Anne Krug

Quantitative Expression of Toll-Like Receptor 1–10 mRNA in Cellular Subsets of Human Peripheral Blood Mononuclear Cells and Sensitivity to CpG Oligodeoxynucleotides

The Toll-like receptor (TLR)9 is critical for the recognition of immunostimulatory CpG motifs but may cooperate with other TLRs. We analyzed TLR1–10 mRNA expression by using quantitative real-time PCR in highly purified subsets of human PBMC and determined the sensitivity of these subsets to CpG oligodeoxynucleotides (ODN). TLR1 and TLR6 were expressed in all cell types examined. TLR10 was highly expressed in B cells and weakly expressed in plasmacytoid dendritic cells (PDC). High expression of TLR2 was characteristic for monocytes. PDC and B cells expressed marked levels of TLR7 and TLR9 and were directly sensitive to CpG ODN. In CpG ODN-stimulated PDC and B cells, TLR9 expression rapidly decreased, as opposed to TLR7, which was up-regulated in PDC and decreased in B cells. In monocytes, NK cells, and T cells, TLR7 was absent. Despite low expression of TLR9, monocytes, NK cells, and T cells did not respond to CpG ODN in the absence of PDC but were activated in the presence of PDC. In conclusion, our studies provide evidence that PDC and B cells, but not monocytes, NK cells, or T cells, are primary targets of CpG ODN in peripheral blood. The characteristic expression pattern of TLR1–10 in cellular subsets of human PBMC is consistent with the concept that TLR9 is essential in the recognition of CpG ODN in PDC and B cells. In addition, selective regulation of TLR7 expression in PDC and B cells by CpG ODN revealed TLR7 as a candidate TLR potentially involved in modulating the recognition of CpG motifs.

Identification of CpG oligonucleotide sequences with high induction of IFN-α/β in plasmacytoid dendritic cells

The immature plasmacytoid dendritic cell (PDC) is identical with the principal type I IFN‐producing cell upon viral infection. Oligodeoxynucleotides which contain unmethylated CpG motifs (CpG ODN) are recognized by the vertebrate immune system. Previously, we described CpG ODN that strongly activate human B cells and human blood dendritic cells. Here we describe distinct CpG‐containing oligonucleotide sequences which, in contrast to previously described CpG ODN, induced high amounts of IFN‐α and IFN‐β in peripheral blood mononuclear cells (PBMC). Intracellular staining for IFN‐α revealed that within PBMC CpG ODN‐induced IFN‐α is produced exclusively by PDC. Unlike IFN‐α, TNF‐α is up‐regulated in PDC by all CpG ODN tested. Purified PDC responded to CpG ODN, demonstrating direct activation of PDC by CpG ODN. The most active sequence induced the production of up to 5 pg IFN‐α per single PDC, resulting in more than 400 ng/ml IFN‐α in the supernatant of PBMC enriched for PDC. The potency of CpG ODN to stimulate IFN‐α correlated with their ability to stimulate NK cell lytic activity, while purified NK cells did not respond to CpG ODN. IFNγ production in PBMC was dependent on CpG ODN‐induced IFN‐α/β as demonstrated by IFN‐α/β blocking antibodies. IFN‐α‐inducing CpG ODN strongly supported IFN‐γ production of TCR‐triggered CD4 T cells but were less active than other CpG ODN in stimulating B cells. In conclusion our results demonstrate that particular CpG ODN sequences exist which, due to high IFN‐α/β induction in PDC, induce a set of immune responses typical for viral infection.

Toll-like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL-12.

Human plasmacytoid dendritic cells (DC) (PDC, CD123+) and myeloid DC (MDC, CD11c+) may be able to discriminate between distinct classes of microbial molecules based on a different pattern of Toll‐like receptor (TLR) expression. TLR1–TLR9 were examined in purified PDC and MDC. TLR9, which is critically involved in the recognition of CpG motifs in mice, was present in PDCbut not in MDC. TLR4, which is required for the response to LPS, was selectively expressed on MDC. Consistent with TLR expression, PDC were susceptible to stimulation by CpG oligodeoxynucleotide (ODN) but not by LPS, while MDC responded to LPS but not to CpG ODN. In PDC, CpG ODN supported survival, activation (CD80, CD86, CD40, MHC class II), chemokine production (IL‐8, IP‐10) and maturation (CD83). CD40 ligand (CD40L) and CpG ODN synergized to activate PDC and to stimulate the production of IFN‐α and IL‐12 including bioactive IL‐12 p70. Previous incubation of PDC with IL‐3 decreased the amount of CpG‐induced IFN‐α and shifted the cytokine response in favor of IL‐12. CpG ODN‐activated PDC showed an increased ability to stimulate proliferation of naive allogeneic CD4 T cells, butTh1 polarization of developing T cells required simultaneous activation of PDC by CD40 ligation and CpG ODN. CpG ODN‐stimulated PDC expressed CCR7, which mediates homing to lymph nodes. In conclusion, our studies reveal that IL‐12 p70 production by PDC is under strict control of two signals, an adequate exogenous microbial stimulus such as CpG ODN, and CD40L provided endogenously by activatedT cells. Thus, CpG ODN acts as an enhancer of T cell help, while T cell‐controlled restriction to foreign antigens is maintained.

Replication of norovirus in cell culture reveals a tropism for dendritic cells and macrophages

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology.

IRF4 Transcription Factor-Dependent CD11b+ Dendritic Cells in Human and Mouse Control Mucosal IL-17 Cytokine Responses

Summary Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24+CD64− DCs and contaminating CSF-1R-dependent CD24−CD64+ macrophages. Functionally, loss of CD24+CD11b+ DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24+CD11b+ DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies.

Peritumoral CpG DNA Elicits a Coordinated Response of CD8 T Cells and Innate Effectors to Cure Established Tumors in a Murine Colon Carcinoma Model

The immune system of vertebrates is able to detect bacterial DNA based on the presence of unmethylated CpG motifs. We examined the therapeutic potential of oligodeoxynucleotides with CpG motifs (CpG ODN) in a colon carcinoma model in BALB/c mice. Tumors were induced by s.c. injection of syngeneic C26 cells or Renca kidney cancer cells as a control. Injection of CpG ODN alone or in combination with irradiated tumor cells did not protect mice against subsequent tumor challenge. In contrast, weekly injections of CpG ODN into the margin of already established tumors resulted in regression of tumors and complete cure of mice. The injection site was critical, since injection of CpG ODN at distant sites was not effective. Mice with two bilateral C26 tumors rejected both tumors upon peritumoral injection of one tumor, indicating the development of a systemic immune response. The tumor specificity of the immune response was demonstrated in mice bearing a C26 tumor and a Renca tumor at the same time. Mice that rejected a tumor upon peritumoral CpG treatment remained tumor free and were protected against rechallenge with the same tumor cells, but not with the other tumor, demonstrating long term memory. Tumor-specific CD8 T cells as well as innate effector cells contributed to the antitumor activity of treatment. In conclusion, peritumoral CpG ODN monotherapy elicits a strong CD8 T cell response and innate effector mechanisms that seem to act in concert to overcome unresponsiveness of the immune system toward a growing tumor.

Cutting Edge: IFN-Producing Cells Respond to CXCR3 Ligands in the Presence of CXCL12 and Secrete Inflammatory Chemokines upon Activation

Human natural IFN-producing cells (IPC) circulate in the blood and cluster in chronically inflamed lymph nodes around high endothelial venules (HEV). Although L-selectin, CXCR4, and CCR7 are recognized as critical IPC homing mediators, the role of CXCR3 is unclear, since IPC do not respond to CXCR3 ligands in vitro. In this study, we show that migration of murine and human IPC to CXCR3 ligands in vitro requires engagement of CXCR4 by CXCL12. We also demonstrate that CXCL12 is present in human HEV in vivo. Moreover, after interaction with pathogenic stimuli, murine and human IPC secrete high levels of inflammatory chemokines. Thus, IPC migration into inflamed lymph nodes may be initially mediated by L-selectin, CXCL12, and CXCR3 ligands. Upon pathogen encounter, IPC positioning within the lymph node may be further directed by CCR7 and IPC secretion of inflammatory chemokines may attract other IPC, promoting cluster formation in lymph nodes.

Mammalian target of rapamycin (mTOR) orchestrates the defense program of innate immune cells.

The mammalian target of rapamycin (mTOR) can be viewed as cellular master complex scoring cellular vitality and stress. Whether mTOR controls also innate immune‐defenses is currently unknown. Here we demonstrate that TLR activate mTOR via phosphoinositide 3‐kinase/Akt. mTOR physically associates with the MyD88 scaffold protein to allow activation of interferon regulatory factor‐5 and interferon regulatory factor‐7, known as master transcription factors for pro‐inflammatory cytokine‐ and type I IFN‐genes. Unexpectedly, inactivation of mTOR did not prevent but increased lethality of endotoxin‐mediated shock, which correlated with increased levels of IL‐1β. Mechanistically, mTOR suppresses caspase‐1 activation, thus inhibits release of bioactive IL‐1β. We have identified mTOR as indispensable component of PRR signal pathways, which orchestrates the defense program of innate immune cells.

Key role of splenic myeloid DCs in the IFN-alphabeta response to adenoviruses in vivo.

The early systemic production of interferon (IFN)-αβ is an essential component of the antiviral host defense mechanisms, but is also thought to contribute to the toxic side effects accompanying gene therapy with adenoviral vectors. Here we investigated the IFN-αβ response to human adenoviruses (Ads) in mice. By comparing the responses of normal, myeloid (m)DC- and plasmacytoid (p)DC-depleted mice and by measuring IFN-αβ mRNA expression in different organs and cells types, we show that in vivo, Ads elicit strong and rapid IFN-αβ production, almost exclusively in splenic mDCs. Using knockout mice, various strains of Ads (wild type, mutant and UV-inactivated) and MAP kinase inhibitors, we demonstrate that the Ad-induced IFN-αβ response does not require Toll-like receptors (TLR), known cytosolic sensors of RNA (RIG-I/MDA-5) and DNA (DAI) recognition and interferon regulatory factor (IRF)-3, but is dependent on viral endosomal escape, signaling via the MAP kinase SAPK/JNK and IRF-7. Furthermore, we show that Ads induce IFN-αβ and IL-6 in vivo by distinct pathways and confirm that IFN-αβ positively regulates the IL-6 response. Finally, by measuring TNF-α responses to LPS in Ad-infected wild type and IFN-αβR−/− mice, we show that IFN-αβ is the key mediator of Ad-induced hypersensitivity to LPS. These findings indicate that, like endosomal TLR signaling in pDCs, TLR-independent virus recognition in splenic mDCs can also produce a robust early IFN-αβ response, which is responsible for the bulk of IFN-αβ production induced by adenovirus in vivo. The signaling requirements are different from known TLR-dependent or cytosolic IFN-αβ induction mechanisms and suggest a novel cytosolic viral induction pathway. The hypersensitivity to components of the microbial flora and invading pathogens may in part explain the toxic side effects of adenoviral gene therapy and contribute to the pathogenesis of adenoviral disease.

Interferon‐regulatory‐factor 1 controls Toll‐like receptor 9‐mediated IFN‐β production in myeloid dendritic cells

Activation of interferon regulatory factor (IRF)‐3 and/or IRF‐7 drives the expression of antiviral genes and the production of α/β IFN, a hallmark of antiviral responses triggered by Toll‐like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF‐3 and/or IRF‐7 but is driven by IRF‐1. IRF‐1 together with myeloid differentiation factor 88 (MyD88) or IL‐1 receptor‐associated kinase (IRAK)‐1 triggered IFN‐β promoter activation. IRF‐1 physically interacted with MyD88 and activation of mDC via TLR‐9 induced IRF‐1‐dependent IFN‐β production paralleled by rapid transcriptional activation of IFN‐stimulated genes. The NF‐κB‐dependent production of pro‐inflammatory cytokines, however, was not influenced by IRF‐1. TLR‐9 signaling through this pathway conferred cellular antiviral resistance while IRF‐1‐deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR‐9 activation of mDC and macrophages contributes to antiviral immunity via IRF‐1.