Transcription factor complex AP-1 mediates inflammation initiated by Chlamydia pneumoniae infection

Abstract

Chlamydia pneumoniae is responsible for a high prevalence of respiratory infections worldwide and has been implicated in atherosclerosis. Inflammation is regulated by transcription factor (TF) networks. Yet, the core TF network triggered by chlamydiae remains largely unknown. Primary human coronary artery endothelial cells were mock-infected or infected with C. pneumoniae to generate human transcriptome data throughout the chlamydial developmental cycle. Using systems network analysis, the predominant TF network involved receptor, binding and adhesion and immune response complexes. Cells transfected with interfering RNA against activator protein-1 (AP-1) members FOS, FOSB, JUN and JUNB had significantly decreased expression and protein levels of inflammatory mediators interleukin (IL)6, IL8, CD38 and tumour necrosis factor compared with controls. These mediators have been shown to be associated with C. pneumoniae disease. Expression of AP-1 components was regulated by MAPK3K8, a MAPK pathway component. Additionally, knock-down of JUN and FOS showed significantly decreased expression of Toll-like receptor (TLR)3 during infection, implicating JUN and FOS in TLR3 regulation. TLR3 stimulation led to elevated IL8. These findings suggest that C. pneumoniae initiates signalling via TLR3 and MAPK that activate AP-1, a known immune activator in other bacteria not previously shown for chlamydiae, triggering inflammation linked to C. pneumoniae disease.

Fig. 1. AP-1 components dominate the Transcription Factor (TF) network initiated by C. pneumoniae infection of HCAEC

A. AP-1 components, FOS, FOSB and JUNB dominate the TF network initiated by C. pneumoniae infection. The TF network was derived from a genome-wide protein interaction network overlapped with transcriptome data from this study (see Material and Methods). Nodes and edges donate proteins and interactions, respectively. The color of the node represents the gene expression level: Red for up-regulation, green for down-regulation and violet for equivocal at 5 mim.. B. The contribution of TFs to network connectivity and to the diameter of the network activated at 5 min post infection. TFs were systematically knocked out in silico, and the alterations in network connectivity (measured as average number of neighbors) and network diameter (measured as average shortest path length) were calculated (see Materials and Methods). The top TFs that contributed most to the network are shown. FOS and MAP3K8 contributed the most to both connectivity and diameter, respectively, suggesting that AP-1 dominates the TF network for C. pneumoniae. C. Summary of the top 10 TFs from our transcriptome data at each time point that contributed the most to the diameter of the network activated at 5 min, 25 min, 2H, 24H, and 60H. From top to bottom, GBP2, SOCS3, STAT1, JUN, UBE2L6, IRF7, and IRF2 predominated in late infection (24 hrs and 60 hrs), while IRF1, NFKIA, and JUNB were featured at 2 hrs, 24 hrs and 60 hrs. SPIN, MDM2, CEBPB, and APC predominated at 25 min. EGR1, NR4A1, IL6, and FOS were primarily activated from 5 min to 24 hrs or to 60 hrs.

Fig. 2. In silico knockout of hubs and bottlenecks in the TF network

A. Hubs and bottleneck and their neighbors for the wild-type network activated at 5 min. The green arrow highlights a hub (FOS). The pink arrow highlights a bottleneck (SFPQ). B. The network was locally disconnected after the combination of knocking out the first 5 genes, FOS, EGR1, MAP3K8, ID1, and ID2, compared to the wild-type network shown in (A).

Fig. 3. AP-1 target gene predictions using the protein-protein interaction network and promoter sequence analysis

A. Predicting AP-1-associated genes using the protein interaction network. The genes were determined from the network assembled from known transcription factor protein–protein interactions and transcriptome data as described in Materials and Methods. This network was organized by the gene ontology database, in which gene functions and localizations were included. AP-1 (nucleus) is predicted to interact with a number of major functional modules, including those for receptor complexes (yellow), immune response (red), and binding and adhesion (green). B. Computational analysis of AP-1 binding sites for inflammatory mediators. These AP-1 binding sites were identified by scanning the 5′ upstream promoter regions of each gene with MotifMogul. For illustration, only the one for IL8 is shown; the rest are shown in Figure 4.

Fig. 4

AP-1 binding sites for inflammatory mediators predicted by MotifMogul. MotifMogul was used to predict AP-1 binding sites for inflammatory mediators as described in detail in Materials and Methods.

Fig. 5. AP-1 regulates gene expression of inflammatory mediators in C. pneumoniae infected HEp-2 and THP-1 cells

A. AP-1 components FOS, FOSB, JUN and JUNB were knocked down by siRNA. The knockdown efficiency was determined by measuring gene expression alteration by qRT-PCR after siRNA knockdown of each AP-1 member (FOS, F−/−; FOSB, FB−/−; JUN, J−/−; JUNB, J−/−; FOS and JUN, F−/−J−/−) in C. pneumoniae infected HEp-2 and THP-1 cells. At least 75% of the gene expression of AP-1 members was knocked down when compared with the scrambled siRNA control. B–E. Knockdown of AP-1 members FOS and JUN during C. pneumoniae infection significantly decreased key pro-inflammatory mediators TNF, IL8, IL6 and CD38, but not IL6, measured by qRT-PCR in both HEp-2 and THP-1 cells compared to wild type (WT). F. Knockdown of AP-1 components JUN and FOS downregulate inflammatory factors TNF and IL8 compared to WT measured during infection. *p<0.045, **p<0.01, ***p<0.001, and ****p<0.0005.

Fig. 6. AP-1 Complex mediates inflammatory signaling via TLR3 and MAP3K8

A. JUN and FOS knockdown by siRNA dramatically alters gene expression of TLR3 in C. pneumoniae infected HCAEC and THP-1 cells as measured by qRT-PCR. B. Expression of AP-1 components correlates with MAPK signaling pathway during C. pneumoniae infection of HCAEC measured by both microarray and qRT-PCR.

Fig. 7. TLR3 mediates NF-κB activation and IL8 expression

A. Control HEK cells (HEK-Null) or HEK cells expressing TLR2, TLR3 or TLR4 were infected with C. pneumoniae (MOI of 1, 5, 10 or 40) or stimulated with the TLR ligands, 5 mg/mL Pam3CSK4 (TLR2 ligand), 500 ng/mL LPS (TLR4 ligand) or 200 ng/mL, 2 mg/mL or 20 mg/mL poly(I:C) (TLR3 ligand) for 48 hrs or 72 hrs. NF-κB activation was assessed by measuring SEAP activity colorimetrically at 630 nm. ( B. HEK cells expressing TLR3 or TLR2 were infected with C. pneumoniae at the indicated MOI or stimulated with poly(I:C), and IL8 expression was measured by qPCR. *p < 0.045, **p < 0.01, ***p < 0.001, and ****p < 0.0005.