Infection of murine bone marrow-derived macrophages (BMMφ) with Chlamydia pneumoniae induces IFN-αβ-dependent IFN-γ secretion that leads to control of the intracellular bacterial growth. Enhanced growth of C. pneumoniae in Toll-like receptor (TLR) 4−/− and myeloid differentiation factor (MyD) 88−/− (but not TLR2−/−, TLR6−/−, or TLR9−/−) BMMφ is shown in this study. Reduced accumulation of IFN-α and IFN-γ mRNA was also observed in TLR4−/−- and MyD88−/−-infected cells. IL-1R and IL-18R signaling did not account for differences between MyD88−/− and wild-type BMMφ. Surprisingly, infection-induced NF-κB activation as well as TNF-α, IL-1, or IL-6 mRNA expression were all normal in TLR4−/− and MyD88−/− cells. Phosphorylation of the transcription factor STAT1 during bacterial infection is IFN-αβ dependent, and necessary for increased IFN-γ mRNA accumulation and chlamydial growth control. Signaling through common cytokine receptor γ-chain and RNA-dependent protein kinase both mediated IFN-αβ-dependent enhancement of IFN-γ mRNA levels. Accumulation of IFN-γ mRNA and control of C. pneumoniae growth required NF-κB activation. Such NF-κB activation was independent of IFN-αβ, STAT1, and RNA-dependent protein kinase. In summary, C. pneumoniae-induced IFN-γ expression in BMMφ is controlled by a TLR4-MyD88-IFN-αβ-STAT1-dependent pathway, as well as by a TLR4-independent pathway leading to NF-κB activation.

STAT1 mediates signaling in response to IFN-α, -β, and -γ, cytokines required for protective immunity against several viral, bacterial, and eukaryotic pathogens. The protective role of STAT1 in the control of intranasal infection with the obligate intracellular bacterium Chlamydia pneumoniae was analyzed. IFN-γ−/− or IFN-γ receptor (R)−/− mice were highly susceptible to infection with C. pneumoniae. We found that STAT1−/− mice were even more susceptible to C. pneumoniae than IFN-γ−/− or IFN-γR−/− mice. Phosphorylation of STAT1 was detected in the lungs of C. pneumoniae-infected wild-type, IFN-γR−/−, and IFN-αβR−/− mice, but not in mice lacking both IFN-αβR and IFN-γR. In line with this, IFN-αβR−/−/IFN-γR−/− mice showed increased susceptibility to infection compared with IFN-γR−/− mice. However, C. pneumoniae-infected IFN-αβR−/− or IFN regulatory factor 3−/− mice showed no increased susceptibility and similar IFN-γ expression compared with wild-type mice. CD4+ or CD8+ cells released IFN-γ in vivo and conferred protection against C. pneumoniae in a STAT1-independent manner. In contrast, STAT1 mediated a nonredundant protective role of nonhemopoietic cells but not of hemopoietic cells. Nonhemopoietic cells accounted for the expression of STAT1-mediated indoleamine 2, 3-dioxygenase and the p47 GTPase LRG-47, but not inducible NO synthase mRNA. In summary, we demonstrate that STAT1 mediates a cooperative effect of IFN-αβ and IFN-γ on nonhemopoietic cells, resulting in protection against C. pneumoniae.

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TLR signal transduction involves a MyD88-mediated pathway, which leads to recruitment of the IL-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Toll/IL-1R translation initiation region domain-containing adaptor-inducing IFN-beta-mediated pathway, resulting in the activation of IFN regulatory factor (IRF)3. Both pathways can lead to expression of IFN-beta. TLR-dependent and -independent signals converge in the TNF receptor-associated factor 6 (TRAF6) adaptor, which mediates the activation of NF-kappaBeta. Infection of murine bone marrow-derived macrophages (BMM) with Chlamydia pneumoniae induces IFN-alpha/beta- and NF-kappaBeta-dependent expression of IFN-gamma, which in turn, will control bacterial growth. The role of IRAK4 and IRF3 in the regulation of IFN-alpha/beta expression and NF-kappaBeta activation was studied in C. pneumoniae-infected BMM. We found that levels of IFN-alpha, IFN-beta, and IFN-gamma mRNA were reduced in infected IRAK4(-/-) BMM compared with wild-type (WT) controls. BMM also showed an IRAK4-dependent growth control of C. pneumoniae. No increased IRF3 activation was detected in C. pneumoniae-infected BMM. Similar numbers of intracellular bacteria, IFN-alpha, and IFN-gamma mRNA titers were observed in C. pneumoniae-infected IRF3(-/-) BMM. On the contrary, IFN-beta(-/-) BMM showed lower IFN-alpha and IFN-gamma mRNA levels and higher bacterial titers compared with WT controls. C. pneumoniae infection-induced activation of NF-kappaBeta and expression of proinflammatory cytokines were shown to be TRAF6-dependent but did not require IRAK4 or IRF3. Thus, our data indicate that IRAK4, but not IRF3, controls C. pneumoniae-induced IFN-alpha and IFN-gamma secretion and bacterial growth. IRAK4 and IRF3 are redundant for infection-induced NF-kappaB activation, which is regulated by TRAF6.