NOD2 pathway gene variants in patients with non-Crohn's-related granulomatous autoinflammatory diseases
Creators: name: Downes, Morgan
Publisher: name: The Australian National University
Year: 2022

description: The presence of granulomas in clinically diverse autoinflammatory diseases suggests an element of shared pathophysiology. NOD2 genetic variants segregate with several granulomatous diseases, including Crohn's disease, Blau syndrome and Yao syndrome. Here, we investigated NOD2 pathway and NOD2 genetic variants in a cohort of sarcoidosis patients and in a case of Yao syndrome. We also examined the influence of canonical NF-kB signalling factors on NOD2-derived proinflammatory cytokine responses using genetic and pharmacological approaches. Genotyping of six NOD2 variants revealed that NOD2 P268S and IVS8+158 were significantly increased in sarcoidosis. To identify genotype/phenotype relationships, cytokine responses in PBMCs were examined by flow cytometry. Patient IVS8+158 carriers had an elevated TNFa response in monocytes and T cells, an effect not observed in healthy controls with the same allele. Post-hoc analysis of a subset of patients revealed that patients who responded well to anti-TNFa treatment carried IVS8+158 and most had an elevated monocyte TNFa response. These findings indicate that NOD2 genotype may be useful to identify patients who will benefit most from anti-TNFa therapy. The role of canonical NF-kB pathway proteins, NF-kB1 and IKKB, in the regulation of NOD2 signalling was investigated. A heterozygous frameshift variant in NFkB1 was identified in two related patients, one diagnosed with CVID and the other with autoimmunity and immune dysregulation. The variant results in haploinsufficiency of NF-kB1 factors (p105 and p50). Flow cytometric analysis revealed that both patients had an elevated proportion of cytokine producing monocytes following NOD2 activation. This effect was not detected in patient lymphocytes after PKC activation, highlighting pathway-specific differences in the sensitivity to NF-kB1 abundance. Paradoxically, the proportion of monocytes displaying p-p65 was reduced in patients. These findings reveal that an expansion of cytokine producing monocytes may manifest in CVID patients with NF-kB1 haploinsufficiency. To generate a phenocopy of the NFKB1K95fs patient phenotype, healthy control PBMCs were treated with a pharmacological IKKB inhibitor, IMD0354. As observed with NF-kB1 haploinsufficiency, IMD0354 treatment increased the proportion of cytokine producing monocytes after NOD2 activation, however, a concurrent increase in NF-kB activation was not detected. IMD0354 diminished NF-kB activation and cytokine responses in monocytes and lymphocytes after PKC activation. Biochemical analyses revealed that IMD0354 not only reduced the amplitude of NOD2-derived NF-kB signalling, but also modified the temporal kinetics of the partial ensuing response. Our findings demonstrate that the NOD2 response is sensitive to changes in NF-kB1 abundance and to temporal changes in signalling kinetics. Finally, NOD2 signalling was examined in a case of Yao syndrome. The patient carried a heterozygous loss of function variant in NOD2, G908R, and a rare heterozygous variant in a NOD2 interactor, RNF31 R353Q (encoding HOIP). We investigated whether RNF31 R353Q modified NOD2 signalling. The patient had a normal monocyte cytokine response to NOD2 activation. Overexpression assays indicated that the NOD2-HOIP interaction was unaffected by the variants. Co-immunoprecipitation experiments revealed that OTULIN dissociation from HOIP R353Q was reduced after L18-MDP stimulation. Additionally, autolinear ubiquitination of HOIP R353Q was reduced after TNFa stimulation. These findings suggest that RNF31 R353Q may alter the regulation of LUBAC activity.