Loss of REST in breast cancer promotes tumor progression through estrogen sensitization, MMP24 and CEMIP overexpression.

BACKGROUND: Breast cancer is the most common malignancy in women, and is both pathologically and genetically heterogeneous, making early detection and treatment difficult. A subset of breast cancers express normal levels of REST (repressor element 1 silencing transcription factor) mRNA but lack functional REST protein. Loss of REST function is seen in ~ 20% of breast cancers and is associated with a more aggressive phenotype and poor prognosis. Despite the frequent loss of REST, little is known about the role of REST in the molecular pathogenesis of breast cancer. METHODS: TCGA data was analyzed for the expression of REST target genes in breast cancer patient samples. We then utilized gene knockdown in MCF-7 cells in the presence or absence of steroid hormones estrogen and/ progesterone followed by RNA sequencing, as well as chromatin immunoprecipitation and PCR in an attempt to understand the tumor suppressor role of REST in breast cancer. RESULTS: We show that REST directly regulates CEMIP (cell migration-inducing and hyaluronan-binding protein, KIAA1199) and MMP24 (matrix metallopeptidase 24), genes known to have roles in invasion and metastasis. REST knockdown in breast cancer cells leads to significant upregulation of CEMIP and MMP24. In addition, we found REST binds to RE-1 sites (repressor element-1) within the genes and influences their transcription. Furthermore, we found that the estrogen receptor (ESR1) signaling pathway is activated in the absence of REST, regardless of hormone treatment. CONCLUSIONS: We demonstrate a critical role for the loss of REST in aggressive breast cancer pathogenesis and provide evidence for REST as an important diagnostic marker for personalized treatment plans.

Distinct Roles of Interferon Alpha and Beta in Controlling Chikungunya Virus Replication and Modulating Neutrophil-Mediated Inflammation.

Type I interferons (IFNs) are key mediators of the innate immune response. Although members of this family of cytokines signal through a single shared receptor, biochemical and functional variation exists in response to different IFN subtypes. While previous work has demonstrated that type I IFNs are essential to control infection by chikungunya virus (CHIKV), a globally emerging alphavirus, the contributions of individual IFN subtypes remain undefined. To address this question, we evaluated CHIKV pathogenesis in mice lacking IFN-ß (IFN-ß knockout [IFN-ß-KO] mice or mice treated with an IFN-ß-blocking antibody) or IFN-α (IFN regulatory factor 7 knockout [IRF7-KO] mice or mice treated with a pan-IFN-α-blocking antibody). Mice lacking either IFN-α or IFN-ß developed severe clinical disease following infection with CHIKV, with a marked increase in foot swelling compared to wild-type mice. Virological analysis revealed that mice lacking IFN-α sustained elevated infection in the infected ankle and in distant tissues. In contrast, IFN-ß-KO mice displayed minimal differences in viral burdens within the ankle or at distal sites and instead had an altered cellular immune response. Mice lacking IFN-ß had increased neutrophil infiltration into musculoskeletal tissues, and depletion of neutrophils in IFN-ß-KO but not IRF7-KO mice mitigated musculoskeletal disease caused by CHIKV. Our findings suggest disparate roles for the IFN subtypes during CHIKV infection, with IFN-α limiting early viral replication and dissemination and IFN-ß modulating neutrophil-mediated inflammation.IMPORTANCE Type I interferons (IFNs) possess a range of biological activity and protect against a number of viruses, including alphaviruses. Despite signaling through a shared receptor, there are established biochemical and functional differences among the IFN subtypes. The significance of our research is in demonstrating that IFN-α and IFN-ß both have protective roles during acute chikungunya virus (CHIKV) infection but do so by distinct mechanisms. IFN-α limits CHIKV replication and dissemination, whereas IFN-ß protects from CHIKV pathogenesis by limiting inflammation mediated by neutrophils. Our findings support the premise that the IFN subtypes have distinct biological activities in the antiviral response.

Interferon Regulatory Factor 1 Protects against Chikungunya Virus-Induced Immunopathology by Restricting Infection in Muscle Cells.

The innate immune system protects cells against viral pathogens in part through the autocrine and paracrine actions of alpha/beta interferon (IFN-α/ß) (type I), IFN-γ (type II), and IFN-λ (type III). The transcription factor interferon regulatory factor 1 (IRF-1) has a demonstrated role in shaping innate and adaptive antiviral immunity by inducing the expression of IFN-stimulated genes (ISGs) and mediating signals downstream of IFN-γ. Although ectopic expression experiments have suggested an inhibitory function of IRF-1 against infection of alphaviruses in cell culture, its role in vivo remains unknown. Here, we infected Irf1 -/- mice with two distantly related arthritogenic alphaviruses, chikungunya virus (CHIKV) and Ross River virus (RRV), and assessed the early antiviral functions of IRF-1 prior to induction of adaptive B and T cell responses. IRF-1 expression limited CHIKV-induced foot swelling in joint-associated tissues and prevented dissemination of CHIKV and RRV at early time points. Virological and histological analyses revealed greater infection of muscle tissues in Irf1 -/- mice than in wild-type mice. The antiviral actions of IRF-1 appeared to be independent of the induction of type I IFN or the effects of type II and III IFNs but were associated with altered local proinflammatory cytokine and chemokine responses and differential infiltration of myeloid cell subsets. Collectively, our in vivo experiments suggest that IRF-1 restricts CHIKV and RRV infection in stromal cells, especially muscle cells, and that this controls local inflammation and joint-associated swelling.IMPORTANCE Interferon regulatory factor 1 (IRF-1) is a transcription factor that regulates the expression of a broad range of antiviral host defense genes. In this study, using Irf1 -/- mice, we investigated the role of IRF-1 in modulating pathogenesis of two related arthritogenic alphaviruses, chikungunya virus and Ross River virus. Our studies show that IRF-1 controlled alphavirus replication and swelling in joint-associated tissues within days of infection. Detailed histopathological and virological analyses revealed that IRF-1 preferentially restricted CHIKV infection in cells of nonhematopoietic lineage, including muscle cells. The antiviral actions of IRF-1 resulted in decreased local inflammatory responses in joint-associated tissues, which prevented immunopathology.

Pathogen-derived oligosaccharides improve innate immune response to intracellular parasite infection.

Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response-altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)-mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40-producing cells from the draining lymph nodes of treated Leishmania major-infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar-treated and control groups was not observed in draining lymph node cells from TLR2(-/-) mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.

Therapy with CTLA4-Ig and an antiviral monoclonal antibody controls chikungunya virus arthritis.

In 2013, chikungunya virus (CHIKV) transmission was documented in the Western Hemisphere, and the virus has since spread throughout the Americas with more than 1.8 million people infected in more than 40 countries. CHIKV targets the joints, resulting in symmetric polyarthritis that clinically mimics rheumatoid arthritis and can endure for months to years. At present, no approved treatment is effective in preventing or controlling CHIKV infection or disease. We treated mice with eight different disease-modifying antirheumatic drugs and identified CLTA4-Ig (abatacept) and tofacitinib as candidate therapies based on their ability to decrease acute joint swelling. CTLA4-Ig reduced T cell accumulation in the joints of infected animals without affecting viral infection. Whereas monotherapy with CTLA4-Ig or a neutralizing anti-CHIKV human monoclonal antibody provided partial clinical improvement, therapy with both abolished swelling and markedly reduced levels of chemokines, proinflammatory cytokines, and infiltrating leukocytes. Thus, combination CTLA4-Ig and antiviral antibody therapy controls acute CHIKV infection and arthritis and may be a candidate for testing in humans.

Immunologic progression of canine leishmaniosis following vertical transmission in United States dogs.

Canine leishmaniosis (CanL) is caused by Leishmania infantum, an obligate intracellular protozoan parasite, endemic in U.S. hunting dog populations. CanL has been found in dogs in 28 states and two Canadian provinces. Previous studies by our group, (Boggiatto et al., 2011), demonstrated that vertical transmission of Leishmania was the predominant means of transmission within U.S. dogs. Very little is known regarding how this alternative means of transmission, alters the long-term immunity and clinical presentation of leishmaniosis in dogs born to a positive bitch. This study follows the immunological progression of CanL in three pups after birth to an infected bitch. During the course of the study, these dogs were tested every six months over the course of six years. Both immunologic (IFN-γ, T cell proliferation, antibody production) and parasitological parameters (qPCR) of vertically-infected dogs were measured. Within the six years after birth to an L. infantum-infected, oligosymptomatic bitch, all dogs had at least one L. infantum PCR-positive test. Interestingly, despite living in the same location for their entire lives and being full siblings, these pups demonstrate three different disease progression patterns of L. infantum infection. One dog progressed to oligosymptomatic disease, maintaining a positive titer and had intermittent positive PCR results. One asymptomatic dog had positive serological titers and demonstrated a robust CD4(+) immune response to infection. The third dog had a negligible response to L. infantum antigen and was healthy. This work demonstrates the biologic variability associated with vertically-transmitted infection similar to the variety of presentations observed during vector-borne leishmaniosis.

Neutralizing Monoclonal Antibodies Block Chikungunya Virus Entry and Release by Targeting an Epitope Critical to Viral Pathogenesis.

We evaluated the mechanism by which neutralizing human monoclonal antibodies inhibit chikungunya virus (CHIKV) infection. Potently neutralizing antibodies (NAbs) blocked infection at multiple steps of the virus life cycle, including entry and release. Cryo-electron microscopy structures of Fab fragments of two human NAbs and chikungunya virus-like particles showed a binding footprint that spanned independent domains on neighboring E2 subunits within one viral spike, suggesting a mechanism for inhibiting low-pH-dependent membrane fusion. Detailed epitope mapping identified amino acid E2-W64 as a critical interaction residue. An escape mutation (E2-W64G) at this residue rendered CHIKV attenuated in mice. Consistent with these data, CHIKV-E2-W64G failed to emerge in vivo under the selection pressure of one of the NAbs, IM-CKV063. As our study suggests that antibodies engaging the residue E2-W64 can potently inhibit CHIKV at multiple stages of infection, antibody-based therapies or immunogens that target this region might have protective value.