Article Figures & Data
Figures
- FIG 1
Diabetic mice were hypersusceptible to A. baumannii sepsis despite having bacterial densities similar to those in nondiabetic mice. (A, B) Survival curves for diabetic and nondiabetic mice. (A) C57BL/6 mice with streptozotocin-induced (STZ) diabetes (severe, type 1 model) developed lethal bloodstream infections due to the hypervirulent A. baumannii strain HUMC1 (7 × 106-CFU inoculum), whereas nondiabetic control mice cleared the infection (n = 8 mice per group). *, P < 0.05. (B) Mice with diet-induced-obesity (DIO) diabetes (mild, type 2 model) also developed lethal infections due to A. baumannii (2 × 107-CFU inoculum), whereas nondiabetic control mice cleared the infection (n = 30 mice per group). *, P < 0.05. (C) The rates of growth for log-phase A. baumannii were identical in DIO diabetic and nondiabetic mice. (D) DIO diabetic mice had blood bacterial burdens nearly identical to those in nondiabetic control mice at 2 h postinfection (n = 10 mice per group; bars display median values).
- FIG 2
Diabetic mice had worse sepsis biomarkers from A. baumannii sepsis despite bacterial densities similar to those in nondiabetic mice. (A) Body temperatures (n = 10 mice per group) and blood sepsis biomarkers demonstrated severe metabolic acidosis (low blood pH, low serum bicarbonate, and high base deficit) consistent with septic shock at 18 h postinfection with A. baumannii (2 × 107-CFU inoculum) in DIO diabetic wild-type mice but not nondiabetic wild-type control mice (n = 18 nondiabetic control mice, and n = 17 DIO diabetic wild-type mice). (B) Inflammatory cytokine levels were markedly elevated in DIO diabetic wild-type mice compared to the levels in nondiabetic wild-type control mice (n = 10 mice per group). *, P < 0.05; †, P = 0.05. (C) During A. baumannii bacteremia, diabetic mice experienced significantly higher ratios of IL-10 to TNF than nondiabetic mice (n = 10 mice per group). Bar graphs show medians and interquartile ranges; bars on dot plots display medians.
- FIG 3
Treatment with the potent immunosuppressive corticosteroid dexamethasone and disruption of TLR4 restored resistance to infection in diabetic mice. (A) DIO diabetic and nondiabetic mice were infected with a normally lethal dose of A. baumannii (2 × 107-CFU inoculum). Mice treated 20 min postinfection with 4 mg/kg of dexamethasone, a potent corticosteroid, were resistant to infection, while those treated with saline (control) succumbed to infection within 48 h (n = 5 mice per group). *, P < 0.05. (B) Streptozotocin-induced diabetic wild-type C3H (C3HeB/Fe) and TLR4 mutant (C3H/He) mice were infected with A. baumannii (2 × 107-CFU inoculum). TLR4 mutant mice had significantly improved survival (n = 10 mice per group). (C) A TLR4 antagonist (E5564) protected mice from lethal A. baumannii bacteremia caused by i.v. infection. E5564 was administered i.v. at 10 mg/kg/day for 4 days (n = 8 mice per group). (D) DIO wild-type and DIO TLR4 KO mice had significantly higher levels of fasting blood glucose than nondiabetic wild-type mice (n = 10 mice per group; median values and interquartile ranges are shown). *, P < 0.05 versus the results for nondiabetic wild-type mice. (E) DIO diabetic TLR4 KO mice were resistant to A. baumannii bacteremia, while DIO diabetic wild-type mice all died (n = 10 mice per group). *, P < 0.05 versus the results for the control.
- FIG 4
TLR4 disruption abrogated the hypersusceptibility of diabetic mice to sepsis from A. baumannii and other Gram-negative bacteria. (A) DIO diabetic mice had bacterial burdens at 24 h postinfection similar to those in nondiabetic mice, whether on a wild-type or TLR4 KO background (n = 8 mice per group). *, P < 0.05 versus the results for DIO diabetic and nondiabetic wild-type mice. (B) DIO diabetic and nondiabetic wild-type and TLR4 KO mice had nearly identical levels of lipopolysaccharide (LPS), measured as endotoxin units (EU) per bacterial CFU in the blood (n = 7 mice for the nondiabetic wild type, and n = 8 mice for all other groups). (C, D) DIO diabetic TLR4 KO mice were protected from severe acidosis/acidemia (C) and cytokine storms (D) that occurred in DIO diabetic wild-type mice infected with A. baumannii (2 × 107-CFU inoculum) (n = 7 mice per group). Bars represent median values. *, P < 0.05 versus the results for the control.
- FIG 5
TLR4 disruption abrogated the hypersusceptibility of diabetic mice to sepsis from other Gram-negative bacteria. (A) Survival curves for diabetic and nondiabetic mice. DIO diabetic wild-type mice were more susceptible than nondiabetic wild-type mice to infections with ESBL E. coli and pan-drug-resistant, KPC-expressing K. pneumoniae (n = 24 mice for E. coli groups, and n = 16 mice for K. pneumoniae groups from 2 and 3 experiments, respectively). *, P < 0.05 versus the results for the control. (B) DIO diabetic wild-type mice had no significant difference in blood bacterial burdens at 2 h postinfection compared to the blood bacterial burdens in nondiabetic wild-type mice (n = 5 mice per group). P > 0.1 for both comparisons. (C) DIO diabetic TLR4 KO mice are rescued from the increased susceptibility of DIO diabetic mice to both organisms (n = 8 per group). *, P < 0.05 versus the results for the control. Bars on dot plots display median values.
- FIG 6
RAGE disruption ameliorates hypersusceptibility of diabetic mice to Gram-negative bacterial sepsis. (A) DIO diabetic and nondiabetic C57BL/6 mice were infected with A. baumannii and treated once daily for 4 days with PBS (control), a small-molecule RAGE antagonist (FPS-ZM1 at 75 μg/day), or a peptide RAGE antagonist (RAP at 300 μg/day). The nondiabetic C57BL/6 mice (n = 6 mice for control group, and n = 7 mice per treated group) were infected with an inoculum of A. baumannii (3 × 107 CFU) that kills only some control mice to maximize the chances of detecting some protective effect of RAGE antagonism; the DIO diabetic wild-type mice (n = 5 mice per group) were infected with an inoculum (2 × 107 CFU) that is 100% lethal for diabetic mice. RAGE antagonism improved the survival of diabetic but not nondiabetic mice. (B) RAGE antagonism did not alter bacterial burdens in DIO diabetic mice. Bars represent median values. *, P < 0.05 versus the results for the control. (C) Sepsis biomarkers improved significantly in DIO diabetic mice infected with A. baumannii (2 × 107-CFU inoculum) upon treatment with the small-molecule RAGE antagonist FPS-ZM1 (n = 5 mice per group). Bars represent median values. *, P < 0.05 versus the results for the control. (D) DIO diabetic mice are protected from cytokine storms after infection with A. baumannii (2 × 107-CFU inoculum) upon treatment with the small-molecule RAGE antagonist FPS-ZM1 (n = 5 mice per group). Bars represent median values. *, P < 0.05 versus the results for the control. (E) During A. baumannii bacteremia, DIO diabetic mice experienced higher ratios of IL-10 to TNF than mice treated with the small-molecule RAGE antagonist FPS-ZM1, similar to the results for the nondiabetic wild-type mice in the experiment whose results are shown in Fig. 2C (n = 10 mice per group). *, P < 0.05. (F) DIO diabetic mice were infected with an inoculum of E. coli (2 × 108 CFU) that kills some diabetic mice. They were treated once daily for 4 days with PBS (control) or the small-molecule RAGE antagonist FPS-ZM1 (250 μg/day). RAGE antagonism significantly improved survival in the diabetic mice (left), even though the bacterial burdens were no different between control and RAGE antagonist-treated mice (right). *, P < 0.05 versus the results for the control. (G) DIO diabetic mice were infected with an inoculum of K. pneumoniae (2 × 108 CFU) that kills all diabetic mice. They were treated once daily for 4 days with PBS (control) or the small-molecule RAGE antagonist FPS-ZM1 (250 μg/day). RAGE antagonism significantly improved survival in the diabetic mice (left), even though the bacterial burdens were no different between control and RAGE antagonist-treated mice (right). *, P < 0.05 versus the results for the control.
- FIG 7
Knocking out RAGE reverts the susceptibility of diabetic mice to A. baumannii sepsis to the level of nondiabetic wild-type mice. (A) DIO diabetic wild-type and DIO diabetic RAGE KO mice had significantly higher levels of fasting blood glucose than nondiabetic wild-type control mice (n = 10 mice per group). *, P < 0.05 versus the results for nondiabetic wild-type mice. (B) DIO diabetic RAGE KO mice were much more resistant to A. baumannii bloodstream infection (2 × 107-CFU inoculum) than DIO diabetic wild-type mice (n = 10 mice per group). *, P < 0.05 versus the results for the control. (C) DIO diabetic RAGE KO mice are protected from cytokine storms that occur in DIO diabetic wild-type mice infected with A. baumannii (2 × 107-CFU inoculum) (n = 10 mice per group). Bars represent median values. *, P < 0.05 versus the results for the control. (D) During A. baumannii bacteremia, diabetic wild-type mice experienced significantly higher ratios of IL-10 to TNF than diabetic RAGE KO mice, similar to the nondiabetic wild-type mice in the experiment whose results are shown in Fig. 2C (n = 10 mice per group). *, P < 0.05.
- FIG 8
MyD88 disruption completely abrogated the susceptibility of diabetic mice to A. baumannii sepsis. (A) DIO diabetic wild-type and DIO diabetic MyD88 KO mice had significantly higher levels of fasting blood glucose than nondiabetic wild-type control mice (n = 5 mice per group). *, P < 0.05 versus the results for nondiabetic wild-type mice. Bar graph show median values and interquartile ranges. (B) DIO diabetic MyD88 KO mice were completely resistant to A. baumannii bloodstream infection (2 × 107-CFU inoculum), whereas all DIO diabetic wild-type mice died (n = 5 mice per group). *, P < 0.05 versus the results for the control. (C) Damage response framework basic curve, adapted from Casadevall and Pirofski (44) (reprinted from Nature Reviews Microbiology with permission of the publisher). The traditional view of diabetes is that it substantially weakens the host’s immune response, predisposing the host to worse outcomes from infection. The current results indicate that diabetes predisposes to worse outcomes from infection by amplifying the innate immune/inflammatory response to Gram-negative bacterial infection.
