Both TLR2 and TRIF Contribute to Interferon-β Production during Listeria Infection

Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression

The C-Terminal Transactivation Domain of STAT1 Has a Gene-Specific Role in Transactivation and Cofactor Recruitment

STAT1 has a key role in the regulation of innate and adaptive immunity by inducing transcriptional changes in response to cytokines, such as all types of interferons (IFN). STAT1 exist as two splice isoforms, which differ in regard to the C-terminal transactivation domain (TAD). STAT1β lacks the C-terminal TAD and has been previously reported to be a weaker transcriptional activator than STAT1α, although this was strongly dependent on the target gene. The mechanism of this context-dependent effects remained unclear. By using macrophages from mice that only express STAT1β, we investigated the role of the C-terminal TAD during the distinct steps of transcriptional activation of selected target genes in response to IFNγ. We show that the STAT1 C-terminal TAD is absolutely required for the recruitment of RNA polymerase II (Pol II) and for the establishment of active histone marks at the class II major histocompatibility complex transactivator (CIIta) promoter IV, whereas it is dispensable for histone acetylation at the guanylate binding protein 2 (Gbp2) promoter but required for an efficient recruitment of Pol II, which correlated with a strongly reduced, but not absent, transcriptional activity. IFNγ-induced expression of Irf7, which is mediated by STAT1 in complex with STAT2 and IRF9, did not rely on the presence of the C-terminal TAD of STAT1. Moreover, we show for the first time that the STAT1 C-terminal TAD is required for an efficient recruitment of components of the core Mediator complex to the IFN regulatory factor (Irf) 1 and Irf8 promoters, which both harbor an open chromatin state under basal conditions. Our study identified novel functions of the STAT1 C-terminal TAD in transcriptional activation and provides mechanistic explanations for the gene-specific transcriptional activity of STAT1β

Tubulohelical membrane arrays: From the initial observation to the elucidation of nanophysical properties and cellular function

Lipids undergo self-assembly to form ordered nonlamellar, nanoperiodic arrays both in vitro and in vivo. While engineering of such membrane arrays for technical devices is envisaged, we know little about their cellular function. Do they represent building blocks of an inherent cellular nanotechnology? Prospects for answering this question could be improved if the nanophysical properties of the membrane arrays could be studied in the context of specific cellular functions. Therefore, we draw attention to exceptional complex membrane arrays found in the renal epithelial cell line PtK2 that could provide perfect conditions for both biophysical and cell functional studies. The so-called tubulohelical membrane arrays (TUHMAs) combine nanoperiodicity of lipid membranes with that of helix-like proteinaceous core structures. Strikingly, they show several characteristics of dynamic, microtubule-associated single organelles. Our initial data indicate that TUHMA formation occurs in the depth of the cytoplasm under participation of cytoplasmic nucleoporins. Once matured, they may fuse with the nuclear membrane in polarized positions, either perpendicularly or in parallel to the nucleus. As a starting point for the initiation of functional studies we found a connection between TUHMAs and primary cilia, indicated by immunolabeling patterns of detyrosynated tubulin and cytoplasmic nucleoporins. We discuss these observations in the context of the ciliary cycle and of the specific requirement of ciliated renal epithelial cells for oriented cell division. Finally, we raise the question of whether putative nanooptical properties of TUHMAs could serve for communicating orientation between dividing cells

Cooperative Transcriptional Activation of Antimicrobial Genes by STAT and NF-κB Pathways by Concerted Recruitment of the Mediator Complex

The transcriptional response to infection with the bacterium Listeria monocytogenes (Lm) requires cooperative signals of the type I interferon (IFN-I)-stimulated JAK-STAT and proinflammatory NF-κB pathways. Using ChIP-seq analysis, we define genes induced in Lm-infected macrophages through synergistic transcriptional activation by NF-κB and the IFN-I-activated transcription factor ISGF3. Using the Nos2 and IL6 genes as prime examples of this group, we show that NF-κB functions to recruit enzymes that establish histone marks of transcriptionally active genes. In addition, NF-κB regulates transcriptional elongation by employing the mediator kinase module for the recruitment of the pTEFb complex. ISGF3 has a major role in associating the core mediator with the transcription start as a prerequisite for TFIID and RNA polymerase II (Pol II) binding. Our data suggest that the functional cooperation between two major antimicrobial pathways is based on promoter priming by NF-κB and the engagement of the core mediator for Pol II binding by ISGF3

The Tyrosine Kinase Btk Regulates the Macrophage Response to <i>Listeria monocytogenes</i> Infection

<div><p>In this study we investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium <i>Listeria monocytogenes</i> (<i>Lm</i>). In response to <i>Lm</i> infection, Btk was activated in bone marrow-derived macrophages (BMMs) and <i>Btk</i>^−/− BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of <i>E. coli</i> fragments, there was no difference between wt and <i>Btk</i>^−/− BMMs in the uptake of <i>Lm</i> upon infection. Moreover, there was no difference in the response to heat-killed <i>Lm</i> between wt and <i>Btk</i>^−/− BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular <i>Lm</i>. Finally, <i>Btk</i>^−/− mice displayed enhanced resistance and an increased mean survival time upon <i>Lm</i> infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in <i>Btk</i>^−/− mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during <i>Lm</i> infection.</p> </div

TLR2 is required for PgdA-mediated IFN-β response to <i>Listeria</i> in peritoneal but not bone-marrow macrophages.

<p>(A) PEM from C57BL/6J or <i>tlr2^−/−</i> mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. (B) BMM from C57BL/6J or <i>tlr2^−/−</i> mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. Data are mean ± SD (NS, non significant; ***, <i>p</i><0.0001, <i>n</i> = 3). PEM from WT C57BL/6J or <i>tlr2</i>^−/− mice were infected with the Δ<i>pgdA</i> mutant (C) or the complemented Δ<i>pgdA</i> strain (D). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (**, <i>p</i><0.01, <i>n</i> = 5).</p

Bacterial internalization and NF-κB are required for TLR2 and TRIF-dependent IFN-β response in peritoneal macrophages.

<p>(A) PEM from WT C57BL/6J mice and pretreated with dynasore were infected with the parental EGDe strain (black bars) or the Δ<i>pgdA</i> mutant (grey bars). 4 h post-infection, IκB induction was measured by qRT-PCR. (B) PEM from WT C57BL/6J, <i>tlr2</i>^−/− and <i>trif</i>^−/− mice were infected with the parental EGDe strain (black bars) or the Δ<i>pgdA</i> mutant (grey bars). 4 h post-infection, mRNA was isolated and IκB induction was measured by qRT-PCR. Data are mean ± SD (***, <i>p</i><0.0001, <i>n</i> = 3).</p

IFN-β response to <i>Listeria</i> is mediated by IRF3 and IRF7 in peritoneal macrophages.

<p>PEM from WT C57BL/6J, <i>irf3</i>^−/− and <i>irf7</i>^−/− mice were infected with the parental EGDe strain (black bars) or the Δ<i>pgdA</i> mutant (grey bars). 4 h post-infection, mRNA was isolated and the IFN-β induction was measured by qRT-PCR. Data are mean ± SD (***, <i>p</i><0.0001, <i>n</i> = 3).</p