Immunological regulation of skeletal muscle adaptation to exercise.

Exercise has long been acknowledged for its powerful disease-preventing, health-promoting effects. However, the cellular and molecular mechanisms responsible for the beneficial effects of exercise are not fully understood. Inflammation is a component of the stress response to exercise. Recent work has revealed that such inflammation is not merely a symptom of exertion; rather, it is a key regulator of exercise adaptations, particularly in skeletal muscle. The purpose of this piece is to provide a conceptual framework that we hope will integrate exercise immunology with exercise physiology, muscle biology, and cellular immunology. We start with an overview of early studies in the field of exercise immunology, followed by an exploration of the importance of stromal cells and immunocytes in the maintenance of muscle homeostasis based on studies of experimental muscle injury. Subsequently, we discuss recent advances in our understanding of the functions and physiological relevance of the immune system in exercised muscle. Finally, we highlight a potential immunological basis for the benefits of exercise in musculoskeletal diseases and aging.

Borrelia burgdorferi initiates early transcriptional re-programming in macrophages that supports long-term suppression of inflammation.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, establishes a long-term infection and leads to disease manifestations that are the result of host immune responses to the pathogen. Inflammatory manifestations resolve spontaneously despite continued bacterial presence, suggesting inflammatory cells become less responsive over time. This is mimicked by in vitro repeated stimulations, resulting in tolerance, a phenotypic subset of innate immune memory. We performed comparative transcriptional analysis of macrophages in acute and memory states and identified sets of Tolerized, Hyper-Induced, Secondary-Induced and Hyper-Suppressed genes resulting from memory induction, revealing previously unexplored networks of genes affected by cellular re-programming. Tolerized gene families included inflammatory mediators and interferon related genes as would be predicted by the attenuation of inflammation over time. To better understand how cells mediate inflammatory hypo-responsiveness, we focused on genes that could mediate maintenance of suppression, such as Hyper-Induced genes which are up-regulated in memory states. These genes were notably enriched in stress pathways regulated by anti-inflammatory modulators. We examined one of the most highly expressed negative regulators of immune pathways during primary stimulation, Aconitate decarboxylase 1 (Acod1), and tested its effects during in vivo infection with Bb. As predicted by our in vitro model, we show its inflammation-suppressive downstream effects are sustained during in vivo long-term infection with Bb, with a specific role in Lyme carditis.

A discrete 'early-responder' stromal-cell subtype orchestrates immunocyte recruitment to injured tissue.

Following acute injury, stromal cells promote tissue regeneration by a diversity of mechanisms. Time-resolved single-cell RNA sequencing of muscle mesenchymal stromal cells (MmSCs) responding to acute injury identified an 'early-responder' subtype that spiked on day 1 and expressed a notable array of transcripts encoding immunomodulators. IL-1ß, TNF-α and oncostatin M each strongly and rapidly induced MmSCs transcribing this immunomodulatory program. Macrophages amplified the program but were not strictly required for its induction. Transfer of the inflammatory MmSC subtype, tagged with a unique surface marker, into healthy hindlimb muscle induced inflammation primarily driven by neutrophils and macrophages. Among the abundant inflammatory transcripts produced by this subtype, Cxcl5 was stroma-specific and highly upregulated with injury. Depletion of this chemokine early after injury revealed a substantial impact on recruitment of neutrophils, a prolongation of inflammation to later times and an effect on tissue regeneration. Mesenchymal stromal cell subtypes expressing a comparable inflammatory program were found in a mouse model of muscular dystrophy and in several other tissues and pathologies in both mice and humans. These 'early-responder' mesenchymal stromal cells, already in place, permit rapid and coordinated mobilization and amplification of critical cell collaborators in response to injury.

Regulatory T cells shield muscle mitochondria from interferon-γ-mediated damage to promote the beneficial effects of exercise.

Exercise enhances physical performance and reduces the risk of many disorders such as cardiovascular disease, type 2 diabetes, dementia, and cancer. Exercise characteristically incites an inflammatory response, notably in skeletal muscles. Although some effector mechanisms have been identified, regulatory elements activated in response to exercise remain obscure. Here, we have addressed the roles of Foxp3+CD4+ regulatory T cells (Tregs) in the healthful activities of exercise via immunologic, transcriptomic, histologic, metabolic, and biochemical analyses of acute and chronic exercise models in mice. Exercise rapidly induced expansion of the muscle Treg compartment, thereby guarding against overexuberant production of interferon-γ and consequent metabolic disruptions, particularly mitochondrial aberrancies. The performance-enhancing effects of exercise training were dampened in the absence of Tregs. Thus, exercise is a natural Treg booster with therapeutic potential in disease and aging contexts.

The potential for Treg-enhancing therapies in tissue, in particular skeletal muscle, regeneration.

Foxp3+CD4+ regulatory T cells (Tregs) are famous for their role in maintaining immunological tolerance. With their distinct transcriptomes, growth-factor dependencies and T-cell receptor (TCR) repertoires, Tregs in nonlymphoid tissues, termed "tissue-Tregs," also perform a variety of functions to help assure tissue homeostasis. For example, they are important for tissue repair and regeneration after various types of injury, both acute and chronic. They exert this influence by controlling both the inflammatory tenor and the dynamics of the parenchymal progenitor-cell pool in injured tissues, thereby promoting efficient repair and limiting fibrosis. Thus, tissue-Tregs are seemingly attractive targets for immunotherapy in the context of tissue regeneration, offering several advantages over existing therapies. Using skeletal muscle as a model system, we discuss the existing literature on Tregs' role in tissue regeneration in acute and chronic injuries, and various approaches for their therapeutic modulation in such contexts, including exercise as a natural Treg modulator.

Tregs in visceral adipose tissue up-regulate circadian-clock expression to promote fitness and enforce a diurnal rhythm of lipolysis.

Regulatory T cells (Tregs) in nonlymphoid organs provide critical brakes on inflammation and regulate tissue homeostasis. Although so-called "tissue Tregs" are phenotypically and functionally diverse, serving to optimize their performance and survival, up-regulation of pathways related to circadian rhythms is a feature they share. Yet the diurnal regulation of Tregs and its consequences are controversial and poorly understood. Here, we profiled diurnal variations in visceral adipose tissue (VAT) and splenic Tregs in the presence and absence of core-clock genes. VAT, but not splenic, Tregs up-regulated their cell-intrinsic circadian program and exhibited diurnal variations in their activation and metabolic state. BMAL1 deficiency specifically in Tregs led to constitutive activation and poor oxidative metabolism in VAT, but not splenic, Tregs. Disruption of core-clock components resulted in loss of fitness: BMAL1-deficient VAT Tregs were preferentially lost during competitive transfers and in heterozygous TregBmal1Δ females. After 16 weeks of high-fat diet feeding, VAT inflammation was increased in mice harboring BMAL1-deficient Tregs, and the remaining cells lost the transcriptomic signature of bona fide VAT Tregs. Unexpectedly, VAT Tregs suppressed adipocyte lipolysis, and BMAL1 deficiency specifically in Tregs abrogated the characteristic diurnal variation in adipose tissue lipolysis, resulting in enhanced suppression of lipolysis throughout the day. These findings argue for the importance of the cell-intrinsic clock program in optimizing VAT Treg function and fitness.

Down-Regulation of a Profibrotic Transforming Growth Factor-ß1/Cellular Communication Network Factor 2/Matrix Metalloprotease 9 Axis by Triamcinolone Improves Idiopathic Subglottic Stenosis.

Idiopathic subglottic stenosis (iSGS) is a progressive fibrotic disease characterized by life-threatening airway narrowing. Although the molecular underpinnings are unknown, previous reports showing that subglottic serial intralesional steroid injections (SILSIs) improve clinical outcomes suggest a steroid-sensitive pathway in iSGS. Herein, a prospective study was conducted to determine the changes in profibrotic markers during SILSI to identify steroid-sensitive profibrotic drivers. Seven newly diagnosed patients with iSGS were recruited for SILSI. Subglottic biopsies before and after SILSI treatments were evaluated for histologic and molecular markers by confocal microscopy and RT-qPCR. At baseline, iSGS subglottises contained abundant vimentin-positive/α-smooth muscle actin-negative fibroblasts, intermingled with a matrix of fibronectin and types I and VI collagen. Transforming growth factor (TGF)-ß1 was up-regulated primarily in glandular epithelium. Cellular communication network factor 2 (CCN2) was mainly up-regulated in stromal fibroblasts surrounding TGF-ß1-positive glandular structures. SILSI improved iSGS by reducing fibroblast infiltration and increasing matrix remodeling. Mechanistically, SILSI counteracted the effects of TGF-ß1 by inducing matrix metalloprotease 9 (MMP9) expression while repressing CCN2 expression, without affecting TGFß1 levels. Treatment of primary iSGS-derived fibroblasts with TGF-ß1 recapitulated aspects of the disease in vivo, demonstrating that the induction in CCN2 and repression of MMP9 are caused by changes in histone acetylation induced by TGF-ß1. Triamcinolone counteracted the coregulation of these genes by impairing SMAD2/3 binding to promoter regions, and not through histone acetylation. In conclusion, this study shows that SILSI counteracts a dysregulated TGF-ß1/CCN2/MMP9 axis involved in iSGS development.

High-intensity ultrasound irradiation promotes the release of extracellular vesicles from C2C12 myotubes.

Skeletal muscle is an important secretory organ in mammals, producing myriad chemical mediators ("myokines") with distinct biological action in different tissues, including anti-inflammatory activity. Extracellular vesicles (EVs) have recently been identified as a mode of myokine transport from muscle, facilitating such anti-inflammatory activity. In this report, we have demonstrated that high-intensity ultrasound (US) strongly induces EV secretion from cultured myotubes without a reduction in cell viability. High-intensity US of 3.0 W/cm2 with 20% duty cycle increased the number of EVs by 2-fold compared to control at 6 h. This effect was specific to EVs in the 100-150 nm size range. Thus, high-intensity US is a novel modality for inducing myocellular EV release and may hold therapeutic value.

Author Correction: Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses.

Macrophages are activated during microbial infection to coordinate inflammatory responses and host defense. Here we find that in macrophages activated by bacterial lipopolysaccharide (LPS), mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) regulates glucose oxidation to drive inflammatory responses. GPD2, a component of the glycerol phosphate shuttle, boosts glucose oxidation to fuel the production of acetyl coenzyme A, acetylation of histones and induction of genes encoding inflammatory mediators. While acute exposure to LPS drives macrophage activation, prolonged exposure to LPS triggers tolerance to LPS, where macrophages induce immunosuppression to limit the detrimental effects of sustained inflammation. The shift in the inflammatory response is modulated by GPD2, which coordinates a shutdown of oxidative metabolism; this limits the availability of acetyl coenzyme A for histone acetylation at genes encoding inflammatory mediators and thus contributes to the suppression of inflammatory responses. Therefore, GPD2 and the glycerol phosphate shuttle integrate the extent of microbial stimulation with glucose oxidation to balance the beneficial and detrimental effects of the inflammatory response.

Dietary protein restriction reduces circulating VLDL triglyceride levels via CREBH-APOA5-dependent and -independent mechanisms.

Hypertriglyceridemia is an independent risk factor for cardiovascular disease. Dietary interventions based on protein restriction (PR) reduce circulating triglycerides (TGs), but underlying mechanisms and clinical relevance remain unclear. Here, we show that 1 week of a protein-free diet without enforced calorie restriction significantly lowered circulating TGs in both lean and diet-induced obese mice. Mechanistically, the TG-lowering effect of PR was due, in part, to changes in very low-density lipoprotein (VLDL) metabolism both in liver and peripheral tissues. In the periphery, PR stimulated VLDL-TG consumption by increasing VLDL-bound APOA5 expression and promoting VLDL-TG hydrolysis and clearance from circulation. The PR-mediated increase in Apoa5 expression was controlled by the transcription factor CREBH, which coordinately regulated hepatic expression of fatty acid oxidation-related genes, including Fgf21 and Ppara. The CREBH-APOA5 axis activation upon PR was intact in mice lacking the GCN2-dependent amino acid-sensing arm of the integrated stress response. However, constitutive hepatic activation of the amino acid-responsive kinase mTORC1 compromised CREBH activation, leading to blunted APOA5 expression and PR-recalcitrant hypertriglyceridemia. PR also contributed to hypotriglyceridemia by reducing the rate of VLDL-TG secretion, independently of activation of the CREBH-APOA5 axis. Finally, a randomized controlled clinical trial revealed that 4-6 weeks of reduced protein intake (7%-9% of calories) decreased VLDL particle number, increased VLDL-bound APOA5 expression, and lowered plasma TGs, consistent with mechanistic conservation of PR-mediated hypotriglyceridemia in humans with translational potential as a nutraceutical intervention for dyslipidemia.

Metabolism Supports Macrophage Activation.

Macrophages are found in most tissues of the body, where they have tissue- and context-dependent roles in maintaining homeostasis as well as coordinating adaptive responses to various stresses. Their capacity for specialized functions is controlled by polarizing signals, which activate macrophages by upregulating transcriptional programs that encode distinct effector functions. An important conceptual advance in the field of macrophage biology, emerging from recent studies, is that macrophage activation is critically supported by metabolic shifts. Metabolic shifts fuel multiple aspects of macrophage activation, and preventing these shifts impairs appropriate activation. These findings raise the exciting possibility that macrophage functions in various contexts could be regulated by manipulating their metabolism. Here, we review the rapidly evolving field of macrophage metabolism, discussing how polarizing signals trigger metabolic shifts and how these shifts enable appropriate activation and sustain effector activities. We also discuss recent studies indicating that the mitochondria are central hubs in inflammatory macrophage activation.

Au-ACRAMTU-PEt3 Alters Redox Balance To Inhibit T Cell Proliferation and Function.

Although T cells play a critical role in protection from viruses, bacteria, and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. Unwanted T cell responses during organ transplant, graft-versus-host disease, and allergies are also major clinical problems. Although drugs are available to suppress unwanted immune responses, they have limited efficacy with serious side effects. Thus, new therapeutics limiting T cell activation, proliferation, and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation, and function, we examined the immunosuppressive activity of gold(I) analogs of platinum-acridine antitumor agents. We found that the gold complex Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4(+) and CD8(+) T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFN-γ, TNF-α, IL-2, and IL-17 by human and murine CD4(+) and CD8(+) T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection, the expansion of virus-specific CD8(+) T cells was decreased 10-fold and viral load was elevated. Taken together, these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.

Gammaherpesvirus latency differentially impacts the generation of primary versus secondary memory CD8+ T cells during subsequent infection.

UNLABELLED: Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8(+) T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4(+) T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8(+) T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8(+) T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8(+) T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. IMPORTANCE: CD8(+) T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8(+) T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.

Increased cell surface free thiols identify effector CD8+ T cells undergoing T cell receptor stimulation.

Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.

Antioxidant treatment regulates the humoral immune response during acute viral infection.

Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.