Lipopolysaccharide-induced interleukin (IL)-4 receptor-α expression and corresponding sensitivity to the M2 promoting effects of IL-4 are impaired in microglia of aged mice
Introduction
Within the central nervous system (CNS) microglia are responsible for the induction of an innate immune response by receiving and propagating inflammatory signals (Nguyen et al., 2002). Even in the absence of inflammatory stimuli, microglia are actively surveying their local microenvironment (Nimmerjahn et al., 2005). Once activated by an immune stimulus, microglia perform several macrophage-like functions including phagocytosis, inflammatory and anti-inflammatory cytokine production, and antigen presentation (Garden and Moller, 2006). This classical activation (M1) profile is transient with microglia returning to a surveying state as the immune stimulus is resolved. Key to this transition is regulation by several anti-inflammatory mediators (Biber et al., 2007) including neuronal factors, hormones, and cytokines that attenuate microglial activation and promote anti-inflammatory or repair (M2) profiles in microglia (Mantovani et al., 2004, Mosser and Edwards, 2008).
In rodent models of normal and non-neurodegenerative aging, there is an increase in “primed or reactive” microglia that have increased expression of a number of M1 and inflammatory markers including CD86 (Downer et al., 2010), CD68 (Wong et al., 2005), MHC II (Frank et al., 2006, Godbout et al., 2005b, Henry et al., 2009), and toll-like receptors (Letiembre et al., 2007). A consequence of a more inflammatory microglial profile is an exaggerated inflammatory response following peripheral immune activation (Godbout et al., 2005b, Perry et al., 2003). In support of this notion, central (Abraham et al., 2008, Huang et al., 2008) or peripheral innate immune challenges (Chen et al., 2008, Godbout et al., 2005a, Henry et al., 2009, Wynne et al., 2010) lead to amplified and prolonged neuroinflammation (oxidative stress and cytokines) mediated, in part, by a hyperactive MHC II+ microglial population (Henry et al., 2009). An exaggerated M1 microglial response in aged mice is relevant because it is coupled with a myriad of complications including cognitive impairment (Barrientos et al., 2006, Barrientos et al., 2009, Chen et al., 2008), exaggerated and prolonged sickness behavior (Abraham et al., 2008, Barrientos et al., 2009, Godbout et al., 2005a, Huang et al., 2008), and protracted depressive-like behavior (Godbout et al., 2008) following an innate immune challenge (for reviews see Dantzer et al., 2008, Godbout and Johnson, 2009, Jurgens and Johnson, 2010). This is paralleled in clinical studies in which elderly patients with peripheral infections or other illnesses have an increased frequency of concomitant neurobehavioral complications including delirium (Lipowski, 1983, Mulsant et al., 1999) and depression (Alexopoulos, 2005, Godbout and Johnson, 2009, Koenig et al., 1988, Yirmiya et al., 2000) compared to younger adults with the same peripheral insults.
The reason aged mice have a reduced capacity to resolve amplified microglial activation after an immune challenge is unknown, but may be related to a reduced sensitivity to anti-inflammatory feedback by neuronal regulators (CD200, CX3CL1) (Lyons et al., 2007a, Wynne et al., 2010) and anti-inflammatory cytokines. Previous studies demonstrate that IL-10 and IL-4, two key anti-inflammatory and M2-promoting cytokines (Mantovani et al., 2004, Strle et al., 2001) are reduced within the brain of older rodents (Nolan et al., 2005, Szczepanik et al., 2001, Ye and Johnson, 2001). In the context of M2 activation profiles, IL-10 promotes an M2c (classical deactivation) profile and IL-4 promotes an M2a (alternative activation) profile in macrophages. Both M2a and M2c phenotypes reduce M1 cytokines and other inflammatory mediators (Mantovani et al., 2004, Mosser and Edwards, 2008). An M2b activation profile has also been shown with expression of both M1 and M2c markers. The degree to which these activation profiles are conserved in microglia is less understood. In our work, peripheral injection of LPS amplified mRNA and intracellular protein expression of both IL-1β and IL-10 in microglia from aged mice (Henry et al., 2009). Despite exaggerated IL-10 production by microglia from aged mice, neuroinflammation persisted and corresponded with prolonged sickness and depressive-like behaviors (Godbout et al., 2005b, Godbout et al., 2008). In addition, other reports indicate that decreased IL-4 in the brain of aged rats was associated with reduced long-term potentiation (LTP) (Maher et al., 2005), impaired neurogenesis in the hippocampus (Ziv et al., 2006), and increased brain inflammation (Maher et al., 2005, Nolan et al., 2005).
The purpose of this study was to determine the degree to which activated microglia from adult and aged mice were sensitive to anti-inflammatory effects of IL-10 and IL-4. Initial studies were completed using adult mice, BV-2, and primary microglia to determine microglial M1 and M2 profiles, expression of the receptors for IL-4 (IL-4Rα) and IL-10 (IL-10R1), and sensitivity to IL-10 and IL-4 following LPS. Collectively, these initial experiments demonstrated that LPS-activated microglia shifted towards an M2b phenotype, markedly upregulated IL-4Rα protein expression, and were re-directed towards a less inflammatory M2a profile following IL-4 post-treatment. These studies also indicated that IL-10R1 was not induced following LPS-associated activation and IL-10 had little effect in re-directing activated microglia towards a less inflammatory phenotype. In age comparisons, LPS injection induced an exaggerated M2b phenotype in microglia from aged mice compared to adults, but IL-4Rα surface expression was not increased. Moreover, when LPS-activated microglia were isolated and treated ex vivo with IL-4, only microglia from adult mice successfully transitioned from an M1 towards an M2 profile. Thus, failure to increase IL-4Rα surface expression on aged microglia was associated with a reduced sensitivity to the M2 promoting effects of IL-4.
Section snippets
Animals
Adult (3–4 month-old) male BALB/c mice were obtained from a breeding colony kept in barrier-reared conditions in a specific-pathogen-free facility at the Ohio State University. Mice were individually housed in polypropylene cages and maintained at 25 °C under a 12 h light/12 h dark cycle with ad libitum access to water and rodent chow. For age comparisons, male BALB/c mice (18–22 mo) were purchased from the National Institute on Aging Specific-Pathogen-Free Colony (maintained at Charles River
Peripheral LPS injection promoted an M1 and M2c mRNA profile in enriched microglia
We have previously reported that peripheral LPS injection causes amplified and prolonged microglial activation (Henry et al., 2009, Wynne et al., 2010) in aged BALB/c mice that corresponds with protracted sickness and depressive-like behaviors (Godbout et al., 2005b, Godbout et al., 2008). We hypothesize that exaggerated microglial activation in aged mice is related to a reduced sensitivity to anti-inflammatory cytokines that impair the transition from an M1 phenotype to an M2 phenotype. To
Discussion
Previous studies from our lab and others indicate that aged but otherwise healthy rodents have prolonged sickness behavior (Godbout et al., 2005b, Huang et al., 2008), cognitive impairment (Abraham and Johnson, 2009, Barrientos et al., 2006, Chen et al., 2008), and depressive-like behavior (Godbout et al., 2008) following peripheral or central immune challenge. These behavioral deficits correspond to exaggerated and protracted microglial activation in aged mice (Henry et al., 2009, Wynne et
Acknowledgements
This work is supported by NIH grant R01-AG-033028 to J.P.G. A.M.F is supported by a Howard Hughes Medical Institute (HHMI) Med into Grad scholarship. A.D. is supported by a Federal Work-Study Grant. The authors thank Dr. John Sheridan (OSU, Dept. of Oral Biology) for the use of a Becton-Dickinson FACSCaliber four color Cytometer and Dr. Ronald Glaser (OSU, Dept. of MVIMG) for the use of an Applied Biosystems PRISM 7300-sequence detection system.
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