Chronic stress boosts systemic inflammation and compromises antiviral innate immunity in Carassius gibel

Abstract

It is generally considered that stress causes decreased immune function and render fish vulnerable to infection and diseases. However, the molecular mechanisms between stress responses and susceptibility to infections, especially viral diseases, in fish remain unknown. Understanding and monitoring the biological consequences and mechanisms underlying stress responses in fish may contribute to the improvement of animal welfare and production efficiency. In this study, long-term exposure to a variety of stressors, including chasing, overcrowding, restraint stress, and air exposure mimicking chronic stresses, in aquaculture practices was conducted in Carassius gibel to investigate the consequences of chronic stress on inflammation and antiviral capability. With the continuation of stimulation, experimental fish gradually became insensitive to the stress of net chasing and feeding with the accompaniment of upregulated gene expressed in the HPI axis and elevated levels of stress hormones. As expected, stress-induced hyperglycaemia with a decrease in the insulin signaling pathway and altered gene expression in glycolysis and gluconeogenesis, suggesting the disturbance of glycometabolism. Importantly, a link between intestinal homoeostasis and systemic low-grade inflammation in stressed C. gibel was observed, implying crosstalk among the brain, intestine, and other organs. Furthermore, the compromised antiviral capability with impaired antiviral innate immunity in stressed fish was confirmed by RNA sequencing and infection with Cyprinid herpesvirus 2 (CyHV-2), promoting the understanding of enhanced susceptibility to viral infection in stressed fish.

Keywords: CyHV-2; antiviral immune; chronic stress; inflammation; stress.

Figure 1

Chronic stress activates the HPI axis in gibel carp. (A) Schematic representation of the chronic stress model and sampling schedule. (B) Quantification of fish struggling and swimming behaviour after 21 days of twice daily stimulation. (C) ELISA analysis of serum C-reactive protein (CRP) level in gibel carp from healthy controls and stressed individuals (n = 6) after 21 days of twice daily stimulation. (D) ELISA analysis of serum cortisol levels in gibel carp from healthy controls (n = 6) and stressed individuals (n = 6). (E) RT-qPCR analysis of CRF mRNA levels in gibel carp hypothalamus from healthy controls (n = 6) and stressed individuals (n = 6). (F) RT-qPCR analysis of POMC mRNA levels in gibel carp hypothalamus from healthy controls (n = 6) and stressed individuals (n = 6). (G, H) RT-qPCR analysis of GR mRNA levels in gibel carp hypothalamus and liver, respectively, from healthy controls (n = 6) and stressed individuals (n = 6). All graphs show the means ± SEMs for individual fish, and significant differences from the control are indicated by asterisks (**P < 0.01, ***P ≤ 0.001). All graphs show the means ± SEMs for individual fish. Blue represents healthy control, and red represents stressed fish.

Figure 2

Chronic stress disturbs the glycometabolism of gibel carp. (A) Body weight of healthy controls and stressed fish. (B) ELISA analysis of fasting blood glucose levels in crucian from healthy controls (n = 6) and stressed individuals (n = 6). (C) ELISA analysis of serum insulin levels in gibel carp from healthy controls (n = 6) and stressed individuals (n = 6). (D) RT-qPCR analysis of insulin receptor mRNA levels in gibel carp livers from healthy controls (n = 6) and stressed individuals (n = 6). (E) RT-qPCR analysis of IRS mRNA levels in gibel carp livers from healthy controls (n = 6) and stressed individuals (n = 6). (F) RT-qPCR analysis of PFK and PK mRNA levels in gibel carp livers from healthy controls (n = 6) and stressed individuals (n = 6). (G) RT-qPCR analysis of PEPCK, FBP, and G6Pase mRNA levels in gibel carp livers from healthy controls (n = 6) and stressed individuals (n = 6). All graphs show the means ± SEMs for individual fish, and significant differences from the control are indicated by asterisks (*P < 0.05, **P < 0.01, ***P ≤ 0.001). Blue represents heathy control and red represents stressed fish.

Figure 3

Chronic stress causes intestinal damage in gibel carp. (A) Clinical pathologica of intestine in healthy controls and stressed individuals (n = 2). On the left is an overall view of intestinal pathology, and the image on the right is the location specified by the black arrow on the left. (B) Serum levels of lipopolysaccharide (LPS) determined by ELISA from healthy controls (n = 6) and stressed individuals (n = 6).(C) Serum levels of intestinal fatty acid-binding protein (iFABP) determined by ELISA from healthy controls (n = 6) and stressed individuals (n = 6). (D) RT-qPCR analysis of occludin mRNA levels in gibel carp gut from healthy controls (n = 6) and stressed individuals (n = 6). (E) RT-qPCR analysis of ZO-1 mRNA levels in gibel carp guts from healthy controls (n = 6) and stressed individuals (n = 6). All graphs show the means ± SEMs for individual fish, and significant differences from the control are indicated by asterisks (*P < 0.05, **P < 0.01). Blue represents healthy control and red represents stressed fish.

Figure 4

Chronic stress causes an imbalance in the intestinal microbiota in gibel carp. (A) The α-diversity comparison of the intestinal microbiota between healthy controls and stressed individuals (n = 10). The Chao1 index, Shannon index and Simpson index were calculated. Significant differences are indicated by asterisks (**P≤ 0.01, ***P≤0.001). (B) Principal component analysis (PCA) based on Bray-curtis distance were performed. (C) Multivariate dispersions (PERMDISP) was conducted to assess the distances from the group centroid. (D) Enriched microbes from healthy controls (Control, left panel, n = 10) and stressed fish (Stress, right panel, n = 10).

Figure 5

Chronic stress triggers systemic inflammation in gibel carp. (A) RT-qPCR analysis of inflammatory-related gene (IL-1β; IL-6; IL-8; TNFα; IFN-γ) mRNA levels in gibel carp trunk kidney, spleen, liver and intestine from healthy controls (n = 6) and stressed individuals (n = 6). (B) RT-qPCR analysis of inflammatory-related gene (IL-1β; IL-6; IL-8; TNFα; IFN-γ) mRNA levels in gibel carp trunk kidney from healthy controls (n = 6) and stressed individuals (n = 6) in different time points. All graphs show the means ± SEMs for individual fish, and significant differences from the control are indicated by asterisks (*P < 0.05, **P < 0.01). Blue represents heathy control and red represents stressed fish.

Figure 6

DEGs and pathway enrichment in the intestine upon chronic stress. (A) Dot depicting the top 20 enriched pathways based on KEGG pathway enrichment analysis of DEGs. The pathway enrichment statistics were performed by Fisher’s exact test, and those with a corrected P value < 0.05 were considered the most significant pathways. (B) Fold changes in the indicated genes related to intestinal homeostasis after CyHV-2 infection. The results are presented as the ratio of DEGs from stressed fish compared with healthy controls. Significantly, many DEGs were related to inflammation (Cluster A), glycolysis-related genes (Cluster B) and gluconeogenesis-related genes (Cluster C). (C) The different catalogues of DEGs in antiviral genes.

Figure 7

Chronic stress compromises the antiviral innate immunity of gibel carp. (A) Schematic representation of the viral infection schedule. Group A, unstressed fish infected with CyHV-2. Group B, stressed fish with mock infection. Group C, stressed fish infected with CyHV-2. (B) Survival curve of gibel carp upon CyHV-2 infection between groups A, B and C. (C) RT-qPCR analysis of CyHV-2 DNA copies from moribund fish with or without stress according to the standard curve based on the series of diluted plasmids containing the DNA polymerase gene. The results were expressed as copies per µg of total DNA (copies/µg DNA). (D) RT-qPCR analysis of CyHV-2 DNA copies from moribund fish and asymptomatic individuals collected from ponds. (E) ELISA analysis of serum cortisol levels in gibel carp from moribund fish and asymptomatic individuals.