An A2A adenosine receptor agonist, ATL313, reduces inflammation and improves survival in murine sepsis models

Abstract

Background: The pathophysiology of sepsis is due in part to early systemic inflammation. Here we describe molecular and cellular responses, as well as survival, in A 2A adenosine receptor (AR) agonist treated and untreated animals during experimental sepsis.

Methods: Sepsis was induced in mice by intraperitoneal inoculation of live bacteria (Escherichia coli or Staphylococcus aureus) or lipopolysaccharide (LPS). Mice inoculated with live bacteria were treated with an A 2A AR agonist (ATL313) or phosphate buffered saline (PBS), with or without the addition of a dose of ceftriaxone. LPS inoculated mice were treated with ATL313 or PBS. Serum cytokines and chemokines were measured sequentially at 1, 2, 4, 8, and 24 hours after LPS was administered. In survival studies, mice were followed until death or for 7 days.

Results: There was a significant survival benefit in mice infected with live E. coli (100% vs. 20%, p = 0.013) or S. aureus (60% vs. 20%, p = 0.02) when treated with ATL313 in conjunction with an antibiotic versus antibiotic alone. ATL313 also improved survival from endotoxic shock when compared to PBS treatment (90% vs. 40%, p = 0.005). The serum concentrations of TNF-alpha, MIP-1 alpha, MCP-1, IFN-gamma, and IL-17 were decreased by ATL313 after LPS injection (p < 0.05). Additionally, ATL313 increased the concentration of IL-10 under the same conditions (p < 0.05). Circulating white blood cell concentrations were higher in ATL313 treated animals (p < 0.01).

Conclusion: Further studies are warranted to determine the clinical utility of ATL313 as a novel treatment for sepsis.

Figure 1

Dose effect. The optimal dose of ATL313 was determined through a dose response curve where PBS vehicle (white diamond), 5 μg/kg ATL313 (black circle), 50 μg/kg ATL313 (black triangle), 100 μg/kg ATL313 (white square), or 200 μg/kg ATL313 (asterix) was injected IP every 6 hours into BALB/c mice (N = 4 per group) after a lethal IP dose of LPS O111:B4 (20 mg/kg).

Figure 2

ATL313 protects mice in three different sepsis models. ATL313 protects mice from live E. coli challenge. Mice were injected with 1 × 10⁸ CFU of live E. coli. Mice (N = 5 per group) were treated with PBS vehicle (white diamond), ATL313 (black circle), the antibiotic ceftriaxone (white triangle), or a combination of ceftriaxone and ATL313 (black square). Ceftriaxone was administered at a single dose of 25 mg/kg eight hours following inoculation. ATL313 (5 μg/kg) or PBS was dosed IP starting 8 hours after E. coli injection then every 6 hours spanning 48 hours. ATL313 + ceftriaxone treatment protects mice better than ceftriaxone treatment alone (* p = 0.013) and treatment with ATL313 alone prolongs life compared to E. coli untreated controls (#p = 0.031) (A). ATL313 protects mice from live S. aureus challenge. Mice were injected IP with 8 × 10⁸ cfu of live S. aureus. Mice (N = 10–24 per group) were treated with PBS vehicle (white diamond), ATL313 (black circle), the antibiotic ceftriaxone (white triangle), or a combination of ceftriaxone and ATL313 (black square). Ceftriaxone was administered at a single dose of 25 mg/kg eight hours following inoculation. ATL313 (5 μg/kg) or vehicle was dosed IP starting at eight hours after S. aureus injection 4 times at 12 hour intervals. ATL313 + ceftriaxone treatment protects mice better than ceftriaxone treatment alone (* p = 0.02) and ATL313 treatment alone increases survival in S. aureus untreated controls (#p = 0.036) (B). ATL313 decreases LPS-induced mouse mortality via A_2A AR-mediated mechanisms. Mice (N = 20–29 per group) were injected IP with LPS from E. coli (O111:B4, 5 mg/kg). One half hour prior to LPS challenge and at 6 hr intervals, PBS vehicle (white triangle) or ATL313 (black circle) was injected (5 μg/kg) IP for a total of 8 doses/48 hr. ATL313 protects the mice compared to LPS-challenged mice in the absence of ATL313 (* p = 0.005) (C).

Figure 3

ATL313 affects circulating cytokines and chemokines. The A_2A AR agonist, ATL313 significantly decreases the concentrations of pro-inflammatory cytokines and chemokines in mice (N = 5–9 per group) after inoculation with E. coli O111:B4 LPS (25 mg/kg). Mean ± standard error of the mean concentrations are shown in pg/mL. In ATL313 treated animals, TNF-α, at t = 1 hour, was significantly lower than in untreated animals (* p = 0.041) (A). Conversely, the anti-inflammatory cytokine, IL-10, had an increase in concentration in ATL 313 treated animals at t = 1 and 2 hours (* p = 0.036; # p = 0.042) (B). Like TNF-α, MIP-1α concentrations are increased early on after LPS exposure and significantly decreased by ATL313 at t = 2 hours (* p = 0.004) (C). Increases in MCP-1 concentrations occur later after exposure to LPS and are significantly decreased by ATL313 at t = 4 hours (* p = 0.05) (D). Both IFN-γ and IL-17 concentrations are maximal at t = 8 hours and are significantly attenuated by ATL313 at that time (* p = 0.001 and 0.043 respectively) (E and F).

Figure 4

ATL313 affects circulating white blood cells. ATL313 (black circle) increases concentrations of circulating white blood cells in mice (N = 6 per group) after inoculation with LPS from E. coli (O111:B4, 5 mg/kg) (A). Additionally, circulating neutrophil (PMN) concentrations are also increased in mice treated with ATL313 (black circle) after LPS inoculation (B). Mean ± standard error of the mean concentrations are shown in K/μL. The difference in concentrations is significantly different at t = 2 hours for both total WBC and PMN concentrations (* WBC p = 0.003; PMN p = 0.007).