|, CD289, toll like receptor 9|
Toll-like receptor 9 is a protein that in humans is encoded by the TLR9 gene. TLR9 has also been designated as CD289 (cluster of differentiation 289). It is a member of the toll-like receptor (TLR) family. TLR9 is an important receptor expressed in immune system cells including dendritic cells, macrophages, natural killer cells, and other antigen presenting cells. TLR9 preferentially binds DNA present in bacteria and viruses, and triggers signaling cascades that lead to a pro-inflammatory cytokine response. Cancer, infection, and tissue damage can all modulate TLR9 expression and activation. TLR9 is also an important factor in autoimmune diseases, and there is active research into synthetic TLR9 agonists and antagonists that help regulate autoimmune inflammation.
- 1 Function
- 2 Role in non-viral cancer
- 3 Expression in oncogenic viral infection
- 4 Clinical relevance of inflammation response
- 5 Role in heart health
- 6 As an immunotherapy target
- 7 Protein interactions
- 8 References
- 9 External links
The TLR family plays a fundamental role in pathogen recognition and activation of innate immunity. TLRs are named for the high degree of conservation in structure and function seen between mammalian TLRs and the Drosophila transmembrane protein Toll. TLRs are transmembrane proteins, expressed on the cell surface and the endocytic compartment and recognize pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents and initiate signalling to induce production of cytokines necessary for the innate immunity and subsequent adaptive immunity. The various TLRs exhibit different patterns of expression.
This gene is preferentially expressed in immune cell rich tissues, such as spleen, lymph node, bone marrow and peripheral blood leukocytes. Studies in mice and humans indicate that this receptor mediates cellular response to unmethylated CpG dinucleotides in bacterial DNA to mount an innate immune response.
TLR9 is usually activated by unmethylated CpG sequences in DNA molecules. Once activated, TLR9 moves from the endoplasmic reticulum to the Golgi apparatus and lysosomes, where it interacts with MyD88, the primary protein in its signaling pathway. TLR9 is cleaved at this stage to avoid whole protein expression on cell surface, which could lead to autoimmunity. CpG sites are relatively rare (~1%) on vertebrate genomes in comparison to bacterial genomes or viral DNA. TLR9 is expressed by numerous cells of the immune system such as B lymphocytes, monocytes, natural killer (NK) cells, keratinocytes, melanocytes, and plasmacytoid dendritic cells. TLR9 is expressed intracellularly, within the endosomal compartments and functions to alert the immune system of viral and bacterial infections by binding to DNA rich in CpG motifs. TLR9 signals leads to activation of the cells initiating pro-inflammatory reactions that result in the production of cytokines such as type-I interferon, IL-6, TNF, IFNα, and IL-12. There is also recent evidence that TLR9 can recognize nucleotides other than unmethylated CpG present in bacterial or viral genomes. TLR9 has been shown to recognized DNA:RNA hybrids, but not ssDNA.
TLR9 expression progression during cancer varies greatly with the type of cancer. TLR9 may even present an exciting new marker for many cancer types. Breast cancer and renal cell carcinoma have both been shown to diminish expression of TLR9. In these cases higher levels correspond with better outcomes. Conversely studies have shown higher levels of TLR9 expression in breast cancer and ovarian cancer patients, and poor prognosis is associated with higher TLR9 expression in prostate cancer. Non-small cell lung cancer and glioma have also been shown to up-regulate the expression of TLR9. While these results are highly variable, it is clear that TLR9 expression increases the capacity for invasion and proliferation. Whether cancer induces modification of TLR9 expression or TLR9 expression hastens the onset of cancer is unclear, but many of the mechanisms that regulate cancer development also play a role in TLR9 expression. DNA damage and the p53 pathway influence TLR9 expression, and the hypoxic environment of tumor cells certainly induces expression of TLR9, further increases proliferation ability of the cancerous cells. Cellular stress has also been shown to relate to TLR9 expression. It is possible that cancer and TLR9 have a feed-forward relationship, where the occurrence of one leads to the up-regulation of the other. Many viruses take advantage of this relationship by inducing certain TLR9 expression patterns to first infect the cell (down-regulate) then trigger the onset of cancer (up-regulate).
Human papilloma virus (HPV)
Human papilloma virus is a deadly disease[clarification needed] that, if left untreated, can lead to epithelial lesions and cervical cancer. HPV infection inhibits the expression of TLR9 in keratinocytes, abolishing the production of IL-8. However inhibition of TLR9 by oncogenic viruses is temporary, and patients with long-lasting HPV actually show higher levels of TLR9 expression in cervical cells. In fact, the increase in expression is so severe that TLR9 could be used as a biomarker for cervical cancer. The relationship between HPV-induced epithelial lesion, cancer progression, and TLR9 expression is still under investigation.
Hepatitis B virus (HBV)
Hepatitis B virus down-regulates the expression of TLR9 in pDCs and B cells, destroying the production of IFNα and IL-6. However, just as in HPV, as the disease progresses TLR9 expression is up-regulated. HBV induces an oncogenic transformation, which leads to a hypoxic cellular environment. This environment causes the release of mitochondrial DNA, which has CpG regions that can bind to TLR9. This induces over-expression of TLR9 in tumor cells, contrary to the inhibitory early stages of infection.
Epstein-Barr virus (EBV)
Epstein-Barr virus, like other oncogenic viruses, decreases the expression of TLR9 in B cells, diminishing production of TNF and IL-6. EBV has been reported to alter expression of TLR9 at the transcription, translation, and protein level.
The viruses of the polyomavirus family destroy expression of TLR9 in keratinocytes, inhibiting the release of IL-6 and IL-8. Expression is regulated at the promoter, where antigen proteins inhibit transcription. Similar to HPV and HBV infection, TLR9 expression increases as the disease progresses, probably due to the hypoxic nature of the solid tumor environment.
Clinical relevance of inflammation response
TLR9 has been identified as a major player in systemic lupus erythematosus (SLE) and erythema nodosum leprosum (ENL). Loss of TLR9 exacerbates progression of SLE, and leads to increased activation of dentritic cells. TLR9 also controls the release of IgA and IFN-a in SLE, and loss of the receptor leads to higher levels of both molecules. In SLE, TLR9 and TLR7 have opposing effects. TLR9 regulates inflammatory response, while TLR7 promotes inflammatory response. TLR9 has an opposite effect in ENL. TLR9 is expressed at high levels on monocytes of ENL patients, and is positively linked to the secretion of proinflammatory cytokines TNF, IL-6, and IL-1β. TLR9 agonists and antagonists may be useful in treatment of a variety of inflammatory conditions, and research in this area is active. Autoimmune thyroid diseases have also been shown to correlate with an increase in expression of TLR9 on peripheral blood mononuclear cells (PBMCs). Patients with autoimmune thyroid diseases also have higher levels of the nuclear protein HMGB1 and RAGE protein, which together act as a ligand for TLR9. HMB1 is released from lysed or damaged cells. HMGB1-DNA complex then binds to RAGE, and activates TLR9. TLR9 can work through MyD88, an adaptor molecule that increases the expression of NF-kB. However autoimmune thyroid diseases also increase sensitivity of MyD88 independent pathways. These pathway ultimately leads to the production of pro-inflammatory cytokines in PMBCs for patients with autoimmune thyroid diseases. Autoimmune diseases can also be triggered by activated cells undergoing apoptosis and being engulfed by antigen presenting cells. Activation of cells leads to de-methylation, which exposes CpG regions of host DNA, allowing an inflammatory response to be activated through TLR9. Although it is possible that TLR9 also recognizes unmethylated DNA, TLR9 undoubtedly has a role in phagocytosis-induced autoimmunity.
Role in heart health
Inflammatory responses mediated by TLR9 pathways can be activated by unmethylated CpG sequences that exist within human mitochondrial DNA. Usually, damaged mitochondria are digested via autophagy in cardiomyocytes, and mitochondrial DNA is digested by the enzyme DNase II. However mitochondria that escape digestion via the lysosome/autophagy pathway can activate TLR9-induced inflammation via the NF-kB pathway. TLR9 expression in hearts with pressure overload leads to increased inflammation due to damaged mitochondria and activation of the CpG binding cite on TLR9.
There is evidence that TLR9 may play a role in heart heath for individuals who have already suffered a myocardial infarction. In murine trials, TLR9-deficient mice had less myofibroblast proliferation, meaning cardiac muscle recovery is connected to TLR9 expression. Furthermore, class B CpG sequences induce proliferation and differentiation of fibroblasts via the NF-kB pathway, the same pathway that initiates pro-inflammatory reactions in the immune responses. TLR9 shows specific activity in post-heart attack fibroblasts, inducing them to differentiate into myofibroblasts and speed repair of left ventricle tissue. In contrast to pre-myocardial infraction, cardiomyocytes in recovering hearts do not induce an inflammation response via TLR9/NF-kB pathway. Instead, the pathway leads to proliferation and differentiation of fibroblasts.
As an immunotherapy target
There are new immunomodulatory treatments undergoing testing which involve the administration of artificial DNA oligonucleotides containing the CpG motif. CpG DNA has applications in treating allergies such as asthma, immunostimulation against cancer, immunostimulation against pathogens, and as adjuvants in vaccines.
- Lefitolimod (MGN1703) has started clinical trials to treat (in combination with ipilimumab) patients with advanced solid malignancies.
- Ongoing studies are investigating SD-101 (Dynavax) in combination with Keytruda (pembrolizumab), an anti-PD-1 therapy developed by Merck. 
- A Phase 1/2 trial is ongoing with tilsotolimod (IMO-2125, Idera, NASDAQ:IDRA) in combination with Yervoy (ipilimumab), an anti-CTLA-4 therapy developed by Bristol-Myers Squibb in anti-PD-1 refractory melanoma patients. FDA also granted fast track designation for tilsotolimod in combination with ipilimumab for treatment of PD-1 refractory metastatic melanoma. A phase 3 global trial in the same population is currently enrolling (Illuminate 301, NCT03445533).
- TLR9 has been shown to interact with RNF216.
- Epidermal growth factor receptor (EGFR) is constitutively bound to TLR9.
- It can be activated by CpG Oligodeoxynucleotides such as Agatolimod.
- GRCh38: Ensembl release 89: ENSG00000239732 - Ensembl, May 2017
- GRCm38: Ensembl release 89: ENSMUSG00000045322 - Ensembl, May 2017
- "Human PubMed Reference:".
- "Mouse PubMed Reference:".
- Du X, Poltorak A, Wei Y, Beutler B (September 2000). "Three novel mammalian toll-like receptors: gene structure, expression, and evolution". European Cytokine Network. 11 (3): 362–71. PMID 11022119.
- Martínez-Campos C, Burguete-García AI, Madrid-Marina V (March 2017). "Role of TLR9 in Oncogenic Virus-Produced Cancer". Viral Immunology. 30 (2): 98–105. doi:10.1089/vim.2016.0103. PMID 28151089.
- Notley CA, Jordan CK, McGovern JL, Brown MA, Ehrenstein MR (February 2017). "DNA methylation governs the dynamic regulation of inflammation by apoptotic cells during efferocytosis". Scientific Reports. 7: 42204. doi:10.1038/srep42204. PMC 5294421. PMID 28169339.
- "Entrez Gene: TLR9 toll-like receptor 9".
- Dias AA, Silva CO, Santos JP, Batista-Silva LR, Acosta CC, Fontes AN, Pinheiro RO, Lara FA, Machado AM, Nery JA, Sarno EN, Pereira GM, Pessolani MC (September 2016). "DNA Sensing via TLR-9 Constitutes a Major Innate Immunity Pathway Activated during Erythema Nodosum Leprosum". Journal of Immunology. 197 (5): 1905–13. doi:10.4049/jimmunol.1600042. PMID 27474073.
- Christensen SR, Shupe J, Nickerson K, Kashgarian M, Flavell RA, Shlomchik MJ (September 2006). "Toll-like receptor 7 and TLR9 dictate autoantibody specificity and have opposing inflammatory and regulatory roles in a murine model of lupus". Immunity. 25 (3): 417–28. doi:10.1016/j.immuni.2006.07.013. PMID 16973389.
- Omiya S, Omori Y, Taneike M, Protti A, Yamaguchi O, Akira S, Shah AM, Nishida K, Otsu K (December 2016). "Toll-like receptor 9 prevents cardiac rupture after myocardial infarction in mice independently of inflammation". American Journal of Physiology. Heart and Circulatory Physiology. 311 (6): H1485–H1497. doi:10.1152/ajpheart.00481.2016. PMC 5206340. PMID 27769998.
- Peng S, Li C, Wang X, Liu X, Han C, Jin T, Liu S, Zhang X, Zhang H, He X, Xie X, Yu X, Wang C, Shan L, Fan C, Shan Z, Teng W (December 2016). "Increased Toll-Like Receptors Activity and TLR Ligands in Patients with Autoimmune Thyroid Diseases". Frontiers in Immunology. 7: 578. doi:10.3389/fimmu.2016.00578. PMC 5145898. PMID 28018345.
- Oka T, Hikoso S, Yamaguchi O, Taneike M, Takeda T, Tamai T, Oyabu J, Murakawa T, Nakayama H, Nishida K, Akira S, Yamamoto A, Komuro I, Otsu K (May 2012). "Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure". Nature. 485 (7397): 251–5. doi:10.1038/nature10992. PMC 3378041. PMID 22535248.
- Nakayama H, Otsu K (November 2013). "Translation of hemodynamic stress to sterile inflammation in the heart". Trends in Endocrinology and Metabolism. 24 (11): 546–53. doi:10.1016/j.tem.2013.06.004. PMID 23850260.
- Kline JN (July 2007). "Eat dirt: CpG DNA and immunomodulation of asthma". Proceedings of the American Thoracic Society. 4 (3): 283–8. doi:10.1513/pats.200701-019AW. PMC 2647631. PMID 17607014.
- Thompson JA, Kuzel T, Bukowski R, Masciari F, Schmalbach T (July 2004). "Phase Ib trial of a targeted TLR9 CpG immunomodulator (CPG 7909) in advanced renal cell carcinoma (RCC)". Journal of Clinical Oncology, 2004 ASCO Annual Meeting Proceedings (Post-Meeting Edition). 22 (14S).
- Klinman DM (2006). "Adjuvant activity of CpG oligodeoxynucleotides". International Reviews of Immunology. 25 (3–4): 135–54. doi:10.1080/08830180600743057. PMID 16818369.
- MOLOGEN AG: First patient recruited in combination study with lefitolimod and Yervoy. July 2016
- "Dynavax Presents Updated Data for SD-101 in Combination with KEYTRUDA(R) (pembrolizumab) Highlighting an ORR in 7 out of 7 Patients Naive to an Anti-PD-1 or Anti-PD-L1 Therapy (NASDAQ:DVAX)". investors.dynavax.com. Retrieved 2017-06-06.
- Intratumoral IMO-2125 in combination with ipilimumab demonstrating an ORR of 44% in melanoma patients refractory to anti-PD1 therapy vs 10-13% response rate with ipilimumab monotherapy
- U.S. FDA Grants Fast Track Designation for Idera Pharmaceuticals’ tilsotolimod in Combination with Ipilimumab for Treatment of PD-1 Refractory Metastatic Melanoma
- Chuang TH, Ulevitch RJ (May 2004). "Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors". Nature Immunology. 5 (5): 495–502. doi:10.1038/ni1066. PMID 15107846.
- Veleeparambil M, Poddar D, Abdulkhalek S, Kessler PM, Yamashita M, Chattopadhyay S, Sen GC (April 2018). "Constitutively Bound EGFR-Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal". Journal of Immunology. 200 (8): 2809–2818. doi:10.4049/jimmunol.1700691. PMC 5893352. PMID 29531172.