Abstract
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
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Data availability
RNA sequence data have been deposited in the Gene Expression Omnibus under the accession code GSE261031. Exome sequencing data will not be made publicly available as they contain information that might compromise research participant privacy/consent. Source data are provided with this paper.
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Acknowledgements
We thank the participants and their family members and the healthy donors for their enthusiastic support during this research study and M. Pasparakis, H. Kashkar and A. Annibaldi (Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases and Center for Molecular Medicine Cologne, University of Cologne, Germany) for logistic support and valuable suggestions. We thank the The Walter and Eliza Hall Institute Histology Core Facility for their support and assistance in this work. This study was supported, in part, by the Intramural Research Programs of the National Human Genome Research Institute, the National Institute of Arthritis and Musculoskeletal and Skin Diseases, the National Institute of Allergy and Infectious Diseases, the National Cancer Institute, the National Heart, Lung, and Blood Institute, the Clinical Center of the NIH and the Peter MacCallum Cancer Foundation. H.O. was supported by the Deutsche Forschungsgemeinschaft (DFG; German Research Foundation), including CRC1403 (414786233) and Germany’s Excellence Strategy (EXC 2030 (390661388)) and by Fritz-Thyssen Stiftung (10.23.1.013MN). K.E.L. is supported by an Australian Research Council Future Fellowship (FT190100266). N.L., J. Silke and H.A. are supported by National Health and Medical Research Council Leadership Investigator Grants (2017929, 1195038 and 1194144). This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government National Health and Medical Research Council IRIISS (GNT9000719). S.B. is supported by France’s National Research Agency, the Investment for the Future Program (ANR-11-LABX-0070_TRANSPLANTEX) as well as Strasbourg’s Interdisciplinary Thematic Institute for Precision Medicine, CNRS and INSERM, funded by IdEx Unistra (ANR-10-IDEX-0002) and SFRI-STRAT’US (ANR-20-SFRI-0012). H.W. is supported by Cancer Research UK (A27323), a Wellcome Trust Investigator Award (214342/Z/18/Z), the Medical Research Council (MR/S00811X/1), the DFG (CRC1399, 413326622; CRC1530, 455784452; CRC1403, 414786233), an Alexander von Humboldt Foundation Professorship Award and CANTAR. The Laboratory of Human Genetics of Infectious Diseases is supported by the Howard Hughes Medical Institute, the Rockefeller University, the St. Giles Foundation, the NIH (R21AI159728 to B.B. & P01AI061093 to J.L.C.), the National Center for Advancing Translational Sciences (NCATS), NIH Clinical and Translational Science Award (CTSA) program (UL1TR001866), the French National Research Agency (ANR) under the ‘Investments for the Future’ program (ANR-10-IAHU-01), the Integrative Biology of Emerging Infectious Diseases Laboratory of Excellence (ANR-10-LABX-62-IBEID), the French Foundation for Medical Research (FRM) (EQU201903007798), the Square Foundation, Grandir – Fonds de solidarité pour l’enfance, William E. Ford, General Atlantic’s Chairman and Chief Executive Officer, Gabriel Caillaux, General Atlantic’s Co-President, Managing Director and Head of Business in EMEA, and the General Atlantic Foundation, Institut National de la Santé et de la Recherche Médicale (INSERM) and the University of Paris Cité.
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H.O., N.L., I.A. and D.L.K. conceived and designed the study, analyzed the data and wrote the manuscript. K.M., P.P.C., E.R., O.V., O.K., C.R., S.N., H.S.K., M. Swart, Y.W., N.I.Ç., A.M., R.C., Q.X., S.P., D.B.B., J.J.C., K.D., C.L.S., H.A., K.E.L., H.Y., D.Y., M.B., D.R., W.L.T., M.G. and J.T. performed experiments. B.M., J.M., J. Stoddard and J.N. analyzed and interpreted the results. K.M., M.N., A.K.O., P.H., T.R., N.T.D., H.K., V.Z., N.M., M. Shahrooei, N. Parvaneh, N.A.-O., R.C., J.D., P.M., M.J.K., B.B., J.-L.C., S.B. and A.P.R. developed the human materials and/or recruited participants. C.T.M., K.I., S.D.R., L.D.N., J. Silke, P.L.S., N. Peltzer and H.W. provided critical scientific input and/or reagents. H.O. and N.L. wrote the initial draft of the paper. All authors contributed to the final review and editing of the paper.
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Extended data
Extended Data Fig. 1 Genetic and molecular investigations of sharpenia.
(a) Sanger sequence electropherograms demonstrating homozygous frameshift variants in the patients. (b) Population allele frequency and CADD score for SHARPIN variants homozygous in public databases. The two SHARPIN variants appear in red. CADD−Mutation Significance Score (MSC) cutoff for SHARPIN (90% confidence interval) was indicated by dashed line. NR: not reported. (c, d) Normalized mRNA levels of SHARPIN in (c) PBMCs and (d) fibroblasts from LUBAC-deficient patients and healthy controls. RNA was extracted from each sample and was measured with technical quadruplicates. Mean value is displayed as a bar. (e, f) Supporting data for Fig. 2e. TNFR1-signaling complex (TNFR1-SC) formation in fibroblasts from P1 and two unrelated healthy controls. Fibroblasts were stimulated with modified tandem affinity purification (moTAP)-tagged TNF (1 μg/ml) for the indicated times. TNFR1-SC was purified with anti-FLAG immunoprecipitation, and analyzed by western blotting. (g) Normal induction of non-canonical NF-κB in P1. Total PBMCs were stimulated with anti-CD3 (aCD3) for the indicated durations, and the expression of NFKB2 p100 (full length) and p52 (active form) was detected by western blot. Sis: P1’s sister carrying a heterozygous p.Leu74ProfsX86 variant. Representative result of two independent experiments.
Extended Data Fig. 2 Cytokine expression studies ex vivo in LUBAC-deficient patients.
(a) Cytokine expression in LUBAC-deficient monocytes. PBMCs from SHARPIN (P1)- and HOIP-deficient patients and two healthy controls were stimulated with IL-1β (10 ng/ml) for 6 h, and the intracellular accumulation of cytokines in CD14+ monocytes was quantified by flow cytometry. (b, c) Cytokine secretion from LUBAC-deficient PBMCs. PBMCs from SHARPIN and HOIP-deficient patients and two healthy controls were stimulated with (b) LPS (1 μg/ml) or (c) IL-1β (10 ng/ml) for 6 h, and secreted cytokines were measured by ELISA. (d, e) Cytokine secretion from LUBAC-deficient fibroblasts. Fibroblasts from SHARPIN (P1)- and HOIP-deficient patients and two healthy controls were stimulated with (d) LPS (1 μg/ml) or (e) IL-1β (10 ng/ml) for 24 h, and secreted cytokines were measured by ELISA. (a-e) The experiments were performed with biological triplicates (a,d,e) or duplicates (b,c), and shown are the representatives of two independent experiments. Mean values ± s.d are displayed.
Extended Data Fig. 3 Cell death induction assays.
(a) Cell death assay using immortalized mouse embryonic fibroblasts from Sharpin-deficient mice stably reconstituted with wild-type SHARPIN or patient-derived SHARPIN mutants (P1 and P2). (b, c) Cell death assay using fibroblasts from a SHARPIN-deficient patient (P1), patients with HOIL1 deficiency, otulipenia and cleavage resistant RIPK1-induced autoinflammation (CRIA), and two unrelated healthy controls. The cells were stimulated with TNF (100 ng/ml) combined with (b) smac mimetic (SM: compound A: 100 nM) or (c) human recombinant TWEAK (50 ng/ml), in the presence or absence of zVAD (pan-caspase inhibitor: 20 μM) or Nec1 (RIPK1 inhibitor: 50 μM). The dead cell percentages after 16 h of treatment are shown. (a-c) The experiments were performed with biological triplicates (a, b) or duplicates (c), and shown are the representatives of two (a, c) or five (b) independent experiments. Mean values ± s.d are displayed. Quantitative data were analyzed using one-way ANOVA followed by Tukey-Kramer test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, N.S., not significant. (d) Supporting data for Fig. 3c. Western blot analysis of caspase-3 cleavage in cell lysates from fibroblasts from a SHARPIN-deficient patient and two unrelated healthy controls. The cells were stimulated with TNF (100 ng/ml) and cycloheximide (CHX 50 μg/ml) for the indicated times. (e, f) Necroptosis induction assay using EBV-immortalized lymphoblastoid cells (e) and fibroblasts (f) from SHARPIN-deficient patient. The fibroblasts were stimulated with TSZ (TNF, smac mimetic (compound A) and zVAD) for the indicated time. HT29 cells were used as a positive control for the phospho-antibodies. Representative result of two independent experiments. (g) Supporting data for Fig. 3d. Complex II immunoprecipitation in fibroblasts from SHARPIN-deficienct P1 compared with an unrelated healthy control. Fibroblasts were stimulated with TNF + zVAD for the indicated times, and the lysates were subjected to immunoprecipitation.
Extended Data Fig. 4 In vivo and in vitro characterization of cell death in human LUBAC deficiency.
(a) Hematoxylin and eosin (H&E) staining of colon biopsy samples from LUBAC-deficient patients. Bars: 0.4 mm. These images are representative of three biopsy specimens per donor. (b, c) Supporting data for Fig. 3g to validate the specificity of p-RIPK1 antibody to detect RIPK1 Ser166 phosphorylation with (b) western blot and (c) immunocytochemistry. HT29 cells were stimulated with TSZ (TNF + smac mimetic (BV6) + zVAD) for 4 h. Note that TSZ-stimulated cells show positive staining of pRIPK1, which was removed by λ-phosphatase treatment. (d, e) Supporting data for Fig. 3h to validate the antibody specificity for cleaved GSDMD (Asp275) with (d) western blot and (e) immunocytochemistry. THP1 cells were pre-incubated with LPS for 3 h and were further stimulated with nigericin for another 1 h. (b-e) These experiments were aimed to confirm the specificity of the antibodies and were not repeated. (f) Cleavage of GSDME in dermal fibroblasts stimulated with TNF (100 ng/ml) + CHX (50 μg/ml) for the indicated times. HeLa cells were used as a positive control. Representative result of two independent experiments.
Extended Data Fig. 5 Characterization of joint inflammation in SHARPIN deficiency.
(a) Multiplex ELISA measurement of chemokines in the sterile synovial fluid from P1 before the initiation of anti-TNF treatment, compared with osteoarthritis (OA) control donors (N = 7). The samples were measured in technical triplicate (P1) or duplicate (OA), respectively. Mean values ± s.d are displayed. (b) Quantification of CD45 positive cells in tendons of shoulder joints of control and Sharpin-deficient mice (n = 4 for each group). Data are represented as mean values + SEM. Significance calculated with a two-tailed Mann-Whitney test. (c) Representative hematoxylin and eosin (H&E) staining sections of elbow joints from Sharpin-deficient mice (N = 2) and wild-type littermate controls (N = 2). The arrowhead indicates an inflamed ligament.
Extended Data Fig. 6 Characterization of secondary lymphoid organs in LUBAC deficiencies.
(a, b) Aberrant formation of lymphoid follicles and paracortex in secondary lymphoid organs from LUBAC deficient patients. (a) Lymph node histology of a HOIP-deficient patient compared with a control specimen from an unrelated donor. (b) Adenoid histology of SHARPIN-deficient P1 compared with a control specimen from an unrelated donor. The immunohistochemistry staining was not repeated due to the limited clinical specimens. (c) Gating strategy for the adenoid spectral flow cytometry analysis.
Extended Data Fig. 7 Spectral flow cytometry analysis of human adenoids.
(a–e) Adenoid single-cell suspensions from SHARPIN-deficient P1 and 10 unrelated pediatric control donors were analyzed. (a) Quantification of CD3+, CD4+, CD8+ and CD20+ populations in adenoid samples. (b, c) Surface immunoglobulin expression in the (b) germinal center B and (c) memory B populations. (d, e) Quantification of T cell subpopulations in the adenoid samples. Mean values ± s.d are displayed.
Extended Data Fig. 8 Normal T cell phenotyping results in the SHARPIN-deficient patient ex vivo.
(a) T cell proliferation assay. PMBCs were incubated with Cell Trace Violet, stimulated with anti-CD3/28 or PHA for 72 h and analyzed by flow cytometer. (b) Intracellular cytokine staining for Th1, Th2 and Th17 populations. PBMCs were stimulated with PMA (100 ng/ml) and ionomycin (1 μM) for 5 h with Brefeldin A. Stimulated cells were surface stained, fixed and permeabilized with BD Cytofix/Cytoperm kit. Cells were further stained for intracellular cytokines and analyzed by flow cytometry. Ctrl: unrelated healthy control, Sister: sister carrying the heterozygous frameshift SHARPIN variant p.Leu74ProfsX86. Representative result of two independent experiments.
Extended Data Fig. 9 Whole blood RNA sequencing.
(a, b) mRNA expression of selected cytokines (a) and chemokines (b) in the pre- and post-anti-TNF treatment P1 whole blood RNA samples as well as four age-matched healthy controls. (c) A heatmap demonstrating the changes of genes representative for type I interferon-stimulated gene signature in pre- and post-treatment samples from the SHARPIN-deficient P1.
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Oda, H., Manthiram, K., Chavan, P.P. et al. Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency. Nat Immunol (2024). https://doi.org/10.1038/s41590-024-01817-w
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DOI: https://doi.org/10.1038/s41590-024-01817-w
