This study demonstrates a distinct pattern of cytokine/chemokine production following focal ischemia in mice with disruptions of MyD88 and TRIF. It also shows that the MyD88 pathway plays a major role in the production of neutrophil chemoattractants following focal ischemia. Patterns of cytokine production in response to focal cerebral ischemia are altered in mice with disruptions of these downstream adaptors. These findings indicate that cytokine/chemokine production following focal cerebral ischemia is mediated by these downstream TLR signaling pathways. Our results also suggest a possible compensatory skew toward the Th2 adaptive immune response at baseline in mice with disruptions of MyD88 and a subsequent paradoxical Th1 switch following focal cerebral ischemia. These findings support our previous supposition that the lack of protection against cerebral ischemia noted in MyD88−/− and TRIF-mutant mice was not due to compensation in the production of downstream effectors by either pathway when the other was disrupted .
Serum and brain IL-6 production during focal cerebral ischemia followed previously reported kinetics [17, 18]. There was no apparent compensation in IL-6 levels by the TRIF pathway. Overall, our current results demonstrate that IL-6 production is MyD88-dependent following focal ischemia. IL-6 levels were similar in mice with disruptions of the TRIF pathway and WT mice. These findings explain, in part, our earlier results which showed that MyD88−/− mice were not protected against cerebral ischemia and are in agreement with previous reports showing no protection in mice with deficiency of IL-6 in models of focal ischemia [18, 19]. In addition, to decreased levels of IL-6, we also observed decreased levels of KC, the murine ortholog of human IL-8 and a neutrophil chemoattractant , in MyD88−/− mice following focal cerebral ischemia. These results concur with reports of KC production via the MyD88 pathway in a model of infection [21–23] and highlights the parallels between the innate immune response to focal ischemia, noted in our study, and the reported innate immune response to infection.
G-CSF, another neutrophil chemoattractant, which was produced in a MyD88-dependent manner in our study, has been shown to increase in the brain following cerebral ischemia in humans . However, the specific downstream TLR adaptors that mediate its production following cerebral ischemia have not previously been studied. G-CSF has been reported to be neuroprotective following cerebral ischemia [25–28]. The production of G-CSF in a MyD88-dependent manner following cerebral ischemia in our current study is supported by findings of significantly reduced G-CSF levels in MyD88−/− mice in models of infection . The observed decrease in brain levels of G-CSF and IL-6 in MyD88−/− mice, along with the reported lack of protection against cerebral ischemia in IL-6-deficient mice, may explain, in part, the absence of protection against cerebral ischemia in these mice reported in our previous studies .
The inclusion of baseline (naïve animals) cytokine/chemokine analysis in our study allowed us to show major perturbations in cytokine levels in mice that did not have surgery. This analysis permitted categorization of cytokines/chemokines into Th1/Th2 groups. Our results reveal a paradoxical Th2 phenotype at baseline in MyD88−/− mice that reverted to a more Th1 phenotype following focal cerebral ischemia. This is in contrast to the normal switch from a Th1- to a Th2-type phenotype associated with cerebral ischemia . In some cases, this abnormal baseline elevation in MyD88−/− mice was found only in serum samples and, in other cases, such as IL-10, also in the brain. These findings are consistent with previous results that have shown tissue specificity in cytokine production following ischemia . Interestingly, the Th2-type phenotype at baseline in MyD88−/− mice is consistent with previous findings that TLRs control activation of the adaptive immune response via their role in dendritic cell maturation . Importantly, these findings seem to indicate that this baseline Th2 phenotype skew in MyD88−/− mice is not protective during acute cellular ischemic stress. These results may have clinical implications for patients with acute focal cerebral ischemia and an underlying chronic inflammatory condition that predisposes them to a Th2 skew at baseline, as observed in MyD88−/− mice.
Overall, it is noteworthy that the cytokine profile obtained from our MyD88−/− mice following cerebral ischemia was similar to the cytokine profile obtained when whole-blood cells from patients with deficiencies of MyD88 were stimulated with specific TLR agonists . This highlights the physiological relevance and possible extrapolation of our current findings, with respect to the cytokine production, during the innate immune response to events occurring during cerebral ischemia in humans.
IP-10 was partially produced in a TRIF-dependent manner in our studies. However, MIP-1α was clearly produced in a predominantly TRIF-dependent manner following pMCAO, as shown by MIP-1α levels that were significantly decreased at baseline in mice with disruptions of the TRIF pathway and not in WT mice or in mice with disruptions of MyD88. Also of note, consistent with disruption of the TRIF pathway was the presence of comparable levels of IL-6 in TRIF-mutant mice and WT mice, which is evidence of a functioning MyD88 pathway. To the best of our knowledge, expression of MIP-1α has not previously been reported to occur via the TRIF signaling pathway following focal cerebral ischemia.
In this study, we have demonstrated a role for the MyD88 pathway in neutrophil migration to the site of ischemia by showing significant decreases in the levels of neutrophil chemoattractants, such as KC and G-CSF, in MyD88−/− mice following focal cerebral ischemia. A study of inflammatory infiltrate with apparently fewer neutrophils in the brains of MyD88−/− mice following focal ischemia provided additional evidence in this regard. These combined results strengthen our conclusion that the MyD88 pathway is involved in neutrophil migration to the site of cerebral ischemia. These findings are in agreement with studies showing involvement of the MyD88 pathway in neutrophil migration to the site of infection in the context of low levels of the neutrophil chemoattractant KC .
In summary, we demonstrate for the first time robust differences in cytokine production by the two different major arms of the TLR signaling pathways, MyD88 and TRIF, following focal cerebral ischemia. The significance of these findings includes the following. (1) Demonstration for the first time of new roles for the downstream TLR adaptors MyD88 and TRIF in the production of specific cytokines and chemokines in a model of focal cerebral ischemia. (2) Indications that the MyD88 pathway plays a major role in the expression of neutrophil chemoattractants, such as G-CSF and KC, which direct neutrophils to the site of tissue injury. (3) Observation of a paradoxical Th1/Th2 cytokine profile when these downstream adaptors are disrupted. These findings have not been described previously and suggest a decreased ability of these mice to mount an effective innate immune response to focal cerebral ischemia. (4) Furthermore, we describe for the first time major perturbations in the baseline cytokine profile in mice with disruptions of MyD88. These findings may explain, in part, the lack of protection against focal cerebral ischemia noted when these downstream adaptors are disrupted. Overall, our findings add to the current knowledge by describing new roles for the downstream adaptors MyD88 and TRIF following focal cerebral ischemia and providing great insights into the complex downstream TLR signaling pathway.
Some of the strengths of our study include the fact that we simultaneously tested for dynamic changes in the levels of 25 cytokines/chemokines at baseline and at different time points in brain and serum samples using multiplex array methodology. This approach allowed us to determine that the innate immune response to ischemic stress has striking similarities to the reported innate immune response to stress from infection. These similarities include the expression of cytokines and chemokines that direct the recruitment of immune cells to the site of injury as well as modulation of the Th1/Th2 response. Studies are underway to determine the differential gene expression profile in the brains of mice with disruptions of MyD88 and TRIF to help shed further light on these downstream TLR signaling events at the level of gene expression.