An atypical role for the myeloid receptor Mincle in central nervous system injury

The C-type lectin Mincle is implicated in innate immune responses to sterile inflammation, but its contribution to associated pathologies is not well understood. Herein, we show that Mincle exacerbates neuronal loss following ischemic but not traumatic spinal cord injury. Loss of Mincle was beneficial in a model of transient middle cerebral artery occlusion but did not alter outcomes following heart or gut ischemia. High functional scores in Mincle KO animals using the focal cerebral ischemia model were accompanied by reduced lesion size, fewer infiltrating leukocytes and less neutrophil-derived cytokine production than isogenic controls. Bone marrow chimera experiments revealed that the presence of Mincle in the central nervous system, rather than recruited immune cells, was the critical regulator of a poor outcome following transient middle cerebral artery occlusion. There was no evidence for a direct role for Mincle in microglia or neural activation, but expression in a subset of macrophages resident in the perivascular niche provided new clues on Mincle's role in ischemic stroke.


INTRODUCTION
Background 3 a. Include sufficient scientific background (including relevant references to previous work) to understand the motivation and context for the study, and explain the experimental approach and rationale. b. Explain how and why the animal species and model being used can address the scientific objectives and, where appropriate, the study's relevance to human biology.

a)Paragraph 1-3 b)Paragraph 3-4
Objectives 4 Clearly describe the primary and any secondary objectives of the study, or specific hypotheses being tested.

Ethical statement 5
Indicate the nature of the ethical review permissions, relevant licences (e.g. Animal [Scientific Procedures] Act 1986), and national or institutional guidelines for the care and use of animals, that cover the research.

Paragraph 1
Study design 6 For each experiment, give brief details of the study design including: a. The number of experimental and control groups. b. Any steps taken to minimise the effects of subjective bias when allocating animals to treatment (e.g. randomisation procedure) and when assessing results (e.g. if done, describe who was blinded and when).
c. The experimental unit (e.g. a single animal, group or cage of animals). A time-line diagram or flow chart can be useful to illustrate how complex study designs were carried out.
a,b) Paragraph 1,3,6,7,9,10 Statistical 13 a. Provide details of the statistical methods used for each analysis. methods b. Specify the unit of analysis for each dataset (e.g. single animal, group of animals, single neuron). c. Describe any methods used to assess whether the data met the assumptions of the statistical approach.

Baseline data 14
For each experimental group, report relevant characteristics and health status of animals (e.g. weight, microbiological status, and drug or test naïve) prior to treatment or testing. (This information can often be tabulated).

Methods paragraph 1
Numbers 15 a. Report the number of animals in each group included in each analysis. analysed Report absolute numbers (e.g. 10/20, not 50% 2 ). b. If any animals or data were not included in the analysis, explain why.

Methods paragraph 2
Outcomes and 16 Report the results for each analysis carried out, with a measure of precision estimation (e.g. standard error or confidence interval).

Figures 1-5
Adverse events 17 a. Give details of all important adverse events in each experimental group. b. Describe any modifications to the experimental protocols made to reduce adverse events. n.a.

DISCUSSION
Interpretation/ 18 a. Interpret the results, taking into account the study objectives and scientific hypotheses, current theory and other relevant studies in the literature. implications b. Comment on the study limitations including any potential sources of bias, any limitations of the animal model, and the imprecision associated with the results 2 .
c. Describe any implications of your experimental methods or findings for the replacement, refinement or reduction (the 3Rs) of the use of animals in research.

Throughout discussion
Generalisability/ 19 Comment on whether, and how, the findings of this study are likely to translation translate to other species or systems, including any relevance to human biology.

Throughout discussion
Funding 20 List all funding sources (including grant number) and the role of the funder(s) in the study.

In vivo experiments
In vitro experiments bleeding or death within 24 h after tMCAO. Table 1 summarizes the planned and actual animal numbers used in this study. 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 1h, 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 MCAO territory.

Quantification of cerebral infarction and neurological deficit assessment. At 3 or 7 days
post-reperfusion, mice were euthanized, the brains removed into PBS (4ºC) for 15 min, and 2 mm coronal sections were obtained. These were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, T8877 Sigma-Aldrich) at 37ºC for 15 min. The stained sections were photographed, and the images digitized. The infarct area of each section was defined as the pale area surrounded by red undamaged tissue. Measurements were done using NIH image 6.1 software. To correct for brain swelling, the infarct area was determined by subtracting the area of undamaged tissue in the left (ipsilateral) hemisphere from that of the whole contralateral hemisphere. Infarct volume was calculated by integration of infarct areas for all slices of each brain, and then expressed as a % of the ipsilateral hemisphere. The functional consequences of tMCAO were evaluated using a 5-point neurological deficit score (0, no deficit; 1, failure to extend right paw; 2, circling to the right; 3, falling to the right; and 4, unable to walk spontaneously 29 ).
Global ischemia model and TUNEL assay. Adult male, age-matched C57BL6/J WT (n=4) and Clec4e  Data were acquired with a LSR II FACS system (BD Biosciences) and analyzed with FlowJo (TreeStar). Doublets were excluded with FSC-A and FSC-H linearity.
Spinal cord injury and assessment of locomotor recovery. Adult, age-and weight-matched female C57BL6/J WT (n=12) and Clec4e -/mice (n=14) were used for these experiments. Order of surgery was randomized based on predesigned lists, with the experimenter conducting the surgery remaining blinded to genotype throughout all aspects of surgery. In brief, mice were anesthetized via intraperitoneal injection with 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 . A force-controlled 70 kilodyne (kd) impact was applied at spinal level T11, using the Infinite Horizon impactor device (Precision Systems and Instrumentation).
Paravertebral muscles were sutured post-impact. SCI mice were then randomly re-assigned to Red (Thermo Fisher Scientific; #F34652). Hoechst 33342 nuclear dye was used for counterstaining. Images were captured on a single plane using a Zeiss Axio Imager and Zen Blue 2012 Software (Zeiss), and analysed with ImageJ software. Section areas were determined by outlining the section boundary on the GFAP + channel (excluding the leptomeninges).
Proportional area measurements were calculated by thresholding the FluoroMyelin Red stained area in ImageJ and dividing it by the total section area. Lesion volumes and/or length were calculated by multiplying fibronectin + areas by the section thickness and 1:5 series count.

Intestinal ischemia and reperfusion, histological analysis and myeloperoxidase (MPO)
quantification. Mice (WT, n = 11; Clec4e -/-, n = 10) were anesthetized with 2% isoflurane in Arumugam and Manzanero, et al  oxygen through a facemask, with spontaneous breathing and body temperature at 37°C. The surgeries were not randomized, but tissues were collected into coded tubes and the analysis performed by an operator blind to genotype. A midline incision was made through the skin and then along the linea alba separating the rectus abdominis muscle. The exposed intestines were After homogenisation and centrifugation, supernatants were assayed with 1.21 mg/ml odianisidine dihydrochloride (D3252, Sigma-Aldrich) and 2.17% hydrogen peroxide, and absorbance read at 460 nm.
Myocardial infarction and echocardiography analysis. All animals in the same cage (siblings) were experimented on in a blinded fashion (for both echocardiography and surgery).
Genotypes were checked after the experiment was finalized. To induce myocardial infarction, the left coronary artery (LDCA) of 10 week-old mice was ligated. For this procedure, animals were anaesthetized using 2% isoflurane and subjected to artificial ventilation through endotracheal cannulation. An incision was made through the muscle of the 4 th and 5 th intercostal space, and an 8-0 polyethylene suture passed under and tied around the LDCA 1 mm below the tip of the left auricle. Buprenorphine analgesic solution was administered subcutaneously (0.05 µg/g) twice a day for 3 days following surgery. For non-invasive Arumugam and Manzanero, et al  MgCl 2 , 3.6 NaHCO 3 , 5 HEPES, pH 7.2, supplemented with gentamycin 10 mg/L), placed in an incubator where the oxygen was displaced with nitrogen to a level of 0.2%, and incubated for 3 hours. Incubation with trehalose dimycolate (Sigma Aldrich) was conducted for 24 hours.