Familial Mediterranean Fever (FMF) Cell Models for Research

Familial Mediterranean Fever (FMF) is the most common autoinflammatory disease, predominantly affecting populations of Mediterranean ancestry, including Armenians, Turks, Arabs, and Sephardic Jews. The global prevalence is estimated at 1-5 per 10,000, with higher rates in endemic regions (e.g., 1 in 500 in Armenia). FMF is characterized by recurrent episodes of fever, serositis (peritonitis, pleuritis, synovitis), and erysipelas-like skin lesions. Without treatment, complications include AA amyloidosis, leading to renal failure. The disease is caused by mutations in the MEFV gene, which encodes pyrin, a key regulator of innate immunity. Early diagnosis and treatment with colchicine have dramatically improved outcomes, but a subset of patients remains resistant or intolerant, highlighting the need for novel therapeutic targets.

FMF serves as an ideal model for studying inflammasome biology, innate immune signaling, and autoinflammation. The well-defined genetic basis (MEFV mutations) allows for precise gene editing to create isogenic cell lines with specific mutations. These models enable mechanistic studies of pyrin function, inflammasome assembly, and cytokine release. Public datasets, such as those from the International FMF Consortium, provide genotype-phenotype correlations. Open questions include the role of modifier genes, the molecular basis of colchicine resistance, and the development of targeted therapies beyond IL-1 inhibition.

FMF is driven by dysregulated innate immune responses, primarily involving the pyrin inflammasome. Key pathways include:

• • Pyrin Inflammasome Activation: Mutations in MEFV lead to constitutive or hyperactive pyrin, which oligomerizes with ASC and pro-caspase-1, forming the inflammasome complex. This triggers caspase-1 activation and cleavage of pro-IL-1β and pro-IL-18 into their active forms.
• • NF-κB Signaling: Pyrin also modulates NF-κB activation, influencing the transcription of pro-inflammatory cytokines.
• • Rho GTPase Regulation: Pyrin interacts with Rho GTPases; bacterial toxins that inactivate RhoA activate pyrin, linking infection to inflammation.
• • Cytokine Release: The release of IL-1β and IL-18 drives systemic inflammation, fever, and serositis.

Data from ClinVar and the Infevers database.

The pyrin inflammasome is central, but other networks are implicated:

• • IL-1β/IL-18 Signaling: Autocrine and paracrine effects amplify inflammation.
• • TNF-α Pathway: TNF-α is elevated and contributes to synovitis.
• • NF-κB and MAPK Pathways: These are activated downstream of inflammasome and cytokine receptors.
• • Autophagy: Impaired autophagy may enhance inflammasome activity.

Key nodes include: pyrin, ASC, caspase-1, IL-1β, IL-18, and NF-κB.

Organoids derived from patient-derived induced pluripotent stem cells (iPSCs) offer a more physiologically relevant model, recapitulating tissue-specific inflammation.

• • Knock-in mouse models: Mice carrying FMF-associated MEFV mutations (e.g., M680I) exhibit spontaneous inflammation, mimicking human disease.
• • Conditional knockout models: Tissue-specific deletion of MEFV to study cell-type-specific roles.
• • Humanized mice: Engraftment of human immune cells to study FMF in vivo.
• • PDX models: Not commonly used for FMF due to its non-oncological nature, but patient-derived xenografts of immune cells can be used for drug testing.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise MEFV mutations. For example:

• • MEFV knockout cell lines: Complete loss of pyrin to study its function.
• • Knock-in lines with specific point mutations (e.g., M694V) to model common FMF variants.
• • Reporter lines (e.g., IL-1β promoter-driven fluorescent reporters) to monitor inflammasome activation.

Commercially available, sequence-verified gene-edited cell lines accelerate research by providing consistent, reproducible models. These are generated using CRISPR-Cas9 with guide RNAs targeting specific exons, followed by selection and validation via Sanger sequencing and functional assays.

Gene-edited cell lines allow for the validation of MEFV mutations and the identification of novel modifiers. For example, CRISPR knockout of pyrin in THP-1 cells abolishes inflammasome activation, confirming its essential role. Knock-in of the M694V mutation recapitulates the hyperactive phenotype, enabling studies of pyrin's interaction with other proteins.

Isogenic pairs (wild-type vs. mutant) are used in high-throughput screens to identify compounds that inhibit inflammasome activation. These models also help study resistance to colchicine and the development of alternative therapies. For instance, screening a library of kinase inhibitors in MEFV-mutant cells can identify new targets.

CRISPR-based synthetic lethality screens can identify genes that, when silenced, are lethal only in MEFV-mutant cells. This approach can uncover novel therapeutic targets and biomarkers for patient stratification.