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, 6 (3), e135
eCollection

Targeting the Pro-Inflammatory Factor CCL2 (MCP-1) With Bindarit for Influenza A (H7N9) Treatment

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Targeting the Pro-Inflammatory Factor CCL2 (MCP-1) With Bindarit for Influenza A (H7N9) Treatment

Stefan Wolf et al. Clin Transl Immunology.

Abstract

Influenza A viruses are important human and animal pathogens. Seasonal influenza viruses cause infections every year, and occasionally zoonotic viruses emerge to cause pandemics with significantly higher morbidity and mortality rates. Three cases of laboratory confirmed human infection with avian influenza A (H7N9) virus were reported in 2013, and there have been several cases reported across South East Asia, and recently in North America. Most patients experience severe respiratory illness, with mortality rates approaching 40%. No vaccine is currently available and the use of antivirals is complicated due to the emergence of drug resistant strains. Thus, there is a need to identify new drugs for therapeutic intervention and disease control. In humans, following H7N9 infection, there is excessive expression of pro-inflammatory factors CCL2, IL-6, IL-8, IFNα, interferon-γ, IP-10, MIG and macrophage inflammatory protein-1β, which has been shown to contribute to fatal disease outcomes in mouse models of infection. In the current study, the potent inhibitor of CCL2 synthesis, Bindarit, was examined as a countermeasure for H7N9-induced inflammation in a mouse model. Bindarit treatment of mice did not have any substantial therapeutic efficacy in H7N9 infection. Consequently, the results suggest that Bindarit may be ill-advised in the treatment of influenza H7N9 infection.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Bindarit reduces CCL2 gene expression in IAV-infected cells. A549 cells were infected with A/Ca (H1N1) at MOI 0.1 and subsequently treated with Bindarit at 100 μm. At 24 h pi, RNA was extracted for host gene expression analysis of CCL2 using RT-qPCR. Expression was normalized to 18 s and compared to non-infected cells. Data are from two independent experiments using three replicates per group.
Figure 2
Figure 2
Effect of Bindarit treatment on weight loss and survival rate following avian IAV H7N9 infection. Mice were infected i.n. with a lethal dose (10 × LD50) of A/Anhui (H7N9). Mice were then treated with either Bindarit or vehicle starting at day 1 pi. Mice were monitored for weight loss (a) and survival (b). Time points represent days pi. Data are from seven mice per group±s.e.m.
Figure 3
Figure 3
Effect of Bindarit on histopathological changes following avian IAV H7N9 infection. Mice were infected i.n. with a lethal dose (10 × LD50) of H7N9 or PBS. Mice were then treated with either Bindarit or vehicle starting at day 1 pi. At day 4 pi, mice were killed and lungs collected for histopathological analysis. Mock-infected methylcellulose (a) and Bindarit-treated (b). Viable epithelial cells line the bronchioles and there is no exudate within the lumen. No peribronchiolar or perivascular infiltrations are present and the parenchyma has no changes. H7N9-infected methylcellulose (c) and Bindarit-treated (d). There is diffuse necrosis and loss of the bronchiolar epithelium with luminal necrotic cellular debris lining the denuded wall. A mild to moderate peribronchiolar and perivascular infiltration of mostly lymphocytes is also present. Parenchymal changes are mild to moderate with mild thickening of the alveolar septa and a few inflammatory cells in alveoli. Data are from three mice per group. B, bronchiole; P, parenchyma; V, vessel.
Figure 4
Figure 4
Effect of Bindarit on cellular infiltration following avian IAV H7N9 infection. Mice were infected i.n. with a lethal dose (10 × LD50) of A/Anhui (H7N9) or PBS. Mice were then treated with either Bindarit or vehicle starting at day 1 pi. At day 5 pi, mice were killed and BAL fluids were collected for analysis with flow cytometry. (a) The total number of BAL leukocytes, (b) the number of macrophages, (c) the number of eosinophils, (d) the number of CD3+ T-cells, (e) the number of CD4+ T cells, and (f) the number of CD8+ T cells were determined. Data are presented as the number of specific type of cells per million total cells. Data are from five mice per group±s.e.m. *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5
Effect of Bindarit treatment on weight loss and virus titer following avian IAV H7N9 infection. Mice were i.n. infected with a sub-lethal dose of H7N9 (102.7 PFU) or PBS. Mice were then orally treated with either Bindarit or vehicle starting at day 1 pi. Mice were monitored for weight loss until day 8 pi (a), when mice were killed and lungs were collected for virus titer analysis with RT-qPCR (b). Virus titers were determined per 5 ng of total RNA extracted from lung homogenates. Time points represent days pi. Data are from five to six mice per group±s.e.m. *P<0.05, **P<0.01.
Figure 6
Figure 6
Effect of Bindarit on pro-inflammatory cytokine gene expression following avian IAV H7N9 infection. Mice were i.n. infected with a sub-lethal dose of H7N9 (102.7 PFU) or PBS. Mice were then treated with either Bindarit or vehicle starting at day 1 pi. On day 8 pi, mice were killed and lungs were collected for gene expression analysis for Ccl2 (a), Il6 (b) and Ifng (c) with RT-qPCR. Expression was normalized to 18S and relative to non-infected mice. Data are from five mice per group±s.e.m. *P<0.05, **P<0.01.
Figure 7
Figure 7
Effect of Bindarit on protein level of pro-inflammatory following avian IAV H7N9 infection. Mice were infected i.n. with a sub-lethal dose of A/Anhui (H7N9) or PBS. Mice were then treated with either Bindarit or vehicle starting at day 1 pi. On day 8 pi, mice were killed and BAL was collected for protein expression analysis of CCL2 (a), IL-6 (b), RANTES (c), IL-15 (d) and TNFα (e) with a multiplex enzyme-linked immunosorbent assay. Data are from five mice per group±s.e.m. *P<0.05.
Figure 8
Figure 8
Effect of Bindarit on pulmonary cell infiltrates following avian IAV H7N9 infection. Mice were i.n. infected with a sub-lethal dose of H7N9 (102.7 PFU) or PBS. Mice were then treated with either Bindarit or vehicle starting on day 1 pi. On day 8 pi mice were killed and BAL fluids collected for analysis with flow cytometry. (a) The total number of leukocytes, (b) the number of macrophages, (c) the number of eosinophils, (d) the number of CD3+ T-cells, (e) the number of CD4+ T cells, and (f) the number of CD8+ T cells were determined. Data are presented as the number of specific type of cells per million total cells. Data are from five mice per group±s.e.m. *P<0.05, **P>0.01, ***P>0.001.

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