Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.

Dysregulation of B Cell Repertoire Formation in Myasthenia Gravis Patients Revealed through Deep Sequencing.

Myasthenia gravis (MG) is a prototypical B cell-mediated autoimmune disease affecting 20-50 people per 100,000. The majority of patients fall into two clinically distinguishable types based on whether they produce autoantibodies targeting the acetylcholine receptor (AChR-MG) or muscle specific kinase (MuSK-MG). The autoantibodies are pathogenic, but whether their generation is associated with broader defects in the B cell repertoire is unknown. To address this question, we performed deep sequencing of the BCR repertoire of AChR-MG, MuSK-MG, and healthy subjects to generate ∼518,000 unique VH and VL sequences from sorted naive and memory B cell populations. AChR-MG and MuSK-MG subjects displayed distinct gene segment usage biases in both VH and VL sequences within the naive and memory compartments. The memory compartment of AChR-MG was further characterized by reduced positive selection of somatic mutations in the VH CDR and altered VH CDR3 physicochemical properties. The VL repertoire of MuSK-MG was specifically characterized by reduced V-J segment distance in recombined sequences, suggesting diminished VL receptor editing during B cell development. Our results identify large-scale abnormalities in both the naive and memory B cell repertoires. Particular abnormalities were unique to either AChR-MG or MuSK-MG, indicating that the repertoires reflect the distinct properties of the subtypes. These repertoire abnormalities are consistent with previously observed defects in B cell tolerance checkpoints in MG, thereby offering additional insight regarding the impact of tolerance defects on peripheral autoimmune repertoires. These collective findings point toward a deformed B cell repertoire as a fundamental component of MG.

Small-molecule fluorophores to detect cell-state switching in the context of high-throughput screening.

A small molecule capable of distinguishing the distinct states resulting from cellular differentiation would be of enormous value, for example, in efforts aimed at regenerative medicine. We screened a collection of fluorescent small molecules for the ability to distinguish the differentiated state of a mouse skeletal muscle cell line. High-throughput fluorescence-based screening of C2C12 myoblasts and myotubes resulted in the identification of six compounds with the desired selectivity, which was confirmed by high-content screening in the same cell states. The compound that resulted in the greatest fluorescence intensity difference between the cell states was used as the screening agent in a pilot screen of 84 kinase inhibitors, each present in four doses, for inhibition of myogenesis. Of the kinase inhibitors, 17 resulted in reduction of fluorescence at one or more concentrations; among the "hits" included known inhibitors of myogenesis, confirming that this compound is capable of detecting the differentiated myotube state. We suggest that the strategy of screening for screening agents reported here may be extended more broadly in the future.

Inhibition of DYRK1A Stimulates Human ß-Cell Proliferation.

Restoring functional ß-cell mass is an important therapeutic goal for both type 1 and type 2 diabetes (1). While proliferation of existing ß-cells is the primary means of ß-cell replacement in rodents (2), it is unclear whether a similar principle applies to humans, as human ß-cells are remarkably resistant to stimulation of division (3,4). Here, we show that 5-iodotubercidin (5-IT), an annotated adenosine kinase inhibitor previously reported to increase proliferation in rodent and porcine islets (5), strongly and selectively increases human ß-cell proliferation in vitro and in vivo. Remarkably, 5-IT also increased glucose-dependent insulin secretion after prolonged treatment. Kinome profiling revealed 5-IT to be a potent and selective inhibitor of the dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) and cell division cycle-like kinase families. Induction of ß-cell proliferation by either 5-IT or harmine, another natural product DYRK1A inhibitor, was suppressed by coincubation with the calcineurin inhibitor FK506, suggesting involvement of DYRK1A and nuclear factor of activated T cells signaling. Gene expression profiling in whole islets treated with 5-IT revealed induction of proliferation- and cell cycle-related genes, suggesting that true proliferation is induced by 5-IT. Furthermore, 5-IT promotes ß-cell proliferation in human islets grafted under the kidney capsule of NOD-scid IL2Rg(null) mice. These results point to inhibition of DYRK1A as a therapeutic strategy to increase human ß-cell proliferation.

An Isochemogenic Set of Inhibitors To Define the Therapeutic Potential of Histone Deacetylases in ß-Cell Protection.

Modulation of histone deacetylase (HDAC) activity has been implicated as a potential therapeutic strategy for multiple diseases. However, it has been difficult to dissect the role of individual HDACs due to a lack of selective small-molecule inhibitors. Here, we report the synthesis of a series of highly potent and isoform-selective class I HDAC inhibitors, rationally designed by exploiting minimal structural changes to the clinically experienced HDAC inhibitor CI-994. We used this toolkit of isochemogenic or chemically matched inhibitors to probe the role of class I HDACs in ß-cell pathobiology and demonstrate for the first time that selective inhibition of an individual HDAC isoform retains beneficial biological activity and mitigates mechanism-based toxicities. The highly selective HDAC3 inhibitor BRD3308 suppressed pancreatic ß-cell apoptosis induced by inflammatory cytokines, as expected, or now glucolipotoxic stress, and increased functional insulin release. In addition, BRD3308 had no effect on human megakaryocyte differentiation, while inhibitors of HDAC1 and 2 were toxic. Our findings demonstrate that the selective inhibition of HDAC3 represents a potential path forward as a therapy to protect pancreatic ß-cells from inflammatory cytokines and nutrient overload in diabetes.

A small-molecule screening strategy to identify suppressors of statin myopathy.

The reduction of plasma low-density lipoprotein levels by HMG-CoA reductase inhibitors, or statins, has had a revolutionary impact in medicine, but muscle-related side effects remain a dose-limiting toxicity in many patients. We describe a chemical epistasis approach that can be useful in refining the mechanism of statin muscle toxicity, as well as in screening for agents that suppress muscle toxicity while preserving the ability of statins to increase the expression of the low-density lipoprotein receptor. Using this approach, we identified one compound that attenuates the muscle side effects in both cellular and animal models of statin toxicity, likely by influencing Rab prenylation. Our proof-of-concept screen lays the foundation for truly high-throughput screens that could help lead to the development of clinically useful adjuvants that can one day be co-administered with statins.

Large-scale chemical dissection of mitochondrial function.

Mitochondrial oxidative phosphorylation (OXPHOS) is under the control of both mitochondrial (mtDNA) and nuclear genomes and is central to energy homeostasis. To investigate how its function and regulation are integrated within cells, we systematically combined four cell-based assays of OXPHOS physiology with multiplexed measurements of nuclear and mtDNA gene expression across 2,490 small-molecule perturbations in cultured muscle. Mining the resulting compendium revealed, first, that protein synthesis inhibitors can decouple coordination of nuclear and mtDNA transcription; second, that a subset of HMG-CoA reductase inhibitors, combined with propranolol, can cause mitochondrial toxicity, yielding potential clues about the etiology of statin myopathy; and, third, that structurally diverse microtubule inhibitors stimulate OXPHOS transcription while suppressing reactive oxygen species, via a transcriptional mechanism involving PGC-1alpha and ERRalpha, and thus may be useful in treating age-associated degenerative disorders. Our screening compendium can be used as a discovery tool both for understanding mitochondrial biology and toxicity and for identifying novel therapeutics.