14-3-3 proteins interact with neurofilament protein-L and regulate dynamic assembly of neurofilaments.

Neurofilament protein-L (NF-L) is the core component of neurofilaments. Recent studies indicate that the NF-L mutations reported in human Charcot-Marie-Tooth (CMT) disease lead to the formation of NF-L aggregates and result in axon degeneration of motor and sensory neurons, which are thought to be the cause of CMT disease type 2E. In the present study, we investigated the dynamic regulation of NF-L assembly and the mechanism of aggregate formation of CMT NF-L mutants. We report that 14-3-3 proteins interact with NF-L in a phosphorylation-dependent manner. Investigation of mutations of phospho-serine sites at the head domain of NF-L revealed that several phosphorylation sites, particularly Ser43 and Ser55, were important for 14-3-3 binding. 14-3-3 overexpression resulted in a significant increase in the dynamic exchange rate of NF-L subunits and induced striking disassembly of neurofilaments. CMT NF-L mutants, particularly those with mutations in the Pro8 and Pro22 sites of the NF-L head domain, led to substantially diminished interaction between 14-3-3 and NF-L, which resulted in the formation of NF-L aggregates and the disruption of the neurofilament co-assembly of NF-L and NF-M. However, aggregate formation in CMT NF-L mutants was downregulated by 14-3-3 overexpression. Taken together, these results suggest the important role of 14-3-3 in the dynamic regulation of NF-L assembly, and in the capacity to prevent the formation of NF-L aggregates. Thus, the 14-3-3 proteins are a possible molecular target for CMT disease therapy.

The Function and Roles of ADAMTS-7 in Inflammatory Diseases.

The ADAMTS proteinases are a group of multidomain and secreted metalloproteinases containing the thrombospondin motifs. ADAMTS-7 is a member of ADAMTS family and plays a crucial role in the pathogenesis of arthritis. Overexpression of ADAMTS-7 gene promotes the breakdown of cartilage oligomeric matrix protein (COMP) matrix and accelerates the progression of both surgically induced osteoarthritis and collagen-induced arthritis. Moreover, ADAMTS-7 and tumor necrosis factor-α (TNF-α) form a positive feedback loop in osteoarthritis. More significantly, granulin-epithelin precursor, a growth factor has important roles in bone development and bone-associated diseases, disturbs the interaction between ADAMTS-7 and COMP, and prevents COMP degradation. This review is based on our results and provides an overview of current knowledge of ADAMTS-7, including its structure, function, gene regulation, and inflammatory diseases involvement.

Scaffold protein Disc large homolog 1 is required for T-cell receptor-induced activation of regulatory T-cell function.

Foxp3(+)CD4(+)CD25(high) regulatory T cell (Treg) suppression of inflammation depends on T-cell receptor-mediated Nuclear Factor of Activated T cells c1 (NFATc1) activation with reduced Akt activity. We investigated the role of the scaffold protein Disc large homolog 1 (Dlgh1) in linking the T-cell receptor to this unique signaling outcome. The Treg immunological synapse (IS) recruited fourfold more Dlgh1 than conventional CD4(+) T-cell IS. Tregs isolated from patients with active rheumatoid arthritis, or treated with tumor necrosis factor-α, displayed reduced function and diminished Dlgh1 recruitment to the IS. Furthermore, Dlgh1 silencing abrogated Treg function, impaired NFATc1 activation, reduced phosphatase and tensin homolog levels, and increased Akt activation. Dlgh1 operates independently of the negative feedback pathway mediated by the related adapter protein Carma1 and thus presents an array of unique targets to selectively manipulate Treg function.

The vimentin intermediate filament network restrains regulatory T cell suppression of graft-versus-host disease.

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.

Increased generation of Foxp3(+) regulatory T cells by manipulating antigen presentation in the thymus.

Regulatory T-cell (Treg) selection in the thymus is essential to prevent autoimmune diseases. Although important rules for Treg selection have been established, there is controversy regarding the degree of self-reactivity displayed by T-cell receptors expressed by Treg cells. In this study we have developed a model of autoimmune skin inflammation, to determine key parameters in the generation of skin-reactive Treg cells in the thymus (tTreg). tTreg development is predominantly AIRE dependent, with an AIRE-independent component. Without the knowledge of antigen recognized by skin-reactive Treg cells, we are able to enhance skin-specific tTreg cell generation using three approaches. First, we increase medullary thymic epithelial cells by using mice lacking osteoprotegerin or by adding TRANCE (RANKL, Tnfsf11). Second, we inject intrathymically peripheral dendritic cells from skin-draining sites. Finally, we inject skin tissue lysates intrathymically. These findings have implications for enhancing the generation of organ-specific Treg cells in autoimmune diseases.

PGRN protects against colitis progression in mice in an IL-10 and TNFR2 dependent manner.

This study was aimed to determine the role and regulation of progranulin (PGRN) in the pathogenesis of inflammatory bowel diseases (IBD). Dextran sulfate sodium (DSS)-, picrylsulfonic acid (TNBS)-induced, bone marrow chimera and CD4+CD45Rb(hi) T cell transfer colitis model were established and analyzed in wild-type and several genetically-modified mice, including PGRN, IL-10 and TNFR2 deficient mice. Elevated levels of PGRN were found in colitis samples from human IBD patients and mouse colitis models in comparison to the corresponding controls. PGRN-deficient mice became highly susceptible to DSS- and TNBS-induced colitis, whereas recombinant PGRN ameliorated the pathology and reduced the histological score in both DSS and TNBS colitis models. In addition, hematopoietic-derived PGRN was critical for protection against DSS-induced colitis, and lack of PGRN signaling in CD4+ T cells also exacerbated experimental colitis. PGRN-mediated protective effect in colitis was compromised in the absence of IL-10 signaling. In addition, PGRN's effect was also largely lost in the TNFR2-deficient colitis model. Collectively, these findings not only provide the new insight into PGRN's anti-inflammatory action in vivo, but may also present PGRN and its derivatives as novel biological agent for treating IBD.