Developing a method of co‐incubating murine adipocytes and macrophages for evaluating cellular communication

Abstract

Adipose tissue, an organ containing both adipocytes and adipose tissue macrophages (ATMs), secretes cytokines known as adipokines. Of these two cell types, ATMs have a larger role in the production and release of proinflammatory adipokines. The objective of this research was to develop a system of co‐incubating adipocytes and macrophages that allowed us to quantitatively measure the cross‐talk between these two cell types. Nitric Oxide (NO) and interleukin‐6 (IL‐6) concentrations were quantified to determine macrophage activation and production of a proinflammatory adipokine, respectively. Leptin concentrations were used as a measure of adipocyte‐specific adipokine production. The cell lines used were RAW 264.7 (murine macrophages) and 3T3‐L1 (murine fibroblasts). 3T3‐L1 fibroblasts were differentiated into adipocytes, which was confirmed by the presence of lipid droplets. Transwell plates with permeable inserts were used to co‐incubate the macrophages and adipocytes; the confluent adipocytes were maintained in the 12‐well plate (lower compartment) and the macrophages were added to the transwell insert (upper compartment). Macrophages were stimulated with 0.01 mg/ml of LPS either before or after the addition of the the macrophages to the insert. The treatments were: 3T3+media, 3T3+media+LPS, 3T3+unstimulatedRAW, 3T3+RAW+LPS, 3T3+stimulatedRAW (in fresh media), and 3T3+stimulatedRAW (in conditioned media). There was no difference in IL‐6 concentration measured in the upper compartment of the transwell insert vs the lower compartment, while measured NO levels were higher in the upper compartment and leptin levels were higher in the lower compartment (p<0.001, matched pairs t‐test). The 3T3+RAW+LPS, 3T3+stimulatedRAW (in fresh media) and 3T3+stimulatedRAW (in conditioned media) co‐incubation protocols resulted in the highest concentration of IL‐6 (p=0.0006). The 3T3+RAW+LPS and 3T3+stimulatedRAW (in conditioned media) co‐incubation methods yielded the highest concentrations of NO in both the upper and bottom compartments of the plate (p<0.001). There was no difference in leptin concentrations due to co‐incubation protocol. There was a positive relationship between NO and IL‐6 in both the top and bottom compartments (p<0.001, linear regression) and between NO and leptin in the top (p=0.03) but not in the bottom compartment. There was no significant relationship between IL‐6 and leptin concentrations in either the top or bottom compartments. For future experiments, our lab will utilize the co‐incubation protocols that resulted in the highest production of NO and IL‐6: 3T3+RAW+LPS and 3T3+stimulatedRAW (in conditioned media).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.