Mice with Homozygous Disruption of the mdr2 P-Glycoprotein Gene A Novel Animal Model for Studies of Nonsuppurative Inflammatory Cholangitis and Hepatocarcinogenesis

The mouse mdr2gene (and its human homologue MDR3, also called MDR2) encodes a P-glycoprotein that is present in high concentration in the bile canalicular membrane of hepatocytes. The 129/OlaHsd mice with a homozygous disruption of the mdr2 gene (-/-mice) lack this P-glycoprotein in the canalicular membrane. These mice are unable to secrete phospholipids into bile, showing an essential role for the mdr2 P-glycoprotein in the transport of phosphatidylcholine across the canalicular membrane. The complete absence of phospholipids from bile leads to a hepatic disease, which becomes manifest shortly after birth and shows progression to an end stage in the course of 3 months. The liver pathology is that of a nonsuppurative inflammatory cholangitis with portal inflammation and ductular proliferation, consistent with toxic in-jury of the biliary system from bile salts unaccompanied by phospholipids. Thus, the mdr2 (-/-) mice can serve as an animal model for studying mechanisms and potential interventions in nonsuppurative inflammatory cholangitis (in a generic sense) in human disease, be it congenital or acquired. When the mice are 4 to 6 months of age, preneoplastic lesions develop in the liver, progressing to metastatic liver cancer in the terminal phase. The mdr2 (-/-) mice therefore also provide a tumor progression model of value for the study of hepatic carcinogenesis. Interestingly, also in this regard, the model mimicks human disease, because chronic inflammation of the biliary system in humans may similarly carry increased cancer risk.     

Role of Gamma Interferon in Helicobacter pylori-Induced Gastric Inflammatory Responses in a Mouse Model

The immune responses to Helicobacter pylori infection play important roles in gastroduodenal diseases. The contribution of gamma interferon (IFN-γ) to the immune responses, especially to the induction of gastric inflammation and to protection from H. pylori infection, was investigated with IFN-γ gene knockout (IFN-γ^−/−) mice. We first examined the colonizing abilities of eight H. pylori strains with a short-term infection test in order to select H. pylori strains which could colonize the mouse stomach. Only three strains (ATCC 43504, CPY2052, and HPK127) colonized C57BL/6 wild-type mice, although all of the strains except for ATCC 51110 could colonize IFN-γ^−/− mice. The number of H. pylori organisms colonizing the stomach in wild-type mice was lower than that in IFN-γ^−/− mice. Oral immunization with the CPY2052 sonicate and cholera toxin protected against infection with strain CPY2052 in both types of mouse. These findings suggested that IFN-γ may play a protective role in H. pylori infection, although the degree of its protective ability was estimated to be low. In contrast, in a long-term infection test done to examine the contribution of IFN-γ to gastric inflammation, CPY2052-infected wild-type mice developed a severe infiltration of mononuclear cells in the lamina propria and erosions in the gastric epithelium 15 months after infection, whereas CPY2052-infected IFN-γ^−/− mice showed no inflammatory symptoms. This result clearly demonstrated that IFN-γ plays an important role in the induction of gastric inflammation caused by H. pylori infection.     

Filifactor alocis Infection and Inflammatory Responses in the Mouse Subcutaneous Chamber Model

Recent microbiome studies have implicated a role for Filifactor alocis in periodontal disease. In this study, we investigated the colonization and survival properties of F. alocis in a mouse subcutaneous chamber model of infection and characterized host innate immune responses. An infection of 10⁹ F. alocis successfully colonized all chambers; however, the infection was cleared after 72 h. F. alocis elicited a local inflammatory response with neutrophils recruited into the chambers at 2 h postinfection along with an increase in levels of the proinflammatory cytokines interleukin 1β (IL-1β), IL-6, and tumor necrosis factor (TNF). F. alocis also induced apoptosis in chamber epithelial cells and neutrophils. Consistent with resolution of infection, neutrophil numbers and cytokine levels returned to baseline by 72 h. Fluorescent in situ hybridization (FISH) and quantitative PCR demonstrated that F. alocis exited the chambers and spread to the spleen, liver, lung, and kidney. Massive neutrophil infiltration was observed in the spleen and lungs, and the recruited neutrophils were in close proximity to the infecting bacteria. Significant epithelial injury was observed in the kidneys. Infection of all tissues was resolved after 7 days. This first in vivo study of the pathogenicity of F. alocis shows that in the chamber model the organism can establish a proinflammatory, proapoptotic local infection which is rapidly resolved by the host concordant with neutrophil influx. Moreover, F. alocis can spread to, and transiently infect, remote tissues where neutrophils can also be recruited.     

Characterization of Clinical and Immune Responses in an Experimental Chronic Autoimmune Uveitis Model

Autoimmune uveitis is a sight-threatening intraocular inflammatory disease. For >30 years, the mouse model of experimental autoimmune uveitis has been employed to investigate disease mechanisms and test immunotherapeutic approaches. However, inflammation in this model is self-limited, and does not replicate the chronic, insidious nature prevalent in the human disease. Herein, a robust and reliable model of chronic autoimmune uveitis was developed and characterized in two strains of wild-type mice by modifying interphotoreceptor retinoid-binding protein dose and peptide fragments from conventional experimental autoimmune uveitis models. In both of these murine strains, immunization with our modified protocols resulted in a slowly progressive uveitis, with retinal scars and atrophy observed in the chronic stage by fundoscopy. Optical coherence tomography demonstrated decreased retinal thickness in chronic autoimmune uveitis mice, and electroretinography showed significantly reduced amplitudes of dark-adapted a- and b-waves and light-adapted b-waves. Histologic examination revealed prominent choroiditis with extensive retinal damage. Flow cytometry analysis showed substantially increased numbers of CD44^hiIL-17⁺IFN-γ⁻ memory T-helper 17 (Th17) cells in the retina, cervical lymph nodes, inguinal lymph nodes, and spleen. These data establish new modified protocols for inducing chronic uveitis in wild-type mice, and demonstrate a predominant memory Th17 cell response, suggesting an important role for memory Th17 cells in driving chronic inflammation in autoimmune uveitis.

Uveitis is a sight-threatening inflammatory condition of the uveal tract of the eye, comprising the iris, ciliary body, and choroid.¹ Responsible for an estimated 30,000 new cases of legal blindness each year in the United States, uveitis and its complications cause approximately 10% of irreversible vision loss.2, 3, 4, 5 The socioeconomic impact of uveitis is notable because as blindness frequently occurs in individuals of working age.⁶ In contrast to acute uveitis, which often resolves after a short course of topical corticosteroids, chronic uveitis is characterized by active inflammation that persists for at least several months, and frequently years. The persistent inflammation and its long-term requirement for corticosteroids lead to a high incidence of complications that endanger vision, including glaucoma, cataract, optic disc atrophy, and chorioretinal scars.⁷Experimental animal models have been used to recapitulate the clinical phenotype of uveitis, and have led to significant advances in our understanding of the cellular and molecular mechanisms that cause autoimmune uveitis.⁸ For example, these studies have established the critical roles of pathogenic effector T-helper 1 (Th1) and T-helper 17 (Th17) cells in the immunopathogenesis of uveitis.8, 9, 10 However, most preclinical research on autoimmune uveitis has investigated acute disease, but not the development and maintenance of chronic autoimmune uveitis (CAU).¹¹ This is because, in the widely used mouse experimental autoimmune uveitis (EAU) model, disease peaks in the first 2 to 3 weeks after immunization before resolving spontaneously.12, 13, 14, 15 Yet, as discussed, it is CAU that is associated with the greatest morbidity and risk of sight-threatening complications in the clinic, underscoring a pressing need for the development of a chronic uveitis animal model.⁷In the present study, a reliable murine model of chronic experimental autoimmune uveitis was established and characterized in two wild-type mouse strains, which exhibit clinical features consistent with noninfectious uveitis observed in humans. Furthermore, in contrast to early acute uveitis in which the immune response is characterized by both effector Th1 and effector Th17 cells, we demonstrate that CAU is primarily characterized by a robust memory Th17 response in both the retina and peripheral lymphoid compartments.     

Detection of Alterations in the Levels of Neuropeptides and Salivary Gland Responses in the Non-Obese Diabetic Mouse Model for Autoimmune Sialoadenitis

The salivary glands of non-obese diabetic (NOD) mice and BALB/c controls were evaluated for the stimulatory effects of the following neuropeptides; substance P (SP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY). Injection of either of the three neuropeptides in combination with the muscarinic–cholinergic agonist pilocarpine increased saliva flow rates in BALB/c mice while there was no observable augmentation to flow rates in pre-diabetic or diabetic NOD mice. Small increases in protein content of the stimulated saliva were observed in the BALB/c group of animals with the injection of any of the above neuropeptides in combination with pilocarpine. In pre-diabetic NOD animals, only VIP and NPY increased the protein content-ratio above pilocarpine alone. Radioimmunoassay determination of neuropeptide concentrations in the submandibular and parotid glands revealed reduced levels of SP with diabetes onset as compared with pre-diabetic NOD or BALB/c mice. The levels of NPY were similar between BALB/c and NOD animals except in the pre-diabetic parotid gland where NPY concentrations were 1.3-fold greater. On the other hand, VIP concentrations were substantially reduced in the submandibular gland of NOD mice, while in the parotid gland neuropeptide levels were evaluated 3.8-fold relative to BALB/c controls. Immunohistochemical staining of the parotid and submandibular glands for SP revealed primarily ductal cell staining which was reduced with diabetes onset in NOD animals. These findings further define the sialoadenitis observed in NOD mice to be due, in part, to a general loss of neurotransmitter responsiveness on the part of salivary gland cells.     

Pattern of Cytokine Responses to Gram-Positive and Gram-Negative Commensal Bacteria is Profoundly Changed when Monocytes Differentiate into Dendritic Cells

Lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their health-promoting effects. In our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with Waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. According to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-Lactobacillus antibodies, human monoclonal antibody like (Ab1) and anti-idiotypic antibody (Ab2). In this study, BALB/ c mice were immunized with two doses of bacteria Lactobacillus acidophilus in complete and incomplete Freund's adjuvant and phosphate-buffered saline (PBS), respectively. Seven days after the last immunization, sera from immunized mice were collected and the presence of Lactobacillus-specific Ab1 and Ab2 were determined by ELISAs. In the sera of immunized mice, antibodies specific to bacteria Lactobacillus acidophilus were shown. The concentration of Lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of IgM DJ) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). Moreover, Ab1 and Ab2 antibodies were detected in the sera of Lactobacillus-hyperimmunized mice. In this study, we have shown the idiotypic network interactions in mice immunized with bacteria Lactobacillus acidophilus .     

Tyrosine Kinase Inhibitors Ameliorate Autoimmune Encephalomyelitis in a Mouse Model of Multiple Sclerosis

Multiple sclerosis is an autoimmune disease of the central nervous system characterized by neuroinflammation and demyelination. Although considered a T cell-mediated disease, multiple sclerosis involves the activation of both adaptive and innate immune cells, as well as resident cells of the central nervous system, which synergize in inducing inflammation and thereby demyelination. Differentiation, survival, and inflammatory functions of innate immune cells and of astrocytes of the central nervous system are regulated by tyrosine kinases. Here, we show that imatinib, sorafenib, and GW2580—small molecule tyrosine kinase inhibitors—can each prevent the development of disease and treat established disease in a mouse model of multiple sclerosis. In vitro, imatinib and sorafenib inhibited astrocyte proliferation mediated by the tyrosine kinase platelet-derived growth factor receptor (PDGFR), whereas GW2580 and sorafenib inhibited macrophage tumor necrosis factor (TNF) production mediated by the tyrosine kinases c-Fms and PDGFR, respectively. In vivo, amelioration of disease by GW2580 was associated with a reduction in the proportion of macrophages and T cells in the CNS infiltrate, as well as a reduction in the levels of circulating TNF. Our findings suggest that GW2580 and the FDA-approved drugs imatinib and sorafenib have potential as novel therapeutics for the treatment of autoimmune demyelinating disease.     

Model Analysis of Tissue Responses to Transient and Chronic Heating

Thermal models are used to analyze responses of muscle and lung tissue to transient (30–45 min) and chronic (4–7 week) heating in vivo. The general bioheat model, which describes one-dimensional temperature dynamics, incorporates heat conductance and perfusion. In general, perfusion changes with time and distance from a heated surface. One of the main objectives of this study was to analyze long-term perfusion change, which reflects tissue adaptation associated with angiogenesis. The database for these models was obtained using heated disks implanted in calves for up to seven weeks. Tissue temperature distributions were obtained repeatedly from thermistors protruding 1 to 10 mm from the heated disk surface. The perfusion parameter was estimated from the transient experiments at least several times each week by nonlinear, least-squares fitting of the model predicted temperature to the measured temperature response.Chronic heating at a heat flux 0.08 W/cm² caused perfusion of muscle tissue to increase with postimplant day (PID). Under the same conditions, lung tissue perfusion increased with chronic heating from early to late PID, but less than that for muscle tissue. During chronic heating above 42 °C and below 50 °C, a decrease in tissue temperature is associated with higher perfusion that develops with time. Over seven weeks, perfusion of muscle tissue near the heated disk surface increased by about 70% at 0.08 W/cm² and 40% at 0.06 W/cm². Furthermore, the model can be used to predict tissue and perfusion changes continuously over weeks for heat fluxes around 0.08 W/cm². © 2003 Biomedical Engineering Society. PAC2003: 8719Pp, 8719Uv, 8710+e, 8719Ff     

Transthyretin Is Dysregulated in Preeclampsia,and Its Native Form Prevents the Onset of Disease in a Preclinical Mouse Model

Preeclampsia is a major pregnancy complication with potential short- and long-term consequences for both mother and fetus. Understanding its pathogenesis and causative biomarkers is likely to yield insights for prediction and treatment. Herein, we provide evidence that transthyretin, a transporter of thyroxine and retinol, is aggregated in preeclampsia and is present at reduced levels in sera of preeclamptic women, as detected by proteomic screen. We demonstrate that transthyretin aggregates form deposits in preeclampsia placental tissue and cause apoptosis. By using in vitro approaches and a humanized mouse model, we provide evidence for a causal link between dysregulated transthyretin and preeclampsia. Native transthyretin inhibits all preeclampsia-like features in the humanized mouse model, including new-onset proteinuria, increased blood pressure, glomerular endotheliosis, and production of anti-angiogenic factors. Our findings suggest that a focus on transthyretin structure and function is a novel strategy to understand and combat preeclampsia.Preeclampsia occurs in 5% to 8% of pregnancies worldwide and is a major cause of fetal and maternal morbidity and mortality.^1–3 It is a heterogeneous disease with varied presentations from mild self-limited hypertension and proteinuria to severe forms with significant end-organ dysfunction and HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets).³ Although the cause of preeclampsia and its appropriate treatment remain elusive, this syndrome has been proposed to reflect at least two stages of complications during pregnancy. These begin with preclinical manifestations at the maternal-fetal interface, followed by systemic clinical symptoms.^1,2 Hypertension, proteinuria, and edema, with a variable degree of fetal growth restriction, are the cardinal features of preeclampsia.³ Because the placenta is the nutritional and immunological gateway to normal fetal development and pregnancy outcome, placenta-related events are believed to be central to the pathogenesis of this disease. Evidence exists for the release of disease-initiating molecules into maternal circulation that triggers the clinical symptoms.^1,4 Placental and systemic anomalies reflected by circulating placental debris, inflammation, impaired remodeling of spiral arteries, placental hypoxia/ischemia, excess production of anti-angiogenic factors [soluble fms-like tyrosine kinase-1 (sFlt-1)], and soluble endoglin (sEng), and angiotensin receptor autoantibodies have all emerged as contributors to the pathophysiological characteristics of preeclampsia.^2,4–14Preeclampsia has remained enigmatic because of lack of well-defined etiology and animal models. Although normal mice do not develop preeclampsia spontaneously, mouse models have been judged to be particularly useful to uterine diseases and pregnancy complications because many similarities in female reproduction and placentation have been identified between the two species.¹⁵ Moreover, their tractable genetics provide an effective way to probe mechanisms more deeply than many other species.^15–17 We recently showed that sera from preeclamptic women could function as a source of novel causative factors that induced hypertension, proteinuria, and kidney pathological characteristics, as well as intrauterine growth restriction (IUGR), in IL-10^−/− mice in a pregnancy-specific manner.¹⁸ IL-10 functions as a potent vascular and anti-inflammatory cytokine and has been shown to be present at significantly reduced levels in preeclampsia placental tissue.^19,20 Preeclampsia serum (PES) was found to disrupt endovascular cross talk between trophoblasts and endothelial cells and to induce placental hypoxia and excess production of sFlt-1 and sEng,¹⁸ soluble factors known to precipitate maternal symptoms.^21,22 These results from our serum-based humanized mouse model suggest that the pathophysiological characteristics of preeclampsia are more complex than previously thought and are likely to involve interactions and dysregulation of multiple factors. By using serum proteomic screening by surface-enhanced laser-desorption ionization-time-of-flight (SELDI-TOF), our results suggest that PES contains a reduced abundance of transthyretin, a plasma transport protein for the thyroid hormone, thyroxine, and retinol-binding protein.²³ More important, transthyretin has been widely studied for its role in amyloid diseases associated with protein misfolding and aggregation, resulting in deposits of toxic, fibrillar aggregates in specific organs.^24–26 Dysregulated or reduced transthyretin has also been implicated in Alzheimer disease, and overexpression of a wild-type human transthyretin transgene has been shown to ameliorate the disease in the transgenic murine model of human Alzheimer disease.^27,28 Transthyretin in its native form assumes a homotetrameric quaternary configuration (approximately 14 kDa per monomer). Post-translational modifications of the monomer result in detection of several isoforms.²⁹ Circulating transthyretin is also a validated marker of malnutrition and has a putative role in oocyte maturation and inflammation.^30–32 Although the presence of transthyretin during implantation in mice and in the placenta and trophoblasts in humans has been reported,^33,34 its functional role in normal pregnancy or adverse pregnancy outcomes has not been recognized. We hypothesize that transthyretin in preeclampsia is structurally and functionally dysregulated and contributes to the onset of this serious pregnancy complication. Herein, we present complementary in vitro and in vivo approaches, which show that endogenously altered transthyretin is a preeclampsia-causing agent and that native transthyretin has the ability to block the onset of preeclampsia-like features.     

Involvement of Innate Lymphoid Cells and Dendritic Cells in a Mouse Model of Chemical-induced Asthma

PurposeExposure to low concentrations of toluene diisocyanate (TDI) leads to immune-mediated chemical-induced asthma. The role of the adaptive immune system has already been thoroughly investigated; nevertheless, the involvement of innate immune cells in the pathophysiology of chemical-induced asthma is still unresolved. The aim of the study is to investigate the role of innate lymphoid cells (ILCs) and dendritic cells (DCs) in a mouse model for chemical-induced asthma.MethodsOn days 1 and 8, BALB/c mice were dermally treated (20 µL/ear) with 0.5% TDI or the vehicle acetone olive oil (AOO; 2:3). On days 15, 17, 19, 22 and 24, the mice received an oropharyngeal challenge with 0.01% TDI or AOO (1:4). One day after the last challenge, airway hyperreactivity (AHR) to methacholine was assessed, followed by an evaluation of pulmonary inflammation and immune-related parameters, including the cytokine pattern in bronchoalveolar lavage fluid, lymphocyte subpopulations of the lymph nodes and their ex vivo cytokine production profile, blood immunoglobulins and DC and ILC subpopulations in the lungs.ResultsBoth DC and ILC2 were recruited to the lungs after multiple airway exposures to TDI, regardless of the prior dermal sensitization. However, prior dermal sensitization with TDI alone results in AHR and predominant eosinophilic airway inflammation, accompanied by a typical type 2 helper T (Th2) cytokine profile.ConclusionsTDI-induced asthma is mediated by a predominant type 2 immune response, with the involvement of adaptive Th2 cells. However, from our study we suggest that the innate ILC2 cells are important additional players in the development of TDI-induced asthma.     

Shorter Dosing Intervals of Sublingual Immunotherapy Lead to More Efficacious Treatment in a Mouse Model of Allergic Inflammation

Current day practice of sublingual immunotherapy (SLIT) includes varying modalities of treatment that differ with regard to formulation, dosing and administration regimens. The aim of this study was to explore the importance of the dosing intervals in SLIT. The immunological effect of increased SLIT dosing frequency was tested in a mouse model of allergic inflammation. Mice sensitized to Phleum pratense (Phl p) were SLIT‐treated with the same weekly cumulative dose administered with different administration frequencies. A SLIT sham‐treated group was also included. All mice were challenged intra‐nasally with Phl p extract following SLIT. Local and systemic cytokine production, eosinophil infiltration into airways and the development of Phl p‐specific antibody responses were determined. Higher frequency of sublingual administration of allergen extract has a profound positive impact on the effect of SLIT, measured as induction of IgG and IgA antibodies. The once daily SLIT was the only treatment regimen being able to reduce all systemic Th2 cytokines and systemic IgE antibody responses when compared to sham‐treated mice after the intra‐nasal challenge period. The group receiving SLIT with the highest frequency of administration had the most pronounced effect of the treatment. In the same group, there was also a higher degree of protection against increase in IgE antibody levels after intra‐nasal challenge with the allergen, our data demonstrate that a once daily regimen is more efficacious than regimens where SLIT, with the same weekly cumulative allergen dose, is administered with longer intervals but higher doses.     

Progesterone Attenuates Brain Inflammatory Response and Inflammation-Induced Increase in Immature Myeloid Cells in a Mouse Model

Inflammation - Progesterone has been shown to regulate immunity during pregnancy, and progesterone administration may reduce inflammation-induced preterm labor. We sought to determine the maternal...