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Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis - PubMed

Temporal overexpression of IL-22 and Reg3γ differentially impacts the severity of experimental autoimmune encephalomyelitis

Ahmet Eken et al. Immunology. 2021 Sep.

Abstract

IL-22 is an alpha-helical cytokine which belongs to the IL-10 family of cytokines. IL-22 is produced by RORγt+ innate and adaptive lymphocytes, including ILC3, γδ T, iNKT, Th17 and Th22 cells and some granulocytes. IL-22 receptor is expressed primarily by non-haematopoietic cells. IL-22 is critical for barrier immunity at the mucosal surfaces in the steady state and during infection. Although IL-22 knockout mice were previously shown to develop experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), how temporal IL-22 manipulation in adult mice would affect EAE course has not been studied previously. In this study, we overexpressed IL-22 via hydrodynamic gene delivery or blocked it via neutralizing antibodies in C57BL/6 mice to explore the therapeutic impact of IL-22 modulation on the EAE course. IL-22 overexpression significantly decreased EAE scores and demyelination, and reduced infiltration of IFN-γ+IL-17A+Th17 cells into the central nervous system (CNS). The neutralization of IL-22 did not alter the EAE pathology significantly. We show that IL-22-mediated protection is independent of Reg3γ, an epithelial cell-derived antimicrobial peptide induced by IL-22. Thus, overexpression of Reg3γ significantly exacerbated EAE scores, demyelination and infiltration of IFN-γ+IL-17A+ and IL-17A+GM-CSF+Th17 cells to CNS. We also show that Reg3γ may inhibit IL-2-mediated STAT5 signalling and impair expansion of Treg cells in vivo and in vitro. Finally, Reg3γ overexpression dramatically impacted intestinal microbiota during EAE. Our results provide novel insight into the role of IL-22 and IL-22-induced antimicrobial peptide Reg3γ in the pathogenesis of CNS inflammation in a murine model of MS.

Keywords: IL-22; Reg3γ; experimental autoimmune encephalomyelitis; microbiota; multiple sclerosis treatment.

© 2021 John Wiley & Sons Ltd.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1

Temporal IL‐22 overexpression via hydrodynamic gene delivery ameliorates experimental autoimmune encephalomyelitis (EAE) pathology and disease scores. (a) IL‐22 plasmid or empty plasmid was injected on day −2, and EAE was induced by MOG35‐55 immunization on day 0. Mice were monitored for 3–4 weeks, and EAE disease was scored. (b) Spinal cords were removed and stained with Luxol fast blue to quantify demyelination 3 weeks after EAE induction (n = 11 mice for both groups). (c) A representative picture of Luxol fast blue staining. (d) Spinal cords and brain tissues from IL‐22 plasmid‐ or empty plasmid‐injected mouse groups were harvested 3 weeks after EAE induction, and infiltrating lymphocytes were quantified based on IFN‐γ, IL‐17A and GM‐CSF production. Absolute number or percentages of indicated cytokine‐producing CD4+ T cells were quantified (n = 11 mice (control), n = 11 mice). (e) Mean fluorescent intensity (MFI) of Foxp3 protein in Treg cells in the brain at 3 weeks after EAE induction, or percentages of Treg cells among CD4+ T cells, or their absolute numbers. (f) Lymph node or splenic IL‐17A production by T cells in IL‐22 plasmid‐ or empty plasmid‐injected mice on day 7 of EAE induction (priming phase of the disease). (g) Treg cell numbers in LN and spleen on day 7 of EAE induction. The experiments were repeated at least three times. (*) indicates p‐value <0·05, (**), p < 0·01

FIGURE 2
FIGURE 2

Neutralization of IL‐22 exacerbates experimental autoimmune encephalomyelitis (EAE) disease scores (a) After EAE was induced by MOG35‐55 immunization, one group of mice received every other day 150 µg anti‐mouse IL‐22, and the other group received isotype antibody until day 10 post‐immunization. The EAE was scored daily. (b) On day 22, spinal cords were removed and stained with Luxol fast blue and scored to quantify demyelination (n = 9–10 per group). (c) Spinal cords and brain tissues from anti‐IL‐22 and isotype antibody (control) groups were harvested 3 weeks after EAE induction, and infiltrating lymphocytes were quantified based on IFN‐γ, IL‐17A and GM‐CSF production. Absolute number or percentages of indicated cytokine‐producing CD4+ T cells were quantified (n = 9–10 per group). The experiments were repeated 3 times. (*) indicates p‐value <0·05

FIGURE 3
FIGURE 3

Temporal Reg3γ overexpression via hydrodynamic gene delivery exacerbates experimental autoimmune encephalomyelitis (EAE) pathology and disease scores (a) Reg3γ plasmid or empty plasmid were injected on day −2, and 3 days later, Reg3γ message was quantified by real‐time qPCR in the liver and small intestine tissue (n = 3). (b) Reg3γ plasmid or empty vector was injected on day −2, and EAE was induced by MOG35‐55 immunization on day 0. Mice were monitored for 3‐4 weeks, and EAE disease was scored (n = 9–10 per group). (c) Spinal cords were removed and stained with Luxol fast blue to quantify demyelination 3 weeks after EAE induction (n = 9–10 per group). (d) Spinal cords and brain tissues from Reg3γ plasmid‐ or empty vector‐injected mouse groups were harvested 3 weeks after EAE induction, and infiltrating lymphocytes were quantified based on IFN‐γ, IL‐17A and GM‐CSF production. Absolute number of indicated cytokine‐producing CD4+ T cells or Treg cells (last panel) was quantified (n = 9–10 mice per group). (e) Absolute number of indicated cytokine‐producing CD4+ T cells in the draining LNs of Reg3γ plasmid‐ or empty plasmid‐injected mouse groups at 3 weeks post‐immunization of EAE. (n = 4 mice per group). (*) indicates p‐value <0·05; (**), p < 0·01; (***), p < 0·001; and (****), p < 0·0001

FIGURE 4
FIGURE 4

Reg3γ blocks Treg expansion in vivo and ex vivo (a) Foxp3‐YFP or B6 mice were injected with 2 ml of saline containing 15 µg/ml plasmid (IL‐22, Reg3γ or empty plasmid as control) on day −2 or −1. Then, on starting day 0, the mouse groups received 100 µl of intraperitoneal injections of 5 ng IL‐2 per gram mouse (in PBS), daily for 7 days. The mice were killed on day 7, and the absolute number of CD4+ T cells and Treg cells and Treg cell percentage in LNs (inguinal and brachial) (a) were quantified. (b) HEK293 T cells were transfected with Reg3γ or empty plasmid or as control GFP vector. The supernatant (conditioned media) was collected on days 2 and 5. (c) Filtered antibiotic‐added conditioned media were added into CD3/CD28‐activated sorted splenocytes obtained from Foxp3‐YFP reporter mice and cultured for 3 days. A representative flow plot of Treg cell expansion. (d) Quantification of the absolute number of Treg cells in ‘C’. (e) Sorted CD4+ Foxp3‐YFP+ Treg cells were cultured for 2 h in Reg3γ or empty plasmid‐conditioned media from HEK293 T cell transfections. Then, cells were stimulated with IL‐2 and STAT5 phosphorylation was quantified by nuclear staining. MFI indicates mean fluorescent intensity. (f) Splenocytes from B6 mice were cultured for 24 h in Reg3γ or empty plasmid‐conditioned media from HEK293 T cell transfections with the addition of CD3/CD28 1 µg/ml each. Both the adherent and suspension cells were collected and used for real‐time qPCR array (Qiagen, PAMM 52Z‐Mouse). A bar graph (top) and list (bottom) of differentially regulated genes (p < 0·05 for all) are given. (*) indicates p‐value <0·05; and (**), p < 0·01. Experiments in (a–e) were repeated three times

FIGURE 5
FIGURE 5

Reg3γ and its putative receptor Extl3’s expression kinetics in the spinal cord and intestinal tissues during experimental autoimmune encephalomyelitis (EAE) and in MS patient CSF cells (a) Spinal cord (SC) and small intestine (SI) tissues from naïve, 7‐ and 20‐day post‐immunization from EAE mice were collected, and real‐time qPCR was performed for Reg1α, Reg3γ and Extl3 (n = 3). (b) The cerebrospinal fluid (CSF) from MS patients or controls was spun, and the cell pellet was used for real‐time qPCR for EXTL1, EXTL3, REG1A, REG1B and REG3A (n = 5 for each group). (*) indicates p‐value <0·05

FIGURE 6
FIGURE 6

Reg3γ overexpression causes dysbiosis in the intestinal microbiota in experimental autoimmune encephalomyelitis (EAE) mice (a) Reg3γ plasmid or empty vector was injected on day −2, and EAE was induced by MOG35‐55 immunization on day 0. Mice were killed on day 20, and ileal faeces were collected for microbiota analyses. Principal co‐ordinate analysis ordinated based on the Unweighted UniFrac beta‐diversity metric plot (n = 7 per group). (b) Genus‐ and phylum‐level abundance profile of ileal microbiota in Reg3γ plasmid‐ or empty vector‐injected EAE mice at the peak of the disease (n = 7 per group). (c) Total microbiota abundance of ileal microbiota in Reg3γ plasmid‐ or empty vector (control)‐injected EAE mice at the peak of the disease (n = 7 per group), p‐value <0·05. (d) Significantly reduced genera of microbes in ileal microbiota of Reg3γ plasmid‐injected compared with empty vector‐injected EAE mice at the peak of the disease (n = 7 per group). All genera plots shown in ‘D’ are significantly altered (p‐value <0·05). (e) Significantly enriched (all plots shown in ‘E’ have p‐value <0·05) genera of microbes in ileal microbiota in Reg3γ plasmid‐injected compared with empty vector‐injected EAE mice at the peak of the disease (n = 7 per group), (*) indicates p‐value <0·05

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