The Expression And Function Of ProBDNF/p75^NTR Signaling In Sepsis-Induced Lung Injury

Background:Sepsis-induced lung injury is the unresolvable lung inflammation caused by cytokine storm during sepsis and then develops into acute lung injury（ALI）or acute respiratory distress syndrome（ARDS）,which causes irreversible damage to lung and high mortality.It is the common clinical complication and cause of death in sepsis.Compared with ALI/ARDS caused by other causes,sepsis-induced ALI/ARDS has a higher mortality rate of 30%to 40%.It is a difficult and important clinical issue in the treatment of sepsis.In response to the immune imbalance characterized by excessive inflammation and immunosuppression in sepsis,immunotherapy is currently a hot topic in research on sepsis-induced lung injury.However,treatment targeting inflammatory cytokines has not shown significant clinical benefit,and there is an urgent need to explore more targeted immunomodulatory targets.Studies have shown that pro-brain-derived neurotrophic factor（proBDNF）activates downstream signaling pathways by binding to its high-affinity receptor,neurotrophic pan receptor p75(p75^NTR),promoting biological effects such as neuronal apoptosis and negative regulation of synaptic plasticity.Our team has revealed that the upregulation of proBDNF/p75^NTR signaling on immune cells under pathological conditions disrupts the immune-inflammatory microenvironment and exacerbates organ damage.Our previous studies have confirmed that upregulated proBDNF in the immune system promoted the pathogenesis of SAE by downregulating the circulating CD4⁺T cells,limiting its infiltration into the meninges and perturbing the meningeal pro-/anti-inflammatory homeostasis.In the present thesis project,we speculate that proBDNF/p75^NTR signaling derived from immune cells plays a critical role in the progression of sepsis-induced lung injury.Objectives:1.To explore the expression of proBDNF and p75^NTR in immune cell subsets in the peripheral blood of patients with sepsis and septic mice models.2.To clarify the effect of neutralizing proBDNF on the disease progression of sepsis-induced lung injury and neutrophil function.3.To analyze the effects of conditional knockout of p75^NTR in neutrophils on sepsis-induced lung injury and neutrophil function.Methods:1.Clinical study:34 sepsis patients from the intensive care unit of the Second Xiangya Hospital of Central South University and 34 healthy volunteers from the Physical Examination Center of the Second Xiangya Hospital were included in the study.The sepsis group collected peripheral blood samples and initial routine blood tests on admission,while the control group also collected peripheral blood samples and routine blood tests provided by the physical examination center.Flow cytometry was used to detect phenotypes/percentages and the expression of proBDNF and p75^NTR on immune cell subpopulations in the peripheral blood of septic patients and healthy volunteers.2.In vivo experiments:（1）Establishment of sepsis mouse model:Eight-week-old C57 male mice are randomly divided into the experimental group and the control group.The mice in the experimental group are intraperitoneally injected with Lipopolysaccharide（LPS）to establish a sepsis mouse model,and the control group is intraperitoneally injected with the same volume of PBS solution.（2）Flow cytometry:Collect peripheral blood from the mice’s eyeball vein at 24 h and 48 h after the injection of LPS.The expressions of proBDNF/p75^NTRon monocytes and neutrophils in the peripheral blood of mice in the sepsis group and control group were detected by flow cytometry.（3）Expression of proBDNF and p75^NTR in the lung tissues of septic mice detected by histological staining and co-location detection with neutrophils:Lung tissues of septic mice were collected at 6 h,24 h and 48h after injection of LPS,and the expression of proBDNF and p75^NTR in lung tissues of septic mice were observed by immunohistochemical staining,then calculate and analyze the numbers of proBDNF-positive cells and p75^NTR-positive cells.Furthermore,the co-location of proBDNF and p75^NTRin lung tissues with Ly6G⁺neutrophils was observed by double-labeling immunofluorescence.（4）The effects of mAb-proB intervention on sepsis-induced lung injury:the sepsis model mice were intervened by intraperitoneal injection of mAb-proB.Observe lung injury through survival rate experiment,lung tissue pathological changes and lung injury score,the m RNA levels of inflammatory factors,calculation of lung wet-dry ratio,detection of myeloperoxidase（MPO）activity,and total protein concentration in alveolar lavage fluid（BALF）.（1）Survival rate experiment:The mice in the sepsis group are intervened by intraperitoneal injection of mAb-proB at 30 minutes before LPS injection and 12 hours after LPS injection,and the control group was injected with the same amount of isotype control IgG antibody at the same time.Observe and record the vital signs and survival rate of the mice daily by double-blind method,including activity changes,taking food,body weight and mental state,and observe for 7 days.（2）The mice in the sepsis group are intervened by intraperitoneal injection of mAb-proB before LPS injection,and the control group was injected with isotype control IgG.At the 24 h and 48 h after LPS injection,collect the lung tissues of the mAb-proB intervention group and the control group at the same time,and observe the pathological changes of lung tissue through HE staining;detect the m RNA levels of inflammatory factors（IL-1β,TNF-α and IL-6）in lung tissues through RT-q PCR;calculate the wet-dry ratio of the lung,detect the MPO activity and the total protein concentration of BALF.（5）The effects of conditional knockout of neutrophil p75^NTR on sepsis-induced lung injury:（1）Establishment and characterization of conditional myeloid lineage knockout p75^NTR(Lyz2^Cre-p75^f/f)mice:Lyz2^Cre-p75^f/fmice were obtained by crossing p75^{NTR flox/flox} transgenic mice and myeloid lineage-specific Cre^+/-transgenic mice(Lyz2^Cre).Collect mice’s tail and identify the genotype by PCR product size.Detect the m RNA levels and protein levels of p75^NTRon monocytes,neutrophils,and lymphocytes in peripheral blood by q PCR and flow cytometry to verify the myeloid-specific knockout of p75^NTR.（2）Lyz2^Cre-p75^f/f mice and p75^f/fmice were intraperitoneally injected LPS to establish a sepsis mouse model.Observe lung injury through survival rate experiment,lung tissue pathological changes and lung injury score,the m RNA levels of inflammatory factors,calculation of lung dry-wet ratio,detection of MPO activity,and total protein concentration in BALF.（the methods were the same as those in mAb-proB intervention）.3.In vitro experiments:（1）The effects of conditional knockout of neutrophil p75^NTR on neutrophil function in sepsis mice model:Lyz2^Cre-p75^f/f mice and p75^f/fmice were intraperitoneally injected LPS to establish sepsis mouse model.Collect the peripheral blood of septic mice and separate neutrophils at 24h after injection,then add LPS to stimulate culture in vitro for 4 hours.Flow cytometry was used to detect and analyze the reactive oxygen species（ROS）content,phagocytic function,and percentage of apoptosis in neutrophils after culture.（2）The effects of mAb-proB intervention on LPS-induced neutrophil function in healthy volunteers:Collect the peripheral blood of healthy volunteers and separate neutrophils,then add LPS or equivolume PBS solution to stimulate culture in vitro for 14 hours.Flow cytometry was used to detect and analyze the expression of proBDNF and p75^NTRon neutrophils after culture.On this basis,respectively add human proBDNF protein,control PBS buffer,mAb-proB and IgG homotypic control,and then culture in vitro for 14 hours.Flow cytometry was used to detect and analyze ROS content,phagocytic function,and percentage of apoptosis in neutrophils after culture.Results:1.Clinical study:（1）Blood routine analysis:White blood cell count,neutrophil count and neutrophil percentage in sepsis patients at admission were significantly higher than the control group;on the contrary,monocyte count and monocyte percentage in sepsis patients were lower than the control group.And the monocytes/lymphocytes ratio in sepsis patients was significantly higher than the control group.（2）Flow cytometry results showed that the percentages of total neutrophils,CD66b⁺CD64⁺neutrophils and CD66b⁺CD64^-neutrophils in the peripheral blood of sepsis patients were significantly higher than the control group.Inactivated CD66b⁺CD64^-neutrophils were the major neutrophil phenotype.Compared with the control group,the percentages of CD3⁺T cells and CD19⁺B cells in the peripheral blood of sepsis patients were significantly reduced.（3）Flow cytometry results showed the following:Compared to the control group,（1）the expression of proBDNF on total neutrophils,CD66b⁺CD64⁺neutrophils and CD66b⁺CD64^-neutrophils in peripheral blood of sepsis patients were increased,and the p75^NTR was highly enriched in CD66b⁺CD64⁺neutrophils;（2）the expression of proBDNF and p75^NTR on intermediate and non-classical monocytes in peripheral blood of sepsis patients were reduced,and the expression of p75^NTR was increased in classical monocytes but not proBDNF;（3）the expression of proBDNF was upregulated on B cells and CD4⁺T cells in peripheral blood of sepsis patients,and the expression of p75^NTR was increased in B cells,CD4⁺T cells,and CD8⁺T cells.2.In vitro experiments:（1）Flow cytometry showed that compared with the control group,the expression of proBDNF and p75^NTR on Ly6G⁺neutrophils in the peripheral blood of septic mice at 24 h and 48 h after LPS injection was significantly increased,while there was no significant difference in the expression of proBDNF and p75^NTR on Ly6C^hi monocytes and Ly6C⁺monocytes.（2）The histological staining showed that compared with the control group,the expression of proBDNF and p75^NTR in the lung tissue of sepsis mice was upregulated and co-localized with Ly6G⁺neutrophils at 24 h and 48 h after LPS injection.（3）Compared to the IgG isotype control group,mAb-proB intervention improved the survival rate of septic mice,improved lung pathological injury and reduced lung injury score,reduced the m RNA levels of pro-inflammatory cytokines（IL-6,IL-1β and TNF-α）,reduced Ly6G positive neutrophil infiltration.Compared to the IgG isotype control group,lung dry/wet ratio,the total protein concentration of BALF,and activity of MPO in the mAb-proB intervention group decreased.（4）Compared to the p75^NTRf/fseptic mice,conditional knockout of neutrophil p75^NTR improved the survival rate of septic mice,improved lung pathological injury and reduced lung injury score,and reduced Ly6G positive neutrophil infiltration.Compared to the IgG isotype control group,the lung dry/wet ratio,the total protein concentration of BALF in the mAb-proB intervention group decreased.3.In vitro experiments:（1）Compared to the control group,after LPS stimulation in vitro culture for 4 hours,the ROS mean fluorescence intensity（MFI）of peripheral blood neutrophils from Lyz2^Cre-p75^f/f mice decreased;and the early and late apoptosis rates of peripheral blood neutrophils in the Lyz2^Cre-p75^f/f mice were lower than the control group but with no statistical difference,and the MFI of the p Hrodo?Green fluorescent dye in peripheral blood neutrophils from Lyz2^Cre-p75^f/f mice was increased but with no statistical difference.（2）Compared to the peripheral blood neutrophils of healthy volunteers treated with PBS,the expression of proBDNF and p75^NTR on neutrophils was increased after LPS stimulation in vitro culture for 14hours.Compared to the PBS control group,after LPS stimulation in vitro culture for 14 hours,the ROS MFI of peripheral blood neutrophils in the human proBDNF protein intervention group significantly increased;the MFI of the p Hrodo?Green fluorescent dye in peripheral blood neutrophils in the human proBDNF protein intervention group significantly decreased;early apoptosis rate of neutrophils in the human proBDNF protein intervention group significantly increased but no statistical difference in late apoptosis rate.Compared with the IgG isotype control group,after LPS stimulation in vitro culture for 14 hours,the ROS MFI of peripheral blood neutrophils in the mAb-proB intervention group significantly decreased,while the MFI of the p Hrodo?Green fluorescent dye in peripheral blood neutrophils and apoptosis percentage had no statistical difference.Conclusion:1.Patients with sepsis were in an immunosuppressive status characterized by a decrease in lymphocyte count and an increase in the proportion of neutrophils.2.The expression of proBDNF/p75^NTR on periphery neutrophils of sepsis patients and septic mice was upregulated,and it may participate in regulating the progression of sepsis-induced lung injury through autocrine pathways.3.Neutralizing proBDNF or conditional knock-out p75^NTR gene of the myeloid lineage can improve the survival rate,improve lung injury and decrease neutrophil infiltration in septic mouse models.This may be related to enhancing the phagocytic function and reducing ROS production of neutrophils.