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Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15–20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

Chronic kidney disease (CKD) is a common cause of morbidity and mortality in domestic cats, but the cause is still largely elusive. While some viruses have been associated with this disease, none have been definitively implicated as causative. Recently, Rodent chaphamaparvovirus 1 was recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. A novel virus belonging to the same genus, Carnivore chaphamaparvovirus 2, was recently identified in the feces of cats with diarrhea. The goal of this study was to investigate the possible role of chaphamaparvoviruses including members of Rodent chaphamaparvovirus 1 and Carnivore chaphamaparvovirus 2 in the development of feline CKD. The presence of these viruses was retrospectively investigated in formalin-fixed paraffin-embedded feline kidney samples using polymerase chain reaction, in situ hybridization, and immunohistochemistry. Cats were divided into 3 groups: normal ( N = 24), CKD ( N = 26), and immunocompromised ( N = 25). None of the kidney tissues from any of the 75 cats revealed the presence of chaphamaparvovirus DNA, RNA, or antigen. We conclude that viruses belonging to the chaphamaparvovirus genus are unlikely to contribute to the occurrence of feline CKD.

BackgroundRNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. To date, most studies have focused on m6A, while other RNA modifications, including 5hmC, remain poorly characterised. The interplay between different RNA modifications that may occur in specific cellular contexts remains similarly unexplored.ResultsWe profiled m6A and 5hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. We observed that decreased expression of m6A and 5hmC writers, METTL3 and TET-enzymes respectively, facilitated monocyte-to-macrophage differentiation. Despite a global trend of m6A and 5hmC loss during macrophage differentiation, enrichment of m6A and/or 5hmC on specific categories of transcripts essential for macrophage differentiation positively correlated with their expression and translation. m6A and 5hmC mark and are associated with the expression of transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discovered the coexistence of m6A and 5hmC marking alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of transcripts with key roles in macrophage biology. In specific examples, RNA 5hmC controls the decay of transcripts independently of m6A.ConclusionsThis study: i) uncovers m6A, 5hmC and their writer enzymes as regulators of monocyte and macrophage gene expression programs and ii) provides a comprehensive dataset to interrogate the role of RNA modifications in a plastic system. Altogether, this work sheds light on the role of RNA modifications as central regulators of effector cells in innate immunity.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

T lymphocytes utilize amoeboid migration to navigate effectively within complex microenvironments. The precise rearrangement of the actin cytoskeleton required for cellular forward propulsion is mediated by actin regulators, including the actin‐related protein 2/3 (Arp2/3) complex, a macromolecular machine that nucleates branched actin filaments at the leading edge. The consequences of modulating Arp2/3 activity on the biophysical properties of the actomyosin cortex and downstream T cell function are incompletely understood. We report that even a moderate decrease of Arp3 levels in T cells profoundly affects actin cortex integrity. Reduction in total F‐actin content leads to reduced cortical tension and disrupted lamellipodia formation. Instead, in Arp3‐knockdown cells, the motility mode is dominated by blebbing migration characterized by transient, balloon‐like protrusions at the leading edge. Although this migration mode seems to be compatible with interstitial migration in three‐dimensional environments, diminished locomotion kinetics and impaired cytotoxicity interfere with optimal T cell function. These findings define the importance of finely tuned, Arp2/3‐dependent mechanophysical membrane integrity in cytotoxic effector T lymphocyte activities., Quantification of cytotoxic effector T lymphocytes (CTLs) morphology using standard biomedical assays and developed image analysis algorithms showed that the reduction of actin‐related protein (Arp)3 levels impaired F‐actin content maintenance, leading to defects in CTL functionality. The knockdown CTL motility mode, instead of the actin‐rich lamellipodia‐based migratory strategy, displays blebbing‐like migration at the cost of reduced migration speed. Thus, the role of the Arp2/3 complex is not redundant among the various mechanochemical coordinators involved in the leading‐edge formation in CTLs.

The ubiquitous intracellular protease dipeptidyl peptidase 9 (DPP9) has roles in antigen presentation and B cell signaling. To investigate the importance of DPP9 in immune regeneration, primary and secondary chimeric mice were created in irradiated recipients using fetal liver cells and adult bone marrow cells, respectively, using wild-type (WT) and DPP9 gene-knockin (DPP9S729A) enzyme-inactive mice. Immune cell reconstitution was assessed at 6 and 16 weeks post-transplant. Primary chimeric mice successfully regenerated neutrophils, natural killer, T and B cells, irrespective of donor cell genotype. There were no significant differences in total myeloid cell or neutrophil numbers between DPP9-WT and DPP9S729A-reconstituted mice. In secondary chimeric mice, cells of DPP9S729A-origin cells displayed enhanced engraftment compared to WT. However, we observed no differences in myeloid or lymphoid lineage reconstitution between WT and DPP9S729A donors, indicating that hematopoietic stem cell (HSC) engraftment and self-renewal is not diminished by the absence of DPP9 enzymatic activity. This is the first report on transplantation of bone marrow cells that lack DPP9 enzymatic activity.

Mouse Kidney Parvovirus (MKPV) was recently recognized as the cause of murine inclusion body nephropathy, a disease reported for over 40 years in laboratory mice. Immunodeficient mice are persistently infected with MKPV, leading to chronic renal disease, morbidity and mortality whereas immunocompetent mice seroconvert with mild renal pathology. Given the high incidence of MKPV infection in wild mice in the New York City area, the first goal of this study was to evaluate the possibility of MKPV involvement in feline chronic kidney disease (CKD) from the same geographic region. As MKPV and related parvoviruses recently described in other animal species appear to have a tropism for kidney tissue, the second goal was to investigate the possible role of a virus of this group, other than MKPV, in the development of feline CKD, Presence of MKPV and related viruses was investigated in feline renal samples using PCR, RNA in situ hybridization (ISH) and immunohistochemistry (IHC). Cats were divided into three groups: normal (N=25), CKD (N=25) and immune suppressed (N=25). None of the kidney tissues from any of the 75 cats revealed the presence of MKPV DNA, RNA or antigen expression. Nor was “fechavirus” detected using PCR in renal tissue from cats with chronic kidney disease. We conclude that MKPV is an unlikely cause or contributor to feline CKD.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

Conventional dendritic cells (cDCs) are continuously replenished by bone marrow-derived precursors called pre-DCs, which traffic through the blood to peripheral tissues. Pre-DCs are a heterogeneous population that includes cDC subset-committed progenitors, namely pre-cDC1 and pre-cDC2, which give rise to mature cDC1 and cDC2, respectively. Regulation of pre-DC subset trafficking is thought to aid the host response to immune challenge. However, the molecular cues regulating pre-cDC1 versus pre-cDC2 trafficking toward peripheral sites during homeostasis and disease remain elusive. Here, we report that pre-cDC1 but not pre-cDC2 express the T helper type 1-associated chemokine receptor CXCR3. Moreover, we identify a cell-intrinsic role for CXCR3 in the trafficking of pre-cDC1 to melanoma tumors but not to non-inflamed organs. We also show that tumor cDC1 numbers can be increased pharmacologically by targeting dipeptidyl peptidase-4 (CD26), a negative regulator of CXCR3 ligands. Our findings demonstrate that pre-cDC1 trafficking is regulated distinctly from pre-cDC2, which is relevant for our understanding of the DC lineage in the context of cancer and inflammation.

© 2018 Elsevier Inc. The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed “mouse kidney parvovirus” (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans. A kidney parvovirus found in multiple laboratory mouse colonies causes spontaneous nephropathy and represents a new tool for studying chronic kidney disease.