Flexing the innate immune arm within the human central nervous system : implications for multiple sclerosis

In the inflammatory brain lesions characteristic of multiple sclerosis (MS), both infiltrating macrophages and their central nervous system counterparts, the resident microglia, are present in large numbers and, along with reactive astrocytes, are implicated as potential antigen presenting cells (APC)s. The demyelinating pathology associated with the infiltration of self-reactive T cells in MS requires local antigenic re-stimulation in the context of major histocompatibility complex (MHC) molecules. Human microglia and, less commonly, astrocytes, have been found to express APC molecules in MS brains. This, combined with the potential for production of immunomodulatory and toxic mediators upon activation, highlights the central role for these glial cells in neuroinflammation and, particularly, in MS. Toll-like receptors (TLR)s were recently identified as key mediators signaling the activation of innate immune responses. We hypothesized that TLR ligation by human microglia and/or astrocytes can trigger an APC-like maturation process in the CNS, tailored to the environmental ligand, which can then dictate and polarize subsequent immune responses. We further hypothesize that oligodendrocytes are particularly sensitive to injury mediated by specific reactive oxygen/nitrogen species produced following the activation of these glial cells. Expression and function of TLRs in the human brain was largely unknown at the onset of this work. Using cells isolated from surgically resected normal appearing white matter, we found that human microglia express mRNA for TLRs 1-9, whereas only TLR3 is expressed robustly by human astrocytes. By flow cytometry, we established that microglia strongly express cell surface TLR2 and intracellular TLR3, while TLR4 is expressed below detection levels. Astrocytes were found to restrict their protein expression and functional responses to TLR3. In contrast, microglia responded to the ligation of TLRs 2, 3 and 4. In line with its unique signaling, TLR3 induced the strongest activation program in microglia, characterized by high level production of classical pro-inflammatory cytokines (TNF-alpha and IL-6), the essential polarizing cytokines IL-12 (concomitant with IL-10) and IL-23, known to result in Th1- and Th17-type CD4+ T cell responses, as well as the prototypical type I IFN response, including IFN-alpha, IFN-beta, and CXCL-10 production. TLR4- and, particularly, TLR2-mediated microglial activation was found to be more selective and less polarizing. Production of type I IFN and associated responses was absent downstream of TLR2. We hereby established that TLR-mediated activation of human microglia can instruct the subsequent mode of adaptive immune response. Both TLR3 and TLR4 ligation increased microglial MHC and classical co-stimulatory (CD80, CD86) molecule expression on the cell surface, while TLR2 led to a more limited response. Only TLR3 ligation induced CD40 expression. While TLR-mediated microglial activation did not result in increased allogeneic CD4+ T cell proliferation, the secretion of the classical Th1-cytokine IFN-gamma was significantly elevated in allogeneic co-cultures of TLR3 pre-activated microglia and CD4+ T cells. TLR3 ligation also induced significantly higher levels of T cell death in these co-cultures. TLR2-mediated pre-activation led to a trend towards Th2-polarized cytokine secretion (IL-5, IL-13) in these same cultures, while ligation of all three TLRs led to high level IL-6 responses, indicative of generalized activation responses. Collectively, these data indicate that TLR-mediated CNS innate immune cellular activation can be tailored to the environmental trigger, which can then shape the profile of the adaptive immune response. Nitric oxide (NO) and its more potent byproduct, peroxynitrite (ONOO), are implicated as microglia and astrocyte-derived toxic mediators in MS pathology. We established that human oligodendrocytes are surprisingly resistant to NO-mediated damage in vitro and, in fact, show increased metabolic activity following repeated exposure to NO. In contrast, oligodendrocytes were highly susceptible to ONOO--mediated cell death. We further establish that oligodendrocytes in MS lesions can display nitrotyrosine residues, indicative of ONOO- production and potential toxicity during the course of disease. In summary, TLR-signaling can dictate the activation profile of human CNS APCs and therefore subsequent local immune responses; furthermore, upon activation in the CNS, the production of ONOO- has the capacity to specifically injure oligodendrocytes, placing microglia and astrocytes at the core of the MS disease process.

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