Susceptible Mouse Strains Research Articles

Inhibition of matrix metalloproteinases protects mice from ascending infection and chronic disease manifestations resulting from urogenital Chlamydia muridarum infection.

Matrix metalloproteinases (MMP) are a family of host-derived enzymes involved in the turnover of extracellular matrix molecules. We have previously reported enhanced expression of matrix metalloproteinases in Chlamydia muridarum urogenital tract infection of female mice. Kinetics and patterns of MMP expression as well as enhanced expression in susceptible strains of mice in the prior study implied a role for MMP in pathogenesis. To explore this further, we infected a susceptible strain of mice (C3H/HeN) with C. muridarum and treated two groups of mice with either one of two chemical inhibitors of MMP (MMPi; captopril and a chemically modified tetracycline) and reserved infected sham-treated mice as controls. Neither of the treatments affected shedding of viable chlamydiae from the lower urogenital tract, but the administration of either MMPi protected mice from the formation of hydrosalpinx-a surrogate marker of oviduct occlusion and infertility. Interestingly, the mechanism of protection for mice treated with chemically modified tetracycline 3, appeared to be related to prevention of ascending upper genital tract infection. These results imply that MMP are involved in pathogenesis of chlamydial infection in this model by mediating ascension of the infection into the upper genital tract.

Cytokine and Chemokine Transcription Profile duringMycoplasma pulmonisInfection in Susceptible and Resistant Strains of Mice: Macrophage Inflammatory Protein 1β (CCL4) and Monocyte Chemoattractant Protein 2 (CCL8) and Accumulation of CCR5+Th Cells

The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1beta (MIP-1beta; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1beta/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1beta- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.

Increased apoptosis during the early phase of experimental paracoccidioidomycosis as a phenotypic marker of resistance

Paracoccidiodomycosis (PCM) is a systemic mycosis that presents a wide spectrum of clinical manifestations caused by Paracoccidiodes brasiliensis. The experimental murine model has been used to approach the disease with susceptible and resistant mouse strains that reproduce most of the main human immunological features. We investigated whether the pattern of apoptosis of peritoneal cells from two polar strains of mice after infection with P. brasiliensis could be associated with the susceptibility or resistance to this pathogen. Apoptosis of A/J mouse cells (resistant), cultured in the presence or absence of LPS as stimuli, was observed as early as on the first day of infection. Cells from the infected susceptible strain BALB/c did not exhibit apoptosis in absence of LPS and persistently at a lesser degree than that observed in resistant mice. The apoptosis induced by the infection in resistant mice was not due to nitric oxide, since its blockage either in vitro or in vivo did not revert it. Analysis of additional strains of polar susceptibilities to PCM assured the dissociation of NO production and apoptosis. Interestingly, IL-6 and IL-10 were secreted in high amounts, by BALB/c cells and might be involved in shielding cells from apoptosis induced by P. brasiliensis. Furthermore, IFNγ −/− mice did not show apoptosis of peritoneal cells while the Wt controls presented levels similar to those of A/J strain that secreted high amounts of IFNγ and IL-1β. The expression of Fas was increased in both strains and in Wt mice, whereas FasL was decreased in the susceptible strain and not significantly modulated in TNFRI and IFNγ KO mice. These results suggest that apoptosis might be a mechanism of control of engagement of cells that could otherwise contribute to the susceptible phenotype observed in some strains of mice.

ChronicSalmonella entericaSerovar Typhimurium-Induced Colitis and Cholangitis in Streptomycin-PretreatedNramp1+/+Mice

Salmonella enterica subspecies 1 serovar Typhimurium is an enteric bacterial pathogen infecting a broad range of hosts. In susceptible Nramp1(-/-) (Slc11alpha1(-/-)) mice, serovar Typhimurium cannot efficiently colonize the intestine but causes a systemic typhoid-like infection. However, after pretreatment with streptomycin, these susceptible (C57BL/6 and BALB/c) mice develop acute serovar Typhimurium-induced colitis (M. Barthel et al., Infect. Immun. 71:2839-2858, 2003). It was not clear whether resistant Nramp1(+/+) (Slc11alpha1(+/+)) mouse strains would similarly develop colitis. Here we compared serovar Typhimurium infection in streptomycin-pretreated susceptible (C57BL/6) and resistant (DBA/2 and 129Sv/Ev) mouse strains: We found that acute colitis (days 1 and 3 postinfection) is strikingly similar in susceptible and resistant mice. In 129Sv/Ev mice we followed the serovar Typhimurium infection for as long as 6 weeks. After the initial phase of acute colitis, these animals developed chronic crypt-destructive colitis, including ulceration, crypt abscesses, pronounced mucosal and submucosal infiltrates, overshooting regeneration of the epithelium, and crypt branching. Moreover, we observed inflammation of the gall duct epithelium (cholangitis) in the 129Sv/Ev mice between days 14 and 43 of infection. Cholangitis was not attributable to side effects of the streptomycin treatment. Furthermore, chronic infection of 129Sv/Ev mice in a typhoid fever model did not lead to cholangitis. We propose that streptomycin-pretreated 129Sv/Ev mice provide a robust murine model for chronic enteric salmonellosis including complications such as cholangitis.

Early Manifestations of NNK-Induced Lung Cancer

A strong correlation exists between smoking and lung cancer; however, susceptibility to lung cancer among smokers is not uniform. Similarly, mice show differential susceptibility to the tobacco carcinogen nitrosamine 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which produces lung tumors in A/J but not in C3H mice. Host immunity may play a role in the susceptibility to cancer, and cigarette smoke/nicotine suppresses the immune system through activation of nicotinic acetylcholine receptors (nAChRs). Mammalian lungs express alpha7-nAChRs, and NNK is a high-affinity agonist for alpha7-nAChRs. To examine whether NNK differentially modulates lung immunity in susceptible and resistant mouse strains, A/J and C3H mice were treated with NNK and/or immunized with sheep red blood cells. Lung tissues and RNA of treated and untreated animals were analyzed by immunohistochemistry and RT-PCR for alpha7-nAChR and COX-2 expression. Spleen- and the lung-associated lymph node cells from control and immunized animals were assessed for immunologic responses, including anti-sheep red blood cell antibody plaque-forming cells, concanavalin A-induced T-cell proliferation, and the anti-CD3/CD28 antibody-induced rise in intracellular calcium. NNK strongly suppressed these responses in A/J but not in C3H mice. Similar NNK-induced immunologic changes were seen in another pair of carcinogen-sensitive (NGP) and relatively carcinogen-resistant (B10.A) mouse strains. Moreover, NNK stimulates a significantly higher expression of COX-2 and alpha7-nAChRs in A/J than in C3H lungs. These results suggest that the susceptibility to chemical carcinogenesis among various mouse strains might be influenced by their immunologic response to the carcinogen.

Anti-CCL2 treatment inhibits Theiler’s murine encephalomyelitis virus-induced demyelinating disease

Theiler's murine encephalomyelitis virus induces a demyelinating disease (TMEV-IDD) of the central nervous system (CNS) in susceptible mouse strains with accompanying histopathology characterized by mononuclear cell infiltrates. In susceptible strains of mice such as SJL, virus establishes a persistent infection in macrophages, induces a CNS infiltration by macrophages, T cells, and B cells, which results in chronic-progressive paralysis. In the present report the authors have investigated the functional role of CCL2 (monocyte chemotactic protein-1) in the induction and progression of demyelinating disease. Treatment of infected mice at day 0, 14, or 28 with anti-CCL2 resulted in a significant decrease in the clinical disease progression. Further analysis of anti-CCL2-treated mice revealed decreased CNS inflammation and mononuclear cell infiltration with an accompanying change in inflammatory cytokine responses. There was an overall decrease in the absolute numbers of CNS-infiltrating CD4+ T cells, macrophages, and B cells. Finally, anti-CCL2 treatment resulted in decreased viral load in the CNS. These data directly demonstrate a role for CCL2 in the pathogenesis of TMEV-IDD.

Genetically Determined Susceptibility to Tuberculosis in Mice Causally Involves Accelerated and Enhanced Recruitment of Granulocytes

Classical twin studies and recent linkage analyses of African populations have revealed a potential involvement of host genetic factors in susceptibility or resistance to Mycobacterium tuberculosis infection. In order to identify the candidate genes involved and test their causal implication, we capitalized on the mouse model of tuberculosis, since inbred mouse strains also differ substantially in their susceptibility to infection. Two susceptible and two resistant mouse strains were aerogenically infected with 1,000 CFU of M. tuberculosis, and the regulation of gene expression was examined by Affymetrix GeneChip U74A array with total lung RNA 2 and 4 weeks postinfection. Four weeks after infection, 96 genes, many of which are involved in inflammatory cell recruitment and activation, were regulated in common. One hundred seven genes were differentially regulated in susceptible mouse strains, whereas 43 genes were differentially expressed only in resistant mice. Data mining revealed a bias towards the expression of genes involved in granulocyte pathophysiology in susceptible mice, such as an upregulation of those for the neutrophil chemoattractant LIX (CXCL5), interleukin 17 receptor, phosphoinositide kinase 3 delta, or gamma interferon-inducible protein 10. Following M. tuberculosis challenge in both airways or peritoneum, granulocytes were recruited significantly faster and at higher numbers in susceptible than in resistant mice. When granulocytes were efficiently depleted by either of two regimens at the onset of infection, only susceptible mice survived aerosol challenge with M. tuberculosis significantly longer than control mice. We conclude that initially enhanced recruitment of granulocytes contributes to susceptibility to tuberculosis.

Stat1 Deficiency Exacerbates Carditis but Not Arthritis During Experimental Lyme Borreliosis

Activation of the transcription factor Stat1 by interferon-gamma (IFN-gamma) is an important step in the development of antimicrobial effector mechanisms against many bacterial pathogens. Susceptibility to murine Lyme arthritis has been correlated with the production of several proinflammatory cytokines, especially IFN-gamma. To determine the role of IFN-mediated effector mechanisms in the development of Lyme borreliosis, we infected Stat1-deficient mice on both resistant (DBA), and susceptible (C3H) genetic backgrounds. Arthritis in Stat1(/) mice was similar to that of wild-type controls in both mouse strains. Spirochete loads in tissues were also unchanged in Stat1(/) mice. C3H Stat1(/) mice exhibited increased inflammation in the heart, whereas carditis was unchanged in DBA Stat1(/) mice. These results demonstrate that inhibition of macrophage activation and responses to IFN-gamma-mediated signaling do not alter the arthritis resistance or susceptibility phenotype; however, they do affect the severity of carditis in susceptible mouse strains.

High resolution mapping of chromosomal regions controlling resistance to gastrointestinal nematode infections in an advanced intercross line of mice

Fine mapping of quantitative trait loci (QTL) associated with resistance to the gastrointestinal parasite Heligmosomoides polygyrus was achieved on F(6)/F(7) offspring (1076 mice) from resistant (SWR) and susceptible (CBA) mouse strains by selective genotyping (top and bottom 20% selected on total worm count in week 6). Fecal egg counts were recorded at weeks 2, 4, and 6, and the average was also analyzed. Blood packed cell volume in weeks 3 and 6 and five immunological traits (mucosal mast cell protease 1, granuloma score, IgG1 against adult worm, IgG1, and IgE to L4 antigen) were also recorded. On Chromosome 1 single-trait analyses identified a QTL with effects on eight traits located at about 24 cM on the F(2) mouse genome database (MGD) linkage map, with a 95% confidence interval (CI) of 20-32 cM established from a multitrait analysis. On Chromosome 17 a QTL with effects on nine traits was located at about 18 cM on the MGD map (CI 17.9-18.4 cM). Strong candidate genes for the QTL position on Chromosome 1 include genes known to be involved in regulating immune responses and on Chromosome 17 genes within the MHC, notably the Class II molecules and tumor necrosis factor.

Babesia microti Primarily Invades Mature Erythrocytes in Mice

Babesia microti is a tick-borne red blood cell parasite that causes babesiosis in people. Its most common vertebrate reservoir is the white-footed mouse. To determine whether B. microti invades reticulocytes, as does the canine pathogen B. gibsoni, we infected the susceptible inbred mouse strains C.B-17.scid and DBA/2 with a clinical isolate of B. microti. Staining of fixed permeabilized red blood cells with 4',6'-diamidino-2-phenylindole or YOYO-1, a sensitive nucleic acid stain, revealed parasite nuclei as large bright dots. Flow cytometric analysis indicated that parasite DNA is primarily found in mature erythrocytes that expressed Babesia antigens but not the transferrin receptor CD71. In contrast, CD71-positive reticulocytes rarely contained Babesia nuclei and failed to express Babesia antigens. Accordingly, the frequency of YOYO-1-positive, CD71-negative cells strongly correlated with parasitemia, defined as the frequency of infected red blood cells assessed on Giemsa-stained blood smears. Importantly, the absolute numbers generated by the two techniques were similar. Parasitemia was modest and transient in DBA/2 mice but intense and sustained in C.B-17.scid mice. In both strains, parasitemia preceded reticulocytosis, but reticulocytes remained refractory to B. microti. In immunocompetent C.B-17 mice, reticulocytosis developed early, despite a marginal and short-lived parasitemia. Likewise, an early reticulocytosis developed in resistant BALB/cBy and B10.D2 mice. These studies establish that B. microti has a tropism for mature erythrocytes. Although reticulocytes are rarely infected, the delayed reticulocytosis in susceptible strains may result from parasite or host activities to limit renewal of the mature erythrocyte pool, thereby preventing an overwhelming parasitemia.

TCRγδ Intraepithelial Lymphocytes Are Required for Self-Tolerance

Neonatal thymectomy (NTX) impairs T cell regulation and leads to organ-specific autoimmune disease in susceptible mouse strains. In the NOD mouse model of spontaneous type 1 diabetes, we observed that NTX dramatically accelerated autoimmune pancreatic beta cell destruction and diabetes. NTX had only a minor effect in NOD mice protected from diabetes by transgenic expression of the beta cell autoantigen proinsulin in APCs, inferring that accelerated diabetes after NTX is largely due to failure to regulate proinsulin-specific T cells. NTX markedly impaired the development of intraepithelial lymphocytes (IEL), the number of which was already reduced in euthymic NOD mice compared with control strains. IEL purified from euthymic NOD mice, specifically CD8alphaalpha TCRgammadelta IEL, when transferred into NTX-NOD mice, trafficked to the small intestinal epithelium and prevented diabetes. Transfer of prototypic CD4+CD25+ regulatory T cells also prevented diabetes in NTX-NOD mice; however, the induction of these cells by oral insulin in euthymic mice depended on the integrity of TCRgammadelta IEL. We conclude that TCRgammadelta IEL at the mucosal interface between self and nonself play a key role in maintaining peripheral tolerance both physiologically and during oral tolerance induction.

Fine mapping of Dyscalc1, the major genetic determinant of dystrophic cardiac calcification in mice

Calcification of severely dystrophic muscle is occasionally observed in targeted mouse models of muscular dystrophy and cardiomyopathy. Intracellular calcium deposition occurs in necrotic myocytes in the absence of plasma calcium and phosphate imbalances. In the heart, this recessive trait is referred to as dystrophic cardiac calcinosis (DCC). We identified previously Dyscalc1, a major genetic determinant of DCC, in a 15.2-Mbp region on proximal chromosome 7. We report now further steps toward the identification of the Dyscalc1 gene by reverse genetics. Transferring the Dyscalc1 locus from susceptible mouse strain C3H/He onto a resistant C57BL/6 strain background, we generated congenic inbred strains B6.C3-(D7Mit56-D7Mit230) and B6.C3-(D7Nds5-D7Mit230). Three days after myocardial freeze-thaw injury, both strains exhibited calcification of necrotic lesions, confirming the pathogenetic relevance of Dyscalc1. Analysis of two (129S1 x C57BL/6) x 129S1 backcrosses allowed mapping of Dyscalc1 more precisely to a region spanning 0.76 Mbp between genes Fgf21 (39.70 Mbp) and Myod1 (40.46 Mbp). This interval contains 31 known and putative genes in three large, ancestral haplotypes shared by susceptible strains C3H/He, 129S1, and DBA/2. Thus we were able to exclude previously proposed candidate genes Bax and Hrc. Instead, a potential candidate may be the gene encoding the ATP-binding cassette C6. Mutations in the orthologous human ABCC6 gene cause pseudoxanthoma elasticum, or Gronblad-Strandberg syndrome, an elastic tissue disorder with cardiovascular calcifications.

Nogo in multiple sclerosis: Growing roles of a growth inhibitor

In recent years, knowledge about the physiological functions of the Nogo-A protein has grown considerably, and this molecule has evolved from being one of the most important axonal regrowth inhibitors present in central nervous system (CNS) myelin, to several other potentially important roles in different areas such as nervous system development, epilepsy, vascular physiology, muscle pathology and CNS tumors. Therapeutically, targeting the Nogo-A protein by means of the immune response has been tried in an attempt to block neurite growth inhibition and promote regeneration in spinal cord injury models; the immune response to Nogo-A, however, has not been extensively studied. We propose to review recent evidence that Nogo-A may also play an important role in autoimmune demyelinating diseases such as experimental autoimmune encephalomyelitis and multiple sclerosis, including that Nogo-66 derived epitopes are encephalitogenic antigens in susceptible mouse strains, and that the immune response to Nogo-66 antigens includes both strong T cell and B cell activation, with epitope spreading of the antibody response to other myelin molecules. In CNS immunotherapy, careful targeting of neural self-antigens is a prerequisite in order to avoid unexpected deleterious effects, and increasing knowledge about the immune response to Nogo-A may provide a safe basis for the development of relevant therapeutic alternatives for several neurological conditions.

Ongoing Coxsackievirus Myocarditis Is Associated with Increased Formation and Activity of Myocardial Immunoproteasomes

A growing body of evidence indicates that viral infections of the heart contribute to ongoing myocarditis and dilated cardiomyopathy. Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the human disease and allow identification of susceptibility factors that modulate the course of viral myocarditis. Susceptible mouse strains develop chronic myocarditis on the basis of restricted viral replication, whereas resistant strains recover after successful virus elimination. In comparative whole-genome microarray analyses of infected hearts, several genes involved in the processing and presentation of viral epitopes were found to be uniformly up-regulated in acutely CVB3-infected susceptible mice compared with resistant animals. In particular, expression of the catalytic subunits LMP2, LMP7, and MECL-1, immunoproteasome proteins important in the generation of major histocom-patibility complex (MHC) class I-restricted peptides, was clearly enhanced in the susceptible host. Increased expression resulted in enhanced formation of immunoproteasomes and altered proteolytic activities of proteasomes in the heart. This was accompanied by a concerted up-regulation of the antigen-presenting machinery in susceptible mice. Thus, we propose that increased formation of immunoproteasomes in susceptible mice affects the generation of antigenic peptides and the subsequent T-cell-mediated immune responses.

Hyperbaric oxygen therapy reduces the size of Leishmania amazonensis-induced soft tissue lesions in mice

In this study we determined whether exposing mice to hyperbaric oxygen (HBO) would alter various disease parameters of a susceptible mouse strain infected with Leishmania amazonensis. BALB/c mice exposed to HBO (100% O 2 at a pressure of 2.5 ATA, 1 h before parasite inoculation and subsequently for 20 days) showed significant delay in lesion development and reduction in lesion parasite burdens compared with HBO-unexposed mice. Circulating levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF-α) were significantly elevated in HBO-exposed as compared to HBO-unexposed mice. Concanavalin A-stimulated lymph nodes cultures from HBO-exposed mice released significantly more IFN-γ and less interleukin 10 (IL-10) than cultures from HBO-unexposed mice, consistent with a skewed Th1 response. These results demonstrate, for the first time, that HBO can play a pathogen control role during leishmaniasis. Further studies are needed to elucidate whether hyperoxia alone or increased atmospheric pressure alone can exert a similar effect.

Immunomodulatory Therapeutic Effect of Glatiramer Acetate on Several Murine Models of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is characterized by detrimental immune reactivity in the gut and imbalance between proinflammatory and anti-inflammatory reactivity. In an attempt to down-regulate colitis, we investigated the effect of the immunomodulator glatiramer acetate (GA, Copaxone, copolymer 1) on two murine models of IBD, chemically induced and spontaneous. Acute experimental colitis of different levels of severity was induced in C57BL/6 mice by dextran sulfate sodium (DSS) administered orally at different concentrations and frequencies. It was manifested in weight loss, intestinal bleeding, and diarrhea, as well as by macroscopic and microscopic colon damage. GA treatment led to amelioration of all of these pathological manifestations, resulting in improved long-term survival. Moreover, even when colitis was induced by three cycles of DSS in this highly susceptible mouse strain, as well as in BALB/c mice that exhibit a chronic disease pattern, a substantial reduction in disease activity and mortality was obtained. GA treatment induced a beneficial effect also in a spontaneous model of colitis developed in the C3H/HeJBir IL-10-deficient mice. The detrimental proinflammatory response manifested by proliferation, tumor necrosis factor-alpha, and interferon-gamma expression was modulated by GA, whereas the regulatory anti-inflammatory transforming growth factor-beta and IL-10 cytokines response was elevated. This was demonstrated on the level of protein secretion in splenocytes and local mesenteric lymphocytes in response to syngeneic colon extract and in the overall response to anti-CD3, as well as on the level of mRNA expression in the colon.

Nanodisk-Associated Amphotericin B Clears Leishmania major Cutaneous Infection in Susceptible BALB/c Mice

Nanometer-scale, apolipoprotein-stabilized phospholipid bilayer disk complexes (nanodisks [ND]) harboring the toxic and poorly soluble antileishmanial agent amphotericin B (AMB) were examined for efficacy in treatment of Leishmania major-infected BALB/c mice (Mus musculus). L. major-infected mice were intraperitoneally (i.p.) treated with AMB-ND in 0-, 1-, and 5-mg/kg doses at 24 h, 48 h, and 4, 7, 14, and 21 days postinfection in two experiments. L. major-infected mice were i.p. treated with phosphate-buffered saline, 5 mg/kg AMB-ND, or 5 mg/kg lipid-associated amphotericin B (liposomal amphotericin B, AmBisome) at 24 h, 48 h, and 10, 20, 30, and 40 days postinfection in one experiment. Parasite numbers, footpad lesion size progression, and development of cytokine responses were assayed at days 7, 15, 30, 50, 140, and 250 or at days 14, 30, 50, 95, and 140 postinfection. Mice administered AMB-ND in 1- or 5-mg/kg doses were significantly protected from L. major, displaying decreases in lesion size and parasite burden, particularly at the 5-mg/kg dosage level. In contrast to the i.p. treated AmBisome group, BALB/c mice treated with i.p. AMB-ND completely cleared an L. major infection by 140 to 250 days postinfection, with no lesions remaining and no parasites isolated from infected animals. Restimulated mixed lymphocyte culture cytokine responses (interleukin-4 [IL-4], IL-12, IL-10, NO, and gamma interferon) were unchanged by AMB-ND administration compared to controls. The marked clearance of Leishmania parasites from a susceptible strain of mice without an appreciable change in the cytokine response suggests that AMB-ND represent a potentially useful formulation for treatment of intrahistiocytic organisms.

Genetic segregation of airway disease traits despite redundancy of calcium-activated chloride channel family members

Complex airway diseases such as asthma and chronic obstructive pulmonary disease exhibit stereotyped traits (especially airway hyperreactivity and mucous cell metaplasia) that are variably expressed in each patient. Here, we used a mouse model for virus-induced long-term expression of these traits to determine whether individual traits can be genetically segregated and thereby linked to separate determinants. We showed that an F2 intercross population derived from susceptible and nonsusceptible mouse strains can manifest individual phenotypic extremes that exhibit one or the other disease trait. Functional genomic analysis of these extremes further indicated that a member of the calcium-activated chloride channel (CLCA) gene family designated mClca3 was inducible with mucous cell metaplasia but not airway hyperreactivity. In confirmation of this finding, we found that mClca3 gene transfer to mouse airway epithelium was sufficient to induce mucous cell metaplasia but not airway hyperreactivity. However, newly developed mClca3(-/-) mice exhibited the same degree of mucous cell metaplasia and airway hyperreactivity as wild-type mice. Bioinformatic analysis of the Clca locus led to the identification of mClca5, and gene transfer indicated that mClca5 also selectively drives mucous cell metaplasia. Thus, in addition to the capacity of CLCA family members to exhibit diverse functional activities, there is also preserved function so that more than one family member mediates mucous cell metaplasia. Nonetheless, Clca expression appears to be a selective determinant of mucous cell metaplasia so that shared homologies between CLCA family members may still represent a useful target for focused therapeutic intervention in hypersecretory airway disease.

The role of Fc-receptors in murine mercury-induced systemic autoimmunity

Inorganic mercury (Hg) in genetically susceptible mouse strains induces a T cell-dependent, systemic autoimmune condition (HgIA) characterized by immunostimulation, anti-nuclear antibodies (ANA) and systemic immune-complex (IC) deposits. The exact phenotypic expression of HgIA in different strains depends on H-2 and non-H-2 genes. Fc receptors (FcRs) are important in the development of many autoimmune diseases. In this study, the effect of targeted mutations for activating and inhibiting FcRs in the BALB/c model of HgIA was examined. Hg-treated BALB/c mice without mutation (wild-type, wt) showed heavy IC deposits in the renal glomerular mesangium, as well as in renal and splenic vessel walls. The renal mesangial IC deposits were severely reduced in Hg-treated BALB/c mice without the gamma-chain (lack of the activating receptors FcgammaRI, FcgammaRIII and FcinRI), but unchanged in mice lacking the inhibitory FcgammaRIIB. The Hg-induced vessel wall IC deposits present in wt mice were abolished and reduced in the FcRgamma and FcgammaRIIB strains, respectively. Hg-treated BALB/c wt mice and mice without the gamma-chain showed an increase in serum IgE, while the increase in IgG1 was attenuated in the latter strain. In contrast, absence of the inhibiting FcgammaRIIB augmented the Hg-induced increase of both serum IgG1 and IgE. In conclusion, FcRs are important mainly for the induction of systmeic IC deposits in the HgIA model, but also affects serum IgG1 and IgE levels.

Studies on LXR- and FXR-mediated effects on cholesterol homeostasis in normal and cholic acid-depleted mice

As previously reported by us, mice with targeted disruption of the CYP8B1 gene (CYP8B1-/-) fail to produce cholic acid (CA), upregulate their bile acid synthesis, reduce the absorption of dietary cholesterol and, after cholesterol feeding, accumulate less liver cholesterol than wild-type (CYP8B1+/+) mice. In the present study, cholesterol-enriched diet (0.5%) or administration of a synthetic liver X receptor (LXR) agonist strongly upregulated CYP7A1 expression in CYP8B1-/- mice, compared to CYP8B1+/+ mice. Cholesterol-fed CYP8B1-/- mice also showed a significant rise in HDL cholesterol and increased levels of liver ABCA1 mRNA. A combined CA (0.25%)/cholesterol (0.5%) diet enhanced absorption of intestinal cholesterol in both groups of mice, increased their liver cholesterol content, and reduced their expression of CYP7A1 mRNA. The ABCG5/G8 liver mRNA was increased in both groups of mice, but cholesterol crystals were only observed in bile from the CYP8B1+/+ mice. The results demonstrate the cholesterol-sparing effects of CA: enhanced absorption and reduced conversion into bile acids. Farnesoid X receptor (FXR)-mediated suppression of CYP7A1 in mice seems to be a predominant mechanism for regulation of bile acid synthesis under normal conditions and, as confirmed, able to override LXR-mediated mechanisms. Interaction between FXR- and LXR-mediated stimuli might also regulate expression of liver ABCG5/G8.