Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice - PubMed (original) (raw)

Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice

Boxiang Gui et al. Virol J. 2017.

Abstract

Background: H9N2 influenza viruses circulate globally and are considered to have pandemic potential. The hyper-inflammatory response elicited by these viruses is thought to contribute to disease severity. Calcitriol plays an important role in modulating the immune response to viral infections. However, its unknown whether calcitriol can attenuate the inflammatory response elicited by H9N2 influenza virus infection.

Methods: Human lung A549 epithelial cells were treated with calcitriol (100 nM) and then infected with an H9N2 influenza virus, or infected and then treated with calcitriol (30 nM). Culture supernatants were collected every 24 h post infection and the viral growth kinetics and inflammatory response were evaluated. Calcitriol (5 mg/kg) was administered daily by intraperitoneal injection to BABL/c mice for 15 days following H9N2 influenza virus infection. Mice were monitored for clinical signs of disease, lung pathology and inflammatory responses.

Results: Calcitriol treatment prior to and post infection with H9N2 influenza significantly decreased expression of the influenza M gene, IL-6, and IFN-β in A549 cells, but did not affect virus replication. In vivo, we found that calcitriol treatment significantly downregulated pulmonary inflammation in mice 2 days post-infection, but increased the inflammatory response 4 to 6 days post-infection. In contrast, the antiviral cytokine IFN-β was significantly higher in calcitriol-treated mice than in the untreated infected mice at 2 days post-infection, but lower than in untreated infected mice on days 4 and 8 post-infection. The elevated levels of pro-inflammatory cytokines and the decreased levels of antiviral cytokine are consistent with the period of maximum body weight loss and the lung damage in calcitriol-treated mice.

Conclusions: These results suggest that calcitriol treatment might have a negative impact on the innate immune response elicited by H9N2 infection in mice, especially at the later stage of influenza virus infection. This study will provide some novel insights into the use of calcitriol to modulate the inflammatory response elicited by influenza virus infection in humans.

Keywords: Calcitriol; Inflammation response; Influenza.

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Figures

Fig. 1

Fig. 1

The effects of calcitriol on A549 cells. a Effect of calcitriol treatment on virus growth kinetic in A549 cells. Cell culture supernatants were harvested every 24 h from infected cells and the viral load was titrated using the TCID50 assay. Real-time PCR was used to measure the effect of calcitriol treatment on the mRNA expression levels of the viral M gene (b), IL-6 (c), and IFN-β (d) in A549 cells. β-actin was used as an internal control. The data are expressed as mean ± SEM of triplicate samples

Fig. 2

Fig. 2

Body weight change in mice. White circles indicate mice inoculated with noninfectious allantoic fluid and treated with sterile saline from days 1 to 15 post-infection; Black circles indicate mice inoculated with noninfectious allantoic fluid and injected with calcitriol (5 mg/kg) intraperitoneally; White triangles indicate mice infected with the H9N2 virus and treated with calcitriol (5 mg/kg) from days 1 to 15 post-infection; Black triangles indicate mice infected with the H9N2 virus and treated with sterile saline from days 1 to 15 post-infection. Data presented are the mean ± SEM for 6 mice per group

Fig. 3

Fig. 3

Changes in lung histopathology of calcitriol treated mice. Lung samples were harvested from calicitriol (5 mg/kg) treated H9N2 infected mice on days 2 (A), 4 (B), 6 (C), and 8 (D) post infection. Lung tissues were harvested from untreated H9N2 infected mice on days 2 (E), 4 (F), 6 (G), and 8 (H) post-infection. Original magnification 100 ×

Fig. 4

Fig. 4

Expression of the H9N2 M gene in the lungs of infected mice after treatment with calcitriol. Mice were inoculated intranasally with 100 μL of allantoic fluid containing influenza A/mallard/Jiangxi/39/2011 (H9N2; 1 × 106 50% egg infection dose). Lung tissues were harvested on the indicated days post-infection. The relative quantification (RQ) values for the H9N2 M gene, are expressed as fold-change. RQ values were obtained using the 2-ΔΔCt method normalizing to the RNA expression levels of the β-actin gene. The average threshold cycle (ΔCt) values of the untreated infected group were used for calibration

Fig. 5

Fig. 5

Expression of pro-inflammatory and antiviral cytokines in calcitriol treated mice. Expression levels of Interleukin (IL)-6 (A), tumor necrosis factor (TNF)-α (B), IL-2 (C), IL-4 (D) and IFN-β (E) were measured by real-time PCR in the lungs of mice treated with calcitriol. The relative quantification (RQ) values for these genes in calcitriol-treated infected mice were calculated, and the average threshold cycle (ΔCt) values of the infected control group were used for calibration

Fig. 6

Fig. 6

VDR expression in the lungs after calcitriol treatment. The relative quantification (RQ) values for vitamin D receptor (VDR) mRNA expression were calculated using the 2-ΔΔCt method normalized to the β-actin gene. The average threshold cycle (ΔCt) values of the infected control group were used for calibration

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