Pattern-recognition receptors (PRRs) of the innate immune system detect infection and tissue injury by binding pathogen-associated molecular patterns (PAMPs) and endogenous "danger" ligands, respectively. We proposed the classification of PRRs into classical PRRs that recognize conserved microbial molecules as ligands and nonclassical PRRs that recognize a proteolytic function. This project focused on the identification of novel mechanisms that regulate the expression and signaling of two transmembrane PRRs: classical PRRs, i.e., Toll-like receptors (TLRs), and a nonclassical PRR, i.e., proteinase-activated receptor 2 (PAR₂). Analysis of basal and lipopolysaccharide (LPS)-induced TLR gene expression in murine macrophages revealed that TLRs 3-6 and 9 were transcriptionally regulated by two key counteracting transcription factors, interferon regulatory factor-1 (IRF-1) and IRF-2. An unexpected role for IRF-2 as a transcriptional activator was identified, in contrast to its well characterized role as a transcriptional repressor, for murine TLR3 gene expression. An examination of signaling interactions between TLRs and PAR₂ identified a novel paradigm for receptor cooperativity between PAR₂ and TLR4 that centers on the shared utilization of TLR adapters. In HEK293T transfectants, PAR₂-induced activation of NF-κB and IRF-3 reporters was TRIF/TRAM-dependent. Although PAR₂ agonist did not directly activate TLR4, co-expression of PAR₂ with TLR4 resulted in MyD88-dependent synergistic enhancement of PAR₂-driven NF-κB activation, whereas PAR₂-induced IRF-3 activation was diminished. We demonstrated a physical interaction between PAR₂ and TLR4, and between PAR₂ and TRIF. Differential, cell-type specific responses were found to occur when PAR₂ and TLRs were simultaneously activated. In murine macrophages, concurrent activation of TLR4 and PAR₂ led to a "T_h2"-like pattern of cytokine expression, findings that were consistent with PAR₂-mediated synergistic enhancement of IL-4-induced alternative activation of macrophages. In epithelial cell lines, PAR₂ activation augmented TLR3-induced NF-κB activation while down-regulating TLR3-induced activation of IRF-3 antiviral pathway. In vivo, PAR₂^-/- and TLR4^-/- mice were both resistant to PAR₂ agonist peptide-induced footpad edema and to mouse-adapted H1N1 influenza virus-induced lethality. These results provide compelling support for the hypothesis that regulating the extracellular proteinase/anti-proteinase balance might represent an effective therapeutic approach to controlling inflammation and suggest that targeting PAR₂ and/or TLR4 receptors and signaling pathways may offer protection against influenza.