Figure 1.
Study protocol.
Figure 2.
IL-8 produced in response to S. aureus supernatant by a co-culture of macrophages and lung epithelial cells is dependent on IL-1 signaling, while IL-8 produced by neutrophils is independent of IL-1.
(A, B) THP-1 macrophages (103 cells), A549 lung epithelial cells (105) were cultured alone or mixed and treated as indicated with HKS, S. aureus CCY broth culture supernatant and Kineret/IL-1Ra. IL-8 was quantified by ELISA at 6 h post-intoxication. (C–E) As indicated, primary human neutrophils were treated with HKS, Kineret/IL-1Ra (E) and rPVL. IL-1β (C) and IL-8 levels were quantified at 6 h PI (E) or at the indicated time post-intoxication (C, D). One experiment representative of three independent experiments with three independent donors is shown.
Figure 3.
PVL is associated with an increase in IL-1β and IL-8 release in a rabbit model of S. aureus-mediated pneumonia.
(A–G) Rabbits were inoculated intratracheally with 1.5×109 cfu of USA300 LAC WT or ΔPVL strains and euthanized 6 h post-infection. IL-1β (A), IL-8 levels in BALF (B) or in lung lysates (C) were quantified by ELISA. (D) Total protein levels in BALF were quantified by a Bradford assay. (E) Gross pathological scoring was performed. (F) The ratio of lung weight to body weight is shown. (G) Bacterial burden as determined by CFU in each rabbit lung is shown. (A–D) Each point represents the value obtained in the BALF from one lobe or (E–G) the value obtained for one rabbit. (A–G) The geometric mean is shown. One experiment (n = 6) representative of two experiments (n = 12) is shown.
Figure 4.
Sequential intratracheal instillation of HKS and rPVL triggers IL-1β and IL-8 release and reproduces some key aspects of S. aureus-mediated pneumonia.
(A–E) Rabbits were inoculated intratracheally with HKS, 3 h later with PBS or with rPVL and euthanized 6 h post-HKS instillation. IL-1β (A) and IL-8 levels in BALF (B) or in lung lysates (C) were quantified by ELISA. (D) Gross pathological scoring was performed. (E) The ratio of lung weight on body weight is shown. (A–C) Each point represents the value obtained in the BALF from one lobe or (D, E) the value obtained for one rabbit. (A–F) The geometric mean is shown. Results from three independent experiments (n = 12) are shown.
Figure 5.
Kineret/IL-1Ra inhibits the IL-1/IL-8 cascade triggered by sequential intratracheal instillation of HKS and rPVL.
(A–E) Rabbits were inoculated intratracheally with HKS, 3 h later with rPVL and euthanized 6 h post-HKS instillation. When applicable, Kineret/IL-1Ra was injected IV at 2 h post-HKS and was co-instillated with rPVL. IL-8 levels in BALF (A) or in lung lysates (B) were quantified by ELISA. (C) Gross pathological scoring was performed. (D) The ratio of lung weight to body weight is shown. (E) Total protein levels in BALF were quantified by Bradford assay. (A–B, E) Each point represents the value obtained in the BALF from one lobe or (C, D) the value obtained for one rabbit. (A–E) The geometric mean is shown. Results from two independent experiments (n = 12) are shown.
Figure 6.
Kineret/IL-1Ra does not reduce IL-8 levels during PVL+ S. aureus-mediated pneumonia but increases lung bacterial load.
(A–F) Rabbits were inoculated intratracheally with USA300 LAC strain and euthanized 6 h post-infection. When applicable, Kineret/IL-1Ra was injected IV 1 h before and 3 h after infection and was co-instilled with S. aureus. IL-8 levels in BALF (A) or in lung lysates (B) were quantified by ELISA. (C) Gross pathological scoring was performed. (D) The ratio of lung weight to body weight is shown. (E) Total protein levels in BALF were quantified by Bradford assay. (F) Bacterial burden as determined by CFU assay is shown. (A, B, E) Each point represents the value obtained in the BALF from one lobe or (C, D, F) the value obtained for one rabbit. (A–F) The geometric mean is shown. Results from two independent experiments (n = 12) are shown.