Volume 146, Issue 7, Pages 1763-1774 (June 2014) Lactate Reduces Liver and Pancreatic Injury in Toll-Like Receptor– and Inflammasome- Mediated Inflammation via GPR81-Mediated Suppression of Innate Immunity  Rafaz Hoque, Ahmad Farooq, Ayaz Ghani, Fred Gorelick, Wajahat Zafar Mehal  Gastroenterology  Volume 146, Issue 7, Pages 1763-1774 (June 2014) DOI: 10.1053/j.gastro.2014.03.014 Copyright © 2014 AGA Institute Terms and Conditions

Figure 1 Lactate suppresses TLR4-mediated inflammatory signaling. Murine peritoneal macrophages assessed for (A) LPS-mediated lactate production, and lactate alteration of (B) LPS-induced Pro-Il1β with dose response, (C) proinflammatory gene transcription, and (D) phospho-p65 NF-κB levels. (E) NF-κB reporter gene luciferase activity in RAW 264.7 cells in response to lactate and LPS. (F) Primary human peripheral blood monocytes assessed for proinflammatory gene transcription in response to LPS and lactate. ∗Significant difference (P < .05) between treatment or bracketed groups. ∗∗Significant difference between 5- and 15-mmol/L lactate groups. PBMC, peripheral blood mononuclear cell. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 2 Lactate suppresses TLR-primed NLRP3 inflammasome activity. Murine peritoneal macrophages were treated with LPS, ATP, and lactate, and then assessed for (A) CASP1 cleavage to the P10 subunit in Western blots, (B) caspase-1 activity, and (C) IL1β release into the supernatant. (D) Human peripheral blood mononuclear cells treated with LPS, ATP, and lactate and assessed for IL1β release. ∗Significant difference (P < .05) between treatment groups. PBMC, peripheral blood mononuclear cell; WB, Western Blot. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 3 Lactate suppression of TLR4 requires GPR81 and ARRB2. (A and D) RAW 264.7 cells were treated with siRNA for Gpr81, Arrb2, or scramble siRNA and assessed for Gpr81 and Arrb2 knockdown. (B and E) Pro-Il1β induction was measured in siRNA-treated cells stimulated with LPS and lactate. (C) Murine peritoneal macrophages were treated with LPS and the GPR81 agonist 3-chloro-5hydroxybenzoic acid 100 μmol/L and the phospho-p65 NF-κB levels was assessed. (F) RAW 264.7 cell were treated with or without lactate, and cell lysates then were immunoprecipitated (IP) with anti-GPR81 antibody or control serum, and immunostained for ARRB2 and GPR81. ∗Significant difference (P < .05) between treatment or bracketed groups when marked. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 4 Lactate pre-injury and post-injury suppresses liver inflammation and liver injury in TLR4-mediated immune hepatitis. (A–C) Lactate or PBS was administered to mice concurrent with LPS and galactosamine and data were collected at 5 hours post-injury. Induction of (A) inflammatory genes in the liver, (B) serum ALT levels, and (C) liver histology scoring and representative histology. Lactate or normal saline was administered post-injury to mice 1 hour after administration of LPS and galactosamine, and data were collected at 5 hours after LPS and galactosamine dosing. Liver histology scoring and (D) representative histology, (E) serum ALT levels, and (F) serum and liver IL1b levels were determined. ∗Significant difference (P < .05) between treatment groups. GalN, D-Galactosamine. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 5 GPR81 is required for dampening proinflammatory responses and for lactate-mediated immunomodulation in vivo. Gpr81 expression in isolated liver cell populations. (A) Inflammatory gene induction in response to LPS, CpG, and lactate in isolated Kupffer cells. (B) Western blot and densitometry from liver lysates of mice treated with siRNA scramble or siRNA for Gpr81 probed with anti-GPR81 antibody. Mice were treated with siRNA for Gpr81 or siRNA scramble, LPS, and galactosamine, and pretreated with lactate or PBS by intraperitoneal injection. Induction of (C) inflammatory genes in the liver, (D) liver histology scoring and representative histology, (E) serum ALT values, and (F) survival data. ∗Significant difference (P < .05) between bracketed groups. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 6 GPR81 and lactate post-injury mitigate inflammation and injury in LPS and cerulein-induced severe acute pancreatitis. (A and B) Mice were treated with siRNA scramble of siRNA for Gpr81 and then administered LPS and 6 hourly injections of cerulein. (C–F) In separate experiments, mice were treated with LPS and cerulein as described previously and then administered subcutaneous lactate or saline concurrent with the third intraperitoneal injection of cerulein. Representative (A and C) pancreatic histology and histology scoring, (B and D) serum amylase and pancreatic trypsin activity, (E) pancreatic caspase-1 activity, and (F) pancreatic myeloperoxidase activity. ∗Significant difference (P < .05) between bracketed groups. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Figure 7 Tissue macrophage induction of NF-κB in vivo is suppressed by lactate pretreatment in experimental acute hepatitis and severe acute pancreatitis. Mice transgenic for the NF-κB GFP reporter gene were treated with lactate or normal saline by subcutaneous or intraperitoneal injection concurrent with administration of (A and D) LPS and galactosamine and (B, C, E, and F) LPS or cerulein, respectively. The latter mice received 2 additional hourly doses of cerulein. Mice were euthanized 3 hours after LPS dosing, and liver or pancreas and spleen cell suspensions were obtained. Cell suspensions were immunostained for F4/80 and assessed for GFP-positive populations on F4/80 gated cells by flow cytometry as described in the Materials and Methods section. ∗Significant difference (P < .05) between bracketed groups. All measured groups were significantly different from untreated groups. Caer, cerulein; FSC, fetal calf serum; GalN, D-Galactosamine; SSC, side scatter. Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 2 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 3 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 4 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 5 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 6 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 7 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 8 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions

Supplementary Figure 9 Gastroenterology 2014 146, 1763-1774DOI: (10.1053/j.gastro.2014.03.014) Copyright © 2014 AGA Institute Terms and Conditions