iNKT Cells Orchestrate a Switch from Inflammation to Resolution of Sterile Liver Injury  Pei Xiong Liew, Woo-Yong Lee, Paul Kubes  Immunity  Volume 47, Issue 4, Pages 752-765.e5 (October 2017) DOI: 10.1016/j.immuni.2017.09.016 Copyright © 2017 Terms and Conditions

Figure 1 iNKT Cell Response to Localized Sterile Injury (A) Stitched image (from 70 different fields of view) of a section of liver lobe of Cxcr6Gfp/+ mice under basal conditions. Scale bar, 300 μm. (B) Representative images from three or more individual experiments of the response of iNKT cells (green, GFP) to sterile injury (dead cells, red, propidium iodide) over time. Scale bars, 200 μm. (C and D) Quantification of iNKT cells (C) within 100 μm of injury and (D) infiltrating the injury over time (n ≥ 3 mice; ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA; ###p < 0.001 by t test; n.d., not determined). All expressed values are displayed as mean ± SEM. Please also see Figure S1. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 2 iNKT Cells Are Restricted from Entering the Localized Sterile Injury at 4 hr (A) iNKT cells infiltrating imaginary sham or real injury under different treatments (n ≥ 3 mice; ∗p < 0.05 by t test). (B) iNKT cells U-turn from imaginary or real injury under different treatments (n ≥ 3 mice; ∗∗p < 0.01, ###p < 0.001 by t test). (C) Representative image from three separate experiments of iNKT cells crawling over imaginary injury under sham conditions. Scale bar, 50 μm. (D) Representative image demonstrating staining of regular sinusoids and sinusoid remnants with PECAM-1 antibodies injected before (red) or after (blue) sterile injury from three separate experiments. Scale bar, 50 μm. (E) Representative still images over time of iNKT cells U-turning specifically at collapsed sinusoids over time from three independent experiments (yellow arrow, iNKT cell approaching collapsed sinusoid; red arrow, iNKT cell U-turning and leaving collapsed sinusoid). Scale bar, 25 μm. (F) Quantification of iNKT cells U-turning at regular or sinusoid remnants (n ≥ 3 mice; ∗∗∗p < 0.001 by t test). All expressed values are displayed as mean ± SEM. Please also see Figure S2. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 3 iNKT Cells Arrest Next to Injured Site at 8 and 24 hr (A) Representative still images from five different experiments demonstrate arrest of iNKT cells (white arrows) over 60 min. Scale bars, 25 μm. (B) Quantification of stationary iNKT cells within 100 μm of injured site at 8 hr (n ≥ 3 mice; ∗∗p < 0.01 by t test). (C) Quantification of stationary iNKT cells within 100 μm of injured site at 24 hr (n ≥ 3 mice; ∗∗p < 0.01 by t test). (D) Velocity profiles of iNKT cells within 100 μm of injury over time (n ≥ 3 mice). (E) Velocity profiles of iNKT cells in sham surgery mice (n = 3). (F) Representative flow cytometry image from three experiments of CD69 expression of iNKT cells (CD45+CD3+CD1d-tetramer+) obtained from liver biopsies in uninjured mice and after sterile injury at 24 hr (n = 3 mice). (G and H) For intracellular staining of cytokines, harvested liver biopsies were incubated 5–10 mins in brefeldin A before mechanical disruption and subsequent antibody staining. (G) Representative flow cytometry plot of intracellular IL-4 staining of iNKT cells in uninjured mice and after sterile injury at 24 and 48 hr from three or more independent experiments. (H) Representative flow cytometry image from two experiments of intracellular IFN-γ staining of iNKT cells in uninjured mice and after sterile injury at 24 hr. (I) Luminex assays of cytokines present in tissue biopsies after sterile injury at 24 hr (n ≥ 6 mice; ∗p < 0.05, #p < 0.05 by t test). All expressed values are displayed as mean ± SEM. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 4 iNKT Cells Arrest and Are Retained Spatially and Temporally around the Injury as a Result of CD1d Presentation of Endogenous Ligands and Subsequent Cytokine-Driven Signaling (A) Representative still images from four experiments of crawling iNKT cells after anti-CD1d antibody blockade at 8 hr. Scale bars, 25 μm. (B) Quantification of stationary iNKT cells within 100 μm of injury after anti-CD1d antibody treatment at 8 hr (n ≥ 3 mice; ∗∗∗p < 0.001 by t test). (C) Number of iNKT cells within 400 μm of the injury without antibody treatment (black bars), with anti-CD1d treatment (white bars), or with isotype treatment (gray bars) at 8 hr (n ≥ 3 mice; ǂp < 0.05, ∗∗p < 0.01 by t test). (D) Quantification of stationary iNKT cells within 100 μm of the injury after anti-CD1d treatment at 24 hr (n = 3 mice). (E) Quantification of stationary iNKT cells within 100 μm of the injury after anti-IL-12 and anti-IL-18 treatment at 24 hr (n ≥ 3 mice; ∗∗p < 0.01 by t test). (F) Percentage of stationary iNKT cells within 100 μm of the injury at 8 hr with or without anti-IL-12 and anti-IL18 treatment (n = 3 mice). (G) Number of iNKT cells within 100 μm of the injury at 8 hr with and without anti-IL12 and anti-IL18 treatment (n = 3 mice). (H) Number of iNKT cells within 100 μm of the injury without antibody treatment (black bars), with anti-CD1d treatment (white bars), or with isotype treatment (gray bars) at 24 hr (n ≥ 3 mice; ∗p < 0.05, by t test). (I) Number of iNKT cells within 100 μm of the injury without antibody treatment (black bars), with anti-IL-12 and anti-IL-18 treatment (white bars), or with isotype treatment (gray bars) at 24 hr (n ≥ 3 mice; ∗p < 0.05 by t test). All expressed values are displayed as mean ± SEM. Please also see Figure S3. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 5 Cells Contributing to iNKT Cell Arrest and Activation after Sterile Injury (A) Representative stitched image of injured site (red) with KCs (magenta) and sinusoids (blue) at 8 hr from nine independent experiments. Scale bar, 50 μm. (B) Quantification of stationary iNKT cells within 100 μm of the injury without treatment (black bars), with CLL treatment (white bars), and after endothelial cell CD1d knockout with (gray bar with diagonal pattern) and without (plain gray bar) CLL treatment at 8 hr (n ≥ 3 mice; ∗∗p < 0.01 by t test). All expressed values are displayed as mean ± SEM. (C) Intravital imaging snapshot of iNKT cells arrested next to KCs (red arrows). Scale bar, 100 μm. Please also see Figure S4. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 6 Functional Role of iNKT Cells in Injury Healing (A) Representative still image of wound size (no sinusoid stain inside injury) 4 hr and 7 days after injury from at least three independent experiments; sinusoids were stained with PECAM-1 (red). Scale bars, 100 μm. (B) Quantification of injury size over time between WT BALB/c and Cd1d−/− mice (n ≥ 3 mice; ∗p < 0.05, ∗∗p < 0.01 by t test). (C) Quantification of hepatocyte cell density between WT BALB/c and CD1d−/− mice 7 days after injury (n ≥ 3 mice; ∗p < 0.05 by t test). (D) Quantification of length of hepatocytes at the injury versus further away (>300 μm) (n ≥ 3 mice). (E) Representative immunofluorescence staining of BrdU+ cells between WT BALB/c and Cd1d−/− mice from three separate experiments. Scale bars, 200 μm (4×) and 40 μm (20×). (F) Quantification of BrdU+ cells between WT BALB/c and Cd1d−/− mice 3 and 7 days after injury (n ≥ 3 mice; ∗∗p < 0.01, ###p < 0.001 by t test). (G) Quantification of injury size 7 days after injury between C57BL/6 and Ja18−/− mice (n ≥ 3 mice; ∗p < 0.05 by t test). (H) Quantification of hepatocyte cell density around injury area between C57BlL6 and Ja18−/− mice (n ≥ 3 mice; ∗∗∗∗p < 0.0001 by t test). (I) Quantification of injury size 7 days after injury in Ja18−/− mice adoptively transferred with WT or Il4−/− iNKT cells (n ≥ 3 mice; ∗p < 0.05 by t test). (J) Quantification of hepatocyte cell density around injury area 7 days after injury in Ja18−/− mice adoptively transferred with WT or Il4−/− iNKT cells (n ≥ 3 mice; ∗p < 0.05 by t test). All expressed values are displayed as mean ± SEM. Please also see Figure S5. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions

Figure 7 iNKT Cells Regulate Transition of Monocytes, and Preventing Activation or IL-4 Production Delays Wound Healing (A) Representative image of inflammatory (red-CCR2hiCX3Cr1lo) and reparative (green-CCR2loCX3Cr1hi) monocytes in Ccr2Rfp/+Cx3cr1Gfp/+ and B6.Cd1d−/−Ccr2Rfp/+Cx3cr1Gfp/+ mice 24 hr after injury from three or more experiments. Scale bars, 100 μm. (B) Analysis of monocytic hues after sterile injury in Ccr2Rfp/+Cx3cr1Gfp/+ mice, B6.Cd1d−/−Ccr2Rfp/+Cx3cr1Gfp/+ mice, or Ccr2Rfp/+Cx3cr1Gfp/+ mice treated with anti-CD1d, anti-IL-12, and anti-IL-18 antibodies 24 hr after injury (n ≥ 3 mice; ∗p < 0.05, ∗∗p < 0.01 by t test). (C) Quantification of the amount of dead cells inside the injury between WT and Cd1d−/− mice 72 hr after injury (n = 3 mice; ∗p < 0.05 by t test). (D) Quantification of the amount of collagen inside and outside the injury in WT BALB/c and Cd1d−/− mice 72 hr after injury (n = 3 mice; ∗p < 0.05 by t test). (E) Quantification of injury size after antibody treatments and in Cd1d−/− mice 7 days after injury (n ≥ 3 mice; ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by one-way ANOVA; #p < 0.05 by t test). (F) Quantification of hepatocyte cell density after antibody treatments and in Cd1d−/− mice 7 days after injury (n ≥ 3 mice; ∗p < 0.05 by t test). (G) Quantification of injury size in WT BALB/c mice, Cd1d−/− mice, and mice treated with anti-IL-4 antibody 7 days after injury (n = 3 mice; ∗∗p < 0.01, ∗∗∗p < 0.001 by one-way ANOVA). (H) Quantification of hepatocyte cell density in WT BALB/c, Cd1d−/− mice, and mice treated with anti-IL-4 antibody 7 days after injury (n ≥ 3 mice; ∗p < 0.05 by t test). All expressed values are displayed as mean ± SEM. Please also see Figure S5. Immunity 2017 47, 752-765.e5DOI: (10.1016/j.immuni.2017.09.016) Copyright © 2017 Terms and Conditions