Kathrin S. Michelsen

Human Intestinal Epithelial Cells Are Broadly Unresponsive to Toll-Like Receptor 2-Dependent Bacterial Ligands: Implications for Host-Microbial Interactions in the Gut

Intestinal epithelial cells (IEC) interact with a high density of Gram-positive bacteria and are active participants in mucosal immune responses. Recognition of Gram-positive organisms by Toll-like receptor (TLR)2 induces proinflammatory gene expression by diverse cells. We hypothesized that IEC are unresponsive to Gram-positive pathogen-associated molecular patterns and sought to characterize the functional responses of IEC to TLR2-specific ligands. Human colonic epithelial cells isolated by laser capture microscopy and IEC lines (Caco-2, T84, HT-29) were analyzed for expression of TLR2, TLR6, TLR1, and Toll inhibitory protein (Tollip) mRNA by RT-PCR and quantitative real-time PCR. Response to Gram-positive bacterial ligands was measured by NF-κB reporter gene activation and IL-8 secretion. TLR2 protein expression was analyzed by immunofluorescence and flow cytometry. Colonic epithelial cells and lamina propria cells from both uninflamed and inflamed tissue demonstrate low expression of TLR2 mRNA compared with THP-1 monocytes. IECs were unresponsive to TLR2 ligands including the staphylococcal-derived Ags phenol soluble modulin, peptidoglycan, and lipotechoic acid and the mycobacterial-derived Ag soluble tuberculosis factor. Transgenic expression of TLR2 and TLR6 restored responsiveness to phenol soluble modulin and peptidoglycan in IEC. In addition to low levels of TLR2 protein expression, IEC also express high levels of the inhibitory molecule Tollip. We conclude that IEC are broadly unresponsive to TLR2 ligands secondary to deficient expression of TLR2 and TLR6. The relative absence of TLR2 protein expression by IEC and high level of Tollip expression may be important in preventing chronic proinflammatory cytokine secretion in response to commensal Gram-positive bacteria in the gut.

Chlamydial Heat Shock Protein 60 Activates Macrophages and Endothelial Cells Through Toll-Like Receptor 4 and MD2 in a MyD88-Dependent Pathway

Active inflammation and NF-κB activation contribute fundamentally to atherogenesis and plaque disruption. Accumulating evidence has implicated specific infectious agents including Chlamydia pneumoniae in the progression of atherogenesis. Chlamydial heat shock protein 60 (cHSP60) has been implicated in the induction of deleterious immune responses in human chlamydial infections and has been found to colocalize with infiltrating macrophages in atheroma lesions. cHSP60 might stimulate, enhance, and maintain innate immune and inflammatory responses and contribute to atherogenesis. In this study, we investigated the signaling mechanism of cHSP60. Recombinant cHSP60 rapidly activated NF-κB in human microvascular endothelial cells (EC) and in mouse macrophages, and induced human IL-8 promoter activity in EC. The inflammatory effect of cHSP60 was heat labile, thus excluding a role of contaminating LPS, and was blocked by specific anti-chlamydial HSP60 mAb. In human vascular EC which express Toll-like receptor 4 (TLR4) mRNA and protein, nonsignaling TLR4 constructs that act as dominant negative blocked cHSP60-mediated NF-κB activation. Furthermore, an anti-TLR4 Ab abolished cHSP60-induced cellular activation, whereas a control Ab had no effect. In 293 cells, cHSP60-mediated NF-κB activation required both TLR4 and MD2. A dominant-negative MyD88 construct also inhibited cHSP60-induced NF-κB activation. Collectively, our results indicate that cHSP60 is a potent inducer of vascular EC and macrophage inflammatory responses, which are very relevant to atherogenesis. The inflammatory effects are mediated through the innate immune receptor complex TLR4-MD2 and proceeds via the MyD88-dependent signaling pathway. These findings may help elucidate the mechanisms by which chronic asymptomatic chlamydial infection contribute to atherogenesis.

β-Defensin-2 Expression Is Regulated by TLR Signaling in Intestinal Epithelial Cells

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of β-defensin expression by IEC. The effect of LPS and peptidoglycan on β-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of β-defensin-2 was assessed using promoter-reporter constructs of the human β-defensin-2 gene. LPS and peptidoglycan stimulated β-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-κB or AP-1 site within the β-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of β-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide β-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.

HMGB1 Mediates Endogenous TLR2 Activation and Brain Tumor Regression

Background Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs. Methods and Findings Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4+ and CD8+ T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1. Conclusions Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.

TLR SIGNALING: AN EMERGING BRIDGE FROM INNATE IMMUNITY TO ATHEROGENESIS

Chronic inflammation and disordered lipid metabolism represent hallmarks of atherosclerosis. Considerable evidence suggests that innate immune defense mechanisms might interact with proinflammatory pathways and contribute to development of arterial plaques. The preponderance of such evidence has been indirect clinical and epidemiologic studies, with some support from experimental animal models of atherosclerosis. However, recent data now directly implicate signaling by TLR4 in the pathogenesis of atherosclerosis, establishing a key link between atherosclerosis and defense against both foreign pathogens and endogenously generated inflammatory ligands. In this study, we briefly review these and closely related studies, highlighting areas that should provide fertile ground for future studies aimed at a more comprehensive understanding of the interplay between innate immune defense mechanisms, atherosclerosis, and related vascular disorders.

TL1A (TNFSF15) Regulates the Development of Chronic Colitis by Modulating Both T-Helper 1 and T-Helper 17 Activation

BACKGROUND & AIMS TL1A is a tumor necrosis factor-like molecule that mediates a strong costimulation of T-helper (T(H)) 1 cells. Expression of TL1A is increased in the mucosa of Crohns disease patients and murine models of ileitis. The aim of this study was to determine the possible role of TL1A in chronic intestinal inflammation. METHODS We used dextran sodium sulfate (DSS)-induced chronic colitis to investigate the effects of TL1A on the development of colitis. The cytokine profile in the gut-associated lymphoid tissue (GALT) was measured. Neutralizing anti-TL1A antibodies were injected intraperitoneally into DSS-induced chronic colitis and G protein alphai2(-/-) T-cell transfer colitis models. Severity of colitis was evaluated by body weight, colon length, histology, and cytokine production. RESULTS DSS-induced chronic colitis was characterized by the infiltration of CD4(+) T cells. TL1A, death receptor 3, interferon (IFN)-gamma, and interleukin (IL)-17 were increased significantly in GALT of DSS-treated mice. TL1A up-regulated both IFN-gamma production from T(H)1 cells and IL-17 production from T(H)17 cells in GALT CD4(+) T cells. Furthermore, IFN-gamma and IL-17 production from CD4(+) T cells, induced by IL-12 and IL-23 respectively, was enhanced synergistically by combination with TL1A. Anti-TL1A antibody prevented chronic colitis and attenuated established colitis by down-regulation of both T(H)1 and T(H)17 activation. CONCLUSIONS Our results reveal that TL1A is an important modulator in the development of chronic mucosal inflammation by enhancing T(H)1 and T(H)17 effector functions. The central role of TL1A represents an attractive, novel therapeutic target for the treatment of Crohns disease patients.

Mycobacterium Tuberculosis Heat Shock Proteins Use Diverse Toll-like Receptor Pathways to Activate Pro-inflammatory Signals

Although the Toll-like receptors used by Mycobacterium tuberculosis membrane and secreted factors are known, the pathways activated by M. tuberculosis heat shock proteins are not. An efficient immune response against the intracellular pathogen M. tuberculosis is critically dependent on rapid detection of the invading pathogen by the innate immune system and coordinated activation of the adaptive immune response. Macrophage phagocytosis of M. tuberculosis is accompanied by activation of the transcription factor NF-κB and secretion of inflammatory mediators that play an important role in granuloma formation and immune protection during M. tuberculosis infection. The interaction between M. tuberculosis and the various Toll-like receptors is complex, and it appears that distinct mycobacterial components may interact with different members of the Toll-like receptor family. Here we show that recombinant, purified, mycobacterial heat shock proteins 65 and 70 induce NF-κB activity in a dose-dependent manner in human endothelial cells. Furthermore, we show that whereas mycobacterial heat shock protein 65 signals exclusively through Toll-like receptor 4, heat shock protein 70 also signals through Toll-like receptor 2. Mycobacterial heat shock protein 65-induced NF-κB activation was MyD88-, TIRAP-, TRIF-, and TRAM-dependent and required the presence of MD-2. A better understanding of the recognition of mycobacterial heat shock proteins and their role in the host immune response to the pathogen may open the way to a better understanding of the immunological processes induced by this important human pathogen and the host-pathogen interactions and may help in the rational design of more effective vaccines or vaccine adjuvants.

Blood-brain barrier invasion by group B Streptococcus depends upon proper cell-surface anchoring of lipoteichoic acid

Group B streptococci (GBSs) are the leading cause of neonatal meningitis. GBSs enter the CNS by penetrating the blood-brain barrier (BBB), which consists of specialized human brain microvascular endothelial cells (hBMECs). To identify GBS factors required for BBB penetration, we generated random mutant libraries of a virulent strain and screened for loss of hBMEC invasion in vitro. Two independent hypo-invasive mutants possessed disruptions in the same gene, invasion associated gene (iagA), which encodes a glycosyltransferase homolog. Allelic replacement of iagA in the GBS chromosome produced a 4-fold decrease in hBMEC invasiveness. Mice challenged with the GBS DeltaiagA mutant developed bacteremia comparably to WT mice, yet mortality was significantly lower (20% vs. 90%), as was the incidence of meningitis. The glycolipid diglucosyldiacylglycerol, a cell membrane anchor for lipoteichoic acid (LTA) and predicted product of the IagA glycosyltransferase, was absent in the DeltaiagA mutant, which consequently shed LTA into the media. Attenuation of virulence of the DeltaiagA mutant was found to be independent of TLR2-mediated signaling, but bacterial supernatants from the DeltaiagA mutant containing released LTA inhibited hBMEC invasion by WT GBS. Our data suggest that LTA expression on the GBS surface plays a role in bacterial interaction with BBB endothelium and the pathogenesis of neonatal meningitis.

CsgA is a pathogen‐associated molecular pattern of Salmonella enterica serotype Typhimurium that is recognized by Toll‐like receptor 2

Knowledge about the origin and identity of the microbial products recognized by the innate immune system is important for understanding the pathogenesis of inflammatory diseases. We investigated the potential role of Salmonella enterica serotype Typhimurium fimbriae as pathogen‐associated molecular patterns (PAMPs) that may stimulate innate pathways of inflammation. We screened a panel of 11 mutants, each carrying a deletion of a different fimbrial operon, for their enteropathogenicity using the calf model of human gastroenteritis. One mutant (csgBA) was attenuated in its ability to elicit fluid accumulation and GROα mRNA expression in bovine ligated ileal loops. The mechanism by which thin curled fimbriae encoded by the csg genes contribute to inflammation was further investigated using tissue culture. The S. Typhimurium csgBA mutant induced significantly less IL‐8 production than the wild type in human macrophage‐like cells. Purified thin curled fimbriae induced IL‐8 expression in human embryonic kidney (HEK293) cells transfected with Toll‐like receptor (TLR) 2/CD14 but not in cells transfected with TLR5, TLR4/MD2/CD14 or TLR11. Fusion proteins between the major fimbrial subunit of thin curled fimbriae (CsgA) and glutathione‐S‐transferase (GST) elicited IL‐8 production in HEK293 cells transfected with TLR2/CD14. Proteinase K treatment abrogated IL‐8 production elicited in these cells by GST–CsgA, but not by synthetic lipoprotein. GST–CsgA elicited more IL‐6 production than GST in bone marrow‐derived macrophages from TLR2+/+ mice, while there was no difference in IL‐6 secretion between GST–CsgA and GST in macrophages from TLR2–/– mice. These data suggested that CsgA is a PAMP that is recognized by TLR2.

The T Cell Costimulator TL1A Is Induced by FcγR Signaling in Human Monocytes and Dendritic Cells

The recently described TL1A/DR3 ligand/receptor pair mediates strong costimulation of Th1 cells. Activation of T and NK cells induces DR3 expression, permitting soluble recombinant TL1A to increase IFN-γ production and proliferation of these cells. Gut T cells and macrophages express TL1A, especially in Crohn’s disease (CD), and there is a strong association between CD and tl1a single nucleotide polymorphisms. Murine studies implicate TL1A in gut inflammation. To determine whether professional T cell-activating cells can express TL1A, fresh blood monocytes and monocyte-derived dendritic cells were stimulated with various activating ligands, including TLR agonists, IFN-γ, and immune complexes. FcγR stimulation strongly induced TL1A mRNA in both cell types, which correlated with the detection of TL1A on the cell surface and in cell culture medium. TLR agonists capable of inducing IL-6 and TNF-α in monocytes and dendritic cells did not induce surface nor soluble TL1A. Furthermore, we demonstrate that TL1A production in monocytes leads to enhancement of T cell responses. The induction of TL1A on APCs via specific pathway stimulation suggests a role for TL1A in Th1 responses to pathogens, and in CD.