ICAM1+ neutrophils promote chronic inflammation via ASPRV1 in B cell–dependent autoimmune encephalomyelitis
Ryder F. Whittaker Hawkins, Alexandre Patenaude, Aline Dumas, Rajiv Jain, Yodit Tesfagiorgis, Steven Kerfoot, Takeshi Matsui, Matthias Gunzer, Patrice E. Poubelle, Catherine Larochelle, Martin Pelletier, Luc Vallières
Ryder F. Whittaker Hawkins, Alexandre Patenaude, Aline Dumas, Rajiv Jain, Yodit Tesfagiorgis, Steven Kerfoot, Takeshi Matsui, Matthias Gunzer, Patrice E. Poubelle, Catherine Larochelle, Martin Pelletier, Luc Vallières
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Research Article Neuroscience

ICAM1+ neutrophils promote chronic inflammation via ASPRV1 in B cell–dependent autoimmune encephalomyelitis

Abstract

Neutrophils contribute to demyelinating autoimmune diseases, yet their phenotype and functions have been elusive to date. Here, we demonstrate that ICAM1 surface expression distinguishes extra- from intravascular neutrophils in the mouse CNS during experimental autoimmune encephalomyelitis (EAE). Transcriptomic analysis of these 2 subpopulations indicated that neutrophils, once extravasated, acquire macrophage-like properties, including the potential for immunostimulation and MHC class II–mediated antigen presentation. In corroboration, super-resolution (3D stimulated emission-depletion [STED]) microscopy revealed neutrophils forming synapses with T and B cells in situ. Further, neutrophils specifically express the aspartic retroviral-like protease ASPRV1, which increases in the CNS during EAE and severe cases of multiple sclerosis. Without ASPRV1, mice immunized with a new B cell–dependent myelin antigen (but not with the traditional myelin oligodendrocyte glycoprotein peptide) develop a chronic phase of EAE that is less severe and even completely fades in many individuals. Therefore, ICAM1+ macrophage–like neutrophils can play both shared and nonredundant roles in autoimmune demyelination, among them perpetuating inflammation via ASPRV1.

Authors

Ryder F. Whittaker Hawkins, Alexandre Patenaude, Aline Dumas, Rajiv Jain, Yodit Tesfagiorgis, Steven Kerfoot, Takeshi Matsui, Matthias Gunzer, Patrice E. Poubelle, Catherine Larochelle, Martin Pelletier, Luc Vallières

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Figure 9

ASPRV1 is required for the chronic phase of EAE induced with a B cell–dependent myelin antigen (bMOG) but not for initial neutrophil recruitment or T cell priming.

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ASPRV1 is required for the chronic phase of EAE induced with a B cell–de...
(A) Kaplan-Meier plot of EAE incidence in WT (Asprv1+/+) and ASPRV1-deficient mice (Asprv1–/–) after immunization with either MOG35–55 or bMOG. Shaded area represents the 95% pointwise CI. For sample size and statistical testing, see incidence in Table 1. (B) Severity of EAE over time by group versus time from appearance of first symptoms. P value shown on graph was calculated by 2-way ANOVA with repeated measures using rank-transformed scores. Stars indicate differences in post-hoc testing by time point (Wilcoxon test, P < 0.0386). Only mice that had developed EAE are included (see incidence in Table 1 for sample size). (C) Flow cytometric counts of immune cells in the spinal cord of Asprv1–/– and WT mice before immunization with bMOG (naive) or after at day 8 (EAE preonset), 13 (EAE peak), or 21 (EAE chronic phase). Counts were normalized to CD45– cells as an internal control. Stars indicate significant differences from Asprv1+/+ mice (Wilcoxon test, P < 0.0454). Mic, CD45loCD11b+ microglia; N, Ly6G+ neutrophils; Mc, CD45hiCD11b+CD11c– macrophages; DC, CD45hiCD11b+CD11c+ DCs; T, CD3ε+ T cells; B, CD19+ B cells. Sample size per group: 4–6 (naive), 7 (preonset), 9–22 (peak), 7–10 (chronic). (D) Counts of the ICAM1– (intravascular) and ICAM1+ (extravascular) subpopulations of macrophages and neutrophils showing no intergroup difference, except for ICAM1+ macrophages at the peak of bMOG EAE. Star indicates a significant difference from Asprv1+/+ mice (Wilcoxon test, P = 0.0374). Sample size as in C. (E) Counts of blood neutrophils showing that bMOG immunization induces a similar mobilization of neutrophils between the genotypes. Sample size per group: 3–5 (naive), 7 (preonset), 7–10 (peak). (F) Proportions of Th17 (IL-17+) and Th1 (IFN-γ+) cells in inguinal lymph nodes (LN) at day 9 after immunization with bMOG, suggesting that there were no intergroup difference in T cell priming. Intracellular staining was performed on freshly collected LN cells after a 4-hour stimulation with phorbol myristate acetate and ionomycin in the presence of brefeldin A. n = 14–17 per group.