Antibody-induced vascular inflammation skews infiltrating macrophages to a novel remodeling phenotype in a model of transplant rejection.

HLA donor-specific antibodies (DSAs) binding to vascular endothelial cells of the allograft trigger inflammation, vessel injury, and antibody-mediated rejection (AMR). Accumulation of intragraft-recipient macrophages is a histological characteristic of AMR, which portends worse outcome. HLA class I (HLA I) DSAs enhance monocyte recruitment by activating endothelial cells and engaging FcγRs, but the DSA-activated donor endothelial influence on macrophage differentiation is unknown. In this study, we explored the consequence of DSA-activated endothelium on infiltrating monocyte differentiation. Here we show that cardiac allografts from murine recipients treated with MHC I DSA upregulated genes related to monocyte transmigration and Fc receptor stimulation. Human monocytes co-cultured with HLA I IgG-stimulated primary human endothelium promoted monocyte differentiation into CD68+ CD206+ CD163+ macrophages (M(HLA I IgG)), whereas HLA I F(ab')2 stimulated endothelium solely induced higher CD206 (M(HLA I F(ab')2 )). Both macrophage subtypes exhibited significant changes in discrete cytokines/chemokines and unique gene expression profiles. Cross-comparison of gene transcripts between murine DSA-treated cardiac allografts and human co-cultured macrophages identified overlapping genes. These findings uncover the role of HLA I DSA-activated endothelium in monocyte differentiation, and point to a novel, remodeling phenotype of infiltrating macrophages that may contribute to vascular injury.

Induction of interferon-stimulated genes by IRF3 promotes replication of Toxoplasma gondii.

Innate immunity is the first line of defense against microbial insult. The transcription factor, IRF3, is needed by mammalian cells to mount innate immune responses against many microbes, especially viruses. IRF3 remains inactive in the cytoplasm of uninfected cells; upon virus infection, it gets phosphorylated and then translocates to the nucleus, where it binds to the promoters of antiviral genes and induces their expression. Such genes include type I interferons (IFNs) as well as Interferon Stimulated Genes (ISGs). IRF3-/- cells support enhanced replication of many viruses and therefore, the corresponding mice are highly susceptible to viral pathogenesis. Here, we provide evidence for an unexpected pro-microbial role of IRF3: the replication of the protozoan parasite, Toxoplasma gondii, was significantly impaired in IRF3-/- cells. In exploring whether the transcriptional activity of IRF3 was important for its pro-parasitic function, we found that ISGs induced by parasite-activated IRF3 were indeed essential, whereas type I interferons were not important. To delineate the signaling pathway that activates IRF3 in response to parasite infection, we used genetically modified human and mouse cells. The pro-parasitic signaling pathway, which we termed PISA (Parasite-IRF3 Signaling Activation), activated IRF3 without any involvement of the Toll-like receptor or RIG-I-like receptor pathways, thereby ruling out a role of parasite-derived RNA species in activating PISA. Instead, PISA needed the presence of cGAS, STING, TBK1 and IRF3, indicating the necessity of DNA-triggered signaling. To evaluate the physiological significance of our in vitro findings, IRF3-/- mice were challenged with parasite infection and their morbidity and mortality were measured. Unlike WT mice, the IRF3-/- mice did not support replication of the parasite and were resistant to pathogenesis caused by it. Our results revealed a new paradigm in which the antiviral host factor, IRF3, plays a cell-intrinsic pro-parasitic role.

Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor.

Virus infection is sensed in the cytoplasm by retinoic acid-inducible gene I (RIG-I, also known as DDX58), which requires RNA and polyubiquitin binding to induce type I interferon (IFN) and activate cellular innate immunity. We show that the human IFN-inducible oligoadenylate synthetases-like (OASL) protein has antiviral activity and mediates RIG-I activation by mimicking polyubiquitin. Loss of OASL expression reduced RIG-I signaling and enhanced virus replication in human cells. Conversely, OASL expression suppressed replication of a number of viruses in a RIG-I-dependent manner and enhanced RIG-I-mediated IFN induction. OASL interacted and colocalized with RIG-I, and through its C-terminal ubiquitin-like domain specifically enhanced RIG-I signaling. Bone-marrow-derived macrophages from mice deficient for Oasl2 showed that among the two mouse orthologs of human OASL, Oasl2 is functionally similar to human OASL. Our findings show a mechanism by which human OASL contributes to host antiviral responses by enhancing RIG-I activation.

Centrobin-centrosomal protein 4.1-associated protein (CPAP) interaction promotes CPAP localization to the centrioles during centriole duplication.

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.

Viral degradasome hijacks mitochondria to suppress innate immunity.

The balance between the innate immunity of the host and the ability of a pathogen to evade it strongly influences pathogenesis and virulence. The two nonstructural (NS) proteins, NS1 and NS2, of respiratory syncytial virus (RSV) are critically required for RSV virulence. Together, they strongly suppress the type I interferon (IFN)-mediated innate immunity of the host cells by degrading or inhibiting multiple cellular factors required for either IFN induction or response pathways, including RIG-I, IRF3, IRF7, TBK1 and STAT2. Here, we provide evidence for the existence of a large and heterogeneous degradative complex assembled by the NS proteins, which we named "NS-degradasome" (NSD). The NSD is roughly ∼300-750 kD in size, and its degradative activity was enhanced by the addition of purified mitochondria in vitro. Inside the cell, the majority of the NS proteins and the substrates of the NSD translocated to the mitochondria upon RSV infection. Genetic and pharmacological evidence shows that optimal suppression of innate immunity requires mitochondrial MAVS and mitochondrial motility. Together, we propose a novel paradigm in which the mitochondria, known to be important for the innate immune activation of the host, are also important for viral suppression of the innate immunity.