Neutrophil-derived IL-1β is sufficient for abscess formation in immunity against Staphylococcus aureus in mice

Abstract

Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.

Figure 1. Gene expression analysis in wt and IL-1R-deficient mice after S. aureus skin infection.

Wt and IL-1R^−/− mice were inoculated intradermally with S. aureus and gene expression analysis was performed on skin specimens taken at 4 hrs and compared with samples from uninfected skin (n = 3 mice per group). Upregulated genes were defined according to the criteria of fold-change (FC) >1.5, p-value<0.05. (A) Top 20 induced genes in wt mice ordered by FC (gene function was assigned using NetAffx Gene Ontology Mining Tool [Affymetrix]). (B) Top four biological functions in wt versus IL-1R^−/− mice generated by Ingenuity Pathway analysis (ranked by p-value). (C) Top four cell types in the Cellular Movement functional group (ranked by p-value). (D) Functional network analysis of genes in the Cell Movement of Neutrophils group in wt mice (left) compared with the same genes in IL-1R^−/− mice (right). The genes in the networks and pie charts are shaded according to FC. Red: >7.0 FC (strongly upregulated), pink: 3.0<FC<7.0 (moderately upregulated), light pink: <3.0 FC (slightly upregulated), or grey: no change or not statistically significant. (E) Real-time Q-PCR confirmation (mean log₁₀ fold change ± SEM) of 2 representative genes that were similarly-induced between wt and IL-1R-deficient mice and 8 representative genes that were differentially-induced in wt mice compared with IL-1R-deficient mice from the Cell Movement of Neutrophils sub-group from microarray analysis in Fig. 1D.

Figure 2. Real-time kinetics of IL-1β production and neutrophil recruitment after S. aureus skin infection.

pIL1-DsRed reporter mice or LysEGFP mice were inoculated intradermally with S. aureus. (A) Representative skin lesions (left) (entire dorsal backs [top, millimeter ruler shown for scale] and close-ups of lesions [bottom]) and mean total lesion size (cm²) ± SEM (right). (B) Representative in vivo S. aureus bioluminescence (left) and mean total flux (photons/s) ± SEM (logarithmic scale) (right). (C) Representative in vivo EGFP-neutrophil fluorescence (left) and mean total radiant efficiency (photons/s)/(µW/cm²) ± SEM (right). (D) Representative in vivo DsRed-IL-1β fluorescence (left) and mean total radiant efficiency (photons/s)/(µW/cm²) ± SEM (right). Data are from 2 experiments with at least 4 mice/group per experiment. *p<0.05; ^†p<0.01, S. aureus-infected mice versus none (sham injection alone) (Student's t-test).

Figure 3. Neutrophils are the predominant cell type that expresses IL-1β after S. aureus skin infection.

pIL1-DsRed mice were infected intradermally with S. aureus and lesional skin specimens were collected at 4 and 24 hrs. Representative photomicrographs of sections labeled with anti-DsRed (IL-1β, red) and anti-MOMA2 (monocytes/macrophages, green) (A) or anti-7/4 (neutrophils, green) (C) and sections analyzed by confocal microscopy. Cells expressing both markers appear yellow (merge). High (left) and low (right) magnification images are shown (Scale bars = 50 µm). Dotted line = dermoepidermal junction. Quantification of co-localization of IL-1β-DsRed fluorescence with MOMA2⁺ monocytes/macrophages (B) or 7/4⁺ neutrophils (D) using the Manders' coefficient for a value range of 0 to 1 in which 0 = no pixels co-localize and 1 = all pixels co-localize. Data are representative from 4 mice per group.

Figure 4. Neutrophil-derived IL-1β is sufficient for abscess formation and bacterial clearance of S. aureus-infected mice.

Neutrophils from IL-1β^−/− or wt donor mice were adoptively transferred into IL-1β^−/− recipient mice. After 2 hrs, these mice and normal wt and IL-1β^−/− mice were infected intradermally with S. aureus. (A) Mean total lesion size (cm²) ± SEM. (B) In vivo bioluminescence quantified by mean total flux (photons/s) ± SEM (logarithmic scale). (C) Representative photomicrographs of sections labeled with H&E stain and anti-7/4 (neutrophils) (immunoperoxidase method) at 1 day after inoculation. Scale bars = 100 µm. (D) Mean myeloperoxidase (MPO) activity (U/mg tissue weight) ± SEM from lesional skin at 1 day after inoculation. Data are representative from 2 independent experiments with at least 4 mice/group. *p<0.05; ^†p<0.01, ^‡p<0.001, IL-1β^−/− mice or mice with adoptively transferred neutrophils versus wt mice (Student's t-test).

Figure 5. The PRRs TLR2, NOD2, and FPR1 contribute to optimal production of IL-1β during infection.

TLR2^−/−, NOD2^−/−, FPR1^−/− and wt mice were inoculated intradermally with S. aureus. (A) Mean total lesion size (cm²) ± SEM. (B) In vivo bioluminescence quantified by mean total flux (photons/s) ± SEM (logarithmic scale). (C) Mean IL-1β protein levels (pg/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. (D) Mean myeloperoxidase (MPO) activity (U/mg tissue weight) ± SEM from lesional skin at 4 hrs after inoculation. *p<0.05, ^†p<0.01, TLR2^−/−, NOD2^−/− or FPR1^−/− mice versus wt mice (Student's t-test). Data are from 2 experiments with at least 4 mice/group per experiment.

Figure 6. PRRs, α-toxin and the ASC/NLRP3 inflammasome contribute to S. aureus- and MRSA-induced IL-1β production by mouse neutrophils.

Neutrophils from mouse bone marrow were infected with live S. aureus (SH1000) or MRSA (USA300 LAC strain) (MOI bacteria∶neutrophils of 5∶1) for a total culture time of 6 hrs and gentamicin was added at 60 min from the start of the infection to prevent bacterial overgrowth. IL-1β protein levels (mean ± SEM) were measured in culture supernatants by ELISA. (A, B) IL-1β protein levels in supernatants from S. aureus-infected neutrophils from TLR2^−/−, NOD2^−/−, FPR1^−/−, ASC^−/− and wt mice. (C, D) IL-1β protein levels in supernatants from wt mouse neutrophils infected with S. aureus (C) or MRSA (D), in the absence and presence of an NLRP3-inhibitor (glibenclamide), a caspase-1 inhibitor (Z-YVAD-FMK) or anti-staphylococcal α-toxin antiserum. The respective vehicle controls (DMSO or normal rabbit IgG) for the inhibitors had IL-1β protein levels that did not differ than media alone (data not shown). Data are from 3–5 mice per group. *p<0.05; ^†p<0.01, ^‡p<0.001, TLR2^−/−, NOD2^−/−, FPR1^−/− or ASC^−/− mice versus wt mice (A, B) or the NLRP3-inhibitor, caspase-1 inhibitor or anti-staphylococcal α-toxin antiserum treatment versus media alone (C, D) (Student's t-test).