Reduction of glutamate neurotoxicity: A novel therapeutic approach for Niemann-Pick disease, type C1.

Niemann-Pick disease, type C1 is a progressive, lethal, neurodegenerative disorder due to endolysosomal storage of unesterified cholesterol. Cerebellar ataxia, as a result of progressive loss of cerebellar Purkinje neurons, is a major symptom of Nieman-Pick disease, type C1. Comparing single cell RNAseq data from control (Npc1+/+) and mutant (Npc1-/-) mice, we observed significantly decreased expression of Slc1a3 in Npc1-/- astrocytes. Slc1a3 encodes a glutamate transporter (GLAST, EAAT1) which functions to decrease glutamate concentrations in the post synaptic space after neuronal firing. Glutamate is an excitatory neurotransmitter and elevated extracellular levels of glutamate can be neurotoxic. Impaired EAAT1 function underlies type-6 episodic ataxia, a rare disorder with progressive cerebellar dysfunction, thus suggesting that impaired glutamate uptake in Niemann-Pick disease, type C1 could contribute to disease progression. We now show that decreased expression of Slc1a3 in Npc1-/- mice has functional consequences that include decreased surface protein expression and decreased glutamate uptake by Npc1-/- astrocytes. To test whether glutamate neurotoxicity plays a role in Niemann-Pick disease, type C1 progression, we treated NPC1 deficient mice with ceftriaxone and riluzole. Ceftriaxone is a ß-lactam antibiotic that is known to upregulate the expression of Slc1a2, an alternative glial glutamate transporter. Although ceftriaxone increased Slc1a2 expression, we did not observe a treatment effect in NPC1 mutant mice. Riluzole is a glutamate receptor antagonist that inhibits postsynaptic glutamate receptor signaling and reduces the release of glutamate. We found that treatment with riluzole increased median survival in Npc1-/- by 12%. Given that riluzole is an approved drug for the treatment of amyotrophic lateral sclerosis, repurposing of this drug may provide a novel therapeutic approach to decrease disease progression in Niemann-Pick disease type, C1 patients.

Single Cell Transcriptome Analysis of Niemann-Pick Disease, Type C1 Cerebella.

Niemann-Pick disease, type C1 (NPC1) is a lysosomal disease characterized by endolysosomal storage of unesterified cholesterol and decreased cellular cholesterol bioavailability. A cardinal symptom of NPC1 is cerebellar ataxia due to Purkinje neuron loss. To gain an understanding of the cerebellar neuropathology we obtained single cell transcriptome data from control (Npc1+/+) and both three-week-old presymptomatic and seven-week-old symptomatic mutant (Npc1-/-) mice. In seven-week-old Npc1-/- mice, differential expression data was obtained for neuronal, glial, vascular, and myeloid cells. As anticipated, we observed microglial activation and increased expression of innate immunity genes. We also observed increased expression of innate immunity genes by other cerebellar cell types, including Purkinje neurons. Whereas neuroinflammation mediated by microglia may have both neuroprotective and neurotoxic components, the contribution of increased expression of these genes by non-immune cells to NPC1 pathology is not known. It is possible that dysregulated expression of innate immunity genes by non-immune cells is neurotoxic. We did not anticipate a general lack of transcriptomic changes in cells other than microglia from presymptomatic three-week-old Npc1-/- mice. This observation suggests that microglia activation precedes neuronal dysfunction. The data presented in this paper will be useful for generating testable hypotheses related to disease progression and Purkinje neurons loss as well as providing insight into potential novel therapeutic interventions.

Unique molecular signature in mucolipidosis type IV microglia.

BACKGROUND: Lysosomal storage diseases (LSD) are a large family of inherited disorders characterized by abnormal endolysosomal accumulation of cellular material due to catabolic enzyme and transporter deficiencies. Depending on the affected metabolic pathway, LSD manifest with somatic or central nervous system (CNS) signs and symptoms. Neuroinflammation is a hallmark feature of LSD with CNS involvement such as mucolipidosis type IV, but not of others like Fabry disease. METHODS: We investigated the properties of microglia from LSD with and without major CNS involvement in 2-month-old mucolipidosis type IV (Mcoln1-/-) and Fabry disease (Glay/-) mice, respectively, by using a combination of flow cytometric, RNA sequencing, biochemical, in vitro and immunofluorescence analyses. RESULTS: We characterized microglia activation and transcriptome from mucolipidosis type IV and Fabry disease mice to determine if impaired lysosomal function is sufficient to prime these brain-resident immune cells. Consistent with the neurological pathology observed in mucolipidosis type IV, Mcoln1-/- microglia demonstrated an activation profile with a mixed neuroprotective/neurotoxic expression pattern similar to the one we previously observed in Niemann-Pick disease, type C1, another LSD with significant CNS involvement. In contrast, the Fabry disease microglia transcriptome revealed minimal alterations, consistent with the relative lack of CNS symptoms in this disease. The changes observed in Mcoln1-/- microglia showed significant overlap with alterations previously reported for other common neuroinflammatory disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Indeed, our comparison of microglia transcriptomes from Alzheimer's disease, amyotrophic lateral sclerosis, Niemann-Pick disease, type C1 and mucolipidosis type IV mouse models showed an enrichment in "disease-associated microglia" pattern among these diseases. CONCLUSIONS: The similarities in microglial transcriptomes and features of neuroinflammation and microglial activation in rare monogenic disorders where the primary metabolic disturbance is known may provide novel insights into the immunopathogenesis of other more common neuroinflammatory disorders. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01067742, registered on February 12, 2010.

Gastrointestinal Tract Pathology in a BALB/c Niemann-Pick Disease Type C1 Null Mouse Model.

BACKGROUND: Niemann-Pick disease, type C (NPC) is a rare lysosomal storage disorder characterized by progressive neurodegeneration, splenomegaly, hepatomegaly, and early death. NPC is caused by mutations in either the NPC1 or NPC2 gene. Impaired NPC function leads to defective intracellular transport of unesterified cholesterol and its accumulation in late endosomes and lysosomes. A high frequency of Crohn disease has been reported in NPC1 patients, suggesting that gastrointestinal tract pathology may become a more prominent clinical issue if effective therapies are developed to slow the neurodegeneration. The Npc1 nih mouse model on a BALB/c background replicates the hepatic and neurological disease observed in NPC1 patients. Thus, we sought to characterize the gastrointestinal tract pathology in this model to determine whether it can serve as a model of Crohn disease in NPC1. METHODS: We analyzed the gastrointestinal tract and isolated macrophages of BALB/cJ cNctr-Npc1m1N/J (Npc1-/-) mouse model to determine whether there was any Crohn-like pathology or inflammatory cell activation. We also evaluated temporal changes in the microbiota by 16S rRNA sequencing of fecal samples to determine whether there were changes consistent with Crohn disease. RESULTS: Relative to controls, Npc1 mutant mice demonstrate increased inflammation and crypt abscesses in the gastrointestinal tract; however, the observed pathological changes are significantly less than those observed in other Crohn disease mouse models. Analysis of Npc1 mutant macrophages demonstrated an increased response to lipopolysaccharides and delayed bactericidal activity; both of which are pathological features of Crohn disease. Analysis of the bacterial microbiota does not mimic what is reported in Crohn disease in either human or mouse models. We did observe significant increases in cyanobacteria and epsilon-proteobacteria. The increase in epsilon-proteobacteria may be related to altered cholesterol homeostasis since cholesterol is known to promote growth of this bacterial subgroup. CONCLUSIONS: Macrophage dysfunction in the BALB/c Npc1-/- mouse is similar to that observed in other Crohn disease models. However, neither the degree of pathology nor the microbiota changes are typical of Crohn disease. Thus, this mouse model is not a good model system for Crohn disease pathology reported in NPC1 patients.

Nrf2 establishes a glutathione-mediated gradient of UVB cytoprotection in the epidermis.

Ultraviolet (UV) B irradiation can severely damage the skin and even induce tumorigenesis. It exerts its effects by direct DNA modification and by formation of reactive oxygen species (ROS). We developed a strategy to genetically activate target gene expression of the transcription factor NF-E2-related factor 2 (Nrf2) in keratinocytes in vivo based on expression of a constitutively active Nrf2 mutant. Activation of Nrf2 target genes strongly reduced UVB cytotoxicity through enhancement of ROS detoxification. Remarkably, the protective effect was extended to neighboring cells. Using different combinations of genetically modified mice, we demonstrate that Nrf2 activates the production, recycling, and release of glutathione and cysteine by suprabasal keratinocytes, resulting in protection of basal cells in a paracrine, glutathione/cysteine-dependent manner. Most importantly, we found that endogenous Nrf2 controls selective protection of suprabasal keratinocytes from UVB-induced apoptosis through activation of cytoprotective genes. This finding explains the preferential UVB-induced apoptosis of basal cells, which is important for elimination of mutated stem cells as well as for preservation of skin integrity. Taken together, our results identify Nrf2 as a key regulator in the UV response of the skin.

Induction of regulatory T cells by a murine ß-defensin.

ß-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ß-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.

CHOP-mediated IL-23 overexpression does not drive colitis in experimental spondyloarthritis.

HLA-B27 is a major risk factor for spondyloarthritis (SpA), yet the underlying mechanisms remain unclear. HLA-B27 misfolding-induced IL-23, which is mediated by endoplasmic reticulum (ER) stress has been hypothesized to drive SpA pathogenesis. Expression of HLA-B27 and human ß2m (hß2m) in rats (HLA-B27-Tg) recapitulates key SpA features including gut inflammation. Here we determined whether deleting the transcription factor CHOP (Ddit3-/-), which mediates ER-stress induced IL-23, affects gut inflammation in HLA-B27-Tg animals. ER stress-mediated Il23a overexpression was abolished in CHOP-deficient macrophages. Although CHOP-deficiency also reduced Il23a expression in immune cells isolated from the colon of B27+ rats, Il17a levels were not affected, and gut inflammation was not reduced. Rather, transcriptome analysis revealed increased expression of pro-inflammatory genes, including Il1a, Ifng and Tnf in HLA-B27-Tg colon tissue in the absence of CHOP, which was accompanied by higher histological Z-scores. RNAScope localized Il17a mRNA to the lamina propria of the HLA-B27-Tg rats and revealed similar co-localization with Cd3e (CD3) in the presence and absence of CHOP. This demonstrates that CHOP-deficiency does not improve, but rather exacerbates gut inflammation in HLA-B27-Tg rats, indicating that HLA-B27 is not promoting gut disease through ER stress-induced IL-23. Hence, CHOP may protect rats from more severe HLA-B27-induced gut inflammation.

Ultraviolet radiation-induced upregulation of antimicrobial proteins in health and disease.

This article reviews recent data on the expression, regulation and activation of antimicrobial peptides (AMP) in human skin, and considers their potential protective and pro-inflammatory roles following upregulation by ultraviolet radiation (UVR). Antimicrobial peptides are small peptides that are key components of the innate immune system, originally identified by their vital role in protecting the body-environment interface from infection. However, it has now become clear that AMP have more extensive actions, including the provision of pivotal links with the adaptive immune system. Moreover, aberrant AMP expression may contribute to immuno-modulated inflammatory dermatoses including psoriasis, eczema and the photoaggravated condition lupus erythematosus. Recent work has demonstrated the direct upregulation of AMP in healthy skin by cutaneous UVR exposure. This may serve to protect the skin from risks imposed by both the biophysical barrier-compromise and the immunosuppression that are attributable to UVR exposure. Furthermore, it is observed that UVR provokes upregulation of AMP in an atypical manner in the photosensitivity disorder polymorphic light eruption. Dysregulated UVR responses of these pro-inflammatory proteins may play a role in the pathogenesis of certain immune-mediated diseases caused or aggravated by sunlight.

VEGF Secretion Drives Bone Formation in Classical MAP2K1+ Melorheostosis.

Patients with classical melorheostosis exhibit exuberant bone overgrowth in the appendicular skeleton, resulting in pain and deformity with no known treatment. Most patients have somatic, mosaic mutations in MAP2K1 (encoding the MEK1 protein) in osteoblasts and overlying skin. As with most rare bone diseases, lack of affected tissue has limited the opportunity to understand how the mutation results in excess bone formation. The aim of this study was to create a cellular model to study melorheostosis. We obtained patient skin cells bearing the MAP2K1 mutation (affected cells), and along with isogenic control normal fibroblasts reprogrammed them using the Sendai virus method into induced pluripotent stem cells (iPSCs). Pluripotency was validated by marker staining and embryoid body formation. iPSCs were then differentiated to mesenchymal stem cells (iMSCs) and validated by flow cytometry. We confirmed retention of the MAP2K1 mutation in iMSCs with polymerase chain reaction (PCR) and confirmed elevated MEK1 activity by immunofluorescence staining. Mutation-bearing iMSCs showed significantly elevated vascular endothelial growth factor (VEGF) secretion, proliferation and collagen I and IV secretion. iMSCs were then differentiated into osteoblasts, which showed increased mineralization at 21 days and increased VEGF secretion at 14 and 21 days of differentiation. Administration of VEGF to unaffected iMSCs during osteogenic differentiation was sufficient to increase mineralization. Blockade of VEGF by bevacizumab reduced mineralization in iMSC-derived affected osteoblasts and in affected primary patient-derived osteoblasts. These data indicate that patient-derived induced pluripotent stem cells recreate the elevated MEK1 activity, increased mineralization, and increased proliferation seen in melorheostosis patients. The increased bone formation is driven, in part, by abundant VEGF secretion. Modifying the activity of VEGF (a known stimulator of osteoblastogenesis) represents a promising treatment pathway to explore. iPSCs may have wide applications to other rare bone diseases. © 2023 American Society for Bone and Mineral Research (ASBMR).

Paradoxical Effects of Endoplasmic Reticulum Aminopeptidase 1 Deficiency on HLA-B27 and Its Role as an Epistatic Modifier in Experimental Spondyloarthritis.

OBJECTIVE: We undertook this study to examine the functional basis for epistasis between endoplasmic reticulum aminopeptidase 1 (ERAP1) and HLA-B27 in experimental spondyloarthritis (SpA). METHODS: ERAP1-knockout rats were created using genome editing and bred with HLA-B27/human ß2 -microglobulin-transgenic (HLA-B27-Tg) rats and HLA-B7-Tg rats. The effects of ERAP1 deficiency on HLA allotypes were determined using immunoprecipitation and immunoblotting, flow cytometry, allogeneic T cell proliferation assays, and gene expression analyses. Animals were examined for clinical features of disease, and tissue was assessed by histology. RESULTS: ERAP1 deficiency increased the ratio of folded to unfolded (ß2 m-free) HLA-B27 heavy chains, while having the opposite effect on HLA-B7. Furthermore, in rats with ERAP1 deficiency, HLA-B27 misfolding was reduced, while free HLA-B27 heavy chain dimers on the cell surface and monomers were increased. The effects of ERAP1 deficiency persisted during up-regulation of HLA-B27 and led to a reduction in endoplasmic reticulum stress. ERAP1 deficiency reduced the prevalence of arthritis in HLA-B27-Tg rats by two-thirds without reducing gastrointestinal inflammation. Dendritic cell abnormalities attributed to the presence of HLA-B27, including reduced allogeneic T cell stimulation and loss of CD103-positive/major histocompatibility complex class II-positive cells, were not rescued by ERAP1 deficiency, while excess Il23a up-regulation was mitigated. CONCLUSION: ERAP1 deficiency reduced HLA-B27 misfolding and improved folding while having opposing effects on HLA-B7. The finding that HLA-B27-Tg rats had partial protection against SpA in this study is consistent with genetic evidence that loss-of-function and/or reduced expression of ERAP1 reduces the risk of ankylosing spondylitis. Functional studies support the concept that the effects of ERAP1 on HLA-B27 and SpA may be a consequence of how peptides affect the biology of this allotype rather than their role as antigenic determinants.

UV radiation induces the release of angiopoietin-2 from dermal microvascular endothelial cells.

In human skin, ultraviolet radiation (UVR)-induced erythema is characterized by the inflammatory and angiogenic activation of dermal endothelial cells. Recently, it has been shown that the release of angiopoietin-2 (Ang-2) from cytoplasmic storages of activated endothelial cells is crucial for the induction of inflammation and angiogenesis. Therefore, we hypothesized that UVR exposure induces the release of Ang-2 from endothelial cells controlling the early steps of erythema formation. In an in vivo study, suction blister fluids generated from UV-irradiated skin showed significantly increased concentrations of Ang-2, vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNFα). Likewise, in vitro UVR exposure of human dermal microvascular endothelial cells (HDMECs) triggered the release of Ang-2 that enhanced the pro-inflammatory response of these cells and facilitated their detachment from smooth muscle cells as evidenced by employing a three-dimensional co-culture spheroid model. These effects were inhibited by angiopoietin-1 (Ang-1), which competes with Ang-2 for binding the endothelial cell Tie2 receptor. Collectively, these observations suggest that UVR triggers the release of endothelial Ang-2 which may promote the destabilization and pro-inflammatory phenotype of the microvascular endothelium. As Ang-1 counteracts UVR-induced effects, stimulating the Ang-1 activity may represent a strategy to stabilize the dermal microcirculatory system, thus protecting against UVR-induced skin damages.

In vivo reprogramming of UV radiation-induced regulatory T-cell migration to inhibit the elicitation of contact hypersensitivity.

BACKGROUND: Regulatory T (Treg) cells induced by UV radiation (UVR) inhibit only the induction and not the elicitation of contact hypersensitivity (CHS) because they migrate into the lymph nodes but not the skin. The tissue-homing receptor expression and migratory behavior of Treg cells can be altered by means of in vitro coincubation with skin-derived antigen-presenting cells. On this in vitro treatment, Treg cells migrate into the skin and thus inhibit the elicitation of CHS. OBJECTIVE: We attempted to determine whether Treg cells can be induced by UVR in sensitized mice and manipulated entirely in vivo in such a way that they suppress the elicitation of immune responses. METHODS: Treg cells were induced by applying contact allergens onto UV-exposed skin in wild-type, langerin diphtheria toxin receptor knock-in, or depletion of Treg cell transgenic mice. RESULTS: UVR-induced Treg cells inhibit the elicitation of CHS in sensitized mice when stimulated by means of an antigen-specific boost through the skin. This requires cutaneous antigen-presenting cells that alter the migratory behavior of Treg cells and drive them out of the lymph nodes into the skin. CONCLUSIONS: The indication is that antigen-specific Treg cells can be induced in sensitized hosts and manipulated in such a way that they suppress the elicitation of specific immune reactions. Because this is achieved entirely in vivo without invasive interventions, our findings might have important implications for strategies aiming to induce and use Treg cells in a therapeutic setting.

Fibroblasts from Patients with Melorheostosis Promote Angiogenesis in Healthy Endothelial Cells through Secreted Factors.

Melorheostosis is a rare sclerosing bone disease with associated vascular abnormalities in skin and bone, which is caused by somatic mosaic single nucleotide variations in the MAP2K1 gene, which encodes MAPK/extracellular signal‒regulated kinase (ERK) kinase 1. However, disease pathogenesis is poorly understood. Using patient-derived cells, we found that affected skin fibroblasts carrying the single nucleotide variations have increased activation of ERK1/2, which results in increased expression and secretion of proangiogenic factors, including VEGF. VEGF secretion was strongly reduced in affected cells after treatment with MAPK/ERK kinase 1 inhibitor trametinib. Treatment of healthy endothelial cells on matrigel with conditioned medium from affected fibroblasts induces the adoption of a proangiogenic phenotype. Direct coculture of fibroblasts and endothelial cells further shows that both secreted factors and extracellular matrix are capable of inducing a proangiogenic phenotype in healthy endothelial cells. Blocking VEGF with bevacizumab reduces the proangiogenic effect of affected fibroblasts in both the matrigel and direct coculture angiogenesis models, indicating that elevated VEGF secretion is a key mediator of increased angiogenesis in melorheostosis tissue. In conclusion, this work identifies the role of several important molecular mediators in the pathogenesis of melorheostosis, including MAPK/ERK kinase 1, phosphorylated ERK1/2, and VEGF, all of which have clinically available pharmacologic inhibitors, which could be further explored as therapeutic targets.

The enigmatic role of HLA-B*27 in spondyloarthritis pathogenesis.

Establishing a clear role for HLA-B*27 in the pathogenesis of spondyloarthritis continues to be challenging. Aberrant properties of the heavy chain as well as a potential role presenting arthritogenic peptides continue to be pursued as plausible mechanisms. Recent studies implicate HLA-B*27 in aberrant bone formation. An unanticipated cell surface interaction between HLA-B*27 and the bone morphogenetic protein pathway receptor subunit ALK2 may augment TGFß superfamily signaling pathways, increasing responsiveness to Activin A and TGFß. This has the potential to increase bone formation as well as Th17 T cell development, presenting an attractive model to explain several aspects of axial and peripheral spondyloarthritis. In a separate study, intracellular effects of misfolded HLA-B*27 implicate this mechanism in increased osteoblast mineralization and bone formation. HLA-B*27 expression in early osteoblasts activates unfolded protein response-mediated X-box binding protein-1 mRNA splicing and induction of the retinoic acid receptor-ß gene, with downstream increases in expression of tissue non-specific alkaline phosphatase. Increased TNAP expression in osteoblasts was linked to increased mineralization in vitro and bone formation in vivo. In the ongoing search for evidence of arthritogenic peptides, high-throughput TCR (T cell receptor) sequencing has provided evidence for reduced clonal expansion and increased TCR diversity in ankylosing spondylitis. In addition to two common CD8+ TCR sequences identified in one study, similar CD8 and CD4 TCR motifs were found in another study. Further work will be needed to shed light on the nature of the peptide-HLA class I complex recognized by these T cells and its role in disease.

UV-B radiation induces the expression of antimicrobial peptides in human keratinocytes in vitro and in vivo.

BACKGROUND: Suppression of the adaptive immune system by UV radiation plays an important role in photocarcinogenesis. Exacerbation of skin infections has been proposed as a further consequence of UV-induced immunosuppression. Clinically bacterial infections are not a problem. For defense against bacteria, the innate immune response including the release of antimicrobial peptides is much more relevant than the adaptive immune response. Keratinocytes have the capacity to release antimicrobial peptides. OBJECTIVE: We asked whether UV radiation induces antimicrobial peptides in vitro and in vivo. METHODS: Antimicrobial peptide expression by normal human keratinocytes was measured by real-time PCR and fluorescence-activated cell sorting analysis. Biopsies taken from human volunteers and skin explants were studied with immunohistochemistry. RESULTS: Real-time PCR of normal human keratinocytes revealed a dose-dependent increase of human beta-defensin-2, -3, ribonuclease 7, and psoriasin (S100A7) after UV radiation. This was confirmed at the protein level by intracellular fluorescence-activated cell sorting and in vitro immunofluorescence analysis. Immunohistochemistry of biopsies taken from healthy volunteers exposed to different UV radiation doses revealed enhanced epidermal expression of antimicrobial peptides after UV exposure. This was also confirmed by exposing human skin explants to UV radiation. CONCLUSION: UV radiation exerts diverse effects on the immune system, suppressing the adaptive but inducing the innate immune response. This may explain why T-cell-mediated immune reactions are suppressed on UV exposure but not host defense reactions against bacterial attacks.

The Role of Autophagy in the Degradation of Misfolded HLA-B27 Heavy Chains.

OBJECTIVE: To determine whether autophagy is involved in the degradation of misfolded HLA-B27 in experimental spondyloarthritis. METHODS: Bone marrow-derived macrophages from HLA-B27/human ß2 -microglobulin (hß2 m)-transgenic rats were incubated in the presence or absence of interferon-γ and proteasome or autophagy inhibitors. Immunoprecipitation, immunoblotting, and immunofluorescence analysis were used to measure HLA-B27 heavy chains and autophagy. Autophagy was induced using rapamycin. Macrophages from HLA-B7/hß2 m-transgenic and wild-type rats were used as controls. RESULTS: HLA-B27-expressing macrophages showed phosphatidylethanolamine-conjugated microtubule-associated protein 1 light chain 3B levels similar to those in both control groups, before and after manipulation of autophagy. Blocking autophagic flux with bafilomycin resulted in the accumulation of misfolded HLA-B27 dimers and oligomers as well as monomers, which was comparable with the results of blocking endoplasmic reticulum-associated degradation (ERAD) with the proteasome inhibitor bortezomib. HLA-B7 monomers also accumulated after blocking each degradation pathway. The ubiquitin-to-heavy chain ratio was 2-3-fold lower for HLA-B27 than for HLA-B7. Activation of autophagy with rapamycin rapidly eliminated ~50% of misfolded HLA-B27, while folded HLA-B27 or HLA-B7 monomeric heavy chains were minimally affected. CONCLUSION: This study is the first to demonstrate that both autophagy and ERAD play roles in the elimination of excess HLA class I heavy chains expressed in transgenic rats. We observed no evidence that HLA-B27 expression modulated the autophagy pathway. Our results suggest that impaired ubiquitination of HLA-B27 may play a role in the accumulation of misfolded disulfide-linked dimers, the elimination of which can be enhanced by activation of autophagy. Manipulation of the autophagy pathway should be further investigated as a potential therapeutic target in spondyloarthritis.

Causes and consequences of endoplasmic reticulum stress in rheumatic disease.

Rheumatic diseases represent a heterogeneous group of inflammatory conditions, many of which involve chronic activation of both innate and adaptive immune responses by multiple genetic and environmental factors. These immune responses involve the secretion of excessive amounts of cytokines and other signalling mediators by activated immune cells. The endoplasmic reticulum (ER) is the cellular organelle that directs the folding, processing and trafficking of membrane-bound and secreted proteins, including many key components of the immune response. Maintaining homeostasis in the ER is critical to cell function and survival. Consequently, elaborate mechanisms have evolved to sense and respond to ER stress through three main signalling pathways that together comprise the unfolded protein response (UPR). Activation of the UPR can rapidly resolve the accumulation of misfolded proteins, direct permanent changes in the size and function of cells during differentiation, and critically influence the immune response and inflammation. Recognition of the importance of ER stress and UPR signalling pathways in normal and dysregulated immune responses has greatly increased in the past few years. This Review discusses several settings in which ER stress contributes to the pathogenesis of rheumatic diseases and considers some of the therapeutic opportunities that these discoveries provide.

The role of HLA-B*27 in spondyloarthritis.

The mechanism by which HLA-B*27 predisposes to spondyloarthritis remains unresolved. Arthritogenic peptides have not been defined in humans and are not involved in experimental models of spondyloarthritis. Aberrant properties of HLA-B*27 can activate the IL-23/IL-17 axis in HLA-B*27 transgenic rats and humans. In HLA-B*27-independent rodent models, spondyloarthritis can be driven by IL-23 triggering entheseal-resident CD4-/CD8- T cells or CD4+ Th17 T cells. These findings point toward noncanonical mechanisms linking HLA-B*27 to the disease and provide a potential explanation for HLA-B*27-negative spondyloarthritis. Gut microbial dysbiosis may be important in the development of spondyloarthritis. HLA-B*27-induced changes in gut microbiota are complex and suggest an ecological model of dysbiosis in rodents. The importance of the IL-23/IL-17 axis in ankylosing spondylitis has been demonstrated by studies showing efficacy of IL-17. Although deciphering the precise role(s) of HLA-B*27 in disease requires further investigation, considerable progress has been made in understanding this complex relationship.

Generation and differentiation of induced pluripotent stem cells reveal ankylosing spondylitis risk gene expression in bone progenitors.

Axial spondyloarthritis (axSpA), which encompasses ankylosing spondylitis, is a complex genetic disease. Aberrant bone formation is a key feature of pathogenesis that can lead to ankylosis of the spine. Our objective is to determine, whether genes whose variants confer susceptibility to AS are expressed in bone progenitors like mesenchymal stem cells (MSCs). Since MSCs from bone marrow is difficult to obtain, we first examined, whether MSCs can be derived from induced pluripotent stem cells (iPSCs). Dermal fibroblasts of two axSpA patients and one healthy control were reprogrammed into iPSCs using a Sendai virus vector encoding pluripotency genes. Pluripotency of iPSCs was examined by embryoid body formation and by testing for stem cell specific gene and protein expression using RT-PCR and immuno fluorescence. iPSCs were differentiated into MSCs by a TGFß inhibitor. MSCs were characterized by flow cytometry using lineage specific antibodies and by their capacity to develop into chondrocytes, adipocytes, and osteoblasts in lineage-specific medium. RNA-seq was applied to determine genome-wide gene expression patterns in MSCs, iPSCs, and blood. We show for the first time, that expression levels of several AS susceptibility genes (EDIL3, ANO6, HAPLN1, ANTXR2) involved in bone formation are significantly elevated in MSCs (2-15-fold; p ≤ 0.05) compared to blood or iPSCs and demonstrate that iPSC-derived MSCs can be differentiated into osteoblasts, chondrocytes, and adipocytes. We conclude, MSCs generated from patient fibroblast-derived iPSC lines are useful tools for studying functional genomics of risk genes associated with bone formation in AS pathogenesis.