Deubiquitinating Enzyme UCH-L1 Promotes Dendritic Cell Antigen Cross-Presentation by Favoring Recycling of MHC Class I Molecules.

The deubiquitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1) is required for the maintenance of axonal integrity in neurons and is thought to regulate the intracellular pool of ubiquitin in the brain. In this study, we show that UCH-L1 has an immunological function in dendritic cell (DC) Ag cross-presentation. UCH-L1 is expressed in mouse kidney, spleen, and bone marrow-derived DCs, and its expression and activity are regulated by the immune stimuli LPS and IFN-γ. UCH-L1-deficient mice have significantly reduced ability to cross-prime CD8 T cells in vivo and in vitro because of a reduced ability of DCs to generate MHC class I (MHC I) peptide complexes for cross-presented Ags. Mechanistically, Ag uptake by phagocytosis and receptor-mediated endocytosis as well as phagosome maturation are unaffected by loss of UCH-L1 in DCs. Rather, MHC I recycling is reduced by loss of UCH-L1, which affects the colocalization of intracellular MHC I with late endosomal/lysosomal compartments necessary for cross-presentation of Ag. These results demonstrate a hitherto unrecognized role of the deubiquitinating enzyme UCH-L1 in DC Ag processing.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

C5a receptor 1-/- mice are protected from the development of IgE-mediated experimental food allergy.

BACKGROUND: Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. METHODS: Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. RESULTS: Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. CONCLUSIONS: Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Ubiquitin C-Terminal Hydrolase L1 is required for regulated protein degradation through the ubiquitin proteasome system in kidney.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is a major deubiquitinating enzyme of the nervous system and associated with the development of neurodegenerative diseases. We have previously shown that UCH-L1 is found in tubular and parietal cells of the kidney and is expressed de novo in injured podocytes. Since the role of UCH-L1 in the kidney is unknown we generated mice with a constitutive UCH-L1-deficiency to determine its role in renal health and disease. UCH-L1-deficient mice developed proteinuria, without gross changes in glomerular morphology. Tubular cells, endothelial cells, and podocytes showed signs of stress with an accumulation of oxidative-modified and polyubiquitinated proteins. Mechanistically, abnormal protein accumulation resulted from an altered proteasome abundance leading to decreased proteasomal activity, a finding exaggerated after induction of anti-podocyte nephritis. UCH-L1-deficient mice exhibited an exacerbated course of disease with increased tubulointerstitial and glomerular damage, acute renal failure, and death, the latter most likely a result of general neurologic impairment. Thus, UCH-L1 is required for regulated protein degradation in the kidney by controlling proteasome abundance. Altered proteasome abundance renders renal cells, particularly podocytes and endothelial cells, susceptible to injury.

Autoantibodies against thrombospondin type 1 domain-containing 7A induce membranous nephropathy.

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, and one-third of patients develop end-stage renal disease (ESRD). Circulating autoantibodies against the podocyte surface antigens phospholipase A2 receptor 1 (PLA2R1) and the recently identified thrombospondin type 1 domain-containing 7A (THSD7A) are assumed to cause the disease in the majority of patients. The pathogenicity of these antibodies, however, has not been directly proven. Here, we have reported the analysis and characterization of a male patient with THSD7A-associated MN who progressed to ESRD and subsequently underwent renal transplantation. MN rapidly recurred after transplantation. Enhanced staining for THSD7A was observed in the kidney allograft, and detectable anti-THSD7A antibodies were present in the serum before and after transplantation, suggesting that these antibodies induced a recurrence of MN in the renal transplant. In contrast to PLA2R1, THSD7A was expressed on both human and murine podocytes, enabling the evaluation of whether anti-THSD7A antibodies cause MN in mice. We demonstrated that human anti-THSD7A antibodies specifically bind to murine THSD7A on podocyte foot processes, induce proteinuria, and initiate a histopathological pattern that is typical of MN. Furthermore, anti-THSD7A antibodies induced marked cytoskeletal rearrangement in primary murine glomerular epithelial cells as well as in human embryonic kidney 293 cells. Our findings support a causative role of anti-THSD7A antibodies in the development of MN.

Alterations in the ubiquitin proteasome system in persistent but not reversible proteinuric diseases.

Podocytes are the key cells affected in nephrotic glomerular kidney diseases, and they respond uniformly to injury with cytoskeletal rearrangement. In nephrotic diseases, such as membranous nephropathy and FSGS, persistent injury often leads to irreversible structural damage, whereas in minimal change disease, structural alterations are mostly transient. The factors leading to persistent podocyte injury are currently unknown. Proteolysis is an irreversible process and could trigger persistent podocyte injury through degradation of podocyte-specific proteins. We, therefore, analyzed the expression and functional consequence of the two most prominent proteolytic systems, the ubiquitin proteasome system (UPS) and the autophagosomal/lysosomal system, in persistent and transient podocyte injuries. We show that differential upregulation of both proteolytic systems occurs in persistent human and rodent podocyte injury. The expression of specific UPS proteins in podocytes differentiated children with minimal change disease from children with FSGS and correlated with poor clinical outcome. Degradation of the podocyte-specific protein α-actinin-4 by the UPS depended on oxidative modification in membranous nephropathy. Notably, the UPS was overwhelmed in podocytes during experimental glomerular disease, resulting in abnormal protein accumulation and compensatory upregulation of the autophagosomal/lysosomal system. Accordingly, inhibition of both proteolytic systems enhanced proteinuria in persistent nephrotic disease. This study identifies altered proteolysis as a feature of persistent podocyte injury. In the future, specific UPS proteins may serve as new biomarkers or therapeutic targets in persistent nephrotic syndrome.

Mitochondrial gene polymorphisms that protect mice from colitis.

BACKGROUND & AIMS: Dysregulated energy homeostasis in the intestinal mucosa frequently is observed in patients with ulcerative colitis (UC). Intestinal tissues from these patients have reduced activity of the mitochondrial oxidative phosphorylation (OXPHOS) complex, so mitochondrial dysfunction could contribute to the pathogenesis of UC. However, little is known about the mechanisms by which OXPHOS activity could be altered. We used conplastic mice, which have identical nuclear but different mitochondrial genomes, to investigate activities of the OXPHOS complex. METHODS: Colitis was induced in C57BL/6J wild-type (B6.B6) and 3 strains of conplastic mice (B6.NZB, B6.NOD, and B6.AKR) by administration of dextran sodium sulfate or rectal application of trinitrobenzene sulfonate. Colon tissues were collected and analyzed by histopathology, immunohistochemical analysis, and immunoblot analysis; we also measured mucosal levels of adenosine triphosphate (ATP) and reactive oxygen species, OXPHOS complex activity, and epithelial cell proliferation and apoptosis. RESULTS: We identified mice with increased mucosal OXPHOS complex activities and levels of ATP. These mice developed less-severe colitis after administration of dextran sodium sulfate or trinitrobenzene sulfonate than mice with lower mucosal levels of ATP. Colon tissues from these mice also had increased enterocyte proliferation and transcription factor nuclear factor-κB activity, which have been shown to protect the mucosal barrier-defects in these processes have been associated with inflammatory bowel disease. CONCLUSIONS: Variants in mitochondrial DNA that increase mucosal levels of ATP protect mice from colitis. Increasing mitochondrial ATP synthesis in intestinal epithelial cells could be a therapeutic approach for UC.

A CD74-dependent MHC class I endolysosomal cross-presentation pathway.

Immune responses are initiated and primed by dendritic cells (DCs) that cross-present exogenous antigen. The chaperone CD74 (invariant chain) is thought to promote DC priming exclusively in the context of major histocompatibility complex (MHC) class II. However, we demonstrate here a CD74-dependent MHC class I cross-presentation pathway in DCs that had a major role in the generation of MHC class I-restricted, cytolytic T lymphocyte (CTL) responses to viral protein- and cell-associated antigens. CD74 associated with MHC class I in the endoplasmic reticulum of DCs and mediated the trafficking of MHC class I to endolysosomal compartments for loading with exogenous peptides. We conclude that CD74 has a previously undiscovered physiological function in endolysosomal DC cross-presentation for priming MHC class I-mediated CTL responses.

Dendritic cell cross-priming is essential for immune responses to Listeria monocytogenes.

Cross-presentation is now recognized as a major mechanism for initiating CD8 T cell responses to virus and tumor antigens in vivo. It provides an elegant mechanism that allows relatively few Dendritic cells (DCs) to initiate primary immune responses while avoiding the consumptive nature of pathogenic infection. CD8 T cells play a major role in anti-bacterial immune responses; however, the contribution of cross-presentation for priming CD8 T cell responses to bacteria, in vivo, is not well established. Listeria monocytogenes (Listeria) is the causative agent of Listeriosis, an opportunistic food-borne bacterial infection that poses a significant public health risk. Here, we employ a transgenic mouse model in which cross-presentation is uniquely inactivated, to investigate cross-priming during primary Listeria infection. We show that cross-priming deficient mice are severely compromised in their ability to generate antigen-specific T cells to stimulate MHC I-restricted CTL responses following Listeria infection. The defect in generation of Listeria-elicited CD8 T cell responses is also apparent in vitro. However, in this setting, the endogenous route of processing Listeria-derived antigens is predominant. This reveals a new experimental dichotomy whereby functional sampling of Listeria-derived antigens in vivo but not in vitro is dependent on cross-presentation of exogenously derived antigen. Thus, under normal physiological circumstances, cross-presentation is demonstrated to play an essential role in priming CD8 T cell responses to bacteria.

MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells.

BACKGROUND: Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing. CONCLUSIONS/SIGNIFICANCE: We conclude that DCs have 'hijacked' and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.

A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery.

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

The 52 000 MW Ro/SS-A autoantigen in Sjögren's syndrome/systemic lupus erythematosus (Ro52) is an interferon-gamma inducible tripartite motif protein associated with membrane proximal structures.

The 52 000 MW Ro/SS-A (Ro52) protein is a major target of autoantibodies in autoimmune conditions such as systemic lupus erythematosus and Sjögren's syndrome. Recent genomic and bioinformatic studies have shown that Ro52 belongs to a large family of related RING/Bbox/coiled-coil (RBCC) tripartite motif proteins sharing overall domain structure and 40-50% identity at the amino acid level. Ro52 also has a B30.2 domain at the C-terminus. Using the human genome draft sequence, the genomic organization of the Ro52 gene on human chromosome 11p15.5 has been deduced and related to the protein domain structure. We show that the steady-state levels of Ro52 mRNA are normally very low but are induced by cell activation with interferon-gamma. In transient transfection of HeLa cells, epitope-tagged Ro52 protein was localized to unidentified membrane proximal rod-like structures. Using in vitro coupled transcription/translation followed by immunoprecipitation, the autoimmune response to Ro52 protein was investigated and two distinct interactions were resolved. The Ro52 C-terminal B30.2 domain interacts with human immunoglobulin independently of antibody specificities. Sera derived from patients with Sjögren's syndrome and systemic lupus erythematosus, in addition, contained specific autoantibodies directed towards the rest of the Ro52 molecule. The majority of these autoimmune sera also immunoprecipitated the Ro52-related molecule RNF15. A possible role for Ro52 protein in alterations of plasma membranes during cellular activation or apoptosis is discussed.