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Comparative Study
. 2014 Mar 25:11:57.
doi: 10.1186/1742-2094-11-57.

Comparison of microglia and infiltrating CD11c⁺ cells as antigen presenting cells for T cell proliferation and cytokine response

Agnieszka Wlodarczyk, Morten Løbner, Oriane Cédile, Trevor Owens  1
Affiliations
Comparative Study

Comparison of microglia and infiltrating CD11c⁺ cells as antigen presenting cells for T cell proliferation and cytokine response

Agnieszka Wlodarczyk et al. J Neuroinflammation. .

Abstract

Background: Tissue-resident antigen-presenting cells (APC) exert a major influence on the local immune environment. Microglia are resident myeloid cells in the central nervous system (CNS), deriving from early post-embryonic precursors, distinct from adult hematopoietic lineages. Dendritic cells (DC) and macrophages infiltrate the CNS during experimental autoimmune encephalomyelitis (EAE). Microglia are not considered to be as effective APC as DC or macrophages.

Methods: In this work we compared the antigen presenting capacity of CD11c⁺ and CD11c⁻ microglia subsets with infiltrating CD11c⁺ APC, which include DC. The microglial subpopulations (CD11c⁻ CD45dim CD11b⁺ and CD11c⁺ CD45dim CD11b⁺) as well as infiltrating CD11c⁺ CD45high cells were sorted from CNS of C57BL/6 mice with EAE. Sorted cells were characterised by flow cytometry for surface phenotype and by quantitative real-time PCR for cytokine expression. They were co-cultured with primed T cells to measure induction of T cell proliferation and cytokine response.

Results: The number of CD11c⁺ microglia cells increased dramatically in EAE. They expressed equivalent levels of major histocompatibility complex and co-stimulatory ligands CD80 and CD86 as those expressed by CD11c⁺ cells infiltrating from blood. CD11c⁺ microglia differed significantly from blood-derived CD11c⁺ cells in their cytokine profile, expressing no detectable IL-6, IL-12 or IL-23, and low levels of IL-1β. By contrast, CD11c⁻ microglia expressed low but detectable levels of all these cytokines. Transforming growth factor β expression was similar in all three populations. Although CNS-resident and blood-derived CD11c⁺ cells showed equivalent ability to induce proliferation of myelin oligodendrocyte glycoprotein-immunised CD4⁺ T cells, CD11c⁺ microglia induced lower levels of T helper (Th)1 and Th17 cytokines, and did not induce Th2 cytokines.

Conclusions: Our findings show distinct subtypes of APC in the inflamed CNS, with a hierarchy of functional competence for induction of CD4⁺ T cell responses.

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Figures

Figure 1
Figure 1
Comparison of microglia and infiltrating cells in the central nervous system during inflammation. Representative flow cytometry profiles of individual central nervous system suspensions prepared from control mice or mice with severe experimental autoimmune encephalomyelitis (EAE) showing staining for CD11c and CD45. Vertical line indicates isotype control. Cytometry profile shows increase of both microglia and infiltrating cells expressing CD11c (A). Flow cytometry analysis from six combined experiments shows a significant increase of CD11c+ microglia presented as a percentage from total microglia (B). CD11c+ microglia showed an increased expression of CD45, but the expression did not reach the level of CD45high expressing cells (C). Quantitative real-time PCR analysis shows significantly lower expression of CCR2 in both population of microglia than in CD45high CD11c+ cells (D). Data are presented as a means ± SEM of six individual experiments (n ≥ 13). ***P < 0.001.
Figure 2
Figure 2
CD11c+ microglia resemble CD45high CD11c+ cells in expression of molecules needed for antigen presentation and T-cell activation. Isolated cells from B6 mice with severe experimental autoimmune encephalomyelitis were stained for major histocompatibility complex (MHC) class I, MHC class II, CD80 and CD86 and analyzed by flow cytometry. For comparison of central nervous system antigen presenting cells, cells were gated as CD45dim CD11b+, CD11c−, CD45dim CD11b+, CD11c+ and CD45high CD11c+. CD11c+ cells from the spleen (spl. DC) were used as positive control. Data are presented as means ± SEM of three individual experiments (n = 10). ns, Non-significant differences (P > 0.05). NetMFI, Median Fluorescent Intensity – background.
Figure 3
Figure 3
Cytokine expression profile in antigen presenting cells from the central nervous system of mice with experimental autoimmune encephalomyelitis. Expression of IL-1β, IL-6, IL-12p35, IL-23p19, and transforming growth factor (TGF)β in fluorescence-activated cell sorted antigen presenting cells (CD11c+ microglia, CD11c− microglia and CD45high CD11c+) from the central nervous system was analyzed by quantitative real-time PCR. Data are presented as means ± SEM of three individual experiments (n ≥ 5). ns, Not significant; *P < 0.05; **P < 0.01. ND, not detected.
Figure 4
Figure 4
Relative efficiency of central nervous system and spleen antigen presenting cells in stimulating proliferation of myelin-specific CD4 T cells. Proliferation of CD4 T cells sorted from B6 mice with severe experimental autoimmune encephalomyelitis (EAE). Splenic CD4+ T cells from myelin oligodendrocyte glycoprotein (MOG)p35–55 immunised B6 mice with severe EAE were isolated using magnetic beads and cultured with antigen presenting cells (CD11c− microglia, CD11c+ microglia, CD45high CD11c+ cells and spleen-derived dendritic cells (spl. DC), with (black bars) or without (white bars) MOGp35–55. Proliferation was measured by CFSE dilution assay. Data are presented as means ± SEM of three individual experiments (n ≥ 9 ). ns, Non-significant differences (P > 0.05).
Figure 5
Figure 5
Cytokine induction by central nervous system antigen presenting cells in primed CD4 T cells. Fluorescence-activated cell sorted CD4+ T cells from myelin oligodendrocyte glycoprotein (MOG)p35–55 immunised B6 mice with severe experimental autoimmune encephalomyelitis were co-cultured with antigen presenting cells (CD11c− microglia, CD11c+ microglia, CD45high CD11c+ cells and spleen-derived dendritic cells (spl. DC) in the presence of MOGp35–55 peptide. The cytokines IFN-γ and IL-17A from supernatants were quantified by cytokine bead array. Data are presented as means ± SEM of three individual experiments (n ≥ 9). ns, Not significant; *P < 0.05; **P < 0.01.
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