Toshimitsu Uede

Penicillium marneffei Causes Osteopontin-Mediated Production of Interleukin-12 by Peripheral Blood Mononuclear Cells

ABSTRACT We investigated the role of osteopontin (OPN) in interleukin-12 (IL-12) production from peripheral blood mononuclear cells (PBMCs) stimulated with Penicillium marneffei. Kinetic studies showed that OPN synthesis preceded that of IL-12 at both mRNA and protein levels when PBMCs were cocultured with P. marneffei. Treatment with anti-OPN monoclonal antibodies (MAb) significantly suppressed IL-12 secretion. Furthermore, native OPN induced a profound level of synthesis of IL-12 from noninfected PBMCs. The major cellular source of OPN was monocytes, because depletion of CD14+ cells resulted in the abrogation of such production. We also examined the regulatory role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in OPN secretion from P. marneffei-stimulated PBMCs. Neutralizing anti-GM-CSF MAb significantly reduced OPN secretion, and treatment with this cytokine induced OPN production from both infected and noninfected PBMCs. Finally, antagonists against the mannose receptor but not the β-glucan receptor almost completely abrogated the production of OPN. Our results demonstrated that OPN secreted from monocytes is involved in the production of IL-12 from PBMCs after stimulation with P. marneffei and that OPN production is regulated by GM-CSF. Our results also indicated the possible involvement of the mannose receptor as a signal-transducing receptor for triggering the secretion of OPN by P. marneffei-stimulated PBMCs.

Tumor-infiltrating DCs suppress nucleic acid-mediated innate immune responses through interactions between the receptor TIM-3 and the alarmin HMGB1

The mechanisms by which tumor microenvironments modulate nucleic acid–mediated innate immunity remain unknown. Here we identify the receptor TIM-3 as key in circumventing the stimulatory effects of nucleic acids in tumor immunity. Tumor-associated dendritic cells (DCs) in mouse tumors and patients with cancer had high expression of TIM-3. DC-derived TIM-3 suppressed innate immune responses through the recognition of nucleic acids by Toll-like receptors and cytosolic sensors via a galectin-9-independent mechanism. In contrast, TIM-3 interacted with the alarmin HMGB1 to interfere with the recruitment of nucleic acids into DC endosomes and attenuated the therapeutic efficacy of DNA vaccination and chemotherapy by diminishing the immunogenicity of nucleic acids released from dying tumor cells. Our findings define a mechanism whereby tumor microenvironments suppress antitumor immunity mediated by nucleic acids.

Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice

Rheumatoid arthritis is one of the most critical diseases that impair the quality of life of patients, but its pathogenesis has not yet been fully understood. Osteopontin (OPN) is an extracellular matrix protein containing Arg-Gly-Asp (RGD) sequence, which interacts with αvβ3 integrins, promotes cell attachment, and cell migration and is expressed in both synovial cells and chondrocytes in rheumatoid arthritis; however, its functional relationship to arthritis has not been known. Therefore, we investigated the roles of OPN in the pathogenesis of inflammatory process in a rheumatoid arthritis model induced by a mixture of anti-type II collagen mAbs and lipopolysaccharide (mAbs/LPS). mAbs/LPS injection induced OPN expression in synovia as well as cartilage, and this expression was associated with joint swelling, destruction of the surface structures of the joint based on scanning electron microscopy, and loss of toluidine blue-positive proteoglycan content in the articular cartilage in wild-type mice. In contrast, OPN deficiency prevented the mice from such surface destruction, loss of proteoglycan in the articular joint cartilage, and swelling of the joints even when the mice were subjected to mAbs/LPS injection. Furthermore, mAbs/LPS injection in wild-type mice enhanced the levels of CD31-positive vessels in synovia and terminal deoxynucleotidyltransferase-mediated UTP end labeling-positive chondrocytes in the articular cartilage, whereas such angiogenesis as well as chondrocyte apoptosis was suppressed significantly in OPN-deficient mice. These results indicated that OPN plays a critical role in the destruction of joint cartilage in the rheumatoid arthritis model in mice via promotion of angiogenesis and induction of chondrocyte apoptosis.

Essential role of the cryptic epitope SLAYGLR within osteopontin in a murine model of rheumatoid arthritis

It has been shown that osteopontin (OPN) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). However, the molecular mechanism of OPN action is yet to be elucidated. Splenic monocytes obtained from arthritic mice exhibited a significant capacity for cell migration toward thrombin-cleaved OPN but not toward full-length OPN. Migratory monocytes expressed alpha9 and alpha4 integrins. Since cleavage of OPN by thrombin exposes the cryptic epitope recognized by alpha9 and alpha4 integrins, we investigated the role of the cryptic epitope SLAYGLR in a murine RA model by using a specific antibody (M5) reacting to SLAYGLR sequence. The M5 antibody could abrogate monocyte migration toward the thrombin-cleaved form of OPN. Importantly, M5 antibody could inhibit the proliferation of synovium, bone erosion, and inflammatory cell infiltration in arthritic joints. Thus, we demonstrated that a cryptic epitope, the SLAYGLR sequence of murine OPN, is critically involved in the pathogenesis of a murine model of RA.

Osteopontin Deficiency Attenuates Atherosclerosis in Female Apolipoprotein E-Deficient Mice

Objective—Osteopontin (OPN), a noncollagenous adhesive protein, is implicated in atherosclerosis, in which macrophages within atherosclerotic plaques express OPN. However, it is not known whether the elevated OPN expression is a cause or result of atherosclerosis. Methods and Results—We generated mice that lacked OPN and crossed them with apolipoprotein (apo) E–deficient mice and analyzed these mice with a mixed C57BL/6×129 background after 36 weeks on a normal chow diet. In female mice, OP+/−E−/− and OP−/−E−/− mice had significantly smaller atherosclerotic and inflammatory lesions compared with OP+/+E−/− mice, and that was reflected by smaller area of MOMA-2–positive staining. In male mice, however, there was no significant difference in the atherosclerosis lesion areas among 3 genotypes. In both OP−/−E−/− and OP+/+E−/− mice, typical atherosclerotic lesions were detected, which include necrotic core, foamy cell collections, and cholesterol clefts. However, we found that vascular mineral-deposited areas in 60-week-old male OP−/−E−/− mice were significantly increased compared with those in OP+/+E−/− male mice. Conclusions—These results suggest that OPN plays a promoting effect in atherosclerosis and inhibitory effect in vascular calcification. The suppression of OPN expression in females should be considered a therapeutic possibility in atherosclerosis.

Role of Osteopontin in Cardiac Fibrosis and Remodeling in Angiotensin II-Induced Cardiac Hypertrophy

Osteopontin (OPN) is upregulated in several experimental models of cardiac fibrosis and remodeling. However, its direct effects remain unclear. We examined the hypothesis that OPN is important for the development of cardiac fibrosis and remodeling. Moreover, we examined whether the inhibitory effect of eplerenone (Ep), a novel aldosterone receptor antagonist, was mediated through the inhibition of OPN expression against cardiac fibrosis and remodeling. Wild-type (WT) and OPN-deficient mice were treated with angiotensin II (Ang II) for 4 weeks. WT mice receiving Ang II were divided into 2 groups: a control group and an Ep treatment group. Ang II treatment significantly elevated blood pressure and caused cardiac hypertrophy and fibrosis in WT mice. Ep treatment and OPN deficiency could reduce the Ang II–induced elevation of blood pressure and ameliorate the development of cardiac fibrosis, whereas Ep-only treatment abolished the development of cardiac hypertrophy. Most compelling, the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and subsequent left ventricular dilatation in Ang II–treated OPN-deficient mice. These results suggest that OPN has a pivotal role in the development of Ang II–induced cardiac fibrosis and remodeling. Moreover, the effect of Ep on the prevention of cardiac fibrosis, but not cardiac hypertrophy, might be partially mediated through the inhibition of OPN expression.

Interleukin-17A Deficiency Accelerates Unstable Atherosclerotic Plaque Formation in Apolipoprotein E-Deficient Mice

Objective—Interleukin(IL)-17A, an inflammatory cytokine, has been implicated in atherosclerosis, in which inflammatory cells within atherosclerotic plaques express IL-17A. However, its role in the development of atheroscelrosis remains to be controversial. Methods and Results—To directly examine the role of IL-17A in atherosclerosis, we generated apolipoprotein E (ApoE)/IL-17A double-deficient (ApoE−/−IL-17A−/−) mice. Mice were fed with high-fat diet (HFD) for either 8 or 16 weeks, both starting at ages of 6 to 8 weeks. We found that splenic CD4+ T-cells produced high amounts of IL-17A in ApoE−/− mice after HFD feeding for 8 weeks. Atherosclerosis was significantly accelerated in HFD-fed ApoE−/−IL-17A−/− mice compared with ApoE−/− mice. Splenic CD4+ T-cells of ApoE−/−IL-17A−/− mice after HFD feeding for 8 weeks, but not for 16 weeks, exhibited increased interferon gamma and decreased IL-5 production. Importantly, formation of vulnerable plaque as evidenced by reduced numbers of vascular smooth muscle cells and reduced type I collagen deposition in the plaque was detected in ApoE−/−IL-17A−/− mice after HFD feeding for 8 weeks. Conclusion—These results suggest that IL-17A regulates the early phase of atherosclerosis development after HFD feeding and plaque stability, at least partly if not all by modulating interferon gamma and IL-5 production from CD4+ T-cells.

Osteopontin/Eta-1 upregulated in Crohn’s disease regulates the Th1 immune response

Background and aims: The pathogenesis of Crohn’s disease (CD), a chronic inflammatory bowel disease characterised by a Th1 immune response, remains unclear. Osteopontin (OPN) is a phosphoprotein known as an adhesive bone matrix protein. Recent studies have shown that OPN plays an important role in lymphocyte migration, granuloma formation, and interleukin 12 (IL-12) production. The present study investigated expression and the pathophysiological role of OPN in CD. Methods: Plasma OPN concentration was measured by enzyme linked immunosorbent assay. Expression of OPN in human intestinal mucosa was determined using reverse transcription-polymerase chain reaction and western blot, and localisation of OPN was examined by immunohistochemistry. Expression of integrin β3, an OPN receptor, on lamina propria mononuclear cells (LPMC) was assessed by flow cytometry. Functional activation of OPN in LPMC was investigated by measuring the production of cytokines. Results: Plasma OPN concentration was significantly higher in patients with CD compared with normal controls or patients with ulcerative colitis (UC). OPN was upregulated in intestinal mucosa from UC and CD patients. OPN producing cells were epithelial or IgG producing plasma cells, or partial macrophages. OPN was detected in areas surrounding granuloma from mucosa in CD. Integrin β3 expressing macrophages infiltrated inflamed mucosa in UC and CD; in contrast, there was no expression of integrin β3 on intestinal macrophages in normal mucosa. OPN induced production of IL-12 from LPMC in CD but not in normal controls or UC. Conclusions: Increased OPN expression facilitates cytokine production and is closely involved in the Th1 immune response associated with CD.

Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway.

Background. Inducible co-stimulator (ICOS) is one of the most recently described members of the CD28 family, and it plays an important role in immune responses. To investigate the role of ICOS in allograft rejection, the authors studied graft survival after cardiac transplantation in mice. Methods. Hearts from BALB/c mice were transplanted into C3H/He mice. Immunohistochemical staining and flow cytometry were performed. Monoclonal antibody to ICOS or ICOS-immunoglobulin (Ig) was injected intraperitoneally. The authors performed mixed lymphocyte reaction (MLR). Results. ICOS was expressed strongly by graft-infiltrating cells during rejection of the allograft. Blockade of the ICOS pathway with anti-ICOS antibody and ICOSIg significantly prolonged graft survival time relative to that in untreated mice; however, all cardiac allografts were eventually rejected by a single treatment. Treatment with both ICOSIg and cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig induced not only long-term acceptance of the cardiac allograft but also donor-specific tolerance, which was shown by acceptance of donor but not third-party skin. Graft arterial intimal hyperplasia in these cardiac allografts was remarkably less than that in cardiac allografts treated with tacrolimus. Addition of anti-ICOS antibody or ICOSIg to MLR resulted in inhibition of T-cell proliferation. Conclusions. Inhibition of T-cell proliferation with ICOSIg and CTLA4Ig was more effective than that with ICOSIg alone. Thus, ICOS appears to be an important regulator of T-cell activation, and may be an effective therapy in clinical cardiac transplantation.

Syndecan-4 Prevents Cardiac Rupture and Dysfunction After Myocardial Infarction

Rationale: Syndecan-4 (Syn4), a cell-surface heparan sulfate proteoglycan, has been detected in the infarct region after myocardial infarction (MI), but its functional significance has not been elucidated. Objective: We examined whether and how Syn4 regulates the cardiac healing process after MI. Methods and Results: Although the heart in Syn4-deficient (Syn4−/−) mice was morphologically and functionally normal, Syn4−/− mice exhibited impaired heart function and increased mortality rate as a result of cardiac ruptures after MI. Cardiac ruptures in Syn4−/− mice were associated with reduced inflammatory reaction and impaired granulation tissue formation during the early phase of MI, as evidenced by reduced numbers of leukocytes, fibroblasts, myofibroblasts, macrophages, and capillary vessels, along with reduced extracellular matrix protein deposition in the infarct region after MI. Transforming growth factor-&bgr;1–dependent cell signaling was preserved, whereas cell migration, fibronectin-induced cell signaling, and differentiation into myofibroblasts were defective in Syn4−/− cardiac fibroblasts. We also found that Syn4 was involved in basic fibroblast growth factor–dependent endothelial cell signaling, cell proliferation, and tube formation. Finally, overexpression of the shed form of Syn4 before MI creation led to an increase in mortality due to cardiac rupture via its action as a dominant-negative inhibitor of endogenous Syn4 signaling, which suggested a protective role of Syn4 signaling in MI. Conclusions: These results suggest that Syn4 plays an important role in the inflammatory response and granulation tissue formation, thereby preventing cardiac rupture and dysfunction after MI.