01.07 TLR4 inhibition after TBI improves cognitive outcomes and increases anti-inflammatory monocytes

Y. Chun1, B. P. Soares2, W. Fulton1, C. Sodhi1, D. J. Hackam1, I. Nasr1  1The Johns Hopkins University School Of Medicine,Pediatric Surgery,Baltimore, MD, USA 2University Of Vermont College Of Medicine / Fletcher Allen Health Care,Radiology,Burlington, VT, USA


Traumatic brain injury (TBI) induces a robust neuroinflammatory response that activates the innate immune system.  Signaling through the Toll-like receptor 4 (TLR4) pathway is one of the key mechanisms by which the innate immune system triggers the neuroinflammatory cascade in response to TBI.  We hypothesize that the pharmacologic inhibition of TLR4 using the novel compound, C34 will lead to a reduction in the acute neuroinflammatory response and result in improved cognitive outcomes.  We propose that the infiltrating monocytes play a role in attenuating the neuroimmune response.

A murine controlled cortical impact TBI model was used.  The experimental groups are: (1) wild-type (WT) control, (2) WT+C34, (3) CX3CR1GFP.  CX3CR1 is a microglial marker. Cell sorting was used to isolate monocytes and microglial cells from the brain.  Real-time PCR quantified cytokine gene expression. Morris Water Maze was used to assess cognitive outcomes. Cell sorting was used to separate microglia and monocytes from the brain parenchyma after TBI.  Statistical analysis: Student’s T-test and One-way ANOVA.

Short-term TLR4 inhibition showed significant improvement in cognitive behavior. This was assessed using the Morris Water Maze which showed improved latency in week 1 and platform entries on week 4 (p=0.026).  The treatment group showed a marked increase in infiltrating monocytes on post-injury day (PID) 7. CX3CR1 positive monocytes were identified in the spleen on PID 7 (n=5) but not on PID 1 (n=5). The cell sorting technique was used to phenotype the infiltrating monocytes as well as the resident microglia in the brain parenchyma.  On PID 1, the infiltrating monocytes had significantly higher gene expression of the anti-inflammatory gene IL-10 (31.66±5.013, n=5) but decreased expression of the pro-inflammatory gene TNF? (512.5±59.35, n=5) compared to the expression in brain microglia: IL-10 (2.748±0.3728, n=5), and TNF? (1220±166.8, n=5). On PID 7, the infiltrating monocytes had higher gene expression of the anti-inflammatory gene  IL-10 (7.068±1.79, n=4) but decreased expression of the pro-inflammatory genes IL-6 (8.898±0.7059, n=5) and TNF? (92.64±20.07, n=4) compared to the expression in brain microglia: IL-10 (1.604±0.4127, n=5), IL-6 (85.68±12.82, n=5), and TNF? (512.6±80.31, n=5).

Our findings demonstrate that infiltrating monocytes play a key role in both the early and the delayed immune responses to TBI.  Infiltrating monocytes displayed an anti-inflammatory gene expression profile in the injured brain on PID 1 and PID 7. These infiltrating monocytes express more anti-inflammatory cytokines compared to the resident microglial population.  The increased number of these infiltrating anti-inflammatory monocytes potentially contributes to the improved cognitive outcomes seen in the C34 treated mice.