Severe influenza infection is associated with inflammatory programmed cell death in infected macrophages

Abstract

Introduction: Influenza A virus (IAV) is one of the leading causes of respiratory tract infections in humans, representing a major public health concern. The various types of cell death have a crucial role in IAV pathogenesis because this virus may trigger both apoptosis and necroptosis in airway epithelial cells in parallel. Macrophages play an important role in the clearance of virus particles, priming the adaptive immune response in influenza. However, the contribution of macrophage death to pathogenesis of IAV infection remains unclear.

Methods: In this work, we investigated IAV-induced macrophage death, along with potential therapeutic intervention. We conducted in vitro and in vivo experiments to evaluate the mechanism and the contribution of macrophages death to the inflammatory response induced by IAV infection.

Results: We found that IAV or its surface glycoprotein hemagglutinin (HA) triggers inflammatory programmed cell death in human and murine macrophages in a Toll-like receptor-4 (TLR4)- and TNF-dependent manner. Anti-TNF treatment in vivo with the clinically approved drug etanercept prevented the engagement of the necroptotic loop and mouse mortality. Etanercept impaired the IAV-induced proinflammatory cytokine storm and lung injury.

Conclusion: In summary, we demonstrated a positive feedback loop of events that led to necroptosis and exacerbated inflammation in IAV-infected macrophages. Our results highlight an additional mechanism involved in severe influenza that could be attenuated with clinically available therapies.

Keywords: TNF; etanercept; influenza virus; macrophages; necroptosis.

Figure 1

IAV and viral HA induce inflammatory programmed cell death in primary macrophages. Murine (BMDM) or human macrophage (MDM) cultures were pretreated with zVAD (10 µM) or Nec-1 (25 µM), infected with IAV at an MOI of 0.25, and exposed to 10 ng/mL HA or 1 ng/mL TNF-α. (A, B) BMDM cell death was also evaluated by flow cytometry analysis of Annexin V/PI-positive cells. Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C) or humans (D). (E) The expression of p-RIPK1, RIPK3 and MLKL was detected in BMDM lysates by Western blotting. β-actin levels were used as a control for protein loading. (F) The expression of p-MLKL was detected in BMDMs by Western blotting. β-actin levels were used as a control for protein loading. (G) Graphs of band densitometry of MLKL and p-MLKL obtained after loading normalization and expressed as fold change over mock control. Data are presented as the mean ± SEM of 3 independent experiments ^* P < 0.05 versus the control group (MOCK); (H) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (I) IL-6 and (J) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus control group (MOCK); ^#P < 0.05 versus the respective untreated infected/stimulated group.

Figure 2

TLR4 engagement is necessary for IAV- and HA-induced inflammatory programmed cell death in macrophages. (A, B) BMDM cultures were pretreated with 2 µM CLI95 for 2 h, infected with IAV at an MOI of 0.25, and exposed to 10 ng/mL HA or LPS. After 24 h, LDH was measured (A), and annexin V/PI staining was performed (B). *P < 0.05 versus the respective control group (mock); ^#P < 0.05 versus the respective untreated infected/stimulated group. (C, D) BMDM cultures from WT, TLR2^-/- and TLR4^-/- mice were infected with IAV at an MOI of 0.25 and exposed to 10 ng/mL HA or LPS. After 24 h, LDH was measured (C), and annexin V/PI staining was performed (D). (E) The expression of p-RIPK1, RIPK3 and MLKL was detected in BMDMs by Western blotting. β-actin levels were used as a control for protein loading. The levels of (F) IL-6, (G) TNF and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. ^* P < 0.05 in comparison to the respective noninfected groups (MOCK) and ^# P < 0.05 versus the respective Wt infected/stimulated group. The graphs are representative of three independent experiments.

Figure 3

IAV- and HA-induced inflammatory programmed cell death is dependent on TNF. Macrophage cultures were pretreated with 1 ng/mL anti-TNF-α antibody and then infected with IAV (MOI of 0.25) or exposed to 10 ng/mL viral HA for 24 h. Cell death analysis was performed by flow cytometry analysis of annexin V/PI-positive cells (A, B). Assessment of cell viability through the measurement of LDH release in the supernatant of BMDMs (C). (D) The expression of p-RIPK1 was detected in BMDMs by Western blotting. β-actin levels were used as a control for protein loading. (E) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock untreated control. (F) Levels of nitrite were measured by the Griess method in the supernatant of BMDM cultures after 24 h of stimulus. The levels of (G) IL-6 and (H) IL-10 were measured by ELISA in the supernatant of BMDM cultures after 24 h of stimulation. Data are presented as the mean ± SEM of 5 independent experiments *P < 0.05 versus untreated control group (MOCK); ^#P < 0.05 versus respective untreated infected/stimulated group. (I) Model of A/HA-triggered necroptosis. IAV/HA triggered necroptosis by a loop of events involving TLR4, TNF-α, and RIPK1, leading to exacerbation of inflammation. Images were created with BioRender.com.

Figure 4

Etanercept reduced weight loss and improved survival during lethal IAV infection in mice. C57Bl/6 mice were inoculated intranasally with 10³ PFU of IAV. Six hours postinfection, animals were treated intraperitoneally with 2.5 mg/kg etanercept (ETN). Mice received a daily dose of ETN for 7 days and were monitored for survival (A) and body-weight loss (B) analysis. Data are shown as the percentage of survival and weight. The graphs are representative of three independent experiments. ^* P < 0.05 in comparison to the IAV-infected untreated group; ^#P < 0.05 in comparison to Mock group.

Figure 5

Etanercept reduced inflammatory programmed cell death in macrophages in vivo. C57Bl/6 mice were inoculated intranasally with 10³ PFU of IAV with and without daily treatment with Etanercep (ETN, 2.5 mg/kg, i.p.). At days 3 and 5 postinfection, animals were euthanized, and BAL and lung were collected. (A, B) Total and differential cell counts in bronchoalveolar lavage (BAL) are represented as the number of differential cell counts at days 3 (A) and 5 (B) postinfection. Total: leukocytes total, Mono: mononuclear leukocytes, PMN: polymorphonuclear leukocytes. (C, D) Mononuclear cell death was evaluated by annexin V/PI labeling. (E) Lung damage was evaluated by measuring LDH release in the bronchoalveolar lavage (BAL); (F–I) The expression of p-RIPK1, MLKL, and cleaved Caspase-8 was detected in the lung tissue homogenates by Western blotting. β-actin levels were used as a control for protein loading. (G–I) Graphs of band densitometry obtained after loading normalization and expressed as fold change over mock control. Data are expressed as the means ± SEMs; one-way ANOVA with Dunnett’s post hoc test. ^* P < 0.05 in comparison to mock and ^# P < 0.05 in comparison to the IAV-infected untreated group. Experiments were performed with 4-6 mice/group.

Figure 6

Etanercept reduced the cytokine storm in IAV-infected mice. C57Bl/6 mice were inoculated intranasally with 10³ PFU of IAV with and without daily treatment with Etanercep (ETN, 2.5 mg/kg, i.p.). At days 3 and 5 postinfection (DPI), animals were euthanized, and bronchoalveolar lavage (BAL) was collected for quantification of total protein (A) and inflammatory mediators. The levels of CXCL1/KC (B), CCL2/MCP1 (C), IL-6 (D), TNF (E), INF-γ (F) and IL-10 (G) were measured by ELISA. Data are expressed as the means ± SEMs; one-way ANOVA with Dunnett’s post hoc test. ^* P < 0.05 in comparison to mock and ^# P < 0.05 in comparison to the IAV-infected untreated group. Experiments were performed with 4-6 mice/group.