Online Data Supplement. Induction of Pulmonary Granuloma Formation by Propionibacterium acnes is regulated by. MyD88 and Nox2

Transcription

1 Online Data Supplement Induction of Pulmonary Granuloma Formation by Propionibacterium acnes is regulated by MyD88 and Nox2 Jessica L. Werner *, Sylvia G. Escolero *, Jeff T. Hewlett *, Tim N. Mak *3, Brian P. Williams *, Yoshinobu Eishi, and Gabriel Núñez * * Department of Pathology and Compreheive Cancer Center, University of Michigan Medical School, Ann Arbor, MI 4819 Department of Human Pathology, Graduate School and Faculty of Medicine, Tokyo Medical and Dental University, , Yushima, Bunkyo-ku, Tokyo , Japan * Address correspondence to Dr. Gabriel Núñez, Department of Pathology and Compreheive Cancer Center, University of Michigan Medical School, 15 East Medical Center Drive, Ann Arbor, MI Phone: Fax: address: gabriel.nunez@umich.edu

2 Online Supplementary Methods Granuloma Quantification Both naïve mice and mice 9 days after P. acnes administration were euthanized and the lungs removed. Individual sectio of the whole lung were fixed and imbedded flat in paraffin in preparation for sectioning. Three lung sectio 2 micro apart were obtained from each mouse and stained with Hematoxylin and Eosin (H&E) by the Histology Core at the University of Michigan Cancer Center. H&E slides were scanned into whole slide images (WSI) at 4X by the University of Michigan Pathology Imaging Core. Granulomas in mouse lungs were quantified from WSI through pattern matching software Spatially Invariant Vector Quantization (SIVQ) that allows for both granuloma enumeration 1 and calculates total granuloma area 2. The background measurements for naïve wild-type, Myd88 -/-, and Cybb -/- mice were averaged and subtracted from individual measurements of granuloma size and number for the corresponding P. acnes-administered mice. Flow gating Neutrophils (CD11b pos Ly6G hi ), alveolar macrophages (CD11c hi CD11b lo ), dendritic cells (CD11c hi CD11b hi ), tissue macrophages/monocytes (NK1.1 neg CD4 neg CD8 neg CD19 neg CD11b pos CD11c neg-mid Gr-1 neg-mid ), CD4 T cells (CD3 pos CD4 pos ), CD8 T cells (CD3 pos CD8 pos ), B cells (B22 pos NK1.1 neg ), NK cells (NK1.1 pos CD19 neg ), and eosinophils (NK1.1 neg CD4 neg CD8 neg CD19 neg CD11b pos CD11c neg-mid Gr-1 neg-mid Siglec-F pos ). Siglec- F, CD3, Ly6G were purchased from Becton, Dickion and Company, B22 and Gr-1 were E2

3 purchased from BioLegend, and CD11c, NK1.1, CD4, CD8, and CD11b were purchased from ebioscience. Intracellular Cytokine Staining Both naïve and mice 9 days after P. acnes administration were euthanized and their lungs were digested as previously described 3 Lymphocytes were concentrated using a Ficoll-Paque gradient (GE Healthcare, Uppsala, Sweden) and then stimulated for 4 hours at 37 C with PMA/I (5ng/1ml PMA (Sigma, St. Louis, MO) and 1ng/1ml Ionomycin (Sigma, St. Louis, MO)) in the presence of Golgi Plug (BD Biosciences). The Fc receptors were blocked with CD16/32 (ebioscience, San Diego, CA, USA), and viable cells were stained with Live/Dead (Invitrogen, Carlsbad, CA, USA). The cells were then stained with surface markers CD4 PE (BD Pharmingen), CD8 FITC (ebioscience), and CD3 APC (ebioscience) and fixed and permeabilized with Cytofix/Cytoperm Plus Kit (BD Bioscience) and then stained for intracellular cytokines IFN-γ PE-Cy7 (BD Pharmingen). ELISA Mice were euthanized at and 6hrs after P. acnes administration, and the lungs were homogenized in 1ml protease inhibitor buffer (Roche, Basel, Switzerland) and centrifuged at 12,g for 1 minutes and then the supernatant was frozen at -8 C until analysis. The supernatants were thawed and analyzed for CXCL1, IL-1α, TNF-α, and MCP-1 by ELISA by the University of Michigan Cancer Center ELISA Core. Neutrophil depletion studies E3

4 Neutrophils were depleted as previously described 4. Briefly mice were intraperitoneally injected with either 1ug of monoclonal neutrophil-specific antibody 1A8, or the isotype control antibody 2A3 (BioXCell, West Lebanon, NH) every other day starting with the day before P. acnes administration. 9 days after P. acnes administration the mice were sacrificed, their lungs were fixed and embedded for H&E staining. Alveolar macrophage TNFα studies Alveolar macrophages were isolated through bronchoalveolar lavage of wild-type and MyD88 KO mice as previously described 5. Briefly the mouse was euthanized and the trachea was cannulated and the lungs were washed with 1ml of cold PBS (Gibco). Alveolar macrophages were counted, plated in HDMI (Gibco) with 1% FBS (Gibco), 2% Penicillin-Streptomycin (Gibco) and 2% L-Glutamin (Gibco) at a deity of 1 5 cells in 2µl, and left un-stimulated or stimulated overnight with viable P. acnes at a ratio of either 1:1 or 1:1. The supernatants were harvested, clarified at 12,g for 1 minutes and frozen until analysis. E4

5 Online Supplementary Methods References 1. Hipp JD, Cheng JY, Toner M, Tompki RG, Balis UJ. Spatially Invariant Vector Quantization: A pattern matching algorithm for multiple classes of image subject matter including pathology. J. Pathol. Inform. 211;2: Hipp J, Cheng J, Daignault S, et al. Automated area calculation of histopathologic features using SIVQ. Anal. Cell. Pathol. Amst. 211;34(5): Werner JL, Gessner MA, Lilly LM, et al. Neutrophils produce interleukin 17A (IL-17A) in a dectin-1- and IL-23-dependent manner during invasive fungal infection. Infect. Immun. 211;79(1): Knight JS, Luo W, O Dell AA, et al. Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Respoes in Murine Models of Atherosclerosis. Circ. Res. 214;114(6): Werner JL, Metz AE, Horn D, et al. Requisite role for the dectin-1 beta-glucan receptor in pulmonary defee agait Aspergillus fumigatus. J. Immunol. Baltim. Md ;182(8): Furukawa A, Uchida K, Ishige Y, et al. Characterization of Propionibacterium acnes isolates from sarcoid and non-sarcoid tissues with special reference to cell invasiveness, serotype, and trigger factor gene polymorphism. Microb. Pathog. 29;46(2):8 87. E5

6 Online Supplementary Figures Supplementary Figure 1. Increased production of IFN-γ in mice administered P. acnes. Lymphocytes were isolated from the lungs of naïve and P. acnes treated mice and stimulated for cytokine production using PMA/I and then stained for CD4 and CD8 surface markers and IFN-γ production. Percentage of IFN- γ positive CD4 and CD8 cells from both naïve and P. acnes treated mice from two studies with an n=4-5 are shown. Statistical significance was determined using a 2 way ANOVA using Sidak s multiple comparison test. **, and **** represents a p<.5, and p<.1, respectively. Supplementary Figure 2. Granuloma Formation by an additional clinical isolate of P. acnes. Wild-type mice were intratracheally administered 1 9 viable P. acnes cultured from an additional clinical isolate from a sarcoidosis patient as previously reported 6 and grown as described in the Methods section. Mice were euthanized after 9 days and their lungs were fixed, embedded, and sectio were stained with H&E for granuloma (black arrows) evaluation. Supplementary Figure 3. Antibody depletion of neutrophils does not effect granuloma formation. Neutrophils were depleted from wild-type mice via 1A8 (neutrophil-specific antibody) before P.acnes administration. 9 days after P.acnes administration the lungs were removed, fixed, sectio were stained for H&E, and granuloma number (A) and area (B) were quantified using SIVQ. Mice were bleed on day 3 post P.acnes administration and stained for neutrophils (CD11b + Ly6G + ) using flow cytometry, (D) representative flow plot shown. Statistical significance was determined using unpaired t tests. **p<.1 E6

7 Supplementary Figure 4. Fold change in Wt, Myd88 -/-, and Cybb -/- immune cells after P. acnes administration. Fold change was calculated from Wt (Fig 1), Myd88 -/- (Fig 3), and Cybb -/- (Fig 5) mice surface staining immunophenotyping experiments. The average for each cell type was calculated for both lymphoid (A) and myeloid (B) cells in naïve mice and the fold change in respoe to P. acnes administration was calculated. Statistical significance was determined using a 2 way ANOVA with Dunnett s multiple comparison test. *p<.5, ****p<.1 Supplementary Figure 5. P.acnes stimulated production of TNFα is dependent on MyD88. Alveolar macrophages were isolated from Wt and Myd88 -/- mice and stimulated overnight with viable P.acnes (Pa) at a ratio of both 1:1 and 1:1. Statistical significance was determined using one-way ANOVA. ***p<.1, ****p<.1 Supplementary Figure 6. Neutrophil recruitment intact in mice deficient in CybB. Three days after P.acnes administration Wt and Cybb -/- lungs were removed and digested into a single cell suspeion as in Figure 1, naïve mice were used as a control. Cells were labeled with neutrophil markers CD11b and Ly6G and neutrophils were gated as CD11b + and Ly6G high. Percentages (A) and total number (B) of neutrophils in the lung were calculated. Levels of CXCL1 (C) and IL-1α (D) were analyzed in lung homogenate of Cybb -/- mice by ELISA (pg/ml). Statistical significance was determined using unpaired t tests. *p<.5, **p<.1 E7

8 Supplementary Figure 1 6 **** ** Naive IFN-! + T cells (% per mouse) 4 2 CD4 CD8 P. acnes treated Supplementary Figure 1. Increased production of IFN-γ in mice administered P. acnes. Lymphocytes were isolated from the lungs of naïve and P. acnes treated mice and stimulated for cytokine production using PMA/I and then stained for CD4 and CD8 surface markers and IFN-γ production. Percentage of IFN- γ positive CD4 and CD8 cells from both naïve and P. acnes treated mice from two studies with an n=4-5 are shown. Statistical significance was determined using a 2 way ANOVA using Sidak s multiple comparison test. **, and **** represents a p<.5, and p<.1, respectively. E8

9 A Supplementary Figure 2 4x 6 μm Supplementary Figure 2. Granuloma formation by an additional clinical isolate of P. acnes. Wild-type mice were intratracheally administered 19 viable P. acnes cultured from an additional clinical isolate from a sarcoidosis patient as previously reported6 and grown as described in the Methods section. Mice were euthanized after 9 days and their lungs were fixed, embedded, and sectio were stained with H&E for granuloma (black arrows) evaluation. E9

10 Supplementary Figure 3 A$ 1 B$ 4 Granuloma (#) Granuloma Area (mm 2 ) C$ Isotype Control 2 1A8 ** D$ Isotype Control 1A8 Isotype$ 1A8$ Neutrophils (%) Ly6G$ Isotype Control 1A8 CD11b$ Supplementary Figure 3. Antibody depletion of neutrophils does not effect granuloma formation. Neutrophils were depleted from wild-type mice via 1A8 (neutrophil-specific antibody) before P.acnes administration. 9 days after P.acnes administration the lungs were removed, fixed, sectio were stained for H&E, and granuloma number (A) and area (B) were quantified using SIVQ. Mice were bleed on day 3 post P.acnes administration and stained for neutrophils (CD11b + Ly6G + ) using flow cytometry, (D) representative flow plot shown. Statistical significance was determined using unpaired t tests. **p<.1 E1

11 Supplementary Figure 4. A Lymphoid cells (fold change from naive) **** * CD4 CD8 B cells * Wt Myd88 -/- Cybb -/- B Myeloid cells (fold change from naive) **** **** Wt Myd88 -/- Cybb -/- AM TM/MO DC PMN Supplementary Figure 4. Fold change in Wt, Myd88 -/-, and Cybb -/- immune cells after P. acnes administration. Fold change was calculated from Wt (Fig 1), Myd88 -/- (Fig 3), and Cybb -/- (Fig 5) mice surface staining immunophenotyping experiments. The average for each cell type was calculated for both lymphoid (A) and myeloid (B) cells in naïve mice and the fold change in respoe to P. acnes administration was calculated. Statistical significance was determined using a 2 way ANOVA with Dunnett s multiple comparison test. *p<.5, ****p<.1 E11

12 Supplementary Figure 5. 6 **** TNF! (pg/ml) 4 2 *** Wt Wt + 1:1 Pa Wt + 1:1 Pa Myd88 -/- Myd88 -/- 1:1 Pa Myd88 -/- 1:1 Pa Supplementary Figure 5. P.acnes stimulated production of TNFα is dependent on MyD88. Alveolar macrophages were isolated from Wt and Myd88 -/- mice and stimulated overnight with viable P.acnes (Pa) at a ratio of both 1:1 and 1:1. Statistical significance was determined using one-way ANOVA. ***p<.1, ****p<.1 E12

13 Supplementary Figure 6 A B 8 6!1 7 * Neutrophils (%) Neutrophils (#) 4!1 7 2!1 7 Wt Naive Cybb -/- Naive Wt Pa Cybb -/- Pa Wt Naive Cybb -/- Naive Wt Pa Cybb -/- Pa C CXCL1 (pg/ml) ** D IL-1! (pg/ml) Wt naive Cybb -/- naive Wt Pa Cybb -/- Pa Wt naive Cybb -/- naive Wt Pa Cybb -/- Pa Supplementary Figure 6. Neutrophil recruitment intact in mice deficient in CybB. Three days after P.acnes administration Wt and Cybb -/- lungs were removed and digested into a single cell suspeion as in Figure 1, naïve mice were used as a control. Cells were labeled with neutrophil markers CD11b and Ly6G and neutrophils were gated as CD11b + and Ly6G high. Percentages (A) and total number (B) of neutrophils in the lung were calculated. Levels of CXCL1 (C) and IL-1α (D) were analyzed in lung homogenate of Cybb -/- mice by ELISA (pg/ml). Statistical significance was determined using unpaired t tests. *p<.5, **p<.1 E13