An atypical role for the myeloid receptor Mincle in CNS injury

Mincle is a C-type lectin known to play a role in innate immune responses to sterile inflammation, but its contribution to pathologies following an ischemic or traumatic injury is not well understood. In the current study we demonstrate a key role for Mincle in ischemic (i.e. transient middle cerebral artery occlusion) but not traumatic central nervous system injury; absence of Mincle also did not significantly alter the extent of tissue damage or functional outcome in peripheral models of ischemic tissue injury. In the stroke model mice lacking Mincle displayed significantly improved functional outcome from focal cerebral ischemia. The functional improvements in Mincle KO animals were accompanied by reduced infiltration of neutrophils and lower levels of proinflammatory cytokines in recruited peripheral blood cells. Bone marrow chimera experiments revealed that presence of Mincle in the central nervous system, but not peripheral immune cells, was the critical regulator of a poor outcome following transient focal cerebral ischemia, however we exclude a direct role for Mincle in microglia or neural activation. We demonstrate that Mincle lacks widespread expression in the brain, but is specifically associated with macrophages resident in the perivascular niche. These findings implicate Mincle in the initiation, extent and severity of local responses to ischemic injury in the brain, but not peripheral tissues. Mincle signalling therefore offers a novel therapeutic target in the quest to limit damage after stroke. Sources of support: Australian National Health & Medical Research Council [1057846, 1060538 and Fellowship to NAR], SpinalCure Australia (Career Development Fellowship to MJR), the Australian Research Council, the State Government of Victoria, the Australian Government and The University of Queensland.


Introduction
Ischemic stroke results in the damage and death of neurons in the perfusion territory of the affected blood vessel; the neurodegenerative mechanisms involve metabolic and oxidative stress, excitotoxicity and apoptosis, as well as neuroinflammation and the infiltration of activated leukocytes 1 . Chronic inflammatory conditions including arteriosclerosis, obesity and infection, also increase the risk of stroke and worsen outcome 2,3 . Sterile inflammation is therefore an important clinical target in tissue injury arising from cerebral ischemia. Molecular models of innate immune signalling suggest that excessive / deregulated inflammation is a confounding factor, in stroke as well as other forms of central nervous system (CNS) trauma. For example, mouse knock-out models focusing on innate immune receptors, such as the Toll-like receptor (TLR) family, suggest that blocking inflammation is beneficial 4,5 to limit tissue damage. In contrast, targeting the TLR adaptor protein, MyD88 provided no benefit for neurological outcomes and worsened neuronal cell death in animals subjected to global or focal ischemic injuries 6,7 . Likewise, in mouse models of spinal cord injury (SCI), blocking Tlr2 or Tlr4 reduces microglia and/or astrocyte activation 8 but worsens tissue damage and functional recovery 9 . The presence of TLRs in the central nervous system indicates pleiotropic, possibly neuroprotective roles in sterile injury, whereas increased expression of TLR2 and 4 in peripheral leukocytes is concordant with higher JCBFM 6 inflammatory markers in clinical stroke, and is predictive of poor outcome in some stroke patients 10 .
Clinical studies have shown that high circulating neutrophil numbers or high neutrophil to lymphocyte ratio are positive predictors of stroke severity 11 . Strategies that reduce neutrophil influx to the site of ischemic injury reduce inflammation, collateral blood vessel occlusion and the severity of injury [12][13][14] . In contrast, blocking the phagocytic clearance of dead cells by microglia will exacerbate injury 15 , and reparative roles of tissue macrophages are necessary for wound healing and functional recovery from sterile inflammation 16 . It is thus becoming increasingly apparent that functional differences in infiltrating myeloid cells (e.g. inflammatory neutrophils vs. monocytes) during the acute phase of sterile injury are important determinants of neuroprotection, blood brain barrier integrity, and recovery from stroke.
Innate immune cells, particularly macrophages and neutrophils, express a variety of receptors for endogenous ligands that are candidate regulators of inflammation during ischemic injury. The C-type lectin, Dectin-1, is a myeloid receptor that antagonises Tlr2 in SCI. Specifically, Dectin-1 knock-out mice were protected from axonal dieback after traumatic injury, and in the wild type (WT) animals, Tlr2 activation reduced the harmful effects of Dectin-1 on axonal damage. 17 Mincle (also designated as Clec4e) is a myeloid receptor closely related to Dectin-1 that has been reported to recognize necrotic cells via JCBFM 7 detection of the nuclear protein Sap130 18 . Mincle was originally identified as a lipopolysaccharide (LPS)-inducible protein in macrophages and has subsequently been shown to stimulate inflammatory responses to fungal and mycobacterial pathogens [19][20][21][22] .
Mincle associates with the Fc receptor common gamma chain (FcRγ) in immune cells, triggering intracellular signaling through the spleen tyrosine kinase (Syk) and the caspase recruitment domain protein Card9, which drives production of inflammatory cytokines such as TNF 23,24 ; inhibition of Syk limits thrombosis and vascular inflammation in a variety of animal models of sterile injury 25 . Therefore, the Mincle/Syk axis is likely to also contribute to the pathophysiology of inflammation in ischemic stroke.
Two previous studies have suggested a deleterious role for Mincle in rodent models of subarachnoid haemorrhage 26 and ischemic stroke 27 . Both used Syk inhibition, which is not selective for Mincle signalling, and as a result the role of Mincle in stroke outcomes remains poorly defined. In the current study, we used Mincle knockout mice (Clec4e -/-) to demonstrate that absence of Mincle, specifically in the central nervous system, significantly improves ischemic stroke outcomes. Mincle does not have the widespread brain expression previously described 26,27 , but instead appears restricted to perivascular macrophages and peripheral leukocytes. Absence of Mincle did not affect outcomes following traumatic spinal cord injury, or of ischemic injuries in other organs, such as the heart or the intestine. The combined data presented here suggest a key role for JCBFM 8 Mincle in ischemic CNS injuries where the integrity of the blood-brain/spinal cord barrier is not compromised by mechanical forces during the initiating event.

Materials and Methods
Animals and reagents. All experimental procedures followed the "Australian code of practice for the care and use of animals for scientific purposes", and were approved by and SBMS/311/12/SPINALCURE). The mouse colony was maintained in conventional or specific pathogen free conditions but, leading up to experiments, all animals spent at least one week in conventional housing conditions, with autoclaved sawdust or corn cob for bedding and a maximum of 5 mice per cage. Mice were kept in a conventional light cycle (12h light/12h dark), with controlled temperature (22-26 ºC) and humidity (40-60%), and had access to normal chow diet and water ad libitum. Autoclaved cardboard boxes were used as environmental enrichment, and animals were checked for health daily. Homozygous null C57Bl/6J Clec4e -/mice were used as previously described 22 , and they were compared to either WT C57BL/6J or cohoused Clec4e +/littermates, which have been shown to display the same immune phenotype as WT C57BL/6J mice 28 .
Four different laboratories conducted the surgeries described herein. In all cases, except where indicated, a randomized experimental design consisted of pre-assigned groups of mice where the surgeon or operator was blind to genotype. were blinded to genotype or treatment group and randomization was based on predesigned lists using colour coded cages and reagents. Exclusion criteria were excessive bleeding or death within 24 h after tMCAO. On these grounds, 1 out of 10 Syk inhibitor pre-treated, 2 out of 16 Clec4e -/and 7 out of 34 WT animals with tMCAO were excluded. Mice were anesthetized with 2% isoflurane in oxygen with spontaneous breathing and body temperature at 37°C. After a midline neck incision, the left external carotid and pterygopalatine arteries were isolated and ligated with 5-0 silk thread. The internal carotid artery was occluded with a clip at the peripheral site of the bifurcation to the pterygopalatine artery and the common carotid artery was then ligated with 5-0 silk thread. The external carotid artery was cut and a 6-0 nylon suture with a blunted tip (0.20 mm) was inserted. The clip at the internal carotid artery was then removed for advancement of the nylon suture into the middle cerebral artery to slightly more than 6 mm from the internal carotid-pterygopalatine artery bifurcation. After 1 h occlusion, the nylon suture and ligatures were removed to initiate reperfusion for 24 h up to 7 days. In the sham group, these arteries were visualized but not disturbed. Animals were subjected to cerebral blood flow (CBF) measurements using a laser Doppler perfusion monitor (Moor Lab) to confirm MCAO. The Doppler laser tip was placed perpendicular to the surface of the right parietal skull (1 mm posterior and 5 mm lateral to the bregma) to monitor blood flow in the middle cerebral artery territory.  Xylazine (10 mg/kg, Ilium) and Zolazepam (50 mg/kg, Virbac) and subjected to a severe contusive SCI. The ninth thoracic (T9) vertebra was identified as described previously 31 , followed by a dorsal laminectomy as described previously 32,33 . This

Mincle deficiency improves functional outcomes and reduces infarct size in mouse models of cerebral ischemia
To directly address a role for Mincle in stroke-induced inflammation and tissue damage,

Mincle expression in the brain is restricted to a specific cell type.
To better understand the unique role of Mincle in ischemic stroke, we performed a careful search for which cell(s) in the CNS express Mincle. Given that Mincle is a

30
In order to verify our observations that Mincle does not have widespread expression in the brain, we examined rat brain tissue after permanent MCAO. For this purpose, we carried out IHC on paraffin-embedded brain sections from rats that had undergone 24 h of permanent focal cerebral ischemia by distal occlusion of the middle cerebral artery 36 .
Throughout the brain, in both infarct and non-ischemic tissue, Mincle + cells were always associated with the vasculature. Monoclonal antibodies 4A9 and 16E3, generated in rat and mouse, respectively, provided the same result ( Figure 6). Mincle was not present in cells positive for the pericyte marker alpha-SMA (Figure 6a,b,d), with the astrocyte marker GFAP (Figure 6a,c) or with the microglial marker Iba1 ( Figure 6c). They always appeared sandwiched between pericytes and astrocytes ( Figure 6b), and showed to be positive for CD163 (Figure 6d), which is considered a marker of rat brain perivascular macrophages 36 . We noted Mincle was readily visible in healthy and spontaneously hypertensive stroke-prone rats. Together the mouse and rat studies confirm that Mincle is not directly regulating microglia or astrocyte responses to sterile inflammation, but is restricted to macrophages associated with the brain vasculature. This pattern of expression may indicate a role for Mincle in regulating cerebral vasculature in response to injury, and thus help explain the apparent differences in phenotype of the Mincle KO mouse responding to brain MCAO or spinal cord neurotrauma.

Discussion
The data presented here offer new insights into the initiation of sterile inflammation in the ischemic brain. Genetic deletion revealed a key role for Mincle in the severity of We also excluded a functional role for Mincle in microglia, which are radio-resistant and thus retained in the recipient animal in bone marrow chimeras 40 . When looking at TNF, a hallmark of the inflammatory phenotype, and in contrast with the dramatic differences between genotypes observed in tissue damage, we observed no differences in microglia-derived TNF production between WT and Clec4e -/animals after tMCAO.
Microglia isolated from sham-operated or tMCAO brains one day after the injury did not express the Clec4e mRNA, and the transcriptional profile of Mincle-deficient microglia did not deviate from that of isogenic controls, after sham surgery or ischemia.
The studies that reported widespread Mincle expression in multiple brain cells also The direct physical association between Mincle + perivascular macrophages and the adventitial plane of alpha-SMA + pericytes further supports a role for these cells in the regulation of the brain microvasculature and/or breakdown of the blood-brain barrier JCBFM 35 following an ischemic event. The dramatic injury reduction observed in Clec4e -/mice may therefore be explained by a key role for Mincle + macrophages at this perivascular location with regards to injury propagation. An alternate and not mutually exclusive explanation would be that Mincle has a role in directing inflammatory cell recruitment across the blood-brain barrier, which may have contributed to the observed reduction in inflammatory infiltrate in the brains of Clec4e -/mice after stroke ( Figure 2).
Although our results leave many unanswered questions about the role of Mincle in stroke, its dramatic influence over the outcome from brain ischemia and reperfusion injury, which mainly emerges from Mincle's presence in the brain itself, warrants further investigation to resolve this aspect of the Clec4e -/phenotype for future therapeutic targeting.        The ARRIVE Guidelines Checklist

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