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C5L2) in spinal cord injury
Abstracts / Immunobiology 221 (2016) 1131–1225
LECs. By flow cytometry, we observed that both C3 and C3aR were predominantly present intracellularly in the BEAS-2B cell line as well as in hTECs. On analyzing subcellular fractions, C3 and C3aR in the BEAS-2B cell line predominantly localized to the intracellular membrane fraction. Conclusion: C3 is endogenously produced and secreted by LECs, and is also present intracellularly in distinct sub-cellular compartments. C3aR is present intracellularly, possibly in association with C3a-containing proteins. These results establish the presence of an intracellular C3-C3aR system that is constitutively active in unstimulated human LECs. These findings form the basis of our ongoing work on identifying the mechanism(s) of C3 and C3aR production and their intracellular shuttling, activation and function. http://dx.doi.org/10.1016/j.imbio.2016.06.056 42 Microglia specific ablation of C1q gene definitively demonstrates microglia as the dominant source of C1q in both normal mouse brain and in models of Alzheimer’s disease Maria I. Fonseca 1,∗ , Shu-Hui Chu 1 , Michael Hernandez 2 , Melody Fang 1 , Lila Modarresi 1 , Andrea J. Tenner 1,2,3 1
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA 2 Department of Pathology and Laboratory Science, University of California, Irvine School of Medicine, Irvine, CA, USA 3 Department of Neurobiology and Behavior and University of California, Irvine, Irvine, CA, USA Alzheimer’s disease (AD) is a dementia characterized by the accumulation of amyloid plaques, neurofibrillary tangles, neuronal loss, and neuroinflammation. C1q (first component of complement cascade) can play a detrimental role in AD progression via activating the complement cascade inducing neuroinflammation and synapse loss. However, in vitro C1q has direct neuroprotective effects which may be beneficial, and thus has implications for therapeutic control of complement activities in neurodegenerative disease. To investigate the source of C1q in brain, C1qaFL/FL mice were crossed to Cx3cr1CreERT and to Thy1CreERT mice to enable inducible cell specific ablation of the C1q gene in either microglia or pyramidal neurons respectively. In brain, C1q staining was localized in the neuropil and inside microglia and a subset of interneurons. After tamoxifen treatment to induce Cre recombinase gene ablation, the C1qaFL/FL :Thy1CreERT mice showed no decrease in C1q expression. In contrast, C1qaFL/FL :Cx3cr1CreERT were devoid of C1q in microglia as well as in the neuropil particularly in the molecular layer of the hippocampus, even in the absence of tamoxifen. However, C1q was still present in a subset of interneurons. Neonatal derived microglia showed comparable levels of C1q in both C1qaFL/FL :Cx3cr1CreERT and WT littermates, but by 1 month of age the decrease in C1q expression in the C1qaFL/FL :Cx3cr1CreERT was near knock out levels at both the protein and mRNA levels. In contrast, no differences in the levels of C1q were detected in either liver or kidney from C1qaFL/FL :Cx3cr1CreERT mice relative to WT or C1qaFL/FL littermates, and C1qaFL/FL :Cx3cr1CreERT mice had only a partial, if any, reduction in C1q plasma levels. These data are interesting given the recent report of a preferential regulation of interneuronal synapse pruning by the complement system. In summary, the deletion of C1q in microglia caused the elimination of C1q in brain with the exception of interneuronal C1q, providing unequivocal evidence that microglia, not
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peripheral sources, are the dominant source of C1q in brain. Finally, in the Cx3cr1CreERT system, perhaps due to high expression of Cre under the Cx3cr1 promotor and the long lifetime of microglia, Cre gains access to the nucleus independently of tamoxifen, inducing deletion of floxed genes, and thus must be used with caution. http://dx.doi.org/10.1016/j.imbio.2016.06.057 43 Investigating the role of complement component C5a receptor 2 (C5aR2/C5L2) in spinal cord injury P.J.C. Biggins 1,∗ , F.H. Brennan 1 , S.M. Taylor 1 , T.M. Woodruff 1 , M.J. Ruitenberg 1,2,3 1 School of Biomedical Sciences, University of Queensland, Brisbane, Australia 2 Queensland Brain Institute, University of Queensland, Brisbane, Australia 3 Trauma, Critical Care & Recovery, Brisbane Diamantina Health Partners, Brisbane, Australia
Traumatic spinal cord injury (SCI) initiates a complex multiphasic neuro-inflammatory response at and in proximity to the lesion site. Activation of the complement cascade is an integral part and one of the earliest acting aspects of the neuroinflammatory response to SCI. This activation of the complement system is mostly thought of as detrimental, particularly during the acute phase of injury, although some time-dependent reparative roles have also been ascribed to select complement activation products. We recently reported that genetic ablation and/or pharmacological targeting of the main signalling receptor for complement component 5a (C5a), C5aR1, attenuates early inflammation and improves the neurological outcome. These findings support a pro-inflammatory, injurious role for the C5a-C5aR1 axis in the (sub-) acute phase of injury. The role of the second C5a receptor, C5aR2/C5L2, has, however, not been studied in SCI to date and its function in general has remained enigmatic, with both pro- and anti-inflammatory roles ascribed in different inflammatory disease models. Taking advantage of C5L2 knock-out (C5l2−/− ) mice, our findings support a mostly antiinflammatory/neuroprotective role for C5aR2/C5L2 in the context of SCI. Specifically, C5l2−/− mice displayed significantly larger lesion volumes, worsened locomotor recovery and less myelin sparing post-SCI relative to wild-type controls. The C5l2−/− phenotype was rescued by C5aR1 antagonism, suggesting that C5aR2/C5L2 may act as a negative modulator of the C5a-C5aR1 axis in this model. Our ongoing studies are focused to further elucidate the exact mechanism via which C5aR2/C5L2 influences SCI outcomes. The authors declare no relevant conflicts of interest. http://dx.doi.org/10.1016/j.imbio.2016.06.058
LECs. By flow cytometry, we observed that both C3 and C3aR were predominantly present intracellularly in the BEAS-2B cell line as well as in hTECs. On analyzing subcellular fractions, C3 and C3aR in the BEAS-2B cell line predominantly localized to the intracellular membrane fraction. Conclusion: C3 is endogenously produced and secreted by LECs, and is also present intracellularly in distinct sub-cellular compartments. C3aR is present intracellularly, possibly in association with C3a-containing proteins. These results establish the presence of an intracellular C3-C3aR system that is constitutively active in unstimulated human LECs. These findings form the basis of our ongoing work on identifying the mechanism(s) of C3 and C3aR production and their intracellular shuttling, activation and function. http://dx.doi.org/10.1016/j.imbio.2016.06.056 42 Microglia specific ablation of C1q gene definitively demonstrates microglia as the dominant source of C1q in both normal mouse brain and in models of Alzheimer’s disease Maria I. Fonseca 1,∗ , Shu-Hui Chu 1 , Michael Hernandez 2 , Melody Fang 1 , Lila Modarresi 1 , Andrea J. Tenner 1,2,3 1
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA, USA 2 Department of Pathology and Laboratory Science, University of California, Irvine School of Medicine, Irvine, CA, USA 3 Department of Neurobiology and Behavior and University of California, Irvine, Irvine, CA, USA Alzheimer’s disease (AD) is a dementia characterized by the accumulation of amyloid plaques, neurofibrillary tangles, neuronal loss, and neuroinflammation. C1q (first component of complement cascade) can play a detrimental role in AD progression via activating the complement cascade inducing neuroinflammation and synapse loss. However, in vitro C1q has direct neuroprotective effects which may be beneficial, and thus has implications for therapeutic control of complement activities in neurodegenerative disease. To investigate the source of C1q in brain, C1qaFL/FL mice were crossed to Cx3cr1CreERT and to Thy1CreERT mice to enable inducible cell specific ablation of the C1q gene in either microglia or pyramidal neurons respectively. In brain, C1q staining was localized in the neuropil and inside microglia and a subset of interneurons. After tamoxifen treatment to induce Cre recombinase gene ablation, the C1qaFL/FL :Thy1CreERT mice showed no decrease in C1q expression. In contrast, C1qaFL/FL :Cx3cr1CreERT were devoid of C1q in microglia as well as in the neuropil particularly in the molecular layer of the hippocampus, even in the absence of tamoxifen. However, C1q was still present in a subset of interneurons. Neonatal derived microglia showed comparable levels of C1q in both C1qaFL/FL :Cx3cr1CreERT and WT littermates, but by 1 month of age the decrease in C1q expression in the C1qaFL/FL :Cx3cr1CreERT was near knock out levels at both the protein and mRNA levels. In contrast, no differences in the levels of C1q were detected in either liver or kidney from C1qaFL/FL :Cx3cr1CreERT mice relative to WT or C1qaFL/FL littermates, and C1qaFL/FL :Cx3cr1CreERT mice had only a partial, if any, reduction in C1q plasma levels. These data are interesting given the recent report of a preferential regulation of interneuronal synapse pruning by the complement system. In summary, the deletion of C1q in microglia caused the elimination of C1q in brain with the exception of interneuronal C1q, providing unequivocal evidence that microglia, not
1149
peripheral sources, are the dominant source of C1q in brain. Finally, in the Cx3cr1CreERT system, perhaps due to high expression of Cre under the Cx3cr1 promotor and the long lifetime of microglia, Cre gains access to the nucleus independently of tamoxifen, inducing deletion of floxed genes, and thus must be used with caution. http://dx.doi.org/10.1016/j.imbio.2016.06.057 43 Investigating the role of complement component C5a receptor 2 (C5aR2/C5L2) in spinal cord injury P.J.C. Biggins 1,∗ , F.H. Brennan 1 , S.M. Taylor 1 , T.M. Woodruff 1 , M.J. Ruitenberg 1,2,3 1 School of Biomedical Sciences, University of Queensland, Brisbane, Australia 2 Queensland Brain Institute, University of Queensland, Brisbane, Australia 3 Trauma, Critical Care & Recovery, Brisbane Diamantina Health Partners, Brisbane, Australia
Traumatic spinal cord injury (SCI) initiates a complex multiphasic neuro-inflammatory response at and in proximity to the lesion site. Activation of the complement cascade is an integral part and one of the earliest acting aspects of the neuroinflammatory response to SCI. This activation of the complement system is mostly thought of as detrimental, particularly during the acute phase of injury, although some time-dependent reparative roles have also been ascribed to select complement activation products. We recently reported that genetic ablation and/or pharmacological targeting of the main signalling receptor for complement component 5a (C5a), C5aR1, attenuates early inflammation and improves the neurological outcome. These findings support a pro-inflammatory, injurious role for the C5a-C5aR1 axis in the (sub-) acute phase of injury. The role of the second C5a receptor, C5aR2/C5L2, has, however, not been studied in SCI to date and its function in general has remained enigmatic, with both pro- and anti-inflammatory roles ascribed in different inflammatory disease models. Taking advantage of C5L2 knock-out (C5l2−/− ) mice, our findings support a mostly antiinflammatory/neuroprotective role for C5aR2/C5L2 in the context of SCI. Specifically, C5l2−/− mice displayed significantly larger lesion volumes, worsened locomotor recovery and less myelin sparing post-SCI relative to wild-type controls. The C5l2−/− phenotype was rescued by C5aR1 antagonism, suggesting that C5aR2/C5L2 may act as a negative modulator of the C5a-C5aR1 axis in this model. Our ongoing studies are focused to further elucidate the exact mechanism via which C5aR2/C5L2 influences SCI outcomes. The authors declare no relevant conflicts of interest. http://dx.doi.org/10.1016/j.imbio.2016.06.058