Autoinflammation in patients with leukocytic CBL loss of heterozygosity is caused by constitutive ERK-mediated monocyte activation
Jonathan Bohlen, … , Jean-Laurent Casanova, Jacinta Bustamante
Jonathan Bohlen, … , Jean-Laurent Casanova, Jacinta Bustamante
Published October 15, 2024
Citation Information: J Clin Invest. 2024;134(20):e181604. https://doi.org/10.1172/JCI181604.
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Research Article Autoimmunity Immunology

Autoinflammation in patients with leukocytic CBL loss of heterozygosity is caused by constitutive ERK-mediated monocyte activation

Abstract

Patients heterozygous for germline CBL loss-of-function (LOF) variants can develop myeloid malignancy, autoinflammation, or both, if some or all of their leukocytes become homozygous for these variants through somatic loss of heterozygosity (LOH) via uniparental isodisomy. We observed an upregulation of the inflammatory gene expression signature in whole blood from these patients, mimicking monogenic inborn errors underlying autoinflammation. Remarkably, these patients had constitutively activated monocytes that secreted 10 to 100 times more inflammatory cytokines than those of healthy individuals and CBL LOF heterozygotes without LOH. CBL-LOH hematopoietic stem and progenitor cells (HSPCs) outgrew the other cells, accounting for the persistence of peripheral monocytes homozygous for the CBL LOF variant. ERK pathway activation was required for the excessive production of cytokines by both resting and stimulated CBL-LOF monocytes, as shown in monocytic cell lines. Finally, we found that about 1 in 10,000 individuals in the UK Biobank were heterozygous for CBL LOF variants and that these carriers were at high risk of hematological and inflammatory conditions.

Authors

Jonathan Bohlen, Ivan Bagarić, Taja Vatovec, Masato Ogishi, Syed F. Ahmed, Axel Cederholm, Lori Buetow, Steicy Sobrino, Corentin Le Floc’h, Carlos A. Arango-Franco, Luis Seabra, Marine Michelet, Federica Barzaghi, Davide Leardini, Francesco Saettini, Francesca Vendemini, Francesco Baccelli, Albert Catala, Eleonora Gambineri, Marinella Veltroni, Yurena Aguilar de la Red, Gillian I. Rice, Filippo Consonni, Laureline Berteloot, Laetitia Largeaud, Francesca Conti, Cécile Roullion, Cécile Masson, Boris Bessot, Yoann Seeleuthner, Tom Le Voyer, Darawan Rinchai, Jérémie Rosain, Anna-Lena Neehus, Lucia Erazo-Borrás, Hailun Li, Zarah Janda, En-Jui Cho, Edoardo Muratore, Camille Soudée, Candice Lainé, Eric Delabesse, Claire Goulvestre, Cindy S. Ma, Anne Puel, Stuart G. Tangye, Isabelle André, Christine Bole-Feysot, Laurent Abel, Miriam Erlacher, Shen-Ying Zhang, Vivien Béziat, Chantal Lagresle-Peyrou, Emmanuelle Six, Marlène Pasquet, Laia Alsina, Alessandro Aiuti, Peng Zhang, Yanick J. Crow, Nils Landegren, Riccardo Masetti, Danny T. Huang, Jean-Laurent Casanova, Jacinta Bustamante

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Figure 2

High levels of cytokine secretion by the monocytes of patients with CBL LOH.

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High levels of cytokine secretion by the monocytes of patients with CBL ...
(A and B) The PBMCs of patients with CBL LOH produce excessively large amounts of inflammatory cytokines. Cytokine levels in the supernatants of PBMCs from the indicated individuals after 24 hours of culture ex vivo without stimulation (A) or with the indicated stimuli (B). Heterozygous individuals are: patients P10, P11, and P12, the father of patients P1–P3, and the grandmother, uncle, and brother of P7. Cytokine levels were assessed by bead-based ELISA. The statistical significance of differences between healthy controls and homozygous patients was assessed in multiple Mann-Whitney tests, corrected for multiple testing. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005. FC, fold change; US, unstimulated. (C) PBMCs or the indicated leukocyte subsets from the indicated individuals, purified by magnetic sorting, were stimulated as indicated for 24 hours, and CCL2 levels were assessed in the supernatant. The statistical significance of differences was assessed in multiple Mann-Whitney tests, corrected for multiple testing. *P < 0.05. (D) Monocytes or mDCs from the indicated individuals were obtained by magnetic sorting and stimulated with the indicated agonists for 24 hours. (E) Variant allele frequencies of CBL variants in the indicated leukocyte subsets of the indicated individuals, as determined by amplicon sequencing. (F) Engineered THP-1 cell lines. Western blot of wild-type (CBLWT) and CBL-knockout (CBLKO) THP-1 cells generated by CRISPR/Cas9 genome editing and stably transduced with constructs: empty vector (EV), wild-type CBL (WT CBL), or Y371C-mutated CBL (Y371C CBL). (G) Cytokine levels in the supernatant were assessed by bead-based ELISA on the THP-1 cells shown after stimulation, as indicated, for 24 hours. *P < 0.05 by Mann-Whitney test, with correction for multiple testing.