Antiapoptotic Mcl-1 is critical for the survival and niche-filling capacity of Foxp3⁺ regulatory T cells.

Foxp3⁺ regulatory T (Treg) cells are a crucial immunosuppressive population of CD4⁺ T cells, yet the homeostatic processes and survival programs that maintain the Treg cell pool are poorly understood. Here we report that peripheral Treg cells markedly alter their proliferative and apoptotic rates to rapidly restore numerical deficit through an interleukin 2-dependent and costimulation-dependent process. By contrast, excess Treg cells are removed by attrition, dependent on the Bim-initiated Bak- and Bax-dependent intrinsic apoptotic pathway. The antiapoptotic proteins Bcl-xL and Bcl-2 were dispensable for survival of Treg cells, whereas Mcl-1 was critical for survival of Treg cells, and the loss of this antiapoptotic protein caused fatal autoimmunity. Together, these data define the active processes by which Treg cells maintain homeostasis via critical survival pathways.

IL-2 consumption by highly activated CD8 T cells induces regulatory T-cell dysfunction in patients with hemophagocytic lymphohistiocytosis.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a severe inflammatory condition driven by excessive CD8(+) T-cell activation. HLH occurs as both acquired and familial hemophagocytic lymphohistiocytosis (FHL) forms. In both conditions, a sterile or infectious trigger is required for disease initiation, which then becomes self-sustaining and life-threatening. Recent studies have attributed the key distal event to excessive IFN-γ production; however, the proximal events driving immune dysregulation have remained undefined. OBJECTIVE: We sought to investigate the role of regulatory T (Treg) cells in the pathophysiology of experimental FHL. METHODS: Because mutation in perforin is a common cause of FHL, we used an experimental FHL mouse model in which disease in perforin-deficient mice is triggered by lymphocytic choriomeningitis virus (LCMV). We assessed Treg and CD8(+) T-cell homeostasis and activation during the changing systemic conditions in the mice. In addition, human blood samples were collected and analyzed during the HLH episode. RESULTS: We found no primary Treg cell defects in perforin-deficient mice. However, Treg cell numbers collapsed after LCMV inoculation. The collapse of Treg cell numbers in LCMV-triggered perforin-deficient, but not wild-type, mice was accompanied by the combination of lower IL-2 secretion by conventional CD4(+) T cells, increased IL-2 consumption by activated CD8(+) T cells, and secretion of competitive soluble CD25. Moreover low Treg cell numbers were observed in untreated patients experiencing HLH flares. CONCLUSION: These results demonstrate that excessive CD8(+) T-cell activation rewires the IL-2 homeostatic network away from Treg cell maintenance and toward feed-forward inflammation. These results also provide a potential mechanistic pathway for the progression of infectious inflammation to persistent inflammation in patients with HLH.

Lpr-induced systemic autoimmunity is unaffected by mast cell deficiency.

The function of mast cells in allergic and organ-specific autoimmune responses is highly controversial. In the current study, we aimed to dissect the role of mast cells in systemic autoimmunity in the B6(lpr/lpr) mouse, a spontaneous model of systemic lupus erythematosus. B6(lpr/lpr) mice were interbred with C57Bl/6-Kit(W-sh/W-sh) (Wsh) mice, resulting in mast cell deficiency. The offspring from this cross (Lpr/Wsh mice) developed symptoms of lupus of the same severity as B6(lpr/lpr) mice. Loss of mast cells on the Lpr background did not alter autoantibody production, proteinuria, the composition of T and B cell populations or autoimmune pathology. Reduced c-Kit expression did drive expanded splenomegaly and impeded interleukin-4 production by CD4(+) cells, suggesting minor functions for mast cells. In general, we conclude that mast cell deficiency and c-Kit deficiency do not play a role in the pathogenesis of lupus in B6(lpr/lpr) mice.

A novel Zap70 mutation with reduced protein stability demonstrates the rate-limiting threshold for Zap70 in T-cell receptor signalling.

Loss of ζ-associated protein 70 (Zap70) results in severe immunodeficiency in humans and mice because of the critical role of Zap70 in T-cell receptor (TCR) signalling. Here we describe a novel mouse strain generated by N-ethyl-N-nitrosourea mutagenesis, with the reduced protein stability (rps) mutation in Zap70. The A243V rps mutation resulted in decreased Zap70 protein and a reduced duration of TCR-induced calcium responses, equivalent to that induced by a 50% decrease in catalytically active Zap70. The reduction of signalling through Zap70 was insufficient to substantially perturb thymic differentiation of conventional CD4 and CD8 T cells, although Foxp3(+) regulatory T cells demonstrated altered thymic production and peripheral homeostasis. Despite the mild phenotype, the Zap70(A243V) variant lies just above the functional threshold for TCR signalling competence, as T cells relying on only a single copy of the Zap70(rps) allele for TCR signalling demonstrated no intracellular calcium response to TCR stimulation. This addition to the Zap70 allelic series indicates that a rate-limiting threshold for Zap70 protein levels exists at which signalling capacity switches from nearly intact to effectively null.

Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases.

Matrix metalloproteinases (MMPs), originally discovered to function in the breakdown of extracellular matrix proteins, have gained the status of regulatory proteases in signaling events by liganding and processing hormones, cytokines, chemokines, adhesion molecules and other membrane receptors. However, MMPs also cleave intracellular substrates and have been demonstrated within cells in nuclear, mitochondrial, various vesicular and cytoplasmic compartments, including the cytoskeletal intracellular matrix. Unbiased high-throughput degradomics approaches have demonstrated that many intracellular proteins are cleaved by MMPs, including apoptotic regulators, signal transducers, molecular chaperones, cytoskeletal proteins, systemic autoantigens, enzymes in carbohydrate metabolism and protein biosynthesis, transcriptional and translational regulators, and proteins in charge of protein clearance such as lysosomal and ubiquitination enzymes. Besides proteolysis inside cells, intracellular proteins may also be modulated by MMPs in the extracellular milieu. Indeed, many intracellular proteins exit cells by non-classical secretion mechanisms or by various conditions of cell death by apoptosis, necrosis and NETosis, and become accessible to extracellular proteases. Intracellular substrate proteolysis by MMPs is involved in innate immune defense and apoptosis, and affects oncogenesis and pathology of cardiac, neurological, protein conformational and autoimmune diseases, including ischemia-reperfusion injury, cardiomyopathy, Parkinson's disease, cataract, multiple sclerosis and systemic lupus erythematosus. Since the same MMP may affect physiology and pathology in different and even opposite ways, depending on its extracellular or subcellular localization, an additional layer of complexity is added to therapeutic MMP inhibition. Hence, further elucidation of intracellular MMP localizations and intracellular substrate proteolysis is a new challenge in MMP research.

Deficiency of gelatinase B/MMP-9 aggravates lpr-induced lymphoproliferation and lupus-like systemic autoimmune disease.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a key enzyme involved in inflammatory, hematological, vascular and neoplastic diseases. In previous studies, we explored the intracellular substrate set or 'degradome' of MMP-9 and found many systemic autoantigens as novel intracellular gelatinase B substrates. Little is known, however, about the functional role of MMP-9 in the development of systemic autoimmunity in vivo. B6(lpr/lpr) mice with defective Fas-mediated apoptosis were used to investigate the functions of MMP-9 in lymphocyte proliferation and in the development of systemic autoimmunity. Combined Fas and gelatinase B deficiency resulted in extreme lymphoproliferative disease with enhanced lymphadenopathy and splenomegaly, and significantly reduced survival compared with single Fas deficiency. At the cellular level, this was corroborated by increased lymph node accumulation of 'double negative' T cells, B cells and myeloid cells. In addition, higher autoantibody titers and more pronounced autoimmune tissue injury were found in the absence of MMP-9, culminating in chronically enhanced systemic lupus erythematosus (SLE)-like autoimmunity. After cleavage by MMP-9 the SLE autoantigens U1snRNP A and ribosomal protein P0 were hardly recognized by plasma samples of both B6(lpr/lpr).MMP-9⁻/⁻ and B6(lpr/lpr).MMP-9+/+ mice, pointing to a destruction of B cell epitopes by MMP-9-mediated proteolysis. In addition, the same loss of immunodominant epitopes was observed with plasma samples from SLE patients, suggesting that MMP-9 suppresses systemic antibody-mediated autoimmunity by clearance of autoepitopes in immunogenic substrates. Thus, new protective functions for MMP-9 were revealed in the suppression of lymphoproliferation and dampening of systemic autoimmunity, cautioning against the long-term use of MMP inhibitors in autoimmune lymphoproliferative syndrome (ALPS) and SLE.

Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9.

Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1 was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.

Truncated recombinant puumala virus nucleocapsid proteins protect mice against challenge in vivo.

In Europe, Puumala virus and Dobrava virus are the major hantaviruses that cause hemorrhagic fever with renal syndrome in humans. As hantaviruses can cause diseases with high morbidity and mortality rates, and as to date there is no specific treatment, efforts are concentrated on the development of vaccines. In this study we characterized the immunogenicity of recombinant nucleocapsid proteins of Puumala virus (PUUV) linked to a carrier protein corresponding to the outer membrane protein A from Klebsiella pneumoniae (rP40). The rP40 molecule is a novel carrier protein that facilitates exogenous antigen uptake by dendritic cells. We cloned and expressed the recombinant PUUV proteins in the E. coli mutant ICONE 200 using the tryptophan promoter-controlled pTEXmp18 expression vector. All recombinant PUUV proteins were found to be highly immunogenic in NMRI mice after three immunizations of 10 microg each of the protein. Only the truncated construct, P40-Puu118, gave high antibody titers after two vaccinations of 0.2 microg each. Likewise in the challenge experiments in NMRI mice, only the truncated construct P40-Puu118 resulted in 100% protection after three immunizations of 10 microg each. The results suggest that P40-Puu118 in particular is a good candidate for a recombinant vaccine against Puumala virus. All recombinant proteins linked to rP40 induced high antibody responses, indicating that rP40 is a carrier protein with potential for use in other vaccines.

Gelatinase B/matrix metalloproteinase-9 is a phase-specific effector molecule, independent from Fas, in experimental autoimmune encephalomyelitis.

Gelatinase B/matrix metalloproteinase-9 (MMP-9) triggers multiple sclerosis (MS) and the animal model of experimental autoimmune encephalomyelitis (EAE) by the breakdown of the blood-brain barrier. Interestingly, MMP-9 is beneficial in systemic autoimmunity caused by Fas-deficiency. Fas-deficient (faslpr) and Fas-ligand-deficient mice are protected against EAE. We here investigated the interaction between Fas and MMP-9 in the setting of induction of EAE and compared short- and long-term effects. We provoked EAE with myelin oligodendrocyte glycoprotein (MOG) peptide and compared EAE development in four genotypes (wild-type (WT), single knockout mmp-9-/-, faslpr, and mmp-9-/-/faslpr) and monitored leukocytes, cytokines and chemokines as immunological parameters. As expected, faslpr mice were resistant against EAE induction, whereas MMP-9 single knockout mice were not. In the double mmp-9-/-/ faslpr mice the effects on disease scores pointed to independent rather than interrelated disease mechanisms. On a short term, after EAE induction leukocytes infiltrated into the brain and cytokine and chemokine levels were significantly higher in all the four genotypes studied, even in the faslpr and mmp-9-/-/faslpr, which did not develop clinical disease. The levels of MMP-9 but not of MMP-2 were increased in the brain and in the peripheral organs after EAE induction. After 40 days all the animals recovered and did not show signs of EAE. However, the absence of MMP-9 in the remission phase suggested a protective role of MMP-9 in the late phase of the disease, because single mmp-9-/- mice presented a delayed remission in comparison with WT animals suggesting a phase-dependent role of MMP-9 in the disease. Nevertheless, the levels of some cytokines and chemokines remained higher than in control animals even 100 days after EAE induction, attesting to a prolonged state of immune activation. We thus yielded new insights and useful markers to monitor this activated immune status. Furthermore, MMP-9 but not MMP-2 levels remained increased in the brains and, to a higher extend, in the spleens of the WT mice even during the remission phase, which is in line with the role of MMP-9 as a useful marker and a protective factor for EAE in the remission phase.

Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations.

The nucleopore is an essential structure of the eukaryotic cell, regulating passage between the nucleus and cytoplasm. While individual functions of core nucleopore proteins have been identified, the role of other components, such as Nup210, are poorly defined. Here, through the use of an unbiased ENU mutagenesis screen for mutations effecting the peripheral T cell compartment, we identified a Nup210 mutation in a mouse strain with altered CD4/CD8 T cell ratios. Through the generation of Nup210 knockout mice we identified Nup210 as having a T cell-intrinsic function in the peripheral homeostasis of T cells. Remarkably, despite the deep evolutionary conservation of this key nucleopore complex member, no other major phenotypes developed, with viable and healthy knockout mice. These results identify Nup210 as an important nucleopore complex component for peripheral T cells, and raise further questions of why this nucleopore component shows deep evolutionary conservation despite seemingly redundant functions in most cell types.

Treatment patterns in moderate-to-severe plaque psoriasis: results from a Belgian cross-sectional study (DISCOVER).

PURPOSE: The present study aimed to evaluate current treatment patterns and achievement of treatment goals in Belgian patients with moderate-to-severe plaque psoriasis. MATERIALS AND METHODS: This cross-sectional observational study (DISCOVER) was conducted in 2011 - 2012 in Belgian dermatology centers. Patient data were collected during a single visit and included information on psoriasis management and severity (PASI and DLQI). Treatment success was defined according to the current European consensus treatment goal algorithm. RESULTS: Of the 556 patients included in the study, 38.1% reported no current treatment or only topicals, 34.2% were being treated with traditional systemics and/or phototherapy, and 29.5% with biologics. Methotrexate (11.7%) was the most commonly prescribed traditional systemic and adalimumab (14.2%) was the most commonly prescribed biologic agent at the time of the study. The percentage of patients achieving treatment goals was significantly higher in biologic-treated patients (73.1%) compared to those using traditional systemics (50.6%), phototherapy (41.1%), or no treatment/only topicals (20.9%; p < .001). CONCLUSIONS: Nearly 40% of Belgian patients with moderate-to-severe psoriasis in the DISCOVER study were undertreated despite the severity of their disease. Undertreatment of psoriasis remains a problem in Belgium and more effective educational strategies are needed to ensure the best treatment outcome for these patients. [Formula: see text].

Inflammation-associated enterotypes, host genotype, cage and inter-individual effects drive gut microbiota variation in common laboratory mice.

BACKGROUND: Murine models are a crucial component of gut microbiome research. Unfortunately, a multitude of genetic backgrounds and experimental setups, together with inter-individual variation, complicates cross-study comparisons and a global understanding of the mouse microbiota landscape. Here, we investigate the variability of the healthy mouse microbiota of five common lab mouse strains using 16S rDNA pyrosequencing. RESULTS: We find initial evidence for richness-driven, strain-independent murine enterotypes that show a striking resemblance to those in human, and which associate with calprotectin levels, a marker for intestinal inflammation. After enterotype stratification, we find that genetic, caging and inter-individual variation contribute on average 19%, 31.7% and 45.5%, respectively, to the variance in the murine gut microbiota composition. Genetic distance correlates positively to microbiota distance, so that genetically similar strains have more similar microbiota than genetically distant ones. Specific mouse strains are enriched for specific operational taxonomic units and taxonomic groups, while the 'cage effect' can occur across mouse strain boundaries and is mainly driven by Helicobacter infections. CONCLUSIONS: The detection of enterotypes suggests a common ecological cause, possibly low-grade inflammation that might drive differences among gut microbiota composition in mammals. Furthermore, the observed environmental and genetic effects have important consequences for experimental design in mouse microbiome research.

Insufficiently defined genetic background confounds phenotypes in transgenic studies as exemplified by malaria infection in Tlr9 knockout mice.

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases. However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential.

Multidimensional degradomics identifies systemic autoantigens and intracellular matrix proteins as novel gelatinase B/MMP-9 substrates.

The action radius of matrix metalloproteinases or MMPs is not restricted to massive extracellular matrix (ECM) degradation, it extends to the proteolysis of numerous secreted and membrane-bound proteins. Although many instances exist in which cells disintegrate, often in conjunction with induction of MMPs, the intracellular MMP substrate repertoire or degradome remains relatively unexplored. We started an unbiased exploration of the proteolytic modification of intracellular proteins by MMPs, using gelatinase B/MMP-9 as a model enzyme. To this end, multidimensional degradomics technology was developed by the integration of broadly available biotechniques. In this way, 100-200 MMP-9 candidate substrates were isolated, of which 69 were identified. Integration of these results with the known biological functions of the substrates revealed many novel MMP-9 substrates from the intracellular matrix (ICM), such as actin, tubulin, gelsolin, moesin, ezrin, Arp2/3 complex subunits, filamin B and stathmin. About 2/3 of the identified candidates were autoantigens described in multiple autoimmune conditions and in cancer (e.g. annexin I, nucleolin, citrate synthase, HMGB1, alpha-enolase, histidyl-tRNA synthetase, HSP27, HSC70, HSP90, snRNP D3). These findings led to the insight that MMPs and other proteases may have novel (immuno)regulatory properties by the clearance of toxic and immunogenic burdens of abundant ICM proteins released after extensive necrosis. In line with the extracellular processing of organ-specific autoantigens, proteolysis might also assist in the generation of immunodominant 'neo-epitopes' from systemic autoantigens. The study of proteolysis of ICM molecules, autoantigens, alarmins and other crucial intracellular molecules may result in the discovery of novel roles for proteolytic modification.

The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) constitute a family of more than 20 endopeptidases. Identification of specific matrix and non-matrix components as MMP substrates showed that, aside from their initial role as extracellular matrix modifiers, MMPs play significant roles in highly complex processes such as the regulation of cell behavior, cell-cell communication, and tumor progression. Thanks to the comprehensive examination of the expanded MMP action radius, the initial view of proteases acting in the soluble phase has evolved into a kaleidoscope of proteolytic reactions connected to the cell surface. Important classes of cell surface molecules include adhesion molecules, mediators of apoptosis, receptors, chemokines, cytokines, growth factors, proteases, intercellular junction proteins, and structural molecules. Proteolysis of cell surface proteins by MMPs may have extremely diverse biological implications, ranging from maturation and activation, to inactivation or degradation of substrates. In this way, modification of membrane-associated proteins by MMPs is crucial for communication between cells and the extracellular milieu, and determines cell fate and the integrity of tissues. Hence, insights into the processing of cell surface proteins by MMPs and the concomitant effects on physiological processes as well as on disease onset and evolution, leads the way to innovative therapeutic approaches for cancer, as well as degenerative and inflammatory diseases.