Nobuko Wakamatsu

Both TRIF- and MyD88-Dependent Signaling Contribute to Host Defense against Pulmonary Klebsiella Infection

Klebsiella pneumoniae causes extensive lung damage. TLR signaling involves adaptors TRIF and MyD88. However, the relative contribution of TRIF and MyD88 signaling in host defense against pulmonary K. pneumoniae infection has not been elucidated. Therefore, we investigated the role of TRIF and MyD88 in K. pneumoniae pneumonia. TRIF−/− mice infected with K. pneumoniae showed impaired survival and reduced bacterial clearance, neutrophil influx, histopathologic evidence of inflammation, and TNF-α, IL-6, KC, MIP-2, but not LIX, expression in the lungs. In addition, K. pneumoniae-induced late NF-κB activation and phosphorylation of MAPKs was attenuated in the lungs of TRIF−/− mice. However, MyD88−/− mice infected with K. pneumoniae showed a much more remarkable phenotype, including impaired survival and reduced bacterial clearance, histopathology, and TNF-α, IL-6, KC, MIP-2, and LIX expression with almost no neutrophil influx in the lungs. In MyD88−/− mice, K. pneumoniae-induced early NF-κB and MAPK activation in the lungs was also reduced. Furthermore, the role of MyD88 is dominant over TRIF because TRIF/MyD88 double knockout mice displayed a more pronounced phenotype than TRIF−/− mice. Moreover, human alveolar macrophages pretreated with MyD88 blocking peptide showed attenuated TNF-α, IL-6, and IL-8 expression. Also, C57BL/6 mice pretreated with MyD88 blocking peptide exhibited attenuation in K. pneumoniae-induced neutrophil influx and enhanced bacterial burden in the lungs and dissemination. Overall, this investigation provides new insights into the TRIF and MyD88 signaling triggered by pulmonary K. pneumoniae infection in the lungs and demonstrate the therapeutic potential of MyD88 in reducing excessive neutrophil influx in human disease during Gram-negative bacterial pneumonia.

Experimental Pathogenesis for Chickens, Turkeys, and Pigeons of Exotic Newcastle Disease Virus from an Outbreak in California during 2002-2003:

Exotic Newcastle disease virus (NDV) isolated from chickens during the 2002-2003 California outbreak (CA exotic Newcastle disease [END] virus) was inoculated into 4-week-old specificpathogen-free (SPF) White Leghorn chickens, 3-week-old SPF Beltsville White turkeys, 6-week-old commercial Broad Breasted White turkeys, and 10- to 20-week-old racing pigeons, and the clinicopathologic features of disease were compared. Birds were monitored clinically and euthanized sequentially with collection of tissues. Tissues were examined by histopathology, by immunohistochemistry to detect viral nucleoprotein, and by in situ hybridization to detect viral mRNA. Clinically, infected chickens and SPF turkeys showed severe depression, and all died or were euthanized because of severe clinical signs by day 5 postinoculation. In these birds, histologic lesions were widespread and virus was detected in multiple organs. All infected commercial turkeys showed mild depression, and incoordination was observed in some birds. Histologic lesions were mild, and viral distribution was limited. In pigeons, only 1 bird showed overt clinical disease, and histologic lesions and viral distribution were present in limited organs. Consequently, susceptibility to highly virulent NDV was shown to vary among chickens, SPF turkeys, commercial turkeys, and pigeons. Additionally, we have evidence of CA END virus subclinical infections that suggest pigeons could be subclinical carriers of other virulent NDV.

NLRC4 Inflammasome-Mediated Production of IL-1β Modulates Mucosal Immunity in the Lung against Gram-Negative Bacterial Infection

Bacterial flagellin is critical to mediate NLRC4 inflammasome-dependent caspase-1 activation. However, Shigella flexneri, a nonflagellated bacterium, and a flagellin (fliC) knockout strain of Pseudomonas aeruginosa are known to activate NLRC4 in bone marrow-derived macrophages. Furthermore, the flagellin-deficient fliC strain of P. aeruginosa was used in a mouse model of peritonitis to show the requirement of NLRC4. In a model of pulmonary P. aeruginosa infection, flagellin was shown to be essential for the induction of NLRC4-dependent caspase-1 activation. Moreover, in all P. aeruginosa studies, IL-1β production was attenuated in NLRC4−/− mice; however, the role of IL-1β in NLRC4-mediated innate immunity in the lungs against a nonflagellated bacterium was not explored. In this article, we report that NLRC4 is important for host survival and bacterial clearance, as well as neutrophil-mediated inflammation in the lungs following Klebsiella pneumoniae infection. NLRC4 is essential for K. pneumoniae-induced production of IL-1β, IL-17A, and neutrophil chemoattractants (keratinocyte cell-derived chemokines, MIP-2, and LPS-induced CXC chemokines) in the lungs. NLRC4 signaling in hematopoietic cells contributes to K. pneumoniae-induced lung inflammation. Furthermore, exogenous IL-1β, but not IL-18 or IL-17A, partially rescued survival, neutrophil accumulation, and cytokine/chemokine expression in the lungs of NLRC4−/− mice following infectious challenge. Furthermore, IL-1R1−/− mice displayed a decrease in neutrophilic inflammation in the lungs postinfection. Taken together, these findings provide novel insights into the role of NLRC4 in host defense against K. pneumoniae infection.

Recombinant vesicular stomatitis virus-based west Nile vaccine elicits strong humoral and cellular immune responses and protects mice against lethal challenge with the virulent west Nile virus strain LSU-AR01

Abstract Vesicular stomatitis virus (VSV) has been extensively utilized as a viral vector system for the induction of protective immune responses against a variety of pathogens. We constructed recombinant VSVs specifying either the Indiana or Chandipura virus G glycoprotein and expressing the West Nile virus (WNV) envelope (E) glycoprotein. Mice were intranasally vaccinated using a prime (Indiana)-boost (Chandipura) immunization approach and challenged with the virulent WNV-LSU-AR01. Ninety-percent (9 of 10) of the vaccinated mice survived as compared to 10% of the mock-vaccinated mice after WNV lethal challenge. Histopathological examination of brain tissues revealed neuronal necrosis in mock-vaccinated mice but not in vaccinated mice, and vaccinated, but not mock-vaccinated mice developed a strong neutralizing antibody response against WNV. Extensive immunological analysis using polychromatic flow cytometry staining revealed that vaccinated, but not mock-vaccinated mice developed robust cellular immune responses as evidenced by up-regulation of CD4+ CD154+ IFNγ+ T cells in vaccinated, but not mock-vaccinated mice. Similarly, vaccinated mice developed robust E-glycoprotein-specific CD8+ T cell immune responses as evidenced by the presence of a high percentage of CD8+ CD62Llow IFNγ+ cells. In addition, a sizeable population of CD8+ CD69+ cells was detected indicating E-specific activation of mature T cells and CD4+ CD25+ CD127low T regulatory (T reg) cells were down-regulated. These results suggest that VSV-vectored vaccines administered intranasally can efficiently induce protective humoral and cellular immune responses against WNV infections.

Prevalence of and risk factors associated with atherosclerosis in psittacine birds.

OBJECTIVE To estimate the prevalence of clinically relevant atherosclerotic lesions in birds and identify epidemiological variables and illness types associated with development of atherosclerosis. DESIGN Retrospective case-control study. SAMPLE Records of 7683 psittacine birds, including 525 with advanced atherosclerosis. PROCEDURES 5 pathology centers provided databases and access to histopathology slides. Age and sex were collected for all birds of the Amazona, Ara, Cacatua, Nymphicus, and Psittacus genera. Databases were searched for atherosclerosis cases, and slides were reviewed for the presence of type IV through VI atherosclerotic lesions. Results were used to build several multiple logistic models to define the association between advanced atherosclerosis and age, sex, genus, illness type, and specific lesions. Prevalence was reported as a function of age, sex, and genus. RESULTS In the first model including 7683 birds, age, female sex, and the genera Psittacus, Amazona, and Nymphicus were significantly associated with clinically relevant atherosclerosis detected via necropsy. Subsequent models of 1,050 cases revealed further associations with reproductive disease, hepatic disease, and myocardial fibrosis, controlling for age, sex, and genus. CONCLUSIONS AND CLINICAL RELEVANCE Age, female sex, and 3 genera appeared to be positively associated with the presence of advanced atherosclerotic lesions in psittacine birds. This information may be useful in clinical assessment of the cardiovascular system and patient management. Reproductive diseases were the only potentially modifiable risk factor identified and could be a target for prevention in captive psittacine birds.

Characterization and classification of psittacine atherosclerotic lesions by histopathology, digital image analysis, transmission and scanning electron microscopy

Atherosclerosis is a degenerative and inflammatory vascular disease characterized in mammals and birds by the accumulation of inflammatory cells, lipids, calcium, and formation of large fibrofatty lesions within the intima of arteries resulting in the disorganization of the arterial wall and stenosis of the lumen. Despite the high incidence of atherosclerosis in parrots and the high number of case reports, there are few pathologic investigations and the ultrastructural study of the lesions has not been documented. Sixty-three major arteries were collected from 24 psittacine birds of 11 species during routine post-mortem examinations. Samples from the major arteries were fixed in 2% paraformaldehyde and 1.25% glutaraldehyde, and processed for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Additional samples were fixed in 10% formalin and embedded in paraffin for histological examination. Additional histochemical stains for calcium, elastic fibres, and lipid were performed. Toluidine blue-stained 0.5 µm-thick resin sections were also obtained. Digital image analysis was performed to provide objective quantitative information on the different lesions. The histopathology and ultrastructure of psittacine atherosclerosis were found to be similar to other avian and mammalian species. Seven lesion types could be described, which were similar to the human classification system. Digital image analysis, TEM, and SEM helped to further describe the lesions and refine the classification system. TEM findings were similar to other avian and mammalian species with the notable presence of macrophage-derived and smooth muscle cell-derived foam cells and extracellular lipid. SEM revealed various stages of endothelial surface defects and, occasionally, adherent blood cells.

Experimental Diet-Induced Atherosclerosis in Quaker Parrots (Myiopsitta monachus)

Spontaneous atherosclerosis is common in psittaciformes, and clinical signs associated with flow-limiting stenosis are encountered in pet birds. Nevertheless, a psittacine model of atherosclerosis has not been developed for research investigations. Sixteen captive-bred Quaker parrots (Myiopsitta monachus) were used in this study. While 4 control birds were fed a maintenance diet, 12 other birds were fed an atherogenic diet composed of 1% cholesterol controlling for a calorie-to-protein ratio for periods ranging from 2 to 8 months. The birds were euthanized at the end of their respective food trial period. Histopathology, transmission electron microscopy, and cholesterol measurement were performed on the ascending aorta and brachiocephalic and pulmonary arteries. Plasma lipoproteins, cholesterol, and triglycerides were also measured on a monthly basis. Significant atherosclerotic lesions were induced within 2 months and advanced atherosclerotic lesions within 4 to 6 months. The advanced lesions were histologically similar to naturally occurring lesions identified in the same parrot species with a lipid core and a fibrous cap. Ultrastructurally, there were extracellular lipid, foam cell, and endothelial changes. Arterial cholesterol content increased linearly over time. Plasma cholesterol and low-density lipoprotein (LDL) significantly increased over time by an average of 5- and 15-fold, respectively, with a shift from high-density lipoprotein to LDL as the main plasma lipoprotein. Quaker parrots also exhibited high plasma cholesteryl ester transfer protein activity that increased, although not significantly, over time. This experiment demonstrates that in Quaker parrots fed 1% cholesterol, advanced atherosclerosis can be induced relatively quickly, and lesions resemble those found in other avian models and humans.

Impact and Regulation of Lambda Interferon Response in Human Metapneumovirus Infection

ABSTRACT Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-λ1), IFN-λ2, IFN-λ3, and IFN-λ4. The induction of IFN-λ expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN-λ reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN-λ response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-λs) play a critical protective role in hMPV infection. IMPORTANCE Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-λ, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN-λ exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

Intermittent Claudication-like Syndrome Secondary to Atherosclerosis in a Yellow-naped Amazon Parrot (Amazona ochrocephala auropalliata)

Abstract A 25-year-old yellow-naped Amazon parrot (Amazona ochrocephala auropalliata) was presented for nasal discharge and sneezing. Physical examination revealed poor feather quality, a mild serous nasal discharge, and a mass on the dorsal surface of the oral cavity. Cytologic examination of a mass aspirate as well as results of a choanal culture revealed squamous metaplasia of the salivary glands and bacterial rhinitis, respectively. Following resolution of the presenting conditions, the patient was presented for hind limb weakness and ataxia. The clinical signs were transient and generally resolved with rest but could be reproduced after stressful episodes, such as restraint for procedures or treatment. Test results from a complete blood count, biochemistry profile, whole-body radiographs, needle electromyography of the leg muscles, and an edrophonium challenge test were within reference limits. Based on the clinical signs and results of the diagnostic workup, the presumptive diagnosis was intermittent claudication, a condition caused by peripheral vascular disease and defined as intermittent weakness and pain in the legs induced by exercise and relieved by rest. Shortly after initiation of treatment with isoxsuprine, the bird died. Postmortem examination and histopathology revealed severe atherosclerotic lesions throughout the vascular system with stenotic lesions present in the abdominal aorta and femoral arteries. Electron microscopic examination of the great arteries was also performed and helped to further characterize the nature of the lesions. This case is the first report, to our knowledge, of an intermittent claudication-like syndrome associated with peripheral atherosclerosis in a psittacine bird. In addition, the distribution and some of the macroscopic and histopathologic features of the lesions differ from previous descriptions of atherosclerosis in psittacine birds.

Interleukin-12p40 Modulates Human Metapneumovirus-Induced Pulmonary Disease in an Acute Mouse Model of Infection

The mechanisms that regulate the host immune response induced by human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, are largely unknown. Cytokines play an important role in modulating inflammatory responses during viral infections. IL-12p40, a known important mediator in limiting lung inflammation, is induced by hMPV and its production is sustained after the resolution phase of infection suggesting that this cytokine plays a role in the immune response against hMPV. In this work, we demonstrated that in mice deficient in IL-12p40, hMPV infection induced an exacerbated pulmonary inflammatory response and mucus production, altered cytokine response, and decreased lung function. However, hMPV infection in these mice does not have an effect on viral replication. These results identify an important regulatory role of IL-12p40 in hMPV infection.