INTRODUCTION
The dysfunctional postnatal immune response may lead to rising morbidity and mortality in the early life [
1]. Inflammatory bowel disease (IBD) is a gut disorder featured by chronic inflammation and considered an immune-mediated disease [
2]. IBD has an increasing incidence in children globally and early-onset disease is more severe in infants and young children [
3‐
7]. Studies show that pediatric colitis present failure to thrive and increased percentage of early death [
8,
9]. Also, pediatric-onset IBD increases the risk of thromboembolism, colon cancer, and depressive disorder, which is responsible for higher mortality during adulthood [
4,
10‐
12], but the underlying mechanisms are complicated.
Natriuretic peptide receptor 1 (NPR1) (also known as NPRA or GC-A) is a membrane-bound receptor for atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which is expressed in many cell types and tissues including heart, vasculature, spleen, and gut [
13‐
15]. Upon activation by ANP or BNP, NPR1 stimulates the production of cyclic guanosine monophosphate (cGMP) that promotes vascular tone and volume homeostasis by activation of protein kinase cGMP-dependent 1 (PKG), thereby controlling blood pressure and maintaining cardiovascular homeostasis [
16,
17]. Mice with homozygous deletion of
Npr1 (
Npr1−/−) have shown hypertension, cardiac hypertrophy, and sudden death [
18]. But how NPR1 contributes to vascular aging and related cardiovascular diseases remains not fully illustrated.
We initially generated
Npr1−/− mouse line to assess its contribution to age-related vascular function and observed very high death rate during postnatal period rather than in the adulthood as reported previously [
18]. When close-up examining these mice, we unexpectedly found the morphological changes in the spleen and colon. Thus, we decided to test the hypothesis that
Npr1 deficiency impairs the immune system that further contributes to colitis in different models.
MATERIALS AND METHODS
Mouse Models
Npr1−/− mice were generated in the C57BL/6 background using CRISPR/Cas9 technique (Bioray Laboratories Inc, China). The genotypes were validated by PCR-based strategies. Mice at the age of 4 weeks were used for spleen and colon tissue collection. DSS-induced colitis mice were fed with 6% dextran sodium sulfate (DSS) (Bmassay, China) in water every other day for 7 days. Simultaneously, the control mice were fed with water only. The study was conducted only with the male mice. All procedures in this study were conducted according to the Guide for the Care and Use of Laboratory Animals from the Human Aging Research Institute of Nanchang University.
RNA Sequencing and Data Analysis
Total RNA was isolated from mouse spleen and colon tissues using Trizol reagent (Invitrogen, USA). mRNA was purified by Oligo(dT)-attached magnetic beads and consequently prepared into single-strand circle DNA as the final library. After amplification, DNA nanoballs were made and then loaded onto the patterned nanoarray for sequencing using BGIseq500 platform (BGI-Shenzhen, China). The sequencing results were filtered and the clean reads were mapped to the Mus musculus genome. Afterward, the data were analyzed by Gene Ontology (GO) enrichment analysis. A heatmap was created using Package “pheatmap” (v1.0.8) based on the gene expression value.
Fluorescence-Activated Cell Sorting (FACS) Analysis
The cells from spleen were harvested from WT and Npr1−/− mice and washed with cold PBS buffer. Then, the cells were made a suspension of 1 × 106 cells per milliliter in PBS and divided into two groups. One group of cells were incubated with mouse anti-CD45, anti-CD3, anti-CD8, anti-CD11B, anti-Ly-6G, and anti-NK1.1 (BD Biosciences, USA) at 4 °C for 15 min. The other group of cells were first incubated with mouse anti-CD3, anti-CD4, and anti-CD8 at 4 °C for 15 min and then blocked with 1 × TF Fix/Perm working solution for 40 min. Subsequently, the cells were incubated with mouse anti-FOXP3 (BD Biosciences, USA). Finally, the cells from both groups were washed with PBS and inspected using FACSVerse flow cytometer (BD Biosciences, USA). The scatter plots were created and analyzed by FlowJo v10.0.
8-Br-cGMP Treatment
For administration of 8-Br-cGMP (MedChemExpress, China), two groups of Npr1−/− mice (n = 4 per group) at the age of 8 weeks were used. The experimental group received 0.1 mmol/L of 8-Br-cGMP in saline by tail-vein injection each week for 4 consecutive weeks. The control group received saline with the same procedure. For DSS-induced colitis mouse model with administration of 8-Br-cGMP, WT mice (8 weeks) were divided into three groups (n = 10 per group) including two experimental groups (DSS + saline and DSS + 8-Br-cGMP) and one control group. Prior to the induction of colitis, mice from each group were injected with one dose of 0.1 mmol/L of 8-Br-cGMP in saline or saline only through tail vein, respectively.
Blood Pressure Monitor
The measurement of systolic blood pressure was performed in mice by non-invasive tail-cuff device IITC (Life Science, USA). Prior to recorded measurement, mice underwent adaptation training for 1 week. Afterward, the blood pressure was measured with the inflation and deflation reading at an interval of 15 s, and 10 consecutive readings were recorded. The monitor was carried out for 3 consecutive days for each mouse.
RNA Isolation and RT-PCR Analysis
The spleen and colon tissues from WT and Npr1−/− mice were isolated and homogenized in TRIzol reagent (Bmassay, China) for extracting RNA. cDNA was obtained from 500 μg of total RNA by reverse transcriptase kit (Zomanbio, China). Levels of mRNA expression of Il6, Gapdh were analyzed by RT-PCR. The primer sequences were as follows:
-
mouse-β-actin-F: 5ʹ-GCCGACAGGATGCAGAAGGAGATCA-3ʹ.
-
mouse-β-actin-R: 5ʹ-AAGCATTTGCGGTGGACGATGGA-3ʹ.
-
mouse-Il6-F: 5ʹ-GTCAGGGGTGGTTATTGC-3ʹ.
-
mouse-Il6-R: 5ʹ-TCATCACTGGTCTTTTGGAG-3ʹ.
Enzyme-Linked Immunosorbent Assay
Serum was collected from WT and Npr1−/− mice at the age of 4 weeks. IL1B and IL6 were measured by duoset enzyme-linked immunosorbent assay kits (Proteintech, China) according to the manufacturer manual.
Histological Analysis
The ascending colon, the most commonly affected tissues [
19], from mice was fixed with 4% paraformaldehyde for 24 h, and then embedded in paraffin. The sample sections were prepared in 5 μm thickness and later analyzed by H&E staining.
Immunofluorescent Staining
Tissue fixation was carried out in 4% paraformaldehyde solution for 15 min, and then permeabilization in PBS with 0.25% Triton X-100 for 10 min. After blocking, the samples were incubated with primary antibodies NPR1 (1:100, Thermo, USA), CD3 (1:200, Proteintech, China), CD4 (1:100, Abcam, USA), CD8 (1:500, Abcam, USA) at 4 °C overnight, and followed by Cyanine3 Donkey anti-rabbit IgG (1:200, Biolegend, USA) for 1 h in the dark at room temperature. Hoechst 33,342 (1:100, Beyotime, China) was used for nuclei staining. Images were taken with Zeiss confocal microscope (LSM800, Zeiss, Germany).
DISCUSSION
In this study, we found that null function of Npr1 disrupts the immune response that further leads to colitis in mice at the early life stages.
Pediatric IBD at very early onset accounts for about 25% of the cases [
21], which results in growth retardation and increased mortality in later life [
10,
22]. The pathogenesis of IBD is multifaceted with the involvement of genetic effect and immune response [
23]. Here, we demonstrate in both
Npr1−/− and DSS mouse models that loss of
Npr1 disturbs the immune response with shifted population of immune cells, which causes experimental colitis during postnatal period.
In our study,
Npr1−/− mouse model was initially utilized for evaluation of vascular aging. Unexpectedly, we found abnormal morphology of spleen and colon. To investigate the underlying mechanism for this phenotype, we conducted RNA sequencing for these two tissue samples. Based on the data analysis, we found a diverse set of DEGs for the spleen and colon of
Npr1−/− mice, which are linked to the signaling pathways of the immune response. Among those up-regulated DEGs, PLA2G1B has been reported to decline the survival rate of CD4
+ T cells
in vitro and induce dysfunction of CD4
+ T cells but CD8
+ T cells, which inhibited the sensitivity of CD4
+ T cells to inflammatory cytokines such as IL2, IL4, and IL7 [
24]. Another gene
LGALS9 is an important molecule involved in the immune response and development. Increased
LGALS9 expression in humans is positively correlated with augmentation of T-cell markers and proinflammatory cytokines such as IL1B and IL6 in the affected intestines, which also has positive correlation to the severity of colitis [
25]. Conversely, lack of
Lgals9 in mice showed less gut inflammation reflected by colonic morphology and histology [
25]. In a cluster of down-regulated DEGs,
Jak3 is the most studied gene involved in immunological function.
Jak3 deficiency exacerbates colonic inflammation with shortened colon, marked mucosal impairment, elevated neutrophil, and increased Il6 [
26,
27], which is consistent to the phenotype observed in our
Npr1−/− mouse model. In mice lacking
Jak3, ENaC (epithelial Na
+ channel) activity is damaged and accompanied by sodium depletion in the colonic epithelium [
28]. Therefore, NPR1 may interplay with these genes during colonic inflammation in the postnatal period of mice.
Spleen is the largest immune organ. It serves as a critical location for immune surveillance and response, while it is not totally developed until the age of 2 years in human [
1,
29], equivalent to 2–3 weeks in mice [
30]. Available evidence indicates that the postnatal life is a critical time frame for immune development and homeostasis [
31]. The impairment of immune response in the early neonatal period may contribute to immune-related or inflammatory disorder [
32]. We found enlarged spleen, increased spleen weight, elevated spleen index, and altered immune cell profiles in
Npr1−/− mice at postnatal stage. An elevated population of leukocytes and neutrophils is a strong evidence for splenomegaly caused by immune cell infiltration. In the early neonatal period, neutrophils undergo maturation in functionality [
1]. Of note, neutrophils are the important population of the innate leukocytes, which release some cytokines such as Il1b [
33]. Loss of granulocyte–macrophage colony-stimulating factor results in highly expressed Il1b and Il6 in the spleen and increased mortality in mice [
34]. Il6 also mediates recruiting leukocytes to the spleen and enhances Il6 level, causing early postnatal death [
35,
36]. Consistent with the results from previous reports, we observed that IL6 level was significantly increased in the spleen of
Npr1−/− mice. Similar to the spleen, increased IL1B and IL6 were found in the colon of
Npr1−/− mice. Our findings support that impaired mucosal immune response promotes local proinflammatory cytokine, causing colonic inflammation [
37,
38].
It is well documented that T cells are essential for the immune response [
39]. The immune response is modulated by T-cell activation pathway [
23,
32]. Through cytofluorimetric analysis, we found that the populations of CD3
+ T cells were elevated but CD4
+ T cells were decreased in the spleen from
Npr1−/− mice. CD3
+ T cells comprise CD4
+ and CD8
+ T cells that are important for assessing immunological function and pathological alterations [
40]. CD3
+ T cell is one of the subsets in the splenic leukocytes, accounting for one-fourth of all splenocytes [
34,
41]. Studies show that dysfunction of CD3
+ mediates gut inflammation with higher Il6 and Il1b levels, while anti-CD3 antibody remarkably decreases inflammatory phenotype by inhibiting the production and secretion of proinflammatory cytokine [
42,
43]. Furthermore, we also found that the population of CD4
+ T cells was reduced in the colon tissue from
Npr1−/− and DSS mice. Administration of 8-Br-cGMP, a downstream activator of NPR1, restored these immune-cell populations disturbed in
Npr1−/− mice. It has been reported that CD4
+ T cells with large population are presented in the lamina propria of gut, which are important for immune system homeostasis and inflammation [
44]. The decline of CD4
+ cell amount and function predisposes inflammation and infection [
45]. Stimulation of CD4
+ T cells to produce IL-10 inhibits the inflammatory response in the process of colitis [
46]. In IBD patients and colitis mice, the population of CD4
+ T cells is significantly decreased [
47,
48]. A previous study has shown that NPR1 is expressed in CD4
+ T cells, which controls the development of Th17 cells by ANP stimulation [
49]. These suggest that NPR1 regulates the immune response mediated by restructuring T-cell subpopulations to promote early development of colitis.
Our data from two mouse models demonstrate that
Npr1 deficiency causes a colonic inflammation phenotype accompanied with growth retardation, resembling pediatric onset colitis [
8,
9,
50]. When administrating PKG activator 8-Br-cGMP, the phenotype of colitis was attenuated in
Npr1−/− and DSS mice. A very recent study has reported that decreased levels of ANP and NPR1 exacerbate ulcerative colitis in humans and the experimental mice. Mechanistically, this inflammatory response is mediated by ANP stimulator of interferon gene (STING) cascade in the process of colitis [
15].
In the clinical perspective, pediatric IBD patients seem to have more serious disease course with adverse consequences including perianal fistulae, pancolitis, growth failure, psychosocial disorder, and resistant to medical treatment [
4,
51,
52]. For the immune-related disease, the biologic therapy has been emerged as an improved therapeutic approach [
53]. Early intervention with biologic therapies provides more effective outcomes for pediatric IBD [
4,
54]. The biologic agents include monoclonal antibodies and fusion proteins, such as anti-TNF-α, anti-integrin, and anti-interleukin 12/23 [
53]. Our study provides more supportive evidence for intervention strategy in IBD. In sum, our findings highlight that loss of
Npr1 possibly disturbs the immune response leading to colitis in the early life. Importantly, NPR1 may become a potential therapeutic target for anti-colonic inflammation beyond regulating vascular homeostasis such as blood pressure.
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