Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis Julie A. Poposki, MS, Anjeni Keswani, MD,

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Tissue proteases convert CCL23 into potent monocyte chemoattractants in patients with chronic rhinosinusitis Julie A. Poposki, MS, Anjeni Keswani, MD, Julie K. Kim, MD, Aiko I. Klingler, PhD, Lydia A. Suh, BS, James Norton, MS, Roderick G. Carter, BS, Anju T. Peters, MD, Kathryn E. Hulse, PhD, Leslie C. Grammer, MD, Bruce K. Tan, MD, David B. Conley, MD, Juan C. Jaen, PhD, Thomas J. Schall, PhD, Robert C. Kern, MD, Atsushi Kato, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 4, Pages 1274-1277.e9 (April 2016) DOI: 10.1016/j.jaci.2015.09.029 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Posttranslational modification by NP trypsin-like serine proteases controls CCL23 activity. A-E, Recombinant CCL23 was incubated with 1 mg/mL NP extracts, 1 mg/mL BSA (control), supernatants from activated mast cells (MC), neutrophils (Neut), eosinophils (Eos), M1 macrophages (M1) and M2 macrophages (M2), tryptase βII, or skin chymase for 6 (Fig 1, A and C-E) or 0 to 24 (Fig 1, B) hours. CCL23 was incubated with NP extract in the presence of DMSO, PIC, aprotinin (Apr), nafamostat mesylate (Naf), leupeptin (Leu), E-64, pepstatin A (PepA), K579, marimastat (Mar), UK370106 (UK), SSR69071 (SSR), TLCK, or tosyl phenylalanyl chloromethyl ketone (TPCK) for 6 hours (Fig 1, C). CCL23, cleaved CCL23, and CCL23 (46-120) were detected by means of Western blotting with an anti-CCL23 antibody. Results are representative of 3 separate experiments with separate donors (Fig 1, B-D). F, THP-1 cells were incubated with NP-treated CCL23 (solid circles), the same concentration of NP extracts without CCL23 (solid squares), or BSA-treated CCL23 (solid triangles) for 2 hours by using a microchemotaxis plate (n = 4). RFU, Relative fluorescence units. *P < .05, 1-way ANOVA, compared with medium control (Fig 1, F). Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 CCL23 (47-117) is an active CCL23 metabolite in NP extracts. NP extract–treated CCL23 proteins were separated by using SDS-PAGE. A, N-terminal protein sequences of each product were detected by using an Edman-based sequencer. B, CCL23 was incubated with NP extracts for 1 to 6 hours. Truncated products were detected by using MALDI-TOF mass spectrometry. C, Summary table of identified truncated products assigned by comparison of measured with predicted m/z values by using MALDI-TOF mass spectrometry and N-terminal sequencing. D, Protein sequence of CCL23. Arrows indicate identified cleavage sites. Red color indicates the sequence of the potential major active metabolite CCL23 (47-117) in NPs. Results are representative of 3 separate experiments with separate donors (Fig 2, A-C). E, THP-1 cells were incubated with CCL23, CCL23 (46-120), or CCL23 (47-117) for 2 hours by using a microchemotaxis plate (n = 4). F, THP-1 cells were incubated with 1 nmol/L CCL23 (47-117) for 2 hours by using a microchemotaxis plate in the presence of 0.02% DMSO (vehicle control) or 100 nmol/L CCX8960 (n = 3). RFU, Relative fluorescence units. *P < .05, 1-way ANOVA, compared with medium control (Fig 2, E). Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Trypsin-like serine protease inhibitor blocks truncation of CCL23 by NP extracts and tryptase. Recombinant CCL23 (2 μg/mL) was preincubated with 1 mg/mL NP extracts (A) or 15 μg/mL recombinant tryptase βII (B) in the presence or absence of 200 μmol/L TLCK or 200 μmol/L TPCK for 3 hours. CCL23 and cleaved CCL23 were detected by means of Western blotting with an anti-CCL23 antibody. Results are representative of 3 separate experiments. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 NP-treated CCL23 has potent chemoattractant activity for PBMCs. CCL23 (50 μg/mL) was preincubated with 1 mg/mL NP extracts for 6 hours. The migration assay was performed in a 96-well microplate format FluoroBlok microchemotaxis system (3-μm pore, BD Biosciences). PBMCs were labeled with Calcein AM dye and resuspended in phenol red–free RPMI-1640 containing 0.1% BSA. Labeled PBMCs were incubated with 200 ng/mL (A) or 10 to 200 ng/mL (B) CCL23 (blue symbols), NP-treated CCL23 (red symbols), the same concentration of NP extracts without CCL23 (open symbols), or medium alone (black symbols) for 0.5 to 4 (Fig E2, A) or 2 (Fig E2, B) hours by using a microchemotaxis plate (n = 5). Migrated cells were analyzed with a SpectraMAX Gemini EM microplate reader (excitation at 494 nm and emission at 517 nm). *P < .05, 1-way ANOVA, compared with medium control. RFU, Relative fluorescence units. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 NP-pretreated CCL23 has potent chemoattractant activity for THP-1 cells through CCR1. A, Recombinant CCL23 (5 μmol/L) was preincubated with 1 mg/mL NP extracts for 0 to 24 hours, and then THP-1 cells were incubated with 1 nmol/L NP-treated CCL23, the same concentration of NP extracts without CCL23, and 1 nmol/L untreated CCL23 for 2 hours by using a microchemotaxis plate (n = 5). B, THP-1 cells were incubated with 1 nmol/L NP-treated CCL23 and 1 nmol/L CCL2 for 2 hours by using a microchemotaxis plate in the presence of 0.02% DMSO (vehicle control) or 100 nmol/L CCX8960 (a CCR1-specific antagonist, n = 3). *P < .05, 1-way ANOVA, compared with medium control. RFU, Relative fluorescence units. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Serine protease inhibitor blocks truncation of CCL23 by NP extracts. CCL23 was incubated with NP extract in the presence of 1% DMSO (vehicle control) or 10 μmol/L nafamostat for 6 hours. Truncated products were detected by using MALDI-TOF mass spectrometry. The x-axis of the mass spectra represents m/z. NP indicates proteins contained in the NP extracts. Numbered peaks refer to corresponding amino acid residues of CCL23. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Synthesis of recombinant CCL23 (47-117). Recombinant CCL23 (47-117) was synthesized at BioLegend. Purity of recombinant CCL23 (47-117) was greater than 98% analyzed by using SDS-PAGE (A) and HPLC (not shown). B, Molecular mass was detected as 8014.9 by using ESI-TOF mass spectrometry. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 CCR1 is expressed on macrophages in NPs. We identified the population of macrophage subsets in NPs with the following steps. We first selected singlets (by using forward scatter [FSC]-A/FSC-W and side scatter [SSC]-A/SSC-W), excluded dead cells (Aqua+), selected the CD45+ population, excluded granulocytes (SSChigh), and then selected CD68+ macrophages. A, Representative flow cytometric plots for M1 macrophages (CD163−CD206−) and M2 macrophages (CD163+CD206+, n = 3). B, Expression of CCR1 was determined on M1 and M2 macrophages. FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 CCR1 is expressed on mDCs in NPs. We identified the population of DC subsets in NPs with the following steps. We first selected singlets (by using forward scatter [FSC]-A/FSC-W and side scatter [SSC]-A/SSC-W), excluded dead cells (Aqua+), selected the CD45+ population, and then removed granulocytes (SSChigh) and B cells (CD19+). We then identified DC subsets by the following markers: mDC1 (CD1c+CD11c+), mDC2 (CD141highCD11c+), and plasmacytoid dendritic cells (pDC; CD303+). CCR1 expression was determined on mDC1s, mDC2s, pDCs, and CD19+ B cells (negative control). FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology 2016 137, 1274-1277.e9DOI: (10.1016/j.jaci.2015.09.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions