Remodeling of metabolism and inflammation by exercise ameliorates tumor-associated anemia

A considerable number of patients with cancer suffer from anemia, which has detrimental effects on quality of life and survival. The mechanisms underlying tumor-associated anemia are multifactorial and poorly understood. Therefore, we aimed at systematically assessing the patho-etiology of tumor-associated anemia in mice. We demonstrate that reduced red blood cell (RBC) survival rather than altered erythropoiesis is driving the development of anemia. The tumor-induced inflammatory and metabolic remodeling affect RBC integrity and augment splenic phagocyte activity promoting erythrophagocytosis. Exercise training normalizes these tumor-associated abnormal metabolic profiles and inflammation and thereby ameliorates anemia, in part, by promoting RBC survival. Fatigue was prevented in exercising tumor-bearing mice. Thus, exercise has the unique potential to substantially modulate metabolism and inflammation and thereby counteracts pathological remodeling of these parameters by the tumor microenvironment. Translation of this finding to patients with cancer could have a major impact on quality of life and potentially survival

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency

BACKGROUND: Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. OBJECTIVE: We explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. METHODS: Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T cell-intrinsic DNA damage tolerance was tested with an ensemble of in vitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type vs. mutant LIG4 were performed in LIG4 knock-out Jurkat T cells and DNA damage tolerance was subsequently assessed. RESULTS: A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naïve CD4$^{+}$ T cells and low TCR-Vα7.2$^{+}$ T cells, while T/B-cell receptor repertoires showed only mild alterations. Cohort screening identified two other non-related patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. CONCLUSION: We provide evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency

Dominantly inherited combined immunodeficiency as a consequence of heterozygous germline mutations in V(D)J recombination enzymes.

Human immunodeficiencies associated with biallelic mutations in genes required for the generation of the B and T lymphocyte antigen receptors are prototypic inborn errors of immunity. We detected novel heterozygous missense mutations in critical genes implicated in the B and T cell receptor generation, termed RAG1/RAG2 and LIG4 and demonstrated that those can be associated with clinically severe immunodeficiency and immune- dysregulation. By analyzing murine models with titrable in vivo RAG activity we experimentally linked Rag gene dosage to lymphocyte development, anti-viral immunity and immune dysregulation. Our study widens the spectrum of immunological and clinical phenotypes associated with RAG and LIG4 deficiency, which might have important implications for future molecular diagnosis and enabling effective targeted therapeutic interventions

Mechanistic basis of the dynamic response of TWIK1 ionic selectivity to pH

Abstract Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5–6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel

Nano-scale microfluidics to study 3D chemotaxis at the single cell level

Directed migration of cells relies on their ability to sense directional guidance cues and to interact with pericellular structures in order to transduce contractile cytoskeletal- into mechanical forces. These biomechanical processes depend highly on microenvironmental factors such as exposure to 2D surfaces or 3D matrices. In vivo, the majority of cells are exposed to 3D environments. Data on 3D cell migration are mostly derived from intravital microscopy or collagen-based in vitro assays. Both approaches offer only limited controllability of experimental conditions. Here, we developed an automated microfluidic system that allows positioning of cells in 3D microenvironments containing highly controlled diffusion-based chemokine gradients. Tracking migration in such gradients was feasible in real time at the single cell level. Moreover, the setup allowed on-chip immunocytochemistry and thus linking of functional with phenotypical properties in individual cells. Spatially defined retrieval of cells from the device allows down-stream off-chip analysis. Using dendritic cells as a model, our setup specifically allowed us for the first time to quantitate key migration characteristics of cells exposed to identical gradients of the chemokine CCL19 yet placed on 2D vs in 3D environments. Migration properties between 2D and 3D migration were distinct. Morphological features of cells migrating in an in vitro 3D environment were similar to those of cells migrating in animal tissues, but different from cells migrating on a surface. Our system thus offers a highly controllable in vitro-mimic of a 3D environment that cells traffic in vivo.ISSN:1932-620

Maturation of the Human Immunoglobulin Heavy Chain Repertoire With Age

B cells play a central role in adaptive immune processes, mainly through the production of antibodies. The maturation of the B cell system with age is poorly studied. We extensively investigated age-related alterations of naïve and antigen-experienced immunoglobulin heavy chain (IgH) repertoires. The most significant changes were observed in the first 10 years of life, and were characterized by altered immunoglobulin gene usage and an increased frequency of mutated antibodies structurally diverging from their germline precursors. Older age was associated with an increased usage of downstream IgH constant region genes and fewer antibodies with self-reactive properties. As mutations accumulated with age, the frequency of germline-encoded self-reactive antibodies decreased, indicating a possible beneficial role of self-reactive B cells in the developing immune system. Our results suggest a continuous process of change through childhood across a broad range of parameters characterizing IgH repertoires and stress the importance of using well-selected, age-appropriate controls in IgH studies

On-chip phenotyping and harvesting of cells.

<p>(A) DCs were loaded localized in the middle compartment of the migration chamber in a collagen gel (1.7 mg/mL) and allowed to migrate for 2h in cell culture medium or in a soluble CCL19 gradient (5 μg/mL). Cells were stained on-chip with an anti-mouse I-A/I-E antibody. Scale bar, 100 μm. (B) Quantification of anti-I-A/I-E (MHC class II) staining of cells. Corrected Total Cell Fluorescence (CTCF) (= Integrated Density–Area of selected cell * mean fluorescence of background readings) is shown relative to Y-position in migration chamber where 0 μm indicates the chemokine source and 1700 μm the sink of the gradient system. (C) Nuclear staining of DCs with DRAQ5, cells are shown in the microfluidic chamber prior harvesting (left panel, scale bar: 100 μm) and in micro well following harvest (right panel, scale bar: 200 μm).</p

Quantification of microfluidic 2D and 3D migration.

<p>(A) Distribution of migration tracks of cells migrating on fibronectin (100 μg/mL) or in a collagen gel (1.7 mg/mL). Rose plot summarizing frequency of directions relative to the starting point. (B) Quantification of migration characteristics of cells migrating on fibronectin (100 μg/mL, FN) or in a collagen gel of various densities (col conc = collagen concentration). Chemotactic Index is a measure of chemotactic efficiency (calculated by dividing the distance from start to end point of cells in axis of the gradient by the total migration distance of every cell). Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. Not significant (ns) ρ ≥ 0.05; * ρ < 0.05; **** ρ < 0.0001. (C) Representative images of DC morphology in microfluidic 2D and 3D environments and mouse ear tissue. Scale bar, 10 μm. (D) Quantification of cell morphology of cells migrating on fibronectin (100 μg/mL), in a collagen gel (1.7 mg/mL) or in mouse ear tissue. Elongation factor = cell length divided by its perpendicular cell width. FN = fibronectin, col = collagen 1.7 mg/mL, crawl-in = mouse ear crawl-in migration assay. Median and interquartile ranges shown, ρ values were calculated by Kruksal-Wallis test with Dunn’s multiple comparison test. ** ρ < 0.01; *** ρ < 0.001.</p

Geometry and functionality of collagen migration chip.

<p>(A) Photograph of the device, control channels are stained in red, flow channels are depicted in blue color. Inset shows air-filled migration chamber. (B) Schematic overview of device, inset shows migration chamber in detail. (C) Schematic overview of the functionality and workflow of the migration device. Insets show localized loading in migration chamber and tracking of dendritic cells migrating along a gradient of CCL19. Scale bar, 100 μm. (D) Comparison of gradient generation across migration chamber coated with fibronectin (100 μg/mL) and filled with cell culture medium or filled with collagen matrix (1.7 mg/mL). Gradient is visualized with a 10 kDa AF647-Dextran.</p