All-trans retinoic acid increases the pathogenicity of the H9N2 influenza virus in mice.

BACKGROUND: The H9N2 virus can infect not only birds but also humans. The pathogenicity of H9N2 virus infection is determined by an excessive immune response in the lung. All-trans retinoic acid (ATRA), the active metabolite of vitamin A, plays an important regulatory role and has been widely used in the clinical practice. This study was aimed to investigate whether ATRA could regulate the immune response to H9N2 virus infection in the lungs of mice, thereby reducing the pathogenicity of the H9N2 virus in mice. METHODS: Mice were infected intranasally with H9N2 virus, and injected intraperitoneally with 0.2 mL of ATRA at low (1 mg/kg), medium (5 or 10 mg/kg), or high therapeutic dose (20 mg/kg), and toxic dose (40, 60, or 80 mg/kg), once per day for 10 days. Clinical signs, survival rates, and lung gross pathology were compared between the ATRA-treated H9N2-infected group, the ATRA group, and the H9N2-infected group, to investigate the effect of different doses of ATRA on the pathogenicity of H9N2 virus. Additionally, the viral load and cytokine concentration of lungs were measured at 3, 5, 7, and 9 days after infection, to investigate the potential mechanism of ATRA in affecting the pathogenicity of the H9N2 virus. Expression levels of cellular retinoic acid-binding protein 1 (CRABP1), cellular retinoic acid-binding protein 2 (CRABP2), and Retinoic acid-inducible gene-I (RIG-I) were detected using Western blotting. RESULTS: The ATRA-treated H9N2-infected mice showed more severe clinical signs compared with the H9N2-infected group. The medium and high therapeutic doses of ATRA reduced the survival rates, aggravated lung tissue damage, decreased the expression of interferon beta (IFN-ß), and increased the concentrations of interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and C-C motif chemokine ligand 2 (CCL2) in the lungs of the H9N2-infected mice. At the same time, the expression patterns of CRABP1, CRABP2, and RIG-I were changed in mice infected by H9N2 and treated with different concentrations of ATRA. CONCLUSIONS: Our findings suggest that the therapeutic dose of ATRA can increase the pathogenicity of the H9N2 virus. Therefore, the consequences of those infected by influenza virus would be more severe after ATRA treatment.

Vitamin D receptor and 1α-hydroxylase are highly expressed in lungs of mice infected with H9N2 avian influenza viruses.

The H9N2 avian influenza viruses infect poultry worldwide, and can potentially cause a human pandemic without adaptation. Vitamin D3 (D3) is increasingly being recognized for its extra-skeletal roles, such as the inflammatory and immune responses to infection. The aim of this study was to analyze the changes in vitamin D metabolizing enzymes and vitamin D receptor (VDR) in the lung tissues of mice infected with H9N2. The mice were intranasally inoculated with the appropriate dose of the virus, and various clinical indices were measured on days 3, 7, 14 and 21 post-infection. H9N2 infection significantly increased the expression levels of 1α-hydroxylase mRNA and protein, which is the activating enzyme of 25-hydroxyvitamin D (25(OH)D3), but had no significant effect on the 25(OH)D3 inactivating enzyme 24-hydroxylase, indicating that inactive D3 might be converted to its active form in the H9N2-infected lungs. Furthermore, a significant increase was also observed in the VDR mRNA and protein levels, suggesting enhanced responsiveness of the lung tissues to 1, 25(OH)2D3 post H9N2 infection. In addition, daily 25(OH)D3 injection from day 2-14 post-infection did not affect the clinical signs, virus replication and cytokine (IL-1ß and TNF-α) production in the lungs of the infected mice. Given that the biological effects of D3 rely on its activation, and the binding of 1, 25(OH)2D3 to VDR in specific tissues, our findings provide novel insights into the possible role of vitamin D in the development and progression of influenza.

Increased Pulmonary Pneumococcal Clearance after Resolution of H9N2 Avian Influenza Virus Infection in Mice.

H9N2 avian influenza virus has been continuously circulating among poultry and can infect mammals, indicating that this virus is a potential pandemic strain. During influenza pandemics, secondary bacterial (particularly pneumococcal) pneumonia usually contributes to excessive mortality. In the present study, we observed the dynamic effect of H9N2 virus infection on host defense against secondary pneumococcal infection in mice. BALB/c mice were intranasally inoculated with 1.2 × 105 PFU of H9N2 virus followed by 1 × 106 CFU of Streptococcus pneumoniae at 7, 14, or 28 days post-H9N2 infection (dpi). The bacterial load, histopathology, body weight, and survival were assessed after pneumococcal infection. Our results showed that H9N2 virus infection had no significant impact on host resistance to secondary pneumococcal infection at 7 dpi. However, H9N2 virus infection increased pulmonary pneumococcal clearance and reduced pneumococcal pneumonia-induced morbidity after secondary pneumococcal infection at 14 or 28 dpi, as reflected by significantly decreased bacterial loads, markedly alleviated pulmonary histopathological changes, and significantly reduced weight loss in mice infected with H9N2 virus followed by S. pneumoniae compared with mice infected only with S. pneumoniae Further, the significantly decreased bacterial loads were observed when mice were previously infected with a high dose (1.2 × 106 PFU) of H9N2 virus. Also, similar to the results obtained in BALB/c mice, improvement in pulmonary pneumococcal clearance was observed in C57BL/6 mice. Overall, our results showed that pulmonary pneumococcal clearance is improved after resolution of H9N2 virus infection in mice.

The active GLP-1 analogue liraglutide alleviates H9N2 influenza virus-induced acute lung injury in mice.

Influenza virus is responsible for significant morbidity and mortality worldwide. Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is the major cause of death in influenza virus infected patients. Recent studies indicated that active glucagon like peptide-1 (GLP-1) encoded by glucagon (GCG) gene exerts anti-inflammatory functions. The aim of this study was to determine the potential role of active GLP-1 in H9N2 influenza virus-induced ALI/ARDS in mice. First, we uncovered that GCG mRNA expression levels and GCG precursor protein levels were significantly increased, but total GLP-1 and active GLP-1 levels were decreased in the lungs of H9N2-infected mice. Next, liraglutide, an active GLP-1 analogue, was used to treat infected mice and to observe its effects on H9N2 virus-induced ALI. Liraglutide treatment ameliorated the declined body weight, decreased food intake and mortality observed in infected mice. It also alleviated the severity of lung injury, including lowering lung index, decreasing inflammatory cell infiltration and lowing total protein levels in bronchoalveolar lavage fluid (BALF). In addition, liraglutide did not influence viral titers in infected lungs, but decreased the levels of interleukin-1ß, interleukin-6 and tumor necrosis factor-α in BALF. These results indicated that liraglutide alleviated H9N2 virus-induced ALI in mice most likely due to lower levels of pro-inflammatory cytokines.

TLR4 deficiency reduces pulmonary resistance to Streptococcus pneumoniae in gut microbiota-disrupted mice.

Streptococcus pneumoniae is a clinically important pathogen responsible for significant morbidity and mortality worldwide. Disruption of the host gut microbiota by antibiotics reduces the pulmonary resistance to S. pneumoniae. The aim of our study was to determine the potential role of TLR4 in the reduced pulmonary resistance to S. pneumoniae following gut microbiota disruption. Wild-type and TLR4-deficient mice were given broad-spectrum antibiotics for 3 weeks by oral gavage to disrupt the gut microbiota, and subsequently inoculated intra-nasally with S. pneumoniae. The extent of the decline in pulmonary resistance in both animal groups was evaluated in terms of the overall survival and pulmonary bacterial clearance. Both survival and pulmonary clearance of S. pneumoniae were lower in the TLR4-deficient mice with disrupted gut microbiota, compared to their intestinally healthy counterparts after pneumococcal infection. However, the degree of decline was much lower in the TLR4-deficient mice compared to the wild-type mice. Our findings indicate that impaired TLR4 function might be the basis of the reduced pulmonary resistance to S. pneumoniae caused by gut microbiota disruption.