Background: Helper T cell activity is dysregulated in a number of diseases including those associated with rheumatic autoimmunity. Treatment options are limited and usually consist of systemic immune suppression, resulting in undesirable consequences from compromised immunity. Hedgehog (Hh) signaling has been implicated in the activation of T cells and the formation of the immune synapse, but remains understudied in the context of autoimmunity. Modulation of Hh signaling has the potential to enable controlled immunosuppression but a potential therapy has not yet been developed to leverage this opportunity. Methods: In this work, we developed biodegradable nanoparticles to enable targeted delivery of eggmanone (Egm), a specific Hh inhibitor, to CD4 + T cell subsets. We utilized two FDA-approved polymers, poly(lactic-co-glycolic acid) and polyethylene glycol, to generate hydrolytically degradable nanoparticles. Furthermore, we employed maleimide-thiol mediated conjugation chemistry to decorate nanoparticles with anti-CD4 F(ab') antibody fragments to enable targeted delivery of Egm. Results: Our novel delivery system achieved a highly specific association with the majority of CD4 + T cells present among a complex cell population. Additionally, we have demonstrated antigen-specific inhibition of CD4 + T cell responses mediated by nanoparticle-formulated Egm. Conclusion: This work is the first characterization of Egm's immunomodulatory potential. Importantly, this study also suggests the potential benefit of a biodegradable delivery vehicle that is rationally designed for preferential interaction with a specific immune cell subtype for targeted modulation of Hh signaling.

Fluorescence recovery after photobleaching (FRAP) has been useful in delineating cardiac myofilament biology, and innovations in fluorophore chemistry have expanded the array of microscopic assays used. However, one assumption in FRAP is the irreversible photobleaching of fluorescent proteins after laser excitation. Here we demonstrate reversible photobleaching regarding the photoconvertible fluorescent protein mEos3.2. We used CRISPR/Cas9 genome editing in human induced pluripotent stem cells (hiPSCs) to knock-in mEos3.2 into the COOH terminus of titin to visualize sarcomeric titin incorporation and turnover. Upon cardiac induction, the titin-mEos3.2 fusion protein is expressed and integrated in the sarcomeres of hiPSC-derived cardiomyocytes (CMs). STORM imaging shows M-band clustered regions of bound titin-mEos3.2 with few soluble titin-mEos3.2 molecules. FRAP revealed a baseline titin-mEos3.2 fluorescence recovery of 68% and half-life of ~1.2 h, suggesting a rapid exchange of sarcomeric titin with soluble titin. However, paraformaldehyde-fixed and permeabilized titin-mEos3.2 hiPSC-CMs surprisingly revealed a 55% fluorescence recovery. Whole cell FRAP analysis in paraformaldehyde-fixed, cycloheximide-treated, and untreated titin-mEos3.2 hiPSC-CMs displayed no significant differences in fluorescence recovery. FRAP in fixed HEK 293T expressing cytosolic mEos3.2 demonstrates a 58% fluorescence recovery. These data suggest that titin-mEos3.2 is subject to reversible photobleaching following FRAP. Using a mouse titin-eGFP model, we demonstrate that no reversible photobleaching occurs. Our results reveal that reversible photobleaching accounts for the majority of titin recovery in the titin-mEos3.2 hiPSC-CM model and should warrant as a caution in the extrapolation of reliable FRAP data from specific fluorescent proteins in long-term cell imaging.

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Circulating levels of oxidized lipoprotein (oxLDL) correlate with myocardial infarction (MI) risk. Previous work demonstrates that oxLDL immune complexes can signal through FcγRs on bone marrow-derived dendritic cells (BMDCs), enhancing their activation and inflammatory cytokine secretion. While global FcγR−/− studies show that activating FcγRs are proatherogenic, the role of inhibitory FcγRIIb is unclear. We sought to determine the role of DC-specific FcγRIIb on atherosclerosis and glucose tolerance, as both are important MI risk factors. Bone marrow chimeras were generated by rescuing lethally irradiated female Ldlr−/− mice with hematopoietic cells from littermate CD11c-Cre+or CD11c-Cre− Fcgr2bfl/fl donors. Four weeks after transplant, recipients were placed on a Western diet for eight weeks. Quantitation of atherosclerosis in the proximal aorta demonstrated a 58% increase CD11c− Cre+ Fcgr2bfl/fl recipients. We discovered that hepatic cholesterol, scavenger receptor CD36, and MHC Class II were increased on CD11c+CD11b+ cells in CD11c-Cre+ Fcgr2bfl/fl recipients. Furthermore, study of aortic draining lymph nodes revealed a 50% reduction in TREGS. To study glucose tolerance, six-week-old CD11c-Cre+ and CD11c-Cre− Fcgr2bfl/fl mice were placed on a 60 kcal% fat diet. Despite similar weight gain, CD11c-Cre+ Fcgr2bfl/fl mice were significantly more glucose intolerant after six weeks. Analysis of white adipose tissue (WAT) demonstrated a two-fold increase in pro-inflammatory AT DCs with increased MHC Class II and CD86. Collectively, these results demonstrate that the absence of FcγRIIb on CD11c+ cells results in increased liver and AT inflammation, influencing atherogenesis and development of glucose intolerance.