Activity-dependent FosB gene expression negatively regulates mast cell functions.

Mast cells are innate immune cells that play a crucial role in numerous physiological processes across tissues by releasing pre-stored and newly synthesized mediators in response to stimuli, an activity largely driven by changes in gene expression. Given their widespread influence, dysfunction in mast cells can contribute to a variety of pathologies including allergies, long COVID, and autoimmune and neuroinflammatory disorders. Despite this, the specific transcriptional mechanisms that control mast cell mediator release remain poorly understood, significantly hindering the development of effective therapeutic strategies. We found that the two proteins encoded by the transcription factor FosB, FOSB and the highly stable variant ΔFOSB, are robustly expressed upon stimulation in both murine and human mast cell progenitors. Motivated by these findings, we generated a novel mouse model with targeted ablation of FosB gene expression specifically in mast cells (MC FosB- ) by crossing a mast cell-specific Cre reporter line (Mcpt5-Cre) with a Cre-dependent floxed FosB mouse lines. We found that mast cell progenitors derived from MC FosB- mice, compared to wild types (WT), exhibit baseline increased histamine content and vesicle numbers. Additionally, they show enhanced calcium mobilization, degranulation, and histamine release following allergy-related IgE-mediated stimulation, along with heightened IL-6 release in response to infection-like LPS stimulation. In vivo experiments with IgE- mediated and LPS challenges revealed that MC FosB- mice experience greater drops in body temperature, heightened activation of tissue-resident mast cells, and increased release of pro-inflammatory mediators compared to their WT counterparts. These findings suggest that FosB products play a crucial regulatory role in moderating stimulus-induced mast cell activation in response to both IgE and LPS stimuli. Lastly, by integrating CUT&RUN and RNAseq data, we identified several genes targeted by ΔFOSB that could mediate these observed effects, including Mir155hg, CLCF1, DUSP4, and Trib1. Together, this study provides the first evidence that FOSB/ΔFOSB modulate mast cell functions and provides a new possible target for therapeutic interventions aimed at ameliorating mast cell-related diseases.

Early weaning and biological sex shape long-term immune and metabolic responses in pigs.

During the early pre and postnatal life, host and environmental factors can impart a major influence on immune development, thus shaping lifelong disease resistance. Two major factors known to influence immune function and mortality in animals and people are early life stress and biological sex. How these two factors interact to shape long-term immune development and later life disease risk is poorly understood. Here we investigated how early weaning, a common early life stressor in pigs, and biological sex impacts long-term systemic inflammatory responses and hypothalamic-pituitary-adrenal axis (HPA axis) activation later in life. Ten-week-old female (F), intact-male (IM) and castrated-male (CM) pigs that were randomly assigned to early weaning (EW) and later weaning (LW) (at 15 or 28 days of age, respectively) were intramuscularly injected with either saline vehicle or lipopolysaccharide (LPS) to induce a systemic inflammatory response. Complete blood counts (CBC), proinflammatory cytokines, cortisol, testosterone, estradiol, and rectal temp were measured at 0 h, 2 h, and 4 h post-LPS challenge. At 4 h post-LPS, peritoneal fluid (PF) and white blood cells (WBC) were collected for differential analysis. LPS challenge significantly increased rectal temp and plasma cortisol level in all treatment groups. Together, the CBC results and immune cell counts in peritoneal cavity indicated that EW-F exhibited greater systemic immune response characterized by increased neutrophils to lymphocytes ratio (NLR) and enhanced neutrophil trafficking to the peritoneal cavity. Early weaning had an opposite effect on IM and CM pigs, which exhibited a suppressed LPS-induced neutrophil migration. Early weaning induced significantly greater cortisol responses only in IM pigs indicating a heightened HPA axis responses in EW-IM. how early weaning and biological sex affect immune and stress responses in pigs. Together, these results demonstrate that early weaning and biological sex and castration shape later life immune responses in pigs and provides insight into potential mechanisms driving sex differences in later life inflammatory disease risk and mortality.

Effects of a multi-strain Bacillus subtilis-based direct-fed microbial on immunity markers and intestinal morphology in diets fed to weanling pigs.

The objective of this experiment was to evaluate the effects of a multi-strain Bacillus subtilis-based direct-fed microbial (DFM) on nursery pig health as indicated by intestinal mucosal and blood plasma immunological markers and intestinal morphology. Eighty pigs, of equal number of barrows and gilts (initial BW: 7.0 ±â€…0.60 kg), weaned at 21 ±â€…1 d of age were randomly allotted to sixteen pens, with five pigs per pen. Two dietary treatments were implemented, a basal control (CON) and a basal control plus DFM (CDFM). Both diets were corn, soybean meal, and distillers dried grains based and were formulated to meet or exceed all nutritional requirements (NRC, 2012) and manufactured on site. Diets were fed for 42 d. On d 21 and 42 of the experiment, one pig per pen was randomly selected and euthanized, with equal number of males and females represented. Blood samples were collected prior to euthanasia for assessment of plasma concentrations of immunoglobulin A (IgA) and intestinal fatty acid binding protein. Segments of the gastrointestinal tract including duodenum, jejunum, ileum, ascending and distal colon were removed for analysis of intestinal morphology, and levels of interleukin 6, interleukin 10 (IL-10), and tumor necrosis factor alpha. Jejunal villus height was greater in the CDFM pigs as compared with CON pigs (P = 0.02) and ascending colon crypt depth tended to be greater on d 21 (P = 0.10). Compared to CON, CDFM significantly increased overall plasma IgA (P = 0.03) (0.58 vs. 0.73 0.05 mg/mL, respectively), while it tended to increase plasma IgA (P = 0.06) on d 21 (0.34 vs. 0.54 ±â€…0.07 mg/mL, respectively) and tended to increase overall IL-10 (P = 0.10) in the jejunum (113 vs. 195 ±â€…35 pg/mL, respectively). Addition of a multi-strain Bacillus subtilis-based DFM may have an early benefit to nursery pig health status, observed through specific changes in morphology and both systemic and localized immunological markers.

Biological sex: an understudied factor driving disease susceptibility in pigs.

Biological sex is a major host factor influencing risk for infectious disease-associated mortality, and chronic inflammatory and metabolic diseases. Research in human and rodent models -has revealed sex differences that exist across organ systems during health and disease that may contribute to sex biases in disease risk. Despite the robust and growing literature on the role of sex as a risk factor in human disease, comparatively little attention has been focused on investigating the role of biological sex in disease susceptibility in agriculturally important animal populations such as the pig. To date, comparisons between sexes have focused on carcass composition, growth rate, and feed efficiency in pigs. However, there is a large gap in the literature regarding the effects of biological sex on other integral aspects of health and disease. The objective of this review is to highlight the available literature reporting sex differences in pig health and biology with an emphasis on sex differences in mortality, immunity, and gastrointestinal (GI) physiology and to address biological sex as a significant biological variable in disease risk and research study design. A basic overview of the biology of sex differences including the major hormonal and genetic/chromosomal mechanisms of sexual differentiation and the developmental periods in which sex differences emerge will be covered. This review will also discuss how production-relevant management and environmental factors (e.g., wean age, castration, stress, and nutrition) interact with biological sex to shape host immune and GI development and function. Perceived gaps in knowledge and areas of future research will also be discussed.

It has become increasingly evident that females and males differ in their susceptibility to disease and mortality. Females typically have higher survivability rates during pandemics and environmental challenges compared with males. In many cases, females mount a greater immune response compared with males which may have survival benefits, but at the same time may predispose them to chronic inflammatory disorders. Despite this accumulated knowledge on the key role that sex plays on immunity and disease outcomes in humans, little attention has been placed on sex differences in agriculturally important species such as the pig. The objective of this review is to highlight the literature on sex differences in swine with a focus on mortality, immunity, and GI health.

Early life adversity drives sex-specific anhedonia and meningeal immune gene expression through mast cell activation.

Exposure to early life adversity (ELA) in the form of physical and/or psychological abuse or neglect increases the risk of developing psychiatric and inflammatory disorders later in life. It has been hypothesized that exposure to ELA results in persistent, low grade inflammation that leads to increased disease susceptibility by amplifying the crosstalk between stress-processing brain networks and the immune system, but the mechanisms remain largely unexplored. The meninges, a layer of three overlapping membranes that surround the central nervous system (CNS)- dura mater, arachnoid, and piamater - possess unique features that allow them to play a key role in coordinating immune trafficking between the brain and the peripheral immune system. These include a network of lymphatic vessels that carry cerebrospinal fluid from the brain to the deep cervical lymph nodes, fenestrated blood vessels that allow the passage of molecules from blood to the CNS, and a rich population of resident mast cells, master regulators of the immune system. Using a mouse model of ELA consisting of neonatal maternal separation plus early weaning (NMSEW), we sought to explore the effects of ELA on sucrose preference behavior, dura mater expression of inflammatory markers and mast cell histology in adult male and female C57Bl/6 mice. We found that NMSEW alone does not affect sucrose preference behavior in males or females, but it increases the dura mater expression of the genes coding for mast cell protease CMA1 (cma1) and the inflammatory cytokine TNF alpha (tnf alpha) in females. When NMSEW is combined with an adult mild stress (that does not affect behavior or gene expression in NH animals) females show reduced sucrose preference and even greater increases in meningeal cma1 levels. Interestingly, systemic administration of the mast cell stabilizer Ketotifen before exposure to adult stress prevents both, reduction in sucrose preference an increases in cma1 expression in NMSEW females, but facilitates stress-induced sucrose anhedonia in NMSEW males and NH females. Finally, histological analyses showed that, compared to males, females have increased baseline activation levels of mast cells located in the transverse sinus of the dura mater, where the meningeal lymphatics run along, and that, in males and females exposed to adult stress, NMSEW increases the number of mast cells in the interparietal region of the dura mater and the levels of mast cell activation in the sagittal sinus regions of the dura mater. Together, our results indicate that ELA induces long-term meningeal immune gene changes and heightened sensitivity to adult stress-induced behavioral and meningeal immune responses and that these effects could mediated via mast cells.

Developmental alterations of intestinal SGLT1 and GLUT2 induced by early weaning coincides with persistent low-grade metabolic inflammation in female pigs.

Early-life adversity (ELA) is linked with the increased risk for inflammatory and metabolic diseases in later life, but the mechanisms remain poorly understood. Intestinal epithelial glucose transporters sodium-glucose-linked transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) are the major route for intestinal glucose uptake but have also received increased attention as modulators of inflammatory and metabolic diseases. Here, we tested the hypothesis that early weaning (EW) in pigs, an established model of ELA, alters the development of epithelial glucose transporters and coincides with elevated markers of metabolic inflammation. The jejunum and ileum of 90-day-old pigs previously exposed to EW (16 days wean age), exhibited reduced SGLT1 activity (by ∼ 30%, P < 0.05) than late weaned (LW, 28 days wean age) controls. In contrast, GLUT2-mediated glucose transport was increased (P = 0.003) in EW pigs than in LW pigs. Reciprocal changes in SGLT1- and GLUT2-mediated transport coincided with transporter protein expression in the intestinal brush-border membranes (BBMs) that were observed at 90 days and 150 days of age. Ileal SGLT1-mediated glucose transport and BBM expression were inhibited by the ß-adrenergic receptor (ßAR) blocker propranolol in EW and LW pigs. In contrast, propranolol enhanced ileal GLUT2-mediated glucose transport (P = 0.015) and brush-border membrane vesicle (BBMV) abundance (P = 0.035) in LW pigs, but not in EW pigs. Early-weaned pigs exhibited chronically elevated blood glucose and C-reactive protein (CRP) levels, and adipocyte hypertrophy and upregulated adipogenesis-related gene expression in visceral adipose tissue. Altered development of intestinal glucose transporters by EW could underlie the increased risk for later life inflammatory and metabolic diseases.NEW & NOTEWORTHY These studies reveal that early-life adversity in the form of early weaning in pigs causes a developmental shift in intestinal glucose transport from SGLT1 toward GLUT2-mediated transport. Early weaning also induced markers of metabolic inflammation including persistent elevations in blood glucose and the inflammatory marker CRP, along with increased visceral adiposity. Altered intestinal glucose transport might contribute to increased risk for inflammatory and metabolic diseases associated with early-life adversity.

Effects of a multi-strain Bacillus subtilis-based direct-fed microbial on weanling pig growth performance and nutrient digestibility.

A study was conducted to evaluate the effects of a multi-strain Bacillus subtilis-based direct-fed microbial (DFM) on growth performance and apparent nutrient digestibility of nursery pigs. Eighty pigs, of equal number of barrows and gilts (initial body weight: 7.0 ± 0.60 kg), were weaned at 21 ± 1 d and randomly allotted to 1 of the 16 pens, with 5 pigs per pen. Two dietary treatments were implemented, a basal control (CON) and a control plus DFM (CDFM). Both diets were corn, soybean meal, and distillers dried grains based. Diets were fed for 42 d and growth performance measures were recorded weekly. On days 21 and 42 of the experiment, one pig per pen, with equal number of males and females, was randomly selected and euthanized. Digestibility of nitrogen (N), amino acids (AA), and energy were evaluated within the duodenum, jejunum, ileum, and ascending and distal colon. Relative to CON, CDFM tended to increase ADG during week 2 (P = 0.08) and significantly increased ADFI during week 2 (P = 0.04) and week 3 (P = 0.02). In addition, CDFM decreased the gain to feed ratio (G:F) during week 6 relative to CON (P = 0.04). Within the jejunum, pigs fed the DFM had greater digestibility of tryptophan (P = 0.04) and cysteine (P = 0.04) and tended to have greater digestibility of lysine (P = 0.07), methionine (P = 0.06), and threonine (P = 0.08), relative to CON. The content pH in the ascending colon did not differ between CDFM and CON. Compared with CON, apparent total tract digestibility (ATTD) of energy did not differ from CDFM, whereas ATTD of nitrogen of CDFM was lower (P = 0.05). The addition of a multi-strain B. subtilis-based DFM appears to impact growth performance, AA, and N digestibility depending upon the location in the gastrointestinal tract, with primary AA differences occurring within the mid-jejunum.

Perinatal androgens organize sex differences in mast cells and attenuate anaphylaxis severity into adulthood.

Mast cell (MC)-associated diseases, including allergy/anaphylaxis and neuroinflammatory pain disorders, exhibit a sex bias, with females at increase risk. While much attention has been directed toward adult sex hormones as drivers of sex differences, that female sex bias in MC-associated diseases is evident in prepubertal children, suggesting early-life origins of sex differences which have yet to be explored. Utilizing rodent models of MC-mediated anaphylaxis, our data here reveal that, 1) compared with females, males exhibit significantly reduced severity of MC-mediated anaphylactic responses that emerge prior to puberty and persist into adulthood, 2) reduced severity of MC-mediated anaphylaxis in males is linked with the naturally high level of perinatal androgens and can be recapitulated in females by perinatal exposure to testosterone proprionate, 3) perinatal androgen exposure guides bone marrow MC progenitors toward a masculinized tissue MC phenotype characterized by decreased concentration of prestored MC granule mediators (e.g., histamine, serotonin, and proteases) and reduced mediator release upon degranulation, and 4) engraftment of MC-deficient Kit W-sh/W-sh mice with adult male, female, or perinatally androgenized female MCs results in MC-mediated anaphylaxis response that reflects the MC sex and not host sex. Together, these data present evidence that sex differences in MC phenotype and resulting disease severity are established in early life by perinatal androgens. Thus, factors affecting levels of perinatal androgens could have a significant impact on MC development and MC-associated disease risk across the life span.

Technical note: Bovine adipocyte and preadipocyte co-culture as an efficient adipogenic model.

Reductionist studies of adipose tissue biology require reliable in vitro adipocyte culturing models. Current protocols for adipogenesis induction in stromal vascular fraction-derived preadipocytes require extended culturing periods and have low adipogenic rates. We compared the adipogenic efficiency of a 7-d co-culture model of visceral (VIS) and subcutaneous (SC) stromal vascular fraction-derived preadipocytes with mature adipocytes with a 14-d standard adipocyte differentiation protocol. We obtained preadipocytes and mature adipocytes from SC and VIS adipose tissue of nonlactating, nongestating Holstein cows (n = 6). Adipogenesis induction was performed using a standard protocol for 7 (SD7; control) or 14 d (SD14), and a co-culture model for 7 d (CC7). Culture conditions, including medium composition, were the same for all treatments. For CC7, 900 primary adipocytes/cm2 were placed in 0.4-µm transwell inserts and co-cultured with preadipocytes for adipogenesis induction. Both CC7 and SD14 similarly stimulated gene expression of adipogenic genes such as ADIPOQ, CEBPA, and CEBPB in VIS and SC. The CC7 increased triacylglycerol accumulation compared with SD14 and SD7. CC7 augmented triacylglycerol accumulation by 40- and 16-fold in SC and VIS compared with 22- and 4-fold increment in SD14, respectively. Lipolytic responses to 2-h ß-adrenergic stimulation with 1 µM isoproterenol were higher in CC7 and SD14 than SD7 in SC; CC7 increased glycerol release compared with SD7 in VIS but SD7 and SD14 had similar responses. Overall, CC7 was more efficient in inducing adipogenesis in preadipocytes from VIS and SC than SD14. Furthermore, CC7 stimulated similar lipolysis and lipogenic responses than SD14 but in a shorter time. The adipogenic approach of co-culturing preadipocytes with mature adipocytes will improve the use of reductionist models to study adipocyte physiology in dairy cows and the assessment of pharmacological or nutritional interventions for enhancing dairy cow health and production.

Perivascular Adipocytes Store Norepinephrine by Vesicular Transport.

Objective- Perivascular adipose tissue (PVAT) contains an independent adrenergic system that can take up, metabolize, release, and potentially synthesize the vasoactive catecholamine norepinephrine. Norepinephrine has been detected in PVAT, but the mechanism of its protection within this tissue is unknown. Here, we investigate whether PVAT adipocytes can store norepinephrine using VMAT (vesicular monoamine transporter). Approach and Results- High-performance liquid chromatography identified norepinephrine in normal male Sprague Dawley rat aortic, superior mesenteric artery, and mesenteric resistance vessel PVATs, and retroperitoneal fat. Real-time polymerase chain reaction revealed VMAT1 and VMAT2 mRNA expression in the adipocytes and stromal vascular fraction of mesenteric resistance vessel PVAT. Immunofluorescence demonstrated the presence of VMAT1 and VMAT2, and the colocalization of VMAT2 with norepinephrine, in the cytoplasm of adipocytes in mesenteric resistance vessel PVAT. A protocol was developed to capture real-time uptake of Mini 202-a functional and fluorescent VMAT probe-in live rat PVAT adipocytes. Mini 202 was taken up by freshly isolated and differentiated adipocytes from mesenteric resistance vessel PVAT and adipocytes from thoracic aortic and superior mesenteric artery PVATs. In adipocytes freshly isolated from mesenteric resistance vessel PVAT, addition of rose bengal (VMAT inhibitor), nisoxetine (norepinephrine transporter inhibitor), or corticosterone (organic cation 3 transporter inhibitor) significantly reduced Mini 202 signal. Immunofluorescence supports that neither VMAT1 nor VMAT2 is present in retroperitoneal adipocytes, suggesting that PVAT adipocytes may be unique in storing norepinephrine. Conclusions- This study supports a novel function of PVAT adipocytes in storing amines in a VMAT-dependent manner. It provides a foundation for future studies exploring the purpose and mechanisms of norepinephrine storage by PVAT in normal physiology and obesity-related hypertension.

Adipogenic potential of perivascular adipose tissue preadipocytes is improved by coculture with primary adipocytes.

Perivascular adipose tissue (PVAT) has the capacity to secrete vasoactive mediators with the potential to regulate vascular function. Given its location adjacent to the vasculature, PVAT dysfunction may be part of the pathophysiology of cardiovascular diseases. To study the mechanisms of PVAT dysfunction, several adipogenic models have been proposed. However, these approaches do not adequately reflect PVAT adipocyte phenotypes variability that depends on their anatomical location. Despite PVAT importance in modulating vascular function, to date, there is not a depot-specific adipogenic model for PVAT adipocytes. We present a model that uses coculturing of PVAT stromal vascular fraction derived preadipocytes with primary adipocytes isolated from the same PVAT. Preadipocytes were isolated from thoracic aorta PVAT and mesenteric resistance artery PVAT (mPVAT). Upon confluency, cells were induced to differentiate for 7 and 14 days using a standard protocol (SP) or standard protocol cocultured with primary adipocytes isolated from the same adipose depots (SPA) for 96, 120, and 144 h. SPA reduced the time for differentiation of stromal vascular fraction derived preadipocytes and increased their capacity to store lipids compared with SP as indicated by lipid accumulation, lipolytic responses, gene marker profile expression, and adiponectin secretion. The coculture system improved adipogenesis efficiency by enhancing lipid accumulation and reducing the time of induction, therefore, is a more efficient method compared to SP alone.

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting.

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the development of hypertension during obesity. The extent of hypertrophy and hyperplasia depends on depot location, sex, and the type of Adipocyte Progenitor Cell (APC) phenotypes present. Techniques used for APC and preadipocytes isolation in the last 10 years have drastically improved the accuracy at which individual cells can be identified based on specific cell surface markers. However, isolation of APC and adipocytes can be a challenge due to the fragility of the cell, especially if the intact cell must be retained for cell culture applications. Magnetic-activated Cell Sorting (MCS) provides a method of isolating greater number of viable APC per weight unit of adipose tissue. APC harvested by MCS can be used for in vitro protocols to expand preadipocytes and differentiate them into adipocytes through use of growth factor cocktails allowing for analysis of the prolific and adipogenic potential retained by the cells. This experiment focused on the aortic and mesenteric PVAT depots, which play key roles in the development of cardiovascular disease during expansion. These protocols describe methods to isolate, expand, and differentiate a defined population of APC. This MCS protocol allows isolation to be used in any experiment where cell sorting is needed with minimal equipment or training. These techniques can aid further experiments to determine the functionality of specific cell populations based on the presence of cell surface markers.

The distribution and adipogenic potential of perivascular adipose tissue adipocyte progenitors is dependent on sexual dimorphism and vessel location.

There are sex associated differences in the risk for cardiovascular comorbidities in obesity and metabolic syndrome. A common clinical finding in these diseases is the expansion of perivascular adipose tissues (PVAT) which is associated with alterations in their role as regulators of vessel function. PVAT hyperplasia and hypertrophy are dependent on the biology of populations of adipocyte progenitor cells (APC). It is currently unclear if PVAT enlargement diverges between males and females and the mechanisms linking APC biology with sexual dimorphism remain poorly understood. This study tested the hypothesis that vessel location and sexual dimorphism affect the distribution and adipogenic capacity of APC in cardiovascular disease risk relevant PVAT sites. PVAT from thoracic aorta (aPVAT) and mesenteric resistance arteries (mPVAT) was collected from 10-week-old female and male Sprague-Dawley rats. Differences in APC distribution in stromal vascular fraction cells from PVAT were determined. APC were defined as cells expressing CD34, CD44, and platelet derived growth factor α In both sexes aPVAT had fewer APC compared to mPVAT and perigonadal adipose tissue (GON). Sex-related differences were observed in the expression of CD34, where females had fewer CD34+ cells in PVATs. APC proliferation and adipogenic capacity in vitro were not affected by sex. However, APC from aPVAT had a lower proliferation capacity compared to mPVAT These data demonstrate that the distribution of APC within PVAT exhibits sexual dimorphism and is affected by anatomical location.

Adipose tissue remodeling in late-lactation dairy cows during feed-restriction-induced negative energy balance.

Excessive rates of demand lipolysis in the adipose tissue (AT) during periods of negative energy balance (NEB) are associated with increased susceptibility to disease and limited lactation performance. Lipolysis induces a remodeling process within AT that is characterized by an inflammatory response, cellular proliferation, and changes in the extracellular matrix (ECMT). The adipose tissue macrophage (ATM) is a key component of the inflammatory response. Infiltration of ATM-forming cellular aggregates was demonstrated in transition cows, suggesting that ATM trafficking and phenotype changes may be associated with disease. However, it is currently unknown if ATM infiltration occurs in dairy cows only during NEB states related to the transition period or also during NEB-induced lipolysis at other stages of lactation. The objective of this study was to evaluate changes in ATM trafficking and inflammatory phenotypes, and the expression of genetic markers of AT remodeling in healthy late-lactation cows during feed restriction-induced NEB. After a 14-d (d -14 to d -1) preliminary period, Holstein cows were randomly assigned to 1 of 2 feeding protocols, ad libitum (AL) or feed restriction (FR), for 4 d (d 1-4). Caloric intake was reduced in FR to achieve a targeted energy balance of -15 Mcal/d of net energy for lactation. Omental and subcutaneous AT samples were collected laparoscopically to harvest stromal vascular fraction (SVF) cells on d -3 and 4. The FR induced a NEB of -14.1±0.62 Mcal/d of net energy for lactation, whereas AL cows remained in positive energy balance (3.2±0.66 Mcal/d of NEL). The FR triggered a lipolytic response reflected in increased plasma nonesterified fatty acids (0.65±0.05 mEq/L on d 4), enhanced phosphorylation of hormone sensitive lipase, and reduced adipocyte diameter. Flow cytometry and immunohistochemistry analysis revealed that on d 4, FR cows had increased numbers of CD172a+, an ATM (M1 and M2) surface marker, cells in SVF that were localized in aggregates. However, FR did not alter the number of SVF cells expressing M1 markers (CD14 and CD11c) or M2 markers (CD11b and CD163). This finding contrasts with the predominately M1 phenotype observed previously in ATM from clinically diseased cows. No changes were observed in the expression of ECMT-related or cell proliferation markers. In summary, an acute 4-d lipolytic stimulus in late-lactation dairy cows led to ATM infiltration with minimal changes in inflammatory phenotype and no changes in ECMT. These results underscore that physiological changes related to parturition, the onset of lactation, extended periods of lipolysis, or a combination of these can induce intense AT remodeling with enhanced ATM inflammatory phenotype expression that may impair the metabolic function of AT in transition dairy cattle.