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Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Ahn DS, Parker D, Planet PJ, Nieto PA, Bueno SM, Prince A - Mucosal Immunol (2014)

Bottom Line: We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide.The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia.Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Columbia University College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide. IL-16 production involved multiple signals including ligation of tumor necrosis factor receptor (TNFR) family members or epidermal growth factor receptor, both receptors for SpA and generation of Ca(2+) fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4(+) cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

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MRSA induction of IL-16 release is calcium and calpain dependent in THP-1sCalcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p<0.05 value as compared to MRSA infected samples, Student's t-test, post-hoc Dunnett's test for multiple comparisons. The above graphs represent data from at least 2 independent experiments.
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Figure 5: MRSA induction of IL-16 release is calcium and calpain dependent in THP-1sCalcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p<0.05 value as compared to MRSA infected samples, Student's t-test, post-hoc Dunnett's test for multiple comparisons. The above graphs represent data from at least 2 independent experiments.

Mentions: Our results suggest that multiple ligands that activate caspase-8 and caspase-3 could participate in the induction of pro-IL-16 processing22. Given the participation of the Ca2+-dependent calpains in caspase activation, we examined the requirement of Ca2+ fluxes in the induction of IL-16. MRSA induced Ca2+ fluxes in Flo-4/AM loaded THP-1s within 10 min of exposure to MRSA with attenuation by pretreatment with BAPTA (Figure 5a and Supplementary Figure S1a-b). THP-1s in media alone showed no baseline Ca2+ fluxes and gave significant fluorescence when treated with thapsigargin (Figure 5b and Supplementary Figure S1c). IL-16 secretion was stimulated by ionomycin alone and significantly decreased in the presence of EDTA (p<0.05 compared to media) (Figure 5c). Pretreatment of THP-1s with calpeptin, a calpain inhibitor, resulted in diminished secretion of IL-16 (p<0.05 compared to DMSO) (Figure 5d), indicating that IL-16 processing in THP-1s is calpain, as well as calcium dependent. Similar differences in Jurkats with calcium and calpain inhibitors were not seen (data not shown). Caspase-3 involvement was demonstrated when THP-1s preloaded with Caspase-3/7 Green Detection Reagent were infected with MRSA, showing an increase in fluorescence over baseline (Figure 5e). We then observed decreased secretion of IL-16 when THP-1s were pretreated with pancaspase inhibitor Z-VAD-FMK (p<0.05 compared to DMSO with MRSA) or a caspase-3 inhibitor (p<0.05 compared to DMSO with MRSA) but not a caspase-1 inhibitor (Figure 5f). Similarly, IL-16 release was reduced in Jurkats pretreated with caspase-3 inhibitor (p<0.05 compared to DMSO with MRSA) (Figure 5g). Staurosporine control, an in vitro inducer of caspase activity30, showed induction of IL-16 secretion in both THP-1s and Jurkats.


Secretion of IL-16 through TNFR1 and calpain-caspase signaling contributes to MRSA pneumonia.

Ahn DS, Parker D, Planet PJ, Nieto PA, Bueno SM, Prince A - Mucosal Immunol (2014)

MRSA induction of IL-16 release is calcium and calpain dependent in THP-1sCalcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p<0.05 value as compared to MRSA infected samples, Student's t-test, post-hoc Dunnett's test for multiple comparisons. The above graphs represent data from at least 2 independent experiments.
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Figure 5: MRSA induction of IL-16 release is calcium and calpain dependent in THP-1sCalcium fluxes were measured by fluorescence activation on an inverted microscope with a GFP mercury laser. THP-1s loaded with AM/Flo-4 (1mM) and PowerLoad concentrate (1mM) 2 hours prior to stimulation with (a) MRSA (MOI 100) with and without BAPTA (6μM), or (b) media alone followed by thapsigargin (1μM) (T) as a positive control. Representative cells were chosen and fluorescence plotted over time. (c) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with EGTA (0.5 or 1mmol) with MRSA (MOI 50) was measured 2 hours after infection. (d) IL-16 levels in supernatants of THP-1s pretreated 1 hour prior to infection with calpeptin (CPEP) (20 or 200 μM) with MRSA (MOI 50) was measured 2 hours after infection. Ionomycin (iono) in DMSO was 5 mM. (e) THP-1s were preloaded with Caspase-3/7 Green Detection agent (5μM) 30 minutes prior to infection and then infected with MRSA (MOI 100). Fluorescence was then plotted over time. (f) IL-16 levels in supernatants of THP-1s were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM, caspase-3 inhibitor (C3 inh) 50 μM, and pan-caspase inhibitor (ZVAD) 100 μM. Staurosporine in DMSO was 1μM. (g) IL-16 levels in supernatants of Jurkats were pretreated 1 hour prior to infection with respective inhibitor with MRSA (MOI 50) for 2 hours; caspase 1 inhibitor (C1 inh) 50 μM and caspase-3 inhibitor (C3 inh) 50 μM. Staurosporine in DMSO was 1μM. *p<0.05 value as compared to MRSA infected samples, Student's t-test, post-hoc Dunnett's test for multiple comparisons. The above graphs represent data from at least 2 independent experiments.
Mentions: Our results suggest that multiple ligands that activate caspase-8 and caspase-3 could participate in the induction of pro-IL-16 processing22. Given the participation of the Ca2+-dependent calpains in caspase activation, we examined the requirement of Ca2+ fluxes in the induction of IL-16. MRSA induced Ca2+ fluxes in Flo-4/AM loaded THP-1s within 10 min of exposure to MRSA with attenuation by pretreatment with BAPTA (Figure 5a and Supplementary Figure S1a-b). THP-1s in media alone showed no baseline Ca2+ fluxes and gave significant fluorescence when treated with thapsigargin (Figure 5b and Supplementary Figure S1c). IL-16 secretion was stimulated by ionomycin alone and significantly decreased in the presence of EDTA (p<0.05 compared to media) (Figure 5c). Pretreatment of THP-1s with calpeptin, a calpain inhibitor, resulted in diminished secretion of IL-16 (p<0.05 compared to DMSO) (Figure 5d), indicating that IL-16 processing in THP-1s is calpain, as well as calcium dependent. Similar differences in Jurkats with calcium and calpain inhibitors were not seen (data not shown). Caspase-3 involvement was demonstrated when THP-1s preloaded with Caspase-3/7 Green Detection Reagent were infected with MRSA, showing an increase in fluorescence over baseline (Figure 5e). We then observed decreased secretion of IL-16 when THP-1s were pretreated with pancaspase inhibitor Z-VAD-FMK (p<0.05 compared to DMSO with MRSA) or a caspase-3 inhibitor (p<0.05 compared to DMSO with MRSA) but not a caspase-1 inhibitor (Figure 5f). Similarly, IL-16 release was reduced in Jurkats pretreated with caspase-3 inhibitor (p<0.05 compared to DMSO with MRSA) (Figure 5g). Staurosporine control, an in vitro inducer of caspase activity30, showed induction of IL-16 secretion in both THP-1s and Jurkats.

Bottom Line: We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide.The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia.Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pediatrics, Columbia University College of Physicians and Surgeons, New York, New York, USA.

ABSTRACT
Staphylococcus aureus is a major cause of severe pneumonia. Multiple mechanisms of proinflammatory signaling are activated to recruit immune cells into the airway in response to S. aureus. We found that interleukin-16 (IL-16), a T cell cytokine that binds CD4, is potently activated by S. aureus, specifically by protein A (SpA), and to a much greater extent than by Gram-negative pathogens or lipopolysaccharide. IL-16 production involved multiple signals including ligation of tumor necrosis factor receptor (TNFR) family members or epidermal growth factor receptor, both receptors for SpA and generation of Ca(2+) fluxes to activate calpains and caspase-3. Although human airway epithelial cells, vascular endothelial cells, THP-1 and Jurkat T cells released IL-16 in response to S. aureus in vitro, in a murine model of pneumonia, CD4(+) cells were the major source of IL-16 suggesting the involvement of an autocrine signaling pathway. The production of IL-16 contributed to lung damage as neutralization of IL-16 enhanced S. aureus clearance and resulted in diminished lung pathology in S. aureus pneumonia. Our results suggest that the ability of S. aureus to activate TNFR1 and Ca(2+)/calpain signaling contribute to T cell activation and excessive inflammation in the setting of acute pneumonia.

Show MeSH
Related in: MedlinePlus