The CBM-opathies—A Rapidly Expanding Spectrum of Human Inborn Errors of Immunity Caused by Mutations in the CARD11-BCL10-MALT1 Complex

The caspase recruitment domain family member 11 (CARD11 or CARMA1)—B cell CLL/lymphoma 10 (BCL10)—MALT1 paracaspase (MALT1) [CBM] signalosome complex serves as a molecular bridge between cell surface antigen receptor signaling and the activation of the NF-κB, JNK, and mTORC1 signaling axes. This positions the CBM complex as a critical regulator of lymphocyte activation, proliferation, survival, and metabolism. Inborn errors in each of the CBM components have now been linked to a diverse group of human primary immunodeficiency diseases termed “CBM-opathies.” Clinical manifestations range from severe combined immunodeficiency to selective B cell lymphocytosis, atopic disease, and specific humoral defects. This surprisingly broad spectrum of phenotypes underscores the importance of “tuning” CBM signaling to preserve immune homeostasis. Here, we review the distinct clinical and immunological phenotypes associated with human CBM complex mutations and introduce new avenues for targeted therapeutic intervention

Regulation of Gonadotropin-Releasing Hormone-(1–5) Signaling Genes by Estradiol Is Age Dependent

Gonadotropin-releasing hormone (GnRH) is a key regulatory molecule of the hypothalamus–pituitary (PIT)–gonadal (HPG) axis that ultimately leads to the downstream release of estradiol (E2) and progesterone (P). These gonadal steroids feed back to the hypothalamus and PIT to regulate reproductive function and behavior. While GnRH is thought to be the master regulator of reproduction, its metabolic product GnRH-(1–5) is also biologically active. Thimet oligopeptidase 1 (also known as EP24.15) cleaves GnRH to form GnRH-(1–5). GnRH-(1–5) is involved in regulation of the HPG axis, exerting its actions through a pair of orphan G protein-coupled receptors, GPR101 and GPR173. The physiological importance of GnRH-(1–5) signaling has been studied in several contexts, but its potential role during reproductive senescence is poorly understood. We used an ovariectomized (OVX) rat model of reproductive senescence to assess whether and how GnRH-(1–5) signaling genes in hypothalamic subnuclei change in response to aging and/or different estradiol replacement regimens designed to model clinical hormone replacement in women. We found that Gpr101 and Gpr173 mRNA expression was increased with age in the arcuate nucleus, while expression of Gpr173 and EP24.15 increased with age in the medial preoptic area. Treatment with E2 in younger OVX animals increased expression of Gpr101, Gpr173, and EP24.15. However, older animals treated with E2 showed decreased expression of these GnRH-(1–5) signaling genes, displaying an age-related decline in responsiveness to E2. To our knowledge, this is the first study to systematically assess the effects of age and different clinically relevant regimens of E2 replacement on GnRH-(1–5) signaling genes

GnRH-(1–5) Inhibits TGF-β Signaling to Regulate the Migration of Immortalized Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons originate outside the central nervous system (CNS) in the nasal placode where their migration to the basal forebrain is dependent on the integration of multiple signaling cues during development. The proper migration and establishment of the GnRH neuronal population within the CNS are critical for normal pubertal onset and reproductive function. The endopeptidase EP24.15 is expressed along the migratory path of GnRH neurons and cleaves the full-length GnRH to generate the metabolite GnRH-(1–5). Using the GN11 cell model, which is considered a pre-migratory GnRH neuronal cell line, we demonstrated that GnRH-(1–5) inhibits cellular migration in a wound closure assay by binding the orphan G protein-coupled receptor 173 (GPR173). In our current experiments, we sought to utilize an in vitro migration assay that better reflects the external environment that migrating GnRH neurons are exposed to during development. Therefore, we used a transwell assay where the inserts were coated with or without a matrigel, a gelatinous mixture containing extracellular matrix (ECM) proteins, to mimic the extracellular environment. Interestingly, GnRH-(1–5) inhibited the ability of GN11 cells to migrate only through ECM mimetic and was dependent on GPR173. Furthermore, we found that GN11 cells secrete TGF-β1, 2, and 3 but only TGF-β1 release and signaling were inhibited by GnRH-(1–5). To identify potential mechanisms involved in the proteolytic activation of TGF-β, we measured a panel of genes implicated in ECM remodeling. We found that GnRH-(1–5) consistently increased tissue inhibitors of metalloproteinase 1 expression, which is an inhibitor of proteinase activity, leading to a decrease in bioactive TGF-β and subsequent signaling. These results suggest that GnRH-(1–5) activating GPR173 may modulate the response of migrating GnRH neurons to external cues present in the ECM environment via an autocrine-dependent mechanism involving TGF-β

Management of atopy with dupilumab and omalizumab in CADINS disease

The caspase activation and recruitment domain 11 (CARD11) gene encodes a scaffold protein required for lymphocyte antigen receptor signaling. Dominant-negative, loss-of-function (LOF) pathogenic variants in CARD11 result in CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) disease. Patients with CADINS suffer with severe atopic manifestations including atopic dermatitis, food allergy, and chronic spontaneous urticaria in addition to recurrent infections and autoimmunity. We assessed the response of dupilumab in five patients and omalizumab in one patient with CADINS for the treatment of severe atopic symptoms. CARD11 mutations were validated for pathogenicity using a T cell transfection assay to assess the impact on activation-induced signaling to NF-κB. Three children and three adults with dominant-negative CARD11 LOF mutations were included. All developed atopic disease in infancy or early childhood. In five patients, atopic dermatitis was severe and recalcitrant to standard topical and systemic medications; one adult suffered from chronic spontaneous urticaria. Subcutaneous dupilumab was initiated to treat atopic dermatitis and omalizumab to treat chronic spontaneous urticaria. All six patients had rapid and sustained improvement in atopic symptoms with no complications during the follow-up period. Previous medications used to treat atopy were able to be decreased or discontinued. In conclusion, treatment with dupilumab and omalizumab for severe, refractory atopic disease in patients with CADINS appears to be effective and well tolerated in patients with CADINS with severe atopy.</p

Multiplexed Functional Assessment of Genetic Variants in CARD11.

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type