title: Toll like receptor 9 antagonism modulates spinal cord neuronal function and survival: Direct versus astrocyte-mediated mechanisms.
abstract: Toll like receptors (TLRs) are expressed by cells of the immune system and mediate the host innate immune responses to pathogens. However, increasing evidence indicates that they are important contributors to central nervous system (CNS) function in health and in pathological conditions involving sterile inflammation. In agreement with this idea, we have previously shown that intrathecal administration of a TLR9 antagonist, cytidine-phosphate-guanosine oligodeoxynucleotide 2088 (CpG ODN 2088), ameliorates the outcomes of spinal cord injury (SCI). Although these earlier studies showed a marked effect of CpG ODN 2088 on inflammatory cells, the expression of TLR9 in spinal cord (SC) neurons and astrocytes suggested that the antagonist exerts additional effects through direct actions on these cells. The current study was undertaken to assess the direct effects of CpG ODN 2088 on SC neurons, astrocytes and astrocyte-neuron interactions, in vitro. We report, for the first time, that inhibition of TLR9 in cultured SC neurons alters their function and confers protection against kainic acid (KA)-induced excitotoxic death. Moreover, the TLR9 antagonist attenuated the KA-elicited endoplasmic reticulum (ER) stress response in neurons, in vitro. CpG ODN 2088 also reduced the transcript levels and release of chemokine (C-X-C) motif ligand 1 (CXCL1) and monocyte chemotactic protein 1 (MCP-1) by astrocytes and it diminished interleukin-6 (IL-6) release without affecting transcript levels in vitro. Conditioned medium (CM) of CpG ODN 2088-treated astroglial cultures decreased the viability of SC neurons compared to CM of vehicle-treated astrocytes. However, this toxicity was not observed when astrocytes were co-cultured with neurons. Although CpG ODN 2088 limited the survival-promoting effects of astroglia, it did not reduce neuronal viability compared to controls grown in the absence of astrocytes. We conclude that the TLR9 antagonist acts directly on both SC neurons and astrocytes. Neuronal TLR9 antagonism confers protection against excitotoxic death. It is likely that this neuroprotection is partly due to the attenuation of the ER stress response provoked by excitotoxicity. Although CpG ODN 2088 limits the supportive effects of astrocytes on neurons, it could potentially exert beneficial effects by decreasing the release of pro-inflammatory cytokines and chemokines by astroglia. These findings highlight the multiple roles of TLR9 in the SC and have implications for pathological conditions including SCI where excitotoxicity and neuroinflammation play a prominent role in neuronal degeneration.
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States; Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Kilis 7 Aralik University, 79000 Kilis, Turkey.
authors.forename: Cigdem
authors.initials: C
authors.lastname: Acioglu
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States; Graduate School of Biomedical Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States.
authors.forename: Ersilia
authors.initials: E
authors.lastname: Mirabelli
authors.affiliation: Department of Medical Biochemistry, School of Medicine, Acibadem University, 34752 Istanbul, Turkey.
authors.forename: Ahmet Tarik
authors.initials: AT
authors.lastname: Baykal
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States.
authors.forename: Li
authors.initials: L
authors.lastname: Ni
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States.
authors.forename: Ayomi
authors.initials: A
authors.lastname: Ratnayake
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Heary
authors.affiliation: Department of Neurological Surgery, Reynolds Family Spine Laboratory, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ 07103, United States. Electronic address: elkabest@njms.rutgers.edu.
authors.forename: Stella
authors.initials: S
authors.lastname: Elkabes
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000187:drug effects*; D002478:Cells, Cultured; D003597:Cytosine Nucleotides / Q000494:pharmacology; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D005260:Female; D006151:Guanosine / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000187:drug effects*; D018696:Neuroprotective Agents / Q000494:pharmacology*; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D011247:Pregnancy; D013116:Spinal Cord / Q000187:drug effects*; D051217:Toll-Like Receptor 9 / Q000037:antagonists & inhibitors*
chemical_list: D003597:Cytosine Nucleotides; D018696:Neuroprotective Agents; D009838:Oligodeoxyribonucleotides; D051217:Toll-Like Receptor 9; D006151:Guanosine
keywords: Chemokine; CpG ODN; Cytokine; DAMP; Endoplasmic reticulum stress; Excitotoxicity; Injury; Innate immunity; Neurodegeneration; Neuroprotection; Pattern recognition receptors