1. Immunology and Inflammation
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miRNA profile is altered in a modified EAE mouse model of multiple sclerosis featuring cortical lesions

  1. Nicola S Orefice  Is a corresponding author
  2. Owein Guillemot-Legris
  3. Rosanna Capasso
  4. Pauline Bottemanne
  5. Philippe Hantraye
  6. Michele Caraglia
  7. Giuseppe Orefice
  8. Mireille Alhouayek
  9. Giulio G Muccioli
  1. CEA, Fundamental Research Division (DRF), Institute of Biology Francois Jacob, Molecular Imaging Research Center (MIRCen), France
  2. Bioanalysis and Pharmacology of Bioactive Lipids Research Group, Louvain Drug Research Institute, UCLouvain, Université catholique de Louvain, Belgium
  3. Department of Precision Medicine, School of Medicine & Surgery - University of Campania "Luigi Vanvitelli", Italy
  4. Department of Neurosciences, Reproductive and Odontostomatological Sciences, "Federico II" University of Naples, Italy
Research Article
Cite this article as: eLife 2020;9:e56916 doi: 10.7554/eLife.56916
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7 figures, 2 tables and 5 additional files

Figures

Figure 1 with 1 supplement
Clinical and immunohistological characterization of the EAE model with high and low clinical scores.

To induce EAE, mice received MOG35-55 and complete Freund’s adjuvant (CFA) with or without pertussis toxin (PTX). (A) Clinical score of EAE mice receiving PTX (PTX group) or no PTX and with a high clinical score (HIS) or low clinical score (LIS). (B) Quantification of fluoromyelin signal in the spinal cord ventral white matter. (C) Quantification of the CD3 positive area in the ventral white matter of the spinal cord. (D) Representative photomicrographs of CD3 positive cells (lymphocytes) infiltrating the spinal cord ventral white matter. The scale bar represents 50 µm. The quantification of the entire cohort is shown in (C). (E) Representative photomicrographs of GFAP positive cells (astrocytes) and Iba-1 positive cells (microglia, monocytes, macrophages) in the spinal cord grey and white matters of mice. The scale bar represents 50 µm. (F-G) Quantification of GFAP positive area and Iba-1 positive area in (F) the grey matter and in (G) the white matter of the spinal cord. The scale bar represents the indicated length. Data are mean ± sem. N = 7–12/group. For A, two-way ANOVA with Sidak’s post-hoc test. For B to F, one-way ANOVA with Sidak’s post-hoc test. §p≤0.05 §§p≤0.01 §§§p≤0.001 vs CTL, #p≤0.05 ### p≤0.001 vs PTX, *p≤0.05 **p≤0.01 ***p≤0.001 vs LIS.

Figure 1—figure supplement 1
Spinal cord lesions are more pronounced in mice with high clinical score (HIS) compared to mice with low clinical score (LIS).

(A) Clinical score of a mouse cohort showing EAE mice with high clinical score (HIS) or low clinical score (LIS) receiving an injection of cytokine (-C) at day 21 post-immunization. (B) Representative photomicrographs of LFB staining (with cresyl violet counterstaining) performed on the spinal cord of HIS-C and LIS-C mice. (C) Representative photomicrographs of MBP immunofluorescence in the spinal cord of HIS-C and LIS-C mice of the same cohort. N = 9/group. Data are mean ± sem. For A, two-way ANOVA with Sidak’s post-hoc test, vs LIS ***p≤0.001. For B, two-tailed t test, ***p≤0.001.

Figure 2 with 1 supplement
Characterization of inflammatory marker and growth factor expression in the EAE model with high and low clinical scores.

To induce EAE, mice received MOG35-55 and complete Freund’s adjuvant (CFA) with pertussis toxin (PTX) or without PTX (HIS and LIS groups). (A) mRNA expression of Il1b, Ccl3, Tnf, Il17a, Cx3cr1, Foxp3, and Rorc was assessed by RT-qPCR in the spinal cord. (B–C) mRNA expression of Il1b, Tnf, Cxcl10, Cd3g, Atf3, and (C) the neurotrophins Bdnf, Gdnf, Ngf, and Ntf3 was assessed by RT-qPCR in the cortex. Data are mean ± sem. The expression of the CTL group was set at 1, except for Il17a that was not detected in the control group (thus, the PTX group was set at 1). N = 7–12/group. One-way ANOVA with Sidak’s post-hoc test. §p≤0.05 §§p≤0.01 §§§p≤0.001 vs CTL, # p≤0.05 vs PTX, *p≤0.05, **p≤0.01 vs LIS.

Figure 2—figure supplement 1
Time-course evaluation of inflammatory plasma cytokines and chemokines.

Blood of mice treated with complete Freund’s adjuvant (CTL) in addition to MOG35-55 with PTX (PTX group) or without PTX (HIS and LIS groups) was harvested 7, 14, and 21 days post MOG35-55/CFA injection. Plasma cytokines and chemokines IL-1β, IL-6, IL-12(p40), IL-17, TNFα, IFNγ, MIP-1α, RANTES, KC and G-CSF were quantified using a Bio-Plex Multiplex kit. Data are mean ± sem. Two-way ANOVA with Dunnett’s post-hoc test. Statistical significance, if present, is shown in the table. *p≤0.05, ** *p≤0.01, *** *p≤0.001.

Cortical lesion extent depends on high and low clinical scores.

(A) Representative photomicrographs of cortices stained with luxol fast blue and counter-stained with cresyl violet (LFB-CV) and showing the presence of CLs. (B) Quantification of LFB-CV unstained area in mice cortices with a high clinical score (HIS) or low clinical score (LIS) receiving either an injection of the cytokine (-C) or of PBS. (C) Representative photomicrographs depicting close-ups of the ipsilateral cortex of HIS and LIS mice displayed in panel A. (D) Representative photomicrographs and (E) quantification of fluoromyelin green intensity in the ipsilateral cortex of HIS-C and LIS-C mice. (D) The scale bar represents 100 µm. (F) Quantification of MBP intensity in the ipsilateral cortex of HIS-C and LIS-C mice. The dotted line represents the intensity of (E) fluoromyelin or (F) MBP immunostaining measured in CTL mice and set at 100%. (G) Representative photomicrographs and quantification of GFAP immunofluorescence in the ipsilateral cortex of HIS and LIS mice. The scale bar represents 20 µm. HIS-C: high clinical score mice injected with the cytokine mixture; HIS-PBS: high clinical score mice injected with PBS; LIS-C: low clinical score mice injected with the cytokine mixture; LIS-PBS: low clinical score mice injected with PBS. Data are mean ± sem. For B, one-way ANOVA with Dunnett’s post-hoc test. For E and G, one-way ANOVA with Sidak’s post-hoc test, **p≤0.01, ***p≤0.001 vs LIS-C and ## p≤0.01, ### p≤0.001 vs HIS-C. For F, two-tailed t-test **p≤0.01.

Figure 4 with 2 supplements
Cortical lesion distribution and extent differ between mice with a high clinical score (HIS-C) and with a low clinical score (LIS-C) after cytokine injection.

Representative photomicrographs and close-ups of the ipsilateral cortex of (A) LIS-C and (B) HIS-C mice immunostained with Iba-1, MBP, and NeuN. The scale bar represents 100 µm. (C) Quantification of the probability of CL presence in the motor cortex (MO), the somatosensory cortex (SS), and the visual cortex (VIS) in the ipsilateral cortex of LIS-C and HIS-C mice (0:never present, 1:always present). Quantification of (D) intracortical, (E) subpial, and (F) total CL size in the ipsilateral cortex of LIS-C and HIS-C mice. (G) Quantification of Iba-1 occupied area in the cortex of HIS-C and LIS-C mice. (H) Quantification of NeuN negative area in the ipsilateral cortex of HIS-C and LIS-C mice. (I) Clinical score for another cohort of EAE mice with HIS and LIS groups that received cytokine injection either at day 21 post-immunization (HIS-C and LIS-C) or at day 35 post-immunization (HIS-C-35). All mice were euthanized three days after cytokine injection. (J) Quantification of LFB unstained area in cortices for the mice in panel I. (K) mRNA expression of Tnf, Il1b, Cd3g, Atf3, Ifng, Ifnb1, Bdnf, Gdnf, Ntf3 and Ngf was measured by RT-qPCR in mice with low clinical score and injected with cytokines on day 21 (LIS-C), mice with high clinical score and injected with cytokines on day 21 (HIS-C), and in mice with high clinical score and injected with cytokines on day 35 (HIS-C-35). The expression of the LIS-C group was set at 1. Data are mean ± sem. N = 8–10/group. For C two-way ANOVA with Sidak’s post-hoc test, between cortical regions for LIS-C mice *p≤0.05, **p≤0.01; and for HIS-C mice #p≤0.05, ##p≤0.01. For D-H, two-tailed t-test *p≤0.05, **p≤0.01. For me, two-way ANOVA with Sidak’s post-hoc test, ***p≤0.001. For J-K, one-way ANOVA with Sidak’s post-hoc test *p≤0.05, ***p≤0.001 vs LIS-C.

Figure 4—figure supplement 1
Lymphocyte infiltration and microglia activation are similar in mice with high clinical score (HIS-C) and with low clinical score (LIS-C) after cytokine injection.

Representative photomicrographs and quantification of CD3 positive area in the cortex of HIS-C (mice with high clinical score receiving a cytokine injection) and LIS-C (mice with low clinical score receiving a cytokine injection). The scale bar represents the indicated length. N = 8–10/group. Data are mean ± sem.

Figure 4—figure supplement 2
Neuronal loss evidenced by toluidine blue is similar in mice with high clinical score (HIS-C) and with low clinical score (LIS-C) after cytokine injection.

Representative photomicrograph, close-ups and quantification of the area with decreased neuron density assessed with toluidine blue in the ipsilateral cortex of HIS-C and LIS-C mice. The red arrows point to a neuron nucleus. (The scale bar represents the indicated length. N = 8–10/group. Data are mean ± sem.

Alterations in mRNA expression in the ipsilateral cortex before and after cytokine injection.

mRNA expression of Il1b, Tnf, Cxcl10, Atf3, Cd3g, Bdnf, Gdnf, Ngf and Ntf3 measured by RT-qPCR in the ipsilateral cortex of mice with a low clinical score before (LIS) and after (LIS-C) cytokine injection and in mice with a high clinical score before (HIS) and after (HIS-C) cytokine injection. Data are plotted as ΔCt between the gene considered and the reference gene used (Rpl19) (left vertical axis) and as the fold-increase of the expression between LIS group (set at 1) and the three remaining groups (right vertical axis). The smaller the ΔCt between the gene considered and Rpl19, the more the gene is expressed, and conversely, the bigger the ΔCt the less the gene is expressed. Data are box (median) and whiskers (min to max). One-way ANOVA, *p≤0.05, **p≤0.01, ***p≤0.001 vs LIS, #p≤0.05, ###p≤0.001 vs HIS.

miRNA profiling and corresponding prediction of signaling pathway alterations in LIS-C and HIS-C mice.

miRNA profile was assessed using TaqMan Microfluidic Array Cards Type A in control mice and HIS or LIS mice receiving a stereotactic injection of cytokines. (A) Variations of miRNAs at the injection site of EAE mice with high clinical score receiving cytokine injection (HIS-C-ipsi) compared to the injection site of EAE mice with low clinical score receiving cytokine injection (LIS-C-ipsi). Two-fold change for upregulation (plotted on the left y-axis) and downregulation (plotted on the right y-axis) are indicated by the dotted lines. Complete miRNA variations between the HIS-C and LIS-C groups are found in Supplementary file 3. (B) miRNAs expressed in HIS-C-ipsi but not in LIS-C-ipsi and inversely. (C) Predicted functional analysis of the top enriched canonical pathways associated with the target genes related to the miRNAs dysregulated in HIS-C-ipsi vs LIS-C-ipsi and found either in HIS-C-ipsi or LIS-C-ipsi. Indicated pathways were found by performing the Kyoto Encyclopedia of Genes and Genomes analysis by miRNet, mirPath, or Cytoscape String app databases. The pathways retrieved by specific databases are indicated by the colored boxes to the left of the graph (unchecked boxes represent pathways not found in the labeled database). The results are expressed as mean ± sem of -log10 adjusted p-value between the three databases; the dotted line designates the threshold of 1.3 (representing p-value at 0.05). (D) Schematic network representation of interactions between the 13 miRNAs that were mostly found in the pathways represented in C (only the pathways found in all three databases) and controlling the most genes and a panel of the targeted genes found in at least two databases.

Alterations in miRNA levels are associated with variations of their regulator and targets.

(A) Mir155-5p, Mir223-3p, Mir410-3p, Mirlet7c-5p, Mir148a-3p and Mir448 expression was measured by RT-qPCR in the injection site and the corresponding contralateral structure of mice receiving cytokine injection with a high clinical score (HIS-C) and low clinical score (LIS-C). U6 was used as a reference. LIS-C ipsilateral (upper panels) or LIS-C contralateral (lower panels) levels were set at 1. N = 4–5/group. Data are presented as mean ± sem. *p≤0.05. (B) Representative confocal photomicrographs and quantification of FOXO3 (green) in HIS-C and LIS-C groups for both ipsilateral and contralateral sides of the cortex detected by immunofluorescence N = 2/group. (C) mRNA expression of Cxcl10, Foxp3, and Rorc was assessed by RT-qPCR in the ipsilateral cortex of HIS-C and LIS-C groups. (D) Representative confocal photomicrographs and quantification of C1q (red) in HIS-C and LIS-C groups for both ipsilateral and contralateral sides of the cortex detected by immunofluorescence N = 2/group. N = 9/group. Data are mean ± sem. The expression of the LIS-C group was set at 1. Two-tailed t-test, *p≤0.05.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (female mice)Mus musculus, female,
C57BL/6JRj
Janvier LabsSC-C57J-F
Antibodyanti-mouse MBP (Chicken polyclonal)AbcamAb123499(1:1000)
Antibodyanti-mouse FOXO3 (rabbit polyclonal)AbcamAb177487(1:300)
Antibodyanti-mouse C1q
(Mouse monoclonal)
AbcamAb71940(1:100)
Antibodyanti-GFAP (Rabbit polyclonal)DakoZ0334(1:1000)
Antibodyanti-mouse Iba-1
(rabbit polyclonal)
Wako019–19741(1:500)
Antibodyanti-mouse CD3 (rabbit polyclonal)DakoA0452(1:300)
Antibodyanti-NeuN
(mouse monoclonal)
MilliporeMAB377(1:500)
Sequence-based reagentATF3-FThis paperPCR primersCGCCATCCAGAATAAACACC
Sequence-based reagentATF3-RThis paperPCR primersCCTTCAGCTCAGCATTCACA
Sequence-based reagentBDNF-FThis paperPCR primersGGTCACAGCGGCAGATAAA
Sequence-based reagentBDNF-RThis paperPCR primersTGGGATTACACTTGGTCTCGT
Sequence-based reagentCD3-FThis paperPCR primersCCAGTCAAGAGCTTCAGACAA
Sequence-based reagentCD3-RThis paperPCR primersGAGTCCTGCTGAGTTCACTTC
Sequence-based reagentCX3CR1-FThis paperPCR primersAGTTCCCTTCCCATCTGCTC
Sequence-based reagentCX3CR1-RThis paperPCR primersCACAATGTCGCCCAAATAAC
Sequence-based reagentCXCL10-FThis paperPCR primersAGCCAAAAAAGGTCTAAAAGGG
Sequence-based reagentCXCL10-RThis paperPCR primersCTAGCCATCCACTGGGTAAAG
Sequence-based reagentDCX-FThis paperPCR primersGTCACCTGTCTCCATGATTTC
Sequence-based reagentDCX-RThis paperPCR primersGACTCTGCATTCATTCTCATCC
Sequence-based reagentGDNF-FThis paperPCR primersGTGACTCCAATATGCCTGAAGA
Sequence-based reagentGDNF-RThis paperPCR primersGCCGCTTGTTTATCTGGTGA
Sequence-based reagentIFNβ-FThis paperPCR primersGTGGGAGATGTCCTCAACTG
Sequence-based reagentIFNβ-RThis paperPCR primersAGGCGTAGCTGTTGTACTTC
Sequence-based reagentIFNγ-FThis paperPCR primersGTTTGAGGTCAACAACCCACAG
Sequence-based reagentIFNγ-RThis paperPCR primersGCTTCCTGAGGCTGGATTC
Sequence-based reagentIL-1β-FThis paperPCR primersTCGCTCAGGGTCACAAGAAA
Sequence-based reagentIL-1β-RThis paperPCR primersCATCAGAGGCAAGGAGGAAAAC
Sequence-based reagentIL-17-FThis paperPCR primersGACTACCTCAACCGTTCCAC
Sequence-based reagentIL-17-RThis paperPCR primersCACTGAGCTTCCCAGATCAC
Sequence-based reagentFoxP3-FThis paperPCR primersGTTCCTTCCCAGAGTTCTTCC
Sequence-based reagentFoxP3-RThis paperPCR primersCATCGGATAAGGGTGGCATAG
Sequence-based reagentMIP-1α -FThis paperPCR primersAGATTCCACGCCAATTCATC
Sequence-based reagentMIP-1α -RThis paperPCR primersCTCAAGCCCCTGCTCTACAC
Sequence-based reagentNGF-FThis paperPCR primersATGCTGGACCCAAGCTCAC
Sequence-based reagentNGF-RThis paperPCR primersCTGCCTGTACGCCGATCAAA
Sequence-based reagentNT3-FThis paperPCR primersTCACCACGGAGGAAACGCTA
Sequence-based reagentNT3-RThis paperPCR primersGTCACCCACAGGCTCTCACT
Sequence-based reagentRORγ -FThis paperPCR primersGGATGAGATTGCCCTCTACAC
Sequence-based reagentRORγ -RThis paperPCR primersCAGATGTTCCACTCTCCTCTTC
Sequence-based reagentRPL19-FThis paperPCR primersGAAGGTCAAAGGGAATGTGTTCA
Sequence-based reagentRPL19-RThis paperPCR primersCCTTGTCTGCCTTCAGCTTGT
Sequence-based reagentTNF-α-FThis paperPCR primersCTACTGAACTTCGGGGTGATC
Sequence-based reagentTNF-α-RThis paperPCR primersTGAGTGTGAGGGTCTGGGC
Sequence-based reagentTRAF3-FThis paperPCR primersCAAAGACAAGGTGTTTAAGGATAA
Sequence-based reagentTRAF3-RThis paperPCR primersGCCTTCATTCCGACAGTAG
Sequence-based reagentTrail -FThis paperPCR primersTTTAATTCCAATCTCCAAGGATG
Sequence-based reagentTrail -RThis paperPCR primersGATGACCAGCTCTCCATTC
Peptide, recombinant proteinMOG35-55Hooke laboratoriesEK-2110The peptide used is prepared as emulsion in CFA and provided as reference EK-2110 by Hooke laboratories.
Peptide, recombinant proteinTNFαPeproTech315-01A250 ng/2 µL
Peptide, recombinant proteinIFNγPeproTech315–05100U/2 µL
Commercial assay or kitGoScript Reverse Transcription kitPromegaA2791
Commercial assay or kitGoTaq qPCR Master MixPromegaA6002
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine IL-1betaBiorad171G5002M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine IL-6Biorad171G5007M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine IL-12p40Biorad171G5010M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine IL-17ABiorad171G5013M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine G-CSFBiorad171G5015M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine IFNγBiorad171G5017M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine KCBiorad71G5018M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine MIP-1alphaBiorad171G5020M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine RANTESBiorad171G5022M
Commercial assay or kitBio-Plex Pro(tm) Mouse Cytokine TNF-alphaBiorad171G5023M
OtherMOG in CFA emulsionHooke laboratoriesEK-2110The MOG35-55 peptide is prepared as an emulsion in CFA and provided as reference EK-2110 by Hooke laboratories.
Other‘Control’ emulsionHooke laboratoriesCK-2110This is the control emulsion without immunizing peptide
OtherFluoromyelin GreenInvitrogenF34651(1:300)
OtherLuxol Fast BlueSigmaS3382
Table 1
Primer sequences.
GeneProductForward primer (5’ to 3’)Reverse primer (5’ to 3’)
Atf3ATF3CGCCATCCAGAATAAACACCCCTTCAGCTCAGCATTCACA
BdnfBDNFGGTCACAGCGGCAGATAAATGGGATTACACTTGGTCTCGT
Cd3gCD3CCAGTCAAGAGCTTCAGACAAGAGTCCTGCTGAGTTCACTTC
Cx3cr1CX3CR1AGTTCCCTTCCCATCTGCTCCACAATGTCGCCCAAATAAC
Cxcl10CXCL10AGCCAAAAAAGGTCTAAAAGGGCTAGCCATCCACTGGGTAAAG
DcxDCXGTCACCTGTCTCCATGATTTCGACTCTGCATTCATTCTCATCC
GdnfGDNFGTGACTCCAATATGCCTGAAGAGCCGCTTGTTTATCTGGTGA
Ifnb1IFNβGTGGGAGATGTCCTCAACTGAGGCGTAGCTGTTGTACTTC
IfngIFNγGTTTGAGGTCAACAACCCACAGGCTTCCTGAGGCTGGATTC
Il1bIL-1βTCGCTCAGGGTCACAAGAAACATCAGAGGCAAGGAGGAAAAC
Il17aIL-17GACTACCTCAACCGTTCCACCACTGAGCTTCCCAGATCAC
Foxp3FoxP3GTTCCTTCCCAGAGTTCTTCCCATCGGATAAGGGTGGCATAG
Ccl3MIP-1αAGATTCCACGCCAATTCATCCTCAAGCCCCTGCTCTACAC
NgfNGFATGCTGGACCCAAGCTCACCTGCCTGTACGCCGATCAAA
Ntf3NT3TCACCACGGAGGAAACGCTAGTCACCCACAGGCTCTCACT
RorcRORγGGATGAGATTGCCCTCTACACCAGATGTTCCACTCTCCTCTTC
Rpl19RPL19GAAGGTCAAAGGGAATGTGTTCACCTTGTCTGCCTTCAGCTTGT
TnfTNF-αCTACTGAACTTCGGGGTGATCTGAGTGTGAGGGTCTGGGC
Traf3TRAF3CAAAGACAAGGTGTTTAAGGATAAGCCTTCATTCCGACAGTAG
Tnfsf10TrailTTTAATTCCAATCTCCAAGGATGGATGACCAGCTCTCCATTC

Additional files

Source data 1

miRNA expression (raw data) used for Supplementary files 1, 2, 3.

https://cdn.elifesciences.org/articles/56916/elife-56916-data1-v2.xlsx
Supplementary file 1

Supplementary Table S1 : List of the miRNA expressed in the different conditions studied.

https://cdn.elifesciences.org/articles/56916/elife-56916-supp1-v2.xlsx
Supplementary file 2

Supplementary Table S2: Changes in miRNA expression relative to the control.

https://cdn.elifesciences.org/articles/56916/elife-56916-supp2-v2.xlsx
Supplementary file 3

Supplementary Table S3: miRNA expression in the HIS group relative to the LIS group.

https://cdn.elifesciences.org/articles/56916/elife-56916-supp3-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/56916/elife-56916-transrepform-v2.docx

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