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. 2016 May:54:128-139.
doi: 10.1016/j.bbi.2016.01.016. Epub 2016 Jan 18.

CXCL13 promotes isotype-switched B cell accumulation to the central nervous system during viral encephalomyelitis

Timothy W Phares  1 , Krista D DiSano  2 , Stephen A Stohlman  3 , Benjamin M Segal  4 , Cornelia C Bergmann  5
Affiliations

CXCL13 promotes isotype-switched B cell accumulation to the central nervous system during viral encephalomyelitis

Timothy W Phares et al. Brain Behav Immun. 2016 May.

Abstract

Elevated CXCL13 within the central nervous system (CNS) correlates with humoral responses in several neuroinflammatory diseases, yet its role is controversial. During coronavirus encephalomyelitis CXCL13 deficiency impaired CNS accumulation of memory B cells and antibody-secreting cells (ASC) but not naïve/early-activated B cells. However, despite diminished germinal center B cells and follicular helper T cells in draining lymph nodes, ASC in bone marrow and antiviral serum antibody were intact in the absence of CXCL13. The data demonstrate that CXCL13 is not essential in mounting effective peripheral humoral responses, but specifically promotes CNS accumulation of differentiated B cells.

Keywords: Antibody secreting cells; B cells; CXCL13; Central nervous system; Memory B cells; Neuroimmunology.

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Figures

Fig. 1
Fig. 1
Microglia are a primary source of CXCL13. (A) Brain, spinal cord and CSF CXCL13 levels from individual mice were assessed by ELISA. Brain and spinal cord data are expressed as the mean CXCL13 per mg of tissue ±SEM (left-hand y axis) of 6–8 mice per time point from two independent experiments. Typical weights for brain and spinal cord were 397 ± 10 mg and 80 ± 2 mg, respectively. CSF data are expressed as the mean CXCL13 per total CSF volume ± SEM (right-hand y axis) of 3–4 mice per time point from one experiment. Total volume of mouse CSF is estimated to be 40 μl (Johanson et al., 2008). Significant differences between naïve and infected samples determined by the unpaired t test are denoted by **(p < 0.01) and ***(p < 0.005). (B) Relative transcript levels of CXCL13 in FACS-purified GFP+CD45- astrocytes (striped bars), CD45hiCD11b+F4/80+ monocyte-derived macrophages (empty bar) or CD45lo microglia (solid bar) derived from pooled spinal cords of 6–8 GFAP-GFP mice per time point collected at 7 and 10 days p.i. were assessed by real-time PCR. Transcript levels are relative to GAPDH.
Fig. 2
Fig. 2
Reduced CNS accumulation of Bmem and ASC in the absence of CXCL13. (A) Relative transcript levels of CXCR5 assessed by real-time PCR in naïve/early-activated B cells (CD138-IgD+), Bmem (CD138−IgD−) and ASC (CD138+) FACS-purified from pooled spinal cords of 6–8 mice per time point collected at 7 (naïve/early-activated B cells) or 21 (Bmem and ASC) days p.i. Data are expressed as the mean ± SEM transcript level relative to GAPDH mRNA and represent two independent experiments. (B) Representative density plot of IgD and IgM expression on spinal cord-derived CD19+CD138− B cells at day 14 p.i. Gated populations are as follows: 1 = naïve/early-activated B cells IgD+IgM+; 2 = Bmem IgD−IgM+/−. (C and D) Numbers of IgD+IgM+ and IgD−IgM+/− B cells within total CD19+CD138- B cells. (E) Numbers of CD138+ ASC within CD19+ B cells. Insert: Representative density plot of CD138 expression (gated cells) on spinal cord-derived CD19+ B cells at day 21 p.i. Data are expressed as the mean ± SEM and represent three independent experiments, each comprising pooled spinal cords of 3–4 mice per time point. ND = not detected. Significant differences between WT and CXCL13−/− mice determined by the unpaired t test are denoted by **(p < 0.01).
Fig. 3
Fig. 3
Reduced CNS accumulation of virus-specific IgG+ ASC in CXCL13−/− mice. Numbers of total and virus-specific IgM (A and B) or IgG-secreting (C and D) ASC in brain (A and C) or spinal cord (B and D) measured by ELISPOT assay (left column). Data are representative of 2–3 independent experiments with similar trends in each experiment. To better reflect relative differences between WT and CXCL13−/− frequencies, data in the right column show the mean percentage changes in frequencies ±SEM calculated relative to levels in WT mice, which were set to a baseline value of 100. Data represent 2–3 independent experiments, each comprising pooled brain or spinal cords of 3–4 mice. Significant differences between WT and CXCL13−/− mice determined by the unpaired t test are denoted by **(p < 0.01) and ***(p < 0.005).
Fig. 4
Fig. 4
Impaired activation of GC B cells and TFH cells in CLN of CXCL13−/− mice. (A) Representative density plot of CD95 and GL7 expression on WT CLN-derived CD19+ B cells at day 14 p.i. Frequencies of CD95+GL7+ cells with total CD19+ B cells determined by flow cytometry. Data are expressed as the mean percentage ± SEM of 6–8 mice per time point from two independent experiments. Relative transcript levels of AID (B), CXCL12, CCL19 and CCL21 (C) in CLN of naive and infected mice assessed by real-time PCR. (D) Representative density plot of CXCR5 and PD-1 expression on WT CLN-derived CD4+ T cells at day 14 p.i. The proportion of CXCR5+PD-1hi cells within total CD4+ T cells determined by flow cytometry. Data are expressed as the mean percentage ± SEM of 6–8 mice per time point from two independent experiments. (E) Relative transcript levels of IL-21 in CLN of naive and infected mice assessed by real-time PCR. All PCR data are expressed as the mean transcript level ±SEM relative to GAPDH mRNA and represent one experiment comprising individual CLN of 3 to 4 mice per time point. Significant differences between WT and CXCL13−/− mice determined by the unpaired t test are denoted by *(p < 0.05), **(p < 0.01) and ***(p < 0.005).
Fig. 5
Fig. 5
GC formation is severely abrogated in CXCL13−/− mice. GC formation in the CLN of WT and CXCL13−/− mice at day 21 p.i. analyzed using DAPI (blue), anti-GL7 (green) and anti-B220 (red) Ab. GL7+ B cells are absent or sparse in naïve WT and CXCL13−/− mice respectively. Merged images are shown. Photomicrographs are presented at a magnification of 40×. Scale bar is 50 μm. White-boxed area shown in bottom row photomicrographs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
Peripheral ASC responses are intact in CXCL13−/− mice. (A) Virus-specific IgG2a and neutralizing Ab in serum of infected WT and CXCL13−/− mice. Data are expressed as the mean ± SEM and represent two independent experiments, each comprising individual sera of 3 mice per time point. Numbers of virus-specific IgG-secreting ASC in CLN (B) and BM (C) measured by ELISPOT assay. Data are representative of 2–3 independent experiments with similar trends in each experiment. To better reflect relative differences between WT and CXCL13−/− frequencies, data in the right column of (B and C) show the mean percentage changes in frequencies ±SEM calculated relative to levels in WT mice, which were set to a baseline value of 100. Data represent 2–3 independent experiments, each comprising pooled CLN or BM from 3 to 4 mice.
Fig. 7
Fig. 7
CXCL13 deficiency does not affect JHMV pathogenesis or viral control. (A) WT and CXCL13−/− infected mice were monitored daily for clinical symptoms. (B) Virus titers in brain supernatants determined by plaque assay and expressed as the mean ± SEM. Dashed line marks the limit of detection. Data are on a log10 scale. Relative transcript levels of viral nucleocapsid (C), IFN-γ (D), IL-10 (E), IL-21 (F), CXCL12 (G), CCL19 (H) and CCL21 (I) in spinal cords of naïve and infected mice assessed by real-time PCR. All PCR data are expressed as the mean transcript level ±SEM relative to GAPDH mRNA of 6–7 mice per time point from two independent experiments. Significant differences between WT and CXCL13−/− mice determined by the unpaired t test are denoted by ***(p < 0.005).
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