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.

Toll-like receptor linked cytokine profiles in cerebrospinal fluid discriminate neurological infection from sterile inflammation.

Rapid determination of an infective aetiology causing neurological inflammation in the cerebrospinal fluid can be challenging in clinical practice. Post-surgical nosocomial infection is difficult to diagnose accurately, as it occurs on a background of altered cerebrospinal fluid composition due to the underlying pathologies and surgical procedures involved. There is additional diagnostic difficulty after external ventricular drain or ventriculoperitoneal shunt surgery, as infection is often caused by pathogens growing as biofilms, which may fail to elicit a significant inflammatory response and are challenging to identify by microbiological culture. Despite much research effort, a single sensitive and specific cerebrospinal fluid biomarker has yet to be defined which reliably distinguishes infective from non-infective inflammation. As a result, many patients with suspected infection are treated empirically with broad-spectrum antibiotics in the absence of definitive diagnostic criteria. To begin to address these issues, we examined cerebrospinal fluid taken at the point of clinical equipoise to diagnose cerebrospinal fluid infection in 14 consecutive neurosurgical patients showing signs of inflammatory complications. Using the guidelines of the Infectious Diseases Society of America, six cases were subsequently characterized as infected and eight as sterile inflammation. Twenty-four contemporaneous patients with idiopathic intracranial hypertension or normal pressure hydrocephalus were included as non-inflamed controls. We measured 182 immune and neurological biomarkers in each sample and used pathway analysis to elucidate the biological underpinnings of any biomarker changes. Increased levels of the inflammatory cytokine interleukin-6 and interleukin-6-related mediators such as oncostatin M were excellent indicators of inflammation. However, interleukin-6 levels alone could not distinguish between bacterially infected and uninfected patients. Within the patient cohort with neurological inflammation, a pattern of raised interleukin-17, interleukin-12p40/p70 and interleukin-23 levels delineated nosocomial bacteriological infection from background neuroinflammation. Pathway analysis showed that the observed immune signatures could be explained through a common generic inflammatory response marked by interleukin-6 in both nosocomial and non-infectious inflammation, overlaid with a toll-like receptor-associated and bacterial peptidoglycan-triggered interleukin-17 pathway response that occurred exclusively during infection. This is the first demonstration of a pathway dependent cerebrospinal fluid biomarker differentiation distinguishing nosocomial infection from background neuroinflammation. It is especially relevant to the commonly encountered pathologies in clinical practice, such as subarachnoid haemorrhage and post-cranial neurosurgery. While requiring confirmation in a larger cohort, the current data indicate the potential utility of cerebrospinal fluid biomarker strategies to identify differential initiation of a common downstream interleukin-6 pathway to diagnose nosocomial infection in this challenging clinical cohort.

Activation of naïve CD4+ T cells re-tunes STAT1 signaling to deliver unique cytokine responses in memory CD4+ T cells.

The cytokine IL-6 controls the survival, proliferation and effector characteristics of lymphocytes through activation of the transcription factors STAT1 and STAT3. While STAT3 activity is an ever-present feature of IL-6 signaling in CD4+ T cells, prior activation via the T cell antigen receptor limits IL-6's control of STAT1 in effector and memory populations. Here we found that phosphorylation of STAT1 in response to IL-6 was regulated by the tyrosine phosphatases PTPN2 and PTPN22 expressed in response to the activation of naïve CD4+ T cells. Transcriptomics and chromatin immunoprecipitation-sequencing (ChIP-seq) of IL-6 responses in naïve and effector memory CD4+ T cells showed how the suppression of STAT1 activation shaped the functional identity and effector characteristics of memory CD4+ T cells. Thus, tyrosine phosphatases induced by the activation of naïve T cells determine the way activated or memory CD4+ T cells sense and interpret cytokine signals.

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.

Regulation of early cartilage destruction in inflammatory arthritis by death receptor 3.

OBJECTIVE: To investigate the role of death receptor 3 (DR-3) and its ligand tumor necrosis factor-like molecule 1A (TL1A) in the early stages of inflammatory arthritis. METHODS: Antigen-induced arthritis (AIA) was generated in C57BL/6 mice deficient in the DR-3 gene (DR3(-/-) ) and their DR3(+/+) (wild-type) littermates by priming and intraarticular injection of methylated bovine serum albumin. The joints were sectioned and analyzed histochemically for damage to cartilage and expression of DR3, TL1A, Ly-6G (a marker for neutrophils), the gelatinase matrix metalloproteinase 9 (MMP-9), the aggrecanase ADAMTS-5, and the neutrophil chemoattractant CXCL1. In vitro production of MMP-9 was measured in cultures from fibroblasts, macrophages, and neutrophils following the addition of TL1A and other proinflammatory stimuli. RESULTS: DR3 expression was up-regulated in the joints of wild-type mice following generation of AIA. DR3(-/-) mice were protected against cartilage damage compared with wild-type mice, even at early time points prior to the main accumulation of Teff cells in the joint. Early protection against AIA in vivo correlated with reduced levels of MMP-9. In vitro, neutrophils were major producers of MMP-9, while neutrophil numbers were reduced in the joints of DR3(-/-) mice. However, TL1A neither induced MMP-9 release nor affected the survival of neutrophils. Instead, reduced levels of CXCL1 were observed in the joints of DR3(-/-) mice. CONCLUSION: DR-3 drives early cartilage destruction in the AIA model of inflammatory arthritis through the release of CXCL1, maximizing neutrophil recruitment to the joint and leading to enhanced local production of cartilage-destroying enzymes.

The death receptor 3/TL1A pathway is essential for efficient development of antiviral CD4⁺ and CD8⁺ T-cell immunity.

Death receptor 3 (DR3, TNFRSF25), the closest family relative to tumor necrosis factor receptor 1, promotes CD4(+) T-cell-driven inflammatory disease. We investigated the in vivo role of DR3 and its ligand TL1A in viral infection, by challenging DR3-deficient (DR3(KO)) mice and their DR3(WT) littermates with the ß-herpesvirus murine cytomegalovirus or the poxvirus vaccinia virus. The phenotype and function of splenic T-cells were analyzed using flow cytometry and molecular biological techniques. We report surface expression of DR3 by naive CD8(+) T cells, with TCR activation increasing its levels 4-fold and altering the ratio of DR3 splice variants. T-cell responses were reduced up to 90% in DR3(KO) mice during acute infection. Adoptive transfer experiments indicated this was dependent on T-cell-restricted expression of DR3. DR3-dependent CD8(+) T-cell expansion was NK and CD4 independent and due to proliferation, not decreased cell death. Notably, impaired immunity in DR3(KO) hosts on a C57BL/6 background was associated with 4- to 7-fold increases in viral loads during the acute phase of infection, and in mice with suboptimal NK responses was essential for survival (37.5%). This is the first description of DR3 regulating virus-specific T-cell function in vivo and uncovers a critical role for DR3 in mediating antiviral immunity.

DR3 signaling protects against cisplatin nephrotoxicity mediated by tumor necrosis factor.

The expression of death receptor 3 (DR3), a member of the tumor necrosis factor (TNF) receptor superfamily, is up-regulated in human tubular epithelial cells (TECs) during renal injury, but its function in this setting remains unknown. We used cisplatin to induce renal injury in wild-type (DR3(+/+)) or congenitally deficient DR3(-/-) mice to examine the in vivo role of DR3. Cisplatin induced the expression of DR3, its ligand, TNF-like ligand 1A (TL1A), and TNF in TECs, as observed in human renal injury. Cisplatin increased apoptotic death of DR3(-/-) TECs by twofold compared with DR3(+/+) TECs, whereas it reduced the number of tubules expressing phospho-NF-κBp65(Ser276) by 50% at 72 hours. Similar degrees of induction of DR3, TL1A, and TNF, and changes in apoptosis and phospho-NF-κBp65(Ser276), were obtained in mouse kidney organ cultures treated with cisplatin for 3 hours, suggesting a direct effect on TECs. TNF was implicated in mediating cisplatin-induced tubular damage given that the in vivo co-administration of GM6001, an inhibitor of TNF maturation and release, significantly reduced TNF production and tubular damage. Moreover, TNF exacerbated, whereas TL1A reduced, cisplatin-induced apoptosis in the DR3(+/+) mouse proximal tubule cell line, TKPTS. Our data demonstrate that cisplatin-induced nephrotoxicity is mitigated by DR3 signaling, suggesting that this occurs by antagonizing pro-apoptotic signals induced by TNF. Therefore, activating DR3 may be beneficial in reducing acute kidney injury.

The role of tumor necrosis factor receptor superfamily members in mammalian brain development, function and homeostasis.

Tumor necrosis factor receptor superfamily (TNFRSF) members were initially identified as immunological mediators, and are still commonly perceived as immunological molecules. However, our understanding of the diversity of TNFRSF members' roles in mammalian physiology has grown significantly since the first discovery of TNFRp55 (TNFRSF1) in 1975. In particular, the last decade has provided evidence for important roles in brain development, function and the emergent field of neuronal homeostasis. Recent evidence suggests that TNFRSF members are expressed in an overlapping regulated pattern during neuronal development, participating in the regulation of neuronal expansion, growth, differentiation and regional pattern development. This review examines evidence for non-immunological roles of TNFRSF members in brain development, function and maintenance under normal physiological conditions. In addition, several aspects of brain function during inflammation will also be described, when illuminating and relevant to the non-immunological role of TNFRSF members. Finally, key questions in the field will be outlined.

Naive and activated T cells display differential responsiveness to TL1A that affects Th17 generation, maintenance, and proliferation.

Tumor necrosis factor (TNF)-like cytokine (TL1A) is a T-cell costimulator that bolsters cytokine-induced activation through death receptor 3 (DR3). To explore the relationship between T-cell activation and TL1A responsiveness, flow cytometry profiled DR3 expression in resting and activated T cells. In human CD4(+) T cells, DR3 was induced rapidly following activation and expressed prominently by interleukin (IL)-17-secreting T cells (Th17). Splenic T cells from wild-type and DR3-deficient mice showed that TL1A activation of DR3 inhibits Th17 generation (81 ± 2.6% at 100 ng/ml TL1A) from naive T cells. This response was not associated with suppression of T-cell proliferation. Using neutralizing antibodies or T cells derived from genetically modified mice, TL1A inhibition of Th17 development was found to be independent of IL-2, IL-27, γIFN, IFNAR1, and STAT1. Under suboptimal TCR activation, TL1A continued to block IL-17A secretion, however, the reduced threshold of TCR engagement was now linked with an increase in TL1A-driven proliferation. In contrast, fully committed Th17 cells displayed an altered TL1A responsiveness and in the absence of TCR costimulation supported the maintenance of T cell IL-17A expression. Consequently, TL1A orchestrates unique outcomes in naive and effector T-helper cells, which may affect the proliferation, differentiation and maintenance of Th17 cells in peripheral compartments and inflamed tissues.

Defective B cell ontogeny and humoral immune response in mice prematurely expressing human complement receptor 2 (CR2, CD21) is similar to that seen in aging wild type mice.

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and humoral immune response. We have previously determined altered tyrosine phosphorylation patterns within hCR2 transgenic mice, suggesting that irreversible changes in B cell signaling pathways had occurred, which could explain the B cell unresponsiveness associated with hCR2 transgene expression. In support of that assertion, we found that increasing antigen dose or addition of adjuvant had a minimal impact on the ability of B cells to respond to antigen. However, analysis of aged hCR2(high) mice (1 year plus) revealed that both B cell numbers, B cell sub-population distribution including expansion of a newly described B regulatory cell subset, and immune responses were comparable with age-matched hCR2 negative mice. Finally, we established that B cell unresponsiveness to antigen in aging wild type mice (1 year plus) was equivalent to that noted in 3-month-old hCR2(high) mice. This data provides evidence that 3-month-old hCR2(high) mice have a humoral immune system resembling aged mice and suggests that further examination of the precise molecular and cellular parallels between aged wild type mice and 3-month-old hCR2(high) mice could provide an important insight into the mechanisms which lead to B cell unresponsiveness in the aging immune system.