Complement propagates visual system pathology following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) is associated with the development of visual system disorders. Visual deficits can present with delay and worsen over time, and may be associated with an ongoing neuroinflammatory response that is known to occur after TBI. Complement system activation is strongly associated with the neuroinflammatory response after TBI, but whether it contributes to vision loss after TBI is unexplored. METHODS: Acute and chronic neuroinflammatory changes within the dorsal lateral geniculate nucleus (dLGN) and retina were investigated subsequent to a moderate to severe murine unilateral controlled cortical impact. Neuroinflammatory and histopathological outcomes were interpreted in the context of behavioral and visual function data. To investigate the role of complement, cohorts were treated after TBI with the complement inhibitor, CR2-Crry. RESULTS: At 3 days after TBI, complement component C3 was deposited on retinogeniculate synapses in the dLGN both ipsilateral and contralateral to the lesion, which was reduced in CR2-Crry treated animals. This was associated with microglia morphological changes in both the ipsilateral and contralateral dLGN, with a less ramified phenotype in vehicle compared to CR2-Crry treated animals. Microglia in vehicle treated animals also had a greater internalized VGlut2 + synaptic volume after TBI compared to CR2-Crry treated animals. Microglia morphological changes seen acutely persisted for at least 49 days after injury. Complement inhibition also reduced microglial synaptic internalization in the contralateral dLGN and increased the association between VGLUT2 and PSD95 puncta, indicating preservation of intact synapses. Unexpectedly, there were no changes in the thickness of the inner retina, retinal nerve fiber layer or retinal ganglion layer. Neuropathological changes in the dLGN were accompanied by reduced visual acuity at subacute and chronic time points after TBI, with improvement seen in CR2-Crry treated animals. CONCLUSION: TBI induces complement activation within the dLGN and promotes microglial activation and synaptic internalization. Complement inhibition after TBI in a clinically relevant paradigm reduces complement activation, maintains a more surveillance-like microglia phenotype, and preserves synaptic density within the dLGN. Together, the data indicate that complement plays a key role in the development of visual deficits after TBI via complement-dependent microglial phagocytosis of synapses within the dLGN.

Mass cytometric analysis of the immune cell landscape after traumatic brain injury elucidates the role of complement and complement receptors in neurologic outcomes.

Following traumatic brain injury (TBI), a neuroinflammatory response can persist for years and contribute to the development of chronic neurological manifestations. Complement plays a central role in post-TBI neuroinflammation, and C3 opsonins and the anaphylatoxins (C3a and C5a) have been implicated in promoting secondary injury. We used single cell mass cytometry to characterize the immune cell landscape of the brain at different time points after TBI. To specifically investigate how complement shapes the post-TBI immune cell landscape, we analyzed TBI brains in the context of CR2-Crry treatment, an inhibitor of C3 activation. We analyzed 13 immune cell types, including peripheral and brain resident cells, and assessed expression of various receptors. TBI modulated the expression of phagocytic and complement receptors on both brain resident and infiltrating peripheral immune cells, and distinct functional clusters were identified within same cell populations that emerge at different phases after TBI. In particular, a CD11c+ (CR4) microglia subpopulation continued to expand over 28 days after injury, and was the only receptor to show continuous increase over time. Complement inhibition affected the abundance of brain resident immune cells in the injured hemisphere and impacted the expression of functional receptors on infiltrating cells. A role for C5a has also been indicated in models of brain injury, and we found significant upregulation of C5aR1 on many immune cell types after TBI. However, we demonstrated experimentally that while C5aR1 is involved in the infiltration of peripheral immune cells into the brain after injury, it does not alone affect histological or behavioral outcomes. However, CR2-Crry improved post-TBI outcomes and reduced resident immune cell populations, as well as complement and phagocytic receptor expression, indicating that its neuroprotective effects are mediated upstream of C5a generation, likely via modulating C3 opsonization and complement receptor expression.

State of the science in inflammation and stroke recovery: A systematic review.

Stroke is a major cause of mortality worldwide, and survivors often have major life-changing disabilities. Annually in the United States, an estimated 795,000 people experience a new or recurrent stroke. All types of stroke involve an inflammatory reaction that follows the initial phase of incidence. However, investigations into any links between inflammatory markers and recovery processes in the context of post-stroke rehabilitation are lacking. In this systematic review, we searched the literature in PubMed, SCOPUS, and CINAHL databases to gather information on inflammatory biomarkers related to stroke and their association with rehabilitation outcomes, according to PRISMA guidelines. Eleven articles (n=1.773 stroke patients) were selected. Immune markers (interleukin 6 [IL-6], C-reactive protein, IL-1α, tumor necrosis factor α, soluble intercellular adhesion molecule 1) and functional status assessments (Modified Rankin Score, National Institutes of Health Stroke Scale, Functional Independence Measure, etc.) were the primary measures used in the reviewed studies. We found preliminary evidence for the evaluation of inflammatory biomarkers post-stroke, including the role of inflammation in functional recovery and the influence of rehabilitation on inflammation. This is the first systematic review of the topic. The review identifies several gaps in the literature that are critical for understanding the potential use of inflammatory markers to improve post-stroke outcomes.

Barcoded viral tracing of single-cell interactions in central nervous system inflammation.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets.

Aryl Hydrocarbon Receptor Activation in Astrocytes by Laquinimod Ameliorates Autoimmune Inflammation in the CNS.

OBJECTIVE: MS is an autoimmune demyelinating disease of the CNS, which causes neurologic deficits in young adults and leads to progressive disability. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor, can drive anti-inflammatory functions in peripheral immune cells and also in CNS-resident cells. Laquinimod is a drug developed for the treatment of MS known to activate AHR, but the cellular targets of laquinimod are still not completely known. In this work, we analyzed the contribution of AHR activation in astrocytes to its beneficial effects in the experimental autoimmune encephalomyelitis (EAE) preclinical model of MS. METHODS: We used conditional knockout mice, in combination with genome-wide analysis of gene expression by RNA-seq and in vitro culture systems to investigate the effects of laquinimod on astrocytes. RESULTS: We found that AHR activation in astrocytes by laquinimod ameliorates EAE, a preclinical model of MS. Genome-wide RNA-seq transcriptional analyses detected anti-inflammatory effects of laquinimod in glial cells during EAE. Moreover, we established that the Delaq metabolite of laquinimod dampens proinflammatory mediator production while activating tissue-protective mechanisms in glia. CONCLUSIONS: Taken together, these findings suggest that AHR activation by clinically relevant AHR agonists may represent a novel therapeutic approach for the treatment of MS.

Anti-inflammatory and Neuroprotective Agents in Clinical Trials for CNS Disease and Injury: Where Do We Go From Here?

Neurological disorders are major contributors to death and disability worldwide. The pathology of injuries and disease processes includes a cascade of events that often involve molecular and cellular components of the immune system and their interaction with cells and structures within the central nervous system. Because of this, there has been great interest in developing neuroprotective therapeutic approaches that target neuroinflammatory pathways. Several neuroprotective anti-inflammatory agents have been investigated in clinical trials for a variety of neurological diseases and injuries, but to date the results from the great majority of these trials has been disappointing. There nevertheless remains great interest in the development of neuroprotective strategies in this arena. With this in mind, the complement system is being increasingly discussed as an attractive therapeutic target for treating brain injury and neurodegenerative conditions, due to emerging data supporting a pivotal role for complement in promoting multiple downstream activities that promote neuroinflammation and degeneration. As we move forward in testing additional neuroprotective and immune-modulating agents, we believe it will be useful to review past trials and discuss potential factors that may have contributed to failure, which will assist with future agent selection and trial design, including for complement inhibitors. In this context, we also discuss inhibition of the complement system as a potential neuroprotective strategy for neuropathologies of the central nervous system.

Complement-Mediated Microglial Phagocytosis and Pathological Changes in the Development and Degeneration of the Visual System.

The focus of this review is the role of complement-mediated phagocytosis in retinal and neurological diseases affecting the visual system. Complement activation products opsonize synaptic material on neurons for phagocytic removal, which is a normal physiological process during development, but a pathological process in several neurodegenerative diseases and conditions. We discuss the role of complement in the refinement and elimination of synapses in the retina and lateral geniculate nucleus, both during development and in disease states. How complement and aberrant phagocytosis promotes injury to the visual system is discussed primarily in the context of multiple sclerosis, where it has been extensively studied, although the role of complement in visual dysfunction in other diseases such as stroke and traumatic brain injury is also highlighted. Retinal diseases are also covered, with a focus on glaucoma and age-related macular degeneration. Finally, we discuss the potential of complement inhibitory strategies to treat diseases affecting the visual system.

Metabolic Control of Astrocyte Pathogenic Activity via cPLA2-MAVS.

Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention.

Microglial control of astrocytes in response to microbial metabolites.

Microglia and astrocytes modulate inflammation and neurodegeneration in the central nervous system (CNS)1-3. Microglia modulate pro-inflammatory and neurotoxic activities in astrocytes, but the mechanisms involved are not completely understood4,5. Here we report that TGFα and VEGF-B produced by microglia regulate the pathogenic activities of astrocytes in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Microglia-derived TGFα acts via the ErbB1 receptor in astrocytes to limit their pathogenic activities and EAE development. Conversely, microglial VEGF-B triggers FLT-1 signalling in astrocytes and worsens EAE. VEGF-B and TGFα also participate in the microglial control of human astrocytes. Furthermore, expression of TGFα and VEGF-B in CD14+ cells correlates with the multiple sclerosis lesion stage. Finally, metabolites of dietary tryptophan produced by the commensal flora control microglial activation and TGFα and VEGF-B production, modulating the transcriptional program of astrocytes and CNS inflammation through a mechanism mediated by the aryl hydrocarbon receptor. In summary, we identified positive and negative regulators that mediate the microglial control of astrocytes. Moreover, these findings define a pathway through which microbial metabolites limit pathogenic activities of microglia and astrocytes, and suppress CNS inflammation. This pathway may guide new therapies for multiple sclerosis and other neurological disorders.

Detection of aryl hydrocarbon receptor agonists in human samples.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important functions in the immune response and cancer. AHR agonists are provided by the environment, the commensal flora and the metabolism. Considering AHR physiological functions, AHR agonists may have important effects on health and disease. Thus, the quantification of AHR agonists in biological samples is of scientific and clinical relevance. We compared different reporter systems for the detection of AHR agonists in serum samples of Multiple Sclerosis (MS) patients, and assessed the influence of transfection methods and cell lines in a reporter-based in vitro assay. While the use of stable or transient reporters did not influence the measurement of AHR agonistic activity, the species of the cell lines used in these reporter assays had important effects on the reporter readings. These observations suggest that cell-specific factors influence AHR activation and signaling. Thus, based on the reported species selectivity of AHR ligands and the cell species-of-origin effects that we describe in this manuscript, the use of human cell lines is encouraged for the analysis of AHR agonistic activity in human samples. These findings may be relevant for the analysis of AHR agonists in human samples in the context of inflammatory and neoplastic disorders.

Dynamic regulation of serum aryl hydrocarbon receptor agonists in MS.

OBJECTIVE: Several factors influence the clinical course of autoimmune inflammatory diseases such as MS and inflammatory bowel disease. Only recently, the complex interaction between the gut microbiome, dietary factors, and metabolism has started to be appreciated with regard to its potential to modulate acute and chronic inflammation. One of the molecular sensors that mediates the effects of these environmental signals on the immune response is the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor with key functions in immune cells. METHODS: In this study, we analyzed the levels of AHR agonists in serum samples from patients with MS and healthy controls in a case-control study. RESULTS: We detected a global decrease of circulating AHR agonists in relapsing-remitting MS patients as compared to controls. However, during acute CNS inflammation in clinically isolated syndrome or active MS, we measured increased AHR agonistic activity. Moreover, AHR ligand levels in patients with benign MS with relatively mild clinical impairment despite longstanding disease were unaltered as compared to healthy controls. CONCLUSIONS: Collectively, these data suggest that AHR agonists in serum are dynamically modulated during the course of MS. These findings may guide the development of biomarkers to monitor disease activity as well as the design of novel therapeutic interventions for MS.

Sphingosine 1-phosphate receptor modulation suppresses pathogenic astrocyte activation and chronic progressive CNS inflammation.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the CNS that causes disability in young adults as a result of the irreversible accumulation of neurological deficits. Although there are potent disease-modifying agents for its initial relapsing-remitting phase, these therapies show limited efficacy in secondary progressive MS (SPMS). Thus, there is an unmet clinical need for the identification of disease mechanisms and potential therapeutic approaches for SPMS. Here, we show that the sphingosine 1-phosphate receptor (S1PR) modulator fingolimod (FTY720) ameliorated chronic progressive experimental autoimmune encephalomyelitis in nonobese diabetic mice, an experimental model that resembles several aspects of SPMS, including neurodegeneration and disease progression driven by the innate immune response in the CNS. Indeed, S1PR modulation by FTY720 in murine and human astrocytes suppressed neurodegeneration-promoting mechanisms mediated by astrocytes, microglia, and CNS-infiltrating proinflammatory monocytes. Genome-wide studies showed that FTY720 suppresses transcriptional programs associated with the promotion of disease progression by astrocytes. The study of the molecular mechanisms controlling these transcriptional modules may open new avenues for the development of therapeutic strategies for progressive MS.