Differential activation of inflammatory pathways in A549 type II pneumocytes by Streptococcus pneumoniae strains with different adherence properties

Abstract

Background: Adherence of Streptococcus pneumoniae bacteria to lung cells is a first step in the progression from asymptomatic carriage to pneumonia. Adherence abilities vary widely among S. pneumoniae patient isolates. In this study, the binding properties of S. pneumoniae isolates and the effects of binding on activation of the Nuclear Factor-Kappa-B (NFkappaB) pathway and cytokine secretion by type II pneumocytes were measured.

Methods: Mechanisms of high- and low-binding S. pneumoniae adherence to A549 cells were investigated by blocking putative receptors on bacteria and host cells with antibody and by eluting choline-binding proteins off of bacterial surfaces. NFkappaB activation was measured by western blot and immunocytochemistry and cytokine secretion was detected by a protein array.

Results: This study shows that S. pneumoniae isolates from pneumonia patients (n = 298) can vary by as much as 1000-fold in their ability to bind to human lung epithelial cells. This difference resulted in differential activation of the NFkappaB pathway. High-, but not low-binding S. pneumoniae used Choline-binding protein A (CbpA) to bind to complement component C3 on epithelial cell surfaces. Interleukin-8 (IL-8) was the only cytokine secreted by cells treated with either low- or high-binding S. pneumoniae.

Conclusion: These results indicate that S. pneumoniae clinical isolates are not homogeneous in their interaction with host epithelial cells. The differential activation of host cells by high- and low-binding S. pneumoniae strains could have implications for the treatment of pneumococcal pneumonia and for vaccine development.

Figure 1

Adherence of high-binders, but not low-binders, is reduced by blocking CbpA or C3. Solid bars, negative control; open bars, bacteria pre-treated with anti-CbpA antibody; dotted bars, A549 cells pre-treated with anti-C3 antibody. Negative control A549 cells pre-treated with normal rabbit serum. All values are mean ± one standard deviation, based on three independent experiments. Mean values are above each bar. The Y-axis units differ on graphs 1A and 1B. 1A. High-binding S. pneumoniae are identified by serotype along X-axis. 6301 evolved, 3 evolved, and 18C evolved are high-binding descendants of low-binding parent strains. Isolates identifed as 6 B all share that serotype but have different PFGE patterns. 1B. Low-binding S. pneumoniae strains are identified by serotype along X-axis. 6301 parent, 3 parent, and 18C parent are initial low-binding strains of 6301 evolved, 3 evolved, and 18C evolved.

Figure 2

High-binders induce prolonged degradation of IκB compared to low-binders. All values are mean ± one standard deviation, based on three independent experiments. 2A. IκB degradation induced by serotype 3 evolved high-binder and 3 low-binding parent strains. Closed squares, IκB level relative to time zero in A549 treated with low-binder. Open triangles, IκB level relative to time zero in A549 treated with high-binder. Representative Western blots for IκB are shown below each line graph. 2B. IκB degradation induced by serotype 9N low-binding clinical isolate and serotype 6B high-binding clinical isolate. Closed squares, IκB level relative to time zero in A549 treated with low-binder. Open triangles, IκB level relative to time zero in A549 treated with high-binder. Representative Western blots for IκB shown below line graph.

Figure 3

NFκB migrates to the nucleus after cell interaction with high-binders, but not low-binders, 4 hours after inoculation with bacteria. NFκB is detected as a dark brown stain against a pale lavender background. 3A, negative control. 3B, A549 incubated with serotype 3 low-binders (Black arrow shows NFκB protein in the cytoplasm). 3C, A549 incubated with serotype 3 high-binders (White arrow shows NFκB protein detected in the nucleus).

Figure 4

Supernatants from A549 cells treated with high- and low-binding S. pneumoniae were evaluated with a RayBio^® Human Cytokine Antibody Array III, which detects 42 different human cytokines. 1A and 2A, positive control; 3A and 4A, negative control; 5A, ENA-78; 6A, GCSF; 7A, GM-CSF, 8A, GRO; 9A, Gro-α; 10A, I-309; 11A, IL-1α; 12A, IL-1β; 1B, IL-2; 2B, IL-3; 3B, IL-4; 4B, IL-5; 5B, IL-6; 6B, IL-7; 7B, IL-8; 8B, IL-10; 9B, IL-12; 10B, IL-13; 11B, IL-15; 12B, IFN-γ; 1C, MCP-1; 2C, MCP-2; 3C, MCP-3; 4C, MCSF; 5C, MDC; 6C, MIG; 7C, MIP-1; 8C, RANTES; 9C, SCF; 10C, SDF-1; 11C, TARC; 12C, TGF-β; 1D, TNF-α; 2D, TNF-β; 3D, EGF; 4D, IGF-1; 5D, angiogenin; 6D, oncostatin M; 7D, thrombopoietin; 8D, VEGF; 9D, PDGF; 10D, leptin; 11D, negative control; 12D, positive control. The positive result for IL-8, in position 7B, with negative results for all other cytokines, was observed for A549 cells treated with both high- and low-binding bacteria. Experiment replicated with four different high-binding, and four different low-binding, S. pneumoniae strains.