Hydrogen Sulfide Is an Antiviral and Antiinflammatory Endogenous Gasotransmitter in the Airways. Role in Respiratory Syncytial Virus Infection

Abstract

Hydrogen sulfide (H₂S) is an endogenous gaseous transmitter whose role in the pathophysiology of several lung diseases has been increasingly appreciated. Our recent studies in vitro have shown, we believe for the first time, that H₂S has an important antiviral and antiinflammatory activity in respiratory syncytial virus (RSV) infection, the leading cause of bronchiolitis and viral pneumonia in children. Our objective was to evaluate the therapeutic potential of GYY4137, a novel slow-releasing H₂S donor, for the prevention and treatment of RSV-induced lung disease, as well as to investigate the role of endogenous H₂S in a mouse model of RSV infection. Ten- to 12-week-old BALB/c mice treated with GYY4137, or C57BL/6J mice genetically deficient in the cystathionine γ-lyase enzyme, the major H₂S-generating enzyme in the lung, were infected with RSV and assessed for viral replication, clinical disease, airway hyperresponsiveness, and inflammatory responses. Our results show that intranasal delivery of GYY4137 to RSV-infected mice significantly reduced viral replication and markedly improved clinical disease parameters and pulmonary dysfunction compared with the results in vehicle-treated control mice. The protective effect of the H₂S donor was associated with a significant reduction of viral-induced proinflammatory mediators and lung cellular infiltrates. Furthermore, cystathionine γ-lyase-deficient mice showed significantly enhanced RSV-induced lung disease and viral replication compared with wild-type animals. Overall, our results indicate that H₂S exerts a novel antiviral and antiinflammatory activity in the context of RSV infection and represent a potential novel pharmacological approach for ameliorating virus-induced lung disease.

Keywords: airway hyperresponsiveness; antiviral; cystathionine γ-lyase; lung injury; paramyxovirus.

Figure 1.

GYY4137 treatment attenuates respiratory syncytial virus (RSV)-induced disease and pulmonary lung function. (A) GYY4137 dose response in vivo. Mice were treated intranasally (i.n.) with different doses of GYY4137 (50 mg, 100 mg, and 200 mg/kg body weight) or an appropriate volume of vehicle (PBS) 1 hour before and 6 and 24 hours after infection. Mice were inoculated with either RSV or PBS. (A) Mice were monitored daily, and body weight was calculated on the basis of the original weight before the infection. Data are expressed as mean ± SEM (n = 3–4 mice/group). (B) Disease parameters—BALB/c mice were infected with 10⁶ plaque-forming units (pfu) RSV and treated with GYY4137 or vehicle as follows: (1) three doses, at 2 hours and at 6 and 24 hours after infection, (2) two doses, one at 6 hours and one at 24 hours after infection, and (3) one dose, at 24 hours before infection. Data are expressed as mean ± SEM (n = 4 mice/group). *P < 0.05 compared with PBS/RSV at Days 1, 2, and 3 postinfection (p.i.). (C, D, and E) Mice were treated i.n. with GYY4137 (50 mg/kg body weight) or an appropriate volume of vehicle (PBS) 1 hour before and 6 and 24 hours after infection. Mice were inoculated with RSV or PBS. (C) Clinical illness scores of GYY4137/RSV (open squares) and RSV vehicle (solid squares) were measured from Day 1 to Day 7 p.i. (data shown for the 50 mg/kg dose). GYY4137/RSV-infected mice were assigned an illness score 0 starting with Day 3 p.i. (data shown as n.d. [not detected]). Sham (mock)-infected mice treated with either vehicle or GYY4137 received a healthy illness score 0 throughout the course of the experiment (data not shown). (D) Unrestrained, whole-body plethysmography (Buxco Electronics, Inc., Sharon, CT) was used to measure the enhanced pause (Penh) to evaluate airway hyperresponsiveness. Baseline and postmethacholine challenge Penh values were determined at Day 5 after infection. Penh values are presented as mean ± SEM (n = 4–6 mice/group). (E) Airway resistance (Day 5 p.i.) measured in mechanically ventilated mice by the Flexivent system. Data are expressed as mean ± SEM (n = 3 mice/group). *P < 0.05, **P < 0.001, and ***P < 0.0001 compared with PBS/RSV group.

Figure 2.

GYY4137 treatment reduces viral replication in RSV-infected mice. (A) Mice were treated with GYY4137 or vehicle and infected with either RSV or PBS and harvested at Day 5 postinfection to determine viral replication by plaque assay, expressed as pfu per gram of lung tissue. The bar graph represents mean ± SEM (n = 4 mice/group), **P < 0.001 compared with the PBS/RSV group. (B) Mice were infected with 10⁶ pfu RSV and treated with GYY4137 after infection as indicated. Data are expressed as mean ± SEM (n = 4 mice/group). At Day 5 postinfection, virus titer in the lung was determined by plaque assay. *P < 0.05 and **P < 0.001 compared with the PBS/RSV group.

Figure 3.

GYY4137 reduces airway neutrophilia and lung inflammation after infection. Mice were treated intranasally with GYY4137 (50 mg/kg body weight) or an appropriate volume of vehicle (PBS) 1 hour before and 6 and 20 hours after infection. Mice were inoculated with either RSV or PBS as described in Material and Methods. Bronchoalveolar lavage (BAL) and lungs were collected at different time points after infection to determine differential cell counts by hematoxylin and eosin staining in (A) BAL and (B) neutrophil (CD11b⁺Gr1⁺) recruitment to the lung by flow cytometry analysis after staining with specific antibodies. (C) Lung samples were harvested at Day 7 postinfection, fixed for slide preparation, and stained with hematoxylin and eosin. Representative stained lung tissue sections from the indicated treatment. (D) Pathology score of prepared slides (scored as described in Materials and Methods). The bar graph represents mean ± SEM (n = 3–4 mice/group). *P < 0.05 and **P <  0.001 compared with PBS/RSV mice. Scale bars, 200 μm. GR-1, granulocyte-differentiation antigen-1.

Figure 4.

GYY4137 inhibits proinflammatory mediator secretion in response to RSV infection. Mice were treated with GYY4137 or vehicle, RSV at the dose of 10⁶ pfu, or sham infected and harvested at Day 1 postinfection to collect bronchoalveolar lavage samples to measure (A) cytokines and (B) chemokines by multi-Plex cytokine detection system, and (C) type I IFN by ELISA. The bar graph represents mean ± SEM (n = 4–6 mice/group). *P < 0.05, **P < 0.001, and ***P < 0.0001 compared with the PBS/RSV group. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte–macrophage colony-stimulating factor; KC, neutrophil chemokine; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage inflammatory protein-1; RANTES, regulated upon activation, normal T-cell expressed and secreted.

Figure 5.

Effect of GYY4137 on cytokines/chemokine secretion in response to ultraviolet (UV)-inactivated RSV. Mice were treated intranasally with GYY4137 or vehicle and inoculated with either UV-inactivated 10⁷ pfu RSV or mock infected. BAL was collected at Day 1 postinfection to measure cytokines and chemokines by a multi-Plex cytokine detection system. The bar graph represents mean ± SEM (n = 4 mice/group). **P < 0.001, ***P < 0.0001 compared with the PBS/UV-RSV group.

Figure 6.

Cystathionine γ-lyase (CSE)-deficient mice have increased disease severity, viral replication, and lung inflammation. C57BL/6 (wild-type [WT]) and CSE^−/− (CSE KO) mice were infected intranasally with 10⁷ pfu of RSV or PBS. (A) Mice were monitored daily, and body weight was calculated on the basis of the original weight before infection. Data are expressed as mean ± SEM (n = 3–4 mice/group). (B) Left panel, unrestrained, whole-body plethysmography (Buxco Electronics, Inc., Sharon, CT) was used to measure the Penh to evaluate airway hyperresponsiveness. Baseline and postmethacholine challenge Penh values were determined at Day 1 p.i. Data are expressed as mean ± SEM (n = 3–4 mice/group). Right panel, airway resistance (Day 1 p.i.) was measured in mechanically ventilated mice by the Flexivent system. Data are expressed as mean ± SEM (n = 3–4 mice/group). (C) Viral replication. At Day 5 p.i., lungs were excised, and viral titers were determined by plaque assay. The bar graph represents mean ± SEM (n = 3–4 mice/group). (D and E) C57BL/6 (WT) and CSE^−/− (CSE KO) mice were given GYY4137 or vehicle at a dose of 50 mg/kg 1 hour before and 6 and 24 hours after infection. Mice were infected intranasally with 10⁷ pfu of RSV or PBS. (D) Mice were monitored daily, and body weight was calculated on the basis of the original weight before the infection. Data are expressed as mean ± SEM (n = 2–3 mice/group). *P < 0.05 CSE KO/GYY4137/RSV versus CSE KO/PBS/RSV and WT/GYY4137/RSV versus WT/PBS/RSV at Day 1 postinfection; CSE KO/GYY4137/RSV versus CSE KO/PBS/RSV and CSE KO/PBS/RSV versus WT/PBS/RSV at Day 2 p.i., WT/GYY4137/RSV versus WT PBS/RSV and CSE KO/PBS/RSV versus WT/PBS/RSV at Day 4 p.i. (E) Unrestrained, whole-body plethysmography (Buxco Electronics, Inc., Sharon, CT) was used to measure the Penh to evaluate airway hyperresponsiveness. Baseline and postmethacholine challenge Penh values were determined at Day 5 p.i. Data are expressed as mean ± SEM (n = 2–3 mice/group). *P < 0.05, **P < 0.001, and ***P < 0.0001 compared with the WT/RSV and the WT/GYY4137/RSV group and between CSE KO/RSV and CSE KO/GYY4137/RSV mice.

Figure 7.

Effect of CSE deficiency on cytokine and chemokine production and pulmonary inflammation after infection. C57BL/6 (WT) and CSE^−/− (CSE KO) mice were infected intranasally with 10⁷ pfu of RSV or PBS. (A) Mice were infected with RSV or mock infected and killed at Day 1 postinfection to collect bronchoalveolar lavage samples. Cytokine/chemokine production was measured by a multi-Plex cytokine detection system. The bar graph represents mean ± SEM (n = 3–4 mice/group). (B) Bronchoalveolar lavage was collected at Day 5 after infection to measure IFN-γ and IL-4 by ELISA. Data are expressed as mean ± SEM (n = 4 mice/group). (C) Pathology score of lungs harvested at Day 7 postinfection, fixed, and hematoxylin and eosin stained. The bar graph represents mean ± SEM (n = 3–4 mice/group). *P < 0.05 compared with WT/RSV group.