title: Detection of muramyl dipeptide-sensing pathway defects in monocytes of patients with Crohn's disease using phospho-specific whole blood flow cytometry.
abstract: Peripheral blood mononuclear cells of Crohn's disease (CD) patients with the common 1007fs mutation of the caspase recruitment domain-containing 15/nucleotide-binding oligomerization domain-containing 2 (CARD15/NOD2) gene show impaired nuclear factor kappa B (NF-κB) activation in response to muramyl dipeptide (MDP), as determined by Western blotting. We applied phospho-specific flow cytometry to examine NF-κB and p38 activation in whole blood monocytes of 16 CD patients with or without the 1007fs and previously described rare mutations of the CARD15 gene, and healthy reference subjects. Aliquots of whole blood were supplemented with MDP (0-1000 ng/mL), incubated for 10-40 min and processed for flow cytometry. Bacterial lipopolysaccharide (LPS) was used as a positive control agonist. We found that NF-κB and p38 phosphorylation induced by MDP was not detectable in monocytes of patients homozygous for the CARD15 1007fs mutation, while those induced by LPS were normal. We also determined MDP-induced NF-κB phosphorylation levels in nuclear extracts of mononuclear cells separated from blood using enzyme-linked immunosorbent assay (ELISA), and observed that the levels decreased in a 1007fs mutation-dose dependent manner. We conclude that phospho-specific whole blood flow cytometry provides a means to study phosphorylation of NF-κB and p38 in clinical samples and can be applied to screening of CD patients homozygous for the CARD15 1007fs mutation.
authors.affiliation: Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki.
authors.forename: Krista
authors.initials: K
authors.lastname: Kuuliala
authors.forename: Maarit
authors.initials: M
authors.lastname: Lappalainen
authors.forename: Ulla
authors.initials: U
authors.lastname: Turunen
authors.forename: Pauli
authors.initials: P
authors.lastname: Puolakkainen
authors.forename: Esko
authors.initials: E
authors.lastname: Kemppainen
authors.forename: Sanna
authors.initials: S
authors.lastname: Siitonen
authors.forename: Heikki
authors.initials: H
authors.lastname: Repo
authors.forename: Harri
authors.initials: H
authors.lastname: Mustonen
mesh_terms: D000119:Acetylmuramyl-Alanyl-Isoglutamine / Q000097:blood*; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D003424:Crohn Disease / Q000097:blood* / Q000175:diagnosis / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D016328:NF-kappa B / Q000097:blood; D053473:Nod2 Signaling Adaptor Protein / Q000235:genetics; D010750:Phosphoproteins / Q000097:blood; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000097:blood
chemical_list: D016328:NF-kappa B; C429018:NOD2 protein, human; D053473:Nod2 Signaling Adaptor Protein; D010750:Phosphoproteins; D000119:Acetylmuramyl-Alanyl-Isoglutamine; D048051:p38 Mitogen-Activated Protein Kinases