M2a macrophages facilitate resolution of chemically-induced colitis in TLR4-SNP mice.

IMPORTANCE: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, impacts millions of individuals worldwide and severely impairs the quality of life for patients. Dysregulation of innate immune signaling pathways reduces barrier function and exacerbates disease progression. Macrophage (Mφ) signaling pathways are potential targets for IBD therapies. While multiple treatments are available for IBD, (i) not all patients respond, (ii) responses may diminish over time, and (iii) treatments often have undesirable side effects. Genetic studies have shown that the inheritance of two co-segregating SNPs expressed in the innate immune receptor, TLR4, is associated with human IBD. Mice expressing homologous SNPs ("TLR4-SNP" mice) exhibited more severe colitis than WT mice in a DSS-induced colonic inflammation/repair model. We identified a critical role for M2a "tissue repair" Mφ in the resolution of colitis. Our findings provide insight into potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.

Protection against influenza-induced Acute Lung Injury (ALI) by enhanced induction of M2a macrophages: possible role of PPARγ/RXR ligands in IL-4-induced M2a macrophage differentiation.

Many respiratory viruses cause lung damage that may evolve into acute lung injury (ALI), a cytokine storm, acute respiratory distress syndrome, and ultimately, death. Peroxisome proliferator activated receptor gamma (PPARγ), a member of the nuclear hormone receptor (NHR) family of transcription factors, regulates transcription by forming heterodimers with another NHR family member, Retinoid X Receptor (RXR). Each component of the heterodimer binds specific ligands that modify transcriptional capacity of the entire heterodimer by recruiting different co-activators/co-repressors. However, the role of PPARγ/RXR ligands in the context of influenza infection is not well understood. PPARγ is associated with macrophage differentiation to an anti-inflammatory M2 state. We show that mice lacking the IL-4Rα receptor, required for M2a macrophage differentiation, are more susceptible to mouse-adapted influenza (A/PR/8/34; "PR8")-induced lethality. Mice lacking Ptgs2, that encodes COX-2, a key proinflammatory M1 macrophage mediator, are more resistant. Blocking the receptor for COX-2-induced Prostaglandin E2 (PGE2) was also protective. Treatment with pioglitazone (PGZ), a PPARγ ligand, increased survival from PR8 infection, decreased M1 macrophage gene expression, and increased PPARγ mRNA in lungs. Conversely, conditional knockout mice expressing PPARγ-deficient macrophages were significantly more sensitive to PR8-induced lethality. These findings were extended in cotton rats: PGZ blunted lung inflammation and M1 cytokine gene expression after challenge with non-adapted human influenza. To study mechanisms by which PPARγ/RXR transcription factors induce canonical M2a genes, WT mouse macrophages were treated with IL-4 in the absence or presence of rosiglitazone (RGZ; PPARγ ligand), LG100754 (LG; RXR ligand), or both. IL-4 dose-dependently induced M2a genes Arg1, Mrc1, Chil3, and Retnla. Treatment of macrophages with IL-4 and RGZ and/or LG differentially affected induction of Arg1 and Mrc1 vs. Chil3 and Retnla gene expression. In PPARγ-deficient macrophages, IL-4 alone failed to induce Arg1 and Mrc1 gene expression; however, concurrent treatment with LG or RGZ + LG enhanced IL-4-induced Arg1 and Mrc1 expression, but to a lower level than in WT macrophages, findings confirmed in the murine alveolar macrophage cell line, MH-S. These findings support a model in which PPARγ/RXR heterodimers control IL-4-induced M2a differentiation, and suggest that PPARγ/RXR agonists should be considered as important tools for clinical intervention against influenza-induced ALI.

Early or Late Bacterial Lung Infection Increases Mortality After Traumatic Brain Injury in Male Mice and Chronically Impairs Monocyte Innate Immune Function.

OBJECTIVES: Respiratory infections in the postacute phase of traumatic brain injury impede optimal recovery and contribute substantially to overall morbidity and mortality. This study investigated bidirectional innate immune responses between the injured brain and lung, using a controlled cortical impact model followed by secondary Streptococcus pneumoniae infection in mice. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Adult male C57BL/6J mice. INTERVENTIONS: C57BL/6J mice were subjected to sham surgery or moderate-level controlled cortical impact and infected intranasally with S. pneumoniae (1,500 colony-forming units) or vehicle (phosphate-buffered saline) at 3 or 60 days post-injury. MAIN RESULTS: At 3 days post-injury, S. pneumoniae-infected traumatic brain injury mice (TBI + Sp) had a 25% mortality rate, in contrast to no mortality in S. pneumoniae-infected sham (Sham + Sp) animals. TBI + Sp mice infected 60 days post-injury had a 60% mortality compared with 5% mortality in Sham + Sp mice. In both studies, TBI + Sp mice had poorer motor function recovery compared with TBI + PBS mice. There was increased expression of pro-inflammatory markers in cortex of TBI + Sp compared with TBI + PBS mice after both early and late infection, indicating enhanced post-traumatic neuroinflammation. In addition, monocytes from lungs of TBI + Sp mice were immunosuppressed acutely after traumatic brain injury and could not produce interleukin-1ß, tumor necrosis factor-α, or reactive oxygen species. In contrast, after delayed infection monocytes from TBI + Sp mice had higher levels of interleukin-1ß, tumor necrosis factor-α, and reactive oxygen species when compared with Sham + Sp mice. Increased bacterial burden and pathology was also found in lungs of TBI + Sp mice. CONCLUSIONS: Traumatic brain injury causes monocyte functional impairments that may affect the host's susceptibility to respiratory infections. Chronically injured mice had greater mortality following S. pneumoniae infection, which suggests that respiratory infections even late after traumatic brain injury may pose a more serious threat than is currently appreciated.

Influenza "Trains" the Host for Enhanced Susceptibility to Secondary Bacterial Infection.

We previously reported that the Toll-like receptor 4 (TLR4) antagonist Eritoran blocks acute lung injury (ALI) therapeutically in mouse and cotton rat models of influenza. However, secondary (2°) bacterial infection following influenza virus infection is associated with excess morbidity and mortality. Wild-type (WT) mice infected with mouse-adapted influenza A/Puerto Rico/8/34 virus (PR8) and, 7 days later, with Streptococcus pneumoniae serotype 3 (Sp3) exhibited significantly enhanced lung pathology and lethality that was reversed by Eritoran therapy after PR8 infection but before Sp3 infection. Cotton rats infected with nonadapted pH1N1 influenza virus and then superinfected with methicillin-resistant Staphylococcus aureus also exhibited increased lung pathology and serum high-mobility-group box 1 (HMGB1) levels, both of which were blunted by Eritoran therapy. In mice, PR8 infection suppressed Sp3-induced CXCL1 and CXCL2 mRNA, reducing neutrophil infiltration and increasing the bacterial burden, all of which were reversed by Eritoran treatment. While beta interferon (IFN-ß)-deficient (IFN-ß-/-) mice are highly susceptible to PR8, they exhibited delayed death upon Sp3 superinfection, indicating that while IFN-ß was protective against influenza, it negatively impacted the host response to Sp3 IFN-ß-treated WT macrophages selectively suppressed Sp3-induced CXCL1/CXCL2 transcriptionally, as evidenced by reduced recruitment of RNA polymerase II to the CXCL1 promoter. Thus, influenza establishes a "trained" state of immunosuppression toward 2° bacterial infection, in part through the potent induction of IFN-ß and its downstream transcriptional regulation of chemokines, an effect reversed by Eritoran.IMPORTANCE Enhanced susceptibility to 2° bacterial infections following infection with influenza virus is a global health concern that accounts for many hospitalizations and deaths, particularly during pandemics. The complexity of the impaired host immune response during 2° bacterial infection has been widely studied. Both type I IFN and neutrophil dysfunction through decreased chemokine production have been implicated as mechanisms underlying enhanced susceptibility to 2° bacterial infections. Our findings support the conclusion that selective suppression of CXCL1/CXCL2 represents an IFN-ß-mediated "training" of the macrophage transcriptional response to TLR2 agonists and that blocking of TLR4 therapeutically with Eritoran after influenza virus infection reverses this suppression by blunting influenza-induced IFN-ß.

Novel role of gastrin releasing peptide-mediated signaling in the host response to influenza infection.

Gastrin-releasing peptide (GRP) is an evolutionarily well-conserved neuropeptide that was originally recognized for its ability to mediate gastric acid secretion in the gut. More recently, however, GRP has been implicated in pulmonary lung inflammatory diseases including bronchopulmonary dysplasia, chronic obstructive pulmonary disease, emphysema, and others. Antagonizing GRP or its receptor mitigated lethality associated with the onset of viral pneumonia in a well-characterized mouse model of influenza. In mice treated therapeutically with the small-molecule GRP inhibitor, NSC77427, increased survival was accompanied by decreased numbers of GRP-producing pulmonary neuroendocrine cells, improved lung histopathology, and suppressed cytokine gene expression. In addition, in vitro studies in macrophages indicate that GRP synergizes with the prototype TLR4 agonist, lipopolysaccharide, to induce cytokine gene expression. Thus, these findings reveal that GRP is a previously unidentified mediator of influenza-induced inflammatory disease that is a potentially novel target for therapeutic intervention.

Host-based lipid inflammation drives pathogenesis in Francisella infection.

Mass spectrometry imaging (MSI) was used to elucidate host lipids involved in the inflammatory signaling pathway generated at the host-pathogen interface during a septic bacterial infection. Using Francisella novicida as a model organism, a bacterial lipid virulence factor (endotoxin) was imaged and identified along with host phospholipids involved in the splenic response in murine tissues. Here, we demonstrate detection and distribution of endotoxin in a lethal murine F. novicida infection model, in addition to determining the temporally and spatially resolved innate lipid inflammatory response in both 2D and 3D renderings using MSI. Further, we show that the cyclooxygenase-2-dependent lipid inflammatory pathway is responsible for lethality in F. novicida infection due to overproduction of proinflammatory effectors including prostaglandin E2. The results of this study emphasize that spatial determination of the host lipid components of the immune response is crucial to identifying novel strategies to effectively address highly pathogenic and lethal infections stemming from bacterial, fungal, and viral origins.

The θ-defensin retrocyclin 101 inhibits TLR4- and TLR2-dependent signaling and protects mice against influenza infection.

Despite widespread use of annual influenza vaccines, seasonal influenza-associated deaths number in the thousands each year, in part because of exacerbating bacterial superinfections. Therefore, discovering additional therapeutic options would be a valuable aid to public health. Recently, TLR4 inhibition has emerged as a possible mechanism for protection against influenza-associated lethality and acute lung injury. Based on recent data showing that rhesus macaque θ-defensins could inhibit TLR4-dependent gene expression, we tested the hypothesis that a novel θ-defensin, retrocyclin (RC)-101, could disrupt TLR4-dependent signaling and protect against viral infection. In this study, RC-101, a variant of the humanized θ-defensin RC-1, blocked TLR4-mediated gene expression in mouse and human macrophages in response to LPS, targeting both MyD88- and TRIF-dependent pathways. In a cell-free assay, RC-101 neutralized the biologic activity of LPS at doses ranging from 0.5 to 50 EU/ml, consistent with data showing that RC-101 binds biotinylated LPS. The action of RC-101 was not limited to the TLR4 pathway because RC-101 treatment of macrophages also inhibited gene expression in response to a TLR2 agonist, Pam3CSK4, but failed to bind that biotinylated agonist. Mouse macrophages infected in vitro with mouse-adapted A/PR/8/34 influenza A virus (PR8) also produced lower levels of proinflammatory cytokine gene products in a TLR4-independent fashion when treated with RC-101. Finally, RC-101 decreased both the lethality and clinical severity associated with PR8 infection in mice. Cumulatively, our data demonstrate that RC-101 exhibits therapeutic potential for the mitigation of influenza-related morbidity and mortality, potentially acting through TLR-dependent and TLR-independent mechanisms.

Enhanced allergic responsiveness after early childhood infection with respiratory viruses: Are long-lived alternatively activated macrophages the missing link?

Early childhood infection with respiratory viruses, including human rhinovirus, respiratory syncytial virus (RSV) and influenza, is associated with an increased risk of allergic asthma and severe exacerbation of ongoing disease. Despite the long recognition of this relationship, the mechanism linking viral infection and later susceptibility to allergic lung inflammation is still poorly understood. We discuss the literature and provide new evidence demonstrating that these viruses induce the alternative activation of macrophages. Alternatively activated macrophages (AAM) induced by RSV or influenza infection persisted in the lungs of mice up to 90 days after initial viral infection. Several studies suggest that AAM contribute to allergic inflammatory responses, although their mechanism of action is unclear. In this commentary, we propose that virus-induced AAM provide a link between viral infection and enhanced responses to inhaled allergens.

The Tick Protein Sialostatin L2 Binds to Annexin A2 and Inhibits NLRC4-Mediated Inflammasome Activation.

Tick saliva contains a number of effector molecules that inhibit host immunity and facilitate pathogen transmission. How tick proteins regulate immune signaling, however, is incompletely understood. Here, we describe that loop 2 of sialostatin L2, an anti-inflammatory tick protein, binds to annexin A2 and impairs the formation of the NLRC4 inflammasome during infection with the rickettsial agent Anaplasma phagocytophilum Macrophages deficient in annexin A2 secreted significantly smaller amounts of interleukin-1ß (IL-1ß) and IL-18 and had a defect in NLRC4 inflammasome oligomerization and caspase-1 activation. Accordingly, Annexin a2-deficient mice were more susceptible to A. phagocytophilum infection and showed splenomegaly, thrombocytopenia, and monocytopenia. Providing translational support to our findings, better binding of annexin A2 to sialostatin L2 in sera from 21 out of 23 infected patients than in sera from control individuals was also demonstrated. Overall, we establish a unique mode of inflammasome evasion by a pathogen, centered on a blood-feeding arthropod.

A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.

Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll/interleukin-1 receptor (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling. Cell-permeable peptides derived from the D helix and the segment immediately N-terminal to the TLR2 TIR domain potently inhibited TLR2-mediated cytokine production. The D-helix peptide, 2R9, also potently inhibited TLR4, TLR7, and TLR9, but not TLR3 or TNF-α signaling. Cell imaging, co-immunoprecipitation, and in vitro studies demonstrated that 2R9 preferentially targets TIRAP. 2R9 diminished systemic cytokine responses elicited in vivo by synthetic TLR2 and TLR7 agonists; it inhibited the activation of macrophages infected with influenza strain A/PR/8/34 (PR8) and significantly improved the survival of PR8-infected mice. Thus, 2R9 represents a TLR-targeting agent that blocks protein interactions downstream of activated TLRs.

Macrophage activation and polarization: nomenclature and experimental guidelines.

Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.

Agents that increase AAM differentiation blunt RSV-mediated lung pathology.

RSV is the most significant cause of serious lower respiratory tract infection in infants and young children worldwide. There is currently no vaccine for the virus, and antiviral therapy (e.g., ribavirin) has shown no efficacy against the disease. We reported that alternatively activated macrophages (AAMs) mediate resolution of RSV-induced pathology. AAM differentiation requires macrophage-derived IL-4 and -13, autocrine/paracrine signaling through the type I IL-4 receptor, and STAT6 activation. Based on these findings, we reasoned that it would be possible to intervene therapeutically in RSV disease by increasing AAM differentiation, thereby decreasing lung pathology. Mice treated with the IL-4/anti-IL-4 immune complexes, shown previously to sustain levels of circulating IL-4, increased the RSV-induced AAM markers arginase-1 and mannose receptor and decreased the lung pathology. Induction of PPARγ, shown to play a role in AAM development, by the PPARγ agonist rosiglitazone or treatment of mice with the macrolide antibiotic AZM, also reported to skew macrophage differentiation to an AAM phenotype, increased the AAM markers and mitigated RSV-induced lung pathology. Collectively, our data suggest that therapeutic manipulation of macrophage differentiation to enhance the AAM phenotype is a viable approach for ameliorating RSV-induced disease.

A recombinant anchorless respiratory syncytial virus (RSV) fusion (F) protein/monophosphoryl lipid A (MPL) vaccine protects against RSV-induced replication and lung pathology.

We previously demonstrated that the severe cytokine storm and pathology associated with RSV infection following intramuscular vaccination of cotton rats with FI-RSV Lot 100 could be completely abolished by formulating the vaccine with the mild TLR4 agonist and adjuvant, monophosphoryl lipid A (MPL). Despite this significant improvement, the vaccine failed to blunt viral replication in the lungs. Since MPL is a weak TLR4 agonist, we hypothesized that its adjuvant activity was mediated by modulating the innate immune response of respiratory tract resident macrophages. Therefore, we developed a new vaccine preparation with purified, baculovirus expressed, partially purified, anchorless RSV F protein formulated with synthetic MPL that was administered to cotton rats intranasally, followed by an intradermal boost. This novel formulation and heterologous "prime/boost" route of administration resulted in decreased viral titers compared to that seen in animals vaccinated with F protein alone. Furthermore, animals vaccinated by this route showed no evidence of enhanced lung pathology upon RSV infection. This indicates that MPL acts as an immune modulator that protects the host from vaccine-enhanced pathology, and reduces RSV replication in the lower respiratory tract when administered by a heterologous prime/boost immunization regimen.

Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase epsilon-dependent induction of Sp1 protein and interleukin 15.

In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.

Sustained generation of nitric oxide and control of mycobacterial infection requires argininosuccinate synthase 1.

Nitric oxide (NO) defends against intracellular pathogens, but its synthesis must be regulated due to cell and tissue toxicity. During infection, macrophages import extracellular arginine to synthesize NO, generating the byproduct citrulline. Accumulated intracellular citrulline is thought to fuel arginine synthesis catalyzed by argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl), which would lead to abundant NO production. Instead, we find that citrulline is exported from macrophages during early stages of NO production with <2% retained for recycling via the Ass1-Asl pathway. Later, extracellular arginine is depleted, and Ass1 expression allows macrophages to synthesize arginine from imported citrulline to sustain NO output. Ass1-deficient macrophages fail to salvage citrulline in arginine-scarce conditions, leading to their inability to control mycobacteria infection. Thus, extracellular arginine fuels rapid NO production in activated macrophages, and citrulline recycling via Ass1 and Asl is a fail-safe system that sustains optimum NO production.

Respiratory syncytial virus fusion protein-induced toll-like receptor 4 (TLR4) signaling is inhibited by the TLR4 antagonists Rhodobacter sphaeroides lipopolysaccharide and eritoran (E5564) and requires direct interaction with MD-2.

UNLABELLED: Respiratory syncytial virus (RSV) is a leading cause of infant mortality worldwide. Toll-like receptor 4 (TLR4), a signaling receptor for structurally diverse microbe-associated molecular patterns, is activated by the RSV fusion (F) protein and by bacterial lipopolysaccharide (LPS) in a CD14-dependent manner. TLR4 signaling by LPS also requires the presence of an additional protein, MD-2. Thus, it is possible that F protein-mediated TLR4 activation relies on MD-2 as well, although this hypothesis has not been formally tested. LPS-free RSV F protein was found to activate NF-κB in HEK293T transfectants that express wild-type (WT) TLR4 and CD14, but only when MD-2 was coexpressed. These findings were confirmed by measuring F-protein-induced interleukin 1ß (IL-1ß) mRNA in WT versus MD-2(-/-) macrophages, where MD-2(-/-) macrophages failed to show IL-1ß expression upon F-protein treatment, in contrast to the WT. Both Rhodobacter sphaeroides LPS and synthetic E5564 (eritoran), LPS antagonists that inhibit TLR4 signaling by binding a hydrophobic pocket in MD-2, significantly reduced RSV F-protein-mediated TLR4 activity in HEK293T-TLR4-CD14-MD-2 transfectants in a dose-dependent manner, while TLR4-independent NF-κB activation by tumor necrosis factor alpha (TNF-α) was unaffected. In vitro coimmunoprecipitation studies confirmed a physical interaction between native RSV F protein and MD-2. Further, we demonstrated that the N-terminal domain of the F1 segment of RSV F protein interacts with MD-2. These data provide new insights into the importance of MD-2 in RSV F-protein-mediated TLR4 activation. Thus, targeting the interaction between MD-2 and RSV F protein may potentially lead to novel therapeutic approaches to help control RSV-induced inflammation and pathology. IMPORTANCE: This study shows for the first time that the fusion (F) protein of respiratory syncytial virus (RSV), a major cause of bronchiolitis and death, particularly in infants and young children, physically interacts with the Toll-like receptor 4 (TLR4) coreceptor, MD-2, through its N-terminal domain. We show that F protein-induced TLR4 activation can be blocked by lipid A analog antagonists. This observation provides a strong experimental rationale for testing such antagonists in animal models of RSV infection for potential use in people.

Transcriptional regulation of murine IL-33 by TLR and non-TLR agonists.

IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of TLR (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IFN regulatory factor-3 (IRF-3) in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3(-/-) macrophages and TBK-1(-/-) mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3 dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-ß(-/-) macrophages, indicating that IL-33 is not dependent on IFN-ß as an intermediate. Epinephrine and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergize with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the protein kinase A inhibitor H-89 but not by tyrosine kinase or protein kinase C inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter "A" species is preferentially induced by all IL-33-inducing agonists except Newcastle disease virus, a RIG-I agonist that induced expression of both "A" and "B" transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.

Members of the Francisella tularensis phagosomal transporter subfamily of major facilitator superfamily transporters are critical for pathogenesis.

Francisella tularensis is the causative agent of tularemia. Due to its aerosolizable nature and low infectious dose, F. tularensis is classified as a category A select agent and, therefore, is a priority for vaccine development. Survival and replication in macrophages and other cell types are critical to F. tularensis pathogenesis, and impaired intracellular survival has been linked to a reduction in virulence. The F. tularensis genome is predicted to encode 31 major facilitator superfamily (MFS) transporters, and the nine-member Francisella phagosomal transporter (Fpt) subfamily possesses homology with virulence factors in other intracellular pathogens. We hypothesized that these MFS transporters may play an important role in F. tularensis pathogenesis and serve as good targets for attenuation and vaccine development. Here we show altered intracellular replication kinetics and attenuation of virulence in mice infected with three of the nine Fpt mutant strains compared with wild-type (WT) F. tularensis LVS. The vaccination of mice with these mutant strains was protective against a lethal intraperitoneal challenge. Additionally, we observed pronounced differences in cytokine profiles in the livers of mutant-infected mice, suggesting that alterations in in vivo cytokine responses are a major contributor to the attenuation observed for these mutant strains. These results confirm that this subset of MFS transporters plays an important role in the pathogenesis of F. tularensis and suggest that a focus on the development of attenuated Fpt subfamily MFS transporter mutants is a viable strategy toward the development of an efficacious vaccine.

Proteinase-activated receptor 2 activation promotes an anti-inflammatory and alternatively activated phenotype in LPS-stimulated murine macrophages.

Proteinase-activated receptor 2 (PAR(2)), a 7-transmembrane G protein-coupled receptor, contributes to inflammation either positively or negatively in different experimental systems. Previously, we reported that concurrent activation of PAR(2) and TLRs in human lung and colonic epithelial cells resulted in a synergistic increase in NF-κB-mediated gene expression, but a down-regulation of IRF-3-mediated gene expression. In this study, the effect of PAR(2) activation on LPS-induced TLR4 signaling was examined in primary murine macrophages. The PAR(2) activation of wild-type macrophages enhanced LPS-induced expression of the anti-inflammatory cytokine, IL-10, while suppressing gene expression of pro-inflammatory cytokines, TNF-α, IL-6, and IL-12. Similar PAR(2)-mediated effects on LPS-stimulated IL-10 and IL-12 mRNA were also observed in vivo. In contrast, PAR 2-/- macrophages exhibited diminished LPS-induced IL-10 mRNA and protein expression and downstream STAT3 activation, but increased KC mRNA and protein. PAR(2) activation also enhanced both rIL-4- and LPS-induced secretion of IL-4 and IL-13, and mRNA expression of alternatively activated macrophage (AA-M) markers, e.g. arginase-1, mannose receptor, Ym-1. Thus, in the context of a potent inflammatory stimulus like LPS, PAR(2) activation acts to re-establish tissue homeostasis by dampening the production of inflammatory mediators and causing the differentiation of macrophages that may contribute to the development of a Th2 response.