Background
Inflammatory mechanisms intrinsic to brain and blood-borne inflammatory mediators are among the major drivers of focal injury following cerebral ischemia. Among the inflammatory cascades, the complement system represents a powerful contributor to ischemic brain injury by several possible mechanisms including anaphylatoxin release, endothelial activation aiding leukocyte adhesion and recruitment, over-activation of the phagocytic system, and direct cellular lysis [
1]. Activation of the complement can be achieved by three distinct pathways: the classical pathway (CP), the alternative pathway (AP), and the lectin pathway (LP). All three pathways encompass tightly regulated sequential activation cascades converging at the cleavage of the abundant complement component C3. Cleavage of C3 releases the small complement anaphylatoxin C3a and the large C3b fragment, which possess pro-inflammatory properties and promote opsonization and direct phagocytosis, respectively. In addition, C3b is also a constituent of the AP C3 convertase (C3bBb), and the deposition of C3b promotes the formation of more AP C3 convertases by binding to native factor B (fB) to form the AP zymogen complex C3bB [
2]. C3b is also an essential component of both C5-cleaving convertase complexes (C3bBb(C3b)
n and C4b2a(C3b)
n, respectively). With the cleavage of C5, the last enzymatic step of complement activation is completed. C5 cleavage releases the potent complement anaphylatoxin C5a and the larger fragment C5b that initiates the formation of the membrane attack complex (MAC) by subsequent recruitment of the terminal cascade components C6-C9. This MAC inserts into cell membranes to form a pore that results in ion flux, causing cell lysis.
The critical contribution of the complement system to the pathophysiology of ischemia-reperfusion injury (IRI) has been demonstrated in several models of ischemia-reperfusion injury [
3‐
7]. It was initially hypothesized that the complement activation pathway responsible for IRI is the CP driven by natural antibodies binding to ischemia-damaged cells [
8]. The involvement of the LP in IRI was first described by Collard et al. in 2000 [
9], a finding that was later underlined by our own work reporting the observation that CP-deficient C1q
−/− mice were not protected in models of cerebral IRI [
6]. The prominent role of the LP over the CP in mediating IRI was underlined by the protective phenotype of mannan-binding lectin (MBL) deficiency and the therapeutic effect of inhibitory molecules against MBL in various mouse models [
7,
10‐
14]. Deficiency or inhibition of MBL achieved long-lasting neuroprotection and improved functional outcome in mouse models of stroke with a wide window for therapeutic intervention (up to 24 h) [
13]. The latter findings are supported by numerous recent publications, which underline the prominent role of the LP in the pathogenesis and progression of brain damage in a clinical context. In stroke patients, the LP was shown to be the relevant pathway in the progression of ischemic brain damage [
12,
15‐
17] with studies highlighting MBL [
18,
19] and ficolin-3 [
15], two different LP recognition molecules, as independent predictors of ischemic stroke outcome.
The initiation of the complement activation via the LP requires the binding of one or more of the five different human recognition subcomponents (i.e., MBL, collectin-11 (CL-11), ficolin-1, ficolin-2, or ficolin-3) or the five different mouse recognition components (i.e., MBL-A, MBL-C, CL-11, ficolin-A, or ficolin-B) to their cognate ligands on activating surfaces [
20‐
22]. The ligands that mediate the binding of LP recognition components on ischemic cells are presently unknown. LP activation complexes are formed when a multimolecular complex composed of oligomers of homotrimeric recognition subunits associated with LP-specific serine proteases, called mannan-binding lectin-associated serine proteases (MASPs), bind to cognate ligands on activator surfaces. Three MASP enzymes named MASP-1, MASP-2, and MASP-3 have been described and are encoded by two different genes. The
MASP1 gene is located on human chromosome 3 (mouse chromosome 16) and encodes MASP-1 and MASP-3. The
MASP2 gene is located on human chromosome 1 (mouse chromosome 4) and encodes the serine protease MASP-2 [
23,
24].
Of the three different MASPs, only MASP-2 is able to cleave both C2 and C4 to form the LP C3 convertase C4bC2a and the C5 convertase C4bC2a(C3b)
n. Indeed, in the absence of MASP-2, but not of MASP-1/MASP-3, a complete inhibition of LP activation was observed [
20,
25]. In addition, targeting MASP-2 by gene disruption or administration of antibodies that inhibit MASP-2 functional activity reduced IRI in models of myocardial, intestinal, or renal IRI [
20,
26].
With respect to MASP-1, previous work suggested that, due to its ability to cleave C2 but not C4, it cannot drive LP activation in the absence of MASP-2 but may facilitate MASP-2-driven LP activation [
20,
27‐
29]. A recent study, however, proposed that MASP-1 has an essential role in driving MASP-2 and LP activation by being an exclusive activator of MASP-2 [
30], analogous to the CP serine proteases where C1r is the exclusive activator of C1s [
31]. Further studies attributed an additional role to MASP-1 and/or MASP-3 in driving AP activation by converting factor D (fD) and/or fB zymogen into their enzymatically active forms [
32,
33].
As for the subsequent activation steps, the activation of complement C3, but not of complement C5, was shown to be instrumental in the development of cerebral IRI, as demonstrated in a mouse model of focal ischemia [
34]. Thus, it appears that C5a is not required to mediate the hallmarks of post-ischemic inflammation such as endothelial cell activation, facilitation of leukocyte adhesion and recruitment, and activation of phagocytic cells.
This study reveals the involvement of MASPs in cerebral IRI by assessing the impact of MASP-2 and of combined MASP-1 and MASP-3 deficiency in gene-targeted mouse strains and in WT mice treated with a MASP-2-specific inhibitor on functional and histopathological damage following cerebral focal ischemia. We directly compared our findings against the phenotypes seen in fB-deficient and C4-deficient mice (fB−/− and C4−/−, respectively) analyzed in parallel.
Methods
Animals
Procedures involving animals and their care for transient middle cerebral artery occlusion (tMCAO) surgery were conducted at the Mario Negri Institute in conformity with institutional guidelines (Quality Management System Certificate-UNI EN ISO 9001:2008, Reg. no. 8576-A) in compliance with national (D.L. n:116,G.U. suppl. 40, February 18, 1992) and international laws and policies (EEC Council Directive 86/609, OJL 358,1; Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996, Eight Edition, 2011). All animal experiments were approved by the Mario Negri Institutional Animal Care Committee. Male 9- to 13-week-old C57Bl/6J (Charles River Laboratories, Italy) and MASP-2
−/− [
20], MASP-1/3
−/− [
27], C4
−/− [
8], and fB
−/− [
35] mice (bred at the Biomedical Services, University of Leicester) were used.
Procedures involving animals and their care for three-vessel occlusion (3VO) surgery were conducted at the University of Leicester, in accordance to the UK Animals (Scientific Procedures) Act, 1986. Female 9- to 13-week-old C57Bl/6J (Charles River Laboratories, UK) and MASP-2−/− mice (Biomedical Services, University of Leicester) were used.
MASP-2 inhibitory antibody treatment
The inhibitory MASP-2 antibody (HG4) used in this study is a derivative of the human MASP-2 inhibitory mAb OMS721 modified for improved LP inhibition in mice. HG4 and isotype control (IC) antibody (ET904, supplied by BioLegend) were administered (10 mg/kg intraperitoneally) twice (7 and 3.5 days) as single injections prior to initiation of ischemia and once (10 mg/kg intravenously), again as a single injection, at time of reperfusion.
Transient middle cerebral artery occlusion
Surgery
tMCAO was induced by a siliconized filament (7-0, Doccol Corporation) introduced into the right carotid artery and advanced to block the origin of MCA for 60 min as described previously [
6,
13]. Surgery-associated mortality rate was 7 %. See also Additional file
1.
Neurological deficits
At 48 h after tMCAO, each mouse was rated on two neurological function scales unique to mouse [
5,
36]. The general deficit scale describes the well-being of the mouse, evaluating hair, ears, eyes, posture, spontaneous activity, and epileptic behavior. The focal deficit scale evaluates body symmetry, gait, climbing on a surface held at 45°, circling behavior, front limb symmetry, compulsory circling, and whisker response to a light touch. In both scales, the mice were scored from 0 (healthy mouse) to 28 (the worst performance in all categories) by a trained investigator blinded to the experimental conditions. See also Additional file
1.
Infarct-volume quantification
After assessment of neurological deficits, the mice were perfused and brains were obtained as described previously [
6]. Twenty-micron coronal brain cryosections were cut serially and stained with cresyl violet (Sigma-Aldrich). Ischemic lesion size was calculated on seven slices by delineating the infarcted area, visualized by the relative paleness of histological staining. Infarct volumes were calculated by the integration of infarcted areas after correction for the percentage of brain swelling due to edema using Analytical Image System (Imaging Research Inc.). See also Additional file
1.
Three-vessel occlusion
Surgery
3VO was conducted as described by Yanamoto et al. 2003 [
37]. The two common carotid arteries (CCAs) were exposed, and the left one was clipped. The left MCA was exposed through a small burr hole in the temporal bone and permanently occluded using a bipolar coagulator. Complete ischemia was induced by clipping the right CCA for 30 min. Then, both clips on CCAs were removed, allowing reperfusion. Surgery-associated mortality rate was 8 %. See Additional file
1.
Infarct-volume quantification
Twenty-four hours after 3VO, the mice were sacrificed by cervical dislocation and then brains were removed, cut serially at 1-mm intervals, and stained with 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) for infarct measurements using Analytical Image System. See also Additional file
1.
Lectin pathway-specific C3 deposition assay
To assess the inhibitory effects of antibody administration on systemic LP functional activity, the sera of the mice treated with HG4 or its corresponding isotype control antibody were analyzed in LP-specific C3 deposition ELISA, as described previously [
20].
Microglia/macrophage and C3 immunofluorescence and confocal analysis
Twenty-micrometer coronal brain sections were incubated with rat anti-mouse CD11b (1:500, kindly provided by Dr. Doni, for microglia/macrophage staining) and rabbit anti-C3 polyclonal (Santa Cruz Biotechnology) primary antibodies followed by Alexa 546 anti-rat and Alexa 488 anti-rabbit (both 1:500, Invitrogen) secondary antibodies [
13,
38]. Images were acquired by confocal microscopy as described previously [
38]. Three-dimensional images were acquired over a 10- to 12-μm
z-axis with a 0.23-μm step size and processed using Imaris software (Bitplane) and Photoshop CS2 (Adobe Systems Europe Ltd). See also Additional file
1.
Microglia/macrophage and C3 immunohistochemical analysis
Immunohistochemistry was performed on 20-μm brain coronal sections using rat anti-mouse CD11b (1:800, kindly provided by Dr. Doni) and rabbit anti-C3 polyclonal (1:50, Santa Cruz Biotechnology) followed by biotinylated anti-rat and anti-rabbit secondary antibody (Vector Laboratories, CA, USA). Positive cells were stained by reaction with 3,3diaminobenzidinetetrahydrochloride (DAB, Vector laboratories). For negative control staining, the primary antibody was omitted, and no staining was observed.
For quantitative analysis of microglia/macrophage, field selection was performed on one brain coronal section (+0.1 mm from the bregma, Additional file
1: Figure S1) using a BX61 Olympus microscope equipped with a motorized stage. Frames were acquired using the software AnalySIS (Olympus) [
38]. Twelve quantification fields at ×40 magnification (pixel size = 0.172 μm) were uniformly distributed over the cortex. Image processing was performed using Fiji software [
39] through the algorithm previously described [
40]. Once segmented, the cells were measured for the following parameters: area, Feret’s diameter (caliper), and solidity. Mean single-cell values for each parameter were used for statistics. See also Additional file
1.
For quantitative analysis of C3 staining, the entire ipsilateral cortex of one coronal section per mouse (+0.1 mm from the bregma) was acquired at ×20 by Olympus BX-61 Virtual Stage microscope, with a pixel size of 0.346 μm. Acquisition was done over 6-μm thick stacks, with a step size of 2 μm. The different focal planes were merged into a single stack by mean intensity projection to ensure consistent focus throughout the sample. The acquired area was analyzed using Fiji software. C3 staining was expressed as positive pixels/total assessed pixels and reported as the percentage of total stained area as previously described [
38].
Blinding and statistical analysis
All experimental procedures including surgery, behavioral tests, infarct-volume quantification, immunofluorescence, immunohistochemical analyses, and biochemical assays were performed by investigators blinded to the experimental conditions. Group size was defined using the following formula: n = 2σ2f(α, β) / Δ2 (sd in groups = σ, type 1 error α = 0.05, type II error β = 0.2, percentage difference between groups Δ = 20). For each measure, the standard deviation between groups was calculated on the basis of previous experiments with the same output parameters (e.g., for lesion volume quantification σ = 17, yielding n = 11.4). p values lower than 0.05 and 0.01 were considered significant and highly significant, respectively. Data are expressed as scatter-dot plots and means (bars). GraphPad Prism 6 software was used for statistical analysis. All the data were checked for normal distribution by Kolmogorov-Smirnov test, and unpaired t test or one-way ANOVA followed by Tukey’s post hoc test was used for statistical comparison among groups.
Discussion
This study provides a comparative analysis of the phenotypes of mouse lines with targeted deficiencies of the LP-specific serine proteases MASP-1, MASP-2, and MASP-3 in a mouse model of stroke. Our analysis includes functional outcomes based on behavior tests (focal deficit and general neurological deficits) and histopathological outcomes (infarct sizes) as well as immunohistochemistry for deposition of the C3 activation products as a parameter for complement activation events within the ischemic areas of the brain and for morphometric endpoints (morphology of CD11b-positive macrophages/microglial cells). An AP-deficient mouse line with a targeted deficiency of the essential AP zymogen fB and a CP-deficient mouse line with a targeted deficiency in C4 were also evaluated. When compared to the WT mice, a strong protective phenotype was observed in the MASP-2-deficient mice at the level of functional and histopathological outcomes. Absence of MASP-2 lead to reduced C3 deposition in the ischemic brain area and less prominent hypertrophic and amoeboid microglial morphology. Similarly, improved outcomes were observed in the WT mice treated with a MASP-2-inhibitory antibody, corroborating the findings observed in the MASP-2−/− mice.
Overall, the CD11b morphological analysis revealed that, in the absence of MASP-2 functional activity, microglial cells within the ischemic areas primarily present as ramified microglia suggestive of an anti-inflammatory polarization state [
41‐
43] as opposed to the hypertrophic microglial morphology associated with phagocytic activity and pro-inflammatory state in the ischemic areas of WT brains. The complement system has a major role in the activation of microglia, which constitutively express receptors for C1q and for C3 cleavage products. As a consequence of complement component binding, microglia activate phagocytosis and cytokine production. Activated microglia in turn contribute to complement component production that feeds autocrine/paracrine signaling [
41]. Since microglia act as a major contributor to post-injury inflammatory responses, it is plausible that complement activation through MASP-2-dependent processes could drive microglia from the ramified, surveying state towards the pro-inflammatory phagocytic phenotype, with visualized morphological changes of amoeboid cell shape, an enlarged cell soma, and retracted processes [
40,
41,
44].
Since in vitro experiments suggested that MASP-1 fulfills a critical role in the activation of MASP-2 [
45], we expected that the mice deficient in MASP-1 would also show protection from cerebral IRI following tMCAO. Surprisingly, the MASP-1/3
−/− mice presented with larger infarct volumes then their WT controls but showed a very similar degree of severity in neurological deficits. Likewise, the degree of C3 deposition within the ischemic areas in the brains of the MASP-1/3
−/− was similar to that seen in the WT mice. The morphometric analysis of microglia revealed no differences between the WT and MASP-1/3
−/− mice. The absence of any degree of protection of the MASP-1 and MASP-3 double-deficient mouse line indicates that neither MASP-1 nor MASP-3 are involved in the pathophysiological processes leading to cerebral IRI.
Several studies have indicated that MASP-1 acts as a rate-limiting protease in the activation of MASP-2 in serum. SFMI-1, a peptide that preferentially inhibits MASP-1, was reported to inhibit the LP to an extent similar to a MASP-2-specific peptide inhibitor, SFMI-2 [
45]. Subsequent studies using second-generation peptide inhibitors of either MASP-1 or MASP-2 with greater specificity corroborated the initial results. These findings led to the hypothesis that, in the LP, MASP-1 is the initiating protease critically required to cleave MASP-2 zymogen into its enzymatically active form, analogous to the CP where C1r is the exclusive activator of C1s [
30]. Separate in vitro studies using serum from a Malpuech–Michels–Mingarelli–Carnevale (3MC) patient lacking both MASP-1 and MASP-3 supported this hypothesis [
46]. We previously reported that serum of mice deficient in both MASP-1 and MASP-3 maintains a reduced but clearly detectable LP functional activity [
20,
27].
The in vivo data presented here demonstrate that targeting MASP-2 reduces cerebral IRI while the absence of MASP-1 and MASP-3 affords no protection. MASP-2 does not require MASP-1 to drive IRI. Thus, targeting MASP-1 is unlikely to disrupt the LP-mediated pathophysiological processes that lead to unfavorable outcomes.
It is undisputed that MASP-1 facilitates the conversion of zymogen MASP-2 into its enzymatically active form, most likely because the relatively low abundance of MASP-2 compared to MASP-1 is a limiting factor during the LP-specific trans-activation events involving the juxtaposition of activating complexes [
47]. The non-protective phenotype of a combined MASP-1 and MASP-3 deficiency in a MASP-2-dependent pathophysiological process underlines that MASP-1 is not an essential activator of MASP-2 functional activity and highlights the fundamental differences between LP and CP activation events.
We demonstrate that targeting MASP-2 with a specific inhibitory antibody is very effective in limiting both the post-stroke neurological deficits as well as the associated ischemic lesion and in reducing the consequent activation of microglia towards the pro-inflammatory amoeboid phenotype.
This study complements our previous work in which we targeted the LP recognition subcomponents MBL-A and MBL-C by either MBL-specific mAbs or through the injection of an excess of a fluid-phase carbohydrate ligand orthologue [
13]. There are advantages to targeting the single, low-abundance LP enzyme that critically drives the pathophysiology of cerebral IRI over targeting one or more of the five different LP recognition components. Mice, for example, do not express an orthologue for the human recognition subcomponent ficolin-3 (alias H-ficolin) [
48]. Our own recent work has demonstrated that ficolin-3 drives LP activation in patients with subarachnoid hemorrhage [
17]. In stroke patients, ficolin-1 (alias M-ficolin) serum levels are dramatically decreased at time point 6 h after the onset of symptoms, suggesting massive ficolin-1 consumption following cerebral ischemia. Ficolin-2 (alias L-ficolin) and ficolin-3 levels also decrease during the acute phase, while serum MBL levels remain unaffected [
49]. These findings are in full agreement with a previously published clinical study reporting the consumption of ficolin-3 and ficolin-2 during the acute phase of stroke [
15]. These reports strongly suggest that in man, ficolins are critically involved in triggering LP activation during the reperfusion phase and that blocking MBL in stroke patients might be therapeutically less effective than what we and others observed when targeting MBL in mouse models of cerebral ischemia. In addition to its low abundance, MASP-2 is exclusively synthesized in the liver [
50], and the effectiveness of systemic MASP-2 inhibitory agents in target organs is not complicated by local biosynthesis.
The assessment of fB
−/− mice in our model of tMCAO suggests that the AP contributes to IRI following cerebral ischemia as indicated by the significant amelioration of general neurological deficits. An even stronger protection has previously been reported by Elvington et al. [
51] in which a reduction in both neurological deficits and in ischemic volume were observed in fB
−/− mice. In our hands, the lack of AP activation does not affect the lesion size as assessed by cresyl violet staining following correction for oedema. The factor B data suggest a role for the AP in cerebral IRI by amplifying complement activation initiated by the LP. The predominant role of the MASP-2-dependent LP over the AP in the mediation of cerebral IRI pathology is underlined by the lack of a protective IRI phenotype in the MASP-1/-3 double-deficient mouse line, which is associated with low to undetectable AP functional activity [
32].
The absence of a protective effect of C4 deficiency in cerebral IRI confirms the non-protective phenotype of C4 deficiency previously reported in models of myocardial and renal IRI and emphasizes the general importance of a MASP-2-dependent C4-bypass activation route [
20,
22,
26].
Acknowledgements
We would like to thank Edward Bampton for his supervision and scientific discussions on the 3VO stroke model and Nicholas Lynch and Youssif Mohammed Ali for the help in breeding the various mouse colonies and in establishing ELISAs. We thank Stefano Fumagalli for his directions in microglia/macrophage morphology characterization and Claire Gibson for her guidance in TTC staining and infarct measurements in the 3VO model. We thank Christi Wood and Larry Tjoelker for the preparation of the HG4 antibody and Robert B. Sim and Peter Lachmann for their comments reading the manuscript. The mouse lines deficient in fB or in C4 were established from breeding pairs provided by Marina Botto.
This work is dedicated to the memory of our co-worker and friend Dr. Minoru Takahashi who passed away on the 8th of May 2016.