Lactate aggravates inflammation through the upregulation of ICAM-1 via GPR81-HSPA12B-mediated signaling in acute lung injury

To the Editor: Acute respiratory distress syndrome (ARDS) ranks among the most prevalent conditions in the intensive care unit (ICU), with mortality rates soaring to 50% in cases progressing to moderate-to-severe ARDS.[1] A central pathogenic mechanism in lung injury involves pulmonary vascular endothelial damage, marked by heightened adhesion molecule expression, neutrophil infiltration, and increased endothelial permeability, culminating in pulmonary vascular endothelial dysfunction and compromised gas exchange. Lactate is frequently elevated under hypoxic or inflammatory conditions, which are prominent features of ARDS.[2] Recent evidence suggests lactate may act as a signaling molecule in the regulation of inflammation; however, the precise role remains underexplored. G-protein-coupled receptor 81 (GPR81) serves as a selective lactate receptor expressed on endothelial cells (ECs). L-lactate binds to GPR81, initiating intracellular signaling that regulates cellular functions (e.g. increase endothelial permeability).[3] Heat shock protein A12B (HSPA12B), a member of the HSP70 family, is essential for angiogenesis. Overexpression of HSPA12B promotes angiogenesis in ECs and mitigates the expression of various adhesion molecules. Recent research also indicates that extracellular lactate can influence the expression of HSPA12B.[4] In this study, our objective was to ascertain whether lactate plays a role in exacerbating acute lung injury (ALI) and to elucidate the underlying mechanisms. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee of Southeast University (No. 20220228002). Lipopolysaccharide (LPS, 5 mg/mL) solution was instilled intra-tracheally (i.t.) in 4-to-6-week-old C57BL/6 mice (1 μg/g) to establish the ALI model. Six hours after LPS instillation, mice in the treatment group received an intraperitoneal (i.p.) lactate (0.5 g/kg) injection. In separate experiments, a subgroup of mice was i.p. injected with 3-hydroxy-butyrate (3-OBA) (120 μmol), a small molecular agent targeting the GPR81 protein, 6 h before LPS instillation [Supplementary Figure 1, https://links.lww.com/CM9/C17]. Lactate levels in mouse lung tissue were determined using a colorimetric assay. Hematoxylin-eosin (H&E) stain was used to evaluate lung histopathological injury. Immunofluorescent stain was used to determine myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) and CD31 protein expressions in lung tissue. Lactate (10 mmol/L) was added to the medium of mouse pulmonary vascular ECs and incubated for 24 h. In separate experiments, ECs were pre-conditioned with 3-OBA (5 mmol/L) for 6 h and then incubated with lactate for an additional 24 h. The small interfering RNAs (siRNA) targeting GPR81 and HSPA12B were transfected and the efficiency of siRNA-mediated inhibition of protein expression was evaluated by Western blotting after 48 h. ICAM-1, HSPA12B, and GPR81 protein in tissue homogenates or lysates were quantified by Western blotting. Data were analyzed using GraphPad Prism (GraphPad Software, Boston, MA, USA). A two-tailed threshold of P <0.05 was considered statistically significant. An elevated level of lactate was detected in lung tissues in the mouse model of ALI induced by i.t. LPS instillation. An additional increase in pulmonary lactate levels was observed after potentiation via i.p. lactate injection. Lactate levels were correlated with the severity of lung injury, with histopathological injury being significantly more severe after lactate i.p. injection compared with i.t. injection alone in H&E staining. Further lactate i.p. injection was linked to heightened histopathological injury and increased MPO activation [Supplementary Figure 2, https://links.lww.com/CM9/C17]. Western blotting indicated that ICAM-1 protein expression was significantly induced in lung tissues in this mouse ALI model. Moreover, an in vitro experiment revealed a marked increase in ICAM-1 protein expression in vascular ECs stimulated by lactate. Immunofluorescence staining for ICAM-1 and CD31 co-expression indicated elevated vascular endothelial ICAM-1 protein levels in the lungs of ALI model mice, with further increases observed after lactate i.p. injection [Supplementary Figure 3, https://links.lww.com/CM9/C17]. GPR81 protein expression was significantly abrogated after siRNA transfection as determined by Western blotting [Supplementary Figure 4, https://links.lww.com/CM9/C17]. Subsequent Western blotting indicated a significant decrease in ICAM-1 protein expression in ECs stimulated by lactate after siRNA-mediated GPR81 knockdown compared with the control group. ECs were also pre-treated with 3-OBA, and the results were consistent with those observed in response to siRNA-mediated GPR81 knockdown [Supplementary Figure 5, https://links.lww.com/CM9/C17]. Western blotting demonstrated a decrease in HSPA12B protein levels in lung tissue after LPS treatment in ALI model mice, and this reduction was even more dramatic after mice were i.p. injected with lactate. We also observed that HSPA12B expression was restored after GPR81 knockdown even when vascular ECs were treated with lactate, suggesting that the lactate/GPR81 axis plays an important role in modulating HSPA12B expression [Figure 1].Figure 1: Lactate promoted inflammation through the upregulation of ICAM-1 via GPR81-HSPA12B-mediated signaling in acute lung injury. (A) Lactate depressed HSPA12B expression mediate by GPR81. NC represented negative control. Si1 and Si2 represented siRNAs targeting GPR81. (B) HSPA12b knock-down further increased ICAM1 expression stimulated by lactate in vascular endothelial cells. NC represented negative control. Si1 and Si2 represented siRNAs targeting HSPA12B. (C) GPR81 blocking prevented against ALI induced by lactate. Lung tissue was stained by hematoxylin-eosin (20× magnification), and also by immunoflurorescence staining (20× magnification). * P <0.05, †P <0.01, ‡P <0.001, §P <0.0001. ALI: Acute lung injury; GPR81: G-protein-coupled receptor 81; HSPA12B: Heat shock protein A12B; ICAM1: Intercellular adhesion molecule 1; i.p.: Intraperitoneal; i.t.: Intra-tracheally; LPS: Lipopolysaccharide.ICAM-1 protein expression was induced by lactate stimulation and further increased after HSPA12B knockdown in ECs [Supplementary Figure 6, https://links.lww.com/CM9/C17], suggesting that inhibiting HSPA12B potentiates lactate-mediated ICAM-1 protein expression. Finally, the results showed that ALI mice pre-conditioned with 3-OBA exhibited restored HSPA12B protein expression in the lungs, leading to a consequent decrease in ICAM-1 expression compared with that associated with ALI alone. Histopathological analyses also revealed attenuated lung tissue injury induced by LPS after GPR81 blockade, and fluorescent staining revealed decreased MPO activity [Figure 1]. In this study, we characterized lactate as a pathogenic mediator that contributes to increased vascular endothelial injury and inflammatory responses associated with the progression of ALI induced by LPS. Elevated pulmonary lactate levels were correlated with lung histopathological injury, increased ICAM-1 expression, and leukocyte infiltration. In addition, lactate supplementation exacerbated tissue damage and the inflammatory responses in ALI model mice. Mechanistically, elevated lactate levels downregulated HSPA12B protein expression, consequently upregulating ICAM-1 via GPR81. HSPA12B knockdown further increased the lactate-induced upregulation of ICAM-1. Inhibiting lactate/GPR81 signaling reversed changes in HSPA12B expression, decreasing ICAM-1 expression and mitigating deleterious effects on pulmonary ECs and lung injury in mice [Supplementary Figure 7, https://links.lww.com/CM9/C17]. Recently, there has been a growing interest in lactate in settings of critical illness because it serves not only as an end-product of glycolysis but also as a signaling molecule that modulates inflammatory and immune responses. Lactate is widely regarded as a hallmark of sepsis and a predictor of mortality. Previous studies have already demonstrated lactate's involvement in the deleterious effects of sepsis. Lactate levels were significantly increased in lung tissue in the LPS-induced ALI mouse model[2] Recent research indicates that the lungs are a major original source of lactate during sepsis, and the accumulated lactate may be a significant contributor to the development of regional organ inflammation. We observed that additional lactate administration exacerbated LPS-induced lung injury by upregulating endothelial ICAM-1 expression. GPR81 is a recently identified lactate-selective receptor expressed by various cell types, including pulmonary vascular ECs. L-lactate can bind to and activate GPR81 to regulate cell functions during inflammatory processes. Recent studies have indicated that the lactate/GPR81 signaling axis increases endothelial permeability in sepsis or under inflammatory conditions. In our present study, we found that the lactate/GPR81 axis could enhance pulmonary endothelial ICAM-1 expression, aggravating leukocyte infiltration during ALI. Pharmacological blockade of GPR81 alleviated LPS-induced ALI and may thus offer value as an alternative treatment for ALI. Recent studies have found that lactate can directly modulate cell functions through post-translational protein modification (lactylation), independent of GPR81.[5] This process has been reported to alter the metabolome during tumorigenesis. As lactate levels are also significantly elevated during infection and sepsis, its role in metabolic alteration and subsequent influences on the process of infection and sepsis require further investigation. Previous studies have suggested a role for HSPA12B in the context of angiogenesis and vascular protection. The suppression of HSPA12B has been implicated in the VE-cadherin pathway, tight junction disruption, and consequent endothelial injury.[4] Here, we detected a novel role for HSPA12B in the setting of lactate-associated endothelial inflammation. In our experiments, HSPA12B acted as a downstream mediator of lactate/GPR81 signaling, inducing the upregulation of ICAM-1 in pulmonary endothelial cells. Lactate was able to decrease HSPA12B expression, which is consistent with previously published papers. Knockdown of HSPA12B increased ICAM-1 expression on the endothelium, while pharmacologically blocking lactate/GPR81 signaling attenuated HSPA12B expression in murine lungs in vivo or endothelial cells in vitro. This phenomenon provides new evidence regarding the role of HSPA12B in pulmonary homeostasis and endothelial function. Our study has several limitations. We only focused on one time point in ALI, and the dynamic changes in lactate and its role in lung injury remain to be investigated. In addition, there is increasing evidence suggesting that lactate manipulates the immune cell response to inflammation. Therefore, it is imperative to investigate whether lactate in the lung can alter immune cell phenotypes or functions, thereby contributing to endothelium injury and lung damage during ALI. In conclusion, our findings demonstrate the pathogenic role of lactate/GPR81 signaling in ALI by downregulating HSPA12B on the endothelium, thereby increasing ICAM-1 expression and enhancing leukocyte infiltration into the lung. Inhibition of the lactate/GPR81-HSPA12B signaling axis is an effective approach to suppressing inflammatory responses in the lung during ALI. Funding This work is partially supported by grants from Jiangsu Province's Key Discipline/Laboratory of Medicine (No. ZDXKA2016025), Jiangsu Provincial Maternal and Child Health Research Project (No. F2021-05), and Nanjing Medical Science and Technology Development Project (No. YKK20234). Conflicts of interest None.