SummaryDendritic cells (DC) are required for the immune response against Listeria monocytogenes and are permissive for infection in vivo and in vitro. However, it is unclear if DC provide a desirable intracellular niche for bacterial growth. To address this issue, we have compared the behaviour of L. monocytogenes in murine bone marrow-derived DC and macrophages (BMM). Similar to BMM, bacteria escaped to the cytosol in DC, replicated, and spread to adjacent cells. However, DC infection was less robust in terms of intracellular doubling time and total increase in bacterial numbers. Immunofluorescence analysis using a strain of L. monocytogenes that expresses green fluorescent protein upon bacterial entry into the cytosol suggested that a subpopulation of DC restricted bacteria to vacuoles, a finding that was confirmed by electron microscopy. In unstimulated DC cultures, L. monocytogenes replicated preferentially in phenotypically immature cells. Furthermore, DC that were induced to mature prior to infection were poor hosts for bacterial growth. We conclude that DC provide a suboptimal niche for L. monocytogenes growth, and this is at least in part a function of the DC maturation state. Therefore, the generation of an effective T cell response may be a net effect of both productive and non-productive infection of DC.
To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α + , CD4 + , and CD8α − CD4 − DC and the B220 + plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8+ T cells at 24hpi. The CD8α + DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4+ DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α − CD4 − DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8 + DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8 + T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α + DC subset.
Rationale ATP-binding cassette transporter A1 (ABCA1) plays a critical role in eliminating excess free cholesterol (FC) from tissues by effluxing cellular FC and phospholipids to lipid-poor apolipoprotein A-I. Macrophage ABCA1 also dampens pro-inflammatory MyD88-dependent Toll-like receptor signaling by reducing cellular membrane FC and lipid raft content, indicating a role of ABCA1 in innate immunity. However, whether ABCA1 expression has a role in regulating macrophage function in vivo is unknown. Objective We investigated whether macrophage ABCA1 expression impacts host defense function, including microbial killing and chemotaxis. Methods and Results Myeloid cell-specific ABCA1 knockout (MSKO) vs. wild type (WT) mice were infected with Listeria monocytogenes (Lm) for 36h or 72h before sacrifice. Lm-induced monocytosis was similar for WT and MSKO mice; however, MSKO mice were more resistant to Lm infection, with significantly less body weight loss, less Lm burden in liver and spleen, and less hepatic damage 3 days post infection. In addition, Lm-infected MSKO mouse livers had: 1) greater MCP-1 and MIP-2 expression, 2) more monocyte/macrophage infiltration, 3) less neutral lipid accumulation, and 4) diminished expression of lipogenic genes. MSKO macrophages showed enhanced chemotaxis toward chemokines in vitro and increased migration from peritoneum in response to LPS in vivo. Lm infection of WT macrophages markedly reduced expression of ABCA1 protein as well as other cholesterol export proteins (such as ABCG1 and apoE). Conclusions Myeloid-specific ABCA1 deletion favors host response to and clearance of Lm. Macrophage Lm infection reduces expression of cholesterol export proteins, suggesting that diminished cholesterol efflux enhances innate immune function of macrophages.
Inactivated influenza vaccines are not approved for use in infants less than 6 months of age due to poor immunogenicity in that population. While the live attenuated influenza vaccine has the potential to be more immunogenic, it is not an option for infants and other vulnerable populations, including the elderly and immunocompromised individuals due to safety concerns. In an effort to improve the immunogenicity of the inactivated vaccine for use in vulnerable populations, we have used an approach of chemically crosslinking the Toll-like receptor (TLR) 7/8 agonist R848 directly to virus particles. We have reported previously that an R848-conjugated, inactivated vaccine is more effective at inducing adaptive immune responses and protecting against lung pathology in influenza challenged neonatal African green monkeys than is the unmodified counterpart. In the current study, we describe a second generation vaccine that utilizes an amide-sulfhydryl crosslinker with different spacer chemistry and length to couple R848 to virions. The new vaccine has significantly enhanced immunostimulatory activity for murine macrophages and importantly for monocyte derived human dendritic cells. Demonstration of the significant differences in stimulatory activity afforded by modest changes in linker impacts our fundamental view of the design of TLR agonist-antigen vaccines.
Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2؉ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2؉ , major histocompatibility complex class II ؉ (MHC-II ؉ ) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response.
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