The Roles Of TLR2 And IFN-? In Phagocytes Against Listeria Monocytogenes Infection

Object:Listeria monocytogenes(L.monocytogenes,Lm)is a foodborne Gram-positive intracellular pathogen capable of invasion and replication in both phagocytic and non-phagocytic cells(e.g.,hepatocytes).After invadation,it disseminates and further replicates in peripheral organs such as the liver and spleen.Overloaded intracellular Lm egresses and enters into the circulation,and finally invades the brain or the placenta,causing meningitis,encephalitis,septicemia,and even death(death rate 20-30%).There are many components in Lm that can be recognized by TLRs.Different TLRs play different roles in Lm recognition:in the host,Lm does not express flagella,so it cannot be recognized by TLR5;and the absence of TLR4 and TLR9 does not affect the immune response against Lm.TLR2 plays an important role in anti-Lm immune response by enhancing the phagocytic ability of macrophages and up-regulating the production of TNF-?,IL-12,NO,as well as the expression of costimulatory molecules CD40 and CD86.As an important member of the innate immune system,macrophages phagocytize Lm and limit bacterial replication in the early phase of infection.As a major regulatory factor of macrophage function,IFN-? is important in host resistance to Lm.IFN-? promotes macrophage-mediated bacterial clearance mainly by upregulating the expression of bactericidal molecules such as inducible nitric oxide synthase(iNOS).Moreover,IFN-? can induce the expression of MHC-?,which enhances antigen presentation and initiates adaptive immune responses.Activated NK cells or T cells are thought to be the main producers of IFN-?.In the liver,Kupffer cells(KCs),the liver resident macrophages,undergo rapid necroptotic cell death induced by the bacterial virulence factor listeriolysin O.The circulating monocytes are recruited into the liver,where they differentiate into monocyte-derived macrophages to compensate for KC loss.In addition to macrophages,neutrophils are another important family member of phagocytes that play a role in the early stage of LM infection.As the most abundant type of leukocytes,neutrophils are the first immune cells to accumulate at the infected site and phagocytize pathogens.However,the role of neutrophils in defense against LM infection is not entirely clear,as the limited research that was conducted reported controversial conclusions.Phagocytes play an important role in acute Lm infection,including phagocytising Lm,restriction of bacterial proliferation,and secretion cytokines and chemokines.TLR2 is important in Lm recognition by phagocytes;while IFN-y promotes the activation and bacterial clearance of phagocytes.However,it is still not clear that whether TLR2 and IFN-? have other functions in anti-Lm immune response,and how phagocytes interact with each other.In this study,we investigated the role and mechanism of TLR2 on monocytes/macrophages migration to the liver.Our study revealed the role and internal mechanism of IFN-y in the interaction between neutrophils and macrophages as well as in the tissue protection during Lm infection.Methods:1.Mice were i.p.infected with Lm.The roles of TLR2,TLR4,TLR9 and IFN-? in the defense against Lm were studied by detecting the survival rate,the weight of body,liver and spleen,bacterial burden in the liver and spleen,and histopathology of liver and spleen.2.The frequency,the number and the Ki-67 level of monocyte/macrophage(Mo/M?)in the liver and spleen of Lm infected WT and Tlr2-/-mice were detected by FACS.CFSE-labeled PECs were adoptively transferred to WT and Tlr2-/-mice followed by Lm infection.The frequency and absolute number of transferred Mo/M? in the liver were detected by FACS.3.Hepatocytes were isolated by in situ hepatic irrigation.The purity of hepatocyte was detected by immunofluorescence and FACS.Hepatocytes were infected with Lm in vitro,and the hepatocyte apoptosis was detected by FACS.4.The expression of cytokines/chemokines in WT and Tlr2-/-hepatocytes were measured using a proteome profiler array,qPCR and ELISA.The expression of chemokine receptors on Mo/M?s were measured by qPCR.5.The role of TLR2 on hepatocytes recruiting Mo/M? was detected by Transwell with stimulation of chemokines,CXCR2 antagonist or TLR2 agonist.6.The role of TLR2/NO/F-actin pathway on macrophage mobility was detected by Transwell with stimulation of NO donor or NO scavenger.F-actin polymerization was detected by rhodamine-phalloidin staining.7.The role of TLR2 on the formation of hepatic microabscesses and limiting bacterial spread were detected by H&E staining and IHC.8.WT and Tlr2-/-mice were treated with clodronate liposomes,adoptive transferred with macrophages,and infected with Lm.The survival was monitored.9.The expression of CD 169,CD204,and CD206 on macrophages in the liver was detected by FACS.The phagocytosis ability of macrophage was measured by neutral red uptake assay and bacterial burden.10.The expression of iNos was measured by qPCR,and the level of ROS was detected by FACS.The role of TLR2 on bacterial clearance by macrophages was detected by bacterial burden assay.11.Serum levels of the cytokines in the WT mice infected with Lm or the control WT mice were determined using CBA or ELISA.12.The frequency of IFN-y-producing immune cell subsets was detected by FACS.Serum IFN-y levels in Lm-infected WT,Rag1-/-and WT mice treated with anti-NKl.1,clodronate liposomes or anti-Ly-6G were measured by ELISA.The splenic neutrophils were sorted,and the supernatant IFN-y level was measured by ELISA.13.The expression of iNos was measured by qPCR,and the level of NO in the supernatants was detected using a Nitric Oxide Synthase Assay Kit.The role of IFN-y on bacterial clearance by neutrophils was detected by bacterial burden assay.14.WT and Ifng-/-mice were infected with Lm.The frequency,the apoptosis rate and the absolute number of splenic neutrophils was determined by FACS.15.Bone marrow neutrophils of WT and Ifng-/-mice were isolated and infected with Lm in vitro.The ROS level was detected by FACS.Bone marrow neutrophils that were not infected,infected after pre-treatment with an ROS scavenger,or infected without any pre-treatment,were transferred to the WT mice.The role of ROS released by apoptotic neutrophils on tissue injury was determined by H&E staining of the spleens.16.The level of ROS,MHC-?,CD86 and CD80 on splenic macrophages from WT and Ifng-/-mice was detected by FACS.The expression of iNos was measured by qPCR.The role of IFN-y on bacterial clearance by macrophages was detected by bacterial burden assay.17.CFSE-labeled bone marrow neutrophils isolated from WT or Ifng-/-mice incubated with macrophages and infected with Lm in vitro.Images were obtained using a fluorescence microscope.For the in vivo experiment,CFSE-labeled bone marrow neutrophils were injected into the corresponding mice the percentage of CFSE-positive macrophages were detected by FACS.18.MPO level in splenic macrophages from control or infected WT or Ifng-/-mice were detected by FACS at 2 dpi.19.TGF-? level in the supernatant obtained from the 12 h co-culture of bone marrow neutrophils and peritoneal macrophages in the presence of Lm was mearsured by ELISA.TGF-? level in the serum of infected WT mice,lfng-/-mice,and infected Ifng-/-mice treated with IFN-y was mearsured by ELISA.Results:1.TLR2 protected mice against Lm infectionWT,Tlr2-/-,Tlr4-/-and Tlr9-/-mice were i.p.infected with 1 × 106 CFU of Lm.Compared with mice in other groups,the survival time of Tlr2-/-mice was significantly shortened.The spleen of Tlr2-/-mice was not enlarged at 2 dpi(days post infection),with low level of IFN-? and IL-6 in serum,suggesting that the immune response in the spleen may be impaired.Moreover,Tlr2-/-mice exhibited a high bacterial loads in both the liver and spleen at 3 dpi.This indicates that TLR2 deficiency makes mice more sensitive to Lm infection,and TLR2 plays an important protective role in mice against Lm infection.2.TLR2 promotes hepatic Mo/M? infiltration in response to acute Lm infectionAt day 2 post-infection,a deficiency in TLR2 resulted in a decreased proportion and absolute number of Mo/M? in the liver and spleen,however,it is not due to the disturbed proliferative capacity.WT and Tlr2-/-mice were transferred with CFSE-1abeled WT-or Tlr2-/--PECs,respectively,and followed by Lm infection.WT recipient liver recruited more Mo/M?s than that of the Tlr2-/-recipient liver.Hepatocytes were isolated and infected with Lm in vitro.The bacterial burden and rate of hepatocyte apoptosis were similar between Lm-infected WT-and Tlr2-/--hepatocytes.The effect of hepatocytes on recruiting Mo/M?s was measured by Transwell.WT-hepatocytes recruited more Mo/M?s than Tlr2-/--hepatocytes,and WT-Mo/M?s exhibited a stronger migratory capacity compared to Tlr2-/--Mo/M?s.This indicates that TLR2 promotes hepatic Mo/M? infiltration in response to acute Lm infection.3.TLR2 signaling promotes the production of CCL2 and CXCL1 by hepatocytes,which recruit Mo/M? into the liverCompared with WT mice,Tlr2-/-mice showed lower levels of CCL2 and CXCL1 in the Lm-infected liver homogenate and in the supernatant of in vitro infected hepatocyte.The infiltration capacity of Mo/M?s was promoted by exogenous CXCL1 treatment,while the ability of WT-hepatocyte-mediated Mo/M? infiltration was obviously inhibited by a CXCR2 antagonist.In addition,the expression of Cxcr2 was decreased on Tlr2-/--Mo/M?s.These demonstrate that Lm-activated TLR2 signaling promotes the production of CCL2 and CXCL1 by hepatocytes,which recruit Mo/M? into the liver;CXCL1-CXCR2 signaling provides a novel and important mechanism for TLR2-induced Mo/M? migration in addition to CCL2.4.TLR2 promotes macrophage mobility via the TLR2/NO/F-actin pathwayUsing a transwell chamber migration assay,obvious migration was observed in Lm-infected WT macrophages;however,few Tlr2-/-macrophages migrated into the lower chamber.The level of F-actin in TLr2-/--Mo/M?s was lower than in WT-Mo/M?s both infected with Lm in vivo and in vitro.Both Lm and the TLR2 agonist,Pam3CSK4,could enhance NO production in the supernatant of WT-Mo/M?s.NO donor(SNP)stimulated WT-Mo/M? mobility and F-actin polymerization,while the mobility and F-actin polymerization in the Mo/M?s induced by TLR2 could be suppressed by the NO scavenger.These indicate that TLR2 triggers macrophage mobility via the TLR2/NO/F-actin pathway.5.TLR2 contributes to hepatic microabscess formationCompared to WT infected mice,Tlr2-/-infected mice showed fewer and smaller hepatic microabscesses at 2 dpi.Both WT-and Tlr2-/-Mo/M?s were scattered evenly throughout the uninfected liver tissues.In response to Lm infection,WT-Mo/M?s aggregated to form hepatic microabscesses,whereas most Tlr2-/-Mo/M?s remained in a distributed manner with few microabscesses.Importantly,Lm was limited in the hepatic microabscesses of WT mice,while Lm diffused and replicated in the livers of the Tlr2-/-mice.These indicate that TLR2 promotes the mobility of Mo/M?,which enhances the formation of hepatic microabscesses,and then limits Lm spread.6.TLR2 induces the recognition,phagocytosis and clearance of Lm by macrophagesThe presence or absence of TLR2 does not affect macrophages phagocytosing granules,but a TLR2 deficiency decreased the recognition and phagocytosis of Lm by macrophages,which was accompanied by the low percentage of macrophages expressing pattern recognition receptors(PRRs)associated with phagocytosis(e.g.,CD 169,CD204,and CD206).The expression of these PRRs were recovered in Tlr2-/-mice by transferring WT-macrophages,and the survival time was prolonged.The expression of inducible nitric oxide synthase(iNOS)in Tlr2-/-macrophages was far below that of the WT-macrophages.The bacterial burden in Tlr2-/--macrophages was higher than that in WT-macrophages.These indicate that TLR2 induces the recognition,phagocytosis and clearance of Lm by macrophages,which plays a critical role in macrophage-mediated anti-Lm responses.7.IFN-? is rapidly produced and plays a critical protective role against Lm infectionVarious cytokines were secreted in response to LM infection.IFN-? was hardly secreted under the resting state,but it was found in abundance after Lm infection.lfng-/-mice were highly sensitive to Lm infection,as demonstrated by short survival time,the drop in body weight,high bacterial load in the liver and spleen,and severer tissue injury.These results demonstrate that IFN-? is quickly produced and plays a critical role in the defense against Lm infection by both bacterial clearance and suppression of Lm-induced tissue injury.8.Neutrophils are an important source of IFN-? during Lm acute infectionAfter Lm infection,neutrophils comprised a large proportion of the IFN-?-producing subpopulations.The level of IFN-y in the neutrophil-depleted mice was maintained at a significantly low level after Lm infection.Splenic neutrophils were sorted from Lm-infected WT mice incubated in vitro,and IFN-? production could be detected in the supernatant of Lm-infected neutrophils.Furthermore,by transferring WT-neutrophils,Ifng-/-mice could produce IFN-? markedly in response to Lm infection and showed prolonged survival time.These indicate that neutrophils are an important source of IFN-? during LM acute infection.9.IFN-y augments the anti-Lm activity of neutrophils and promotes neutrophil survivalAfter Lm infection,the mRNA level of iNos and production of nitric oxide(NO)in Ifng-/-neutrophils was significantly lower than that in WT neutrophils,accompanied with a higher bacterial load.Lm induced neutrophil apoptosis in a dose-dependent manner.Compared with WT mice,both the apoptosis rate and the absolute number of apoptotic splenic neutrophils in the Ifng-/-mice were significantly aggravated at 2 dpi.These demonstrate that IFN-y promotes NO production and augments the anti-Lm activity of neutrophils,and augments the survival of neutrophils by protecting them against Lm infection-induced apoptosis.10.Lm-induced apoptosis of neutrophils contributes to tissue injury via release of ROSLm infection in vitro induced neutrophil apoptosis.The ROS level was very high in non-necrotic and early apoptotic neutrophils,while it was very low in late apoptotic and necrotic neutrophils.There was a higher percentage of ROSlow neutrophils among the Ifrng-/-neutrophils than among the WT neutrophils.Bone marrow neutrophils were undergone apoptosis after Lm infection,and transferred into WT mice.The spleens from these WT mice showed obvious tissue damage,which was alleviated by pre-treatment with the ROS inhibitor.Importantly,compared to lfng-/-mice transferred with Ifng-/--neutrophils,Ifng-/-mice transferred with WT-neutrophils displayed an alleviative injury.These indicate that IFN-? protects tissues from damage by Lm infection by preventing the apoptosis of neutrophils as well as the release of ROS.11.IFN-? promotes macrophage-mediated phagocytosis of apoptotic neutrophils and alleviates tissue injuryBoth in vivo and in vitro macrophage phagocytosis assay showed that WT macrophages could effectively induce the phagocytosis of neutrophils,while Ifng-/-macrophages exhibited very low phagocytic efficiency.The percentage of MPO+macrophages in the spleen of Ifng-/-mice was lower that in WT mice at 2dpi.Moreover,IFN-y deficiency decreased the TGF-? production in the supernatant obtained from co-culture of macrophages and neutrophils,and in the serum of mice after Lm infection.These indicate that IFN-? promotes macrophage-mediated phagocytosis of apoptotic neutrophils,accompanied with phagocytizing MPO and producing TGF-? by macrophages.Both of these contribute to alleviating tissue injury.Conclusions:1.TLR2 plays an important protective role in mice against Lm infection.TLR2 promotes hepatic Mo/M? infiltration in response to acute Lm infection.TLR2 signaling promotes the production of CCL2 and CXCL1 by hepatocytes,and CXCL1-CXCR2 signaling provides a novel and important mechanism for TLR2-induced Mo/M? migration in addition to CCL2.Futhermore,TLR2 triggers macrophage mobility via the TLR2/NO/F-actin pathway and promotes the mobility of Mo/M?,which enhances the formation of hepatic microabscesses,and then limits Lm spread.TLR2 induces the recognition,phagocytosis and clearance of Lm by macrophages,which plays a critical role in macrophage-mediated anti-Lm responses.2.Neutrophils are an important source of IFN-? during Lm acute infection.IFN-? is quickly produced and plays a critical role in the defense against Lm infection by both bacterial clearance and suppression of Lm-induced tissue injury.IFN-?.promotes NO production,augments the anti-Lm activity of neutrophils and the survival of neutrophils by protecting them against Lm infection-induced apoptosis.IFN-? protects Lm infection-induced tissue damage by preventing the release of ROS by the apoptosis of neutrophils as well as.In addition,IFN-? promotes macrophage-mediated phagocytosis of apoptotic neutrophils,accompanied with phagocytizing MPO and producing TGF-? by macrophages.Both of these contribute to alleviating tissue injury.