Prevention of LPS-induced microglia activation, cytokine production and sickness behavior with TLR4 receptor interfering peptides

Abstract

The innate immune receptor Toll-like 4 (TLR4) is the receptor activated by lipopolysaccharide (LPS), and TLR4-LPS interaction is well known to induce an innate immune response, triggering sickness behavior. Within the brain, TLR4 is highly expressed in brain microglia, and excessive inflammation resulting from activation of this pathway in the brain has been implicated in depressive disorders and neurodegenerative pathologies. We hypothesized that blocking LPS-induced activation of TLR4 would prevent downstream immune signaling in the brain and suppress the induction of sickness behavior. We used interfering peptides to block TLR4 activation and confirmed their efficacy in preventing second messenger activation and cytokine production normally induced by LPS treatment. Further, these peptides blocked morphological changes in microglia that are typically induced by LPS. We also demonstrated that intraperitoneal (i.p.) injection of Tat-TLR4 interfering peptides prevented LPS-induced sickness behavior, as assessed in home cage behavior and with the intracranial self-stimulation paradigm. These newly synthesised peptides inhibit TLR4 signaling thereby preventing changes in behavior and motivation caused by inflammatory stimuli. These peptides highlight the roll of TLR4 and microglia morphology changes in sickness behavior, and thus may be of therapeutic value in limiting the deleterious impact of excessive inflammation in specific CNS pathologies.

Figure 1. Schematic diagram showing the mechanism of action of the Tat-MyD88 and Tat-TLR4 peptides and their efficacy in preventing protein interactions in vivo.

A. The peptides are directed against regions of the TLR4 receptor and MyD88 TIR domain, thereby interfering with the interaction of these two proteins. LPS treatment has been shown to increase TLR4 and MyD88 binding leading to the activation of MAP kinases and NFκβ modulation of TNF-α. Thus the peptides may be effective in blocking downstream signalling to MAP kinases and TNF-α. B,C. 2-photon images of hippocampal tissue following intraperitoneal (i.p.) injection in the mouse reveals that dansylated Tat peptide can be observed in brain cells. D When i.p. injected, Tat-MyD88 but not Tat-scram reduced co-immunoprecipitation of TLR4 and MyD88 from brain tissue. E Densitometry quantification of co-immunoprecipitated protein normalized to immunoprecipitated protein.

Figure 2. Time course of kinase activation and TNF-α formation following LPS treatment, and the inhibition by Tat-MyD88 and Tat-TLR4.

A. Representative blots showing P-p38 MAPK and P-JNK rapidly increased in brain tissue following LPS treatment. GAPDH was monitored as a loading control. B,C. Quantification of the increased P-p38 MAPK and P-JNK levels over 60 minutes following LPS treatment. D-F. P-p38 MAP kinase and P-JNK increases from LPS were attenuated by Tat-MyD88 and Tat-TLR4. D. Representative blots of kinase activation following various treatments. E. Quantification of P-p38 MAPK normalized to GAPDH levels. F. Quantification of P-JNK normalized to GAPDH levels. G,H. LPS treatment increased TNF-α levels, and this increase was blocked by Tat-TLR4 and Tat-MyD88. Quantification of TNF-α levels using ELISA in acute brain slice (G) parallels results found in whole brain lysates of injected animals (H).

Figure 3. Time course of LPS-induced microglia morphology changes visualized using 2-photon imaging and the block by Tat-TLR4 and Tat-MyD88.

A. Series of images at 0, 40 and 80 minutes following application of LPS showing the progression to amoeboid shape in microglia. B. Series of images at 0, 40 and 80 minutes showing Tat-MyD88 blocked the amoeboid shape change normally induced by LPS. C. Representative images of single microglia following individual treatments. D. Quantification of the number of branches of microglia following treatment.

Figure 4. LPS induced sickness behavior was blocked by Tat-MyD88 and Tat-TLR4 as assessed in mice using a modified SHIRPA screen and in the novel home cage following various treatments.

A. Cumulative score of sickness obtained using the SHIRPA screen. B. Representative paths of mice over 30 min in the home cage showing decreased exploration induced by LPS, and effective block by Tat-MyD88 and Tat-TLR4 but not Tat-scram. Average speed travelled (C), cumulative distance travelled (D), number of rears in the home cage (E). F, G. Assessment of sickness behavior in rats using intracranial self stimulation. F. Number of responses per minute during baseline, LPS treatment, and following LPS treatment. G. Number of responses per minute during baseline, LPS plus Tat-MyD88 treatment, and following LPS plus Tat-MyD88.