Targeted disruption of the MHC class II Aa gene in C57BL/6 mice

The MHC class II gene Aa was disrupted by targeted mutation in embryonic stem (ES) cells derived from C57BL/6 mice to prevent expression of MHC class II molecules. Contrary to previous reports, the effect of the null-mutation on T cell development was investigated in C57BL/6 mice, which provide a defined genetic background. The complete lack of cell surface expression of MHC class II molecules in B6-Aa0/Aa0 homozygous mutant mice was directly demonstrated by cytofiuorometric analysis using anti-Ab and anti-la specific mAbs. Development of CD4+CD8− T cells in the thymus was largely absent except for a small population of thymocytes expressing high levels of CD4 together with low amounts of CD8. The majority of these cells express the TCR at high density. Although mature CD4+CD8− T cells were undetectable in the thymus, some T cells with a CD4+CD8−TCRhigh phenotype were found in lymph nodes and spleen. Peripheral T cells from themutant mice can be polyclonally activated in vitro with the mitogen concanavalin A. However, they could not be stimulated with staphylococcal enterotoxin B in autologous lymphocyte reactions, thereby demonstrating the absence of MHC class II expression in these mice. Peripheral B cells in B6-Aa0/Aa0 mutants were functional and responded to the T cell independent antigen levan by the production of antigenspecific IgM antibodies similar to wild-type cells. The B6-Aa0/Aa0 mutant mice described in this study represent an important tool to investigate the involvement of MHC class II molecules in lymphocyte maturation and the immune respons

The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal

BACKGROUND: Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. RESULTS: Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr (-/-) mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. CONCLUSION: Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance

Analysis of a wild mouse promoter variant reveals a novel role for FcγRIIb in the control of the germinal center and autoimmunity.

Genetic variants of the inhibitory Fc receptor FcγRIIb have been associated with systemic lupus erythematosus in humans and mice. The mechanism by which Fcgr2b variants contribute to the development of autoimmunity is unknown and was investigated by knocking in the most commonly conserved wild mouse Fcgr2b promoter haplotype, also associated with autoimmune-prone mouse strains, into the C57BL/6 background. We found that in the absence of an AP-1-binding site in its promoter, FcγRIIb failed to be up-regulated on activated and germinal center (GC) B cells. This resulted in enhanced GC responses, increased affinity maturation, and autoantibody production. Accordingly, in the absence of FcγRIIb activation-induced up-regulation, mice developed more severe collagen-induced arthritis and spontaneous glomerular immune complex deposition. Our data highlight how natural variation in Fcgr2b drives the development of autoimmune disease. They also show how the study of such variants using a knockin approach can provide insight into immune mechanisms not possible using conventional genetic manipulation, in this case demonstrating an unexpected critical role for the activation-induced up-regulation of FcγRIIb in controlling affinity maturation, autoantibody production, and autoimmunity

Enterocyte-specific A20 deficiency sensitizes to tumor necrosis factor–induced toxicity and experimental colitis

A20 is a nuclear factor κB (NF-κB) target gene that encodes a ubiquitin-editing enzyme that is essential for the termination of NF-κB activation after tumor necrosis factor (TNF) or microbial product stimulation and for the prevention of TNF-induced apoptosis. Mice lacking A20 succumb to inflammation in several organs, including the intestine, and A20 mutations have been associated with Crohn’s disease. However, ablation of NF-κB activity, specifically in intestinal epithelial cells (IECs), promotes intestinal inflammation. As A20 deficiency sensitizes cells to TNF-induced apoptosis yet also promotes NF-κB activity, it is not clear if A20 deficiency in IECs would exacerbate or ameliorate intestinal inflammation. We generated mice lacking A20 specifically in IECs. These mice did not show spontaneous intestinal inflammation but exhibited increased susceptibility to experimental colitis, and their IECs were hypersensitive to TNF-induced apoptosis. The resulting TNF-driven breakdown of the intestinal barrier permitted commensal bacterial infiltration and led to systemic inflammation. These studies define A20 as a major antiapoptotic protein in the intestinal epithelium and further indicate that defects in A20 might contribute to inflammatory bowel disease in humans

CIN85 drives B cell responses by linking BCR signals to the canonical NF-κB pathway

CIN85 transduces B cell receptor signals to IKK-β, and its expression in B cells is essential for T cell–independent type II antibody responses in mice

MHC class II–restricted antigen presentation by plasmacytoid dendritic cells inhibits T cell–mediated autoimmunity

Although plasmacytoid dendritic cells (pDCs) express major histocompatibility complex class II (MHCII) molecules, and can capture, process, and present antigens (Ags), direct demonstrations that they function as professional Ag-presenting cells (APCs) in vivo during ongoing immune responses remain lacking. We demonstrate that mice exhibiting a selective abrogation of MHCII expression by pDCs develop exacerbated experimental autoimmune encephalomyelitis (EAE) as a consequence of enhanced priming of encephalitogenic CD4+ T cell responses in secondary lymphoid tissues. After EAE induction, pDCs are recruited to lymph nodes and establish MHCII-dependent myelin-Ag–specific contacts with CD4+ T cells. These interactions promote the selective expansion of myelin-Ag–specific natural regulatory T cells that dampen the autoimmune T cell response. pDCs thus function as APCs during the course of EAE and confer a natural protection against autoimmune disease development that is mediated directly by their ability to present of Ags to CD4+ T cells in vivo

IKK-induced NF-kappa B1 p105 proteolysis is critical for B cell antibody responses to T cell-dependent antigen

The importance of IκB kinase (IKK)–induced proteolysis of NF-κB1 p105 in B cells was investigated using Nfkb1SSAA/SSAA mice, in which this NF-κB signaling pathway is blocked. Nfkb1SSAA mutation had no effect on the development and homeostasis of follicular mature (FM) B cells. However, analysis of mixed bone marrow chimeras revealed that Nfkb1SSAA/SSAA FM B cells were completely unable to mediate T cell–dependent antibody responses. Nfkb1SSAA mutation decreased B cell antigen receptor (BCR) activation of NF-κB in FM B cells, which selectively blocked BCR stimulation of cell survival and antigen-induced differentiation into plasmablasts and germinal center B cells due to reduced expression of Bcl-2 family proteins and IRF4, respectively. In contrast, the antigen-presenting function of FM B cells and their BCR-induced migration to the follicle T cell zone border, as well as their growth and proliferation after BCR stimulation, were not affected. All of the inhibitory effects of Nfkb1SSAA mutation on B cell functions were rescued by normalizing NF-κB activation genetically. Our study identifies critical B cell-intrinsic functions for IKK-induced NF-κB1 p105 proteolysis in the antigen-induced survival and differentiation of FM B cells, which are essential for T-dependent antibody responses

CLUH regulates mitochondrial metabolism by controlling translation and decay of target mRNAs

Mitochondria are essential organelles that host crucial metabolic pathways and produce adenosine triphosphate. The mitochondrial proteome is heterogeneous among tissues and can dynamically change in response to different metabolic conditions. Although the transcriptional programs that govern mitochondrial biogenesis and respiratory function are well known, posttranscriptional regulatory mechanisms remain unclear. In this study, we show that the cytosolic RNA-binding protein clustered mitochondria homologue (CLUH) regulates the expression of a mitochondrial protein network supporting key metabolic programs required under nutrient deprivation. CLUH exerts its function by controlling the stability and translation of target messenger RNAs. In the absence of Cluh, mitochondria are severely depleted of crucial enzymes involved in catabolic energy-converting pathways. CLUH preserves oxidative mitochondrial function and glucose homeostasis, thus preventing death at the fetal–neonatal transition. In the adult liver, CLUH ensures maximal respiration capacity and the metabolic response to starvation. Our results shed new light on the posttranscriptional mechanisms controlling the expression of mitochondrial proteins and suggest novel strategies to tailor mitochondrial function to physiological and pathological conditions.Peer reviewe

Neoplastic Transformation of T Lymphocytes through Transgenic Expression of a Virus Host Modification Protein

Virus host evasion genes are ready-made tools for gene manipulation and therapy. In this work we have assessed the impact in vivo of the evasion gene A238L of the African Swine Fever Virus, a gene which inhibits transcription mediated by both NF-κB and NFAT. The A238L gene has been selectively expressed in mouse T lymphocytes using tissue specific promoter, enhancer and locus control region sequences for CD2. The resulting two independently derived transgenic mice expressed the transgene and developed a metastasic, angiogenic and transplantable CD4+CD8+CD69– lymphoma. The CD4+CD8+CD69– cells also grew vigorously in vitro. The absence of CD69 from the tumour cells suggests that they were derived from T cells at a stage prior to positive selection. In contrast, transgenic mice similarly expressing a mutant A238L, solely inhibiting transcription mediated by NF-κB, were indistinguishable from wild type mice. Expression of Rag1, Rag2, TCRβ-V8.2, CD25, FoxP3, Bcl3, Bcl2 l14, Myc, IL-2, NFAT1 and Itk, by purified CD4+CD8+CD69– thymocytes from A238L transgenic mice was consistent with the phenotype. Similarly evaluated expression profiles of CD4+CD8+ CD69– thymocytes from the mutant A238L transgenic mice were comparable to those of wild type mice. These features, together with the demonstration of (mono-)oligoclonality, suggest a transgene-NFAT-dependent transformation yielding a lymphoma with a phenotype reminiscent of some acute lymphoblastic lymphomas