Figure 7.
Transcriptional changes in the hippocampus in cGVHD and attenuation of neuroinflammation by donor MHC II KO. (A) Heatmap representing log2 counts-per-million (logCPM) values of 381 differentially expressed genes (false discovery rate [FDR], <0.05) based on RNA-seq analysis of isolated hippocampi from TCD and GVHD mice 70 days after transplant (n = 3 per group). Expression across each gene has been scaled so that mean expression is 0 and standard deviation is 1. (B-C) Heatmap of logCPM values from TCD and GVHD mouse hippocampi for genes related to the IFN-γ response hallmark gene set (B) and antigen presentation (C) as identified by ingenuity pathway analysis of RNA-seq data (n = 3 per group). (D) Heatmap of logCPM values for genes involved in synaptic signaling as identified by Gene Ontology analysis (0045202). Enrichment plot from gene set enrichment analysis demonstrating downregulation of genes associated with the neuron-to-neuron synapse in GVHD compared with TCD. (E) Quantification of synaptophysin puncta expression in the hippocampus at days 35 (n = 8 mice per group; pooled from 2 independent experiments) and 70 (n = 6-11 mice per group) after transplant. (F) Schematic for the transplantation of WT (PTPxC57Bl/6) or MHC II KO.B6 (H2b) bone marrow depleted of T-cells into irradiated DBA2xF1 (H2Dd) recipients. (G) Clinical scores of transplant recipients (n = 11-12 mice per group; data pooled from 2 independent experiments). (H) Representative images of hematoxylin and eosin staining of skin from recipient mice at day 70 after transplant showing dermal thickening and loss of subcutaneous fat as evidence of scleroderma. Original magnification ×20; scale bar, 300 μm. (I) Time spent floating (immobile) and swimming (mobile) in the forced swim test (n = 4 mice per group) at day 70. (J) Representative confocal images of Iba1+ cells in the hippocampus at day 70 after transplant. Resident microglia express GFP and are indicated with white arrows. Original magnification ×20; scale bar, 50 μm. (K) Representative confocal images demonstrating morphological similarities in the resident microglia phenotype of recipients of MHC II KO TCD BM and WT TCD BM. Original magnification ×100; scale bar, 10 μm. (L-M) Messenger RNA expression of selected genes detected by quantitative real-time polymerase chain reaction in the coronal brain section (L) and hippocampus (M) of transplant recipients at day 70 after transplant. Reported as the fold change of the expression from the WT TCD BM group, calculated relative to the Hprt gene (n = 3-6 mice per group). Data are presented as mean ± standard error of the mean (SEM). Significant differences calculated using 2-way analysis of variance (G) or unpaired Student t test (E,M). *P < .05, ***P < .001. DAPI, 4’,6-diamidino-2-phenylindole; FOV, field of view; NES, normalized enrichment score.

Transcriptional changes in the hippocampus in cGVHD and attenuation of neuroinflammation by donor MHC II KO. (A) Heatmap representing log2 counts-per-million (logCPM) values of 381 differentially expressed genes (false discovery rate [FDR], <0.05) based on RNA-seq analysis of isolated hippocampi from TCD and GVHD mice 70 days after transplant (n = 3 per group). Expression across each gene has been scaled so that mean expression is 0 and standard deviation is 1. (B-C) Heatmap of logCPM values from TCD and GVHD mouse hippocampi for genes related to the IFN-γ response hallmark gene set (B) and antigen presentation (C) as identified by ingenuity pathway analysis of RNA-seq data (n = 3 per group). (D) Heatmap of logCPM values for genes involved in synaptic signaling as identified by Gene Ontology analysis (0045202). Enrichment plot from gene set enrichment analysis demonstrating downregulation of genes associated with the neuron-to-neuron synapse in GVHD compared with TCD. (E) Quantification of synaptophysin puncta expression in the hippocampus at days 35 (n = 8 mice per group; pooled from 2 independent experiments) and 70 (n = 6-11 mice per group) after transplant. (F) Schematic for the transplantation of WT (PTPxC57Bl/6) or MHC II KO.B6 (H2b) bone marrow depleted of T-cells into irradiated DBA2xF1 (H2Dd) recipients. (G) Clinical scores of transplant recipients (n = 11-12 mice per group; data pooled from 2 independent experiments). (H) Representative images of hematoxylin and eosin staining of skin from recipient mice at day 70 after transplant showing dermal thickening and loss of subcutaneous fat as evidence of scleroderma. Original magnification ×20; scale bar, 300 μm. (I) Time spent floating (immobile) and swimming (mobile) in the forced swim test (n = 4 mice per group) at day 70. (J) Representative confocal images of Iba1+ cells in the hippocampus at day 70 after transplant. Resident microglia express GFP and are indicated with white arrows. Original magnification ×20; scale bar, 50 μm. (K) Representative confocal images demonstrating morphological similarities in the resident microglia phenotype of recipients of MHC II KO TCD BM and WT TCD BM. Original magnification ×100; scale bar, 10 μm. (L-M) Messenger RNA expression of selected genes detected by quantitative real-time polymerase chain reaction in the coronal brain section (L) and hippocampus (M) of transplant recipients at day 70 after transplant. Reported as the fold change of the expression from the WT TCD BM group, calculated relative to the Hprt gene (n = 3-6 mice per group). Data are presented as mean ± standard error of the mean (SEM). Significant differences calculated using 2-way analysis of variance (G) or unpaired Student t test (E,M). *P < .05, ***P < .001. DAPI, 4’,6-diamidino-2-phenylindole; FOV, field of view; NES, normalized enrichment score.

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