Dissection of host cell signal transduction during Acinetobacter baumannii-triggered inflammatory response

Abstract

Infected airway epithelial cells up-regulate the expression of chemokines, chiefly IL-8, and antimicrobial molecules including beta-defensins (BD). Acinetobacter baumannii is a cause of hospital-acquired pneumonia. We examined whether A. baumannii induced the expressions of IL-8 and BD2 by airway epithelial cells and the receptors implicated in bacterial detection. A549 and human primary airway cells released IL-8 upon infection. A. baumannii-infected cells also increased the expression of BD2 which killed A. baummannii strains. IL-8 induction was via NF-kappaB and mitogen-activated kinases p38 and p44/42-dependent pathways. A. baumannii engaged Toll-like receptor (TLR) 2 and TLR4 pathways and A549 cells could use soluble CD14 as TLRs co-receptor. A. baumannii lipopolysaccharide stimulated IL-8 release by A549 cells and sCD14 facilitated the recognition of the lipopolysaccharide. Mass spectrometry analysis revealed that A. baumannii lipid A structure matches those with endotoxic potential. These results demonstrate that airway epithelial cells produce mediators important for A. baumannii clearance.

Figure 1. Acinetobacter baumannii induces the secretion of IL-8 by airway epithelial cells.

(A–B) ELISA of IL-8 released by A549 (panel A) or NHBE (panel B) cells left untreated (CON) or infected for different time points with A. baumannii ATCC 17978 (n = 3). *, P<0.05 (for the indicated comparisons; one-way ANOVA). (C) ELISA of IL-8 released by A549 left untreated (CON) or infected for different time points with different A. baumannii strains (n = 3). *, P<0.05 (results are significantly different from the results for untreated cells; one-way ANOVA).

Figure 2. Acinetobacter baumannii up-regulates the expression of human β-defensin 2 (BD2) and it is susceptible to this defensin.

(A) A549 cells were transfected with BD2 luciferase reporter gene and Renilla luciferase plasmid. Cells were infected with A. baummannii strains and BD2 promoter activation was measured 6 h post-infection. Activity is normalized by correction of Renilla expression and is presented relative to the cells untreated. CON, non infected cells. Bars represent mean ± SD (n = 3) *, results are significantly different (P<0.05; one-tailed t test) from the results for non infected cells (CON). (B) Survival of bacteria (percentage of cell colonies with respect to not exposed to agents) in presence of 3 µg/ml of BD2. Error bars display standard deviation from the mean of three experiments, each one run in duplicate.

Figure 3. Activation of NF-κB and MAPKs is required for Acinetobacter baumannii-induced IL-8 expression.

(A) Activation of a NF-κB luciferase reporter plasmid in A549 cells left non treated (CON) or infected for 6 h with A. baumannii ATCC 17978. Activity is normalized by correction of Renilla expression and is presented relative to the cells untreated (the data are means and SD.; n = 3). (B) Immunoblot analysis of IκBα levels in lysates of A549 cells left untreated (time 0) or infected for different time points with A. baumannii ATCC 17978. Data are representative of three independent experiments. (C) Upper panels, immunoblots showing p65 levels in nuclear and cytoplasmic extracts of A549 cells infected with A. baumannii ATCC 17978 for different time points. Asterisk denotes a protein recognized by the antibody which is only present in nuclear extracts and also serves as a loading control. Blots of cytoplasmic extracts were reprobed with polyclonal antibody anti human tubulin to control that equal amounts of proteins were loaded in each lane. Lower panels, immunoblots showing phospho-p65 and tubulin levels in lysates of A549 infected with A. baumannii ATCC 17978 for different time points. IL-1β (20 ng/ml for 30 min) was used as a positive control for NF-κB activation. The results are representative of three independent experiments. (D) ELISA of IL-8 released by A549 cells left untreated (CON, white bar) or infected for 4 h with A. baumannii ATCC 17978 in the absence or presence of different concentrations of CAPE, an inhibitor of NF-κB, which was added 1 h before infecting the cells (n = 3). (E) Immunoblots showing phospho-p38 (P-p38), phospho p44/42 (P-p44/42), phospho JNK (P-JNK) The results are representative of three independent experiments. (F) ELISA of IL-8 released by A549 cells left untreated (CON, white bar) or infected for 4 h with A. baumannii ATCC 17978 in the absence or presence of different concentrations of SB203580 (5 and 10 µM; p38 MAPK inhibitor), U0126 (1 and 10 µM; p44/42 MAPK inhibitor) or SP600125 (5 and 10 µM; JNK MAPK inhibitor) which were added 2 h before infecting the cells (n = 3). *, P<0.05 (results are significantly different from the results for untreated cells; one-way ANOVA). Δ, P<0.05 (results are significantly different from the results for infected cells in the absence of inhibitor).

Figure 4. Role of TLR2, TLR4 and sCD14 in Acinetobacter baumannii-induced IL-8 expression.

(A) ELISA of IL-8 secreted by A549 cells transfected with either control, TLR2, or TLR4 siRNAs, which were left untreated (white bars) or infected for 4 h with A. baumannii ATCC 17978 (data are means and SD.; n = 3). *, P<0.05 (one-way ANOVA). (B) siRNA efficiency was quantified by RT-qPCR in samples from the same experiment shown in panel A. mRNA level was normalized to GADPH and then relative mRNA levels in cells transfected with control siRNA or specific siRNA were compared. mRNA levels in cells transfected with control siRNA were set to 100% (the data are means and SD.; n = 3). (C) ELISA of IL-8 secreted by A549 left untreated (CON, white bar) or infected with A. baumannii ATCC 17978 for 4 h in the absence or presence of different amounts of sCD14 (n = 3). *, P<0.05 (results are significantly different from the results for untreated cells; one-way ANOVA). Δ, P<0.05 (results are significantly different from the results for infected cells in the absence of sCD14). (D) ELISA of IL-8 secreted by A549 cells transfected with either control, TLR2, or TLR4 siRNAs, which were left untreated (white bars) or infected for 4 h with A. baumannii ATCC 17978 in the presence or absence of sCD14 (1 ng/ml) (data are means and SD.; n = 3). *, P<0.05 (one-way ANOVA).

Figure 5. Acinetobacter baumannii and Escherichia coli LPSs-induced IL-8 from airway epithelial cells.

A549 cells were challenged with medium alone (dashed line) or with 0.001–1 µg/ml of each LPS preparation in the absence (black symbols) or presence (white symbols) of sCD14 (1 ng/ml). Supernatant IL-8 levels were measured at 18 h. Results are presented as means and SD.; (n = 3). ▵, ▴ A. baumannii LPS; ○, • E. coli LPS.

Figure 6. Analysis of Acinetobacter baumannii lipid A.

(A) Negative ion MALDI-TOF mass spectra of lipid A isolated from Acinetobacter baumannii ATCC 17978. (B) Proposed structures of the main molecular species present in Acinetobacter baumannii ATCC 17978 lipid A.