MAVS signaling is required for preventing persistent chikungunya heart infection and chronic vascular tissue inflammation.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

The γδ IEL effector API5 masks genetic susceptibility to Paneth cell death.

Loss of Paneth cells and their antimicrobial granules compromises the intestinal epithelial barrier and is associated with Crohn's disease, a major type of inflammatory bowel disease1-7. Non-classical lymphoid cells, broadly referred to as intraepithelial lymphocytes (IELs), intercalate the intestinal epithelium8,9. This anatomical position has implicated them as first-line defenders in resistance to infections, but their role in inflammatory disease pathogenesis requires clarification. The identification of mediators that coordinate crosstalk between specific IEL and epithelial subsets could provide insight into intestinal barrier mechanisms in health and disease. Here we show that the subset of IELs that express γ and δ T cell receptor subunits (γδ IELs) promotes the viability of Paneth cells deficient in the Crohn's disease susceptibility gene ATG16L1. Using an ex vivo lymphocyte-epithelium co-culture system, we identified apoptosis inhibitor 5 (API5) as a Paneth cell-protective factor secreted by γδ IELs. In the Atg16l1-mutant mouse model, viral infection induced a loss of Paneth cells and enhanced susceptibility to intestinal injury by inhibiting the secretion of API5 from γδ IELs. Therapeutic administration of recombinant API5 protected Paneth cells in vivo in mice and ex vivo in human organoids with the ATG16L1 risk allele. Thus, we identify API5 as a protective γδ IEL effector that masks genetic susceptibility to Paneth cell death.

RAGE impairs murine diabetic atherosclerosis regression and implicates IRF7 in macrophage inflammation and cholesterol metabolism.

Despite advances in lipid-lowering therapies, people with diabetes continue to experience more limited cardiovascular benefits. In diabetes, hyperglycemia sustains inflammation and preempts vascular repair. We tested the hypothesis that the receptor for advanced glycation end-products (RAGE) contributes to these maladaptive processes. We report that transplantation of aortic arches from diabetic, Western diet-fed Ldlr-/- mice into diabetic Ager-/- (Ager, the gene encoding RAGE) versus WT diabetic recipient mice accelerated regression of atherosclerosis. RNA-sequencing experiments traced RAGE-dependent mechanisms principally to the recipient macrophages and linked RAGE to interferon signaling. Specifically, deletion of Ager in the regressing diabetic plaques downregulated interferon regulatory factor 7 (Irf7) in macrophages. Immunohistochemistry studies colocalized IRF7 and macrophages in both murine and human atherosclerotic plaques. In bone marrow-derived macrophages (BMDMs), RAGE ligands upregulated expression of Irf7, and in BMDMs immersed in a cholesterol-rich environment, knockdown of Irf7 triggered a switch from pro- to antiinflammatory gene expression and regulated a host of genes linked to cholesterol efflux and homeostasis. Collectively, this work adds a new dimension to the immunometabolic sphere of perturbations that impair regression of established diabetic atherosclerosis and suggests that targeting RAGE and IRF7 may facilitate vascular repair in diabetes.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

Autophagy protein ATG16L1 prevents necroptosis in the intestinal epithelium.

A variant of the autophagy gene ATG16L1 is associated with Crohn's disease, an inflammatory bowel disease (IBD), and poor survival in allogeneic hematopoietic stem cell transplant recipients. We demonstrate that ATG16L1 in the intestinal epithelium is essential for preventing loss of Paneth cells and exaggerated cell death in animal models of virally triggered IBD and allogeneic hematopoietic stem cell transplantation. Intestinal organoids lacking ATG16L1 reproduced this loss in Paneth cells and displayed TNFα-mediated necroptosis, a form of programmed necrosis. This cytoprotective function of ATG16L1 was associated with the role of autophagy in promoting mitochondrial homeostasis. Finally, therapeutic blockade of necroptosis through TNFα or RIPK1 inhibition ameliorated disease in the virally triggered IBD model. These findings indicate that, in contrast to tumor cells in which autophagy promotes caspase-independent cell death, ATG16L1 maintains the intestinal barrier by inhibiting necroptosis in the epithelium.

The TGF-ß/Smad4 Signaling Pathway in Pancreatic Carcinogenesis and Its Clinical Significance.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human cancers due to its complicated genomic instability. PDAC frequently presents at an advanced stage with extensive metastasis, which portends a poor prognosis. The known risk factors associated with PDAC include advanced age, smoking, long-standing chronic pancreatitis, obesity, and diabetes. Its association with genomic and somatic mutations is the most important factor for its aggressiveness. The most common gene mutations associated with PDAC include KRas2, p16, TP53, and Smad4. Among these, Smad4 mutation is relatively specific and its inactivation is found in more than 50% of invasive pancreatic adenocarcinomas. Smad4 is a member of the Smad family of signal transducers and acts as a central mediator of transforming growth factor beta (TGF-ß) signaling pathways. The TGF-ß signaling pathway promotes many physiological processes, including cell growth, differentiation, proliferation, fibrosis, and scar formation. It also plays a major role in the development of tumors through induction of angiogenesis and immune suppression. In this review, we will discuss the molecular mechanism of TGF-ß/Smad4 signaling in the pathogenesis of pancreatic adenocarcinoma and its clinical implication, particularly potential as a prognostic factor and a therapeutic target.

Netrin-1 is highly expressed and required in inflammatory infiltrates in wear particle-induced osteolysis.

OBJECTIVE: Netrin-1 is a chemorepulsant and matrix protein expressed during and required for osteoclast differentiation, which also plays a role in inflammation by preventing macrophage egress. Because wear particle-induced osteolysis requires osteoclast-mediated destruction of bone, we hypothesised that blockade of Netrin-1 or Unc5b, a receptor for Netrin-1, may diminish this pathological condition. METHODS: C57BL/6 mice, 6-8 weeks old, had 3 mg of ultrahigh-molecular-weight polyethylene particles implanted over the calvaria and then received 10 µg of monoclonal antibodies for Netrin-1 or its receptors, Unc5b and deleted in colon cancer (DCC), injected intraperitoneally on a weekly basis. After 2 weeks, micro-computed tomography and histology analysis were performed. Netrin-1 expression was analysed in human tissue obtained following primary prosthesis implantation or after prosthesis revision for peri-implant osteolysis and aseptic implant loosening. RESULTS: Weekly injection of anti-Netrin-1 or anti-Unc5b-antibodies significantly reduced particle-induced bone pitting in calvaria exposed to wear particles (46±4% and 49±3% of control bone pitting, respectively, p<0.001) but anti-DCC antibody did not affect inflammatory osteolysis (80±7% of control bone pitting, p=ns). Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibody treatment markedly reduced the inflammatory infiltrate and the number of tartrate resistance acid phosphatase (TRAP)-positive osteoclasts (7±1, 4±1 and 14±1 cells/high power field (hpf), respectively, vs 12±1 cells/hpf for control, p<0.001), with no significant changes in alkaline phosphatase-positive osteoblasts on bone-forming surfaces in any antibody-treated group. Netrin-1 immunostaining colocalised with CD68 staining for macrophages. The peri-implant tissues of patients undergoing prosthesis revision surgery showed an increase in Netrin-1 expression, whereas there was little Netrin-1 expression in soft tissues removed at the time of primary joint replacement. CONCLUSIONS: These results demonstrate a unique role for Netrin-1 in osteoclast biology and inflammation and may be a novel target for prevention/treatment of inflammatory osteolysis.

Synergy of topical toll-like receptor 7 agonist with radiation and low-dose cyclophosphamide in a mouse model of cutaneous breast cancer.

PURPOSE: This study tested the hypothesis that topical Toll-like receptor (TLR) 7 agonist imiquimod promotes antitumor immunity and synergizes with other treatments in a model of skin-involving breast cancer. EXPERIMENTAL DESIGN: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice. Imiquimod 5% or placebo cream was applied topically on the shaved skin overlying tumors three times/wk. In some experiments, local ionizing radiation therapy (RT) was delivered to the tumor in three fractions of 8 Gy, given on consecutive days. Cyclophosphamide was given intraperitoneally (i.p.) in one dose of 2 mg/mouse. Mice were followed for tumor growth and survival. RESULTS: Treatment with imiquimod significantly inhibited tumor growth, an effect that was associated with increased tumor infiltration by CD11c(+), CD4(+), and CD8(+) cells, and abolished by depletion of CD8(+) cells. Administration of imiquimod in combination with RT enhanced significantly tumor response compared with either treatment alone (P < 0.005), and 11% to 66% of irradiated tumors completely regressed. Importantly, the addition of topical imiquimod also resulted in growth inhibition of a secondary tumor outside of the radiation field. Low-dose cyclophosphamide given before start of treatment with imiquimod and RT further improved tumor inhibition and reduced tumor recurrence. Mice that remained tumor-free rejected a tumorigenic inoculum of TSA cells, showing long-term immunologic memory. CONCLUSIONS: Topical imiquimod inhibits tumor growth and synergizes with RT. Addition of cyclophosphamide further increases the therapeutic effect and induces protective immunologic memory, suggesting that this combination is a promising strategy for cutaneous breast cancer metastases.

Fractionated but not single-dose radiotherapy induces an immune-mediated abscopal effect when combined with anti-CTLA-4 antibody.

PURPOSE: This study tested the hypothesis that the type of dose fractionation regimen determines the ability of radiotherapy to synergize with anti-CTLA-4 antibody. EXPERIMENTAL DESIGN: TSA mouse breast carcinoma cells were injected s.c. into syngeneic mice at two separate sites, defined as a "primary" site that was irradiated and a "secondary" site outside the radiotherapy field. When both tumors were palpable, mice were randomly assigned to eight groups receiving no radiotherapy or three distinct regimens of radiotherapy (20 Gy x 1, 8 Gy x 3, or 6 Gy x 5 fractions in consecutive days) in combination or not with 9H10 monoclonal antibody against CTLA-4. Mice were followed for tumor growth/regression. Similar experiments were conducted in the MCA38 mouse colon carcinoma model. RESULTS: In either of the two models tested, treatment with 9H10 alone had no detectable effect. Each of the radiotherapy regimens caused comparable growth delay of the primary tumors but had no effect on the secondary tumors outside the radiation field. Conversely, the combination of 9H10 and either fractionated radiotherapy regimens achieved enhanced tumor response at the primary site (P < 0.0001). Moreover, an abscopal effect, defined as a significant growth inhibition of the tumor outside the field, occurred only in mice treated with the combination of 9H10 and fractionated radiotherapy (P < 0.01). The frequency of CD8+ T cells showing tumor-specific IFN-gamma production was proportional to the inhibition of the secondary tumor. CONCLUSIONS: Fractionated but not single-dose radiotherapy induces an abscopal effect when in combination with anti-CTLA-4 antibody in two preclinical carcinoma models.

Critical role for TSLC1 expression in the growth and organ infiltration of adult T-cell leukemia cells in vivo.

Adult T-cell leukemia (ATL) is associated with human T-cell leukemia virus type 1 infection. The tumor suppressor lung cancer 1 (TSLC1) gene was previously identified as a novel cell surface marker for ATL, and this study demonstrated the involvement of TSLC1 expression in tumor growth and organ infiltration of ATL cells. In experiments using NOD/SCID/gamma c(null) mice, both leukemia cell lines and primary ATL cells with high TSLC1 expression caused more tumor formation and aggressive infiltration of various organs of mice. Our results suggest that TSLC1 expression in ATL cells plays an important role in the growth and organ infiltration of ATL cells.

Overexpressed NF-kappaB-inducing kinase contributes to the tumorigenesis of adult T-cell leukemia and Hodgkin Reed-Sternberg cells.

The nuclear factor-kappaB (NF-kappaB) transcription factors play important roles in cancer development by preventing apoptosis and facilitating the tumor cell growth. However, the precise mechanisms by which NF-kappaB is constitutively activated in specific cancer cells remain largely unknown. In our current study, we now report that NF-kappaB-inducing kinase (NIK) is overexpressed at the pretranslational level in adult T-cell leukemia (ATL) and Hodgkin Reed-Sternberg cells (H-RS) that do not express viral regulatory proteins. The overexpression of NIK causes cell transformation in rat fibroblasts, which is abolished by a super-repressor form of IkappaBalpha. Notably, depletion of NIK in ATL cells by RNA interference reduces the DNA-binding activity of NF-kappaB and NF-kappaB-dependent transcriptional activity, and efficiently suppresses tumor growth in NOD/SCID/gammac(null) mice. These results indicate that the deregulated expression of NIK plays a critical role in constitutive NF-kappaB activation in ATL and H-RS cells, and suggest also that NIK is an attractive molecular target for cancer therapy.

Inhibition of IkappaB kinase beta restrains oncogenic proliferation of pancreatic cancer cells.

PURPOSE: Pancreatic cancer is characterized by an extremely poor prognosis due to the aggressive disease course and lack of effective therapeutic intervention. IkappaB kinase (IKK), a central kinase for nuclear factor-kappaB (NF-kappaB) activation, is often constitutively activated in pancreatic cancer cells, playing a crucial role in the malignant phenotype and resistance to anti-cancer agents. This study explored how specific inhibition of IKKbeta suppresses oncogenic proliferation of pancreatic cancer cells. EXPERIMENTAL DESIGN: We employed two different approaches, RNA interference-mediated depletion of IKKbeta (IKKbetai) and use of a novel molecularly designed IKKbeta inhibitor IMD-0354 to investigate the effects on the in vitro and in vivo growth and apoptotic response of pancreatic cancer cells. RESULTS: IKKbetai and IMD-0354 efficiently suppressed constitutive NF-kappaB activity and the growth of pancreatic cancer cells in monolayer and soft agar. IMD-0354 induced Annexin V expression, a typical apoptotic cell response. Notably, daily administration of IMD-0354 significantly suppressed tumor growth in NOD/SCID/gamma c(null) (NOG) mice without any deleterious side effect. CONCLUSIONS: These results identify IKKbeta as an attractive molecular target for pancreatic cancer therapy.

Hematopoietic stem cell-engrafted NOD/SCID/IL2Rgamma null mice develop human lymphoid systems and induce long-lasting HIV-1 infection with specific humoral immune responses.

Critical to the development of an effective HIV/AIDS model is the production of an animal model that reproduces long-lasting active replication of HIV-1 followed by elicitation of virus-specific immune responses. In this study, we constructed humanized nonobese diabetic/severe combined immunodeficiency (NOD/SCID)/interleukin-2 receptor gamma-chain knockout (IL2Rgamma(null)) (hNOG) mice by transplanting human cord blood-derived hematopoietic stem cells that eventually developed into human B cells, T cells, and other monocytes/macrophages and 4 dendritic cells associated with the generation of lymphoid follicle-like structures in lymphoid tissues. Expressions of CXCR4 and CCR5 antigens were recognized on CD4+ cells in peripheral blood, the spleen, and bone marrow, while CCR5 was not detected on thymic CD4+ T cells. The hNOG mice showed marked, long-lasting viremia after infection with both CCR5- and CXCR4-tropic HIV-1 isolates for more than the 40 days examined, with R5 virus-infected animals showing high levels of HIV-DNA copies in the spleen and bone marrow, and X4 virus-infected animals showing high levels of HIV-DNA copies in the thymus and spleen. Furthermore, we detected both anti-HIV-1 Env gp120- and Gag p24-specific antibodies in animals showing a high rate of viral infection. Thus, the hNOG mice mirror human systemic HIV infection by developing specific antibodies, suggesting that they may have potential as an HIV/AIDS animal model for the study of HIV pathogenesis and immune responses.

Efficient intervention of growth and infiltration of primary adult T-cell leukemia cells by an HIV protease inhibitor, ritonavir.

Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.

Rapid tumor formation of human T-cell leukemia virus type 1-infected cell lines in novel NOD-SCID/gammac(null) mice: suppression by an inhibitor against NF-kappaB.

We established a novel experimental model for human T-cell leukemia virus type 1 (HTLV-1)-induced tumor using NOD-SCID/gammac(null) (NOG) mice. This model is very useful for investigating the mechanism of tumorigenesis and malignant cell growth of adult T-cell leukemia (ATL)/lymphoma, which still remains unclear. Nine HTLV-1-infected cell lines were inoculated subcutaneously in the postauricular region of NOG mice. As early as 2 to 3 weeks after inoculation, seven cell lines produced a visible tumor while two transformed cell lines failed to do so. Five of seven lines produced a progressively growing large tumor with leukemic infiltration of the cells in various organs that eventually killed the animals. Leukemic cell lines formed soft tumors, whereas some transformed cell lines developed into hemorrhagic hard tumors in NOG mice. One of the leukemic cell lines, ED-40515(-), was unable to produce visible tumors in NOD-SCID mice with a common gamma-chain after 2 weeks. In vivo NF-kappaB DNA binding activity of the ED-40515(-) cell line was higher and the NF-kappaB components were changed compared to cells in vitro. Bay 11-7082, a specific and effective NF-kappaB inhibitor, prevented tumor growth at the sites of the primary region and leukemic infiltration in various organs of NOG mice. This in vivo model of ATL could provide a novel system for use in clarifying the mechanism of growth of HTLV-1-infected cells as well as for the development of new drugs against ATL.