Live Vaccine Strain Infection Research Articles

ANATOMY OF THE IMMUNE RESPONSE TO FRANCISELLA TULARENSISLVS INFECTION IN THE LUNGS.

Abstract The respiratory mucosa is under constant immune surveillance because of its vulnerability to infectious diseases. Infection of the lungs with the live vaccine strain (LVS) derived from Francisella tularensis (Ft) subspecies holarctica models pulmonary tularaemia-like disease in mice. Current evidence suggest many immune cells and cytokines respond to Ft LVS infection, but how these various immune system components are integrated to mount a protective response remains unclear. Hence, in a transcriptomics study at single cell resolution, we characterized the acute immune landscape in the lungs of C57BL/6 mice at day 0 and day 7 post intranasal inoculation with Ft LVS. Defining features of the immune response include a robust type 1 immune response characterized by the accumulation of inflammatory neutrophils and the expansion of innate-like effector lymphocytes, primarily interferon-γ producing NKT1, NK, and effector CD8 +T cells. Increased accumulation of MAIT17 over MAIT1 cells is another feature, which is in line with previous findings linking MAIT cells and IL-17 to LVS immune response. Surprisingly, a significant proportion of MAIT17 cells either maintained or upregulated type 1 inflammatory markers. Similarly, MAIT1 cells maintained or upregulated Type 3 inflammatory markers. Additionally, a highly active MAIT cell subset has increased Nr4a1 expression, which encodes Nur77, suggesting a T cell receptor-mediated activation. By contrast, NKT1 cells poorly upregulated Nr4a1 expression but induced Il18r1 expression suggesting, a cytokine-mediated activation. Consequently, we predict that unconventional NKT & MAIT cells integrate innate cues to control tularaemia-like disease caused by Ft LVS infection in the mouse. VUSM MSTP NIGMS of the National Institutes of Health T32GM007347 (GDO); IO1 BX001444, BX001610, BX00xxx, IK6 BX004595 & RO1 AI137082; IBX000915A (HMA)

Chronically Hypertensive Transgenic Mice Expressing Human AT1R Haplotype‐I Exhibit Increased Susceptibility To Francisella Tularensis

Angiotensin receptor type 1 (AT1R), a G-protein coupled receptor mediates the effect of angiotensin-II and contributes to the pathophysiological consequences of metabolic disorders leading to vascular remodeling, hypertension, and end-organ damage. Genetic variations that increase human AT1R (hAT1R) expression can cause above pathological outcomes due to renin-angiotensin system overactivity. Previously we have shown that transgenic (TG) mice containing haplotype-I (Hap-I, hypertensive genotype) of human AT1R gene are more prone to develop the metabolic syndrome (MetS) related disorders as compared to the TG mice containing haplotype-II (Hap-II, normotensive genotype). We have also shown that as compared to Hap-II, the aged Hap-I TG mice overexpress the hAT1R gene resulting in an increase in blood pressure, inflammatory molecules, and renal dysfunction. Aging together with an increased risk of hypertension, can affect multiple organ systems in a complex manner, including susceptibility to various infections. The current study, therefore, was designed to examine the potential effect of aging and associated MetS disorders in our hAT1R containing TG mice model, in response to the infection by Francisella tularensis live vaccine strain (LVS) inhalation. Our results show that Hap-I TG mice have a significantly high mortality rate two weeks post-infection as compared to the Hap-II animals. Histochemical analysis of lung tissues from adult and aged animals show a comparatively elevated pathology in mice containing Hap-I of hAT1R. The lung tissue and plasma from Hap-I TG mice also show a relatively higher bacterial load five days post-infection. Preliminary RT-PCR experiments show increased inflammatory markers like IL-6, TGF-β, TNF-α, CRP and redox markers like NOX1, NOX4 in lung tissues of Hap-I TG mice. These studies suggest that haplotype dependent over-expression of hAT1R gene, leads to an enhanced mortality and lung pathology to F. tularensis LVS infection, which gets aggravated in aged animals. Clinically, our results may help in exploring the role of AT1R induced hypertension in pulmonary infections.

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.

Guanylate-Binding Proteins Are Critical for Effective Control of Francisella tularensis Strains in a Mouse Co-Culture System of Adaptive Immunity.

Francisella tularensis is a Select Agent that causes the severe disease tularemia in humans and many animal species. The bacterium demonstrates rapid intracellular replication, however, macrophages can control its replication if primed and activation with IFN-γ is known to be essential, although alone not sufficient, to mediate such control. To further investigate the mechanisms that control intracellular F. tularensis replication, an in vitro co-culture system was utilized containing splenocytes obtained from naïve or immunized C57BL/6 mice as effectors and infected bone marrow-derived wild-type or chromosome-3-deficient guanylate-binding protein (GBP)-deficient macrophages. Cells were infected either with the F. tularensis live vaccine strain (LVS), the highly virulent SCHU S4 strain, or the surrogate for F. tularensis, F. novicida. Regardless of strain, significant control of the bacterial replication was observed in co-cultures with wild-type macrophages and immune splenocytes, but not in cultures with immune splenocytes and GBP chr3-deficient macrophages. Supernatants demonstrated very distinct, infectious agent-dependent patterns of 23 cytokines, whereas the cytokine patterns were only marginally affected by the presence or absence of GBPs. Levels of a majority of cytokines were inversely correlated to the degree of control of the SCHU S4 and LVS infections, but this was not the case for the F. novicida infection. Collectively, the co-culture assay based on immune mouse-derived splenocytes identified a dominant role of GBPs for the control of intracellular replication of various F. tularensis strains, regardless of their virulence, whereas the cytokine patterns markedly were dependent on the infectious agents, but less so on GBPs.

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.

2888. STAT4 Mutation in Three Generations with Disseminated Coccidioidomycosis (DCM) also Exhibits Increased Susceptibility to Coccidioidal Infection in Transfected Mice

BackgroundReported coccidioidomycosis has increased with case rates of 198/100,000 in Arizona (2012). In California alone, 2000–2011 hospitalizations were $2.2B. Dissemination occurs in 8% of reports with significant morbidity and occasional deaths. DCM was found in 3 generations: grandmother (skin), mother (skin), and son (bone). Whole exome sequencing identified a heterozygous (het) STAT4 mutation (p.E626G) in all three. This mutation alters the phosphotyrosine binding pocket and is predicted to impair STAT4 function, interfering with (i) receptor binding and phosphorylation, (ii) nuclear localization, and/or (iii) transcription. Expression profiling of antigen-stimulated peripheral blood mononuclear cells from one patient showed dampening of known STAT4 targets compared with controls.MethodsSTAT4 p.E626G was generated and confirmed in C57BL/6NJ (WT) mice using CRISPR-Cas9. With continued breeding, neither homozygous (hom) nor het mice had gross abnormalities. There were normal spleen and lung lymphoid cell numbers. Thymus and bone marrow had normal development of lymphoid subsets. We performed intranasal infection with reduced virulence C. posadasii strain 1038 or with F. tularensis live vaccine- strain (LVS). Naïve or Δcps1-vaccinated mice were tested for resistance to C. posadasii strain Silveira.ResultsAt day 21 post Cp 1038 infection, hom, het, and WT mice had similar lung fungal burdens (~104.7 cfu). All p.E626G mice died between days 31 and 39 with lung burden significantly higher (~9 × 106 cfu) than WT sacrificed on day 44 (7 × 105 cfu, P = 0.015). After LVS infection, p.E626G mice had increased lung bacterial cfu and all had dissemination to the spleen compared with WT lung bacterial burden and no splenic dissemination. Immunized het and WT mice all had significantly reduced lung cfu 14 days following C. posadasii infection compared with unvaccinated WT mice.ConclusionThe STAT4 p.E626G mutated mouse recapitulated patients’ increased susceptibility to coccidioidal infection. The decreased fungal burdens seen in Δcps1-vaccinated mice suggest that vaccination may be effective in those persons genetically susceptible to DCM. Given the increasing frequency and economic burdens of coccidioidomycosis, pursuit of vaccination strategies should continue. Disclosures All Authors: No reported Disclosures.

AR-13, a Celecoxib Derivative, Directly Kills Francisella In Vitro and Aids Clearance and Mouse Survival In Vivo.

Francisella tularensis (F. tularensis) is the causative agent of tularemia and is classified as a Tier 1 select agent. No licensed vaccine is currently available in the United States and treatment of tularemia is confined to few antibiotics. In this study, we demonstrate that AR-13, a derivative of the cyclooxygenase-2 inhibitor celecoxib, exhibits direct in vitro bactericidal killing activity against Francisella including a type A strain of F. tularensis (SchuS4) and the live vaccine strain (LVS), as well as toward the intracellular proliferation of LVS in macrophages, without causing significant host cell toxicity. Identification of an AR-13-resistant isolate indicates that this compound has an intracellular target(s) and that efflux pumps can mediate AR-13 resistance. In the mouse model of tularemia, AR-13 treatment protected 50% of the mice from lethal LVS infection and prolonged survival time from a lethal dose of F. tularensis SchuS4. Combination of AR-13 with a sub-optimal dose of gentamicin protected 60% of F. tularensis SchuS4-infected mice from death. Taken together, these data support the translational potential of AR-13 as a lead compound for the further development of new anti-Francisella agents.

Increased Resistance to Intradermal Francisella tularensis LVS Infection by Inactivation of the Sts Phosphatases.

The Suppressor of TCR signaling proteins (Sts-1 and Sts-2) are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic lineages, including T lymphocytes. Mice lacking Sts expression are characterized by enhanced T cell responses. Additionally, a recent study demonstrated that Sts-/- mice are profoundly resistant to systemic infection by Candida albicans, with resistance characterized by enhanced survival, more rapid fungal clearance in key peripheral organs, and an altered inflammatory response. To investigate the role of Sts in the primary host response to infection by a bacterial pathogen, we evaluated the response of Sts-/- mice to infection by a Gram-negative bacterial pathogen. Francisella tularensis is a facultative bacterial pathogen that replicates intracellularly within a variety of cell types and is the causative agent of tularemia. Francisella infections are characterized by a delayed immune response, followed by an intense inflammatory reaction that causes widespread tissue damage and septic shock. Herein, we demonstrate that mice lacking Sts expression are significantly resistant to infection by the live vaccine strain (LVS) of F. tularensis Resistance is characterized by reduced lethality following high-dose intradermal infection, an altered cytokine response in the spleen, and enhanced bacterial clearance in multiple peripheral organs. Sts-/- bone marrow-derived monocytes and neutrophils, infected with F. tularensis LVS ex vivo, display enhanced restriction of intracellular bacteria. These observations suggest the Sts proteins play an important regulatory role in the host response to bacterial infection, and they underscore a role for Sts in regulating functionally relevant immune response pathways.

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells.

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.

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.

T-bet Regulates Immunity to Francisella tularensis Live Vaccine Strain Infection, Particularly in Lungs

Upregulation of the transcription factor T-bet is correlated with the strength of protection against secondary challenge with the live vaccine strain (LVS) of Francisella tularensis. Thus, to determine if this mediator had direct consequences in immunity to LVS, we examined its role in infection. Despite substantial in vivo gamma interferon (IFN-γ) levels, T-bet-knockout (KO) mice infected intradermally (i.d.) or intranasally (i.n.) with LVS succumbed to infection with doses 2 log units less than those required for their wild-type (WT) counterparts, and exhibited significantly increased bacterial burdens in the lung and spleen. Lungs of LVS-infected T-bet-KO mice contained fewer lymphocytes and more neutrophils and interleukin-17 than WT mice. LVS-vaccinated T-bet-KO mice survived lethal LVS intraperitoneal secondary challenge but not high doses of LVS i.n. challenge, independently of the route of vaccination. Immune T lymphocytes from the spleens of i.d. LVS-vaccinated WT or KO mice controlled intracellular bacterial replication in an in vitro coculture system, but cultures with T-bet-KO splenocyte supernatants contained less IFN-γ and increased amounts of tumor necrosis factor alpha. In contrast, immune T-bet-KO lung lymphocytes were greatly impaired in controlling intramacrophage growth of LVS; this functional defect is the likely mechanism underpinning the lack of respiratory protection. Taken together, T-bet is important in host resistance to primary LVS infection and i.n. secondary challenge. Thus, T-bet represents a true, useful correlate for immunity to LVS.

Infection with Francisella tularensis LVSclpBLeads to an Altered yet Protective Immune Response

Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo. We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB, can serve as a model for the rational design of attenuated vaccines.

Differing Effects of Interleukin-10 on Cutaneous and Pulmonary Francisella tularensis Live Vaccine Strain Infection

We investigated the role of interleukin-10 (IL-10) in cutaneous and pulmonary infection with Francisella tularensis. We found that after intradermal challenge of mice with the live vaccine strain (LVS) of F. tularensis, splenic IL-10 levels increased rapidly and reached a peak 5 days after infection. However, IL-10 expression after infection was detrimental, since IL-10(-/-) mice showed increased bacterial clearance and were resistant to an infectious dose (>10(6) CFU/mouse) that was uniformly lethal for IL-10(+/+) mice. Furthermore, IL-10(+/+) mice treated with neutralizing anti-IL-10R monoclonal antibody were able to survive lethal cutaneous LVS challenge. The presence of IL-10 appeared to restrain the expression of IL-17, since high levels of splenic IL-17 were observed after intradermal LVS infection only in IL-10(-/-) mice. Furthermore, treatment with neutralizing anti-IL-17R antibody ablated the enhanced survival observed in IL-10(-/-) mice. However, neutralization of IL-10 activity in IL-17R(-/-) mice failed to provide protection. Thus, IL-10 suppresses a protective IL-17 response that is necessary for resistance to cutaneous LVS infection. Surprisingly, however, IL-10(-/-) mice were significantly more susceptible to pulmonary infection with LVS. Finally, although IL-10 is a critical and novel regulator of immunity to F. tularensis LVS infection, its effects were masked during infection with the highly virulent SchuS4 strain. Taken together, these findings suggest that differentially regulating expression of the IL-10 pathway in various tissues could ultimately have prophylactic and therapeutic benefits for protection against tularemia.

Assessment of antimicrobial peptide LL-37 as a post-exposure therapy to protect against respiratory tularemia in mice

Early activation of the innate immune response is important for protection against infection with Francisella tularensis live vaccine strain (LVS) in mice. The human cathelicidin antimicrobial peptide LL-37 is known to have immunomodulatory properties, and therefore exogenously administered LL-37 may be suitable as an early post-exposure therapy to protect against LVS infection. LL-37 has been evaluated for immunostimulatory activity in uninfected mice and for activity against LVS in macrophage assays and protective efficacy when administered post-challenge in a mouse model of respiratory tularemia. Increased levels of pro-inflammatory cytokine IL-6, chemokines monocyte chemoattractant protein 1 (MCP-1) and CXCL1 with increased neutrophil influx into the lungs were observed in uninfected mice after intranasal administration of LL-37. Following LVS challenge, LL-37 administration resulted in increased IL-6, IL-12 p70, IFNγ and MCP-1 production, a slowing of LVS growth in the lung, and a significant extension of mean time to death compared to control mice. However, protection was transient, with the LL-37 treated mice eventually succumbing to infection. As this short course of nasally delivered LL-37 was moderately effective at overcoming the immunosuppressive effects of LVS infection this suggests that a more sustained treatment regimen may be an effective therapy against this pathogen.

Interleukin-6 is essential for primary resistance to Francisella tularensis live vaccine strain infection.

We employed Francisella tularensis live vaccine strain (LVS) to study mechanisms of protective immunity against intracellular pathogens and, specifically, to understand protective correlates. One potential molecular correlate identified previously was interleukin-6 (IL-6), a cytokine with pleotropic roles in immunity, including influences on T and B cell functions. Given its role as an immune modulator and the correlation with successful anti-LVS vaccination, we examined the role IL-6 plays in the host response to LVS. IL-6-deficient (IL-6 knockout [KO]) mice infected with LVS intradermally or intranasally or anti-IL-6-treated mice, showed greatly reduced 50% lethal doses compared to wild-type (WT) mice. Increased susceptibility was not due to altered splenic immune cell populations during infection or decreased serum antibody production, as IL-6 KO mice had similar compositions of each compared to WT mice. Although LVS-infected IL-6 KO mice produced much less serum amyloid A and haptoglobin (two acute-phase proteins) than WT mice, there were no other obvious pathophysiological differences between LVS-infected WT and IL-6 KO mice. IL-6 KO or WT mice that survived primary LVS infection also survived a high-dose LVS secondary challenge. Using an in vitro overlay assay that measured T cell activation, cytokine production, and abilities of primed splenocytes to control intracellular LVS growth, we found that IL-6 KO total splenocytes or purified T cells were slightly defective in controlling intracellular LVS growth but were equivalent in cytokine production. Taken together, IL-6 is an integral part of a successful immune response to primary LVS infection, but its exact role in precipitating adaptive immunity remains elusive.

Francisella tularensis Alters Human Neutrophil Gene Expression: Insights into the Molecular Basis of Delayed Neutrophil Apoptosis

We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

MTOR modulates host immune responses to Francisella tularensis LVS infection (112.7)

Abstract Francisella tularensis (Ft) is the cause of tularemia, a disease with a high morbidity and mortality rate. The Ft live vaccine strain (LVS) has been used to study disease pathogenesis since infection of mice with LVS resembles tularemia in humans. It has been reported that Ft LVS is a TLR2 agonist and involves PI3K/Akt signaling. However, the involvement of mTOR, a downstream molecule of the PI3K/Akt pathway, has not been determined in Ft LVS infection, nor has the interplay between TLR2 and the mTOR cascade. In the present study, we provide evidence that mTOR exerts a differential regulation on both cytokine mRNA and secreted protein in murine peritoneal macrophages stimulated with Ft LVS. IL-6, IL-10 and IFN-β mRNA production increased with inhibition of the mTOR pathway, while levels of IFN-γ mRNA were independent of mTOR signaling. Secreted IL-6 and IFN-γ but not IFN-β and TNFα was dependent of mTOR regulation. Furthermore, mTOR signaling was critical for the DNA binding activity of CREB, but not of NF-κB p65. Upregulation of the interferon regulatory factors (IRF)-1, IRF-3 and IRF-5 in nuclear fractions of cell lysates was observed upon inhibition of mTOR, and correlated with increased levels of the pro-apoptotic proteins Bax, caspase 3 and caspase 8, indicating mTOR modulation of apoptosis via IRF downreguation. Overall, these studies identify mTOR as a critical regulator of the host immune response to Ft LVS infection.

The role of PKCθ in Francisella tularensis infection in mice (164.4)

Abstract Francisella tularensis is a facultative intracellular bacteria that causes tularemia in animals and humans. F. tularensis is highly virulent and can be lethal. T cells play a critical role in Francisella immunity. It has been shown that mice succumb to sublethal infections when all T cells are depleted; however individual subsets including CD4, CD8 and an unusual CD4-CD8-NK1.1- population can be protective. While T cells control infection by producing IFNγ and TNFα, other less well-characterized mechanisms also play a role. PKCθ is a protein kinase involved in T cell activation; PKCθ is important in IFNγ production and PKCθ-/- mice are protected against both TH1 and TH2 mediated diseases. To investigate the role of PKCθ in F. tularensis infection, we infected wild-type C57Bl/6 (WT) mice and B6.PKCθ-/- mice with a lethal dose of LVS (live vaccine strain) which is commonly used to study F. tularensis infection. While WT mice died within 2 weeks of infection, surprisingly, over 50% of the PKCθ-/- mice survived past 3 weeks. We found that infected PKCθ-/- mice had more CD4-CD8- T cells in liver and lung than WT mice. In contrast, PKCθ-/- mice had fewer CD4+ T cells in lymph nodes, spleen, liver, and lung. We found no difference in numbers of activated T cells in liver and lung of infected WT and PKCθ-/- mice. These results show that PKCθ may be important in the expansion of CD4+ and CD4-CD8- T cell subtypes and this shift in the absence of PKCθ may protect against LVS infection.

Phosphatidylinositol 3-kinase activation attenuates the TLR2-mediated macrophage proinflammatory cytokine response to Francisella tularensis live vaccine strain.

An inadequate innate immune response appears to contribute to the virulence of Francisella tularensis following pulmonary infection. Studies in mice suggest that this poor response results from suppression of proinflammatory cytokine production early during infection, but the mechanisms involved are not understood. PI3K is known to regulate proinflammatory cytokine expression, but its exact role (positive versus negative) is controversial. We sought to clarify the role of PI3K in regulating proinflammatory signaling and cytokine production during infection with F. tularensis live vaccine strain (LVS). In this study, we demonstrate that the induction of TNF and IL-6 expression by LVS in mouse bone marrow-derived macrophages was markedly enhanced when PI3K activity was inhibited by either of the well-known chemical inhibitors, wortmannin or LY294002. The enhanced cytokine expression was accompanied by enhanced activation of p38 MAPK and ERK1/2, both of which were critical for LVS-induced expression of TNF and IL-6. LVS-induced MAPK activation and cytokine production were TLR2- and MyD88- dependent. PI3K/Akt activation was MyD88-dependent, but was surprisingly TLR2-independent. LVS infection also rapidly induced MAPK phosphatase-1 (MKP-1) expression; PI3K and TLR2 signaling were required. Peak levels of MKP-1 correlated closely with the decline in p38 MAPK and ERK1/2 phosphorylation. These data suggest that infection by LVS restrains the TLR2-triggered proinflammatory response via parallel activation of PI3K, leading to enhanced MKP-1 expression, accelerated deactivation of MAPKs, and suppression of proinflammatory cytokine production. This TLR2-independent inhibitory pathway may be an important mechanism by which Francisella suppresses the host's innate immune response.