The MYD88-Independent Pathway Is Not Mobilized in Human Neutrophils Stimulated via TLR4

Abstract

LPS activates both MyD88-dependent and -independent signaling via TLR4, but the extent to which each cascade is operative in different cell types remains unclear. This prompted us to revisit the intriguing issue of CXCL10 production, which we previously showed to be inducible in neutrophils stimulated with LPS and IFN-γ but not with either stimulus alone, contrary to other myeloid cells. We now report that in neutrophils the MyD88-independent pathway is not activated by LPS. Indeed, microarray and real-time PCR experiments showed that neither IFNβ nor IFNβ-dependent genes (including CXCL10) are inducible in LPS-treated neutrophils, in contrast to monocytes. Further investigation into the inability of LPS to promote IFNβ expression in neutrophils revealed that the transcription factors regulating the IFNβ enhanceosome, such as IFN-regulatory factor-3 and AP-1, are not activated in LPS-treated neutrophils as revealed by lack of dimerization, nuclear translocation, confocal microscopy, and inducible binding to DNA. Moreover, we show that the upstream TANK-binding kinase-1 is not activated by LPS in neutrophils. A lack of IFNβ/CXCL10 mRNA expression and IFN-regulatory factor 3 activation was also observed in myeloid leukemia HL60 cells differentiated to granulocytes and then stimulated with LPS, indicating that the inability of neutrophils to activate the MyD88-independent pathway represents a feature of their terminal maturation. These results identify a disconnected activation of the two signaling pathways downstream of TLR4 in key cellular components of the inflammatory and immune responses and help us to better understand the primordial role of neutrophils in host defense against nonviral infections.

Molecular mechanisms underlying the synergistic induction of CXCL10 by LPS and IFN‐γ in human neutrophils

“…In keeping with the inability of neutrophils to produce IFN-b, we have already shown that the activation of IRF-3 is not inducible in IFN-c plus LPS-treated neutrophils [11]. We have also shown that neutrophils express little or no c-Jun, a key component of AP-1 complexes, and that consequently, AP-1 is not inducible by LPS and/or IFN-c in these cells [11,18]. Consequently, we reasoned that a possible mechanism leading to the stronger induction of CXCL10 gene by the LPS plus IFN-c combination in neutrophils could be a synergistic enhancement of IFN-c-induced STAT1 tyrosine phosphorylation and, in turn, STAT1-containing DNAbinding activities exerted by LPS.…”

“…Finally, our data do not exclude that the induction of CXCL10 mRNA expression by IFN-c and LPS in neutrophils might also be favored because it is not counter regulated by attenuators of cytokine-induced responses, such as the SOCS proteins [29]. Indeed, our previous observations have shown that in contrast to monocytes, neither SOCS-1 nor SOCS-3 protein expression is induced by LPS in neutrophils [11,30].Conversely, it appears that monocyte responses are attenuated by LPS-activated SOCS-1/SOCS-3, since the induction of CXCL10 mRNA expression by IFN-c plus LPS is much lower than that exerted by IFN-c alone [6]. Interestingly, the reason why SOCS-1 is not inducible by LPS in neutrophils derives from the lack of mobilization of the MyD88-independent pathway, which controls SOCS-1 gene via endogenous .…”

“…Conversely, pharmacological inhibition of NF-jB activation by seven different NF-jB blockers markedly decreased the induction of CXCL10 mRNA in IFN-c plus LPS-treated neutrophils, without affecting the induction of MX1 or CD64, whose induction is known to be jB independent. In view of our findings that LPS (but not IFN-c) activates NF-jB in neutrophils [11,19], our data indicate that the synergy between LPS and IFN-c toward CXCL10 gene expression must reflect the cooperative induction of the NF-jB and STAT1 transcription factors by LPS and IFN-c, respectively (Fig. 6).…”

“…Indeed, CXCL10 expression occurs in vitro only if However, the molecular bases of the particular inducibility of CXCL10 in human neutrophils have only been partially elucidated thus far. In this regard, we have recently clarified that the reason explaining the inability of LPS to induce CXCL10 mRNA expression in neutrophils reflects the fact that in this cell type, LPS is unable to mobilize the intracellular MyD88-independent pathway upon TLR4 binding [11]. As a result, LPS fails to induce IFN-b production and release in neutrophils, thereby preventing the autocrine/paracrine induction of STAT1 tyrosine phosphorylation that is otherwise typically triggered by the MyD88-independent pathway in other cell types, such as autologous monocytes, and which is ultimately responsible for the activation of a host of antimicrobial and antiviral genes, including CXCL10 [10].…”

“…This, however, proved to be wrong, since IFN-c alone already triggers the tyrosine phosphorylation of p91 STAT1a (and to a much lesser extent, that of the modestly expressed p84 STAT1b) (Fig. 4A), and already induces STAT1-containing DNA-binding activities at optimal levels ( Moreover, no significant NF-jB activation was observed in IFN-c-treated neutrophils [11,19]. However, Fig.…”