IL-12p40 deletion aggravates lipopolysaccharide-induced cardiac dysfunction in mice

Abstract

Background: Cardiac dysfunction is one of the most common complications of sepsis and is associated with the adverse outcomes and high mortality of sepsis patients. IL-12p40, the common subunit of IL-12 and IL-23, has been shown to be involved in a variety of inflammation-related diseases, such as psoriasis and inflammatory bowel disease. However, the role of IL-12p40 in lipopolysaccharide (LPS)-induced cardiac dysfunction remains obscure. This study aimed to explore the role of IL-12p40 in LPS-induced cardiac dysfunction and its potential mechanisms.

Methods: In this study, mice were treated with LPS and the cardiac expression of IL-12p40 was determined. Then, IL-12p40^-/- mice were used to detect the role and mechanisms of IL-12p40 in LPS-induced cardiac injury. In addition, monocytes were adoptively transferred to IL-12p40^-/- mice to explore their effects on LPS-induced cardiac dysfunction.

Results: The results showed that cardiac IL-12p40 expression was significantly increased after treated with LPS. In addition, IL-12p40 deletion significantly aggravated LPS-induced cardiac dysfunction, evidenced by the increased serum levels of cardiomyocyte injury markers and heart injury scores, as well as by the deteriorated cardiac function. Moreover, IL-12p40 deletion increased LPS-induced monocyte accumulation and cardiac expression of inflammatory cytokines, as well as enhanced the activation of the NF-κB and MAPK pathways. Furthermore, adoptive transfer WT mouse monocytes to IL-12p40^-/- mice alleviated LPS-induced cardiac dysfunction and decreased the phosphorylation of p65.

Conclusion: IL-12p40 deletion significantly aggravated LPS-induced cardiac injury and cardiac dysfunction in mice by regulating the NF-κB and MAPK signaling pathways, and this process was related to monocytes. Therefore, IL-12p40 show a protective role in SIC, and IL-12p40 deficiency or anti-IL-12p40 monoclonal antibodies may be detrimental to patients with SIC.

Keywords: IL-12p40 deletion; LPS; cardiac dysfunction; monocytes; sepsis.

Figure 1

LPS treatment increases cardiac IL-12p40 expression in mice. (A) Western blot analysis of IL-12p40 protein levels in the hearts of each group (n = 5). (B) qRT-PCR analysis of IL-12p40 mRNA expression levels in the hearts of each group (n = 6). *P < 0.05 compared with the Saline group.

Figure 2

IL-12p40 deletion aggravates LPS-induced cardiac injury and cardiac dysfunction in mice. (A,B) The levels of LDH and CK-MB in serum of mice in each group (n = 6). (C,D) qRT-PCR analysis of ANP and BNP mRNA expression levels in the hearts of mice in each group (n = 6). (E,F) Echocardiography analysis of LVEF and LVFS of mice in each group (n = 6). (G) HE and masson's trichrome stainings and the quantitative results of heart tissues in each group (n =6; scale bar, 100 μm). *P < 0.05 compared with the Saline group. ^#P < 0.05 compared with the LPS+WT group.

Figure 3

IL-12p40 deletion increases the phosphorylation of NF-κB and MAPK signaling pathways and aggravates cardiac inflammation in mice treated with LPS. (A) Western blot analysis of T-ERK, p-ERK, T-p38, p-p38, T-JNK, p-JNK, T-STAT1, p-STAT1, T-P65, and p-P65 protein levels in the hearts of mice and the ratios of p-ERK/T-ERK, p-p38/ T-p38, p-JNK/T-JNK, p-P65/T-P65, and p-STAT1/T-STAT1 in each group (n = 5). (B) qRT-PCR analysis of IL-1β, IL-6, IL-17, TNF-α and INF-γ mRNA expression levels in the hearts of mice in each group (n =6). *P < 0.05 compared with the Saline group. ^#P < 0.05 compared with the LPS+WT group.

Figure 4

IL-12p40 deletion increases monocytes infiltration. (A) The immunofluorescence analysis of CD14 and CD16 in heart sections of each group (n = 6; scale bar, 50 μm). (B) Flow cytometry analysis of CD14++CD16+monocyte percents in spleen tissues of mice in each group (n = 6). *P < 0.05 compared with the Saline group. ^#P < 0.05 compared with the LPS+WT group.

Figure 5

IL-12p40 deletion aggravates LPS-induced myocardial apoptosis. (A) Western blot analysis of Bax, Bcl-2 and C-caspase-3 protein levels in heart tissues of each group (n = 5). (B) TUNEL staining and the quantitative results of heart tissues in each group (n = 6; scale bar, 50 μm). *P < 0.05 compared with the Saline group. ^#P < 0.05 compared with the LPS+WT group.

Figure 6

WT monocyte adoptive transfer alleviates cardiac injury in LPS-treated IL-12p40^−/− mice. (A,B) The levels of LDH and CK-MB in serum of mice in each group (n = 6). (C,D) qRT-PCR analysis of ANP and BNP mRNA expression levels in the hearts of mice in each group (n = 6). (E,F) Echocardiography analysis of LVEF and LVFS of mice in each group (n = 6). (G) HE and masson's trichrome stainings and the quantitative results of heart tissues in each group (n =6; scale bar, 100 μm). *P < 0.05 compared with the KO+WT Mono group. ^#P < 0.05 compared with the KO+WT Mono+LPS group.

Figure 7

WT monocyte adoptive transfer alleviates myocardial apoptosis in LPS-treated IL-12p40^−/− mice. (A) Western blot analysis of Bax, Bcl-2 and C-caspase-3 protein levels in heart tissues of each group (n = 5). (B) TUNEL staining and the quantitative results of heart tissues in each group (n =6; scale bar, 50 μm). *P < 0.05 compared with the KO+WT Mono group. ^#P < 0.05 compared with the KO+WT Mono+LPS group.

Figure 8

WT monocyte adoptive transfer reduces cardiac inflammation in LPS-treated IL-12p40^−/− mice. (A) The immunofluorescence analysis of p-p65 in heart sections of each group (n = 6; scale bar, 50 μm). (B) qRT-PCR analysis of IL-1β, IL-6, IL-17, TNF-α, and INF-γ mRNA expression levels in the hearts of mice in each group (n =6). *P < 0.05 compared with the KO+WT Mono group. ^#P < 0.05 compared with the KO+WT Mono+LPS group.