Oleg Varlamov

The fetal immune system develops within a tightly regulated environment that balances immune tolerance with readiness for postnatal antigen exposure. However, limited fetal tissue access constrains understanding of immune ontogeny across compartments. Here we present a multi-tissue single-cell transcriptional atlas of the late-gestation (GD130-135) rhesus macaque fetal immune system, profiling leukocytes from lung, spleen, and umbilical cord blood (UCBMC) spanning myeloid, lymphoid, innate lymphoid, and hematopoietic stem cell lineages. Fetal lung is enriched in myeloid cells and ILC2s and shows a proinflammatory bias. Spleen is dominated by T and B cells, with B cells showing enhanced signatures of V(D)J recombination and isotype switching and CD4 T cells showing increased activation and Treg frequency. UCBMC is predominantly composed of T cells with a regulatory transcriptional landscape. Spectral flow cytometry and functional assays corroborate these tissue-specific differences revealing elevated secretion of inflammatory mediators in fetal lung at rest, while spleen and UCBMC show greater inducible responses following stimulation. Together, these data provide a comprehensive cross-tissue atlas of late-gestation primate fetal immunity, revealing tissue-specific immune specialization, functional capacity, and intercellular communication networks that shape immune readiness prior to birth.

Background: Maternal obesity is a global health challenge with profound consequences for offspring health. While its impact on metabolic programming has been widely studied, far less is known about how maternal obesity shapes the fetal immune system. The fetal bone marrow (FBM) is the central site of hematopoietic stem and progenitor cell (HSPC) development, and disruptions in this niche can have lifelong effects on immunity, infection susceptibility, and inflammatory disease risk. In this study, we examined FBM hematopoiesis in a nonhuman primate model of spontaneous maternal obesity. Methods: Using spectral flow cytometry, single-cell RNA sequencing, and functional differentiation assays, we mapped progenitor composition, lineage trajectories, and immune function in offspring exposed to maternal obesity compared with lean controls. These complementary approaches allowed us to capture cellular frequencies and transcriptional programs, while trajectory and signaling analyses provided insight into how progenitor maturation and intercellular communication are disrupted by maternal obesity. Results: Our findings reveal that maternal obesity decreases CD34+ HSPCs and common lymphoid progenitor populations, while expanding megakaryocyte-erythroid and granulocyte-monocyte progenitors. Pseudotime analysis demonstrated altered maturation, with cells accumulating at early differentiation states. Transcriptional profiling uncovered a strong inflammatory bias, with myeloid progenitors upregulating alarmins, interferon-stimulated genes, and proinflammatory mediators. Functionally, monocytes derived from obese FBM showed impaired migratory and colony-stimulating capacity, coupled with exaggerated TNFα responses to LPS stimulation. Conclusion: Together, these results demonstrate that maternal obesity, even in the absence of obesogenic diet, disrupts fetal bone marrow hematopoiesis by altered HSPC maturation, reprogramming lineage trajectories, and inducing inflammatory bias.

The microprotein adipogenin (Adig) is predominantly expressed in adipose tissues. Here, we found that Adig interacts with seipin to form a stable, rigid complex. We present the structure of the seipin-Adig complex at an overall resolution of ~3.0 angstroms. The structure revealed that mammalian seipin assembles into two distinct oligomeric forms: undecamers and dodecamers. Adig selectively bound to the dodecameric form and enhanced seipin assembly by bridging and stabilizing adjacent subunits. Functionally, this complex promoted lipid droplet development at both early and late stages. In transgenic mice, adipocyte-specific overexpression of Adig increased fat mass and enlarged lipid droplets, whereas Adig deletion disrupted triglyceride accumulation in brown adipose tissues. Thus, Adig can modulate lipid storage through its structural and functional interactions with seipin.

Maternal obesity, often linked to the consumption of a high-fat Western-style diet (WSD), poses significant risks to both maternal and fetal health. This review explores the impact of maternal obesity on fetal hematopoietic stem and progenitor cells (HSPCs), highlighting how metabolic and inflammatory shifts in the maternal environment affect HSPC proliferation, differentiation, and long-term immune system development. Maternal obesity leads to hormonal imbalances, increased inflammatory cytokines, placental insufficiency, and altered nutrient availability that disrupt normal HSPC function, potentially predisposing offspring to immune dysfunction, metabolic disorders, and cardiovascular diseases later in life. Notably, maternal obesity skews HSPC differentiation toward the myeloid lineage, which can impair adaptive immune responses and increase the risk of autoimmune diseases and infections. Furthermore, maternal diet-driven epigenetic and transcriptional reprogramming of fetal HSPCs exacerbates chronic inflammation, reinforcing a pro-inflammatory phenotype in downstream progeny that persists into postnatal stages. The review also emphasizes the need for further research to clarify the mechanisms underlying these effects across different species and developmental stages, as well as the potential for targeted interventions to mitigate the adverse impacts of maternal obesity on fetal hematopoiesis and lifelong health outcomes.

Current antiretroviral therapy (ART) regimens efficiently limit HIV replication, thereby improving the life expectancy of people living with HIV; however, they also cause metabolic side effects. The ongoing obesity epidemic has resulted in more people with metabolic comorbidities at the time of HIV infection, yet the effect of preexisting metabolic dysregulation on infection sequelae and response to ART is unclear. Here, to investigate the impact of preexisting obesity and insulin resistance on acute infection and subsequent long-term ART, we infected a cohort of lean and obese adult male macaques with SIV and administered ART. The responses of lean and obese macaques to SIV and ART were similar with respect to plasma and cell-associated viral loads, ART drug levels in plasma and tissues, SIV-specific immune responses, adipose tissue and islet morphology, and colon inflammation, with baseline differences between lean and obese groups largely maintained. Both groups exhibited a striking depletion of CD4 ⁺ T cells from adipose tissue that did not recover with ART. However, differential responses to SIV and ART were observed for body weight, omental adipocyte size, and the adiponectin/leptin ratio, a marker of cardiometabolic risk. Thus, obesity and insulin resistance had limited effects on multiple responses to acute SIV infection and ART, while several factors that underlie long-term metabolic comorbidities were influenced by prior obesity and insulin resistance. These studies provide the foundation for future investigations into the efficacy of adjunct therapies such as metformin and glucagon-like peptide-1 receptor agonists in the prevention of metabolic comorbidities in people living with HIV.

Objective: The pathological expansion of white adipose tissue (WAT) in obesity involves adipocyte hypertrophy accompanied by expansion of the collagen‐rich pericellular extracellular matrix (ECM) and development of crown‐like structures (CLS). Traditionally, WAT morphology is assessed through immunohistochemical analysis of WAT sections. However, manual analysis of large histological sections is time‐consuming, and the available digital tools for analyzing adipocyte size and pericellular ECM are limited. To address this gap, the authors developed the Adipose Tissue Analysis Toolkit (ATAT), an ImageJ plugin facilitating analysis of adipocyte size, WAT ECM, and CLS.Methods and Results: ATAT utilizes local and image‐level differentials in pixel intensity to independently threshold image background, distinguishing adipocyte‐free tissue without user input. It accurately captures adipocytes in histological sections stained with common dyes and automates the analysis of adipocyte cross‐sectional area, total‐field, and localized region‐of‐interest ECM. ATAT allows fully automated batch analysis of histological images using default or user‐defined adipocyte detection parameters.Conclusions: ATAT provides several advantages over existing WAT image analysis tools, enabling high‐throughput analyses of adipocyte‐specific parameters and facilitating the assessment of ECM changes associated with WAT remodeling due to weight changes and other pathophysiological alterations that affect WAT function.

Objective The pathological expansion of white adipose tissue (WAT) in obesity involves adipocyte hypertrophy accompanied by expansion of collagen-rich pericellular extracellular matrix (ECM) and the development of crown-like structures (CLS). Traditionally, WAT morphology is assessed through immunohistochemical analysis of WAT sections. However, manual analysis of large histological sections is time-consuming, and available digital tools for analyzing adipocyte size and pericellular ECM are limited. To address this gap, we developed the Adipose Tissue Analysis Toolkit (ATAT), an ImageJ plugin facilitating analysis of adipocyte size, WAT ECM and CLS. Methods and Results ATAT utilizes local and image-level differentials in pixel intensity to independently threshold background, distinguishing adipocyte-free tissue without user input. It accurately captures adipocytes in histological sections stained with common dyes and automates the analysis of adipocyte cross-sectional area, total-field, and localized region-of-interest ECM. ATAT allows fully automated batch analysis of histological images using default or user-defined adipocyte detection parameters. Conclusions ATAT provides several advantages over existing WAT image analysis tools, enabling high-throughput analyses of adipocyte-specific parameters and facilitating the assessment of ECM changes associated with WAT remodeling due to weight changes and other pathophysiological alterations that affect WAT function. What is already known about this subject?The manual analysis of large WAT histological sections is very time-consuming, while digital tools for the analysis of WAT are limited. What are the new findings in your manuscript? - ATAT enables fully automated analysis of batches of histological images using either default or user-defined adipocyte detection parameters - ATAT allows high-throughput analyses of adipocyte-specific parameters and pericellular extracellular matrix - ATAT enables the assessment of fibrotic changes associated with WAT remodeling and crown-like structures How might your results change the direction of research or the focus of clinical practice? - ATAT is designed to work with histological sections and digital images obtained using a slide scanner or a microscope. - This tool will help basic and clinical researchers to conduct automated analyses of adipose tissue histological sections.

Problem: Anovulatory infertility is commonly associated with hyperandrogenemia (elevated testosterone, T), insulin resistance, obesity, and white adipose tissue (WAT) dysfunction associated with adipocyte hypertrophy. However, whether hyperandrogenemia and adipocyte hypertrophy per se induce a proinflammatory response is unknown.Method of study: Young adult female rhesus macaques were exposed to an obesogenic Western‐style diet (WSD) in the presence of elevated circulating testosterone (T+WSD) or a low‐fat control diet with no exogenous T. Immune cells residing in visceral omental white adipose tissue (OM‐WAT), corpus luteum and the contralateral ovary, endometrium, lymph nodes, bone marrow, and peripheral blood mononuclear cells were characterized by flow cytometry during the luteal phase of the reproductive cycle.Results: Following one year of treatment, T+WSD animals became more insulin‐resistant and exhibited increased body fat and adipocyte hypertrophy compared to controls. T+WSD treatment did not induce macrophage polarization toward a proinflammatory phenotype in the tissues examined. Additionally, T+WSD treatment did not affect TNFα production by bone marrow macrophages in response to toll‐like receptor agonists. While the major lymphoid subsets were not significantly affected by T+WSD treatment, we observed a significant reduction in the frequency of effector memory CD8+ T‐cells (Tem) in OM‐WAT, but not in other tissues. Notably, OM‐WAT Tem frequencies were negatively correlated with insulin resistance as assessed by the Homeostatic Model Assessment for Insulin Resistance (HOMA‐IR).Conclusion: This study shows that short‐term T+WSD treatment induces weight gain, insulin resistance, and adipocyte hypertrophy, but does not have a significant effect on systemic and tissue‐resident proinflammatory markers, suggesting that adipocyte hypertrophy and mild hyperandrogenemia alone are not sufficient to induce a proinflammatory response.

Fluorescence‐activated cell sorting enables separation and analysis of heterogeneous cell populations based on size, granularity, and fluorescence intensity. Cell sorting has been widely used for isolation of cells that are ∼10 to 25 μm in diameter. By contrast, cell sorting of unilocular adipocytes isolated from white adipose tissue imposes a significant technological challenge. The combination of their large size (up to 200 μm) and the fragile nature of lipid‐laden adipocytes requires the use of specialized flow cytometers equipped with a large nozzle and capable of using low pressure to reduce shear forces during the cell sorting process. Furthermore, isolation of single adipocytes is rarely performed due to the lack of specialized cell sorters that can dispense single adipocytes into individual wells. Conducting cell sorting on single adipocytes would enable analyses of the cell‐autonomous heterogeneity in nutrient uptake and metabolism observed in white adipose tissue. In this protocol, we describe single‐cell sorting of rhesus macaque adipocytes labeled with fluorescent fatty acid and live‐cell indicators using large‐particle flow cytometry. This methodology represents a valuable resource for basic and translational studies aimed at understanding the development and function of adipocytes. © 2021 Wiley Periodicals LLC.Basic Protocol: Single‐cell flow sorting of adipocytes