Bacterial flagellin elicits widespread innate immune defense mechanisms, apoptotic signaling, and a sepsis-like systemic inflammatory response in mice.
BACKGROUND
Systemic inflammation in sepsis is initiated by interactions between pathogen molecular motifs and specific host receptors, especially toll-like receptors (TLRs). Flagellin is the main flagellar protein of motile microorganisms and is the ligand of TLR5. The distribution of TLR5 and the actions of flagellin at the systemic level have not been established. Therefore, we determined TLR5 expression and the ability of flagellin to trigger prototypical innate immune responses and apoptosis in major organs from mice.


METHODS
Male Balb/C mice (n = 80) were injected intravenously with 1-5 μg recombinant Salmonella flagellin. Plasma and organ samples were obtained after 0.5 to 6 h, for molecular investigations. The expression of TLR5, the activation state of nuclear factor kappa B (NFκB) and mitogen-activated protein kinases (MAPKs) [extracellular related kinase (ERK) and c-jun-NH2 terminal kinase (JNK)], the production of cytokines [tumor necrosis alpha (TNFα), interleukin-1β (IL-1β), interleukin-6 (IL-6), macrophage inhibitory protein-2 (MIP-2) and soluble triggering receptor expressed on myeloid cells (TREM-1)], and the apoptotic cleavage of caspase-3 and its substrate Poly(ADP-ribose) polymerase (PARP) were determined in lung, liver, gut and kidney at different time-points. The time-course of plasma cytokines was evaluated up to 6 h after flagellin.


RESULTS
TLR5 mRNA and protein were constitutively expressed in all organs. In these organs, flagellin elicited a robust activation of NFκB and MAPKs, and induced significant production of the different cytokines evaluated, with slight interorgan variations. Plasma TNFα, IL-6 and MIP-2 disclosed a transient peak, whereas IL-1β and soluble TREM-1 steadily increased over 6 h. Flagellin also triggered a marked cleavage of caspase-3 and PARP in the intestine, pointing to its ability to promote significant apoptosis in this organ.


CONCLUSIONS
Bacterial flagellin elicits prototypical innate immune responses in mice, leading to the release of multiple pro-inflammatory cytokines in the lung, small intestine, liver and kidney, and also activates apoptotic signalling in the gut. Therefore, this bacterial protein may represent a critical mediator of systemic inflammation and intestinal barrier failure in sepsis due to flagellated micro-organisms.
Rolli
Joëlle
J
Department of Intensive Care Medicine, University Hospital Medical Center and Faculty of Biology and Medicine, rue du Bugnon 46, Lausanne 1010, Switzerland. j.rolli@urbanet.ch
Loukili
Noureddine
N
Levrand
Sandra
S
Rosenblatt-Velin
Nathalie
N
Rignault-Clerc
Stéphanie
S
Waeber
Bernard
B
Feihl
François
F
Pacher
Pal
P
Liaudet
Lucas
L
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005408:Flagellin / Q000494:pharmacology*; D007113:Immunity, Innate / Q000187:drug effects*; D053583:Interleukin-1beta / Q000096:biosynthesis; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D016328:NF-kappa B / Q000096:biosynthesis; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011971:Receptors, Immunologic / Q000096:biosynthesis; D018746:Systemic Inflammatory Response Syndrome / Q000139:chemically induced*; D051216:Toll-Like Receptor 6 / Q000096:biosynthesis; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
D054426:Chemokine CXCL2; D053583:Interleukin-1beta; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011971:Receptors, Immunologic; C517262:TREM1 protein, mouse; C495369:Tlr6 protein, mouse; D051216:Toll-Like Receptor 6; D000073871:Triggering Receptor Expressed on Myeloid Cells-1; D014409:Tumor Necrosis Factor-alpha; D005408:Flagellin; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D053148:Caspase 3