Materials and Methods br Results br Discussion Activation of

Materials and Methods
Results
Discussion Activation of TLRs by damage-associated patterns is a well-recognized trigger for inflammation. TLR ligation results in a large increase in glycolytic metabolism in macrophages and other immune cells,12, 27 and we hypothesized that the glycolytic metabolite lactate could provide negative regulatory feedback and suppress TLR and NLRP3-mediated signaling in immune agomelatine through the recently identified lactate receptor GPR81. Previous investigation of potential immunomodulatory effects of lactate on macrophages showing proinflammatory gene induction were limited to cell lines, a high concentration of lactate for many hours, or reduced pH, which independently can alter TLR signaling.22, 23, 28 We confirmed that TLR signaling could induce lactate production in macrophages (Figure 1A), and showed that lactate could suppress TLR4- and TLR9-mediated proinflammatory responses such as pro-Il1β via down-regulation of the NF-κB pathway (Figure 1 and Supplementary Figure 1). The dose dependence of 5–15 mmol/L is consistent with the low affinity of GPR81 for lactate, with a median effective concentration for lactate of approximately 5 mmol/L. Serum lactate concentrations at rest in human beings and mice are 1–3 mmol/L, can increase transiently to about 15 mmol/L with intense exercise, with sustained levels of 5–10 mmol/L in severe ischemic pathology.29, 30 The reduction in TLR-induced pro-Il1β and inflammasome components Nlrp3 and Casp1 by lactate predicts that lactate also would reduce CASP1 activity, and IL1β release, which is the case (Figure 2). The recently identified lactate receptor GPR81 was required for the suppressive effects (Figure 3), but unexpectedly the lactate-induced immunosuppressive effects were independent of cyclic adenosine monophosphate signaling (Supplementary Figure 3). Instead, the cytosolic signaling molecule ARRB2 was required for these immunosuppressive effects, with a direct interaction between ARRB2 and GPR81 (Figure 3). ARRB2 signaling is known to antagonize both TLR and NLRP3 pathways and therefore may account for these GPR81 effects in macrophages.18, 19 IL-1β is an important proximal proinflammatory cytokine in a wide range of inflammatory conditions and if lactate was inducing a significant reduction it would have significant therapeutic potential. Pretreatment and post-injury treatment with lactate strongly suppressed liver inflammation and organ injury in a Kupffer cell–driven model of fulminant hepatitis mediated by LPS and d-galactosamine (Figure 4). Of note, this model is also strongly dependent on caspase-1 (Supplementary Figure 4), and thus allowed interrogation of the effects of lactate on TLR4 and caspase-1–mediated liver injury. Kupffer cells were the predominant Gpr81-expressing cell type in the liver and displayed lactate-mediated suppression of TLR4 and TLR9 proinflammatory signaling (Figure 5). GPR81 knockdown strongly augmented hepatic Pro-Il1β and Nlrp3, increased hepatocyte apoptosis and liver hemorrhage, and promoted mortality, highlighting a critical role for GPR81 in endogenous dampening of innate immune response (Figure 5). Of note, lactate treatment in the GPR81 knockdown background did not alter hepatic inflammation, hepatocyte apoptosis, or rescue from lethality, providing evidence that the protective effect of lactate was strongly GPR81-dependent in vivo. Although the lactate concentrations involved in autocrine and paracrine loops remain unclear, the serum lactate concentrations in this hepatitis model are reported to be about 0.2 mmol/L. GPR81 knockdown also increased the severity of severe acute pancreatitis induced by LPS and cerulein, suggesting a significant endogenous role for GPR81 in dampening inflammation and limiting tissue injury in the pancreas as well (Figure 6). Lactate treatment post-injury mitigated pancreatic inflammation and organ injury, including necrosis, in this severe model (Figure 6). This is especially interesting because lactate therapy has been found to be superior to saline in the management of severe acute pancreatitis and results in less systemic inflammation in a recent well-controlled clinical trial.