Relative Contributions of Extracellular and Internalized Bacteria to Early Macrophage Proinflammatory Responses to Streptococcus pneumoniae.

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to The capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with Experiments using purified capsule material and a mutant expressing an serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated and those with unencapsulated , largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking internalization using cytochalasin D had minimal effects on the inflammatory response to Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated that could contribute to disease pathogenesis. Multiple extra- and intracellular innate immune receptors have been identified that recognize , but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.