Replication Of Live Vaccine Strain Research Articles

Itaconate deficiency results in prolonged bacterial burdens and enhanced T cell responses during Francisella tularensis infection

Abstract Shifts in cellular metabolism and associated metabolites regulate immune cell function. Recently, it has been shown that the metabolite itaconate plays a critical role in modulating inflammatory responses. Using animals that cannot synthesize itaconate due to an absence of aconitate decarboxylase 1 (AOCD KO), we previously established the lack of itaconate increased susceptibility to high dose challenge with the live vaccine strain (LVS) of Francisella tularensis in terms of both bacterial clearance and survival. However, ACOD KO mice that survived sublethal LVS infection had improved survival against a lethal challenge with virulent F. tularensis (Ftt). Since appropriate coordination of both macrophage and T cell function is essential for surviving Ftt, we next determined if the improved survival of ACOD KO mice was due to alterations in macrophage and/or T cell function. LVS immune ACOD KO mice had increased numbers of lung resident and splenic effector CD4+ and CD8+ T cells compared to WT mice following resolution of infection. Utilizing an in vitro macrophage-T cell co-culture system we did not observe any difference in the ability of WT and ACOD KO T cells to control LVS replication in macrophages when T cells were co-cultured with genetically matched macrophages. However, when macrophage sources were mixed, we observed that regardless of the source of LVS immune T cells, WT macrophages controlled bacterial replication significantly better than ACOD derived macrophages. Together, these findings suggest that the lack of itaconate results in increased and prolonged bacterial burdens in vivo, which leads to greater expansion of effector T cells that confer enhanced protection during Ftt challenge.

Immune lymphocytes halt replication of Francisella tularensis LVS within the cytoplasm of infected macrophages

Francisella tularensis is a highly infectious intracellular bacterium that causes tularemia by invading and replicating in mammalian myeloid cells. Francisella primarily invades host macrophages, where it escapes phagosomes within a few hours and replicates in the cytoplasm. Less is known about how Francisella traffics within macrophages or exits into the extracellular environment for further infection. Immune T lymphocytes control the replication of Francisella within macrophages in vitro by a variety of mechanisms, but nothing is known about intracellular bacterial trafficking in the face of such immune pressure. Here we used a murine model of infection with a Francisella attenuated live vaccine strain (LVS), which is under study as a human vaccine, to evaluate the hypothesis that immune T cells control intramacrophage bacterial growth by re-directing bacteria into toxic intracellular compartments of infected macrophages. We visualized the interactions of lymphocytes and LVS-infected macrophages using confocal microscopy and characterized LVS intramacrophage trafficking when co-cultured with immune lymphocytes. We focused on the late stages of infection after bacteria escape from phagosomes, through bacterial replication and the death of macrophages. We found that the majority of LVS remained cytosolic in the absence of immune pressure, eventually resulting in macrophage death. In contrast, co-culture of LVS-infected macrophages with LVS-immune lymphocytes halted LVS replication and inhibited the spread of LVS infection between macrophages, but bacteria did not return to vacuoles such as lysosomes or autophagosomes and macrophages did not die. Therefore, immune lymphocytes directly limit intracellular bacterial replication within the cytoplasm of infected macrophages.

Microbial co-infection alters macrophage polarization, phagosomal escape, and microbial killing

Macrophages are important innate immune cells that respond to microbial insults. In response to multi-bacterial infection, the macrophage activation state may change upon exposure to nascent mediators, which results in different bacterial killing mechanism(s). In this study, we utilized two respiratory bacterial pathogens, Mycobacterium bovis (Bacillus Calmette Guẻrin, BCG) and Francisella tularensis live vaccine strain (LVS) with different phagocyte evasion mechanisms, as model microbes to assess the influence of initial bacterial infection on the macrophage response to secondary infection. Non-activated (M0) macrophages or activated M2-polarized cells (J774 cells transfected with the mouse IL-4 gene) were first infected with BCG for 24–48 h, subsequently challenged with LVS, and the results of inhibition of LVS replication in the macrophages was assessed. BCG infection in M0 macrophages activated TLR2-MyD88 and Mincle-CARD9 signaling pathways, stimulating nitric oxide (NO) production and enhanced killing of LVS. BCG infection had little effect on LVS escape from phagosomes into the cytosol in M0 macrophages. In contrast, M2-polarized macrophages exhibited enhanced endosomal acidification, as well as inhibiting LVS replication. Pre-infection with BCG did not induce NO production and thus did not further reduce LVS replication. This study provides a model for studies of the complexity of macrophage activation in response to multi-bacterial infection.

IL-23 p19 knockout mice exhibit minimal defects in responses to primary and secondary infection with Francisella tularensis LVS.

Our laboratory’s investigations into mechanisms of protective immunity against Francisella tularensis Live Vaccine Strain (LVS) have uncovered mediators important in host defense against primary infection, as well as those correlated with successful vaccination. One such potential correlate was IL-12p40, a pleiotropic cytokine that promotes Th1 T cell function as part of IL-12p70. LVS-infected IL-12p40 deficient knockout (KO) mice maintain a chronic infection, but IL-12p35 KO mice clear LVS infection; thus the role that IL-12p40 plays in immunity to LVS is independent of the IL-12p70 heterodimer. IL-12p40 can also partner with IL-23p19 to create the heterodimeric cytokine IL-23. Here, we directly tested the role of IL-23 in LVS resistance, and found IL-23 to be largely dispensable for immunity to LVS following intradermal or intranasal infection. IL-23p19 KO splenocytes were fully competent in controlling intramacrophage LVS replication in an in vitro overlay assay. Further, antibody responses in IL-23p19 KO mice were similar to those of normal wild type mice after LVS infection. IL-23p19 KO mice or normal wild type mice that survived primary LVS infection survived maximal doses of LVS secondary challenge. Thus p40 has a novel role in clearance of LVS infection that is unrelated to either IL-12 or IL-23.

Models derived from in vitro analyses of spleen, liver, and lung leukocyte functions predict vaccine efficacy against the Francisella tularensis Live Vaccine Strain (LVS).

ABSTRACTCurrently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e.g., tumor necrosis factor alpha [TNF-α], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and quantifying correlative T cell responses.

Alternative Activation of Macrophages and Induction of Arginase Are Not Components of Pathogenesis Mediated by Francisella Species

Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS) nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

Comparison of bone marrow-derived and mucosal mast cells in controlling intramacrophage Francisella tularensis replication

Although the importance of mast cells (MCs) in response to allergens has been characterized extensively, the contribution of these cells in host defense against bacterial pathogens is not well understood. Previously, we have demonstrated that the release of interleukin-4 by bone marrow-derived MCs inhibits intramacrophage replication of Francisella tularensis live vaccine strain (LVS). Because pneumonic tularemia is one of the several manifestations of infection by Francisella, it is important to determine whether MCs present in mucosal tissues, i.e. the lung, exhibit similar effects on LVS replication. On the basis of this rationale, we phenotypically compared mucosal mast cells (MMCs) to traditional bone marrow-derived MCs. Both cell types exhibited similar levels of cell surface expression of fragment crystal epsilon receptor I (FcεRI), mast/stem cell growth factor receptor (c-Kit) and major histocompatibility complex I (MHCI), as well as patterns of granulation. MMCs exhibited a comparable, but somewhat greater uptake of fluorescent-labeled beads compared with MCs, suggesting an increased phagocytic ability. MCs and MMCs co-cultured with primary macrophages exhibited comparable significant decreases in LVS replication compared with macrophages cultured alone. Collectively, these results suggest that MMCs are phenotypically similar to MCs and appear equally effective in the control of intramacrophage F. tularensis LVS replication.

Perforin- and Granzyme-Mediated Cytotoxic Effector Functions Are Essential for Protection against Francisella tularensis following Vaccination by the Defined F. tularensis subsp. novicida ΔfopCVaccine Strain

A licensed vaccine against Francisella tularensis is currently not available. Two Francisella tularensis subsp. novicida (herein referred to by its earlier name, Francisella novicida) attenuated strains, the ΔiglB and ΔfopC strains, have previously been evaluated as potential vaccine candidates against pneumonic tularemia in experimental animals. F. novicida ΔiglB, a Francisella pathogenicity island (FPI) mutant, is deficient in phagosomal escape and intracellular growth, whereas F. novicida ΔfopC, lacking the outer membrane lipoprotein FopC, which is required for evasion of gamma interferon (IFN-γ)-mediated signaling, is able to escape and replicate in the cytosol. To dissect the difference in protective immune mechanisms conferred by these two vaccine strains, we examined the efficacy of the F. novicida ΔiglB and ΔfopC mutants against pulmonary live-vaccine-strain (LVS) challenge and found that both strains provided comparable protection in wild-type, major histocompatibility complex class I (MHC I) knockout, and MHC II knockout mice. However, F. novicida ΔfopC-vaccinated but not F. novicida ΔiglB-vaccinated perforin-deficient mice were more susceptible and exhibited greater bacterial burdens than similarly vaccinated wild-type mice. Moreover, perforin produced by natural killer (NK) cells and release of granzyme contributed to inhibition of LVS replication within macrophages. This NK cell-mediated LVS inhibition was enhanced with anti-F. novicida ΔfopC immune serum, suggesting antibody-dependent cell-mediated cytotoxicity (ADCC) in F. novicida ΔfopC-mediated protection. Overall, this study provides additional immunological insight into the basis for protection conferred by live attenuated F. novicida strains with different phenotypes and supports further investigation of this organism as a vaccine platform for tularemia.

Non-FcεR bearing mast cells secrete sufficient interleukin-4 to control Francisella tularensis replication within macrophages

Mast cells have classically been implicated in the triggering of allergic and anaphylactic reactions. However, recent findings have elucidated the ability of these cells to selectively release a variety of cytokines leading to bacterial clearance through neutrophil and dendritic cell mobilization, and suggest an important role in innate host defenses. Our laboratory has established a primary bone marrow derived mast cell–macrophage co-culture system and found that mast cells mediated a significant inhibition of Francisella tularensis live vaccine strain (LVS) uptake and replication within macrophages through contact and the secreted product interleukin-4 (IL-4). In this study, we utilized P815 mast cells and J774 macrophages to further investigate whether mast cell activation by non-FcεR driven signals could produce IL-4 and control intramacrophage LVS replication. P815 supernatants collected upon activation by the mast cell activating peptide MP7, as well as P815 cells co-cultured with J774 macrophages, exhibited marked inhibition of bacterial uptake and replication, which correlated with the production of IL-4. The inhibition noted in vitro was titratable and preserved at ratios relevant to cellular infiltration events following pulmonary challenge. Collectively, our data suggest that both primary mast cell and P815 mast cell (lacking FcεR) secreted IL-4 can control intramacrophage Francisella replication.

Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules.

Francisella tularensis live vaccine strain (LVS) was grown in culture with nonadherent resident, starch-elicited, or Proteose Peptone-elicited peritoneal cells. Numbers of bacteria increased 4 logs over the input inoculum in 48 to 72 h. Growth rates were faster in inflammatory cells than in resident cells: generation times for the bacterium were 3 h in inflammatory cells and 6 h in resident macrophages. LVS-infected macrophage cultures treated with lymphokines did not support growth of the bacterium, although lymphokines alone had no inhibitory effects on replication of LVS in culture medium devoid of cells. Removal of gamma interferon (IFN-gamma) by immunoaffinity precipitation rendered lymphokines ineffective for induction of macrophage anti-LVS activity, and recombinant IFN-gamma stimulated both resident and inflammatory macrophage populations to inhibit LVS growth in vitro. Inflammatory macrophages were more sensitive to effects of IFN-gamma: half-maximal activity was achieved at 5 U/ml for inflammatory macrophages and 20 U/ml for resident macrophages. IFN-gamma-induced anti-LVS activity correlated with the production of nitrite (NO2-), an oxidative end product of L-arginine-derived nitric oxide (NO). Anti-LVS activity and nitrite production were both completely inhibited by the addition of either the L-arginine analog NG-monomethyl-L-arginine or anti-tumor necrosis factor antibodies to activated macrophage cultures. Thus, macrophages can be activated by IFN-gamma to suppress the growth of F. tularensis by generation of toxic levels of NO, and inflammatory macrophages are substantially more sensitive to activation activities of IFN-gamma for this effector reaction than are more differentiated resident cells.