Card9 Broadly Regulates Host Immunity against Experimental Pulmonary Cryptococcus neoformans 52D Infection.

The ubiquitous soil-associated fungus Cryptococcus neoformans causes pneumonia that may progress to fatal meningitis. Recognition of fungal cell walls by C-type lectin receptors (CLRs) has been shown to trigger the host immune response. Caspase recruitment domain-containing protein 9 (Card9) is an intracellular adaptor that is downstream of several CLRs. Experimental studies have implicated Card9 in host resistance against C. neoformans; however, the mechanisms that are associated with susceptibility to progressive infection are not well defined. To further characterize the role of Card9 in cryptococcal infection, Card9em1Sq mutant mice that lack exon 2 of the Card9 gene on the Balb/c genetic background were created using CRISPR-Cas9 genome editing technology and intratracheally infected with C. neoformans 52D. Card9em1Sq mice had significantly higher lung and brain fungal burdens and shorter survival after C. neoformans 52D infection. Susceptibility of Card9em1Sq mice was associated with lower pulmonary cytokine and chemokine production, as well as reduced numbers of CD4+ lymphocytes, neutrophils, monocytes, and dendritic cells in the lungs. Histological analysis and intracellular cytokine staining of CD4+ T cells demonstrated a Th2 pattern of immunity in Card9em1Sq mice. These findings demonstrate that Card9 broadly regulates the host inflammatory and immune response to experimental pulmonary infection with a moderately virulent strain of C. neoformans.

The IL-1 Receptor Is Required to Maintain Neutrophil Viability and Function During Aspergillus fumigatus Airway Infection.

Aspergillus fumigatus airway infections are associated with increased rates of hospitalizations and declining lung function in patients with chronic lung disease. While the pathogenesis of invasive A. fumigatus infections is well studied, little is known about the development and progression of airway infections. Previous studies have demonstrated a critical role for the IL-1 cytokines, IL-1α and IL-1ß in enhancing pulmonary neutrophil recruitment during invasive aspergillosis. Here we use a mouse model of A. fumigatus airway infection to study the role of these IL-1 cytokines in immunocompetent mice. In the absence of IL-1 receptor signaling, mice exhibited reduced numbers of viable pulmonary neutrophils and increased levels of neutrophil apoptosis during fungal airway infection. Impaired neutrophil viability in these mice was associated with reduced pulmonary and systemic levels of G-CSF, and treatment with G-CSF restored both neutrophil viability and resistance to A. fumigatus airway infection. Taken together, these data demonstrate that IL-1 dependent G-CSF production plays a key role for host resistance to A. fumigatus airway infection through suppressing neutrophil apoptosis at the site of infection.

Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians.

OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.

Gram-Negative Pneumonia Augments Non-Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4 Activation.

INTRODUCTION: Surgery is essential for cure of early-stage non-small cell lung cancer (NSCLC). Rates of postoperative bacterial pneumonias, however, remain high, and clinical data suggests that post-operative infectious complications confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory response to infection by recognizing evolutionarily conserved bacterial structures at the surface of numerous pulmonary cell types; yet, little is known about how host TLR activation influences NSCLC metastasis. TLR4 recognizes gram-negative bacterium lipopolysaccharide activating the innate immune system. METHODS: C57BL/6 and TLR4 knockout murine airways were inoculated with Escherichia coli or lipopolysaccharide. Hepatic metastasis assays and intravital microscopy were performed. Bronchoepithelial conditioned media was generated through coincubation of bronchoepithelial cells with TLR4 activating Escherichia coli or lipopolysaccharide. Subsequently, H59 NSCLC were stimulated with conditioned media and subject to various adhesion assays. RESULTS: We demonstrate that gram-negative Escherichia coli pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through TLR4 activation. Additionally, infected C57BL/6 mice demonstrate increased H59 NSCLC in vivo hepatic sinusoidal adhesion compared with negative controls, a response that is significantly diminished in TLR4 knockout mice. Similarly, intratracheal injection of purified TLR4 activating lipopolysaccharide increases in vivo adhesion of H59 cells to murine hepatic sinusoids. Furthermore, H59 cells incubated with bronchoepithelial conditioned medium show increased cell adhesion to in vitro extracellular matrix proteins and in vivo hepatic sinusoids through a mechanism dependent on bronchoepithelial TLR4 activation and interleukin-6 secretion. CONCLUSION: TLR4 is a viable therapeutic target for NSCLC metastasis augmented by gram-negative pneumonia.

Resistance and Tolerance to Cryptococcal Infection: An Intricate Balance That Controls the Development of Disease.

Cryptococcus neoformans is a ubiquitous environmental yeast and a leading cause of invasive fungal infection in humans. The most recent estimate of global disease burden includes over 200,000 cases of cryptococcal meningitis each year. Cryptococcus neoformans expresses several virulence factors that may have originally evolved to protect against environmental threats, and human infection may be an unintended consequence of these acquired defenses. Traditionally, C. neoformans has been viewed as a purely opportunistic pathogen that targets severely immune compromised hosts; however, during the past decade the spectrum of susceptible individuals has grown considerably. In addition, the closely related strain Cryptococcus gattii has recently emerged in North America and preferentially targets individuals with intact immunity. In parallel to the changing epidemiology of cryptococcosis, an increasing role for host immunity in the pathogenesis of severe disease has been elucidated. Initially, the HIV/AIDS epidemic revealed the capacity of C. neoformans to cause host damage in the absence of adaptive immunity. Subsequently, the development and clinical implementation of highly active antiretroviral treatment (HAART) led to recognition of an immune reconstitution inflammatory syndrome (IRIS) in a subset of HIV+ individuals, demonstrating the pathological role of host immunity in disease. A post-infectious inflammatory syndrome (PIIRS) characterized by abnormal T cell-macrophage activation has also been documented in HIV-negative individuals following antifungal therapy. These novel clinical conditions illustrate the highly complex host-pathogen relationship that underlies severe cryptococcal disease and the intricate balance between tolerance and resistance that is necessary for effective resolution. In this article, we will review current knowledge of the interactions between cryptococci and mammalian hosts that result in a tolerant phenotype. Future investigations in this area have potential for translation into improved therapies for affected individuals.

Rel-Dependent Immune and Central Nervous System Mechanisms Control Viral Replication and Inflammation during Mouse Herpes Simplex Encephalitis.

Herpes simplex encephalitis (HSE), caused by HSV type 1 (HSV-1) infection, is an acute neuroinflammatory condition of the CNS and remains the most common type of sporadic viral encephalitis worldwide. Studies in humans have shown that susceptibility to HSE depends in part on the genetic make-up of the host, with deleterious mutations in the TLR3/type I IFN axis underlying some cases of childhood HSE. Using an in vivo chemical mutagenesis screen for HSV-1 susceptibility in mice, we identified a susceptible pedigree carrying a causal truncating mutation in the Rel gene (RelC307X ), encoding for the NF-κB transcription factor subunit c-Rel. Like Myd88-/- and Irf3-/- mice, RelC307X mice were susceptible to intranasal HSV-1 infection. Reciprocal bone marrow transfers into lethally irradiated hosts suggested that defects in both hematopoietic and CNS-resident cellular compartments contributed together to HSE susceptibility in RelC307X mice. Although the RelC307X mutation maintained cell-intrinsic antiviral control, it drove increased apoptotic cell death in infected fibroblasts. Moreover, reduced numbers of CD4+CD25+Foxp3+ T regulatory cells, and dysregulated NK cell and CD4+ effector T cell responses in infected RelC307X animals, indicated that protective immunity was also compromised in these mice. In the CNS, moribund RelC307X mice failed to control HSV-1 viral replication in the brainstem and cerebellum, triggering cell death and elevated expression of Ccl2, Il6, and Mmp8 characteristic of HSE neuroinflammation and pathology. In summary, our work implicates c-Rel in both CNS-resident cell survival and lymphocyte responses to HSV-1 infection and as a novel cause of HSE disease susceptibility in mice.

The TLR4-TRIF pathway can protect against the development of experimental allergic asthma.

The Toll-like receptor (TLR) adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll, interleukin-1 receptor and resistance protein (TIR) domain-containing adaptor inducing interferon-ß (TRIF) comprise the two principal limbs of the TLR signalling network. We studied the role of these adaptors in the TLR4-dependent inhibition of allergic airway disease and induction of CD4+ ICOS+ T cells by nasal application of Protollin™, a mucosal adjuvant composed of TLR2 and TLR4 agonists. Wild-type (WT), Trif-/- or Myd88-/- mice were sensitized to birch pollen extract (BPEx), then received intranasal Protollin followed by consecutive BPEx challenges. Protollin's protection against allergic airway disease was TRIF-dependent and MyD88-independent. TRIF deficiency diminished the CD4+ ICOS+ T-cell subsets in the lymph nodes draining the nasal mucosa, as well as their recruitment to the lungs. Overall, TRIF deficiency reduced the proportion of cervical lymph node and lung CD4+ ICOS+ Foxp3- cells, in particular. Adoptive transfer of cervical lymph node cells supported a role for Protollin-induced CD4+ ICOS+ cells in the TRIF-dependent inhibition of airway hyper-responsiveness. Hence, our data demonstrate that stimulation of the TLR4-TRIF pathway can protect against the development of allergic airway disease and that a TRIF-dependent adjuvant effect on CD4+ ICOS+ T-cell responses may be a contributing mechanism.

Gram-positive pneumonia augments non-small cell lung cancer metastasis via host toll-like receptor 2 activation.

Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.

Immune Recognition of Fungal Polysaccharides.

The incidence of fungal infections has dramatically increased in recent years, in large part due to increased use of immunosuppressive medications, as well as aggressive medical and surgical interventions that compromise natural skin and mucosal barriers. There are relatively few currently licensed antifungal drugs, and rising resistance to these agents has led to interest in the development of novel preventative and therapeutic strategies targeting these devastating infections. One approach to combat fungal infections is to augment the host immune response towards these organisms. The polysaccharide-rich cell wall is the initial point of contact between fungi and the host immune system, and therefore, represents an important target for immunotherapeutic approaches. This review highlights the advances made in our understanding of the mechanisms by which the immune system recognizes and interacts with exopolysaccharides produced by four of the most common fungal pathogens: Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum. Work to date suggests that inner cell wall polysaccharides that play an important structural role are the most conserved across diverse members of the fungal kingdom, and elicit the strongest innate immune responses. The immune system senses these carbohydrates through receptors, such as lectins and complement proteins. In contrast, a greater diversity of polysaccharides is found within the outer cell walls of pathogenic fungi. These glycans play an important role in immune evasion, and can even induce anti-inflammatory host responses. Further study of the complex interactions between the host immune system and the fungal polysaccharides will be necessary to develop more effective therapeutic strategies, as well as to explore the use of immunosuppressive polysaccharides as therapeutic agents to modulate inflammation.

Contribution of IL-1RI Signaling to Protection against Cryptococcus neoformans 52D in a Mouse Model of Infection.

Interleukin-1 alpha (IL-1α) and interleukin-1 beta (IL-1ß) are pro-inflammatory cytokines that are induced after Cryptococcus neoformans infection and activate the interleukin-1 receptor type I (IL-1RI). To establish the role of IL-1RI signaling in protection against cryptococcal infection, we analyzed wild-type (WT) and IL-1RI-deficient (IL-1RI-/-) mice on the BALB/c background. IL-1RI-/- mice had significantly reduced survival compared to WT mice after intratracheal challenge with C. neoformans 52D. Microbiological analysis showed a significant increase in the lung and brain fungal burden of IL-1RI-/- compared to WT mice beginning at weeks 1 and 4 postinfection, respectively. Histopathology showed that IL-1RI-/- mice exhibit greater airway epithelial mucus secretion and prominent eosinophilic crystals that were absent in WT mice. Susceptibility of IL-1RI-/- mice was associated with significant induction of a Th2-biased immune response characterized by pulmonary eosinophilia, M2 macrophage polarization, and recruitment of CD4+ IL-13+ T cells. Expression of pro-inflammatory [IL-1α, IL-1ß, TNFα, and monocyte chemoattractant protein 1 (MCP-1)], Th1-associated (IFNγ), and Th17-associated (IL-17A) cytokines was significantly reduced in IL-1RI-/- lungs compared to WT. WT mice also had higher expression of KC/CXCL1 and sustained neutrophil recruitment to the lung; however, antibody-mediated depletion of these cells showed that they were dispensable for lung fungal clearance. In conclusion, our data indicate that IL-1RI signaling is required to activate a complex series of innate and adaptive immune responses that collectively enhance host defense and survival after C. neoformans 52D infection in BALB/c mice.

Regulation of ULK1 Expression and Autophagy by STAT1.

Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy.

Innate Immune B Cell Activation by Leishmania donovani Exacerbates Disease and Mediates Hypergammaglobulinemia.

Participation of B cells in the immune response by various antibody-independent mechanisms has recently been uncovered. B cells producing cytokines have been described for several infections and appear to regulate the adaptive immune response. B cell activation by Leishmania donovani results in disease exacerbation. How Leishmania activates B cells is still unknown. We show that L. donovani amastigotes activate B cells by triggering endosomal TLRs; this activation leads to the induction of various cytokines. Cytokine expression is completely abrogated in B cells from Ifnar(-/-) mice upon exposure to L. donovani, suggesting an involvement of IFN-I in a positive feedback loop. IFN-I also appears to enhance the expression of endosomal TLRs following exposure to L. donovani. Cell-specific ablation of endosomal TLR signaling in B cells revealed that innate B cell activation by L. donovani is responsible for disease exacerbation through IL-10 and IFN-I production and for the promotion of hypergammaglobulinemia.

Divergent impact of Toll-like receptor 2 deficiency on repair mechanisms in healthy muscle versus Duchenne muscular dystrophy.

Injury to skeletal muscle, whether acute or chronic, triggers macrophage-mediated innate immunity in a manner which can be either beneficial or harmful for subsequent repair. Endogenous ligands for Toll-like receptor 2 (TLR2) are released by damaged tissues and might play an important role in activating the innate immune system following muscle injury. To test this hypothesis, we compared macrophage behaviour and muscle repair mechanisms in mice lacking TLR2 under conditions of either acute (cardiotoxin-induced) or chronic (mdx mouse genetic model of Duchenne muscular dystrophy; DMD) muscle damage. In previously healthy muscle subjected to acute damage, TLR2 deficiency reduced macrophage numbers in the muscle post-injury but did not alter the expression pattern of the prototypical macrophage polarization markers iNOS and CD206. In addition, there was abnormal persistence of necrotic fibres and impaired regeneration in TLR2-/- muscles after acute injury. In contrast, TLR2 ablation in chronically diseased muscles of mdx mice not only resulted in significantly reduced macrophage numbers but additionally modified their phenotype by shifting from inflammatory (iNOS(pos) CD206(neg) ) to more anti-inflammatory (iNOS(neg) CD206(pos) ) characteristics. This decrease in macrophage-mediated inflammation was associated with ameliorated muscle histopathology and improved force-generating capacity of the dystrophic muscle. Our results suggest that the role of TLR2 in macrophage function and skeletal muscle repair depends greatly upon the muscle injury context, and raise the possibility that inhibition of TLR2 could serve as a useful therapeutic measure in DMD.

Type I interferon restricts type 2 immunopathology through the regulation of group 2 innate lymphoid cells.

Viral respiratory tract infections are the main causative agents of the onset of infection-induced asthma and asthma exacerbations that remain mechanistically unexplained. Here we found that deficiency in signaling via type I interferon receptor led to deregulated activation of group 2 innate lymphoid cells (ILC2 cells) and infection-associated type 2 immunopathology. Type I interferons directly and negatively regulated mouse and human ILC2 cells in a manner dependent on the transcriptional activator ISGF3 that led to altered cytokine production, cell proliferation and increased cell death. In addition, interferon-γ (IFN-γ) and interleukin 27 (IL-27) altered ILC2 function dependent on the transcription factor STAT1. These results demonstrate that type I and type II interferons, together with IL-27, regulate ILC2 cells to restrict type 2 immunopathology.

The Cnes2 locus on mouse chromosome 17 regulates host defense against cryptococcal infection through pleiotropic effects on host immunity.

The genetic basis of natural susceptibility to progressive Cryptococcus neoformans infection is not well understood. Using C57BL/6 and CBA/J inbred mice, we previously identified three chromosomal regions associated with C. neoformans susceptibility (Cnes1, Cnes2, and Cnes3). To validate and characterize the role of Cnes2 during the host response, we constructed a congenic strain on the C57BL/6 background (B6.CBA-Cnes2). Phenotypic analysis of B6.CBA-Cnes2 mice 35 days after C. neoformans infection showed a significant reduction of fungal burden in the lungs and spleen with higher pulmonary expression of gamma interferon (IFN-γ) and interleukin-12 (IL-12), lower expression of IL-4, IL-5, and IL-13, and an absence of airway epithelial mucus production compared to that in C57BL/6 mice. Multiparameter flow cytometry of infected lungs also showed a significantly higher number of neutrophils, exudate macrophages, CD11b(+) dendritic cells, and CD4(+) cells in B6.CBA-Cnes2 than in C57BL/6 mice. The activation state of recruited macrophages and dendritic cells was also significantly increased in B6.CBA-Cnes2 mice. Taken together, these findings demonstrate that the Cnes2 interval is a potent regulator of host defense, immune responsiveness, and differential Th1/Th2 polarization following C. neoformans infection.

Unc93b1 -Dependent Endosomal Toll-Like Receptor Signaling Regulates Inflammation and Mortality during Coxsackievirus B3 Infection.

Coxsackievirus strain B serotype 3 (CVB3)-induced myocarditis is an important human disease that causes permanent tissue damage and can lead to death from acute infection or long-term morbidity caused by chronic inflammation. The timing and magnitude of immune activation following CVB3 infection can mediate a positive host outcome or increase tissue pathology. To better elucidate the role of endosomal Toll-like receptor (TLR) signaling in acute CVB3 infection, we studied mice with a loss-of-function mutation, known as Letr for 'loss of endosomal TLR response', in Unc93b1, which is a chaperone protein for TLR3, TLR7 and TLR9. Using Unc93b1(Letr/)(Letr) mice, we determined that Unc93b1-dependent TLR activation was essential for the survival of acute CVB3-induced myocarditis. We also determined that a lack of endosomal TLR signaling was associated with a higher viral load in target organs and that it increased inflammation, necrosis and fibrosis in cardiac tissue. Loss of Unc93b1 function was also associated with increased cardiac expression of Ifn-b and markers of tissue injury and fibrosis including Lcn2 and Serpina3n early after CVB3 infection. These observations establish a significant role for Unc93b1 in the regulation of the host inflammatory response to CVB3 infection and also reveal potential mediators of host tissue damage that merit further investigation in acute viral myocarditis.

Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation.

Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll-like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non-small cell lung cancer (NSCLC) cells with heat-inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

Varying concentrations of lipopolysaccharide (LPS) in ovalbumin (OVA) may influence the airway response to allergic sensitization and challenge. We assessed the contribution of LPS to allergic airway inflammatory responses following challenge with LPS-rich and LPS-free commercial OVA. BALB/c mice were sensitized with LPS-rich OVA and alum and then underwent challenge with the same OVA (10 µg intranasally) or an LPS-free OVA. Following challenge, bronchoalveolar lavage (BAL), airway responsiveness to methacholine and the lung regulatory T cell population (Treg) were assessed. Both OVA preparations induced BAL eosinophilia but LPS-rich OVA also evoked BAL neutrophilia. LPS-free OVA increased interleukin (IL)-2, IL-4 and IL-5 whereas LPS-rich OVA additionally increased IL-1ß, IL-12, IFN-γ, TNF-α and KC. Both OVA-challenged groups developed airway hyperresponsiveness. TLR4-deficient mice challenged with either OVA preparation showed eosinophilia but not neutrophilia and had increased IL-5. Only LPS-rich OVA challenged mice had increased lung Tregs and LPS-rich OVA also induced in vitro Treg differentiation. LPS-rich OVA also induced a Th1 cytokine response in human peripheral blood mononuclear cells.We conclude that LPS-rich OVA evokes mixed Th1, Th2 and innate immune responses through the TLR-4 pathway, whereas LPS-free OVA evokes only a Th2 response. Contaminating LPS is not required for induction of airway hyperresponsiveness but amplifies the Th2 inflammatory response and is a critical mediator of the neutrophil, Th1 and T regulatory cell responses to OVA.

Other viral pneumonias: coronavirus, respiratory syncytial virus, adenovirus, hantavirus.

Severe viral pneumonia is an increasing problem among adults. The incidence and number of viruses known to cause pneumonia and respiratory failure have also expanded in recent years. This article provides an overview of severe respiratory disease caused by coronavirus, respiratory syncytial virus, adenovirus, and hantavirus. These emerging pathogens are easily overlooked and timely diagnosis requires a high index of suspicion and confirmation by molecular testing. Management of individual cases is mainly supportive and requires institution of appropriate infection control measures. Vaccines and effective therapeutics for these potentially devastating respiratory viruses are urgently required.

IL-33 signaling regulates innate and adaptive immunity to Cryptococcus neoformans.

Susceptibility to progressive infection with the fungus Cryptococcus neoformans is associated with an allergic pattern of lung inflammation, yet the factors that govern this host response are not clearly understood. Using a clinically relevant mouse model of inhalational infection with virulent C. neoformans H99, we demonstrate a role for IL-33-dependent signaling in host immune defense. Infection of BALB/c mice with 10(4) CFU of C. neoformans H99 caused a time-dependent induction of IL-33 with accumulation of type 2 pulmonary innate lymphoid cells and alternatively activated macrophages in the lungs as well as Th2-polarized CD4(+) T cells in draining lymph nodes. IL-33R subunit T1/ST2-deficient (T1/ST2(-/-)) mice infected with C. neoformans H99 had improved survival with a decreased fungal burden in the lungs, spleen, and brain, compared with wild-type mice. Signaling through T1/ST2 was required for the accumulation and early production of IL-5 and IL-13 by lung type 2 pulmonary innate lymphoid cells. Further analysis of T1/ST2(-/-) mice revealed increased fungicidal exudate macrophages in the lungs and decreased C. neoformans-specific Th2 cells in the mediastinal lymph nodes. T1/ST2 deficiency also diminished goblet cell hyperplasia, mucus hypersecretion, bronchoalveolar lavage eosinophilia, alternative activation of macrophages, and serum IgE. These observations demonstrate that IL-33-dependent signaling contributes to the expansion of innate type 2 immunity and subsequent Th2-biased lung immunopathology that facilitates C. neoformans growth and dissemination.