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. 2015 Sep 17:12:173.
doi: 10.1186/s12974-015-0392-7.

Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

Lusine Bozoyan  1 , Aline Dumas  2 , Alexandre Patenaude  3 , Luc Vallières  4   5
Affiliations

Interleukin-36γ is expressed by neutrophils and can activate microglia, but has no role in experimental autoimmune encephalomyelitis

Lusine Bozoyan et al. J Neuroinflammation. .

Abstract

Background: Experimental autoimmune encephalomyelitis (EAE) is a model of inflammatory demyelinating diseases mediated by different types of leukocytes. How these cells communicate with each other to orchestrate autoimmune attacks is not fully understood, especially in the case of neutrophils, whose importance in EAE is newly established. The present study aimed to determine the expression pattern and role of different components of the IL-36 signaling pathway (IL-36α, IL-36β, IL-36γ, IL-36R) in EAE.

Methods: EAE was induced by either active immunization with myelin peptide, passive transfer of myelin-reactive T cells or injection of pertussis toxin to transgenic 2D2 mice. The molecules of interest were analyzed using a combination of techniques, including quantitative real-time PCR (qRT-PCR), flow cytometry, Western blotting, in situ hybridization, and immunohistochemistry. Microglial cultures were treated with recombinant IL-36γ and analyzed using DNA microarrays. Different mouse strains were subjected to clinical evaluation and flow cytometric analysis in order to compare their susceptibility to EAE.

Results: Our observations indicate that both IL-36γ and IL-36R are strongly upregulated in nervous and hematopoietic tissues in different forms of EAE. IL-36γ is specifically expressed by neutrophils, while IL-36R is expressed by different immune cells, including microglia and other myeloid cells. In culture, microglia respond to recombinant IL-36γ by expressing molecules involved in neutrophil recruitment, such as Csf3, IL-1β, and Cxcl2. However, mice deficient in either IL-36γ or IL-36R develop similar clinical and histopathological signs of EAE compared to wild-type controls.

Conclusion: This study identifies IL-36γ as a neutrophil-related cytokine that can potentially activate microglia, but that is only correlative and not contributory in EAE.

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Figures

Fig. 1
Fig. 1
IL-36γ and IL-36R are transcriptionally upregulated in 3 EAE models. Quantification of the mRNAs encoding IL-36γ and IL-36R by qRT-PCR in different tissues from mice killed at the indicated time points after induction of EAE by either active immunization with MOG peptide, passive transfer of encephalitogenic T cells, or injection of PTX to 2D2 mice. Control non-transgenic mice were injected with PBS, PTX, or CFA only. Stars indicate significant differences from the PBS group only (white star) or all the corresponding control groups (black star), as determined by Wilcoxon tests (P ≤ 0.038). Sample size: 11–13 (PBS), 5–13 (PTX), 4–8 (CFA), 7–8 (active EAE), 5–8 (passive EAE), 6 (2D2 with EAE), or 5 (2D2 without [w/o] EAE)
Fig. 2
Fig. 2
IL-36γ is selectively expressed by neutrophils, while IL-36R is expressed by different leukocytes such as monocytic cells. a Quantification of IL-36γ or IL36R mRNA by qRT-PCR in different cells purified from the spinal cord or spleen of EAE mice by FACS using the following gating strategies: neutrophils, Ly6G+CD11b+CD45+CD3−CD19−; microglia, CD11b+CD45lowLy6G−CD3−CD19−; monocyte-derived cells (MDC; comprising macrophages and CD11c+ dendritic cells), CD11b+CD45+CD11c+/−Ly6G−CD3−CD19−; other intraspinal leukocytes, CD45+CD3+/−CD19+/−CD11b−Ly6G−CD11c−; B cells, CD19+CD45+CD11b−Ly6G−CD3−; splenic macrophages, CD11b+CD45+CD11c−Ly6G−CD3−CD19−; other splenic leukocytes, CD45+CD3+/−CD11b+/−CD11c+/−Ly6G−CD19−. Sample size: spinal cord, one pooled sample of sorted cells from four mice; spleen, five non-pooled samples from individual mice. b Western blotting showing the full-length form of IL-36γ (~22 kDa) in splenic neutrophils from a wild-type EAE mouse (IL-36γ+/+), but not from an IL-36γ-deficient EAE mouse (IL-36γ−/−). Data are representative of at least four mice per group. The recombinant (truncated) form of IL-36γ was used as a control (right lane). β-actin (lower panels) was used to control for protein loading. Asterisks indicate non-specific bands. c Autoradiograms showing in situ hybridization signals (arrows) for IL-36γ or IL-36R mRNA in the spinal cord of mice killed on day 12 after active EAE induction, but not in naïve mice. Note the submeningeal distribution of the signals (representative of at least five mice). Scale bar = 500 μm. d Double labeling for IL-36γ or IL-36R mRNA (black grains, in situ hybridization) and cell type-specific markers (red brown, immunohistochemistry) in CNS sections from EAE mice. Arrows show examples of double-labeled cells. Scale bar = 20 μm
Fig. 3
Fig. 3
IL-36γ stimulates the expression of inflammatory genes in microglial cultures. a Genes that were upregulated ≥2.5 times in BV2 microglia treated for 6 h with recombinant IL-36γ (100 ng/ml) compared to PBS, as determined using Affymetrix DNA microarrays (Student’s t test, P < 0.04). b, c Quantitative PCR analysis confirming that Csf3, IL-1β, and Cxcl2 mRNAs were upregulated by IL-36γ in BV2 cells (b) and primary microglia from IL-36R+/+ mice (c) (Wilcoxon test, P < 0.005). Note in c the absence of upregulation in primary microglia from IL-36R−/− mice, confirming the specificity of the results
Fig. 4
Fig. 4
Neither IL-36γ nor IL-36R is required for EAE development. a Kaplan-Meier curves showing EAE incidence in mice expressing (black squares) or lacking (white squares) IL-36γ (upper graph) or IL-36R (bottom graph) after immunization with MOG. The graphs include all the mice tested. No significant intergenotype difference was detected (Wilcoxon tests, P ≥ 0.41). Sample size: 20 (IL-36γ+/+), 20 (IL-36γ−/−), 6 (IL-36R+/+), or 6 (IL-36R−/−). b EAE severity in mice expressing (black squares) or lacking (white squares) IL-36γ (upper graph) or IL-36R (bottom graph). The graphs include only mice that had developed clinical signs of EAE at the end of the study (i.e., 21 days). No significant intergenotype difference was detected (Wilcoxon tests, P ≥ 0.12). Sample size: 17 (IL-36γ+/+), 17 (IL-36γ−/−), 5 (IL-36R+/+), or 5 (IL-36R−/−). c Additional statistics for EAE in mice expressing (+/+) or not (−/−)IL-36γ or IL-36R. No significant intergenotype difference was detected in any of these parameters (Wilcoxon tests, P ≥ 0.34). Sample size: 20 (IL-36γ+/+), 20 (IL-36γ−/−), 6 (IL-36R+/+), or 6 (IL-36R−/−)
Fig. 5
Fig. 5
No difference in leukocyte recruitment in the spinal cord of EAE mice expressing or not IL-36γ or IL-36R. a Gating strategy used for flow cytometric analysis. Dead cells and doublets were excluded. The data shown are from a representative wild-type EAE mouse. b, c Counts of immune cells in the spinal cord of IL-36γ−/−, IL-36R−/−, or wild-type mice killed 21 days after immunization with MOG peptide. The counts were normalized to CD45− cells used as an internal control. When the cells were collected, the animals had clinical scores ranging from 0.5 to 3. No significant difference was found between the genotypes (Wilcoxon tests, P ≥ 0.07). Sample size: 6 (b) or 9 (c) mice per group
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References

    1. Goverman J. Autoimmune T cell responses in the central nervous system. Nat Rev Immunol. 2009;9:393–407. doi: 10.1038/nri2550. - DOI - PMC - PubMed
    1. King IL, Dickendesher TL, Segal BM. Circulating Ly-6C+ myeloid precursors migrate to the CNS and play a pathogenic role during autoimmune demyelinating disease. Blood. 2009;113:3190–7. doi: 10.1182/blood-2008-07-168575. - DOI - PMC - PubMed
    1. Serafini B, Rosicarelli B, Magliozzi R, Stigliano E, Capello E, Mancardi GL, et al. Dendritic cells in multiple sclerosis lesions: maturation stage, myelin uptake, and interaction with proliferating T cells. J Neuropathol Exp Neurol. 2006;65:124–41. - PubMed
    1. Fischer HG, Reichmann G. Brain dendritic cells and macrophages/microglia in central nervous system inflammation. J Immunol. 2001;166:2717–26. doi: 10.4049/jimmunol.166.4.2717. - DOI - PubMed
    1. Serafini B, Columba-Cabezas S, Di Rosa F, Aloisi F. Intracerebral recruitment and maturation of dendritic cells in the onset and progression of experimental autoimmune encephalomyelitis. Am J Pathol. 2000;157:1991–2002. doi: 10.1016/S0002-9440(10)64838-9. - DOI - PMC - PubMed

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