The flagellin/Toll-like receptor 5 axis elicits diffuse proinflammatory signaling and innate immune defenses in vivo

Background

The development of septic shock is related to the activation of nonspecific (innate) immune responses, triggered by the interactions between molecules released by pathogens and specific cellular receptors in the host, termed Toll-like receptors (TLRs). Flagellin is a 55 kDa protein isolated from the flagellum of Gram-negative bacteria, which may activate such responses through its recognition by TLR5. The tissue distribution of TLR5, as well as the actions of flagellin on various organs in vivo, has not been previously established. We therefore conducted the present study to determine the presence of TLR5 receptor in major organs from mice, and to evaluate whether flagellin could trigger prototypical innate immune responses through activation of NFκB and mitogen-activated protein kinase (MAPK) signaling pathways in these organs.

Methods

Mice were injected intravenously with 1 μg recombinant Salmonella flagellin. At selected timepoints (30 minutes to 6 hours), the mice were sacrificed and the major organs (lung, liver, gut and kidney) were harvested for expression of the flagellin receptor TLR5; for the activation state of NFκB (monitored by the degree of phosphorylation and degradation of its inhibitor IκBα and by the NFκB-DNA-binding activity); for the activation state of MAPK (monitored by the degree of phosphorylation of JNK, p38 and ERK); for the expression of inflammatory cytokines; and for the activation of apoptotic pathways (monitored by the degree of caspase-3 and poly(ADP-ribose) polymerase cleavage). Plasma was obtained for the measurements of cytokine levels.

Results

TLR5 protein was constitutively expressed in all organs. The injection of flagellin activated NFκB and MAPKs at 30 minutes, and markedly enhanced the generation of the cytokines TNFα, IL-1β, IL-6, TREM-1, and MIP-2 at 1 hour and 3 hours. Similarly, these cytokines significantly increased in the plasma from 1 hour to 6 hours after flagellin. Flagellin also triggered prototypical apoptotic changes in all organs.

Conclusion

Bacterial flagellin activates inflammatory signaling and apoptosis in most major organs in vivo, and thus may represent a critical mediator of multiple organ failure during Gram-negative septic shock.