Volume 24, Issue 1, Pages 79-91 (January 2006) MyD88 Mediates Neutrophil Recruitment Initiated by IL-1R but Not TLR2 Activation in Immunity against Staphylococcus aureus  Lloyd S. Miller, Ryan M. O'Connell, Miguel A. Gutierrez, Eric M. Pietras, Arash Shahangian, Catherine E. Gross, Ajaykumar Thirumala, Ambrose L. Cheung, Genhong Cheng, Robert L. Modlin  Immunity  Volume 24, Issue 1, Pages 79-91 (January 2006) DOI: 10.1016/j.immuni.2005.11.011 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 MyD88-Deficient Mice Have Markedly Larger Skin Lesions and Higher Bacterial Counts Compared with Wild-Type Mice MyD88−/− and wt mice were inoculated subcutaneously with S. aureus (2.5 × 106 CFUs in 100 μl PBS) (bioluminescent strain SH1000). (A) Representative photographs. (B) Mean total lesion size (cm2) ± SEM. (C) Representative photographs of in vivo bioluminescence (Xenogen IVIS). (D) Mean total flux (photons/s) ± SEM. (E) Representative photographs of bacterial culture plates after overnight culture ± bioluminescence and colony-forming units (CFUs) of S. aureus recovered from 8 mm lesional punch biopsies on days 3 and 7. ∗p < 0.05, †p < 0.01, ‡p < 0.001 MyD88−/− versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 MyD88-Deficient Mice Have a Marked Decrease in Neutrophil Recruitment and in Production of Cytokines and Chemokines after Infection with S. aureus MyD88−/− and wt mice were inoculated subcutaneously with S. aureus (2.5 × 106 CFUs in 100 μl PBS). (A) Representative photomicrographs of sections labeled with H&E stain, anti-Gr-1 mAb (immunoperoxidase method), and gram stain of lesional skin at 1 and 2 days after inoculation. (B) Mean myeloperoxidase (MPO) activity (units/mg tissue) ± SEM. (C) Mean level of mRNA in arbitrary units (A.U.) ± SEM of IL-1β, KC, and TNFα at 6 hr after inoculation with S. aureus or PBS determined by Q-PCR and normalized to the housekeeping gene L32. (D) Mean protein levels (pg/mg tissue) ± SEM of IL-1β, KC, and MIP2 from tissue homogenates at 0, 6, and 24 hr after infection with S. aureus. ∗p < 0.05, †p < 0.01, ‡p < 0.001 MyD88−/− versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 TLR2-Deficient Mice Have Larger Skin Lesions with Higher In Vivo Bioluminescence but Have No Defect in Neutrophil Recruitment Compared with Wild-Type Mice after Infection with S. aureus TLR2−/− and wt mice were inoculated subcutaneously with S. aureus (2.5 × 106 CFUs in 100 μl PBS). (A) Mean total lesion size (cm2) ± SEM. (B) Mean total flux (photons/s) ± SEM. (C) Representative photomicrographs of sections labeled with H&E stain, anti-Gr-1 mAb, and gram stain of lesional skin at 1 and 2 days after inoculation. (D) Mean myeloperoxidase (MPO) activity (units/mg tissue) ± SEM. (E) Mean level of mRNA in arbitrary units (A.U.) ± SEM of IL-1β, KC, and TNFα at 6 hr after inoculation with S. aureus or PBS determined by Q-PCR and normalized to the housekeeping gene L32. (F) Mean protein levels (pg/mg tissue) ± SEM of IL-1β, KC, and MIP2 from tissue homogenates at 0, 6, and 24 hr after infection with S. aureus. ∗p < 0.05, †p < 0.01, ‡p < 0.001 TLR2−/− versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 IL-1R- but Not TNFαR-Deficient Mice Have Markedly Larger Skin Lesions with Higher Bacterial Counts and Decreased Neutrophil Recruitment Compared with Wild-Type Mice after Subcutaneous Inoculation with S. aureus IL-1R−/−, TNFαR−/−, and wt control mice were inoculated subcutaneously with S. aureus (2.5 × 106 CFUs in 100 μl PBS). (A and G) Mean total lesion size (cm2) ± SEM. (B and H) Mean total flux (photons/s) ± SEM. (C and I) Representative photomicrographs of sections labeled with H&E stain, anti-Gr-1 mAb, and gram stain of lesional skin at 1 and 2 days after inoculation. (D and J) Mean myeloperoxidase (MPO) activity (units/mg tissue) ± SEM. (E) Mean level of mRNA in arbitrary units (A.U.) ± SEM of IL-1β, KC, and TNFα at 6 hr after inoculation with S. aureus or PBS determined by Q-PCR and normalized to the housekeeping gene L32. (F and K) Mean protein levels (pg/mg tissue) ± SEM of IL-1β, KC, and MIP2 from tissue homogenates at 0, 6, and 24 hr after inoculation. ∗p < 0.05, †p < 0.01, ‡p < 0.001 IL-1R−/− mice or TNFαR−/− mice versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Neutrophil Recruitment in Response to S. aureus Infection Is Not Dependent upon MyD88 Expression by Recruited Bone Marrow-Derived Cells Wild-type recipient mice were lethally irradiated (1000 Rads) and reconstituted with bone marrow (BM) from wt or MyD88−/− donor mice (wt BM→wt and MyD88−/− BM→wt, respectively). After 8 weeks, BM reconstituted mice and nonirradiated/nonreconstituted MyD88−/− and wt control mice were inoculated subcutaneously with S. aureus (5 × 106 CFUs in 100 μl PBS). (A) Mean total lesion size (cm2) ± SEM. (B) Mean total flux (photons/s) ± SEM. (C) 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) Mean myeloperoxidase (MPO) activity (units/mg tissue) ± SEM. (E) BM-derived macrophages (BMMs) from all mice were assessed for: MyD88 mRNA expression by Q-PCR, MyD88 protein expression by immunoblotting, and MyD88 function by measuring mRNA levels of IL-1β and IL-6 by Q-PCR after stimulation of BMMs with Pam3Cys (1 μg/ml) and CpG (100 nM). ∗p < 0.05, †p < 0.01, ‡p < 0.001 MyD88−/− mice or BM-reconstituted mice versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Neutrophil Recruitment in Response to S. aureus Infection Is Dependent upon IL-1R/MyD88 Signaling by Resident Skin Cells and Not by Bone Marrow-Derived Recruited Cells Wild-type and IL-1R−/− recipient mice were lethally irradiated (1000 Rads) and reconstituted with bone marrow (BM) cells from wt or IL-1R−/− donor mice: (A–D) wt BM→wt and IL-1R−/− BM→wt and (E and F) wt BM→IL-1R−/− and IL-1R−/− BM→IL-1R−/−. After 8 weeks, BM-reconstituted mice and nonirradiated/nonreconstituted IL-1R−/− and wt control mice were inoculated subcutaneously with S. aureus (2.5 × 106 CFUs in 100 μl PBS). (A and E) Mean total lesion size (cm2) ± SEM. (B and F) Mean total flux (photons/s) ± SEM. (C and G) Representative photomicrographs of sections labeled with H&E, anti-Gr-1 mAb, and gram stain of lesional skin at 1 day after inoculation. (D and H) Mean myeloperoxidase (MPO) activity (units/mg tissue) ± SEM. (I) BM-derived macrophages from BM-reconstituted mice and nonirradiated/nonreconstituted IL-1R−/− and wt mice were assessed for IL-1R activity by measuring mRNA levels of IL-6 by Q-PCR after stimulating BMMs with rIL-1β (50 ng/ml). ∗p < 0.05, †p < 0.01, ‡p < 0.001 IL-1R−/− mice or BM-reconstituted mice versus wt mice (Student's t test). Immunity 2006 24, 79-91DOI: (10.1016/j.immuni.2005.11.011) Copyright © 2006 Elsevier Inc. Terms and Conditions