Kento Yoshioka

Mechanism for p38α-mediated experimental autoimmune encephalomyelitis

Background: p38 signaling pathway plays a key role in inflammatory diseases. Results: A single copy disruption of the p38α gene or a p38α inhibitor markedly reduced the pathogenesis of EAE by decreasing IL-17 production. Conclusion: p38α regulates the pathogenesis of EAE through transcriptional regulation of IL-17 production. Significance: Anti-p38α strategy achieves therapeutic benefit for the treatment of multiple sclerosis. One of the mitogen-activated protein kinases, p38, has been found to play a crucial role in various inflammatory responses. In this study, we analyzed the roles of p38α in multiple sclerosis, using an animal model, experimental autoimmune encephalomyelitis (EAE). p38α+/− mice (p38α−/− showed embryonic lethality) showed less severe neurological signs than WT mice. Adoptive transfer of lymph node cells (LNC) from sensitized WT mice with MOG(35–55) to naive WT-induced EAE was much more severe compared with the case using LNC from sensitized p38α+/− mice. Comprehensive analysis of cytokines from MOG(35–55)-challenged LNC by Western blot array revealed that production of IL-17 was significantly reduced by a single copy disruption of the p38α gene or a p38 inhibitor. Likewise, by a luciferase reporter assay, an electrophoresis mobility shift assay, and characterization of the relationship between p38 activity and IL-17 mRNA expression, we confirmed that p38 positively regulates transcription of the Il17 gene. Furthermore, oral administration of a highly specific p38α inhibitor (UR-5269) to WT mice at the onset of EAE markedly suppressed the progression of EAE compared with a vehicle group. These results suggest that p38α participates in the pathogenesis of EAE through IL-17 induction.

Therapeutic effect of lung mixed culture-derived epithelial cells on lung fibrosis

Cell-based therapy is recognized as one of potential therapeutic options for lung fibrosis. However, preparing stem/progenitor cells is complicated and not always efficient. Here, we show easily prepared cell populations having therapeutic capacity for lung inflammatory disease that are named as ‘lung mixed culture-derived epithelial cells’ (LMDECs). LMDECs expressed surfactant protein (SP)-C and gave rise to type I alveolar epithelial cells (AECs) in vitro and in vivo that partly satisfied type II AEC-like characteristics. An intratracheal delivery of not HEK 293 cells but LMDECs to the lung ameliorated bleomycin (BLM)-induced lung injury. A comprehensive analysis of bronchoalveolar fluid by western blot array revealed that LMDEC engraftment could improve the microenvironment in the BLM-instilled lung in association with stromal cell-derived factor-1 (SDF-1)/CXC chemokine receptor 4 signaling axis. SDF-1 enhanced both migration activity and differentiating efficiency of LMDECs. Further classification of LMDECs by flow cytometric study showed that a major population of LMDECs (LMDECMaj, 84% of total LMDECs) was simultaneously SP-C+, CD44+, CD45+, and hematopoietic cell lineage+ and that LMDECs included bronchioalveolar stem cells (BASCs) showing SP-C+Clara cell secretory protein+stem cell antigen (Sca)1+ as a small population (1.8% of total LMDECs). CD44+-sorted LMDECMaj and Sca1+-sorted LMDECs equally ameliorated fibrosis induced by BLM like LMDECs did. However, infiltrated neutrophils were observed in Sca1+-sorted LMDEC-treated alveoli that was not typical in LMDECMaj- or LMDEC-treated alveoli. These findings suggest that the protective effect of LMDECs against BLM-induced lung injury depends greatly on that of LMDECMaj. Furthermore, the cells expressing both alveolar epithelial and hematopoietic cell lineage markers (SP-C+CD45+) that have characteristics corresponding to LMDECMaj were observed in the alveoli of lung and increased approximately threefold in response to BLM instillation. Taken together, LMDECs newly classified in the present study are easily culture expanded and have a potential role in future regenerative cell therapy for pulmonary fibrosis.

Endothelin regulates function of IL-17-producing T cell subset.

AIMS Although endothelin (ET) is known to play pleiotropic roles in various pathological conditions, its relation to autoimmune disease has not been elucidated. Here, we focused on interleukin (IL)-17, which is closely related to the pathogenesis of multiple sclerosis, and investigated the effect of ET receptor blockers on the production of IL-17 by T lymphocytes. MAIN METHODS Lymph node cells from mice at 8 days post-immunization with MOG35-55 were stimulated in vitro with MOG35-55 in the presence or absence of an ET receptor blocker (BQ123 for ETA or BQ788 for ETB). Naïve T cells from mice were subjected to an in vitro model of Th17 differentiation, and ET-mediated IL-17 production was investigated under the states of Th17 differentiation and activation. KEY FINDINGS ELISA revealed that MOG35-55-induced IL-17 production was significantly inhibited by BQ123 but not BQ788. Consistent with the ELISA results for IL-17, the frequency of CD4(+) T cells producing IL-17 but not IFN-γ was reduced by BQ123. Under the differentiating state from naïve T cells to Th17 cells, the spontaneous release of IL-17 from CD4(+) T cells was increased, which was insensitive to BQ123, indicating that ET/ETA signaling did not affect Th17 differentiation. After the time period of Th17 differentiation, however, the increase in IL-17 production by restimulation of the cells with anti-CD3 plus anti-CD28 antibodies was significantly inhibited by BQ123. SIGNIFICANCE We demonstrated that ET/ETA signaling plays a crucial role in IL-17 production by Th17. BQ123 might be expected to be a future therapeutic drug for multiple sclerosis.

Hepatocyte nuclear factor 1β induced by chemical stress accelerates cell proliferation and increases genomic instability in mouse liver.

The liver has a considerable capacity of regeneration against the damage. The regulatory factors and molecular mechanism for the capacity are not fully appreciated. In developmental processes, hepatocyte nuclear factor 1β (HNF1β) is a cooperative factor for HNF6, which is a known stimulatory factor for hepatocyte proliferation after partial hepatectomy. We showed that carbon tetrachloride (CCl4)-induced liver injury up-regulated HNF1β, whereas the expression of HNF6 was not affected by the chemical stress, indicating unknown physiological roles of HNF1β against the chemical stress, not in cooperation with HNF6. To determine whether HNF1β has a novel function in the liver regeneration, we overexpressed HNF1β in the mouse liver by adenoviral gene delivery. We revealed that overexpression of HNF1β resulted in accelerated cell proliferation with the protein level up-regulation of plasminogen and plasmin, a converted active form of plasminogen, which play a pivotal role in liver regeneration inducing hepatocyte proliferation. Despite this stimulatory effect for the liver regeneration, HNF1β overexpression significantly increased genomic instability with decreased protein level of mediator of DNA damage checkpoint 1 (MDC1) and dephosphorylation of SP1 transcription factor. The increased expression of HNF1β is associated with several types of hepatocyte carcinomas, indicating possible involvement of the factor in carcinogenesis. Our data extend the current understanding of the mechanism underlying liver regeneration against chemical stress, and identified HNF1β as a novel regulatory factor in this mechanism and as a potential initiator for carcinogenesis.

p38 Mitogen-activated protein kinase accelerates emphysema in mouse model of chronic obstructive pulmonary disease.

Abstract Context: There are few short-term mouse models of chronic obstructive pulmonary disease (COPD) mimicking the human disease. In addition, p38 is recently recognized as a target for the treatment of COPD. However, the precise mechanism how p38 contributes to the pathogenesis of COPD is still unknown. Objective: We attempted to create a new mouse model for COPD by intra-tracheal administration of a mixture of lipopolysaccharide (LPS) and cigarette smoke solution (CSS), and investigated the importance of the p38 mitogen-activated protein kinase (p38) pathway in the pathogenesis of COPD. Methods: Mice were administered LPS + CSS once a day on days 0–4 and 7–11. Thereafter, CSS alone was administered to mice once a day on days 14–18. On day 28, histopathological changes of the lung were evaluated, and bronchoalveolar lavage fluid (BALF) was subjected to western blot array for cytokines. Transgenic (TG) mice expressing a constitutive-active form of MKK6, a p38-specific activator in the lung, were subjected to our experimental protocol of COPD model. Results: LPS + CSS administration induced enlargement of alveolar air spaces and destruction of lung parenchyma. BALF analyses of the LPS + CSS group revealed an increase in expression levels of several cytokines involved in the pathogenesis of human COPD. These results suggest that our experimental protocol can induce COPD in mice. Likewise, histopathological findings of the lung and induction of cytokines in BALF from MKK6 c.a.-TG mice were more marked than those in WT mice. Conclusion: In a new experimental COPD mouse model, p38 accelerates the development of emphysema.

p38α controls self-renewal and fate decision of neurosphere-forming cells in adult hippocampus

Neural stem cells (NSC) from the adult hippocampus easily lose their activityin vitro. Efficientin vitro expansion of adult hippocampus‐derived NSC is important for generation of tools for research and cell therapy. Here, we show that a single copy disruption or pharmacological inhibition of p38α enables successful long‐term neurosphere culture of adult mouse hippocampal cells. Expanded neurospheres with high proliferative activity differentiated into the three neuronal lineages under differentiating conditions. Thus, inhibition of p38α can maintain adult hippocampal NSC activityin vitro.

Endothelin B receptor-mediated encephalopathic events in mouse sepsis model.

AIMS We evaluated whether pathophysiological events in the brain in sepsis are mediated by ET-1/ETB receptor axis. MAIN METHODS We prepared raw fecal fluid from soft stool of mice. Mice were randomly divided into three groups: pre-PBS+raw fecal fluid group (Sepsis, easy stool method (ESM) group); pre-BQ788+raw fecal fluid group (BQ group); and pre-BQ788+PBS group (PBS group). According to each experimental condition, PBS or BQ788 was intravenously injected into mice prior to intraperitoneal administration of fecal fluid or PBS. All groups of mice were sacrificed at 8h after administration, and then brain samples were prepared. KEY FINDINGS In the ESM group, an increase of apoptotic neuroblasts was demonstrated in the subgranular zone of the hippocampal dentate gyrus, enhanced expression of c-FOS was observed in arginine-vasopressin-containing neurons in the hypothalamic paraventricular nucleus, and various cytokines involving TNF-α were upregulated in the brain, compared with those in the PBS group. In the region corresponding to their findings, the number of reactive microglia and vascular leakage was markedly increased. BQ788 inhibited the induction of c-FOS expression, neuroblast apoptosis, cytokine upregulation and reactive microglia without affecting vascular leakage. SIGNIFICANCE We demonstrated that BQ788 could protect the brain from the following sepsis-associated pathophysiological output: neural cell death, inflammatory response and the Hans Selyes environmental stress reaction.

Adaptive gene regulation of pyruvate dehydrogenase kinase isoenzyme 4 in hepatotoxic chemical-induced liver injury and its stimulatory potential for DNA repair and cell proliferation

The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl4)-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl4-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl4-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.

Genetic and Pharmacological Inhibition of p38α Improves Locomotor Recovery after Spinal Cord Injury

One of the mitogen-activated protein kinases, p38α plays a crucial role in various inflammatory diseases and apoptosis of various types of cells. In this study, we investigated the pathophysiological roles of p38α in spinal cord injury (SCI), using a mouse model. Lateral hemisection at T9 of the SC was performed in wild type (WT) and p38α+/- mice (p38α-/- showed embryonic lethality). p38α+/- mice showed a better functional recovery from SCI-associated paralyzed hindlimbs compared to WT mice at 7 days post-injury (dpi), which remained until 28 dpi (an end time point of monitoring the behavior). In histopathological analysis at 28 dpi, there was more axonal regeneration with remyelination on the caudal side of the lesion epicenter in p38α+/- mice than in WT mice. At 7 dpi, infiltration of inflammatory cells into the lesion and expression of cytokines in the lesion were reduced in p38α+/- mice compared with WT mice. At the same time point, the number of apoptotic oligodendrocytes in the white matter at the caudal boarder of the lesion of p38α+/- mice was lower than that of WT mice. At 14 dpi, more neural and oligodendrocyte precursor cells in the gray matter and white matter, respectively, were observed around the lesion epicenter of p38α+/- mice compared with the case of WT mice. At the same time point, astrocytic scar formation was less apparent in p38α+/- than in WT mice, while compaction of inflammatory immune cells associated with the wound contraction was more apparent in p38α+/- than in WT mice. Furthermore, we verified the effectiveness of oral administration of SB239063, a p38α inhibitor on the hindlimb locomotor recovery after SCI. These results suggest that p38α deeply contributes to the pathogenesis of SCI and that inhibition of p38α is a beneficial strategy to recovery from SCI.