Glycolipid Metabolite ß-Glucosylceramide Is a Neutrophil Extracellular Trap-Inducing Ligand of Mincle Released during Bacterial Infection and Inflammation.

Neutrophil extracellular traps (NETs) are implicated in host defense and inflammatory pathologies alike. A wide range of pathogen- and host-derived factors are known to induce NETs, yet the knowledge about specific receptor-ligand interactions in this response is limited. We previously reported that macrophage-inducible C-type lectin (Mincle) regulates NET formation. In this article, we identify glycosphingolipid ß-glucosylceramide (ß-GlcCer) as a specific NET-inducing ligand of Mincle. We found that purified ß-GlcCer induced NETs in mouse primary neutrophils in vitro and in vivo, and this effect was abrogated in Mincle deficiency. Cell-free ß-GlcCer accumulated in the lungs of pneumonic mice, which correlated with pulmonary NET formation in wild-type, but not in Mincle-/-, mice infected intranasally with Klebsiella pneumoniae Although leukocyte infiltration by ß-GlcCer administration in vivo did not require Mincle, NETs induced by this sphingolipid were important for bacterial clearance during Klebsiella infection. Mechanistically, ß-GlcCer did not activate reactive oxygen species formation in neutrophils but required autophagy and glycolysis for NET formation, because ATG4 inhibitor NSC185058, as well as glycolysis inhibitor 2-deoxy-d-glucose, abrogated ß-GlcCer-induced NETs. Forced autophagy activation by tamoxifen could overcome the inhibitory effect of glycolysis blockage on ß-GlcCer-mediated NET formation, suggesting that autophagy activation is sufficient to induce NETs in response to this metabolite in the absence of glycolysis. Finally, ß-GlcCer accumulated in the plasma of patients with systemic inflammatory response syndrome, and its levels correlated with the extent of systemic NET formation in these patients. Overall, our results posit ß-GlcCer as a potent NET-inducing ligand of Mincle with diagnostic and therapeutic potential in inflammatory disease settings.

Sialylation on O-linked glycans protects von Willebrand factor from macrophage galactose lectin-mediated clearance.

Terminal sialylation determines the plasma half-life of von Willebrand factor (VWF). A role for macrophage galactose lectin (MGL) in regulating hyposialylated VWF clearance has recently been proposed. In this study, we showed that MGL influences physiological plasma VWF clearance. MGL inhibition was associated with a significantly extended mean residence time and 3-fold increase in endogenous plasma VWF antigen levels (P<0.05). Using a series of VWF truncations, we further demonstrated that the A1 domain of VWF is predominantly responsible for enabling the MGL interaction. Binding of both full-length and VWF-A1-A2-A3 to MGL was significantly enhanced in the presence of ristocetin (P<0.05), suggesting that the MGL-binding site in A1 is not fully accessible in globular VWF. Additional studies using different VWF glycoforms demonstrated that VWF O-linked glycans, clustered at either end of the A1 domain, play a key role in protecting VWF against MGLmediated clearance. Reduced sialylation has been associated with pathological, increased clearance of VWF in patients with von Willebrand disease. Herein, we demonstrate that specific loss of α2-3 linked sialylation from O-glycans results in markedly increased MGL-binding in vitro, and markedly enhanced MGL-mediated clearance of VWF in vivo. Our data further show that the asialoglycoprotein receptor (ASGPR) does not have a significant role in mediating the increased clearance of VWF following loss of O-sialylation. Conversely however, we observed that loss of N-linked sialylation from VWF drives enhanced circulatory clearance predominantly via the ASGPR. Collectively, our data support the hypothesis that in addition to regulating physiological VWF clearance, the MGL receptor works in tandem with ASGPR to modulate enhanced clearance of aberrantly sialylated VWF in the pathogenesis of von Willebrand disease.

Klebsiella pneumoniae infection of murine neutrophils impairs their efferocytic clearance by modulating cell death machinery.

Neutrophils are the first infiltrating cell type essential for combating pneumoseptic infections by bacterial pathogens including Klebsiella pneumoniae (KPn). Following an infection or injury, removal of apoptotic infiltrates via a highly regulated process called efferocytosis is required for restoration of homeostasis, but little is known regarding the effect of bacterial infection on this process. Here we demonstrate that KPn infection impedes the efferocytic uptake of neutrophils in-vitro and in-vivo in lungs by macrophages. This impaired efferocytosis of infected neutrophils coincides with drastic reduction in the neutrophil surface exposure of apoptosis signature phospholipid phosphatidyserine (PS); and increased activity of phospholipid transporter flippases, which maintain PS in the inner leaflet of plasma membrane. Concomitantly, pharmacological inhibition of flippase activity enhanced PS externalization and restored the efferocytosis of KPn infected neutrophils. We further show that KPn infection interferes with apoptosis activation and instead activates non-apoptotic programmed cell death via activation of necroptosis machinery in neutrophils. Accordingly, pharmacological inhibition of necroptosis by RIPK1 and RIPK3 inhibitors restored the efferocytic uptake of KPn infected neutrophils in-vitro. Importantly, treatment of KPn infected mice with necroptosis inhibitor improved the disease outcome in-vivo in preclinical mouse model of KPn pneumonia. To our knowledge, this is the first report of neutrophil efferocytosis impairment by KPn via modulation of cell death pathway, which may provide novel targets for therapeutic intervention of this infection.

A novel role for the macrophage galactose-type lectin receptor in mediating von Willebrand factor clearance.

Previous studies have shown that loss of terminal sialic acid causes enhanced von Willebrand factor (VWF) clearance through the Ashwell-Morrell receptor (AMR). In this study, we investigated (1) the specific importance of N- vs O-linked sialic acid in protecting against VWF clearance and (2) whether additional receptors contribute to the reduced half-life of hyposialylated VWF. α2-3-linked sialic acid accounts for <20% of total sialic acid and is predominantly expressed on VWF O-glycans. Nevertheless, specific digestion with α2-3 neuraminidase (α2-3Neu-VWF) was sufficient to cause markedly enhanced VWF clearance. Interestingly, in vivo clearance experiments in dual VWF-/-/Asgr1-/- mice demonstrated enhanced clearance of α2-3Neu-VWF even in the absence of the AMR. The macrophage galactose-type lectin (MGL) is a C-type lectin that binds to glycoproteins expressing terminal N-acetylgalactosamine or galactose residues. Importantly, the markedly enhanced clearance of hyposialylated VWF in VWF-/-/Asgr1-/- mice was significantly attenuated in the presence of an anti-MGL inhibitory antibody. Furthermore, dose-dependent binding of human VWF to purified recombinant human MGL was confirmed using surface plasmon resonance. Additionally, plasma VWF:Ag levels were significantly elevated in MGL1-/- mice compared with controls. Collectively, these findings identify MGL as a novel macrophage receptor for VWF that significantly contributes to the clearance of both wild-type and hyposialylated VWF.

Galectin-3 in M2 Macrophages Plays a Protective Role in Resolution of Neuropathology in Brain Parasitic Infection by Regulating Neutrophil Turnover.

Macrophages/microglia with M2-activation phenotype are thought to play important anti-inflammatory and tissue reparative functions in the brain, yet the molecular bases of their functions in the CNS remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti, we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3-deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in vitro Furthermore, adoptive transfer of M2 macrophages from galectin-3-sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3-/- mice. Together, these results demonstrate, for the first time, a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell-protective functions in the CNS microenvironment are undefined. The current study reports that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models in vivo and in vitro, they show that galectin-3 in M2 macrophages functions to clear neutrophils accumulated in the CNS. Importantly, galectin-3 in M2 macrophages plays a central role in the containment of neuropathology and disease severity. These results provide a direct mechanistic evidence of the protective function of M2 macrophages in the CNS.

Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H2O2) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H2O2. This TRPM2-dependent H2O2-mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2-/- mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2-/- mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Macrophage Galactose-Type Lectin-1 Deficiency Is Associated with Increased Neutrophilia and Hyperinflammation in Gram-Negative Pneumonia.

C-type lectin receptors (CLRs), the carbohydrate-recognizing molecules, orchestrate host immune response in homeostasis and in inflammation. In the present study we examined the function of macrophage galactose-type lectin-1 (MGL1), a mammalian CLR, in pneumonic sepsis, a deadly immune disorder frequently associated with a nonresolving hyperinflammation. In a murine model of pneumonic sepsis using pulmonary infection with Klebsiella pneumoniae, the expression of MGL1 was upregulated in the lungs of K. pneumoniae-infected mice, and the deficiency of this CLR in MGL1(-/-) mice resulted in significantly increased mortality to infection than in the MGL1-sufficient wild-type mice, despite a similar bacterial burden. The phagocytic cells from MGL1(-/-) mice did not exhibit any defects in bacterial uptake and intracellular killing and were fully competent in neutrophil extracellular trap formation, a recently identified extracellular killing modality of neutrophils. Instead, the increased susceptibility of MGL1(-/-) mice seemed to correlate with severe lung pathology, indicating that MGL1 is required for resolution of pulmonary inflammation. Indeed, the MGL1(-/-) mice exhibited a hyperinflammatory response, massive pulmonary neutrophilia, and an increase in neutrophil-associated immune mediators. Concomitantly, MGL1-deficient neutrophils exhibited an increased influx in pneumonic lungs of K. pneumoniae-infected mice. Taken together, these results show a previously undetermined role of MGL1 in controlling neutrophilia during pneumonic infection, thus playing an important role in resolution of inflammation. To our knowledge, this is the first study depicting a protective function of MGL1 in an acute pneumonic bacterial infection.

Mincle-Mediated Neutrophil Extracellular Trap Formation by Regulation of Autophagy.

BACKGROUND: Neutrophil extracellular traps (NETs) constitute antimicrobial function of neutrophils but have also been linked to perpetuation of inflammation. Despite this evident physiological relevance, mechanistic understanding of NET formation is poor. In this study, we examined the mechanism by which Mincle, a C-type lectin receptor, regulates NET formation. METHODS: NET formation, reactive oxygen species, autophagy activation and intracellular signaling pathways were analyzed in Mincle-sufficient and -deficient neutrophils stimulated in vitro with various stimuli and in vivo during Klebsiella infection. RESULTS: We found that Mincle mediates NET formation in response to several activation stimuli in vitro and in vivo during pneumoseptic infection with Klebsiella pneumoniae, indicating its regulatory role in NET formation. Mechanistically, we show that attenuated NET formation in Mincle-/- neutrophils correlates with an impaired autophagy activation in vitro and in vivo, whereas reactive oxygen species (ROS) formation in these neutrophils remained intact. The requirement of autophagy in Mincle-mediated NET formation was further supported by exogenous treatment with autophagy inducer tamoxifen, which rescued the NET formation defect in Mincle-/- neutrophils. CONCLUSIONS: Our findings identify a previously unrecognized role of Mincle as a regulator of autophagy, which mediates NET formation without affecting ROS generation. Our study addresses a major challenge in the field by positing this pathway to be targeted for modulation of NETs while preserving ROS production, an important innate immune defense.

Protective role of Mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis and extracellular trap formation.

BACKGROUND: Nosocomial infections with Klebsiella pneumoniae are a frequent cause of Gram-negative bacterial sepsis. To understand the functioning of host innate immune components in this disorder, we examined a previously uninvestigated role of the C-type lectin receptor Mincle in pneumonic sepsis caused by K. pneumoniae. METHODS: Disease progression in wild-type and Mincle(-/-) mice undergoing pulmonary infection with K. pneumoniae was compared. RESULTS: Whereas the wild-type mice infected with a sublethal dose of bacteria could resolve the infection with bacterial clearance and regulated host response, the Mincle(-/-) mice were highly susceptible with a progressive increase in bacterial burden, despite their ability to mount an inflammatory response that turned to an exaggerated hyperinflammation with the onset of severe pneumonia. This correlated with severe lung pathology with a massive accumulation of neutrophils in their lungs. Importantly, Mincle(-/-) neutrophils displayed a defective ability to phagocytize nonopsonic bacteria and an impaired ability to form extracellular traps (NETs), an important neutrophil function against invading pathogens, including K. pneumoniae. CONCLUSION: Our results demonstrate protective role of Mincle in host defense against K. pneumoniae pneumonia by coordinating bacterial clearance mechanisms of neutrophils. A novel role for Mincle in the regulation of neutrophil NET formation may have implications in chronic disease conditions characterized by deregulated NET formation.

Inhibition of store-operated calcium entry in microglia by helminth factors: implications for immune suppression in neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a disease of the central nervous system (CNS) caused by the cestode Taenia solium. The infection exhibits a long asymptomatic phase, typically lasting 3 to 5 years, before the onset of the symptomatic phase. The severity of the symptoms is thought to be associated with the intensity of the inflammatory response elicited by the degenerating parasite. In contrast, the asymptomatic phase shows an absence of brain inflammation, which is presumably due to immunosuppressive effects of the live parasites. However, the host factors and/or pathways involved in inhibiting inflammation remain largely unknown. Recently, using an animal model of NCC in which mice were intracranially inoculated with a related helminth parasite, Mesocestoides corti, we reported that Toll-like receptor (TLR)-associated signaling contributes to the development of the inflammatory response. As microglia shape the initial innate immune response in the CNS, we hypothesized that the negative regulation of a TLR-induced inflammatory pathway in microglia may be a novel helminth-associated immunosuppressive mechanism in NCC. METHODS AND RESULTS: Here we report that helminth soluble factors (HSFs) from Mesocestoides corti inhibited TLR ligation-induced production of inflammatory cytokines in primary microglia. This was correlated with an inhibition of TLR-initiated upregulation of both phosphorylation and acetylation of the nuclear factor κB (NF-κB) p65 subunit, as well as phosphorylation of JNK and ERK1/2. As Ca2+ influx due to store-operated Ca2+ entry (SOCE) has been implicated in induction of downstream signaling, we tested the inhibitory effect of HSFs on agonist-induced Ca2+ influx and specific Ca2+ channel activation. We discovered that HSFs abolished the lipopolysaccharide (LPS)- or thapsigargin (Tg)-induced increase in intracellular Ca2+ accumulation by blocking the ER store release and SOCE. Moreover, electrophysiological recordings demonstrated HSF-mediated inhibition of LPS- or Tg-induced SOCE currents through both TRPC1 and ORAI1 Ca2+ channels on plasma membrane. This was correlated with a decrease in the TRPC1-STIM1 and ORAI1-STIM1 clustering at the plasma membrane that is essential for sustained Ca2+ entry through these channels. CONCLUSION: Inhibition of TRPC1 and ORAI1 Ca2+ channel-mediated activation of NF-κB and MAPK pathways in microglia is likely a novel helminth-induced immunosuppressive mechanism that controls initiation of inflammatory response in the CNS.

Poly(ADP-Ribose) Polymerase-1 Regulates Pyroptosis Independent Function of NLRP3 Inflammasome in Neutrophil Extracellular Trap Formation.

Neutrophil extracellular traps (NETs) function to control infectious agents as well as to propagate inflammatory response in a variety of disease conditions. DNA damage associated with chromatin decondensation and NACHT domain-leucine-rich repeat-and pyrin domain-containing protein 3 (NLRP3) inflammasome activation have emerged as crucial events in NET formation, but the link between the two processes is unknown. In this study, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair enzyme, regulates NET formation triggered by NLRP3 inflammasome activation in neutrophils. Activation of mouse neutrophils with canonical NLRP3 stimulants LPS and nigericin induced NET formation, which was significantly abrogated by pharmacological inhibition of PARP-1. We found that PARP-1 is required for NLRP3 inflammasome assembly by regulating post-transcriptional levels of NLRP3 and ASC dimerization. Importantly, this PARP-1-regulated NLRP3 activation for NET formation was independent of inflammasome-mediated pyroptosis, because caspase-1 and gasdermin D processing as well as IL-1ß transcription and secretion remained intact upon PARP-1 inhibition in neutrophils. Accordingly, pharmacological inhibition or genetic ablation of caspase-1 and gasdermin D had no effect on NLRP3-mediated NET formation. Mechanistically, PARP-1 inhibition increased p38 MAPK activity, which was required for downmodulation of NLRP3 and NETs, because concomitant inhibition of p38 MAPK with PARP-1 restored NLRP3 activation and NET formation. Finally, mice undergoing bacterial peritonitis exhibited increased survival upon treatment with PARP-1 inhibitor, which correlated with increased leukocyte influx and improved intracellular bacterial clearance. Our findings reveal a noncanonical pyroptosis-independent role of NLRP3 in NET formation regulated by PARP-1 via p38 MAPK, which can be targeted to control NETosis in inflammatory diseases.

Current bacteriological profile of tonsillar surface versus core tissue in patients undergoing tonsillectomy.

PURPOSE: The current study explored the bacteriological profile in the tonsillar core in patients undergoing tonsillectomy and compared it with tonsillar surface organisms. The antibiotic sensitivity and resistance patterns were also studied. MATERIALS AND METHODS: This prospective and observational study was conducted in the Department of Otorhinolaryngology, Head and Neck surgery in a tertiary care institute, on a total of sixty-one patients clinically diagnosed as chronic tonsillitis and undergoing tonsillectomy. Tonsillar surface culture swabs and core tissue specimen were sent in separate vials. The culture isolates and their antibiotic sensitivity were analysed and compared. RESULTS: Bacterial growth was detected in 61% of the cases, in cultures from the surface or the core tissue or both. 50.8% specimens of core tissue versus 31.1% of tonsillar surface samples revealed bacterial growth. Of the 13 cases in which surface and core both had bacterial culture growth, the same pathogens were identified in 8 cases. Pseudomonas aeruginosa was the most commonly reported organism in both tonsillar core as well as surface swab cultures. CONCLUSION: Our study shows that surface swab culture is not always a reliable indicator of organisms present in core of tonsils in cases of chronic infection. The surface swabs did not always match the pathogens in the core. The higher prevalence and the variability of pathogenic organisms in the core tissue as compared to the surface suggests that a targeted antibiotic treatment based on surface culture swabs is unlikely to eradicate them.

Defect in efferocytosis leads to alternative activation of macrophages in Francisella infections.

The macrophage is a versatile cell type that can sense and respond to a particular need based on the conditions of the microenvironment. Some studies have recently suggested that pathogens can directly influence the polarization of macrophages. As Francisella infections are characterized by intense necrotic infiltrates in the lung as well as in distal sites of infection, we sought to investigate whether pulmonary Francisella infections could cause the polarization of alternatively activated macrophages (M2/aaMs). Our results indicate that Francisella infections can cause the polarization of M2/aaM in vivo and that macrophages can be polarized toward an M2/aaM phenotype more potently if dead cell debris is used for stimulation in the presence and absence of Francisella infections. Finally, we also demonstrate that efferocytosis is inhibited in macrophages infected with Francisella, thus providing a potential explanation for the lack of clearance and eventual accumulation of dead cell debris associated with this disease.

TLR4-dependent activation of inflammatory cytokine response in macrophages by Francisella elongation factor Tu.

The bacterial determinants of pulmonary Francisella induced inflammatory responses and their interaction with host components are not clearly defined. In this study, proteomic and immunoblot analyses showed presence of a cytoplasmic protein elongation factor Tu (EF-Tu) in the membrane fractions of virulent Francisella novicida, LVS and SchuS4, but not in an attenuated F. novicida mutant. EF-Tu was immunodominant in mice vaccinated and protected from virulent F. novicida. Moreover, recombinant EF-Tu induced macrophages to produce inflammatory cytokines in a TLR4 dependent manner. This study shows immune stimulatory properties of a cytoplasmic protein EF-Tu expressed on the membrane of virulent Francisella strains.

Features of sepsis caused by pulmonary infection with Francisella tularensis Type A strain.

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain Francisella novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB1. This study shows that pulmonary tularemia caused by the Type A strain of F. tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen.

Helminth derived factors inhibit neutrophil extracellular trap formation and inflammation in bacterial peritonitis.

Despite their protective antimicrobial function, neutrophil extracellular traps (NETs) have been implicated in propagation of inflammatory responses in several disease conditions including sepsis. Highly diffusible exogenous ROS produced under such inflammatory conditions, can induce exuberant NETs, thus making inhibition of NETs desirable in inflammatory diseases. Here we report that helminth parasite excretory/secretory factors termed as parasitic ligands (PL) inhibit ROS-induced NETs by blocking the activation of nonselective calcium permeable channel Transient Receptor Potential Melastatin 2 (TRPM2). Therapeutic implication of PL mediated blockage of NET formation was tested in preclinical model of septic peritonitis, where PL treatment regulated neutrophil cell death modalities including NET formation and mitigated neutrophil mediated inflammatory response. This translated into improved survival and reduced systemic and local bacterial load in infected mice. Overall, our results posit PL as an important biological regulator of neutrophil functions with implications to a variety of inflammatory diseases including peritonitis.

Vaccination with an attenuated strain of Francisella novicida prevents T-cell depletion and protects mice infected with the wild-type strain from severe sepsis.

Francisella tularensis is the causative agent of zoonotic tularemia, a severe pneumonia in humans, and Francisella novicida causes a similarly severe tularemia in mice upon inhalation. The correlates of protective immunity, as well as the virulence mechanisms of this deadly pathogen, are not well understood. In the present study, we compared the host immune responses of lethally infected and vaccinated mice to highlight the host determinants of protection from this disease. Intranasal infection with an attenuated mutant (Mut) of F. novicida lacking a 58-kDa hypothetical protein protected C57BL/6 mice from a subsequent challenge with the fully virulent wild-type strain U112 via the same route. The protection conferred by Mut vaccination was associated with reduced bacterial burdens in systemic organs, as well as the absence of bacteremia. Also, there was reduced lung pathology and associated cell death in the lungs of vaccinated mice. Both vaccinated and nonvaccinated mice displayed an initial 2-day delay in upregulation of signature inflammatory mediators after challenge. Whereas the nonvaccinated mice developed severe sepsis characterized by hypercytokinemia and T-cell depletion, the vaccinated mice displayed moderated cytokine induction and contained increased numbers of alphabeta T cells. The recall response in vaccinated mice consisted of a characteristic Th1-type response in terms of cytokines, as well as antibody isotypes. Our results show that a regulated Th1 type of cell-mediated and humoral immunity in the absence of severe sepsis is associated with protection from respiratory tularemia, whereas a deregulated host response leading to severe sepsis contributes to mortality.

Lethal pulmonary infection with Francisella novicida causes depletion of alphabeta T cells from lungs.

Respiratory Francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. In this study, mice infected intranasally with Francisella novicida showed a depletion of alphabeta T cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. The depleted T cells were predominantly CD4(+). The alphabeta T cells in infected mice showed significantly higher levels of Annexin V binding than those in mock control mice suggesting increased apoptosis of T cells. These results suggest that lack of transition from an innate to adaptive host response is associated with lethality of respiratory tularemia.

Function of SLAM-Associated Protein (SAP) in Acute Pneumoseptic Bacterial Infection.

Sepsis resulting from acute pneumonic infections by Gram-negative bacteria is often characterized by dysfunction of innate immune components. Here we report a previously unrecognized innate protective function of SAP, an adaptor protein primarily reported in T cells, NK cells, and NKT cells, during acute pneumonic infection with Klebsiella pneumoniae (KPn). SAP-deficient mice were highly susceptible to this infection with elevated systemic bacterial spread and increased lung damage. While the overall influx of infiltrating cells in the lungs remained largely intact, increased mortality of SAP-deficient mice correlated with increased accumulation of large NK1.1+ cells harboring bacteria and an impairment of neutrophil extracellular trap formation in vivo during KPn pneumonia, which likely facilitated bacterial outgrowth. Neutrophils were found to express SAP; however, adoptive transfer experiment supported a neutrophil-extrinsic function of SAP in neutrophil extracellular trap formation. Collectively, these data present the first report depicting innate protective function of SAP in an acute pulmonary infection.