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.

Transcriptome sequencing and metabolome analysis reveal the metabolic reprogramming of partial hepatectomy and extended hepatectomy.

Surgical resection remains a critical treatment option for many patients with primary and secondary hepatic neoplasms. Extended hepatectomy (eHx) may be required for some patients with large tumors, which may cause liver failure and death. Partial hepatectomy (pHx) and eHx mouse models were constructed, liver tissues were sampled at 18, 36, and 72 h posthepatectomy. Transcriptome and metabolome analyses were employed to explore the different potential mechanisms in regeneration and injury between pHx and eHx. The results showed that eHx was associated with more severe liver injury and lower survival rates than pHx. Transcriptomics data showed there were 1842, 2129, and 1277 differentially expressed genes (DEGs) in eHx and 962, 1305, and 732 DEGs in pHx at 18, 36, and 72 h posthepatectomy, respectively, compared with the those in the sham groups. Compared with pHx, the number of DEGs in the eHx group reached a maximum of 230 at 18 h after surgery and decreased sequentially to 87 and 43 at 36 and 72 h. Metabolomics analysis identified a total of 1399 metabolites, and 48 significant differentially produced metabolites (DPMs) were screened between eHx and pHx. Combined analysis of DEGs and DPMs indicated that cholesterol metabolism and insulin resistance may be two important pathways for liver regeneration and mouse survival postextended hepatectomy. Our results showed the global influence of pHx and eHx on the transcriptome and metabolome in mouse liver, and revealed cholesterol metabolism and insulin resistance pathways might be involved in regeneration post-pHx and -eHx.

Complement inhibition alleviates donor brain death-induced liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase signaling.

Brain death (BD) donors are the primary source of donor organs for liver transplantation. However, the effects of BD on donor livers and outcomes after liver transplantation remain unclear. Here, we explored the role of complement and the therapeutic effect of complement inhibition in BD-induced liver injury and posttransplantation injury in a mouse BD and liver transplantation model. For complement inhibition, we used complement receptor 2 (CR2)-Crry, a murine inhibitor of C3 activation that specifically targets sites of complement activation. In the mouse model, BD resulted in complement activation and liver injury in donor livers and a cascade liver injury posttransplantation, mediated in part through the C3a-C3aR (C3a receptor) signaling pathway, which was ameliorated by treatment with CR2-Crry. Treatment of BD donors with CR2-Crry improved graft survival, which was further improved when recipients received an additional dose of CR2-Crry posttransplantation. Mechanistically, we determined that complement inhibition alleviated BD-induced donor liver injury and posttransplant cascade injury by regulating phosphoinositide 3-kinase (PI3K) signaling pathways. Together, BD induced donor liver injury and cascade injury post-transplantation, which was mediated by complement activation products acting on PI3K signaling pathways. Our study provides an experimental basis for developing strategies to improve the survival of BD donor grafts in liver transplantation.

A role for P-selectin and complement in the pathological sequelae of germinal matrix hemorrhage.

BACKGROUND: Germinal matrix hemorrhage is a devastating disease of pre-term infancy commonly resulting in post-hemorrhagic hydrocephalus, periventricular leukomalacia, and subsequent neurocognitive deficits. We demonstrate vascular expression of the adhesion molecule P-selectin after GMH and investigate a strategy to specifically target complement inhibition to sites of P-selectin expression to mitigate the pathological sequelae of GMH. METHODS: We prepared two fusion proteins consisting of different anti-P-selectin single chain antibodies (scFv's) linked to the complement inhibitor Crry. One scFv targeting vehicle (2.12scFv) blocked the binding of P-selectin to its PSGL-1 ligand expressed on leukocytes, whereas the other targeting vehicle (2.3scFv) bound P-selectin without blocking ligand binding. Post-natal C57BL/6 J mice on day 4 (P4) were subjected to collagenase induced-intraventricular hemorrhage and treated with 2.3Psel-Crry, 2.12Psel-Crry, or vehicle. RESULTS: Compared to vehicle treatment, 2.3Psel-Crry treatment after induction of GMH resulted in reduced lesion size and mortality, reduced hydrocephalus development, and improved neurological deficit measurements in adolescence. In contrast, 2.12Psel-Crry treatment resulted in worse outcomes compared to vehicle. Improved outcomes with 2.3Psel-Crry were accompanied by decreased P-selectin expression, and decreased complement activation and microgliosis. Microglia from 2.3Psel-Crry treated mice displayed a ramified morphology, similar to naïve mice, whereas microglia in vehicle treated animals displayed a more ameboid morphology that is associated with a more activated status. Consistent with these morphological characteristics, there was increased microglial internalization of complement deposits in vehicle compared to 2.3Psel-Crry treated animals, reminiscent of aberrant C3-dependent microglial phagocytosis that occurs in other (adult) types of brain injury. In addition, following systemic injection, 2.3Psel-Crry specifically targeted to the post-GMH brain. Likely accounting for the unexpected finding that 2.12Psel-Crry worsens outcome following GMH was the finding that this construct interfered with coagulation in this hemorrhagic condition, and specifically with heterotypic platelet-leukocyte aggregation, which express P-selectin and PSGL-1, respectively. CONCLUSIONS: GMH induces expression of P-selectin, the targeting of which with a complement inhibitor protects against pathogenic sequelae of GMH. A dual functioning construct with both P-selectin and complement blocking activity interferes with coagulation and worsens outcomes following GMH, but has potential for treatment of conditions that incorporate pathological thrombotic events, such as ischemic stroke.

Complement Drives Chronic Inflammation and Progressive Hydrocephalus in Murine Neonatal Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is a pathology that occurs in infancy, with often devastating long-term consequences. Posthemorrhagic hydrocephalus (PHH) can develop acutely, while periventricular leukomalacia (PVL) is a chronic sequala. There are no pharmacological therapies to treat PHH and PVL. We investigated different aspects of the complement pathway in acute and chronic outcomes after murine neonatal GMH induced at postnatal day 4 (P4). Following GMH-induction, the cytolytic complement membrane attack complex (MAC) colocalized with infiltrating red blood cells (RBCs) acutely but not in animals treated with the complement inhibitor CR2-Crry. Acute MAC deposition on RBCs was associated with heme oxygenase-1 expression and heme and iron deposition, which was reduced with CR2-Crry treatment. Complement inhibition also reduced hydrocephalus and improved survival. Following GMH, there were structural alterations in specific brain regions linked to motor and cognitive functions, and these changes were ameliorated by CR2-Crry, as measured at various timepoints through P90. Astrocytosis was reduced in CR2-Crry-treated animals at chronic, but not acute, timepoints. At P90, myelin basic protein and LAMP-1 colocalized, indicating chronic ongoing phagocytosis of white matter, which was reduced by CR2-Crry treatment. Data indicate acute MAC-mediated iron-related toxicity and inflammation exacerbated the chronic effects of GMH.

Complement Drives Synaptic Degeneration and Progressive Cognitive Decline in the Chronic Phase after Traumatic Brain Injury.

Cognitive deficits following traumatic brain injury (TBI) remain a major cause of disability and early-onset dementia, and there is increasing evidence that chronic neuroinflammation occurring after TBI plays an important role in this process. However, little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation after TBI. Here, we identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response that is associated with cognitive decline. Three months after an initial insult, there is ongoing complement activation in the injured brain of male C57BL/6 mice, which drives a robust chronic neuroinflammatory response extending to both hemispheres. This chronic neuroinflammatory response promotes synaptic degeneration and predicts progressive cognitive decline. Synaptic degeneration was driven by microglial phagocytosis of complement-opsonized synapses in both the ipsilateral and contralateral brain, and complement inhibition interrupted the degenerative neuroinflammatory response and reversed cognitive decline, even when therapy was delayed until 2 months after TBI. These findings provide new insight into our understanding of TBI pathology and its management; and whereas previous therapeutic investigations have focused almost exclusively on acute treatments, we show that all phases of TBI, including at chronic time points after TBI, may be amenable to therapeutic interventions, and specifically to complement inhibition.SIGNIFICANCE STATEMENT There is increasing evidence of a chronic neuroinflammatory response after traumatic brain injury (TBI), but little is known about the molecular mechanisms responsible for triggering and maintaining chronic inflammation. We identify complement, and specifically complement-mediated microglial phagocytosis of synapses, as a pathophysiological link between acute insult and a chronic neurodegenerative response, and further that this response is associated with cognitive decline. Complement inhibition interrupted this response and reversed cognitive decline, even when therapy was delayed until 2 months after injury. The data further support the concept that TBI should be considered a chronic rather than an acute disease condition, and have implications for the management of TBI in the chronic phase of injury, specifically with regard to the therapeutic application of complement inhibition.

Impact of inter-vendor variability on evaluation of left ventricular mechanical dispersion.

PURPOSE: Left ventricular mechanical dispersion (LVMD) is a novel speckle tracking parameter for prognostic assessment of arrhythmic risk prediction. There is growing evidence to support its use in a variety of cardiomyopathic processes. There is paucity of data addressing any presence of inter-vendor discrepancies for LVMD. The aim of this study was to assess inter-vendor variability of LVMD in vendor specific software (VSS) and vendor independent software (VIS) in subjects with preserved and reduced left ventricular function. METHODS: Fifty-nine subjects (14 normal subjects and 45 subjects with cardiac disease) were recruited and 2D speckle tracking echocardiographic images were acquired on two different ultrasound machines (GE and Philips). LVMD was measured by two different VSS (EchoPac GE and QLAB Philips) and one VIS (TomTec Arena). RESULTS: There was significant bias and wide limits of agreement (LOA) in the overall cohort observed between two different VSS (17.6 ms; LOA: -29.6 to 64.8; r: .47). There was acceptable bias and narrower LOA with good agreement for LVMD between images obtained on different vendors when performed on VIS (-3.1 ms; LOA: -27.6 to 21.4; r: .75). QLAB LVMD was consistently higher than GE LVMD and TomTec LVMD in both preserved and reduced left ventricular function. LVMD measurements have high intra-vendor reproducibility with excellent inter and intra-observer agreement. CONCLUSIONS: There was acceptable bias and narrower LOA for LVMD assessment on a VIS. Inter-vendor variability exists for LVMD assessment between VSS. Serial measurements of LVMD should be performed using a single vendor for consistent and reliable results.

A Role of Complement in the Pathogenic Sequelae of Mouse Neonatal Germinal Matrix Hemorrhage.

Germinal matrix hemorrhage (GMH) is a devastating disease of infancy that results in intraventricular hemorrhage, post-hemorrhagic hydrocephalus (PHH), periventricular leukomalacia, and neurocognitive deficits. There are no curative treatments and limited surgical options. We developed and characterized a mouse model of GMH based on the injection of collagenase into the subventricular zone of post-natal pups and utilized the model to investigate the role of complement in PHH development. The site-targeted complement inhibitor CR2Crry, which binds deposited C3 complement activation products, localized specifically in the brain following its systemic administration after GMH. Compared to vehicle, CR2Crry treatment reduced PHH and lesion size, which was accompanied by decreased perilesional complement deposition, decreased astrocytosis and microgliosis, and the preservation of dendritic and neuronal density. Complement inhibition also improved survival and weight gain, and it improved motor performance and cognitive outcomes measured in adolescence. The progression to PHH, neuronal loss, and associated behavioral deficits was linked to the microglial phagocytosis of complement opsonized neurons, which was reversed with CR2Crry treatment. Thus, complement plays an important role in the pathological sequelae of GMH, and complement inhibition represents a novel therapeutic approach to reduce the disease progression of a condition for which there is currently no treatment outside of surgical intervention.

Complement-Dependent Synaptic Uptake and Cognitive Decline after Stroke and Reperfusion Therapy.

Despite the success of reperfusion therapy in significantly reducing the extent of infarct expansion after stroke, the effect of revascularization on poststroke neuroinflammation and the role of anti-inflammatory strategies in postreperfusion era are yet to be explored. Here, we investigate whether the neuroinflammatory response may still contribute to neurologic deficits after reperfused stroke by using targeted complement inhibition to suppress poststroke neuroinflammation in mice with or without concurrent reperfusion therapy. Complement inhibition was achieved using B4Crry, an injury site-targeted inhibitor of C3 activation. Following embolic stroke in male C57bl/6 mice, thrombolysis using tissue-plasminogen activator (t-PA) reduced injury and improved motor deficits, but did not improve cognitive outcomes. After both reperfused and non-reperfused stroke, complement activation and opsonization of hippocampal synapses directed ongoing microglia-dependent phagocytosis of synapses for at least 30 d after stroke, leading to a loss of synaptic density that was associated with cognitive decline. B4Crry treatment, alone or in combination with tPA, limited perilesional complement deposition, reduced microgliosis and synaptic uptake, and improved cognitive outcome without affecting regenerative responses. Furthermore, complement inhibition improved the safety, efficacy, and treatment window of reperfusion therapy with t-PA by limiting hemorrhagic transformation. This work thus demonstrates that poststroke neuroinflammation contributes to hemorrhagic transformation and progression of neurodegenerative responses in the brain even following early and successful revascularization.SIGNIFICANCE STATEMENT This study addresses two major challenges facing the treatment of stroke in the era of reperfusion therapy: hemorrhagic transformation and the disconnect between successful revascularization and functional outcomes. We studied how complement-dependent neuroinflammation drives the pathophysiology behind these challenges using a translationally relevant strategy. Complement inhibition was achieved using B4Crry, an injury site-targeted inhibitor of C3 activation. Following embolic stroke, pharmacological thrombolysis limited infarct size, but did not prevent complement activation. In reperfused and non-reperfused stroke, complement activation and opsonization of hippocampal synapses resulted in synaptic phagocytosis and subsequent cognitive decline. B4Crry treatment limited perilesional complement deposition, reduced microgliosis and synaptic uptake, and improved cognitive outcomes. Complement inhibition also improved the safety, efficacy, and treatment window of thrombolytic therapy.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

Natural immunoglobulin M-based delivery of a complement alternative pathway inhibitor in mouse models of retinal degeneration.

PURPOSE: Age-related macular degeneration is a slowly progressing disease. Studies have tied disease risk to an overactive complement system. We have previously demonstrated that pathology in two mouse models, the choroidal neovascularization (CNV) model and the smoke-induced ocular pathology (SIOP) model, can be reduced by specifically inhibiting the alternative complement pathway (AP). Here we report on the development of a novel injury-site targeted inhibitor of the alternative pathway, and its characterization in models of retinal degeneration. METHODS: Expression of the danger associated molecular pattern, a modified annexin IV, in injured ARPE-19 cells was confirmed by immunohistochemistry and complementation assays using B4 IgM mAb. Subsequently, a construct was prepared consisting of B4 single chain antibody (scFv) linked to a fragment of the alternative pathway inhibitor, fH (B4-scFv-fH). ARPE-19 cells stably expressing B4-scFv-fH were microencapsulated and administered intravitreally or subcutaneously into C57BL/6 J mice, followed by CNV induction or smoke exposure. Progression of CNV was analyzed using optical coherence tomography, and SIOP using structure-function analyses. B4-scFv-fH targeting and AP specificity was assessed by Western blot and binding experiments. RESULTS: B4-scFv-fH was secreted from encapsulated RPE and inhibited complement in RPE monolayers. B4-scFv-fH capsules reduced CNV and SIOP, and western blotting for breakdown products of C3α, IgM and IgG confirmed a reduction in complement activation and antibody binding in RPE/choroid. CONCLUSIONS: Data supports a role for natural antibodies and neoepitope expression in ocular disease, and describes a novel strategy to target AP-specific complement inhibition to diseased tissue in the eye. PRECIS: AMD risk is tied to an overactive complement system, and ocular injury is reduced by alternative pathway (AP) inhibition in experimental models. We developed a novel inhibitor of the AP that targets an injury-specific danger associated molecular pattern, and characterized it in disease models.

Left atrial reservoir strain provides incremental value to left atrial volume index for evaluation of left ventricular filling pressure.

BACKGROUND: Left atrial analysis is employed in diastolic assessment with left atrial volume index (LAVI) incorporated in the 2016 ASE/EACVI diastology guideline algorithm. LAVI has sub-optimal correlation with invasive left ventricular filling pressure (LVFP) and incorporation of left atrial reservoir strain (LASr) may improve diastolic assessment. METHODS: A cross-sectional prospective study of 139 patients was undertaken with all patients undergoing transthoracic echocardiography immediately prior to cardiac catheterization with invasive evaluation of LVFP. LASr by speckle tracking echocardiography and conventional echocardiographic parameters were assessed in relation to invasive LVFP. Modification of the 2016 guideline algorithm was performed with incorporation of LASr in place of LAVI (LASr ≤23% indicating elevated LVFP). Accuracy of the modified and conventional algorithm were assessed for predicting invasive LVFP. RESULTS: The mean age was 63±12 years with 27% female. LASr demonstrated superior correlation and receiver operator characteristic for predicting LVFP than LAVI (LASr: r -.46 (p < 0.01), AUC: .82 vs LAVI: r .19 (p 0.02), AUC: .66). LASr of ≤23% was the optimal cut-off for discriminating elevated LVFP (sensitivity 80%, specificity 77%). Modification of the 2016 algorithm with incorporation of LASr in place of LAVI reclassified 12% of the patient cohort and improved concordance of echocardiographic and invasive LVFP assessment (modified algorithm κ .47 vs 2016 algorithm κ: .33). No patients were incorrectly reclassified by modified algorithm assessment. CONCLUSIONS: LASr better predicts invasive LVFP than LAVI. Modification of the 2016 guideline algorithm with incorporation of LASr in place of LAVI improves accuracy of echocardiographic assessment of LVFP.

New therapeutic and diagnostic opportunities for injured tissue-specific targeting of complement inhibitors and imaging modalities.

Despite substantial opportunity and commercial interest in developing drugs that modulate the complement system in a broad range of non-orphan indications, several obstacles remain to be overcome. Among these issues is the biophysical nature of complement proteins, whose circulating levels are typically very high and whose turnover rates are relatively rapid, especially in the setting of chronic inflammatory conditions. This situation necessitates the use of very high levels of therapeutic compounds in order to achieve both multi-pathway and multiple effector mechanism inhibition. In addition, one must avoid infectious complications or the systemic impairment of the other important physiological functions of complement. Herein we focus on the development of a novel therapeutic strategy based on injured tissue-specific targeting of complement inhibitors using the antigen-combining domains of a small subset of natural IgM antibodies, which as endogenous antibodies specifically recognize sites of local damage across a broad range of tissues and locally activate complement C3, resulting in C3 fragment covalent fixation. Because the use of such recombinant tissue-targeting inhibitors precludes the utility of measuring systemic levels of complement biomarkers or function, since a goal of this targeting strategy is to leave those processes intact and unimpeded, we also briefly describe a new method designed to quantitatively measure using imaging modalities the inhibition of generation of fixed C3 fragments at sites of inflammation/injury. In addition to the ability to determine whether complement activation is locally constrained with the use of inhibitors, there is also a broader application of this imaging approach to inflammatory and autoimmune diseases characterized by local complement activation.

Injury site-specific targeting of complement inhibitors for treating stroke.

Cumulative evidence indicates a role for the complement system in both pathology and recovery after ischemic stroke. Here, we review the current understanding of the dual role of complement in poststroke injury and recovery, and discuss the challenges of anti-complement therapies. Most complement directed therapeutics currently under investigation or development systemically inhibit the complement system, but since complement is important for immune surveillance and is involved in various homeostatic activities, there are potential risks associated with systemic inhibition. Depending on the target within the complement pathway, other concerns are high concentrations of inhibitor required, low efficacy and poor bioavailability. To overcome these limitations, approaches to target complement inhibitors to specific sites have been investigated. Here, we discuss targeting strategies, with a focus on strategies developed in our lab, to specifically localize complement inhibition to sites of tissue injury and complement activation, and in particular to the postischemic brain. We discuss various injury site-specific targeted complement inhibitors as potential therapeutic agents for the treatment of ischemic stroke treatment, as well as their use as investigative tools for probing complement-dependent pathophysiological processes.

Identifying the Role of Complement in Triggering Neuroinflammation after Traumatic Brain Injury.

The complement system is implicated in promoting acute secondary injury after traumatic brain injury (TBI), but its role in chronic post-traumatic neuropathology remains unclear. Using various injury-site targeted complement inhibitors that block different complement pathways and activation products, we investigated how complement is involved in neurodegeneration and chronic neuroinflammation after TBI in a clinically relevant setting of complement inhibition. The current paradigm is that complement propagates post-TBI neuropathology predominantly through the terminal membrane attack complex (MAC), but the focus has been on acute outcomes. Following controlled cortical impact in adult male mice, we demonstrate that although inhibition of the MAC (with CR2-CD59) reduces acute deficits, inhibition of C3 activation is required to prevent chronic inflammation and ongoing neuronal loss. Activation of C3 triggered a sustained degenerative mechanism of microglial and astrocyte activation, reduced dendritic and synaptic density, and inhibited neuroblast migration several weeks after TBI. Moreover, inhibiting all complement pathways (with CR2-Crry), or only the alternative complement pathway (with CR2-fH), provided similar and significant improvements in chronic histological, cognitive, and functional recovery, indicating a key role for the alternative pathway in propagating chronic post-TBI pathology. Although we confirm a role for the MAC in acute neuronal loss after TBI, this study shows that upstream products of complement activation generated predominantly via the alternative pathway propagate chronic neuroinflammation, thus challenging the current concept that the MAC represents a therapeutic target for treating TBI. A humanized version of CR2fH has been shown to be safe and non-immunogenic in clinical trials.SIGNIFICANCE STATEMENT Complement, and specifically the terminal membrane attack complex, has been implicated in secondary injury and neuronal loss after TBI. However, we demonstrate here that upstream complement activation products, generated predominantly via the alternative pathway, are responsible for propagating chronic inflammation and injury following CCI. Chronic inflammatory microgliosis is triggered by sustained complement activation after CCI, and is associated with chronic loss of neurons, dendrites and synapses, a process that continues to occur even 30 d after initial impact. Acute and injury-site targeted inhibition of the alternative pathway significantly improves chronic outcomes, and together these findings modify the conceptual paradigm for targeting the complement system to treat TBI.

Acute Complement Inhibition Potentiates Neurorehabilitation and Enhances tPA-Mediated Neuroprotection.

Because complement activation in the subacute or chronic phase after stroke was recently shown to stimulate neural plasticity, we investigated how complement activation and complement inhibition in the acute phase after murine stroke interacts with subsequent rehabilitation therapy to modulate neuroinflammation and neural remodeling. We additionally investigated how complement and complement inhibition interacts with tissue plasminogen activator (tPA), the other standard of care therapy for stroke, and a U.S. Food and Drug Administration preclinical requirement for translation of an experimental stroke therapy. CR2fH, an injury site-targeted inhibitor of the alternative complement pathway, significantly reduced infarct volume, hemorrhagic transformation, and mortality and significantly improved long-term motor and cognitive performance when administered 1.5 or 24 h after middle cerebral artery occlusion. CR2fH interrupted a poststroke inflammatory process and significantly reduced inflammatory cytokine release, microglial activation, and astrocytosis. Rehabilitation alone showed mild anti-inflammatory effects, including reduced complement activation, but only improved cognitive recovery. CR2fH combined with rehabilitation significantly potentiated cognitive and motor recovery compared with either intervention alone and was associated with higher growth factor release and enhanced rehabilitation-induced neuroblast migration and axonal remodeling. Similar outcomes were seen in adult, aged, and female mice. Using a microembolic model, CR2fH administered in combination with acute tPA therapy improved overall survival and enhanced the neuroprotective effects of tPA, extending the treatment window for tPA therapy. A human counterpart of CR2fH has been shown to be safe and nonimmunogenic in humans and we have demonstrated robust deposition of C3d, the CR2fH targeting epitope, in ischemic human brains after stroke.SIGNIFICANCE STATEMENT Complement inhibition is a potential therapeutic approach for stroke, but it is not known how complement inhibition would interact with current standards of care. We show that, after murine ischemic stroke, rehabilitation alone induced mild anti-inflammatory effects and improved cognitive, but not motor recovery. However, brain-targeted and specific inhibition of the alternative complement pathway, when combined with rehabilitation, significantly potentiated cognitive and motor recovery compared with either intervention alone via mechanisms involving neuroregeneration and enhanced brain remodeling. Further, inhibiting the alternative pathway of complement significantly enhanced the neuroprotective effects of thrombolytic therapy and markedly expanded the therapeutic window for thrombolytic therapy.

Acid sphingomyelinase deficiency protects mitochondria and improves function recovery after brain injury.

Traumatic brain injury (TBI) is one of the leading causes of disability worldwide and a prominent risk factor for neurodegenerative diseases. The expansion of nervous tissue damage after the initial trauma involves a multifactorial cascade of events, including excitotoxicity, oxidative stress, inflammation, and deregulation of sphingolipid metabolism that further mitochondrial dysfunction and secondary brain damage. Here, we show that a posttranscriptional activation of an acid sphingomyelinase (ASM), a key enzyme of the sphingolipid recycling pathway, resulted in a selective increase of sphingosine in mitochondria during the first week post-TBI that was accompanied by reduced activity of mitochondrial cytochrome oxidase and activation of the Nod-like receptor protein 3 inflammasome. TBI-induced mitochondrial abnormalities were rescued in the brains of ASM KO mice, which demonstrated improved behavioral deficit recovery compared with WT mice. Furthermore, an elevated autophagy in an ASM-deficient brain at the baseline and during the development of secondary brain injury seems to foster the preservation of mitochondria and brain function after TBI. Of note, ASM deficiency attenuated the early stages of reactive astrogliosis progression in an injured brain. These findings highlight the crucial role of ASM in governing mitochondrial dysfunction and brain-function impairment, emphasizing the importance of sphingolipids in the neuroinflammatory response to TBI.

Natural immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice.

Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of self-reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger-associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody-deficient Rag1-/- mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4-specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1-/- mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild-type (WT) mice. A single-chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post-IR in WT mice, although, interestingly, B4scFv did not alter regeneration post-PHx, indicating that anti-Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross-species injury-specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721-735).

Complement C3-Targeted Gene Therapy Restricts Onset and Progression of Neurodegeneration in Chronic Mouse Glaucoma.

Dysregulation of the complement system is implicated in neurodegeneration, including human and animal glaucoma. Optic nerve and retinal damage in glaucoma is preceded by local complement upregulation and activation, but whether targeting this early innate immune response could have therapeutic benefit remains undefined. Because complement signals through three pathways that intersect at complement C3 activation, here we targeted this step to restore complement balance in the glaucomatous retina and to determine its contribution to degeneration onset and/or progression. To achieve this, we combined adeno-associated virus retinal gene therapy with the targeted C3 inhibitor CR2-Crry. We show that intravitreal injection of AAV2.CR2-Crry produced sustained Crry overexpression in the retina and reduced deposition of the activation product complement C3d on retinal ganglion cells and the inner retina of DBA/2J mice. This resulted in neuroprotection of retinal ganglion cell axons and somata despite continued intraocular pressure elevation, suggesting a direct restriction of neurodegeneration onset and progression and significant delay to terminal disease stages. Our study uncovers a damaging effect of complement C3 or downstream complement activation in glaucoma, and it establishes AAV2.CR2-Crry as a viable therapeutic strategy to target pathogenic C3-mediated complement activation in the glaucomatous retina.

Natural IgM antibodies that bind neoepitopes exposed as a result of spinal cord injury , drive secondary injury by activating complement.

BACKGROUND: Natural IgM antibodies (Abs) function as innate immune sensors of injury via recognition of neoepitopes expressed on damaged cells, although how this recognition systems function following spinal cord injury (SCI) exposes various neoepitopes and their precise nature remains largely unknown. Here, we investigated the role of two natural IgM monoclonal Abs (mAbs), B4 and C2, that recognize post-ischemic neoepitopes following ischemia and reperfusion in other tissues. METHODS: Identification of post-SCI expressed neoepitopes was examined using previously characterized monoclonal Abs (B4 and C2 mAbs). The role of post-SCI neoepitopes and their recognition by natural IgM Abs in propagating secondary injury was examined in Ab-deficient Rag1-/- or wild type C57BL/6 mice using Ab reconstitution experiments and neoepitope-targeted therapeutic studies, respectively. RESULTS: Administration of B4 or C2 mAb following murine SCI increased lesion size and worsened functional outcome in otherwise protected Ab-deficient Rag1-/- mice. Injury correlated with colocalized deposition of IgM and C3d in injured spinal cords from both mAb reconstituted Rag1-/- mice and untreated wild-type mice. Depletion of peritoneal B1 B cells, a source of natural Abs, reduced circulating levels of IgM with B4 (annexin-IV) and C2 (subset of phospholipids) reactivity, reduced IgM and complement deposition in the spinal cord, and protected against SCI. We therefore investigated whether the B4 neoepitope represents a therapeutic target for complement inhibition. B4-Crry, a fusion protein consisting of a single-chain Ab derived from B4 mAb, linked to the complement inhibitor Crry, significantly protected against SCI. B4-Crry exhibited a dual function in that it inhibited both the binding of pathogenic IgM and blocked complement activation in the spinal cord. CONCLUSIONS: This study identifies important neoepitopes expressed within the spinal cord after injury. These neoepitopes are recognized by clonally specific natural IgM Abs that activate complement and drive pathology. We demonstrate that these neoepitopes represent novel targets for the therapeutic delivery of a complement inhibitor, and possibly other payload, to the injured spinal cord.