Data on sulforaphane treatment mediated suppression of autoreactive, inflammatory M1 macrophages

Any chronic, inflammatory, autoimmune disease (e.g. arthritis) associated pathogenesis directs uncontrolled accumulation of both soluble forms of collagens in the synovial fluids and M1 macrophages around inflamed tissues. Despite of few studies demonstrating efficiency of Sulforaphane (SFN) in suppressing arthritis associated collagen restricted T cells or fibroblasts, its effects on macrophage polarity and plasticity are less understood. Recently, we reported regulation of phenotypic and functional switching by SFN in induced and spontaneously differentiating human monocytes [1]. Here, flow cytometry, western blot and ELISA derived data demonstrated that SFN inhibited in vitro inflammatory responses developed by soluble human collagens (I–IV) induced auto-reactive M1 type monocyte/macrophage model.


Value of the data
These in vitro data provide the information on soluble form of human collagen induced functional M1 polarization in PMA differentiating THP1 monocytes.
These data indicate the efficiency of SFN to bring about phenotypic and functional changes in autoreactive M1 cells.

Generation of conventional M1, auto-reactive M1 and immune-suppressive (M2) macrophages
Human monocytic cell line, THP1 was selected for the experiments due to its ability to differentiate into M1 or M2 type macrophages depending on external stimuli [8][9][10]. About 0.5 Â 10 6 THP1 monocytes were treated with 20 ng/ml PMA alone for initial 6 h and then treated (in presence of PMA) with LPS (100 ng/ml) and IFNγ (20 ng/ml) together (to derive conventional inflammatory M1 cells) or 1 mg/ml soluble collagens I-IV (biomarker of chronic arthritic diseases [11,12]) alone or with SFN alone for another 24-48 h to polarize THP1 monocytes differentiation. The work plan is described in Scheme 1.
(Biorad,USA). β-actin was also detected in all the experimental conditions to ensure uniform protein loading.

Cytokine measurement
Using sandwich ELISA, M1 and M2 specific cytokine production was measured in the supernatants isolated from each experimental matured macrophages. For this purpose, BD OptEIA™ (BD biosciences) containing sets for both IL-12p70 (M1 type, Cat no. 555183) and IL-10 (M2 type, Cat no. 555157) were used as per the manufacturer instructions to quantify the cytokine levels simultaneously. The optical densities at 450 nm were recorded using Biorad plate reader and compared with a standard curve generated for each cytokine to determine the concentration in each of the experimental samples.

Surface markers expression
Surface marker expressions on the cells belong to each experimental set up (in presence and absence of indicated external agents) were analyzed by flow cytometry using FACS CaliburFlow Cytometer (BD Biosciences). Following treatment, the cells were harvested, washed and a single-cell suspension were prepared using ice cold PBS. Cells were then washed before and after staining with 500 mL of PBS containing 0.1% BSA and 0.02% sodium azide (FACS buffer). These cells were then labeled with specific monoclonal antibodies for 30 min in 50 mL of FACS buffer. About 10,000 events per sample were acquired and the mean fluorescence intensity (MFI) of cells staining positive for each surface protein was determined by comparing test samples with unstained, negative control samples.