Ryotaro Sakamori

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell-based approaches could be beneficial to infants with this often lethal condition.

Natural killer cell and hepatic cell interaction via NKG2A leads to dendritic cell-mediated induction of CD4+ CD25+ T cells with PD-1-dependent regulatory activities

Summary Natural killer (NK) cells have the ability to control dendritic cell (DC)‐mediated T cell responses. However, the precise mechanisms by which NK receptor‐mediated regulation of NK cells determines the magnitude and direction of DC‐mediated T cell responses remain unclear. In the present study, we applied an in vitro co‐culture system to examine the impact of NK cells cultured with hepatic cells on DC induction of regulatory T cells. We found that interaction of NK cells and non‐transformed hepatocytes (which express HLA‐E) via the NKG2A inhibitory receptor resulted in priming of DCs to induce CD4+ CD25+ T cells with regulatory properties. NKG2A triggering led to characteristic changes of the cytokine milieu of co‐cultured cells; an increase in the transforming growth factor (TGF)‐β involved in the generation of this specific type of DC, and a decrease in the tumour necrosis factor‐α capable of antagonizing the effect of TGF‐β. The regulatory cells induced by NK cell‐primed DCs exert their suppressive actions through a negative costimulator programmed death‐1 (PD‐1) mediated pathway, which differs from freshly isolated CD4+ CD25+ T cells. These findings provide new insight into the role of NK receptor signals in the DC‐mediated induction of regulatory T cells.

Signal transducer and activator of transcription 3 signaling within hepatocytes attenuates systemic inflammatory response and lethality in septic mice

Sepsis is an infection‐induced syndrome with systemic inflammatory response leading to multiorgan failure and occasionally death. During this process, signal transducer and activator of transcription 3 (STAT3) is activated in the liver, but the significance of this molecule has not been established. We generated hepatocyte‐specific STAT3‐deficient mice (L‐STAT3 KO) and examined the susceptibility of these mice to cecal ligation and puncture–induced peritonitis, a well‐established septic model. L‐STAT3 KO mice showed significantly higher mortality and produced lesser amounts of various acute phase proteins than control littermates. Although blood bacterial infection did not differ between L‐STAT3 KO mice and control mice, the former showed deterioration of the systemic inflammatory response as evidenced by a significant increase in various cytokines such as tumor necrosis factor α, IFN‐γ, IL‐6, IL‐10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β. A similar hyperinflammatory response was observed in another septic model caused by lipopolysaccharide (LPS) injection. In vitro analysis revealed that soluble substances derived from hepatocytes and dependent on STAT3 were critical for suppression of cytokine production from LPS‐stimulated macrophage and splenocytes. Conclusion: STAT3 activation in hepatocytes can attenuate a systemic hyperinflammatory response and lethality in sepsis, in part by suppressing immune cell overactivation, implying a critical role of hepatocyte STAT3 signaling in maintaining host homeostasis. (HEPATOLOGY 2007.)

Immunotherapy of murine colon cancer using receptor tyrosine kinase EphA2-derived peptide-pulsed dendritic cell vaccines

Further optimization of dendritic cell (DC)‐based vaccines is required clinically against advanced stage cancer. Given the broad range of expression levels observed in the recently defined tumor antigen EphA2 in a diverse types of cancers, especially in advanced stage or metastatic cancers, the authors evaluated the effectiveness of vaccination using DCs pulsed with EphA2‐derived peptides (Eph‐DCs) in a murine colon cancer model.

Injection of IL-12 gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity.

Dendritic cell (DC)-based vaccines have been applied clinically in the setting of advanced-stage cancer. To date, the clinical efficacy of these vaccines has been limited, possibly owing to the impairment of transferred DC function in cancer-bearing patients. In this study, we examined the therapeutic efficacy of interleukin-12 (IL-12) gene-transfected DCs isolated from tumor-bearing hosts against liver tumor. The endogenous DCs isolated from subcutaneous (s.c.) CMS4 tumor-bearing mice (CMS4DC) exhibited decreased expression levels of antigen-presenting molecules and low-allostimulatory capacity. CMS4DC produced less IL-12p70 than DCs isolated from normal mice. Adenoviral transfection of IL-12 gene into CMS4DC (AdIL12DC) restored the expression of antigen-presenting molecules and allostimulatory capacity. Intratumoral (i.t.) delivery of AdIL12DC resulted in complete rejection of intrahepatic CMS4 tumors and activation of innate and acquired immune cells. Antibody depletion studies revealed that both CD4+ and CD8+ T cells as well as natural killer cells play critical roles in mediating liver tumor rejection. I.t. treatment of AdIL12DC resulted in long-term protection against s.c. rechallenge with CMS4 tumor cells. These results revealed that IL-12 gene transfer is capable of improving the impaired functions of DC isolated from tumor-bearing hosts, and support the preclinical therapeutic efficacy of intrahepatic injection of AdIL12DC.

Intrahepatic delivery of α -galactosylceramide -pulsed dendritic cells suppresses liver tumor

Alpha‐galactosylceramide, a glycosphingolipid, mediates interaction of dendritic cells (DCs) and NKT cells, leading to activation of both innate and acquired immunity. For cancer treatment, conventional DC‐based vaccine has been tried, but its clinical efficacy is limited against liver cancer. Intrahepatic injection of α‐Galactosylceramide‐pulsed DCs (αGCDC) has not yet been tested in the liver that contains abundant immune cells such as NK, NKT, and T cells. In the present study, we examined the efficacy of αGCDC administration in comparison with p53 peptide‐pulsed DCs using a well‐established murine CMS4 tumor model. Injection of αGCDC into CMS4 liver tumors resulted in complete tumor rejection and established long‐term survival of the animals, while injection of p53232‐240 peptide‐pulsed DCs (pepDC) only partially suppressed tumor growth in the liver. The levels of IFN‐γ in sera of αGCDC‐treated mice were significantly higher than those of pepDC‐treated mice. Hepatic NK cells were efficiently activated by αGCDC injection and played a critical role in liver tumor rejection as evidenced by an in vivo antibody‐mediated NK cell depletion study. Injection of αGCDC into liver tumor led to higher p53232‐240 peptide‐specific CD8+ T cell response than that of pepDC. The mice that had been protected from CMS4 liver tumor by αGCDC injection became resistant to subcutaneous CMS4 rechallenge, but not to Colon26 rechallenge. Conclusion: These results demonstrate that αGCDC injection into the liver can efficiently activate NK cells that in turn reject liver tumors to establish potent acquired immunity against the original tumor. (HEPATOLOGY 2007;45:22–30.)

Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It encompasses a spectrum ranging from simple steatosis to fatty liver with hepatocellular injury, termed nonalcoholic steatohepatitis. Recent studies have demonstrated hepatic autophagy being impaired in NAFLD. In the present study, we investigated the impact of Rubicon, a Beclin1‐interacting negative regulator for autophagosome‐lysosome fusion, in the pathogenesis of NAFLD. In HepG2 cells, BNL‐CL2 cells, and murine primary hepatocytes, Rubicon was posttranscriptionally up‐regulated by supplementation with saturated fatty acid palmitate. Up‐regulation of Rubicon was associated with suppression of the late stage of autophagy, as evidenced by accumulation of both LC3‐II and p62 expression levels as well as decreased autophagy flux. Its blockade by small interfering RNA attenuated autophagy impairment and reduced palmitate‐induced endoplasmic reticulum stress, apoptosis, and lipid accumulation. Rubicon was also up‐regulated in association with autophagy impairment in livers of mice fed a high‐fat diet (HFD). Hepatocyte‐specific Rubicon knockout mice generated by crossing Rubicon floxed mice with albumin‐Cre transgenic mice did not produce any phenotypes on a normal diet. In contrast, on an HFD, they displayed significant improvement of both liver steatosis and injury as well as attenuation of both endoplasmic reticulum stress and autophagy impairment in the liver. In humans, liver tissues obtained from patients with NAFLD expressed significantly higher levels of Rubicon than those without steatosis. Conclusion: Rubicon is overexpressed and plays a pathogenic role in NAFLD by accelerating hepatocellular lipoapoptosis and lipid accumulation, as well as inhibiting autophagy. Rubicon may be a novel therapeutic target for regulating NAFLD development and progression. (Hepatology 2016;64:1994‐2014).

STAT3 signaling within hepatocytes is required for anemia of inflammation in vivo.

BackgroundAnemia of inflammation, commonly observed in patients with chronic diseases, is associated with decreased serum iron. Hepcidin, mainly produced by hepatocytes in a STAT3- and/or SMAD-dependent manner, is involved in iron homeostasis. What remains to be established is whether or not the hepatic IL-6/STAT3 signal has a role in anemia of inflammation in vivo.MethodsTurpentine oil was subcutaneously injected into wild-type mice or hepatocyte-specific STAT3-deficient mice (L-STAT3KO) to induce inflammation.ResultsTurpentine injection increased serum IL-6 levels. It activated liver STAT3 in wild-type mice, but not in L-STAT3KO mice. In chronic inflammation, wild-type mice showed decreased serum iron levels and anemia with up-regulation of hepcidin levels in the liver. In contrast, L-STAT3KO mice showed no increase in hepatic hepcidin levels or anemia.ConclusionsLiver STAT3 is critically involved in the development of anemia of inflammation via the expression of hepcidin. The liver regulates anemia of inflammation through STAT3 signaling.

TLR sorting by Rab11 endosomes maintains intestinal epithelial‐microbial homeostasis

Compartmentalization of Toll‐like receptors (TLRs) in intestinal epithelial cells (IECs) regulates distinct immune responses to microbes; however, the specific cellular machinery that controls this mechanism has not been fully identified. Here we provide genetic evidences that the recycling endosomal compartment in enterocytes maintains a homeostatic TLR9 intracellular distribution, supporting mucosal tolerance to normal microbiota. Genetic ablation of a recycling endosome resident small GTPase, Rab11a, a gene adjacent to a Crohns disease risk locus, in mouse IECs and in Drosophila midgut caused epithelial cell‐intrinsic cytokine production, inflammatory bowel phenotype, and early mortality. Unlike wild‐type controls, germ‐free Rab11a‐deficient mouse intestines failed to tolerate the intraluminal stimulation of microbial agonists. Thus, Rab11a endosome controls intestinal host‐microbial homeostasis at least partially via sorting TLRs.

CDC42 Inhibition Suppresses Progression of Incipient Intestinal Tumors

Mutations in the APC or β-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer xenograft models, we show that incipient intestinal tumor cells activate CDC42, an APC-interacting small GTPase, as a crucial step in malignant progression. In the mouse, Cdc42 ablation attenuated the tumorigenicity of mutant intestinal cells carrying single APC or β-catenin mutations. Similarly, human colorectal cancer with relatively higher levels of CDC42 activity was particularly sensitive to CDC42 blockade. Mechanistic studies suggested that Cdc42 may be activated at different levels, including at the level of transcriptional activation of the stem cell-enriched Rho family exchange factor Arhgef4. Our results indicate that early-stage mutant intestinal epithelial cells must recruit the pleiotropic functions of Cdc42 for malignant progression, suggesting its relevance as a biomarker and therapeutic target for selective colorectal cancer intervention.