Abstract
BackgroundLipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT) that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement.ResultsHere we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK.Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation.ConclusionsOn the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune response.
Highlights
Lipopolysaccharide (LPS)-triggered Toll-like receptor (TLR) 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity
Pasteurella multocida toxin (PMT) modulates LPS-triggered cytokine release of human blood-derived monocytes Antigen presenting cells regulate lymphocyte-mediated immune responses through presentation of antigens to lymphocytes via the major histocompatibility complex (MHC) on their surface, costimulatory or co-inhibitory signals that are delivered by ligands of the B7 family and via the release of cytokines [19]
Our study aimed to clarify whether the LPS-induced activation of human blood-derived monocytes (hBDMs) through TLR4 is modulated by the heterotrimeric G protein activator PMT and whether the toxin itself activates monocytes
Summary
PMT modulates LPS-triggered cytokine release of human blood-derived monocytes Antigen presenting cells regulate lymphocyte-mediated immune responses through presentation of antigens to lymphocytes via the major histocompatibility complex (MHC) on their surface, costimulatory or co-inhibitory signals that are delivered by ligands of the B7 family and via the release of cytokines [19]. Mixed lymphocyte reactions revealed that the COX-2 inhibitors did not restore the T cell-activating ability of LPS-treated monocytes after PMTwt-stimulation (Figure 2D) Based on these data we exclude a prominent impact of the COX2/PGE2 pathway on the PMTwt-mediated effect on endotoxin-stimulated hBDMs. PMT-induced modulation of LPS-triggered IL-12 release involves the Gαi/cAMP pathway In order to characterize the PMT-induced signalling pathway that modulates cytokine production of LPSactivated monocytes, we focused on the heterotrimeric G protein Gαi that is directly activated by PMT [10]. Simultaneous inhibition of JNK and Gαi activation through Ptx treatment abolished the PMT effect (Figure 4C) and restored the T cellactivating ability of LPS-treated cells after PMTwt-stimulation (Figure 4D). PMT inhibits the production of IL-12p40 through Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation as well as by PI3kinase/Akt and JNK activation mediated by the dissociated Gβγ subunit (Figure 6)
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