LL-37 Degradation by GCF: A Role for Porphyromonas gingivalis Proteinases?

Background: LL-37, composed of 37 amino acid residues, is an innate host defence peptide of the cathelicidin family. It is expressed by neutrophils, monocytes and epithelial cells and exhibits both anti-bacterial and immunomodulatory properties. LL-37 is however prone to proteolytic degradation by proteinases, thus potentially limiting its inherent host defence properties in the inflammatory milieu. Objectives: The present study was designed to determine whether LL-37 was degraded by components of gingival crevicular fluid (GCF) from healthy subjects or those with periodontitis. In addition, we aimed to deduce whether degradation of the peptide was accelerated in GCF samples which were determined to be positive for the periodontopathic bacterium Porphyromonas gingivalis. Methods: GCF and bacterial plaque samples, pre- and post non-surgical periodontal treatment, were collected from 4 individual sites in patients presenting with advanced periodontitis. In healthy subjects, GCF samples only were collected. Plaque samples were analysed by QPCR for the presence or absence of P. gingivalis. Pooled GCF samples from healthy sites; periodontitis sites which were P. gingivalis negative (Pg-); or periodontitis sites which were P. gingivalis positive (Pg+), were incubated with synthetic LL-37 for 0 – 180 min. The degradation products were then analysed by matrix-assisted laser desorption/ionisation time of flight mass spectrometry (MALDI-TOF MS). Results: LL-37 was not degraded when incubated with GCF from healthy subjects. In contrast, LL-37 was degraded after 30 min when incubated with Pg- GCF. However degradation of LL-37 was apparent after only 2 min incubation with Pg+ GCF and the parent molecule was almost completely degraded after 30 min. Conclusions: The rapid degradation of LL-37, particularly in Pg+ sites, highlights the limited role which this host defence peptide may play in the presence of biologically active proteinases. It also underscores a potent virulence mechanism of P. gingivalis used to circumvent innate host responses.

IL-1α and inflammasome-independent IL-1β promote neutrophil infiltration following alum vaccination

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin-1 (IL-1) secretion in vitro in dendritic cells and macrophages via Nucleotide-binding domain and leucine-rich repeat-containing (NLR) family, pyrin domain-containing 3 (NLRP3) inflammasome activation. However, the contribution of IL-1 to alum-induced innate and adaptive immune responses is controversial and the role of IL-1α following alum injection has not been addressed. This study shows that IL-1 is dispensable for alum-induced antibody and CD8 T cell responses to ovalbumin. However, IL-1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL-1 independent. Both IL-1α and IL-1β are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3-inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum-induced IL-1β at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL-1β secretion, show that inflammasome formation is dispensable for alum-induced innate immunity and reveal that IL-1α and IL-1β are both necessary for alum-induced neutrophil influx in vivo.

Low-dose salinomycin induces anti-leukemic responses in AML and MLL

Development of anti-cancer drugs towards clinical application is costly and inefficient. Large screens of drugs, efficacious for non-cancer disease, are currently being used to identify candidates for repurposing based on their anti-cancer properties. Here, we show that low-dose salinomycin, a coccidiostat ionophore previously identified in a breast cancer screen, has anti-leukemic efficacy. AML and MLLr cell lines, primary cells and patient samples were sensitive to submicromolar salinomycin. Most strikingly, colony formation of normal hematopoietic cells was unaffected by salinomycin, demonstrating a lack of hemotoxicity at the effective concentrations. Furthermore, salinomycin treatment of primary cells resulted in loss of leukemia repopulation ability following transplantation, as demonstrated by extended recipient survival compared to controls. Bioinformatic analysis of a 17-gene signature identified and validated in primary MLLr cells, uncovered immunomodulatory pathways, hubs and protein interactions as potential transducers of low dose salinomycin treatment. Additionally, increased protein expression of p62/Sqstm1, encoded for by one of the 17 signature genes, demonstrates a role for salinomycin in aggresome/vesicle formation indicative of an autophagic response.
Together, the data support the efficacy of salinomycin as an anti-leukemic at non-hemotoxic concentrations. Further investigation alone or in combination with other therapies is warranted for future clinical trial.