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

https://doi.org/10.1016/j.bbi.2011.10.003Get rights and content

Abstract

In several models of aging, microglia become more inflammatory and reactive to immune challenges. For example, peripheral LPS injection causes exaggerated microglial activation associated with prolonged sickness and depressive-like behavior in aged BALB/c mice. Therefore, the purpose of this study was to determine the extent to which age-related amplified microglial activation was associated with reduced sensitivity to the anti-inflammatory and M2 promoting cytokines interleukin (IL)-10 and IL-4. In initial studies with adult mice, LPS induced a time-dependent increase in M1 and M2 mRNA profiles in microglia. Furthermore, peripheral LPS injection markedly increased surface expression of IL-4 receptor-alpha (IL-4Rα), but not IL-10 receptor-1 (IL-10R1) on microglia. In BV-2 cells, IL-4, but not IL-10, re-directed LPS-activated microglia towards an M2 phenotype. Based on these findings, comparisons of M1 and M2 activation profiles, induction of IL-4Rα, and sensitivity to IL-4 were determined in microglia from adult (3–4 mo) and aged (18–22 mo) mice. In aged microglia, LPS promoted an exaggerated and prolonged M1 and M2 profile compared to adults. Moreover, IL-4Rα protein was not increased on aged microglia following LPS injection. To determine the consequence of impaired IL-4Rα upregulation, adult and aged mice were injected with LPS and activated microglia were then isolated and treated ex vivo with IL-4. While ex vivo IL-4 induced an M2 profile in activated microglia from adult mice, activated microglia from aged mice retained a prominent M1 profile. These data indicate that activated microglia from aged mice are less sensitive to the anti-inflammatory and M2-promoting effects of IL-4.

Highlights

Microglia from aged mice injected with LPS failed to increase surface expression of IL-4Rα corresponding with a reduced sensitivity to the M2 promoting effects of IL-4.

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.

References (81)

  • E.J. Downer et al.

    A novel anti-inflammatory role of NCAM-derived mimetic peptide, FGL

    Neurobiol. Aging

    (2010)
  • M. Frank et al.

    MRNA up-regulation of MHC II and pivotal pro-inflammatory genes in normal brain aging

    Neurobiol. Aging

    (2006)
  • J.P. Godbout et al.

    Age and neuroinflammation: a lifetime of psychoneuroimmune consequences

    Immunol. Allergy Clin.

    (2009)
  • J.P. Godbout et al.

    α-Tocopherol reduces lipopolysaccharide-induced peroxide radical formation and interleukin-6 secretion in primary murine microglia and in brain

    J. Neuroimmunol.

    (2004)
  • J. Godbout et al.

    [alpha]-Tocopherol attenuates NF[kappa]B activation and pro-inflammatory cytokine production in brain and improves recovery from lipopolysaccharide-induced sickness behavior

    J. Neuroimmunol.

    (2005)
  • P.J. Haley

    Species differences in the structure and function of the immune system

    Toxicology

    (2003)
  • S.J. Haque et al.

    Protein-tyrosine phosphatase Shp-1 Is a negative regulator of IL-4- and IL-13-dependent signal transduction

    J. Biol. Chem.

    (1998)
  • C. Henry et al.

    Peripheral lipopolysaccharide (LPS) challenge promotes microglial hyperactivity in aged mice that is associated with exaggerated induction of both pro-inflammatory IL-1[beta] and anti-inflammatory IL-10 cytokines

    Brain Behav. Immun.

    (2009)
  • Y. Huang et al.

    Exaggerated sickness behavior and brain proinflammatory cytokine expression in aged mice in response to intracerebroventricular lipopolysaccharide

    Neurobiol. Aging

    (2008)
  • S. Ito et al.

    Interleukin-10 inhibits expression of both interferon-alpha and interferon-gamma induced genes by suppressing tyrosine phosphorylation of STAT1

    Blood

    (1999)
  • M. Letiembre et al.

    Innate immune receptor expression in normal brain aging

    Neuroscience

    (2007)
  • A. Mantovani et al.

    The chemokine system in diverse forms of macrophage activation and polarization

    Trends Immunol.

    (2004)
  • Y. Nolan et al.

    Role of interleukin-4 in regulation of age-related inflammatory changes in the hippocampus

    J. Biol. Chem.

    (2005)
  • P.G. Popovich et al.

    Manipulating neuroinflammatory reactions in the injured spinal cord: back to basics

    Trends Pharmacol. Sci.

    (2003)
  • A.F. Richwine et al.

    Cognitive deficits in interleukin-10-deficient mice after peripheral injection of lipopolysaccharide

    Brain Behav. Immun.

    (2009)
  • R. Sandhir et al.

    Exacerbated glial response in the aged mouse hippocampus following controlled cortical impact injury

    Exp. Neurol.

    (2008)
  • M. Schwartz et al.

    Protective autoimmunity and neuroprotection in inflammatory and noninflammatory neurodegenerative diseases

    J. Neurol. Sci.

    (2005)
  • A.M. Szczepanik et al.

    IL-4, IL-10 and IL-13 modulate A[beta](1–42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line

    J. Neuroimmunol.

    (2001)
  • A.M. Wong et al.

    Macrosialin increases during normal brain aging are attenuated by caloric restriction

    Neurosci. Lett.

    (2005)
  • A. Wynne et al.

    Protracted downregulation of CX3CR1 on microglia of aged mice after lipopolysaccharide challenge

    Brain Behav. Immun.

    (2010)
  • J. Abraham et al.

    Consuming a diet supplemented with resveratrol reduced infection-related neuroinflammation and deficits in working memory in aged mice

    Rejuvenation Res.

    (2009)
  • J.B. Allen et al.

    Suppression of monocyte function and differential regulation of IL-1 and IL-1ra by IL-4 contribute to resolution of experimental arthritis

    J. Immunol.

    (1993)
  • W.S. Begolka et al.

    Differential expression of inflammatory cytokines parallels progression of central nervous system pathology in two clinically distinct models of multiple sclerosis

    J. Immunol.

    (1998)
  • E. Bell

    MicroRNAs and the immune response

    Nat. Rev. Immunol.

    (2007)
  • E.N. Benveniste

    Role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis

    J. Mol. Med.

    (1997)
  • C.C. Chao et al.

    Neuroprotective role of IL-4 against activated microglia

    J. Immunol.

    (1993)
  • W. Cho et al.

    Membrane-protein interactions in cell signaling and membrane trafficking

    Annu. Rev. Biophys. Biomol. Struct.

    (2005)
  • R. Dantzer et al.

    From inflammation to sickness and depression: when the immune system subjugates the brain

    Nat. Rev. Neurosci.

    (2008)
  • N.C. Derecki et al.

    Regulation of learning and memory by meningeal immunity: a key role for IL-4

    J. Exp. Med.

    (2010)
  • K. Frei et al.

    Antigen presentation in the central nervous system. The inhibitory effect of IL-10 on MHC class II expression and production of cytokines depends on the inducing signals and the type of cell analyzed

    J. Immunol.

    (1994)
  • Cited by (0)

    View full text