Predominant role of toll-like receptor 2 versus 4 in Chlamydia pneumoniae-induced activation of dendritic cells.

Chlamydia pneumoniae is an obligate intracellular human pathogen causing diseases such as pneumonia, bronchitis, and pharyngitis. Because of its intracellular replication, cell-mediated immune responses are needed to mediate successful defenses of the host. Because dendritic cells play a central role in linking innate immunity and Ag-specific cell-mediated immune responses we asked whether dendritic cells are activated upon contact with C. pneumoniae and whether known Toll like receptors (TLR) are involved in this process. Here we show that C. pneumoniae was taken up by bone marrow-derived murine dendritic cells. Ingested C. pneumoniae appeared to be unable to develop mature inclusion inside dendritic cells. Furthermore, upon contact with C. pneumoniae dendritic cells were potently stimulated because NF-kappaB was activated and translocated to the nucleus, cytokines like IL-12p40 and TNF-alpha were secreted, and expression of MHC class II molecules, CD40, CD80, and CD86 was up-regulated. Importantly, secretion of cytokines as well as translocation of NF-kappaB were dependent on the presence of TLR2 and independent from TLR4 with the exception of IL-12p40 secretion, which was attenuated in the absence of either a functional TLR2 or 4. In conclusion, we show here that recognition of the Gram-negative bacterium C. pneumoniae depends largely on TLR2 and only to a minor extent on TLR4.

Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition.

The Toll-like receptor (TLR) family consists of phylogenetically conserved transmembrane proteins, which function as mediators of innate immunity for recognition of pathogen-derived ligands and subsequent cell activation via the Toll/IL-1R signal pathway. Here, we show that human TLR9 (hTLR9) expression in human immune cells correlates with responsiveness to bacterial deoxycytidylate-phosphate-deoxyguanylate (CpG)-DNA. Notably "gain of function" to immunostimulatory CpG-DNA is achieved by expressing TLR9 in human nonresponder cells. Transfection of either human or murine TLR9 conferred responsiveness in a CD14- and MD2-independent manner, yet required species-specific CpG-DNA motifs for initiation of the Toll/IL-1R signal pathway via MyD88. The optimal CpG motif for hTLR9 was GTCGTT, whereas the optimal murine sequence was GACGTT. Overall, these data suggest that hTLR9 conveys CpG-DNA responsiveness to human cells by directly engaging immunostimulating CpG-DNA.

Bacterial CpG-DNA triggers activation and maturation of human CD11c-, CD123+ dendritic cells.

Human plasmacytoid precursor dendritic cells (ppDC) are a major source of type I IFN upon exposure to virus and bacteria, yet the stimulus causing their maturation into DCs is unknown. After PBMC activation with immunostimulatory bacterial DNA sequences (CpG-DNA) we found that ppDC are the primary source of IFN-alpha. In fact, either CpG-DNA or dsRNA (poly(I:C)) induced IFN-alpha from purified ppDC. Surprisingly, only CpG-DNA triggered purified ppDC survival, maturation, and production of TNF, GM-CSF, IL-6, and IL-8, but not IL-10 or IL-12. Known DC activators such as CD40 ligation triggered ppDC maturation, but only IL-8 production, while bacterial LPS was negative for all activation criteria. An additional finding was that only CpG-DNA could counteract IL-4-induced apoptosis in ppDC. Therefore, CpG-DNA represents a pathogen-associated molecular pattern for ppDC. In contrast to these finding, CpG-DNA, like LPS, caused TNF, IL-6, and IL-12 release from PBMC and purified monocytes; however, differentiation of monocytes into DCs with GM-CSF and IL-4 unexpectedly resulted in refractoriness to CpG-DNA, but not LPS. Taken together, these results suggest that within a DC subset a multiplicity of responses can be generated by distinct environmental stimuli and that responses to a given stimulus may be dissimilar between DC subsets.

Interaction of Chlamydia pneumoniae and human alveolar macrophages: infection and inflammatory response.

The obligate intracellular pathogen Chlamydia pneumoniae is associated with chronic respiratory, atherosclerotic, and rheumatic disease. The alveolar macrophage (AM) is a potential target cell for the pathogen and may contribute to respiratory immunopathology. We therefore investigated in vitro the interaction between chlamydiae and macrophages with cocultures of C. pneumoniae and AM from 12 healthy volunteers. Inflammatory responses were evaluated through lucigenin-amplified chemiluminescence; secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin 8 (IL-8); and expression of intercellular adhesion molecule-1 (ICAM-1) and human leukocyte antigen-DR (HLA-DR). C. pneumoniae readily induced productive infection in the AM. Inclusions containing replicating pathogens could be maintained for up to 120 h. Morphologically similar infection patterns were seen ex vivo in AM collected from six patients with known C. pneumoniae pneumonia. AM responded to the infection with a marked, dose-dependent release of reactive oxygen species, TNF-alpha, IL-1beta, and IL-8. ICAM-1 expression remained unchanged, but HLA-DR was significantly upregulated. Our data indicate that the release of antimicrobial mediators cannot prevent chlamydial infection and replication in AM, but may be involved in amplification of the local inflammatory response in C. pneumoniae pneumonia.