Osteopontin aggravates acute lung injury in influenza virus infection by promoting macrophages necroptosis

Abstract

Infection with influenza A virus (IAV) can trigger pulmonary inflammation and lung damage. Osteopontin (OPN) is an essential regulator of cell death and immunity. However, the role and underlying mechanism of OPN in cell death in IAV-induced pulmonary injury remain poorly understood. Here, we demonstrated that OPN-deficient (OPN^-/-) mice were insensitive to IAV, exhibiting decreased viral loads and attenuated lung injury after IAV infection compared to those in wild-type (WT) mice. Moreover, macrophage necroptosis was significantly reduced in OPN^-/- mice infected with IAV compared to that in infected WT mice. OPN increased the expression of necroptosis-related genes and exacerbated macrophage necroptosis in IAV-infected THP1 cells. Notably, adoptive transfer of WT bone marrow-derived macrophages (BMDMs) or OPN^-/- BMDMs into mice restored resistance to influenza infection, and the rescue effect of OPN^-/- BMDMs was better than that of WT BMDMs. Collectively, these results suggest that OPN deficiency in macrophages reduces necroptosis, which leads to a decrease in viral titers and protects against IAV infection. Therefore, OPN is a potential target for the treatment of IAV infection.

Fig. 1. Positive correlation between Osteopontin (OPN) levels and the levels of necroptosis-related genes in influenza A virus (IAV) patients.

A OPN mRNA expression in blood samples from healthy control (HC, n = 82) and IAV patients (IAV, n = 85). B RIPK1, RIPK3, and MLKL mRNA expression in blood samples from healthy control and IAV patients. C Spearman correlation analysis of RIPK1, RIPK3, or MLKL mRNA levels with OPN mRNA levels. Data was analyzed by Student’s t test (two-tailed) and expressed as mean ± SEM. Spearman rank correlation was used to test for correlations.*p < 0.05; **p < 0.01; ****p < 0.0001.

Fig. 2. OPN deficiency reduces virus replication and decreases IAV-induced lung inflammation.

A, B WT mice were infected with PR8 (1.5 × 10³ PFU/animal). Analysis was performed on data collected 24 h post infection. A OPN protein levels in BALF supernatants. B OPN mRNA levels in the lung homogenates. C–H WT mice or OPN^−/− mice were infected with PR8 (1.5 × 10³ PFU/animal). Analysis was performed on data collected 24 h post infection. C Serial lung sections of representative lungs stained with hematoxylin and eosin, scale bar = 100 μm. D The level of tissue injury was quantified from the LDH levels in serum. E BALF was obtained and inflammatory factor (IL-6, MCP-1, and TNF-α) levels in the BALF supernatant were measured using ELISA. F Levels of inflammatory factors (IL-6, MCP-1, and IFN-γ) in lung homogenates were measured using qPCR. G Total viral titers in the lung homogenates were quantified using qPCR. H Lung homogenates were collected and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. Data was analyzed by Student’s t test (two-tailed) (A, B, G) or one-way ANOVA (D, E, F, H) and expressed as mean ± SEM. n = 3-7/group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 3. OPN promotes IAV-induced macrophage necroptosis in the lung.

A, B WT and OPN^−/− mice were infected with PR8 (1.5 × 10³ PFU/animal). Analysis was performed on data collected 24 h post infection. A mRNA levels of necroptosis-related genes (RIPK1, RIPK3, and MLKL) in lung homogenates were measured using qPCR. B P-MLKL levels in the lung homogenates were measured using western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. C–F WT mice were intraperitoneally administered clodronate liposomes (CLs) or the same volume of sterile PBS for 24 h. C Serial lung sections of representative lungs stained by immunohistochemistry, scale bar = 50 μm (above) or 100 μm (below). Red arrows indicate F4/80 positive cells. D Serial lung sections of representative lungs of mice 24 h after PR8 (1.5 × 10³ PFU/animal) infection were stained with hematoxylin and eosin, scale bar = 100 μm. E Serum LDH levels were measured 24 h after PR8 (1.5 × 10³ PFU/animal) infection. F Lung homogenates were collected 24 h after PR8 (1.5 × 10³ PFU/animal) infection and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. G WT mice or OPN^−/− mice were infected with PR8 (1.5 × 10³ PFU/animal). Macrophages in BALF were collected 24 h post infection and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. Data was analyzed by Student’s t test (two-tailed) (E, F) or one-way ANOVA (A, B, G) and expressed as mean ± SEM. n = 3-7/group. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4. OPN knockdown inhibits necroptosis in THP1 cells infected with IAV.

PMA was added to the THP1 culture for 24 h to stimulate THP1 cells adherence and differentiation into macrophage-like cells. A, B THP1 cells were infected with 3 MOI PR8 for 24 h. A OPN protein levels in the cell culture supernatant. B OPN mRNA levels in the cell lysate. C THP1 cells were infected with GFP-PR8 (green) for 24 h and stained with DAPI (blue). Red arrows indicate positive cells. D THP1 cells were infected with 3 MOI PR8 for 0, 6, 12, and 24 h. Cell lysates were collected and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. E–G OPN expression in THP1 cells was silenced using OPN-specific small interfering RNA (siRNA) or nonspecific siRNA (siNC). THP1 cells were infected with 3 MOI PR8 for 24 h. E OPN gene silencing efficiency in THP1 cells. F RIPK1, RIPK3, and MLKL mRNA levels in THP1 cells. G Cell lysates were collected and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. H Total viral titers in cell lysates were quantified by qPCR. Data was analyzed by Student’s t test (two-tailed) (A, B, E, H) or one-way ANOVA (D, F, G) and expressed as mean ± SEM. n = 3-7/group. **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 5. Adoptive transfer of OPN^−/− BMDM reduces tissue injury in mice induced by subsequent PR8 infection.

A–E WT or OPN^−/− BMDMs were intranasally transferred to WT recipient mice. One day after transfer, mice were infected with PR8 (1.5 × 10³ PFU/animal) and lung tissues were collected 24 h after infection. A Diagrammatic representation of adoptive transfer. B Serial lung sections of representative lungs stained with hematoxylin and eosin, scale bar = 100 μm. C Inflammatory factor (IL-6, MCP-1, and IFN-γ) levels in lung homogenates were measured using qPCR. D Representative images of immunofluorescence staining for P-MLKL in the lung. E Lung homogenates were collected and subjected to western blot analysis. Each lane corresponds to an individual mouse. Lane-loading differences were normalized by levels of ACTIN. Data was analyzed by one-way ANOVA and expressed as mean ± SEM. n = 3-4/group. *p < 0.05; ****p < 0.0001; ns denote no statistical significance.