Microparticles Released Bylisteriamono-Cytogenesinfected Macrophages Are Required For Dendritic Cell-elicited Protective Immunity

[Objective] The mutual interaction between innate and adaptive immune responses plays a crucial role in the optimal clearance of invading pathogens. Innate immune cells respond first to infection but are frequently insufficient to overcome the virulence mechanisms of pathogens; thus, the adaptive immune responses are activated. Macrophages and dendritic cells are two key innate immune cell types involved in phagocytosis and presentation of antigen respectively, upon bacterial infection. It is accepted that both macrophages and dendritic cells contribute to the activation of T cells; however, the interplay between these cells in the processing and presentation of bacterial antigens for the goal of activating T cells remains poorly understood. Using a Listeria monocytogenes (Lm) infection model, investigating a cross-talk between these two cell types. DCs play a critical role in priming adaptive immunity against Lm, whereas, macrophages fail to initiate anti-Listeria CTL responses in the absence of DC. However, DCs were not effective at directly capturing Lm, compared with macrophages. Such discrepancies suggest that a mutual interaction might exist between macrophages and dendritic cells in the processing, and presentation of antigens to T cells in the induction of protective immune response. Recent studies show that, microparticles play a important role in cell communications. It is not clear that the effection of microparticles in adaptive immune responses. In the present study, we show that both macrophages and DCs are essential for the induction of Lm-specific T cell responses but with different responsibilities. Macrophages phagocytose and release Lm antigens-containing MPs; which are subsequently captured by DCs leading to priming T cell responses.[Methods]1. To investigate the role of macrophages in eliciting the adaptive immunity against Lm infection.(1) To detect the the proliferation of adoptively transferred T cells in depleting macrophages mice in vivo.BALB/c nude mice (n=6) were i.p. injected with clodrolip or anti-F4/80depleting mAb for macrophage depletion. Then mice were adoptively transferred with CFSE-labeled T cells isolated from the spleens of Lm-infected mice or naive mice (control), and1.0×103viable Lm were injected into these mice after6h.60h later, the proliferation of adoptively transferred T cells in the spleen was determined by flow cytometry.(2) To investigate the role of macrophages in the proliferation of anti-Lm T cells in vitro.Bone marrow-derived DCs (BALB/c background) were incubated with viable Lm (100μg/ml gentamycin added) in the presence or absence of peritoneal macrophages for various time intervals (6,12, and18h).1×105splenic T cells, isolated from the spleens of Lm-infected BALB/c mice, were co-cultured with those DCs (1×104) or Lm-infected macrophages.[3H] thymidine was added after cuturing72h, then T cell proliferation was measured by liquid scintillation analyser18h later.(3) Supernatants from Lm-infected macrophage cell cultures stimulated the proliferation of T cells.T cells, isolated from the spleens of Lm-infected BALB/c mice, were incubated with DCs that had been treated with the supernatants of Lm-infected macrophages, Lm-infected DCs, or untreated DCs.[3H] thymidine was added after cuturing72h, then T cell proliferation was measured by liquid scintillation analyser18h later.2. To detect MPs from Lm-infected macrophage cell(1) Isolation of microparticlesSupernatants of cultured macrophages were used to isolate MPs as described before.16Briefly, supernatants were centrifuged at300gx5min,500gx5min,1500gx5min and5000gx5min to remove of cells and debris. The supernatant was passed through a1.2μm filter in order to remove bacteria, and then further centrifuged for60min at14000g to pellet MPs.(2) Labelling of microparticlesBacteria were stained with CFSE and used to infect macrophages. MPs isolated from macrophages were labeled with a red-fluorescent cell linker (PKH26, Sigma) according to the manufacturer’s protocol. Such fluorescent MPs were observed under two-photon fluorescent microscopy or analyzed by flow cytometr.3. Functions of microparticles(1) MPs drived from Lm-infected macrophage stimulated DCs activation.DCs were incubated with PBS, MPs from Lm infected or control macrophages for24h and were stained with CD80, CD86or MHC class Ⅱ mAb. and analyzed by flow cytometry. Bone marrow-derived DCs were stimulated with MPs from Lm-infected or control macrophages for various time intervals (0-60min). Western blot was performed for analysis of MAPK ERK and IkB phosphorylation.(2) MPs drived from Lm-infected macrophage stimulated Tcell proliferation.Macrophages were treated with different MOIs, and the isolated MPs were used to treat DCs for T-cell proliferation. 4. In vivo Listeria monocytogenes protection assay5×106macrophages in2ml culture media were treated with PBS or5x107viable Lm (100μg/ml gentamycin added30min later) for48h. Additionally,5×107viable Lm were incubated in2ml culture media alone with gentamycin. Each2ml supernatants were used for MP isolation and the quantity was used for one mouse injection. Mice were immunized subcutaneously with MPs mixed with rehydragel adjuvants for7days and challenged by i.v. injection of1×105viable Lm. Survival was monitored for10days. Six mice were used per group.[Results]1. Macrophages are required for DC-elicited anti-Lm T cell response in vivoSplenic macrophages, but not DCs, were depleted in mice by i.p. injecting liposomal clodronate or anti-F4/80antibody. Under such condition, BALB/c nude mice were adoptively transferred with T cells isolated from the spleens of Lm-infected mice. We found that the depletion of macrophages abrogated the in vivo proliferative response of adoptively transferred T cells60h after the i.v. injection of1.0×103viable Lm. To confirm these results, we transferred the Lm-infected macrophages into naive C57BL/6mice for7days and the spleen cells were cultured with killed bacteria for measurement of cytokine production by ELISA assay. As expected, the inoculation of Lm-infected macrophages into C57BL/6mice strongly induced the production of IFN-y and IL-22, two potent mediators of cellular inflammatory responses against bacterial pathogens. In addition, IFN-y-producing CD4+T cells were analyzed by FACS. These findings suggested that the initial infection of macrophages by Lm is required for the generation of protective immune responses. Thus, although macrophages do not directly present Lm peptides, they seem to participate in the induction of Lm specific T cell responses.2. Supernatants from Lm-infected macrophage cell cultures confer DC maturation and presentation of Lm antigensMacrophages were infected with viable Lm (gentamycin was added30min later) for24h and supernatants were harvested by centrifugation and filtration. And then the supernatants were incubated with DCs for24h. The expression of CD80, CD86, and MHC class II on DCs was upregulated by the supernatants from Lm-infected macrophages compared to cells incubated with supernatants from uninfected macrophages. Such DC activation was not ascribed to the bacterial contamination, since the supernatant from the above Lm alone did not affect DC maturation. Moreover, we found that DCs treated with supernatants of Lm-infected macrophages effectively induced T cell proliferation. These findings suggested that factors released from Lm-infected macrophages are capable of eliciting the maturation and conferring the immunogenicity of DCs against Lm infection.3. Microparticles shed by Lm-infected macrophages are the source for DC immunogenicityMacrophages were infected with CFSE-labeled Lm and the released MPs were isolated from the supernatants. The fluorescence was observed in MPs via both flow cytometry (27.4%CFSE positive MPs) and under the microscope, indicating the presence of Lm-derived bacterial components in MPs. Consistently, Lm component-containing MPs stimulated DC maturation and resulted in T cell proliferation. Here, we also used Lm with different multiplicity of infection (MOI) to treat macrophages and assayed the effect of isolated MPs on T cell proliferation. We found that even low numbers of Lm could result in T cell proliferationvia DC antigen presentation and that increased numbers of Lm further promoted T cell proliferation. To further analyze the effect of MPs on DCs, we examined the activation of MAP kinases and NF-κB, two critical signaling pathways involved in DC activation. DCs were stimulated with MPs from Lm-infected or control macrophages for various time intervals (10,30,60min). The activation of MAP kinase and NF-κB by MPs from Lm-infected macrophages was confirmed by the induction of ERK and IκB phosphorylation.4. Macrophages/microparticles/DCs form an axis to transfer Lm antigenicityNext, we wondered how MPs by Lm-infected macrophages transferred Lm antigenicity to DCs. By using PKH26membrane dye to stain MPs, we found that35%DCs presented red fluorescence. DCs are known to have the capacity to take up apoptotic cells. Here, we also found that phosphatidylserine was translocated to the outer layer of the membrane of MPs, the marker of apoptosis. In addition, we wanted to clarify DCs acquiring antigens pathway. MHC class I-deficient macrophages were infected with Lm to generate MHC class I-deficient MPs. These MPs significantly reduced the presentation of Lm antigen by DCs to CD8+T cells. We also found that MyD88deficiency in macrophages did not affect the activation of T cells, but MHC class II deficiency in DCs significantly reduced the presentation of MP-derived Lm antigen by DCs to CD4+T cells. These data also suggested that DCs may capture MPs through different pathways, including the membrane fusion between DCs and MPs and DC uptake and processing.5. Actin filament is required for the generation of microparticles containing Lm components by macrophagesIn this regard, we wondered whether cytoskeleton was required for the production of Lm-induced MPs by macrophages. Thus, we treated macrophages with cytochalasin D, an inhibitor of F-actin polymerization, and found that the impairment of actin filament formation resulted in the decreased of MP release by Lm-treated macrophages. We then further treated macrophages with blebbistatin, an inhibitor of myosin II ATPase activity. Similarly, the inhibition of actin filament motility also led to the decreased MP release. Consistently, it was found that after the cytochalasin D or blebbistatin treatment MPs attenuated the effect of Lm components on DCs eliciting T cell proliferation. Therefore, these findings suggest that myosin Ⅱ-triggered, actin filament-generated tension might mediate the production of Lm component-containing MPs by macrophages.6. Generation of microparticles containing Lm components by macrophages and uptake by DCs in vivoNext, we validated the in vivo generation of MPs by macrophages under the condition of Lm infection. Using a peritoneal infection model, we i.p. injected1x107CFU CFSE-labeled Lm to mice.12h later, the peritoneal lavage was applied to isolate the MPs. As expected, we found that13%MPs contained Lm components by flow cytometry. However, if we previously depleted peritoneal macrophages we found that Lm infection only resulted in0.6%CFSE positive MPs, suggesting that MPs containing Lm components are mainly generated by macrophages after peritoneal Lm infection. To clarify DCs taking up these MPs, MPs were isolated from peritoneal lavage12h after CFSE-labeled Lm peritoneal infection, and then i.p. injected into naive mice.6hours later, We harvested peritoneal cells and found that3～6%of peritoneal cells were CD11c+DCs and～40%of these cells were CFSE positive. Consistently, the result of confocal microscopy also showed that DCs took up Lm components. These findings suggested that during Lm infection in vivo, macrophages phagocytose Lm and release Lm component-packaging MPs, leading to the subsequent uptake of the released MPs by DCs.7. Microparticles from Lm-infected macrophages elicit protective immunityFinally, we wondered whether the Lm antigenicity of MPs is able to elicit protective immune response in vivo. To verify this, Mice were immunized subcutaneously with MPs from Lm-infected macrophages mixed with rehydragel adjuvant, for7days followed by challenge by i.v. injection of1×105CFU viable Lm. The results showed that most mice immunized with MPs from Lm-infected macrophages survived, as opposed to the mice immunized with control MPs. However, such protective immunity could not be ascribed to the contamination of Lm in MPs, since after the filtration of the supernatants of single Lm incubation, the centrifugated pellets had no protective effect against Lm challenge. Furthermore, in these in vivo experiments, we also used gentamicin to treat mice concomitant with MP injection. Together, these data suggested that MPs containing Lm components elicit protective immune responses.[Conclusions] In the present study, we demonstrated that DCs require the participation of macrophages to generate protective immune responses against Lm infection. MPs were released from Lm-infected macrophages, might contain Lm components. DCs may capture Lm-MPs through different pathways, including the membrane fusion between DCs and MPs and DC uptake and processing. DCs directly present MHC class Ⅰ-peptide complexes derived from Lm-MPs or process Lm antigens and assemble MHC class Ⅱ-peptidecomplexes to T cells.In conclusion, Microparticles released by Listeria Monocytogenes infected macrophages are required for dendritic cell-elicited protective immunity.