Toll-Like Receptor-mediated Innate Immune Functions of Rodent Microglia Ex vivo

Microglia are the only significant population of immune cells residing within the healthy CNS parenchyma, which phenotypically and functionally resemble macrophages. Macrophages are important cellular elements of the innate immune system because they can rapidly respond to invading pathogens through TLR-mediated recognition of antigenic PAMPs. Macrophages are also necessary for the processing and presentation of antigen to cells of the adaptive immune system and serve to bridge the innate and adaptive immune responses. TLR engagement leads to intracellular signaling, cellular activation, and production of a variety of pro-inflammatory molecules including the chemical mediator NO. Production of NO at immunologically relevant levels is the responsibility of iNOS. iNOS expression requires activation of both the NF-B and STAT-1 signal transduction pathways. Our laboratory has previously shown that microglia isolated from the adult murine CNS have a reduced capacity to produce NO when stimulated through TLR4, as compared to those isolated from neonatal mice. Here we have expanded these findings by comparing the proinflammatory cytokine and chemokine responses of adult and neonatal murine microglia to stimulation through TLR2, 3, and 4. We observed that although adult murine microglia exhibited a reduced NO response compared to neonatal cells, they were capable of producing similar levels of pro-inflammatory cytokines. In addition, our data suggest that iNOS is regulated primarily at the posttranslational level, which results in the observed differential NO response. We also observed that NO production by acutely isolated microglia began to diminish as early as 12 days postpartum in the mouse. We extended these findings to iii the rat, demonstrating that microglia from adult and neonatal rat exhibit a similar differential NO response to TLR4 stimulation in vitro. However, TLR4-mediated NO responses of rat microglia in vitro appeared to be less dependent than murine cells on exogenous IFN-. Since both neonatal and adult rat microglia were capable of upregulating significant levels of IFN- mRNA in response to TLR4 stimulation, these data suggest that rat microglia can activate their STAT-1 pathway through paracrine and autocrine signaling. Finally, we identified in vitro culture conditions that significantly increased the subsequent NO response to TLR stimulation.

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