UC Riverside

Specialized glial cells called microglia serve as the resident immunocompetent cells of the central nervous system (CNS). Though many describe microglia as having a "resting" state in the healthy brain, their processes are highly mobile and constantly survey their microenvironment (Nimmerjahn et al. 2005). In response to trauma, inflammation or infection, microglia become activated and peripheral macrophages are recruited to the CNS. Both microglial morphology and the collection of receptors that they express changes with their activation state. In the adult CNS, the type and magnitude of microglial activation and macrophage influx triggered by inflammation are well characterized. However, little is known about if and how CNS immunity changes during critical periods of postnatal brain development associated with synaptogenesis and oligodendrocyte maturation. This dissertation primarily focuses on understanding how microglial phenotype changes in the healthy brain during the early postnatal period and how their phenotype and the degree of macrophage influx changes following systemic inflammation.

A variety of receptors throughout the body have roles in mediating immune responses. Recently, a family of receptors called the Triggering Receptors Expressed on Myeloid cells (TREMs) have been implicated in regulating both innate and adaptive immune responses. TREM1 expression has been shown to amplify TLR mediated pro-inflammatory immune responses (Bouchon et al. 2001). TREM2 expression in the brain is known to promote neuroprotective anti-inflammatory immune responses. However, lack of a functional TREM2 signaling pathway has been shown to cause a rare genetic disorder called Nasu-Hakola disease (Madry et al. 2007). This disease is characterized by bone cysts, presenile cognitive dementia and death in the 40's or 50's (Madry et al. 2007). Using dual in situ hybridization/immunohistochemistry, quantitative RT-PCR and flow cytometric studies, we show that several TREMs are expressed in the brain in a cell type specific manner. In situ hybridization/immunohistochemistry analysis of 3 month old unmanipulated murine brain illustrates that TREM4 is expressed by subsets of neurons and is not regulated by inflammation. qRT-PCR studies of 3 month old mice sacrificed 24 hours post intracerebroventricular (ICV) of lipopolysaccharide (LPS) demonstrate that TREM1 mRNA is higher in CD45lo microglia than CD45hi CNS infiltrating macrophages, TREM2 mRNA is higher in microglia and TREM3 mRNA is higher in infiltrating macrophages. Lastly, using flow cytometry, we have found the microglia upregulate TREM2 following an ICV LPS injection, while CNS infiltrating macrophages TREM1 and a small population of macrophages express TREM3. Together, these data demonstrate that TREMs are differentially expressed by cells within the brain and along with their roles in innate and adaptive immune responses, TREMs may also play roles in healthy brain and cognitive development as well as in CNS diseases.

Inflammation that is experienced during the perinatal period has been implicated in playing a role in the development and/or pathogenesis of several neurodevelopmental disorders (Chew et al., 2006; Doorduin et al., 2008; Pardo et al., 2005). Though microglial activation has been observed in the brains of patients with cerebral palsy, schizophrenia and autism, the potential roles of these cells in the pathogenesis of these disorders are unknown. Using flow cytometry, we show that during the early postnatal period, microglia are in a pre-activated state that is non-polarized. This pre-activated state is lost as the brain matures. We also examined microglial phenotypes and responses to systemic inflammation and the degree of macrophage influx as a function of normal development. Here we demonstrate that systemic inflammation induced by an intraperitoneal injection of LPS leads to widespread activation of microglia and the transient influx of peripheral macrophages into the brain in an age specific manner. Interestingly, systemic inflammation led to the induction of TREM2, PD-L1 and CX3CR1 which are known to promote anti-inflammatory microglial activation. This data contributes to the literature illustrating that the brain may display age specific susceptibilities to insults.

Interestingly, the aged brain is also characterized by changes in microglial phenotype. By examining morphology and the expression of activation markers, several studies have shown that microglia become activated in the brain with healthy aging. Moreover, many have shown that microglia display exaggerated immune responses following inflammatory events. Though several studies have found that both the CNS microenvironment becomes more pro-inflammatory in the aged brain and that microglia produce the pro-inflammatory molecules CD40, B7.2 and IL-1β in during this period, many have only begun to examine whether anti-inflammatory molecules are produced as well (Carson lab unpublished data, Henry et al. 2009). Here we confirm that following intracerebroventricular injection of LPS, the anti-inflammatory molecule TREM2 is more highly induced by microglia isolated from the young adult murine brain than those isolated from the aged murine brain. We also show that in the 3 month old murine brain, the anti-inflammatory molecules CX3CR1 and TREM2 binding activity are present and induced following inflammation. This is not observed in the 15 month old murine brain.

Overall these studies suggest that (1) TREM receptors are expressed on a variety of cell types in the brain and their contribution to immune responses depends on the compartment (CNS intrinsic versus CNS extrinsic) in which they are expressed and (2) CNS immune responses in the healthy and inflamed brain are age-specific.