Systemic interleukin-6 inhibition ameliorates acute neuropsychiatric phenotypes in a murine model of acute lung injury

Abstract

Acute neuropsychiatric impairments occur in over 70% of patients with acute lung injury. Mechanical ventilation is a well-known precipitant of acute lung injury and is strongly associated with the development of acute delirium and anxiety phenotypes. In prior studies, we demonstrated that IL-6 mediates neuropathological changes in the frontal cortex and hippocampus of animals with mechanical ventilation-induced brain injury; however, the effect of systemic IL-6 inhibition on structural and functional acute neuropsychiatric phenotypes is not known. We hypothesized that a murine model of mechanical ventilation-induced acute lung injury (VILI) would induce neural injury to the amygdala and hippocampus, brain regions that are implicated in diverse neuropsychiatric conditions, and corresponding delirium- and anxiety-like functional impairments. Furthermore, we hypothesized that these structural and functional changes would reverse with systemic IL-6 inhibition. VILI was induced using high tidal volume (35 cc/kg) mechanical ventilation. Cleaved caspase-3 (CC3) expression was quantified as a neural injury marker and found to be significantly increased in the VILI group compared to spontaneously breathing or anesthetized and mechanically ventilated mice with 10 cc/kg tidal volume. VILI mice treated with systemic IL-6 inhibition had significantly reduced amygdalar and hippocampal CC3 expression compared to saline-treated animals and demonstrated amelioration in acute neuropsychiatric behaviors in open field, elevated plus maze, and Y-maze tests. Overall, these data provide evidence of a pathogenic role of systemic IL-6 in mediating structural and functional acute neuropsychiatric symptoms in VILI and provide preclinical justification to assess IL-6 inhibition as a potential intervention to ameliorate acute neuropsychiatric phenotypes following VILI.

Keywords: Anxiety; Cleaved caspase-3; Delirium; IL-6; Neural injury; Neuroinflammation; VILI.

Fig. 1

Cleaved caspase-3 is significantly elevated in the amygdala and hippocampus of VILI mice. A Schematic of experimental design and representative regions of interest (ROIs) of measured brain regions. B Quantification of lung inflammation (%PMN in BALF). C–D Quantification of cleaved caspase-3 (CC3) percent area in the amygdala and the hippocampus where each dot represents one animal. E Representative amygdalar sections stained for CC3 (positive signal displayed in red) overlaid on DAPI nuclear stain (gray). Magnified regions of the indicated area of amygdala from the micrograph directly above. F Representative hippocampal sections stained for CC3 (positive signal displayed in red) overlaid on DAPI nuclear stain (gray). Quantitative data are expressed in mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig. 2

VILI increases amygdalar neuronal activity, neuronal stress response, and both amygdalar and hippocampal IL-6 and TNF-α. A–B Quantified levels of cellular stress response (HSP90) and of neuronal activity (c-fos) within the amygdala and hippocampus. C–H IL-6 and TNF-α are significantly increased in VILI brains compared to SB controls in amygdala but not in the hippocampus, while there is no significant change in IL-1β in both areas. I Representative micrographs stained for IL-6 (positive signal displayed in red); cell nuclei are revealed by DAPI staining (gray). Quantitative data are expressed in mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001

Fig. 3

Lung inflammation, cerebral IL-6, and cerebral TNF-α positively and significantly correlate with both amygdalar and hippocampal CC3. A–B Regression analysis between the percent of PMNs in bronchoalveolar lavage fluid (BALF) with amygdalar and hippocampal CC3, IL-6, or TNF-α. SB group is indicated in blue and VILI in red. Significance (p) and fitness (r²) values are indicated. C Regression analysis demonstrating significant positive relationships between amygdalar CC3 with IL-6 and TNF-α. D Micrographs of amygdalar cortical neurons stained for IL-6 (displayed in green) and CC3 (displayed in red). Magnified areas of individual neurons and glial cells are inset. Note that CC3-positive neurons also co-stain for IL-6 in VILI amygdala. E Regression analysis demonstrates a significant positive relationship between hippocampal CC3 with IL-6 but not with TNF-α

Fig. 4

IL-6 inhibition significantly reduces amygdalar or hippocampal CC3 expression. A PMNs in BALF after VILI were significantly different from SB regardless of the three intervention groups (ANOVA, F = 9.861, dF = 21, p = 0.0005). There are no significant differences in oxygen saturations between the three VILI intervention groups; VILI + Saline, VILI + α-IL-6, and VILI + α-IL-6-receptor antibody. B–C Amygdalar (ANOVA, F = 8.738, dF = 21, p = 0.0009) and hippocampal (ANOVA, F = 9.806, dF = 21, p = 0.0005) CC3 expressions are significantly increased in the VILI + Saline group compared to SB control, but significantly reduced in both IL-6 inhibited groups. D–E IL-6 (ANOVA, F = 5.160, dF = 21, p = 0.0095) and TNF-α (ANOVA, F = 5.655, dF = 21, p = 0.0066) are significantly increased in the VILI + Saline group compared to SB control, but significantly reduced in both VILI + IL-6 inhibited groups. F There is no significant difference in percent area of amygdalar IL-1β between all groups. G–I Similar to amygdalar cytokines, IL-6 (ANOVA, F = 8.658, dF = 21, p = 0.0009) and TNF-α (ANOVA, F = 4.210, dF = 21, p = 0.0202) are significantly increased in the VILI + Saline group compared to SB control and significantly reduced in both VILI + IL-6 inhibiter groups; however, there is no difference in the percent area of IL-1β between all groups

Fig. 5

Lung inflammation, cerebral IL-6, and cerebral TNF-α positively and significantly correlate with amygdalar and hippocampal CC3 after IL-6 inhibition. A Regression analysis between the percent of PMNs in bronchoalveolar lavage fluid with amygdalar CC3. SB group is indicated in blue and VILI + Saline in green, VILI + α-IL-6 in red, and VILI + α-IL-6-receptor in brown. Significance (p) and fitness (r²) values are indicated. B Regression analysis between the percent of PMNs in bronchoalveolar lavage fluid with hippocampal CC3. C–D Regression analysis shows a direct and significant relationship between IL-6 and CC3, and TNF-α and CC3 in amygdala and hippocampus

Fig. 6

Systemic IL-6 inhibition significantly improves acute delirium- and anxiety-like neuropsychiatric functional impairments following VILI. A Schema of the apparatus and assessment of behavioral function in the center and peripheral zones of the Open Field. B–C In the open-field behavioral test, VILI + Saline mice spent significantly less time and traveled less distance in the center zone compared to the VILI + α-IL-6 group, indicating more anxiety behavior in the VILI + Saline group compared to the α-IL-6 treated group. D Schema of the elevated plus maze for behavioral assessment in open and closed arms. E–F In the elevated plus maze, the VILI + Saline mice entered and spent significantly less time in the open arms as compared to VILI + α-IL-6 mice, indicating increased anxiety and avoidance behaviors in the VILI + Saline group. G Schema of the Y-maze paradigm. H–I VILI + Saline mice had significantly less spontaneous alternations in the Y-maze compared to the α-IL-6 treated group, indicating impaired short-term memory. Quantitative data are expressed in mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001