Pathological Pain and the Neuroimmune Interface - Nri3621

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Pain has been associatewith the immune system since

the time of Celsus, who ientifiepain (dolor) as a cari-

nal sign of acute inflammation. Though acute pain mayoccur in relative isolation from broaer synromes, it

was ientifiein the mi-twentieth century as one of a

constellation of aaptive behaviours, collectively termethe sickness respo nse.

The iea that pain animmunity might be associ-

atebeyonan acute response first arose from clinicalobservations in the 1970s that patients with chronicpain exhibiteother symptoms, in aition to hyper-

algesia , that parallel the classical systemic sicknessresponse — incluing lethargy,epression ananxiety.The concomitance of sickness behaviours with chronic

pain is therefore suggestive of unerlying immuneactivity. Efforts to ientify the origin annature of

the immune meiators involvesoon followe, lea-

ing to the iscovery that elevateperipheral levelsof interleukin-1β (IL-1β) both inucehyperalgesia

perse anmeiatesickness-inucehyperalgesia1,2.Although periphera l sen sitization of pain fibres at local

tissue sites of inflammation has a key role in heighten-ing pain from those regions, these peripheral observa-tions were soon extenewith theiscovery of a central

nervous system (CNS) mechanism of action for IL-1β another cytokines3,4. This research was accompanieby a growing appreciation that the release of cytokinesin the CNS, anthe behavioural effects that occursubsequent to this, might not be solelyepenent on

peripheral signalling (for example, sensory vagal nerve

stimulation an /o r cytokin e release from periph-

eral immune cells), but coulalso be ue to the local

release of cytokines by glia resiing within the CNS5.Glia were first implicatewhen increaseexpression of

astrocyte- anmicroglia-associateactivation markers

was observein the lumbar spinal cors of rats withperipheral nerve injury6,7. The contribut ion of spinal

glia antheir pro-inflammatory proucts to allodynia

anhyperalgesia was then emonstratewhen spi-nal intrathecal injection of inhibitors of gliosis or IL-1receptor antagonist (IL-1Ra) was founto attenuate

the pain behaviours that are associatewith iversepainmoels8–11.

Since these seminal observations, there have been

major avances in unerstaning how glia animmunecells in the CNS responto pain stimuli ancontrib-

ute to pathological pain. In this Review, weetail these

avances in the context of the neuroimmune interface ,which is integral to an appreciation of central immune

signalling. It shoulbe notethat neuroimmune inter-actions are crucial to inflammation-inuceperiph-

eral sensitization anpathophysiological changes at thesite of peripheral nerve injury. However, this Reviewis restricteto the composition of the neuroimmune

interface in the CNS, anhow its constituent cellsbecome reactive ancontribute to chronic pain. The

protective role of immune signalling in neuropathicpain, together with methos to pharm acologicallytarget the neuroimmune interface for superior pain

control are also iscusse.

Celsus

Aulus Cornelius Celsus was

afirst century Roman

encyclopaedist who gathered

extensive writings from the

Greek emp ire and t ranslated

them into Latin. In his great

work De Medicina, he

characterized the four cardinal

signs of inflammation: heat,

pain, swelling and redness.

Pathological pain and theneuroimmune interfacePeter M.Grace 1 ,2 , Ma rk R.Hutchinson1 ,2 , Ste ven F.Maier1 a nd Linda R.Watkins 1

Abstract | Reciprocal signalling between immunocompetent cells in the central nervous

system (CNS) has emerged as a key phenomenon underpinning pathological and chronic

pain mechanisms. Neuronal excitability can be powerfully enhanced both by classical

neurotransmitters derived from neurons, and by immune mediators released from

CNS-resident microglia and astrocytes, and from infilt rating cells such as Tcells. In thisReview, we discuss the current understanding of the contribution of central immune

mechanisms to pathological pain, and how the heterogeneous immune functions of different

cells in the CNS could be harnessed to develop new therapeutics for pain control. Given the

prevalence of chronic pain and the incomplete efficacy of current drugs — which focus on

suppressing aberrant neuronal activity — new strategies to manipulate neuroimmune pain

transmission hold considerable promise.

1Department of Psychology

and Neuroscience, and

TheCenter for Neuroscience,

University of Colorad o

Boulder, Boulder

803 09–0 345 , USA.2School of Medical Sciences,

University of Adelaide,

Adelaide 50 05 , Australia.

Correspondence to P.M.G.

e-mail:

[email protected]

doi:10.1038/nri3621

Published online

28 February 2014

REVIEWS

NATURE REVIEWS | IMMUNOLOGY VOLUME 14 | APRIL 2014 | 217



Sickness resp onse

A defence mechanism

triggered by the recognition

of anything foreign to the host.

An organized constellation

of responses initiate d by

the immune system but

co-ordinate d a nd p artially

created by the brain,

includingphysiological

responses (for example,

fever,increased s leep,

hyperalgesia and allodynia),behavioural responses (for

example, decreased social

interaction, sexual activity,

an dfood and water intake),

and hormonal responses

(increased release of classic

hypothalamo–pituitary–

adrenal and sympathetic

hormones).

Hyperalgesia

Increased pain from a

stimulusthat normally

provokes p ain. A form o f

nociceptive hypersensitivity.

Physiological pain processingPain (either nociceptive pain or inflammatory pain) is pro-tective anaaptive, warning the iniviual to escape

the pain-inucing stimulus anto protect the injure

tissue site uring healing. The basic scientific uner-staning of sensory processing anmoulation hasbeen ramatically improveby the evelopment of

pain assays that recreate some elements of clinical painsynromes (BOX1 ).

Painful stimuli (for example, mechanical, thermal

anchemical) are initially transuceinto neuronalelectrical activity anconuctefrom the peripheral

stimulus site to the CNS along a series of well-character-

izeperipheral nociceptive sensory neurons (first-orderprimary afferent neurons). The nociceptive signal is then

transmitteat central synapses through the release of avariety of neurotransmitters that have the potential to

excite second-order nociceptive projection neurons in thespinal dorsal horn or hindbrain (FIG.1). This process ofnoci-

ception can occur through several mechanisms involving

glutamate anneuropepties (for example, substance Por calcitonin gene-relatepeptie (CGRP)). Glutamate

activates postsynaptic glutamate AMPA (α-amino-3-hyroxy-5-methyl-4-isoxazole proprionic aci) ankainate receptors on secon-orer nociceptive projec-

tion neurons. Interestingly, these receptor systems are

not all engageequally in response to ifferent types of

pain. Moification of the nociceptive signal can occur

at the level of the spinal corthrough activation of localGABAergic (that prouce γ-aminobutyric aci) angly-

cinergic inhibitory interneurons.

Secon-orer nociceptive projection neurons projectto supra-spinal sites, which further project to cortical

ansubcortical regions via third-order neurons, enabling

the encoing anperception of the multiimensionalpain experience. Secon-orer nociceptive projectionneurons can be further moulatethrough the acti-

vation of escening serotonergic annorarenergicprojections to the spinal cor, which can influence theresponse to anperception of pain (FIG.1). For recent

reviews that escribe these processes in etail, seeREFS12,13 .

Pathological pain processingPain can extenbeyonits protective usefulness, last-

ing for a perioof weeks to years — well beyontheresolution of the initial injury. In this case, pain is mala-

aptive anis believeto result from abnormal func-tioning of the nervous system. In the UniteStates alone,such persistent pain is estimateto affect ~37% of the

population; representing an economic buren of up toUS$635billion per year14. Perhaps the most well-stuieexample is neuropathic pain, which is relatively commonanarises as a irect consequence of a lesion or iseaseaffecting the somatosensorysystem.

Intense, repeateansustaineactivity of first-orer neurons elicits well-characterizechanges inneuronal anbiochemical processing at centr al syn-

apses anescening projections, transitioning these

sites into a pain-facilitatory state12,13. In the spinalorsal horn , these changes are collectively known as

centra l sensitization anwindup . These processes involve

the phosphorylation of a range of receptors, incluingNMDA (N-methyl-d-aspartate), AMPA an /or kainate

receptors, which increases synaptic efficacy by altering

channel opening time, increasing burst firing, remov-ing the Mg2+-meiatechannel blockae at the NMDAreceptor, anpromoting trafficking of receptors to

the synaptic membrane15. Uner such circumstances,low-thresholsensory (Aβ) fibres that are activatebyinnocuous stimuli are able to activate high-threshol

nociceptive neurons, owing to either a strengtheneexcitatory input or the lowereexcitation thresholof

nociceptive projection neurons15. Central sensitization

is maintaineby ongoing stimuli, such as spontaneousactivity arising from sensory fibres or locally releaseimmune meiators (see below), which are responsi-ble for the persistence anspreaof neuropathic pain

beyonthe initial injurysite.Although the importance of these neuronal pain

facilitation mechanisms is unispute, not all symptoms

anmechanisms can be explainesolely by such neu-ronal mechanisms. These neuronal pathophysiological

mechanisms are being supplementeby an apprecia-tion for the role of central immune signalling, such thatneuropathic pain is now consiereas a neuroimmune

isorer16.

Box 1 | Precl ini cal pain assays and measures

The development of preclinical pain assays — the experimental procedures by which

pain is induced in the subject — has typically occurred over several distinct phases154.

Init ial studies of pain used acute assays, involving t he application of a noxious stimulus

(which may be thermal, mechanical, electr ical or chemical) to an accessible body part

(usually the hindpaws, tail or abdomen); or inflammatory assays that directly activate

nociceptors (for example, treatment with formalin or capsaicin) or the immune system

(for example, treatment with complete Freund’s adjuvant or carrageenan). In orderto study the unique pathophysiological mechanisms of neuropathic pain that arise

owing to dif fering adaptations resulting from injury to nervous — compared wit h

other somatic — tissues, as well as increased pain duration, three main assays were

developed: first, chronic constriction injury, in which loose ligatures are tied around the

sciatic nerve to produce damage to some of the axons by inducing swelling and then

strangulation155; second, partial sciatic nerve ligation, in which a tight ligature is applied

through approximately one-half of t he proximal sciatic nerve156; and finally, spinal nerve

ligation, involving tight ligation of the lumbar spinal nerves (L4 and L5), close to the

dorsal root ganglion157. These assays have undergone continual development, and have

been adapted to study orofacial pain and heterogeneous pain thresholds154,158. The

recognition that existing assays can model ext remely rare pain syndromes, but lack face

validit y for t he more common pain syndromes, has led to more direct attempts to mimic

the pain associated with specific disease states, often by attempting t o induce the

disease, injury or the physiological state it self (for example, chemotherapy-induced

neuropathic pain or multiple sclerosis159,160)154.Furthermore, modern studies of chronic pain use acute assays to quantify

hypersensit ivit y, which is the most common preclinical end point. The pain measures

typically used are evoked spinal reflexes (for example, the von Frey test for mechanical

allodynia and the Hargreaves test for thermal hyperalgesia), spino-bulbospinal reflexes

(for example, jumping or abdominal stretching), or simple innate behaviours (for

example, licking, guarding or vocalization). As part of the general crit ique of pain

models, the validit y of current pain measures is presently being re-examined, and

alternative measures are being developed to reflect supraspinal pain processing,

such as operant measures (for example, conditioned place aversion161), spontaneously-

emitted behaviours (for example, facial grimacing or suppressed voluntary wheel

running162,163), as well as complex states affected by chronic pain (for example,

altered social interactions or sleep disruptions164)154,165.

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218 | APRIL 2014 | VOLUME 14 www.nature.com/reviews/immunol





oglial cell

T cell

CNSparenchyma

Systemiccirculation

P2X4R7

P2Y13RP2Y12R

TLR2 oTLR4

Fn

IL-1R1

CCC

CCR7

CX3CR1

MMP2,MMP9

IL-1β orIL-18

TNF,BDNF

TLR4

CCR2 orCX3CR1

TN

CCR2

CX3CR1

CCR2

LFA1

ICAM1

REVIEWS




Gliosis

The transition from a role

inphysiological maintenance or

surveillance to a reactive

phenotype that is characterized

by changes incell number,

morphology, phenotype,

motility, protein expression

and by the release o f

immunoregulatory prod ucts.

Neuroimmune interface

Proposed to de scribe the

bidirectional, modulatory

signalling between immune

cells and neurons. We a rgue

that su ch interfaces are formed

in the central nervous system

and can exp lain the sensoryada ptations unde rlying

pathological pain.

Central immune signalling

The process and

consequencesof immune

mediator release byreactive

immunocompetent cells in

the cen tral nervous system.

Nociceptive pain

Physiological pain produced

byintense noxious stimuli

thatactivate high-threshold

nociceptor neurons.

anregional heterogeneity throughout the parenchyma,presumably to coorinate iverse responses to insult18.

Uner a basal surveillance state, the cytoarchitecture

of microglia allows them to continuously sample theextracellular space for perturbations19. Transition to a

state of reactive gliosis involves changes in cell number,

morphology, phenotype anmotility, the expressionof membrane-bounanintracellular signalling pro-

teins (for example, mitogen-activateprotein kinases

(MAPKs)), anthe release of immunoregulatory pro-ucts, such as cytokines anchemokines. A commonmarker of astrogliosis is increaseexpression of glial

fibrillary aciic protein (GFAP), which is inicative ofalteremorphology. Microgliosis is often correlatewithincreaseexpression of CD11b anallograft inflamma-

tory factor1 (AIF1; also known as IBA1)20,21. It shoulbe notethat the functional relationship between the

expression of such markers annociceptive hypersensi-

tivity is currently unclear, anin certain circumstancesthere seems to be no connection between thetwo22.

Enothelial cells responto chemokines such asCC-chemokine ligan2 (CCL2; also known as MCP1)

anCX3C-chemokine ligan1 (CX

3CL1; also known

as fractalkine) by increasing expression of receptorsantethering proteins that facilitate transenothelial

migration of monocytes anTcells across the bloo–CNS barrier. CCL2 anCX

3CL1 also attract monocytes

anTcells to the CNS, where they interact with localimmunocompetent cells to facilitate the propagationanifferentiation of the immune response, which

eventually leas to moification of neuronal activity23,24.

The presence of specific Tcell subpopulations at the

neuroimmune interface is not well establisheanhas

primarily been efineon the basis of cytokine expres-sion. For example, the respective T helper 1 (T

H1), T

H2

anTH17-associatecytokines interferon-γ (IFNγ), IL-4

anIL-17 have been etectein roent lumbar spinalcors after peripheral nerve injury or inflammation25–28.

A specific role for regulatory T (TReg

) cells at the neuro-

immune interface has not yet been establishe, butsystemic expansion of T

Reg cells was founto attenuate

nerve injury-inucenociceptive hypersensitivity29.

Although not systematically characterize, the natureof the precipitating injury or insult is likely to influencethe cell populations that are recruiteto the spinal or-

sal horn. For example, pain is associatewith both spi-nal corinjury anperipheral ner ve injury. However,

infiltration of neutrophils into the spinal coris onlyassociatewith spinal corinjury, which coulbe ueto ifferential expression of cell ahesion molecules30,31.

Interestingly, nociceptive threshols have been suc-cessfully preicteon the basis of peripheral immune

cell activity in preclinical stuies, anin both healthyvolunteers anpatients with chronic pain 32–35. Finally,neurons not only responto neurotransmitterserive

from other neurons, but also express a wie range ofconstitutive aninucible classical immune signalling

receptors anligans, anare capable of moulatinglocal immune activity via both contact-epenent ancontact-inepenent mechanisms36,37.

Transition of immunocompetent cells in the spinalorsal horn to a reactive phenotype has been implicatein neuropathic pain8–11,23,38–42. Consierable investiga-

tion has aressekey questions of how such central

immune signalling is initiateafter a peripheral nerveinjury, anthe mechanisms by which it contributes to

central sensitization.

The neuroimmune interf ace in pathological painFollowing peripheral nerve injury, high-threshol

activity or amage of first-orer neurons initiates cen-tral immune signalling at the level of innervation43–45.Although astrocytes anTcells may respon to

neuronal signals, microglia have been ientifieas thefirst responers to a host of neuronally-erivemeia-tors, incluing matr ix metalloproteinase 9 (MMP9),

neuregulin 1, chemokines, ATP anenogenous angersignals (amage-associatemolecular patterns (DAMPs;

also known as alarmins))46,47. Upon etection of such

signals, microglia transition into a state of reactive gliosis.Peripheral immune cell infiltration anastrogliosis soon

follow, owing to the release of cytokines, chemokines,DAMPs aninflammatory meiators from reactive

microglia23,42 (FIG.2).

Chemokines, ATP and initiation of central immune sig-

nalling. Following the transuction of noxious stimuli,ATP anchemokines — such as CCL2, CX

3CL1 an

CCL21 (also known as SLC an6Ckine) — are releasefrom central terminals in the spinal orsal horn, aninuce signalling via G protein-couple receptors

(GPCRs) anligan-gateion channels. The expression

Figure 2 | Init iat ion of central immune signall ing. Damage to or high-thresholdactivation of first-order neurons by noxious stimuli induces the release of ATP,CC-chemokine ligand 2 (CCL2), CCL21 and neuregulin 1 (NRG1), as well as endogenousdanger signals to initiate central immune signalling in the dorsal horn of the spinal cord.CCL2 is released and rapidly upregulated by neurons following depolarization, andsignals via its cognate receptor CC-chemokine receptor 2 (CCR2) on microglia.CX

3C-chemokine ligand 1 (CX

3CL1) is liberated from the neuronal membrane by matrix

metalloproteinase 9 (MMP9) and MMP2 and by microglial cell-derived cathepsin S (CatS)

and signals via CX3C-chemokine receptor 1 (CX3CR1), which is expressed by microglia.CCR2 and CX3CR1 signalling also induces the expression of integrins (such as

intercellular adhesion molecule 1 (ICAM1) and platelet endothelial cell adhesionmolecule (PECAM1)) by endothelial cells, allowing the transendothelial migration ofTcells. Neuronal cell release of CCL21 stimulates local microglia via CCR7, andinfilt rating Tcells are activated via CXCR3. ATP induces microgliosis via the purinergicreceptors P2X

4R, P2X

7R, P2Y

12R and P2Y

13R. Microglia express Toll-like receptor 2 (TLR2)

and TLR4 leading to activation of the MYD88 pathway after detection of endogenousdanger signals, such as heat shock protein 60 (HSP60), HSP90, high mobility group box 1(HMGB1) and fibronectin. Following the detection of such signals, many int racellularpathways are recruited including SRC family kinases (SRC, LYN and FYN), MAPKs(extracellular signal-regulated kinase (ERK), p38 and JUN N-terminal kinase (JNK))and the inflammasome. This leads to phenotypic changes, increased cell motility andproli feration, altered receptor expression, the activation of transcript ion factors,such as nuclear factor-κ B (NF-κ B), and the production of inflammatory mediators

(such as interleukin-1β, (IL-1β), IL-18, tumour necrosis factor (TNF) and brain-derivedneurotrophic factor (BDNF)). Interleukin-1 receptor (IL-1R1) signalling reduces microglialcell expression of G protein-coupled receptor kinase 2 (GRK2), which is a negativeregulator of G protein-coupled receptors (GPCRs), including chemokine receptors.This leads to sustained GPCR signalling, and the exaggerated and prolonged productionof pro-inflammatory mediators. The release of soluble mediators also provides afeedback mechanism by which further immune cells are activated.

◀

REVIEWS




Inflammatory pain

Occurs in response to tissue

injury and the subsequent

inflammatory response.

First-order primary

afferent neurons

Sensory neurons the cell

bodies of which lie in the

trigeminal or spinal dorsal

root ganglia, with projections

that transmit nociceptive

signals from the periphery

(the p eripheral terminal) to

the spinal cord (the central

terminal).

Central synapse s

The synapses between

first-order neurons, whichformthe p resynaptic terminal,

and nociceptive projection

neurons, which form the

postsynap tic terminal.

Second-order nociceptive

projection neuron s

Neurons p rojecting from the

medullary dorsal horn or

the superficial laminae of the

spinal dorsal horn to the

brainstem or th alamic nuclei.

Spinal dorsal horn

Two longitu dina l sub divisions

of grey matter in the p osterior

part of the spinal cord that

receive terminals from afferent

fibres originating from each

side of the body that encode

several types of sensory

informa tion, including

nociception.

Hindbrain

Also known as the

rhombencephalon. An area

rostral to the spinal cord that

gives rise to the cerebellum,

pons and medulla.

Nociception

The neural process o fencodingnoxious mechanical,

thermal and /or chemical

stimuli that occurs in afferent

fibres, which send signals to

the cen tral nervous system.

Pain is expressed as the

complex emotional and

beha vioural response to the

central integration of

nociceptive signals.

Third-order ne urons

Sensory ne uronal projections

originat ing from th alamic

nuclei.

of CCL2 anCX3CL1 is not only inucible, but these

chemokines can also be constitutively expresseby first-

orer neurons48–52. Genetic or pharmacological manipu-lation of the receptors for CCL2 anCX

3CL1 attenuates

microgliosis anthe ensuing evelopment of nocicep-

tive hypersensitivity after peripheral nerve injury24,48–53.CX

3CL1 may be a seconary signal to microgliosis, as

cleavage of this chemokine from neuronal membranes

typically requires the release of cathepsin S (a lysosomalprotease), which is prouceby P2X

7 receptor (P2X

7R)-

stimulatemicroglia51,54. MMP9 anMMP2 prouce

by neurons anastrocytes may also promote CX3CL1

liberation55. Interestingly, the pro-inflammatory role ofCX

3CL1 has largely beenefinewithin the pain fiel, as

this neuronally tetherechemokine is commonly vieweas a homeostatic regulator that maintains a ‘surveillance’

phenotype in microglial cells. However, it has been pos-itethat CX

3CL1 can haveifferent functions in its teth-

ereancleavestates56. CCL21 has also been implicatein nociceptive hypersensitivity. It is releasefrom thecentral terminals of injureneurons, anstimulates an

attracts local microglia aninfiltrating Tcells57,58.Resient microglia also express a range of ionotropic

anmetabotropic purinergic receptors — P2X anP2Y

purinoceptors, respectively — that shape microgliosis59.Hence, neuronal release of ATP, either as a neurotransmit-

ter or as a consequence of cellamage, is a crucial molecu-lar substrate for early microgliosis60,61. P2X

4R anP2X

7R

have been implicatein neuropathic pain by experiments

that blockereceptor function pharmacologically anbygenetic ablation22,40,62–64. However, certain evelopmentalfactors shoulbe consierewhen interpreting knock-

out ata anhypo-functional variants. For example,

nitric oxie prouction by enothelial cells is impaireinP2X

4

R-eficient mice65. This makes itifficult to interpret

the irect effects of P2X4R activation on microglia using

these animals, asisruption of enothelial cell functionwill perturb the cellular milieu in the CNS after periph-

eral nerve injury. Furthermore, some P2X7R-eficient

mouse strains express a splice variant, which allows themto retain P2X

7R function66. Intrathecal aoptive transfer of

ATP-stimulatemicroglia into naive rats, which inuce

robust alloynia, showethat these effects are local-izeto microglia39,40. Furthermore, activation of P2X

7R,

expresseby microglia, inucethe release of IL-1β an

cathepsin S, which are key contributors to nociceptivehypersensitivity54,67. The evelopment of alloynia also

correlates temporally with an increase in spinal micro-

glial cell expression of P2X4R22,40. P2Y12receptor (P2Y12R)

anP2Y13

R expression by microglia rapily increases after

peripheral nerve injury, anpharmacological or geneticisruption of signalling through these receptors prevents

the evelopment of alloynia68,69. These ata proviecompelling evience that P2 receptor activation is crucialto microgliosis anthe aetiology of neuropathicpain.

Toll-like receptors and the initiation of central immune

signalling. Damage first-orer sensory neuronsmay also release extracellular matrix molecules anDAMPs, which are etecteby Toll-like receptors

(TLRs) expresseby immunocompetent cells within the

CNS70,71. TLRs responto iverse invaing pathogens

anDAMPs, anrely on receptor imerization, such as

that of the co-receptor CD14 with myeloiifferentia-tion protein 2 (MD2; also known as LY96), to achieve

specificity in agonist recognition71. Spinal DAMPs

implicatein roent pain moels inclue high mobilitygroup box 1 (HMGB1), fibronectin anthe heat shock

proteins HSP60 anHSP90. Neuronal HMGB1 expres-

sion is increasein the spinal corafter peripheral nerveinjury, antemporally correlates with the evelopmentof alloynia in bone cancer aniabetic neuropathy

assays72–74. Such pain is attenuateby intrathecal aminis-tration of an HMGB1-specific neutralizing antiboy73,74.Fibronectin has been shown to upregulate spinal micro-

glial cell expression of P2X4R59. After peripheral nerve

injury, HSP60 is upregulatein the spinal coranintrathecal HSP90 inhibition reverses alloynia75,76.

To ate, only TLR2 anTLR4 have been implicatein gliosis after peripheral nerve injury. IncreaseTLR4

expression correlates temporally with the evelopmentof nociceptive hypersensitivity after peripheral nerve

injury77. In aition, spinal expression of CD11b anGFAP is ecreasein nerve-injuremice with geneticisruption of Tlr4 or Cd14, which is inicative of attenu-

ategliosis in these animals72,77,78. This is not simply a con-sequence ofisrupteTLR4 signalling at the site of nerve

injury, as nerve injury-inucealloynia can be preventeor reverseby intrathecal aministration of TLR4 antago-nists79,80. Furthermore, gliosis annociceptive hypersensi-

tivity is attenuatein nerve-injureTLR2-eficient mice,anpro-inflammatory cytokine expression is abolisheinTLR2-eficient microglia treatewith conitionemeia

from amagesensory neurons43. Activation of alterna-

tive immune signalling pathways in the absence of TLRsshoulalso be consiere. For example, comparewith

wil-type controls, TLR4-eficient mice have elevate

levels of TH1- anT

H2-type cytokines uring infection81.

Furthermore, TLR4-eficient mice stillevelop alloynia,

although unlike in wil-type mice it can no longer be

inhibiteby IL-1Ra82. These reports inicate that cau-tion must be exercisewhen interpreting the influenceof TLRs on nociception when the conclusions are solely

rawn from knockoutata.Provocative ata also suggest that neurons may be

capable of irectly sensing DAMPs, owing to the expres-

sion of a wie range of TLRs anTLR aaptor proteinsby primary sensory afferents, such as trigeminal sensory

neurons andorsal root ganglia (DRG) neurons in both

humans anroents (for a review, see REF.71 ). At present,expression of TLRs in spinal orsal horn secon-orer

projection neurons remains to be shown. Neuronal TLRexpression has not only beenemonstrateat the mRNA

level anthrough the use of antiboies (which currentlylack suitable specificity, making antiboy-baseetec-tion suspect), but also by bining assays anmeasures

of function, such as patch clamp electrophysiology71,83,84.In DRG neurons, TLR3 expression increaseafter injury,

anactivation of TLR4, TLR7 anTLR9 in DRG neuronseliciteintracellular calcium accumulation, inwarcur-rents, sensitization of transient receptor potential cation

channel subfamily V member 1 (TRPV1), anprouction

REVIEWS




Central sensitization

A period o f facilitate d

transmission in nociceptive

projection neurons th at is

characterized by a decreased

activation threshold and an

increase d responsiveness to

nocicep tive stimuli.

Windup

Cumulative increases in

memb rane dep olarization

elicited by repeated C-fibre

stimulation.

Masseter

A facial muscle that has a

ma jor role in chewing.

of IL-1β, CCL5, CXCL10 anPGE2 (REFS71 , 83 , 84 ).

Although neuronal TLR7 has been shown to signal

through MYD88, the intracellular signalling events thatare ownstream of other neuronal TLRs, as well as their

consequences for action potential generation, have not yet

been fully characterize85. However, it is clear that the TLRsignalling pathways requireby innate immune cells are

not necessarily ientical to those of neuronal TLRs. For

example, in mice, DRG neurons require myeloiiffer-entiation factor 1 (MD1; also known as LY86) anCD14for functional TLR4 signalling, whereas TLR4 signalling

by innate immune cells requires MD2 anCD14 (REF.86).

Intra cellular signalling pathways underlying gliosis.

Given the iverse array of neuronal animmune cell-

erivesignals escribeabove, it is no surprise that

a wie range of intracellular signalling pathways canbe activate, leaing to gliosis anthe prouction ofimmune meiators41. MAPK signalling pathways (such

as those involving extracellular signal-regulatekinase(ERK), p38 anJUN N-terminal kinases (JNK)) in

glia have an important role in intracellular signalling,

leaing to the activation of transcription factors, such

as nuclear factor-κ B (NF-κ B), that promote the expres-

sion of a wie array of genes encoing pro-inflammatoryproucts (such as cytokines anbrain-derived neurotrophic

factor (BDNF)). In various moels of peripheral pain,

spinal microglia show increasephosphorylation of SRCfamily kinases (namely, SRC, LYN anFYN), which are

upstream of ERK28,87,88. Furthermore, specific inhibition of

SRC family kinases in microglia attenuates nerve injury-inucealloynia by preventing long-term potentiationin spinal orsal C-fibres88,89. Aaptations in intracellular

signalling may also contribute to the tr ansition fromacute to chronic pain (BOX2 ).

Pain enhancement at the neuroimmune interfaceGlia animmune cells exert their influence on neural

pain processing circuitry via soluble meiators that arereleasefor months after injury as a consequence ofgliosis90. A recent stuy has also raisethe intr iguing

possibility that neurons may be autonomous ‘immunesensors’, acting inepenently of glial animmune cell

influences. Pain inuceby Staphylococcus aureus wasshown not to be initiateby an immune cell intermei-ary, but rather by irect activation of first-orer noci-

ceptive neurons by bacterially eriveN-formylatepepties signalling at formyl peptie receptors, as well as

byα-haemolysin91. Nonetheless, meiatorserivefromglia animmune cells iffuse anbinto receptors onpresynaptic anpostsynaptic terminals in the spinal or-

sal horn to moulate excitatory aninhibitory synaptictransmission. This results in nociceptive hypersensitiv-ity that is characteristic of central sensitization (FIG.3).

Notably, comparewith classical neurotransmitters,

immune meiators can moulate spinal corsynaptictransmission at substantially lower concentrations41.

Enhanced excitatory synaptic transmission. Immunemeiators, such as tumour necrosis factor (TNF),

IL-1β, IFNγ, CCL2 anreactive oxygen species (ROS)

can irectly moulate excitatory synaptic transmissionat central terminals, principally by enhancing gluta-mate release92–96. Such effects are meiateby a func-

tional coupling between IL-1 receptors anpresynapticNMDA receptors, anpresynaptic TRPV1 antransientreceptor potential cation channel subfamily A member 1

(TRPA1) activation that leas to Ca2+-epenent gluta-mate release95–98. Immune meiators such as TNF, IL-1β,

IL-17, prostaglanin E2 (PGE2), CCL2 anCXCL1 also

irectly sensitize postsynaptic terminals of central syn-apses by several key mechanisms. These inclue the

increasetrafficking ansurface expression of AMPAreceptors (incluing those permeable to Ca2+), which

reners neurons vulnerable to excitotoxicity, as well asthe phosphorylation of the NR1 anNR2A or NR2Bsubunits of the NMDA receptor27,99–105. Excitatory syn-

aptic transmission is also inirectly enhancefollowingastrogliosis, as the spinal astrocyte glutamate transport-

ers excitatory amino acitransporter 1 (EAAT1) anEAAT2 are persistently ownregulateafter peripheralnerve injury, leaing to excitotoxicity annociceptive

hypersensitivity106,107.

Box 2 | Acute to chronic pain t ransit ion and t he neuroimmune interf ace

The neurobiological mechanisms underlying the transit ion f rom adaptive acute pain

to maladaptive chronic pain are not yet well understood, as most people recover

from a lesion or disease affect ing the somatosensory system without going on t o

develop chronic neuropathic pain. For example, only ~9% of pat ients (uncorrected

for age) with acute herpes zoster virus infect ion continue to experience pain 1year

after the initial infection166. This raises an intriguing question: what makes these

patient subsets different?

One hypothesis is that unregulated gliosis may sustain central sensit ization. For

instance, G protein-coupled receptor kinase 2 (GRK2; also known asβ-adrenergic

receptor kinase 1) is an enzymatic regulator of t he homologous desensit ization of many

G protein-coupled receptors (GPCRs) that are involved in pain signalling (for example,receptors for chemokines and prostaglandins and some glutamate receptors). Such

desensitization of GPCRs by GRK2 protects cells against overstimulation. Peripheral

inflammation or nerve injury induces a 35–40% reduction in GRK2 expression in lumbar

dorsal horn microglia, as a secondary adaptation of interleukin-1 receptor 1

(IL-1R1)-mediated signalling in these cells167–169. This decrease in GRK2 expression

prolongs nociceptive signalling, as shown by several studies in which peripherally

induced hyperalgesia was prolonged in mice with a specific deletion of GRK2 in

microglia, macrophages and granulocytes169. Downregulation of GRK2 may lead to

uncontrolled stimulation of microglia and, therefore, to the exaggerated and prolonged

production of pro-inflammatory mediators.

Another answer may lie in the immunological history of patients with chronic

neuropathic pain. There is emerging evidence that after a primary immune challenge,

microglia may retain enhanced transcript ional activit y or epigenetic modifications that

confer a heightened response to subsequent immune challenges20. Such immune

priming (also called immune training or postactivation) by stress, ageing, il lness or injuryfollowed by a somatosensory lesion or disease (or vice versa) may lead to chronic pain in

clinical populations, and has been modelled in several preclinical studies. Prior

treatment with corticosterone potentiated lipopolysaccharide (LPS)-induced allodynia

and increased spinal cord levels of interleukin-1β (IL-1β) and IL-6 (REFS170,171). Such

potentiated allodynia was inhibited by co-administration of the IL-1 receptor antagonist

protein (IL-1Ra)170. In a similar manner, prior exploratory abdominal surgery (laparotomy)

also potentiated LPS-induced allodynia, which was attenuated by minocycline (a

broad-spectrum tetracycline antibiotic and anti-inflammatory agent) administered

either at the time of surgery or LPS administration172. Prior t reatment with morphine

(which induces microgliosis; BOX3 ) has also been shown to potentiate allodynia induced

by hindpaw or masseter inflammation, as well as peripheral nerve injury173. Although it is

likely that such primed microglia retain enhanced transcript ional activity, these changes

may only be subtle and the intracellular mechanisms involved are not well understood.

REVIEWS




Blood– CNS ba rrier

A barrier formed by ast rocyte

end-feet and the tight junctions

between endothelial cells lining

blood vessels that excludes

constituents of the syste mic

circulation from entry into the

central nervous system (CNS).

It forms an important

boundary between the

sensitive microenvironment

of the CNS and the relatively

volatile environment of the

systemic circulation.

Trigemina l sens ory neuron s

Neurons found in the

trigeminal nerve tha t med iate

facial sensation and motor

functions, including biting and

chewing.

Dorsal root ganglia

(DRG).The cell bodies of

sensory neurons are collected

together in pa ired ganglia tha tlie alongside the spinal cord.

Each cell body is enca psulated

by satellite glia, with the entire

ganglia being surrounded by a

capsule of connective tissue

and a perineurium.

Brain-derived

neurotrophicfactor

(BDNF). A neurotrophin

expressed at high levels in the

central nervous system tha t

is vital for the growth and

survival of neurons, and has

been implicated in many

forms of synaptic plasticity.

C-fibres

Small diameter, unmyelinated

primary afferent sensory fibres,

with small cell bod ies in the

dorsal root ganglion. Some

C-fibres are m echa nically

insensitive, but most a re

polymodal, responding to

noxious, t hermal, me chanical

and chemical stimuli.

Figure 3 | Neuroimmune enhancement of noci cept ion. Soluble mediators released by reactive immunocompetentcells in the central nervous system diffuse and bind to receptors on presynaptic and postsynaptic terminals in thespinaldorsal horn to modulate excitatory and inhibitory synaptic transmission, result ing in nociceptive hypersensit ivity.Tumour necrosis factor (TNF), interleukin-1β (IL-1β), CC-chemokine ligand 2 (CCL2), reactive oxygen species (ROS) andinterferon-γ (IFNγ) increase glutamate (Glu) release from central terminals, part ly due to the activationof transientreceptor potential channel subtypes (TRPV1 and TRPA1), and functional coupling between interleukin-1 receptor 1(IL-1R1) and presynaptic ionot ropic glutamate receptors (NMDAR). TNF, IL-1β, IL-6 and ROS decrease GABA(γ-aminobutyric acid) and glycine (Gly) release by inhibitory interneurons. IL-1β and CCL2 also increase AMPA

(α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid) signalling, and TNFR1 signalling increases the expressionof Ca2+-permeable AMPA receptors (AMPARs). TNF increases NMDAR activity through the phosphorylation (P) ofextracellular signal-regulated kinases (ERKs), while IL-17 and ROS induce phosphorylat ion of the NR1 receptor subunitin spinal cord neurons. IL-1β increases the calcium permeability of NMDAR through activation of SRC family kinases andalso via phosphorylation of the NR1, NR2A and NR2B subunits. CXCL1 signalling via CXCR2 induces rapid activation ofneuronal ERK and CREB. Brain-derived neurot rophic factor (BDNF) signall ing via TRKB downregulates thepotassium-chloride cotransporter KCC2, increasing t he intracellular Cl – concentration and weakening the inhibitoryGABA

A and glycine channel hyperpolarization of second-order nociceptive projection neurons. Prostaglandin E2 (PGE2)

signalling at the EP2 receptor activates protein kinase A (PKA), thereby inhibiting glycinergic neurotransmission viaGlyR3. IL-1R1 signalling reduces neuronal expression of Gprotein-coupled receptor kinase 2 (GRK2), a negativeregulator of G protein-coupled receptors (GPCRs), including chemokine and prostaglandin receptors, leading tosustained neuronal GPCR signalling. TNF and IL-1β downregulate astrocyte expression of the Glu transporters excitatoryamino acid transporter 1 (EAAT1) and EAAT2, leading to enhanced glutamatergic transmission. CCR2, CC-chemokinereceptor 2; GABAR, GABA receptor.

IL-1R1 NMDARTRPV1TRPA1

Second-order painprojection neuron

First-orderneuron

Inhibitoryinterneuron

TNFR1

CCR2

Ca2+

Ca2+

AMPAR

CXCL1

TRKB

BDNF

NMDARGlyR3GABAR

EP2

PGE2

IL-1R1

NR2B

CCL2

REVIEWS




Lamina I neuron s

The most s uperficial aspe ct

of the sp inal dorsal horn

fromwhich second-order

nociceptive projection

neuronsoriginate.

Reversal poten tial

The membrane potential at

which chemical and electrical

drive are eq ual and oppo site,

such that there is no ne t flow

of ions across the me mbrane.

The direction of flow reverses

above a nd be low this potential.

Diminished inhibitory synaptic transmission. The noci-

ceptive pathway is regulateat multiple levels through

numerous inhibitory systems. Interestingly, a reuctionor loss of inhibitory synaptic transmission (known as

isinhibition) in the spinal corpain circuit has also

been implicatein the genesis of central sensitizationanchronic pain15. TNF, IL-1β, IL-6, CCL2, IFNγ anROS ecrease inhibitory signalling in the spinal or-

sal horn by reucing the release of GABA anglycinefrom interneurons aninhibitory escening projec-tions92,108–110. Such immune meiators also contribute to

isinhibition at the postsynaptic membrane of centralsynapses. BDNF is releaseas a consequence of micro-gliosis an, on activation of the neuronal cognate recep-

tor TRKB (also known as NTRK2), ownregulates theprincipal neuronal potassium-chlorie cotransporter,

KCC2, thereby increasing the intracellular Cl− concen-tration in lamina I neurons38,39. This positive shift of theanion reversal pote ntial weakens GABA

A anglycine

channel hyperpolarization, aneven causes GABA-evokeepolarization in a minority of neurons38,39. The

ultimate consequence of suchisinhibition is an increasein nociceptive hypersensitivity, anan unmasking ofresponses to innocuous peripheral inputs38,39,111.

Protective role of central immune signall ingA complex network of regulatory mechanisms activelycontrols central immune signalling after neuronal insult.These mechanisms inclue the prouction of anti-

inflammatory meiators anthe polarization of special-izecells with an anti-inflammatory phenotype to preventuncontrolleinflammation, as well as coorination of

temporal aniverse classic pro-inflammatory mecha-

nisms. An imbalance in such regulatory mechanismsmight contribute to the evelopment of chronic pain,

presenting therapeutic opportunities to enhance neuro-

protection anneuroregeneration while suppressingestructive inflammation.

Protective anti-inflammatory mechanisms and pheno-types. A reactive phenotype is not synonymous witha pro-inflammatory anpro-nociceptive phenotype.

That is,epening on the stimulating signal, there aresubpopulations of reactive immune cells with an anti-inflammatory phenotype. Although the existence of a

parallel anti-inflammatory microglial cell phenotyperemains controversial, these anti-inflammatory popula-

tions inclue alternatively activatemacrophages (also

known as M2 macrophages), anTH2 anTReg cellsthat contribute to the resolution of nociceptive hyper-

sensitivity after nerve injury18,23,26,29,112,113. A hallmark ofsuch anti-inflammatory cell types is their prouction of

anti-inflammatory meiators, such as IL-1Ra, IL-4 anIL-10 in the spinal orsal horn. The factors riving therecruitment anifferentiation of such anti-inflamma-

tory phenotypes are not well unerstoo. Nociceptivehypersensitivity associatewith spinal gliosis is attenu-

ateby intrathecal aministration of IL-1Ra, elevationof spinal IL-10 levels, anby systemic aministration ofglatiramer acetate, which increases expression of IL-4+

anIL-10+ Tcells in the spinal orsal horn8,26,114,115 .

Asie from the effects of IL-1Ra on excitatory post-

synaptic current (EPSC) frequency anNR1 subunit

phosphorylation, it remains to be shown whether otheranti-inflammatory cytokines irectly regulate central

sensitization96,101. However, the anti-nociceptive effects

may be inirect, as IL-4 anIL-10 inhibit the synthesisof pro-inflammatory cytokines anchemotactic factors

by microglia anTcells, anregulate cell phenotype

anthe expression of MHC classII anco-stimulatorymolecules116,117. In aition to these meiators, cellswith an anti-inflammatory phenotype express enzymes

that promote extracellular matrix repair anMAPKphosphatases, as well as receptors that suppress pro-inflammatory signalling (for example, IL-1R2 anIL-10R). Microglia also express scavenger receptors anco-stimulatory molecules (such as CD86) that can pro-

mote the evelopment of regulatory aaptive immuneresponses18,23,116,118,119 .

Protection mediated by pro-inflammatory mechanisms.The isplay of pro-inflammatory phenotypes by reac-

tive innate anaaptive immune cells is not necessarilysynonymous with nociceptive hypersensitivity antissueestruction; a rapi, well-regulateimmune response to

neuronal insult is important for the regulation of gliosisanclearance of tissue ebris to promote neuroregen-

eration. TNF seems to have a more prominent role thanIL-1β in promoting neuronal regeneration in the CNS,whereas both cytokines have crucial roles in peripheral

neuronal regeneration120,121. In a moel of nitric oxie-inuceneurotoxicity, neuronal celleath anemyeli-nation were increasein TNF-eficient mice relative to

wil-type controls120. Furthermore, although microgliosis

was attenuatewithin 6hours in these mice, it was exac-erbate4ays later. Hence, TNF perse may be requirefor the control of seconary amage, or the sequence of

the early reactive cascae in general may be crucial forsuch control. In aition to temporal action, several other

factors may influence neuroprotection in general, inclu-

ing the receptor subtype involve(for example, TNFR1or TNFR2, IL-1R1 or IL-1R2) another stimulatingsignals that accompany insult etection63,64,67,122.

Finally, after neuronal insult, reactive microgliaphagocytose apoptotic cells anebris, aneliminate thesource of DAMPs, leaving healthy tissue intact. Such a

nuanceanregulatereactive response may be coor-

inatethough microglial cell recognition of particu-

lar inhibitory molecules that are expresseby healthy

cells within the CNS. The molecules involvein inhib-iting innate immune cells may be humoral (for exam-

ple, semaphorin) or membrane-boun(for example,CD22, CD47, CD200 anCD200R)36,37. Cells that have

unergone apoptosis lose expression of these inhibitorymolecules anincrease their expression of ‘altere-selfmolecular patterns’ (for example, nucleic acis, sugars,

oxiizelow-ensity lipoproteins analteremembraneelectrical charge), allowing reactive microglia to selec-

tively phagocytose such cells37. These mechanisms havenot been irectly implicatein nerve injury-inucecentral immune signalling, but may be ysregulatein

persistentpain.

REVIEWS




Anti-allodynic and

anti-hyperalgesic

Compounds that op pose

allodynia a nd hyperalgesia

torestore basal sensory

thresholds.

The neuroimmune interf ace and pain controlThe funamental role of neurons in neuropathic pain isunispute. However, the strongest pain managementstrategies currently in use focus on suppressing aberrant

neuronal activity anlack suitable efficacy123. Given thekey role of the neuroimmune interface in persistent pain,there is interest in targeting these mechanisms for pain

management. There are several strategies that coulbeuseto target the neuroimmune interface: first,irect inhi-

bition of local pro-inflammatory signalling; secon, stimu-

lation of local protective anti-inflammatory mechanisms;thir, inhibition of specific immune meiators; anfinally,

antagonism of specific cytokine or chemokine receptors.Cytokines anchemokines in the CNS have been

targetepreclinically using the TNF inhibitor etaner-cept anIL-1Ra to successfully attenuate nociceptivehypersensitivity8,124. However, clinical translation of

such therapies may be limiteby potential isruptionof the beneficial neuroprotective anneuroregenerative

effects of TNF anIL-1, anby the inability of such mol-ecules to penetrate the bloo–CNS barrier. Although thebeneficial effects of pro-inflammatory meiators coulbe left intact by selectively targeting receptor subtypes

that meiate amaging pro-inflammatory processes

(for example, TNFR1 or IL-1R1), such approaches havenot yet been emonstratefor pain, but are likely to betoo specific to aress the wiespreaaaptations an

possible reunancy of cytokines anpain process-ing pathways33. Hence, the first two cellular approachespresent the greatest potential for clinical translation an

are focuseon below (summarizein TABLE1).

Inhibition of pro-inflammatory signalling at the neuro-

immune interface. Extensive preclinical stuies of theanti-inflammatory rugs minocycline, propentofyl-

line, ibuilast (MN-166; MeiciNova) anmethotrex-ate (which inhibit gliosis anTcell activation through

various mechanisms; TABLE1), have shown anti-allodynic

and anti-hyperalgesic efficacy in neuropathic pain moels,however, some of these rugs have hamixeclinical

success23,42,125–129. Ibuilast has shown signs of efficacy inneuropathic pain anmultiple sclerosis126. Ibuilast is in

PhaseII of clinical evelopment for meication overuseheaache (ClinicalTrials.gov ientifier: NCT01317992)anprogressive multiple sclerosis with neuropathic

pain as a seconary enpoint (NCT01982942).

Table 1 | Modulators of the neuroimmune interface

Drug name Cellular targets Mechanism of act ion Clinical use

Amitriptyline Microglia Disrupts TLR4 signalling by binding to MD2 Depression

ATL313 (Santen Pharmaceutical) Microglia and astrocytes • Adenosine A2A

receptor agonist• PKA and PKC activation

NA

BAY 60–6583 (Bayer HealthCare) Microglia and astrocytes Adenosine A2B

receptor agonist NA

Ceftriaxone Astrocytes Increases EAAT2 expression by inhibit ing NF-κ B activity • Bacterial meningitis• Lyme disease

Dilmapimod Microglia Selective p38 MAPK inhibition NA

FP-1 Microglia TLR4 antagonist NA

Glatiramer acetate Tcells Promotes generation of anti-inflammatory Tcell phenotypes Multiple sclerosis

Ibudilast(MN-166; MediciNova)

Microglia, astrocytesand Tcells

Non-selective phosphodiesterase inhibitor • Asthma• Post-stroke dizziness

Methotrexate Tcells Suppresses expression of cell adhesion molecules • Breast cancer• Rheumatoid arthrit is

Minocycline Microglia, Tcells andneurons

• Inhibits NF-κ B translocation to the nucleus• Inhibits NFAT1

Acne vulgaris

Paroxetine Microglia P2X4R antagonist Depression

Propentofylline(Aventis Pharma)

Microglia, astrocytesand neurons

• Inhibits cAMP and cGMP phosphodiesterases• Adenosine reuptake inhibitor

Ischaemic stroke

Resolvin D1(ResolvyxPharmaceuticals)

Microglia and neurons • Attenuates pro-inflammatory cytokine release• Inhibits TRPV1• Reverses NMDAR subunit phosphorylation

NA

Resolvin E1(ResolvyxPharmaceuticals)

Microglia and neurons • Attenuates pro-inflammatory cytokine release• Inhibits TRPV1• Attenuates glutamate release• Reverses NMDAR subunit phosphorylation

NA

Rifampin Microglia Disrupts TLR4 signalling by binding to MD2 Tuberculosis

(+)-naloxone Microglia Disrupts TLR4 signalling by binding to MD2 NA

(+)-naltrexone Microglia Disrupts TLR4 signalling by binding to MD2 NA

A2A

, adenosine receptor 2A; A2B

, adenosine receptor 2B; cAMP, cyclic AMP; cGMP, cyclic GMP; EAAT2, excitatory amino acid t ransporter 2; MAPK,mitogen-activated protein kinase; MD2, myeloid differentiation protein 2; NA, not applicable (no current clinical application); NFAT1, nuclear factor of activatedTcells 1; NF-κ B, nuclear factor-κ B; NMDAR, ionotropic glutamate receptor; P2X4R, P2X purinoceptor 4; PKA, protein kinase A; PKC, protein kinase C; TLR4, Toll-likereceptor 4; TRPV1, transient receptor potential cation channel subfamily V member 1

REVIEWS




RadiculopathyA condition a rising from

acompressed spinal nerve

rootthat is ass ociated with

pain, numbness, tingling or

weakness along the course

of the ne rve.

Carpal tunnel syndrome

A syndrome arising from

compression of the median

nerve — which runs from the

forearm into the palm of the

hand — that is associated with

itching numbness, burning

and /or tingling.

There are several possible reasons for these mixe

results, such as the inherent esign flaws that have beenhighlightefor trials involving propentofylline anminocycline130,131. It is therefore important that prior

failures are taken into consieration anthat a consen-sus is reacheon clinical trial esign for rugs target-

ing the neuroimmune interface. It is also possible thatcurrent neuroimmune moulators retain confoun-ing off-target effects that limit efficacy. For example,

minocycline has been shown to increase the expressionanphosphorylation of Ca2+-permeable AMPA recep-tors in mice, which will oppose anti-alloynic ananti-

hyperalgesic efficacy132. Furthermore, neuroimmune

moulators such as minocycline, propentofylline anibuilast may lack selectivity for specific glial animmune cell phenotypes, leaing to suppression of

neuroprotective anneuroregenerative effects. Theresults of an ear ly stuy suggestethat minocycline

hanon-selective actions on microglial cell subpopu-

lations as, espite attenuating alloynia inucebyintrathecal aministrat ion of HIV-1 gp120 envelopeprotein, minocycline also suppressethe expression

of IL1RN (which encoes IL-1Ra) anIL10 mRNA10.However, only a single stuy to ate has irectly char-acterizethe effect of neuroimmune moulators on

the phenotype of microglia, in which minocyclineselectively inhibiteexpression of M1, but not M2,

microglia markers in the mutant superoxie is-

mutase1 (SOD1G93A) mouse moel of amyotrophiclateral sclerosis (ALS)133. Hence, it is necessary that

further stuies are unertaken to efine the selectivityof phenotype-specific effects of neuroimmune mou-

lators, as broa-spectrum inhibition may be etrimen-tal to recovery.

The ambitious but attractive, approach is to antago-

nize specific stimulatory signals that rive estructiveanpro-nociceptive processes, while simultaneously

allowing for the coorination of a normal immuneresponse to insult via anti-inflammatory anregulatepro-inflammatory phenotypes. Preclinical ata sug-

gest that the TLR4 anpurinergic receptor signalling

cascaes in microglia may fulfil such criteria59,71.

Several novel TLR4 antagonists have shown efficacy

in preclinical neuropathic pain moels, incluingrifampin, FP-1 anthe opioireceptor-inactive isomers

(+)-naloxone an(+)-naltrexone79,80,134–136. In aition,

TLR4 antagonism may be exploiteto improve theefficacy anaverse effect profile of opiois (BOX3 ).

Given the observepreclinical efficacy, the purinergic

receptors P2X4R or P2X

7R are also potential therapeu-

tic targets for neuropathic pain22,40,63,64. Interestingly,some antiepressants current ly inicatefor neuro-

pathic pain, such as amitriptyline anparoxetine, pos-sess significant TLR4 anP2X

4R inhibitory activity,

respectively137,138. An alternative irect approach may

be to target ownstream signalling pathways, such asp38 MAPK, which rives microglia towars a reac-

tive state. A recent trial emonstrateoral analgesicefficacy for a selective p38 inhibitor, ilmapimo(SB681323; GlaxoSmithKline), in patients with nerve

trauma, radiculopathy or carpal tunnel syndrome139.Pro-resolution lipi meiators, such as the

resolvins, protectins an lipoxins, are emerging aspotent ial therapies that have irect effects on patho-logical neuroimmune transmission. Biosynthesize

uring the resolution of acute inflammation, suchmeiators were or iginally isolatefrom exuates in

both roents anhumans140. Pro-resolution meia-tors signal at their cognate GPCRs, such as CHEMR23(also known as CMKLR1; which is activate by

resolvin E1) anN-formyl peptie receptor 2 (FPR2;also known as ALX) which is activateby resolvin D1anlipoxin A4), anare wiely expresseby neurons,

glia anperipheral immune cells140. These meiators

have markeeffects on nociceptive hypersensitivityin iverse pain moels, not by moifying basal pain

threshols like analgesics, but by restoring normal sen-

sitivity through moification of aberrant neuroimmunetransmission140,141.

Stimulation of anti-inflammatory signalling at theneuroimmune interface. There are several potentialtherapeutics that may attenuate pathological neuro-

immune signalling by stimulating anti-inflammatorymechanisms to restore homeostasis in the spinal orsalhorn microenvironment. The aenosine GPCRs A

2A

anA2B

are expresseby peripheral immune cells anglia, anpharmacologically targeting these receptors

with a single intrathecal injection of specific agonists

reverses establisheperipheral nerve injury-nociceptivehypersensitivity for more than 4weeks114,115. This thera-

peutic effect is epenent on the activation of proteinkinase A (PKA) anprotein kinase C (PKC), leaing

to elevateIL-10 anecreaseTNF levels. Withoutexcluing a contribution from recruiteor other resi-ent cells, these pathways were specifically ientifie

in resient microglia anastrocytes. In aition, ceftri-axone anglatiramer acetate may restore homeostasis

either by normalizing astrocyte expression of EAAT2or by increasing the recruitment of IL-4-proucinganIL-10-proucing Tcells to the spinal orsal horn,

respectively26,107.

Box 3 | Opioids and central immune signall ing

Opioids remain the gold standard therapy for acute and chronic pain. However, their

clinical use is limit ed by issues with tolerance, paradoxical hyperalgesia and their

abuse potential. Although these adverse effects can be partly explained by neuronal

mechanisms, the emerging role of central immune signalling is revolut ionizing opioid

pharmacology136,174. In addit ion to stereoselectively signalling at classical opioid

receptors, morphine is now known to non-stereoselectively signal at Toll- like receptor 4

(TLR4) by binding t o myeloid differentiation factor 2 (MD2)136,174,17

. Hence, in addit ion tobeing activated by opioid receptor active (-)-isomers, TLR4 is also non-stereoselectively

activated by opioid receptor inactive isomers, such as (+)-morphine and the metabolite

morphine-3-glucuronide, and may be selectively antagonized by opioid receptor

inactive (+)-naloxone and (+)-nalt rexone80,136,174. The initiation of central immune

signalling by TLR4 activation has marked consequences for opioid pharmacodynamics,

including opposit ion of analgesia, probably by induction of central sensit ization, and

also opioid reward, craving, reinstatement and withdrawal174,176. A recent study

has also shown that µ-opioid receptor signalling on microglia induced brain-derived

neurotrophic factor (BDNF) release, downregulating the potassium-chloride

cotransporter KCC2 and disrupting the anion reversal potential177. Such alterations

were found to contribute to hyperalgesia, but not tolerance.

REVIEWS




Complex regional pain

syndrome

An idiopathic chronic pain

condition that usually affects

an a rm or leg and typically

develops after an injury,

surgery, stroke or hea rt atta ck,

but the pain is out of

proportion to the se verity of

the initial injury, if any.

Capsaicin

The active component of chilli

pep pers. It selectively binds to

transient receptor pote ntial

cat ion chann el subfamily V

me mbe r 1 (TRPV1) on

nociceptive a nd heat-sensing

neurons.

Postherp etic neuralgia

Neuropathic pain that occurs

in some patients following

infection with t he varicella

zoster virus, predominantlyaffecting the face.

Peripheral benzodiazepine

binding site

GABAA receptors in the central

nervous system (CNS)

represent the primary site of

action for benzodiazepines,

such as diazepam. The

peripheral benzodiazepine

binding site was originally

discovered in the periphery

asecondary binding site, but

has since b ee n identified in

the CNS.

Anti-inflammatory cytokine gene therapy with

either nakeDNA, or a range of vectors, is yet another

approach142. IL-4 anIL-10 gene therapies have proveneffective in moels of peripheral inflammation annerve injury, spinal corinjury, paclitaxel neuropathy

anexperimental encephalomyelitis42,142. Such efficacyis correlatewith reucelevels of phosphorylatep38, TNF, IL-1β anPGE2 in spinal microglia, as well

as ecreaseGFAP expression by astrocytes. Avoiingsome of the safety issues associatewith viral vectors,long-term reversal of peripheral nerve injury-inuce

nociceptive hypersensitivity has been achieveusingsynthetic polymer vectors to encapsulate anreleasethe IL-10 transgene upon int rathecal injection142,143.

Clinical evidence for the neuroimmune interf aceSeveral lines of correlative aninirect evience existin support of glial cell involvement in clinical chronicpain conitions. In a post-mortem stuy, astrocyte but

not microglial cell activation markers were upregulatein the orsal spinal cors of patients with HIV who ha

been experiencing chronic pain, anastrocytes fromthese iniviuals also showean increaseensity anhypertrophic morphology144. The patients also isplaye

increasephosphorylation of ERK, p38 anJNK, as wellas elevatelevels of TNF anIL-1β, in the orsal spinal

cor144. Another post-mortem stuy reporteincreasemicrogliosis anastrogliosis in the spinal corgrey matterof a patient with longstaning complex regional pain syn-

drome, although this stuy may have been confounebythe effects of prolongeopioiuse by the patient145 (BOX3).However, these finings are supporteby observations

that the levels of IL-1β anIL-6 are increasein the cer-

ebrospinal fluiof some patients with complex regionalpain synrome146. Clearly, there is a paucity of human

ata on pathological anpharmacological glial responses,

as highlighteaniscusseby several groups41,129,130.Eneavours to characterize the functional ifferences

between roent anhuman glia are of paramount

importance to inform translational pharmacology.P2X7R is expresseby all myeloicells, inclu-

ing microglia, ansingle nucleotie polymorphisms in

P2RX7 that affect pore formation anchannel functionare associatewith variability in chronic pain in two clini-cal populations62. Hyper- or hypo-functional variants

were associatewith increaseor ecreasepain ratings,respectively, in cohorts of patients with post-mastectomy

anosteoarthritis pain compareto pain-free controls62.

However, as impaireP2X7R function is also associatewith reucebone mineral content anensity in mice,

ifferences in pathology might also explain the correlationbetween osteoarthritis pain anP2RX7 polymorphism147.

In a recent stuy in healthy volunteers, intravenouspretreatment with a low ose of enotoxin was shownto enhance flare, hyperalgesia analloynia inuce

by intraermal capsaicin, ancorrelate with increaseplasma levels of IL-6 (REF.148). The observelarge effect

size anthe fact that alloynia anhyperalgesia are cen-trally meiate, suggests the inuction of central sensi-tization, probably ue to an inirect effect of enotoxin

meiatethrough a peripheral cytokine cascae.

Asescribeabove, microglia have been pharmaco-

logically targetein clinical chronic pain populations. A

recent stuy showethat minocycline attenuatehyperal-gesia in a small cohort of patients with sciatica128. Ibuilast

also showesigns of efficacy in relieving pain associate

with painful iabetic neuropathy ancomplex regionalpain synrome126. In contrast to these promising stu-

ies, propentofylline faileto improve self-reportepain

scores, comparewith placebo, in patients with posther-

petic neuralgia129. Trials such as these have been unertakenon the assumption that possible functional ifferences

between human anroent glia woulnot limit the effi-cacy ofrugs such as propentofylline in attenuating gliosisin humans. In aition to this assumption, severalesign

flaws were inherent in the propentofylline trial, incluingselection of a pain synrome for which no ata exist to

support glial cell involvement, ana lack of confirmationof whether the rug reachetherapeutic concentrationsat the target site. Furthermore, as preclinical stuies ini-

cate that it is easier to prevent than reverse neuropathicpain, propentofylline osing may have been prematurely

concluebefore reversal of long-staning pain coulhave been achieve130. The CC-chemokine receptor 2(CCR2) antagonist AZD2423 (AstraZeneca) showea

trentowars reuction in paroxysmal pain anpares-thesia anysesthesia, suggesting efficacy for some sen-

sory components of pain149. As with many clinical trialsfor pain, intra- aninter-iniviual variability was high,which may be overcome by enriching for a specific subset

of patients. Furthermore, target site efficacy was not evalu-ate. These issues further highlight the neeto evelopmethos to enableetection of glial responses inhumans.

Imaging may be one approach to etect real-time

gliosis in patients with chronic pain. Although not com-pletely specific for these cells, the peripheral b enzodiaz-

epine binding site is expresseby reactive microglia an

allows them to be imagein magnetic resonance imagingscans using the selective raioligan[11C](R)-PK11195.

Bining of [11C](R)-PK11195 to microglia in the human

thalamus is increasefor many years after peripheralnerve injury150, however, further investigation is requireto valiate these finings.

Finally, it is worth noting that neuropathic pain is oftencomorbiwithiseases in which ysregulation of centralimmune signalling is better establishe, such as multiple

sclerosis anALS151,152. In aition, epression, anxietyanrug abuse — common comorbiities of chronic pain

— have a central immune basis153. These converging lines

of evience provie further inirect support forysfunc-tional central immune signalling as a major contributor

to chronic pain in clinical populations.

Conclusions and future directionsMore than two ecaes of research has seen the accumu-lation of a wealth of ata supporting a major role for cen-

tral immune signalling in preclinical moels of pain, withmicroglia anastrocytes being among the best character-

izemembers of the neuroimmune interface. However,future stuies are likely to uncover prominent roles forother resient aninfiltrating immunocompetent cells in

the spinalorsal horn. Although the early stuy of central

REVIEWS




immune signalling focuseon the pro-nociceptive effects

of reactive phenotypes animmune meiators, the fiel

is beginning to appreciate pleiotropic roles for such ‘pro-inflammatory’ phenotypes, in aition to those of special-

izeanti-inflammatory reactive phenotypes. Mirroring

the general trenwithin preclinical pain research, mostinvestigations have exclusively focuseon the contribution

of central immune signalling to the sensory component of

pain. Given that patients with chronic pain experiencemany other symptoms, incluing isability, anxiety,epression, cognitiveysfunction ansleep loss (some of

which can be preclinically moelle), investigation of therespective neuroimmune correlates will yielgreat insight.

The existence anprecise mechanisms of cen-

tral immune signalling in humans have not yet been

conclusively establishe. A consierable challengefor the future is to etermine whether pathological

neuroimmune signalling contributes to chronic pain

in clinical populations. However, clinical tr ials oftherapeutics that target the neuroimmune interface

are a possible approach. A further challenge for rug

evelopment is to integrate the basic scientific ata onnuancecentral immune signalling in orer to evelopagents that normalize pathological neuroimmune

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AcknowledgementsFunding source s: P.M.G. is a CJ Mart in Fellow of the Austra lian

Government National Health and Medical Research Council

and an American Australian Association Sir Keith Murdoch

Fellow. M.R.H. is an Australian Resea rch Council Fellow.

Compet ing interests s ta tementThe authors declare no competing interests.

DATABASESClinicalTrials.gov: http://clinicaltrials.gov/

NCT01317992 |NCT01982942

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Pathological Pain and the Neuroimmune Interface - Nri3621