Toll-like receptor 4 stimulation before or after Streptococcus pneumoniae induced sepsis improves survival and is dependent on T-cells

Abstract

Introduction: Endotoxin tolerance improves outcomes from gram negative sepsis but the underlying mechanism is not known. We determined if endotoxin tolerance before or after pneumococcal sepsis improved survival and the role of lymphocytes in this protection.

Methods: Mice received lipopolysaccharide (LPS) or vehicle before or after a lethal dose of Streptococcus pneumoniae. Survival, quantitative bacteriology, liver function, and cytokine concentrations were measured. We confirmed the necessity of Toll-like receptor 4 (TLR4) for endotoxin tolerance using C3H/HeN (TLR4 replete) and C3H/HeJ (TLR4 deficient) mice. The role of complement was investigated through A/J mice deficient in C5 complement. CBA/CaHN-Btk(xid/)/J mice with dysfunctional B cells and Rag-1 knockout (KO) mice deficient in T and B cells delineated the role of lymphocytes.

Results: Endotoxin tolerance improved survival from pneumococcal sepsis in mice with TLR4 that received LPS pretreatment or posttreatment. Survival was associated with reduced bacterial burden and serum cytokine concentrations. Death was associated with abnormal liver function and blood glucose concentrations. Endotoxin tolerance improved survival in A/J and CBA/CaHN-Btk(xid/)/J mice but not Rag-1 KO mice.

Conclusions: TLR4 stimulation before or after S. pneumoniae infection improved survival and was dependent on T-cells but did not require an intact complement cascade or functional B cells.

Figure 1. Survival curves for control (N = 18) and LPS pretreated mice (N = 29) following intravenous bacterial challenge with 2×10⁶ CFU of S. pneumoniae culture, *log-rank test, p<0.001.

Figure 2. (A) Survival curves for C57BL/6 mice treated with LPS with or without ceftriaxone after bacterial challenge.

Mice were treated with LPS 3(N = 7) or 6 hours (N = 5) and then every 12 hours after bacterial challenge for 2 days and compared with PBS treated controls (N = 10); (B) Survival curves for C57BL/6 mice treated with LPS and ceftriaxone (N = 20) 6 hours after bacterial challenge compared to controls (N = 18) that received ceftriaxone and PBS 6 hours after bacterial challenge, *log-rank test, p<0.001.

Figure 3. Bacterial clearance in (A) blood (N = 5–8 per group), and (B) lung homogenates (N = 3 per group) after S. pneumoniae challenge in LPS pretreated and PBS treated control mice.

The results are expressed as the mean and standard deviations (SDs) are indicated by error bars, *t-test, p<0.05.

Figure 4. Mean ± SD serum (A) AST,(B) ALT, and (C) glucose concentrations at 6 hours after pneumococcal challenge in mice that received LPS pretreatment compared to controls (N = 4–6 per group), *t-test, p<0.05.

Figure 5. Survival curves for (A) C3H/HeN (N = 8) and (B) TLR4-deficient C3H/HeJ (N = 17) mice challenged with S. pneumoniae after pretreatment with LPS compared to PBS treated controls, *log-rank test, p<0.05.

Figure 6. Serum concentrations of (A) TNF-α, (B) IL-1β, (C) IL-6, and (D) IFN-γ at 6 hours after bacterial challenge in LPS pretreated and PBS pretreated control C3H/HeN and C3H/HeJ mice (n = 5 per group), *t-test, p<0.05.

Figure 7. Survival curves for LPS pretreated and PBS control (A) A/J, (B) CBA/CaHN-Btk^xid//J, and (C) Rag-1 KO mice after challenge with S. pneumoniae (n = 4–14 mice per group), *log-rank test, p<0.001.