HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

The cytokine receptor DR3 identifies and promotes the activation of thymic NKT17 cells.

Invariant natural killer T (iNKT) cells correspond to a population of thymus-generated T cells with innate-like characteristics and effector functions. Among the various iNKT subsets, NKT17 is the only subset that produces the proinflammatory cytokine IL-17. But, how NKT17 cells acquire this ability and what would selectively trigger their activation remain incompletely understood. Here, we identified the cytokine receptor DR3 being specifically expressed on thymic NKT17 cells and mostly absent on other thymic iNKT subsets. Moreover, DR3 ligation promoted the in vivo activation of thymic NKT17 cells and provided costimulatory effects upon agonistic α-GalCer stimulation. Thus, we identified a specific surface marker for thymic NKT17 cells that triggers their activation and augments their effector functions both in vivo and in vitro. These findings provide new insights for deciphering the role and function of murine NKT17 cells and for understanding the development and activation mechanisms of iNKT cells in general.

Quantitative proteomic analysis of SARS-CoV-2 infection of primary human airway ciliated cells and lung epithelial cells demonstrates the effectiveness of SARS-CoV-2 innate immune evasion.

Background: Quantitative proteomics is able to provide a comprehensive, unbiased description of changes to cells caused by viral infection, but interpretation may be complicated by differential changes in infected and uninfected 'bystander' cells, or the use of non-physiological cellular models. Methods: In this paper, we use fluorescence-activated cell sorting (FACS) and quantitative proteomics to analyse cell-autonomous changes caused by authentic SARS-CoV-2 infection of respiratory epithelial cells, the main target of viral infection in vivo. First, we determine the relative abundance of proteins in primary human airway epithelial cells differentiated at the air-liquid interface (basal, secretory and ciliated cells). Next, we specifically characterise changes caused by SARS-CoV-2 infection of ciliated cells. Finally, we compare temporal proteomic changes in infected and uninfected 'bystander' Calu-3 lung epithelial cells and compare infection with B.29 and B.1.1.7 (Alpha) variants. Results: Amongst 5,709 quantified proteins in primary human airway ciliated cells, the abundance of 226 changed significantly in the presence of SARS-CoV-2 infection (q <0.05 and >1.5-fold). Notably, viral replication proceeded without inducing a type-I interferon response. Amongst 6,996 quantified proteins in Calu-3 cells, the abundance of 645 proteins changed significantly in the presence of SARS-CoV-2 infection (q < 0.05 and > 1.5-fold). In contrast to the primary cell model, a clear type I interferon (IFN) response was observed. Nonetheless, induction of IFN-inducible proteins was markedly attenuated in infected cells, compared with uninfected 'bystander' cells. Infection with B.29 and B.1.1.7 (Alpha) variants gave similar results. Conclusions: Taken together, our data provide a detailed proteomic map of changes in SARS-CoV-2-infected respiratory epithelial cells in two widely used, physiologically relevant models of infection. As well as identifying dysregulated cellular proteins and processes, the effectiveness of strategies employed by SARS-CoV-2 to avoid the type I IFN response is illustrated in both models.

SARS-CoV-2 host-shutoff impacts innate NK cell functions, but antibody-dependent NK activity is strongly activated through non-spike antibodies.

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

An Optimized CRISPR/Cas9 Adenovirus Vector (AdZ-CRISPR) for High-Throughput Cloning of sgRNA, Using Enhanced sgRNA and Cas9 Variants.

Recombinant adenovirus vectors enable highly efficient gene delivery in vitro and in vivo. As a result, they are widely used in gene therapy, vaccination, and anticancer applications. We have previously developed the AdZ vector system, which uses recombineering to permit high-throughput cloning of transgenes into Adenovirus vectors, simplifies alteration of the vector backbone, and enables rapid recovery of infectious virus, even if a transgene is incompatible with vector replication. In this study, we adapt this vector system to enable high-throughput cloning of sequences for CRISPR/Cas9 editing. Vectors were optimized to ensure efficient cloning, and high editing efficiency using spCas9 and single guide RNA (sgRNA) sequences in a single vector. Using a multiplicity of infection of 50, knockout efficiencies of up to 80% could be achieved with a single sgRNA. Vectors were further enhanced by altering the spCas9 sequence to match that of SniperCas9, which has reduced off-target activity, but maintains on-target efficiency, and by applying modifications to the sgRNA sequence that significantly enhance editing efficiency. Thus, the AdZ-CRISPR vectors offer highly efficient knockout, even in hard to transfect cells, and enables large-scale CRISPR/Cas9 projects to be undertaken easily and quickly.

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.

A prenylated dsRNA sensor protects against severe COVID-19.

Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.

CD57+ Memory T Cells Proliferate In Vivo.

A central paradigm in the field of lymphocyte biology asserts that replicatively senescent memory T cells express the carbohydrate epitope CD57. These cells nonetheless accumulate with age and expand numerically in response to persistent antigenic stimulation. Here, we use in vivo deuterium labeling and ex vivo analyses of telomere length, telomerase activity, and intracellular expression of the cell-cycle marker Ki67 to distinguish between two non-exclusive scenarios: (1) CD57+ memory T cells do not proliferate and instead arise via phenotypic transition from the CD57- memory T cell pool; and/or (2) CD57+ memory T cells self-renew via intracompartmental proliferation. Our results provide compelling evidence in favor of the latter scenario and further suggest in conjunction with mathematical modeling that self-renewal is by far the most abundant source of newly generated CD57+ memory T cells. Immunological memory therefore appears to be intrinsically sustainable among highly differentiated subsets of T cells that express CD57.

Human Cytomegalovirus Long Non-coding RNA1.2 Suppresses Extracellular Release of the Pro-inflammatory Cytokine IL-6 by Blocking NF-κB Activation.

Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides that are not translated into functional proteins. Cellular lncRNAs have been shown to act as regulators by interacting with target nucleic acids or proteins and modulating their activities. We investigated the role of RNA1.2, which is one of four major lncRNAs expressed by human cytomegalovirus (HCMV), by comparing the properties of parental virus in vitro with those of deletion mutants lacking either most of the RNA1.2 gene or only the TATA element of the promoter. In comparison with parental virus, these mutants exhibited no growth defects and minimal differences in viral gene expression in human fibroblasts. In contrast, 76 cellular genes were consistently up- or down-regulated by the mutants at both the RNA and protein levels at 72 h after infection. Differential expression of the gene most highly upregulated by the mutants (Tumor protein p63-regulated gene 1-like protein; TPRG1L) was confirmed at both levels by RT-PCR and immunoblotting. Consistent with the known ability of TPRG1L to upregulate IL-6 expression via NF-κB stimulation, RNA1.2 mutant-infected fibroblasts were observed to upregulate IL-6 in addition to TPRG1L. Comparable surface expression of TNF receptors and responsiveness to TNF-α in cells infected by the parental and mutant viruses indicated that activation of signaling by TNF-α is not involved in upregulation of IL-6 by the mutants. In contrast, inhibition of NF-κB activity and knockdown of TPRG1L expression reduced the extracellular release of IL-6 by RNA1.2 mutant-infected cells, thus demonstrating that upregulation of TPRG1L activates NF-κB. The levels of MCP-1 and CXCL1 transcripts were also increased in RNA1.2 mutant-infected cells, further demonstrating the presence of active NF-κB signaling. These results suggest that RNA1.2 plays a role in manipulating intrinsic NF-κB-dependent cytokine and chemokine release during HCMV infection, thereby impacting downstream immune responses.

Downregulation of HLA-I by the molluscum contagiosum virus mc080 impacts NK-cell recognition and promotes CD8+ T-cell evasion.

Molluscum contagiosum virus (MCV) is a common cause of benign skin lesions in young children and currently the only endemic human poxvirus. Following the infection of primary keratinocytes in the epidermis, MCV induces the proliferation of infected cells and this results in the production of wart-like growths. Full productive infection is observed only after the infected cells differentiate. During this prolonged replication cycle the virus must avoid elimination by the host immune system. We therefore sought to investigate the function of the two major histocompatibility complex class-I-related genes encoded by the MCV genes mc033 and mc080. Following insertion into a replication-deficient adenovirus vector, codon-optimized versions of mc033 and mc080 were expressed as endoglycosidase-sensitive glycoproteins that localized primarily in the endoplasmic reticulum. MC080, but not MC033, downregulated cell-surface expression of endogenous classical human leucocyte antigen (HLA) class I and non-classical HLA-E by a transporter associated with antigen processing (TAP)-independent mechanism. MC080 exhibited a capacity to inhibit or activate NK cells in autologous assays in a donor-specific manner. MC080 consistently inhibited antigen-specific T cells being activated by peptide-pulsed targets. We therefore propose that MC080 acts to promote evasion of HLA-I-restricted cytotoxic T cells.

Oestrogen-deficiency induces bone loss by modulating CD14+ monocyte and CD4+ T cell DR3 expression and serum TL1A levels.

BACKGROUND: Oestrogen-deficiency induced by menopause is associated with reduced bone density and primary osteoporosis, resulting in an increased risk of fracture. While the exact etiology of menopause-induced primary osteoporotic bone loss is not fully known, members of the tumour necrosis factor super family (TNFSF) are known to play a role. Recent studies have revealed that the TNFSF members death receptor 3 (DR3) and one of its ligands, TNF-like protein 1A (TL1A) have a key role in secondary osteoporosis; enhancing CD14+ peripheral blood mononuclear cell (PBMC) osteoclast formation and bone resorption. Whether DR3 and TL1A contribute towards bone loss in menopause-induced primary osteoporosis however, remains unknown. METHODS: To investigate this we performed flow cytometry analysis of DR3 expression on CD14+ PBMCs isolated from pre- and early post-menopausal females and late post-menopausal osteoporotic patients. Serum levels of TL1A, CCL3 and total MMP-9 were measured by ELISA. In vitro osteoclast differentiation assays were performed to determine CD14+ monocyte osteoclastogenic potential. In addition, splenic CD4+ T cell DR3 expression was investigated 1 week and 8 weeks post-surgery, using the murine ovariectomy model. RESULTS: In contrast to pre-menopausal females, CD14+ monocytes isolated from post-menopausal females were unable to induce DR3 expression. Serum TL1A levels were decreased approx. 2-fold in early post-menopausal females compared to pre-menopausal controls and post-menopausal osteoporotic females; no difference was observed between pre-menopausal and late post-menopausal osteoporotic females. Analysis of in vitro CD14+ monocyte osteoclastogenic potential revealed no significant difference between the post-menopausal and post-menopausal osteoporotic cohorts. Interestingly, in the murine ovariectomy model splenic CD4+ T cell DR3 expression was significantly increased at 1 week but not 8 weeks post-surgery when compared to the sham control. CONCLUSION: Our results reveals for the first time that loss of oestrogen has a significant effect on DR3; decreasing expression on CD14+ monocytes and increasing expression on CD4+ T cells. These data suggest that while oestrogen-deficiency induced changes in DR3 expression do not affect late post-menopausal bone loss they could potentially have an indirect role in early menopausal bone loss through the modulation of T cell activity.

Inhibitory killer cell immunoglobulin-like receptors strengthen CD8+ T cell-mediated control of HIV-1, HCV, and HTLV-1.

Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell-mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1-, hepatitis C virus (HCV)-, and human T cell leukemia virus type 1 (HTLV-1)-infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

HCMV-Encoded NK Modulators: Lessons From in vitro and in vivo Genetic Variation.

Human cytomegalovirus (HCMV) is under constant selective pressure from the immune system in vivo. Study of HCMV genes that have been lost in the absence of, or genetically altered by, such selection can focus research toward findings of in vivo significance. We have been particularly interested in the most pronounced change in the highly passaged laboratory strains AD169 and Towne-the deletion of 13-15 kb of sequence (designated the UL/b' region) that encodes up to 22 canonical genes, UL133-UL150. At least 5 genes have been identified in UL/b' that inhibit NK cell function. UL135 suppresses formation of the immunological synapse (IS) by remodeling the actin cytoskeleton, thereby illustrating target cell cooperation in IS formation. UL141 inhibits expression of two activating ligands (CD155, CD112) for the activating receptor CD226 (DNAM-1), and two receptors (TRAIL-R1, R2) for the apoptosis-inducing TRAIL. UL142, ectopically expressed in isolation, and UL148A, target specific MICA allotypes that are ligands for NKG2D. UL148 impairs expression of CD58 (LFA-3), the co-stimulatory cell adhesion molecule for CD2 found on T and NK cells. Outside UL/b', studies on natural variants have shown UL18 mutants change affinity for their inhibitory ligand LIR-1, while mutations in UL40's HLA-E binding peptide differentially drive NKG2C+ NK expansions. Research into HCMV genomic stability and its effect on NK function has provided important insights into virus:host interactions, but future studies will require consideration of genetic variability and the effect of genes expressed in the context of infection to fully understand their in vivo impact.

High-Definition Analysis of Host Protein Stability during Human Cytomegalovirus Infection Reveals Antiviral Factors and Viral Evasion Mechanisms.

Human cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors.

CCL3 and MMP-9 are induced by TL1A during death receptor 3 (TNFRSF25)-dependent osteoclast function and systemic bone loss.

Reduced bone density and secondary osteoporosis, resulting in increased risk of fracture, is a significant complicating factor in the inflammatory arthritides. While the exact etiology of systemic bone loss is not fully elucidated, recent insights into the tumor necrosis factor super family (TNFSF) revealed a potential role for death receptor 3 (DR3/TNFRSF25) and one of its ligands, TNF-like protein 1A (TL1A/TNFSF15). The mechanisms by which DR3/TL1A signalling modulates bone loss are unclear. We investigated the effect of DR3/TL1A signalling upon osteoclast-dependent chemokine and MMP production to unravel novel mechanisms whereby this pathway regulates OC formation and OC-dependent bone resorption. Collagen induced arthritis (CIA) was established in DR3wt and DR3ko mice, joints were sectioned and analysed histologically for bone damage while systemic trabecular bone loss distal to the affected joints was compared by micro-CT. Ablation of DR3 protected DBA/1 mice against the development and progression of CIA. In DR3ko, joints of the ankle and mid-foot were almost free of bone erosions and long bones of mice with CIA were protected against systemic trabecular bone loss. In vitro, expression of DR3 was confirmed on primary human CD14+ osteoclast precursors by flow cytometry. These cells were treated with TL1A in osteoclast differentiation medium and TRAP+ osteoclasts, bone resorption, levels of osteoclast-associated chemokines (CCL3, CCL2 and CXCL8) and MMP-9 measured. TL1A intensified human osteoclast differentiation and bone resorption and increased osteoclast-associated production of CCL3 and MMP-9. Our data reveals the DR3 pathway as an attractive therapeutic target to combat adverse bone pathology associated with inflammatory arthritis. We demonstrate that DR3 is critical in the pathogenesis of murine CIA and associated secondary osteoporosis. Furthermore, we identify a novel mechanism by which the DR3/TL1A pathway directly enhances human OC formation and resorptive activity, controlling expression and activation of CCL3 and MMP-9.

Death Receptor 3 Promotes Chemokine-Directed Leukocyte Recruitment in Acute Resolving Inflammation and Is Essential for Pathological Development of Mesothelial Fibrosis in Chronic Disease.

Death receptor 3 (DR3; TNFRSF25) and its tumor necrosis factor-like ligand TL1A (TNFSF15) control several processes in inflammatory diseases through the expansion of effector T cells and the induction of proinflammatory cytokines from myeloid and innate lymphoid cells. Using wild-type (DR3+/+) and DR3-knockout (DR3-/-) mice, we show that the DR3/TL1A pathway triggers the release of multiple chemokines after acute peritoneal inflammation initiated by a single application of Staphylococcus epidermidis supernatant, correlating with the infiltration of multiple leukocyte subsets. In contrast, leukocyte infiltration was not DR3 dependent after viral challenge with murine cytomegalovirus. DR3 expression was recorded on connective tissue stroma, which provided DR3-dependent release of chemokine (C-C motif) ligand (CCL) 2, CCL7, CXCL1, and CXCL13. CCL3, CCL4, and CXCL10 production was also DR3 dependent, but quantitative RT-PCR showed that their derivation was not stromal. In vitro cultures identified resident macrophages as a DR3-dependent source of CCL3. Whether DR3 signaling could contribute to a related peritoneal pathology was then tested using multiple applications of S. epidermidis supernatant, the repetitive inflammatory episodes of which lead to peritoneal membrane thickening and collagen deposition. Unlike their DR3+/+ counterparts, DR3-/- mice did not develop fibrosis of the mesothelial layer. Thus, this work describes both a novel function and essential requirement for the DR3/TL1A pathway in acute, resolving, and chronic inflammation in the peritoneal cavity.

Characterization of death receptor 3-dependent aortic changes during inflammatory arthritis.

Murine collagen-induced arthritis (mCIA) is characterized by decreased vascular constriction responses and increased MMP-9. Here, we describe additional histological alterations within the aorta and surrounding perivascular adipose tissue (PVAT), study the role of PVAT in constriction response, and investigate the potential involvement of death receptor 3 (DR3). mCIA was induced in wild-type (WT) and DR3-/- mice with nonimmunized, age-matched controls. Vascular function was determined in isolated aortic rings ±PVAT, using isometric tension myography, in response to cumulative serotonin concentrations. Cellular expression of F4/80 (macrophages), Ly6G (neutrophils), DR3, and MMP-9 was determined using immunohistochemistry. In WTs, arthritis-induced vascular dysfunction was associated with increased F4/80+ macrophages and increased DR3 expression in the aorta and PVAT. MMP-9 was also up-regulated in PVAT, but did not correlate with alterations of PVAT intact constriction. DR3-/- mice inherently showed increased leukocyte numbers and MMP-9 expression in the PVAT, but retained the same nonarthritic constriction response as DR3WT mice ±PVAT. Arthritic DR3-/- mice had a worsened constriction response than DR3WT and showed an influx of neutrophils to the aorta and PVAT. Macrophage numbers were also up-regulated in DR3-/- PVAT. Despite this influx, PVAT intact DR3-/- constriction responses were restored to the same level as DR3WT. Impaired vascular constriction in inflammatory arthritis occurs independently of total MMP-9 levels, but correlates with macrophage and neutrophil ingress. Ablating DR3 worsens the associated vasculature dysfunction, however, DR3-/- PVAT is able to protect the aorta against aberrant vasoconstriction caused in this model.