Neutrophil-related factors as biomarkers in EAE and MS

Abstract

A major function of T helper (Th) 17 cells is to induce the production of factors that activate and mobilize neutrophils. Although Th17 cells have been implicated in the pathogenesis of multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE), little attention has been focused on the role of granulocytes in those disorders. We show that neutrophils, as well as monocytes, expand in the bone marrow and accumulate in the circulation before the clinical onset of EAE, in response to systemic up-regulation of granulocyte colony-stimulating factor (G-CSF) and the ELR(+) CXC chemokine CXCL1. Neutrophils comprised a relatively high percentage of leukocytes infiltrating the central nervous system (CNS) early in disease development. G-CSF receptor deficiency and CXCL1 blockade suppressed myeloid cell accumulation in the blood and ameliorated the clinical course of mice that were injected with myelin-reactive Th17 cells. In relapsing MS patients, plasma levels of CXCL5, another ELR(+) CXC chemokine, were elevated during acute lesion formation. Systemic expression of CXCL1, CXCL5, and neutrophil elastase correlated with measures of MS lesion burden and clinical disability. Based on these results, we advocate that neutrophil-related molecules be further investigated as novel biomarkers and therapeutic targets in MS.

Figure 1.

Neutrophils and monocytes expand in the bone marrow after EAE induction. (A–C) WT mice were immunized with MOG_35-55 in CFA and injected with PTx on days 0 and 2 p.i. Bone marrow cells were flushed from femurs and tibiae of representative mice at serial time points and analyzed by flow cytometry to enumerate neutrophils (CD31⁻Ly6C^intLy6G⁺, black bars/closed circles), monocytes (CD31⁺Ly6C^hiLy6G⁻, gray bars and circles), and lymphocytes (CD31⁺Ly6C⁻, diagonal stripes/open triangles). (A) Percentage of leukocyte subsets within bone marrow cells. Data are representative of six experiments (n ≥ 3 per time point). (B and C) Absolute numbers of neutrophils (B) and monocytes and lymphocytes (C) recovered per mouse. Data are representative of nine experiments (n ≥ 3 per time point). All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.01 compared with unimmunized mice.

Figure 2.

Serum G-CSF and CXCL1 are up-regulated and myeloid cells are mobilized into the circulation during EAE. (A–D) Peripheral blood cells and sera were collected from mice that had been primed with MOG_35-55 or OVA_323-339 in CFA or MOG_35-55 in IFA, with or without administration of PTx. (A) Percentage of circulating neutrophils (CD11b⁺Ly6C^intLy6G⁺, black bars) and monocytes (CD11b⁺Ly6C^hiLy6G⁻, white bars) on day 7 p.i. Shown is a representative of three experiments (n ≥ 6 mice per group). (B) Numbers of neutrophils (top panels, closed squares) and monocytes (bottom panels, open circles) per ml of blood or per spleen at serial time points after active immunization with MOG_35-55 in CFA. Data are pooled from 10 experiments (blood, n ≥ 6 per time point) or 5 experiments (spleen, n ≥ 3 per time point). (C and D) Serum levels of G-CSF (C) and CXCL1 (D) were measured by ELISA. Data were pooled from 10 experiments (n ≥ 3 mice per time point). (E and F) G-CSF (E) and CXCL1 (F) were measured in tissue homogenates and normalized to total protein. Shown is a representative of two experiments (n = 4 mice per time point). All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.01 compared with unimmunized mice. ^#, P < 0.05; ^##, P < 0.01 between groups. ND = not detectable.

Figure 3.

EAE is dependent on G-CSF signaling in hematopoietic cells. (A–E) WT (closed triangles, black bars) and Csf3r^−/− (open triangles, white bars) mice were actively immunized with MOG_35-55 in CFA. (A) Mean clinical scores (n = 25 WT, 21 Csf3r^−/− pooled from five independent experiments). (B) Representative paraffin sections of spinal cords stained with H&E. (C) Cell subsets recovered from spinal cords at peak of disease, shown as a percentage of total CD45⁺ cells. Cell types were defined as follows: neutrophil (CD45^hi, CD11b⁺, Ly6G⁺), monocyte (CD45^hi, CD11b⁺, CD11c⁻, Ly6G⁻), DC (CD45^hi, CD11b⁺, CD11c⁺), CD3⁺ (CD45^hi, CD3⁺), and microglia (CD45^mid CD11b⁺). Data are representative of two experiments (n ≥ 4 mice/group). (D and E) Circulating and splenic neutrophils (D) and monocytes (E) were enumerated by flow cytometry. Data were pooled from two independent experiments (n ≥ 5 per group). (F) MOG-specific cytokine production by draining lymph node cells measured by ELIspot. Data are representative of three experiments (n = 3–5 mice per group). In the experiment shown there were 2.4 × 10⁵ total cells/well. (G) Mean clinical scores of WT to WT (closed triangles, n = 10) or Csf3r^−/− to WT (open triangles, n = 9) bone marrow chimeric mice after active immunization with MOG_35-55 in CFA. Data are representative of three experiments. All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bars, 100 µm.

Figure 4.

Neutrophils accumulate at onset of disease in adjuvant-free models of EAE. (A–D) OSE mice were sacrificed when healthy (n = 5; white bars), within 2 d of the onset of clinical EAE (n = 5; black bars) or during the chronic stages of EAE (n = 4; gray bars). Peripheral blood cells (A), splenocytes (B), BM cells (C) and spinal cord–infiltrating cells (D) were collected. Neutrophils and monocytes were enumerated by flow cytometry. (E and F) WT mice were injected with IL-12–polarized (Th1; black bars) or IL-23–polarized (Th17; gray bars) MOG-specific T cells. At day 7 after transfer, blood (E) and spleens (F) were collected and neutrophils and monocytes were enumerated by flow cytometry. Data are pooled from four (Th17 transfers) or two (Th1 transfers) experiments (n ≥ 10 mice per group). All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.005; ***, P = 0.001; ****, P < 0.0001, by two-way ANOVA, correcting for multiple comparisons.

Figure 5.

Adoptive transfer of encephalitogenic Th17 cells induces the systemic up-regulation of G-CSF and neutrophil mobilization. (A–F) WT mice were injected with IL-23 polarized, MOG-specific CD4⁺ Th17 cells. (A) Circulating neutrophils (closed circles) and monocytes (white squares) were enumerated by flow cytometry at serial time points. Data were pooled from three experiments (n ≥ 7 mice per group). (B) Serum G-CSF levels were measured by ELISA. Data were pooled from three experiments (n ≥ 10 mice per group). (C) G-CSF levels in tissue homogenates obtained from naive mice or from host mice on day 7 after transfer, measured by ELISA and normalized to total protein (n = 5 mice per group). (D–F) Number of monocytes and neutrophils in brain (D), spinal cord (E), and spleen (F), determined by flow cytometry. Data were pooled from two experiments (n ≥ 6 per group). All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.01 compared with naive or day 3 after transfer.

Figure 6.

ELR⁺ CXC chemokines partially compensate for loss of G-CSF signaling in Csf3r^−/− adoptive transfer recipients. (A–G) WT (closed triangles, black bars) and Csf3r^−/− (open triangles, white bars) mice were injected with MOG-specific Th17 cells. (A) Mean clinical scores, representative of seven independent experiments (n ≥ 7 mice per group). (B) Absolute number of neutrophils, monocytes, and microglia recovered from the spinal cord on day 7 after transfer, assessed by flow cytometry (n ≥ 7 per group, pooled from two experiments). (C) Number of neutrophils per milliliter of blood or per spleen at baseline and on day 7 d after transfer, assessed by flow cytometry (n ≥ 7 per group, pooled from two experiments). (D) Fold change in the number of circulating and splenic neutrophils over baseline on day 7 after transfer. (E) Proportion of donor cells expressing IL-17 and IFN-γ immediately before adoptive transfer, and after isolation from the spinal cords of WT or Csf3r^−/− hosts on day 7 after transfer (n = 5 per group). (F) Levels of CXCL1 in sera from naive mice and adoptive transfer recipients, measured by ELISA (n ≥ 9, pooled from three experiments). (G) CXCL1 levels in spinal cord homogenates were measured by ELISA and normalized to total protein (n ≥ 4). (H) Csf3r^−/− recipients of WT Th17 cells were treated with control serum (n = 7) or anti-CXCR2 (n = 6) every other day from days 0– 8 (arrows). Data are representative of two independent experiments. All graphs indicate means; error bars denote SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 7.

Plasma CXCL5, but not CCL2 or CXCL10, levels increase in association with new MS lesion formation. Patients with relapsing MS were classified as having “active” or “inactive” disease based on clinical course, neurological examination, and MRI scanning. Plasma levels of CXCL5, CCL2, and CXCL10 were measured by multiplex assay. Patients with active disease had enhancing MRI lesions and patients with inactive disease had no enhancing lesions on the day of phlebotomy. Box plots show median, interquartile range, sample minimum, and maximum. Circles show outliers.