Opioid Exacerbation of Gram-positive sepsis, induced by Gut Microbial Modulation, is Rescued by IL-17A Neutralization

Abstract

Sepsis is the predominant cause of mortality in ICUs, and opioids are the preferred analgesic in this setting. However, the role of opioids in sepsis progression has not been well characterized. The present study demonstrated that morphine alone altered the gut microbiome and selectively induced the translocation of Gram-positive gut bacteria in mice. Using a murine model of poly-microbial sepsis, we further demonstrated that morphine treatment led to predominantly Gram-positive bacterial dissemination. Activation of TLR2 by disseminated Gram-positive bacteria induced sustained up-regulation of IL-17A and IL-6. We subsequently showed that overexpression of IL-17A compromised intestinal epithelial barrier function, sustained bacterial dissemination and elevated systemic inflammation. IL-17A neutralization protected barrier integrity and improved survival in morphine-treated animals. We further demonstrated that TLR2 expressed on both dendritic cells and T cells play essential roles in IL-17A production. Additionally, intestinal sections from sepsis patients on opioids exhibit similar disruption in gut epithelial integrity, thus establishing the clinical relevance of this study. This is the first study to provide a mechanistic insight into the opioid exacerbation of sepsis and show that neutralization of IL-17A might be an effective therapeutic strategy to manage Gram-positive sepsis in patients on an opioid regimen.

Figure 1. Opioids increase mortality rates in poly-microbial sepsis induced by CLP.

(a) Kaplan–Meier plots of sham-operated or CLP mice treated with placebo or 25 mg morphine pellet. ** p < 0.01 compared with placebo-treated mice subjected to CLP (Mantel-Cox log rank test) (b) Kaplan–Meier plots of sham-operated or CLP mice injected with saline or 15 mg/kg methadone. * p < 0.05 compared with saline-treated mice subjected to CLP (Mantel-Cox log rank test) (c) Kaplan–Meier plots of morphine-treated CLP mice treated with placebo or 30mg naltrexone pellet. ** p < 0.01 compared with placebo-treated mice (Mantel-Cox log rank test) (d) Kaplan–Meier plots of methadone-treated CLP mice treated with placebo or 30 mg naltrexone pellet. * p < 0.05 compared with placebo-treated mice (Mantel-Cox log rank test) Numbers of mice used for each condition are shown in the frame.

Figure 2. Morphine inhibits bacterial clearance and promotes bacterial dissemination during sepsis.

Wild type mice were treated with 25 mg morphine pellets following CLP procedure. (a) Peritoneal lavage was collected at different time points and cultured on blood agar plates overnight. Bacterial colonies were quantified and described as colony-forming unit (CFU). (b) Bacterial colonies of MLN homogenates (c) Bacterial colonies of liver homogenates. (d) Bacterial colonies of spleen homogenates. (e) Bacterial colonies of whole blood. *P < 0.05 compared with placebo-treated animals ** p < 0.01 compared with placebo-treated animals (Mann-Whitney U test) (Each dot represents one animal).

Figure 3. Morphine promotes Gram-positive bacterial translocation and modulates gut microbiome.

(a) Bacterial families in MLN, spleen, and liver isolates from sham-operated or CLP mice treated with placebo or 25 mg morphine pellet. (b)–(c) Bacterial species identified in fecal contents from placebo or 25 mg morphine pellet-treated mice. *p < 0.05 compared with placebo-treated animals and morphine + naltrexone-treated animals (Each dot represents one animal).

Figure 4. Morphine up-regulates IL-17A production during sepsis.

(a) IL-17A concentrations in peritoneal lavage at different time points following CLP. *p < 0.05 (Student’s t test) (n = 5) (b) IL-17A concentrations in serum at different time points following CLP. *p < 0.05 (Student’s t test) (n = 5) (c) IL-17A expression in MLN CD3 + CD4 + cells. (d) Frequencies of IL-17A positive cells in CD3 + CD4 + cells. *p < 0.05, **p < 0.01 (ANOVA followed by Bonferroni’s t test) (Each dot represents one animal) (e) IL-17A expression in MLN CD3-cells (f) Frequencies of IL-17A positive cells in CD3- cells. (Each dot represents one animal).

Figure 5

Neutralization of IL-17A improves survival rate and attenuates sustained inflammation in CLP mice treated with morphine (a) Kaplan–Meier plots of morphine-treated CLP mice injected with isotype control IgG or anti-IL17A antibody. p < 0.01 compared with anti-IL17A-treated mice subjected to CLP (Mantel-Cox log rank test) (b) Bacterial colonies of peritoneal lavage (c) Bacterial colonies of MLN (d) Bacterial colonies of liver *p < 0.05 (Mann-Whitney U test) (Each dot represents one animal) (e) IL-6 concentrations in serum at different time points following CLP. *p < 0.05, **p < 0.01 (Student’s t test) (n = 4) (f) IL-6 concentrations in serum in morphine-treated CLP mice injected with isotype control IgG or anti-IL-17A antibody. **p < 0.01 (Student’s t test) (n = 4).

Figure 6. High levels of IL-17A compromises gut epithelial barrier function and increases gut permeability.

(a) Morphine treatment increased FITC-dextran diffusion across the gut epithelium. The right panel was quantification of FITC intensity. *p < 0.05 (Student t test) (b) Quantification of FITC intensity in peritoneal lavage and whole blood *p < 0.05 (Student’s t test) (c) Anti-IL-17A injection reduced FITC-dextran diffusion across the gut epithelium in morphine-treated CLP animals. The right panel was quantification of FITC intensity. *p < 0.05 (Student’s t test) (d) Quantification of FITC intensity in peritoneal lavage and whole blood *p < 0.05 (Student’s t test) (e) H&E sections of small intestines from sham-operated or CLP animals treated with morphine or placebo. (f) H&E sections of small intestines from morphine-treated CLP animals injected with isotype control and anti-IL-17A. (g) TER was decreased by IL-17A in IEC-6 cell monolayer (H) The permeability of IEC-6 cell monolayer was increased in transwell system **p < 0.01 (ANOVA followed by Bonferroni’s t test) (n = 3) (i) ZO-1 organization in IEC6-cell monolayer treated by vehicle or 100 ng/ml IL-17A. Blue:DAPI Red:F-actin Green:ZO-1 White arrow indicates ZO-1 disruption.(j) H&E sections of small intestines from human patients.

Figure 7. Gram-positive bacteria stimulate MLN to produce IL-17A in a TLR2-dependent manner.

IL-6 (a) and IL-17A (b) concentrations of MLN cell supernatant stimulated by Gram-positive (G+) or Gram-negative (G-) bacteria. *p < 0.05 (ANOVA followed by Bonferroni’s t test) (n = 3) (c) IL-17A concentrations of MLN supernatant treated with morphine or Gram-positive (G+) bacterial mixture ***p < 0.001 (ANOVA followed by Bonferroni’s t test) (n = 3) (d) IL-17A expression in MLN cells from TLR2KO mice. The right panel is the frequencies of IL-17A positive cells in MLN from TLR2KO mice. PC: placebo + CLP; MC: morphine + CLP (e) IL-17A concentrations in serum in TLR2KO mice. PC: placebo + CLP; MC: morphine + CLP (n = 3) Dash line: Serum IL-17A concentration of wild type CLP mice treated with morphine (f) IL-17A concentrations in peritoneal lavage in TLR2KO mice. PC: placebo + CLP MC: morphine + CLP (n = 3) Dash line: Peritoneal lavage IL-17A concentration of wild type CLP mice treated with morphine (g) IL-17A concentrations in supernatant of adherent and non-adherent cells from MLN following Gram-positive (G + ) bacteria stimulation. (n = 3) (h) IL-17A concentrations in supernatant of non-adherent cells from MLN of WT or TLR2KO mice co-cultured with dendritic cells from blood of WT of TLR2KO mice. WT: wild type; TLR2KO: TLR2 Knock; D: dendritic cells; T: non-adherent T cells ***p < 0.001 (Two-way ANOVA followed by Bonferroni’s t test) (n = 3)

Figure 8. IL-1β and IL-23 promotes IL-17A production by MLN Cells

(a)–(d) IL-6, TGF-β, IL-1β, and IL-23 concentrations of MLN adherent cell supernatant following Gram-positive (G+) bacterial stimulation ***p < 0.001 **p < 0.01 (ANOVA followed by Bonferroni’s t test) (n = 3) (e)–(i) IL-17A concentrations of MLN supernatant following G+ bacterial stimulation in the presence of isotype control, anti-IL-6, anti-TGF-β, anti-IL-1β or anti-IL-23p19 antibodies. ***p < 0.001 (ANOVA followed by Bonferroni’s t test) (n = 3) (j) IL-17A concentrations of MLN non-adherent cell supernatant following IL-1β or IL-23 stimulation. ***p < 0.001 (ANOVA followed by Bonferroni’s t test) (n = 3).