Background and Aims NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human‐induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. Approach and Results We used isogenic hiPSC lines with either a knockout (PNPLA3KO) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M) to model PNPLA3‐associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD‐like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid‐induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol‐induced and methotrexate‐induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild‐type and PNPLA3KO cells. Conclusions Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.

Regulatory B cells (Breg cells) that secrete IL-10 or IL-35 (i35-Breg) play key roles in regulating immunity in tumor microenvironment or during autoimmune and infectious diseases. Thus, loss of Breg function is implicated in development of autoimmune diseases while aberrant elevation of Breg prevents sterilizing immunity, exacerbates infectious diseases, and promotes cancer metastasis. Breg cells identified thus far are largely antigen-specific and derive mainly from B2-lymphocyte lineage. Here, we describe an innate-like IL-27–producing natural regulatory B-1a cell (i27-Breg) in peritoneal cavity and human umbilical cord blood. i27-Bregs accumulate in CNS and lymphoid tissues during neuroinflammation and confers protection against CNS autoimmune disease. i27-Breg immunotherapy ameliorated encephalomyelitis and uveitis through up-regulation of inhibitory receptors (Lag3, PD-1), suppression of Th17/Th1 responses, and propagating inhibitory signals that convert conventional B cells to regulatory lymphocytes that secrete IL-10 and/or IL-35 in eye, brain, or spinal cord. Furthermore, i27-Breg proliferates in vivo and sustains IL-27 secretion in CNS and lymphoid tissues, a therapeutic advantage over administering biologics (IL-10, IL-35) that are rapidly cleared in vivo. Mutant mice lacking irf4 in B cells exhibit exaggerated increase of i27-Bregs with few i35-Bregs, while mice with loss of irf8 in B cells have abundance of i35-Bregs but defective in generating i27-Bregs, identifying IRF8/BATF and IRF4/BATF axis in skewing B cell differentiation toward i27-Breg and i35-Breg developmental programs, respectively. Consistent with its developmental origin, disease suppression by innate i27-Bregs is neither antigen-specific nor disease-specific, suggesting that i27-Breg would be effective immunotherapy for a wide spectrum of autoimmune diseases.

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