Mini Review Mechanisms of post-ischemic inflammation
Inflammation and Regeneration

Vol.33 No.3

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150

Mini Review
Peroxiredoxin triggers cerebral post-ischemic
inflammation
Takashi Shichita1, 2), Ryota Sakaguchi1), Minako Ito1),＊)
Taisuke Kondo1), and Akihiko Yoshimura1,＊
1)

Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan
Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency,
Tokyo, Japan

2)

Post-ischemic inflammation is an essential step in the progression of ischemic stroke. However, it has not been sufficiently clarified how post-ischemic inflammation begins. Brain is asterile organ, but Toll-like receptor (TLR) 2 and TLR4 have a pivotal role in the initiation of inflammation. Some endogenous danger associated molecular patterns (DAMPs) are released frominjured brain cells. Among them, high mobility group box 1 (HMGB1) and peroxiredoxin (Prx)family proteins are key initiators of post-ischemic inflammation. HMGB1 induces blood brain
barrier breakdown at the hyperacute phase; on the other hand, Prxs directly activate infiltrating
macrophage through TLR2/4 and induce inflammatory cytokines production at the acute phase.
Thus, there is a time lag as well as functional differences between HMGB1 and Prxs. Novelneuroprotective treatment could be developed through more detailed elucidation of the regulation of post-ischemic inflammation.
Rec.12/10/2012, Acc.1/29/2013, pp150-155＊
Correspondence should be addressed to:
Akihiko Yoshimura, PhD, Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi,
Shinjuku-ku, Tokyo 160-8582, Japan. Phone: +81-3-5363-3483, Fax: +81-3-5360-1508, E-mail: yoshimura@a6.keio.jp

Key w
ords
wo

brain ischemia, inflammation, macrophage, TLR, DAMPs

Introduction

sis or occlusion of cerebral artery. The deprivation of oxy-

Stroke is a major cause of death and disability in Japan,

gen, glucose, and other nutrients caused by ischemia re-and about 70 % of all cases are ischemic stroke. Post-

sults in the failure to maintain the neuronal microenviron-ischemic inflammation is an essential step in ischemic brain

ment which consists of glial cells, endothelial cells, andinjury and is correlated with patient’s prognosis. More de-neurons. The dysfunction of cerebrovascular units leads

tailed understanding of inflammatory processes enables

to blood-brain barrier (BBB) breakdown and subsequent

us to develop a more effective treatment with a prolonged

inflammation caused by the infiltration of hematopoietic

therapeutic time window

1, 2)

.

immune cells3).

Brain infarction is a brain tissue death caused by insuffi-

Hematopoietic cells, such as macrophages and T cells

cient blood supply (ischemia) which is due to severe steno-

are implicated in post-ischemic inflammation and play aMini Review Mechanisms of post-ischemic inflammation
Inflammation and Regeneration

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151

role as effectors of innate immunity4). To prevent the pri-damages. Thus, HMGB1 is an essential DAMP in the hy-

mary and secondary progression of an infarct lesion, it isperacute phase of ischemic brain injury.

indispensable to clarify the detailed mechanism of post-

The number of infiltrating macrophage increases fromischemic inflammation evoked by these immune cells. De-

day 1 to 3 after stroke onset; on the other hand, the extra-

spite intensive studies, the complexity of the brain inflam-cellular release of HMGB1 seems too early to have signifi-

mation mechanisms has thus far remained insufficiently

cant effects on infiltrating macrophages directly12-14). It isclarified.

possible that another DAMP in the ischemic brain triggers
post-ischemic inflammation by activating immune cells and

The initiation of inflammatory process inischemic brain injury

such a molecule may be one of the ideal therapeutic target
for ischemic stroke.

In the acute phase of brain ischemia, infiltrating macroph-

We have investigated other potential DAMPs in brain

ages produce various cytokines and promote inflamma-

homogenate lysates and determined peroxiredoxin (Prx)tion. However, circulating intravascular blood cells do notfamily proteins as an essential initiator of cerebral post-produce inflammatory cytokines much. These hematopoi-

ischemic inflammation12). We have discovered that brain

etic immune cells should be activated by the infiltration into

homogenate lysate has an inflammatory cytokines-induc-the ischemic brain.

ing activity in cultured dendritic cells. Because such an

Toll-like receptor (TLR) is one of the most important mol-

activity was almost completely diminished by heat or pro-ecules for the activation of macrophages. Originally, TLR

teinase treatment, some specific proteins were presumed

functions as a pathogen sensor which recognizes the lipo-

to function as DAMPs. With various fractionation methods

protein, lipopolysaccharide, and nucleotides derived from

including sucrose density gradient centrifugation, we havebacteria and virus. It has been reported that TLR2 and TLR4

detected high activity to induce inflammatory cytokines inare implicated in post-ischemic inflammation, although there

15-25 kDa fractions. After further analysis by LC/MS and

5, 6)

.

the recombinant protein assay, we have identified Prx family

It remains clarified what molecule stimulates TLR in is-proteins (Prx1, Prx2, Prx5, and Prx6) as strong inducers ofchemic brain.

inflammatory cytokines in cultured dendritic cells.

are no pathogens in both the normal and ischemic brain

Recently, it has attracted attention that some endogenous
trating immune cells. Such endogenous molecules are

Peroxiredoxin as a previously unknown
DAMP

called danger-associated molecular patterns (DAMPs).

Compared with HMGB1, it may take more time for Prxs

Heat shock proteins (HSPs), β-amyloid (Aβ), hyaluronic

to function in the ischemic brain. Prxs expression is in-acid, formyl peptides, oxidized low-density lipoproteins

duced by ischemic stress and becomes evident 12 hours(oxLDL) have been considered as possible DAMPs in theafter stroke onset12). The highly expressed Prx is released

ischemic brain7-9). However, it has been insufficiently eluci-

into the extracellular compartment around injured brain cells

dated what molecule triggers post-ischemic inflammationand co-localizes with the membranes of infiltrating mac-in brain.

rophages. Such an extracellular release of Prxs directly

molecules released from the injured tissues activate infil-

activates infiltrating macrophages. In fact, the neutraliza-

Potential DAMPs in ischemic braintion of extracellular Prxs decreases the expression of in-Among these DAMPs, high mobility group box 1 (HMGB1)

flammatory cytokines in infiltrating inflammatory cells, andis an important DAMP10). HMGB1, which is localized in cell

thereby attenuates ischemic damage in mice. Interestingly,

nuclei in normal condition, translocates into the cytosol

an increased amount of Prx proteins in the extracellular

under ischemic condition, and is released into the extra-

fluid of the ischemic brain has been also reported in rats

cellular compartment. The extracellular release of HMGB1

and stroke patients15, 16). Thus, the extracellularly released

occurs within 2 to 4 hours after the stroke onset and in-Prxs from injured brain cells appears to activate infiltrating

11)

duces the breakdown of blood brain barrier (BBB) . The

macrophages and initiates post-ischemic inflammation.

administration of HMGB1 neutralizing antibody improves

Prxs have been well-known as ubiquitous anti-oxidant

the vascular permeability and attenuates ischemic brain

enzymes, which are more abundant in brain than in otherMini Review Mechanisms of post-ischemic inflammation
Inflammation and Regeneration

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Fig.1 Two functions of Prx, one inside and one outside brain cells
The expression of Prxs within brain cells is induced by ischemic stress. Intracellular Prxs contribute to the survival ofbrain cells by catabolizing reactive oxygen species (ROS).
When ischemic phenomena finally result in necrosis,
intracellularlly expressed Prxs are released into the extracellular compartment. Such extracellular Prxs function asstrong TLR2 and TLR4 stimulators (DAMPs) and activate
infiltrating macrophages in ischemic brain.

tissues. Prx proteins contain one (1-cysteine (1-Cys): Prx6)

through TLR pathway induces strong inflammatory re-or two (2-Cys: Prx1-5) cysteine residues to scavenge ROS

sponses. The clinical relevance of TLR2 and TLR4 in stroke17)

in cooperation with thioredoxin (Trx) . Intracellular induc-patients has been also demonstrated22).

tion of Prxs are observed in various brain tissue injuries

Both leukocytes and brain cells express TLRs and thisand are considered to diminish ROS and protect from tis-fact makes it difficult to fully understand the complexity ofsue damages18). However, another function of extracellu-

inflammatory mechanisms in ischemic brain. The function

larly released Prxs as DAMPs has not been reported. We

of TLRs on brain has not been sufficiently clarified. Brain

have demonstrated that Prx family members are strongly

cells, which are activated by ischemic injury probably via

induced inside the injured ischemic cells to improve their

TLRs, also contribute to the initiation of post-ischemic in-survival. Thereafter, they are released into the extracellu-

flammation by producing inflammatory mediators. TNF-α,

lar compartment once the cells are about to die (ischemic

IL-1β, nitric oxide, and MMPs which are produced fromnecrosis), and such an extracellular release of Prxs acti-

brain cells regulate the cerebrovascular permeability andvates infiltrating macrophages directly and triggers the pro-exaggerate brain edema23, 24). Microglia, resident macroph-duction of inflammatory cytokines. Thus, Prxs have two

age in the brain, is also activated via TLRs, and not only

opposing functions, one inside and one outside the brain

acts as an inflammatory mediator by producing neurotoxic

cells (Fig.1).

cytokines, but also produces neurotrophic factors for tis-

To investigate the common structure of Prxs for the in-sue repair25).

duction of inflammatory cytokines, we have developed

We have tried to investigate the function of TLRs in brain

mutant recombinant proteins of Prxs and elucidated thatcells by using bone marrow (BM) chimeric mice12). Although

the cysteine residues which are important for anti-oxidant

microglia, which are radiation-resistant, are a rapid effec-

activity do not function as DAMPs. On the other hand, if α3

tor in ischemic brain, TLR2- or TLR4-deficient microglia

helix region of Prxs are deleted, their cytokine-inducing

transferred with wild-type BM reveals no improvement in

activity is diminished . The α3 helix region of Prxs exists

ischemic brain injury26). Mice lacking MyD88, which is thein the relatively sequestered position on the molecular sur-adaptor protein required for almost all TLR signaling cas-

face and is located almost in the opposite side from thecades (other than TLR3), showed either no improvement

location of anti-oxidant activity, according to the previous

or exaggeration27). These results have indicated that the12). Targeting this region of Prxs,

effect of TLRs is dependent of the cell types in ischemica specific anti-inflammatory therapy may be developed forbrain and the timing of their activation. Due to such a com-ischemic stroke.

plexity, more detailed knowledge about TLRs function in

19-21)

crystal structure analysis

ischemic brain is needed for the development of the thera-

TLRs as DAMPs receptors

peutic potential of this strategy.

TLR2 and TLR4 have been recently reported to contribbrain injury. Indeed, HMGB1 and Prxs can stimulate TLR2

Inflammatory cytokines and infiltrating
immune cells

and TLR410, 12). The activation of macrophages and T cells

Macrophages are the main inflammatory effectors. The

ute to non-infectious sterile organ injury, including ischemic

Mini Review Mechanisms of post-ischemic inflammation
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MAY 2013

Fig.2 Post-ischemic inflammation is triggered by
DAMPs
At the hyperacute phase of brain ischemia (within 6 hours
after stroke onset), high mobility group box 1 (HMGB1) isreleased from injured brain cells and promotes BBB breakdown. Thereafter, circulating macrophages begin to infiltrate
into the ischemic brain during the acute phase of ischemia(12 to 24 hours after stroke onset). In this phase, the expression of Prxs is induced within the wounded brain cellsby ischemic stress for their survival. When Prxs are released
into the extracellular compartment from necrotic brain cells,
extracellularly released Prxs induce the various inflammatory cytokine expression from infiltrating macrophages
through TLR2 and TLR4. IL-1β and TNF-α directly promote neuronal cell death. On the other hand, IL-23 acts onγδT cells, which infiltrate during the delayed phase (morethan 24 hours after stroke onset) and induces the IL-17 production which enhances ischemic brain damages.

infiltration of macrophages becomes evident from 12 hours
to 24 hours after stroke onset and produces various in-

Conflict of interest
Noneflammatory cytokines, such as IL-1β and IL-23. These
cytokines induces the subsequent inflammation caused by
T cells (Fig.2).
T cells are also important effectors in the delayed phaseof brain ischemia. The number of infiltrating T cells increases over 24 hours after stroke onset and reaches thepeak around day 328, 29). T cells appear to be localized toReferences
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