Increased inflammation and impaired resistance to Chlamydophila pneumoniae infection in Dusp1(-/-) mice: critical role of IL-6.

The MAPK phosphatase DUSP1 is an essential negative regulator of TLR-triggered innate immune activation. Here, we have investigated the impact of DUSP1 on inflammatory and antimicrobial host responses to the intracellular pathogen Chlamydophila pneumoniae. Following nasal infection, DUSP1-deficient mice mounted an enhanced pulmonary cytokine (IL-1beta, IL-6) and chemokine response (CCL3, CCL4, CXCL1, CXCL2), leading to increased leukocyte infiltration. Of interest, the increased inflammatory response, in the absence of DUSP1, was associated with higher bacterial numbers in the lungs, although the expression of IFN-gamma and critical antichlamydial effector molecules, such as iNOS, was intact. Blockade of IL-6 trans-signaling by injection of a soluble gp130-Fc fusion protein corrected the overshooting chemokine production as well as the increased chlamydial load in Dusp1(-/-) mice. Furthermore, IL-6 enhanced the replication of C. pneumoniae in embryonic fibroblasts in vitro. These data show that DUSP1 is required to achieve a balanced response to chlamydial infection and identify IL-6 as critical for amplifying inflammation and benefiting chlamydial growth through direct effects on infected cells.

Adjuvanticity of a synthetic cord factor analogue for subunit Mycobacterium tuberculosis vaccination requires FcRgamma-Syk-Card9-dependent innate immune activation.

Novel vaccination strategies against Mycobacterium tuberculosis (MTB) are urgently needed. The use of recombinant MTB antigens as subunit vaccines is a promising approach, but requires adjuvants that activate antigen-presenting cells (APCs) for elicitation of protective immunity. The mycobacterial cord factor Trehalose-6,6-dimycolate (TDM) and its synthetic analogue Trehalose-6,6-dibehenate (TDB) are effective adjuvants in combination with MTB subunit vaccine candidates in mice. However, it is unknown which signaling pathways they engage in APCs and how these pathways are coupled to the adaptive immune response. Here, we demonstrate that these glycolipids activate macrophages and dendritic cells (DCs) via Syk-Card9-Bcl10-Malt1 signaling to induce a specific innate activation program distinct from the response to Toll-like receptor (TLR) ligands. APC activation by TDB and TDM was independent of the C-type lectin receptor Dectin-1, but required the immunoreceptor tyrosine-based activation motif-bearing adaptor protein Fc receptor gamma chain (FcRgamma). In vivo, TDB and TDM adjuvant activity induced robust combined T helper (Th)-1 and Th-17 T cell responses to a MTB subunit vaccine and partial protection against MTB challenge in a Card9-dependent manner. These data provide a molecular basis for the immunostimulatory activity of TDB and TDM and identify the Syk-Card9 pathway as a rational target for vaccine development against tuberculosis.

A genome-wide analysis of LPS tolerance in macrophages.

Among the multiple mechanisms that control the intensity and duration of macrophage activation, the development of a state of refractoriness to a second stimulation in cells treated with lipopolysaccharide (LPS) has long been recognized. Release of inhibitory cytokines and alterations in intracellular signaling pathways may be involved in the development of LPS tolerance. Although a number of molecules have been implicated, a detailed picture of the molecular changes in LPS tolerance is still missing. We have used a genome-wide gene expression analysis approach to (i) define which fraction of LPS target genes are subject to tolerance induction and (ii) identify genes that are expressed at high levels in tolerant macrophages. Our data show that in LPS-tolerant macrophages the vast majority of LPS-induced gene expression is abrogated. The extent of tolerance induction varies for individual genes, and a small subset of genes appears to be exempted. Compared to other negative control mechanisms of macrophages, e.g., IL-10-induced deactivation, LPS tolerance inhibits a much wider range of transcriptional targets. Some previously described negative regulators of TLR signaling (e.g. IRAK-M) were confirmed as expressed at higher levels in LPS-tolerant macrophages. In addition, we discuss other potential players in LPS tolerance identified in this group of genes.

MyD88-dependent changes in the pulmonary transcriptome after infection with Chlamydia pneumoniae.

Chlamydia pneumoniae, an intracellular bacterium, causes pneumonia in humans and mice. Toll-like receptors and the key adaptor molecule myeloid differentiation factor-88 (MyD88) play a critical role in inducing immunity against this microorganism and are crucial for survival. To explore the influence of MyD88 on induction of immune responses in vivo on a genome-wide level, wildtype (WT) or MyD88(-/-) mice were infected with C. pneumoniae on anesthesia, and the pulmonary transcriptome was analyzed 3 days later by microarrays. We found that the infection caused pulmonary cellular infiltration in WT but not MyD88(-/-) mice. Furthermore, it induced the transcription of 360 genes and repressed 18 genes in WT mice. Of these, 221 genes were not or weakly induced in lungs of MyD88(-/-) mice. This cluster contains primarily genes encoding for chemokines and cytokines like MIP-1alpha, MIP-2, MIP-1gamma, MCP-1, TNF, and KC and other immune effector molecules like immunoresponsive gene-1 and TLR2. Arginase was highly induced after C. pneumoniae infection and was MyD88 dependent. Genes induced by interferons were abundant in a cluster of 102 genes that were only partially MyD88 dependent. Also, lcn2 (lipocalin-2) and timp1 were represented within this cluster. Interestingly, a set of 37 genes including sprr1a was induced more strongly in MyD88(-/-) mice, and most of them are involved in the regulation of cellular replication. In summary, ex vivo analysis of the pulmonary transcriptome on infection with C. pneumoniae demonstrated a major impact of MyD88 on inflammatory responses but not on interferon-type responses and identified MyD88-independent genes involved in cellular replication.

Dual specificity phosphatase 1 (DUSP1) regulates a subset of LPS-induced genes and protects mice from lethal endotoxin shock.

Activation of the mitogen-activated protein kinase (MAPK) cascade after Toll-like receptor stimulation enables innate immune cells to rapidly activate cytokine gene expression. A balanced response to signals of infectious danger requires that cellular activation is transient. Here, we identify the MAPK phosphatase dual specificity phosphatase 1 (DUSP1) as an essential endogenous regulator of the inflammatory response to lipopolysaccharide (LPS). DUSP1-deficient (DUSP1-/-) bone marrow-derived macrophages showed selectively prolonged activation of p38 MAPK and increased cytokine production. Intraperitoneal challenge of DUSP1-/- mice with LPS caused increased lethality and overshooting production of interleukin (IL)-6 and tumor necrosis factor alpha. Transcriptional profiling revealed that DUSP1 controls a significant fraction of LPS-induced genes, which includes IL-6 and IL-10 as well as the chemokines CCL3, CCL4, and CXCL2. In contrast, the expression of the important mediators of endotoxin lethality, interferon gamma and IL-12, was not significantly altered by the absence of DUSP1. These data together demonstrate a specific regulatory role of DUSP1 in controlling a subset of LPS-induced genes that determines the outcome of endotoxin shock.

Control of dual-specificity phosphatase-1 expression in activated macrophages by IL-10.

Ligation of Toll-like receptors (TLR) on macrophages induces cytokines and mediators important for the control of pathogens. Macrophage activation has to be tightly controlled to prevent hyper-inflammation. Accordingly, the hallmarks of TLR-triggered signaling, nuclear translocation of NF-kappaB and phosphorylation of mitogen-activated protein kinases (MAPK), are transient events. We have mined microarray datasets for changes in the expression of phosphatases in resting and TLR-activated macrophages. Several members of the dual-specificity phosphatases (DUSP) were induced upon triggering TLR4 with LPS. Up-regulation of DUSP1 mRNA was transient after stimulation with LPS alone, but addition of the immunosuppressive cytokine IL-10 resulted in robust, continued DUSP1 expression. IL-10 also synergized with the anti-inflammatory glucocorticoid dexamethasone in the induction of DUSP1 mRNA expression in activated macrophages, as well as in the inhibition of IL-6 and IL-12 production. Increased expression of DUSP1 in IL-10-treated activated macrophages was correlated with a faster down-regulation of p38 MAPK activation. Thus, these data suggest an operational link between IL-10 and inhibition of p38 MAPK via sustained expression of DUSP1.

The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily.

Loxoribine (7-allyl-7,8-dihydro-8-oxo-guanosine) acts as synthetic adjuvant in anti-tumor responses. Here we first demonstrate that loxoribine activates cells of the innate immune system selectively via the Toll-like receptor (TLR) 7/MyD88-dependent signaling pathway. TLR7- and MyD88-deficient immune cells fail to proliferate or produce cytokines in response to loxoribine, and genetic complementation of TLR7-deficient cells with murine or human TLR7 confers responsiveness. Subsequently we show that cellular activation by loxoribine and resiquimod (R-848), a stimulus for TLR7 and TLR8, depends on acidification and maturation of endosomes and targets MyD88 to vesicular structures with lysosomal characteristics. This mode of TLR7 and TLR8 action resembles CpG-DNA-driven TLR9 activation. We thus conclude that TLR7, 8 and 9 form a functional subgroup within the TLR family that recognizes pathogen-associated molecular patterns in endosomal/lysosomal compartments.