Ann Lin

Proapoptotic Bak and Bax guard against fatal systemic and organ-specific autoimmune disease

Dysregulation of the “intrinsic” apoptotic pathway is associated with the development of cancer and autoimmune disease. Bak and Bax are two proapoptotic members of the Bcl-2 protein family with overlapping, essential roles in the intrinsic apoptotic pathway. Their activity is critical for the control of cell survival during lymphocyte development and homeostasis, best demonstrated by defects in thymic T-cell differentiation and peripheral lymphoid homeostasis caused by their combined loss. Because most bak−/−bax−/− mice die perinatally, the roles of Bax and Bak in immunological tolerance and prevention of autoimmune disease remain unclear. We show that mice reconstituted with a Bak/Bax doubly deficient hematopoietic compartment develop a fatal systemic lupus erythematosus-like autoimmune disease characterized by hypergammaglobulinemia, autoantibodies, lymphadenopathy, glomerulonephritis, and vasculitis. Importantly, these mice also develop a multiorgan autoimmune disease with autoantibodies against most solid glandular structures and evidence of glandular atrophy and necrotizing vasculitis. Interestingly, similar albeit less severe pathology was observed in mice containing a hematopoietic compartment deficient for only Bak, a phenotype reminiscent of the disease seen in patients with point mutations in BAK. These studies demonstrate a critical role for Bak and an ancillary role for Bax in safeguarding immunological tolerance and prevention of autoimmune disease. This suggests that direct activators of the intrinsic apoptotic pathway, such as BH3 mimetics, may be useful for treatment of diverse autoimmune diseases.

Impact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK.

It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok−/−Bax−/−Bak−/− triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax−/−Bak−/− double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.

The NF-κB transcription factor RelA is required for the tolerogenic function of Foxp3+ regulatory T cells

The properties of CD4(+) regulatory T cell (Treg) subsets are dictated by distinct patterns of gene expression determined by FOXP3 and different combinations of various transcription factors. Here we show the NF-κB transcription factor RelA is constitutively active in naïve and effector Tregs. The conditional inactivation of Rela in murine FOXP3(+) cells induces a rapid onset, multi-focal autoimmune disease that depends on RelA being expressed in conventional T cells. In addition to promoting Treg lineage stability, RelA determines the size of the effector Treg population, a function influenced by the presence or absence of RelA in conventional T cells. These findings showing that RelA controls Treg stability and promotes the competitive fitness of effector Tregs highlight the importance of RelA activity in peripheral Treg induced tolerance.

Impact of loss of NF-κB1, NF-κB2 or c-REL on SLE-like autoimmune disease and lymphadenopathy in Fas lpr/lpr mutant mice

Defects in apoptosis can cause autoimmune disease. Loss‐of‐function mutations in the ‘death receptor’ FAS impair the deletion of autoreactive lymphocytes in the periphery, leading to progressive lymphadenopathy and systemic lupus erythematosus‐like autoimmune disease in mice (Faslpr/lpr (mice homozygous for the lymphoproliferation inducing spontaneous mutation)) and humans. The REL/nuclear factor‐κB (NF‐κB) transcription factors regulate a broad range of immune effector functions and are also implicated in various autoimmune diseases. We generated compound mutant mice to investigate the individual functions of the NF‐κB family members NF‐κB1, NF‐κB2 and c‐REL in the various autoimmune pathologies of Faslpr/lpr mutant mice. We show that loss of each of these transcription factors resulted in amelioration of many classical features of autoimmune disease, including hypergammaglobulinaemia, anti‐nuclear autoantibodies and autoantibodies against tissue‐specific antigens. Remarkably, only c‐REL deficiency substantially reduced immune complex‐mediated glomerulonephritis and extended the lifespan of Faslpr/lpr mice. Interestingly, compared with the Faslpr/lpr animals, Faslpr/lprnfkb2−/− mice presented with a dramatic acceleration and augmentation of lymphadenopathy that was accompanied by severe lung pathology due to extensive lymphocytic infiltration. The Faslpr/lprnfkb1−/− mice exhibited the combined pathologies caused by defects in FAS‐mediated apoptosis and premature ageing due to loss of NF‐κB1. These findings demonstrate that different NF‐κB family members exert distinct roles in the development of the diverse autoimmune and lymphoproliferative pathologies that arise in Faslpr/lpr mice, and suggest that pharmacological targeting of c‐REL should be considered as a strategy for therapeutic intervention in autoimmune diseases.

Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA

Evidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA

Chemical chaperone TUDCA prevents apoptosis and improves survival during polymicrobial sepsis in mice

Sepsis-induced lymphopenia is a major cause of morbidities in intensive care units and in populations with chronic conditions such as renal failure, diabetes, HIV and alcohol abuse. Currently, other than supportive care and antibiotics, there are no treatments for this condition. We developed an in vitro assay to understand the role of the ER-stress-mediated apoptosis process in lymphocyte death during polymicrobial sepsis, which was reproducible in in vivo mouse models. Modulating ER stress using chemical chaperones significantly reduced the induction of the pro-apoptotic protein Bim both in vitro and in mice. Furthermore, in a ‘two-hit’ pneumonia model in mice, we have been able to demonstrate that administration of the chemical chaperone TUDCA helped to maintain lymphocyte homeostasis by significantly reducing lymphocyte apoptosis and this correlated with four-fold improvement in survival. Our results demonstrate a novel therapeutic opportunity for treating sepsis-induced lymphopenia in humans.

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1–3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), FasLΔs/Δs mice, or the membrane-bound form of FasL (mFasL), FasLΔm/Δm mice. After PVIgG treatment, blisters are only visible in FasLΔm/Δm animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the FasLΔs/Δs animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.