Cxcr3-expressing leukocytes are necessary for neurofibroma formation in mice

Abstract

Plexiform neurofibroma is a major contributor to morbidity in patients with neurofibromatosis type I (NF1). Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Antiinflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in Nf1-mutant nerves, which invariably form neurofibroma, and show disruption of neuron-glial cell interactions and immune cell infiltration to mouse models, which rarely progresses to neurofibroma with or without disruption of neuron-glial cell interactions. We find that the chemokine Cxcl10 is uniquely upregulated in NF1 mice that invariably develop neurofibroma. Global deletion of the CXCL10 receptor Cxcr3 prevented neurofibroma development in these neurofibroma-prone mice, and an anti-Cxcr3 antibody somewhat reduced tumor numbers. Cxcr3 expression localized to T cells and DCs in both inflamed nerves and neurofibromas, and Cxcr3 expression was necessary to sustain elevated macrophage numbers in Nf1-mutant nerves. To our knowledge, these data support a heretofore-unappreciated role for T cells and DCs in neurofibroma initiation.

Keywords: Cancer; Chemokines; Inflammation; Mouse models; Neuroscience.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. Gene expression reveals differential expression of Cxcl10 in neurofibroma development.

(A) Gene expression in control nerves compared with Nf1-mutant GEMM and related EGFR and HRas mouse models; 2,028 genes were differentially expressed (ANOVA, P < 0.05, Benjamini-Hochberg FDR), forming 6 distinct gene expression clusters. Relative levels of gene expression are shown as fold change (left); red means high and blue means low gene expression. Clusters were refined using K-means clustering (n = 6) for subsequent gene ontology (GO) analyses (the colored column to the right of the heatmap labeled C1–C6 represents K-means clusters). The pattern of gene expression in clusters C1 and C6 was associated with the presence of nerve disruption, a common pattern of axon-glial dissociation, fibrosis, and inflammation occurring in plexiform neurofibroma mouse models and CNPase-hEGFR nerves, which is designated by a green bar under the heatmap. (B) An independent analysis of 1-month and 2-month Nf1fl/fl (control), CNPase-hEGFR/ CNPase-hEGFR, and Dhh-Cre Nf1fl/fl nerves identified 1,339 genes as differentially expressed between these groups at the 2-month but not the 1-month time point (ANOVA, P < 0.05, Benjamini-Hochberg FDR; n = 4 for the 2-month Nf1fl/fl control, n = 3 other groups). CNPase-hEGFR/CNPase-hEGFR and Dhh-Cre Nf1fl/fl nerves at the 2-month time point shared a common pattern of gene regulation distinct from maturation-associated changes in 2-month controls. Consistent with the presence of the nerve disruption phenotype in these 2-month experimental nerves, many of these differentially expressed genes were associated with the nerve disruption phenotype identified in A (indicated by the column to the right of the heatmap in B). (C) Genes differentially expressed between Dhh-Cre Nf1fl/fl and CNPase-hEGFR/CNPase-hEGFR nerve/DRG. Only 38 genes were significantly upregulated or downregulated greater than 2-fold in 2-month Dhh-Cre Nf1fl/fl nerve/DRG relative to 2-month Nf1fl/fl controls that were not similarly upregulated or downregulated in CNPase-hEGFR/CNPase-hEGFR nerve/DRG (P < 0.05, Benjamini-Hochberg FDR) in Dhh-Cre Nf1fl/fl nerve/DRG but not CNPase-hEGFR nerve/DRG (relative to their respective controls). The 7 most upregulated genes are shown. Cxcl10 was the only cytokine uniquely upregulated in Dhh-Cre Nf1fl/fl nerve/DRG. (D) Cxcl10 upregulation in 2-month Dhh-Cre Nf1fl/fl (n = 3 all groups) nerve/DRG was validated by quantitative PCR (**P < 0.01, Dunnett’s multiple-comparisons test [MCT]). Cxcl10 was also upregulated in neurofibroma (****P < 0.0001, Dunnett’s MCT). (E–G) Its receptor, Cxcr3 (****P < 0.0001, Dunnett’s MCT), and its alternative ligands, Cxcl9 and Cxcl11 (**P < 0.01, Dunnett’s MCT), were overexpressed in neurofibroma but not in 2-month Dhh-Cre Nf1fl/fl nerve/DRG (n = 3 all groups). Symbols represent individual mice; horizontal bars indicate the mean ± SD.

Figure 2. Cxcl10 and associated gene expression in single-cell sequencing from 2-month Dhh-Cre Nf1fl/fl nerve/DRG.

(A) Iterative Clustering and Guide-gene Selection (ICGS) performed with the MarkerFinder analysis option initially identified 9 primary cell populations and associated population-specific genes. Results are shown following multiplet cell and cluster (C8) exclusion. The cell types of these clusters were then inferred on the basis of their associated population-specific genes relative to published references and the GO. (B) Visualization of cell populations by t-SNE (arbitrary units). Fabp7 (Blbp), an immature SC marker gene, is localized to SC cluster C9 (SC-2). Cxcl10 is also predominantly localized to this cluster. The pattern of Nf1 localization is different from that of Cxcl10. (C) Single cells in SC-1 (blue dots) and SC-2 (red dots), plotted by normalized expression of Cxcl10 and Nf1. Nf1-expressing cells in SC-1 largely lack Cxcl10 and are compressed on the y axis, while 73.8% of Cxcl10-expressing cells in SC-2 lack Nf1 expression and are compressed on the x axis. (D) Unsupervised analysis of SCs in cluster C9 by ICGS partitions these cells into 3 subclusters, distinguished by localization of Fabp7 (cluster C1) and Cxcl10 (cluster C3).

Figure 3. Global deletion of Cxcr3 prevents plexiform neurofibroma and reduces nerve pathology in Dhh-Cre Nf1fl/fl mice.

(A) Dhh-Cre Nf1fl/flCxcr3-null (n = 25) mice survived significantly longer than Dhh-Cre Nf1fl/fl (n = 11) mice (****P < 0.0001, log-rank test). All of the Dhh-Cre Nf1fl/fl and none of the Dhh-Cre Nf1fl/flCxcr3-null mice developed neurofibromas. Censored data points represent Dhh-Cre Nf1fl/flCxcr3-null mice collected at 18 to 20 months of age for dissection, pathological analyses, and histological analyses. (B) Loss of Cxcr3 did not prevent Nf1 recombination in Dhh-Cre Nf1fl/flCxcr3-null sciatic nerve. (C–E) Representative images of age-matched (10-month) spinal cords and associated spinal nerve/DRG are shown; such dissections were performed on all study mice (n > 10 each group). White arrows indicate plexiform neurofibromas. (F–H) Close-up of C–E; white arrows indicate bilateral plexiform neurofibromas compressing the spinal cord. (I–K) Representative electron micrographs from saphenous nerves (n = 3 examined for each genotype). (I) Electron micrograph of a normal 7-month saphenous nerve. (J) Black arrowhead indicates a disrupted Remak bundle in Dhh-Cre Nf1fl/fl saphenous nerve. Dark gray particulate (black asterisks) indicates deposited collagen. (K) Remak bundle disruption and collagen deposition were absent in 7-month Dhh-Cre Nf1fl/flCxcr3-null saphenous nerves. Original magnification, 4000× (I, J, and K).

Figure 4. The effects of Cxcr3 deletion on mast cell and macrophage infiltration of Dhh-Cre Nf1fl/fl nerves (n ≥ 4 all groups).

(A) Toluidine blue staining in 2-month and 7-month sciatic nerves (original magnification, ×40. Scale bar: 50 μm). (B) Mast cell infiltration of nerves was not significantly affected by loss of Cxcr3 (NS, 2-way ANOVA). HPF, high-power field. (C) Iba-1 (macrophage) staining in sciatic nerves (original magnification, ×40. Scale bar: 50 μm). (D) Loss of Cxcr3 did not affect the initial recruitment of macrophages to nerves but resulted in the resolution of macrophage inflammation by 7 months (**P < 0.01, ***P < 0.001, ****P < 0.0001, 2-way ANOVA with Tukey’s MCT). Symbols represent individual mice; horizontal bars indicate the mean ± SD. (E) Cxcr3 expression does not colocalize to IBA-1+ macrophages (original magnification, ×20. Scale bar: 50 μm), but (F) Cxcr3-expressing cells are CD45+ hematopoietic cells (original magnification, ×20. Scale bar: 50 μm).

Figure 5. Identification of Cxcr3-expressing cells in Dhh-Cre Nf1fl/fl nerve/DRG and neurofibroma.

(A) Cxcr3 expression colocalizes with CD3+ T cells and CD11c+ DCs in 2-month nerve/DRG (original magnification, ×60. Scale bar: 20 μm) and (B) in 7-month neurofibroma (original magnification, ×60. Scale bar: 20 μm). (C) Cxcr3-expressing cells are rare in WT nerves (n = 4) and are predominantly T cells and DCs in Dhh-Cre Nf1fl/fl nerve/DRG (n = 9) and neurofibroma (n = 7) (****P < 0.0001, 2-way ANOVA with Tukey’s MCT). The box plot depicts the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. (D) T cells were rare in 2-month (n ≥ 4 all groups) sciatic nerves, but loss of Cxcr3 reduced T cell accumulation in 7-month Dhh-Cre Nf1fl/fl sciatic nerves (**P < 0.01, ***P < 0.001, 2-way ANOVA with Tukey’s MCT). Symbols represent individual mice; horizontal bars indicate the mean ± SD. (E) DC numbers in sciatic nerves were unaffected by loss of Cxcr3 (NS, 2-way ANOVA). (F and G) Colorimetric (DAB) staining of CD3+ T cells and CD11c+ DCs in human peripheral nerve and plexiform neurofibroma (original magnification, ×40. Scale bar: 50 μm). (F) T cells are absent in normal human peripheral nerves (n = 7) but numerous in human plexiform neurofibroma (n = 19). (G) DCs are absent in human peripheral nerves (n = 7) but detectable in some human plexiform neurofibromas (n = 15). Arrowhead points to CD11c+ (brown) cell. (H) Quantification of T cells (*P < 0.01, unpaired t test) and (I) quantification of CD11c+ cells (NS, P = 0.056, unpaired t test) in human tissue sections. (J) Box-and-whisker plot showing relative CXCR3 mRNA expression in human plexiform neurofibroma (pNF) versus normal human nerve. The box plot depicts the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range.

Figure 6. The proposed role of Cxcr3-expressing T cells and DCs in neurofibroma development.

(A) In Dhh-Cre Nf1fl/fl DRG/nerve, SCs express chemokines and growth factors that can recruit macrophages, mast cells, and T cells/DCs. This likely drives the initial immune cell infiltration at the site of neurofibroma formation, consistent with our finding that loss of Cxcr3 does not prevent macrophage or mast cell recruitment to 2-month Dhh-Cre Nf1fl/flCxcr3-null DRG/nerve. CXCR3 is predominantly expressed by T cells and DCs in Dhh-Cre Nf1fl/fl peripheral nerves and neurofibroma, and we hypothesize that cytokine production or other functions of T cells or DCs recruited through CXCR3 facilitate chronic inflammation and neurofibroma development. (B) Macrophage inflammation resolves by 7 months in Dhh-Cre Nf1fl/flCxcr3-null peripheral nerves, and neurofibromas do not form in these mice. We suggest that loss of Cxcr3 reduces T cell and DC recruitment to these nerves, allowing for the resolution of nerve inflammation and the prevention of neurofibroma development.