Supplementary Material for: Synthetic Immunomodulatory Peptide IDR-1002 Enhances Monocyte Migration and Adhesion on Fibronectin

Regulation of the immune system by immunomodulatory agents, such as the synthetic innate defense regulator (IDR) peptides, has been proposed as a potential strategy to strengthen host immune responses against infection. IDR peptides confer protection in vivo against a range of bacterial infections and have been developed as components of single-dose vaccine adjuvants due to their ability to modulate innate immunity, correlating with an increased recruitment of monocytes to sites of infection or immunization. However, the mechanisms by which IDR peptides augment monocyte recruitment remain poorly defined. Anti-infective peptide IDR-1002 was demonstrated here to lack direct monocyte chemoattractive activity yet enhance, by up to 5-fold, the ability of human monocytes to migrate on fibronectin towards chemokines. This effect correlated with an increased adhesion of monocytes and THP-1 cells to fibronectin by IDR-1002 and other IDR peptides and the adhesion of THP-1 cells to fibronectin occurred in a β₁-integrin-dependent manner, corresponding with an increased activation of β₁-integrins and the phosphoinositide 3-kinase (PI3K)-Akt pathway. PI3K- and Akt-specific inhibitors abrogated IDR-1002-induced adhesion and activation of β₁-integrins, whereas p38 and MEK1 inhibitors did not affect, or moderately inhibited, adhesion, respectively. Furthermore, IDR-1002 enhancement of monocyte migration towards chemokines and activation of β₁-integrins was abrogated in the presence of PI3K- and Akt-specific inhibitors. In summary, IDR-1002 enhanced monocyte migration on fibronectin through promotion of β₁-integrin-mediated interactions regulated by the PI3K-Akt pathway, revealing a mechanism by which IDR-1002 promotes monocyte recruitment

Supplementary Material for: Signaling Pathways Mediating Chemokine Induction in Keratinocytes by Cathelicidin LL-37 and Flagellin

Cathelicidin LL-37 is a multifunctional immunomodulatory and antimicrobial host defense peptide that has an important role in the immune defenses of the skin and other epithelial barriers. We have previously demonstrated that at physiological concentrations LL-37 synergistically augments the production of immune mediators in response to microbial compounds in human primary keratinocytes. Here we define the signaling mechanisms responsible for this activity. We demonstrate that inhibition of Src family kinases (SFKs) strongly inhibited the synergistic chemokine production in response to LL-37 and flagellin in keratinocytes. SFK activation was induced by LL-37 stimulation and was required for the downstream activation of Akt (protein kinase B) and the transcription factors CREB and ATF1. In cells stimulated with LL-37 and flagellin together, Akt activation was primarily induced by LL-37, while both flagellin and LL-37 contributed to the activation of CREB and ATF1 and consequently chemokine induction. The purinergic receptor P2X₇ was identified as the receptor upstream of SFK activation in LL-37-stimulated keratinocytes. Overall, these findings established the P2X₇–SFK–Akt–CREB/ATF1 signaling pathway activated by LL-37 in primary keratinocytes. These signaling mechanisms mediated the synergistic effects of LL-37 on chemokine production in flagellin-stimulated keratinocytes, and thus might have a role in the immune defenses of the skin and possibly other epithelial barriers