Soluble CEACAM8 secreted by human granulocytes inhibits TLR2-dependent proinflammatory immune response in human bronchial epithelium Bernhard B. Singer*, Lena Opp*, Luis Berrocal, Kerstin Heyl, Annina Heinrich, Hortense Slevogt
Septomics Research Center, Jena University Hospital and Friedrich Schiller University Jena Albert-Einstein-Str. 10, 07745 Jena, Germany, www.septomics.dewww.septomics.de
*) contributed equally
Introduction III V
Chronic obstructive lung disease (COPD) is characterized by a b c d neutrophilic inflammation in the human airways that is associated with pathological bacterial colonization in the lower airways of affected patients. In recent unpublished work we demonstrated that the soluble form of CEACAM8, solely expressed on human granulocytes, binds to CEACAM1 on human bronchial epithelium. We hypothesized that binding of soluble CEACAM8 to CEACAM1 is able to inhibit TLR2 receptor signaling on bronchial epithelium as it has been recently demonstrated for the CEACAM1 binding pathogens Moraxella catarrhalis or Neisseria meningitides1. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine- based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated TLR2–dependent Fig.5) Co-stimulation of Pam3cys and sCEACAM8 leads to a CEACAM1-dependent activation of the phosphatidylinositol 3-OH kinase-Akt kinase pathway. downregulation of TLR2-triggered immune response in A549 cells (a) (b) IL-8 released into the supernatants of A549 cells preincubated for 1 h with anti-CEACAM1 (Ag18/20) or left The aim of our study was to investigate the regulation of CEACAM8 untreated or stimulated for 16h with Pam3Cys alone or with Pam3Cys and sCEACAM8 (both expression and secretion by human granulocytes as well as the role of 100 ng/ml) measured by ELISA. (b) Viability of A549 cells 16 h after infection with Pam3Cys or the sCEACAM8 - CEACAM1 interaction on human bronchial with Pam3Cys and sCEACAM8 or left untreated, assessed by annexin V and propidium iodide staining and presented as fold of the viability of uninfected control cells. (c) Expression of epithelium. CEACAM1 after gene-silencing in A549 cells left transfected for 96h with CEACAM1-targeting or control siRNA (csi). (d) IL-8 measured by ELISA in supernatants of A549 cells transfected for 96 h CEACAM1-specific or with control siRNA, and infected for 16 h with Pam3Cys alone or a combination of Pam3Cys and sCEACAM8 (both 100 ng/ml). (f) IL-8 in Data are from one representative of three independent experiments. Data presented are mean ± s.e.m. of three different experiments performed in triplicates Data are from one representative of three independent experiments.*, P <0.05.
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I Slevogt H. Nature Immunology 2008
Granulocytes [%] Fig.3) CEACAM8 expression on human granulocytes is regulated by TLR7/8 whereas Fig. 6) sCEACAM8 reduces the TLR2-dependent M. catarrhalis and H. influenzae- of all mononuclear 0,5-3,9% 4,0-14,9 % 15,0-95,0 % soluble CEACAM8 secretion is TLR9-dependent. (a) Expression of CEACAM8 on the induced epithelial immune response by interacting with CEACAM1 (a) IL-8 in cells in the BALF surface of granulocytes measured by flow cytometry and then treated for 14 hours with supernatants of A549 left untreated or incubated for 16h with wildtype non-typeable H. 20ng/ml PMA, 5ng/ml TNFα, 10 µg/ml Pam3Cys (P3C), 100 ng/ml Poly(I:C) (P:IC), 1 µg/ml influenzae strain (NTHi1128.wt) and with the P5 deficient mutant NTHi1128f- alone (that does Flagellin (Fl), 10 µg/ml Resiquimod-848 (R848), 100 µg/ml unmethylated CpG (CpG), 10 not interact with CEACAM1) or a combination of NTHi1128f- and 100ng/ml sCEACAM8 Presence of sCC8 in µg/ml Peptidoglycan (PGN) und 10 µg/ml Muramyl Dipeptide (MDP). (b) ELISA of soluble measured by ELISA. (b) IL-8 in supernatants of A549 infected for 16h with M. catarrhalis 31,3 / 68,7 41,9 / 58,1 82,1 / 17,9 p < 0,001 CEACAM8 in supernatants of granulocytes treated as described in (a). (c) IL8 ELISA from BALF (yes / no) in % wildtype strain (M.cat.wt) and the USPA1-deficient mutant strain M.cat.∆UspA1 alone that supernatants of granulocytes treated the same way as in (a). (d) Flow cytometry of the does not interact with CEACAM1 or in combination with sCEACAM8 (100ng/ml). Data expression of CEACAM8 on the surface of granulocytes incubated with M. catarrhalis wildtype presented are mean ± s.e.m. of three different experiments performed in triplicates*, P <0.05 strain (M.cat. wt.) BBH18 und Non-typable H. influenza wildtype strain (NtHi wt) 1128. (e) Fig.1) In human bronchial lavage fluid (BALF) soluble CEACAM8 occurs in relation to sCEACAM8 ELISA assessed in supernatants of granulocytes treated the same way as in the amount of granulocytes. Human bronchial lavage fluid was tested for the presence of figure 2d. Data presented are mean ± s.e.m. of three different experiments performed in cells by FACS analysis and then analyzed for soluble CEACAM8 by ELISA. Data demonstrate duplicates (a,b,c) . *, P <0.05. the percentage of a positive sCEACAM8 in dependence of low (0,5-3,9%), medium (4,0- VII 14,9%) and high (≥ 15%) amount of granulocytes in the human BALF. a b
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Fig.7) sCEACAM8-CEACAM1 interaction leads to tyrosine phosphorylation of the ITIM of CEACAM1 and recruitment of SHP-1, which negatively regulated TLR2–dependent activation of PI3K–Akt kinase pathway in human bronchial epithelial cells. (a) Co- immunoprecipitation of A549 cells treated with Pam3Cys alone, with Pam3Cys and sCEACAM8 (each 100 ng/ml), with 4mM pervanadate (PV) used as a positive control or left untreated. Tyrosine phosphorylation of CEACAM1, recruitment of SHP1 and precipitated CEACAM1 were detected. (b) Immunoblot analysis of Akt phosphorylation in A549 cells treated with Pam3Cys or with Pam3Cys and sCEACAM8 (100 ng/ml each) or left untreated for 30 min. ß-Tubulin was used as a loading control. (c) ChIP analysis of the binding of p65 and polymerase II (Pol II) to the IL8 promoter in A549 cells after stimulation with Pam3Cys alone , Pam3Cys and sCEACAM8 (100 ng/ml each) or left untreated. Data show one representative of three independent experiments (a,b,c)
Conclusion
Herein we have identified a CEACAM8-dependent strategy of human granulocytes to interfere with airway inflammation by interacting with CEACAM1 on human pulmonary epithelium. Our Fig.2) Expression analysis of CEACAM8 and sCEACAM8 on PMA-stimulated human Fig.5) The Soluble CEACAM8 - CEACAM1-interaction down regulates the TLR2 - granulocytes. Granulocytes were pre-incubated with 50ng/ml GM-CSF for 90 min in all triggered immune response on primary bronchial epithelial cells (a) Expression of data are suggesting a new mechanism how neutrophils may reduce experiments. Flow cytometry of the expression of CEACAM8 (a) or CD62L (b) on the cell CEACAM1 and TLR2 (black lines) on the surface of normal bronchial epithelial cells (NHBEs) pro-inflammatory immune responses by the secretion of soluble surface was assessed by fluorescence intensity after treatment with 20ng/ml PMA for 1 h. (c) cells by FACS analysis. The same results we found for A549 cells. Grey lines represent CEACAM8 in neutrophil-driven infections in the human airways. Viability of human granulocytes stimulated with PMA 20ng/ml PMA for 1 h or left untreated. isotype-matched control. (b) IL-8 in supernatants of NHBE cells incubated with Pam3Cys Cell death was measured with annexin V and propidium iodide staining. Soluble CEACAM8 alone, with Pam3Cys and CEACAM8 (100 ng/ml each) or left untreated for 16 h. (c) IL-6 in Soluble CEACAM8 may thus functions as resolution-associated ELISA (d) and IL8-ELISA (e) of the supernatants of granulocytes incubated for 1 h with supernatants of NHBE cells incubated with Pam3Cys, with Pam3Cys and CEACAM8 (100 molecular pattern (RAMP) on human bronchial epithelial cells. This 20ng/ml PMA. Untreated cells served as a control. Data presented are mean ± s.e.m. of ng/ml each) or left untreated for 16 h. Data are from one representative of three independent interaction may also play a role for the reduced capacity to clear the three different experiments performed in duplicates (a,d,e) or one of three essentially identical experiments (a), are mean ± s.e.m. of three different experiments performed in triplicates experiments (b,c). * P < 0.05 (b,c), *, P <0.05. lower respiratory tract from bacterial colonization in the airways of COPD-patients.