(D-144) Development of responsive element luciferase probes to measure real-time macrophage polarization

Introduction:: Alternatively activated macrophage or M2 macrophage, in response to inflammatory stimuli, plays a pivotal role in wound healing, immunosuppression and tumor progression. STAT6-mediated signaling pathway, activated by interleukin 4(IL-4)/IL-13, drives the alternative macrophage polarization. STAT6 functions by forming a phosphorylated homodimer and binding onto STAT6-responsive element within promoters to regulate anti-inflammatory associated genes. Measuring STAT6 activity can be a proxy for measuring macrophage polarization. However, cellular probes that enable real-time measurement of macrophage polarization are needed. Specifically, we developed a STAT6-RE THP1 reporter that allows us to access STAT6 activation in response to inflammatory stimuli. Combined with bioluminescence temporal spectroscopy (BTS) we can quantitively measure macrophage polarization. In this study, THP-1 monocytes were lentivirally transduced to express the STAT6-RE-FLuc-GFP. We demonstrate that reporter is activated by only by phosphorylated STAT6 via immunohistochemistry and ELISA. Importantly, the STAT6-RE can accurately report the dynamics of endogenous STAT6 activity. The results indicated a novel probe that can be used to assess macrophage alternative polarization. These experiments allow for a better understanding of the association between molecular mechanisms and macrophage polarization, facilitating the development of therapeutic strategies on manipulating macrophages for attenuating inflammation and tumor progression as well as promoting wound healing.


Materials and Methods:: (1) (1) Vector construct and molecular cloning: stat6-responsive element(RE) lentivector is constructed through PCR cloning (2) Lentiviral particle production and transduction: lentivirus containing stat6-RE vector was generated through co-transfecting packaging plasmids pCMV-p8.9, pMDG-VSVG and shuttle plasmid pLV- STAT6-RE-rFluc-GFP into HEK 293T cells. After lentiviral transduction on wildtype THP1 cells, puromycin selection was performed to screen GFP positive cells (3) Bioluminescence temporal spectrometry: to measure the bioluminescence signal from THP1 STAT6-RE macrophage activation, cells were seeded in a white clear bottom 96-well plate with 10ng/ml PMA. After the media was changed to 1mM D-luciferin supplemented RPMI complete media, bioluminescence signals were measured in the multimode microplate reader (Spark) at a 12-hour interval. (4) Immunocytochemistry: ICC was performed to visualize the distribution of p-stat6 in STAT6-RE THP1 cells with tofacitinib or leptomycin during IL4-induced activation. (5) ELISA: p-stat6 ELISA was performed to quantitively measure the p-stat6 level during IL-4 activation with or without inhibitor treatments. (6) Western blotting: p-stat6 protein expression level was measured between WT and stat6-RE THP1 during IL4 activation. (6) BTS on STAT6-RE THP-1 cell line can be used for studying tumor associated macrophage development.

Results, Conclusions, and Discussions:: Result:

1.STAT6-RE THP1 cell line has comparable transcriptomic profile and unimpaired stat6 signaling pathway compared to the wildtype THP1 cell line

2.Bioluminescence temporal spectrometry on STAT6-RE THP1 cell line can indicate STAT6-dependent M2 polarization

3.STAT6-RE THP-1 cell line can assess the inhibitors that target the STAT6 signaling pathways

4. The reporter cell is used for measuring TAM M2 polarization in A549 lung cancer models and interruption of STAT6 signaling attenuated the M2 polarization.

5. STAT6-RE luciferase reporter system can be applied on primary macrophages.

Conclusion and Discussion:

Due to the dynamic and sensory nature of macrophage, it is a great challenge to characterize macrophage polarization states. Current phenotyping techniques such as flow cytometry to techniques such as RNA sequencing and proteomics fail to characterize macrophage polarization in the equal dynamic way. In our study, we established the STAT6-RE luciferase reporter cells that have comparable transcriptomic profiles and intact molecular machinery as the wildtype THP1 cells. By performing BTS on STAT6-RE THP-1 cells in different biological contexts, we can measure the M2 polarization in real time. Therefore, STAT6-RE THP-1 cell line provides a promising platform for characterizing macrophage behaviors in diseased models and screening chemical compounds that target M2 macrophages.


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