Post-Treatment Of ISO-1 Attenuates the Expression of MIF And Inflammatory Response to Influenza A (H9N2) Infection on A549 Cells

Post-Treatment Of ISO-1 Attenuates the Expression of MIF And Inflammatory Response to Influenza A (H9N2) Infection on Cells. Abstract H9N2 Avian influenza viruses generally cause mild to moderate infections, and occasionally cause clinical respiratory diseases in humans. Macrophage Migration Inhibitory Factor (MIF), has been evaluated as a pro-inflammatory cytokine, anterior pituitary-derived hormone, and glucocorticoid-induced immune-modulator, associated with inflammatory and immunology reactions of lung diseases. ISO-1[(S, R)-3-(4-hydroxyphenyl)-4-5-isoxazole acetic acid methyl ester] is a specific antagonist of macrophage Migration Inhibition Factor (MIF). The question, whether ISO-1-treatment could modulate the inflammatory response induced by H9N2 avian influenza virus, is unclear. This study was undertaken to investigate the chemokine /cytokine response to influenza A infection by post-treatment of ISO-1 on human lung alveolar epithelial cells (A549 cells). The avian influenza virus H9N2 (10-4.46TCID50/100μl) was incubated with A549 cells for 24h, then treated with 100ng/μl ISO-1 and incubated with the A549 cells together at 37℃ under 5% CO2 for 1h. The cell culture was collected and subjected to RNA extraction. The virus titer, mRNA expression of MIF and other cytokines were analyzed by real-time quantitative-PCR (qRT-PCR). Treatment with ISO-1 after H9N2 infection could attenuate mRNA expression levels of MIF; tumor-necrosis-factor-alpha (TNF-α), interferon-β (IFN-β), nucleus–κB (NF-κB), leukocyte interleukin (CXCL8), interleukin-6 (IL-6), and influenza M gene significantly, which were up-regulated by H9N2 infection. This study demonstrates that MIF small-molecule antagonist ISO-1 treatment has a significant anti-inflammatory effect on H9N2virus-induced A549 cell inflammation, which further supports the investigation of ISO-1 to address the inflammatory disease induced by the avian influenza viruses.


Introduction
The H9N2 subtype of influenza viruses have continues to be the most abundant influenza viruses isolated from chickens in China.
Multiple studies show that the H9N2 viruses have undergone continuous and rapid evolution as well as wide re-assortment with other subtypes of avian influenza viruses, such as the H9N2 viruses might be the donors of the "internal" genes of the lethal H5N1 viruses, H5N6, H10N8, and H7N9 [1][2][3][4][5][6]. Furthermore, the pressure of vaccine and natural immunity may contribute to substantial virus evolution, and the rapid evolution also leads to enhanced pathogenicity for the virus in mammals and poultry [7]. Mouseadapted H9N2 influenza viruses could replicate efficiently and be transmitted among mice through both contact and respiratory droplet routes [8]. Events show that H9N2 influenza viruses are capable of infecting mammals including humans from 1999 [1].

Influenza virus infections lead to the interaction of virus and host
immune responses including excessive inflammatory responses in the form of cytokine storm. The increased expression of proinflammatory cytokines has an important role both in the adaptive and innate immune responses [9]. Macrophage Migration Inhibitory Factor (MIF) is known to be an important regulator of the innate and adaptive immune systems and inflammatory responses, which has been implicated in several inflammatory diseases, such as sepsis, arthritis, diabetes, and lung injury [10].
MIF is produced by a variety of inflammatory and immune cells; its expression is regulated by several different stimuli such as LPS or virus infection [11]. Recent findings demonstrated that MIF modulates inflammatory and immunological responses which functions as an initiator by regulating production of several cytokines, including tumor necrosis factor (TNF-a), interleukin (IL)-1 and IL-6 [12]. As such, a promising therapeutic approach to diminish pathological immune inflammation is to inhibit the production and/or biological activity of MIF. There are well-documented that anti-MIF antibody could attenuate the inflammatory cascade in sepsis and other diseases [13]. ISO-1[(S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester)], was synthesized from 1997 as a non-toxic, specific antagonist of MIF [14]. ISO-1 inhibits TNF-α release from macrophages isolated from LPS treated wild type mice but has no effect on cytokine release from MIF deficient macrophages [13].
It is well documented that modulation of inflammatory response through treatment with an anti-inflammatory agent can effectively reduce clinical complications and optimize recovery in virusinfected patients [12,15]. In this study, we hypothesis that ISO-1 could regulate the inflammatory responses on A549 cells induced by the H9N2 virus infection, which might provide mechanistic insights into the anti-inflammatory efficacy of ISO-1.

Virus
The avian influenza virus A/mallard/Jiangxi/39/2011 (H9N2) was isolated from the wild duck from Poyang Lake in Jiangxi province of China in 2011 [16]. Phylogenetic analysis showed

Cells and reagents
The human alveolar epithelial cell line A549 was purchased

Dose-response and time-course effects of ISO-1 on A549 cells
The dose-response and time-course effects of ISO-1 acted on the A549 cells were tested. In brief, the A549 cells were grown to confluence, different concentrations of ISO-1 (10, 30, 100, 200, and 400ng/μl) were added and incubated with the cultured A549 cells in triplicate, and then the cell culture were harvested at 0.5h, 1h, 4h, 6h, 16h, 21h, 24h after the incubation. The total cellular RNA was extracted according to a previous protocol. MIF and other cytokines expression were analyzed by real-time quantitative-PCR (qRT-PCR).
All quantifications were normalized to the housekeeping GAPDH gene, which showed a very stable expression in A549 cells. Each sample was amplified in fourfold, and all data was analyzed using Sequence Detector Systems software (Applied Biosystems, USA).
Fold changes in expression were calculated according to the 2-ΔΔCt method, in which the ΔCt values were compared with the value of the control groups.

Statistical analysis
All data are expressed as means±Standard Deviation (SD).
Statistical analysis was performed using SPSS for Windows version 19.0 (SPSS Inc, USA) and Sigma plot version 12.5 (Systat Software, US). The one-way analysis of variance (ANOVA) followed by a posthoc Tukey test or unpaired two-tailed t-test was used to evaluate the statistical significance of differences between two groups. In all statistical analyses, significance was accepted at P<0.05.

Effects on MIF expression after H9N2 influenza infection
MIF has been well characterized as a mediator of numerous pulmonary inflammatory disease [12], and the elevated concentrations of MIF may highly positively link to the disease's severity. The present study reveals that MIF reached a peak 24h after H9N2 influenza virus infection (Figure 1), and then decreased after that. The result is in line with previous reports showing that the MIF mRNA and protein levels were increased in the lungs of H5N1 influenza virus infection [16].

Optimization of the time and concentration of ISO-1
ISO-1, as a small molecule antagonist of MIF, can inhibited its activity [14]. To determine the dose-response and time-course effects of ISO-1 on A549 cells, firstly different ISO-1 concentrations ranging from 10 to 400ng/μl were used according to the previous studies [14,[18][19][20]. We found that ISO-1 treatment leaded to reduce the expression of MIF, especially the dosage of 100ng/μl of ISO-1, the expression level of MIF was significantly reduced about 6 times compared with that of the control group (p<0.05) (Figure 2A).
Then, A549 cells were treated with 100ng/μl ISO-1 and incubated for 0.5h, 1h, 4h, 6h, 16h, 21h, and 24h respectively. Interestingly, a significant reduction in the expression of MIF were only observed at the early stage of the incubation from 0.5 to 4h and could not be detected at the later time points ( Figure 2B). We speculate that with the incubation time prolonged, ISO-1 might lose the ability to attenuate the mRNA expression of MIF gradually. According to the results, it was identified that intracellular MIF expression was inhibited by ISO-1, but the expression of MIF in A549 cell was not in a time-dependent and dose-dependent manner. We select the 100ng/μl ISO-1 and an hour incubation time as the optimization of the time and concentration of ISO-1 in these following studies.

Effects of ISO-1 on the expression of MIF after H9N2 virus infection
To determine whether ISO-1 could inhibit MIF expression on

Am J Biomed Sci & Res
Copy@ Dr Guimei He, Chuanxia Hu 110 expression of MIF at mRNA levels in A549 cells. Compared with the two non-infected control groups, H9N2-challenged group had a higher MIF expression level, while ISO-1 treatment could significantly [17] inhibit the MIF expression in ISO-1-treatedinfected group and the ISO-1-treated control group (Figure 3).
In addition, we also tested the effect of ISO-1 pre-treatment on the expression of MIF after H9N2 virus infection but found only slightly decrease of MIF expression (data no shown). These results confirmed that post-treatment with ISO-1 in H9N2 infected cells could significantly reduce the MIF expression levels after H9N2 virus infection.  A549 cells were exposed to H9N2 and then challenged with ISO-1(100ng/μl, 1h). MIF expression levels were normalized to β-actin and expressed as fold increases relative to control groups. **P<0.01, ***P<0.001, data were calculated using the 2-△△Ct method. Representative results of three independent experiments are shown.

Figure 4:
Effect of post-treatment of ISO-1 on cytokines expression after H9N2 infection. A549 cells were exposed to H9N2 and then challenged with ISO-1(100ng/μl, 1h). The genes TNF-a, IL-6, IFN-b, NF-kB, and CXCL8 ( Figure 4A-E) mRNA expression levels were normalized to β-actin and expressed as fold increases relative to control groups. **P<0.01. Data were calculated using the 2-△△Ct method.

Am J Biomed Sci & Res
Copy@ Dr Guimei He, Chuanxia Hu

Effect of ISO-1 on viral replication after H9N2 virus infection
Seeing the downregulation of cytokines on treatment of ISO-1, we then focused our attention on comparing the virus titer of H9N2 and M gene expression in the two infected groups with or without ISO-1 treatment. Figure 5B showed that the expression levels of M gene were significantly decreased after ISO-1 treatment.
Accordingly, we reasoned that ISO-1 inhibition of MIF might suppress the virus replication in A549. We compared the virus titer between the two groups, found that the virus titer was also lower in the ISO-1 treated group than the virus control group ( Figure   5A

Conclusion
We