skin immune response to S. aureus infection. Figure 4. View largeDownload slide Recombinant tumor necrosis factor (rTNF)-α restores host defense against cutaneous Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 mice per group) were intradermally injected with 100 µL mouse rTNF-α (200 ng) or normal saline (NS) along with 100 µL S. aureus (3 × 107 colony-forming units [CFU]). (A) Representative photographs of lesions at day 1, 3, 5, and 7 after infection. (B) Lesion area (mm2). (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E) and myeloperoxidase (MPO) staining of skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained slides 3 days after infection. (F) Transcriptional levels of interleukin (IL)-17A and cathelicidin-related antimicrobial peptide (CRAMP) were detected using quantitative reverse-transcription polymerase chain reaction 3 days after infection.*, P < .05 and **, P < .01. Abbreviation: mRNA, messenger ribonucleic acid. Figure 4. View largeDownload slide Recombinant tumor necrosis factor (rTNF)-α restores host defense against cutaneous Staphylococcus aureus infection in KitW-sh/W-sh mice. Wild-type (WT) and KitW-sh/W-sh mice (n = 5–10 mice per group) were intradermally injected with 100 µL mouse rTNF-α (200 ng) or normal saline (NS) along with 100 µL S. aureus (3 × 107 colony-forming units [CFU]). (A) Representative photographs of lesions at day 1, 3, 5, and 7 after infection. (B) Lesion area (mm2). (C) Staphylococcus aureus counts in the skin lesions at day 3 after infection. Individual values and median are shown for each group. (D) Representative hematoxylin-eosin (H&E) and myeloperoxidase (MPO) staining of skin tissue sections at day 3 after infection. Magnification, ×50 or ×400; scale bar, 200 μm. (E) Neutrophil counts in H&E-stained slides 3 days after infection. (F) Transcriptional levels of interleukin (IL)-17A and cathelicidin-related antimicrobial peptide (CRAMP) were detected using quantitative reverse-transcription polymerase chain reaction 3 days after infection.*, P < .05 and **, P < .01. Abbreviation: mRNA, messenger ribonucleic acid. Staphylococcus aureus-Induced Mast Cell Activation Is Dependent on the Expression of c-Kit Receptor Previous studies have showed the importance of c-kit receptor in mediating MC activity [27, 28]. To assess the role of c-kit receptor in controlling S. aureus infection, the expression of kinds of inflammatory mediators in BMMCs was investigated (Figure 5A). It was found that Masitinib, the c-kit receptor inhibitor, could markedly reduce the levels of IL-6 and TNF-α from MCs after infection. Moreover, the data were consistent with those obtained from immortalized P815 MCs after incubation with S. aureus (Figure 6A), confirming that c-kit receptor activation is involved in the functional regulation of MCs against bacterial infection. Di Nardo et al [16] demonstrated that activated murine MCs produced peptides such as CRAMP (the murine homolog of human LL-37), and they showed potent antimicrobial activity. The current data also revealed that the levels of CRAMP in MCs (BMMCs and P815) were significantly increased after exposure to S. aureus. Furthermore, Masitinib remarkably reduced the bacteria-induced expression of CRAMP, along with the attenuated antimicrobial potency of P815 cells against S. aureus (Figure 5B and Figure 6B and C). These results reveal that c-kit-related signaling participates in the antibacterial immune response of MCs. Figure 5. View largeDownload slide Staphylococcus aureus induces bone marrow mast cell (BMMC) activation dependent of C-kit receptor. Bone marrow mast cells pretreated or not pretreated with Masitinib for 30 minutes were incubated with S. aureus. (A) The transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α in BMMCs were analyzed using quantitative reverse-transcription polymerase chain reaction at 4 hours and 8 hours after infection. (B) The transcriptional levels of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) 4 hours and 8 hours after infection *, P < .05 and **, P < .01. Abbreviations: mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 5. View largeDownload slide Staphylococcus aureus induces bone marrow mast cell (BMMC) activation dependent of C-kit receptor. Bone marrow mast cells pretreated or not pretreated with Masitinib for 30 minutes were incubated with S. aureus. (A) The transcriptional levels of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α in BMMCs were analyzed using quantitative reverse-transcription polymerase chain reaction at 4 hours and 8 hours after infection. (B) The transcriptional levels of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) 4 hours and 8 hours after infection *, P < .05 and **, P < .01. Abbreviations: mRNA, messenger ribonucleic acid; PBS, phosphate-buffered saline. Figure 6. View largeDownload slide Staphylococcus aureus induces P815 cell activation via the c-kit receptor. P815 cells were pretreated or not pretreated with Masitinib for 30 minutes before S. aureus stimulation. (A) The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) detection of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed in P815 cells at 4 hours and 8 hours after bacterial incubation. (B) The qRT-PCR detection of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) mRNA 4 hours and 8 hours after infection. (C) The antimicrobial activity of the conditional medium from P815 cells after S. aureus infection. *, P < .05 and **, P < .01; ∆P < .05 vs phosphate-buffered saline (PBS) group; #P < .05 vs S. aureus + Masitinib group. Abbreviation: CFU, colony-forming units. Figure 6. View largeDownload slide Staphylococcus aureus induces P815 cell activation via the c-kit receptor. P815 cells were pretreated or not pretreated with Masitinib for 30 minutes before S. aureus stimulation. (A) The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) detection of interleukin (IL)-1β, IL-6, IL-17A, keratinocyte-derived cytokine (KC), macrophage inflammatory protein (MIP)-2, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed in P815 cells at 4 hours and 8 hours after bacterial incubation. (B) The qRT-PCR detection of cathelicidin-related antimicrobial peptide (CRAMP) and regenerating islet-derived IIIγ (RegIIIγ) mRNA 4 hours and 8 hours after infection. (C) The antimicrobial activity of the conditional medium from P815 cells after S. aureus infection. *, P < .05 and **, P < .01; ∆P < .05 vs phosphate-buffered saline (PBS) group; #P < .05 vs S. aureus + Masitinib group. Abbreviation: CFU, colony-forming units. C-kit/PI3K/AKT/p65-NF-κB Signaling Mediates Antimicrobial Activity and Inflammation Response of Mast Cells to Staphylococcus aureus Infection Finally, the underlying molecular mechanisms by which MCs produced TNF-α and CRAMP after S. aureus infection were investigated. The present data in Figure 7A and B revealed that S. aureus profoundly induced the phosphorylation of PI3K, AKT, and P65-NF-κB in MCs, which was remarkably attenuated by pretreatment with the c-kit receptor inhibitor Masitinib. However, Masitinib alone had no effect on the phosphorylation of these molecules in non-infected cells (Supplementary Figure 1). The S. aureus-induced production of TNF-α and CRAMP was significantly reduced by pretreatment with the specific PI3K inhibitor Wortmannin or the specific NF-κB inhibitor PDTC (Figure 7C and D). Therefore, S. aureus-induced MC activation was dependent on c-kit receptor-activated PI3K/AKT/P65-NF-κB signaling. Figure 7. View largeDownload slide The c-kit/phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway mediates immune response in mast cells upon Staphylococcus aureus challenge. (A and B) Mast cells P815 were pretreated or not pretreated with Masitinib for 30 minutes. The phosphorylation of PI3K, AKT, and P65-NF-ĸB was detected by Western blotting at 15, 30, and 60 minutes postinfection. (C) Mast cells P815 were incubated with dimethyl sulfoxide (DMSO), Wortmannin, and pyrrolidine dithiocarbamate (PDTC), respectively, for 30 minutes before 4 hours exposure to S. aureus. The quantitative reverse-transcription polymerase chain reaction analysis of cathelicidin-related antimicrobial peptide (CRAMP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed. (D) The enzyme-linked immunosorbent assay of CRAMP, IL-6, and TNF-α was performed at 4 hours after infection. *, P < .05 and **, P < .01. Figure 7. View largeDownload slide The c-kit/phosphoinositide 3-kinase (PI3K)/AKT/P65-nuclear factor (NF-κB) pathway mediates immune response in mast cells upon Staphylococcus aureus challenge. (A and B) Mast cells P815 were pretreated or not pretreated with Masitinib for 30 minutes. The phosphorylation of PI3K, AKT, and P65-NF-ĸB was detected by Western blotting at 15, 30, and 60 minutes postinfection. (C) Mast cells P815 were incubated with dimethyl sulfoxide (DMSO), Wortmannin, and pyrrolidine dithiocarbamate (PDTC), respectively, for 30 minutes before 4 hours exposure to S. aureus. The quantitative reverse-transcription polymerase chain reaction analysis of cathelicidin-related antimicrobial peptide (CRAMP), interleukin (IL)-6, and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) was performed. (D) The enzyme-linked immunosorbent assay of CRAMP, IL-6, and TNF-α was performed at 4 hours after infection. *, P < .05 and **, P < .01. DISCUSSION Mast cells have been shown to contribute to innate immune response against pathogens in addition to the role in allergic responses [16, 17]. However, the roles of MCs in protecting the skin against bacterial infection remain to be defined. This study utilized a cutaneous infection model of MC-deficient (KitW-sh/W-sh) mice to assess the effects of MCs on S. aureus infection. The present data revealed that MCs play an essential role in the host defense against cutaneous S. aureus infection. In particular, upon S. aureus infection, MCs released TNF-α to mediate neutrophil recruitment to the infection site, resulting in the promotion of bacterial clearance. Furthermore, MC-derived TNF-α was regulated by the activation of the c-kit-mediated PI3K/AKT/p65-NF-κB signaling pathway. During skin infection, a variety of immune cells and cytokines could coordinate and orchestra host skin immunity [29, 30]. Previous studies have shown that the activation of MCs is evolved in host defense against bacteria. Siebenhaar et al [17] found that MCs are important for the clearance for P. aeruginosa through the upregulation of endothelin-1 and the accumulation of neutrophil infiltrations in P. aeruginosa- infected skin. Di Nardo et al [16] demonstrated that MCs were able to trigger the anti-group A Streptococcus immunity in the skin, in part, by releasing CRAMP. The antimicrobial peptide CRAMP released from skin MCs was shown to inactivate vaccinia virus infectivity [18]. Furthermore, MCs have been shown to play an essential role in defending against Chlamydia pneumoniae lung infection by recruiting neutrophils and lymphocytes into the airspace [31]. However, peritoneal MCs have no effect on S. aureus growth after intraperitoneal infection, although they could be activated in vitro [32]. Lê et al [33] found that the activation of MCs induced by P. aeruginosa contributes to the increase in alveolar-capillary permeability. Therefore, the precise role of MCs in the host immunity could be dependent on the types of pathogens and sites of infection. In the current study, using KitW-sh/W-sh mice, we demonstrated that MC-deficient mice were significantly more susceptible to S. aureus skin infection than WT mice, along with larger skin lesions and higher bacterial loads. To rule out the broader effect from loss of c-Kit in KitW-sh/W-sh mice, an adoptive transfer of BMMCs into the skin of KitW-sh/W-sh mice before S. aureus infection was further performed. Compared with controls, the knockout mice that received WT-derived BMMCs had significantly smaller lesions and markedly decreased bacterial burden 3 days after the infection. Consistently, the impaired recruitment of neutrophils and suppressed inflammatory response were largely reconstituted in KitW-sh/W-sh mice with adoptive BMMC administration. Therefore, the present study revealed that MC deficiency definitely leads to impaired host defense against skin S. aureus infection. However, it should be noted that the Kit-independent MC-deficient Mcpt5-Cre+ × R-DTA mice would be an additional ideal mouse model for the verification of the role of MCs in diverse models of diseases [32, 34, 35]. Instant and appropriate inflammatory response is essential for cutaneous immunity against pathogens. Tumor necrosis factor-α is an early response cytokine that facilitates neutrophil infiltration into the lesion site and clears pathogens [36, 37]. The bacterial load was significantly higher in the S. aureus-induced experimental brain abscess in TNF-α−/− mice, when compared with WT mice [38]. Mast cell-derived TNF enhanced Th17 cell-dependent inflammatory response with rich neutrophil infiltration in the airway [25], and IL-17 from resident epidermal γδT cells promoted neutrophil recruitment into the site of skin S. aureus infection [26]. The blockade of TNF-α by infliximab was able to reduce the expression of inflammation cytokines including IL-17 [39]. The present study revealed that TNF-α was rapidly produced after S. aureus infection, and involved in neutrophil recruitment into the skin lesion. This finding was further supported by the fact that the administration of rTNF-α restored host resistance to S. aureus in MC-deficient mice. Previous studies have supported that CRAMP produced by MCs contributed to host defense against pathogens in skin lesions infected by group A Streptococcus and vaccinia virus [16, 18]. Our data revealed that upregulated expression of IL-17A and CRAMP occurred in skin lesions injected with rTNF-α, which enhanced host antibacterial immunity. Taken together, these results demonstrate that MC-facilitated inflammatory response, which is mainly mediated by TNF-α, would be beneficial to the ultimate outcome of skin S. aureus infection. Staphylococcus aureus can cause serious skin infections due to the expression of diverse toxins such as PVL, Hla, Hld, and other cytolytic toxins, which are able to initiate the innate immune system. Previous studies have shown that PVL and Hla contribute to the pathology of tissues through the activation of macrophages and recruitment of excessive neutrophils [5, 9]. Furthermore, the host immune system, as a target for invading pathogens, recognizes them through pattern recognition receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [10, 22]. Thus, understanding the host response to bacteria and their elements would be helpful for developing novel therapeutic strategies. TLRs sense pathogen-associated molecular patterns from pathogens and primarily mediate signaling via its interaction with adaptor proteins MyD88 to activate the NF-κB pathways [22, 40, 41]. NOD2 stimulation also leads to direct activation of NF-κB and increases expression of its target genes. During skin infection, S. aureus-induced IL-6 response depends on the activation of NOD2 [10]. Blocking TLR or NOD with specific antibodies abolished peptidoglycan-induced activation of MCs [42]. In addition to TLRs and NOD, other receptors are also involved in response to pathogens. FimH receptor CD48 has been found to be able to detect the presence of Escherichia coli and S. aureus [43, 44]. CD48 triggered the TNF-α release in MCs after E. coli infection through FimH fimbrial adhesion [43]. Similarly, infection of human cord blood-derived MCs with S. aureus induced CD48 activation and TNF-α release [44]. The c-kit has recently been shown to be essential in the mediation of MC maturation and survival. Stem cell factor stimulation led to secondary signaling events, including the phosphorylation of PI3K, AKT, and p65-NF-κB proteins in cells [45, 46]. Our previous studies have defined that the activation of AKT and NF-κB by S. aureus was a critical molecular mechanism for maintaining the balance between host antibacterial immunity and tissue injury [22, 24]. Oviedo-Boyso et al [47] reported that PI3K-AKT-P65 pathway was also important for the internalization of S. aureus in endothelial cells. Our current data showed that, upon S. aureus infection, skin MCs were able to produce and secrete TNF-α and CRAMP through the activation of the c-kit-dependent PI3K-AKT-p65-NF-κB signaling pathway. CONCLUSIONS In conclusion, we have demonstrated that MCs facilitate neutrophil recruitment to the infection site and prompt cutaneous host defense against S. aureus. The c-kit-mediated TNF-α production of MCs initiates this critical early innate immune response. The present study provides a novel therapeutic strategy to augment and regulate the function of MCs to combat S. aureus infection. Supplementary Data Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. Notes Financial support. This work was supported by grants from the National Natural Science Foundation of China (81770008, 81570005, and 81370176) and Major Science and Technology Special Project of Zhejiang Province (2014C03033). Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. References 1. Gibson S , Miller HR . Mast cell subsets in the rat distinguished immunohistochemically by their content of serine proteinases . Immunology 1986 ; 58 : 101 – 4 . Google Scholar PubMed 2. Caughey GH . Mast cell proteases as protective and inflammatory mediators . Adv Exp Med Biol 2011 ; 716 : 212 – 34 . Google Scholar CrossRef Search ADS PubMed 3. da Silva EZ , Jamur MC , Oliver C . Mast cell function: a new vision of an old cell . J Histochem Cytochem 2014 ; 62 : 698 – 738 . Google Scholar CrossRef Search ADS PubMed 4. Caughey GH . Mast cell proteases as pharmacological targets . 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Journal

The Journal of Infectious DiseasesOxford University Press

Published: May 8, 2018

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