Hans Häcker

Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments.

Recognition by innate immune cells of the pathogen associated molecular patterns (PAMP) lipopolysaccharide (LPS) from Gram‐negative bacteria and bacterial CpG‐DNA depends on Toll‐like receptor4 (TLR4) and TLR9, respectively. To define differences in the response to these distinct PAMP we compared a key intracellular event, namely recruitment of myeloid differentiation marker 88 (MyD88) to the respective PAMP‐initiated TLR signaling. Using MyD88‐GFP fusion protein expressing macrophages we demonstrate that LPS and CpG‐DNA trigger signaling from two different cellular locations: theformer at the cell membrane and the latter at the lysosomal compartment. While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG‐DNA to activate TLR9. In support of these data TLR9 is not localized at the cell surface, but intracellularily. These data stress the need to characterize individual TLR at the very beginning of signal initiation in order to understand their diverse biological functions.

CpG‐DNA‐specific activation of antigen‐presenting cells requires stress kinase activity and is preceded by non‐specific endocytosis and endosomal maturation

Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40‐CD40 ligand‐independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG‐DNA induces phosphorylation of Jun N‐terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein‐1 (AP‐1) via phosphorylation of its constituents c‐Jun and ATF2. Moreover, stress kinase activation is essential for CpG‐DNA‐induced cytokine release of tumor necrosis factor α (TNFα) and interleukin‐12 (IL‐12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non‐CpG‐DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG‐ODN‐triggered activation of antigen‐presenting cells.

Cell type-specific activation of mitogen-activated protein kinases by CpG-DNA controls interleukin-12 release from antigen-presenting cells.

Activation of antigen‐presenting cells (APCs) by invariant constituents of pathogens such as lipopolysaccharide (LPS) or bacterial DNA (CpG‐DNA) initiates immune responses. We have analyzed the mitogen‐activated protein kinase (MAPK) pathways triggered by CpG‐DNA and their significance for cytokine production in two subsets of APCs, i.e. macrophages and dendritic cells (DCs). We found that CpG‐DNA induced extracellular signal‐regulated kinase (ERK) activity in macrophages in a classic MEK‐dependent way. This pathway up‐regulated tumor necrosis factor production but down‐regulated interleukin (IL)‐12 production. However, in DCs, which produce large amounts of IL‐12, CpG‐DNA and LPS failed to induce ERK activity. Consistent with a specific negative regulatory role for ERK in macrophages, chemical activation of this pathway in DCs suppressed CpG‐DNA‐induced IL‐12 production. Overall, these results imply that differential activation of MAP kinase pathways is a basic mechanism by which distinct subsets of innate immune cells regulate their effector functions.

CpG-DNA Activates In Vivo T Cell Epitope Presenting Dendritic Cells to Trigger Protective Antiviral Cytotoxic T Cell Responses

MHC class I-restricted T cell epitopes lack immunogenicity unless aided by IFA or CFA. In an attempt to circumvent the known inflammatory side effects of IFA and CFA, we analyzed the ability of immunostimulatory CpG-DNA to act as an adjuvant for MHC class I-restricted peptide epitopes. Using the immunodominant CD8 T cell epitopes, SIINFEKL from OVA or KAVYNFATM (gp33) from lymphocytic choriomeningitis virus glycoprotein, we observed that CpG-DNA conveyed immunogenicity to these epitopes leading to primary induction of peptide-specific CTL. Furthermore, vaccination with the lymphocytic choriomeningitis virus gp33 peptide triggered not only CTL but also protective antiviral defense. We also showed that MHC class I-restricted peptides are constitutively presented by immature dendritic cells (DC) within the draining lymph nodes but failed to induce CTL responses. The use of CpG-DNA as an adjuvant, however, initiated peptide presenting immature DC progression to professional licensed APC. Activated DC induced cytolytic CD8 T cells in wild-type mice and also mice deficient of Th cells or CD40 ligand. CpG-DNA thus incites CTL responses toward MHC class I-restricted T cell epitopes in a Th cell-independent manner. Overall, these results provide new insights into CpG-DNA-mediated adjuvanticity and may influence future vaccination strategies for infectious and perhaps tumor diseases.

Activation of the immune system by bacterial CpG-DNA

The past decade has seen a remarkable process of refocusing in immunology. Cells of the innate immune system, especially macrophages and dendritic cells, have been at the centre of this process. These cells had been regarded by some scientists as non‐specific, sometimes perhaps even confined to the menial job of serving T cells by scavenging antigen and presenting it to the sophisticated adaptive immune system. Only over the last few years has it become unequivocally clear that cells of the innate immunity hold, by variation of context and mode of antigen presentation, the power of shaping an adaptive immune response. The innate immune response, in turn, is to a significant degree the result of stimulation by so‐called pathogen‐associated molecular patterns (PAMPs). One compound with high stimulatory potential for the innate immune system is bacterial DNA. Here we will review recent evidence that bacterial DNA should be ranked with other PAMPs such as lipopolysaccharide (LPS) and lipoteichoic acid. We will further review our present knowledge of DNA recognition and DNA‐dependent signal transduction in cells of the immune system.

Caspase-9/-3 Activation and Apoptosis Are Induced in Mouse Macrophages upon Ingestion and Digestion of Escherichia coli Bacteria

A number of highly virulent, intracellular bacteria are known to induce cell death by apoptosis in infected host cells. In this work we demonstrate that phagocytosis of bacteria from the Escherichia coli laboratory strain K12 DH5α is a potent cell death stimulus for mouse macrophages. RAW264.7 mouse macrophages took up bacteria and digested them within 2–4 h as investigated with green fluorescent protein-expressing bacteria. No evidence of apoptosis was seen at 8 h postexposure, but at 24 h ∼70% of macrophages displayed an apoptotic phenotype by a series of parameters. Apoptosis was blocked by inhibition of caspases or by forced expression of the apoptosis-inhibiting protein Bcl-2. Processing of caspase-3 and caspase-9 but not caspase-8 was seen suggesting that the mitochondrial branch of the apoptotic pathway was activated. Active effector caspases could be detected in two different assays. Because the adapter molecule myeloid differentiation factor 88 (MyD88) has been implicated in apoptosis, involvement of the Toll-like receptor pathway was investigated. In RAW264.7 cells, heat-treated bacteria were taken up poorly and failed to induce significant apoptosis. However, cell activation was almost identical between live and heat-inactivated bacteria as measured by extracellular signal-regulated kinase activation, generation of free radicals, and TNF secretion. Furthermore, primary bone marrow-derived macrophages from wild-type as well as from MyD88-deficient mice underwent apoptosis upon phagocytosis of bacteria. These results show that uptake and digestion of bacteria leads to MyD88-independent apoptosis in mouse macrophages. This form of cell death might have implications for the generation of the immune response.

Signal Transduction Pathways Activated by CpG-DNA

While more and more attention has been paid to CpG-DNA with respect to its usefulness as an adjuvant, its molecular mechanism of action is less well defined. Over the last few years, at least two major signalling pathways have been shown to be utilized by CpG-DNA: the NF-kappa B activation pathway and the stress-kinase pathway. Direct downstream events of these pathways are induction of transcriptional activity of NF-kappa B and transcriptional activity of AP-1. As far as investigated, CpG-DNA uses signal transduction pathways originally described for other stimuli, such as LPS, IL-1 or TNF. Therefore, to us, the prime question is: where does CpG-DNA-induced signalling enter these known pathways? This raises questions about the existence of a CpG-DNA-sequence-specific receptor. Several points of evidence support the probability of the existence of a cellular receptor: There is a strong motif (unmethylated CpG) dependency for CpG-DNA-induced signalling. There is cell-type specificity. Dendritic cells, macrophages and B cells respond to CpG-DNA, but other cell types, such as fibroblasts and T cells, do not. In addition, classic signal-transduction pathways are rapidly switched on in a parallel manner, as is known for other receptors. Using competing non-CpG ODNs and inhibitors of endosomal acidification, some evidence has been obtained that CpG ODNs are taken up into endosomes by a CpG-independent receptor, followed by a pH-dependent step before signalling starts. A model based on these findings is proposed in Fig. 4. Nevertheless, other receptor-independent activities of CpG-DNA cannot yet be ruled out. Although unlikely, we should consider the possibility that CpG-DNA directly interacts with cellular nucleic acids either by direct hybridization with complementary nucleotides or by formation of DNA triplexes (VASQUEZ and WILSON 1998). While these possibilities have been explored by antisense technology, using a huge variety of ODNs, there is no experimental evidence that such interactions are important for the activity of CpG-DNA. In this context, it is important to note that DNA, especially phosphothioate-stabilized ODNs with poly-G stretches, have substantial CpG-independent activities, although these activities seem not to depend on specific, antisense-like DNA-DNA interactions (PISETSKY 1996). One good example comes from experiments using ODNs on primary T cells. Co-stimulation of CD3-primed T cells with CpG ODN leads to a significant increase of IL-2 secretion and proliferation; however, these effects are CpG independent (K. Heeg, personal communication). Remarkably, these poly-G stretches seem to be inactive when transferred to double-stranded DNAs, such as plasmid DNA (WLOCH et al. 1998). In contrast, to my knowledge, no immune-stimulatory effect of bacterial DNA has been described that can not be abolished by CpG-specific methylation. Taken together, CpG-dependent and CpG-independent activities must be distinguished from one another. Among these effects, CpG-dependent signalling is better defined. Much effort is going into the investigation of the pharmacological applications of CpG-DNA. Once CpG-receptor-like structures are known, the question of the physiological role of CpG-DNA can be tackled.

IL-4 regulates IL-12 p40 expression post-transcriptionally as well as via a promoter-based mechanism

IL‐12 and IL‐4, respectively, dominate Th1 versus Th2 polarization. Additionally IL‐4 can inhibit IL‐12 p40 production in DC. Here we show that macrophages respond to bacterial CpG‐DNA with IL‐12 p40 production in an IL‐4‐sensitive manner. Analysis of the molecular mechanism of this inhibition shows that IL‐4 acts by reducing the stability of IL‐12 p40 mRNA as well as by affecting promoter activity. IL‐4 did not affect early CpG‐DNA‐induced signal transduction. However, IL‐4 reduced the activity of the IL‐12 p40 promoter and when de novo transcription of IL‐12 p40 mRNA was blocked,IL‐4 led to acceleration of IL‐12 p40 mRNA degradation. These data show that IL‐4 regulates IL‐12 p40 expression by influencing promoter activity and by interfering with mRNA stability.