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The Journal of Immunology, 2007, 179, 6933 -6942
Copyright © 2007 by The American Association of Immunologists, Inc.
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Inflammasome-Mediated Production of IL-1β Is Required for Neutrophil Recruitment against Staphylococcus aureus In Vivo1

Lloyd S. Miller2,*, Eric M. Pietras2,{dagger}, Lawrence H. Uricchio*, Kathleen Hirano*, Shyam Rao*, Heping Lin{ddagger}, Ryan M. O’Connell§, Yoichiro Iwakura, Ambrose L. Cheung{ddagger}, Genhong Cheng3,{dagger} and Robert L. Modlin3,*,{dagger}

* Division of Dermatology and {dagger} Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095; {ddagger} Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, NH 03755; § Department of Biology, California Institute of Technology, Pasadena, CA 91125; and Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IL-1R activation is required for neutrophil recruitment in an effective innate immune response against Staphylococcus aureus infection. In this study, we investigated the mechanism of IL-1R activation in vivo in a model of S. aureus infection. In response to a S. aureus cutaneous challenge, mice deficient in IL-1β, IL-1{alpha}/IL-1β, but not IL-1{alpha}, developed larger lesions with higher bacterial counts and had decreased neutrophil recruitment compared with wild-type mice. Neutrophil recruitment and bacterial clearance required IL-1β expression by bone marrow (BM)-derived cells and not by non-BM-derived resident cells. In addition, mice deficient in the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) had the same defects in neutrophil recruitment and host defense as IL-1β-deficient mice, demonstrating an essential role for the inflammasome in mediating the production of active IL-1β to promote neutrophil recruitment in host defense against S. aureus. This finding was further supported by the ability of recombinant active IL-1β to control the infection and promote bacterial clearance in IL-1β-deficient mice. These studies define a key host defense circuit where inflammasome-mediated IL-1β production by BM-derived cells signals IL-1R on non-BM-derived resident cells to activate neutrophil recruitment in the innate immune response against S. aureus in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Staphylococcus aureus is a Gram-positive bacterium that is responsible for the vast majority of skin and soft tissue infections in humans, such as impetigo, folliculitis/furunculosis, and cellulitis, which result in 11.6 million outpatient and emergency room visits and 464,000 hospital admissions per year in the United States (1). Although S. aureus infections usually originate in the skin, invasive and frequently life-threatening infections are common sequelae, including lymphangitis, septic arthritis, abscesses of various organs, osteomyelitis, bacteremia, pneumonia, meningitis, endocarditis, and sepsis (2, 3, 4). In addition, many community- and hospital-acquired S. aureus infections have been complicated by virulent antibiotic resistant strains such as methicillin-resistant S. aureus and multidrug-resistant strains (5, 6, 7, 8, 9).

Neutrophil recruitment and abscess formation is a hallmark of S. aureus infections and is required for elimination of the pathogen (10, 11). IL-1R activation plays a critical host defense role against S. aureus brain abscesses, septic arthritis, and systemic infections (12, 13, 14). Using a cutaneous mouse infection model, we previously reported that IL-1R-deficient mice developed larger lesions with higher bacterial counts compared with wild-type (wt)4 mice. Moreover, the lesions of IL-1R-deficient mice had severely decreased neutrophil recruitment and defective induction of the neutrophil chemokines KC and MIP2 (15). In a similar S. aureus cutaneous infection model, neutrophil-depleted mice (using an anti-Gr-1 mAb (clone RB6-8C5)) developed large nonhealing skin lesions and failed to clear S. aureus from these lesions (10). Thus, IL-1R-deficient mice and neutrophil-depleted mice share a similar phenotype, suggesting that IL-1R-mediated neutrophil recruitment is crucial for host defense against S. aureus skin infection (16).

IL-1{alpha} and IL-1β are the known primary ligands that activate IL-1R signaling (17, 18, 19, 20). IL-1{alpha} is constitutively expressed by epithelial cells, including keratinocytes, and is released upon nonspecific injury or infection (17, 20, 21). IL-1β is predominantly produced by activated immune cells such as monocytes/macrophages, dendritic cells, and Langerhans cells (17, 18, 19, 20, 21). Previous studies involving the skin have demonstrated a key role for IL-1{alpha} in the pathogenesis of contact dermatitis, inflammatory dermatitis, and in protection against chemical-induced skin carcinomas (22, 23, 24). IL-1β activity in the skin has been implicated in the pathogenesis of contact dermatitis and psoriasis (25, 26, 27, 28). The inflammasome, which facilitates caspase-1 activation, has been shown to be important in posttranslational processing of IL-1β into its active form (29, 30, 31, 32, 33, 34, 35). Furthermore, the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is required for the generation of active IL-1β in S. aureus-stimulated bone marrow (BM)-derived macrophages in vitro (36). In the present study, we investigated the requirement and differential contribution of IL-1{alpha}, IL-1β, and ASC to IL-1R-mediated neutrophil recruitment and host defense against a cutaneous S. aureus challenge in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
S. aureus strains

All strains used were derived from S. aureus strain RN6390, which has a known 11-bp deletion in the rsbU gene within the sigB operon (37). Strain SH1000 is a derivative of RN6390 with the rsbU gene restored (38). All data presented were obtained using the S. aureus bioluminescent strain ALC2906, which is strain SH1000 containing the shuttle plasmid pSK236 with the penicillin-binding protein 2 (pbp2) promoter fused to the luxABCDE reporter cassette from Photorhabdus luminescens as previously described (15). In control experiments, strain ALC2506, a nonbioluminescent control SH1000 strain containing pSK236 with a promoterless luxABCDE reporter cassette, was used (data not shown).

Preparation of S. aureus for skin inoculation

The S. aureus bioluminescent strain ALC2906 and nonbioluminescent strain ALC2506 have a chloramphenicol resistance plasmid selection marker and all cultures were performed in the presence of chloramphenicol (10 µg/ml; Sigma-Aldrich). S. aureus was streaked onto tryptic soy agar (tryptic soy broth (TSB) plus 1.5% bacto agar plus chloramphenicol; BD Biosciences/Sigma-Aldrich) and grown overnight at 37°C in a bacterial incubator. Colonies of S. aureus were grown overnight at 37°C in a shaking incubator (240 rpm) in TSB plus chloramphenicol. Mid-logarithmic phase bacteria were obtained after a 3-h subculture of a 1/50 dilution of the overnight culture. Bacterial cells were pelleted, resuspended, and washed three times in PBS. Bacterial concentrations were estimated by measuring the absorbance at 600 nm (A600; DU 640B spectrophotometer; Beckman Coulter). CFUs were verified by plating dilutions of the inoculum onto TSB agar ± chloramphenicol overnight.

Mice

All mice have a C57BL/6J genetic background. IL-1{alpha}-(F4), IL-1β-(F8), and IL-1{alpha}/IL-1β-(F11) deficient mice were generated as previously described (39). ASC-deficient mice were generated as previously described (40). Mice deficient in IL-1RI (IL1rtm1Imx) (F8) and wt control mice were obtained from The Jackson Laboratory. All of these mouse colonies at University of California (Los Angeles, CA (UCLA)) are pathogen free and are maintained in autoclaved cages.

Mouse model of cutaneous S. aureus infection

All procedures were approved by the UCLA Animal Research Committee. The mice were shaved on the back and inoculated s.c. with 100 µl of mid-logarithmic growth phase S. aureus strain ALC2906 (~2 x 106 CFUs/100 µl = 1/10 dilution of A600 of 0.5/ml) in sterile pharmacy grade saline (0.9%) using a 27-gauge tuberculin syringe (Abbott Laboratories). Groups of four to five mice were used in each experiment followed by one to two repeats to confirm results. Measurements of total lesion size (cm2) were made by analyzing digital photographs (Nikon Coolpix 5400) of mice taken every 1–3 days using the software program "Image J" (National Institutes of Health Research Services Branch (http://rsbweb.nih.gov/ij/)) and a millimeter ruler as a reference.

Quantification of in vivo S. aureus (in vivo bioluminescence)

In vivo bioluminescence was performed using the Xenogen IVIS imaging system at the Crump Institute for Molecular Imaging (UCLA) as previously described (15). Mice were anesthetized via an i.p. injection of a mix of ketamine and xylazine (100 and 20 mg/kg body weight, respectively). Data are presented on color scale overlaid on a grayscale photograph of mice and quantified as total flux (photons per second) within a circular region of interest (1 x 103 pixels) using Living Image software (Xenogen) (lower limit of detection: 1 x 104 photons/s).

Tissue embedding and staining

For histological analysis, lesional 8-mm punch biopsy (Acuderm) specimens were bisected and one half was fixed in formalin (10%) and embedded in paraffin and the other half was embedded in Tissue-Tek OTC compound (Sakura Finetek) and frozen in liquid nitrogen. H&E and Gram stains were performed on paraffin sections (4 µm) by the Tissue Procurement and Histology Core Laboratory and Histopathology Laboratory (UCLA), according to guidelines for clinical samples.

Immunoperoxidase labeling

Detection of Gr-1 (Ly-6G)-positive cells, Mac-3-positive cells, or IL-1β expression on frozen cryostat specimens of lesional skin punch biopsy specimens were performed with the following biotinylated mAbs: rat anti-Gr-1 mAb (clone RB6-8C5; IgG2b isotype; 1–5 µg/ml; BD Pharmingen), rat anti-Mac-3 mAb (clone CI:A3-1, IgG1 isotype; 1 µg/ml; Biolegend), or mouse anti-mouse IL-1β mAb (20 µg/ml; clone 1400.24.17; Pierce-Endogen) and corresponding biotinylated isotype control mAbs using the immunoperoxidase method as previously described (15, 41).

Myeloperoxidase (MPO) assay

MPO activity from lesional skin was obtained from tissue homogenates (Tissue-tearer; Biospec Products) of 8-mm punch biopsy specimens (Acuderm) using a MPO assay kit according to the manufacturer’s recommendations (Cytostore).

ELISAs

Protein levels of IL-1{alpha}, IL-1β, KC, and MIP2 (picograms per milligram of tissue) from lesional skin were obtained from tissue homogenates (Tissue-tearer; Biospec Products) of 8-mm punch biopsy specimens performed at various time points after S. aureus skin inoculation in 0.01% Triton using commercially available ELISA kits (KC and MIP2 obtained from R&D systems; IL-1{alpha} and IL-1β obtained from BD Pharmingen).

BM reconstitution

BM reconstitution experiments were performed as previously described (15, 42). BM was flushed from tibias and femurs from donor mice, RBC-depleted using ACK/RBC lysis buffer (0.15 M ammonium chloride (NH4Cl), 1 mM potassium bicarbonate (KHCO3), 0.1 mM EDTA (pH 7.3)), and washed twice in PBS. A total of 1 x 107 BM cells were injected into the tail veins of lethally irradiated (1000 rad) recipient mice. Reconstituted mice were maintained in autoclaved cages and were administered sulfamethoxazole and trimethoprim oral suspension (48 mg/ml in drinking water) for the first 3 wk postirradiation. All experiments were performed 8 wk after BM reconstitution. To verify the efficiency of reconstitution, BM was harvested from euthanized mice and differentiated into BM-derived macrophages (BMMs) as previously described (43). BMMs were stimulated with LPS (0.1 µg/ml) for 24 h and the presence or absence of IL-1β protein expression was determined by immunoblotting (data not shown).

Immunoblotting

IL-1β protein expression and processing in skin homogenates and in BMMs was assayed by immunoblotting using polyclonal Abs against the cleaved and active form of IL-1β (Asp116; rabbit anti-mouse 17-kDa cleaved IL-1β peptide; Cell Signaling Technology) and pro-IL-1β (goat-anti-mouse IL-1β; R&D Systems) as previously described (15, 44).

Administration of recombinant murine IL-1β

In some experiments (see Fig. 6), one dose of active recombinant murine IL-1β (rIL-1β; 50 ng/100 µl (R&D Systems)) in sterile pharmacy grade saline plus 0.1% endotoxin-free BSA (Sigma-Aldrich) as a carrier protein) or vehicle alone (saline plus 0.1% endotoxin-free BSA) was administered along with the s.c. inoculum of S. aureus (2 x 106 CFUs/100 µl) to IL-1β-deficient mice. This s.c. dose of rIL-1β (50 ng/100 µl) was previously shown in other models to be biologically active in vivo (45, 46). Previous studies have also demonstrated that rIL-1β does not have any direct antimicrobial activity against S. aureus (47, 48, 49).

Statistical analyses

Data were compared using a Student t test. All data are expressed as mean ± SEM where indicated. Values of p < 0.05, p < 0.01, and p < 0.001 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-1β- and IL-1{alpha}/IL-1β-, but not IL-1{alpha}-, deficient mice develop markedly larger skin lesions with higher bacterial counts compared with wt mice after skin inoculation with S. aureus

Using a mouse model of a localized cutaneous infection with S. aureus, we previously reported that IL-1R-deficient mice developed larger lesions with higher bacterial counts compared with wt mice (15). To investigate the differential contribution of the known activating ligands to IL-1R (i.e., IL-1{alpha} and IL-1β) to the IL-1R-deficient mouse phenotype, we used the same S. aureus cutaneous infection model and inoculated wt mice and IL-1{alpha}-, IL-1β-, and IL-1{alpha}/IL-1β-deficient mice with a bioluminescent strain of S. aureus (SH1000 strain, 2 x 106 CFUs/100 µl) (15). Lesion sizes and in vivo bioluminescence of live, actively metabolizing bacteria within the lesions over time were evaluated (Fig. 1). wt mice developed visible skin lesions by day 3, which had a maximum size of 0.55 ± 0.13 cm2, and healed by day 14 (Fig. 1, A and B). IL-1{alpha}-deficient mice developed skin lesions that did not significantly differ from lesion sizes of wt mice. In contrast, IL-1β- and IL-1{alpha}/IL-1β-deficient mice developed >3-fold larger lesions than wt mice (or IL-1{alpha}-deficient mice) that failed to completely heal by day 14 after the inoculation.


Figure 1
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  		FIGURE 1. IL-1β- and IL-1{alpha}/IL-1β- but not IL-1{alpha}-deficient mice develop markedly larger skin lesions with higher bacterial counts compared with wt mice after skin inoculation with S. aureus. IL-1{alpha}–/–, IL-1β–/–, IL-1{alpha}/IL-1β–/–, and wt mice were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS) (bioluminescent strain SH1000). A, Mean total lesion size (cm2) ± SEM. B, Representative photographs. C, Mean total flux (photons per second) ± SEM. D, Representative photographs of in vivo bioluminescence (Xenogen IVIS); *, p < 0.05; {dagger}, p < 0.01; {ddagger}, p < 0.001; IL-1{alpha}–/–, IL-1β–/–, or IL-1{alpha}/β–/– mice vs wt mice (Student’s t test).

 
To determine whether the larger lesions of IL-1β- and IL-1{alpha}/IL-1β-deficient mice were associated with a defect in bacterial clearance, we anesthetized mice and determined bacterial counts within the lesions over time using in vivo bioluminescence (Xenogen IVIS) (Fig. 1, C and D). We previously demonstrated that in vivo bioluminescence closely estimates bacterial CFUs harvested from the skin lesions at various time points after infection (15). Following infection with S. aureus, wt and IL-1{alpha}-deficient mice had similar bioluminescent signals that decreased over 14 days. In contrast, IL-1β- and IL-1{alpha}/IL-1β-deficient mice had increased bioluminescent signals (color scale) that were up to 10-fold higher (logarithmic scale) compared with wt mice at all time points following inoculation. Thus, IL-1β- and IL-1{alpha}/IL-1β-deficient mice have a defect in bacterial clearance, which likely explains the increased size and persistence of the skin lesions. Taken together, the phenotype of IL-1β-deficient mice closely resembled that of IL-1{alpha}/IL-1β-deficient mice, whereas the phenotype of IL-1{alpha}-deficient mice did not significantly differ from that of wt mice. These data suggest that IL-1β, and not IL-1{alpha}, is the key IL-1R ligand involved in IL-1R-mediated host defense against a cutaneous S. aureus challenge. To confirm this result, in a separate experiment wt mice and IL-1β- and IL-1{alpha}/IL-1β-, along with IL-1R- deficient mice were inoculated with S. aureus and lesion size and in vivo bioluminescence were measured as in Fig. 1 (data not shown). We found that IL-1β-, IL-1{alpha}/IL-1β-, and IL-1R-deficient mice all developed similarly increased lesion sizes and bioluminescent signals that were significantly greater than those of wt mice (data not shown), providing further evidence that IL-1β is the key mediator of IL-1R-dependent host defense against a S. aureus cutaneous challenge.

IL-1β-, but not IL-1{alpha}-, deficient mice have a defect in neutrophil recruitment compared with wt mice after skin inoculation with S. aureus

We previously reported that lesions of IL-1R-deficient mice have severely decreased neutrophil recruitment to the site of infection and decreased induction of neutrophil chemokines KC and MIP2 compared with wt mice. Because IL-1β-deficient mice (but not IL-1{alpha}-deficient mice) had a similar phenotype to IL-1R-deficient mice, namely increased lesion sizes and bioluminescent signals compared with wt mice, we evaluated the histology of lesional skin of wt, IL-1{alpha}-, and IL-1β-deficient mice at 1 day after inoculation with S. aureus (Fig. 2A). Lesions of wt and IL-1{alpha}-deficient mice had large neutrophilic abscesses in both H&E and anti-Gr-1 (neutrophil marker) mAb-labeled histologic sections. In addition to Gr-1-positive neutrophils, there were scattered monocytes/macrophages throughout the abscesses that were detected with anti-Mac-3 (monocyte/macrophage marker) mAb (50). These monocytes/macrophages were negative for F4/80 (data not shown), consistent with the monocyte/macrophage phenotype observed during skin inflammation (51, 52). In addition, S. aureus bacteria were barely detectable by Gram stain in wt mice and IL-1{alpha}-deficient mice, likely due to phagocytosis and clearance of the bacteria by the neutrophils within the abscess (Fig. 2A).


Figure 2
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  		FIGURE 2. IL-1β- and IL-1{alpha}/IL-1β- but not IL-1{alpha}-deficient mice have a marked decrease in neutrophil recruitment and in production of cytokines and chemokines after infection with S. aureus. IL-1{alpha}–/–, IL-1β–/–, IL-1{alpha}/IL-1β–/–, and wt mice were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS). A, Representative photomicrographs of sections labeled with H & E stain and anti-Gr-1 mAb, anti-Mac-3 mAb, or isotype control mAb (immunoperoxidase method), and Gram stain of lesional skin at 1 day after inoculation. B, Mean MPO activity (units per milligram of tissue) ± SEM of lesional skin. C and D, Mean protein levels (picograms per milligram of tissue) ± SEM of IL-1β, KC, MIP2, and IL-1{alpha} from lesional skin homogenates of skin biopsies performed at 0, 6, and 24 h after infection with S. aureus (nd, not detected). *, p < 0.05 IL-1{alpha}–/–, IL-1β–/–, or IL-1{alpha}/ IL-1β–/– mice vs wt mice (Student’s t test).

 
In contrast, lesions of IL-1β-deficient mice had severely decreased numbers of neutrophils and Mac-3+ monocytes/macrophages with no observable abscess formation and contained a blue-staining band of Gram-positive bacteria spanning the entire horizontal length of the section. Thus, IL-1β-deficient mice but not IL-1{alpha}-deficient mice have a severe defect in the recruitment of neutrophils to the site of infection with S. aureus. Furthermore, the readily detectable Gram-positive bacteria in lesions of IL-1β-deficient mice corroborate the results obtained with in vivo bioluminescence, demonstrating that IL-1β-deficient mice have impaired bacterial clearance. There was also significantly less MPO activity, a marker of phagocytic function, in homogenized lesional punch biopsy specimens of IL-1β-deficient mice compared with wt mice at 1 day after inoculation with S. aureus (Fig. 2B). In contrast, there was no difference in MPO activity in lesions of IL-1{alpha}-deficient mice compared with wt mice. Taken together, these data demonstrate that IL-1β-deficient mice have decreased neutrophil recruitment to the site of infection, suggesting that IL-1β is the predominant ligand that mediates IL-1R-dependent neutrophil recruitment to the site of infection.

IL-1β-, but not IL-1{alpha}-, deficient mice have impaired production of cytokines and chemokines involved in neutrophil recruitment in vivo after skin inoculation with S. aureus

The induction of cytokines and chemokines involved in neutrophil recruitment was evaluated in lesions of wt, IL-1{alpha}-, and IL-1β-deficient mice. Protein levels of IL-1β and neutrophil chemokines KC and MIP2 were determined by performing ELISAs on homogenized 8-mm lesional punch biopsies performed at 0, 6, and 24 h after inoculation with S. aureus (Fig. 2C). Lesions of IL-1β-deficient mice had no detectable protein levels of IL-1β and significantly decreased protein levels of KC and MIP2 compared with lesions of wt mice at 6 h but not at 24 h after inoculation. In contrast, there was no difference in protein levels of IL-1β, KC, and MIP2 between lesions of IL-1{alpha}-deficient mice and wt mice. Thus, the decreased neutrophil recruitment in IL-1β-deficient mice may in part be due to an early decreased production of cytokines and chemokines involved in neutrophil recruitment. In addition, we also evaluated protein levels of IL-1{alpha} by ELISA from homogenized lesional skin and found that wt and IL-1β-deficient mice had constitutive expression of IL-1{alpha} at 0 h (which has been previously reported; Refs. 17 , 20 , 21) and was not further induced at 6 and 24 h after skin inoculation with S. aureus (Fig. 2D). As expected, IL-1{alpha}-deficient mice had virtually undetectable levels of IL-1{alpha} at all time points after infection. However, the presence or absence of IL-1{alpha} had little or no effect on IL-1R-dependent neutrophil recruitment since the phenotype of IL-1{alpha}-deficient mice did not significantly differ from that of wt mice after S. aureus cutaneous challenge.

IL-1β protein is detected within the neutrophilic abscess after skin inoculation with S. aureus

The presence and distribution of IL-1β within the skin lesions was evaluated by immunoperoxidase labeling of IL-1β in frozen histologic sections of punch biopsies from lesional skin of wt, IL-1{alpha}-, and IL-1β-deficient mice performed at 1 day after s.c. inoculation with S. aureus (Fig. 3). In histologic sections of lesions of wt and IL-1{alpha}-deficient mice, IL-1β was expressed exclusively within the s.c. neutrophilic abscess, which contained cells expressing the neutrophil marker Gr-1. As an Ag control, we also performed immunoperoxidase labeling for IL-1β in histologic sections from lesions of IL-1β-deficient mice and, as expected, no IL-1β was detected. These results demonstrate that IL-1β protein is expressed within the neutrophilic abscesses of lesional skin after inoculation with S. aureus. In contrast to histologic sections from S. aureus inoculated lesional skin, no neutrophilic infiltrate was seen and virtually no Gr-1 or IL-1β was expressed in histologic sections of skin biopsies performed at 1 day after a sham injection of vehicle/saline alone (data not shown).


Figure 3
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  		FIGURE 3. IL-1β protein is detected within the neutrophilic abscess after skin inoculation with S. aureus. IL-1{alpha}–/–, IL-1β–/–, and wt mice were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS). Representative photomicrographs (x200) of sections labeled with anti-Gr-1 mAb, anti-IL-1β mAb, or isotype control mAb (immunoperoxidase method) of frozen sections lesional skin at 1 day after skin inoculation with S. aureus.

 
Neutrophil recruitment in response to S. aureus infection is dependent upon IL-1β expression by BM-derived cells and not resident skin cells

Because the lesions of S. aureus-infected mice are comprised of both resident skin cells and recruited BM-derived cells, we wanted to determine which population of cells was important in promoting IL-1β-dependent neutrophil recruitment and host defense. To address this question, we used a lethal dose of irradiation (1000 rad) to effectively eliminate all BM cells in the recipient mice. Lethally irradiated recipient wt mice were reconstituted with BM from donor wt or IL-1β-deficient mice to generate two groups: 1) wt mice reconstituted with wt BM (wt BM->wt mice) and 2) wt mice reconstituted with IL-1β-deficient BM (IL-1β–/– BM->wt mice). At 8 wk postreconstitution, these BM-reconstituted mice and normal nonirradiated/nonreconstituted wt and IL-1β–/– mice were s.c. inoculated with S. aureus as in Figs. 1 and 2 (Fig. 4, A–E). IL-1β–/– BM->wt mice and IL-1β–/– mice developed 3-fold larger lesion sizes with at least 10-fold higher bioluminescent signals compared with wt BM->wt mice or wt control mice (Fig. 4, A and B). In addition, lesions of IL-1β–/– BM->wt mice and IL-1β–/– mice had markedly decreased neutrophilic abscess formation, abundant Gram-positive bacteria, and decreased MPO activity, and virtually undetectable levels of IL-1β protein compared with wt BM->wt mice or wt control mice (Fig. 4, C–E). These findings suggest that IL-1β expression by BM-derived cells is required for neutrophil recruitment and immunity against S. aureus.


Figure 4
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  		FIGURE 4. Neutrophil recruitment and host defense against S. aureus infection is dependent upon IL-1β expression by BM-derived cells and not resident skin cells. wt and IL-1β–/–recipient mice were lethally irradiated (1000 rad) and reconstituted with BM from wt or IL-1β–/– donor mice: (A–E) wt BM->wt and IL-1β–/– BM->wt mice and (F–J) wt BM->IL-1β–/– and IL-1β–/– BM->IL-1β–/– mice, respectively. After 8 wk, BM-reconstituted mice and nonirradiated/nonreconstituted IL-1β–/– and wt control mice were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS). A and F, Mean total lesion size (cm2) ± SEM. B and I, Mean total flux (photons per second) ± SEM. C and G, Representative photomicrographs of sections labeled by H & E stain, anti-Gr-1 mAb, and Gram stain of lesional skin at 1 day after inoculation. D and H, Mean MPO activity (units per milligram of tissue) ± SEM of lesional skin. E and J, Mean protein levels (picograms per milligram of tissue) ± SEM of IL-1β from lesional skin homogenates of skin biopsies performed at 0, 6, and 24 h after infection with S. aureus. *, p < 0.05; {dagger}, p < 0.01; {ddagger}, p < 0.001 IL-1β–/– or BM-reconstituted mice vs wt mice (Student’s t test).

 
To corroborate the requirement for BM-derived cells in promoting IL-1β-dependent neutrophil recruitment and host defense, IL-1β-deficient recipient mice were reconstituted with either wt or IL-1β-deficient donor BM to generate two groups: 1) wt BM->IL-1β–/– mice and 2) IL-1β–/– BM->IL-1β–/– mice. At 8 wk postreconstitution, these BM-reconstituted mice and normal nonirradiated/nonreconstituted wt and IL-1β–/– mice were s.c. inoculated with S. aureus (Fig. 4, F–J). wt BM->IL-1β–/– mice had similar lesion sizes and bioluminescence signals as wt control mice, which were dramatically less than those of IL-1β–/– BM->IL-1β–/– mice or IL-1β–/– mice (Fig. 4, F and G). In addition, lesions of wt BM->IL-1β–/– mice had similar neutrophilic abscess formation, MPO activity, and levels of IL-1β protein as wt mice, which were substantially greater than those of IL-1β–/– BM->IL-1β–/– mice or IL-1β–/– mice (Fig. 4, H–J). Taken together, these data demonstrate that IL-1β expression by BM-derived cells is required for adequate neutrophil recruitment and host defense against a cutaneous S. aureus challenge.

ASC-deficient mice developed larger lesions with increased bacterial counts and had defective neutrophil recruitment compared with wt mice after skin inoculation with S. aureus

A key event in the production of active IL-1β has been shown to be the proteolytic processing of pro-IL-1β by caspase-1. This processing is dependent upon the inflammasome, which facilitates caspase-1 activation and subsequent cleavage of pro-IL-1β into its active form (30, 31, 32, 33, 35). Recently, in S. aureus-stimulated BM-derived macrophage cultures, caspase-1 activation and subsequent generation of active IL-1β was found to be dependent upon the inflammasome component ASC (36). Because we found that BM-derived cells are required for production of IL-1β in response to a cutaneous S. aureus challenge, we hypothesized that ASC may be important in inflammasome-mediated production of active IL-1β during the infection in vivo. ASC-deficient, IL-1β-deficient, and wt mice were s.c. inoculated with S. aureus as in Figs. 1 and 2 (Fig. 5). ASC- and IL-1β-deficient mice developed up to 3-fold larger lesions with increased bioluminescent signals (up to 12-fold higher) compared with wt mice (Fig. 5, A and B). In addition, lesions of ASC- and IL-1β-deficient mice had markedly decreased neutrophilic abscess formation, abundant Gram-positive bacteria, decreased MPO activity, and significantly decreased levels of KC and MIP2 compared with wt mice (Fig. 5, C–E). Furthermore, we assessed the cleavage of pro-IL-1β into its active p17 form in tissue homogenates of lesional skin from wt and ASC-deficient mice at 1 day after inoculation with S. aureus. The active 17-kDa cleaved form of IL-1β (p17) was detected in lesional skin of wt but not ASC-deficient mice whereas pro-IL-1β was detected in lesions of both wt and ASC-deficient mice (Fig. 5F), demonstrating that ASC is required for processing of IL-1β in vivo. Taken together, these findings demonstrate that ASC-deficient mice have a similar phenotype as IL-1β-deficient mice and suggest that the inflammasome component ASC plays a key role in promoting the processing of IL-1β in vivo against a S. aureus cutaneous challenge.


Figure 5
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  		FIGURE 5. ASC-deficient mice, similar to IL-1β-deficient mice, develop markedly larger skin lesions with higher bacterial counts and decreased neutrophil recruitment compared with wt mice after skin inoculation with S. aureus. ASC–/–, IL-1β–/–, and wt mice were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS). A, Mean total lesion size (cm2) ± SEM. B, Mean total flux (photons per second) ± SEM. C, Representative photomicrographs of sections labeled with H & E stain, anti-Gr-1 mAb (immunoperoxidase method), and Gram stain of lesional skin at 1 day after inoculation. D, Mean MPO activity (units per milligram of tissue) ± SEM of lesional skin. E, Mean protein levels (picograms per milligram of tissue) ± SEM of KC and MIP2 from lesional skin homogenates of skin biopsies performed at 0, 6, and 24 h after infection with S. aureus. F, Immunoblot of expression of the active p17 form of IL-1β and pro-IL-1β within lesional skin homogenates at 1 day after inoculation with S. aureus. *, p < 0.05; {dagger}, p < 0.01; {ddagger}, p < 0.001; IL-1β–/– or ASC–/– mice vs wt mice (Student’s t test).

 
Administration of active p17 rIL-1β with the S. aureus inoculum rescues IL-1β-deficient mice

Given the critical role of IL-1β and ASC-mediated processing of IL-1β in promoting neutrophil recruitment and host defense against cutaneous S. aureus challenge, we evaluated whether the addition of active recombinant p17 IL-1β (rIL-1β) could reduce the lesion sizes and bacterial counts observed in IL-1β-deficient mice. Administration of one dose of rIL-1β (50 ng/100 µl PBS) given with the S. aureus inoculum resulted in lesion sizes and bioluminescent signals that were significantly less than those of IL-1β-deficient mice inoculated with S. aureus plus vehicle alone and closely resembled the lesion sizes and bioluminescent signals observed in wt mice (Fig. 6). Thus, administration of active rIL-1β rescued IL-1β-deficient mice, demonstrating that the active form of IL-1β is required for IL-1R-dependent host defense and bacterial clearance against a cutaneous S. aureus challenge and further highlights the key role of ASC and the inflammasome in the generation of active IL-1β in vivo.


Figure 6
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  		FIGURE 6. Administration of rIL-1β with the S. aureus inoculum rescues IL-1β-deficient mice. IL-1β–/– mice and wt were inoculated s.c. with S. aureus (2 x 106 CFUs in 100 µl of PBS) along with administration of one dose of recombinant murine IL-1β (rIL-1β) (50 ng/100 µl) or vehicle alone to IL-1β–/– mice. A, Mean total lesion size (cm2) ± SEM. B, Mean total flux (photons per second) ± SEM; *, p < 0.05; {dagger}, p < 0.01; {ddagger}, p < 0.001; IL-1β–/– mice plus rIL-1β or IL-1β–/– mice plus vehicle alone vs wt mice (Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Neutrophil recruitment is an essential innate immune response in host defense against S. aureus infections and is required for bacterial clearance (10, 11). Using a model of S. aureus infection in the skin, we previously reported that activation of IL-1R-signaling by non-BM-derived resident skin cells is critical for neutrophil recruitment to the site of infection (15). In the present study, we investigated the mechanism of IL-1R-mediated neutrophil recruitment against a cutaneous S. aureus challenge. We found that inflammasome-mediated production of active IL-1β, and not IL-1{alpha}, by BM-derived cells is required for IL-1R-dependent neutrophil recruitment in host defense against S. aureus in vivo.

Although both IL-1{alpha} and IL-1β use IL-1R to mediate their activity (17, 18, 19, 20), there are key differences between these ligands, including the cellular source and posttranslational processing of these cytokines. IL-1{alpha} is constitutively expressed by epithelial cells (including keratinocytes) and endothelial cells (17, 18, 19, 20, 53). In contrast, IL-1β production is induced by activated immune cells such as monocytes/macrophages, dendritic cells, and Langerhans cells (17, 18, 19, 20). Despite the presence of detectable IL-1{alpha} in the skin before and during the infection (Fig. 2D), our findings demonstrate that IL-1β induced after the infection, and not constitutively expressed IL-1{alpha}, is the predominant mediator of IL-1R-dependent neutrophil recruitment in host defense against a S. aureus cutaneous challenge in vivo.

There is currently great interest in the mechanism of IL-1β processing in immune responses, with focus on the inflammasome as a key intracellular mediator for such processing (30, 31, 32, 33, 35). IL-1{alpha} and IL-1β are products of different genes and are translated into distinct 31-kDa proteins (pro-IL-1{alpha} and pro-IL-1β) (18, 20, 54, 55). Pro-IL-1{alpha} is fully active in its precursor form and is believed to be released from intracellular stores upon cell death or lysis (18, 20, 21, 23). After release, pro-IL-1{alpha} is cleaved into mature IL-1{alpha} by cell membrane-associated calpain proteases as well as extracellular proteases (18, 20, 56). However, IL-1{alpha} did not play a major role in host defense or neutrophil recruitment against a S. aureus cutaneous challenge. In contrast to pro-IL-1{alpha}, pro-IL-1β is an inactive precursor that requires cleavage by caspase-1 to generate 17-kDa active and secreted IL-1β (57, 58). Caspase-1 activation is dependent upon activation of the inflammasome (29, 30, 31, 32, 33, 34, 35). Moreover, generation of active IL-1β by S. aureus-stimulated BM-derived macrophages in vitro was found to be dependent upon the inflammasome component ASC (36). In the present study, ASC was also found to be required for the generation of active IL-1β in vivo and subsequent neutrophil recruitment and host defense against S. aureus (Fig. 5). This requirement for the inflammasome in generating active IL-1β in vivo was further supported by the ability of the administration of active rIL-1β to rescue the impaired host defense and bacterial clearance observed in IL-1β-deficient mice (Fig. 6). Thus, neutrophil recruitment in host defense against S. aureus cutaneous challenge requires inflammasome processing of IL-1β in vivo.

In the present study, we identified that BM-derived cells are required for production of IL-1β within the infected lesion, which subsequently activates IL-1R signaling. We previously described that the function of IL-1R-signaling on non-BM-derived resident cells is to promote neutrophil recruitment to the site of S. aureus infection in the skin (15). Taken together, these data define a host defense circuit in which BM-derived cells are required for production of IL-1β, which subsequently activates IL-1R expressed on non-BM-derived resident cells to promote neutrophil recruitment in host defense against a S. aureus cutaneous challenge. Our discovery that active IL-1β is required for IL-1R-dependent neutrophil recruitment in host defense against S. aureus in vivo is consistent with previous work demonstrating the importance of IL-1R activation in host defense against S. aureus brain abscesses, septic arthritis, and systemic infections (12, 13, 14). Thus, the IL-1β/IL-1R host defense circuit involving the interplay between BM- and non-BM-derived cells is likely a key innate immune mechanism for neutrophil recruitment against different types of S. aureus infections and perhaps other microbial pathogens. These other microbial pathogens may include Salmonella typhimurium and Francisella tularensis where the inflammasome has previously been shown to important in host defense and control of the infection (59, 60, 61).

Matsukawa et al. (62, 63, 64, 65) previously demonstrated in acute inflammatory arthritis models in rabbits that IL-1β was responsible for optimal production of the neutrophil CXC chemokines, IL-8 and growth-related oncogene, which corresponded to maximal neutrophil recruitment. Mice, unlike humans and rabbits, do not have an ortholog to IL-8, but do produce KC and MIP2, which are related to growth-related oncogene chemokines (66, 67, 68). Similar to the previous studies using rabbit arthritis models, we found that IL-1β was also expressed within the neutrophilic abscess and played a major role in neutrophil recruitment and in the induction of chemokines (i.e., KC and MIP2) at the site of S. aureus infection in the skin of mice. Although the studies in rabbits used arthritis models and there are differences between the species, both sets of studies demonstrate that IL-1β plays a key role in neutrophil recruitment.

In another study by the same group, using a S. aureus-induced arthritis model in rabbits, blockade of IL-1β and/or TNF-{alpha} inhibited early but not later leukocyte infiltration or subsequent inflammation and joint damage/arthritis (69). We previously demonstrated that TNF-{alpha} does not play a major role in host defense or in neutrophil recruitment to a site of S. aureus infection in the skin of mice (15). In the present study and in previous studies, neutrophil recruitment to a site of S. aureus infection in the skin has been shown to be an event required for bacterial clearance (10, 11, 15). Therefore, from a clinical point of view, augmentation of the IL-1β/IL-1R host defense circuit may provide a potential basis for novel therapeutic strategies aimed at enhancing the host’s own immune responses in the treatment of S. aureus infections in the skin.


   Acknowledgments
 
We thank Dr. Vishva Dixit (Vice President of the Molecular Oncology Department, Genentech, San Francisco, CA) for the gift of ASC-deficient mice. We also thank Ping Fu and Christopher Creencia (UCLA Tissue Procurement and Histology Core Laboratory) and Saeedeh Shapourifar-Tehrani, MPH (UCLA Histopathology Laboratory) for their expertise with embedding, cutting, and H & E and Gram staining of tissue sections.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grants R01 AI22553, R01 AI47868, R01 AR40312 (to R.L.M.), R01 AI056154, R01 CA87924, and R01 AI052359 (to G.C.), K08 AI62985 (to L.S.M.) and in part by the University of California Los Angeles Small Animal Imaging Resource Program National Institutes of Health (NIH)-National Cancer Institute 2U24 CA092865 Cooperative Agreement from the NIH. Ruth L. Kirschstein Research Service Award GM 007185 also supported this work (to E.M.P.). Back

2 L.S.M. and E.M.P. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Robert L. Modlin, David Geffen School of Medicine, Center for Health Sciences, UCLA, 52-121, 10833 Le Conte Avenue, Los Angeles, CA 90095; E-mail address: rmodlin{at}mednet.ucla.edu or Dr. Genhong Cheng, David Geffen School of Medicine, Center for Health Sciences, UCLA, 52-121, 10833 Le Conte Avenue, Los Angeles, CA 90095; E-mail address: genhongc{at}microbio.ucla.edu Back

4 Abbreviations used in this paper: wt, wild type; ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain; BM, bone marrow; BMM, BM-derived macrophage; TSB, tryptic soy broth; MPO, myeloperoxidase. Back

Received for publication June 14, 2007. Accepted for publication August 29, 2007.


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 Materials and Methods
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