Hortense Slevogt

Listeria monocytogenes-infected human peripheral blood mononuclear cells produce IL-1beta, depending on listeriolysin O and NLRP3.

Different NOD-like receptors, including NLRP1, NLRP3, and NLRC4, as well as the recently identified HIN-200 protein, AIM2, form multiprotein complexes called inflammasomes, which mediate caspase-1–dependent processing of pro-IL-1β. Listeria monocytogenes is an intracellular pathogen that is actively phagocytosed by monocytes/macrophages and subsequently escapes from the phagosome into the host cell cytosol, depending on its pore-forming toxin listeriolysin O (LLO). In this study, we demonstrate that human PBMCs produced mature IL-1β when infected with wild-type L. monocytogenes or when treated with purified LLO. L. monocytogenes mutants lacking LLO or expressing a noncytolytic LLO as well as the avirulent Listeria innocua induced strongly impaired IL-1β production. RNA interference and inhibitor experiments in human PBMCs as well as experiments in Nlrp3 and Rip2 knockout bone marrow-derived macrophages demonstrated that the Listeria-induced IL-1β release was dependent on ASC, caspase-1, and NLRP3, whereas NOD2, Rip2, NLRP1, NLRP6, NLRP12, NLRC4, and AIM2 appeared to be dispensable. We found that L. monocytogenes-induced IL-1β production was largely dependent on phagosomal acidification and cathepsin B release, whereas purified LLO activated an IL-1β production independently of these mechanisms. Our results indicate that L. monocytogenes-infected human PBMCs produced IL-1β, largely depending on an LLO-mediated phagosomal rupture and cathepsin B release, which is sensed by Nlrp3. In addition, an LLO-dependent but cathepsin B-independent NLRP3 activation might contribute to some extent to the IL-1β production in L. monocytogenes-infected cells.

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.

CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells.

Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2–initiated transcription factor NF-κB–dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2–dependent activation of the phosphatidylinositol 3-OH kinase–Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.

Statins Control Oxidized LDL-Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells

Objective—Activation of the endothelium by oxidized low-density lipoprotein (oxLDL) has been implicated in the development of atherosclerosis. Histone modifications impact on the transcriptional activity state of genes. We tested the hypothesis that oxLDL-induced inflammatory gene expression is regulated by histone modifications and experienced the effect of statins on these alterations. Methods and Results—OxLDL-related interleukin-8 (IL-8) and monocyte-chemoattractant protein-1 (MCP-1) secretion in endothelial cells was reduced by statins but enhanced by histone deacetylase inhibitors. OxLDL induced lectin-like oxidized LDL receptor-1 (LOX-1) and extracellular regulated kinases (ERK1/2)-dependent acetylation of histone H3 and H4 as well as phosphorylation of histone H3, both globally and on the promoters of il8 and mcp1. Pretreatment of oxLDL-exposed cells with statins reduced the above mentioned histone modification, as well as recruitment of CREB binding protein (CBP) 300, NF-&kgr;B, and of RNA polymerase II but prevented loss of binding of histone deacetylase (HDAC)-1 and -2 at the il8 and mcp1 gene promoters. OxLDL reduced HDAC1 and 2 expression, and statins partly restored global HDAC-activity. Statin-related effects were reverted with mevalonate. In situ experiments indicated decreased expression of HDAC2 in endothelial cells in atherosclerotic plaques of human coronary arteries. Conclusions—Histone modifications seem to play an important role in atherosclerosis.

Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response

Moraxella catarrhalis is an important pathogen in patients with chronic obstructive lung disease (COPD). While M.u2003catarrhalis has been categorized as an extracellular bacterium so far, the potential to invade human respiratory epithelium has not yet been explored. Our results obtained by electron and confocal microscopy demonstrated a considerable potential of M.u2003catarrhalis to invade bronchial epithelial (BEAS‐2B) cells, type II pneumocytes (A549) and primary small airway epithelial cells (SAEC). Moraxella invasion was dependent on cellular microfilament as well as on bacterial viability, and characterized by macropinocytosis leading to the formation of lamellipodia and engulfment of the invading organism into macropinosomes, thus indicating a trigger‐like uptake mechanism. In addition, the cells examined expressed TLR2 as well as NOD1, a recently found cytosolic protein implicated in the intracellular recognition of bacterial cell wall components. Importantly, inhibition of TLR2 or NOD1 expression by RNAi significantly reduced the M.u2003catarrhalis‐induced IL‐8 secretion. The role of TLR2 and NOD1 was further confirmed by overexpression assays in HEK293 cells. Overall, M.u2003catarrhalis may employ lung epithelial cell invasion to colonize and to infect the respiratory tract, nonetheless, the bacteria are recognized by cell surface TLR2 and the intracellular surveillance molecule NOD1.

IFNβ responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI)

Intracellular bacteria and cytosolic stimulation with DNA activate type I IFN responses independently of Toll‐like receptors, most Nod‐like receptors and RIG‐like receptors. A recent study suggested that ZBP1 (DLM‐1/DAI) represents the long anticipated pattern recognition receptor which mediates IFNα/β responses to cytosolic DNA in mice. Here we show that Legionella pneumophila infection, and intracellular challenge with poly(dA‐dT), but not with poly(dG‐dC), induced expression of IFNβ, full‐length hZBP1 and a prominent splice variant lacking the first Zα domain (hZBP1ΔZα) in human cells. Overexpression of hZBP1 but not hZBP1ΔZα slightly amplified poly(dA‐dT)‐stimulated IFNβ reporter activation in HEK293 cells, but had no effect on IFNβ and IL‐8 production induced by bacteria or poly(dA‐dT) in A549 cells. We found that mZBP1 siRNA impaired poly(dA‐dT)‐induced IFNβ responses in mouse L929 fibroblasts at a later time point, while multiple hZBP1 siRNAs did not suppress IFNβ or IL‐8 expression induced by poly(dA‐dT) or bacterial infection in human cells. In contrast, IRF3 siRNA strongly impaired the IFNβ responses to poly(dA‐dT) or bacterial infection. In conclusion, intracellular bacteria and cytosolic poly(dA‐dT) activate IFNβ responses in different human cells without requiring human ZBP1.

Histone Acetylation and Flagellin Are Essential for Legionella pneumophila-Induced Cytokine Expression

Legionella pneumophila causes severe pneumonia. Acetylation of histones is thought to be an important regulator of gene transcription, but its impact on L. pneumophila-induced expression of proinflammatory cytokines is unknown. L. pneumophila strain 130b induced the expression of the important chemoattractant IL-8 and genome-wide histone modifications in human lung epithelial A549 cells. We analyzed the IL-8-promoter and found that histone H4 was acetylated and H3 was phosphorylated at Ser10 and acetylated at Lys14, followed by transcription factor NF-κB. Recruitment of RNA polymerase II to the IL-8 promoter corresponded with increases in gene transcription. Histone modification and IL-8 release were dependent on p38 kinase and NF-κB pathways. Legionella-induced IL-8 expression was decreased by histone acetylase (HAT) inhibitor anacardic acid and enhanced by histone deacetylase (HDAC) inhibitor trichostatin A. After Legionella infection, HATs p300 and CREB-binding protein were time-dependently recruited to the IL-8 promoter, whereas HDAC1 and HDAC5 first decreased and later reappeared at the promoter. Legionella specifically induced expression of HDAC5 but not of other HDACs in lung epithelial cells, but knockdown of HDAC1 or 5 did not alter IL-8 release. Furthermore, Legionella-induced cytokine release, promoter-specific histone modifications, and RNA polymerase II recruitment were reduced in infection with flagellin-deletion mutants. Legionella-induced histone modification as well as HAT-/HDAC-dependent IL-8 release could also be shown in primary lung epithelial cells. In summary, histone acetylation seems to be important for the regulation of proinflammatory gene expression in L. pneumophila infected lung epithelial cells. These pathways may contribute to the host response in Legionnaires’ disease.

Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

β-PIX and Rac1 GTPase Mediate Trafficking and Negative Regulation of NOD2

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-κB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (β-PIX) plays a role in this negative regulation, because knock-down of β-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with β-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of β-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that β-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1’s positive regulatory role in TLR2 signaling.

Comparison of six immunohistochemical markers for the histologic diagnosis of neoplasia in Barrett’s esophagus

In esophageal neoplasms, the histopathologic differentiation between Barrett’s esophagus with or without intraepithelial neoplasia and adenocarcinoma is often challenging. Immunohistochemistry might help to differentiate between these lesions. The expression of CDX2, LI-cadherin, mucin 2 (MUC2), blood group 8 (BG8, Lewisy), claudin-2, and villin was investigated in normal gastroesophageal (nu2009=u200923) and in Barrett’s (nu2009=u200917) mucosa, in low-grade (nu2009=u200912) and high-grade (nu2009=u20099) intraepithelial neoplasia (IEN) as well as in esophageal adenocarcinoma (nu2009=u200916), using immunohistochemistry. For CDX2 and LI-cadherin, the immunoreactivity score was highest in IEN while for MUC2, BG8, and villin, it dropped gradually from Barrett’s via IEN to adenocarcinoma, and expression of Claudin-2 was only weak and focal in all lesions. The expression of MUC2 and LI-cadherin differed significantly between all examined lesions except between low-grade and high-grade IEN. MUC2 and LI-cadherin are useful immunohistochemical markers for the differentiation between normal glandular mucosa, Barrett’s mucosa, IEN, and invasive carcinoma of the esophagus; however, none of the examined markers was helpful for the differentiation between low-grade and high-grade IEN.