Pyroptosis is a caspase-1 dependent cell death, associated with proinflammatory cytokine production, and is considered to play a crucial role in sepsis. Pyroptosis is induced by the two distinct stimuli, microbial PAMPs (pathogen associated molecular patterns) and endogenous DAMPs (damage associated molecular patterns). Importantly, cathelicidin-related AMPs (antimicrobial peptides) have a role in innate immune defense. Notably, human cathelicidin LL-37 exhibits the protective effect on the septic animal models. Thus, in this study, to elucidate the mechanism for the protective action of LL-37 on sepsis, we utilized LPS (lipopolysaccharide) and ATP (adenosine triphosphate) as a PAMP and a DAMP, respectively, and examined the effect of LL-37 on the LPS/ATP-induced pyroptosis of macrophage-like J774 cells. The data indicated that the stimulation of J774 cells with LPS and ATP induces the features of pyroptosis, including the expression of IL-1β mRNA and protein, activation of caspase-1, inflammasome formation and cell death. Moreover, LL-37 inhibits the LPS/ATP-induced IL-1β expression, caspase-1 activation, inflammasome formation, as well as cell death. Notably, LL-37 suppressed the LPS binding to target cells and ATP-induced/P2X7-mediated caspase-1 activation. Together these observations suggest that LL-37 potently inhibits the LPS/ATP-induced pyroptosis by both neutralizing the action of LPS and inhibiting the response of P2X7 to ATP. Thus, the present finding may provide a novel insight into the modulation of sepsis utilizing LL-37 with a dual action on the LPS binding and P2X7 activation.

LL-37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL-37 can modulate various inflammatory reactions. We previously revealed that LL-37 suppresses the LPS/ATP-induced pyroptosis of macrophages in vitro by both neutralizing the action of LPS and inhibiting the response of P2X7 (a nucleotide receptor) to ATP. Thus, in this study, we further evaluated the effect of LL-37 on pyroptosis in vivo using a cecal ligation and puncture (CLP) sepsis model. As a result, the intravenous administration of LL-37 improved the survival of the CLP septic mice. Interestingly, LL-37 inhibited the CLP-induced caspase-1 activation and pyroptosis of peritoneal macrophages. Moreover, LL-37 modulated the levels of inflammatory cytokines (IL-1β, IL-6 and TNF-α) in both peritoneal fluids and sera, and suppressed the activation of peritoneal macrophages (as evidenced by the increase in the intracellular levels of IL-1β, IL-6 and TNF-α). Finally, LL-37 reduced the bacterial burdens in both peritoneal fluids and blood samples. Together, these observations suggest that LL-37 improves the survival of CLP septic mice by possibly suppressing the pyroptosis of macrophages, and inflammatory cytokine production by activated macrophages and bacterial growth. Thus, the present findings imply that LL-37 can be a promising candidate for sepsis because of its many functions, such as the inhibition of pyroptosis, modulation of inflammatory cytokine production and antimicrobial activity.

Upon exposure to invading microorganisms, neutrophils undergo NETosis, a recently identified type of programmed cell death, and release neutrophil extracellular traps (NETs). NETs are described as an antimicrobial mechanism, based on the fact that NETs can trap microorganisms and exhibit bactericidal activity through the action of NET-associated components. In contrast, the components of NETs have been recognized as damage-associated molecular pattern molecules (DAMPs), which trigger inflammatory signals to induce cell death, inflammation and organ failure. In the present study, to clarify the effect of NETs on cytokine production by macrophages, mouse macrophage-like J774 cells were treated with NETs in combination with lipopolysaccharide (LPS) as a constituent of pathogen-associated molecular patterns. The results revealed that NETs significantly induced the production of interleukin (IL)-1β by J774 cells in the presence of LPS. Notably, the NET/LPS-induced IL-1β production was inhibited by both caspase-1 and caspase-8 inhibitors. Furthermore, nucleases and serine protease inhibitors but not anti-histone antibodies significantly inhibited the NET/LPS-induced IL-1β production. Moreover, we confirmed that caspase-1 and caspase-8 were activated by NETs/LPS, and the combination of LPS, DNA and neutrophil elastase induced IL-1β production in reconstitution experiments. These observations indicate that NETs induce the production of IL-1β by J774 macrophages in combination with LPS via the caspase-1 and caspase-8 pathways, and NET-associated DNA and serine proteases are involved in NET/LPS-induced IL-1β production as essential components.

LL‑37 is the only known member of the cathelicidin family of antimicrobial peptides in humans. In addition to its broad spectrum of antimicrobial activities, LL‑37 may modulate various inflammatory reactions. The authors previously revealed that LL‑37 improves the survival of a murine cecal ligation and puncture (CLP) sepsis model. In the present study, the mechanism for the protective action of LL‑37 was elucidated using the CLP model, focusing on the effect of LL‑37 on the release of neutrophil extracellular traps (NETs). The results indicated that the intravenous administration of LL‑37 suppressed the increase of damage-associated molecular patterns (DAMPs), including histone‑DNA complex and high‑mobility group protein 1, in addition to interleukin‑1β, tumor necrosis‑α and soluble triggering receptor expressed on myeloid cells (TREM)‑1 in plasma and peritoneal fluids. Notably, LL‑37 significantly suppressed the decrease of mononuclear cell number in blood, and the increase of polymorphonuclear cell (neutrophil) number in the peritoneal cavity during sepsis. Furthermore, LL‑37 reduced the bacterial burden in blood and peritoneal fluids. Notably, LL‑37 increased the level of NETs (myeloperoxidase‑DNA complex) in plasma and peritoneal fluids. In addition, it was verified that LL‑37 induces the release of NETs from neutrophils, and NETs possess the bactericidal activity. Overall, these observations suggest that LL‑37 improves the survival of CLP septic mice by possibly suppressing the inflammatory responses as evidenced by the inhibition of the increase of cytokines, soluble TREM‑1 and DAMPs (host cell death) and the alteration of inflammatory cell numbers, and bacterial growth via the release of NETs with bactericidal activity.

The liver is a major organ that removes waste substances from the blood, and liver sinusoidal endothelial cells (LSECs) are professional scavenger cells, which incorporate and degrade various endogenous and exogenous molecules including pathogenic factor LPS. Mammalian cells express a number of peptide antibiotics that function as effectors in the innate host defense systems. LL-37, a human cathelicidin antimicrobial peptide, has a potent LPS-neutralizing activity and exhibits protective actions on various infection models. However, the effect of LL-37 on the LPS clearance has not been clarified. In this study, to further understand the host-protective mechanism of LL-37, we evaluated the effect of LL-37 on the LPS clearance in vitro. LL-37 enhanced the LPS uptake by human LSECs. Of interest, LL-37 was similarly incorporated into LSECs both in the presence and the absence of LPS, and the incorporated LPS and LL-37 were colocalized in LSECs. Importantly, the uptake of LPS and LL-37 was inhibited by endocytosis inhibitors, heparan sulfate proteoglycan analogs, and glycosaminoglycan lyase treatment of the cells. Moreover, the uptake of LL-37-LPS did not activate TLR4 signaling in both MyD88-dependent and -independent pathways. In addition, the incorporated LL-37-LPS was likely transported to the lysosomes in LSECs. Together these observations suggest that LL-37 enhances the LPS uptake by LSECs via endocytosis through the complex formation with LPS and the interaction with cell-surface heparan sulfate proteoglycans, thereby facilitating the intracellular incorporation and degradation of LPS without cell activation. In this article, we propose a novel function of LL-37 in enhancing LPS clearance.