Inactivated Whole Cells Induced Memory Alveolar Macrophages Play An Important Role In Its Rapid Protection Against Acinetobacter Baumannii Infection

Background:In 2017,the WHO listed 12 drug-resistant bacteria that posed a serious threat to human health,with Acinetobacter baumannii at the top of the list.A.baumannii mainly causes infection in hospitalized patients,especially those with severe diseases.It mainly causes lung infections.Multi-drug resistant A.baumannii is prevalent worldwide,which makes the treatment of A.baumannii induced infection face serious challenge.As an economical and effective means of prevention and treatment,vaccines can control the infection and spread of drug-resistant bacteria at the source.A.baumannii vaccination mainly focuses on hospitalized patients.The time window of vaccination is very short,which requires rapid reaction within 3to 7 days after inoculation for early immune protection.In the previous study,our group found that intranasal immunization with a single dose of inactivated whole cell(IWC)vaccine could provide 100%rapid protection for mice against the lethal dose of A.baumannii.The rapid immune protection of IWC could also be induced in Rag1-/-mice,whose T and B cells were deficient.And the host response dynamics results showed that the challenge at 7 days after immunization triggered a memory response,suggesting that the rapid protection is dependent on innate immune memory.Innate immunity memory means the innate immune cells can be reprogrammed by metabolism and epigenetics for immune memory after the initial immune exposure,which leads to an enhanced host defense response upon the second stimulation.The mTOR-HIF-1?signaling pathway regulates a variety of metabolic processes,mediating epigenetic reprogramming,and thus induces innate immune memory.Alveolar macrophages(AMs)are the first line of defense against pathogens invading in the lung.AMs renew slowly and can self-sustain,creating favorable conditions for the formation and maintenance of immune memory.Therefore,we speculate that A.baumannii IWC immunity may induce AMs to form innate immune memory,which plays an important role in rapid protection of vaccines.Macrophages are highly heterogenous.However,the characteristics of memory macrophage subset remain unclear.Our research used single cell RNA sequencing(scRNA-seq)to clarify the characteristics of AM subsets with IWC-induced memory and verify its function in the rapid protection,which provides theoretical basis for quick reaction of A.baumannii vaccines.Objectives:1.To establish a lethal mouse pneumonia model for vaccine evaluation.2.To clarify the role of alveolar macrophages in the rapid protection of A.baumannii IWC vaccine.3.To identify the subsets of memory alveolar macrophages and their characteristics after vaccine immunization.4.To explore the role of mTOR pathway in the rapid protection of A.baumannii IWC vaccine mediated by alveolar macrophages.Methods:1.Thirty clinical isolates of A.baumannii were used to intratracheally infect mice,and the mortality of mice was observed to identify their virulence.The sequence type(ST)of clinical strain was identified by multilocus-sequence typing(MLST),and then screened representative genotypes of A.baumannii.Mice were infected with hypervirulent A.baumannii strain to establish pneumonia model.And the survival rate,bacterial burden,and histopathology of infected mice were tested,cytokines were detected by real-time PCR,and the inflammatory cell infiltration in BALF was detected by flow cytometry.Mice were immunized using IWC of A.baumannii strains with different STs and infected after 7 days,survival rate of mice was monitored.2.B-NDG mice were intranasally immunized with IWC and then intratracheally challenged to determine the survival rate and confirm the role of innate immune cells in the rapid protection of IWC vaccine.Donor mice were intranasally immunized with IWC,after 7days,CD11C+AMs of donor mice were isolated with immunomagnetic beads and adopted into the lung of acceptor mice.Then acceptor mice were challenged with A.baumannii.At 24hours post infection,clinical score and bacterial burden were determined.In vivo and in vitro AMs training models with IWC were established,and real-time PCR was used to detect the cytokine levels of AMs at 7 days after immunization.Two hours after IWC restimulation,TNF-?level in cell culture supernatant was detected by ELISA.IWC immunized mice wereintraperitoneally injected with TNF-?antibody and then challenged to determine the survival rate.The expression of phagocytic and bactericidal genes of AMs was detected by real-time PCR at 7 days after IWC immunization,the phagocytic and bactericidal capacity of AMs after1 hour and 2 hours of challenge were determined by flow cytometry.3.Flow cytometry was used to detect the dynamic changes of AMs in BALF of mice at 0,7,14,and 30 days after IWC immunization.ScRNA-seq was performed to study the lung tissues of the unimmunized mice and the mice after 7 days of IWC immunization,the lung cells were clustered,the changes of number and proportion of lung cell clusters after immunization were determined and the effects of IWC immunization on the lung cells were analyzed.Cluster,definition,pesudotime of mononuclear macrophages,and the expression of specific genes of different cell clusters were analyzed.According to the results of scRNA-seq,real-time PCR was used to detect the expressions of specific genes of the unique AM cluster.4.Seven days after single dose immunization of IWC,mTOR activation in lung tissues and AMs were detected by WB,real-time PCR,and flow cytometry,respectively.Rapamycin was intraperitoneally injected to inhibit the mTOR pathway of mice at the immune stage of IWC,the survival rate of mice was determined.Bacterial burden in lung tissue and blood and the lung histopathology were evaluated at 24 hours after challenge.ELISA was used to detect the levels of cytokines in lung homogenate and serum.Results:Part i:Isolation and identification of A.baumannii and establishment of pneumonia model1.Twenty-three of the 30 clinical strains could cause death of mice,among which 6strains were hypervirulent with a mortality of 100%.And SJZ24 was the most virulent.2.MLST genotyping results showed that 21 of the 30 isolates were ST2 and belonged to CC2,and 5 hypervirulent strains belonged to ST2.3.The pneumonia model of A.baumannii was established with the hypervirulent strain SJZ24.Infection with gradient dose of SJZ24 showed that 5×10~7 CFU was the lowest 100%lethal dose.Mice were infected with 5×10~7 CFU SJZ24,from 4 to 24 hours after infection,the bacterial burden of lung was maintained at 8 log10 CFU,and the burden of blood and other organs increased.After infection,the lung tissue damage,cytokine levels,and inflammatory cell infiltration of the mice were increased after infection(P<0.05),suggesting the mouse lethal pneumonia model was successfully established.4.Mice were immunized with IWC of clinical strains with different STs and then challenged.The survival rates were significantly higher in IWC vaccinated groups than control group(P<0.05),suggesting that the mouse models of clinical strains can be used for A.baumannii pneumonia vaccine evaluation.Part ii:Function of alveolar macrophages in the rapid immune protection of A.baumannii IWC vaccine1.Survival rate of IWC-immunized B-NDG mice was 60%,indicating that innate immune cells,except for T,B and NK cells,played an important role in IWC-induced fast protection.2.The adoption the IWC immunized AMs led to significantly lower clinical score and bacterial burden in mice at 24 hpi compared with control mice(P<0.05),which indicated that AMs played an important role in the IWC-mediated immune protection.3.Seven days after IWC immunization,the levels of TNF-?,IL-6 and IL-1?in AMs of IWC-immunized group were significantly higher than those in the unimmunized control group(P<0.05).In the ex vitro and in vitro models of AMs,after restimulated with IWC for 2hours,the TNF-?level of IWC-immunized AMs was significantly higher than that in the unimmunized group(P<0.05).AMs maintained a higher transcription level of cytokines after IWC immunization and produced a higher level of cytokines when restimulation,suggesting that IWC could induce the formation of memory AMs.4.The survival of mice in the IWC-immunized group blocked by TNF-?antibody was significantly lower than the group without antibody(P<0.05),indicating that AMs might play an important role in the IWC-mediated protection through the production of TNF-?.5.Compared with unimmunized AMs,the transcription levels of phagocytic and bactericidal genes CYBB,CAMP and S100A8 in IWC-immunized AMs were significantly increased(P<0.05)after 7 days of IWC immunization.Mice were challenged with GFP-A.baumannii after 7 days of immunization.After 1 hour and 2 hours of challenge,the phagocytic ability of AMs in IWC-immunized group was higher compared with the unimmunized group.It indicated that IWC-immunized AMs showed stronger ability to devour bacteria after challenge,so as to play an immune protection role.Part iii:Exploration of the characteristics of memory alveolar macrophages subset after A.baumannii vaccine immunization1.After IWC immunization,a unique group of CD11C+CD11B+cells were significant increased and persisted for a long time,indicating that IWC vaccine induced the production of a new group of AMs,which may be related to innate immune memory of AMs.This group of CD11C+CD11B+cells could persist 14 and 30 days after vaccination,suggesting the persistence of this innate immune memory.2.The scRNA-seq results of lung tissue showed that all cells were divided into 41clusters.The number and percentage of mononuclear macrophages was significantly higher than that of the control group at 7 days after IWC immunization.3.The scRNA-seq results of mononuclear macrophages were analyzed to obtain 16 cell clusters,which were mainly divided into three states by pseudotime trajectory analysis.Compared with the control group,the IWC-7d immunization group had a unique Cluster 7.GO and KEGG showed that Cluster 7 might have stronger cellular chemotaxis,phagocytosis,inflammatory factor response,and inflammatory signal transduction abilities than other clusters,and its mTOR signaling pathway was significantly up-regulated.4.Real-time PCR was used to verify the scRNA-seq results,the expression of Cluster 7specific genes Gpnmb,Anpep,Syngr1 and Mmp14 in AMs of IWC immunized group was significantly higher than that in the unimmunized control group(P<0.01).Part iv:Role of mTOR pathway in the fast protection of A.baumannii IWC mediated by alveolar macrophages1.The phosphorylation and expression of proteins in mTOR pathway were significantly increased in IWC-immunized group compared to unimmunized group at 7 days after immunization(P<0.05),suggesting mTOR signal can be activated and maintained a higher level after IWC immunization.2.When immunized with IWC,the survival of mice treated with rapamycin in immunization stage was significantly lower than that of mice without rapamycin(P<0.05).At24 hours after challenge,the bacterial burden,tissue damage degree,and cytokine production of the rapamycin treated group were significantly higher than that of the untreated group(P<0.05).It shows that mTOR signal plays an important role in the rapid protection mediated by IWC vaccine.Conclusions:1.The lethal mouse pneumonia model established by hypervirulent clinical strains of A.baumannii can be used to evaluate the universality of the rapid protective effect of IWC vaccine.2.Alveolar macrophages can be induced with memory by A.baumannii IWC vaccine and play an important role in its rapid immune protection.3.Immunization with A.baumannii IWC vaccine induces a unique cluster of memory alveolar macrophages in mice.4.mTOR pathway may play an important role in the rapid protective effect of A.baumannii IWC vaccine mediated by alveolar macrophages.Significance:This study confirmed that alveolar macrophages formed innate immune memory with training of IWC,and presented a unique memory AMs cluster.Moreover,memory AMs play an important role in the rapid protection of vaccine.The role of mTOR pathway in AMs-mediated rapid protection was preliminarily elucidated.These results provide a theoretical basis for the rapidly protective vaccine and enrich the understanding of innate immune memory.