Membrane and cytosolic fractions were isolated from THP1 M0 cells using a Mem-PERTM Plus membrane protein extraction kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. To precipitate proteins, 15% (v/v) TCA was added to conditioned media samples and centrifuged, and the protein pellet was washed with 100% acetone. initiated secretion of tumor necrosis factor (TNF) and CXCL8 (IL8) from macrophages. We also demonstrated that WRS is a potent monocyte chemoattractant. Of note, AZD4017 WRS increased matrix metalloproteinase (MMP) activity in AZD4017 the conditioned medium of macrophages in a TNF-dependent manner. Using purified recombinant proteins and LC-MS/MS to identify proteolytic cleavage sites, we demonstrated that multiple MMPs, but primarily macrophage MMP7 and neutrophil MMP8, cleave secreted WRS at several sites. Loss of the WHEP domain following cleavage at Met48 generated a WRS proteoform that also results from alternative splicing, designated 1C47 WRS. The MMP-cleaved WRS lacked TLR signaling and proinflammatory activities. Thus, our results suggest that moonlighting WRS promotes IFN proinflammatory activities, and these responses can be dampened by MMPs. proteomics techniques for the analysis of AZD4017 proteolysis, with quantification enabled by isotope-coded affinity tags (ICAT) (43), isobaric tags for relative and absolute quantification (iTRAQ) (44), or terminal amine isotopic labeling of substrates (TAILS) that identifies the cleavage sites themselves (38, 40). Here, we report the effects of IFN on WRS expression and secretion from macrophages as well as proteolytic processing of WRS by MMPs. We describe how cleavage of the N terminus of WRS to the 1C47 WRS proteoform by MMP7 and MMP8 abrogates its proinflammatory functions, aligning with other well-documented anti-inflammatory activities of MMPs. Results Secretion of WRS is induced by IFN To investigate potential inflammatory roles of WRS, we profiled the secretion of WRS in response to cytokine stimulation of macrophages. THP1 monocytes were differentiated to a macrophage-like phenotype (THP1 M0) using phorbol 12-myristate 13-acetate (PMA). After treatment with IFN (20 ng/ml), TNF (40 ng/ml), or IL4 (40 ng/ml), we collected cytosolic, membrane, and conditioned medium fractions. Immunoblot analysis using antibodies recognizing the N or C terminus of WRS (N-WRS and C-WRS, respectively) showed that only the proinflammatory IFN increased cytosolic WRS levels (= 3; Fig. 1= 3; Fig. S1and = 3 independent experiments, = 2 for fibroblasts. Original uncut immunoblots can be seen in Fig. S6, = 3; Fig. 1, and = 2; Fig. 1= 3; Fig. S1= 3; Fig. 2, and for images of the uncut original immunoblots). Relative band densities were plotted as means S.D. of = 3 independent experiments. and = 2 independent experiments) in the conditioned media of THP1 M0 cells treated for 3 h with increasing concentrations of WRS (scatter plots with mean S.D. shown, = 4) (1C47 WRS (100 nm each), heat-denatured WRS AZD4017 (100 C WRS), or buffer (scatter plots with means S.D., = 4) (= 4) of = 2 independent experiments. and = 3) of = 2 and = 3 independent experiments for and and using a one-way Rabbit Polyclonal to GJC3 ANOVA with Dunnett’s multiple comparison posttests. *, 0.05; **, 0.01; ***, 0.001; represent S.D. By ELISA, we showed that WRS stimulated TNF release from THP1 M0 cells, with maximal release at 100 nm WRS (= 2; Fig. 2= 3; Fig. 2= 2; Fig. 2= 2; Fig. 2= 3; Fig. 2= 2; Fig. 3and and = 4) of = 2 independent experiments; TLR9, = 1. shown, = 4) of = 2 independent experiments. Statistical significance was determined as follows: test. ***, 0.001; represent S.D. The NF-B pathway inhibitor BAY11-7082 (10 m); the IB kinase inhibitor (47, 48) C29 (100 m), a specific inhibitor of TLR2 signaling that disrupts the intracellular recruitment of myeloid differentiation primary response gene 88 to TLR2 (49); and CLI-095 (1 g/ml), a cyclohexane derivative that disrupts the intracellular domain of TLR4 (50, 51) all blocked TLR activation in the WRS-treated reporter cell AZD4017 lines (= 2; Fig. 3, and = 2; Fig. 3, and = 2; Fig. 4= 2; Fig. 4shown, = 4 of = 2 independent experiments. Statistical significance was determined against vehicle using a one-way ANOVA with Dunnett’s multiple comparison posttests in and between each isotype control and antibody using a two-tailed unpaired Student’s test in 0.05; ***, 0.001; represent S.D. We had removed lipopolysaccharide (LPS), a potent heat-stable proinflammatory TLR stimulator, from our ClearColi?.