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.

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.

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.

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.

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.

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.

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.

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.

Dose-dependent effects of IL-17 on IL-13-induced airway inflammatory responses and airway hyperresponsiveness.

The Th2 cytokine IL-13 regulates several aspects of the asthmatic phenotype, including airway inflammation, airway hyperresponsiveness, and mucus production. The Th17 cytokine IL-17A is also implicated in asthma and has been shown to both positively and negatively regulate Th2-dependent responses in murine models of allergic airways disease. Our objective in this study was to better understand the role of IL-17 in airway inflammation by examining how IL-17 modifies IL-13-induced airway inflammatory responses. We treated BALB/c mice intranasally with IL-13 or IL-17 alone or in combination for 8 consecutive days, after which airway hyperresponsiveness, inflammatory cell influx into the lung, and lung chemokine/cytokine expression were assessed. As expected, IL-13 increased airway inflammation and airway hyperresponsiveness. IL-13 also increased numbers of IL-17-producing CD4(+) and γδ T cells. Treating mice with a combination of IL-13 and IL-17 reduced infiltration of IL-17(+) γδ T cells, but increased the number of infiltrating eosinophils. In contrast, coadministration of IL-13 with a higher dose of IL-17 decreased all IL-13-induced inflammatory responses, including infiltration of both IL-17(+)CD4(+) and γδ T cells. To examine the inhibitory activity of IL-17-expressing γδ T cells in this model, these cells were adoptively transferred into naive recipients. Consistent with an inhibitory role for γδ T cells, IL-13-induced infiltration of eosinophils, lymphocytes, and IL-17(+)CD4(+) T cells was diminished in recipients of the γδ T cells. Collectively, our data indicate that allergic airway inflammatory responses induced by IL-13 are modulated by both the quantity and the cellular source of IL-17.

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.

Inhaled birch pollen extract induces airway hyperresponsiveness via oxidative stress but independently of pollen-intrinsic NADPH oxidase activity, or the TLR4-TRIF pathway.

Oxidative stress in allergic asthma may result from oxidase activity or proinflammatory molecules in pollens. Signaling via TLR4 and its adaptor Toll-IL-1R domain-containing adapter inducing IFN-ß (TRIF) has been implicated in reactive oxygen species-mediated acute lung injury and in Th2 immune responses. We investigated the contributions of oxidative stress and TLR4/TRIF signaling to experimental asthma induced by birch pollen exposure exclusively via the airways. Mice were exposed to native or heat-inactivated white birch pollen extract (BPEx) intratracheally and injected with the antioxidants, N-acetyl-L-cysteine or dimethylthiourea, prior to sensitization, challenge, or all allergen exposures, to assess the role of oxidative stress and pollen-intrinsic NADPH oxidase activity in allergic sensitization, inflammation, and airway hyperresponsiveness (AHR). Additionally, TLR4 signaling was antagonized concomitantly with allergen exposure, or the development of allergic airway disease was evaluated in TLR4 or TRIF knockout mice. N-acetyl-L-cysteine inhibited BPEx-induced eosinophilic airway inflammation and AHR except when given exclusively during sensitization, whereas dimethylthiourea was inhibitory even when administered with the sensitization alone. Heat inactivation of BPEx had no effect on the development of allergic airway disease. Oxidative stress-mediated AHR was also TLR4 and TRIF independent; however, TLR4 deficiency decreased, whereas TRIF deficiency increased BPEx-induced airway inflammation. In conclusion, oxidative stress plays a significant role in allergic sensitization to pollen via the airway mucosa, but the pollen-intrinsic NADPH oxidase activity and TLR4 or TRIF signaling are unnecessary for the induction of allergic airway disease and AHR. Pollen extract does, however, activate TLR4, thereby enhancing airway inflammation, which is restrained by the TRIF-dependent pathway.

ICOS-expressing CD4 T cells induced via TLR4 in the nasal mucosa are capable of inhibiting experimental allergic asthma.

Modulation of adaptive immune responses via the innate immune pattern recognition receptors, such as the TLRs, is an emerging strategy for vaccine development. We investigated whether nasal rather than intrapulmonary application of Protollin, a mucosal adjuvant composed of TLR2 and TLR4 ligands, is sufficient to elicit protection against murine allergic lower airway disease. Wild-type, Tlr2(-/-), or Tlr4(-/-) BALB/c mice were sensitized to a birch pollen allergen extract (BPEx), then received either intranasal or intrapulmonary administrations of Protollin or Protollin admixed with BPEx, followed by consecutive daily BPEx challenges. Nasal application of Protollin or Protollin admixed with BPEx was sufficient to inhibit allergic lower airway disease with minimal collateral lung inflammation. Inhibition was dependent on TLR4 and was associated with the induction of ICOS in cells of the nasal mucosa and on both CD4+Foxp3+ and CD4+Foxp3- T cells of the draining lymph nodes (LNs), as well as their recruitment to the lungs. Adoptive transfer of cervical LN CD4+ICOS+, but not CD4+ICOS-, cells inhibited BPEx-induced airway hyperresponsiveness and bronchoalveolar lavage eosinophilia. Thus, our data indicate that expansion of resident ICOS-expressing CD4+ T cells of the cervical LNs by nasal mucosal TLR4 stimulation may inhibit the development of allergic lower airway disease in mice.

Inhibition of mammalian target of rapamycin augments lipopolysaccharide-induced lung injury and apoptosis.

Acute lung injury during bacterial infection is associated with neutrophilic inflammation, epithelial cell apoptosis, and disruption of the alveolar-capillary barrier. TLR4 is required for lung injury in animals exposed to bacterial LPS and initiates proinflammatory responses in part via the transcription factor NF-κB. Ligation of TLR4 also initiates a proapoptotic response by activating IFN-ß and STAT1-dependent genes. We recently demonstrated that mammalian target of rapamycin (mTOR), a key controller of cell growth and survival, can physically interact with STAT1 and suppress the induction of STAT1-dependent apoptosis genes. We therefore hypothesized that the mTOR inhibitor rapamycin would increase LPS-induced apoptosis and lung injury in vivo. Rapamycin increased lung injury and cellular apoptosis in C57BL/6J mice exposed to intratracheal LPS for 24 h. Rapamycin also augmented STAT1 activation, and the induction of STAT1-dependent genes that mediate cellular apoptosis (i.e., Fas, caspase-3). LPS-induced lung injury was attenuated in STAT1 knockout mice. In addition, LPS and IFN-ß-induced apoptosis was absent in cultured cells lacking STAT1, and, unlike in wild-type cells, a permissive effect of rapamycin was not observed. In contrast to its effect on STAT1, rapamycin inhibited NF-κB activation in vivo and reduced selected markers of inflammation (i.e., neutrophils in the bronchoalveolar lavage fluid, TNF-α). Therefore, although it inhibits NF-κB and neutrophilic inflammation, rapamycin augments LPS-induced lung injury and apoptosis in a mechanism that involves STAT1 and the induction of STAT1-dependent apoptosis genes.

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.

Susceptibility to progressive Cryptococcus neoformans pulmonary infection is regulated by loci on mouse chromosomes 1 and 9.

Genetic factors that regulate the pathogenesis of pneumonia caused by the fungus Cryptococcus neoformans are poorly understood. Through a phenotypic strain survey we observed that inbred C3H/HeN mice develop a significantly greater lung fungal burden than mice of the resistant CBA/J strain 4 weeks following intratracheal infection with C. neoformans ATCC 24067. The aim of the present study was to characterize the inflammatory response of C3H/HeN mice following C. neoformans pulmonary infection and to identify genetic loci that regulate host defense. Following cryptococcal infection, C3H/HeN mice demonstrated a Th2 immune response with heightened airway and tissue eosinophilia, goblet cell metaplasia, and significantly higher lung interleukin-5 (IL-5) and IL-13 protein expression relative to CBA/J mice. Conversely, CBA/J mice exhibited greater airway and tissue neutrophilia that was associated with significantly higher pulmonary expression of gamma interferon, CXCL10, and IL-17 proteins than C3H/HeN mice. Using the fungal burden at 4 weeks postinfection as a phenotype, genome-wide quantitative trait locus (QTL) analysis among 435 segregating (C3H/HeN × CBA/J)F2 (C3HCBAF2) hybrids identified two significant QTLs on chromosomes 1 (Cnes4) and 9 (Cnes5) that control susceptibility to cryptococcal pneumonia in an additive manner. Susceptible C3H/HeN mice carry a resistance allele at Cnes4 and a susceptibility allele at Cnes5. These studies reveal additional genetic complexity of the host response to C. neoformans that is associated with divergent patterns of pulmonary inflammation.

N-ethyl-N-nitrosourea-induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αß signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium.

To deepen our knowledge of the natural host response to pathogens, our team undertook an in vivo screen of mutagenized 129S1 mice with Salmonella Typhimurium. One mutation affecting Salmonella susceptibility was mapped to a region of 1.3 Mb on chromosome 6 that contains 15 protein-coding genes. A missense mutation was identified in the Usp18 (ubiquitin-specific peptidase 18) gene. This mutation results in an increased inflammatory response (IL-6, type 1 IFN) to Salmonella and LPS challenge while paradoxically reducing IFN-gamma production during bacterial infection. Increased STAT1 phosphorylation correlated with impaired STAT4 phosphorylation, resulting in overwhelming IL-6 secretion but reduced IFN-gamma production during infection. The reduced IFN-gamma levels, along with the increased inflammation, rationalize the S. Typhimurium susceptibility in terms of increased bacterial load in target organs and cytokine-induced septic shock and death.

Urotensin II receptor knockout mice on an ApoE knockout background fed a high-fat diet exhibit an enhanced hyperlipidemic and atherosclerotic phenotype.

RATIONALE: Expression of the vasoactive peptide Urotensin II (UII) is elevated in a number of cardiovascular diseases. OBJECTIVE: Here, we sought to determine the effect of UII receptor (UT) gene deletion in a mouse model of atherosclerosis. METHODS AND RESULTS: UT knockout (KO) mice were crossed with ApoE KO mice to generate UT/ApoE double knockout (DKO) mice. Mice were placed on a high-fat Western-type diet for 12 weeks. We evaluated the degree of atherosclerosis and hepatic steatosis by histology. In addition, serum glucose, insulin, and lipids were determined. DKO mice exhibited significantly increased atherosclerosis compared to ApoE KO mice (P<0.05). This was associated with a significant increase in serum insulin and lipids (P<0.001) but a decrease in hepatic steatosis (P<0.001). UT gene deletion led to a significant increase in systolic pressure and pulse pressure. RT-PCR and immunoblot analyses showed significant reductions in hepatic scavenger receptors, nuclear receptors, and acyl-CoA:cholesterol acyltransferase (ACAT1) expression in DKO mice. UII induced a significant increase in intracellular cholesteryl ester formation in primary mouse hepatocytes, which was blocked by the MEK inhibitor, PD98059. Hepatocytes of UTKO mice showed a significant reduction in lipoprotein uptake compared to wild-type mice. CONCLUSIONS: We propose that UT gene deletion in an ApoE-deficient background promotes downregulation of ACAT1, which in turn attenuates hepatic lipoprotein receptor-mediated uptake and lipid transporter expression. As the liver is the main organ for uptake of lipoprotein-derived lipids, DKO leads to an increase in hyperlipidemia, with a concomitant decrease in hepatic steatosis, and consequently increased atherosclerotic lesion formation. Furthermore, the hypertension associated with UT gene deletion is likely to contribute to the increased atherosclerotic burden.

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.

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.