Diagram of the proposed mechanism of LL-37-induced cell death and protection from GM-0111.

<p>Nasal epithelial cells subjected to LL-37 demonstrate a pro-inflammatory response, characterized by increased ATP, IL-6, and -8 production and pyroptosis and/or necrosis via caspase-1 and -8 but not caspase-3 or -7 activation. These changes are prevented by GM-0111 treatment. IL-6 and -8 promote an inflammatory response <i>in vivo</i> through the recruitment of neutrophils and further inflammatory signaling in a positive feedback loop. The process of pro-inflammatory cell death is propagated to nearby cells due to these changes in the local environment, resulting in an unchecked cytotoxic response initiated by LL-37.</p

In cells committed to terminal cell death, caspase-1 and -8 activity is increased with LL-37 treatment and reduced with GM-0111.

<p><b>A</b>. The percentage of total HNEpCs and J774.2 cells with active caspase-1 and 7-AAD (late cell death marker) is significantly increased with LL-37 treatment, and this is dose-dependently reversed with GM-0111 treatment. <b>B</b>. The percentage of total HNEpCs and J774.2 cells with active caspase-8 and 7-AAD is significantly increased with LL-37 treatment and dose-dependently decreased with GM-0111. ****P ≤ 0.0001, *P ≤ 0.05, ns (not significant) P > 0.05. The data represent the means ± SD (n = 2–5). Note the differences in y-axis scale (panel B y-axis maximum only 1.5%), adjusted to allow visual resolution but heights of bars are not comparable between panels. <b>C</b>. There is a statistically significant though minimal (<1%) increase in caspase-3 or -7 activity in HNEpCs also positive for 7-AAD after LL-37 treatment, and the effect is reversed with GM-0111. There is no significant change in caspase-3 or -7 activity with LL-37 or GM-0111 in J774.2 cells.</p

LL-37 increases cell death, which is reduced by GM-0111.

<p><b>A</b>. Treatment with LL-37 alone results in a dose-dependent increase in late cell death (Annexin V⁺/7-AAD⁺) in HNEpCs. <b>B</b>. The percentage of total HNEpCs and J774.2 cells that are viable (Annexin V^-/7-AAD^-) is significantly reduced with LL-37 treatment, and this is dose-dependently reversed with GM-0111. <b>C</b>. The percentage of cells undergoing early death (Annexin V⁺/7-AAD^-) is significantly increased in HNEpCs treated with LL-37, and the effect is reversed with GM-0111. <b>D</b>. The percentage of HNEpCs and J774.2 in late cell death (Annexin V⁺/7-AAD⁺) is also significantly increased with LL-37 treatment and dose-dependently reversed with GM-0111. ****<i>P</i> ≤ 0.0001, ***<i>P</i> ≤ 0.001, **<i>P</i> ≤ 0.01, *<i>P</i> ≤ 0.05, ns (not significant) <i>P</i> > 0.05. The data represent the means ± SD (n = 4).</p

LL-37 induces morphologic change in HNEpCs (upper two rows) and J774.2 cells (bottom two rows), which are prevented with GM-0111 treatment.

<p>All images are at 40x magnification.</p

LL-37 causes a dose-dependent increase in ATP release from both HNEpC and J774.2 cells (A), which is blocked by GM-0111 in a dose-dependent manner (B).

<p>Each column represents the mean ± SD (n = 4). ****<i>P</i> ≤ 0.0001, ***<i>P</i> ≤ 0.001, *<i>P</i> ≤ 0.05, ns (not significant) <i>P</i> > 0.05.</p

LL-37 causes cell death of human nasal epithelial cells, which is inhibited with a synthetic glycosaminoglycan - Fig 2

<p><b>Treatment of HNEpCs with LL-37 for 60 min results in a dose-dependent increase in cytokines IL-6 and -8 (A)</b>. Pre-treatment with GM-0111 causes significant dose-dependent reductions in LL-37-induced cytokine release (<b>B</b>). There is no significant effect of GM-0111 alone on IL-6 or -8 levels. ****<i>P</i> ≤ 0.0001.</p