Efficient skeletal muscle regeneration requires the accumulation of Foxp3+CD4+ regulatory T (Treg) cells. Muscle Tregs have a transcriptome and T cell receptor repertoire distinct from Tregs found in other tissues. The gene, Areg, was enriched in muscle Tregs and this molecule enhanced regeneration at the level of the satellite cell, a myogenic precursor. Il1rl1, which encodes for the interleukin(IL)-33 receptor, ST2, was also enriched in muscle Tregs. Our findings showed that muscle Tregs required expression of ST2 to both accumulate in injured muscle and potentiate repair. When we examined the producers of IL-33, we uncovered IL-33 was primarily produced by fibro/adipocyte progenitor cells, often in close association with neural structures, e.g. nerve tracts, bundles and muscle spindles, a muscle structure specialized for proprioception. Aging severely impairs skeletal muscle regeneration. Interestingly, although the Treg fraction in lymphoid organs increased with age, we found Treg accumulation in the injured muscle from aged mice was severely limited. Diminished Treg accumulation was due to impaired immigration from circulation, defective proliferation, and less efficient retention within muscle. Additionally, aging was accompanied by a decrease in IL-33-producing cells. When we administered exogenous IL-33, the aged muscle Treg population was restored to levels found in young mice, and regeneration was enhanced. In summary, these studies expand our understanding of this numerically small, but potent immune cell population. We uncover how aging impairs the Treg regenerative response, and in turn, highlight how IL-33 may potentially address age-related sacropenia and other muscle regeneration defects. / Medical Sciences

Epidemiological and experimental data implicate branched chain amino acids (BCAAs) in the development of insulin resistance, but the mechanisms underlying this link remain unclear. Insulin resistance in skeletal muscle stems from excess accumulation of lipid species, a process that requires blood-borne lipids to first traverse the blood vessel wall. Little is known, however, of how this trans-endothelial transport occurs or is regulated. Here, we identify 3-hydroxy-isobutyrate (3-HIB), a catabolic intermediate of the BCAA valine, as a novel paracrine regulator of trans-endothelial transport of fatty acids. PGC-1α, a transcriptional co-activator that regulates broad programs of fatty acid consumption, induces the secretion from muscle of 3-HIB, which then triggers fatty acid uptake and transport in endothelial cells. Conversely, inhibiting the synthesis of 3-HIB in muscle cells blocks the promotion of endothelial fatty acid uptake. Providing animals with 3-HIB in drinking water, or inducing 3-HIB levels in skeletal muscle by over-expressing PGC-1α, stimulates muscle to take up fatty acids in vivo, leading to muscle lipid accumulation, and systemic glucose intolerance. 3-HIB levels are elevated in muscle from patients with diabetes. These data thus unveil a novel mechanism that regulates trans-endothelial flux of fatty acids, revealing 3-HIB as a new bioactive signaling metabolite that links the regulation of fatty acid flux to BCAA catabolism and provides a mechanistic explanation for how increased BCAA catabolic flux can cause diabetes. / Medical Sciences

DNA interstrand crosslinks (ICLs) can be induced by multiple crosslinking agents and are a severe form of DNA damage that prevents strand separation during DNA replication and transcription. Failure to repair endogenous ICLs in S phase is thought to underlie the bone marrow failure syndrome Fanconi anemia, and up-regulation of ICL repair is one of the causes of tumor chemoresistance against widely used cancer drugs. In metazoans, a major pathway of ICL repair is coupled to DNA replication and requires the Fanconi anemia pathway. In most current models, collision of a single DNA replication fork with an ICL is sufficient to initiate repair. In contrast, we show that in Xenopus egg extracts, two DNA replication forks must converge on an ICL to trigger repair. When only one fork reaches the ICL, the replicative DNA helicase, CMG, fails to unload from the stalled fork, and repair is blocked. Arrival of a second fork, even when substantially delayed, rescues repair. We conclude that ICL repair requires a replication-induced X-shaped DNA structure surrounding the lesion, and we speculate how this requirement helps maintain genomic stability in S phase. Next, we focus on how psoralen-induced ICLs are repaired. Although psoralen-ICL repair still requires replication fork convergence, downstream repair events are completely different. Unlike cisplatin-ICLs, which are resolved via incisions in the parental strands with formation of double-strand breaks (DSBs), psoralen-ICLs are resolved via cleavage of a N-glycosyl bond that forms part of the ICL. Unlike cisplatin-ICL repair, psoralen-ICL repair does not require CMG helicase unloading or FANCI-FANCD2. When N-glycosyl bond cleavage is inhibited, the psoralen-ICL is repaired via FANCI-FANCD2-dependent incisions. Our work identifies a novel S phase ICL repair mechanism that is independent of the Fanconi anemia pathway and avoids DSBs. / Medical Sciences

M. tuberculosis is the causative agent of a global health epidemic that kills ~1.5 million people each year. The outcomes of infection with Mtb are highly variable. Although many patients are able to control the infection in a quiescent state, others develop active disease. Furthermore, the progression of TB lesions has been shown to vary within a single individual. This phenotypic variability in the infecting population of Mtb may be responsible for the high rate of treatment failures, which can exceed 20% in some endemic areas (World Health Organization, 2015). Although genetic mutation can drive a portion of the observed phenotypic variability, mutation rates in mycobacteria are exceedingly low. Epigenetic factors are therefore likely to be responsible for the majority of observed diversity in infection and treatment outcomes. Here, we investigated epigenetic drivers of phenotypic variability and survival in mycobacteria. We found evidence of high rates of phenotypic variability in response to drug treatment of M. smegmatis, a non-pathogenic, model for TB. Specifically, we found that two distinct subpopulations are able to grow in the presence of drug. These subpopulations exhibited heritability of their transcriptional profiles, growth properties, and ability to grow on drug across generations. We next found that hupB, a histone-like protein, is critical for the formation of these epigenetically regulated subpopulations. We also show that hupB regulates gene expression and is post-translationally modified, and that modification of hupB may drive the formation of one of these phenotypically drug-resistant subpopulations. These findings suggest that modification of a histone-like protein may drive epigenetic inheritance and phenotypic variability in mycobacteria, which allows it to withstand antibiotic treatment. Finally, we also investigated alternative post-transcriptional mechanisms of hupB regulation. / Biological Sciences in Public Health

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin+ cells using cellular reprogramming. Here, I describe the antral stomach as a previously unrecognized source highly amenable to conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic β cells. Expression of β-cell reprogramming factors (Ngn3, Pdx1, and Mafa) in vivo converts antral cells efficiently into insulin+ cells with close molecular and functional resemblance to endogenous β cells. My data further indicate that Cdx2, an intestine-specific transcription factor, acts as a molecular barrier for β-cell conversion. Induced GI insulin+ cells can suppress hyperglycemia over at least 6 months, and they regenerate rapidly after ablation from the native stem-cell compartment. Transplantation of bioengineered stomach mini-organs also produced insulin+ cells and suppressed hyperglycemia. These studies demonstrate the potential of developing engineered stomach tissue as a renewable source of functional β cells for glycemic control. / Medical Sciences

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