James A Ankrum

Abstract Description
 
We investigated whether exposure to polychlorinated biphenyls (PCBs) induces macrophage phenotypic switching toward an inflammatory phenotype. Human exposure to PCBs, an environmental toxicant, has been linked to an increased risk of diabetes, obesity, and metabolic syndrome. Despite the key role of adipose macrophages in metabolic disease and the known accumulation of PCBs in adipose, little is known about how PCBs impact macrophages. Here, we exposed anti-inflammatory macrophages (i.e. polarized with IL-4 or dexamethasone) to PCBs and then profiled phenotypic shifts by assessing surface marker expression, protein secretion, and energy metabolism. Our results show that PCB-exposed macrophages exhibited decreased expression of anti-inflammatory surface markers CD163 and CD206, while expression of inflammatory marker CD86 remained unchanged. PCB exposure also resulted in a marked 400x-1000x increase in the inflammatory cytokine IL-8 as well as a significant reduction in the anti-inflammatory cytokine IL-10. Additionally, we found that PCB-exposed cells had a greater dependence on aerobic glycolysis and a reduced ability to utilize fatty acid and amino acid oxidation as fuel – both metabolic features of more inflammatory macrophages. Collectively, these results demonstrate that PCBs promote macrophage plasticity toward a more inflammatory phenotype. More broadly, our work suggests that PCBs amplify metabolic diseases by altering the inflammatory environment of adipose tissue.

Adipose tissue is a potential source of cells for several types of cell therapy including MSCs, adipocytes, and macrophages. However, adipose tissue cellular composition and the function of the cells within adipose is heavily influenced by the metabolic health of the individual. With obesity rates reaching epidemic levels in the United States, it is important to understand how changes in metabolic state impact the function of adipose derived cells. To facilitate this work, we sought to create an in vitro model of adipose that incorporates adipocytes, MSCs, and macrophages. Our aim was to develop a co-culture technique to identify the influence of macrophage-adipocyte crosstalk on mature adipocyte function, adipose-derived MSC potential, and adipocyte influences on macrophage phenotype.
Stromal vascular fraction from surgical adipose tissue was culture expanded and characterized for MSC markers. MSC organoids were formed via agarose micromolds containing 35 microwells to form 10,000 cell organoids. The organoids were differentiated with adipogenic media for 14 days. Human monocytes were isolated from leukocyte reduction cones, differentiated into naïve macrophages, and polarized to either pro-inflammatory (M1-like) or anti-inflammatory (M2-like) states. To study mature adipocyte-macrophage cross-talk, micromolds were transferred to macrophage wells, cultured within agarose punchouts to control for macrophage seeding density and cell ratios. Media was collected for secretome analysis, and spheroids were stained and imaged through confocal microscopy for lipid content.
The secretome of macrophage/adipocyte co-cultures show distinct profiles between each cell type, and therefore, inflammatory environment. While both macrophage types caused diminished adiponectin production by adipocytes along with induced lipolysis without the presence of catecholamines, the presence of adipocytes lead to a decrease of pro-inflammatory cytokines with M2-like macrophages, yet an increase with M1s.
This work demonstrates the impact of adipose health on the quality of cells for cell therapies. Chronic inflammation impairs adipogenesis and impacts all the cells within adipose. Future studies need to be performed to identify the influence of inflammatory environment on MSC health, along with repeating further studies with additional donors.

The immunosuppressive activity of human mesenchymal stromal cells (MSCs) is central to the approved and investigational use of MSC therapy in inflammatory conditions such as GvHD. External factors like cytokines and other molecules encountered in the disease microenvironment significantly influence MSC immunosuppressive function. The obese microenvironment including elevated levels of serum free fatty acids, specifically palmitate, have the potential to affect MSC therapy. For T cell suppression, it has already been shown that exposure to palmitate impairs the ability of MSCs to carry out their function. However, we do not yet understand the effects of palmitate on MSC immunosuppression of macrophages. This study focused on investigating the influence of palmitate on MSC functional capacity to suppress macrophages and to elucidate the mechanisms of action at play.
We used peripheral blood from healthy control and patients with obesity to investigate the immunosuppressive capacity for MSCs or MSCs exposed to plamitate in vitro. Following exposure to palmitate, the capacity for MSCs to alter human monocyte derived macrophage (MDM) production of TNFa and IL-10 and phenotype following LPS stimulation were investigated. Moreover, chemical anatagonists, neutralising antibodies and a biologically active, cell-permeable ceramide analog were used to uncover the mechanisms of action.
Palmitate exposure significantly increased expression of the MSC immunomodulatory factors ptgs2, il-6, ccl2 and angptl4. Palmitate exposed MSCs had significantly modulated LPS stimulated human MDMs leading to decreased TNFα and increased IL-10 production by MDMs. Using a neutralising antibody, we identified that enhanced suppression mediated by palmitate exposed MSCs involved CCL2. Using the biologically active, cell-permeable ceramide analog C2 as well as the cermide synthases inhibitor (fumonisin B1), we showed that palmitate enhanced MSC immunomodulation of MDMs via activating ceramide de novo synthesis.
Palmitate has a beneficial effect on macrophage immunomodulation by MSCs, suggesting that a high concentration of palmitate in the microenvironment likely does not negatively affect MSC therapy in conditions where MSC -macrophage interactions are central to MSC mode of action.

Interest in macrophages as a cell therapy is growing rapidly for the treatment of stroke, cardiomyopathy, and other indications. These therapies rely on in vitro polarization of macrophages before administration into the patient. While in vitro polarization toward an inflammatory or anti-inflammatory phenotype is effective, macrophages remain plastic and able to adapt to their environment upon transplantation. In this work, we aimed to uncover how in vitro polarized human macrophages would respond to exposure to a mixture of common environmental toxicants, Aroclor 1254. Aroclor 1254 is a mixture of polychlorinated biphenyls that were manufactured and used heavily in building materials and electrical equipment globally until its production was banned in the late 1970s. As a forever chemical, signatures of Aroclor 1254 are still commonly found in buildings, food, and people, where it can significantly alter biological processes.
Primary human monocytes were isolated from leukocyte reduction cones and purified using a monocyte negative selection kit. The monocytes were then differentiated into macrophages using M-CSF and then polarized to M1 (IFNg+LPS), M2a (IL-4), or M2c (Dexamethasone) macrophages. After in vitro polarization, each type of macrophage was challenged with exposure to either vehicle (DMSO) or 10 uM of Aroclor 1254 for 48 hours. Macrophages were then analyzed for changes to surface marker expression, cytokine production, and metabolic profile using a SCENITH assay.
Our results show that acute exposure of in vitro polarized macrophages to Aroclor 1254 significantly alter macrophage phenotype toward an inflammatory state. Specifically, levels of IL-10, CD163, and CD206 (anti-inflammatory markers) dropped significantly while production of inflammatory cytokines, such as IL-8, increased dramatically (Fig. 1A). When we analyzed the metabolic profile of macrophages after exposure to Aroclor 1254, we found they were more dependent on glycolytic processes (Fig. 1B) and less able to adapt to fatty acids or amino acid oxidation as a fuel source (Fig. 1C).
This work demonstrates that in vitro polarized macrophages are highly plastic and the signals they encounter post-transplantation can significantly alter their phenotype. In this work, we specifically showed that acute exposure to a common part of the exposome, polychlorinated biphenyls, can significantly alter the phenotype and secretome of macrophages by inducing an immunometabolic shift.

Perilipin 2 (PLIN2) is the major structural protein of triglycerides (TG) storing lipid droplets (LD) and increased in lipid loaded and type 2 diabetic pancreatic beta cells. PLIN2 is protective for beta cells since its knockdown increases fatty acid (FA) flux to mitochondria and impairs mitochondrial function. Here, we addressed a pathway by which PLIN2 deficiency compromises mitochondrial integrity in beta cells. When the incorporation of Bodipy C12 (C12, a fluorescent long chain FA analog) into organelles was followed in INS1 cells over 8 h, C12 incorporation into LD was seen in both control and shPLIN2 cells during the first hour indicating that PLIN2 downregulation does not affect the initial triglycerides (TG) synthesis and its incorporation into LD. C12 started to accumulate into mitochondria after 4 to 8 h only in shPLIN2 cells indicating that FA transfer to mitochondria occurs after LD formation under PLIN2 deficiency. Treatment with a lysosomal lipase inhibitor Lali2 increased TG level and prevented C12 transfer into mitochondria in shPLIN2 cells, indicating that lysosomal degradation of LD precedes FA transfer to mitochondria. Microlipophagy appears to mediate LD degradation in shPLIN2 cells since a macroautophagy blocker 3MA did not prevent FA flux to mitochondria. Importantly, Lali2 rescued mitochondrial fragmentation (p<0.05 vs DMSO control) and glucose-stimulated insulin secretion (p<0.05) in shPLIN2 INS1 cells. Mitochondrial fragmentation and its rescue by Lali2 were also seen in shPLIN2 treated human beta cells (p<0.05). As for a mechanism mediating FA transfer to mitochondria, the direct contact between lysosome and mitochondria was increased (p<0.01) before FA flux to mitochondria occurs in shPLIN2 cells. Collectively, PLIN2 deficiency in beta cells impairs mitochondrial integrity through lysosomal TG degradation followed by FA delivery to mitochondria. These data highlight the importance of regulation of microlipophagy by PLIN2 for beta cell function. Disclosure S. Liu: None. A. Joglekar: None. A. Freshly: None. S. Peachee: None. I.J. Wipf: None. C. Bovee: None. A. Vikram: None. M. Giedt: None. B. Fink: None. W. Sivitz: None. J.A. Ankrum: None. T. Tootle: None. Y. Imai: None. Funding R01 DK090490 (NIH) I01 BX005107 (VA)

Lipid droplets (LDs) , organelles important for intracellular lipid metabolism, are actively formed in rat and human adult beta cells and affect islet function and health. Acute mobilization of LDs by adipose triglyceride lipase in beta cells is known to support insulin secretion. As LD degradation by lysosomal acid lipase (LIPA) through lipophagy is an alternative pathway for LD catabolism, we addressed whether LIPA regulates beta cell function. LDs in human beta cells are associated with autophagy (LC3) and lysosomal (LAMP1) markers. LIPA suppression via siRNA and LIPA inhibitor (lalistat2) in INS1 cells increased LD size (DMSO 0.5 vs. Lali2 1.5 µm2/cell, p<0.05, p value by Student's t test unless specified otherwise) and number (Scr 2/cell vs. siLIPA 14/cell, p<0.05; DMSO 4.5/cell vs. Lali2 16/cell, p<0.05) . Rat and human beta cells treated with Lali2 also showed increased in LD area and/or number of LDs per cell (p<0.05) . In all three models, LIPA suppression increased TG contents confirming constitutively active lipophagy. While acute inhibition of LIPA by Lali2 had little effect on glucose-stimulated insulin secretion (GSIS) , chronic (over 48 h) LIPA suppression by Lali2 or siRNA reduced GSIS from INS1 cells, rat islets, and human islets. Lentiviral shRNA transduction in human pseudoislets decreased insulin secretion in response to glucose (41 ± 10.5% of Scr, p<0.05) and to KCl (61.3 ±16.1% of Scr, p<0.05) in vitro. When Scr and shLIPA human pseudoislets were transplanted under kidney capsules of NSG mice on Western diet, human insulin secretion in response to intraperitoneal glucose load was blunted in mice carrying shLIPA islets compared with shScr control (2-Way ANOVA, p<0.05) . In summary, lipophagy is constitutively active in human and rat beta cells and serves as a pathway for LD catabolism that is acutely insensitive to glucose. However, LIPA mediated LD catabolism is needed for beta cell function in a long-term.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.