Gaucher disease (GD) is an autosomal recessive disorder caused by mutations in the acid β-glucocerebrosidase gene. To model GD, we generated human induced pluripotent stem cells (hiPSC), by reprogramming skin fibroblasts from patients with type 1 (N370S/N370S), type 2 (L444P/RecNciI), and type 3 (L444P/L444P) GD. Pluripotency was demonstrated by the ability of GD hiPSC to differentiate to all three germ layers and to form teratomas in vivo. GD hiPSC differentiated efficiently to the cell types most affected in GD, i.e., macrophages and neuronal cells. GD hiPSC-macrophages expressed macrophage-specific markers, were phagocytic, and were capable of releasing inflammatory mediators in response to LPS. Moreover, GD hiPSC-macrophages recapitulated the phenotypic hallmarks of the disease. They exhibited low glucocerebrosidase (GC) enzymatic activity and accumulated sphingolipids, and their lysosomal functions were severely compromised. GD hiPSC-macrophages had a defect in their ability to clear phagocytosed RBC, a phenotype of tissue-infiltrating GD macrophages. The kinetics of RBC clearance by types 1, 2, and 3 GD hiPSC-macrophages correlated with the severity of the mutations. Incubation with recombinant GC completely reversed the delay in RBC clearance from all three types of GD hiPSC-macrophages, indicating that their functional defects were indeed caused by GC deficiency. However, treatment of induced macrophages with the chaperone isofagomine restored phagocytosed RBC clearance only partially, regardless of genotype. These findings are consistent with the known clinical efficacies of recombinant GC and isofagomine. We conclude that cell types derived from GD hiPSC can effectively recapitulate pathologic hallmarks of the disease.Gaucher model | Gaucher macrophages | lipid storage disease | glucosylsphingolipids | erythrophagocytosis

Inflammation underlying immune pathology and tissue damage involves an intricate interplay between multiple immunological and biochemical mediators. Cytokines represent the key immune mediators that trigger a cascade of reactions that drive processes such as angiogenesis and proteolytic damage to tissues. IL-17 has now been shown to be a pivotal cytokine in many autoimmune diseases, supplanting the traditional Th1-Th2 paradigm. Also, the dual role of proinflammatory IFN-␥ has unraveled new complexities in the cytokine biology of such disorders. A major hurdle in fully understanding the effector pathways in these disorders is the lack of information regarding the temporal kinetics of the cytokines during the course of the disease, as well as the interplay among the key cytokines. Using an experimental model of arthritic inflammation, we demonstrate that the temporal expression of cytokines during the incubation phase is a critical determinant of disease susceptibility. The susceptible rats raised a vigorous IL-17 response early, followed by IFN-␥ and IL-27 response in that sequence, whereas the resistant rats displayed an early and concurrent response to these three cytokines. Accordingly, treatment with exogenous IFN-␥/IL-27 successfully controlled arthritic inflammation and inhibited the defined mediators of inflammation, angiogenesis, cell survival, apoptosis, and tissue damage. Furthermore, IFN-␥ enhanced IL-27 secretion, revealing a cooperative interplay between the two cytokines. Our results offer a novel immunobiochemical perspective on the pathogenesis of autoimmune arthritis and its therapeutic control.

IL-33, a member of the IL-1 family of cytokines, is produced by many cell types, including macrophages, yet its regulation is largely unknown. Treatment of primary murine macrophages with a panel of Toll-like receptor (TLR) (e.g., TLR2, TLR3, TLR4, and TLR9) agonists and non-TLR (e.g., MDA5, RIG-I) agonists revealed a pattern of gene and protein expression consistent with a role for IRF-3 in the expression of IL-33. Accordingly, induction of IL-33 mRNA was attenuated in IRF-3−/− macrophages and TBK1−/− mouse embryonic fibroblasts. Despite the fact that all IL-33 agonists were IRF-3-dependent, LPS-induced IL-33 mRNA was fully inducible in IFN-β−/− macrophages, indicating that IL-33 is not dependent on IFN-β as an intermediate. Epinephrine (EPI) and Bordetella pertussis adenylate cyclase toxin (ACT), cAMP-activating agents, activate CREB and greatly synergized with LPS to induce IL-33 mRNA in macrophages. Both LPS-induced and ACT/LPS-enhanced expression of IL-33 mRNA was partially, but significantly, inhibited by the Protein Kinase A (PKA) inhibitor, H-89, but not by tyrosine kinase or PKC inhibitors. Two IL-33 mRNA species derived from two alternative promoters encode full-length IL-33; however, the shorter “A” species is preferentially induced by all IL-33-inducing agonists except Newcastle Disease Virus (NDV), a RIG-I agonist, that induced expression of both “A” and “B” transcripts. Together, these studies greatly extend what is currently known about the regulation of IL-33 induction in macrophages stimulated by bacterial and viral agonists that engage distinct innate immune signaling pathways.

Nicotine, a main addictive compound in tobacco smoke, has been linked to promotion and progression of lung, head and neck, pancreatic, and breast cancers, but the detailed mechanisms of cancer progression remain elusive. Here we show that nicotine induces the expression of galectin-3 (an anti-apoptotic β-galactoside-binding lectin) in breast cancer cell line and in primary tumors from breast cancer patients. Nicotine-induced up regulation of galectin-3 is due to an increased expression of α9 isoform of nicotinic acetylcholine receptor (α9nAChR), which activates transcription factor STAT3 that in turn, physically binds to galectin-3 (LGALS3) promoter and induces transcription of galectin-3. Intracellular galectin-3 increased mitochondrial integrity and suppressed chemotherapeutic-induced apoptosis of breast cancer cell. Moreover, nicotine induced enrichment of side population cells with cancer stem cell-like properties was modulated by galectin-3 expression and could be significantly reduced by transient knock down of LGALS3 and its upstream signaling molecules STAT3 and α9nAChR. Thus, galectin-3 or its upstream signaling molecule STAT3 or α9nAChR could be a potential target to prevent nicotine-induced chemoresistance in breast cancer.

Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, while other genes are induced or unchanged in expression. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies have reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-Mφ or M2), we show herein that LPS pretreatment of IL-4Rα−/− and STAT6−/− macrophages, that fail to develop into AA-Mφ, resulted in tolerance of proinflammatory cytokines as well as molecules and chemokines previously associated with AA-Mφ (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) macrophages pretreated with IL-4, the prototype inducer of AA-Mφ, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-Mφ-associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs, and conversely, IL-13−/− macrophages were tolerized equivalently to WT macrophages by LPS pretreatment. Further, IL-4Rα-deficiency did not affect the reversal of endotoxin tolerance exerted by histone deacetylase inhibitor, TSA. Like WT mice, 100% of LPS-tolerized IL-4Rα-deficient mice survived LPS + D-galactosamine-induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-Mφ-associated chemokines induced by LPS challenge when compared to non-tolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-Mφ are dissociable.