Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury

Abstract

Mortality from sepsis has remained high despite recent advances in supportive and targeted therapies. Toll-like receptors (TLRs) sense bacterial products and stimulate pathogenic innate immune responses. Mice deficient in the common adapter protein MyD88, downstream from most TLRs, have reduced mortality and acute kidney injury (AKI) from polymicrobial sepsis. However, the identity of the TLR(s) responsible for the host response to polymicrobial sepsis is unknown. Here, we show that chloroquine, an inhibitor of endocytic TLRs (TLR3, 7, 8, 9), improves sepsis-induced mortality and AKI in a clinically relevant polymicrobial sepsis mouse model, even when administered 6 h after the septic insult. Chloroquine administration attenuated the decline in renal function, splenic apoptosis, serum markers of damage to other organs, and prototypical serum pro- and anti-inflammatory cytokines TNF-alpha and IL-10. An oligodeoxynucleotide inhibitor (H154) of TLR9 and TLR9-deficient mice mirror the actions of chloroquine in all functional parameters that we tested. In addition, chloroquine decreased TLR9 protein abundance in spleen, further suggesting that TLR9 signaling may be a major target for the protective actions of chloroquine. Our findings indicate that chloroquine improves survival by inhibiting multiple pathways leading to polymicrobial sepsis and that chloroquine and TLR9 inhibitors represent viable broad-spectrum and targeted therapeutic strategies, respectively, that are promising candidates for further clinical development.

Figure 1. Chloroquine decreases mortality after polymicrobial sepsis, even when administered after 6 hr

(A) Survival curves from mice given vehicle (dashed line, N = 13) or chloroquine (50 mg/kg, p.o.) 3 hr before CLP surgery (solid line, N = 13). (B) Survival curves from mice given vehicle (dashed line, N = 20) or chloroquine (50 mg/kg, p.o.) 6 hr after CLP surgery (solid line, N = 20). #, P < 0.05 vs. CLP plus vehicle.

Figure 2. Chloroquine improves renal dysfunction, and tubular damage from polymicrobial sepsis

(A) Blood urea nitrogen and serum creatinine as an index of kidney function at 24 hr after sham surgery (white bar, N = 6), CLP surgery plus vehicle (black bar, N = 8-11), chloroquine 3 hr before CLP (gray bar, N = 11, left panel), or chloroquine 6 hr after CLP (gray bar, N = 8, right panel). (B); original magnification, 400X. (C) Kidney histology (periodic acid-Schiff stain) at 24 hr after surgery was scored (see Methods) for tubules in the renal cortex and outer stripe of the outer medulla (OSOM) at 24 hr after surgery (N = 5-6 per group). Values are mean ± SE. *, P < 0.05 vs. CLP plus vehicle.

Figure 3. Chloroquine inhibits multiple organ damage after polymicrobial sepsis

Serum chemistry 24 hr after sham surgery (white bars, N = 5), CLP (black bars, N = 6-9), or CLP after chloroquine treatment (gray bars, N = 6-9). Chloroquine treatment 3 hr before CLP is shown on the left and chloroquine treatment 6 hr after CLP is shown on the right. (A) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (B) amylase (C) creatine kinase (CK) and lactate dehydrogenase (LDH). Values are mean ± SE. *, P < 0.05 vs. CLP given vehicle.

Figure 4. Chloroquine inhibits spleen apoptosis and serum cytokines after polymicrobial sepsis

(A) Number of active caspase 3 positive cells in the spleen at 24 hr after CLP. (N = 5 per group). (B) Serum TNF-alpha and IL-10 at 24 hr after CLP (N = 6-12 per group). Values are mean ± SE. *, P < 0.05 vs. CLP given vehicle.

Figure 5. TLR9 deficiency improves survival, acute kidney injury, splenic apoptosis, and circulating cytokines after polymicrobial sepsis

(A) Survival curves after CLP for wild-type (dashed line, N = 19) and TLR9-deficient (solid line, N = 16) mice. #, P < 0.05 vs wild-type. Mice were subjected to sham surgery (white bars) or CLP in wild-type (black bars) or TLR9-deficient (gray bars) mice. (B) Number of active caspase3 positive cells in the spleen 24 hr after sham surgery (wild-type, N = 4; TLR9-deficient, N = 4) or CLP (wild-type, N = 7; TLR9-deficient, N = 5). (C) Kidney function 24 hr after sham surgery (wild-type, N = 4; TLR9-deficient, N = 4) or CLP in wild-type (N = 10) and TLR9-deficient (N = 11) mice. (D) Kidney histology scores at 24 hr after surgery (see Methods) for tubules in the renal cortex and outer stripe of the outer medulla (OSOM) at 24 hr after surgery (N = 4-6 per group). (E) Serum TNF-alpha and IL-10 at 24 hr after CLP (N = 6-11 per group). Values are mean ± SE. *, P < 0.05 vs. wild-type.

Figure 6. TLR9 inhibition by phosphorothioate oligodeoxynucleotides improves acute kidney injury, multiple organ damage, and serum cytokines

Mice were subjected to sham surgery (white bars, N = 5) or CLP (black bars, N = 8), and CLP was treated with control (A151, hatched bars, N = 8) or TLR9-selective (H154, gray bars, N = 8) phosphorothioate oligodeoxynucleotides, and evaluated at 24 hr for (A) kidney function (B) ALT, AST (C) amylase, CK, LDH (D) Number of active caspase3 positive cells in the spleen (E) TNF-alpha and IL-10. Values are mean ± SE. *, P < 0.05 vs. wild-type.