Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Abstract

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.

Fig 1. S. pneumoniae serotypes differentially induce PLY- and caspase-1-dependent production of IL-1β by PBMCs as well as cell death.

(A-C, H-L) PBMCs were infected with S. pneumoniae strains (MOI = 1) as indicated or (D, E) were infected with S. pneumoniae D39 (MOI = 0.01) and treated with 10 μM Z-YVAD. Production of IL-1β (A, D, H) and IL-8 (C, E, J) was quantified by ELISA after 16 h. (D, E) 100% were defined as the amount of IL-1β or IL-8 released by S. pneumoniae-infected PBMCs. (B, I) Relative expression of Il1b was determined by quantitative RT-PCR after 5 h. (F) Human blood was incubated with pneumococcal lysates of S. pneumoniae serotypes 3, 6B, 7F, 9N, 1 and 8, and haemolytic activity was assessed. (G) Comparative protein model of allele 5 PLY. PLY domains are colour coded and mutations are marked in rose. (K) PBMCs were infected with S. pneumoniae D39 and D39Δply. (L) PBMCs were infected with serotypes 3, 6B, 7F, 9N, 1 and 8 pneumococci. (M) PBMCs were stimulated with allele 1 and allele 5 PLY for 16 h. (K-M) LDH release was quantified by cytotoxicity assay. Data are shown as mean ± SEM of three (D, E, M), five (B, I) or seven (A, C, H, J, K, L) independent experiments carried out in duplicates or triplicates. Significance is indicated by asterisks * = p<0.05, ** = p<0.01, *** = p<0.001.

Fig 2. The production of IL-1β in S. pneumoniae-infected human lung tissue is dependent on PLY and caspase-1.

(A-E) Human lung tissue was infected with 1x10⁶ CFU/mL S. pneumoniae serotype 2 D39 or D39Δply and treated with 100 ng/ml Z-YVAD 1 hour before infection where indicated. After 24 h, production of IL-1β (A, B, E) was quantified by ELISA, relative expression of Il1b was determined by qRT-PCR (C), or expression of pro-IL-1β was assessed by immunoblotting (D). Data are shown as mean ± SEM of three (A), five (E) or six (B, C) independent experiments carried out in duplicates. (D) One representative of four independent experiments is shown. Significance is indicated by asterisks ** = p<0.01, *** = p<0.001.

Fig 3. Pneumococci expressing haemolytic but not non-haemolytic PLY stimulate IL-1β production in human lung tissue.

(A) Human lung tissue was infected with 1x10⁶ CFU/mL S. pneumoniae serotypes 3, 6B, 9N, 1 and 8, or (B) were stimulated with 1μg/ml allele 1 and allele 5 PLY for 24 h. IL-1β secretion was quantified by ELISA. Data are shown as mean ± SEM of eight (A) or six (B) independent experiments carried out in duplicates. Significance is indicated by asterisks *** = p<0.001.

Fig 4. NLRP3 expression is up-regulated in human lung tissue during S. pneumoniae infection.

(A, B) Human lung tissue was infected with 1x10⁶ CFU/mL S. pneumoniae serotype 2 D39 or D39Δply and expression of NLRP3 was assessed by immunoblotting. (C) NLRP3 inhibitor glybenclamide efficiently reduced IL-1β secretion in human lung tissue during S. pneumoniae infection. Human lung tissue was preincubated with the inhibitor for 2 h and infected with 1x10⁶ CFU/mL S. pneumoniae D39 for 16 h. IL-1β production was quantified by ELISA. Data are shown as mean ± SEM of three (A, B) or six (C) independent experiments carried out in duplicates. Significance is indicated by asterisks *** = p<0.001.