Inhibition of mannan-binding lectin associated serine protease (MASP)-2 reduces the cognitive deficits in a mouse model of severe traumatic brain injury.

The lectin pathway (LP) of complement mediates inflammatory processes linked to tissue damage and loss of function following traumatic brain injury (TBI). LP activation triggers a cascade of proteolytic events initiated by LP specific enzymes called MASPs (for Mannan-binding lectin Associated Serine Proteases). Elevated serum and brain levels of MASP-2, the effector enzyme of the LP, were previously reported to be associated with the severity of tissue injury and poor outcomes in patients with TBI. To evaluate the therapeutic potential of LP inhibition in TBI, we first conducted a pilot study testing the effect of an inhibitory MASP-2 antibody (α-MASP-2), administered systemically at 4 and 24 h post-TBI in a mouse model of controlled cortical impact (CCI). Treatment with α-MASP-2 reduced sensorimotor and cognitive deficits for up to 5 weeks post-TBI. As previous studies by others postulated a critical role of MASP-1 in LP activation, we conducted an additional study that also assessed treatment with an inhibitory MASP-1 antibody (α-MASP-1). A total of 78 mice were treated intraperitoneally with either α-MASP-2, or α-MASP-1, or an isotype control antibody 4 h and 24 h after TBI or sham injury. An amelioration of the cognitive deficits assessed by Barnes Maze, prespecified as the primary study endpoint, was exclusively observed in the α-MASP-2-treated group. The behavioral data were paralleled by a reduction of the lesion size when evaluated histologically and by reduced systemic LP activity. Our data suggest that inhibition of the LP effector enzyme MASP-2 is a promising treatment strategy to limit neurological deficits and tissue loss following TBI. Our work has translational value because a MASP-2 antibody has already completed multiple late-stage clinical trials in other indications and we used a clinically relevant treatment protocol testing the therapeutic mechanism of MASP-2 inhibition in TBI.

Plasma-derived and recombinant C1 esterase inhibitor: Binding profiles and neuroprotective properties in brain ischemia/reperfusion injury.

C1 esterase inhibitor (C1INH) is known to exert its inhibitory effect by binding to several target proteases of the contact and complement systems. One of C1INH's targets comprise mannose-binding lectin (MBL), a critical player in post-stroke pathophysiology. We therefore explored the effects of recombinant human (rh) and plasma derived (pd) C1INH in C57BL/6J mice subjected to transient occlusion of the middle cerebral artery (tMCAo), receiving 15U/mouse of pd or rhC1INH intravenously, at reperfusion. We analyzed the compounds' (i)neuroprotective effects, (ii) plasma presence, (iii)effects on circulating and brain MBL, (iv)time course of endothelial deposition, and (v) effects on the formation of active complement products. rhC1INH-treated mice had neuroprotective effects, including reduced behavioral deficits and neuronal loss, associated with decreased MBL brain deposition and decreased formation of complement C4b active fragments. In contrast, pdC1INH did not show these neuroprotective effects despite its longer plasma residence time. We also analyzed the response to tMCAo in C1INH-deficient mice, observing a poorer ischemic outcome compared to the wild type mice, which could be partially prevented by rhC1INH administration. In conclusion, we show that rhC1INH exhibits stronger neuroprotective effects than the corresponding plasma-derived protein after experimental ischemia/reperfusion injury in the brain, placing it as a promising drug for stroke. Differential effects are likely related to more effective MBL inhibition which further confirms it as a useful pharmacological target for stroke.

Ficolin-2 serum levels predict the occurrence of acute coronary syndrome in patients with severe carotid artery stenosis.

BACKGROUND AND PURPOSE: erosion of vulnerable atherosclerotic plaques may cause life-threatening thromboembolic complications. There is indeed an urgent need to recognize a clear-cut biomarker able to identify vulnerable plaques. Here, we focused on circulating proteins belonging to the lectin pathway (LP) of complement activation. METHODS: we analyzed mannose-binding lectin (MBL), ficolin-1, -2 and -3 (LP initiators) levels by ELISA in sera from n = 240 of an already published cohort of patients undergoing endarterectomy for severe carotid stenosis and followed-up until 18 months after surgery. Immunofluorescence followed by confocal and polarized light microscopy was used to detect LP initiator intraplaque localization. Spearman's rank test was drawn to investigate correlation between serum LP levels and circulating inflammatory proteins or intraplaque components. Survival analyses were then performed to test the predictive role of LP on long-term adverse outcome. RESULTS: ficolins, but not MBL, correlated positively with 1) high circulating levels of inflammatory markers, including MPO, MMP-8, MMP-9, ICAM-1, osteopontin, neutrophil elastase, and; 2) immune cell intraplaque recruitment. Immunofluorescence showed ficolins in calcified plaques and ficolin-2 in cholesterol-enriched plaque regions in association with macrophages. In the multivariate survival analysis, ficolin-2 serum levels predicted a major adverse cardiovascular event during the follow-up, independently of symptomatic status and inflammatory markers (hazard ratio 38.6 [95 % CI 3.9-385.2]). CONCLUSIONS: ficolins support intraplaque immune cell recruitment and inflammatory processes ultimately leading to plaque vulnerability. Especially for ficolin-2 a strong predictive value toward adverse cardiovascular events was demonstrated. This evidence offers potentially new pharmacological target to dampen the inflammatory mechanisms leading to plaque vulnerability.

The CCL2/CCL7/CCL12/CCR2 pathway is substantially and persistently upregulated in mice after traumatic brain injury, and CCL2 modulates the complement system in microglia.

Traumatic brain injury (TBI) is the leading cause of death in the global population. Disturbed inflammatory processes after TBI exacerbate secondary brain injury and contribute to unfavorable outcomes. Multiple inflammatory events that accompany brain trauma, such as glial activation, chemokine release, or the initiation of the complement system cascade, have been identified as potential targets for TBI treatment. However, the participation of chemokines in the complement activation remains unknown. Our studies sought to determine the changes in the expression of the molecules involved in the CCL2/CCL7/CCL12/CCR2 pathway in the injured brain and the effect of CCL2, CCL7, and CCL12 (10, 100, and 500 ng/mL) on the classic and lectin complement pathways and inflammatory factors in microglial cell cultures. Brain injury in mice was modeled by controlled cortical impact (CCI). Our findings indicate a time-dependent upregulation of CCL2, CCL7, and CCL12 at the mRNA and protein levels within the cortex, striatum, and/or thalamus beginning 24 h after the trauma. The analysis of the expression of the receptor of the tested chemokines, CCR2, revealed its substantial upregulation within the injured brain areas mainly on the mRNA level. Using primary cortical microglial cell cultures, we observed a substantial increase in the expression of CCL2, CCL7, and CCL12 after 24 h of LPS (100 ng/mL) treatment. CCL2 stimulation of microglia increased the level of IL-1ß mRNA but did not influence the expression of IL-18, IL-6, and IL-10. Moreover, CCL2 significantly increased the expression of Iba1, a marker of microglia activation. CCL2 and CCL12 upregulated the expression of C1qa but did not influence the expression of C1ra and C1s1 (classical pathway); moreover, CCL2 increased ficolin A expression and reduced collectin 11 expression (lectin pathway). Additionally, we observed the downregulation of pentraxin 3, a modulator of the complement cascade, after CCL2 and CCL12 treatment. We did not detect the expression of ficolin B, Mbl1, and Mbl2 in microglial cells. Our data identify CCL2 as a modulator of the classical and lectin complement pathways suggesting that CCL2 may be a promising target for pharmacological intervention after brain injury. Moreover, our study provides evidence that CCL2 and two other CCR2 ligands may play a role in the development of changes in TBI.

Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury-Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model.

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein-GFAP), microglia/macrophages (allograft inflammatory factor 1-IBA-1), and microglia (transmembrane protein 119-TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Combined Genetic Deletion of IL (Interleukin)-4, IL-5, IL-9, and IL-13 Does Not Affect Ischemic Brain Injury in Mice.

Background and Purpose- After ischemic injury, microglia and infiltrated macrophages may acquire different polarization phenotypes promoting inflammation and injury (M1) or repair and protection (M2). There is evidence that immunomodulation, via type 2 helper T-cells (Th2) cytokines, exerts neuroprotection after ischemia. We investigated the consequences of simultaneous genetic deletion of Th2 cytokines (IL [interleukin]-4, IL-5, IL-9, IL-13) on the histopathologic outcome, microglia and infiltrated macrophages markers, and ischemic microenvironment at different time points after ischemic injury in mice subjected to permanent occlusion of the middle cerebral artery. Methods- Wild-type and Th2 cytokine-deficient mice (4KO) were subjected to permanent occlusion of the middle cerebral artery by electrocoagulation and followed up to 5 weeks after permanent occlusion of the middle cerebral artery. Neuropathologic outcome was assessed at 24 hours (n=6), 7 days (n=6), and 5 weeks (n=6-7) by examination of the ischemic lesion, neuronal count, microglia and infiltrated macrophages markers, brain atrophy, collagen deposition, and GFAP (glial fibrillary acidic protein) immunohistochemistry. Selected gene expression was investigated at 7 days (n=6). Results- 4KO mice showed no difference in lesion and neuronal count 7 days and up to 5 weeks after permanent occlusion of the middle cerebral artery compared with wild type. Ischemic 4KO mice had lower CD16/32 expression at 24 hours, lower CD11b and CD16/32 expression at 7 days than wild type. They had higher CD206 expression at 24 hours, higher CD206 and arginase1 at 7 days, and increased mRNA for CXCL9 (chemokine [C-X-C motif] ligand 9) compared with wild type. Additional histopathologic analysis, including brain atrophy, gliotic scar, and collagenous scar confirmed no difference between genotypes at 5 weeks. Conclusions- This study casts light on the proposed neuroprotective function of Th2 cytokines, showing that combined IL-4, IL-5, IL-9, IL-13 deletion does not affect the neuropathologic response to ischemic stroke in the subacute and chronic phases. Our findings indicate that Th2 cytokines are not an essential neuroimmunological cue able to drive the brain's ischemic outcome.

The phagocytic state of brain myeloid cells after ischemia revealed by superresolution structured illumination microscopy.

BACKGROUND: Phagocytosis is a key function of myeloid cells and is highly involved in brain ischemic injury. It has been scarcely studied in vivo, thus preventing a deep knowledge of the processes occurring in the ischemic environment. Structured illumination microscopy (SIM) is a superresolution technique which helps study phagocytosis, a process involving the recruitment of vesicles sized below the resolution limits of standard confocal microscopy. METHODS: Mice underwent permanent occlusion of the middle cerebral artery and were sacrificed at 48 h or 7 days after insult. Immunofluorescence for CD11b, myeloid cell membrane marker, and CD68, lysosomal marker was done in the ischemic area. Images were acquired using a SIM system and verified with SIM check. Lysosomal distribution was measured in the ischemic area by the gray level co-occurrence matrix (GLCM). SIM dataset was compared with transmission electron microscopy images of macrophages in the ischemic tissue at the same time points. Cultured microglia were stimulated with LPS to uptake 100 nm fluorescent beads and imaged by time-lapse SIM. GLCM was used to analyze bead distribution over the cytoplasm. RESULTS: SIM images reached a resolution of 130 nm and passed the quality control diagnose, ruling out possible artifacts. After ischemia, GLCM applied to the CD68 images showed that myeloid cells at 48 h had higher angular second moment (ASM), inverse difference moment (IDM), and lower entropy than myeloid cells at 7 days indicating higher lysosomal clustering at 48 h. At this time point, lysosomal clustering was proximal (< 700 nm) to the cell membrane indicating active target internalization, while at 7 days, it was perinuclear, consistent with final stages of phagocytosis or autophagy. Electron microscopy images indicated a similar pattern of lysosomal distribution thus validating the SIM dataset. GLCM on time-lapse SIM from phagocytic microglia cultures revealed a temporal decrease in ASM and IDM and increase in entropy, as beads were uptaken, indicating that GLCM informs on the progression of phagocytosis. CONCLUSIONS: GLCM analysis on SIM dataset quantitatively described different phases of macrophage phagocytic behavior revealing the dynamics of lysosomal movements in the ischemic brain indicating initial active internalization vs. final digestion/autophagy.

Mannose-Binding Lectin Drives Platelet Inflammatory Phenotype and Vascular Damage After Cerebral Ischemia in Mice via IL (Interleukin)-1α.

Objective- Circulating complement factors are activated by tissue damage and contribute to acute brain injury. The deposition of MBL (mannose-binding lectin), one of the initiators of the lectin complement pathway, on the cerebral endothelium activated by ischemia is a major pathogenic event leading to brain injury. The molecular mechanisms through which MBL influences outcome after ischemia are not understood yet. Approach and Results- Here we show that MBL-deficient (MBL-/-) mice subjected to cerebral ischemia display better flow recovery and less plasma extravasation in the brain than wild-type mice, as assessed by in vivo 2-photon microscopy. This results in reduced vascular dysfunction as shown by the shift from a pro- to an anti-inflammatory vascular phenotype associated with MBL deficiency. We also show that platelets directly bind MBL and that platelets from MBL-/- mice have reduced inflammatory phenotype as indicated by reduced IL-1α (interleukin-1α) content, as early as 6 hours after ischemia. Cultured human brain endothelial cells subjected to oxygen-glucose deprivation and exposed to platelets from MBL-/- mice present less cell death and lower CXCL1 (chemokine [C-X-C motif] ligand 1) release (downstream to IL-1α) than those exposed to wild-type platelets. In turn, MBL deposition on ischemic vessels significantly decreases after ischemia in mice treated with IL-1 receptor antagonist compared with controls, indicating a reciprocal interplay between MBL and IL-1α facilitating endothelial damage. Conclusions- We propose MBL as a hub of pathogenic vascular events. It acts as an early trigger of platelet IL-1α release, which in turn favors MBL deposition on ischemic vessels promoting an endothelial pro-inflammatory phenotype.

Induction of a transmissible tau pathology by traumatic brain injury.

Traumatic brain injury is a risk factor for subsequent neurodegenerative disease, including chronic traumatic encephalopathy, a tauopathy mostly associated with repetitive concussion and blast, but not well recognized as a consequence of severe traumatic brain injury. Here we show that a single severe brain trauma is associated with the emergence of widespread hyperphosphorylated tau pathology in a proportion of humans surviving late after injury. In parallel experimental studies, in a model of severe traumatic brain injury in wild-type mice, we found progressive and widespread tau pathology, replicating the findings in humans. Brain homogenates from these mice, when inoculated into the hippocampus and overlying cerebral cortex of naïve mice, induced widespread tau pathology, synaptic loss, and persistent memory deficits. These data provide evidence that experimental brain trauma induces a self-propagating tau pathology, which can be transmitted between mice, and call for future studies aimed at investigating the potential transmissibility of trauma associated tau pathology in humans.

Targeting Extracellular Cyclophilin A Reduces Neuroinflammation and Extends Survival in a Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation is a major hallmark of amyotrophic lateral sclerosis (ALS), which is currently untreatable. Several anti-inflammatory compounds have been evaluated in patients and in animal models of ALS, but have been proven disappointing in part because effective targets have not yet been identified. Cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), as a foldase is beneficial intracellularly, but extracellularly has detrimental functions. We found that extracellular PPIA is a mediator of neuroinflammation in ALS. It is a major inducer of matrix metalloproteinase 9 and is selectively toxic for motor neurons. High levels of PPIA were found in the CSF of SOD1G93A mice and rats and sporadic ALS patients, suggesting that our findings may be relevant for familial and sporadic cases. A specific inhibitor of extracellular PPIA, MM218, given at symptom onset, rescued motor neurons and extended survival in the SOD1G93A mouse model of familial ALS by 11 d. The treatment resulted in the polarization of glia toward a prohealing phenotype associated with reduced NF-κB activation, proinflammatory markers, endoplasmic reticulum stress, and insoluble phosphorylated TDP-43. Our results indicates that extracellular PPIA is a promising druggable target for ALS and support further studies to develop a therapy to arrest or slow the progression of the disease in patients.SIGNIFICANCE STATEMENT We provide evidence that extracellular cyclophilin A, also known as peptidylprolyl cis-/trans-isomerase A (PPIA), is a mediator of the neuroinflammatory reaction in amyotrophic lateral sclerosis (ALS) and is toxic for motor neurons. Supporting this, a specific extracellular PPIA inhibitor reduced neuroinflammation, rescued motor neurons, and extended survival in the SOD1G93A mouse model of familial ALS. Our findings suggest selective pharmacological inhibition of extracellular PPIA as a novel therapeutic strategy, not only for SOD1-linked ALS, but possibly also for sporadic ALS. This approach aims to address the neuroinflammatory reaction that is a major hallmark of ALS. However, given the complexity of the disease, a combination of therapeutic approaches may be necessary.

Critical Role and Therapeutic Control of the Lectin Pathway of Complement Activation in an Abortion-Prone Mouse Mating.

The abortion-prone mating combination CBA/J × DBA/2 has been recognized as a model of preeclampsia, and complement activation has been implicated in the high rate of pregnancy loss observed in CBA/J mice. We have analyzed the implantation sites collected from DBA/2-mated CBA/J mice for the deposition of the complement recognition molecules using CBA/J mated with BALB/c mice as a control group. MBL-A was observed in the implantation sites of CBA/J × DBA/2 combination in the absence of MBL-C and was undetectable in BALB/c-mated CBA/J mice. Conversely, C1q was present in both mating combinations. Searching for other complement components localized at the implantation sites of CBA/J × DBA/2, we found C4 and C3, but we failed to reveal C1r. These data suggest that complement is activated through the lectin pathway and proceeds to completion of the activation sequence as revealed by C9 deposition. MBL-A was detected as early as 3.5 d of pregnancy, and MBL-A deficiency prevented pregnancy loss in the abortion-prone mating combination. The contribution of the terminal complex to miscarriage was supported by the finding that pregnancy failure was largely inhibited by the administration of neutralizing Ab to C5. Treatment of DBA/2-mated CBA/J mice with Polyman2 that binds to MBL-A with high affinity proved to be highly effective in controlling the activation of the lectin pathway and in preventing fetal loss.

Internalization of nanopolymeric tracers does not alter characteristics of placental cells.

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA-NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV-MSC). We report that PMMP-NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP-NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP-NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS-treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP-NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP-NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV-MSC in preclinical models of inflammatory-driven diseases.

Macrophages are essential for maintaining a M2 protective response early after ischemic brain injury.

Resident microglia and recruited macrophages are major contributors to the post-ischemic inflammatory response. Initially considered functionally homogeneous populations, data now suggest distinct but still controversial roles after brain injury. Using a model of conditional monocyte/macrophage depletion we studied the contribution of these myeloid cells to brain lesion progression after ischemia, and their influence on the ischemic inflammatory environment. Male CD11b-DTR transgenic mice, expressing the human diphtheria toxin receptor under the control of the CD11b promoter, were treated with diphtheria toxin to induce monocyte/macrophage depletion. Twenty four hours later the middle cerebral artery was permanently occluded. The ischemic lesion was measured 24h after injury. At the same time microglia and macrophage activation and polarization were assessed by quantitative immunohistochemistry and confocal microscopy for CD45high, CD11b, CD68, CD16/32, iNOS, Arg1, Ym1, and CD206, and gene expression was investigated on CD11b+ sorted cells. Depletion of monocytes/macrophages worsened the ischemic lesion within 24h after the ischemic insult. This effect was associated with higher M1/M2 polarization ratio in the ischemic lesion. Moreover, depletion increased the expression of M1 phenotypic markers on CD11b positive cells. Gene expression on CD11b+ sorted cells indicated a selective increase of iNOS and lower Arg1 mRNA expression than in non depleted mice. Depletion of monocytes/macrophages increases the ischemic lesion, an effect accompanied by an increase in the M1/M2 polarization ratio of microglia and macrophages in the ischemic area. Thus in ischemic injury recruited monocytes/macrophages may control an excessive M1 pro-inflammatory response, suggesting their ability to drive M2 protective polarization.

Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.

BACKGROUND: Complement activation via the lectin activation pathway (LP) has been identified as the key mechanism behind post-ischemic tissue inflammation causing ischemia-reperfusion injury (IRI) which can significantly impact the clinical outcome of ischemic disease. This work defines the contributions of each of the three LP-associated enzymes-mannan-binding lectin-associated serine protease (MASP)-1, MASP-2, and MASP-3-to ischemic brain injury in experimental mouse models of stroke. METHODS: Focal cerebral ischemia was induced in wild-type (WT) mice or mice deficient for defined complement components by transient middle cerebral artery occlusion (tMCAO) or three-vessel occlusion (3VO). The inhibitory MASP-2 antibody was administered systemically 7 and 3.5 days before and at reperfusion in WT mice in order to assure an effective MASP-2 inhibition throughout the study. Forty-eight hours after ischemia, neurological deficits and infarct volumes were assessed. C3 deposition and microglia/macrophage morphology were detected by immunohistochemical, immunofluorescence, and confocal analyses. RESULTS: MASP-2-deficient mice (MASP-2(-/-)) and WT mice treated with an antibody that blocks MASP-2 activity had significantly reduced neurological deficits and histopathological damage after transient ischemia and reperfusion compared to WT or control-treated mice. Surprisingly, MASP-1/3(-/-) mice were not protected, while mice deficient in factor B (fB(-/-)) showed reduced neurological deficits compared to WT mice. Consistent with behavioral and histological data, MASP-2(-/-) had attenuated C3 deposition and presented with a significantly higher proportion of ramified, surveying microglia in contrast to the hypertrophic pro-inflammatory microglia/macrophage phenotype seen in the ischemic brain tissue of WT mice. CONCLUSIONS: This work demonstrates the essential role of the low-abundant MASP-2 in the mediation of cerebral ischemia-reperfusion injury and demonstrates that targeting MASP-2 by an inhibitory therapeutic antibody markedly improved the neurological and histopathological outcome after focal cerebral ischemia. These results contribute to identifying the key lectin pathway component driving brain tissue injury following cerebral ischemia and call for a revision of the presently widely accepted view that MASP-1 is an essential activator of the lectin pathway effector component MASP-2.

Protection of Brain Injury by Amniotic Mesenchymal Stromal Cell-Secreted Metabolites.

OBJECTIVES: To define the features of human amniotic mesenchymal stromal cell secretome and its protective properties in experimental models of acute brain injury. DESIGN: Prospective experimental study. SETTING: Laboratory research. SUBJECTS: C57Bl/6 mice. INTERVENTIONS: Mice subjected to sham or traumatic brain injury by controlled cortical impact received human amniotic mesenchymal stromal cells or phosphate-buffered saline infused intracerebroventricularly or intravenously 24 hours after injury. Organotypic cortical brain slices exposed to ischemic injury by oxygen-glucose deprivation were treated with human amniotic mesenchymal stromal cells or with their secretome (conditioned medium) in a transwell system. MEASUREMENTS AND MAIN RESULTS: Traumatic brain injured mice receiving human amniotic mesenchymal stromal cells intravenously or intracerebroventricularly showed early and lasting functional and anatomical brain protection. cortical slices injured by oxigen-glucose deprivation and treated with human amniotic mesenchymal stromal cells or conditioned medium showed comparable protective effects (neuronal rescue, promotion of M2 microglia polarization, induction of trophic factors) indicating that the exposure of human amniotic mesenchymal stromal cells to the injured tissue is not necessary for the release of bioactive factors. Using sequential size-exclusion and gel-filtration chromatography, we identified a conditioned medium subfraction, which specifically displays these highly protective properties and we found that this fraction was rich in bioactive molecules with molecular weight smaller than 700 Da. Quantitative RNA analysis and mass spectrometry-based peptidomics showed that the active factors are not proteins or RNAs. The metabolomic profiling of six metabolic classes identified a list of molecules whose abundance was selectively elevated in the active conditioned medium fraction. CONCLUSIONS: Human amniotic mesenchymal stromal cell-secreted factors protect the brain after acute injury. Importantly, a fraction rich in metabolites, and containing neither proteic nor ribonucleic molecules was protective. This study indicates the profiling of protective factors that could be useful in cell-free therapeutic approaches for acute brain injury.

Scaffold Optimisation of Tetravalent Antagonists of the Mannose Binding Lectin.

Antagonists of mannose binding lectin (MBL) have shown a protective role against brain reperfusion damage after acute ischemic stroke. Here we describe the design and streamlined synthesis of glycomimetic MBL antagonists based on a new tetravalent dendron scaffold. The dendron was developed by optimisation of a known polyester structure previously demonstrated to be very efficient for ligand presentation to MBL. Replacement of a labile succinyl ester bond with a more robust amide functionality, use of a longer and more hydrophilic linker, fast modular synthesis and orthogonal functionalisation at the focal point are the main features of the new scaffold. The glycoconjugate constructs become stable to silica gel chromatography and to water solutions at physiological pH, while preserving water solubility and activity in an SPR assay against the murine MBL-C isoform. Higher-order constructs were easily assembled, as demonstrated by the synthesis of a 16-valent dendrimer, which leads to two orders of magnitude increase in activity over the tetravalent version against MBL-C.

Mannose-binding lectin is expressed after clinical and experimental traumatic brain injury and its deletion is protective.

OBJECTIVE: Mannose-binding lectin protein is the activator of the lectin complement pathway. Goals were (1) to investigate mannose-binding lectin expression after human and experimental traumatic brain injury induced by controlled cortical impact and (2) to evaluate whether mannose-binding lectin deletion is associated with reduced sequelae after controlled cortical impact. DESIGN: Translational research, combining a human/experimental observational study and a prospective experimental study. SETTING: University hospital/research laboratory. PATIENTS AND SUBJECTS: Brain-injured patients, C57Bl/6 mice, and mannose-binding lectin-A and mannose-binding lectin-C double-knockout (-/-) mice. INTERVENTIONS: Using anti-human mannose-binding lectin antibody, we evaluated mannose-binding lectin expression in tissue samples from six patients who underwent surgery for a cerebral contusion. Immunohistochemistry was also performed on tissues obtained from mice at 30 minutes; 6, 12, 24, 48, and 96 hours; and 1 week after controlled cortical impact using anti-mouse mannose-binding lectin-A and mannose-binding lectin-C antibodies. We evaluated the effects of mannose-binding lectin deletion in wild-type and mannose-binding lectin-A and mannose-binding lectin-C double-knockout mice. Functional outcome was evaluated using the neuroscore and beam walk tests for 4 weeks postinjury (n = 11). Histological injury was evaluated by comparing neuronal cell counts in the cortex adjacent to the contusion (n = 11). MEASUREMENTS AND MAIN RESULTS: Following human traumatic brain injury, we observed mannose-binding lectin-positive immunostaining in the injured cortex as early as few hours and up to 5 days postinjury. Similarly in mice, we observed mannose-binding lectin-C-positive immunoreactivity in the injured cortex beginning 30 minutes and persisting up to 1 week postinjury. The extent of mannose-binding lectin-A expression was lower when compared with that of mannose-binding lectin-C. We observed attenuated sensorimotor deficits in mannose-binding lectin (-/-) mice compared with wild-type mice at 2-4 weeks postinjury. Furthermore, we observed reduced cortical cell loss at 5 weeks postinjury in mannose-binding lectin (-/-) mice compared with wild-type mice. CONCLUSIONS: Mannose-binding lectin expression was documented after traumatic brain injury. The reduced sequelae associated with mannose-binding lectin absence suggest that mannose-binding lectin modulation might be a potential target after traumatic brain injury.

Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.

BACKGROUND: The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. METHODS AND RESULTS: Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL(-/-) mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. CONCLUSIONS: Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice.

The studies on fractalkine and its unique receptor CX3CR1 in neurological disorders yielded contrasting results. We have explored the consequences of CX3CR1 deletion in ischemic (30' MCAo) mice on: (1) brain infarct size; (2) microglia dynamism and morphology; (3) expression of markers of microglia/macrophages (M/M) activation and polarization. We observed smaller infarcts in cx3cr1(-/-) (26.42 ± 7.41 mm(3) , mean ± sd) compared to wild type (36.29 ± 11.57) and cx3cr1(-/+) (34.49 ± 8.91) mice. We longitudinally analyzed microglia by in vivo two-photon microscopy before, 1 and 24 h after transient ischemia. Microglia were stationary in both cx3cr1(-/-) and cx3cr1(-/+) mice throughout the study. In cx3cr1(-/-) mice, they displayed a significantly higher number of ramifications >10 µm at baseline and at 24 h after ischemia compared to cx3cr1(-/+) mice, indicating that CX3CR1 deficiency impaired the development of microglia hypertrophic/amoeboid morphology. At 24 h after ischemia, we performed post mortem quantitative immunohistochemistry for different M/M markers. In cx3cr1(-/-) immunoreactivity for CD11b (M/M activation) and for CD68 (associated with phagocytosis) were decreased, while that for CD45(high) (macrophage and leukocyte recruitment) was increased. In addition, immunoreactivity for Ym1 (M2 polarization) was enhanced, while that for iNOS (M1) was decreased. Our data show that in cx3cr1(-/-) mice protection from ischemia at early time points after injury is associated with a protective inflammatory milieu, characterized by the promotion of M2 polarization markers.