BCG vaccination stimulates integrated organ immunity by feedback of the adaptive immune response to imprint prolonged innate antiviral resistance.

Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted ∼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.

Visceral adipose microbial and inflammatory signatures in metabolically healthy and unhealthy nonhuman primates.

OBJECTIVE: Obesity is a key risk factor for metabolic syndrome (MetS); however, >10% of lean individuals meet MetS criteria. Visceral adipose tissue (VAT) disproportionately contributes to inflammation and insulin resistance compared with subcutaneous fat depots. The primary aim of this study was to profile tissue microbiome components in VAT over a wide range of metabolic statuses in a highly clinically relevant model. METHODS: VAT was profiled from nonhuman primates that naturally demonstrate four distinct health phenotypes despite consuming a healthy diet, namely metabolically healthy lean and obese and metabolically unhealthy lean and obese. RESULTS: VAT biopsied from unhealthy lean and obese nonhuman primates demonstrated upregulation of immune signaling pathways, a tissue microbiome enriched in gram-negative bacteria including Pseudomonas, and deficiencies in anti-inflammatory adipose tissue M2 macrophages. VAT microbiomes were distinct from fecal microbiomes, and fecal microbiomes did not differ by metabolic health group, which was in contrast to the VAT bacterial communities. CONCLUSIONS: Immune activation with gram-negative VAT microbial communities is a consistent feature in elevated MetS risk in both lean and obesity states.

Long-term dasatinib plus quercetin effects on aging outcomes and inflammation in nonhuman primates: implications for senolytic clinical trial design.

Cellular senescence increases with aging and results in secretion of pro-inflammatory factors that induce local and systemic tissue dysfunction. We conducted the first preclinical trial in a relevant middle-aged nonhuman primate (NHP) model to allow estimation of the main translatable effects of the senolytic combination dasatinib (D) and quercetin (Q), with and without caloric restriction (CR). A multi-systemic survey of age-related changes, including those on immune cells, adipose tissue, the microbiome, and biomarkers of systemic organ and metabolic health are reported. Age-, weight-, sex-, and glycemic control-matched NHPs (D + Q, n = 9; vehicle [VEH] n = 7) received two consecutive days of D + Q (5 mg/kg + 50 mg/kg) monthly for 6 months, where in month six, a 10% CR was implemented in both D + Q and VEH NHPs to induce equal weight reductions. D + Q reduced senescence marker gene expressions in adipose tissue and circulating PAI-1 and MMP-9. Improvements were observed in immune cell types with significant anti-inflammatory shifts and reductions in microbial translocation biomarkers, despite stable microbiomes. Blood urea nitrogen showed robust improvements with D + Q. CR resulted in significant positive body composition changes in both groups with further improvement in immune cell profiles and decreased GDF15 (p = 0.05), and the interaction of D + Q and CR dramatically reduced glycosylated hemoglobin A1c (p = 0.03). This work indicates that 6 months of intermittent D + Q exposure is safe and may combat inflammaging via immune benefits and improved intestinal barrier function. We also saw renal benefits, and with CR, improved metabolic health. These data are intended to provide direction for the design of larger controlled intervention trials in older patients.

Delayed effects of radiation in adipose tissue reflect progenitor damage and not cellular senescence.

The pathogenesis of many age-related diseases is linked to cellular senescence, a state of inflammation-inducing, irreversible cell cycle arrest. The consequences and mechanisms of age-associated cellular senescence are often studied using in vivo models of radiation exposure. However, it is unknown whether radiation induces persistent senescence, like that observed in ageing. We performed analogous studies in mice and monkeys, where young mice and rhesus macaques received sub-lethal doses of ionizing radiation and were observed for ~ 15% of their expected lifespan. Assessments of 8-hydroxy-2' -deoxyguanosine (8-OHdG), senescence-associated beta-galactosidase (SAß-gal), and p16Ink4a and p21 were performed on mitotic and post-mitotic tissues - liver and adipose tissue - 6 months and 3 years post-exposure for the mice and monkeys, respectively. No elevations in 8-OHdG, SA-ßgal staining, or p16 Ink4a or p21 gene or protein expression were found in mouse and monkey liver or adipose tissue compared to control animals. Despite no evidence of senescence, progenitor cell dysfunction persisted after radiation exposure, as indicated by lower in situ CD34+ adipose cells (p = 0.03), and deficient adipose stromal vascular cell proliferation (p < 0.05) and differentiation (p = 0.04) ex vivo. Our investigation cautions that employing radiation to study senescence-related processes should be limited to the acute post-exposure period and that stem cell damage likely underpins the dysfunction associated with delayed effects of radiation.

Macrophage Phenotypes and Gene Expression Patterns Are Unique in Naturally Occurring Metabolically Healthy Obesity.

Obesity impacts 650 million individuals globally, often co-occurring with metabolic syndrome. Though many obese individuals experience metabolic abnormalities (metabolically unhealthy obese [MUO]), ~30% do not (metabolically healthy obese [MHO]). Conversely, >10% of lean individuals are metabolically unhealthy (MUL). To evaluate the physiologic drivers of these phenotypes, a 44-animal African green monkey cohort was selected using metabolic syndrome risk criteria to represent these four clinically defined health groups. Body composition imaging and subcutaneous adipose tissue (SQ AT) biopsies were collected. Differences in adipocyte size, macrophage subtype distribution, gene expression, vascularity and fibrosis were analyzed using digital immunohistopathology, unbiased RNA-seq, endothelial CD31, and Masson's trichrome staining, respectively. MHO AT demonstrated significant increases in M2 macrophages (p = 0.02) and upregulation of fatty acid oxidation-related terms and transcripts, including FABP7 (p = 0.01). MUO AT demonstrated downregulation of these factors and co-occurring upregulation of immune responses. These changes occurred without differences in AT distributions, adipocyte size, AT endothelial cells, collagen I deposition, or circulating cytokine levels. Without unhealthy diet consumption, healthy obesity is defined by an increased SQ AT M2/M1 macrophage ratio and lipid handling gene expression. We highlight M2 macrophages and fatty acid oxidation as targets for improving metabolic health with obesity.

Diet impacts triple-negative breast cancer growth, metastatic potential, chemotherapy responsiveness, and doxorubicin-mediated cardiac dysfunction.

Anthracyclines are standard-of-care chemotherapy for the treatment of triple-negative breast cancer (TNBC). However, high anthracyclines cumulative doses increase heart failure risk. Designing therapeutic strategies that ameliorate cardiac toxicities without compromising oncologic efficacy are important to improve TNBC outcomes and survivorship. The purpose of this study was to determine the impact of diet on TNBC chemotherapeutic responsiveness and development of chemotherapy-induced cardiac damage. Female BALB/c mice fed a control, Western, Mediterranean, or Western + fish oil diet were injected with 1 × 106 4T1-luciferase TNBC into the mammary fat pad. Tumors grew for 21 days before surgical tumor resection, then mice were treated with 3.3 mg/kg i.v. doxorubicin for 3 weeks. Vevo (R) cardiac ultrasound was performed. Female nu/nu mice were placed on diets before 1 × 105  MDA-MB-231-luciferase TNBC were injected via the tail vein to induce the development of lung metastases. Mice were treated with saline or 3.3 mg/kg i.v. doxorubicin for 3 weeks, and the development of metastases visualized by IVIS (R). Consumption of a high-fat diet increased TNBC growth regardless of dietary pattern. Western diet-fed mice developed lung metastases sooner and displayed increased lung metastatic lesion formation, which was not observed in Mediterranean diet-fed mice. Western diet-fed animals displayed worse cardiac function when compared with Mediterranean diet-fed animals. Hearts from Western diet-fed animals displayed increased fibrosis. Diet represents a modifiable component directly impacting tumor growth, antitumor chemotherapy efficacy, and cardiac toxicities. Our data suggest that the Mediterranean diet may reduce lung metastatic lesions formation and prevent the development of cardiac toxicities.

Hypertension promotes microbial translocation and dysbiotic shifts in the fecal microbiome of nonhuman primates.

Accumulating evidence indicates a link between gut barrier dysfunction and hypertension. However, it is unclear whether hypertension causes gut barrier dysfunction or vice versa and whether the gut microbiome plays a role. To understand this relationship, we first cross-sectionally examined 153 nonhuman primates [NHPs; Chlorocebus aethiops sabaeus; mean age, 16 ± 0.4 yr; 129 (84.3%) females] for cardiometabolic risk factors and gut barrier function biomarkers. This analysis identified blood pressure and age as specific factors that independently associated with microbial translocation. We then longitudinally tracked male, age-matched spontaneously hypertensive NHPs (Macaca mulatta) to normotensives (n = 16), mean age of 5.8 ± 0.5 yr, to confirm hypertension-related gut barrier dysfunction and to explore the role of microbiome by comparing groups at baseline, 12, and 27 mo. Collectively, hypertensive animals in both studies showed evidence of gut barrier dysfunction (i.e., microbial translocation), as indicated by higher plasma levels of lipopolysaccharide-binding protein (LBP)-1, when compared with normotensive animals. Furthermore, plasma LBP-1 levels were correlated with diastolic blood pressure, independent of age and other health markers, suggesting specificity of the effect of hypertension on microbial translocation. In over 2 yr of longitudinal assessment, hypertensive animals had escalating plasma levels of LBP-1 and greater bacterial gene expression in mesenteric lymph nodes compared with normotensive animals, confirming microbes translocated across the intestinal barrier. Concomitantly, we identified distinct shifts in the gut microbial signature of hypertensive versus normotensive animals at 12 and 27 mo. These results suggest that hypertension contributes to microbial translocation in the gut and eventually unhealthy shifts in the gut microbiome, possibly contributing to poor health outcomes, providing further impetus for the management of hypertension.NEW & NOTEWORTHY Hypertension specifically had detrimental effects on microbial translocation when age and metabolic syndrome criteria were evaluated as drivers of cardiovascular disease in a relevant nonhuman primate model. Intestinal barrier function exponentially decayed over time with chronic hypertension, and microbial translocation was confirmed by detection of more microbial genes in regional draining lymph nodes. Chronic hypertension resulted in fecal microbial dysbiosis and elevations of the biomarker NT-proBNP. This study provides insights on the barrier dysfunction, dysbiosis, and hypertension in controlled studies of nonhuman primates. Our study includes a longitudinal component comparing naturally occurring hypertensive to normotensive primates to confirm microbial translocation and dysbiotic microbiome development. Hypertension is an underappreciated driver of subclinical endotoxemia that can drive chronic inflammatory diseases.

Type-2 Diabetes as a Risk Factor for Severe COVID-19 Infection.

The current outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), termed coronavirus disease 2019 (COVID-19), has generated a notable challenge for diabetic patients. Overall, people with diabetes have a higher risk of developing different infectious diseases and demonstrate increased mortality. Type 2 diabetes mellitus (T2DM) is a significant risk factor for COVID-19 progression and its severity, poor prognosis, and increased mortality. How diabetes contributes to COVID-19 severity is unclear; however, it may be correlated with the effects of hyperglycemia on systemic inflammatory responses and immune system dysfunction. Using the envelope spike glycoprotein SARS-CoV-2, COVID-19 binds to angiotensin-converting enzyme 2 (ACE2) receptors, a key protein expressed in metabolic organs and tissues such as pancreatic islets. Therefore, it has been suggested that diabetic patients are more susceptible to severe SARS-CoV-2 infections, as glucose metabolism impairments complicate the pathophysiology of COVID-19 disease in these patients. In this review, we provide insight into the COVID-19 disease complications relevant to diabetes and try to focus on the present data and growing concepts surrounding SARS-CoV-2 infections in T2DM patients.

Adipose Tissue Macrophage Polarization in Healthy and Unhealthy Obesity.

Over 650 million adults are obese (body mass index ≥ 30 kg/m2) worldwide. Obesity is commonly associated with several comorbidities, including cardiovascular disease and type II diabetes. However, compiled estimates suggest that from 5 to 40% of obese individuals do not experience metabolic or cardiovascular complications. The existence of the metabolically unhealthy obese (MUO) and the metabolically healthy obese (MHO) phenotypes suggests that underlying differences exist in both tissues and overall systemic function. Macrophage accumulation in white adipose tissue (AT) in obesity is typically associated with insulin resistance. However, as plastic cells, macrophages respond to stimuli in their microenvironments, altering their polarization between pro- and anti-inflammatory phenotypes, depending on the state of their surroundings. The dichotomous nature of MHO and MUO clinical phenotypes suggests that differences in white AT function dictate local inflammatory responses by driving changes in macrophage subtypes. As obesity requires extensive AT expansion, we posit that remodeling capacity with adipose expansion potentiates favorable macrophage profiles in MHO as compared with MUO individuals. In this review, we discuss how differences in adipogenesis, AT extracellular matrix deposition and breakdown, and AT angiogenesis perpetuate altered AT macrophage profiles in MUO compared with MHO. We discuss how non-autonomous effects of remote organ systems, including the liver, gastrointestinal tract, and cardiovascular system, interact with white adipose favorably in MHO. Preferential AT macrophage profiles in MHO stem from sustained AT function and improved overall fitness and systemic health.

Biomarkers of senescence in non-human primate adipose depots relate to aging.

Forty-three female African green monkeys (Chlorocebus aethiops sabaeus) were selected to represent young adult to advanced geriatric ages (7-24 years) to exhibit a wide range of obesity status (8-53% body fat) and diverse metabolic syndrome criteria such as diabetes, dyslipidemia, and hypertension. Subcutaneous and visceral adipose tissues were collected and evaluated for the presence of senescence cells in both whole tissue and single-cell isolates from subcutaneous sources, utilizing senescence-associated ß-galactosidase (SAß-gal) staining. Plasma samples were analyzed for selected metabolic and inflammatory biomarkers related to the senescence-associated secretory profile. Our results indicated that tissue staining scores did not differ between subcutaneous and intra-abdominal visceral depots and were highly related within individuals. Tissue staining was significantly associated with chronological age; however, no associations with fatness or metabolic syndrome criteria were observed. Associations with age were unchanged when obesity status was included in regression models. Isolated cell staining did positively relate to age but not tissue staining, suggesting some of the SAß-gal-positive cells were stromal vascular cells or small adipocytes, but that mature large adipocytes, filtered out in the cell isolation process, are also likely to exhibit positive SAß-gal staining. Plasminogen activator inhibitor-1 (PAI-1) concentration in circulation was the sole inflammation-related biomarker that positively associated with age and is considered to be a marker of senescent cell burden. Our study is the largest, most comprehensive assessment of adipose SAß-gal staining in a relevant animal model of human aging, and confirms that this senescence-associated biomarker specifically indicates an age-related process.

Skeletal muscle extracellular matrix remodeling with worsening glycemic control in nonhuman primates.

Type 2 diabetes (T2D) development may be mediated by skeletal muscle (SkM) function, which is responsible for >80% of circulating glucose uptake. The goals of this study were to assess changes in global- and location-level gene expression, remodeling proteins, fibrosis, and vascularity of SkM with worsening glycemic control, through RNA sequencing, immunoblotting, and immunostaining. We evaluated SkM samples from health-diverse African green monkeys (Cholorcebus aethiops sabaeus) to investigate these relationships. We assessed SkM remodeling at the molecular level by evaluating unbiased transcriptomics in age-, sex-, weight-, and waist circumference-matched metabolically healthy, prediabetic (PreT2D) and T2D monkeys (n = 13). Our analysis applied novel location-specific gene differences and shows that extracellular facing and cell membrane-associated genes and proteins are highly upregulated in metabolic disease. We verified transcript patterns using immunohistochemical staining and protein analyses of matrix metalloproteinase 16 (MMP16), tissue inhibitor of metalloproteinase 2 (TIMP2), and VEGF. Extracellular matrix (ECM) functions to support intercellular communications, including the coupling of capillaries to muscle cells, which was worsened with increasing blood glucose. Multiple regression modeling from age- and health-diverse monkeys (n = 33) revealed that capillary density was negatively predicted by only fasting blood glucose. The loss of vascularity in SkM co-occurred with reduced expression of hypoxia-sensing genes, which is indicative of a disconnect between altered ECM and reduced endothelial cells, and known perfusion deficiencies present in PreT2D and T2D. This report supports that rising blood glucose values incite ECM remodeling and reduce SkM capillarization, and that targeting ECM would be a rational approach to improve health with metabolic disease.

Advanced maternal age impacts physiologic adaptations to pregnancy in vervet monkeys.

The trend to delay pregnancy in the USA has resulted in the number of advanced maternal age (AMA) pregnancies to also increase. In humans, AMA is associated with a variety of pregnancy-related pathologies such as preeclampsia (PE). While AMA is known to be a factor which contributes to the development of pregnancy-induced diseases, the molecular and cellular mechanisms giving rise to this phenomenon are still very limited. This is due in part to lack of a preclinical model which has physiologic relevance to human pregnancy while also allowing control of environmental and genetic variability inherent in human studies. To determine potential physiologic relevance of the vervet/African green monkey (Chlorocebus aethiops sabaeus) as a preclinical model to study the effects of AMA on adaptations to pregnancy, thirteen age-diverse pregnant vervet monkeys (3-16 years old) were utilized to measure third trimester blood pressure (BP), complete blood count, iron measurements, and hormone levels. Significant associations were observed between third trimester diastolic BP and maternal age. Furthermore, the presence of leukocytosis with enhanced circulating neutrophils was observed in AMA mothers compared to younger mothers. Moreover, we observed a negative relationship between maternal age and estradiol, progesterone, and cortisol levels. Finally, offspring born to AMA mothers displayed a postnatal growth retardation phenotype. These studies demonstrate physiologic impairment in the adaptation to pregnancy in AMA vervet/African green monkeys. Our data indicate that the vervet/African green monkey may serve as a useful preclinical model and tool for deciphering pathological mediators of maternal disease in AMA pregnancy.

High Diversity of Testate Amoebae (Amoebozoa, Arcellinida) Detected by HTS Analyses in a New England Fen using Newly Designed Taxon-specific Primers.

Testate (shell-building) amoebae, such as the Arcellinida (Amoebozoa), are useful bioindicators for climate change. Though past work has relied on morphological analyses to characterize Arcellinida diversity, genetic analyses revealed the presence of multiple cryptic species underlying morphospecies. Here, we design and deploy Arcellinida-specific primers for the SSU-rDNA gene to assess the community composition on the molecular level in a pilot study of two samplings from a New England fen: (1) 36-cm horizontal transects and vertical cores; and (2) 26-m horizontal transects fractioned into four size classes (2-10, 10-35, 35-100, and 100-300 µm). Analyses of these data show the following: (1) a considerable genetic diversity within Arcellinida, much of which comes from morphospecies lacking sequences on GenBank; (2) communities characterized by DNA (i.e. active + quiescent) are distinct from those characterized by RNA (i.e. active, indicator of biomass); (3) active communities on the surface tend to be more similar to one another than to core communities, despite considerable heterogeneity; and (4) analyses of communities fractioned by size find some lineages (OTUs) that are abundant in disjunct size categories, suggesting the possibility of life-history stages. Together, these data demonstrate the potential of these primers to elucidate the diversity of Arcellinida communities in diverse habitats.

Whole Body Irradiation Induces Diabetes and Adipose Insulin Resistance in Nonhuman Primates.

PURPOSE: Diabetes mellitus is a delayed effect of radiation exposure in human and nonhuman primates. Diabetes mellitus is characterized by peripheral tissue insulin resistance, and as a result, irradiation exposure may cause important changes in insulin-sensitive tissues such as muscle and adipose. METHODS AND MATERIALS: We prospectively investigated changes in response to irradiation (4 Gy whole body exposure) in 16 male rhesus macaques. We evaluated changes in body composition and glycemic control for 2 years. Insulin responsiveness, lipolysis, inflammation, and fibrosis were evaluated at study end. RESULTS: Irradiated animals accumulate less fat and significantly increased percent glycation of hemoglobin A1c over time, such that 40% of irradiated monkeys had values that define them as diabetic at 2 years. Subcutaneous (SQ) adipose tissue was insulin resistant, as evidenced by reduced phosphorylation of the insulin receptor substrate-1 in response to insulin challenge and had increased basal lipolysis despite comparable insulin exposures to control animals. Irradiated SQ adipose tissue had more macrophage infiltration and adipocytes were larger. The observed hypertrophy was associated with decreased glycemic control and macrophage infiltration correlated with decreased adiponectin, signifying that inflammation is associated with worsening health. No evidence of SQ adipose fibrosis was detected. CONCLUSIONS: Our study is the first to prospectively illustrate that sublethal irradiation exposures directly propagate metabolic disease in the absence of obesity in nonhuman primates and implicate SQ adipose dysfunction as a target tissue.