Thiostrepton-Nanomedicine, a TLR9 Inhibitor, Attenuates Sepsis-Induced Inflammation in Mice

Abstract

Sepsis is a life-threatening clinical condition caused by infection and transposition of pathogens and pathogen-associated molecular patterns (PAMPs) into the host bloodstream. During sepsis, activation of toll-like receptors (TLRs) on immune cells triggers the release of pro-inflammatory cytokines and overstimulates the production of vasodilatory mediators such as nitric oxide (NO). These vascular changes lead to widespread inflammation, tissue damage, multiple organ failure, and often death. New therapeutic options are urgently needed. To this end, thiostrepton (TST) has emerged as a candidate for sepsis treatment due to its action as an antibiotic and anti-inflammatory molecule (TLR7-9 inhibitor). Reports in the literature suggest that TLR9 inhibition substantially suppresses the excessive host inflammatory response and attenuates sepsis-induced mortality in the cecal ligation and puncture (CLP) murine model of sepsis. However, to the best of our knowledge, TST has never been directly tested as a therapeutic option for the management of sepsis, possibly due to its low water solubility and drug delivery issues. These facts prompted us to test the central hypothesis that TST encapsulated in phospholipid sterically stabilized micelles (TST-SSM) could be developed into a novel treatment for sepsis. Thus, using our published method of encapsulating the hydrophobic antibiotic TST-SSM, we evaluated the in vivo efficacy of TST-SSM nanomedicine in the murine model of polymicrobial sepsis. We found that TST-SSM increased the median survival of CLP-induced septic mice from 31 to 44 hr by reducing the bacterial burden in the blood and peritoneal lavage. Moreover, plasma levels of pro-inflammatory cytokines (interleukin 6 and tumor necrosis factor-alpha) and NO derivatives were also reduced, whereas renal and hepatic function biomarkers creatinine and aspartate transferase were significantly improved. In conclusion, we identified that TST-SSM nanomedicine has significant potential as a therapeutic agent for sepsis management, primarily due to its anti-inflammatory and antibiotic properties.

Figure 1

Experimental groups and survival study of mice with CLP-induced sepsis. (a, b) C57BL/6 mice (12 per group) underwent cecal ligation and puncture (CLP) or SHAM surgery (time 0). At 6 hr, CLP mice were either treated with empty micelles (SSM) or 20 mg/kg thiostrepton nanomedicine (TST-SSM) intraperitoneally and monitored for survival up to 7 days (168 hr). Survival rates were compared using the log-rank (Mantel-cox) test. P < 0.05 was considered statistically significant.

Figure 2

Cytokine profile and nitric oxide derivatives (NO_x) levels in the plasma of mice with CLP-induced sepsis. C57BL/6 mice (3–6 per group) underwent cecal ligation and puncture (CLP) or SHAM surgery. CLP animals received empty micelles (SSM) or thiostrepton nanomedicine (TST-SSM) intraperitoneally at 6 hr, and all animals were euthanized at 24 hr. TNF-α (a), IL-6 (b), IL-10 (c), TGF-β (d), and nitrite/nitrate levels (NO_x) (e) were quantified from heparinized plasma samples using ELISA kits (cytokines) and Griess Reaction Kit (NO_x). Values are mean ± standard deviation. One-way ANOVA with Tukey's multiple comparison test was used to analyze the results. P < 0.05 was considered statistically significant. NS, not significant,  ^∗P < 0.05,  ^∗∗∗P < 0.001, and  ^∗∗∗∗P < 0.0001.

Figure 3

Biomarkers of hepatic and renal injury. C57BL/6 mice (5–6 per group) underwent CLP or SHAM surgery. CLP animals received empty micelles (SSM) or thiostrepton nanomedicine (TST-SSM) i.p. at 6 hr, and all animals were euthanized at 24 hr. (a, b) Hepatic biomarkers (AST and ALT) and (c, d) renal biomarkers (BUN and creatinine) were measured from heparinized plasma samples. Values are mean ± standard error mean. One-way ANOVA analyzed results with Tukey's multiple comparisons. Ns, nonsignificant; AST, aspartate transferase,  ^∗P < 0.05,  ^∗∗∗P < 0.001, and  ^∗∗∗∗P < 0.0001.

Figure 4

Bacterial burden in blood and peritoneal lavage fluid. C57BL/6 mice (5–6 per group) were subjected to CLP surgery or SHAM surgery and received empty micelles (SSM) or thiostrepton nanomedicine (TST-SSM) via intraperitoneal injection at 6 hr after surgery. All animals were euthanized at 24 hr, and fresh blood (a) and peritoneal lavage fluid (b) were collected for microbiological culture. Results were log-transformed and analyzed by a one-tailed Mann–Whitney test. P < 0.05 was considered statistically significant.  ^∗P < 0.05,  ^∗∗P < 0.01. (c) Summary of research results. CFU, colony forming units; TST-SSM, TST nanomedicine; IL-6, interleukin 6; TNF-α, tumor necrosis factor-alpha; TLR9, toll-like receptor 9; hrs, hours.