CD4+ T cells reactive to a hybrid peptide from insulin-chromogranin A adopt a distinct effector fate and are pathogenic in autoimmune diabetes.

T cell-mediated islet destruction is a hallmark of autoimmune diabetes. Here, we examined the dynamics and pathogenicity of CD4+ T cell responses to four different insulin-derived epitopes during diabetes initiation in non-obese diabetic (NOD) mice. Single-cell RNA sequencing of tetramer-sorted CD4+ T cells from the pancreas revealed that islet-antigen-specific T cells adopted a wide variety of fates and required XCR1+ dendritic cells for their activation. Hybrid-insulin C-chromogranin A (InsC-ChgA)-specific CD4+ T cells skewed toward a distinct T helper type 1 (Th1) effector phenotype, whereas the majority of insulin B chain and hybrid-insulin C-islet amyloid polypeptide-specific CD4+ T cells exhibited a regulatory phenotype and early or weak Th1 phenotype, respectively. InsC-ChgA-specific CD4+ T cells were uniquely pathogenic upon transfer, and an anti-InsC-ChgA:IAg7 antibody prevented spontaneous diabetes. Our findings highlight the heterogeneity of T cell responses to insulin-derived epitopes in diabetes and argue for the feasibility of antigen-specific therapies that blunts the response of pathogenic CD4+ T cells causing autoimmunity.

Deletion of Trps1 regulatory elements recapitulates postnatal hip joint abnormalities and growth retardation of Trichorhinophalangeal syndrome in mice.

Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.

AXIN1 bi-allelic variants disrupting the C-terminal DIX domain cause craniometadiaphyseal osteosclerosis with hip dysplasia.

Sclerosing skeletal dysplasias result from an imbalance between bone formation and resorption. We identified three homozygous, C-terminally truncating AXIN1 variants in seven individuals from four families affected by macrocephaly, cranial hyperostosis, and vertebral endplate sclerosis. Other frequent findings included hip dysplasia, heart malformations, variable developmental delay, and hematological anomalies. In line with AXIN1 being a central component of the ß-catenin destruction complex, analyses of primary and genome-edited cells harboring the truncating variants revealed enhanced basal canonical Wnt pathway activity. All three AXIN1-truncating variants resulted in reduced protein levels and impaired AXIN1 polymerization mediated by its C-terminal DIX domain but partially retained Wnt-inhibitory function upon overexpression. Addition of a tankyrase inhibitor attenuated Wnt overactivity in the AXIN1-mutant model systems. Our data suggest that AXIN1 coordinates the action of osteoblasts and osteoclasts and that tankyrase inhibitors can attenuate the effects of AXIN1 hypomorphic variants.

A Systems-Level Study Reveals Regulators of Membrane-less Organelles in Human Cells.

Membrane-less organelles (MLOs) are liquid-like subcellular compartments that form through phase separation of proteins and RNA. While their biophysical properties are increasingly understood, their regulation and the consequences of perturbed MLO states for cell physiology are less clear. To study the regulatory networks, we targeted 1,354 human genes and screened for morphological changes of nucleoli, Cajal bodies, splicing speckles, PML nuclear bodies (PML-NBs), cytoplasmic processing bodies, and stress granules. By multivariate analysis of MLO features we identified hundreds of genes that control MLO homeostasis. We discovered regulatory crosstalk between MLOs, and mapped hierarchical interactions between aberrant MLO states and cellular properties. We provide evidence that perturbation of pre-mRNA splicing results in stress granule formation and reveal that PML-NB abundance influences DNA replication rates and that PML-NBs are in turn controlled by HIP kinases. Together, our comprehensive dataset is an unprecedented resource for deciphering the regulation and biological functions of MLOs.

A structural basis of T cell cross-reactivity to native and spliced self-antigens presented by HLA-DQ8.

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease that has a strong HLA association, where a number of self-epitopes have been implicated in disease pathogenesis. Human pancreatic islet-infiltrating CD4+ T cell clones not only respond to proinsulin C-peptide (PI40-54; GQVELGGGPGAGSLQ) but also cross-react with a hybrid insulin peptide (HIP; PI40-47-IAPP74-80; GQVELGGG-NAVEVLK) presented by HLA-DQ8. How T cell receptors recognize self-peptide and cross-react to HIPs is unclear. We investigated the cross-reactivity of the CD4+ T cell clones reactive to native PI40-54 epitope and multiple HIPs fused at the same N-terminus (PI40-54) to the degradation products of two highly expressed pancreatic islet proteins, neuropeptide Y (NPY68-74) and amyloid polypeptide (IAPP23-29 and IAPP74-80). We observed that five out of the seven selected SKW3 T cell lines expressing TCRs isolated from CD4+ T cells of people with T1D responded to multiple HIPs. Despite shared TRAV26-1-TRBV5-1 gene usage in some T cells, these clones cross-reacted to varying degrees with the PI40-54 and HIP epitopes. Crystal structures of two TRAV26-1+-TRBV5-1+ T cell receptors (TCRs) in complex with PI40-54 and HIPs bound to HLA-DQ8 revealed that the two TCRs had distinct mechanisms responsible for their differential recognition of the PI40-54 and HIP epitopes. Alanine scanning mutagenesis of the PI40-54 and HIPs determined that the P2, P7, and P8 residues in these epitopes were key determinants of TCR specificity. Accordingly, we provide a molecular basis for cross-reactivity towards native insulin and HIP epitopes presented by HLA-DQ8.

Heterozygous LRP1 deficiency causes developmental dysplasia of the hip by impairing triradiate chondrocytes differentiation due to inhibition of autophagy.

Developmental dysplasia of the hip (DDH) is one of the most common congenital skeletal malformations; however, its etiology remains unclear. Here, we conducted whole-exome sequencing in eight DDH families followed by targeted sequencing of 68 sporadic DDH patients. We identified likely pathogenic variants in the LRP1 (low-density lipoprotein receptor-related protein 1) gene in two families and seven unrelated patients. All patients harboring the LRP1 variants presented a typical DDH phenotype. The heterozygous Lrp1 knockout (KO) mouse (Lrp1+/-) showed phenotypes recapitulating the human DDH phenotypes, indicating Lrp1 loss of function causes DDH. Lrp1 knockin mice with a missense variant corresponding to a human variant identified in DDH (Lrp1R1783W) also presented DDH phenotypes, which were milder in heterozygotes and severer in homozygotes than those of the Lrp1 KO mouse. The timing of triradiate cartilage development was brought forward 1 or 2 wk earlier in the LRP-deficient mice, which leads to malformation of the acetabulum and femoral head. Furthermore, Lrp1 deficiency caused a significant decrease of chondrogenic ability in vitro. During the chondrogenic induction of mice bone marrow stem cells and ATDC5 (an inducible chondrogenic cell line), Lrp1 deficiency caused decreased autophagy levels with significant ß-catenin up-regulation and suppression of chondrocyte marker genes. The expression of chondrocyte markers was rescued by PNU-74654 (a ß-catenin antagonist) in an shRNA-Lrp1-expressed ATDC5 cell. Our study reveals a critical role of LRP1 in the etiology and pathogenesis of DDH, opening an avenue for its treatment.

Genomic heterozygosity is associated with a lower risk of osteoarthritis.

BACKGROUND: Genomic heterozygosity has been shown to confer a health advantage in humans and play a protective role in complex diseases. Given osteoarthritis (OA) is a highly polygenic disease, we set out to determine if an association exists between OA and genomic heterozygosity. RESULTS: End-stage knee and hip OA patients and healthy controls were recruited from the Newfoundland and Labrador (NL) population. The Arthritis Research UK Osteoarthritis Genetics (arcOGEN) consortium database was utilized as a replication cohort. DNA was extracted from blood samples and genotyped. Individual rates of observed heterozygosity (HetRate) and heterozygosity excess (HetExcess) relative to the expected were mathematically derived, and standardized to a z-score. Logistic regression modeling was used to examine the association between OA and HetRate or HetExcess. A total of 559 knee and hip OA patients (mean age 66.5 years, body mass index (BMI) 33.7 kg/m2, and 55% females) and 118 healthy controls (mean age 56.4 years, BMI 29.5 kg/m2, and 59% female) were included in the NL cohort analysis. We found that OA had an inverse relationship with HetRate and HetExcess with odds ratios of 0.64 (95% CI: 0.45-0.91) and 0.65 (95% CI: 0.45-0.93) per standard deviation (SD), respectively. The arcOGEN data included 2,019 end-stage knee and hip OA patients and 2,029 healthy controls, validating our findings with HetRate and HetExcess odds ratios of 0.60 (95% CI: 0.56-0.64) and 0.44 (95% CI: 0.40-0.47) per SD, respectively. CONCLUSIONS: Our results are the first to clearly show evidence, from two separate cohorts, that reduced genomic heterozygosity confers a risk for the future development of OA.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Long-term outcomes of avascular necrosis in sickle cell disease using joint-specific patient-reported outcome measures: Results from a multicentre study.

Avascular necrosis (AVN) is a prevalent and progressive complication in young patients with sickle cell disease (SCD), but no study evaluated the long-term subjective and objective outcome measures. Oxford hip score (OHS) and Oxford shoulder scores (OSS) are validated joint-specific patient-reported outcome measures (PROMs). In this prospective multicentre study, 47 SCD patients with pre-existing diagnosis of AVN occurred at a median age of 35.9 (24.2-47.6) filled out the OHS and OSS at median follow-up of 9.4 years (4.5-12.9). No patient died after diagnosis of AVN. Hip AVN was present in 34 (72%) patients, with bilateral involvement in 25 (74%); 26 (59%) underwent total hip arthroplasty (THA) at a median age of 34.6 (22.6-49.5); and 4 (15%) required re-surgery. OHS revealed moderate to severe impairment both in patients underwent THA and no hip surgery. Shoulder AVN was present in 13 (6%) patients and OSS revealed mild to moderate impairment. A high rate of compromised joint function and pain was observed 10 years after diagnosis of AVN regardless of the type of treatment, outlying the need to improve the management of this sickle-related complication. OHS and OSS are validated joint-specific PROMs easy to use in all SCD centres.

Metabolomic profiles of cartilage and bone reflect tissue type, radiography-confirmed osteoarthritis, and spatial location within the joint.

Osteoarthritis is the most common chronic joint disease, characterized by the abnormal remodeling of joint tissues including articular cartilage and subchondral bone. However, there are currently no therapeutic drug targets to slow the progression of disease because disease pathogenesis is largely unknown. Thus, the goals of this study were to identify metabolic differences between articular cartilage and subchondral bone, compare the metabolic shifts in osteoarthritic grade III and IV tissues, and spatially map metabolic shifts across regions of osteoarthritic hip joints. Articular cartilage and subchondral bone from 9 human femoral heads were obtained after total joint arthroplasty, homogenized and metabolites were extracted for liquid chromatography-mass spectrometry analysis. Metabolomic profiling revealed that distinct metabolic endotypes exist between osteoarthritic tissues, late-stage grades, and regions of the diseased joint. The pathways that contributed the most to these differences between tissues were associated with lipid and amino acid metabolism. Differences between grades were associated with nucleotide, lipid, and sugar metabolism. Specific metabolic pathways such as glycosaminoglycan degradation and amino acid metabolism, were spatially constrained to more superior regions of the femoral head. These results suggest that radiography-confirmed grades III and IV osteoarthritis are associated with distinct global metabolic and that metabolic shifts are not uniform across the joint. The results of this study enhance our understanding of osteoarthritis pathogenesis and may lead to potential drug targets to slow, halt, or reverse tissue damage in late stages of osteoarthritis.