Impact of Large Granular Lymphocyte Leukemia on Blood DNA Methylation and Epigenetic Clock Modeling in Fischer 344 Rats.

Age-dependent differences in methylation at specific cytosine-guanine (CpG) sites have been used in "epigenetic clock" formulas to predict age. Deviations of epigenetic age from chronological age are informative of health status and are associated with adverse health outcomes, including mortality. In most cases, epigenetic clocks are performed on methylation from DNA extracted from circulating blood cells. However, the effect of neoplastic cells in the circulation on estimation and interpretation of epigenetic clocks is not well understood. Here, we explored this using Fischer 344 (F344) rats, a strain that often develops large granular lymphocyte leukemia (LGLL). We found clear histological markers of LGLL pathology in the spleens and livers of 27 out of 61 rats aged 17-27 months. We assessed DNA methylation by reduced representation bisulfite sequencing with coverage of 3 million cytosine residues. Although LGLL broadly increased DNA methylation variability, it did not change epigenetic aging. Despite this, the inclusion of rats with LGLL in clock training sets significantly altered predictor selection probability at 83 of 121 commonly utilized CpG sites. Furthermore, models trained on rat samples that included individuals with LGLL had greater absolute age error than those trained exclusively rats free of LGLL (39% increase; p < .0001). We conclude that the epigenetic signals for aging and LGLL are distinct, such that LGLL assessment is not necessary for valid measures of epigenetic age in F344 rats. The precision and architecture of constructed epigenetic clock formulas, however, can be influenced by the presence of neoplastic hematopoietic cells in training set populations.

A rat epigenetic clock recapitulates phenotypic aging and co-localizes with heterochromatin.

Robust biomarkers of aging have been developed from DNA methylation in humans and more recently, in mice. This study aimed to generate a novel epigenetic clock in rats-a model with unique physical, physiological, and biochemical advantages-by incorporating behavioral data, unsupervised machine learning, and network analysis to identify epigenetic signals that not only track with age, but also relates to phenotypic aging. Reduced representation bisulfite sequencing (RRBS) data was used to train an epigenetic age (DNAmAge) measure in Fischer 344 CDF (F344) rats. This measure correlated with age at (r = 0.93) in an independent sample, and related to physical functioning (p=5.9e-3), after adjusting for age and cell counts. DNAmAge was also found to correlate with age in male C57BL/6 mice (r = 0.79), and was decreased in response to caloric restriction. Our signatures driven by CpGs in intergenic regions that showed substantial overlap with H3K9me3, H3K27me3, and E2F1 transcriptional factor binding.

Commensal bacteria contribute to insulin resistance in aging by activating innate B1a cells.

Aging in humans is associated with increased hyperglycemia and insulin resistance (collectively termed IR) and dysregulation of the immune system. However, the causative factors underlying their association remain unknown. Here, using "healthy" aged mice and macaques, we found that IR was induced by activated innate 4-1BBL+ B1a cells. These cells (also known as 4BL cells) accumulated in aging in response to changes in gut commensals and a decrease in beneficial metabolites such as butyrate. We found evidence suggesting that loss of the commensal bacterium Akkermansia muciniphila impaired intestinal integrity, causing leakage of bacterial products such as endotoxin, which activated CCR2+ monocytes when butyrate was decreased. Upon infiltration into the omentum, CCR2+ monocytes converted B1a cells into 4BL cells, which, in turn, induced IR by expressing 4-1BBL, presumably to trigger 4-1BB receptor signaling as in obesity-induced metabolic disorders. This pathway and IR were reversible, as supplementation with either A. muciniphila or the antibiotic enrofloxacin, which increased the abundance of A. muciniphila, restored normal insulin response in aged mice and macaques. In addition, treatment with butyrate or antibodies that depleted CCR2+ monocytes or 4BL cells had the same effect on IR. These results underscore the pathological function of B1a cells and suggest that the microbiome-monocyte-B cell axis could potentially be targeted to reverse age-associated IR.

Use of an aquarium as a novel enrichment item for singly housed rhesus macaques (Macaca mulatta).

Locomotor stereotypies are behaviors often seen in singly housed rhesus macaques (Macaca mulatta) and are considered to represent a maladaptive response to captive environments. Active and passive enrichment items are commonly used to mitigate these and other abnormal behaviors. Active enrichment items allow physical manipulation and may be temporarily successful in reducing stereotypies, but their beneficial effects usually are confined to relatively short periods of active use. Passive enrichment items that do not involve physical manipulation are less well studied, and the results are mixed. This study evaluated an aquarium with live fish for use as a novel passive enrichment item in a common facility setting as a means to decrease locomotor stereotypy. We hypothesized that the introduction of the aquarium would decrease the frequency of locomotor stereotypy in a group of singly housed rhesus macaques (n = 11) with a known history of abnormal behaviors. Unexpectedly, locomotor stereotypy increased with the introduction of the aquarium and then decreased over time. Furthermore, when the aquarium was removed, the frequency of stereotypy decreased to below baseline levels. These unexpected results are best explained by neophobia, a common phenomenon documented in many animal species. The increase in abnormal behavior is likely to result from the addition of a novel object within the environment. This study demonstrates that, in the context of reducing abnormal behavior, presumably innocuous enrichment items may have unexpected effects and should be evaluated critically after their introduction to a captive population.

Characterization of rat pinworm (Syphacia muris) epidemiology as a means to increase detection and elimination.

Rodent pinworms persist in many institutions, suggesting deficiencies in eradication and diagnostic processes. When pinworms are detected, treatment success is common, but false-negative test results during health surveillance or after treatment likely contribute to the continued presence of this parasite. PCR testing is not always practical, and increased information regarding the life cycle and general epidemiology of pinworm infestations could improve the sensitivity of traditional nonPCR detection methods and improve eradication efforts. We therefore investigated a pinworm (Syphacia muris) infestation in Sprague-Dawley rats (Rattus norvegicus) to develop a more accurate testing strategy. In addition, we sought to determine the duration of egg viability by using an in vitro hatching protocol to assess environmental persistence. Finally, we tested the ovicidal efficacy of a disinfectant used at our institution. Eggs were shed in higher numbers in the midafternoon as compared with other times of the day, and the sex of the host had no consistent effect on egg shedding. Egg shedding showed periodicity over time, with shedding decreasing to 0 at 2- to 3-wk intervals. Neither cecal examination nor tape tests alone reliably predicted pinworm infestation, and results of the 2 tests did not necessarily coincide. Eggs aged for as long as 7 mo remained viable, indicating a potential for recontamination from the environment. Finally, gaseous chlorine dioxide was an effective ovicidal agent, with a kill rate of 99.7%. These results suggest that strategies for S. muris eradication can be optimized to increase detection and elimination.