Masahiro Kaneshige

Enterovirus Infection, CXC Chemokine Ligand 10 (CXCL10), and CXCR3 Circuit: A Mechanism of Accelerated β-Cell Failure in Fulminant Type 1 Diabetes

OBJECTIVE Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated β-cell failure are unclear. RESEARCH DESIGN AND METHODS Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2–5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR. RESULTS Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor–bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-γ and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including β-cells and α-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells. CONCLUSIONS These results strongly suggest the presence of a circuit for the destruction of β-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-γ and CXCL10 in β-cells. CXCL10 secreted from β-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-γ in the islets, not only damaging β-cells but also accelerating CXCL10 generation in residual β-cells and thus further activating cell-mediated autoimmunity until all β-cells have been destroyed.

RIG-I– and MDA5-Initiated Innate Immunity Linked With Adaptive Immunity Accelerates β-Cell Death in Fulminant Type 1 Diabetes

OBJECTIVE The contribution of innate immunity responsible for aggressive β-cell destruction in human fulminant type 1 diabetes is unclear. RESEARCH DESIGN AND METHODS Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid–inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes. RESULTS RIG-I was strongly expressed in β-cells in all three pancreata infected with enterovirus. Melanoma differentiation–associated gene-5 was hyperexpressed in islet cells, including β- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-β were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid–receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in β-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes. CONCLUSIONS These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive β-cell toxicity in fulminant type 1 diabetes.

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

Background: One goal of diabetic regenerative medicine is to convert mature pancreatic acinar cells into insulin-producing cells. Results: Ligand-bound thyroid hormone receptor α (TRα), which interacts with p85α, induces phosphatidylinositol 3-kinase (PI3K) signaling and insulin expression. Conclusion: PI3K signaling must be activated for TRα-induced reprogramming of pancreatic acinar cells. Significance: TRα is critical for postnatal expansion of the β-cell mass. One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

Impaired oxidative endoplasmic reticulum stress response caused by deficiency of thyroid hormone receptor α

Background: Endoplasmic reticulum (ER) stress is involved in β-cell failure and apoptotic death. Results: Upon endogenous TRα knockdown, ER stress significantly enhanced apoptosis in pancreatic β-cells. Conclusion: TRα facilitates reduced apoptosis in pancreatic β-cells under ER stress. Significance: TRα is coupled to stress response modulation and improved survival of pancreatic β-cells. Thyroid hormone receptor α (TRα) is critical to postnatal pancreatic β-cell maintenance. To investigate the association between TRα and the survival of pancreatic β-cells under endoplasmic reticulum (ER) stress, the expression of endogenous TRα was inhibited by infection with an adenovirus expressing double-stranded short hairpin RNA against TRα (AdshTRα). In control adenovirus-infected pancreatic β-cells, palmitate enhanced the expression of activating transcription factor 4 (ATF4) and heme oxygenase 1, which facilitates adaptation to oxidative ER stress. However, in AdshTRα-infected pancreatic β-cells, palmitate did not induce ATF4-mediated integrated stress response, and oxidative stress-associated apoptotic cell death was significantly enhanced. TRα-deficient mice or wild-type mice (WT) were fed a high fat diet (HFD) for 30 weeks, and the effect of oxidative ER stress on pancreatic β-cells was analyzed. HFD-treated TRα-deficient mice had high blood glucose levels and low plasma insulin levels. In HFD-treated TRα-deficient mice, ATF4 was not induced, and apoptosis was enhanced compared with HFD-treated WT mice. Furthermore, the expression level of 8-hydroxydeoxyguanosine, an oxidative stress marker, was enhanced in the β-cells of HFD-treated TRα-deficient mice. These results indicate that endogenous TRα plays an important role for the expression of ATF4 and facilitates reduced apoptosis in pancreatic β-cells under ER stress.

Measurement of soluble C-type lectin-like receptor 2 in human plasma

Abstract Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and β-thromboglobulin (β-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab′)2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and β-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and β-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97 ± 55 pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149 ± 260 pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.

The ligand-bound thyroid hormone receptor in macrophages ameliorates kidney injury via inhibition of nuclear factor-κB activities

In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor α (TRα) in macrophages and examined the role of ligand-bound TRα in macrophage polarization-mediated anti-inflammatory effects. TRα-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRα displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRα-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1β levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRα on macrophages plays a protective role in kidney inflammation through the inhibition of NF-κB pathways, possibly by affecting the pro- and anti-inflammatory balance that controls the development of CKD.