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Virus and host innate immune system interaction plays a significant role in forming the outcome of viral diseases. Host innate immunity initially recognizes the viral invasion and induces a rapid inflammatory response, and this recognition activates signaling cascades that trigger the release of antiviral mediators. This chapter aims to explore the mechanisms by which newly emerged coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activates the host immune system. Since SARS-CoV-2 shares similarities with SARS-CoV that caused the epidemic of SARS in 2003, the pathogenesis of both viruses could be at least very similar. For this, this chapter provides a synthesis of literature concerning antiviral immunity in SARS-CoV and SARS-CoV-2. It includes the presentation of epitopes linked to SARS-CoV-2 as well as the ability of SARS-CoV-2 to cause proteolytic activation and interact with angiotensin-converting enzyme 2 (ACE2) via molecular mimicry. This chapter characterizes various mechanisms that this virus may engage in escaping the host immunity, ended by a discussion of humoral immune responses against SARS-CoV-2.

Lung transplant is a definitive treatment for several end-stage lung diseases. However, the high incidence of allograft rejection limits the overall survival following lung transplantation. Traditionally, alloimmunity directed against human leukocyte antigens (HLA) has been implicated in transplant rejection. Recently, the clinical impact of non-HLA lung-restricted antibodies (LRA) has been recognized and extensive research has demonstrated that they may play a dominant role in the development of lung allograft rejection. The immunogenic lung-restricted antigens that have been identified include amongst others, collagen type I, collagen type V, and k-alpha 1 tubulin. Pre-existing antibodies against these lung-restricted antigens are prevalent in patients undergoing lung transplantation and have emerged as one of the predominant risk factors for primary graft dysfunction which limits short-term survival following lung transplantation. Additionally, LRA have been shown to predispose to chronic lung allograft rejection, the predominant cause of poor long-term survival. This review will discuss ongoing research into the mechanisms of development of LRA as well as the pathogenesis of associated lung allograft injury.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Primary graft dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular nonclassical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flow cytometry revealed that depletion of donor NCM abrogated CM recruitment, single cell RNA sequencing identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine KOs and chimeras indicated that IL-1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL-1β production by NCM was NLRP3 inflammasome dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL-1R-dependent activation of the PKC and NF-κB pathway. Accordingly, we show that IL-1β-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL-1β, or IL-1R antagonism and inflammasome inhibition abrogated recruitment of CM and PGD and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.

Monocytes are one of the most abundant immune cells infiltrating the inflamed organs. However, the majority of studies on monocytes focus on circulating cells, rather than those in the tissue. Here, we identify and characterize an intravascular (i.v.) and extravascular (e.v.) synovial population (Syn Ly6C- cells) which lack cell surface markers of classical monocytes (Ly6C and CD62) or tissue macrophages (CD64 and Tim4), are transcriptionally distinct and conserved in RA patients. e.v. Syn Ly6C- cells are independent of NR4A1 and CCR2, long-lived and embryonically derived while the i.v. Syn Ly6C- cells are dependent on NR4A1, short lived and derived from circulating NCM. e.v. Syn Ly6C- cells undergo increased proliferation and reverse diapedesis dependent on LFA1 in response to arthrogenic stimuli and are required for the development of RA-like disease. These findings uncover a new facet of mononuclear cell biology and are imperative to understanding tissue-resident myeloid cell function in RA.

Summary The processes underlying synchronous multiple organ fibrosis in systemic sclerosis (SSc) remain poorly understood. Age-related pathologies are associated with organismal decline in nicotinamide adenine dinucleotide (NAD+) that is due to dysregulation of NAD+ homeostasis and involves the NADase CD38. We now show that CD38 is upregulated in patients with diffuse cutaneous SSc, and CD38 levels in the skin associate with molecular fibrosis signatures, as well as clinical fibrosis scores, while expression of key NAD+-synthesizing enzymes is unaltered. Boosting NAD+ via genetic or pharmacological CD38 targeting or NAD+ precursor supplementation protected mice from skin, lung, and peritoneal fibrosis. In mechanistic experiments, CD38 was found to reduce NAD+ levels and sirtuin activity to augment cellular fibrotic responses, while inhibiting CD38 had the opposite effect. Thus, we identify CD38 upregulation and resulting disrupted NAD+ homeostasis as a fundamental mechanism driving fibrosis in SSc, suggesting that CD38 might represent a novel therapeutic target., Graphical Abstract, Highlights • CD38 shows elevated expression in skin biopsies of patients with systemic sclerosis • Elevated CD38 is associated with reduced NAD+ and augmented fibrotic responses • Genetic loss of CD38 is associated with increased NAD+ levels and attenuated fibrosis • NAD+ boosting via CD38 inhibition or NR supplementation prevents multi-organ fibrosis, Human Metabolism; Molecular Biology; Immunology

Regulatory T (Treg) cells require Foxp3 expression and induction of a specific DNA hypomethylation signature during development, after which Treg cells persist as a self-renewing population that regulates immune system activation. Whether maintenance DNA methylation is required for Treg cell lineage development and stability and how methylation patterns are maintained during lineage self-renewal remain unclear. Here, we demonstrate that the epigenetic regulator Uhrf1 is essential for maintenance of methyl-DNA marks that stabilize Treg cellular identity by repressing effector T cell transcriptional programs. Constitutive and induced deficiency of Uhrf1 within Foxp3+ cells resulted in global yet non-uniform loss of DNA methylation, derepression of inflammatory transcriptional programs, destabilization of the Treg cell lineage, spontaneous inflammation, and enhanced tumor immunity. These findings support a paradigm in which maintenance DNA methylation is required in distinct regions of the Treg cell genome for both lineage establishment and stability of identity and suppressive function.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Since the first publications coining the term RNA-seq (RNA sequencing) appeared in 2008, the number of publications containing RNA-seq data has grown exponentially, hitting an all-time high of 2,808 publications in 2016 (PubMed). With this wealth of RNA-seq data being generated, it is a challenge to extract maximal meaning from these datasets, and without the appropriate skills and background, there is risk of misinterpretation of these data. However, a general understanding of the principles underlying each step of RNA-seq data analysis allows investigators without a background in programming and bioinformatics to critically analyze their own datasets as well as published data. Our goals in the present review are to break down the steps of a typical RNA-seq analysis and to highlight the pitfalls and checkpoints along the way that are vital for bench scientists and biomedical researchers performing experiments that use RNA-seq.

Intermittent hypoxia (IH) induces activation of the integrated stress response (ISR), but its role in IH-induced visceral white adipose tissue (vWAT) insulin resistance is unknown. CHOP is activated by chronic ISR, whereas GADD34 dephosphorylates the subunit of translation initiation factor 2 (eIF2α), leading to termination of the ISR. We hypothesized that CHOP/Gadd34 null mice would not manifest evidence of insulin resistance after IH exposures. Eight-week-old CHOP/GADD34−/− (double mutant [DM]) and wild-type (WT) littermates were randomly assigned to IH or room air (RA) exposures for 6 weeks. Glucose and insulin tolerance tests were performed, and regulatory T cells (Tregs) and macrophages in vWAT were assessed. Phosphorylated eIF2α:total eIF2α, ATF4, XBP1 expression, and insulin-induced pAKT/AKT expression changes were examined in vWATs. Single GADD34−/− and PERK+/− mice were also evaluated. Body weight and vWAT mass were reduced in DM and WT mice after IH. M1/M2 macrophages and inflammatory macrophages (Ly-6chigh) were significantly increased in WT vWAT but remained unchanged in DM mice. Tregs were significantly decreased in WT vWAT but not in DM mice. Systemic insulin and glucose tolerance tests revealed insulin resistance in IH-WT but not in IH-DM mice. Similarly, decreased pAKT/AKT responses to exogenous insulin emerged in IH-WT compared with RA-WT mice, whereas no significant differences emerged in IH-DM compared with DM-RA. Chronic ISR activation appears to contribute to the insulin resistance and vWAT inflammation that characteristically emerge after long-term IH exposures in a murine model of obstructive sleep apnea.

Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAMs) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor NCMs. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAMs. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or coreceptor CD14 on donor TRAMs prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia/reperfusion-primed donor TRAMs.

Lung transplantation is a life-saving therapy for several end-stage lung diseases. However, lung allografts suffer from the lowest survival rate predominantly due to rejection. The pathogenesis of alloimmunity and its role in allograft rejection has been extensively studied and multiple approaches have been described to induce tolerance. However, in the context of lung transplantation, dysregulation of mechanisms, which maintain tolerance against self-antigens, can lead to lung-restricted autoimmunity, which has been recently identified to drive the immunopathogenesis of allograft rejection. Indeed, both preexisting as well as de novo lung-restricted autoimmunity can play a major role in the development of lung allograft rejection. The three most widely studied lung-restricted self-antigens include collagen type I, collagen type V, and k-alpha 1 tubulin. In this review, we discuss the role of lung-restricted autoimmunity in the development of both early as well as late lung allograft rejection and recent literature providing insight into the development of lung-restricted autoimmunity through the dysfunction of immune mechanisms which maintain peripheral tolerance.

BACKGROUND: Cytomegalovirus (CMV) infection has been associated with poor outcomes following solid organ transplantation. However, the long-term impact of donor and recipient CMV serological status on lung transplant outcomes remains unclear. Accordingly, we evaluated the impact of donor and recipient CMV status on long-term patient as well as allograft survival following single (SLT) and double lung transplantation (BLT). METHODS: The Scientific Registry of Transplant Recipients was used to track all adult lung transplants in United States from May 2005 to June 2016. Patient mortality and bronchiolitis obliterans syndrome were determined up to 5 years using Cox proportional hazards modeling. Additionally, landmark analysis was performed conditional on survival at 1-year. RESULTS: Compared to donor negative-recipient CMV-IgG negative (D-R-), donor positive–recipient negative (D+R-) and donor positive-recipient positive (D+R+) groups had increased mortality at 1- and 5-years following BLT with the former demonstrating highest risk. While mortality was not increased with CMV seropositive donors following SLT at 1-year, both (D+R-) and (D+R+) groups demonstrated greater mortality at 5-year. Risk of bronchiolitis obliterans syndrome was not affected by CMV serological status. Conditional landmark analysis confirmed that lungs from CMV seropositive donors conferred highest risk for long-term mortality. CONCLUSIONS: CMV seronegative recipients undergoing either BLT or SLT from CMV seropositive donors have the highest risk of long-term mortality which extends beyond the first year. Further studies are needed to determine the causes of higher mortality observed in the CMV seronegative recipients and risks benefits of extension of CMV prophylaxis, particularly in the high-risk group.

Ischemia-reperfusion injury, a form of sterile inflammation, is the leading risk factor for both short-term mortality following pulmonary transplantation and chronic lung allograft dysfunction. While it is well recognized that neutrophils are critical mediators of acute lung injury, processes that guide their entry into pulmonary tissue are not well understood. Here, we found that CCR2+ classical monocytes are necessary and sufficient for mediating extravasation of neutrophils into pulmonary tissue during ischemia-reperfusion injury following hilar clamping or lung transplantation. The classical monocytes were mobilized from the host spleen, and splenectomy attenuated the recruitment of classical monocytes as well as the entry of neutrophils into injured lung tissue, which was associated with improved graft function. Neutrophil extravasation was mediated by MyD88-dependent IL-1β production by graft-infiltrating classical monocytes, which downregulated the expression of the tight junction-associated protein ZO-2 in pulmonary vascular endothelial cells. Thus, we have uncovered a crucial role for classical monocytes, mobilized from the spleen, in mediating neutrophil extravasation, with potential implications for targeting of recipient classical monocytes to ameliorate pulmonary ischemia-reperfusion injury in the clinic.

Study Objective The presence of obstructive sleep apnea (OSA) in patients with cancer appears to be accompanied by poorer outcomes. However, the mechanisms underlying such association are unknown. Tumor infiltrating lymphocytes (TILs), including CD8+ T cells, function as cytotoxic T lymphocytes (CTLs) and mount immune responses to cancer by the release of cytolytic enzymes, including granzyme B (GzmB), perforin (Prf), and cytokines such as interferon (IFN)-γ. Methods Using established in vivo mouse models, we investigated CD8+ T cells and cancer stem cells (CSCs) in intermittent hypoxia (IH) and sleep fragmentation (SF) in the context of tumor environment. Results Both IH and SF promoted increased tumor growth and invasion toward adjacent tissues compared to controls. The number and frequency of GzmB-producing CD8+ T cells per milligram of tumor tissue was significantly reduced in IH-exposed mice with impaired cytolytic function in both the groups and correlated with tumor weight. We also found that Oct4+ and CD44+CD133+ expressing CSCs were considerably increased in IH and SF tumors, respectively. Conclusions Reductions in GzmB in intratumoral CD8+ T cells in combination with the changes in tumor microenvironment that maintain the ability of CSCs to self-renew and even confer this capability to the nonstem population are compatible with reduced immunosurveillance and adverse tumor outcomes in animal models of OSA.

"A major complication of factor replacement therapy for haemophilia is the development of anti-factor neutralizing antibodies (inhibitors). Here we show that liver gene therapy by lentiviral vectors (LVs) expressing factor IX (FIX) strongly reduces pre-existing anti-FIX antibodies and eradicates FIX inhibitors in haemophilia B mice. Concomitantly, plasma FIX levels and clotting activity rose to 50-100% of normal. The treatment was effective in 75% of treated mice. FIX-specific plasma cells (PCs) and memory B cells were reduced, likely because of memory B-cell depletion in response to constant exposure to high doses of FIX. Regulatory T cells displaying FIX-specific suppressive capacity were induced in gene therapy treated mice and controlled FIX-specific T helper cells. Gene therapy proved safer than a regimen mimicking immune tolerance induction (ITI) by repeated high-dose FIX protein administration, which induced severe anaphylactoid reactions in inhibitors-positive haemophilia B mice. Liver gene therapy can thus reverse pre-existing immunity, induce active tolerance to FIX and establish sustained FIX activity at therapeutic levels. These data position gene therapy as an attractive treatment option for inhibitors-positive haemophilic patients."

The use of lentiviral vectors (LV)s for in vivo gene therapy is an ideal platform for treating many types of disease. Since LVs can transduce a wide array of cells, support long-term gene expression, and be modified to enhance cell targeting, LVs are a powerful modality to deliver life-long therapeutic proteins. A major limitation facing the use of LVs for in vivo gene therapy is the induction of immune responses, which can reduce the transduction efficiency of LV, eliminate the transduced cells, and inhibit the effect of the therapeutic protein. LV strategies designed to restrict transgene expression to the liver to exploit its naturally tolerogenic properties have proven to significantly reduce the induction of pathogenic immune responses and increase therapeutic efficacy. In this review, we outline the immunological hurdles facing in vivo LV gene therapy and highlight the advantages and limitations of using liver-directed LV gene therapy.

Study objectives Sleep fragmentation (SF) is highly prevalent and has emerged as an important contributing factor to obesity and metabolic syndrome. We hypothesized that SF-induced increases in protein tyrosine phosphatase-1B (PTP-1B) expression and activity underlie increased food intake, inflammation, and leptin and insulin resistance. Methods Wild-type (WT) and ObR-PTP-1b-/- mice (Tg) were exposed to SF and control sleep (SC), and food intake was monitored. WT mice received a PTP-1B inhibitor (RO-7d; Tx) or vehicle (Veh). Upon completion of exposures, systemic insulin and leptin sensitivity tests were performed as well as assessment of visceral white adipose tissue (vWAT) insulin receptor sensitivity and macrophages (ATM) polarity. Results SF increased food intake in either untreated or Veh-treated WT mice. Leptin-induced hypothalamic STAT3 phosphorylation was decreased, PTP-1B activity was increased, and reduced insulin sensitivity emerged both systemic and in vWAT, with the latter displaying proinflammatory ATM polarity changes. All of the SF-induced effects were abrogated following PTP-1B inhibitor treatment and in Tg mice. Conclusions SF induces increased food intake, reduced leptin signaling in hypothalamus, systemic insulin resistance, and reduced vWAT insulin sensitivity and inflammation that are mediated by increased PTP-1B activity. Thus, PTP-1B may represent a viable therapeutic target in the context of SF-induced weight gain and metabolic dysfunction.

Obstructive sleep apnea (OSA) affects 8–10% of the population, is characterized by chronic intermittent hypoxia (CIH), and causally associates with cardiovascular morbidities. In CIH-exposed mice, closely mimicking the chronicity of human OSA, increased accumulation and proliferation of pro-inflammatory metabolic M1-like macrophages highly expressing CD36, emerged in aorta. Transcriptomic and MeDIP-seq approaches identified activation of pro-atherogenic pathways involving a complex interplay of histone modifications in functionally-relevant biological pathways, such as inflammation and oxidative stress in aorta macrophages. Discontinuation of CIH did not elicit significant improvements in aorta wall macrophage phenotype. However, CIH-induced aorta changes were absent in CD36 knockout mice, Our results provide mechanistic insights showing that CIH exposures during sleep in absence of concurrent pro-atherogenic settings (i.e., genetic propensity or dietary manipulation) lead to the recruitment of CD36(+)high macrophages to the aortic wall and trigger atherogenesis. Furthermore, long-term CIH-induced changes may not be reversible with usual OSA treatment.

International audience; Sickle cell anaemia (SCA) is the most frequent genetic haemoglobinopathy, which exhibits a highly variable clinical course characterized by hyper-coagulable and pro-inflammatory states, as well as endothelial dysfunction. Extracellular microvesicles are released into biological fluids and play a role in modifying the functional phenotype of target cells. We hypothesized that potential differences in plasma-derived extracellular microvesicles (EV) function and cargo from SCA patients may underlie divergent clinical trajectories. Plasma EV from SCA patients with mild, intermediate and severe clinical disease course were isolated, and primary endothelial cell cultures were exposed. Endothelial cell activation, monocyte adhesion, barrier disruption and exosome cargo (microRNA microarrays) were assessed. EV disrupted the endothelial barrier and induced expression of adhesion molecules and monocyte adhesion in a SCA severity-dependent manner compared to healthy children. Microarray approaches identified a restricted signature of exosomal microRNAs that readily distinguished severe from mild SCA, as well as from healthy children. The microRNA candidates were further validated using quantitative real time polymerase chain reaction assays, and revealed putative gene targets. Circulating exosomal microRNAs may play important roles in predicting the clinical course of SCA, and in delineation of individually tailored, mechanistically-based clinical treatment approaches of SCA patients in the near future.

// Abdelnaby Khalyfa 1 , Isaac Almendros 1, 4 , Alex Gileles-Hillel 1 , Mahzad Akbarpour 1 , Wojciech Trzepizur 1 , Babak Mokhlesi 2 , Lei Huang 3 , Jorge Andrade 3 , Ramon Farre 4 , David Gozal 1 1 Section of Pediatric Sleep Medicine, Department of Pediatrics, Pritzker School of Medicine, Biological Sciences Division, The University of Chicago, Chicago, IL, USA 2 Department of Medicine, Section of Pulmonary and Critical Care, Sleep Disorders Center, The University of Chicago, Chicago, IL, USA 3 Center for Research Informatics, The University of Chicago, Chicago, IL, USA 4 Unitat de Biofisica i Bioenginyeria, Facultat de Medicina, Universitat de Barcelona-Institut Investigacions Biomediques August Pi Sunyer-CIBER Enfermedades Respiratorias, Barcelona, Spain Correspondence to: David Gozal, email: dgozal@uchicago.edu Keywords: sleep fragmentation, obstructive sleep apnea, tumors, microenvironment, cancer biology Received: April 16, 2016 Accepted: June 30, 2016 Published: July 13, 2016 ABSTRACT Background: Chronic sleep fragmentation (SF) increases cancer aggressiveness in mice. Exosomes exhibit pleiotropic biological functions, including immune regulatory functions, antigen presentation, intracellular communication and inter-cellular transfer of RNA and proteins. We hypothesized that SF-induced alterations in biosynthesis and cargo of plasma exosomes may affect tumor cell properties. Results: SF-derived exosomes increased tumor cell proliferation (~13%), migration (~2.3-fold) and extravasation (~10%) when compared to exosomes from SC-exposed mice. Similarly, Pre exosomes from OSA patients significantly enhanced proliferation and migration of human adenocarcinoma cells compared to Post . SF-exosomal cargo revealed 3 discrete differentially expressed miRNAs, and exploration of potential mRNA targets in TC1 tumor cells uncovered 132 differentially expressed genes that encode for multiple cancer-related pathways. Methods: Plasma-derived exosomes from C57/B6 mice exposed to 6 wks of SF or sleep control (SC), and from adult human patients with obstructive sleep apnea (OSA) before ( Pre ) and after adherent treatment for 6 wks ( Post ) were co-cultured with mouse lung TC1 or human adenocarcinoma tumor cell lines, respectively. Proliferation, migration, invasion, endothelial barrier integrity and extravasation assays of tumor cells were performed. Plasma mouse exosomal miRNAs were profiled with arrays, and transcriptomic assessments of TC1 cells exposed to SF or SC exosomes were conducted to identify gene targets. Conclusions: Chronic SF induces alterations in exosomal miRNA cargo that alter the biological properties of TC1 lung tumor cells to enhance their proliferative, migratory and extravasation properties, and similar findings occur in OSA patients, in whom SF is a constitutive component of their sleep disorder. Thus, exosomes could participate, at least in part, in the adverse cancer outcomes observed in OSA.

Study Objective Increased visceral white adipose tissue (vWAT) mass results in infiltration of inflammatory macrophages that drive inflammation and insulin resistance. Patients with obstructive sleep apnea (OSA) suffer from increased prevalence of obesity, insulin resistance, and metabolic syndrome. Murine models of intermittent hypoxia (IH) mimicking moderate-severe OSA manifest insulin resistance following short-term IH. We examined in mice the effect of long-term IH on the inflammatory cellular changes within vWAT and the potential effect of normoxic recovery (IH-R). Methods Male C57BL/6J mice were subjected to IH for 20 weeks, and a subset was allowed to recover in room air (RA) for 6 or 12 weeks (IH-R). Stromal vascular fraction was isolated from epididymal vWAT and mesenteric vWAT depots, and single-cell suspensions were prepared for flow cytometry analyses, reactive oxygen species (ROS), and metabolic assays. Results IH reduced body weight and vWAT mass and IH-R resulted in catch-up weight and vWAT mass. IH-exposed vWAT exhibited increased macrophage counts (ATMs) that were only partially improved in IH-R. IH also caused a proinflammatory shift in ATMs (increased Ly6c(hi)(+) and CD36(+) ATMs). These changes were accompanied by increased vWAT insulin resistance with only partial improvements in IH-R. In addition, ATMs exhibited increased ROS production, altered metabolism, and changes in electron transport chain, which were only partially improved in IH-R. Conclusion Prolonged exposures to IH during the sleep period induce pronounced vWAT inflammation and insulin resistance despite concomitant vWAT mass reductions. These changes are only partially reversible after 3 months of normoxic recovery. Thus, long-lasting OSA may preclude complete reversibility of metabolic changes.