Novel Chitohexaose Analog Protects Young and Aged mice from CLP Induced Polymicrobial Sepsis

Abstract

In Gram-negative bacterial sepsis, production of excess pro-inflammatory cytokines results in hyperinflammation and tissue injury. Anti-inflammatory cytokines such as IL-10 inhibit inflammation and enhance tissue healing. Here, we report a novel approach to treat septicemia associated with intra-abdominal infection in a murine model by delicately balancing pro- and anti-inflammatory cytokines. A novel oligosaccharide compound AVR-25 selectively binds to the TLR4 protein (IC₅₀ = 0.15 µM) in human peripheral blood monocytes and stimulates IL-10 production. Following the cecal ligation and puncture (CLP) procedure, intravenous dosing of AVR-25 (10 mg/kg, 6-12 h post-CLP) alone and in combination with antibiotic imipenem protected both young adult (10-12 week old) and aged (16-18 month old) mice against polymicrobial infection, organ dysfunction, and death. Proinflammatory cytokines (TNF-α, MIP-1, i-NOS) were decreased significantly and restoration of tissue damage was observed in all organs. A decrease in serum C-reactive protein (CRP) and bacterial colony forming unit (CFU) confirmed improved bacterial clearance. Together, these findings demonstrate the therapeutic ability of AVR-25 to mitigate the storm of inflammation and minimize tissue injury with high potential for adjunctive therapy in intra-abdominal sepsis.

Figure 1

Structure of chitohexaose and AVR-25.

Figure 2

Effect of AVR-25 in human PBMCs as shown by ELISA assay with or without LPS treatment. (a) Binding of AVR-25 to TLR4 at different concentrations. (b) AVR-25 inhibited LPS (100 ng/mL) induced IL-6 and (c) TNF-α. (d) AVR-25 induced IL-10 production in PMBCs. Control indicates untreated cells in media. N = 4; *p < 0.05, **p < 0.005, ***p < 0.0001.

Figure 3

AVR-25 increases survival in both young adult and aged mice. (a) Two doses of AVR-25 (10 mg/kg; 16 h, 24 h) were injected IV, into 10–12-week-old young adult mice alone, or in combination with imipenem (5 mg/kg), and survival was followed up to 15 days. Following CLP, the AVR-25 + imipenem group demonstrated better survival (>80%) than the group treated with imipenem alone or AVR-25 alone. There was no mortality in the sham group, while in the CLP group there was >90% mortality within 72 h of the procedure, N = 5–15, *p < 0.05. (b) AVR-25 (10 mg/kg) was injected into aged female mice (16–18 months old) 6 h post CLP and q12h followed by imipenem injection SC (25 mg/kg) every 12 h for 7 consecutive days. N = 8–10; *p < 0.05. Sham-Black; CLP-orange; CLP + imipenem: Purple; CLP + AVR-25: Blue; CLP + AVR-25 + imipenem: Green. Sham groups and AVR-25 alone were not done for (b and c). (c) AVR-25 (10 mg/kg) was injected into aged female mice (16–18 months old) 6 h post CLP, q8h followed by imipenem injection SC (25 mg/kg) every 12 h for 7 consecutive days. In the aged group, AVR-25 + imipenem demonstrated improved survival (42%) as compared to imipenem alone (~20%). Administration of AVR-25 alone was not done in this group. N = 8–10.

Figure 4

AVR-25 restores normal organ pathology. Representative H/E staining showing restoration of normal tissue morphology in vital organs of both male (a) and female (b) young adult mice in AVR-25 + imipenem treated mice compared to the CLP group. CLP mice showed microthrombi and congestion (arrows) in the heart, lungs, liver, kidney, and brain, increased germinal center size in spleen, necrosis of villi in the gut and loss of testicular and ovarian epithelium. Upon treatment with AVR-25 + imipenem, the histology showed a marked improvement in controlling the damage and the vascular changes. These changes appeared to be superior to those of the imipenem only treated group, N = 15, X100.

Figure 5

Compound AVR-25 recovered CLP induced tissue damage. The graph shows histological scoring (summarized in supplemental section, Table S3) in CLP-induced (a) male and (b) female mice. The recovery of all tissue damage was maximum when treated with AVR-25 + imipenem than with AVR-25 alone or imipenem alone. Vascular damage and tissue congestion induced by CLP in heart, lungs, liver, spleen, kidney, gut, brain, testis and ovary is restored after drug treatment. In all the tissues studied P < 0.01 (CLP vs imipenem alone, AVR-25 alone); P < 0.001 (imipenem alone or AVR-25 alone vs AVR-25 + imipenem in both males and females. Higher scores indicate more injury. In both (a and b), Sham is indicated by grey color; CLP-Red; CLP + imipenem alone-Blue; CLP + AVR-25 alone-Orange; CLP + AVR-25 + imipenem -Green. As the average score for the sham group is 0, the grey color is not visible in the bar graphs. MD: Myocardial Damage; Heart VCH: Heart Vascular Congestion & Hemorrhage; AD: Alveolar Damage; Lung VCH: Lung Vascular Congestion & Hemorrhage; HI: Hepatocyte Injury; Liver VCH: Liver Vascular Congestion & Hemorrhage; LFD: Lymphoid Follicles Damage; Spleen VCH: Spleen Vascular Congestion & Hemorrhage; GI: Glomeruli Injury; TD: Tubular Damage (kidney); Kidney VCH: Kidney Vascular Congestion & Hemorrhage; MVD: Mucosal Villi Damage; Gut VCH: Gut Vascular Congestion & Hemorrhage; ND: Neuronal Damage; Brain VCH: Brain Vascular Congestion & Hemorrhage; TED: Tubular Epithelial Damage; Testis VCH: Testis Vascular Congestion & Hemorrhage; OVD: Ovarian Follicular damage; Ovarian VCH: Ovary Vascular Congestion & Hemorrhage.

Figure 6

Changes in pro-inflammatory markers with AVR-25 treatment. (a–c) Pro-inflammatory cytokines in serum after 15 days in young adult mice (male and female, N = 10). (d,e) Dynamic changes in pro-inflammatory cytokines after 4, 8, 12, 24, and 48 h of treatment with AVR-25 + imipenem in CLP young adult mice. (f,g) CRP and MIP-1 levels measured after 48 h. (h) Dynamic changes in pro-inflammatory cytokines after 24, 48, 72, and 192 h of AVR-25 and imipenem treatment in aged CLP mice. i-k) i-NOS, CRP, and MIP-1 levels measured after 192 h. *p < 0.005 for (a–c). *p < 0.01, **p < 0.001, ****p < 0.0001 for Figs d–k were found between the CLP + imipenem and CLP + AVR-25 + imipenem groups as well as between sham and CLP + AVR-25. There was no difference between results obtained from male and female mice.

Figure 7

(a) Change in anti-inflammatory cytokine IL-10 in serum of young adult mice (male and female, N = 10) after 4, 8, 12, 24, and 48 h of treatment with AVR-25 + imipenem post CLP. (b) Change in IL-10 in serum of aged mice (male and female, N = 8) after 24, 48, 72, and 192 h of treatment with AVR-25 + imipenem post CLP. ***p < 0.0001 was found between CLP + imipenem and CLP + AVR-25 + imipenem groups as well as between sham and CLP + AVR-25. There was no difference between results obtained from male and female mice.