Ah-Mee Park

Heat Shock Protein 27 Plays a Pivotal Role in Myofibroblast Differentiation and in the Development of Bleomycin-Induced Pulmonary Fibrosis

Heat shock protein 27 (HSP27) is a member of the small molecular weight HSP family. Upon treatment with transforming growth factor β1 (TGF-β1), we observed upregulation of HSP27 along with that of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation, in cultured human and mouse lung fibroblasts. Furthermore, by using siRNA knockdown, we demonstrated that HSP27 was involved in cell survival and upregulation of fibronectin, osteopontin (OPN) and type 1 collagen, all functional markers of myofibroblast differentiation, in TGF-β1-treated MRC-5 cells. In lung tissues of bleomycin-treated mice, HSP27 was strongly upregulated and substantially co-localized with α-SMA, OPN and type I collagen but not with proSP-C (a marker of type II alveolar epithelial cells), E-cadherin (a marker of epithelial cells) or F4/80 (a marker of macrophages). A similar co-localization of HSP27 and α-SMA was observed in lung tissues of patients with idiopathic pulmonary fibrosis. Furthermore, airway delivery of HSP27 siRNA effectively suppressed bleomycin-induced pulmonary fibrosis in mice. Collectively, our findings indicate that HSP27 is critically involved in myofibroblast differentiation of lung fibroblasts and may be a promising therapeutic target for lung fibrotic diseases.

IL-35 Suppresses Lipopolysaccharide-Induced Airway Eosinophilia in EBI3-Deficient Mice.

EBI3 functions as the subunit of immune-regulatory cytokines, such as IL-27 and IL-35, by pairing with p28 and p35, respectively. We treated wild-type and EBI3-deficient mice with intratracheal administration of LPS and obtained bronchoalveolar lavage fluid (BALF) 24 h later. Although neutrophils were the predominant cells in BALF from both groups of mice, eosinophils were highly enriched and there was increased production of eosinophil-attracting chemokines CCL11 and CCL24 in BALF of EBI3-deficient mice. The bronchial epithelial cells and alveolar macrophages were the major producers of CCL11 and CCL24. Because no such increases in eosinophils were seen in BALF of p28/IL-27–deficient mice or WSX-1/IL-27Rα subunit-deficient mice upon intratracheal stimulation with LPS, we considered that the lack of IL-35 was responsible for the enhanced airway eosinophilia in EBI3-deficient mice. In vitro, IL-35 potently suppressed production of CCL11 and CCL24 by human lung epithelial cell lines treated with TNF-α and IL-1β. IL-35 also suppressed phosphorylation of STAT1 and STAT3 and induced suppressor of cytokine signaling 3. In vivo, rIL-35 dramatically reduced LPS-induced airway eosinophilia in EBI3-deficient mice, with concomitant reduction of CCL11 and CCL24, whereas neutralization of IL-35 significantly increased airway eosinophils in LPS-treated wild-type mice. Collectively, our results suggest that IL-35 negatively regulates airway eosinophilia, at least in part by reducing the production of CCL11 and CCL24.

Helicobacter pylori and gut microbiota in multiple sclerosis versus Alzheimer's disease: 10 pitfalls of microbiome studies

Alteration of microbiota has been associated with intestinal, inflammatory and neurological diseases. An abundance of “good bacteria,” such as Bifidobacterium, or their products has been generally believed to be beneficial for any diseases, whereas “bad bacteria,” such as pathogenic Helicobacter pylori, are assumed to be always detrimental for hosts. However, this is not the case when we compare and contrast the association of the gut microbiota with two neurological diseases, multiple sclerosis and Alzheimers disease. After H. pylori infection, pro‐inflammatory T helper (Th)1 and Th17 immune responses are initially induced to eradicate bacteria. However, H. pylori evades the host immune response by inducing Th2 cells and regulatory T cells that produce anti‐inflammatory interleukin‐10. Suppression of anti‐bacterial Th1/Th17 cells by regulatory T cells might enhance gastric H. pylori propagation, followed by a cascade reaction involving vitamin B12 and folic acid malabsorption, plasma homocysteine elevation, and reactive oxygen species induction. This can damage the blood‐brain barrier, leading to accumulation of amyloid‐β in the brain, a hallmark of Alzheimers disease. In contrast, this suppression of pro‐inflammatory Th1/Th17 responses to H. pylori has protective effects on the hosts, as it prevents uncontrolled gastritis as well as suppresses the induction of encephalitogenic Th1/Th17 cells, which can mediate neuroinflammation in multiple sclerosis. The above scenario might explain why chronic H. pylori infection is positively associated with Alzheimers disease, whereas it is negatively associated with multiple sclerosis. Finally, we list “10 pitfalls of microbiota studies,” which will be useful for evaluating and designing clinical and experimental microbiota studies.

Three immune-mediated disease models induced by Theiler's virus: Multiple sclerosis, seizures and myocarditis

Theilers murine encephalomyelitis virus (TMEV) infection has been used as a viral model for multiple sclerosis (MS), as TMEV can induce chronic inflammatory demyelinating lesions with viral persistence in the spinal cord of SJL/J mice. In contrast, when C57BL/6 mice are infected with TMEV, the mice can clear the virus from the central nervous system (CNS), without viral persistence or demyelination, but develop seizures and hippocampal sclerosis, which has been used as a viral model for seizures/epilepsy. In the two TMEV‐induced CNS disease models, not only viral infection, but also immune responses contribute to the pathogenesis. Interestingly, acquired immunity plays an effector role in the MS model, whereas innate immunity appears to contribute to the development of seizures. Recently, we have established the third TMEV‐induced disease model, a mouse model for viral myocarditis, using C3H mice. TMEV‐induced myocarditis is a triphasic disease, which mimics human myocarditis; phase I, mediated by viral replication in the heart and innate immunity; phase II, mediated by acquired immunity; and phase III, resulted from cardiac fibrosis. The genetic susceptibility to the aforementioned three models (MS, seizures and myocarditis) differs among mouse strains. We have compared and contrasted the three models induced by one single pathogen, TMEV, particularly in regard to the roles of T helper cells and natural killer T cells, which will give an insight into how interactions between the immune system and the hosts genetic background determine the tissue tropism of virus and the development of virus‐induced organ‐specific immunopathology.

Murine γ-Herpesvirus 68 Induces Severe Lung Inflammation in IL-27–Deficient Mice with Liver Dysfunction Preventable by Oral Neomycin

IL-27 is an immunoregulatory cytokine consisting of p28 and EBI3. Its receptor also has two subunits, WSX1 and gp130. Although IL-27 promotes Th1 differentiation in naive T cells, it also induces IL-10 expression in effector Th1 cells to curtail excessive immune responses. By using p28-deficient mice and WSX1-deficient mice (collectively called IL-27–deficient mice), we examined the role of IL-27 in primary infection by murine γ-herpesvirus 68 (MHV68), a murine model of EBV. Upon airway infection with MHV68, IL-27–deficient mice had more aggravated lung inflammation than wild-type mice, although MHV68 infection per se was better controlled in IL-27–deficient mice. Although epithelial cells and alveolar macrophages were primarily infected by MHV68, interstitial macrophages and dendritic cells were the major producers of IL-27. The lung inflammation of IL-27–deficient mice was characterized by more IFN-γ–producing CD8+ T cells and fewer IL-10–producing CD8+ T cells than that of wild-type mice. An infectious mononucleosis–like disease was also aggravated in IL-27–deficient mice, with prominent splenomegaly and severe hepatitis. Infiltration of IFN-γ–producing effector cells and upregulation of the CXCR3 ligand chemokines CXCL9, CXCL10, and CXCL11 were noted in the liver of MHV68-infected mice. Oral neomycin effectively ameliorated hepatitis, with decreased production of these chemokines in the liver, suggesting that the intestinal microbiota plays a role in liver inflammation through upregulation of these chemokines. Collectively, IL-27 is essential for the generation of IL-10–producing effector cells in primary infection by MHV68. Our findings may also provide new insight into the mechanism of hepatitis associated with infectious mononucleosis.

Forensic luminol reaction for detecting fecal occult blood in experimental mice

Fecal occult blood (FOB) is a sign of gastrointestinal diseases, such as intestinal ulcers and colorectal cancer. In experimental animal studies, there is no standard method to detect FOB. Here, we present a simple protocol to detect FOB in mice, using the Luminol Reaction Experiment Kit® that was originally designed to detect bloodstains at a crime scene in criminal forensics. To obtain positive control bloody feces, we used an indomethacin-induced intestinal ulcer model in mice. By mixing small pieces of feces with a luminol solution, the fecal solution emitted visible blue–white chemiluminescence in dark field when feces contained hemoglobin. We also established a method for semi-quantification of hemoglobin content in the fecal solution, using a luminometer. This method is simple, quick, economical and semi-quantitative, allowing researchers to detect FOB in experimental mice.

T-bet, but not Gata3, overexpression is detrimental in a neurotropic viral infection

Intracerebral Theiler’s murine encephalomyelitis virus (TMEV) infection in mice induces inflammatory demyelination in the central nervous system. Although C57BL/6 mice normally resistant to TMEV infection with viral clearance, we have previously demonstrated that RORγt-transgenic (tg) C57BL/6 mice, which have Th17-biased responses due to RORγt overexpression in T cells, became susceptible to TMEV infection with viral persistence. Here, using T-bet-tg C57BL/6 mice and Gata3-tg C57BL/6 mice, we demonstrated that overexpression of T-bet, but not Gata3, in T cells was detrimental in TMEV infection. Unexpectedly, T-bet-tg mice died 2 to 3 weeks after infection due to failure of viral clearance. Here, TMEV infection induced splenic T cell depletion, which was associated with lower anti-viral antibody and T cell responses. In contrast, Gata3-tg mice remained resistant, while Gata3-tg mice had lower IFN-γ and higher IL-4 production with increased anti-viral IgG1 responses. Thus, our data identify how overexpression of T-bet and Gata3 in T cells alters anti-viral immunity and confers susceptibility to TMEV infection.

Bioinformatics analyses determined the CNS and peripheral lymphoid surrogate biomarker candidates between two distinct EAE models for progressive multiple sclerosis

SJL Spleen Heat maps of most highly upor down-regulated genes in the brains and spleens of mice during the disease peak of PP-EAE. Heat maps were drawn based on brain microarray data of A.SW mice (A) or SJL/J mice (B), or spleen microarray data of A.SW mice (C) or SJL/J mice (D). Overall, heat maps between A.SW and SJL/J mice were similar in the brains and spleens. Inflammation-related genes, such as Lcn2, Cxcl13, and Ccl3, were upregulated in the brains of both mouse strains, while erythrocyterelated genes, such as Gypa, Kel, and Rhd, were downregulated in the spleens. Heat map: the expression patterns of highly upor down-regulated genes are similar between the two strains and different between the brains and spleens

Viral infection activates myelin-specific T cells, triggering MS-like CNS inflammatory demyelination

Background: Multiple sclerosis (MS) has been suggested to be triggered by microbial infections in genetically susceptible hosts harboring anti-myelin autoimmune T cells. Myelin oligodendrocyte glycoprotein (MOG)-specific T cell receptor (TCR) transgenic (tg) 2D2 mice develop experimental autoimmune encephalomyelitis (EAE) following MOG sensitization. In contrast, only 4% of 2D2-tg mice develop spontaneous EAE with mild inflammation in the central nervous system (CNS) after 3 months of age. Objective: To determine whether microbial infection can activate MOG-specific T cells as an adjuvant and confer susceptibility to EAE in 2D2-tg mice. Materials and Methods: We injected 6-week-old 2D2-tg or wild-type C57BL/6 mice intraperitoneally with the following microbes and microbe mimics: Theiler’s murine encephalomyelitis virus (TMEV, RNA virus), poly(I:C) (RNA virus mimic), murine cytomegalovirus (MCMV, DNA virus), and curdlan (bacteria/fungus component, Th17 inducer). Results: During the 2-months observation period, intraperitoneal TMEV injection induced hind-limb paralysis in 43% of 2D2-tg mice (mean onset 36.7 ± 9.6 days) with severe inflammatory demyelination and axonal degeneration in the CNS. Among other groups, only a few 2D2-tg mice injected with MCMV or curdlan had mild CNS involvement. Intracerebral injection of TMEV was more effective for inducing CNS disease; 83% of 2D2-tg mice developed severe inflammatory demyelination with earlier onset time (13.1 ± 1.7 days). No wild-type mice developed CNS demyelination following any treatment (Note: C57BL/6 mice are TMEV-resistant, while susceptible SJL/J mice develop demyelination 1 month after intracerebral, but not intraperitoneal, TMEV infection). Conclusions: Virus infection may activate anti-myelin T cells as an adjuvant, triggering CNS inflammatory demyelination. Abstract

Influx and Efflux of Immune Cells in the Central Nervous System

The influx and efflux of immune cells in the central nervous system (CNS) have long remained to be unclear. In this regard, we have addressed this issue using animal models of CNS tumors and experimental autoimmune encephalitis (EAE) that is relevant to multiple sclerosis (MS) in human. For immune cells to migrate into brain parenchyma, chemokines play central roles. In contrast, when immune cells exit the brain parenchyma, lymphatic system plays central roles. Most recently, the relationship between the CNS immunology and gut microbiome is being addressed using the same systems.