Targeting inflammatory monocytes in sepsis-associated encephalopathy and long-term cognitive impairment

Abstract

Sepsis-associated encephalopathy manifesting as delirium is a common problem in critical care medicine. In this study, patients that had delirium due to sepsis had significant cognitive impairments at 12-18 months after hospital discharge when compared with controls and Cambridge Neuropsychological Automated Test Battery-standardized scores in spatial recognition memory, pattern recognition memory, and delayed-matching-to-sample tests but not other cognitive functions. A mouse model of S. pneumoniae pneumonia-induced sepsis, which modeled numerous aspects of the human sepsis-associated multiorgan dysfunction, including encephalopathy, also revealed similar deficits in spatial memory but not new task learning. Both humans and mice had large increases in chemokines for myeloid cell recruitment. Intravital imaging of the brains of septic mice revealed increased neutrophil and CCR2+ inflammatory monocyte recruitment (the latter being far more robust), accompanied by subtle microglial activation. Prevention of CCR2+ inflammatory monocyte recruitment, but not neutrophil recruitment, reduced microglial activation and other signs of neuroinflammation and prevented all signs of cognitive impairment after infection. Therefore, therapeutically targeting CCR2+ inflammatory monocytes at the time of sepsis may provide a novel neuroprotective clinical intervention to prevent the development of persistent cognitive impairments.

Keywords: Bacterial infections; Inflammation; Innate immunity; Neuroscience.

Figure 1. Behavioral tests and serum cytokines levels in ICU patients.

ICU sepsis survivors were evaluated at 12 months after hospital discharge in (A) pattern recognition memory (control n = 10, sepsis n = 11), (B) spatial recognition memory (control n = 10, sepsis n = 10), and (C) delayed match to sample (control n = 10, sepsis n = 11). Data represent mean ± SEM. *P < 0.05, ***P < 0.001 vs. controls, unpaired 2-tailed t test. (D) The level of blood cytokines was determined at 24 hours after hospital admission in ICU controls (n = 16) and septic patients (n = 34). Data represent mean ± SEM. *P < 0.05 vs. ICU controls, Mann-Whitney U test.

Figure 2. S.pneumoniae–infected mice have increased blood and cerebrospinal fluid cytokines.

Mice were infected with S. pneumoniae, and blood was extracted at 4 hours and 24 hours (S. pneumoniae 4h or 24h serum) or cerebrospinal fluid (CSF) was extracted at 24 hours after infection to measure a large cytokine array. Several cytokines are shown in (A) TNF-α, (B) KC, (C) IL-6, (D) G-CSF, (E) MCP-1, (F) MIP-1α, (G) IP-10 levels, and (H) IL-10. Data in A–H represent mean ± SEM of n = 5–10 in blood serum control, n = 5–6 in blood serum S. pneumoniae 4 hours, n = 18–20 in blood serum S. pneumoniae 24 hours, n = 5–10 in CSF control, and n = 12–15 in CSF S. pneumoniae. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control, 1-way ANOVA followed by Dunnett’s multiple comparison’s test for the blood serum samples and unpaired t test for the CSF samples. Mice recovered from infection showed cognitive impairment in Morris water maze. Mice were infected with S. pneumoniae and allowed to recover for 2 weeks and, at this time, the Morris water maze test was assessed (see Methods). (I) Path length during training, (J) latency during training, (K) percentage of distance traveled in the target quadrant, and (L) percentage of time in target quadrant both on the fourth day after the platform was removed and the mouse memory strength were evaluated. Data in I–L represent mean ± SEM of n = 10 in control and n = 8 in S. pneumoniae–infected mice group. *P < 0.05 vs. control mice, unpaired 2-tailed t test.

Figure 3. Morris water maze at 9 weeks after infection.

Mice were infected with S. pneumoniae and allowed to recover for 2 weeks. At this time, the Morris water maze was assessed. Then, at 9 weeks after infection, the Morris water maze was assessed again in a different pool and different room. (A) Path length during the learning days. (B) Percentage of distance spent in the target quadrant on the fourth day when platform was removed to assess memory strength in mice. Data in A and B represent mean ± SEM of n = 10 in control and n = 8 in S. pneumoniae recovered from infection. *P < 0.05 vs. control mice, unpaired 2-tailed t test. Hippocampus but not amygdala function is altered in mice recovered from S. pneumoniae infection. Mice were infected with S. pneumoniae for 18 weeks, and, at this time, the behavioral tests were conducted. (C) Immediate shock deficit contextual freezing. (D) Cued fear conditioning data in C and D represent mean ± SEM of n = 10 in control and n = 8 in S. pneumoniae–infected mice. **P < 0.01 vs. control mice, 2-way ANOVA followed by Sidak’s multiple comparisons test. MRI brain images of mice recovered from S. pneumoniae infection. (E) Representative multislice images of a mouse brain after recovery from S. pneumoniae infection at 18 weeks after infection. (F) Hippocampus volume was assessed by analyzing voxel based segmentation of the MRI stacks. Data represent mean ± SEM of n = 3. Effects of S. pneumoniae on blood-brain barrier permeability. Mice were treated with S. pneumoniae for 24 hours, and, at this time, permeability was assessed with Evans Direct Blue. (G) Quantification of brain permeability. Data represent mean ± SEM of n = 3. **P < 0.01 vs. control mice, 1-way ANOVA followed by Tukey’s multiple comparisons test. (H) Permeability image in control, B. pertussis toxin– (PTX, used as a positive control for permeability), and S. pneumoniae–infected mice for 24 hours.

Figure 4. S. pneumoniae–infected mice have increased neutrophil recruitment into the brain.

Mice were infected with S. pneumoniae for 24 hours. (A) Intravital microscopy image of the brain vasculature of control mice. Scale bar: 20 μm. (B) Intravital microscopy image of the brain vasculature in S. pneumoniae–infected mice at 24 hours after infection. Neutrophils are visualized in blue, labeled with anti-Ly6G mAb (clone 1A8). Endothelium is visualized in red, labeled with anti-CD31 mAb. Scale bar: 20 μm. (C) Quantification of neutrophil rolling flux (n = 5). (D) Quantification of neutrophil adhesion (n = 5). Data in C and D represent mean ± SEM. ***P < 0.001 vs. control, unpaired 2-tailed t test. (E) Coronal brain section image showing adherent neutrophils in the inset (arrows showing neutrophils visualized in blue). Neutrophils were labeled with anti-Ly6G mAb (clone 1A8) and endothelium was labeled with anti-CD31 mAb. (F) Quantification of neutrophil adhesion in coronal sections at 4 hours (S. pneumoniae 4h) or 24 hours (S. pneumoniae 24h) after S. pneumoniae infection (n = 3 ). Data represent mean ± SEM. *P < 0.05 vs. control, 1-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 5. Inhibition of neutrophil recruitment into the brain does not have an effect cognitive impairment.

Mice were infected with S. pneumoniae and immediately received anti–P selectin mAb (S. pn + anti-P-sel) or at 4 hours after infection received anti-KC mAb (S. pn + anti-KC). (A) Neutrophil rolling flux at 24 hours (n = 4–5). (B) Neutrophil adhesion at 24 hours after infection (n = 4–5). (C) Neutrophil adhesion in coronal brain section (n = 3). Data in A–C represent mean ± SEM. *P < 0.05, ***P < 0.001 vs. control, ^#P < 0.05, ^###P < 0.001 vs. S. pneumoniae, 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) Morris water maze at 2 weeks after infection. Percentage of distance spent in the target quadrant on the fourth day after the platform was removed in control (n = 10), S. pneumoniae–infected (n = 10), and S. pneumoniae–infected mice treated with anti-KC mAb (n = 6) or anti–P selectin (n = 8). Data represent mean ± SEM. *P < 0.05, **P < 0.01 vs. control, 1-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 6. S. pneumoniae infection increases CCR2⁺ monocyte recruitment.

Wild-type reporter mice CX3CR1^GFP/WTCCR2^RFP/WT were infected with S. pneumoniae. (A) Intravital microscopy image of control mouse brain vasculature. Scale bar: 20 μm. (B) Intravital microscopy image of S. pneumoniae–infected brain vasculature at 24 hours. Scale bar: 20 μm. (C) Quantification of CCR2⁺ monocyte rolling flux at 24 hours. (D) Quantification of CCR2⁺ monocyte adhesion at 24 hours. Data in C and D represent mean ± SEM of n = 5. *P < 0.05, ***P < 0.001 vs. control, unpaired 2-tailed t test. (E) Quantification of monocyte adhesion in the coronal brain section at 4 and 24 hours after infection. Data represent mean ± SEM of n = 3. *P < 0.05 vs. control, 1-way ANOVA followed by Dunnett’s multiple comparisons test. (F) Coronal brain section image by confocal microscopy of a control mouse. Scale bar: 1 mm. (G) Coronal brain section image by confocal microscopy of a S. pneumoniae–infected mouse at 24 hours after infection. Scale bar: 1 mm. In the images neutrophils are visualized as blue, detected by anti-Ly6G mAb; CCR2⁺ monocytes are visualized as red (shown by arrows in B and G), by the knockin fluorescent marker CCR2^RFP/WT; microglia are visualized as green, detected by the knockin fluorescent marker CX3CR1^GFP/WT; and endothelium is visualized as red, by anti-CD31 mAb (in A and B).

Figure 7. Effects of S. pneumoniaeinfection on CCR2⁺ monocyte recruitment and Morris water maze in CCR2^def mice.

Wild-type reporter mice CX3CR1^GFP/WTCCR2^RFP/WT and CX3CR1^GFP/WTCCR2^RFP/RFP (CCR2^def) mice were infected with S. pneumoniae for 24 hours. (A) Quantification of CCR2⁺ monocyte rolling flux. (B) Quantification of CCR2⁺ monocyte adhesion. (C) Quantification of CCR2⁺ monocyte counts in the coronal brain section by confocal microscopy. Data in A–C represent mean ± SEM of n = 3–5. **P < 0.01, ***P < 0.001 vs. control, ^#P < 0.05 vs. WT + S. pneumoniae, ^##P < 0.01 vs. WT + S. pneumoniae, 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) Coronal brain image of CCR2^def mice infected with S. pneumoniae for 24 hours. (E) Morris water maze and percentage of distance spent in the target quadrant on the fourth day. (F) Morris water maze and percentage of time in target quadrant. Wild-type mice, CCR2^def mice, and mice treated with anti-CCR2 mAb (see Methods) were infected with S. pneumoniae and allowed to recover for 2 weeks, and at this time, the Morris water maze test was assessed. Data represent mean ± SEM of n = 14 in control, n = 7 in WT + S. pneumoniae, n = 11 in CCR2^def + S. pneumoniae, and n = 5 in WT + anti-CCR2 + S. pneumoniae mice. *P < 0.05 vs. control mice, 1-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 8. Effects of S. pneumoniae infection on microglia activation.

Wild-type reporter mice CX3CR1^GFP/WTCCR2^RFP/WT and CX3CR1^GFP/WTCCR2^RFP/RFP (CCR2^def) mice were infected with S. pneumoniae for 24 hours. (A) Image of microglia Z-stacks, shown as the maximum intensity projection of 20-μm stack obtained by multiphoton microscopy of a S. pneumoniae–infected CX3CR1^GFP/WTCCR2^RFP/WT mouse at 24 hours. Scale bar: 50 μm. (B) Inactivated microglia, characterized by a soma area of 0–50 μm². Scale bar: 10 μm. (C) Image of activated microglia 24 hours after infection in a CX3CR1^GFP/WTCCR2^RFP/WT mouse. Scale bar: 10 μm. (D) Quantification of microglia activation. Data represent mean ± SEM of n = 3. ***P < 0.001 vs. control, unpaired 2-tailed t test. (E) Microglia C11b⁺CD45^intermediate expression by flow cytometry. Brain cells from control and S. pneumoniae–infected mice for 24 hours were isolated as previously described and labeled with CD11b and CD45. The CD11b⁺ cells were gated and analyzed for CD45^intermediate expression. The representative histogram (n = 3) shows the increase in CD45^intermediate expression in brain microglia from S. pneumoniae pneumonia-infected mice versus controls.