Klaus Heeg

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG‐oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “natures adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG‐ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen‐presenting cell (APC) function. Unfractionated murine bone marrow‐derived DC and FACS®‐fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG‐ODN. Similar to lipopolysaccharide, i.s. CpG‐ODN caused up‐regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL‐12, IL‐6 and TNF‐α. CpG‐ODN‐activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B‐driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG‐ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

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.

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Suppressors of Cytokine Signaling (SOCS)-1 and SOCS-3 Are Induced by CpG-DNA and Modulate Cytokine Responses in APCs

During infection, the functional status of the innate immune system is tightly regulated. Although signals resulting in activation have been well characterized, counterregulative mechanisms are poorly understood. Suppressor of cytokine signaling (SOCS) proteins have been characterized as cytokine-inducible negative regulators of Janus kinase/STAT signaling in cells of hemopoietic origin. To analyze whether SOCS proteins could also be induced by pathogen-derived stimuli, we investigated the induction of SOCS-1 and SOCS-3 after triggering of macrophage cell lines, bone marrow-derived dendritic cells, and peritoneal macrophages with CpG-DNA. In this study, we show that CpG-DNA, but not GpC-DNA, induces expression of mRNA for SOCS-1 and SOCS-3 in vitro and in vivo. SOCS mRNA expression could be blocked by chloroquine and was independent of protein synthesis. Inhibitors of the mitogen-activated protein kinase pathway triggered by CpG-DNA were able to impede induction of SOCS mRNA. CpG-DNA triggered synthesis of SOCS proteins that could be detected by Western blotting. SOCS proteins were functional because they inhibited IFN-γ as well as IL-6- and GM-CSF-induced phosphorylation of STAT proteins. Furthermore, IFN-γ-induced up-regulation of MHC class II molecules was also prevented. The same effects could be achieved by overexpression of SOCS-1. Hence, the results indicate a substantial cross-talk between signal pathways within cells. They provide evidence for regulative mechanisms of Janus kinase/STAT signaling after triggering Toll-like receptor signal pathways.

PD-L1 expression on tolerogenic APCs is controlled by STAT-3

During infection, TLR agonists are released and trigger mature as well as differentiating innate immune cells. Early encounter with TLR agonists (R848; LPS) blocks conventional differentiation of CD14+ monocytes into immature dendritic cells (iDCs) resulting in a deviated phenotype. We and others characterized these APCs (TLR‐APC) by a retained expression of CD14 and a lack of CD1a. Here, we show in addition, expression of programmed death ligand‐1 (PD‐L1). TLR‐APCs failed to induce T‐cell proliferation and furthermore were able to induce CD25+Foxp3+ T regulatory cells (Tregs). Since PD‐L1 is described as a key negative regulator and inducer of tolerance, we further analyzed its regulation. PD‐L1 expression was regulated in a MAPK/cytokine/STAT‐3‐dependent manner: high levels of IL‐6 and IL‐10 that signal via STAT‐3 were produced by TLR‐APCs. Blocking of STAT‐3 activation prevented PD‐L1 expression. Moreover, chromatin immunoprecipitation revealed direct binding of STAT‐3 to the PD‐L1 promoter. Those findings indicate a pivotal role of STAT‐3 in regulating PD‐L1 expression. MAPKs were indirectly engaged, as blocking of p38 and p44/42 MAPKs decreased IL‐6 and IL‐10 thus reducing STAT‐3 activation and subsequent PD‐L1 expression. Hence, during DC differentiation TLR agonists induce a STAT‐3‐mediated expression of PD‐L1 and favor the development of tolerogenic APCs.

DNA activates human immune cells through a CpG sequence-dependent manner

While bacterial DNA and cytosine–guanosine‐dinucleotide‐containing oligonucleotides (CpG ODN) are well described activators of murine immune cells, their effect on human cells is inconclusive. We investigated their properties on human peripheral blood mononuclear cells (PBMC) and subsets thereof, such as purified monocytes, T and B cells. Here we demonstrate that bacterial DNA and CpG ODN induce proliferation of B cells, while other subpopulations, such as monocytes and T cells, did not proliferate. PBMC mixed cell cultures, as well as purified monocytes, produced interleukin‐6 (IL‐6), IL‐12 and tumour necrosis factor‐α upon stimulation with bacterial DNA; however, only IL‐6 and IL‐12 secretion became induced upon CpG ODN stimulation. We conclude that monocytes, but not B or T cells, represent the prime source of cytokines. Monocytes up‐regulated expression of antigen‐presenting, major histocompatibility complex class I and class II molecules in response to CpG DNA. In addition, both monocytes and B cells up‐regulate costimulatory CD86 and CD40 molecules. The activation by CpG ODN depended on sequence motifs containing the core dinucleotide CG since destruction of the motif strongly reduced immunostimulatory potential.

Differential Recognition of TLR-Dependent Microbial Ligands in Human Bronchial Epithelial Cells

Bronchial epithelial cells represent the first line of defense against invading airborne pathogens. They are important contributors to innate mucosal immunity and provide a variety of antimicrobial effectors. However, mucosal surfaces are prone to contact with pathogenic, as well as nonpathogenic microbes, and therefore, immune recognition principles have to be tightly controlled to avoid uncontrolled permanent activation. TLRs have been shown to recognize conserved microbial patterns and to mediate inducible activation of innate immunity. Our experiments demonstrate that bronchial epithelial cells express functional TLR1–6 and TLR9 and thus make use of a common principle of professional innate immune cells. Although it was observed that TLR2 ligands dependent on heterodimeric signaling either with TLR1 or TLR6 were functional, other ligands like lipoteichoic acid were not. Additionally, it was found that bronchial epithelial cells could be stimulated only marginally by Gram-positive bacteria bearing known TLR2 ligands while Gram-negative bacteria were easily recognized. This correlated with low expression of TLR2 and the missing expression of the coreceptor CD36. Transgenic expression of both receptors restored responsiveness to the complete set of TLR2 ligands and Staphylococcus aureus. Additional gene-array experiments confirmed hyporesponsiveness to this bacterium while Pseudomonas aeruginosa and respiratory syncytial virus induced common, as well as pathogen-specific, sets of genes. The findings indicate that bronchial epithelium regulates its sensitivity to recognize microbes by managing receptor expression levels. This could serve the special needs of controlled microbial recognition in mucosal compartments.

Bacterial DNA as immune cell activator

Pattern recognition receptors of the innate and adaptive immune systems apparently recognize unmethylated CpG motifs of bacterial DNA. Cells of the innate immune system are activated directly by CpG motifs, and the resulting response dictates a Th1 bias to the developing adaptive immune response. Interestingly, antigen receptor occupancy of cells of the adaptive immune system augments their responsiveness to CpG motifs, suggesting that co-stimulatory mechanisms are operative.

Immunostimulatory CpG-DNA Activates Murine Microglia

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-DNA) triggers innate immune cells through the pattern recognition receptor Toll-like receptor 9 (TLR-9). CpG-DNA possesses potent immunostimulatory effects on macrophages, dendritic cells, and B lymphocytes. Therefore, CpG-DNA contributes to inflammation during the course of bacterial infections. In contrast to other TLR-dependent microbial patterns, CpG-DNA is a strong inductor of IL-12. Thus, it acts as a Th1-polarizing agent that can be utilized as potent vaccine adjuvant. To assess the role of CpG-DNA in immune reactions in the CNS, we analyzed the effects of CpG-DNA on microglial cells in vitro and in vivo. Primary microglial cells as well as microglial cell lines express TLR-9 mRNA. Consequently, CpG-DNA activated microglial cells in vitro and induced TNF-α, IL-12p40, IL-12p70, and NO. Furthermore, MHC class II, B7-1, B7-2, and CD40 molecules were up-regulated. In addition, phagocytic activity of microglia was enhanced. After intracerebroventricular injection of CpG-DNA, microglial cells were activated and produced TNF-α and IL-12p40 transcripts, as shown by in situ hybridization. These results indicate that microglia is sensitive to CpG-DNA. Thus, bacterial DNA containing CpG motifs could not only play an important role during infections of the CNS, but also might trigger and sustain Th1-dominated immunopathogenic reactions.

CpG‐oligodeoxynucleotides co‐stimulate primary T cells in the absence of antigen‐presenting cells

CpG‐containing oligodeoxynucleotides (CpG‐ODN) act as powerful adjuvant during in vivo induction of T cell responses. While CpG‐ODN directly activate antigen‐presenting cells (APC) and thus exert an extrinsic activity on T cells, it is unclear whether they directly affect T cells (intrinsic activity). Here we analyze the effects of CpG‐ODN on T cells in an APC‐free cell culture. We report that CpG‐ODN co‐stimulate T cells provided they were triggered via their TCR. CpG‐ODN induced IL‐2 production, IL‐2 receptor expression and thus proliferation. Proliferation was blocked by cyclosporin A or anti‐IL‐2 monoclonal antibodies (mAb) but not by anti‐IL‐4 mAb. Moreover, CpG‐co‐stimulated T cells differentiated into cytolytic T lymphocytes in vitro. Of note, IL‐2‐driven growth of primed T cells was not affected by CpG‐ODN. Co‐stimulation was also operative in T cells from CD28− / − mice and in TCR‐transgenic T cells stimulated with peptide. CpG‐ODN‐mediated co‐stimulation of T cells in vitro may thus explain part of the potent adjuvant effects of CpG‐ODN in vivo.