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Charlie C Hsu Jisun Paik Piper M Treuting Audrey Seamons Stacey M Meeker Thea L Brabb Lillian Maggio-Price 《Comparative medicine》2014,64(4):256-263
Infection of laboratory mice with murine noroviruses (MNV) is widely prevalent. MNV alters various mouse models of disease, including the Helicobacter bilis-induced mouse model of inflammatory bowel disease (IBD) in Mdr1a−/− mice. To further characterize the effect of MNV on IBD, we used mice deficient in the immunoregulatory cytokine IL10 (Il10−/− mice). In vitro infection of Il10−/− bone marrow-derived macrophages (BMDM) with MNV4 cocultured with H. bilis antigens increased the gene expression of the proinflammatory cytokines IL1β, IL6, and TNFα as compared with that of BMDM cultured with H. bilis antigens only. Therefore, to test the hypothesis that MNV4 infection increases inflammation and alters disease phenotype in H. bilis-infected Il10−/− mice, we compared the amount and extent of inflammation in Il10−/− mice coinfected with H. bilis and MNV4 with those of mice singly infected with H. bilis. IBD scores, incidence of IBD, or frequency of severe IBD did not differ between mice coinfected with H. bilis and MNV4 and those singly infected with H. bilis. Mice infected with MNV4 only had no appreciable IBD, comparable to uninfected mice. Our findings suggest that, unlike in Mdr1a−/− mice, the presence of MNV4 in Il10−/− mouse colonies is unlikely to affect the IBD phenotype in a Helicobacter-induced model. However, because MNV4 altered cytokine expression in vitro, our results highlight the importance of determining the potential influence of MNV on mouse models of inflammatory disease, given that MNV has a tropism for macrophages and dendritic cells and that infection is widely prevalent.Abbreviations: BMDM, bone marrow-derived macrophages; IBD, inflammatory bowel disease; MLN, mesenteric lymph node; MNV, murine norovirusInflammatory bowel disease (IBD), which includes both ulcerative colitis and Crohn disease, is a chronic and relapsing inflammatory disorder of the gastrointestinal tract. In addition, patients with IBD may be at increased risk of developing colorectal cancer.15,46 Although the exact mechanisms of disease are still not understood fully, the pathogenesis of disease is likely multifactorial, with components of the innate and adaptive immune systems, host genetics, and environmental factors (for example, the commensal gut microflora) all playing a role.4,37,55Animal models of IBD have been used to advance our knowledge and understanding of IBD pathogenesis and treatment.16,20,37,38,52 One such model that has been widely used to elucidate the mechanisms of IBD is the interleukin10–deficient (Il10−/−) mouse.3,5,6,20,21,29,33,57 The antiinflammatory cytokine IL10 modulates both innate and adaptive immune responses.41 Produced mainly by dendritic cells, monocytes, macrophages, and T regulatory cells, IL10 exerts its immunomodulatory effects by various mechanisms including decreasing secretion of proinflammatory cytokines (for example, interferon γ, IL1, IL2, IL6, IL12 and TNFα) and downregulating important components of innate immune responses and T-cell activation (for example, MHC class II, costimulatory molecules, and nitric oxide production) in antigen presenting cells.14,41 As a consequence, Il10−/− mice, which lack the suppressive effects of IL10, develop IBD in response to their commensal gut microflora or to certain microbial triggers such as Helicobacter infections.5,6,11,21,29,52,57Antigen-presenting cells such as macrophages and dendritic cells play key roles in the inflammatory responses in IBD.32,47,50 In 2003, a newly discovered murine norovirus (MNV) in laboratory mice was shown to infect macrophages and dendritic cells.27,53 Subsequent studies indicated widespread MNV infection in laboratory mice used for biomedical research, with a serologic prevalence as high as 32%.25,43 Members of the genus Norovirus are regarded as gastrointestinal pathogens in humans and animals, eliciting both innate and adaptive immune responses.19 Therefore, in light of the cellular (macrophages and dendritic cells) and tissue (gastrointestinal) tropisms of MNV as well as the high prevalence of MNV infection in laboratory mice, we hypothesized that MNV infection could be a potential confounder in mouse models of inflammatory diseases including IBD. In support of this idea, our laboratory recently reported that MNV infection in Mdr1a−/− mice (FVB.129P2-Abcb1atm1Bor) accelerated weight loss and exacerbated IBD progression initiated by H. bilis infection.31 This effect potentially was mediated in part through modulating dendritic cell and cytokine responses. In addition, others have reported gastrointestinal abnormalities as a result of MNV infection in some strains of mice,7,26,36 whereas others have described the importance of both innate and adaptive immune responses during MNV infection.8,9,10,28,34,36,48 Collectively, these data indicate that MNV could alter inflammatory responses in laboratory mice.Here we extended our studies of MNV beyond Mdr1a−/− mice to Il10−/− mice, another common animal model of IBD, to further examine the potential effect of MNV on IBD research. Disease was initiated in Il10−/− mice with H. bilis, and we determined whether coinfection with MNV altered disease development, incidence, and severity and the production of cytokines. We demonstrated that although MNV stimulates a Th1 skewing of cytokines in Il10−/− bone marrow-derived macrophages (BMDM) in vitro, MNV does not alter the development, incidence, or severity of disease in vivo. Therefore, although MNV may not affect disease in Il10−/− mouse models, the virus may influence in vitro cytokine phenotypes and thus complicate interpretation of such data. To our knowledge, this report is the first to describe the evaluation of MNV infection in the Helicobacter-induced Il10−/− mouse model of IBD. 相似文献
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Jisun Paik Fiona Kwok Audrey Seamons Thea Brabb Jinkyu Kim Brittany Sullivan Charlie Hsu Kevin D O'Brien Lillian Maggio-Price 《Comparative medicine》2015,65(2):114-122
We previously reported that murine norovirus (MNV), a virus prevalent in United States research institutions, increased atherosclerotic lesion size in Ldlr−/− mice when the mice were infected 8 wk after feeding an atherogenic diet. To determine whether the timing of MNV infection relative to atherosclerosis development altered the disease phenotype and to examine potential mechanisms by which MNV influences the disease process, we fed Ldlr−/− mice an atherogenic diet for 16 wk. Three days after initiating the atherogenic diet, half of the mice received MNV4 and the other half vehicle only (clarified cell-culture lysate; controls). Both groups of mice developed large aortic sinus lesions (control compared with MNV4: 133 ± 8 × 103 µm2 compared with 140 ± 7 × 103 µm2) that were not significantly different in size. Because the timing of MNV infection relative to atherosclerosis development and hypercholesterolemia differed between our previous and the current studies, we examined whether hypercholesterolemia altered MNV4-induced changes in bone-marrow–derived macrophages. MNV4 infection increased the potential of macrophages to take up and store cholesterol by increasing CD36 expression while suppressing the ABCA1 transporter. Thus, the effects of MNV4 infection on atherosclerotic lesion size appear to be dependent on the timing of the infection: MNV4 infection promotes only established lesions. This effect may be due to MNV4’s ability to increase cholesterol uptake and decrease efflux by regulating CD36 and ABCA1 protein expression.Abbreviations: ABCA1, ATP-binding cassette A1; BMDM, bone-marrow–derived macrophage; iNOS, inducible nitric oxide synthase; MNV, murine norovirusChronic viral infection, such as occurs with HIV and hepatitis C virus, has been associated with an increased risk for atherosclerosis,2,19,46,48 although the mechanisms by which this occurs are not clearly defined. Our laboratory has been studying the effects of murine norovirus (MNV), which chronically infects immunocompetent mice, on murine models of inflammatory diseases, including atherosclerosis. MNV is a single-stranded RNA intestinal virus that belongs to the family Caliciviridae and has shown tropism toward antigen-presenting cells such as dendritic cells and macrophages.54 Whereas human norovirus is a major cause of nonbacterial acute gastroenteritis,52 MNV does not cause clinical disease in immunocompetent mice.55 However, the high prevalence of MNV in biomedical research facilities throughout the world,42,55 combined with its tropism for antigen-presenting cells, has prompted concern regarding potential effects on disease phenotypes in murine models of human diseases. Therefore, we previously examined 2 diseases, obesity and atherosclerosis, where macrophages have critical roles.41,42 We found that MNV infection did not influence glucose metabolism and weight gain,41 but it significantly increased the size and macrophage content of aortic sinus lesions in Ldlr−/− mice fed an atherogenic diet.42 These findings suggest that MNV might be a potential tool to determine how viral infection alters the risk of atherosclerosis.Many factors influence the progression of atherosclerosis. Accordingly, we examined whether the timing of MNV infection relative to the stage of atherosclerosis progression influenced disease phenotype and evaluated potential mechanisms by which MNV could affect the disease process. To this end, we modeled the infection of macrophages by using in vitro cultures of bone-marrow–derived macrophages (BMDM). 相似文献
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Karen Chase Lencioni Audrey Seamons Piper M Treuting Lillian Maggio-Price Thea Brabb 《Comparative medicine》2008,58(6):522-533
Murine norovirus (MNV) has recently been recognized as a widely prevalent viral pathogen in mouse colonies and causes disease and mortality in mice with impaired innate immunity. We tested the hypothesis that MNV infection would alter disease course and immune responses in mice with inflammatory bowel disease (IBD). FVB.129P2-Abcb1atm1Bor N7 (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis. As compared with controls, Mdr1a−/− mice coinfected with MNV4 and H. bilis showed greater weight loss and IBD scores indicative of severe colitis, demonstrating that MNV4 can modulate the progression of IBD. Compared with controls, mice inoculated with MNV4 alone had altered levels of serum biomarkers, and flow cytometric analysis of immune cells from MNV4-infected mice showed changes in both dendritic cell (CD11c+) and other nonT cell (CD4− CD8−) populations. Dendritic cells isolated from MNV4-infected mice induced higher IFNγ production by polyclonal T cells in vitro at 2 d after infection but not at later time points, indicating that MNV4 infection enhances antigen presentation by dendritic cells early after acute infection. These findings indicate that acute infection with MNV4 is immunomodulatory and alters disease progression in a mouse model of IBD.Abbreviations: DC, dendritic cell; IBD, inflammatory bowel disease; IP, IFNγ–inducible protein; MCP, macrophage chemotactic protein; MLN, mesenteric lymph node; MNV, murine norovirus; TNF, tumor necrosis factorThe genus Norovirus of the family Caliciviridae contains a large number of single-stranded, positive-sense RNA viruses that infect vertebrates, and strains have been identified in humans, cattle, swine, and (most recently) mice.19,29,34 Murine noroviruses (MNV) are recently recognized pathogens that can cause lethal infection in immunocompromised mice that lack innate immunity.19 However, MNV did not cause clinical disease in wild-type mice or many other strains of immunodeficient mice, including those lacking the recombination-activating gene (Rag−/−) and inducible nitric oxide synthase deficient mice.19,35,37 MNV was reported recently to be widespread in laboratory mice and may persist in immunocompetent animals, depending on the strain of MNV used.15,16,25 Studies in Rag−/− mice and B-cell–deficient strains showed that the acquired immune system plays an important role in the clearance of MNV.6,19,37 MNV has tropism for dendritic cells (DCs),36 which are important in the presentation of antigens to T cells in draining lymph nodes and in the pathogenesis of inflammatory bowel disease (IBD). Therefore, MNV is a potential confounder for in vivo immunology studies, including murine models of IBD.Idiopathic IBD, which encompasses both ulcerative colitis and Crohn disease, is a widely studied disorder that affects approximately 1.4 million people in the United States.20 Although the precise cause of human IBD has not been elucidated, studies with mouse models have demonstrated that abnormal host responses of the innate and adaptive immune systems to intestinal microbiota are important in the pathogenesis of IBD.28,38 DCs are the sentinels of the intestinal mucosal barrier and have a pivotal role in the initiation of IBD in response to microbial ligands.39 Alterations in DC responses could lead to persistence of bacterial infection, aberrant activation of the acquired immune system, and (ultimately) tissue damage.38Viral stimulation of DCs leads to activation of adaptive immune responses,17 including effector T cells, and as demonstrated with murine coronavirus (mouse hepatitis virus), intercurrent viral infections in mice can alter the phenotype of mouse models of human disease.10 Additional evidence suggests that intercurrent viral infection may enhance disease in human IBD patients.12,18 Whether infection with MNV alters DC function and, therefore, influences the progression of IBD in mouse models is unclear.Many mouse models of intestinal inflammation develop IBD that is driven by bacterial flora.9,28 Helicobacter spp. have been shown to drive this process in several mouse models including IL10-deficient, SMAD3-deficient, severe combined immunodeficiency and T-cell–deficient mice.4,5,13,23 FVB.129P2-Abcb1atm1Bor (Mdr1a−/−) mice develop spontaneous IBD that is accelerated by infection with Helicobacter bilis.21,22 In this report, we tested the hypothesis that infection with MNV can modulate IBD in this mouse model of bacterial-induced disease. We demonstrate that intercurrent MNV4 infection accelerates the progression of bacterial-induced IBD in the Mdr1a−/− mouse and alters the immune responses in this mouse model of IBD. 相似文献
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Yong-Liang Han Hai-Xing Song Qiong Liao Yin Yu Shao-Fen Jian Joe Eugene Lepo Qiang Liu Xiang-Min Rong Chang Tian Jing Zeng Chun-Yun Guan Abdelbagi M. Ismail Zhen-Hua Zhang 《Plant physiology》2016,170(3):1684-1698
Enhancing nitrogen use efficiency (NUE) in crop plants is an important breeding target to reduce excessive use of chemical fertilizers, with substantial benefits to farmers and the environment. In Arabidopsis (Arabidopsis thaliana), allocation of more NO3− to shoots was associated with higher NUE; however, the commonality of this process across plant species have not been sufficiently studied. Two Brassica napus genotypes were identified with high and low NUE. We found that activities of V-ATPase and V-PPase, the two tonoplast proton-pumps, were significantly lower in roots of the high-NUE genotype (Xiangyou15) than in the low-NUE genotype (814); and consequently, less vacuolar NO3− was retained in roots of Xiangyou15. Moreover, NO3− concentration in xylem sap, [15N] shoot:root (S:R) and [NO3−] S:R ratios were significantly higher in Xiangyou15. BnNRT1.5 expression was higher in roots of Xiangyou15 compared with 814, while BnNRT1.8 expression was lower. In both B. napus treated with proton pump inhibitors or Arabidopsis mutants impaired in proton pump activity, vacuolar sequestration capacity (VSC) of NO3− in roots substantially decreased. Expression of NRT1.5 was up-regulated, but NRT1.8 was down-regulated, driving greater NO3− long-distance transport from roots to shoots. NUE in Arabidopsis mutants impaired in proton pumps was also significantly higher than in the wild type col-0. Taken together, these data suggest that decrease in VSC of NO3− in roots will enhance transport to shoot and essentially contribute to higher NUE by promoting NO3− allocation to aerial parts, likely through coordinated regulation of NRT1.5 and NRT1.8.China is the largest consumer of nitrogen (N) fertilizer in the world; however, the average N use efficiency (NUE) in fertilizer is only around 35%, suggesting considerable potential for improvements (Shen et al., 2003; Wang et al., 2014). With the high amounts of N-fertilizer being used, crop yields are declining in some areas, where application is exceeding the optimum required for local field crops (Shen et al., 2003; Miller and Smith, 2008; Xu et al., 2012). The extremely low NUE results in waste of resources and environmental contamination, and also presents serious hazards for human health (Xu et al., 2012; Chen et al., 2014). Consequently, exploiting the maximum potential for improving NUE in crop plants will have practical significance for agriculture production and the environment (Zhang et al., 2010; Schroeder et al., 2013; Wang et al., 2014). Elucidating the genetic and physiological regulatory mechanisms governing NUE in plants will allow breeding crops and varieties with higher NUE.Ammonium (NH4+) and nitrate (NO3−) are the main N species absorbed and utilized by crops, and NO3− accumulation and utilization are of major emphasis for N nutrient studies in dry land crops, such as Brassica napus. Several studies revealed the close relationship between NO3− content and NUE in plant tissues (Shen et al., 2003; Zhang et al., 2012; Tang et al., 2013; Han et al., 2015a). When plants are sufficiently illuminated, NO3− assimilation efficiency significantly increase in shoots compared with roots (Smirnoff and Stewart, 1985; Tang et al., 2013). Consequently, under daytime with optimal illumination, higher proportion of NO3− in plant tissue is transported from root to shoot, as an advantageous physiological adaptation that reduces the cost of energy for metabolism (Tang et al., 2013). NO3− assimilation in plant shoots can therefore take advantage of solar energy while improving NUE (Smirnoff and Stewart, 1985; Andrews, 1986; Tang et al., 2012, 2013).The NO3− long-distance transport and distribution between root and shoot is regulated by two genes encoding long transport mechanisms. NRT1.5 is responsible for xylem NO3− loading, while NRT1.8 is responsible for xylem NO3− unloading (Lin et al., 2008; Li et al., 2010). Expression of the two genes is influenced by NO3− concentration. NRT1.5 is strongly induced by NO3− (Lin et al., 2008), while NRT1.8 expression is extremely up-regulated in nrt1.5 mutants (Chen et al., 2012). A negative correlation between the extents of expression of the two genes was observed when plants are subjected to abiotic stresses (Chen et al., 2012). Moreover, expression of NRT1.5 is strongly inhibited by 1-aminocyclopropane-1-carboxylic acid (ACC) and methyl jasmonate (MeJA), whereas the expression of NRT1.8 is significantly up-regulated (Zhang et al., 2014). Based on these studies, we argue that the expression and functioning of NO3− long-distance transport genes NRT1.5 and NRT1.8 are regulated by cytosolic NO3− concentration. In addition, the vacuolar and cytosolic NO3− distribution is likely regulated by proton pumps located within the tonoplast (V-ATPase and V-PPase; Granstedt and Huffaker, 1982; Glass et al., 2002; Krebs et al., 2010). Therefore, NO3− use efficiency must be affected by NO3− long-distant transport (between shoot and root) and short-distant transport (between vacuole and cytosol). However, the physiological mechanisms controlling this regulation are still obscure.Previous studies showed that the chloride channel protein (CLCa) is mainly responsible for vacuole NO3− short-distance transport, as it is the main channel for NO3− movement between the vacuoles and cytosol (De Angeli et al., 2006; Wege et al., 2014). The vacuole proton-pumps (V-ATPase and V-PPase) located in the tonoplast supply energy for active transport of NO3− and accumulation within the vacuole (Gaxiola et al., 2001; Brüx et al., 2008; Krebs et al., 2010). Despite the fact about 90% of the volume of mature plant cells is occupied by vacuoles, vacuolar NO3− cannot be efficiently assimilated because the enzyme nitrate reductase (NR) is cytosolic (Shen et al., 2003; Han et al., 2015a). However, retranslocation of NO3− from the vacuole to the cytosol will permit its immediate assimilation and utilization.Generally, NO3− concentrations in plant cell vacuoles and the cytoplasm are in the range of 30–50 mol m−3 and 3–5 mol m−3, respectively (Martinoia et al., 1981, 2000). Because vacuoles are obviously the organelle for high NO3− accumulation and storage in plant tissues, their function in NO3− use efficiency cannot be ignored (Martinoia et al., 1981; Zhang et al., 2012; Han et al., 2015b). NO3− assimilatory system in the cytoplasm is sufficient for its assimilation when it is transported out of the vacuoles. Therefore, NO3− use efficiency could in part be dependent on vacuolar-cytosolic NO3− short-distance transport in plant tissues (Martinoia et al., 1981; Shen et al., 2003; Zhang et al., 2012; Han et al., 2015a).Evidently, NO3− use efficiency is regulated by both NO3− long-distance transport from root to shoot and short-distance transport and distribution between vacuoles and cytoplasm within cells (Glass et al., 2002; Dechorgnat et al., 2011; Han et al., 2015a). Although vacuoles compartment excess NO3− that accumulates in plant cells (Granstedt and Huffaker, 1982; Krebs et al., 2010), neither NO3− inducible NR genes (NIA1 and NIA2; Fan et al., 2007; Han et al., 2015a) nor the NO3− long-distance transport gene NRT1.5 (Lin et al., 2008) are regulated by vacuolar NO3−, even though they are essential for NO3− assimilation. Only NO3− transported from the vacuole to the cytosol can play a role in regulating NO3− inducible genes. Consequently, we argue that both NO3− assimilation in cells and its long-distance transport from root to shoot are regulated by cytosolic NO3− concentration. However, this hypothesis needs to be substantiated. The mechanisms underlying both NO3− short-distance (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010) and long-distance transport (Lin et al., 2008; Li et al., 2010) have been previously investigated, yet the underlying mechanisms regulating the flux of NO3− and the obvious relationship between the two transport pathways, as well as their relation to NUE, are not well understood.The NRT family of genes play a partial role in vacuolar NO3− accumulation in petioles (Chiu et al., 2004) and seed tissues (Chopin et al., 2007), whereas the proton pumps and CLCa system in the tonoplast play a major role in accumulating NO3− in vacuoles (Gaxiola et al., 2001; De Angeli et al., 2006; Brüx et al., 2008; Krebs et al., 2010). The vacuolar NO3− short-distance transport system is spread throughout the plant tissues and is the principal means by which vacuolar NO3− short-distance transport and distribution is controlled (De Angeli et al., 2006; Krebs et al., 2010).The NRT genes seem to work synergistically to control NO3− long-distance transport between roots and shoots. NRT1.9 is responsible for NO3− loading into the phloem (Wang and Tsay, 2011), whereas NO3− loading and unloading into xylem are regulated by NRT1.5 and NRT1.8, respectively (Lin et al., 2008; Li et al.; 2010). Phloem transport mainly involves organic N; the inorganic-N (NO3−) concentrations in the phloem sap are typically very low, ranging from one-tenth to one-hundredth of that of the inorganic-N in xylem sap (Lin et al., 2008; Fan et al., 2009). Therefore, this study focused on NO3− short-distance transport mediated through the tonoplast proton pumps and the CLCa system and the long-distant transport mechanisms responsible for xylem NO3− loading and unloading via NRT1.5 and NRT1.8, respectively.Questions related to how long- and short-distance transport of NO3− are coupled in plant tissues and their role in determining NUE were addressed using a pair of high- and low-NUE
B. napus genotypes and Arabidopsis (Arabidopsis thaliana). Application of proton pump inhibitors and ACC in the former, and use of mutants with defective proton pumps in the latter, allowed experimental distinction of the physiological mechanisms regulating these processes. Data presented here provide strong evidence from both model plants supporting this linkage and strongly suggest that cytosolic NO3− concentration in roots regulates NO3− long-distance transport from roots to shoots. We also investigated how NO3− concentration in plant tissues would be affected by NO3− long-distance transport, vacuolar NO3− sequestration, and the ensuing relationship with NO3− use efficiency. We also proposed the physiological mechanisms likely to be important for enhancing NO3− use efficiency in plants. These findings will provide scientific rationales for improving NUE in important industrial and food crops. 相似文献
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Katherine A Shuster Gerald A Hish Lindsi A Selles Mahboob A Chowdhury Roger C Wiggins Robert C Dysko Ingrid L Bergin 《Comparative medicine》2013,63(1):55-61
Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich–Wistar–Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.Abbreviations: GBS, Group B Streptococcus; MWF, Munich Wistar Frömter; hDTR, human diphtheria toxin receptorStreptococci are gram-positive, coccoid bacteria that typically are classified according to their hemolytic capacity. α-hemolytic streptococci produce a zone of partial hemolysis that appears greenish on blood agar, whereas β-hemolytic streptococci produce a zone of complete hemolysis, and γ-hemolytic organisms produce no hemolysis on blood agar.24 The β-hemolytic streptococci are further subdivided into Lancefield groups (A through G), according to cell-wall carbohydrate antigens.24,29,39 The group B β-hemolytic Streptococcus (GBS) have been speciated as Streptococcus agalactiae.28,39 It was first isolated as a causative agent of mastitis in cattle.29 This organism has since been recognized as a cause of severe infection in human neonates.28,39 In humans, GBS is harbored asymptomatically in the maternal genitourinary tract.24,28 Infants can be infected and present with serious systemic disease in the first week of life (early-onset GBS) or from 1 wk to 3 mo of age (late-onset GBS).39 In laboratory animals, rats have been used experimentally as models for neonatal1,6,7,20,37,38,43,44,47,50,51 or adult45 GBS infection, but to our knowledge, GBS has not been associated with spontaneous disease in rats. 相似文献
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John P Dowling Yubo Cai John Bertin Peter J Gough Jianke Zhang 《Cell death & disease》2016,7(9):e2379
The death receptor, Fas, triggers apoptotic death and is essential for maintaining homeostasis in the peripheral lymphoid organs. RIP1 was originally cloned when searching for Fas-binding proteins and was later shown to associate also with the signaling complex of TNFR1. Although Fas exclusively induces apoptosis, TNFR1 primarily activates the pro-survival/pro-inflammatory NF-κB pathway. Mutations in Fas lead to lymphoproliferative (lpr) diseases, and deletion of TNFR1 results in defective innate immune responses. However, the function of RIP1 in the adult lymphoid system has not been well understood, primarily owing to perinatal lethality in mice lacking the entire RIP1 protein in germ cells. This current study investigated the requirement for RIP1 in the T lineage using viable RIP1 mutant mice containing a conditional and kinase-dead RIP1 allele. Disabling the kinase activity of RIP1 had no obvious impact on the T-cell compartment. However, T-cell-specific deletion of RIP1 led to a severe T-lymphopenic condition, owing to a dramatically reduced mature T-cell pool in the periphery. Interestingly, the immature T-cell compartment in the thymus appeared intact. Further analysis showed that mature RIP1−/− T cells were severely defective in antigen receptor-induced proliferative responses. Moreover, the RIP1−/− T cells displayed greatly increased death and contained elevated caspase activities, an indication of apoptosis. In total, these results revealed a novel, kinase-independent function of RIP1, which is essential for not only promoting TCR-induced proliferative responses but also in blocking apoptosis in mature T cells.The pro-survival signaling pathways provide protection against cell death responses at various stages during T lymphopoiesis as well as maintenance of the mature population.1, 2 Apoptosis is a major programmed cell death pathway, which can be induced through either intrinsic or extrinsic signals.3 Under normal circumstances, the pro-survival and apoptosis signaling pathways are tightly regulated, which ensures generation of diverse T-cell repertoires, while avoiding autoimmunity. For instance, the Bcl-2 and Bcl-XL genes, which inhibit the intrinsic apoptotic pathway, are essential for both T-cell development and peripheral maintenance.4, 5 However, lack of cell death, as in the case of inactivation of Bim, a pro-apoptotic protein of the Bcl-2 family, results in lymphoproliferative and autoimmune diseases.6 The extrinsic pathway of apoptosis is triggered through cell receptors, including Fas/Apo-1 and tumor necrosis factor receptor 1 (TNFR1). Whereas Fas is a professional death receptor, TNFR1 primarily signals the pro-survival pathway by activating NF-κB, which also promotes inflammation.7, 8Receptor-interacting protein (RIP or RIP1) was originally cloned as a potential Fas-interacting protein.9 However, later studies found that lack of RIP1 has no effect on Fas-induced apoptosis.10, 11 Subsequently, RIP1 was also found to associate with the signaling complex of TNFR1.12 It was shown that RIP1 deficiency disrupts NF-κB activation induced by TNFR1 in primary mouse embryonic fibroblast cells or human Jurkat T lymphoma cells.10, 11 Several functional domains of RIP1 have been defined. In particular, RIP1 contains a serine/threonine kinase domain (KD) at the amino-terminus and a death domain (DD) at the carboxy-terminus. The intermediate domain, but not the protein serine/threonine KD of RIP1, is required for the activation of NF-κB.13 The DD of RIP1 interacts with the DD of TNFR1-associated death domain (TRADD) protein, a signaling adaptor, leading to both apoptosis and NF-κB activation.12 Therefore, RIP1 may serve as a scaffold protein in addition to being a protein serine/threonine kinase.The function of the KD of RIP1 remained unknown until the landmark work by Holler et al.,14 implicating a novel function for RIP1 in a caspase-independent cell death process with certain characteristics of necrosis, namely necroptosis. Importantly, mutations targeting the kinase activity of RIP1 abolish necroptotic cell death induced by TNFR1. The in vivo role of RIP1-mediated necroptosis was first revealed by analysis of the embryonic defect displayed by mice lacking the Fas-associated death domain (FADD) protein.15 The FADD adaptor protein relays exclusively apoptotic signals in the pathways triggered by Fas, TNFR1, and TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs or DR4/5).16, 17, 18 Whereas none of the DRs are essential for mouse development, FADD deficiency resulted in midgestation death of mouse embryos.19, 20 Interestingly, when RIP1 is absent, normal embryonic development is restored in FADD−/− mice,15 indicating that FADD−/− embryonic lethality is caused by RIP1-dependent necroptosis.Although normal during embryogenesis, RIP1−/− FADD−/− double knockout (DKO) mice display perinatal lethality,15 similar to the phenotype of RIP1−/− single knockout mice.10 In contrast, deletion of a RIP1-related protein kinase, RIP3, fully restores normal embryonic as well as postnatal development in FADD−/− mice.21 Recent studies demonstrated that RIP1−/− mice can only reach adulthood when both FADD and RIP3 are absent, indicating that RIP1 protects neonatal cells from FADD-mediated apoptosis and RIP3-dependent necroptosis.22, 23, 24, 25 Importantly, FADD−/− RIP3−/− DKO mice and RIP1−/− FADD−/− RIP3−/− triple knockout mice develop age-dependent lymphadenopathy and splenomegaly, reminiscent of the lymphoproliferative (lpr) disease displayed by Fas−/− mice. Therefore, both apoptosis and necroptosis are essential for homeostasis in the peripheral lymphoid organs.Previous studies have indicated that RIP1 is essential for T-cell development, because RIP1-deficient fetal liver cells fail to reconstitute the T-cell compartment in immunodeficient recipient mice.15, 26 A recent study showed that lack of RIP1 in hematopoietic stem cells and progenitors (HSCs/Ps) leads to a severe defect in hematopoiesis.27 However, the temporal requirement for RIP1, particularly at postlineage commitment stages, remains unclear. In the current study, T lineage-specific deletion of RIP1 revealed a novel stage-specific requirement for RIP1 to protect T cells from apoptosis as well as to allow normal proliferative responses. 相似文献
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L Dorstyn J Puccini A Nikolic S Shalini C H Wilson M D Norris M Haber S Kumar 《Cell death & disease》2014,5(8):e1383
Caspase-2 has been implicated in various cellular functions, including cell death by apoptosis, oxidative stress response, maintenance of genomic stability and tumor suppression. The loss of the caspase-2 gene (Casp2) enhances oncogene-mediated tumorigenesis induced by E1A/Ras in athymic nude mice, and also in the Eμ-Myc lymphoma and MMTV/c-neu mammary tumor mouse models. To further investigate the function of caspase-2 in oncogene-mediated tumorigenesis, we extended our studies in the TH-MYCN transgenic mouse model of neuroblastoma. Surprisingly, we found that loss of caspase-2 delayed tumorigenesis in the TH-MYCN neuroblastoma model. In addition, tumors from TH-MYCN/Casp2−/− mice were predominantly thoracic paraspinal tumors and were less vascularized compared with tumors from their TH-MYCN/Casp2+/+ counterparts. We did not detect any differences in the expression of neuroblastoma-associated genes in TH-MYCN/Casp2−/− tumors, or in the activation of Ras/MAPK signaling pathway that is involved in neuroblastoma progression. Analysis of expression array data from human neuroblastoma samples showed a correlation between low caspase-2 levels and increased survival. However, caspase-2 levels correlated with clinical outcome only in the subset of MYCN-non-amplified human neuroblastoma. These observations indicate that caspase-2 is not a suppressor in MYCN-induced neuroblastoma and suggest a tissue and context-specific role for caspase-2 in tumorigenesis.The caspase family of cysteine proteases are highly conserved regulators of cell death by apoptosis.1 In addition to their pro-apoptotic function, many caspases also have non-apoptotic roles in other physiological processes, such as inflammation, necrosis and tumor suppression.2, 3, 4 The most highly conserved caspase, caspase-2, has recently been demonstrated to function in the cellular stress response, protection against ageing, maintenance of genome stability and in tumor suppression.2, 5, 6, 7, 8The tumor suppressor function of caspase-2 was first demonstrated using E1A/Ras-transformed caspase-2-deficient mouse embryonic fibroblasts (MEFs), which showed an increased tumorigenic potential in athymic nude mice.7 Further supporting evidence came from experiments demonstrating that caspase-2 deficiency enhances B-cell lymphoma development in Eμ-Myc transgenic mice7 and mammary carcinomas in MMTV/c-neu mice,9 suggesting that caspase-2 prevents oncogene-induced lymphomas and epithelial tumors. Importantly, tumor suppression by caspase-2 is also evident in the non-oncogene-driven Atm−/− thymoma mouse model.10Given its role in apoptosis, the tumor suppression function of caspase-2 was thought to be associated with this role, via the elimination of mutagenic or potentially tumorigenic cells. Recent studies have now indicated that the role of caspase-2 may extend beyond apoptosis and that its tumor suppression function may, in part, be mediated by maintaining genomic stability and/or the oxidative stress response. Caspase-2-deficient MEFs and tumor cells from Eμ-Myc/Casp2−/−, MMTV/c-neu/Casp2−/− and Atm−/−;Casp2−/− mice all display aberrant proliferation, and increased genomic instability6, 9, 10 and indicate that caspase-2 is important for the maintenance of genome stability. Importantly, the role of caspase-2 in maintaining genomic stability in primary cells appears to be required for its tumor suppressor function.10Genomic instability is a hallmark of cancer11 and the overexpression of Myc family oncoproteins is commonly associated with genomic instability and a wide spectrum of human cancers.12, 13, 14 Interestingly, a common feature of the oncogene-induced tumor models used in the study of caspase-2 tumor suppressor function is the overexpression of c-Myc15 or aberrant c-Myc signaling.16, 17, 18 Given the role of Myc proteins as key mediators of genomic instability as well as cell proliferation, cell growth and DNA damage, we were interested in further assessing whether caspase-2 can promote tumor suppression in other MYC-dependent mouse tumor models. We used the MYCN mouse model of neuroblastoma (TH-MYCN mouse), in which MYCN is constitutively expressed under the control of the rat tyrosine hydroxylase (TH) promoter leading to neural crest cell-specific expression and early-onset neuroblastoma.19 Amplification of MYCN occurs in ∼20% of human neuroblastomas and high MYCN protein levels are strongly associated with tumor progression and poor clinical outcome.20, 21 Thus, the TH-MYCN transgenic mouse model recapitulates many clinical features of aggressive neuroblastomas in humans and provides a powerful model of preclinical neuroblastoma.19, 22MYCN-mediated neuroblastoma onset and progression is commonly associated with additional genetic events, including the expression of the key genes including Odc1, Mrp1, SirT1 and Ras.23, 24, 25 A recent study has found that caspase-8 is in fact a potent suppressor of neuroblastoma, with the loss of caspase-8 expression occurring in ∼70% of neuroblastoma patients.26, 27 Interestingly, the loss of caspase-8 also promotes bone marrow metastasis in the TH-MYCN neuroblastoma mouse model.26, 27 The role of other caspases in neuroblastoma has not previously been examined, and given the function of caspase-2 in tumor suppression, provided additional relevance in assessing its role in this model.This study shows that caspase-2 is not able to suppress neuroblastoma development in TH-MYCN mice. In contrast to a role for caspase-2 as a tumor suppressor, our findings demonstrate that loss of caspase-2 somewhat delays neuroblastoma onset in mice. Interestingly, expression array data from human neuroblastoma show a strong correlation between low caspase-2 levels and improved outcome. Our data demonstrate that the tumor suppressor function of caspase-2 is not specific to Myc-mediated oncogenesis and that its role is likely to be tissue- and/or context-specific. 相似文献
14.
NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species 总被引:3,自引:0,他引:3
Jian Sun Shaoliang Chen Songxiang Dai Ruigang Wang Niya Li Xin Shen Xiaoyang Zhou Cunfu Lu Xiaojiang Zheng Zanmin Hu Zengkai Zhang Jin Song Yue Xu 《Plant physiology》2009,149(2):1141-1153
Using the scanning ion-selective electrode technique, fluxes of H+, Na+, and Cl− were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na+ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na+ efflux and a correspondingly increased H+ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na+/H+ exchange in root tissues and cells was inhibited by amiloride (a Na+/H+ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H+-ATPase inhibitor). These results indicate that the Na+ extrusion in stressed P. euphratica roots is the result of an active Na+/H+ antiport across the plasma membrane. In comparison, the Na+/H+ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na+ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl− exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H+, Na+, and Cl− differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. When plant roots are subjected to saline environments with high NaCl content, external Na+ and Cl− establish a large electrochemical gradient favoring the passive entry of salt ions through a variety of cation and anion channels and/or transporters in the plasma membrane (PM; Blumwald et al., 2000; Hasegawa et al., 2000; White and Broadley, 2001; Roberts, 2006; Demidchik and Maathuis, 2007). The entry and accumulation of toxic ions lead to disruption of ion homeostasis and finally cause secondary stress, e.g. oxidative bursts (Zhu, 2001, 2003). Accordingly, the maintenance of low salt concentration in the cytosol is of great importance for salt adaptation of plants (Greenway and Munns, 1980; Munns and Tester, 2008).Active Na+ extrusion to the apoplast or external environment is essential for sustaining Na+ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na+/H+ antiporters have been widely considered to play a crucial role in active Na+ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na+/H+ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na+/H+ antiporter gene AtSOS1 decreases the accumulation of Na+ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na+/H+ antiporters depend on electrochemical H+ gradients, which are generated by PM H+-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H+-ATPase in the Na+/H+ antiport according to H+ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H+ pumping is fundamental to Na+/H+ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na+/H+ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.Munns and Tester (2008) claimed that Cl− toxicity is more important than Na+ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl− uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na+ (Chen et al., 2001, 2002, 2003). The differences in Cl− tolerance exhibited by plants are usually related to the ability to restrict Cl− transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl− from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl−, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl− flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl− fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na+ and Cl− in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H+, Na+, and Cl− in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na+ and H+ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na+ exclusion under saline conditions. 相似文献
15.
Lyudmila Y. Kadyrova Basanta K. Dahal Vaibhavi Gujar James M. Daley Patrick Sung Farid A. Kadyrov 《The Journal of biological chemistry》2022,298(4)
The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction. 相似文献
16.
17.
Jennifer L Booth Todd M Umstead Sanmei Hu Kevin F Dybvig Timothy K Cooper Ronald P Wilson Zissis C Chroneos 《Comparative medicine》2014,64(6):424-439
Mycoplasmosis is a frequent causative microbial agent of community-acquired pneumonia and has been linked to exacerbation of chronic obstructive pulmonary disease. The macrophage class A scavenger receptor (SRA) facilitates the clearance of noxious particles, oxidants, and infectious organisms by alveolar macrophages. We examined wildtype and SRA−/− mice, housed in either individually ventilated or static filter-top cages that were cycled with fresh bedding every 14 d, as a model of gene–environment interaction on the outcome of pulmonary Mycoplasma pulmonis infection. Intracage NH3 gas measurements were recorded daily prior to infection. Mice were intranasally infected with 1 × 107 cfu M. pulmonis UAB CT and evaluated at 3, 7, and 14 d after inoculation. Wildtype mice cleared 99.5% of pulmonary M. pulmonis by 3 d after infection but remained chronically infected through the study. SRA−/− mice were chronically infected with 40-fold higher mycoplasma numbers than were wildtype mice. M. pulmonis caused a chronic mixed inflammatory response that was accompanied with high levels of IL1β, KC, MCP1, and TNFα in SRA−/− mice, whereas pulmonary inflammation in WT mice was represented by a monocytosis with elevation of IL1β. Housing had a prominent influence on the severity and persistence of mycoplasmosis in SRA−/− mice. SRA-/- mice housed in static cages had an improved recovery and significant changes in surfactant proteins SPA and SPD compared with baseline levels. These results indicate that SRA is required to prevent chronic mycoplasma infection of the lung. Furthermore, environmental conditions may exacerbate chronic inflammation in M. pulmonis-infected SRA−/− mice.Abbreviations: BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; KC, keratinocyte-derived chemokine (CXCL1); MCP1, monocyte chemotactic protein 1; SPA, surfactant protein A (SFTPA1); SPB, surfactant protein B (SFTPB); SPD, surfactant protein D (SFTPD); SRA, class A scavenger receptor (MSR1); WT, wildtypeThere are numerous options for the housing and husbandry of rodents in the laboratory setting. Various available choices in caging, bedding material, and cage-change frequency have the potential to effect physiologic values and thus experimental outcomes.20,108 In many facilities, current practices involve performing cage changes every 1 to 2 wk, with some facilities exploring the possibility of extending these practices to every 4 wk.97 Cage-change frequency practices are established at various institutions after consideration of several variables that affect animal health, welfare, and cost. Ideally, an appropriate sanitation program provides clean and dry bedding, adequate air quality, and clean cage surfaces and accessories.44 When establishing performance standards for a sanitation program that are different from those which are recommended in the Guide for the Care and Use of Animals in Research,44 microenvironmental conditions, including intracage humidity, temperature, animal behavior and appearance, microbiologic loads, and levels of pollutants such as CO2 and NH3, should be evaluated and verified. Although there are currently no established NH3 exposure limits for laboratory animals, the human occupational exposure limit of 25 ppm as an 8-h time-weighted average, established by the National Institute for Occupational Safety and Health, is often referenced as a guideline for animals.95 Multiple factors, such as animal cage density, sex, age, bedding type, reusable compared with disposable caging, static caging compared with IVC, and cage-change frequency, influence intracage and ambient NH3 levels.82,83,97 Only limited information is available that addresses the effect of natural intracage NH3 levels on respiratory function in experimental rodents and whether exposure to high NH3 levels under current standard practices affects the results of respiratory disease research.Ammonia is an alkaline, corrosive, and irritant gas that is very water soluble. It reacts with the moisture of the mucous membranes of the eyes, mouth, and respiratory tract to form ammonium hydroxide in an exothermic reaction, resulting in thermal and chemical burns.68 Clinical symptoms in humans exposed to high levels of NH3 include eye irritation, headaches, and multiple acute and chronic respiratory symptoms, such as irritation of the nose, pharynx, and sinuses, and in severe cases, development of bronchitis and hyper-reactive airway disease.79 Animals are similarly susceptible to NH3-induced pulmonary disease.23,31,48Mice exposed to naturally increasing levels of intracage NH3 can develop lesions in the rostral nasal cavity, with decreasing severity of the lesions moving caudally into the nasopharynx, and no lesions in the lung.97 However, dust is another common environmental pollutant that is often present in animal settings. Dust particles readily absorb NH3, which then serve as a source of NH3 deposition into the lower respiratory tract. Dust particulate can range from large (300 µm), minimally respirable particles to very fine (< 50 µm) particulate matter, which can settle deep within the alveoli.10,102 The mucociliary system of the respiratory tract is the first line of defense against inspired noxious stimuli and pathogens. Exposure of the ciliated respiratory epithelium to the damaging effects of NH3 are known to cause decreased mucociliary beating.56 Disruption of the respiratory mucociliary escalator initiated by NH3 exposure can then promote establishment of chronic infections and inflammation of the airway mucosa.11,87 Therefore, NH3 potentially can cause pathophysiologic changes of the lung in the absence of histopathologic lesions.Our primary goal was to analyze the effect of 2 housing modalities, which result in different intracage NH3 concentrations, on mice that were challenged with a respiratory pathogen. Mycoplasma pulmonis was chosen as a model because it is a well-established model in rodents which causes chronic mycoplasmosis and reproduces the features of M. pneumoniae in humans.22,41
M. pneumoniae infection is a frequent and contagious etiology of community-acquired pneumonia causing tracheobronchitis, sneezing, cough, and inflammation of the respiratory tract.8,12,47,63 Moreover, atypical and difficult-to-detect respiratory pathogens such as Chlamydophila pneumoniae and Mycoplasma pneumoniae that can establish chronic asymptomatic infections may contribute to both the development and exacerbation of COPD26,45,57,58,62,63,66,72,96,103 and asthma.8,51,65 Infection with M. pulmonis in rodents causes rhinitis, otitis media, tracheitis, and pneumonia, which can be exacerbated by housing conditions and genetic background.14,32,85 The mechanism of pathogenicity of mycoplasmas continues to be an area of interest in the research.The innate host factors protecting against pulmonary mycoplasmosis include the secreted surfactant protein opsonins SPA and SPD, surfactant phospholipids, and the molecular pattern-recognition receptor TLR2.15,16,54,74 Therefore, compared with their wildtype (WT) counterparts, SPA-deficient mice infected with either M. pulmonis or M. pneumoniae develop more severe inflammation and have decreased capacity to clear these infections from the lungs.43 In addition, TLR2-deficient mice exhibit decreased clearance and increased inflammation in response to mycoplasma infection.60,104Second, we wanted to study the effects of SRA deficiency in mycoplasmosis. The class A scavenger receptor (SRA) modulates inflammatory responses and mediates the clearance of airborne oxidants, particulates, and respiratory pathogens.3,17,18,49,88,101 Inhibition of SRA expression in alveolar macrophages in an elastase–LPS model of COPD was associated with decreased clearance of Haemophilus influenzae.33 Lack of SRA similarly impaired alveolar macrophage-mediated clearance of Streptococcus pneumoniae,5 environmental particles,6 and ozone-oxidized lipids18 by alveolar macrophages. Absence of SRA also enhanced hyperoxia-induced lung injury49 and exacerbated inflammation in response to Staphylococcus aureus infection.88 SRA appears to have antiinflammatory properties with the capacity to modify macrophage phenotype and suppress polarization toward the M1 alternative macrophage activation state.13 The SRA gene (MSR1) is polymorphic in both mice and humans.19,29,105 Genetic association studies in humans, however, showed that subjects with truncations or point mutations in MSR1 have significantly increased risk for the development of pulmonary diseases such as COPD33,38,71,94 and asthma.5 Our understanding of the immune factors that contribute to mycoplasmosis is far from complete.In the present study, by investigating the role of SRA in mycoplasmosis jointly with the effects of housing, we demonstrated that genetic and environmental factors both serve as critical players in disease progression. We show that SRA-deficient mice are susceptible to chronic colonization with M. pulmonis and development of chronic mycoplasma-induced bronchopneumonia characterized by persistent multicellular inflammation. Furthermore, we show that housing conditions influence the effect of SRA deficiency on the severity of mycoplasmosis. Taken together, these results indicate that lack of SRA function impairs host protection against both infectious and environmental insults. 相似文献
18.
Jisun Paik Yvette Fierce Rolf Drivdahl Piper M Treuting Audrey Seamons Thea Brabb Lillian Maggio-Price 《Comparative medicine》2010,60(3):189-195
Murine norovirus (MNV) is prevalent in SPF mouse facilities in the United States, and we currently lack sufficient data to determine whether it should be eliminated. It is generally accepted that the virus does not cause clinical symptoms in immunocompetent mice. However, we previously reported that MNV infection alters the phenotype of a mouse model of bacteria-induced inflammatory bowel disease in part through its effects on dendritic cells. The tropism of MNV toward macrophages and dendritic cells makes MNV a potential intercurrent variable in murine models of macrophage-driven inflammatory diseases, such as obesity, insulin resistance, and atherosclerosis. Therefore, we determined whether MNV infection altered obesity and insulin resistance phenotypes in C57BL/6 mice, a widely used model of diet-induced obesity. We found that MNV did not alter weight gain, food intake, and glucose metabolism in this model, but it did induce subtle changes in lymphoid tissue. Further studies using other models of metabolic diseases are needed to provide additional information on the potential role this ‘subclinical’ virus might have on disease progression in mouse models of inflammatory diseases.Abbreviations: HFD, high-fat diet; IPGTT, intraperitoneal glucose tolerance test; IPITT, intraperitoneal insulin tolerance test; MLN, mesenteric lymph node; MNV, murine norovirusMurine norovirus (MNV) is endemic in many SPF mouse colonies across North America,5 creating considerable potential for this virus to interfere with mouse models of human diseases. In addition, the presence of MNV in some mouse colonies and not in others may help explain phenotypic variability in mouse models across institutions. This virus is related to the human Norwalk virus that causes gastrointestinal inflammation in humans. Although MNV does not cause any overt illness in immunocompetent mice, significant inflammation and mortality can be induced in mice with abnormal innate immunity.7 Previously, we investigated the influence of MNV on the development of bacteria-induced inflammatory bowel disease in FVB.129P2-PAbcb1atm1Bor (Mdr1a−/−) mice.8 We found that infection with MNV accelerated the progression of inflammatory bowel disease in this mouse model when mice were coinfected with Helicobacter bilis. In addition, infection with MNV alone altered the immune response, probably through changes in dendritic cells.8 These findings suggest that MNV may induce subtle changes in immune responses even in immunocompetent mice, given that MNV is known to preferentially infect macrophages and dendritic cells.22Obesity has been defined as a disease of chronic inflammation, and in recent years, the prominent role that macrophages play in this process has been recognized.9,10,21,24 Obesity is a risk factor for various chronic diseases that share inflammation as a critical component of the disease process, such as metabolic syndrome, diabetes, and atherosclerosis.3 Because MNV has tropism for macrophages, we wished to determine whether MNV infection influences the development of obesity and insulin resistance in a widely used animal model of diet-induced obesity. C57BL/6 mice are the most frequently used ‘wild-type’ strain and are prone to develop insulin resistance as obesity develops during high-fat feeding.1 We hypothesized that MNV may accelerate inflammation by stimulating macrophage accumulation in adipose tissue, resulting in a more severe obesity or insulin resistance phenotype when mice are fed a high-fat diet. 相似文献
19.
20.
M Shen L Wang B Wang T Wang G Yang L Shen T Wang X Guo Y Liu Y Xia L Jia X Wang 《Cell death & disease》2014,5(11):e1528
Endoplasmic reticulum (ER) stress occurring in stringent conditions is critically involved in cardiomyocytes apoptosis and cardiac contractile dysfunction (CCD). However, the molecular machinery that mediates cardiac ER stress and subsequent cell death remains to be fully deciphered, which will hopefully provide novel therapeutic targets for these disorders. Here, we establish tunicamycin-induced model of cardiomyocyte ER stress, which effectively mimicks pathological stimuli to trigger CCD. Tunicamycin activates volume-sensitive outward rectifying Cl− currents. Blockade of the volume-sensitive outwardly rectifying (VSOR) Cl− channel by 4,4''-diisothiocya-natostilbene-2,2''-disulfonic acid (DIDS), a non-selective Cl− channel blocker, and 4-(2-butyl-6,7-dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid (DCPIB), a selective VSOR Cl− channel blocker, improves cardiac contractility, which correlates with suppressed ER stress through inhibiting the canonical GRP78/eIF2α/ATF4 and XBP1 pathways, and promotes survival of cardiomyocytes by inverting tunicamycin-induced decrease of Wnt through the CHOP pathway. VSOR activation of tunicamycin-treated cardiomyocytes is attributed to increased intracellular levels of reactive oxygen species (ROS). Our study demonstrates a pivotal role of ROS/VSOR in mediating ER stress and functional impairment of cardiomyocytes via the CHOP-Wnt pathway, and suggests the therapeutic values of VSOR Cl− channel blockers against ER stress-associated cardiac anomalies.The endoplasmic reticulum (ER) is characterized as an organelle that participates in the folding of membrane and secretory proteins.1,2 Efficient functioning of the endoplasmic reticulum is important for cell function and survival. Perturbations of ER homeostasis by energy deprivation and glucose,3 viral infections4 and accumulation of misfolded and/or unfolded proteins2 interfere with ER function, leading to a state of ER stress.5, 6, 7 A cohort of chemicals, for example, tunicamycin and thapsigargin, also trigger ER stress.8, 9, 10 Thapsigargin disrupts the calcium storage of ER by blocking calcium reuptake into the ER lumen, thus by depleting calcium from the organelle.11 In particular, tunicamycin is a highly specific ER stress inducer by inhibiting N-linked glycosylation of protein, representing a well-documented method to artificially elicit unfolded protein response.8 In response to ER stress, ER chaperones such as glucose-regulated protein 78 kDa (GRP78) and glucose-regulated protein 94 kDa (GRP94) are upregulated to facilitate the recovery of unfolded or misfolded proteins.12 ER stress may act as a defense mechanism against external insults; however, prolonged and/or severe ER stress may ultimately trigger apoptosis.8 The C/EBP homologous protein (CHOP) has been defined as a pivotal mediator of cell death signaling in ER stress.13, 14 Accumulating evidence has demonstrated that ER stress-induced cell death is an essential step in the pathogenesis of a wide variety of cardiovascular diseases such as ischemia reperfusion heart diseases,15 atherosclerosis,5, 16, 17, 18 myocardial infarction,19 hypertension20, 21 and heart failure.8, 22, 23 Inhibiting ER stress has great therapeutic values for cardiac anomalies. However, the precise mechanism involved in ER stress-induced cardiovascular diseases has not been well identified, which impedes the translation of our understanding of ER stress-induced cardiovascular anomalies into effective therapeutic strategies. Apoptosis induction requires persistent cell shrinkage, named apoptotic volume decrease (AVD).24, 25, 26, 27 It is an early prerequisite for the activation of caspases.24 In various types of cells including cardiomyocytes, AVD process is accomplished by the activation of volume-sensitive outwardly rectifying (VSOR) Cl− channel and is concomitant with the egress of water from the cells undergoing mitochondrion-initiated or death receptor-induced apoptosis.25, 28, 29, 30 Although inhibition of VSOR Cl− channel by DIDS (4,4''-diisothiocyanatostilbene-2,2''-disulphonic acid) and DCPIB (4-(2-butyl-6,7- dichlor-2-cyclopentyl-indan-1-on-5-yl) oxybutyric acid) blocked AVD and rescued cardiomyocytes from mitochondrial and death receptor pathway-induced apoptosis,31, 32 it remains largely unknown concerning the role of VSOR Cl− channel and how it is regulated in ER stress-induced apoptotic cardiomyocyte death.Emerging evidence indicates that Wnt signal pathways are found to be anti-apoptotic in the cardiovascular diseases,33, 34, 35 regulating crucial aspects of cardiovascular biology. However, up to now, its activity in ER stress-induced apoptosis and in the process of AVD in cardiomyocytes remains elusive.In the present study, we probed the role of VSOR Cl− channel in ER stress-induced apoptosis of cardiomyocytes, which intimately correlates with cardiac contractile dysfunction (CCD). We hypothesized that VSOR Cl− channel controls the process of AVD occurring concomitantly with ER stress-induced apoptosis of cardiomyocytes. To test this hypothesis, we investigated VSOR Cl− currents in cardiomyocytes treated with the ER stress inducer tunicamycin. The pathophysiological role of VSOR Cl− channel and the potential signaling mechanisms in the development of ER stress-induced apoptosis in CCD were also dissected. 相似文献