Null Function of Npr1 Disturbs Immune Response in Colonic Inflammation During Early Postnatal Stage

Abstract

Natriuretic peptide receptor 1 (NPR1) is conventionally known as a regulator of vascular homeostasis. Here, we generated an Npr1 knockout mouse model with CRISPR/Cas9 technology and found that homozygous mice (Npr1^-/-) exhibited weight loss and poor survival rate during early postnatal stage. Careful examination revealed unexpectedly that Npr1^-/- mice developed colitis characterized by shortened colon, evident colonic mucosal damage, increased histopathological score, and higher colonic expression of proinflammatory cytokines interleukin-1B (IL1B) and -6 (IL6). RNA-sequencing analysis revealed that differentially expressed genes were prominently enriched in the biological pathways related to immune response in both spleen and colon of Npr1^-/- mice. Cytofluorimetric analysis demonstrated that leukocytes in the spleen were significantly increased, particularly, the populations of neutrophil and CD3⁺ T cell were elevated but CD4⁺ T cells were decreased in Npr1^-/- mice. Administration of 8-Br-cGMP, a downstream activator of NPR1, restored these immune-cell populations disturbed in Npr1^-/- mice and lessened the colitis-related phenotypes. To validate the involvement of Npr1 in colitis, we examined another mouse model induced by dextran sodium sulfate (DSS) and found a decreased Npr1 expression and shifted immune-cell populations as well. Importantly, 8-Br-cGMP treatment exhibited a similar effect in the restoration of immune-cell populations and attenuation of colonic inflammation in DSS mice. Our data indicate that loss of Npr1 possibly interrupts immune response, which is critical to the pathogenesis of colitis in the early life.

Keywords: CD4+ T cells; Npr1; colitis; colonic inflammation; immune response.

Fig. 1

Characteristics of Npr1^−/− mice. a Schematic diagram of the construction of Npr1^−/− mice. b PCR analysis for mouse genotyping and DNA sequencing. DNA bands at 488 and 331 bp represent normal and targeted Npr1 allele, respectively. c Immunofluorescent staining for NPR1 expression (red) and nuclei (blue) in the colon tissue sections from WT and Npr1^−/− mice (4 weeks, n = 4–5). Quantitative data obtained by mean fluorescence intensity of each sample. d Body weight curves in WT and Npr1^−/− mice (n = 7). e Blood pressure in WT and Npr1^−/− mice (n = 4). f Survival curves of WT and Npr1^−/− mice (n = 31–35). Values are mean ± S.D. *** p < 0.001.

Fig. 2

Early onset colitis in Npr1^−/− mice at the age of 4 weeks. a The colon length from WT and Npr1^−/− mice (n = 4–6). b IL1B and IL6 expressions in the colon from WT and Npr1^−/− mice (n = 4). c Histopathological changes in the colon tissue sections from WT and Npr1^−/− mice (n = 4–5). d The colon length from Npr1^−/− mice treated with 8-Br-cGMP or saline as a control (n = 4). e IL1B and IL6 expression in the colon from Npr1^−/− mice treated with 8-Br-cGMP or saline (n = 4). f Histopathological changes in the colon tissue sections from from Npr1^−/− mice treated with 8-Br-cGMP or saline (n = 4). Values are mean ± S.D. * p < 0.05; *** p < 0.001.

Fig. 3

Identification of DEGs in the spleen and colon from Npr1^−/− mice at the age of 4 weeks. a GO enrichment analysis of DEGs (log2 fold-change > 1.5 and p < 0.05) in the spleen from WT and Npr1^−/− mice (n = 3). The rich ratio is the ratio of the DEG number and the number of all genes in a certain enrichment pathway. The dot size denotes the number of DEGs, the colors denotes the adjusted Q-value range. b A heat map for immune-related DEGs in the spleen from WT and Npr1^−/− mice (n = 3). The color intensity indicates the relative expression levels of up-regulated (red) and down-regulated (blue) DEGs. c GO enrichment analysis of DEGs in the colon from WT and Npr1^−/− mice (n = 3). d A heat map for immune-related DEGs in the colon of WT and Npr1^−/− mice (n = 3).

Fig. 4

Changes of immune cell composition in the spleen from Npr1^−/− mice at the age of 4 weeks. a The spleen weight and index from WT and Npr1^−/− mice (n = 7). Spleen index was generated as spleen weight (mg)/body weight (g). b Expression of Il6 mRNA in the spleen from WT and Npr1^−/− mice (n = 3). c Leukocytes defined as CD45⁺ in the splenic immune cells from WT and Npr1^−/− mice (n = 4–6). Population of neutrophils featured by d CD11B⁺LY-6G⁺, e NK cells, and f T cells featured by CD3⁺ from leukocytes in the spleen from WT and Npr1^−/− mice (n = 4–6). g Population of CD3⁺ T cells in the splenic cells from WT and Npr1^−/− mice (n = 4–5). h Population of CD4⁺ T and CD8⁺ T differentiated from CD3⁺ T cells from WT and Npr1^−/− mice. i Population of Treg cells from CD4⁺ T cells in WT and Npr1^−/− mice. Values are mean ± S.D. * p < 0.05; *** p < 0.001; ns, nonsignificant.

Fig. 5

Restructuring of T-cell subpopulation in the colon tissue from Npr1^−/− mice. Immunofluorescent staining for CD3⁺, CD4⁺, and CD8⁺ in a WT and Npr1^−/− mice (4 weeks, n = 4–5) and b Npr1^−/− mice treated with 8-Br-cGMP or saline as a control (12 weeks, n = 4). Values are mean ± S.D. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, nonsignificant.

Fig. 6

NPR1 expression in DSS-induced colitis mouse model. a The colon length from control and DSS-treated mice (8 weeks, n = 3–4). b Npr1 mRNA expression in the colon from control and DSS-treated mice (8 weeks, n = 3–4). c Immunofluorescent staining for NPR1 expression (red) and nuclei (blue) in the colon tissue sections from control and DSS-treated mice (8 weeks, n = 3). Quantitative data obtained by mean fluorescence intensity of each sample. d Histopathological changes in the colon tissue sections from control, DSS-treated mice, and DSS-treated mice with 8-Br-cGMP administration (9 weeks, n = 4–7). Values are mean ± S.D. * p < 0.05; *** p < 0.001.

Fig. 7

Restructuring of T-cell subpopulation in the colon tissue from DSS-treated mice. Immunofluorescent staining for CD3⁺, CD4⁺, and CD8⁺ in control, DSS-treated mice, and DSS-treated mice with 8-Br-cGMP administration (9 weeks, n = 7). Values are mean ± S.D. * p < 0.05; ** p < 0.01; ns, nonsignificant.