In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen causes a rapidly lethal disease in and that heat-inactivated and bacterial flagellin can activate a reconstituted Nv-TLR-to-NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR-expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.

During injury and infection, inflammation is a response by macrophages to effect healing and repair. The kinetics of the responses of proinflammatory TNF α, anti‐inflammatory IL‐10, and inflammatory master regulator NF‐κB elicited by lipopolysaccharide (LPS) may be critical determinants of the inflammatory response by macrophages; however, there is a lack of homogeneous kinetic data in this pathway. To address this gap, we used the RAW 264.7 macrophage cell line to define intracellular signaling kinetics and cytokine expression in cells treated with LPS for 15 min to 72 h. The abundance of IκBα was maximally reduced 45‐min following LPS treatment, but expression increased at 10‐h, reaching a maximum at 16 h. NF‐κB phosphorylation was significantly increased 45‐min following LPS treatment, maximal at 2‐h, and decreased to basal levels by 6‐h. Nuclear NF‐κB expression was elevated 30‐min following LPS treatment, maximal by 45‐min, and returned to basal levels by 24‐h. Binding of nuclear NF‐κB to consensus oligonucleotide sequences followed a similar pattern to that observed for p‐NF‐κB, but lasted slightly longer. Following LPS treatment, TNF α mRNA expression began at 1‐h, was maximal at 6‐h, and decreased starting at 10‐h. TNF α protein secretion in conditioned growth medium began at 4‐h and was maximal by 16‐h. IL‐10 mRNA expression was induced by LPS at 10‐h, and was maximal at 16‐h. IL‐10 protein secretion was induced at 16‐h and was maximal at 24‐h. Our data reveal the temporal kinetics of pro‐ and anti‐inflammatory signaling events that may be important therapeutic targets for inflammatory diseases.

Macrophages play an important role in the response to infection and/or repair of injury in tissues. To examine the NF‐κB pathway in response to an inflammatory stimulus, we used wild‐type bone‐marrow‐derived macrophages (BMDMs) or BMDMs with knockout (KO) of myeloid differentiation primary response 88 (MyD88) and/or Toll/interleukin‐1 receptor domain‐containing adapter‐inducing interferon‐β (TRIF) via CRISPR/Cas9. Following treatment of BMDMs with lipopolysaccharide (LPS) to induce an inflammatory response, translational signalling of NF‐κB was quantified via immunoblot and cytokines were measured. Our findings reveal that MyD88 KO, but not TRIF KO, decreased LPS‐induced NF‐κB signalling, and 10% expression of basal MyD88 expression was sufficient to partially rescue the abolished inflammatory cytokine secretion observed upon MyD88 KO.