Volume 18, Issue 10, Pages 2415-2426 (March 2017) Circulating NOD1 Activators and Hematopoietic NOD1 Contribute to Metabolic Inflammation and Insulin Resistance  Kenny L. Chan, Theresa H. Tam, Parastoo Boroumand, David Prescott, Sheila R. Costford, Nichole K. Escalante, Noah Fine, YuShan Tu, Susan J. Robertson, Dilshaayee Prabaharan, Zhi Liu, Philip J. Bilan, Michael W. Salter, Michael Glogauer, Stephen E. Girardin, Dana J. Philpott, Amira Klip  Cell Reports  Volume 18, Issue 10, Pages 2415-2426 (March 2017) DOI: 10.1016/j.celrep.2017.02.027 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Circulating NOD1 Activators Increase with Obesity and High Fat Diet (A) Serum from mice fed low (LFD) or high fat diet (HFD) for 14 weeks was assessed for NOD1 stimulatory activity. (B) Serum NOD1 stimulatory activity plotted against body weight with linear regression shown. (C) Serum NOD1 stimulatory activity plotted against fasting blood glucose with linear regression shown. Results are means ± SEM, n = 8–9 mice per group. Unpaired Student’s t test. ∗p < 0.05. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Hematopoietic NOD1 Deletion Prevents HFD-Induced Glucose and Insulin Intolerance Mice with wild-type (WT→WT) or Nod1−/− (KO→WT) immune systems were fed LFD or HFD for 18 weeks. (A) Body weight measured weekly. (B) Food intake measured weekly. (C) Epididymal white adipose tissue (EWAT) and inguinal white adipose tissue (IWAT) weight. (D) EWAT adipocyte cross-sectional area. (E) Blood glucose levels following a 4 hr fast measured bi-weekly. (F) Glucose tolerance test 16 weeks into diets. (G) Insulin tolerance test 17 weeks into diets. (H) Akt phosphorylation in EWAT following i.p. saline or insulin injections. Results are means ± SEM, n = 8–11 (A–C, E–G), n = 3–5 (D), n = 2 (saline), or n = 3 (insulin) (H) mice per group. Two-way ANOVA, Tukey post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (WT→WT – LFD versus WT→WT – HFD), #p < 0.05, ##p < 0.01, ###p < 0.001 (KO→WT – LFD versus KO→WT – HFD), †p < 0.05 (WT→WT – HFD versus KO→WT – HFD). n.s., not significant. See also Figure S3. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Hematopoietic NOD1 Regulates Adipose Tissue Neutrophil and Pro-inflammatory Macrophage Count EWAT was isolated following 18 weeks on LFD or HFD. Representative flow cytometry plots and quantification as a percentage of SVF cells are shown. (A) CD11b+ F4/80+ cells (total macrophages). (B) F4/80+ CD11c+ cells (pro-inflammatory macrophages). (C) CD11b+ Ly6c+ cells (monocytes). (D) CD11c+ MHC-II+ cells (dendritic cells). (E) CD11b+ Gr-1+ cells (neutrophils). (F) Itgam/CD11b, Emr1/F4/80, Itgax/CD11c, Elane/neutrophil elastase expression in whole EWAT by qPCR. Results are means ± SEM, n = 4–9 (A–E) or n = 8–9 (F) mice per group. Two-way ANOVA, Tukey post-test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (WT→WT – LFD versus WT→WT – HFD), #p < 0.05, ##p < 0.01 (KO→WT – LFD versus KO→WT – HFD), †p < 0.05, †††p < 0.001 (WT→WT – HFD versus KO→WT – HFD). n.s., not significant. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 4 NOD1-Deficient Neutrophils Do Not Have Impaired Migratory Capacity (A) Following 18 weeks of feeding, CD11b+ Ly6G+ neutrophil percentages in blood were counted by flow cytometry. Representative plot shown. (B) Transwell migration assay using WT or Nod1−/− bone marrow-derived neutrophils (BMDN) migrating toward CXCL1. (C) Expression of Cxcr2 and Cxcr4 in WT or Nod1−/− BMDN by qPCR. Results are means ± SEM, n = 4–6 (A) or n = 3–4 (B and C) mice per group. Two-way ANOVA, Tukey post-test. ∗p < 0.05 (versus 0 μM, WT), #p < 0.05 (versus 0 μM, Nod1−/−). n.s., not significant. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Hematopoietic NOD1 Knockout Diminishes Expression of Neutrophil Chemoattractants by Adipose Tissue Macrophages (A) NF-κB p65 phosphorylation in EWAT. (B) Expression of pro-inflammatory and anti-inflammatory genes in whole EWAT. (C) Expression of Cxcl1 and Cxcl2 in EWAT separated into adipocyte-rich and stromal vascular fractions (SVF). (D) EWAT sections were stained for colocalization between Caveolin-1 (adipocyte marker), F4/80 (macrophage marker), and CXCL1. Images have been adjusted to have equal color balance to show colocalization, not intensity. Scale bar, 50 μm. Results are means ± SEM, n = 4–6 (A and C), n = 4–9 (B), or n = 3–5 (D) mice per group. Two-way ANOVA, Tukey post-test, ∗p < 0.05 (WT→WT – LFD versus WT→WT – HFD), †p < 0.05 (WT→WT – HFD versus KO→WT – HFD). n.s., not significant. See also Figure S5. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions

Figure 6 NOD1 Mediates CXCL1 Production and Neutrophil Attraction by Macrophages Bone marrow-derived macrophages (BMDM) were isolated from LFD-fed WT→WT or KO→WT mice and treated with 10 μg/mL peptidoglycan (PGN) for 18 hr. (A) Cxcl1 mRNA expression. (B) Secreted CXCL1 protein. (C) Neutrophil migration toward supernatant from vehicle (SN-Veh) or PGN-treated (SN-PGN) BMDMs. (D) Neutrophil migration toward PGN. (E) M1 and M2 macrophage marker expression. (F) Tlr2 and Tlr4 mRNA expression. (G and H) BMDM were treated with 0.5 mM palmitate (PA) or 100 ng/ml lipopolysaccharide (LPS) for 18 hr. (G) Cxcl1 mRNA expression. (H) Secreted CXCL1 protein. Results are means ± SEM, n = 6–9 (A–C, E–H) or n = 2 (D) mice per group. Two-way ANOVA, Tukey post-test, ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001. Cell Reports 2017 18, 2415-2426DOI: (10.1016/j.celrep.2017.02.027) Copyright © 2017 The Authors Terms and Conditions