Thymic Senescence

Thymic senescence develops in every person, although at different pace. Thymic senescence significantly lowers the production of naive T cells, leading to increased incidence of infections, cancer and autoimmune diseases. Certain external factors can accelerate thymic senescence. These include chemicals (copper-chelators), hormones (androgens), infections (viruses, fungi, protozoa). Others may slow the aging process of the thymus including perturbations to the hormonal (sex-steroid) system, genetic alterations (PPARgamma deficiency) or chemical compounds (PPARgamma antagonists). Thymic senescence research may provide insight to underlying molecular events and potentially appoint novel therapeutic targets for senescence intervention strategies. These hold promise to postpone thymus senescence and enhance T cell production. That would result in a decreased incidence of infections, cancer and autoimmune diseases, currently affecting the elderly. The attributed drop in healthcare costs and gain in quality of life share tremendous economic and social interest

Central Immune Senescence, Reversal Potentials

Ageing is a complex process that affects all living organisms. Senescence is not only conceivable in multicellular organisms, but also in unicellulars. Unlike certain diseases that have specific morbidity rates, ageing is a physiological process that affects all individuals that live long enough (unaffected by i.e. predation or famine) to experience senescence

Chapter 31 Central Immune Senescence, Reversal Potentials

Ageing is a complex process that affects all living organisms. Senescence is not only conceivable in multicellular organisms, but also in unicellulars. Unlike certain diseases that have specific morbidity rates, ageing is a physiological process that affects all individuals that live long enough (unaffected by i.e. predation or famine) to experience senescence

Immunosenescence and the Ageing Lung

Ageing is generally defined as the progressive decline of homeostasis that occurs after the reproductive phase of life is complete and the “soma becomes disposable” and death is inevitable according to one theory of ageing. The complexity of the ageing process becomes strikingly evident in the lung where tissue maintenance and repair suffer from damage at the genetic level as well as tissue level. Moreover, lung function declines steadily in adulthood and if data for older adults are extrapolated, the outcome suggests an upper age limit beyond which life becomes impossible. In this review we cover the main changes to lung structure and function with age and the impact on respiratory health. We also describe the role that an aged immune system may play in the age-related decline in lung function and the major involvement of altered signalling through developmental pathways with special focus on PPARγ

Species-specific restriction of cell surface expression of mouse MARCO glycoprotein in murine cell lines

The MARCO (macrophage receptor with collagenous structure) glycoprotein belongs to the scavenger receptor type family of pat- tern-recognition molecules produced by a subset of marginal zone macrophages in the spleen. Stimulation with LPS leads to its appear- ance on macrophages located at other tissue compartments. In the present work, we report its in vitro expression by various cell lines using transient and stable (lentiviral) gene delivery aimed at investigating the signaling properties of this receptor and its analysis using a novel rat monoclonal antibody against the SRCR-domain of mouse MARCO. When trying to establish stable mouse MARCO-trans- fectants using lentiviral transduction and other methods, we consistently found that MARCO accumulated intracellularly in various murine host cells. In contrast, such a phenomenon was not observed in non-murine cell lines. Our observations indicate the presence of an unexpected limitation of the in vitro expression of mouse MARCO glycoprotein in murine cell lines. We believe that the failure to express MARCO on the cell surface of the many murine cell lines is likely due to the absence of endoplasmic reticulum molecular chaperones needed for the correct folding and assembly of the trimeric MARCO molecule

Transgenic Exosomes for Thymus Regeneration

During senescence, Wnt4 expression is down-regulated (unlike their Frizzled receptors), while PPARgamma expression increases in the thymus. Together, these changes allow for thymic degeneration to occur, observed as adipose involution. However, when restored, Wnt4 can efficiently counteract PPARgamma and prevent thymic senescence from developing. The Wnt-pathway activator miR27b has also been reported to inhibit PPARgamma. Our goal was to evaluate the Wnt4 and miR27b levels of Wnt4-transgenic thymic epithelial cell (TEC)-derived exosomes, show their regenerative potential against age-related thymic degeneration, and visualize their binding and distribution both in vitro and in vivo. First, transgenic exosomes were harvested from Wnt4 over-expressing TECs and analyzed by transmission electron microscopy. This unveiled exosomes ranging from 50 to 100 nm in size. Exosomal Wnt4 protein content was assayed by ELISA, while miR27b levels were measured by TaqMan qPCR, both showing elevated levels in transgenic exosomes relative to controls. Of note, kit-purified TEI (total exosome isolate) outperformed UC (ultracentrifugation)-purified exosomes in these parameters. In addition, a significant portion of exosomal Wnt4 proved to be displayed on exosomal surfaces. For functional studies, steroid (Dexamethasone or DX)-induced TECs were used as cellular aging models in which DX-triggered cellular aging was efficiently prevented by transgenic exosomes. Finally, DiI lipid-stained exosomes were applied on the mouse thymus sections and also iv-injected into mice, for in vitro binding and in vivo tracking, respectively. We have observed distinct staining patterns using DiI lipid-stained transgenic exosomes on sections of young and aging murine thymus samples. Moreover, in vivo injected DiI lipid-stained transgenic exosomes showed detectable homing to the thymus. Of note, Wnt4-transgenic exosome homing outperformed control (Wnt5a-transgenic) exosome homing. In summary, our findings indicate that exosomal Wnt4 and miR27b can efficiently counteract thymic adipose involution. Although extrapolation of mouse results to the human setting needs caution, our results appoint transgenic TEC exosomes as promising tools of immune rejuvenation and contribute to the characterization of the immune-modulatory effects of extracellular vesicles in the context of regenerative medicine

Transgenic Exosomes for Thymus Regeneration

During senescence, Wnt4 expression is down-regulated (unlike their Frizzled receptors), while PPARgamma expression increases in the thymus. Together, these changes allow for thymic degeneration to occur, observed as adipose involution. However, when restored, Wnt4 can efficiently counteract PPARgamma and prevent thymic senescence from developing. The Wnt-pathway activator miR27b has also been reported to inhibit PPARgamma. Our goal was to evaluate the Wnt4 and miR27b levels of Wnt4-transgenic thymic epithelial cell (TEC)-derived exosomes, show their regenerative potential against age-related thymic degeneration, and visualize their binding and distribution both in vitro and in vivo. First, transgenic exosomes were harvested from Wnt4 over-expressing TECs and analyzed by transmission electron microscopy. This unveiled exosomes ranging from 50 to 100 nm in size. Exosomal Wnt4 protein content was assayed by ELISA, while miR27b levels were measured by TaqMan qPCR, both showing elevated levels in transgenic exosomes relative to controls. Of note, kit-purified TEI (total exosome isolate) outperformed UC (ultracentrifugation)-purified exosomes in these parameters. In addition, a significant portion of exosomal Wnt4 proved to be displayed on exosomal surfaces. For functional studies, steroid (Dexamethasone or DX)-induced TECs were used as cellular aging models in which DX-triggered cellular aging was efficiently prevented by transgenic exosomes. Finally, DiI lipid-stained exosomes were applied on the mouse thymus sections and also iv-injected into mice, for in vitro binding and in vivo tracking, respectively. We have observed distinct staining patterns using DiI lipid-stained transgenic exosomes on sections of young and aging murine thymus samples. Moreover, in vivo injected DiI lipid-stained transgenic exosomes showed detectable homing to the thymus. Of note, Wnt4-transgenic exosome homing outperformed control (Wnt5a-transgenic) exosome homing. In summary, our findings indicate that exosomal Wnt4 and miR27b can efficiently counteract thymic adipose involution. Although extrapolation of mouse results to the human setting needs caution, our results appoint transgenic TEC exosomes as promising tools of immune rejuvenation and contribute to the characterization of the immune-modulatory effects of extracellular vesicles in the context of regenerative medicine

Cigarette Smoke-Induced Pulmonary Inflammation Becomes Systemic by Circulating Extracellular Vesicles Containing Wnt5a and Inflammatory Cytokines

Chronic obstructive pulmonary disease (COPD) is a devastating, irreversible pathology affecting millions of people worldwide. Clinical studies show that currently available therapies are insufficient, have no or little effect on elevated comorbidities and on systemic inflammation. To develop alternative therapeutic options, a better understanding of the molecular background of COPD is essential. In the present study, we show that non-canonical and pro-inflammatory Wnt5a is up-regulated by cigarette smoking with parallel up-regulation of pro-inflammatory cytokines in both mouse and human model systems. Wnt5a is not only a pro-inflammatory Wnt ligand but can also inhibit the anti-inflammatory peroxisome proliferator-activated receptor gamma transcription and affect M1/M2 macrophage polarization. Both Wnt5a and pro-inflammatory cytokines can be transported in lipid bilayer sealed extracellular vesicles that reach and deliver their contents to every organ measured in the blood of COPD patients, therefore, demonstrating a potential mechanism for the systemic nature of this crippling disease

Artificial Neural Network Correlation and Biostatistics Evaluation of Physiological and Molecular Parameters in Healthy Young Individuals Performing Regular Exercise

Studies support that regular physical activity (PA) decelerates senescence-related decline of physiological and molecular parameters in the elderly. We have addressed the other end of this spectrum: healthy and young, inactive individuals participated in a 6- month long personal trainer-guided lifestyle program. We have measured physiological and molecular parameters (differentiating high- and low responders) and their correlation with PA (sedentary status). Cluster analysis helped to distinguish individuals with high- or low PA and differentiate high- and low-responders of each parameter. The assessed cardiovascular parameters (heart rate, blood pressure, 6-min walking distance, relative VO2max), body composition parameters (body fat and muscle mass percentage) metabolic parameters (glucose, insulin, HDL, LDL), immune parameters (cortisol, CRP, lymphocyte counts, hTREC) all showed improvement. Artificial neural network analysis (ANN) showed correlation efficiencies of physiological and molecular parameters using a concept-free approach. ANN analysis appointed PA as the mastermind of molecular level changes. Besides sedentary status, insulin and hTREC showed significant segregation. Biostatistics evaluation also supported the schism of participants for their sedentary status, insulin concentration and hTREC copy number. In the future ANN and biostatistics, may predict individual responses to regular exercise. Our program reveals that high responder individuals of certain parameters may be low responders of others. Our data show that moderate regular PA is essential to counteract senescence in young and healthy individuals, despite individual differences in responsiveness. Such PA may not seem important in the everyday life of young and healthy adults, but shall become the base for healthy aging

Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells

Zeta-chain-associated protein kinase of 70kDa (ZAP-70) kinase is a key regulator in the early steps of TCR signaling but some aspects of its fine regulation are still unclear. From its 31 tyrosine (Y) residues, 11 phosphorylation sites have been identified, some with activator (Y315 and Y493) or inhibitory (Y292 and Y492) and others with unknown function (Y069, Y126 and Y178). In our present work, we aimed to elucidate the role of different Y residues of ZAP-70, especially those with unknown function, in calcium signaling and the autoregulation of the kinase. ZAP-70-deficient Jurkat cells (P116) were stably reconstituted with point-mutated ZAP-70 constructs where tyrosine residues 069, 126, 178, 238, 292, 315, 492 or 493 were replaced with phenylalanine (F). The anti-CD3-elicited calcium signal increased in F069-, F292- and F492-ZAP-70-expressing cell lines but decreased in the F126-, F315- and F493-ZAP-70-expressing cell lines. ZAP-70 point mutations led to phosphorylation changes predominantly in SH2 domain containing leukocyte protein of 76kDa (SLP-76) but not linker of activated T cells (LAT) during CD3-activation; moreover, we detected basal hyperphosphorylation of SLP-76 Y128 in the F126-, F178- and F492-ZAP-70-expressing cell lines. In summary, Y069, Y178, Y292 and Y492 have inhibitory, while Y126, Y315 and Y493 activator role in anti-CD3-induced T-cell activation. Phosphorylation changes in LAT and SLP-76 suggest that fine regulation of ZAP-70 on calcium signaling is rather transmitted through SLP-76 not LAT. Additionally, negative or positive autoregulatory function of Y292 and Y493 or Y315, respectively, was revealed in ZAP-70. These data indicate that previously not characterized Y069, Y126 and Y178 in ZAP-70 participate in the fine regulation of TCR signaling