An amyloidogenic hexapeptide derived from amylin attenuates inflammation and acute lung injury in murine sepsis

<div><p>Although the accumulation of amyloidogenic proteins in neuroinflammatory conditions is generally considered pathologic, in a murine model of multiple sclerosis, amyloid-forming fibrils, comprised of hexapeptides, are anti-inflammatory. Whether these molecules modulate systemic inflammatory conditions remains unknown. We hypothesized that an amylin hexapeptide that forms fibrils can attenuate the systemic inflammatory response in a murine model of sepsis. To test this hypothesis, mice were pre-treated with either vehicle or amylin hexapeptide (20 μg) at 12 hours and 6 hours prior to intraperitoneal (i.p.) lipopolysaccharide (LPS, 20 mg/kg) administration. Illness severity and survival were monitored every 6 hours for 3 days. Levels of pro- (IL-6, TNF-α, IFN-γ) and anti-inflammatory (IL-10) cytokines were measured via ELISA at 1, 3, 6, 12, and 24 hours after LPS (i.p.). As a metric of lung injury, pulmonary artery endothelial cell (PAEC) barrier function was tested 24 hours after LPS administration by comparing lung wet-to-dry ratios, Evan’s blue dye (EBD) extravasation, lung histology and caspase-3 activity. Compared to controls, pretreatment with amylin hexapeptide significantly reduced mortality (p<0.05 at 72 h), illness severity (p<0.05), and pro-inflammatory cytokine levels, while IL-10 levels were elevated (p<0.05). Amylin pretreatment attenuated LPS-induced lung injury, as demonstrated by decreased lung water and caspase-3 activity (p<0.05, versus PBS). Hence, in a murine model of systemic inflammation, pretreatment with amylin hexapeptide reduced mortality, disease severity, and preserved lung barrier function. Amylin hexapeptide may represent a novel therapeutic tool to mitigate sepsis severity and lung injury.</p></div

Pretreatment with amylin reduces illness severity and mortality in an LPS-induced systemic inflammatory mouse model.

<p>Adult female B6 mice were pretreated with 20 μg amylin at 12 h and 6 h prior to i.p. administration of LPS; control mice received no pretreatment. All mice were subsequently inoculated with LPS (i.p.) at a dose of 20 mg/kg at time 0. Illness severity (A) and mortality (B), which were regularly assessed over a 3-day period, were significantly reduced in the pretreatment group. Aggregate data are presented as mean ± SEM, n = 13–15 mice per data point, * p<0.05 ** p<0.01 *** p<0.001 by Student’s t-test between groups.</p

Pretreatment with amylin reduces TNF-α, INF-γ, and IL-6 while sustaining IL-10 levels in an LPS-induced systemic inflammatory mouse model.

<p>Adult female B6 mice, with or without amylin pretreatment, underwent induction of systemic inflammation as described prior. Cytokine Elisa assays for TNF-α (A), IL-6 (B), IFN-γ (C), and IL-10 (D) were performed on plasma collected at 1 h, 3 h, 6 h, 12 h, and 24 h after LPS (i.p.) administration. Pretreatment with amylin reduced pro-inflammatory cytokines (TNF-α, IFN-γ, and IL-6), while sustaining IL-10 levels at 3 h after LPS-endotoxemia induction. Aggregate data are presented as mean ± SEM, n = 10 mice per data bar, * p<0.05 ** p<0.01 *** p<0.001 by Student’s t-test between groups.</p

Pretreatment with amylin attenuates lung injury in an LPS-induced systemic inflammatory mouse model.

<p>Adult female B6 mice, with or without amylin pretreatment, underwent induction of systemic inflammation as described prior. At 24 hours after LPS (i.p.) administration, lung tissues were inflated, fixed and evaluated for lung injury. (A) Evans Blue Dye (EBD) incorporation in lung tissue was determined by retro-orbital injection of EBD 30 min prior to the 24h experimental endpoint in PBS-only, LPS, and LPS+amylin treated mice. (B) Excessive lung water accumulation was measured using the wet/dry weight ratios of lung tissues. All values presented are mean ± SEM, with n = 10 mice/group. *p<0.05 **p<0.005 ***p<0.001 vs. PBS, via 1-way ANOVA and Bonferroni’s multiple comparisons test. Both EBD and lung water accumulation were significantly elevated in the LPS group and did not differ between PBS and LPS+AMY groups. (C) Representative H and E staining of lungs at 20X magnification from mice treated with PBS+AMY, LPS+PBS, or LPS+AMY. In animals treated with LPS, air space disease was evident with fluid in the alveoli, increased interstitial prominence, and hyaline membrane formation. In animals that received amylin pretreatment, air space disease and lung water was decreased, compared to LPS. n = 5 mice/group.</p

Determination of optimal LPS dose for the induction of systemic inflammation in mice.

<p>Adult female B6 mice were inoculated with LPS (i.p.) at doses ranging from 5 mg/kg to 40 mg/kg at time 0; control mice received vehicle (PBS). Mice were assessed every 6 hours for up to 60 hours. Percent survival was determined for each group and presented in Kaplan-Meier format. Each group started with 10 mice.</p

DataSheet_2_Transcriptomic profiles of multiple organ dysfunction syndrome phenotypes in pediatric critical influenza.xlsx

BackgroundInfluenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection.MethodsWe measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR qResultsComparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation (RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways (RPL3, MRPL3, HLA-DMB, EEF1G, CD8A) were associated with MODS recovery within a week.ConclusionThus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome.</p

DataSheet_1_Transcriptomic profiles of multiple organ dysfunction syndrome phenotypes in pediatric critical influenza.pdf

BackgroundInfluenza virus is responsible for a large global burden of disease, especially in children. Multiple Organ Dysfunction Syndrome (MODS) is a life-threatening and fatal complication of severe influenza infection.MethodsWe measured RNA expression of 469 biologically plausible candidate genes in children admitted to North American pediatric intensive care units with severe influenza virus infection with and without MODS. Whole blood samples from 191 influenza-infected children (median age 6.4 years, IQR: 2.2, 11) were collected a median of 27 hours following admission; for 45 children a second blood sample was collected approximately seven days later. Extracted RNA was hybridized to NanoString mRNA probes, counts normalized, and analyzed using linear models controlling for age and bacterial co-infections (FDR qResultsComparing pediatric samples collected near admission, children with Prolonged MODS for ≥7 days (n=38; 9 deaths) had significant upregulation of nine mRNA transcripts associated with neutrophil degranulation (RETN, TCN1, OLFM4, MMP8, LCN2, BPI, LTF, S100A12, GUSB) compared to those who recovered more rapidly from MODS (n=27). These neutrophil transcripts present in early samples predicted Prolonged MODS or death when compared to patients who recovered, however in paired longitudinal samples, they were not differentially expressed over time. Instead, five genes involved in protein metabolism and/or adaptive immunity signaling pathways (RPL3, MRPL3, HLA-DMB, EEF1G, CD8A) were associated with MODS recovery within a week.ConclusionThus, early increased expression of neutrophil degranulation genes indicated worse clinical outcomes in children with influenza infection, consistent with reports in adult cohorts with influenza, sepsis, and acute respiratory distress syndrome.</p