Chapter 5. Results and general discussion
5.4. Pivotal role of S. aureus PSM toxins in stimulation of IL-1β release by infected
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Figure 22: Caspase-1 is required for secretion of IL-1β in S. aureus-infected MG-63 cells.
WT MG-63 cells were infected or not with S. aureus SA113 at MOI 50:1 and compared with (CASP1–/–
MG-63 clone B9) and (CASP1–/– MG-63 clone C3) infected or not with S. aureus SA113 at MOI 50: 1.
IL-1β production was measured in the cell-free supernatant by ELISA. Data show monitoring for a period of 6 h to 9 days post-infection. Tukey’s Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD).
Experiments are performed in triplicate and are representative of data obtained in three independent experiments. Asterisk represent statistically significant difference: *p < 0.05 by two-way ANOVA.
5.4. Pivotal role of S. aureus PSM toxins in stimulation of IL-1β release by
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enterocolitica, Salmonellae species, Pseudomonas aeruginosa, among others, use strategies that manipulate inflammasome activation, in most cases, interfere in the production or recognition of bacterial ligands that trigger inflammasomes (BRODSKY et al., 2010).
The quorum-sensing system in S. aureus known as the accessory gene regulator (Agr) regulates the expression of many virulence factors. Agr regulates main S. aureus toxins, among which regulation of Phenol-soluble modulins (PSMs) reviewed (PESCHEL; OTTO, 2013) contain five α-peptides (δ-toxin and PSMα1-4) and two β-peptides (PSMβ1-2), which all share an amphipathic α-helical structure, thereby acting as biological detergents. Thus, PSMs are regarded as a new class of Staphylococcal leukocidins (KRETSCHMER et al., 2010;
NAKAMURA et al., 2013; PESCHEL; OTTO, 2013; RICHARDSON et al., 2019). PSMs first attract innate immune cells like neutrophils, macrophages, and dendritic cells (DCs) by binding to the formyl peptide receptor 2 (FPR2) (RICHARDSON et al., 2019; SCHREINER et al., 2013;
WANG et al., 2017). Was been reported that PSMs can activate inflammasome-like signaling and trigger IL-1β and IL-18 secretion (MELEHANI; DUNCAN, 2016). In addition, was demonstrated that PSMs stimulate the production of inflammatory cytokines by infected keratinocytes (SYED et al., 2015), thus it is essential that this role is unraveled in our experimental model.
Furthermore, our results on strain dependence of IL-1β stimulation in osteoblasts suggested an involvement of Agr, as the low levels of IL-1β in cells infected with strain SA113 as opposed to those infected with strains MW2 and LAC correlate with levels of Agr functionality in those strains. SA113 is functionally Agr-defective, while MW2 and LAC show high Agr activity. These considerations prompted us to investigate the role of PSMs in IL-1β production by infected osteoblasts.
First, we analyzed LAC (USA300) wild-type and its isogenic mutant LACΔpsmαβhld (S. aureus strain lacking PSMα, PSMβ and δ-toxin) for their ability to stimulate the release of IL-1β. As shown in Fig. 23-A, the level of IL-1β was significantly decreased in the supernatants of WT MG-63 cells exposed to LAC Δpsmαβhld compared to WT MG-63 cells exposed to LAC wild- type on days 5 and 9 post-infection. To further investigate which PSM were involved in the simulation of IL-1β release, we used the PSM deletion strain LACΔpsmαβhld and complemented strains, expressing either the four PSMα peptides (LACΔpsmαβhld-pTXΔα1- 4), the two PSMβ peptides (LACΔpsmαβhld-pTXΔβ1-2), or the δ-toxin (LACΔpsmαβhld- pTXΔhld) and monitored IL-1β levels in cell exposed to those strains up to 9 days post- infection. As shown in Fig. 23-B, there was a significant decrease in the IL-1β released by cells exposed to the LACΔpsmαβhld strain compared to LAC. Exposure to complemented
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mutants demonstrated that the release was partially restored when the strains were complemented with PSMα (LACΔpsmαβhld-pTXΔα1-4), PSMβ (LACΔpsmαβhld pTXΔβ1-2) or δ-toxin (LACΔpsmαβhld-pTXΔhld). However, the difference was statistically significant only when strains were complemented with PSMβ or, at earlier time points, δ-toxin (LACΔpsmαβhld-pTXΔhld) (Fig. 23).
Employment of deletion and complemented PSMs mutants demonstrated a pivotal role of S.
aureus toxins PSMs in inflammasomes related IL-1β production by infected osteoblastic cells.
We would like to highlight that IL-1β production by infected osteoblasts appears to be specifically dependent on PSM betas among PSMs. This is noteworthy, as we don’t know any specific phenotypes attributable to those PSMs so far. In addition to PSMs the action of other factors cannot be excluded since complemented mutants did not restore entirely the level of secreted IL-1β.
Figure 23: S. aureus phenol-soluble modulins stimulate IL-1β release from infected osteoblasts.
A. WT MG-63 cells were exposed to wild type LAC (USA300) and its isogenic mutant LAC ∆psmαβhld at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various time post-infection (2 days and 5 days) cell supernatants were collected, centrifuged and the level of IL- 1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The IL-1β values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA).
P-values < 0.05 ( ⃰ ) were considered to be significant. Tukey’s Honestly Significant Difference test was
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applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).
B. MG-63 cells were exposed to USA300 LAC (pTX∆16), which carries the control plasmid, the deletion mutant LAC∆psmαβhld (pTX∆16) and the complemented strains expressing the four PSMα peptides (LAC∆psmαβhld-pTX∆α1-4), the two PSMβ peptides (LAC∆psmαβhld-pTX∆β1-2), or the δ-toxin (LAC∆psmαβhld-pTX∆hld) at MOI 1:50 for 2 h followed by antibiotic treatment as described in Material and Methods. After various times post-infection (2 days, 5 days, 7 days and 9 days) cell supernatants were collected, centrifuged and the level of IL-1β was determined by a sandwich-ELISA (Thermofischer Life Technologies) as described in Material and Methods. The values are presented as concentrations in pg/ml. Three independent assays were performed. The differences among the groups were assessed by analysis of variance (ANOVA). P-values< 0.05 (*) were considered to be significant. Tukey’s Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as means ± standard deviation (±SD).
In addition, we evaluated the secretion of IL-1β in osteoblast cells exposed to S. aureus strains obtained from patients with osteomyelitis. We analyzed MG-63 cells infected by six S. aureus human clinical isolates. The level of IL-1β was measured by ELISA. Three pairs of S. aureus strains used were obtained from patients with staphylococcal BJI provided by CNF Staphylococcus (National Reference Center for Staphylococci) of Lyon. Three couples of isolates were selected from patients P1, P2, P3 who were diagnosed with initial acute (i) and recurrent (r) staphylococcal BJI: isolates from the same patient were named 45i and 46r (P1), 47i and 48r (P2), 51i and 53r (P3) for initial and recurrent BJI correspondently. (Supplementary Material). Previous studies conducted by our group have demonstrated that the eukaryotic cell cycle delay induced by S. aureus human clinical isolates is associated with the production of PSMα1 that reveals a new mechanism to promote infection (DEPLANCHE et al., 2015).
The analysis of the kinetics of IL-1β release by WT MG-63 cells exposed to either six clinical isolates or S. aureus USA400 strain (MW2) demonstrated an increase of the level of IL-1β in the supernatants from the 2nd day post-infection up to 9 days post-infection. Furthermore, we found different levels of IL-1β production induced by clinical strains collected from three patients. When comparing its acute (i) and recurrent (r) forms, we find that IL-1β production induced by the strains (45i and 46r) was similar (Fig. 24-A). The IL-1β production induced by the acute strain (47i) was significantly higher then induced by the recurrent strain (48r) (Fig.
24-B). A comparison of strains (51i) and (52r) showed that strain (52r) induced higher IL-1β levels compared to strain (51i) (Fig. 24-C). These results demonstrated that clinical strains of S. aureus obtained from patients with staphylococcal BJI induce IL-1β secretion in infected
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MG-63 cells and suggest that S. aureus virulence factors may be involved in inflammasome activation and IL-1β secretion.
Figure 24: kinetics of IL-1β production between 6 h and 9 days post-infection in MG-63 cells by S. aureus clinical strains.
A. S. aureus clinical strain (45i) vs S. aureus clinical strain (46r). B. S. aureus clinical strain (47i) vs S.
aureus clinical strain 48r. C. S. aureus clinical strain (51i) vs S. aureus clinical strain (52r). IL-1β production was measured by ELISA in supernatants of WT MG-63 cells. Tukey’s Honestly Significant Difference test was applied for comparison of means between the groups. The values are expressed as mean ± standard deviation (±SD). Experiments are performed in triplicate and are representative of data obtained in three independent experiments. Significance is denoted as thus: **p < 0.01 by two-way ANOVA.