Toll-Like Receptor 4 Deficiency Increases Disease and Mortality after Mouse Hepatitis Virus Type 1 Infection of Susceptible C3H Mice

Severe acute respiratory syndrome (SARS) is characterized by substantial acute pulmonary inflammation with a high mortality rate. Despite the identification of SARS coronavirus (SARS-CoV) as the etiologic agent of SARS, a thorough understanding of the underlying disease pathogenesis has been hampered by the lack of a suitable animal model that recapitulates the human disease. Intranasal (i.n.) infection of A/J mice with the CoV mouse hepatitis virus strain 1 (MHV-1) induces an acute respiratory disease with a high lethality rate that shares several pathological similarities with SARS-CoV infection in humans. In this study, we examined virus replication and the character of pulmonary inflammation induced by MHV-1 infection in susceptible (A/J, C3H/HeJ, and BALB/c) and resistant (C57BL/6) strains of mice. Virus replication and distribution did not correlate with the relative susceptibilities of A/J, BALB/c, C3H/HeJ, and C57BL/6 mice. In order to further define the role of the host genetic background in influencing susceptibility to MHV-1-induced disease, we examined 14 different inbred mouse strains. BALB.B and BALB/c mice exhibited MHV-1-induced weight loss, whereas all other strains of H-2^b and H-2^d mice did not show any signs of disease following MHV-1 infection. H-2^k mice demonstrated moderate susceptibility, with C3H/HeJ mice exhibiting the most severe disease. C3H/HeJ mice harbor a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts TLR4 signaling. C3H/HeJ mice exhibit enhanced morbidity and mortality following i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.Severe acute respiratory syndrome (SARS) is a disease that was initially observed in 2002 and led to approximately 8,000 affected individuals in multiple countries with over 700 deaths (1, 24, 47, 48). The causative agent of SARS was subsequently identified as a novel coronavirus (CoV) termed SARS-CoV (8, 17, 22, 27, 32, 37). Although SARS-CoV infections following the initial outbreak in 2002 and 2003 have been limited primarily to laboratory personnel, the identification of an animal reservoir for the virus raises concern about the potential for future outbreaks (25).The pathogenesis of SARS has been difficult to study, in part because no animal model is able to fully recapitulate the morbidity and mortality observed in infected humans (35). Infection of a number of inbred mouse strains, including BALB/c, C57BL/6, and 129S, with primary human isolates of SARS-CoV results in the replication of the virus within the lung tissue without the subsequent development of readily apparent clinical disease (11, 16, 41). Infection of aged BALB/c mice results in clinically apparent disease that more closely mimics some aspects of SARS in humans (36). However, immune responses in aged mice are known to be altered (5, 15), and thus, the mechanisms that control the induction of disease may differ between adult and aged mice. Recent work has demonstrated that serial passage of SARS-CoV in mice results in a mouse adaptation that leads to more profound replication of the virus in the lung (28, 34). However, the time to death from this mouse-adapted SARS-CoV is 3 to 5 days, which is much more rapid than the time to mortality observed in fatal cases of SARS in humans.Phylogenetic analysis has revealed that SARS-CoV is most closely related to group 2 CoVs, which include the mouse hepatitis virus (MHV) family (39). Thus, information gathered by infection of mice with closely related members of the group 2 CoVs may further contribute to our understanding of SARS-CoV pathogenesis in humans. While many strains of MHV induce primarily hepatic and central nervous system diseases (6, 7, 12, 18, 21, 23, 40), a recent study demonstrated that intranasal (i.n.) infection of A/J mice with MHV type 1 (MHV-1) induces pulmonary injury that shares several pathological characteristics with SARS-CoV infection of humans (2, 3, 9, 29, 43).In the current study, we examined the relationship between MHV-1 replication in the lungs and the severity of disease in four inbred strains of mice: A/J, BALB/c, C57BL/6, and C3H/HeJ. Our results demonstrate that MHV-1 replicates to similar levels in the lung in each of these inbred strains of mice regardless of their relative levels of susceptibility, as measured by weight loss and clinical illness. Both A/J and C3H/HeJ mice exhibited enhanced weight loss and clinical illness following i.n. MHV-1 infection compared to BALB/c and C57BL/6 mice. Analysis of many different inbred mouse strains confirmed A/J and C3H/HeJ mice as the most susceptible to i.n. MHV-1 infection. Interestingly, C3H/HeJ mice harboring a natural mutation in the gene that encodes Toll-like receptor 4 (TLR4) that disrupts its normal function exhibited greatly increased morbidity and mortality after i.n. MHV-1 infection compared to wild-type C3H/HeN mice. Our results indicate that TLR4 plays an important role in respiratory CoV pathogenesis.     

Evaluation of Borrelia burgdorferi BbHtrA Protease as a Vaccine Candidate for Lyme Borreliosis in Mice

Borrelia burgdorferi synthesizes an HtrA protease (BbHtrA) which is a surface-exposed, conserved protein within Lyme disease spirochetes with activity toward CheX and BmpD of Borrelia spp, as well as aggrecan, fibronectin and proteoglycans found in skin, joints and neural tissues of vertebrates. An antibody response against BbHtrA is observed in Lyme disease patients and in experimentally infected laboratory mice and rabbits. Given the surface location of BbHtrA on B. burgdorferi and its ability to elicit an antibody response in infected hosts, we explored recombinant BbHtrA as a potential vaccine candidate in a mouse model of tick-transmitted Lyme disease. We immunized mice with two forms of BbHtrA: the proteolytically active native form and BbHtrA ablated of activity by a serine to alanine mutation at amino acid 226 (BbHtrA^S226A). Although inoculation with either BbHtrA or BbHtrA^S226A produced high-titer antibody responses in C3H/HeJ mice, neither antigen was successful in protecting mice from B. burgdorferi challenge. These results indicate that the search for novel vaccine candidates against Lyme borreliosis remains a challenge.     

Impaired host defense to infection and Toll-like receptor 2-independent killing of Borrelia burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice

To investigate the role of Toll-like receptor 2 (TLR2)-mediated signaling in host innate defense and development of Lyme disease, the pathogenicity of Borrelia burgdorferi sensu stricto clinical isolates representing two distinct genotypes (RST1 and RST3A) was assessed in TLR2(-/-) C3H/HeJ mice. All TLR2(-/-) mice infected with a B. burgdorferi RST1 isolate developed severe arthritis. The numbers of spirochetes in heart, joint and ear biopsy specimens were significantly higher in TLR2(-/-) mice than in wild-type mice similarly infected as determined by real-time quantitative polymerase chain reaction. Interestingly, despite the higher spirochete levels in heart tissues, milder carditis was observed in TLR2(-/-) than in wild-type mice infected with this RST1 isolate (P=0.02). By contrast, no positive cultures were obtained from any of the blood and tissue specimens from TLR2(-/-) mice inoculated with two RST3A clinical isolates. The data suggest that there is impaired host innate defense against infection and TLR2-independent killing of B. burgdorferi clinical isolates in TLR2-deficient C3H/HeJ mice.     

Development of Autoimmune Hair Loss Disease Alopecia Areata Is Associated with Cardiac Dysfunction in C3H/HeJ Mice

Alopecia areata (AA) is a chronic autoimmune hair loss disease that affects several million men, women and children worldwide. Previous studies have suggested a link between autoimmunity, stress hormones, and increased cardiovascular disease risk. In the current study, histology, immunohistology, quantitative PCR (qPCR) and ELISAs were used to assess heart health in the C3H/HeJ mouse model for AA and heart tissue response to adrenocorticotropic hormone (ACTH) exposure. Mice with AA exhibited both atrial and ventricular hypertrophy, and increased collagen deposition compared to normal-haired littermates. QPCR revealed significant increases in Il18 (4.6-fold), IL18 receptor-1 (Il18r1; 2.8-fold) and IL18 binding protein (Il18bp; 5.2-fold) in AA hearts. Time course studies revealed a trend towards decreased Il18 in acute AA compared to controls while Il18r1, Il18bp and Casp1 showed similar trends to those of chronic AA affected mice. Immunohistochemistry showed localization of IL18 in chronic AA mouse atria. ELISA indicated cardiac troponin-I (cTnI) was elevated in the serum and significantly increased in AA heart tissue. Cultures of heart atria revealed differential gene expression between AA and control mice in response to ACTH. ACTH treatment induced significant increase in cTnI release into the culture medium in a dose-dependent manner for both AA and control mice. In conclusion, murine AA is associated with structural, biochemical, and gene expression changes consistent with cardiac hypertrophy in response to ACTH exposure.     

Induction by Fructose Force-Feeding of Histone H3 and H4 Acetylation at Their Lysine Residues around the Slc2a5 Gene and Its Expression in Mice

It has been reported that fructose force-feeding rapidly induced jejunal Slc2a5 gene expression in rodents. We demonstrate in this study that acetylation at lysine (K) 9 of histone H3 and acetylation at K5 and K16 of histone H4 were more enhanced in the promoter/enhancer to transcribed regions of the Slc2a5 gene in fructose force-fed mice than in glucose force-fed mice. However, fructose force-feeding did not induce acetylation at K14 of histone H3, or at K8 and K12 of histone H4 around the Slc2a5 gene. These results suggest that fructose force-feeding induced selective histone acetylation, particularly of H3 and H4, around the jejunal Slc2a5 gene in mice.     

H9724, a Monoclonal Antibody to Borrelia burgdorferi's Flagellin, Binds to Heat Shock Protein 60 (HSP60) Within Live Neuroblastoma Cells: A Potential Role for HSP60 in Peptide Hormone Signaling and in an Autoimmune Pathogenesis of the Neuropathy of Lyme Disease

Although Borrelia burgdorferi, the causative agent of Lyme disease, is found at the site of many disease manifestations, local infection may not explain all its features. B. burgdorferi's flagellin cross-reacts with a component of human peripheral nerve axon, previously identified as heat shock protein 60 (HSP60). The cross-reacting epitopes are bound by a monoclonal antibody to B. burgdorferi's flagellin, H9724. Addition of H9724 to neuroblastoma cell cultures blocks in vitro spontaneous and peptide growth-factor–stimulated neuritogenesis. Withdrawal of H9724 allows return to normal growth and differentiation. Using electron microscopy, immunoprecipitation and immunoblotting, and FACS analysis we sought to identify the site of binding of H9724, with the starting hypotheses that the binding was intracellular and not identical to the binding site of II-13, a monoclonal anti-HSP60 antibody. The current studies show that H9724 binds to an intracellular target in cultured cells with negligible, if any, surface binding. We previously showed that sera from patients with neurological manifestations of Lyme disease bound to human axons in a pattern identical to H9724's binding; these same sera also bind to an intracellular neuroblastoma cell target. II-13 binds to a different HSP60 epitope than H9724; II-13 does not modify cellular function in vitro. As predicted, II-13 bound to mitochondria, in a pattern of cellular binding very different from H9724, which bound in a scattered cytoplasmic, nonorganelle-related pattern. H9724's effect is the first evidence that HSP60 may play a role in peptide-hormone–receptor function and demonstrates the modulatory potential of a monoclonal antibody on living cells.     

Targeted Disruption of the Pemphigus Vulgaris Antigen (Desmoglein 3) Gene in Mice Causes Loss of Keratinocyte Cell Adhesion with a Phenotype Similar to Pemphigus Vulgaris

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell–cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 −/− mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8–10 d weighed less than DSG3 +/− or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and “tombstoning” of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 −/− mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.     

Characterization of a Spontaneous Novel Mutation in the NPC2 Gene in a Cat Affected by Niemann Pick Type C Disease

Niemann-Pick C disease (NPC) is an autosomal recessive lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids within the lysosomes due to mutation in NPC1 or NPC2 genes. A feline model of NPC carrying a mutation in NPC1 gene has been previously described. We have identified two kittens affected by NPC disease due to a mutation in NPC2 gene. They manifested with tremors at the age of 3 months, which progressed to dystonia and severe ataxia. At 6 months of age cat 2 was unable to stand without assistance and had bilaterally reduced menace response. It died at the age of 10 months. Post-mortem histological analysis of the brain showed the presence of neurons with cytoplasmic swelling and vacuoles, gliosis of the substantia nigra and degeneration of the white matter. Spheroids with accumulation of ubiquitinated aggregates were prominent in the cerebellar cortex. Purkinje cells were markedly reduced in number and they showed prominent intracytoplasmic storage. Scattered perivascular aggregates of lymphocytes and microglial cells proliferation were present in the thalamus and midbrain. Proliferation of Bergmann glia was also observed. In the liver, hepatocytes were swollen because of accumulation of small vacuoles and foamy Kupffer cells were also detected. Foamy macrophages were observed within the pulmonary interstitium and alveoli as well. At 9 months cat 1 was unable to walk, developed seizures and it was euthanized at 21 months. Filipin staining of cultured fibroblasts showed massive storage of unesterified cholesterol. Molecular analysis of NPC1 and NPC2 genes showed the presence of a homozygous intronic mutation (c.82+5G>A) in the NPC2 gene. The subsequent analysis of the mRNA showed that the mutation causes the retention of 105 bp in the mature mRNA, which leads to the in frame insertion of 35 amino acids between residues 28 and 29 of NPC2 protein (p.G28_S29ins35).     

Negative Regulation of Pulmonary Th17 Responses by C3a Anaphylatoxin during Allergic Inflammation in Mice

Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4⁺ T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4⁺ T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3⁺ regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3⁺ regulatory T cell population in the periphery.     

Macrophage-mediated mitogenic suppression induced in mice of the C3H lineage by a vaccine strain of Salmonella typhimurium

Salmonella typhimurium, strain SL3235, an avirulent organism, has been used as a live vaccine in mice of the C3H lineage and has been found to confer high levels of protection. In the present study, it was found that intraperitoneal injection of approximately 5 X 10(5) live SL3235 induced potent suppression of spleen cell mitogenic responses to a panel of B- and T-cell mitogens in the Salmonella-hypersusceptible C3H/HeJ and C3HeB/FeJ, and the inherently resistant C3H/HeNCrlBR mice. Maximal suppression (greater than 99%) was seen at 1 week, and was still significant but waning (50%) at 3 weeks postimmunization. In contrast, cells of mice receiving acetone-killed cells were not suppressed. Removal of macrophages, but not T or B cells, restored responsiveness, indicating that suppression was macrophage mediated. Prostaglandins were not the major mediator of suppression, as in vitro administration of indomethacin failed to abrogate suppression. As mitogenic suppression occurred in mice with high levels of Salmonella immunity, the suppression is interpreted as a marker of a powerful immunomodulatory process induced by live cells, rather than as an indication of poor immune status of the host.     

Progression of Alport Kidney Disease in Col4a3 Knock Out Mice Is Independent of Sex or Macrophage Depletion by Clodronate Treatment

Alport syndrome is a genetic disease of collagen IV (α3, 4, 5) resulting in renal failure. This study was designed to investigate sex-phenotype correlations and evaluate the contribution of macrophage infiltration to disease progression using Col4a3 knock out (Col4a3KO) mice, an established genetic model of autosomal recessive Alport syndrome. No sex differences in the evolution of body mass loss, renal pathology, biomarkers of tubular damage KIM-1 and NGAL, or deterioration of kidney function were observed during the life span of Col4a3KO mice. These findings confirm that, similar to human autosomal recessive Alport syndrome, female and male Col4a3KO mice develop renal failure at the same age and with similar severity. The specific contribution of macrophage infiltration to Alport disease, one of the prominent features of the disease in human and Col4a3KO mice, remains unknown. This study shows that depletion of kidney macrophages in Col4a3KO male mice by administration of clodronate liposomes, prior to clinical onset of disease and throughout the study period, does not protect the mice from renal failure and interstitial fibrosis, nor delay disease progression. These results suggest that therapy targeting macrophage recruitment to kidney is unlikely to be effective as treatment of Alport syndrome.     

Absence of a dose-rate effect in the transformation of C3H 10T1/2 cells by alpha-particles

The findings of Hill et al. (1984) on the greatly enhanced transformation frequencies at very low dose rates of fission neutrons induced us to perform an analogous study with alpha-particles at comparable dose rates. Transformation frequencies were determined with gamma-rays at high dose rate (0.5 Gy/min), and with alpha-particles at high (0.2 Gy/min) and at low dose rates (0.83-2.5 mGy/min) in the C3H 10T1/2 cell system. alpha-particles were substantially more effective than gamma-rays, both for cell inactivation and for neoplastic transformation at high and low dose rates. The relative biological effectiveness (RBE) for cell inactivation and for neoplastic transformation was of similar magnitude, and ranged from about 3 at an alpha-particle dose of 2 Gy to values of the order of 10 at 0.25 Gy. In contrast to the experiments of Hill et al. (1984) with fission neutrons, no increased transformation frequencies were observed when the alpha-particle dose was protracted over several hours.     

Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.     

Interconversion between Parallel and Antiparallel Conformations of a 4H RNA junction in Domain 3 of Foot-and-Mouth Disease Virus IRES Captured by Dynamics Simulations

RNA junctions are common secondary structural elements present in a wide range of RNA species. They play crucial roles in directing the overall folding of RNA molecules as well as in a variety of biological functions. In particular, there has been great interest in the dynamics of RNA junctions, including conformational pathways of fully base-paired 4-way (4H) RNA junctions. In such constructs, all nucleotides participate in one of the four double-stranded stem regions, with no connecting loops. Dynamical aspects of these 4H RNAs are interesting because frequent interchanges between parallel and antiparallel conformations are thought to occur without binding of other factors. Gel electrophoresis and single-molecule fluorescence resonance energy transfer experiments have suggested two possible pathways: one involves a helical rearrangement via disruption of coaxial stacking, and the other occurs by a rotation between the helical axes of coaxially stacked conformers. Employing molecular dynamics simulations, we explore this conformational variability in a 4H junction derived from domain 3 of the foot-and-mouth disease virus internal ribosome entry site (IRES); this junction contains highly conserved motifs for RNA-RNA and RNA-protein interactions, important for IRES activity. Our simulations capture transitions of the 4H junction between parallel and antiparallel conformations. The interconversion is virtually barrier-free and occurs via a rotation between the axes of coaxially stacked helices with a transient perpendicular intermediate. We characterize this transition, with various interhelical orientations, by pseudodihedral angle and interhelical distance measures. The high flexibility of the junction, as also demonstrated experimentally, is suitable for IRES activity. Because foot-and-mouth disease virus IRES structure depends on long-range interactions involving domain 3, the perpendicular intermediate, which maintains coaxial stacking of helices and thereby consensus primary and secondary structure information, may be beneficial for guiding the overall organization of the RNA system in domain 3.     

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined from data for survival and neoplastic transformation were comparable.