Enhanced lactate accumulation upregulates PD‐L1 expression to delay neutrophil apoptosis in sepsis

Malfunction of neutrophil apoptosis and elevated serum lactate levels are the key cellular mechanism of immune suppressive status in septic patients. However, whether increased lactate affects apoptosis of neutrophils and aggravates sepsis development, and the molecular mechanism remain unknown. In this study, first, we analyzed the transcriptional profiles of blood cells in sepsis patients (n = 39) and healthy volunteers (n = 40) to reveal that there is close correlation between the lactate‐related gene expression changes associated with lactate production and immune function in leukocytes, especially in neutrophils. Further, we explored the close relationship between lactate and delayed neutrophil apoptosis in human neutrophils. Programmed cell death 1 legand (PD‐L1) was highly expressed in septic patients compared with healthy volunteers. Then, we indicated that elevated levels of lactate in human neutrophils decreased neutrophil apoptosis (P < .001) by upregulating PD‐L1 expression. Inhibition of monocarboxylate transporter 1 (MCT1) significantly attenuated neutrophil apoptosis caused by lactate (P < .001). We further performed in vivo experiments in sepsis mice model and determined that increased lactate decreased neutrophil apoptosis (P < .05) and reduces mice survival rate (P < .001), which could also be rescued by MCT1 inhibitor (P < .05). This study revealed that elevated level of lactate in sepsis upregulates PD‐L1 expression to decrease apoptosis through MCT1 in neutrophils, which provides new insight into sepsis treatment strategy by reducing lactate accumulation.


INTRODUCTION
Sepsis is a highly prevalent condition and is defined as lifethreatening organ dysfunction caused by a dysregulated host response to infection. 1 At least 20 million patients have sepsis with severe organ dysfunction each year worldwide, 2 and the mortality rate is as high as 30%∼70%. 3etabolic acidosis caused by elevated serum lactate levels is one of the most common symptoms in sepsis, which is mainly caused by severe hypotension and insufficient tissue perfusion in the body, causing microcirculation disorders, thereby leading to decreased oxidative phosphorylation of cells and increased anaerobic glycolysis. 4ncreased lactate in the microenvironment leads to bacterial diffusion and poor response to antibiotics in sepsis. 5he Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3) indicated that increased serum lactate positively correlates with organ dysfunction and mortality in septic patients. 6When the blood lactate level is higher than 2 mmol/L, the mortality of sepsis patients is significantly increased. 6However, how lactate participates in the regulatory mechanism of sepsis development is still unknown.
Lactate can be transported into cells by monocarboxylate transporters (MCTs) or act as a signaling pathway regulatory molecule by serving as an agonist of the Gprotein-coupled receptor GPR81 to play an important role in regulating immune system function. 7Moreover, neutrophils mainly express monocarboxylate transporter 1 (MCT1) and monocarboxylate transporter 4 (MCT4), but not GPR81.MCT1 has a low affinity for lactate and is often expressed in cells with high glycolysis, and is mainly responsible for the transfer of lactate from cells.Therefore, in neutrophils, MCT1 is the main channel for lactate to enter cells.It has been reported that lactate into cells can induce the lactylation of METTL3, which causes the immunosuppressive function of tumorinfiltrating myeloid cells and promotes tumor immune escape. 8Lactate induces ubiquitin-proteasome system activation to inhibit the autophagy of mitochondria in red blood cells to stimulate inflammatory reactions in systemic lupus erythematosus. 9Immune dysfunction is the central link in the occurrence and development of sepsis. 10owever, whether lactate induces dysfunction of immune-related regulatory function in sepsis and the mechanism remain largely unknown.
As an important regulator of the immune system, leukocyte dysfunction is considered to be closely related to the occurrence and development of sepsis. 11Leukocyte activation and leukocyte phenotype and function were significantly more predictive of the clinical course. 11,12eutrophils, the main components of leukocytes are the first immune cells recruited in response to infection and inflammation. 13Neutrophils have a very short lifespan of only approximately 8-20 h.Neutrophils undergo constitutive or spontaneous apoptosis to maintain constant numbers in the circulation, which is a mechanism to preserve immune homeostasis. 14However, spontaneous neutrophil apoptosis in severe sepsis patients was significantly reduced in comparison to that in healthy individuals. 15he delayed neutrophil apoptosis in sepsis results in an increase of migration and extensive infiltration of neutrophils to organs, and inflammatory factor and reactive oxygen and nitrogen species release, 16 eventually causing organ dysfunction and even death. 17Programmed cell death 1 ligand 1 (PD-L1), a coinhibitory molecule expressed on macrophage, dendritic cell, neutrophils, and so on, negatively regulates the activation of T-cell and mediates lymphocyte death by binding to PD-1.Anti-PD-L1 monoclonal antibody and PD-L1-In-1 can inhibit the activity of PD-L1, which has been used in the immunotherapy of tumors. 18Upregulated PD-L1 was reported in sepsis, 19 and high expression of PD-L1 in neutrophils can lead to neutrophil extracellular traps release, leading to organ dysfunction.However, whether the PD-L1 expression is upregulated by elevated lactate accumulation to mediate delayed neutrophil apoptosis in sepsis are still unclear.
Herein, we performed transcriptomics analysis of blood cells from sepsis patients and healthy volunteers to found a strong correlation between lactate and the immune function of blood cells, and this correlation was evidently observed in neutrophils.Then, we further found that lactate can upregulate the expression of PD-L1 through MCT1 to decrease neutrophil apoptosis in a sepsis mouse model.Inhibition MCT1 significantly reduced the mortality rate of the sepsis mouse model.This study revealed that the accumulation of lactate in sepsis regulates MCT1/PD-L1 signaling to induce a delay in neutrophil apoptosis.Our study provides a new idea and method for the prevention and immunotherapy of sepsis in the future.The workflow of this study is illustrated in Figure 1.

Ethics statement
The animal protocol was approved by the Standing Committee on Animals at Tongji University (protocol number: TJBH07022105).The collection of human neutrophils and the related experimental protocol were approved by the institutional research ethics committee of the Shanghai Fourth People's Hospital (protocol number: 2022170-001).

Data collection and preprocessing
The datasets used in the study were obtained from public databases and are listed in geo/). 20The expression profiles of 39 sepsis samples (20 sepsis and 19 septic shock patients) and 40 control samples (healthy controls) in the dataset were extracted to compare gene expression differences between the two groups.The limma package for the R environment was used to detect differentially expressed genes (DEGs) between the sepsis and control samples. 21DEGs were screened with the following cutoff criteria: absolute log2-fold change > 1 and adjusted P value < .05.The lactate-related gene sets and immune-related gene sets were collected from the Molecular Signatures Database (MSigDB, https://www.gseamsigdb.org/gsea/msigdb). 22Correlations between differentially expressed lactate-related genes (lrDEGs) and differentially expressed immune-related genes (irDEGs) were evaluated with Pearson correlation analysis.The E-MTAB-5273 dataset, containing the leukocyte mRNA expression profiles from 227 sepsis and 10 control patients, was downloaded from the EBI database (https://www.ebi.ac.uk/biostudies/) to estimate the relationship between irDEGs and lrDEGs in leukocytes. 23The GSE157344 dataset, containing the mRNA expression for single blood cells from 4 healthy donors, 10 mild SARS-CoV-2 patients, and 19 severe COVID-19 patients admitted to the intensive care unit (ICU), was downloaded from GEO datasets to explore the correlation between lactate and immunity in neutrophils from sepsis patients. 24The Pagoda2 pipeline (https://github.com/hms-dbmi/pagoda2/)was used to execute data analysis. 25In brief, the read.10x.matricesfunction was used to load the UMI table .The following strategies were used to filter low-quality cells: cells with <500 UMIs and >20% of mitochondrial genes were removed.Principal component analysis (PCA) reduction is then computed using the calculate Pca reduction function.The number of computed PCs was changed in each analysis owing to the variable number of cells and cellular heterogeneity.To obtain high-quality cell clusters, we used Leiden community detection implemented in the R package leiden, a wrapper of the Python package leidenalg.The leiden function was applied to the k-nearest neighbor (KNN) graphs with default parameters for each analysis.Marker genes were detected by using getting differential genes function.Uniform Manifold Approximation and Projection (UMAP) low-dimensional embedding was computed using the uwot R package and, more precisely, the UMAP function with the neighbors parameter set to 30 and the metric parameter set to "cosine."To group clusters of cells in the first round of analysis, the mean gene expression of the most variable genes was computed using the aggregate function.Correlations between lactate-related genes and immune-related genes in neutrophils were performed with Pearson correlation analysis.

DEGs functional enrichment analysis
Gene enrichment analysis of DEGs between sepsis patients and healthy volunteers was performed on the web-based portal Metascape (http://metascape.org/), an online bioinformatics pipeline for multiple gene lists that allows effective gene function annotation and data-driven gene prioritization decisions. 26The results of the gene ontology (GO) biological process enriched by DEGs were displayed to explore the mechanism of the influence of lactate on immunity during sepsis. 27Correlations between lrDEGs and significant biological processes were performed with Pearson correlation analysis.

Induction of cecal ligation and puncture
Male mice aged 8 weeks were purchased from Jihui Company and raised in the laboratory animal center at Tongji University.All animals were acclimated under a 12-h light/12-h dark cycle for 1 week before the experiments.Mice were randomly divided into the sham group, CLP group, CLP+lactate (Macklin, China) group, and CLP+AZD (AZD3965: MCT1 inhibitor, purchased from Sellck, USA) group.After isoflurane general anesthesia, a 1.5-cm incision was made in the abdomen, and the abdominal skin and peritoneum were cut in turn.Then, the probe was used for abdominal exploration to expose the cecum.The intestinal feces were gently squeezed into the cecum.A 2-0 sterile silk was used to ligate the cecum, after which a 22 G needle was used to make a single puncture in the tip of the cecum, and 0.5 mm of feces was exposed.After surgery, 50 mL/kg body weight normal saline was immediately injected subcutaneously to prevent shock.The CLP+lactate group was intraperitoneally injected with 0.5 g/kg lactate 6 h after the operation, and the CLP+AZD group was intraperitoneally injected with 50 mg/kg AZD3965 6 h after the operation.
In the sham group, only the cecum was exposed without ligation, and puncture served as a control.All of the mice were given free access to food and water after recovery from anesthesia.

Purification and treatment of neutrophils
Blood samples (20 mL) from healthy volunteers were collected in heparinized tubes.Circulating neutrophils were isolated by 3% dextran sedimentation (Sigma, USA) and centrifugation through a discontinuous Ficoll (GE Healthcare, UK).Neutrophils were suspended at a concentration of 10 6 cells/mL in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% serum (Invitrogen, Canada) and 1% penicillin-streptomycin (Invitrogen).

Survival studies
Mice (n = 30 mice/group) were administered different treatments after the operation and were followed for 7 days to determine the survival rates.

Histology
Twenty-four hours after CLP, 20 mL of phosphate buffered saline (PBS) was injected into the right ventricle of the mouse heart under anesthesia, and then, the hepatic artery was severed to allow the fluid to drain.After tissue perfusion, 1 mL of 10% formalin was used to infuse the lungs through the trachea until they were fully inflated and incubated for 5 min.Then, the lungs were removed and soaked in formalin for 48-72 h.Formalin-fixed paraffinembedded tissue was stained with hematoxylin-eosin staining using a standard protocol.According to a previous study, 13a edema, alveolar wall thickening, vasocongestion, and leukocyte infiltration were used for histological scores.
A score of 0 indicates no inflammation, and a score of 4 indicates severe inflammation and lung damage.

Flow cytometry
Cells were seeded at a density of 5 × 10 5 in DMEM and subsequently incubated for 24 h with different doses of lactate.Untreated cells were used as a control.Cells were collected and cell surface stained utilizing anti-PD-L1-PE antibody according to the manufacturer's instructions.IgG2a was used as an isotype control.Blood samples were collected 24 h after mouse surgery and treated with red cell lysis solution (Biosharp, China).Neutrophils were identified by labeling with anti-LY6G-APC.For PD-L1 expression, we stained with anti-PD-L1-PE to observe the changes in the PD-L1 level of neutrophils in the different groups.Antibodies for flow cytometry were purchased from BioLegend.Neutrophil apoptosis was determined by annexin V and propidium iodide staining (Elabscience, China).Cells positive for annexin V and negative for propidium iodide were considered apoptotic.Stained cells were analyzed with a BD-LSRFortessa flow cytometer (BD Biosciences).Data were analyzed using FlowJo software.

Western blot
Different treatment groups of cells were lysed with protein lysis buffer (Thermo Fisher Scientific, USA) with protease inhibitors (Bimake, China).Protein concentrations were quantified using a Pierce BCA protein assay kit (Beyotime, China).Based on the protein concentration results, 10 μg of total protein was used for detection.Whole proteins were transferred onto PVDF membranes (Bio-Rad, USA).The primary antibodies included a β-actin antibody (1:1000 dilution, #4967, Cell Signaling Technology, USA), a caspase-3 antibody (1:1000 dilution, #9662, Cell Signaling Technology), a cleaved caspase-3 (C-caspase-3) antibody (1:1000 dilution, #9664, Cell Signaling Technology), and a PD-L1 antibody (1:1000 dilution, 14-5982-82, Thermo Fisher, USA).The secondary antibodies were a rabbit IgG for horse radish peroxidase (HRP)-linked antibody and a rat IgG for HRP-linked antibody purchased from Cell Signaling Technology.The bands were visualized using western chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and then quantified using a ChemiDoc XRS + system (Bio-Rad).β-Actin was used to normalize the target protein levels and control for loading differences in the total protein amount.

Lactate measurement
Serum was collected by centrifugation of clotted whole blood at 12,000 rpm for 10 min at 4

Cell Counting Kit-8 (CCK-8) measurement
The viability of neutrophils was detected by Cell Counting Kit 8 (Elabscience, China) according to the manufacturer's instructions.The cells were incubated in 96-well plates and cultured for 24 h.The absorbance at 450 nm was measured by a spectrophotometer.Independent experiments were repeated at least six times.

Myeloperoxidase activity measurement
The myeloperoxidase (MPO) activity was determined by an MPO activity kit (Elabscience, China) according to the manufacturer's instructions at 24 h.Whole lungs were homogenized and sonicated in an extraction buffer to obtain 10% homogenate.The enzymatic activity was determined by measuring the change in absorbance at 460 nm using a spectrophotometer.

Statistical analysis
Transcriptomics data analysis was performed in the R version 4.2.2 environment.Partial packages were applied to analyze the data described in the data collection and preprocessing section.In addition, dplyr, reshape2, and tidyverse were applied to conduct data conversion and analysis; ggplot2, ggpubr, ggstatsplot, pheatmap, RColor-Brewer, VennDiagram, and viridis were applied to visualize the data analysis results.Normally distributed data were analyzed using Student's t test or one-way analysis of variance (one-way ANOVA), and the results are shown as the mean ± SD.Survival studies were analyzed with the log-rank test.P < .05 was considered statistically significant.Statistical analysis was conducted by using GraphPad Prism software (version 8.0; GraphPad Software, USA).

DEGs are enriched in the leukocyte apoptotic process
The biological processes of 998 DEGs were mainly associated with the immune system (Figure 5A).The top 10 biological processes are shown in Figure 5B, which revealed that the DEGs play a critical role in the function of leukocytes.Then, we collected the biological processes associated with leukocytes, which are displayed in Table 2.In sepsis, the function of leukocytes (such as homeostasis, differentiation, proliferation, activation, tethering or rolling, cell-cell adhesion, aggregation, leukocyte-mediated cytotoxicity, degranulation, and apoptotic process) was disturbed (Figure 5C).We carried out correlation analysis between lrDEGs and DEGs enriched in the different functions of leukocytes to explore whether leukocyte dysfunction is related to lactate.There was a strong correlation between lactate and white leukocyte dysfunction, in which there was a significant negative correlation with the apoptotic process (Figure 5D, R 2 = −0.54,P < .0001).The expression of DEGs enriched in the leukocyte apoptosis process is displayed in Figure 5E.CD274 (gene of PD-L1) was highly expressed in sepsis patients compared with healthy volunteers.

Lactate decreases neutrophil apoptosis via MCT1-dependent pathway
Neutrophils, the main components of leukocytes, are the first immune cells to respond to infection and have been found to be the main cells involved in sepsis. 13To determine whether decreased neutrophil apoptosis in sepsis is associated with elevated lactate levels, we isolated healthy human peripheral blood neutrophils for in vitro experiments.Under normal conditions, the survival rate of unstimulated neutrophils was approximately 65% after 24 h of in vitro culture.We treated neutrophils with different doses of lactate (5, 10, and 20 mmol/L) and found that lactate inhibited the apoptosis of neutrophils in a concentration-dependent manner (Figure 6A).Lactate significantly reduced the activation of caspase-3 in neutrophils (Figure 6B,C).Flow cytometry analysis showed that neutrophils cultured with 20 mmol/L lactate displayed less apoptosis than that of the control group (Figure 6D,E).We further applied the MCT1 inhibitor AZD3965 (5 μmol/L) and determined that inhibition of lactate translocation into cells increased the apoptosis of neutrophils, attenuated the lactate-induced enhancement of neutrophil viability, and decreased caspase-3 activation in neutrophils (Figure 6D-H).These results determined that lactate can decrease neutrophil apoptosis via MCT1 dependent pathway.

Lactate upregulates PD-L1 expression to inhibit neutrophil apoptosis
Next, we used different doses of lactate to treat neutrophils in vitro and found that the expression of PD-L1 increased significantly with increasing lactate levels (Figure 7A,B).Inhibition MCT1 by AZD3965 attenuated the PD-L1 upregulation caused by lactate (Figure 7C,D).Moreover, the administration of 10 μmol/L PD-L1 inhibitors (PD-L1-IN-1) significantly reduce the effect of lactate on neutrophils and promote the apoptosis of neutrophils.However, PD-L1-IN-1 alone had no effect on the apoptosis of neutrophils (Figure 7E,F).These results suggested that lactate may inhibit neutrophil apoptosis by upregulating PD-L1 expression via MCT1.

Lactate induces PD-L1 upregulation to decrease neutrophil apoptosis and promote sepsis severity
In this study, we generated a mouse cecal ligation and puncture (CLP) model to simulate clinical sepsis.The lactate levels in the CLP group at 24 h after the operation were obviously elevated compared with those in the sham group (in which the cecum was exposed without ligation and puncture) (Figure 8A).Then, we examined the effect of lactate on regulating the survival rate and organ damage in sepsis model mice.The sepsis model mouse survival rates were 86%, 65%, and 47% at days 1, 3, and 7, respectively.By comparison, lactate administration strongly decreased the survival rate of septic mice.In contrast, MCT1 inhibitor supplementation by intraperitoneal injection increased the survival rate, and only 30% of sepsis model mice died over the 7-day experimental process (Figure 8B), which showed that lactate plays an important role in sepsis-increased mortality.
Since the lung is the first organ to fail during sepsis and is where neutrophils mainly accumulate, acute lung injury has been linked to death during sepsis.Thus, we evaluated the severity of lung injury by histopathology.As expected, CLP sepsis showed obvious signs of inflammation and injury in lung tissues, and lung tissue histology was worse in septic mice with lactate injection.In contrast, MCT1 inhibitor supplementation by intraperitoneal injection showed much better preservation of lung tissue injury (Figure 8C,D).MPO activity is a marker for the lung neutrophil burden, which was investigated in our study.As shown in Figure 8E, after CLP, MPO activity in the lung was significantly increased compared with that in sham mice, and it was worse in septic mice treated with lactate.However, this change was significantly reduced by AZD3965.Next, we used flow cytometry to detect the expression of PD-L1 in the peripheral neutrophils of septic mice and found that PD-L1 expression in the peripheral neutrophils of septic mice was significantly increased (Figure 8F,G).In contrast, the expression of PD-L1 in neutrophils was further increased in septic mice with lactate supplementation.The PD-L1 upregulation in peripheral neutrophils of mice treated with AZD3965 was alleviated (Figure 8F,G).We also used flow cytometry to detect the apoptosis of neutrophils in septic mice and found that the neutrophil apoptosis level was significantly reduced in septic mice compared with the sham group.In contrast, there was less neutrophil apoptosis in septic mice treated with lactate supplementation than in septic mice (Figure 8H,I).The MCT1 inhibitor AZD3965 significantly attenuated the neutrophil apoptosis delay in septic mice (Figure 8H,I).These data suggest that lactate accumulation in sepsis upregulates PD-L1 expression by MCT1 to further induce a decrease in neutrophil apoptosis, which further indicates that lactate accumulation is an important cause of organ injury and death.

DISCUSSION
In this study, we uncovered that lactate accumulation in sepsis may induce a decrease in neutrophil apoptosis by regulating the MCT1/PD-L1 pathway.Elevation of the serum lactate concentration is the most common symptom in sepsis and is closely correlated with severity and even death, 4a,28 higher lactate means higher sepsis mortality. 29Here, we not only further confirmed the studies but also uncovered that the MCT1 inhibitor AZD3965, which can inhibit the transportation of lactate into cells, significantly reduced mortality in septic mice.Our study suggests that lactate is an important risk regulator aggravating the severity of sepsis.Accumulation of lactate is an essential feature of inflammatory diseases. 30Immune responses can be controlled by lactate. 31Lactate treatment reduces effector T-cell function. 32Lactate accumulation in melanomas inhibits tumor surveillance by T cells and NK cells. 33GABA signaling that promotes tumor metastasis 34 can also be regulated by lactate. 35Tumor-derived lactate hinders NK cell-mediated responses. 36We found and confirmed a significant correlation between lactate-related and immunerelated gene expression in both normal people and sepsis patients.
Neutrophils represent the greatest proportion of leukocyte cells and the primary immune cellular barrier.However, neutrophils present double-edged sword of both inflammation and immunosuppression effects in sepsis.Delayed neutrophil apoptosis exacerbates inflammation in sepsis, eventually promoting organ dysfunction and even death.17a,17b, 37 We analyzed the apoptosis-related gene expression panel in leukocytes of sepsis patients and uncovered that PD-L1 is upregulated.The expression of PD-L1 in the kidney can be upregulated by lactate to cause immunosuppression in septic acute renal injury. 38ere, we found that human neutrophils present decreased apoptosis after treatment with lactate in vitro, which was restored by supplementation with an MCT1 inhibitor.PD-L1 expression was also upregulated.A previous study showed that upregulated PD-L1 delays human neutrophil apoptosis. 19Here, we found that lactate upregulates PD-L1 expression in human neutrophils, which further confirmed that lactate/MCT1/PD-L1 signaling regulates the apoptosis delay of neutrophils in sepsis.Our study suggests that uncovering the regulatory mechanism of the delay in neutrophil apoptosis regulated by lactate would be one of the most important steps in the delineation of sepsis immunosuppression.Additionally, effectively reducing the lactate accumulation will be one of the key points in the future clinical treatment of sepsis.

F I G U R E 1
The workflow of this study.

F I G U R E 2
DEGs, lrDEGs, and irDEGs in blood between sepsis patients and healthy control volunteers.(A) Expression profile heatmap of 998 DEGs in blood between the two groups, in which 460 DEGs were upregulated and 538 DEGs were downregulated in sepsis patients compared with healthy control volunteers.(B) Eleven lrDEGs were detected among the 998 DEGs.(C) Expression profile heatmap of the 11 lrDEGs in blood between the two groups.All lrDEGs were highly expressed in sepsis patients.(D) A total of498 irDEGs were detected among 998 DEGs.(E) Expression profile heatmap of the 498 irDEGs in blood between the two groups, in which 276 irDEGs were upregulated and 222 irDEGs were downregulated in sepsis patients compared with healthy control volunteers.(F) Two groups of samples can be obviously selected with thetSNE algorithm based on the lrDEGs and irDEGs.DEGs, differentially expressed genes; lrDEGs, lactate-related differentially expressed genes; irDEGs, immune-related differentially expressed genes.

F I G U R E 6
Lactate decreases neutrophil apoptosis via MCT1.(A) The CCK-8 assay was performed to assess the viability of human neutrophils cultured in vitro with different concentrations of lactate after 24 h.For each group, n = 6.(B) Representative pictures of western blot analysis indicated that different concentrations of lactate treatment for 24 h induced activation of caspase-3 in neutrophils.(C) Statistics of the relative gray value of western blot protein bands indicate the relative protein expression in B, n = 3. (D) Representative pictures of the flow cytometry gating strategy to identify the rate of neutrophil apoptosis.Q3 represents early apoptosis, and Q2 represents late apoptosis.The MCT1 inhibitor AZD3965 attenuates the decrease in neutrophil apoptosis caused by treatment with 20 mmol/L lactate.(E) Statistics of the neutrophil apoptosis rate (Q2+Q3) in D. For each group, n = 3. (F) AZD3965 inhibits the viability of neutrophils as assessed by the CCK-8 assay, n = 6.(G) AZD3965 increased the cleaved caspase-3 level, as detected by western blotting.(H) Statistics of caspase-3 activation represented by the ratio of C-caspase-3 and caspase-3 expression (C-caspase-3/caspase-3) relative tothe gray value indicating the protein expression in G, n = 3. * or # means P < .05,** or ## means P < .01,*** or ### means P < .001.F I G U R E 7 Lactate upregulates PD-L1 expression to inhibit neutrophil apoptosis.(A) Western blot analysis indicates the upregulation of PD-L1 in neutrophils treated with different concentrations of lactate for 24 h.(B) Statistics of the relative gray value indicating the protein expression in A, n = 3. (C) Western blot analysis indicated that AZD3965 attenuated the PD-L1 upregulation induced by 20 mmol/L lactate.(D) Statistics of the relative gray value of western blot protein bands indicate the relative protein expression in A, n = 3. (E) The PD-L1 inhibitor PD-L1-IN-1 attenuated the viability of neutrophils that was enhanced by lactate (20 mmol/L), n = 6.(F) Western blot analysis indicated that AZD3965 increased the C-caspase-3 level.* or # means P < .05,** or ## means P < .01,*** or ### means P < .001.

F I G U R E 8
PD-L1 upregulation caused by lactate decreases neutrophil apoptosis to promote sepsis severity.(A) Serum lactate levels were measured by a lactate assay kit.CLP mice had higher serum lactate levels.For each group, n = 6.(B) The survival rates among the sham, CLP, CLP + Lac, and CLP + AZD mice were compared by the log-rank test.For each group, n = 30.CLP indicates the cecal ligation and puncture group; Lac indicates the lactate intraperitoneal injection group; AZD indicates the intraperitoneal injection of AZD3965.The CLP+lactate group was intraperitoneally injected with 0.5 g/kg lactate 6 h after operation.The CLP+AZD group was intraperitoneally injected with 50 mg/kg AZD3965 6 h after the operation.(C) Representative histological images of tissue cross-sections.(D) Histological analysis of inflammatory changes in the lung tissue was performed.The histological score parameters were as follows: alveolar space closure and collapse, alveolar wall thickening and fibrosis, edema, and leukocyte infiltration.A score of 0-4 indicates different inflammation severity levels.For each group, n = 3. (E) MPO activity in the lung was measured by an MPO activity kit, n = 6.(F) Representative pictures of the flow cytometry gating strategy that identified the expression of PD-L1 in neutrophils in mouse blood among the four groups.(G) Statistics of the expression of PD-L1 in neutrophils in Panel F. For each group, n = 3. (H) Representative flow cytometry gating strategy to identify the rate of neutrophil apoptosis in mouse blood.(I) Statistics of the rate of neutrophil apoptosis in Panel H.For each group, n = 3. * or # means P < .05,** or ## means P < .01,*** or ### means P < .001.