Interleukin-1β regulates CXCL8 release and influences disease outcome in response to Streptococcus pneumoniae, defining intercellular cooperation between pulmonary epithelial cells and macrophages

Abstract

The success of Streptococcus pneumoniae (the pneumococcus) as a pulmonary pathogen is related to its restriction of innate immune responses by respiratory epithelial cells. The mechanisms used to overcome this restriction are incompletely elucidated. Pulmonary chemokine expression involves complex cellular and molecular networks, involving the pulmonary epithelium, but the specific cellular interactions and the cytokines that control them are incompletely defined. We show that serotype 2 or 4 pneumococci induce only modest levels of CXCL8 expression from epithelial cell lines, even in the absence of a polysaccharide capsule. In contrast, coculture of A549 cells with the macrophage-like THP-1 cell line, differentiated with vitamin D, or monocyte-derived macrophages enhanced CXCL8 release. Supernatants from the THP-1 cell line prime A549 cells to release CXCL8 at levels similar to cocultures. Interleukin-1Ra (IL-1Ra) inhibits CXCL8 release from cocultures and reduces the activity of macrophage-conditioned media, but inhibition of tumor necrosis factor alpha (TNF-α) had only a minimal effect on CXCL8 release. Release of IL-1β but not TNF-α was upregulated in cocultures. IL-1 type 1 receptor knockout C57BL/6 and BALB/c mice confirmed the importance of IL-1 signaling in CXC chemokine expression and neutrophil recruitment in vivo. In fulminant disease, increased IL-1 signaling resulted in increased neutrophils in the airway and more invasive disease. These results demonstrate that IL-1 is an important component of the cellular network involving macrophages and epithelial cells, which facilitates CXC chemokine expression and aids neutrophil recruitment during pneumococcal pneumonia. They also highlight a potential clinical role for anti-IL-1 treatment to limit excessive neutrophilic inflammation in the lung.

Fig 1

Presence of macrophages enhances epithelial cell CXCL8 production. (A and B) A549 epithelial cells were either mock infected (white bars) or challenged with encapsulated (pale-gray bars) or unencapsulated (dark-gray bars) nonopsonized serotype 2 (A) and serotype 4 (B) S. pneumoniae (MOI of 10) or IL-1β (black bars). CXCL8 levels in the cell culture supernatants were measured by ELISA at the indicated time points (n = 4 to 6 independent experiments). (C and D) A549 epithelial cells alone (EC), THP-1 cells alone (MC), and A549-THP-1 cocultures (Co) were challenged with opsonized S. pneumoniae for up to 24 h. CXCL8 levels in the cell culture supernatants were measured by ELISA at 2-, 6-, and 24-h time points. (C) Encapsulated (D39) and unencapsulated (FP22) serotype 2 pneumococci (n = 7); (D) encapsulated (TIGR4) and unencapsulated (FP23) serotype 4 pneumococci (n = 5). Mock-infected (MI) and IL-1β data at the 24-h time point is shown for comparison. One-way ANOVA (all P values are <0.05) with Bonferroni's posttest: *, P < 0.05; **, P < 0.01; #, P < 0.05 for CXCL8 level in coculture versus the sum of CXCL8 levels from A549 and THP-1 cells alone).

Fig 2

Physical separation of macrophages from epithelial cells does not impair priming of CXCL8 production. A549 epithelial cells alone (EC), THP-1 cells alone (MC), A549-THP-1 cocultures (Co), and A549 and THP-1 cells separated by transwells (TW) were challenged with D39 pneumococci (MOI of 10). CXCL8 levels in the cell culture supernatants were measured by ELISA at 6 h. IL-1β (positive control) data are shown for comparison (IL-1β). One-way ANOVA with Bonferroni's posttest (n = 6). ns, not significant.

Fig 3

Conditioned media from THP-1 cells challenged with pneumococci primes epithelial cell CXCL8 production. THP-1 cells were exposed to either mock infection or D39 S. pneumoniae (MOI of 100) for 24 h. Mock-infected macrophage-conditioned media (MCM-MI) and D39-challenged macrophage-conditioned media (MCM-D) were collected at 24 h. Monolayers of A549 epithelial cells were then incubated with medium alone (EC), MCM-MI, THP-1 cells and D39 (MOI 10) (Co+D), MCM-D, MCM-D plus IL-1Ra (MCM-D/IL-1Ra), MCM-D plus sTNF type 1 receptor (MCM-D/TNFR1), MCM-D plus both inhibitors (MCM-D/Both), or IL-1β. CXCL8 levels in the cell culture supernatants were measured by ELISA at 6 h (n = 3); one-way ANOVA (P < 0.001) with Bonferroni's posttest: *, P < 0.05; **, P < 0.01.

Fig 4

IL-1Ra blocks enhancement of CXCL8 production in cocultures. Monolayers of A549 epithelial cells alone (EC), THP-1 cells alone (MC), A549-THP-1 cocultures (Co), cocultures with IL-1Ra (Co/IL-1Ra), cocultures with sTNF type 1 receptor (Co/TNFR1), and cocultures with both inhibitors (Co/both) were challenged with encapsulated D39 (A) and unencapsulated FP22 serotype 2 (B) pneumococci and encapsulated TIGR4 (C) and unencapsulated FP23 serotype 4 (D) pneumococci. IL-1β-stimulated cocultures are also shown for comparison. CXCL8 levels in the cell culture supernatants were measured by ELISA at 6 h (n = 3 to 4); one-way ANOVA (all P values are <0.002) with Bonferroni's posttest: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

Enhanced epithelial cell CXCL8 production is also seen in cocultures containing primary monocyte-derived macrophages. A549 epithelial cells alone (EC), monocyte-derived macrophages alone (MDM), and A549-MDM cocultures (Co) were challenged with opsonized S. pneumoniae D39 (A) or TIGR4 (B) (MOI of 10), with or without IL-1 receptor antagonist (IL-1Ra) for 6 h, and CXCL8 levels in the cell culture supernatants were measured by ELISA and normalized for cell number (data show release/1 × 10⁵ A549 cells). Mock-infected (MI) and IL-1β data are shown for comparison. One-way ANOVA (all P values are <0.05) with Bonferroni's posttest: *, P < 0.05; ***, P < 0.01. P values of <0.01 for CXCL8 levels in coculture versus the sum of CXCL8 levels from A549 cells and MDM alone (n = 3 to 5) from 3 independent experiments.

Fig 6

IL-1β but not TNF-α levels are enhanced in cocultures following challenge with pneumococci. A549 epithelial cells (EC) were exposed to media alone (−) or D39 pneumococci (+), as were THP-1 cells (MC) and A549-THP-1 cocultures (Co) and IL-1β (A) and TNF-α (B) levels measured by ELISA at 6 and 24 h (n = 3); one-way ANOVA (all P values are <0.02) with Bonferroni's post test: *, P < 0.05. A549 epithelial cells were exposed to conditioned media from D39-challenged THP-1 cells, and IL-1β (C) and TNF-α (D) levels were measured by ELISA at 0, 2, 6 and 24 h.

Fig 7

Modulation of IL-1 signaling alters production of CXC chemokines and polymorphonuclear cell (PMN) recruitment during S. pneumoniae infection in vivo. (A) Percentage of PMNs in bronchial alveolar lavage (BAL) fluid from C57BL/6 control mice and mice deficient in IL-1 type 1 receptor on a C57BL/6 background (IL-1R1^−/−) 24 h after intratracheal instillation of 5 × 10⁵ CFU type 4 pneumococci. Levels of CXCL1 (B) and CXCL2 (C) in BAL fluid from the same experiment as described for panel A. (D) Percentage of PMNs in BAL fluid from BALB/c control mice and mice deficient in IL-1 type receptor on a BALB/c background (IL-1R^−/−) challenged with pneumococci as described for panel A. Levels of CXCL1 (E) and CXCL2 (F) in BAL fluid from the same experiment as that described for panel D. *, P < 0.05; **, P < 0.01, unpaired t test.

Fig 8

Modulation of IL-1 signaling can reduce the severity of invasive disease in fulminant pneumococcal infection. (A) Percentage of neutrophils (PMN) in bronchial alveolar lavage (BAL) fluid from BALB/c control mice and mice deficient in IL-1 type 1 receptor on a BALB/c background (IL-1R1^−/−) 24 h after intratracheal instillation of 1 × 10⁷ CFU type 4 pneumococci. Levels of CXCL1 (B), CXCL2 (C), and TNF-α (D) in BAL fluid from the same experiment as that described for panel A. CFU of bacteria in lung homogenates (E) and blood (F) in the same experiments as that described for panel A. *, P < 0.05, unpaired t test; **, P < 0.01, Mann-Whitney test.