Autophagy and toll-like receptors: A new link in cancer cells

In addition to its clean-up function, autophagy is considered as an innate immunity mechanism due to its role in the removal of intracellular pathogens. Toll-like receptors (TLRs) are crucial components of innate immunity involved in the recognition of a diverse array of microbial products. Recent works demonstrated that different pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and single-strand RNA are able to induce autophagy via different TLRs in immune cells. In a recent report, we showed that bacterial CpG motifs, another PAMP, can induce autophagy in rodent and human tumor cell lines and that this process is TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after the intratumoral injection of CpG motifs. These results extend the link between TLRs and autophagy to non-immune tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues. In this addendum, we discuss the potential mechanisms and the consequences of the CpG-induced autophagy in tumor cells.

Addendum to: Bertin S, Samson M, Pons C, Guigonis JM, Gavelli A, Baque P, Brossette N, Pagnotta S, Ricci JE, Pierrefite-Carle V. Comparative proteomics study reveals that bacterial CpG motifs induce tumor cell autophagy in vitro and in vivo. Mol Cell Proteomics 2008; In press.     

Autophagy and toll-like receptors: a new link in cancer cells

In addition to its clean-up function, autophagy is considered as an innate immunity mechanism due to its role in the removal of intracellular pathogens. Toll-like receptors (TLRs) are crucial components of innate immunity involved in the recognition of a diverse array of microbial products. Recent works demonstrated that different pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and single-strand RNA are able to induce autophagy via different TLRs in immune cells. In a recent report, we showed that bacterial CpG motifs, another PAMP, can induce autophagy in rodent and human tumor cell lines and that this process is TLR9-dependent. In addition, an increase in the number of autophagosomes can also be observed in vivo after the intratumoral injection of CpG motifs. These results extend the link between TLRs and autophagy to non-immune tumor cells and may be relevant for cancer treatment and more generally for gene therapy approaches in TLR9-positive tissues. In this addendum, we discuss the potential mechanisms and the consequences of the CpG-induced autophagy in tumor cells.     

Cancer immunotherapy with CpG-ODN

Bacterial DNA and synthetic oligodeoxynucleotides containing CpG motifs (CpG-ODN) are the ligands for the Toll-like receptor 9 (TLR9), which is expressed by B-lymphocytes and a subset of dendritic cells. CpG-ODN are strong activators of both innate and specific immunity, and drive the immune response towards the Th1 phenotype. Given the promising results obtained in several experimental models of allergies or infections, CpG-ODN are now entering clinical trials for these diseases. In cancer, promising approaches combined CpG-ODN with tumor antigens, monoclonal antibodies or dendritic cells. When no relevant tumor antigen is known, CpG-ODN can be used alone to activate locally the innate immunity and trigger a tumor-specific immune response, overcoming the need for the identification of a tumoral antigen. Preclinical models have shown impressive results and several clinical trials are on-going worldwide in melanoma, lymphoma, renal carcinoma, breast cancer and glioblastoma.     

A threshold level of TLR9 mRNA predicts cellular responsiveness to CpG-ODN in haematological and non-haematological tumour cell lines

The human toll like receptor 9 (TLR9) detects differences between microbial and host DNA, based on unmethylated deoxycytidyl deoxyguanosine dinucleotide (CpG) motifs, leading to activation of both innate and adaptive immune mechanisms. The synthetic TLR9 agonist, CpG-ODN, can substitute for microbial DNA in these responses, and is in clinical trials as an immunomodulatory agent in diseases as diverse as infections, cancer and allergic disorders. Human TLR9 is expressed on cells of haematopoietic origin (principally plasmacytoid dendritic cells and B cells), but has also been described as being expressed on a number of other cell types. In order to clarify the expression and function of TLR9 in a range of cells of both haematopoietic and non-haematopoietic origin, we investigated the level of expression of TLR9 mRNA, and the ability of the cells to respond to CpG-ODN by upregulation of cell surface markers, cytokine production, cellular proliferation and activation of NFκB. Our data show that the cellular response to CpG-ODN depended on a threshold level of expression of TLR9. TLR9 was widely expressed amongst B cell tumours (with the exception of myeloma cell lines), but we did not find either threshold levels of expression of TLR9 or responses to CpG-ODN in several myeloma or myeloid tumour cell lines or any non-haematological tumour cell lines tested in our study. TLR9-positive cells varied significantly in their responses to CpG-ODN, and the level of TLR9 expression beyond the threshold did not correlate with the magnitude of the response to CpG-ODN. Finally, CpG-ODN induced NFκB activation and increased cellular proliferation in Hek293 cells that had been stably transfected with hTLR9, but did not affect the expression of surface markers or synthesis of IL-6, IL-10 or TNF-α. Thus both haematological and non-haematological cells expressing appropriate levels of TLR9 respond to CpG-ODN, but the nature of the TLR9-mediated response is dependent on cell type.     

Direct evidence that toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-guanine motif recognition

Cytosine-phosphate-guanine (CpG) motifs in bacterial DNA are known to activate the mammalian immune system, and this activation is thought to depend on the Toll-like receptor 9 (TLR9) signaling pathway. Previous studies strongly suggested that TLR9 is involved as the specific receptor for CpG motifs but did not provide direct evidence of their interaction. In this study, we demonstrate for the first time that murine TLR9 binds an unmethylated CpG-containing plasmid. This interaction is sequence-specific and is influenced by the methylation status of the plasmid. Furthermore, we demonstrate that this interaction leads to the activation of the NF-kappaB pathway in mTLR9-expressing cells. Our results provide a molecular basis for the interaction between CpG-DNA and TLR9.     

Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes

Bacterial DNA acts as an alert signal for eukaryotic cells through immunostimulatory CpG motifs. These sequences have therapeutic properties promoting protective immune TH1 responses and are recognized by a membrane protein belonging to the Toll-like receptor (TLR) family, named TLR-9. The aim of this study was to test the capability of murine hepatocytes to sense bacterial DNA and to develop antibacterial mechanisms against Salmonella typhimurium. We show that hepatocyte cell lines and mRNA extracts from murine liver constitutively express TLR-9, which is down-regulated by LPS and the mix of IFNgamma, IL-1beta and LPS. Also, we have found that hepatocyte cell lines can sense the presence of bacterial DNA and respond to it by increasing the pool of intracellular peroxides. This results in inhibition of intracellular growth of S. typhimurium when infected cells were incubated in the presence of CpG synthetic oligonucleotides (CpG-ODN). Expression of hepatocyte Mn-SOD is also induced by stimulation with CpG-oligodeoxynucleotides, LPS, and the mix of IFNgamma, IL-1beta and LPS. These results reinforce the prominent role of hepatocytes as a microbial product-responsive cell and the capabilities of CpG-ODN sequences as potent inducers of the innate immune response through the activation of a broad range of cell types.     

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo

Background

Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-α) and interleukin-1β (IL-1β). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo.

Methods

Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured.

Results

In WT mice, CpG-ODN induced a strong activation of pulmonary NFκB as well as a significant increase in pulmonary TNF-α and IL-1β mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice.

Conclusion

This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.     

Lymphoid follicle destruction and immunosuppression after repeated CpG oligodeoxynucleotide administration

DNA containing unmethylated cytidyl guanosyl (CpG) sequences, which are underrepresented in mammalian genomes but prevalent in prokaryotes, is endocytosed by cells of the innate immune system, including macrophages, monocytes and dendritic cells, and activates a pathway involving Toll-like receptor-9 (TLR9). CpG-containing oligodeoxynucleotides (CpG-ODN) are potent stimulators of innate immunity, and are currently being tested as adjuvants of antimicrobial, antiallergic, anticancer and antiprion immunotherapy. Little is known, however, about the consequences of repeated CpG-ODN administration, which is advocated for some of these applications. Here we report that daily injection of 60 microg CpG-ODN dramatically alters the morphology and functionality of mouse lymphoid organs. By day 7, lymphoid follicles were poorly defined; follicular dendritic cells (FDC) and germinal center B lymphocytes were suppressed. Accordingly, CpG-ODN treatment for > or =7 d strongly reduced primary humoral immune responses and immunoglobulin class switching. By day 20, mice developed multifocal liver necrosis and hemorrhagic ascites. All untoward effects were strictly dependent on CpG and TLR9, as neither the CpG-ODN treatment of Tlr9(-/-) mice nor the repetitive challenge of wild-type mice with nonstimulatory ODN (AT-ODN) or with the TLR3 agonist polyinosinic:cytidylic acid (polyI:C) were immunotoxic or hepatotoxic.     

G3139 and other CpG-containing immunostimulatory phosphorothioate oligodeoxynucleotides are potent suppressors of the growth of human tumor xenografts in nude mice

Several phosphorothioate antisense oligodeoxynucleotides (ODN) are developed to target factors potentially involved in tumor growth and apoptosis suppression. Among them, the 18-mer G3139 (Oblimersen), which targets Bcl-2, is currently being tested in phase II and phase III clinical trials for various tumors in combination with chemotherapy. On the other hand, ODNs containing CpG dinucleotides (CpG-ODN) within specific-sequence contexts (CpG motifs) have been shown to activate rodent or primate immune cells via toll-like receptor 9 (TLR9) and have demonstrated remarkable T cell-dependent antitumor efficacy in a series of murine tumor models. However, immune cell activation by CpG-ODN is largely diminished upon C-5 methylation at CpG cytosine. As G3139 contains CpG motifs, we questioned whether the antitumor effects seen in human tumor xenografts might be abrogated by cytosine C-5 methylation of G3139, which retained the ability of G3139 to suppress Bcl-2 expression in tissue culture, or by similar derivatization of other phosphorothioate ODNs developed for the immune activation of rodent or human cells. The in vivo antitumor efficacy of the immunostimulatory H1826 and H2006 ODNs was compared with that of G3139. Bcl-2 suppression achieved by G3139 purportedly sensitizes tumor cells toward cytotoxic agents, and some of the experiments employed combinations of ODN with such drugs as cisplatin or etoposide. H1826, H2006, and G3139 all produced similar, striking, growth inhibitory effects on either H69 SCLC, A2780 ovarian carcinoma, or A549 lung adenocarcinoma human tumor xenografts at doses of 0.3 mg/kg and 1 mg/kg (H1826, H2006) or 12 mg/kg (G3139) per day. In contrast, the H2006-mC (1 mg/kg) or G3139-mC (12 mg/kg) derivatives demonstrated no significant antitumor effects. The combination of G3139 (12 mg/kg) with cisplatin produced some additive antitumor efficacy, which was not seen in combinations of G3139-mC (12 mg/kg) or H1826 (1 mg/kg) with cisplatin. G3139, at a dose of 12 mg/kg, alone induced extensive enlargement of the spleen. Immunostimulation was evaluated in vitro by flow cytometric measurements of the CD80 and CD86 activation markers found on CD19+ murine splenocytes. The CpG-ODN producing strong antitumor effects in vivo also induced these activation markers in vitro, in contrast to the in vivo inactive G3139-mC. Our data indicate a significant contribution of the immunostimulatory properties of CpG-ODN (including G3139) to the antitumor effects observed in nude mouse xenograft models. This is in contrast to previous data presented by other authors indicating that the activity of G3139 in human tumor xenografts was Bcl-2 specific. Furthermore, as nude mice are devoid of T cells, a T cell-mediated immune response apparently is not required for the potent antitumor responses observed here; innate immune responses are sufficient.     

Newly identified CpG ODNs,M5-30 and M6-395, stimulate mouse immune cells to secrete TNF-α and enhance Th1-mediated immunity

Bacterial CpG motifs are known to induce both innate and adaptive immunity in infected hosts via toll-like receptor 9 (TLR9). Because small oligonucleotides (ODNs) mimicking bacterial CpG motifs are easily synthesized, they have found use as immunomodulatory agents in a number of disease models. We have developed a novel bioinformatics approach to identify effective CpG ODN sequences and evaluate their function as TLR9 ligands in a murine system. Among the CpG ODNs we identified, M5-30 and M6-395 showed significant ability to stimulate TNF-α and IFN-γ production in a mouse macrophage cell line and mouse splenocytes, respectively. We also found that these CpG ODNs activated cells through the canonical NF-κ B signaling pathway. Moreover, both CpG ODNs were able to induce Th1-mediated immunity in Mycobacterium tuberculosis (Mtb)-infected mice. Our results demonstrate that M5-30 and M6-395 function as TLR9-specific ligands, making them useful in the study of TLR9 functionality and signaling in mice.     

TLR ligands in the local treatment of established intracerebral murine gliomas

Local TLR stimulation is an attractive approach to induce antitumor immunity. In this study, we compared various TLR ligands for their ability to affect murine GL261 cells in vitro and to eradicate established intracerebral murine gliomas in vivo. Our data show that GL261 cells express TLR2, TLR3, and TLR4 and respond to the corresponding TLR ligands with increasing MHC class I expression and inducing IL-6 secretion in vitro, while TLR5, TLR7, and TLR9 are essentially absent. Remarkably, CpG-oligonucleotides (CpG-ODN, TLR9) appeared to inhibit GL261 cell proliferation in a cell-type specific, but CpG-motif and TLR9-independent manner. A single intratumoral injection of CpG-ODN most effectively inhibited glioma growth in vivo and cured 80% of glioma-bearing C57BL/6 mice. Intratumoral injection of Pam3Cys-SK4 (TLR1/2) or R848 (TLR7) also produced a significant survival benefit, whereas poly(I:C) (TLR3) or purified LPS (TLR4) stimulation alone was not effective. Additional studies using TLR9(+/+) wild-type and TLR9(-/-) knockout mice revealed that the efficacy of local CpG-ODN treatment in vivo required TLR9 expression on nontumor cells. Additional experiments demonstrated increased frequencies of tumor-infiltrating IFN-gamma producing CD4(+) and CD8(+) effector T cells and a marked increase in the ratio of CD4(+) effector T cells to CD4(+)FoxP3(+) regulatory T cells upon CpG-ODN treatment. Surviving CpG-ODN treated mice were also protected from a subsequent tumor challenge without further addition of CpG-ODN. In summary, this study underlines the potency of local TLR treatment in antiglioma therapy and demonstrates that local CpG-ODN treatment most effectively restores antitumor immunity in a therapeutic murine glioma model.     

CpG DNA: recognition by and activation of monocytes

Unmethylated CpG motifs present in bacterial DNA rapidly trigger an innate immune response characterized by the activation of Ig- and cytokine-secreting cells. Synthetic oligonucleotides (ODNs) containing CpG motifs mimic this activity, triggering monocytes to proliferate, secrete and/or differentiate. Analysis of hundreds of novel ODNs led to the identification of two structurally distinct classes of CpG motif that differentially activate human monocytes. ODNs of the "K"-type interact with Toll-like receptor 9 and induce monocytes to proliferate and secrete IL-6. In contrast, "D"-type ODNs trigger monocytes to differentiate into mature dendritic cells.     

Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses

The TLRs 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. U-rich RNA sequences were recently discovered that stimulate human TLR7/8-mediated or murine TLR7-mediated immune effects. In this study we identified single-stranded RNA sequences containing defined sequence motifs that either preferentially activate human TLR8-mediated as opposed to TLR7- or TLR7/8-mediated immune responses. The identified TLR8 RNA motifs signal via TLR8 and fail to induce IFN-alpha from TLR7-expressing plasmacytoid dendritic cells but induce the secretion of Th1-like and proinflammatory cytokines from TLR8-expressing immune cells such as monocytes or myeloid dendritic cells. In contrast, RNA sequences containing the TLR7/8 motif signal via TLR7 and TLR8 and stimulate cytokine secretion from both TLR7- and TLR8-positive immunocytes. The TLR8-specific RNA sequences are able to trigger cytokine responses from human and bovine but not from mouse, rat, and porcine immune cells, suggesting that these species lack the capability to respond properly to TLR8 RNA ligands. In summary, we describe two classes of single-stranded TLR7/8 and TLR8 RNA agonists with diverse target cell and species specificities and immune response profiles.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

Toll‐like receptor signalling cross‐activates the autophagic pathway to restrict Salmonella Typhimurium growth in macrophages

It has been long recognised that activation of toll‐like receptors (TLRs) induces autophagy to restrict intracellular bacterial growth. However, the mechanisms of TLR‐induced autophagy are incompletely understood. Salmonella Typhimurium is an intracellular pathogen that causes food poisoning and gastroenteritis in humans. Whether TLR activation contributes to S. Typhimurium‐induced autophagy has not been investigated. Here, we report that S. Typhimurium and TLRs shared a common pathway to induce autophagy in macrophages. We first showed that S. Typhimurium‐induced autophagy in a RAW264.7 murine macrophage cell line was mediated by the AMP‐activated protein kinase (AMPK) through activation of the TGF‐β‐activated kinase (TAK1), a kinase activated by multiple TLRs. AMPK activation led to increased phosphorylation of Unc‐51‐like autophagy activating kinase (ULK1) at S317 and S555. ULK1 phosphorylation at these two sites in S. Typhimurium‐infected macrophages overrode the inhibitory effect of mTOR on ULK1 activity due to mTOR‐mediated ULK1 phosphorylation at S757. Lipopolysaccharide (LPS), flagellin, and CpG oligodeoxynucleotide, which activate TLR4, TLR5, and TLR9, respectively, increased TAK1 and AMPK phosphorylation and induced autophagy in RAW264.7 cells and in bone marrow‐derived macrophages. However, LPS was unable to induce TAK1 and AMPK phosphorylation and autophagy in TLR4‐deficient macrophages. TAK1 and AMPK‐specific inhibitors blocked S. Typhimurium‐induced autophagy and xenophagy and increased the bacterial growth in RAW264.7 cells. These observations collectively suggest that activation of the TAK1–AMPK axis through TLRs is essential for S. Typhimurium‐induced autophagy and that TLR signalling cross‐activates the autophagic pathway to clear intracellular bacteria.     

Incidence of asthma and mortality in a cohort of young adults: a 7-year prospective study

Background

CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.

Methods

The expression of TLR9 in HOPE-fixed non-small lung cancer, non-malignant tissue and tumor cell lines was assessed using immunohistochemistry, confocal microscopy, in situ hybridization, RT-PCR and DNA-sequencing. Apoptosis and chemokine expression was detected by FACS analysis and the Bio-Plex system.

Results

We found high TLR9 signal intensities in the cytoplasm of tumor cells in the majority of lung cancer specimens as well as in all tested tumor cell lines. In contrast to this non-malignant lung tissues showed only sporadically weak expression. Stimulation of HeLa and A549 cells with CpG-ODN induced secretion of monocyte chemoattractant protein-1 and reduction of spontaneous and tumor necrosis factor-alpha induced apoptosis.

Conclusions

Here we show that TLR9 is expressed in a selection of human lung cancer tissues and various tumor cell lines. The expression of functionally active TLR9 in human malignant tumors might affect treatment approaches using CpG-ODN and shows that malignant cells can be regarded as active players in tumor-immunology.     

Cutting edge: Role of Toll-like receptor 9 in CpG DNA-induced activation of human cells

Unmethylated CpG motifs present in bacterial DNA stimulate a rapid and robust innate immune response. Human cell lines and PBMC that recognize CpG DNA express membrane-bound human Toll-like receptor 9 (hTLR9). Cells that are not responsive to CpG DNA become responsive when transfected with hTLR9. Expression of hTLR9 dramatically increases uptake of CpG (but not control) DNA into endocytic vesicles. Upon cell stimulation, hTLR9 and CpG DNA are found in the same endocytic vesicles. Cells expressing hTLR9 are stimulated by CpG motifs that are active in primates but not rodents, suggesting that evolutionary divergence between TLR9 molecules underlies species-specific differences in the recognition of bacterial DNA. These findings indicate that hTLR9 plays a critical role in the CpG DNA-mediated activation of human cells.