Neurofibromin 1 controls metabolic balance and Notch-dependent quiescence of murine juvenile myogenic progenitors.

Patients affected by neurofibromatosis type 1 (NF1) frequently show muscle weakness with unknown etiology. Here we show that, in mice, Neurofibromin 1 (Nf1) is not required in muscle fibers, but specifically in early postnatal myogenic progenitors (MPs), where Nf1 loss led to cell cycle exit and differentiation blockade, depleting the MP pool resulting in reduced myonuclear accretion as well as reduced muscle stem cell numbers. This was caused by precocious induction of stem cell quiescence coupled to metabolic reprogramming of MPs impinging on glycolytic shutdown, which was conserved in muscle fibers. We show that a Mek/Erk/NOS pathway hypersensitizes Nf1-deficient MPs to Notch signaling, consequently, early postnatal Notch pathway inhibition ameliorated premature quiescence, metabolic reprogramming and muscle growth. This reveals an unexpected role of Ras/Mek/Erk signaling supporting postnatal MP quiescence in concert with Notch signaling, which is controlled by Nf1 safeguarding coordinated muscle growth and muscle stem cell pool establishment. Furthermore, our data suggest transmission of metabolic reprogramming across cellular differentiation, affecting fiber metabolism and function in NF1.

Activation of gp130 signaling in T cells drives TH17-mediated multi-organ autoimmunity.

The IL-6-gp130-STAT3 signaling axis is a major regulator of inflammation. Activating mutations in the gene encoding gp130 and germline gain-of-function mutations in STAT3 (STAT3GOF) are associated with multi-organ autoimmunity, severe morbidity, and adverse prognosis. To dissect crucial cellular subsets and disease biology involved in activated gp130 signaling, the gp130-JAK-STAT3 axis was constitutively activated using a transgene, L-gp130, specifically targeted to T cells. Activating gp130 signaling in T cells in vivo resulted in fatal, early onset, multi-organ autoimmunity in mice that resembled human STAT3GOF disease. Female mice had more rapid disease progression than male mice. On a cellular level, gp130 signaling induced the activation and effector cell differentiation of T cells, promoted the expansion of T helper type 17 (TH17) cells, and impaired the activity of regulatory T cells. Transcriptomic profiling of CD4+ and CD8+ T cells from these mice revealed commonly dysregulated genes and a gene signature that, when applied to human transcriptomic data, improved the segregation of patients with transcriptionally diverse STAT3GOF mutations from healthy controls. The findings demonstrate that increased gp130-STAT3 signaling leads to TH17-driven autoimmunity that phenotypically resembles human STAT3GOF disease.

Eosinophils promote pulmonary matrix destruction and emphysema via Cathepsin L.

Patients with chronic obstructive pulmonary disease (COPD) who exhibit elevated blood eosinophil levels often experience worsened lung function and more severe emphysema. This implies the potential involvement of eosinophils in the development of emphysema. However, the precise mechanisms underlying the development of eosinophil-mediated emphysema remain unclear. In this study, we employed single-cell RNA sequencing to identify eosinophil subgroups in mouse models of asthma and emphysema, followed by functional analyses of these subgroups. Assessment of accumulated eosinophils unveiled distinct transcriptomes in the lungs of mice with elastase-induced emphysema and ovalbumin-induced asthma. Depletion of eosinophils through the use of anti-interleukin-5 antibodies ameliorated elastase-induced emphysema. A particularly noteworthy discovery is that eosinophil-derived cathepsin L contributed to the degradation of the extracellular matrix, thereby leading to emphysema in pulmonary tissue. Inhibition of cathepsin L resulted in a reduction of elastase-induced emphysema in a mouse model. Importantly, eosinophil levels correlated positively with serum cathepsin L levels, which were higher in emphysema patients than those without emphysema. Expression of cathepsin L in eosinophils demonstrated a direct association with lung emphysema in COPD patients. Collectively, these findings underscore the significant role of eosinophil-derived cathepsin L in extracellular matrix degradation and remodeling, and its relevance to emphysema in COPD patients. Consequently, targeting eosinophil-derived cathepsin L could potentially offer a therapeutic avenue for emphysema patients. Further investigations are warranted to explore therapeutic strategies targeting cathepsin L in emphysema patients.

Analysis of Circulating Food Antigen-Specific T-Cells in Celiac Disease and Inflammatory Bowel Disease.

To demonstrate and analyze the specific T-cell response following barrier disruption and antigen translocation, circulating food antigen-specific effector T-cells isolated from peripheral blood were analyzed in patients suffering from celiac disease (CeD) as well as inflammatory bowel disease (IBD). We applied the antigen-reactive T-cell enrichment (ARTE) technique allowing for phenotypical and functional flow cytometric analyses of rare nutritional antigen-specific T-cells, including the celiac disease-causing gliadin (gluten). For CeD, patient groups, including treatment-refractory cases, differ significantly from healthy controls. Even symptom-free patients on a gluten-free diet were distinguishable from healthy controls, without being previously challenged with gluten. Moreover, frequency and phenotype of nutritional antigen-specific T-cells of IBD patients directly correlated to the presence of small intestinal inflammation. Specifically, the frequency of antigen specific T-cells as well as pro-inflammatory cytokines was increased in patients with active CeD or Crohn's disease, respectively. These results suggest active small intestinal inflammation as key for the development of a peripheral food antigen-specific T-cell response in Crohn's disease and celiac disease.

Expression and function of α4ß7 integrin predict the success of vedolizumab treatment in inflammatory bowel disease.

Inflammatory bowel diseases are medically intractable and require constant therapy in many cases. While a growing number of biologicals and small molecules is available for treatment, a substantial portion of patients experiences primary non-response to these compounds and head-to-head evidence for therapy selection is scarce. Thus, approaches to predict treatment success in individual patients are a huge unmet need. We had previously suggested that the expression and function of α4ß7 integrin on T cells in the peripheral blood correlate to outcomes of therapy with the anti-α4ß7 integrin antibody vedolizumab. Here, we conducted a translational multicenter trial to prospectively evaluate this hypothesis. In a cohort of 89 patients with inflammatory bowel disease undergoing regular therapy with vedolizumab, lower baseline expression of α4ß7 was associated with short-term clinical response. Consistently, low α4ß7 expression in patients achieving remission predicted sustained remission in week 30. Moreover, high dynamic adhesion of CD4+ T cells to MAdCAM-1 and high reduction of adhesion by vedolizumab in vitro at baseline were associated with clinical remission. These data substantiate the potential of α4ß7 integrin function and expression to forecast outcomes of vedolizumab therapy. Further translational efforts are necessary to improve the performance of the assays and to implement the concept in clinical practice.

Effects of caloric restriction on the gut microbiome are linked with immune senescence.

BACKGROUND: Caloric restriction can delay the development of metabolic diseases ranging from insulin resistance to type 2 diabetes and is linked to both changes in the composition and metabolic function of the gut microbiota and immunological consequences. However, the interaction between dietary intake, the microbiome, and the immune system remains poorly described. RESULTS: We transplanted the gut microbiota from an obese female before (AdLib) and after (CalRes) an 8-week very-low-calorie diet (800 kcal/day) into germ-free mice. We used 16S rRNA sequencing to evaluate taxa with differential abundance between the AdLib- and CalRes-microbiota recipients and single-cell multidimensional mass cytometry to define immune signatures in murine colon, liver, and spleen. Recipients of the CalRes sample exhibited overall higher alpha diversity and restructuring of the gut microbiota with decreased abundance of several microbial taxa (e.g., Clostridium ramosum, Hungatella hathewayi, Alistipi obesi). Transplantation of CalRes-microbiota into mice decreased their body fat accumulation and improved glucose tolerance compared to AdLib-microbiota recipients. Finally, the CalRes-associated microbiota reduced the levels of intestinal effector memory CD8+ T cells, intestinal memory B cells, and hepatic effector memory CD4+ and CD8+ T cells. CONCLUSION: Caloric restriction shapes the gut microbiome which can improve metabolic health and may induce a shift towards the naïve T and B cell compartment and, thus, delay immune senescence. Understanding the role of the gut microbiome as mediator of beneficial effects of low calorie diets on inflammation and metabolism may enhance the development of new therapeutic treatment options for metabolic diseases. TRIAL REGISTRATION: NCT01105143 , "Effects of negative energy balance on muscle mass regulation," registered 16 April 2010. Video Abstract.

Fatty Acid Metabolism in Myeloid-Derived Suppressor Cells and Tumor-Associated Macrophages: Key Factor in Cancer Immune Evasion.

The tumor microenvironment (TME) comprises various cell types, soluble factors, viz, metabolites or cytokines, which together play in promoting tumor metastasis. Tumor infiltrating immune cells play an important role against cancer, and metabolic switching in immune cells has been shown to affect activation, differentiation, and polarization from tumor suppressive into immune suppressive phenotypes. Macrophages represent one of the major immune infiltrates into TME. Blood monocyte-derived macrophages and myeloid derived suppressor cells (MDSCs) infiltrating into the TME potentiate hostile tumor progression by polarizing into immunosuppressive tumor-associated macrophages (TAMs). Recent studies in the field of immunometabolism focus on metabolic reprogramming at the TME in polarizing tumor-associated macrophages (TAMs). Lipid droplets (LD), detected in almost every eukaryotic cell type, represent the major source for intra-cellular fatty acids. Previously, LDs were mainly described as storage sites for fatty acids. However, LDs are now recognized to play an integral role in cellular signaling and consequently in inflammation and metabolism-mediated phenotypical changes in immune cells. In recent years, the role of LD dependent metabolism in macrophage functionality and phenotype has been being investigated. In this review article, we discuss fatty acids stored in LDs, their role in modulating metabolism of tumor-infiltrating immune cells and, therefore, in shaping the cancer progression.

Pleiotropic Effects of the Protease-Activated Receptor 1 (PAR1) Inhibitor, Vorapaxar, on Atherosclerosis and Vascular Inflammation.

BACKGROUND: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. METHODS AND RESULTS: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E-knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. CONCLUSIONS: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.

Programmed PPAR-α downregulation induces inflammaging by suppressing fatty acid catabolism in monocytes.

Inflammaging is associated with an increased risk of chronic disease. Monocytes are the principal immune cells for the production of inflammatory cytokines and contribute to inflammaging in the elderly. However, the underlying mechanisms remain largely unknown. Here, we found that monocytes from aged individuals contained high levels of lipid droplets (LDs), and this increase was correlated with impaired fatty acid oxidation. Downregulated peroxisome proliferator-activated receptor (PPAR)-α may be responsible for the pro-inflammatory phenotype of monocytes in aged individuals, as it was positively correlated with LD accumulation and increasing TNF-α concentration. Interestingly, interventions that result in PPAR-α upregulation, such as fenofibrate treatment, TNF-α neutralization, or calorie restriction, reversed the effect of aging on monocytes. Thus the downregulation of PPAR-α and LD levels in monocytes represents a novel biomarker for inflammaging. Furthermore, PPAR-α activation in the elderly may also alleviate long-term inflammaging, preventing the development of life-limiting chronic diseases.

Histone Deacetylases in the Inflamed Intestinal Epithelium-Promises of New Therapeutic Strategies.

The intestinal epithelium is a complex, dynamic barrier that separates luminal contents from the immune compartment while mediating nutrient absorption and controlled passage of antigens to convey oral tolerance. A compromised epithelial barrier often leads to inflammation because immune cells in the lamina propria come into direct contact with luminal antigens. Defects in epithelial cell function were also shown to be involved in the etiology of inflammatory bowel diseases. These are severe, chronically relapsing inflammatory conditions of the gastrointestinal tract that also increase the risk of developing colorectal cancer. Despite major efforts of the scientific community, the precise causes and drivers of these conditions still remain largely obscured impeding the development of a permanent cure. Current therapeutic approaches mostly focus on alleviating symptoms by targeting immune cell signaling. The protein family of histone deacetylases (HDACs) has gained increasing attention over the last years, as HDAC inhibitors were shown to be potent tumor cell suppressors and also alleviate morbid inflammatory responses. Recent research continuously identifies new roles for specific HDACs suggesting that HDACs influence the cell signaling network from many different angles. This makes HDACs very interesting targets for therapeutic approaches but predicting effects after system manipulations can be difficult. In this review, we want to provide a comprehensive overview of current knowledge about the individual roles of HDACs in the intestinal epithelium to evaluate their therapeutic potential for inflammatory conditions of the gut.

Fatty Acid-Driven Polarization of Suppressive Bone Marrow-Derived Macrophages Including Metabolic and Functional Analysis.

Macrophages represent not only the first line of defense against pathogens and are the main drivers of inflammation but are also involved in the initiation, immune evasion as well as metastasis of tumors. Therefore, it has been suggested that diminishing the immune regulatory function of macrophages would support the natural immune surveillance or antitumor therapies, respectively. However, the plasticity of macrophages represents an obstacle in understanding and manipulating the role of macrophages in tumor tissue or the tumor microenvironment. Here, we describe a protocol to differentiate macrophages, based on changing their metabolic environment, from bone marrow precursors to tumor-associated macrophage-like cells of an immune suppressive phenotype. Based on these protocols, the inhibitory functional phenotype of macrophages can be manipulated and therefore further analyzed as described, by interrupting metabolic pathways.

Recognition of food antigens by the mucosal and systemic immune system: Consequences for intestinal development and homeostasis.

The impact of nutrition on systemic and intestinal immune responses remains controversially discussed and yet not fully understood. The majority of studies investigating the effects of dietary antigens focused to understand how local and systemic unresponsiveness is induced by innocuous food antigens. Moreover, it has been shown that both, microbial and dietary antigens are essential for the normal development of the mucosal immune system. Based on experimental findings from animals and IBD patients, we propose a model how the intestinal immune system performs the balancing act between recognition and tolerance of dietary antigens at the same time: In the healthy gut, repetitive uptake of dietary antigens by Peyer's patches leads to increasing activation of CD4+ T cells till hyper-activated lymphocytes undergo apoptosis. In contrast to healthy controls, this mechanism was disturbed in Crohn's disease patients. This observation might help to better understand beneficial effects of dietary intervention therapy.

COVID-19-from mucosal immunology to IBD patients.

Viral infections with SARS-CoV-2 can cause a multi-facetted disease, which is not only characterized by pneumonia and overwhelming systemic inflammatory immune responses, but which can also directly affect the digestive system and infect intestinal epithelial cells. Here, we review the current understanding of intestinal tropism of SARS-CoV-2 infection, its impact on mucosal function and immunology and summarize the effect of immune-suppression in patients with inflammatory bowel disease (IBD) on disease outcome of COVID-19 and discuss IBD-relevant implications for the clinical management of SARS-CoV-2 infected individuals.

Level of Tumor Necrosis Factor Production by Stimulated Blood Mononuclear Cells Can Be Used to Predict Response of Patients With Inflammatory Bowel Diseases to Infliximab.

BACKGROUND & AIMS: A substantial proportion patients with inflammatory bowel disease (IBD) have a primary non-response to infliximab; markers are needed to identify patients most likely to respond to treatment. We investigated whether production of tumor necrosis factor (TNF) by peripheral blood mononuclear cells (PBMCs) can be used as a marker to predict response. METHODS: We performed a prospective study of 41 adults with IBD (mean age, 38 years; 21 male; 21 with Crohn's disease and 20 with ulcerative colitis) not treated with a biologic agent within the past 6 months; patients were given their first infusion of infliximab at a hospital or clinic in Berlin, Germany. We collected data on clinical scores, levels of C-reactive protein, and ultrasound results (Limberg scores) at baseline (before the first infusion) and after 6 weeks (3rd infliximab infusion). PMBCs were obtained from patients at baseline and 10 healthy individuals (controls) and incubated with lipopolysaccharide. We measured production of cytokines (TNF, interleukin 1 [IL1], IL6, IL8, IL10, IL12p70, and IL22) by ELISA and performed cytometric bead array and flow cytometry analyses. The primary endpoint was clinical response (decrease in Harvey Bradshaw Index scores of 2 or more or decrease in partial Mayo scores of 3 or more at week 6) in patients with PBMCs that produced high vs low levels of TNF. RESULTS: Responders had a shorter median disease duration (P = .018) and higher median Limberg score (P = .021), than nonresponders. Baseline PBMCs from responders produced significantly more TNF (P = .049) and IL6 (P = .028) than from nonresponders; a level of 500 pg/ml TNF identified responders with 82% sensitivity and 78% specificity. In patients with Crohn's disease, this cutoff value (500 pg/ml TNF) identified responders with 100% sensitivity and 82% specificity; TNF levels above this level were independently associated with response to infliximab in multivariate analysis (odds ratio, 16.2; 95% CI, 1.8-148.7; P = .014). The percentage of TNF-positive cells was higher among CD14+ monocytes than lymphocytes after stimulation. CONCLUSIONS: Production of a high level of TNF by PBMCs (specifically CD14+ cells) from patients with IBD can identify those most likely to have a clinical response to infliximab therapy. In patients with Crohn's disease, a cutoff value of 500 pg/ml TNF identified responders with 100% sensitivity and 82% specificity.

Pro-inflammatory TNF-α and IFN-γ Promote Tumor Growth and Metastasis via Induction of MACC1.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Early stage CRC patients have a good prognosis. If distant metastasis occurs, the 5-year survival drops below 10%. Despite treatment success over the last decades, treatment options for metastatic disease are still limited. Therefore, novel targets are needed to foster therapy of advanced stage CRC patients and hinder progression of early stage patients into metastasis. A novel target is the crucial oncogene Metastasis-Associated in Colon Cancer 1 (MACC1) involved in molecular pathogenesis of CRC metastasis. MACC1 induces cell proliferation and motility, supports cellular survival and rewires metabolism resulting in increased metastasis in vivo. MACC1 is a prognostic biomarker not only for CRC but for more than 20 solid cancer entities. Inflammation plays a pivotal role in tumorigenesis, tumor progression and metastasis. For CRC, inflammatory bowel disease and ulcerative colitis are important inflammation associated risk factors. Certain cytokines, such as TNF-α and IFN-γ, are key factors in determining the contribution of the inflammatory process to CRC. Knowledge of the connection between inflammation and MACC1 driven tumors remains unclear. Gene expression analysis of CRC cells after cytokine stimulation was analyzed by qRT-PCR and Western blot. Cellular motility was assessed by Boyden chamber assays. MACC1 promoter activity after stimulation with pro-inflammatory cytokines was measured using promoter-luciferase constructs. To investigate signal transduction from receptor to effector molecules, blocking experiments using neutralizing antibodies and knockdown experiments were performed. Following TNF-α stimulation, MACC1 and c-Jun expression were significantly increased at the mRNA and protein level. Knockdown of c-Jun reduced MACC1 inducibility following TNF-α stimulation. TNF-α promoted MACC1-induced cell migration that was reverted following MACC1 knockdown. Moreover, MACC1 and c-Jun expression were downregulated by blocking TNFR1, but not TNFR2. Knock down of the NF-κB subunit, p65, reduced basal MACC1 and c-Jun mRNA expression levels. Adalimumab, a clinically approved monoclonal anti-TNF-α antibody, hindered MACC1 induction. The present study highlights that TNF-α regulates the induction of MACC1 via the NF-κB subunit p65 and the transcription factor c-Jun in CRC cells. This finding unravels a novel signaling pathway upstream of MACC1 and provides a potential therapeutic target for the treatment of CRC patients with an associated inflammation.

IL-4 induces M2 macrophages to produce sustained analgesia via opioids.

IL-4 is a pleiotropic antiinflammatory cytokine, which can be neuroprotective after nervous system injury. The beneficial actions of IL-4 are thought to result from the blunting of action of inflammatory mediators, such as proinflammatory cytokines. Here, we demonstrate that IL-4 induces M2 macrophages to continuously produce opioid peptides and ameliorate pain. IL-4 application at injured nerves in mice shifted F4/80+ macrophages from the proinflammatory M1 to the antiinflammatory M2 phenotype, which synthesized opioid peptides (Met-enkephalin, ß-endorphin, and dynorphin A 1-17). These effects were accompanied by a long-lasting attenuation of neuropathy-induced mechanical hypersensitivity, beyond the IL-4 treatment. This IL-4-induced analgesia was decreased by opioid peptide antibodies and opioid receptor (δ, µ, κ) antagonists applied at injured nerves, which confirms the involvement of the local opioid system. The participation of M2 macrophages was supported by analgesia in recipient mice injected at injured nerves with F4/80+ macrophages from IL-4-treated donors. Together, IL-4-induced M2 macrophages at injured nerves produced opioid peptides, which activated peripheral opioid receptors to diminish pain. Fostering the opioid-mediated actions of intrinsic M2 macrophages may be a strategy to tackle pathological pain.

Metabolic Control of Epigenetics and Its Role in CD8+ T Cell Differentiation and Function.

Epigenetic programs that control posttranslational modifications of histone proteins and DNA itself tightly regulate transcriptional networks determining the identity and function of CD8+ T cells. Chromatin-modifying enzymes such as histone acetyltransferases and deacetylases, represent key molecular determinants of the epigenetic imprinting of CD8+ T cells. The functions of these enzymes highly depend on the availability of key products of cellular metabolism pathways such as acetyl-CoA, NAD (Nicotinamide adenine dinucleotide) and SEM (S-adenosylmethionine), suggesting that there is a close crosstalk between the metabolic and the epigenetic regulation of CD8+ T cells. In this review, we will discuss the metabolic regulation of CD8+ T cell epigenetics during activation and differentiation. We will furthermore summarize how metabolic signals from the tumor microenvironment (TME) shape the epigenetic landscape of CD8+ T cells to better understand the mechanism underlying CD8+ T cell exhaustion in anti-tumor and anti-viral immunity, which might help to overcome limitations of current CD8+ T cell-based therapies.