Lactate reduces liver and pancreatic injury in Toll-like receptor- and inflammasome-mediated inflammation via GPR81-mediated suppression of innate immunity

Abstract

Background & aims: The NACHT, LRR, and pyrin domain-containing protein 3 (NLRP3) inflammasome induces inflammation in response to organ injury, but little is known about its regulation. Toll-like receptors (TLRs) provide the first signal required for activation of the inflammasome and stimulate aerobic glycolysis to generate lactate. We examined whether lactate and the lactate receptor, Gi-protein-coupled receptor 81 (GPR81), regulate TLR induction of signal 1 and limit inflammasome activation and organ injury.

Methods: Primary mouse macrophages and human monocytes were incubated with TLR4 agonists and lactate and assayed for levels of pro-interleukin (IL)1β, NLRP3, and caspase-1 (CASP1); release of IL1β; and activation of nuclear factor-κB (NF-κB) and caspase-1. Small interfering RNAs were used to reduce levels of GPR81 and arrestin β-2 (ARRB2), and an NF-κB luciferase reporter transgene was transfected in RAW 264.7 cells. Cell lysates were analyzed by immunoprecipitation with an antibody against GPR81. Acute hepatitis was induced in C56BL/6N mice by administration of lipopolysaccharide and D-galactosamine. Acute pancreatitis was induced by administration of lipopolysaccharide and cerulein. Some mice were given intraperitoneal injections of sodium lactate or small interfering RNA against Gpr81. Activation of NF-κB in tissue macrophages was assessed in mice that expressed a reporter transgene.

Results: In macrophages and monocytes, increasing concentrations of lactate reduced TLR4-mediated induction of Il1B, Nlrp3, and Casp1; activation of NF-κB; release of IL1β; and cleavage of CASP1. GPR81 and ARRB2 physically interacted and were required for these effects. The administration of lactate reduced inflammation and organ injury in mice with immune hepatitis; this reduction required Gpr81 dependence in vivo. Lactate also prevented activation of NF-κB in macrophages of mice, and, when given after injury, reduced the severity of acute pancreatitis and acute liver injury.

Conclusions: Lactate negatively regulates TLR induction of the NLRP3 inflammasome and production of IL1β, via ARRB2 and GPR81. Lactate could be a promising immunomodulatory therapy for patients with acute organ injury.

Keywords: Immune Regulation; Innate Immune Response; Mouse Model; Pancreas.

Figure 1

Lactate suppresses TLR4 mediated inflammatory signaling. Murine peritoneal macrophages assessed for LPS mediated lactate production (A), and lactate alteration of LPS induced Pro-Il1β with dose response (B), pro-inflammatory gene transcription (C) and phospho-p65 NF-κB levels (D). NF-κB reporter gene luciferase activity in RAW 264.7 cells in response to lactate and LPS (E). Primary human peripheral blood monocytes assessed for pro-inflammatory gene transcription in response to LPS and lactate (F). Asterisks denote significant difference (P<0.05) between treatment or bracketed groups. Double asterisk denotes significant difference between 5 and 15 mM lactate groups.

Figure 2

Lactate suppresses TLR primed NLRP3 inflammasome activity. Murine peritoneal macrophages were treated with LPS, ATP, and lactate, and then assessed for CASP1 cleavage to the P10 subunit in Western blots (A), caspase 1 activity (B), and IL1b release into the supernatant (C). Human peripheral blood mononuclear cells treated with LPS, ATP, and lactate and assessed for IL1brelease (D). Asterisks denote significant difference (P<0.05) between treatment groups.

Figure 3

Lactate suppression of TLR4 requires GPR81 and ARRB2. RAW 264.7 cells were treated with siRNA for Gpr81, Arrb2, or scramble siRNA and assessed for Gpr81 and Arrb2 knockdown (A and D).Pro-Il1β induction was measured in siRNA treated cells stimulated with LPS and lactate (B and E). Murine peritoneal macrophages were treated with LPS and the GPR81 agonist 3-chloro-5hydroxybenzoic acid 100 mM and phospho-p65 NF-κB levels assessed (C). RAW 264.7 cell were treated with or without lactate, and cell lysates then immunoprecipitated (IP) with anti-GPR81 antibody or control serum, and immunostained for ARRB2 and GPR81 (F). Asterisks denote significant difference (P<0.05) between treatment or bracketedgroups when marked. NS, not significant.

Figure 4

Lactate pre-injury and post-injury suppresses liver inflammation and liver injury in TLR4 mediated immune hepatitis. Lactate or PBS was administered to mice concurrent with LPS and galactosamine and data collected at 5 hours post-injury (A–C). Induction of inflammatory genes in the liver (A),serum ALT levels (B), and liver histology scoring and representative histology (C). Lactate or normal saline was administered as post-injury to mice one hour after administration of LPS and galactosamine and data collected at 5 hours after LPS and galactosamine dosing. Liver histology scoring and representative histology (D),serum ALT levels (E), and serum and liver IL1b levels (F) were determined. Asterisks denote significant difference (P<0.05) between treatment groups.

Figure 5

GPR81 is required for dampening pro-inflammatory 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 (A). Western blot and densitometry from liver lysates of mice treated with siRNA scramble or siRNA for Gpr81 probed with anti-GPR81 antibody (B). Mice were treated with siRNA for Gpr81 or siRNA scramble, LPS and galactosamine, and pretreated with lactate or PBS by intraperitoneal injection. Induction of inflammatory genes in the liver (C), liver histology scoring and representative histology (D), serum ALT values (E), and survival data (F). Asterisks denote significant difference (P<0.05) between bracketed groups. NS, not significant.

Figure 6

GPR81 and lactate post-injury mitigate inflammation and injury in LPS and caerulein induced severe acute pancreatitis. Mice were treated with siRNA scramble of siRNA for Gpr81 and then administered LPS and six hourly injections of caerulein (A,B). In separate experiments, mice were treated with LPS and caerulein as above and then administeredsubcutaneous lactate or salineconcurrent with thethird intraperitoneal injection of caerulein (C–F). Representative pancreatic histology and histology scoring (A, C), serum amylase and pancreatic trypsin activity (B, D), pancreatic caspase1 activity (E), and pancreatic myeloperoxidase activity (F). Asterisks denote significant difference (P<0.05) between bracketed groups.

Figure 7

Tissue macrophage induction of NF-kB 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 LPS and galactosamine (A,D) and LPS or caerulein, (B,C,E,F) respectively. The latter mice received two additional hourly doses of caerulein. Mice were euthanized three hours after LPS dosing, and liveror pancreas and spleen cell suspensions obtained, respectively. Cell suspensions were immunostained for F4/80 and assessed for GFP positive populations on F4/80 gated cells by flow cytometry as described in methods. Asterisks denote significant difference (P<0.05) between bracketed groups. All measured groups were significantly different from untreated groups.