Disruption of circadian rhythm by alternating light-dark cycles aggravates atherosclerosis development in APOE*3-Leiden.CETP mice.

Disruption of circadian rhythm by means of shift work has been associated with cardiovascular disease in humans. However, causality and underlying mechanisms have not yet been established. In this study, we exposed hyperlipidemic APOE*3-Leiden.CETP mice to either regular light-dark cycles, weekly 6 hours phase advances or delays, or weekly alternating light-dark cycles (12 hours shifts), as a well-established model for shift work. We found that mice exposed to 15 weeks of alternating light-dark cycles displayed a striking increase in atherosclerosis, with an approximately twofold increase in lesion size and severity, while mice exposed to phase advances and delays showed a milder circadian disruption and no significant effect on atherosclerosis development. We observed a higher lesion macrophage content in mice exposed to alternating light-dark cycles without obvious changes in plasma lipids, suggesting involvement of the immune system. Moreover, while no changes in the number or activation status of circulating monocytes and other immune cells were observed, we identified increased markers for inflammation, oxidative stress, and chemoattraction in the vessel wall. Altogether, this is the first study to show that circadian disruption by shifting light-dark cycles directly aggravates atherosclerosis development.

Co-Expression of TIGIT and Helios Marks Immunosenescent CD8+ T Cells During Aging.

Aging leads to alterations in the immune system that result in ineffective responsiveness against pathogens. Features of this process, collectively known as immunosenescence, accumulate in CD8+ T cells with age and have been ascribed to differentiation of these cells during the course of life. Here we aimed to identify novel markers in CD8+ T cells associated with immunosenescence. Furthermore, we assessed how these markers relate to the aging-related accumulation of highly differentiated CD27-CD28- cells. We found that co-expression of the transcription factor Helios and the aging-related marker TIGIT identifies CD8+ T cells that fail to proliferate and show impaired induction of activation markers CD69 and CD25 in response to stimulation in vitro. Despite this, in blood of older adults we found TIGIT+Helios+ T cells to become highly activated during an influenza-A virus infection, but these higher frequencies of activated TIGIT+Helios+ T cells associate with longer duration of coughing. Moreover, in healthy individuals, we found that TIGIT+Helios+ CD8+ T cells accumulate with age in the highly differentiated CD27-CD28- population. Interestingly, TIGIT+Helios+ CD8+ T cells also accumulate with age among the less differentiated CD27+CD28- T cells before their transit into the highly differentiated CD27-CD28- stage. This finding suggests that T cells with immunosenescent features become prominent at old age also within the earlier differentiation states of these cells. Our findings show that co-expression of TIGIT and Helios refines the definition of immunosenescent CD8+ T cells and challenge the current dogma of late differentiation stage as proxy for T-cell immunosenescence.

Increased Frequency of CD4+ Follicular Helper T and CD8+ Follicular T Cells in Human Lymph Node Biopsies during the Earliest Stages of Rheumatoid Arthritis.

Follicular T helper cells (Tfh cells) provide key B-cell help and are essential in germinal center formation and (auto) antibody generation. To gain more insight into their role during the earliest phase of rheumatoid arthritis (RA), we analyzed their frequencies, phenotypes, and cytokine profiles in peripheral blood and lymph node biopsies of healthy controls (HCs), autoantibody-positive individuals at risk for developing RA (RA-risk individuals), and early RA patients. Subsequently, we confirmed their presence in lymph nodes and synovial tissue of RA patients using immunofluorescence microscopy. In the blood, the frequency of Tfh cells did not differ between study groups. In lymphoid and synovial tissues, Tfh cells were localized in B-cell areas, and their frequency correlated with the frequency of CD19+ B cells. Compared to lymphoid tissues of healthy controls, those of RA patients and RA-risk individuals showed more CD19+ B cells, CD4+CXCR5+ follicular helper T cells, and CD8+CXCR5+ follicular T cells. These Tfh cells produced less IL-21 upon ex vivo stimulation. These findings suggest that Tfh cells may present a novel rationale for therapeutic targeting during the preclinical stage of RA to prevent further disease progression.

Regulatory KIR+ RA+ T cells accumulate with age and are highly activated during viral respiratory disease.

Severe respiratory viral infectious diseases such as influenza and COVID-19 especially affect the older population. This is partly ascribed to diminished CD8+ T-cell responses a result of aging. The phenotypical diversity of the CD8+ T-cell population has made it difficult to identify the impact of aging on CD8+ T-cell subsets associated with diminished CD8+ T-cell responses. Here we identify a novel human CD8+ T-cell subset characterized by expression of Killer-cell Immunoglobulin-like Receptors (KIR+ ) and CD45RA (RA+ ). These KIR+ RA+ T cells accumulated with age in the blood of healthy individuals (20-82 years of age, n = 50), expressed high levels of aging-related markers of T-cell regulation, and were functionally capable of suppressing proliferation of other CD8+ T cells. Moreover, KIR+ RA+ T cells were a major T-cell subset becoming activated in older adults suffering from an acute respiratory viral infection (n = 36), including coronavirus and influenza virus infection. In addition, older adults with influenza A infection showed that higher activation status of their KIR+ RA+ T cells associated with longer duration of respiratory symptoms. Together, our data indicate that KIR+ RA+ T cells are a unique human T-cell subset with regulatory properties that may explain susceptibility to viral respiratory disease at old age.

Compromised DNA Repair Promotes the Accumulation of Regulatory T Cells With an Aging-Related Phenotype and Responsiveness.

Decline of immune function during aging has in part been ascribed to the accumulation of regulatory T cells (Tregs) and decreased T-cell responses with age. Aside from changes to T cells that occur over a lifetime, the impact of intracellular aging processes such as compromised DNA repair on T cells remains incompletely defined. Here we aimed to define the impact of compromised DNA repair on T-cell phenotype and responsiveness by studying T cells from mice with a deficiency in their DNA excision-repair gene Ercc1. These Ercc1 mutant (Ercc1 -/Δ7 ) mice show accumulation of nuclear DNA damage resulting in accelerated aging. Similarly to wild-type aged mice, Ercc1 -/Δ7 mice accumulated Tregs with reduced CD25 and increased PD-1 expression among their naive T cells. Ercc1-deficiency limited the capacity of Tregs, helper T cells, and cytotoxic T cells to proliferate and upregulate CD25 in response to T-cell receptor- and IL-2-mediated stimulation. The recent demonstration that the mammalian target of rapamycin (mTOR) may impair DNA repair lead us to hypothesize that changes induced in the T-cell population by compromised DNA repair may be slowed down or reversed by blocking mTOR with rapamycin. In vivo dietary treatment of Ercc1 -/Δ7 mice with rapamycin did not reduce Treg levels, but highly increased the proportion of CD25+ and PD-1+ memory Tregs instead. Our study elucidates that compromised DNA repair promotes the accumulation of Tregs with an aging-related phenotype and causes reduced T-cell responsiveness, which may be independent of mTOR activation.

Response kinetics reveal novel features of ageing in murine T cells.

The impact of ageing on the immune system results in defects in T cell responsiveness. The search for ageing hallmarks has been challenging due to the complex nature of immune responses in which the kinetics of T cell responsiveness have largely been neglected. We aimed to unravel hallmarks of ageing in the kinetics of the murine T cell response. To this end, we assessed ageing-related T-cell response kinetics by studying the effect of the duration and strength of in vitro stimulation on activation, proliferation, and cytokine secretion by T cells of young and aged mice. Collectively, our data show that stimulatory strength and time kinetics of cytokine secretion, activation markers, and proliferation of Th, Tc, and Treg cells are crucial in understanding the impact of ageing on T cells. Despite low proliferative capacity, T cell subsets of aged mice do respond to stimulation by upregulation of activation markers and secretion of cytokines. These findings therefore indicate that replicative senescence of aged T cells is not a measure of unresponsiveness per se, but rather stress that ageing influences the kinetics of proliferation, upregulation of activation markers and cytokine secretion each to a different extent.

Diversity of aging of the immune system classified in the cotton rat (Sigmodon hispidus) model of human infectious diseases.

Susceptibility and declined resistance to human pathogens like respiratory syncytial virus (RSV) at old age is well represented in the cotton rat (Sigmodon hispidus). Despite providing a preferred model of human infectious diseases, little is known about aging of its adaptive immune system. We aimed to define aging-related changes of the immune system of this species. Concomitantly, we asked whether the rate of immunological alterations may be stratified by physiological aberrations encountered during aging. With increasing age, cotton rats showed reduced frequencies of T cells, impaired induction of antibodies to RSV, higher incidence of aberrations of organs and signs of lipemia. Moreover, old animals expressed high biological heterogeneity, but the age-related reduction of T cell frequency was only observed in those specimens that displayed aberrant organs. Thus, cotton rats show age-related alterations of lymphocytes that can be classified by links with health status.

The involvement of the Toll-like receptor signaling and Nrf2-Keap1 pathways in the in vitro regulation of IL-8 and HMOX1 for skin sensitization.

In vitro gene profiling studies have associated the molecular pathways of Nrf2-Keap1 and Toll-like receptor (TLR) signaling with skin sensitization. In this study, the role of these pathways in the regulation of protein biomarkers for skin sensitization was further elucidated using transient gene knock-down of key components of the signaling cascades in HaCaT cells after exposure to dinitrochlorobenzene (DNCB). The effect of targeting these pathways was established through evaluation of heme oxygenase1 (HMOX1) and interleukin (IL)-8 production. These experiments showed that Nrf2 is not involved in regulating HMOX1 after exposure to DNCB, but that activation of TLR signaling moderates the expression of HMOX1. The regulation of IL-8 depended on Nrf2, but also on the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-ß (TRIF) adaptor protein in TLR signaling. This study provides new insights into the regulation of HMOX1 and IL-8, but the exact regulating mechanisms remain to be further elucidated.