11ß-HSD1 determines the extent of muscle atrophy in a model of acute exacerbation of COPD.

Muscle atrophy is an extrapulmonary complication of acute exacerbations (AE) in chronic obstructive pulmonary disease (COPD). The endogenous production and therapeutic application of glucocorticoids (GCs) have been implicated as drivers of muscle loss in AE-COPD. The enzyme 11 ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates GCs and contributes toward GC-induced muscle wasting. To explore the potential of 11ßHSD1 inhibition to prevent muscle wasting here, the objective of this study was to ascertain the contribution of endogenous GC activation and amplification by 11ßHSD1 in skeletal muscle wasting during AE-COPD. Emphysema was induced by intratracheal (IT) instillation of elastase to model COPD in WT and 11ßHSD1/KO mice, followed by vehicle or IT-LPS administration to mimic AE. µCT scans were obtained prior and at study endpoint 48 h following IT-LPS, to assess emphysema development and muscle mass changes, respectively. Plasma cytokine and GC profiles were determined by ELISA. In vitro, myonuclear accretion and cellular response to plasma and GCs were determined in C2C12 and human primary myotubes. Muscle wasting was exacerbated in LPS-11ßHSD1/KO animals compared with WT controls. RT-qPCR and western blot analysis showed elevated catabolic and suppressed anabolic pathways in muscle of LPS-11ßHSD1/KO animals relative to WTs. Plasma corticosterone levels were higher in LPS-11ßHSD1/KO animals, whereas C2C12 myotubes treated with LPS-11ßHSD1/KO plasma or exogenous GCs displayed reduced myonuclear accretion relative to WT counterparts. This study reveals that 11ß-HSD1 inhibition aggravates muscle wasting in a model of AE-COPD, suggesting that therapeutic inhibition of 11ß-HSD1 may not be appropriate to prevent muscle wasting in this setting.

Evaluation of the Sensitivity of Metabolic Profiling by Rapid Evaporative Ionization Mass Spectrometry: Toward More Radical Oral Cavity Cancer Resections.

Radical resection for patients with oral cavity cancer remains challenging. Rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical vapors has been reported for real-time classification of normal and tumor tissues for numerous surgical applications. However, the infiltrative pattern of invasion of oral squamous cell carcinomas (OSCC) challenges the ability of REIMS to detect low amounts of tumor cells. We evaluate REIMS sensitivity to determine the minimal amount of detected tumors cells during oral cavity cancer surgery. A total of 11 OSCC patients were included in this study. The tissue classification based on 185 REIMS ex vivo metabolic profiles from five patients was compared to histopathology classification using multivariate analysis and leave-one-patient-out cross-validation. Vapors were analyzed in vivo by REIMS during four glossectomies. Complementary desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) was employed to map tissue heterogeneity on six oral cavity sections to support REIMS findings. REIMS sensitivity was assessed with a new cell-based assay consisting of mixtures of cell lines (tumor, myoblasts, keratinocytes). Our results depict REIMS classified tumor and soft tissues with 96.8% accuracy. In vivo REIMS generated intense mass spectrometric signals. REIMS detected 10% of tumor cells mixed with 90% myoblasts with 83% sensitivity and 82% specificity. DESI-MSI underlined distinct metabolic profiles of nerve features and a metabolic shift phosphatidylethanolamine PE(O-16:1/18:2))/cholesterol sulfate common to both mucosal maturation and OSCC differentiation. In conclusion, the assessment of tissue heterogeneity with DESI-MSI and REIMS sensitivity with cell mixtures characterized sensitive metabolic profiles toward in vivo tissue recognition during oral cavity cancer surgeries.

Pulmonary inflammation-induced alterations in key regulators of mitophagy and mitochondrial biogenesis in murine skeletal muscle.

BACKGROUND: Both mitophagy, a selective mechanism for clearance of mitochondria, and mitochondrial biogenesis are key processes determining mitochondrial content and oxidative capacity of the musculature. Abnormalities in these processes could therefore contribute to deterioration of peripheral muscle oxidative capacity as observed in e.g. chronic obstructive pulmonary disease. Although it has been suggested that inflammatory mediators can modulate both mitophagy and mitochondrial biogenesis, it is unknown whether acute pulmonary inflammation affects these processes in oxidative and glycolytic skeletal muscle in vivo. Therefore, we hypothesised that molecular signalling patterns of mitochondrial breakdown and biogenesis temporally shift towards increased breakdown and decreased biogenesis in the skeletal muscle of mice exposed to one single bolus of IT-LPS, as a model for acute lung injury and pulmonary inflammation. METHODS: We investigated multiple important constituents and molecular regulators of mitochondrial breakdown, biogenesis, dynamics, and mitochondrial content in skeletal muscle over time in a murine (FVB/N background) model of acute pulmonary- and systemic inflammation induced by a single bolus of intra-tracheally (IT)-instilled lipopolysaccharide (LPS). Moreover, we compared the expression of these constituents between gastrocnemius and soleus muscle. RESULTS: Both in soleus and gastrocnemius muscle, IT-LPS instillation resulted in molecular patterns indicative of activation of mitophagy. This coincided with modulation of mRNA transcript abundance of genes involved in mitochondrial fusion and fission as well as an initial decrease and subsequent recovery of transcript levels of key proteins involved in the molecular regulation of mitochondrial biogenesis. Moreover, no solid differences in markers for mitochondrial content were found. CONCLUSIONS: These data suggest that one bolus of IT-LPS results in a temporal modulation of mitochondrial clearance and biogenesis in both oxidative and glycolytic skeletal muscle, which is insufficient to result in a reduction of mitochondrial content.

Skeletal muscle unloading results in increased mitophagy and decreased mitochondrial biogenesis regulation.

INTRODUCTION: Physical inactivity significantly contributes to loss of muscle mass and performance in bed-bound patients. Loss of skeletal muscle mitochondrial content has been well-established in muscle unloading models, but the underlying molecular mechanism remains unclear. We hypothesized that apparent unloading-induced loss of muscle mitochondrial content is preceded by increased mitophagy- and decreased mitochondrial biogenesis-signaling during the early stages of unloading. METHODS: We analyzed a comprehensive set of molecular markers involved in mitochondrial-autophagy, -biogenesis, -dynamics, and -content, in the gastrocnemius muscle of C57BL/6J mice subjected to 0- and 3-days hind limb suspension, and in biopsies from human vastus lateralis muscle obtained before and after 7 days of one-leg immobilization. RESULTS: In both mice and men, short-term skeletal muscle unloading results in molecular marker patterns indicative of increased receptor-mediated mitophagy and decreased mitochondrial biogenesis regulation, before apparent loss of mitochondrial content. DISCUSSION: These results emphasize the early-onset of skeletal muscle disuse-induced mitochondrial remodeling.

Pulmonary inflammation-induced loss and subsequent recovery of skeletal muscle mass require functional poly-ubiquitin conjugation.

BACKGROUND: Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation. METHODS: Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice. RESULTS: Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass. CONCLUSION: Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.

Regulation of muscle atrophy by microRNAs: 'AtromiRs' as potential target in cachexia.

PURPOSE OF REVIEW: To provide an overview and describe the mode of action of miRNAs recently implicated in muscle atrophy, and discuss the challenges to explore their potential as putative therapeutic targets in cachexia. RECENT FINDINGS: Recent work showed differentially expressed miRNAs in skeletal muscle of patients with cachexia-associated diseases. Studies using experimental models revealed miRNA regulation of the anabolic IGF-1 and catabolic TGF- ß/myostatin pathways, and downstream protein synthesis and proteolysis signaling in control of muscle mass. SUMMARY: Cachexia is a complex metabolic condition associated with progressive body weight loss, wasting of skeletal muscle mass and decrease in muscle strength. MiRNAs play a central role in post-transcriptional gene regulation by targeting mRNAs, thereby coordinating and fine-tuning many cellular processes. MiRNA expression profiling studies of muscle biopsies have revealed differentially expressed miRNAs in patients with low muscle mass or cachexia. Evaluation in experimental models has revealed muscle atrophy, inhibition of protein synthesis and activation of proteolysis in response to modulation of specific miRNAs, termed 'atromiRs' in this review. These exciting findings call for further studies aimed at exploring the conservation of differentially expressed miRNAs across diseases accompanied by cachexia, identification of miRNA clusters and targets involved in muscle atrophy, and probing whether these miRNAs might be potential therapeutic targets for cachexia.

Differential regulation of muscle protein turnover in response to emphysema and acute pulmonary inflammation.

BACKGROUND: Exacerbations in COPD are often accompanied by pulmonary and systemic inflammation, and associated with increased susceptibility to and prevalence of weight loss and muscle wasting. Muscle mass loss during disease exacerbations may contribute to emphysema-associated muscle atrophy. However, whether pulmonary inflammation in presence of emphysema differentially affects skeletal muscle, including protein synthesis and degradation signaling pathways has not previously been addressed. The aims of this study were to 1) develop a mouse model of disease exacerbation-associated muscle wasting, 2) evaluate whether emphysema and muscle wasting can be monitored non-invasively and 3) assess alterations in muscle protein turnover regulation. METHODS: Emphysema was induced by three, weekly intra-tracheal (IT) elastase (E) or vehicle control (vc) instillations, followed by one single IT-LPS bolus (L) or vc instillation to mimic pulmonary inflammation-driven disease exacerbation. Consequently, four experimental groups were defined: vc/vc ('C'), E/vc ('E'), vc/LPS ('L'), E/LPS ('E + L'). Using micro cone-beam CT-scans, emphysema development and muscle mass changes were monitored, and correlated to muscle weight 48 h after LPS instillation. Protein turnover signaling was assessed in muscle tissue collected 24 h post LPS instillation. RESULTS: Micro-CT imaging correlated strongly with established invasive measurements of emphysema and muscle atrophy. Pulmonary inflammation following LPS instillation developed irrespective of emphysema and body and muscle weight were similarly reduced in the 'L' and 'E + L' groups. Accordingly, mRNA and protein expression levels of genes of the ubiquitin-proteasome pathway (UPS) and the autophagy-lysosomal pathway (ALP) were upregulated in skeletal muscle following IT-LPS ('L' and 'E + L'). In contrast, mTOR signaling, which controls ALP and protein synthesis, was reduced by pulmonary inflammation ('L' and 'E + L') as well as emphysema as a single insult ('E') compared to control. CONCLUSION: Changes in lung tissue density and muscle mass can be monitored non-invasively to evaluate emphysema and muscle atrophy longitudinally. Acute loss of muscle mass evoked by pulmonary inflammation is similar in control and emphysematous mice. Although muscle atrophy cues in response to pulmonary inflammation are not altered by emphysema, emphysema itself affects protein synthesis and ALP signaling, which may interfere with muscle mass recovery and impair maintenance of muscle mass in emphysema.

Muscle-specific GSK-3ß ablation accelerates regeneration of disuse-atrophied skeletal muscle.

Muscle wasting impairs physical performance, increases mortality and reduces medical intervention efficacy in chronic diseases and cancer. Developing proficient intervention strategies requires improved understanding of the molecular mechanisms governing muscle mass wasting and recovery. Involvement of muscle protein- and myonuclear turnover during recovery from muscle atrophy has received limited attention. The insulin-like growth factor (IGF)-I signaling pathway has been implicated in muscle mass regulation. As glycogen synthase kinase 3 (GSK-3) is inhibited by IGF-I signaling, we hypothesized that muscle-specific GSK-3ß deletion facilitates the recovery of disuse-atrophied skeletal muscle. Wild-type mice and mice lacking muscle GSK-3ß (MGSK-3ß KO) were subjected to a hindlimb suspension model of reversible disuse-induced muscle atrophy and followed during recovery. Indices of muscle mass, protein synthesis and proteolysis, and post-natal myogenesis which contribute to myonuclear accretion, were monitored during the reloading of atrophied muscle. Early muscle mass recovery occurred more rapidly in MGSK-3ß KO muscle. Reloading-associated changes in muscle protein turnover were not affected by GSK-3ß ablation. However, coherent effects were observed in the extent and kinetics of satellite cell activation, proliferation and myogenic differentiation observed during reloading, suggestive of increased myonuclear accretion in regenerating skeletal muscle lacking GSK-3ß. This study demonstrates that muscle mass recovery and post-natal myogenesis from disuse-atrophy are accelerated in the absence of GSK-3ß.

Targeted nutritional intervention attenuates experimental lung cancer cachexia.

BACKGROUND: Cachexia, a syndrome with high prevalence in non-small cell lung cancer patients, impairs quality of life and reduces tolerance and responsiveness to cancer therapy resulting in decreased survival. Optimal nutritional care is pivotal in the treatment of cachexia and a recommended cornerstone of multimodal therapy. Here, we investigated the therapeutic effect of an intervention diet consisting of a specific combination of high protein, leucine, fish oil, vitamin D, galacto-oligosaccharides, and fructo-oligosaccharides on the development and progression of cachexia in an orthotopic lung cancer mouse model. METHODS: Eleven-week-old male 129S2/Sv mice were orthotopically implanted with 344P lung epithelial tumour cells or vehicle (control). Seven days post-implantation tumour-bearing (TB) mice were allocated to either intervention- or isocaloric control diet. Cachexia was defined as 5 days of consecutive body weight loss, after which mice were euthanized for tissue analyses. RESULTS: TB mice developed cachexia accompanied by significant loss of skeletal muscle mass and epididymal fat mass compared with sham operated mice. The cachectic endpoint was significantly delayed (46.0 ± 15.2 vs. 34.7 ± 11.4 days), and the amount (-1.57 ± 0.62 vs. -2.13 ± 0.57 g) and progression (-0.26 ± 0.14 vs. -0.39 ± 0.11 g/day) of body weight loss were significantly reduced by the intervention compared with control diet. Moreover, systemic inflammation (pentraxin-2 plasma levels) and alterations in molecular markers for proteolysis and protein synthesis, indicative of muscle atrophy signalling in TB-mice, were suppressed in skeletal muscle by the intervention diet. CONCLUSIONS: Together, these data demonstrate the potential of this multinutrient intervention, targeting multiple components of cachexia, as integral part of lung cancer management.

Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy.

The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food intake in response to hypoxia. Mice were subjected to normoxic, hypoxic (8% oxygen), or pair-fed conditions for 2, 4, and 21 days. Cell-autonomous effects of hypoxia on skeletal muscle were also assessed in differentiated C2C12 myotubes. Hypoxia induced an initial rapid loss of body and muscle weight, which remained decreased during chronic hypoxia and could only in part be explained by the hypoxia-induced reduction of food intake (semistarvation). Regulatory steps of protein synthesis (unfolded protein response and mammal target of rapamycin signaling) remained active in response to acute and sustained hypoxia but not to semistarvation. Activation of regulatory signals for protein degradation, including increased expression of Murf1, Atrogin-1, Bnip3, and Map1lc3b mRNAs, was observed in response to acute hypoxia and to a lesser extent following semistarvation. Conversely, the sustained elevation of Atrogin-1, Bnip3, and Map1lc3b mRNAs and the increased activity of their upstream transcriptional regulator Forkhead box O1 were specific to chronic hypoxia because they were not observed in response to reduced food intake. In conclusion, altered regulation of protein turnover during hypoxia-induced muscle atrophy resulted from an interaction of semistarvation and a hypoxia-specific component. The finding that food restriction but not hypoxia-induced semistarvation inhibited regulatory steps in protein synthesis suggests a hypoxia-specific impairment of the coordination between protein-synthesis signaling and protein-degradation signaling in skeletal muscle.

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy. METHODS: Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors. RESULTS: Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors. CONCLUSIONS: In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.

An optimization and refinement of the whole-gut transit assay in mice.

BACKGROUND: Gastrointestinal motility measurements in mice are currently performed under suboptimal conditions, as these nocturnal animals are measured during light conditions. In addition, other stressors, like individual housing, placement in a new cage during observation, and lack of bedding and cage enrichment cause animal discomfort and might contribute to higher variability. Here we aimed to develop a refined method of the widely-used whole-gut transit assay. METHODS: Wildtype mice (N = 24) were subjected to the standard or refined whole-gut transit assay, either with or without a standardized slowing in gastrointestinal motility induced by loperamide. The standard assay consisted of a gavage with carmine red, observation during the light period and individual housing in a new cage without cage enrichment. For the refined whole-gut transit assay, mice were gavaged with UV-fluorescent DETEX®, observed during the dark period, while pairwise housed in their home cage with cage enrichment. Time until excretion of the first colored fecal pellet was assessed, and pellets were collected to assess number, weight, and water content. KEY RESULTS: The DETEX®-containing pellets were UV-detectable, allowing to measure the mice in their active period in the dark. The refined method caused less variation (20.8% and 16.0%) compared to the standard method (29.0% and 21.7%). Fecal pellet number, weight, and water content was significantly different between the standard and refined method. CONCLUSIONS & INFERENCES: This refined whole-gut transit assay provides a reliable approach to measure whole-gut transit time in mice in a more physiological context, with reduced variability compared to the standard method.

The Modulatory Effects of Fatty Acids on Cancer Progression.

Cancer is the second leading cause of death worldwide and the global cancer burden rises rapidly. The risk factors for cancer development can often be attributed to lifestyle factors, of which an unhealthy diet is a major contributor. Dietary fat is an important macronutrient and therefore a crucial part of a well-balanced and healthy diet, but it is still unclear which specific fatty acids contribute to a healthy and well-balanced diet in the context of cancer risk and prognosis. In this review, we describe epidemiological evidence on the associations between the intake of different classes of fatty acids and the risk of developing cancer, and we provide preclinical evidence on how specific fatty acids can act on tumor cells, thereby modulating tumor progression and metastasis. Moreover, the pro- and anti-inflammatory effects of each of the different groups of fatty acids will be discussed specifically in the context of inflammation-induced cancer progression and we will highlight challenges as well as opportunities for successful application of fatty acid tailored nutritional interventions in the clinic.

A novel orthotopic mouse model replicates human lung cancer cachexia.

INTRODUCTION: Cancer cachexia, highly prevalent in lung cancer, is a debilitating syndrome characterized by involuntary loss of skeletal muscle mass and is associated with poor clinical outcome, decreased survival and negative impact on tumour therapy. Various lung tumour-bearing animal models have been used to explore underlying mechanisms of cancer cachexia. However, these models do not simulate anatomical and immunological features key to lung cancer and associated muscle wasting. Overcoming these shortcomings is essential to translate experimental findings into the clinic. We therefore evaluated whether a syngeneic, orthotopic lung cancer mouse model replicates systemic and muscle-specific alterations associated with human lung cancer cachexia. METHODS: Immune competent, 11 weeks old male 129S2/Sv mice, were randomly allocated to either (1) sham control group or (2) tumour-bearing group. Syngeneic lung epithelium-derived adenocarcinoma cells (K-rasG12D ; p53R172HΔG ) were inoculated intrapulmonary into the left lung lobe of the mice. Body weight and food intake were measured daily. At baseline and weekly after surgery, grip strength was measured and tumour growth and muscle volume were assessed using micro cone beam CT imaging. After reaching predefined surrogate survival endpoint, animals were euthanized, and skeletal muscles of the lower hind limbs were collected for biochemical analysis. RESULTS: Two-third of the tumour-bearing mice developed cachexia based on predefined criteria. Final body weight (-13.7 ± 5.7%; P < 0.01), muscle mass (-13.8 ± 8.1%; P < 0.01) and muscle strength (-25.5 ± 10.5%; P < 0.001) were reduced in cachectic mice compared with sham controls and median survival time post-surgery was 33.5 days until humane endpoint. Markers for proteolysis, both ubiquitin proteasome system (Fbxo32 and Trim63) and autophagy-lysosomal pathway (Gabarapl1 and Bnip3), were significantly upregulated, whereas markers for protein synthesis (relative phosphorylation of Akt, S6 and 4E-BP1) were significantly decreased in the skeletal muscle of cachectic mice compared with control. The cachectic mice exhibited increased pentraxin-2 (P < 0.001) and CXCL1/KC (P < 0.01) expression levels in blood plasma and increased mRNA expression of IκBα (P < 0.05) in skeletal muscle, indicative for the presence of systemic inflammation. Strikingly, RNA sequencing, pathway enrichment and miRNA expression analyses of mouse skeletal muscle strongly mirrored alterations observed in muscle biopsies of patients with lung cancer cachexia. CONCLUSIONS: We developed an orthotopic model of lung cancer cachexia in immune competent mice. Because this model simulates key aspects specific to cachexia in lung cancer patients, it is highly suitable to further investigate the underlying mechanisms of lung cancer cachexia and to test the efficacy of novel intervention strategies.

Precision Medicine for More Oxygen (P4O2)-Study Design and First Results of the Long COVID-19 Extension.

Introduction: The coronavirus disease 2019 (COVID-19) pandemic has led to the death of almost 7 million people, however, with a cumulative incidence of 0.76 billion, most people survive COVID-19. Several studies indicate that the acute phase of COVID-19 may be followed by persistent symptoms including fatigue, dyspnea, headache, musculoskeletal symptoms, and pulmonary functional-and radiological abnormalities. However, the impact of COVID-19 on long-term health outcomes remains to be elucidated. Aims: The Precision Medicine for more Oxygen (P4O2) consortium COVID-19 extension aims to identify long COVID patients that are at risk for developing chronic lung disease and furthermore, to identify treatable traits and innovative personalized therapeutic strategies for prevention and treatment. This study aims to describe the study design and first results of the P4O2 COVID-19 cohort. Methods: The P4O2 COVID-19 study is a prospective multicenter cohort study that includes nested personalized counseling intervention trial. Patients, aged 40-65 years, were recruited from outpatient post-COVID clinics from five hospitals in The Netherlands. During study visits at 3-6 and 12-18 months post-COVID-19, data from medical records, pulmonary function tests, chest computed tomography scans and biological samples were collected and questionnaires were administered. Furthermore, exposome data was collected at the patient's home and state-of-the-art imaging techniques as well as multi-omics analyses will be performed on collected data. Results: 95 long COVID patients were enrolled between May 2021 and September 2022. The current study showed persistence of clinical symptoms and signs of pulmonary function test/radiological abnormalities in post-COVID patients at 3-6 months post-COVID. The most commonly reported symptoms included respiratory symptoms (78.9%), neurological symptoms (68.4%) and fatigue (67.4%). Female sex and infection with the Delta, compared with the Beta, SARS-CoV-2 variant were significantly associated with more persisting symptom categories. Conclusions: The P4O2 COVID-19 study contributes to our understanding of the long-term health impacts of COVID-19. Furthermore, P4O2 COVID-19 can lead to the identification of different phenotypes of long COVID patients, for example those that are at risk for developing chronic lung disease. Understanding the mechanisms behind the different phenotypes and identifying these patients at an early stage can help to develop and optimize prevention and treatment strategies.

NF-κB activation is required for the transition of pulmonary inflammation to muscle atrophy.

Disease exacerbations and muscle wasting comprise negative prognostic factors of chronic obstructive pulmonary disease (COPD). Transient systemic inflammation and malnutrition have been implicated in skeletal muscle wasting after acute exacerbations of COPD. However, the interactions between systemic inflammation and malnutrition in their contributions to muscle atrophy, as well as the molecular basis underlying the transition of systemic inflammation to muscle atrophy, remain unresolved. Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS to model acute disease exacerbation. Systemic inflammation, nutritional intake, and body and muscle weights were determined. Muscle inflammatory signaling and atrophy signaling were examined, and the effect of the muscle-specific inactivation of NF-κB on muscle atrophy was assessed in genetically modified mice. The intratracheal LPS instillation was followed by markedly elevated circulating cytokine concentrations and NF-κB activation in extrapulmonary tissues, including skeletal muscle. The administration of intratracheal LPS increased the expression of muscle E3 ubiquitin ligases, which govern muscle proteolysis, in particular MuRF1, and caused a rapid loss of muscle mass. Reduced food intake only partly accounted for the observed muscle atrophy, and did not activate NF-κB in muscle. Rather, plasma transfer experiments revealed the presence of NF-κB-signaling and atrophy-signaling properties in the circulation of intratracheal LPS-treated mice. The genetic inhibition of muscle NF-κB activity suppressed intratracheal LPS-induced MuRF1 expression and resulted in a significant sparing of muscle tissue. Systemic inflammation and malnutrition contribute to the muscle wasting induced by acute pulmonary inflammation via distinct mechanisms, and muscle NF-κB activation is required for the transition from inflammatory to muscle atrophy signaling.

Palmitate-induced skeletal muscle insulin resistance does not require NF-κB activation.

Palmitate activates the NF-κB pathway, and induces accumulation of lipid metabolites and insulin resistance in skeletal muscle cells. Little information is available whether and how these processes are causally related. Therefore, the objectives were to investigate whether intra-cellular lipid metabolites are involved in FA-induced NF-κB activation and/or insulin resistance in skeletal muscle and to investigate whether FA-induced insulin resistance and NF-κB activation are causally related. Inhibiting DGAT or CPT-1 by using, respectively, amidepsine or etomoxir increased DAG accumulation and sensitized myotubes to palmitate-induced insulin resistance. While co-incubation of palmitate with etomoxir increased NF-κB transactivation, co-incubation with amidepsine did not, indicating that DAG accumulation is associated with insulin resistance but not with NF-κB activation. Furthermore, pharmacological or genetic inhibition of the NF-κB pathway could not prevent palmitate-induced insulin resistance. In conclusion, we have demonstrated that activation of the NF-κB pathway is not required for palmitate-induced insulin resistance in skeletal muscle cells.

Fatty Acids as a Tool to Boost Cancer Immunotherapy Efficacy.

Although immunotherapy represents one of the most potent therapeutic anti-cancer approaches, only a limited number of patients shows clinical benefit. Recent evidence suggests that patients' nutritional status plays a major role in immunotherapy outcome. Fatty acids are essential in a balanced diet and well-known to influence the immune response. Moreover, short-chain fatty acids (SCFAs) show beneficial effects in metabolic disorders as well as in cancer and polyunsaturated fatty acids (PUFAs) contribute to body weight and fat free mass preservation in cancer patients. In line with these data, several studies imply a role for SCFAs and PUFAs in boosting the outcome of immunotherapy. In this review, we specifically focus on mechanistic data showing that SCFAs modulate the immunogenicity of tumor cells and we discuss the direct effects of SCFAs and PUFAs on the immune system in the context of cancer. We provide preclinical and clinical evidence indicating that SCFAs and PUFAs may have the potential to boost immunotherapy efficacy. Finally, we describe the challenges and address opportunities for successful application of nutritional interventions focusing on SCFAs and PUFAs to increase the therapeutic potential of immunotherapeutic approaches for cancer.

Physical exercise at the crossroad between muscle wasting and the immune system: implications for lung cancer cachexia.

Cachexia is a syndrome characterized by involuntary weight loss and wasting of skeletal muscle mass. It is associated with worse overall survival and quality of life. The cancer-induced systemic inflammation and the consequent host derived catabolic stimuli, trigger cachexia by inhibiting muscle protein synthesis and enhancing muscle catabolism. The muscle itself may further promote chronic inflammation, introducing a vicious catabolic circle. Nutritional support alone plays a limited role in the treatment of cancer cachexia and should be combined with other interventions. Physical exercise lowers systemic inflammation and promotes muscle anabolism. It also attenuates the age-related physical decline in elderly and it might counteract the muscle wasting induced by the cancer cachexia syndrome. This review describes how cancer-induced systemic inflammation promotes muscle wasting and whether physical exercise may represent a suitable treatment for cancer-induced cachexia, particularly in patients with non-small cell lung cancer. We summarized pre-clinical and clinical studies investigating whether physical exercise would improve muscle performance and whether this improvement would translate in a clinically meaningful benefit for patients with cancer, in terms of survival and quality of life. Moreover, this review describes the results of studies investigating the interplay between physical exercise and the immune system, including the role of the intestinal microbiota.

Effects of a nutritional intervention on impaired behavior and cognitive function in an emphysematous murine model of COPD with endotoxin-induced lung inflammation.

One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.