Lifetime modulation of the pain system via

neuroimmune and neuroendocrine interactions

Ihssane Zouikr1*, Bianka Karshikoff2, 3

1
RIKEN Brain Science Institute, Japan, 2Department of Clinical Neuroscience, Division
for Psychology, Karolinska Institutet, Sweden, 3Stress Research Institute Stockholm
University, Sweden
Submitted to Journal:
Frontiers in Immunology

Specialty Section:
Multiple Sclerosis and Neuroimmunology

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ISSN:

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1664-3224

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Article type:

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Review Article

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Received on:
07 Oct 2016

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Accepted on:

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24 Feb 2017

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Provisional PDF published on:
24 Feb 2017

Frontiers website link:

www.frontiersin.org

Citation:
Zouikr I and Karshikoff B(2017) Lifetime modulation of the pain system via neuroimmune and
neuroendocrine interactions. Front. Immunol. 8:276. doi:10.3389/fimmu.2017.00276

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 1! Lifetime modulation of the pain system via neuroimmune and neuroendocrine
2! interactions
3! *
Zouikr I 1, Karshikoff B 2,3
4! 1
Laboratory for Molecular Mechanisms of Thalamus Development. RIKEN BSI, Wako, Japan
5! 2
Department of Clinical Neuroscience, Division for Psychology, Karolinska Institutet, Nobels vg 9, 171 65
6! Solna, Stockholm, Sweden
7! 3
Stress Research Institute, Stockholm University, Frescati Hagvg 16A, 106 91 Stockholm, Sweden
8!
* To whom correspondence should be addressed: Ihssane.zouikr@uon.edu.au
9!
10!
11! ABSTRACT
12!
13! Chronic pain is a debilitating condition that still is challenging both clinicians and researchers.
14! Despite intense research, it is still not clear why some individuals develop chronic pain while
15! others do not, or how to heal this disease. In this review, we argue for a multisystem approach
16! to understand chronic pain. Pain is not to be viewed simply as a result of aberrant neuronal
17! activity, but also as a result of adverse early life experiences that impact an individuals
18! endocrine, immune and nervous systems, changes which in turn program the pain system.

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19! First, we will give an overview of the ontogeny of the central nervous system (CNS),
20!
21!

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endocrine, and immune systems and their windows of vulnerability. Thereafter, we
summarize human and animal findings from our labs and others that point to an important

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22! role of the endocrine and immune system in modulating pain sensitivity. Taking early life
23!
24!
25!
26!
27!
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history into account, together with the past and current immunological and endocrine status

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of chronic pain patients, is a necessary step to understand chronic pain pathophysiology and
assist clinicians in tailoring the best therapeutic approach.

Keywords: Psychoneuroimmunology, LPS, inflammation, pain, chronic pain,

28! neuroimmunology, neuroendocrinology, HPA axis, stress.
29!
30! I- INTRODUCTION
31!
32! The pain system is modulated by neuroimmune and neuroendocrine mechanisms from
33! embryonic development, throughout life until old age. Unlike the traditional reductionist view
34! that posits that pain is solely due to aberrant spinal and supraspinal neuronal activity, we now
35! understand pain in the context of a complex multi-system comprising well organized
36! interactions between neuroendocrine and neuroimmune systems [1]. The changes in the
37! nervous system induced by the immune system and the endocrine system are of both
38! structural and functional character, and are a part of the normal, adaptive development of the
39! pain system. However, an adaptation that is advantageous in one situation, may pose a risk
40! factor in another. Exposure to a wide range of stressors, from physical injury (such as

! 1!
41! incision) to infection and inflammation (as induced by e.g. lipopolysaccharide (LPS)),
42! activates the hypothalamo-pituitary-adrenal (HPA) axis as well as peripheral and central
43! immune responses, and reorganizes the sensitivity of the pain system [2; 3; 4; 5]. The HPA
44! axis and neuroimmune activation are of importance in determining long-term pathological
45! states such as chronic pain.
46!
47! Treating chronic pain is complicated by the wide individual differences in symptoms
48! and treatment response. Chronic pain is also associated with a high incidence of psychiatric
49! comorbidity [6] and is often present with other primary diagnoses, like inflammatory disease.
50! Furthermore, stress is often directly targeted in behavioural treatment strategies for chronic
51! pain [7], as part of an integrated treatment approach [8; 9]. Here, we explore some of the
52! biological mechanisms that may form the foundations of the complexity seen in clinical pain.
53!
54! This review will focus on some of the mechanisms involved in the maturation of the

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55! nervous system, that define the function of the pain system later in life. We will highlight the
56!
57!

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importance of neuroimmune and neuroendocrine interactions very early in life in the
programming of the pain system. We will also discuss how the immune system and the

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58! endocrine system continue to modulate pain processing throughout life, and the significance
59!
60!
61!
62!
63!
64!
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of these interactions for chronic pain.

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II- ONTOGENY OF THE CNS DURING THE PRENATAL AND POSTNATAL
PERIOD

Neuronal circuits are forged by sensory experiences. Exposure to environmental

65! stressors during a critical period of brain ontogeny, when neuronal circuits are particularly
66! sensitive to modification by experience, can have long-term consequences on neural circuits,
67! ultimately affecting behaviour [10]. Although our genetic make-up determines much of the
68! structure and function of the nervous system, the environment where the individual is born, as
69! well as the environmental conditions that will accompany the individual throughout his/her
70! life, plays a crucial role in tailoring the neuronal properties. The postnatal developing nervous
71! system responds to the external world to shape its neural circuits in order to subserve a
72! particular function (i.e. vision, auditory, touch etc.). In normal conditions (i.e. in the absence
73! of any adverse events), non-stressful early experience specifies a neural trajectory to the best
74! possible circuits of connectivity. In other words, non-efficient connections are eliminated and
75! those that are functionally stable remain. However, if exposed to stress - whether it is of

! 2!
 76! physical, physiological, psychological or viral/bacterial nature - during a time when the brain
77! is still undergoing fine-tuned maturation, the process of synaptic plasticity or synaptic tuning
78! can go seriously wrong, affecting the behavioural outcome.
79!
80! II-1. Early development of the human brain
81! During the prenatal period, the brain produces approximately 250000 cells per minute
82! [11]. Neuronal migration occurs between gestational week (GW) 8 and 16 forming the
83! subventricular zone [12]. Around GW 16, neurons reach their final target and begin to form
84! connections among brain regions [13]. Synapse formation in both the auditory and prefrontal
85! cortices begins around GW 27 [14]. During the beginning of the third trimester,
86! synaptogenesis occurs with a rate of approximately 40000 synapses per minute [15].
87! Subsequently, myelination as well as proliferation and differentiation of oligodendrocytes
88! (cells that produce myelin) take place. After birth, the size of the brain continues to increase
89! dramatically, with intense metabolic changes associated with synapse formation and axonal

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90! growth during the first three months of postnatal life [16]. The way the complex human brain
91!
92!

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develops and matures is through a significant increase in volume due to overproduction of
synapses, myelination and connections during infancy, followed by the elimination of less

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93! efficient synapses via pruning [17]. Most importantly, the developmental trajectory of the
94!
95!
96!
97!
98!
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neocortex is different depending on brain regions. For instance, the primary visual cortex

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undergoes significant maturation during the first three months of life, whereas the primary
auditory cortex continues to mature over the first three years of life [18]. The bilateral
thalamic connectivity to the prefrontal cortex (PFC) is increased gradually from childhood to
late teens [19], and synaptic pruning in the PFC continues to occur in mid-adolescence [14].
99! The relatively late maturation of thalamo-prefrontal cortex synaptic connections implies that
100! key connections involved in complex cognitive functions, including pain, are still undergoing
101! fine-tuned maturation in early postnatal life. Consequently, exposure to stressful events such
102! as viral/bacterial infections during postnatal life, is likely to be able to alter key neural circuits
103! involved in pain processing. This may lead to altered pain responses later in life. At present,
104! there is a paucity of research tackling this question and further studies investigating the
105! impact of early life stress on neural circuits involved in pain processing are needed.
106!
107!
108!
109!

! 3!
110! II-2. What animal models reveal about neurogenesis and synaptic plasticity
111! The traditional dogma posits that the postnatal brain (including adult brain) possesses a
112! fixed number of neurons that are generated from birth and that no neurogenesis or synaptic
113! plasticity is possible in the adult brain [20]. However, it is now clear that neurogenesis and
114! synaptic plasticity continue to occur in the adult brain, although at a lower rate. Findings from
115! studies that used standard neuronal markers such as NeuN or BrdU, have detected postnatal
116! neurogenesis both in primates and rodents. NeuN+/BrdU+-cells were detected particularly in
117! two regions: the subventricular zone (SVZ)- olfactory bulb and the subgranular zone (SGZ)-
118! hippocampal granule cell layer [21; 22; 23; 24; 25]. Regarding synaptic plasticity in the adult
119! brain, pioneer studies by Merzenich et al. demonstrated that amputation of one finger in adult
120! monkeys resulted in deafference of the devoted territory within the somatosensory cortex, and
121! that this region compensated by receiving inputs from neighbouring fingers [26]. Later on,
122! Robertson and Irvine showed that similar compensatory mechanisms and cortical
123! rearrangement occurred in the auditory cortex following lesion of the cochlea [27].

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124!
125!
126!

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In rats, PFC neural circuits undergo significant changes during the perinatal period. The
myelination of the medial PFC (mPFC) is very low at P7, increases gradually over the period

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127! P21-P50 and reaches peak level at P90 [28]. The ontogenic development of the PFC implies
128!
129!
130!
131!
132!
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that this region, which plays a critical role in cognitive functioning and pain processing [29],

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is particularly susceptible to environmental stimuli during the neonatal period. Consequently,
exposure to stressful events during this period is likely to alter the neural circuits within the
PFC - and consequently pain processing later in life. Indeed, sensory, painful or stressful
experience has been shown to change the dendritic and spine morphology in this area. A
133! combination of prenatal stress (E14-21) and maternal separation (P2-P21) resulted in
134! increased c-Fos expression in the mPFC and reduced dendritic length and dendritic spines of
135! mPFC neurons [30]. A recent study has found that pyramidal neurons from the mPFC of
136! spared nerve injury (SNI) rats are characterised by longer basal dendrites and increased spine
137! density compared to sham-operated animals [29]. Electrophysiological recording of mPFC
138! pyramidal neurons from SNI rats revealed increased NMDA/AMPA ratio in currents evoked
139! by stimulation of layer 5 [29]. However, convincing data linking directly altered PFC neural
140! circuits following early life stress to future pain responses is still lacking.
141!
142!
143!

! 4!
144! Taken together, these data suggest that the perinatal, up to and including the early
145! childhood period, is a time of high plasticity for the brain and adverse events occurring during
146! this critical period of cellular proliferation, differentiation, and maturation can interfere with
147! the normal developmental trajectory of the brain, resulting in structural and/or functional
148! changes in cells, tissues, or organ systems. These changes are proposed to potentially lead to
149! increased susceptibility to neurodevelopmental disorders in later life [31; 32; 33], and may
150! also be critical for determining adult pain responses, and potentially the susceptibility to
151! develop chronic pain.
152!
153! II-3. Early life development of the pain system
154! One of the neuronal systems that undergo significant malleability during the perinatal
155! period is the nociceptive system. For instance, at embryonic day (E) 15-17 myelinated A-
156! fibres are the first to penetrate the spinal lumbar cord before the subsequent projection of C
157! fibers into the substantia gelatinosa (Lamina II, superficial dorsal horn that contains

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158! nociceptive-specific neurons) at E19 [34]. During the neonatal period, lamina II is innervated
159!
160!

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by both A- and C-fibers. During the first three weeks of postnatal age, a withdrawal of A-fiber
primary afferents into deeper laminae is noticed, and C fibres exclusively innervate lamina II

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161! at the adult stage [35]. This developmental pattern of nociceptive fibers is of particular
162!
163!
164!
165!
166!
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relevance to the concept that early life insults are able to alter the neuroanatomical

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components of nociception (including nociceptive fibers), leading to altered pain responses
later in life. For instance, skin wound during the neonatal period is associated with
hyperinnervation of the wounded area by both A and C-fibers [36; 37]. This
hyperinnervation of nociceptive fibers can lead to peripheral sensitization and increased pain
167! sensitivity (i.e. hyperalgesia). Despite the apparent lack of maturity of the nociceptive system,
168! overall, younger animals are markedly more sensitive to noxious stimuli than their adult
169! counterparts [38]. Their behavioral output may however differ from adult animals. The
170! withdrawal threshold from heat stimuli is lower in young animals compared to adults and
171! neonatal rats are significantly more (i.e. tenfold higher) sensitive to formalin injection than
172! preadolescent rats who require higher formalin doses to elicit the formalin-induced
173! behavioural responses [38]. For example, until P10, injection of formalin into the hindpaw
174! elicits predominantly nonspecific whole body movement (i.e. jerking), whereas the formalin-
175! induced specific behaviours such as hindpaw shaking, flexion or licking appears only after
176! P10 [39]. Of particular interest, recent studies predominantly from Hathway et al. elegantly
177! demonstrated that the descending inhibitory control of spinal nociceptive reflexes from the

! 5!
178! periaqueductal grey (PAG) to rostroventral medullar (RVM) in rats undergoes an important
179! developmental switch from facilitatory in young rats to inhibitory in adult rats [40; 41; 42].
180! This developmental switch was found to be driven by opioid actions on RVM, as
181! microinjection of the -opioid agonist [D-Ala2, N-MePh4, Glycol]-enkephalin (DAMGO)
182! into RVM facilitates spinal nociceptive reflexes in preadolescent rats (P21), but elicited anti-
183! nociceptive actions in adult rats [42], and similar response pattern has also been recently
184! shown to occur at the PAG level [41].
185!
186! Overall, a number of neural systems, including those involved in pain modulation, are
187! characterized by significant malleability illustrated by major structural and functional
188! rearrangements in neural circuits following insult. This injury-induced plasticity renders the
189! nociceptive system more vulnerable to future challenges. Why certain patients develop
190! chronic pain while others dont might in fact result from different early life experience in
191! these patients, that may have programmed the pain system differently later in life. Therefore,

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192! taking early life history into account is a necessary step to understand chronic pain
193!
194!

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pathophysiology, and developing individual-based therapeutic strategies [43].

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195! III- ONTOGENY OF THE HPA-AXIS DURING THE PERINATAL PERIOD
196!
197!
198!
199!
200! P o v
III-1. Prenatal development

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The experience of stress, from an evolutionary perspective, is very important in
promoting survival of the organism. A fundamental system that is subjected to programming
by early life events is the neuroendocrine axis that mediates the stress response, the HPA axis
[44; 45]. Activation of this system starts with the recruitment of neurons within the
201! paraventricular nucleus of the hypothalamus (PVN) and the end product is the release from
202! the adrenal cortex of corticosterone for rodents or cortisol for humans, via the release of
203! corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) (the HPA
204! axis has been extensively reviewed elsewhere, please see [46]). During pregnancy, there is an
205! increase in CRH production in the placenta and fetal membranes. The gradual increase in
206! maternal HPA axis activity during this period leads to maternal hypercortisolemia [47; 48].
207! The fetus has much lower levels of glucocorticoids than its mother although endogenous
208! glucocorticoids can cross the placenta easily. 10-20% of cortisol present in the amniotic liquid
209! is from maternal origin, while the remaining 80-90% gets converted into inactive cortisone by
210! an enzyme, 11-HSD2 to protect the fetuss brain from excess in glucocorticoids, which can
211! be neurotoxic [49]. During the third trimester, fetal 11-HSD2 levels decrease and the fetus is

! 6!
212! exposed to high levels of CRH and cortisol. This rise in CRH and cortisol levels is thought to
213! play an important role in the maturation of organs and for preparing the fetus to the ex utero
214! environment [50]. The hippocampus plays a key role in regulating homeostatic levels of
215! glucocorticoids under conditions of stress, and CRF has been shown to modulate the electrical
216! activity of hippocampal neurons [51]. Glucocorticoid receptors (GR) mRNA were detected in
217! human fetal hippocampus at 24 gestational weeks (GW) [52]. Additionally, fibers expressing
218! CRH have been detected in humans by gestational week 16 [53] and the release of CRH into
219! the pituitary has been reported to occur at GW11.5 [54]. At the pituitary, a basic
220! adenohypophysis can be detected at GW6 [55] and by GW8, the pituitary reaches mature
221! stage and can release ACTH [56].
222!
223! In rodents, GR mRNA can be detected in the telencephalon as early as E12.5 with high
224! expression seen in the anterior hypothalamus, pons, spinal cord and pituitary gland [57]. At
225! E14.5, the expression of GR mRNA expression significantly increases in the ventral spinal

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226! cord and the thalamus, and undergoes a moderate decrease in these regions by E15.5. An
227!
228!

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increase in GR mRNA levels is observed at the same time-point in other regions including the
neocortex, cerebellum, and basal ganglia [57]. In the PVN, GR mRNA can be visible at E16,

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229! although whether this PVN GR is functional at this stage is not clear [58]. During the late
230!
231!
232!
233!
234!
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gestation (E17-19), GR mRNA is localized in the hippocampus, thalamus, and the amygdala

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[58]. Mineralocorticoid receptors (MR) ontogenetic expression, however, follows a different
pattern in rodents. MR mRNA expression cannot be detected before E15.5 when a moderate
expression is observed in pituitary gland, brain stem, tegmentum, and neuroepithelium of the
septum and pallidum [57]. MR mRNA expression is first seen in the hypothalamus at E17.5
235! and by E19.5 there is a dramatic increase in MR mRNA expression in the hippocampus,
236! septum, anterior hypothalamus, periaqueductal grey area and brainstem neuroepithelium [57].
237! Regarding the ontogeny of 11-HSD2 mRNA (encoding an enzyme that convert
238! corticosterone into its inactive form) in rodents, the expression of 11-HSD2 mRNA is
239! observed at E11.5 on hippocampal and subicular regions, neocortex, septum, and posterior
240! hypothalamic area. At E14.5, the expression intensity of 11-HSD2 mRNA starts to decline
241! in the neocortex, pallidal area, and spinal cord and by E15.5, 11-HSD2 mRNA is restricted
242! to the thalamus, midbrain, striatum, cerebellum, hypothalamus, medulla, and pallidum [57].
243!
244!
245!

! 7!
246! III-2. Postnatal modulation
247! There is a particular period called the stress hyporesponsive period (SHRP) from P4
248! to P14 in rats and from P2 to P12 in mice during which corticosterone levels, as well as
249! ACTH, are maintained at low levels even in the presence of mild stress [59]. Although, it is
250! generally accepted that pups do not respond to stress with an elevated HPA axis activity
251! during the SHRP period, it has been reported that 12 days-old pups that were separated from
252! their mothers for 24h with no access to food or water, showed a significant increase of both
253! basal and stress-induced corticosterone and ACTH secretion [59; 60]. These results indicate
254! that the HPA axis is particularly sensitive to maternal care even during the SHRP. During this
255! period, high expression of CRH are observed in the PVN whereas hippocampal GR
256! expression is low at birth and increases gradually during the SHRP [61]. In situ hybridization
257! studies in marmoset showed that the ontogenetic profile of MR and GR is different during the
258! postnatal period. Whilst GR mRNA expression in the dentate gyrus is higher in 4-6 weeks old
259! marmoset than in neonates (P1-P2), juveniles (4-5 months), and adult (3-6 years), MR mRNA

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260! expression was developmentally consistent in the hippocampus and PVN throughout life [62].
261!
262!

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Although we need to proceed with caution when extrapolating from animal studies to

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263! humans, the development of the brain in terms of synapse formation and brain growth rate in
264!
265!
266!
267!
268!
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a P6 rat is relatively equivalent to 38-40 weeks of gestation in humans [63; 64]. For obvious

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ethical and methodological reasons, human data regarding the ontogenetic development of
HPA axis is lacking. However, we can conclude from the above-mentioned animal data that
the prenatal period together with the first two weeks of postnatal life constitute a window of
significant plasticity for the neuroendocrine system. Homeostasis of the neuroendocrine
269! function, and consequently any physiological system that is under the influence of this system
270! (e.g. pain), is needed for normal neuroendocrine development. Excessive stress that may
271! challenge or perturb the neuroendocrine system when it is still developing could potentially
272! have far-reaching consequences. This way, early life stress may alter pain, neuroimmune and
273! neuroendocrine responses for life [4; 65; 66; 67; 68; 69].
274!
275!
276!
277!
278!
279!
280!

! 8!
281! IV- EARLY DEVELOPMENT OF THE IMMUNE SYSTEM
282!
283! IV-1. Immaturity of the neonatal immune system and susceptibility to infection
284!
285! Infant mortality due to infection is high particularly in developing countries with a high
286! prevalence of infection during the neonatal period [70]. This high susceptibility of neonates
287! and preterm infants to infection is thought to be due to immaturity of the neonatal immune
288! system. Analysis of umbilical cord from preterm infants revealed fewer nave CD8+ T cells
289! and regulatory CD31 expression compared to full-term neonates [71]. T cells play an
290! important role in the control of intracellular infections. Both human and murine neonates lack
291! mucosally distributed memory CD8+T cells. Although T cells and cytokines mRNA levels
292! (i.e. IL-1, IL-6, IFN-) can be detected in the thymus of mice from GD15 [72], neonatal
293! mouse macrophages do not react in a an adequate way early in life. For example, T-cells are
294! characterized by lower IFN- responses following stimulation [73; 74]. Ex vivo stimulation
295! with the bacterial mimetic LPS in mice produced much less pro-inflammatory and anti-

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296! inflammatory cytokines response in neonates compared to adult mice [75]. The same trend
297!
298!

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was observed in a human study whereby neonatal monocytes and dendritic cells produced less
TNF-alpha, IL-12, and IL-6 following LPS stimulation [76]. When stimulated with an anti-

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299! CD3 antibody, neonatal T cell proliferation was significantly decreased compared to adult T
300!
301!
302!
303!
304!
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cell proliferation. This attenuation of proliferation in neonatal T cells was restored to adult

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levels following the addition of exogenous IL-2 [77]. Furthermore the total cell number of T
cell subtypes (CD4+, CD8+, Thy1+) is markedly lower in the spleen and lymphoid nodes in
P4 mice compared to adult mice [78]. Similarly, the function of antigen presenting cells
(APCs) is markedly decreased in human and murine neonates compared to adults [78].
305! Treatment of both immunocompetent and immunodeficient mice with IL-12, a cytokine
306! produced by APCs [79], prior to inoculation with the parasite Cryptosporidium parvum
307! oocysts markedly reduced the severity of infection [80]. Additionally, neonatal mice exhibited
308! reduced levels of peripheral IL-12 and mice treated with IL-12 24h after birth displayed
309! increased levels of IFN- and IL-10 mRNA in the spleen [81]. Adult humans exhibited much
310! higher levels of granzyme B+ effector differentiated memory CD8+T cells, which are thought
311! to be the first responders to infections [82], than human neonates [83].
312!
313! The incidence of sepsis, defined as a systemic inflammatory condition that occurs
314! following exposure to pathogenic microorganisms or their toxin, is more than 25 times higher
315! in infants less than one year compared to children from 1 to 14 years of age, and constitutes a

! 9!
316! major risk of mortality and morbidity in the paediatric population [84]. The incidence of
317! infections is particularly high during the first postnatal weeks, and rapidly decreases thereafter
318! [85]. Common causes of infections in neonates include commensal bacteria such as
319! Escherichia coli [85]. Both adaptive and innate neonatal immune responses are relatively
320! immature as indicated by a lack of pre-existing memory and decreased Th1-type responses
321! [86; 87] as well as impaired production of TNF following exposure to LPS [88; 89].
322! Neonatal monocyte dendritic cells (moDC) also showed decreased production of interferon-
323! (IFN-) in response to in vitro stimulation with LPS compared to mature adult moDC [89;
324! 90]. Additionally, whole blood neutrophils concentrations in one-month children are shown to
325! be lower than those in adults [91].
326!
327! This immaturity of the immune system during neonatal life may thus predispose the
328! neonatal immune system to infection, both of intra- and extracellular types. Overall, bacterial
329! infection is considered the number one cause of perinatal infection in newborns worldwide

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330! [92; 93], which results in increased infant mortality particularly in developing countries [92;
331!
332!

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94]. In the coming sections, we will argue that this sensitivity of the immune system early in
life may have long-lasting effects in the adult organism.

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333!

334!
335!
336!
337!
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IV-II. Infection as a perinatal stressor

PExposure to pathogens early in life is a common event and is considered to play a

crucial role in priming the neuroendocrine-neuroimmune interface [95]. An infection may not
only be life threatening to an infant, but may also reorganize the function of the nervous
338! system, due to the tight interplay between the nervous and immune system. Human and
339! animal studies have demonstrated that perinatal exposure to an immune challenge can
340! produce changes in the CNS structure and function, leading to an increased risk of developing
341! behavioural and psychopathological alterations later in life [66; 96; 97; 98; 99; 100]. For
342! instance, offspring from mothers exposed to infections such as influenza, LPS or viral RNA
343! (Poly I:C) during pregnancy have higher risk of developing schizophrenia and autism [101;
344! 102; 103; 104; 105; 106]. A significant number of human and animal studies have also
345! indicated that perinatal infection can alter immune [97; 107; 108; 109; 110], metabolic [111;
346! 112], reproductive [113; 114], endocrine [95; 115; 116], neurological [117; 118], and
347! cognitive and behavioural responses later in life [98; 119; 120]. Interestingly, exposure to
348! LPS in rodents and humans can also cause pain facilitation such as thermal hyperalgesia,

! 10!
349! mechanical allodynia or hyperalgesia [121; 122; 123; 124; 125]. Such behavioural findings
350! appear to be the result of altered peripheral and central cytokine activity [122; 126; 127; 128].
351! Increased levels of pro-inflammatory cytokines including interleukin (IL)-1, tumor necrosis
352! factor (TNF)-, or IL-6 produced by the maternal or fetal immune system, have been linked to
353! abnormal brain development and increased risk of developing psychopathology [96; 98; 99;
354! 100]. Moreover, higher amount of IL-6 in the amniotic fluid following bacterial infection
355! during pregnancy have been previously reported to strongly correlate with increased mortality
356! rates and brain injury [129].
357!
358! Taken together, these findings highlight the fundamental role of the microbial
359! environment in programming behavioural and neural responses. In order to understand the
360! mechanisms of perinatal neuroendocrine-neuroimmune interaction, researchers employ
361! experimental models that mimic the antigenic actions of infection.
362!

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363! V- LIPOPOLYSACCHARIDE AS AN EXPERIEMENTAL IMMUNOLOGICAL

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364! STRESSOR

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365!
366!
367!

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Lipopolysaccharide (LPS), a complex glycolipid that is the major component of Gram-
negative cell wall usually derived from Salmonella enteritidis or Escherichia coli (E.coli), is a

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368! powerful activator of innate immune responses and induces behavioural symptomatology in
369!
370!
371!
372!
373!
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the host largely identical to those induced by live bacterial infection [130; 131]. LPS-induced
inflammation model presents well-known advantages, the primary one being that LPS does
not replicate, allowing tight control of dosage and limiting the confounding nature of infection
as compared to live bacteria models. LPS is commonly used to understand the complexities of
the neuroimmune-neuroendocrine relationship, and has been demonstrated to be a reliable
374! activator of innate immune responses [97; 108] and HPA axis [66; 95; 108; 116; 132]. Thus,
375! LPS acts as an experimental systemic immunological stressor [133].
376!
377! LPS activates toll-like receptors (TLRs), and initiates a cascade of signalization leading
378! to cytokine production that is crucial for infection clearance [134]. Monocytes, neutrophils,
379! macrophages, dendritic cells, and mast cells all express TLR4 at their surface membrane [135;
380! 136; 137]. Upon activation of the TLR4/MD2 complex by LPS, a series of phosphorylation
381! steps are activated, leading to the phosphorylation of inhibitory (I)B, which releases nuclear
382! factor (NF)-B from its complex [138]. NF-B is subsequently translocated into the nucleus
383! where it activates the transcription of pro-inflammatory cytokines such as IL-1, TNF, and

! 11!
384! IL-6, as well as anti-inflammatory cytokines such as IL-1 receptor antagonist (IL-1ra) and IL-
385! 10 [139; 140]. Cytokines released in the blood stream are able to activate the release of
386! cyclooxygenase (COX)-2 from the hypothalamus to induce hyperalgesia [141]. COX-2 also
387! stimulates the conversion of arachidonic acid into prostaglandins (PGE2), which acts in the
388! vascular organ of the lamina terminalis and in the ventromedial preoptic area of the anterior
389! hypothalamus to stimulate heat conservation via cutaneous vasoconstriction and attenuation
390! of sweating, and heat production via increases in the metabolism of brown adipose tissue
391! [142]. Circulating IL-1 is also known to directly activate hypothalamic PVN to stimulate the
392! release of corticosterone from adrenal cortex [143; 144]. LPS activation of Kuppfer cells in
393! the liver is also known to activate the release of IL-1 that can contribute to hyperalgesia via
394! vagal afferences [145], as vagotomy abolishes the LPS-induced hyperalgesia [145].
395!
396! V-1. Neonatal LPS exposure changes immune responses later in life
397!
398! Several lines of evidence from clinical and animal work suggest that exposure to LPS
399!
400!

n al
during the neonatal period is associated with altered immune responses later in life [66; 97;
109; 146; 147; 148; 149; 150]. Most importantly, long-term inflammatory responses within

o
si
401! the CNS are greatly influenced by immunological stressors early in life. Incubation of cord
402!
403!

r o vi
blood from one-month old children with LPS for 5h resulted in increased mRNA expression
of IL-6 and TNF compared to cord blood from the same age incubated with medium [146].

P
404! In rats, neonatal LPS exposure produces immediate upregulation of chemokines and cytokines
405! gene expression within the neonatal brain, as indicated by upregulation of mRNA levels of
406! Ccl7, Cxcl1, Cxcl10, IL-1, and IL-6 in the hippocampus two hours following LPS exposure
407! in rat pups at PND 4 [151]. The effect of neonatal LPS exposure on cytokines levels in limbic
408! areas can persist into adulthood. Our laboratory has previously shown that neonatal LPS
409! exposure at PNDs 3 and 5 results in increased IL-1 and TNF protein levels in the
410! hippocampus following exposure to restraint stress in adulthood [66]. Recent investigations
411! point toward a critical role played by the hippocampus in modulating pain via upregulation of
412! IL-1 expression [152]. Del Rey et al. documented a strong correlation between increased
413! hippocampal IL-1 transcripts and mechanical allodynia in chronic constriction injury (CCI)
414! and spare nerve injury (SNI) models [152]. However, it is not known whether changes in
415! protein levels of IL-1 in the hippocampus contribute to increased pain sensitivity in
416! inflammatory pain models (i.e. formalin test). Neonatal immune challenge has been also
417! reported to alter febrile responses later in life [147; 148; 150]. Fever is considered an

! 12!
418! important component of the innate immune response and is thought to play a crucial role in
419! survival through its ability to efficiently clear the pathogen while limiting the extent of
420! inflammatory damage [153; 154]. Animals prevented from developing fever have higher risk
421! of morbidity and mortality than animals that are allowed to develop fever [155]. Rats exposed
422! to LPS at P14 exhibited attenuated fever responses following a subsequent LPS challenge
423! [147; 149] or stress [150] in adulthood. The effect of neonatal LPS exposure on adult febrile
424! responses is thought to be mediated by pro-inflammatory cytokines, as neonatally LPS-treated
425! rats displayed significantly reduced plasma levels of TNF and IL-6 following subsequent
426! LPS exposure in adulthood. This reduction in turn was strongly correlated with the observed
427! attenuated febrile responses in LPS animals [147]. Interestingly, basal maintenance of body
428! temperature in adult rats was not affected by neonatal LPS administration [110]. This finding
429! implies that a single LPS exposure is not able to alter febrile responses later in life, but that a
430! second hit is necessary to unmask the altered febrile responses following a neonatal
431! immune challenge. Central levels of PGE2 and specifically in the preoptic region, a region

l
432! involved in the febrigenic thermoeffector pathways [156; 157], have also been targeted as
433!
434!

sio n a
potential mechanisms mediating the attenuated febrile responses following a neonatal immune
challenge. For instance, PGE2 levels in the preoptic area were increased in rats exposed to

i
435! LPS at P14 [150]. Additionally, glucocorticoids play a critical role in inducing the febrile
436!
437!
438!
439!
440!
o v
response, as adrenalectomy or blockade of glucocorticoid receptors using the glucocorticoid

P r
receptor antagonist RU-486 abolished the fever induced by neonatal exposure to LPS [147].
Finally, our laboratory has previously demonstrated that rats exposed to LPS at PNDs 3 and 5
displayed increased susceptibility to tumor and lung metastases following exposure to stress
in adulthood [97; 108]. Moreover, neonatal immune challenge produced reduced NK cell
441! activity and increased neuroendocrine responsivity to restraint stress in adulthood [97; 108].
442!
443! Taken together, an early immunological stressor has profound effects on the
444! immunological reaction pattern later in life, leading to altered neuroimmune function at
445! subsequent exposures to immunological challenges. This implies that what the immune
446! system of an organism has been exposed to very early in life, will in fact define its capacity to
447! defeat pathogens later in life.!
448! !
449!

! 13!
450! V-2. Impact of neonatal LPS exposure on endocrine function
451! Microbial microbiota can affect the postnatal development of HPA axis, and an
452! increasing body of evidence has demonstrated that neonatal exposure to LPS is associated
453! with long-term alterations in HPA axis activity [66; 97; 116; 149; 158]. Neonatal exposure to
454! LPS during P3 and 5 has been reported to increase circulating levels of corticosterone at both
455! time-points [66; 132; 159], suggesting that neonatal LPS exposure is capable of altering HPA
456! axis function during the SHRP. This alteration in HPA axis function following a neonatal
457! immune challenge persists throughout the life of the animal. Adult rats treated with LPS as
458! neonates displayed enhanced plasma corticosterone and ACTH levels in response to restraint
459! stress, noise stress, or in response to a second LPS hit in adulthood [66; 95; 97; 116; 132].
460! This altered peripheral endocrine response was also accompanied by central neuroendocrine
461! changes, as indicated by increased CRH mRNA levels in the PVN and decreased GR density
462! in the hypothalamus, hippocampus, and frontal cortex following exposure to stress in
463! adulthood [95]. These structures are known to mediate the inhibitory effects of

l
464! glucocorticoids on CRH synthesis in the PVN and the release of ACTH following stress [160;
465!
466!

sio n a
161], suggesting a decreased negative feedback sensitivity to glucocorticoids, and thus an
enhanced HPA responsiveness to stress following a neonatal immune challenge. We have

i
467! demonstrated in our lab that dual exposure to LPS during P3 and P5 in rats is associated with
468!
469!
470!
471!
472!
o v
increased circulating corticosterone at P7 and P22, but not P13, 1h following injection of

P r
formalin into the hindpaw [68]. P22 rats neonatally treated with LPS also exhibited a trend
toward decreased GR mRNA in the hypothalamus [68].

Overall, these data suggest that exposure to LPS during the neonatal period can
473! reprogram the neuroendocrine axis. This reprogramming increases the reactivity of animals to
474! a second physiological challenge later in life. Pain is an aversive experience and therefore
475! capable of activating the HPA axis [162]. Given that neonatal LPS exposure has been
476! associated to increased release of peripheral and central pro-inflammatory cytokines later in
477! life [66; 151] and considering the well-established role of pro-inflammatory cytokines in
478! producing hyperalgesia [145], it is reasonable to assume that neonatal LPS exposure is likely
479! to be associated with increased pain sensitivity later in life.
480!
481!
482!
483!
484!

! 14!
485! V-3. Impact of neonatal exposure to LPS on nociceptive responses
486!
487! The first postnatal week (P7-10) of rodents life is equivalent to the last trimester in
488! humans (36-40 GW) in terms of brain growth, gliogenesis, axonal and dendritic density, as
489! well as consolidation of the immune system [11; 163; 164; 165]. Preterm infants are, as
490! discussed earlier, at high risk of infection during the neonatal period. Early-life infections in
491! turn are known to be the cause of attenuated neurodevelopmental outcomes in these
492! vulnerable infants [166]. It is therefore important to address the impact of immune challenge
493! on pain sensitivity later in life. Boisse et al study found that administration of LPS at P14 in
494! rats produced thermal and mechanical hyperalgesia that paralleled the enhanced expression of
495! COX2 protein levels in the lumbar spinal cord [141]. Although this study did not directly
496! demonstrated that the increased level of COX in the spinal cord contributed to the observed
497! hyperalgesia in LPS-treated animals, it suggested a potential role of prostaglandins in
498! mediating the LPS-induced hyperalgesia. Increased COX mRNA levels were also observed
499! 4h following LPS injection in P3 and P21 rats (P0 is birth) [167]. A number of studies from
500!
501!

o n al
our laboratory have indicated that dual exposure of LPS during P3 and 5 in rats produced
long-term alterations in inflammatory pain responses later in life. Neonatal LPS

si
502! administration evoked increased formalin-induced behavioural responses (i.e. flinching and
503!
504!

r o vi
licking) in P13, 22 and adult rats [4; 68; 168]. The LPS-induced hyperalgesia observed in P22
rats coincided with altered HPA axis activity, as indicated by increased circulating
505!
506!
507!
508!
509!
P
corticosterone and decreased GR hypothalamic mRNA 1h post formalin injection, as well as
altered immune responses following formalin injection as indicated by increased mast cell
degranulation and increased circulating IL-1 [4; 68]. Moreover, the LPS-induced
hyperalgesia in preadolescent rats was accompanied by altered spinal dorsal horn intrinsic
properties, as well as decreased neuronal activity (i.e. Fos expression) in the PAG [68; 168].
510! LPS-treated adult rats exhibited hyperalgesia that coincided with central neuroimmune
511! changes, as indicated by increased IL-1 in the hippocampus 1h post formalin injection. No
512! differences were observed in peripheral IL-1 release or mast cell degranulation [4]. Although
513! we reported enhanced hippocampal IL in LPS-treated adult rats, we do not know which
514! immune cell releases this proinflammatory cytokine following neonatal immune challenge
515! and subsequent inflammatory challenge. Of particular interest, hippocampal parenchyma
516! astrocytes have been recently shown to produce the cytokine CCL2 24h post LPS injection in
517! adult mice [169], suggesting an important role of astrocytes in the neuroinflammation
518! produced by systemic LPS injection.

! 15!
519! Taken together, these data challenge the traditional concept that pain is originating
520! solely from activation of neurons, and suggest that components of the immune system play an
521! imminent role in modulating pain sensitivity. Using LPS as a model of infection, LPS-
522! induced hyperalgesia arises by both peripheral and central mechanisms. Peripherally, LPS
523! triggers e.g. macrophages to release proinflammatory cytokines, which sensitize nociceptors
524! [145; 170; 171]. In fact, LPS can directly activate TRPA1-expressing neurons independent of
525! TLR4 [172]. Centrally, LPS can activate microglial cells in the spinal cord and astrocytes in
526! brain regions such as the hippocampus and produce hyperalgesia [169; 173].
527!
528! VI- THE NEUROIMMUNE INTERFACE IN PAIN IN THE ADULT ORGANISM
529!
530! As discussed so far, the exposure to immunological stressors very early in development
531! of an individual have far reaching effects on neural structure and function, as well as on the
532! the immune and HPA axis activity. We have also pointed to defining changes for the adult
533! pain system. In the mature body, the systems are fully developed and less malleable.
534!
535!

n al
However, the immune system continues to affect the function of the nervous system in a
manner that drives pain sensitivity, by inducing functional changes. In this section, we will

o
si
536! describe some acute neuroimmune interactions in pain perception. Such neuroimmune
537!
538!

r o vi
interaction may potentially be of importance for the transition from acute to chronic pain in a
long-term perspective.

P
539!
540! VI-1. Animal studies demonstrate inflammation-induced pain sensitivity
541! The role of the immune system was traditionally viewed as protecting the organism
542! from invading pathogens. However, it is now well established that the bidirectional
543! interaction between the immune and nervous system plays a crucial role in pain modulation
544! [125; 174; 175; 176; 177]. Proinflammatory cytokines play an important role in this immune
545! to brain bi-directional interaction [121; 145]. When exposed to LPS, immune cells such as
546! macrophages, monocytes and mast cells release many proinflammatory cytokines such as IL-
547! 1, TNF-, and IL-6 into the circulation creating an inflammatory soup condition that
548! enhances pain sensitivity by sensitizing nociceptors [178; 179; 180]. These proinflammatory
549! cytokines also signal to the brain to induce a set of physiological responses including fever,
550! lethargy, decreased social interaction, decreased sexual activity, or decreased food and water
551! intake, increased circulating corticosterone, collectively known as sickness behavior [181;
552! 182; 183]. Importantly, pain facilitation or hyperalgesia is considered to be an integral part of
553! sickness behavior [121; 125]. Peripheral inflammation can lead to central neuroinflammation

! 16!
554! via many different ways. Firstly, through vagal afferences since subdiaphragmatic vagotomy
555! reversed the hyperalgesia induced by IL-1 or LPS [145]. Alternatively, cytokines can access
556! the brain through areas that lack the blood-brain barrier (BBB) such as the organum
557! vasculosum lamina terminalis (OVLT) [184]. LPS produces IL-1 in the brain which is
558! initially restricted to choroid plexus and circumventricular organs, then diffuse to the brain
559! side of BBB [185]. Cytokines have also been suggested to enter the brain via active transport
560! systems across the BBB [186; 187].
561!
562! The first report on the impact of LPS exposure on pain responses was the study by
563! Mason, who demonstrated that i.p. administration of LPS in adult rats significantly decreased
564! tail flick latency, an effect that peaked at 1h post LPS administration [123]. The LPS-induced
565! thermal hyperalgesia observed in adult rats was reversed following the administration of IL-
566! 1ra [188], indicating that IL-1 is an important mediator of this hyperalgesia.
567! Proinflammatory cytokines released by immune cells are known to induce hyperalgesia when

l
568! administered both peripherally or centrally, particularly IL-1 [145; 170; 189; 190]. For
569!
570!

sio n a
instance, intracerebroventricular (ICV) administration of the recombinant human IL-1 (rhIL-
1) in rats induced thermal hyperalgesia [170], while ICV injection of the IL-1 antagonist

i
571! IL-1ra abolished this hyperalgesia [170]. Intraplantar injection of IL-1 has been associated
572!
573!
574!
575!
576!
o v
with increased discharge of spinal dorsal horn neurons in response to non-noxious stimuli

P r
[190]. Local administration of IL-1ra decreased the LPS-induced hyperalgesia [171].

IL-1 is also known to contribute to flinching responses in the formalin test given that
an intraplantar injection in rats of anti-sera anti-IL-1 prior to formalin injection significantly
577! attenuated flinching responses in the formalin test [191]. We have previously shown that rats
578! exposed to LPS during the neonatal period displayed increased circulating IL-1 at P22 in
579! response to formalin injection [4]. Adult rats previously subjected to neonatal immune
580! challenge also displayed enhanced hippocampal IL-1 that coincides with the LPS-induced
581! hyperalgesia at this age [4]. The source of this hippocampal IL-1 is not known, but it is
582! highly probable that it is originating from astrocytes or microglial cells within the
583! hippocampus. Interestingly, at the same age (i.e. PND 22), and at the same time-point
584! following formalin injection (i.e. 1h post formalin injection), we observed altered intrinsic
585! properties of spinal dorsal horn (SDH, laminae I and II) neurons in LPS-treated rats as
586! indicated by lower input resistance compared to saline-treated rats [68].
587!

! 17!
588! SDH neurons are the first component of the central nervous system to receive incoming
589! noxious sensory information, and their output is determined by a combination of their
590! synaptic inputs and intrinsic neuronal properties [192]. Formalin injection is known to
591! activate peripheral nerves, which results in turn in activation of dorsal horn neurons [193;
592! 194; 195]. Hindpaw injection of formalin is associated with the release of numerous
593! substances in the spinal cord, including prostaglandin E2 [196]. Bath application of
594! prostaglandin E2 results in changes of intrinsic properties of dorsal horn neurons including
595! decreased input resistance [197]. Since this change was only observed in LPS-treated
596! preadolescent rats, it is possible that the neonatal exposure to LPS resulted in either an
597! increase of proinflammatory cytokines within the spinal cord, or an increased susceptibility of
598! SDH neurons to proinflammatory cytokines. This assumption is confirmed by the fact that
599! intrathecal administration of IL-1ra has been reported to block formalin-induced hyperalgesia
600! [198]. The source of spinal hyperalgesia seems to involve microglia and astrocytes since
601! intrathecal administration of Fluocitrate, an inhibitor of glial metabolic function, blocked the

l
602! formalin-induced hyperalgesia [198].
603!
604!

sio n a
Additionally, IL-1 has been documented to act supraspinally to induce hyperalgesia.

i
605! For instance, microinjection of IL-1 into the preoptic area of the hypothalamus is sufficient
606!
607!
608!
609!
610!
o v
to induce thermal hyperalgesia [199]. Of particular interest is the observation that IP or ICV

P r
administration of IL-1 has been documented to produce an increase in plasma levels of
corticosterone and ACTH, an action that is mediated by the release of CRH from the PVN
[144; 200]. The neonatal immune challenge is likely to influence the generation of new
neurons in the hippocampus. This assumption is confirmed by the fact that an intraplantar
611! injection of the nociceptive inflammatory agent Complete Freunds Adjuvant (CFA) at P8
612! results in more Bromodeoxyuridine (BrdU) and doublecortin-labeled cells, both measures of
613! newborn neurons, in the subgranular zone of the dentate gyrus [201]. Whether such neurons
614! release IL-1 in response to neonatal LPS exposure remains to be determined.
615!
616! At the peripheral level, the enhanced IL-1 plasma levels observed at PND 22 in LPS-
617! treated rats coincides with higher degree of mast cell degranulation, which was also
618! accompanied by increased formalin-induced nociception [4]. Mast cells are located in the
619! vicinity of primary nociceptive neurons and vasculature and their degranulation has been
620! reported to regulate the excitability of nociceptive nerve endings [202]. Mast cell
621! degranulation can also produce thermal hyperalgesia via the production of nerve growth

! 18!
622! factor (NGF) [203]. Previous studies have documented an important role of mast cells in
623! formalin-induced nociception. Blocking mast cell activity using the mast cell stabilizer
624! Cromolyn abolished formalin-induced pain responses in the late phase [204]. Interestingly,
625! mast cells are also known to express receptor for IL-1 and to produce IL-1 following
626! inflammation [205].
627!
628! VI-2. Inflammation-induced pain sensitivity in humans
629! The human physiology is much more sensitive to LPS provocation than that of rodents.
630! To avoid the risk of sepsis, very low doses of LPS are used in humans (usually 0.2-4.0 ng/kg),
631! the highest doses often requiring additional antipyretic pharmacological treatment. The most
632! common dose for psychological research is around 0.4-1ng/kg LPS from E. Coli, which
633! induces a clear rise of pro-inflammatory cytokines TNF, IL-1, IL-6 and IL-8 in the blood
634! [206; 207; 208]. Human studies can also benefit from vaccinations of healthy individuals as
635! an inflammatory model, and patients undergoing immunotherapy can be studied. The

l
636! behavioural outcomes of experimental immune activation are very similar to sickness
637!
638!

sio n a
behavior exhibited by experimental animals; individuals report increased anxiety, worsened
mood, and increased pain sensitivity [205; 209; 210]. Appetite is reduced and fatigue and

i
639! anhedonia increase parallel to decreased social interest [126]. The immune activation also
640!
641!
642!
643!
644!
o v
disrupts memory and cognition, and changes motivation [6; 211; 212]. In human studies with

P r
the lowest LPS doses, the effects can in fact be so subtle that blinding can be maintained.

VI-2.1. Pain sensitivity during immune provocation

So far, only LPS-stimulations have been used to study the pain system specifically in
645! humans, and several studies have shown that experimental immune activation increases pain
646! sensitivity in humans, too. Deep (muscular and visceral) pain is more readily affected than
647! superficial (cutaneous and mechanical) pain [207; 213; 214]. Also, the change in pain
648! sensitivity usually correlates with peripheral cytokine levels. As in all experimental pain
649! research, the mode of pain stimulation as well as the pain intensity applied may affect the
650! outcome. Threshold pain is not processed exactly the same way as suprathreshold (intense)
651! pain, and pain from within the body is relayed to the brain in pathways partly distinct form
652! those used to relay cutaneous pain [215]. Also, the nociceptive effect may depend on the
653! immunological pressure, i.e. the LPS dose in experimental models. Two studies show that
654! threshold pressure pain sensitivity is affected the same way in men and women, despite the
655! generally higher cytokine levels found in women during LPS-stimulation [207; 216].

! 19!
656! Interestingly, no sex differences in psychological outcomes, such as anxiety or perceived
657! health, are seen either despite the sex differences in cytokine release [207; 208]. One study
658! has however shown that women are indeed more affected by inflammation with regard to pain
659! perception [207]. In this study, the descending pain inhibition of women was weakened
660! during LPS-stimulation, while men remained unaffected. In parallel, women were more pain
661! sensitive to intense cutaneous pain, too, while men only changed their perception of deep
662! pain. Furthermore, one study using a high LPS-dose (2.0 ng/kg) has in fact shown increased
663! pain sensitivity to intense cutaneous pain in men. Sex differences in inflammation-induced
664! pain sensitivity need further exploration. An intriguing mechanism for a potential sex
665! difference was recently suggested in a murine study [217], where female mice did not require
666! microglia activation to develop pain hypersensitivity, but appeared to have alternative routes
667! via the adaptive immune system. This alternative route did not seem accessible to males.
668! Future research will have to establish if these mechanisms are relevant for humans as well,
669! and their role in immune-driven pain sensitivity. Furthermore, sex-dependent alterations in

l
670! neuroendocrine function in human subjects following LPS provocation have been shown
671!
672!

sio n a
[218]. Healthy humans exhibited enhanced circulating levels of cortisol (peak response at 5h
post LPS injection) after LPS injection [208; 219]. The effect appears to be more pronounced

i
673! in women [208], but the data is inconclusive [219]. On a final note, experimental pain is
674!
675!
676!
677!
678!
o v
sensitive to stress, which could potentially be a confounder in LPS-studies on pain. Perhaps

P r
surprisingly however, stress levels generally remain low amongst the participants throughout
the studies [207]. Our experience is that because LPS-stimulations, due to ethical
considerations using bacterial endotoxin injections in healthy subjects, require very clear
participant information and a hospital environment with experienced personnel and constant
679! supervision, participants describe a feeling of safety and control even at higher, quite
680! uncomfortable doses (such as 2.0 ng/kg).
681!
682! VI-2.2. Brain activity during experimental immune activation
683! Although the cytokines released during immune activation may affect and sensitize
684! peripheral nerve endings, the main effect by which the immune system changes the function
685! of the nervous system during sickness is believed to occur centrally via induced sickness
686! behavior. It is reasonable to assume that changes in the emotional circuitries underlying the
687! increased anxiety and depressed mood seen during immune activation, may also lead to
688! increased pain sensitivity due to overlapping function with the medial (affective) pain
689! network [215], such as the amygdala, the cingulate and prefrontal cortices. Also, as sickness

! 20!
690! is per definition an interoceptive signal, i.e. a signal of the internal state of the body [220],
691! areas involved in interoception and homeostasis such as the insular cortex, which is also part
692! of the pain network, could potentially be affected. Several studies have attempted to elucidate
693! the neural correlates of sickness behavior in the human brain. Most studies have used
694! functional magnetic resonance imaging (fMRI) with cognitive and emotional paradigms. The
695! main methodological limitation for this type of research is the fact that only the lower LPS
696! doses used in humans are compatible with a brain scanning protocol, i.e. those that do not
697! induce nausea or shivering.
698!
699! Only two studies have explored pain perception directly during brain imaging so far, one
700! using visceral pain stimuli (deep pain measurement) and mechanical pin-prick pain
701! (cutaneous pain measurement) [221], and the other using pressure pain (deep pain) [222].
702! Benson et al. showed increased activation within the posterior insula, dorsolateral prefrontal
703! (dlPFC), anterior midcingulate (aMCC) and somatosensory cortices for visceral pain

l
704! stimulation, but not mechanical pain provocation. These areas are involved in pain and
705!
706!

sio n a
affective processing, interoception and homeostatic regulation. Karshikoff et al. [222]
described decreased activity after LPS injection in the lateral prefrontal cortex and rostral

i
707! ACC, areas involved in descending pain inhibition, which may point to an increase in
708!
709!
710!
711!
712!
o v
inflammation-induced pain sensitivity via diminished endogenous pain regulation.

P r
Additionally, the LPS group showed increased pain-dependent activity in the anterior insular
cortex compared to placebo.

Emotional and cognitive fMRI paradigms corroborate the involvement of the cingulate, insula
713! and prefrontal cortices when the brain adapts to immune activation [221; 223; 224; 225; 226;
714! 227], which are core areas in affective pain processing and pain regulation. Using a
715! vaccination protocol as experimental immune provocation, Harrison et al. have shown
716! increased activity in the subgenual anterior cingulate cortex (ACC) during emotional stimuli,
717! and increased activity in areas involved to interoceptive function during a Stoop task, such as
718! the brain stem, the cingulate and anterior insula [225; 226]. To maintain the same level of
719! performance during peripheral inflammatory activity, regions of the prefrontal cortex appear
720! to be required [224; 225] areas implicated in pain regulation and processing of affective
721! components of pain. In several studies the increased BOLD-activity in these areas correlates
722! with peripheral cytokine levels [210; 222; 226; 228; 229].

! 21!
723! Immune challenge affects the levels of neurotransmitters in the brain [6; 230]. The expression
724! of sickness behavior can potentially be manipulated by drugs affecting neurotransmitter levels
725! such as serotonin-reuptake inhibitors, which are compounds often used to ameliorate chronic
726! pain. Hannestad et al. [231] have for example shown that the effects on fatigue are
727! ameliorated by pre-treatment of serotonin reuptake inhibitors, but not by dopamine and
728! noradrenaline reuptake inhibitor. Peripherally induced inflammation has also been shown to
729! activate microglia directly [232; 233]. This is of special importance for chronic pain, as
730! microglia have been implicated in the establishment of chronic pain [121].
731!
732! In the past decade is has thus been shown that acute inflammation induces pain sensitivity in
733! humans as well. Most importantly, acute inflammation has a global effect on brain function,
734! modulating the neural function in several brain areas involved in pain perception. Although
735! the experimental models used are of an acute character, similar mechanisms are likely to be
736! involved when the organisms is subdued to long-term inflammatory activity.

l
737!
738!
739!
VII- THE HPA-SYSTEM AND PAIN IN ADULT ORGANISMS

sio n a
i
740! Pain is not only modulated by immunological stressors, but also through activation of

v
741! the HPA-axis. Pain is a sensory as well as an emotional experience. It is by nature a stressful
742!
743!
744!
745!
746!
r o
event and therefore capable of activating the HPA axis. As we have mentioned, there is a

P
large individual variability in developing chronic pain. One possible mechanism that may
account for this individual variability in pain responses is how each individual responds to
stressful events. Exaggeration or maladaptive response following stress may lead to altered
pain responses. The HPA axis involves a defined neural circuit that comprises many brain
747! regions including the amygdala, the mPFC, and the hippocampus. These areas are also
748! important in pain modulation [234; 235; 236; 237]. In other words, a non-painful stressful
749! stimulus is able to recruit parts of the same neural network involved in the pain response.
750! Therefore, under conditions of stress, pain sensitivity may be exaggerated. Indeed, activation
751! of CRH receptors in the amygdala facilitated pain responses through increased excitatory
752! postsynaptic current in the parabrachio-amygdaloid synapse in rodents [238]. Furthermore,
753! administration of CRH into the CeA increased visceral nociception, as indicated by
754! exaggerated number of abdominal muscle contractions in response to colorectal distension
755! [239]. On the other hand, the contribution of acute stress in analgesia commonly known as
756! stress-induced analgesia has been traditionally well documented [240; 241], and at this

! 22!
757! point in time the exact contribution of cortisol in modulating pain is still a matter of debate
758! within the scientific community.
759!
760! In human clinical samples, some researchers have found that low back pain and
761! enhanced musculoskeletal pain are often associated with hypocortisolemia [242; 243], while
762! others demonstrated that patients suffering from chronic back pain displayed higher levels of
763! cortisol compared to control group [244]. This hypercortisolemia was associated with smaller
764! hippocampal volume and higher pain-evoked response in the anterior parahippocampal gyrus
765! [244]. This variability in cortisolemia in pain condition may be due to the intensity of the
766! stress response [245], but may also well depend on the neural circuit recruited following the
767! stress stimulus, as the neural circuits within PVN is quite complex and the final outcome
768! depends on the nature of the stressor (for review, please see [237]). In inflammatory pain
769! model such as the formalin test in rodents, LPS-induced hyperalgesia in infant and
770! preadolescent rats coincided with increased circulating corticosterone one hour following

l
771! intraplantar injection of formalin [68]. However, a recent study demonstrated that elevated
772!
773! responses [246].

sio n a
levels of plasma corticosterone produced analgesia via an attenuated c-fiber mediated spinal

i
774!
775!
776!
777!
778!
779!
o v
Overall, the above animal and human studies suggest that changes in HPA axis activity can

P r
contribute to pain. Although more studies are needed to confirm the exact contribution of
cortisol (in humans) or corticosterone (in rodents) in modulating pain responses, the
involvement of neuroendocrine response in pain is evident. Therefore, new therapeutic
approaches, which not only target neural activity but also the neuroendocrine axis, are needed
780! to treat chronic pain patients.
781!
782! VIII- A LIFETIME PERSPECTIVE
783! Whereas the acute effects of immune provocation on pain sensitivity are fairly well
784! documented by now, as described in the previous sections, long-term inflammatory effects are
785! not as well understood. At this point in time, the most research on long-term effects of
786! inflammatory activity on behavior has focused on depression. In humans, one incentive to
787! study the mechanisms of sickness behavior came from clinical observations of
788! immunotherapy eliciting side effects that resemble sickness behavior, like depressive
789! symptoms, fatigue and aches. In for example hepatitis C patients undergoing IFN- therapy,
790! up to 45% of the patients develop depression [247]. The typical signs of sickness behavior

! 23!
791! appear at the commencement of immunotherapy, whereas the establishment of depression
792! requires time, and potentially, persistent inflammatory input during this time. It is now argued
793! that depression is in part an inflammatory disease [248], and that a subgroup of clinically
794! depressed patients suffers from a chronic low-grade systemic inflammation. Childhood
795! trauma has also been shown to predispose persons to depression, but potentially not only via
796! learning and HPA-dysregulation as traditionally suggested, but also via inflammation.
797! Depressed patients with a history of traumatic events have higher low-grade inflammatory
798! activity [249]. Most interestingly, these patients benefit from pharmacological treatments that
799! combine anti-inflammatory compounds and traditional anti-depressants [249]. Suggested
800! mechanisms between inflammatory activity and depression include cytokines, serotonin,
801! HPA-dysregulation, GABA and glutamate, all of which are neuroimmune pathways also
802! implicated in pain (for extensive reviews see e.g. [6; 250]). Recent research is now shifting
803! the focus towards similar mechanisms for chronic pain and fatigue [6; 230].
804!

l
805! VIII-1. Inflammatory disease and pain
806!
807!

sio n a
Chronic pain is a common comorbid symptom to many inflammatory diseases [251].
Moreover, coronary heart disease [252], metabolic disorders [253] and life stress [254]

i
808! increase the risk of developing chronic pain. It has been suggested that one of the underlying
809!
810!
811!
812!
813!
o v
mechanisms for these association, is indeed inflammation [252; 253; 254]. Furthermore,

P r
chronic pain has been associated with low-grade inflammation [255]. Mechanistically,
peripheral chronic inflammation may become chronic within the CNS via changes in the
central immune responses, by means of mechanism previously discussed. In animals, transient
peripheral infections and inflammations or chronic exposure to low level (sub-clinical)
814! inflammations, can either activate microglia directly [256; 257] or prime the cells so that a
815! recurrent inflammatory provocation becomes more severe [258]. A systemic inflammatory
816! challenge leads to an exaggerated fever response and sickness behavior in the presence of
817! primed microglia in rodents [259; 260]. Priming of immune components, or the
818! requirement of a second immunological hit to reveal susceptibility as discussed previously,
819! is exemplified in a recent clinical study. Obesity has been associated with chronic pain and is
820! considered a chronic low-grade inflammatory state [253]. Obesity did not predict post-
821! surgical pain intensity or inflammatory levels [255]. BMI did however correlate with the
822! increased immune response of leukocytes after LPS stimulation, suggesting sensitivity to
823! inflammatory development in the obese patients that could results in complications associated
824! with inflammation further down the road, like chronic pain. Another study points to

! 24!
825! differences in pharmacological treatment strategies on pain after surgery, depending on prior
826! inflammatory disease. Nonsteroidal anti-inflammatory drugs (NSAIDs) had a better protective
827! effect against the development of long-term pain after surgery in patients with a background
828! of inflammatory disease, than opioids [261].
829!
830! VIII-2. The immune system develops throughout life
831!
832! As discussed previously, the immune system carries the imprint of early-life
833! inflammatory events. However, the function of immune system in fighting previously un-
834! encountered pathogens, and protect the organism on re-infection, relies on the ability to adapt
835! and learn throughout life. Immune functioning is determined partly by genetics [262], and
836! varies greatly between individuals. Individuals differ in their susceptibility to different types
837! of infections, such as bacterial, viral or fungal [262], and several single nucleotide
838! polymorphisms (SNPs) related to immune pathways have been described [262]. For example,
839! the IL-6 and IL-8 pathways appear to have large genetic variations between individuals, while
840!
841!

n al
IL-1 pathway has remained more conserved throughout evolution [262]. Recent research
emphasizes the importance of experience in shaping the adult immune response, similar to

o
si
842! what has been described for infants in the previous sections. In fact, most of the individual
843!
844!

r o vi
differences seen in immune function in adult humans stem from non-heritable changes [263;
264]. The immune system activates distinct cytokine patterns depending on the type of

P
845! infection, cytokines act in far-reaching cascades, and the adaptive immune system
846! continuously learns from experience via the adaptive immune system [265]. In theory, each
847! person thus possesses an immune system that is a product of the types, strengths and number
848! of infections, diseases and injuries encountered throughout life. Prior experience should thus
849! impact future immunological reaction patterns. Epidemiological studies on comorbidity and
850! risk factors for common disease give support to the idea that life-time immune challenges
851! affects disease susceptibility. A recent study shows that in patients with multimorbidity (in
852! this specific study more than 10 disease diagnoses), the incidence of lifetime infections,
853! inflammation, injuries and tumours was 7-10 times as common as in a primary health care
854! population [266]. Life-time accumulation of strong immune activation may thus potentially
855! lead to increased general disease susceptibility and comorbidity [266]. Furthermore, lifetime
856! inflammatory disease is a risk factor for developing neurodegenerative disease [267; 268;
857! 269]. A plausible mechanism is that the accumulation of inflammatory activity in the body
858! induces neuroinflammation in the brain, which in turn affect the function of the CNS [267].

! 25!
859! VIII-3. When adaptation becomes a liability
860! The process of perinatal programming posits that exposure to environmental factors
861! during a sensitive window of development, is able to program or have long-term
862! consequences on physiological systems later in life. A fundamental aspect of perinatal
863! programming is that developing organisms sense the early life environment and use this
864! information to establish homeostatic set points [270; 271]. This process of perinatal
865! programming has evolved as an adaptive mechanism enabling the foetus to constantly interact
866! with the maternal environment (via the placenta) and use this information as a forecast of the
867! environmental conditions it will eventually face postnatally. As such, preparing it to adjust its
868! physiological and behavioral need to match the requirements of the ex utero world [272]. In
869! this perspective, foetal programming is an example of predictive adaptive responses where the
870! foetus uses present cues to shape an adaptive phenotype to future environmental stimuli [31;
871! 273]. However, this adjustment can become maladaptive in the case where a mismatch
872! exists between the expected ex utero environment and the actual circumstances. More

l
873! importantly, when adverse events occur during a critical window of vulnerability of
874!
875!

sio n a
physiological systems that are still undergoing fine-tuning and plasticity, an individual may
become predisposed to high susceptibility and exaggerated sensitivity to environmental

i
876! stimuli later in life.
877!
878!
879!
880!
881!
P r o v
Correspondingly, the immune system and the HPA system adapt and change
according to the stressors that the individual encounters throughout life, in order to maintain
health and homeostasis. Pain is one of the most important survival signals available to us, and
a life without pain perception is often a short one, as can be seen in individuals with
882! congenital insensitivity to pain [274]. However, when the imprint of the different stressors
883! throughout life accumulate, interact and/or become prolonged, the consequence may be
884! detrimental for the pain system. For diseases like chronic pain, with such wide individual
885! variability in symptomology and treatment efficacy [8; 9], not only should comorbid disease
886! and stressful life events (i.e. concurrent with the pain) be considered when exploring the
887! pathophysiology, but past stressors as well. Here, we want to increase the awareness of the
888! profound effect of the immune system on the pain system from birth to old age, via
889! neuroimmune and neuroendocrine interactions. In other words, the faith of the pain system
890! starts in utero.
891!
892!

! 26!
893! IX- CONCLUSIONS
894!
895! In this review, we argue that the individual differences in the susceptibility to chronic
896! pain and success of treatment thereof may be the result of the persons prenatal history,
897! combined with childhood as well as lifetime experience. We have highlighted the biological
898! underpinnings and potential consequences on the pain system induced by the stress and
899! infectious/inflammatory load an individual is subjected to. The neuroimmune and
900! neuroendocrine interactions that affect the pain system start in the womb and modulate the
901! pain system throughout life. The modulations may be of both structural and functional nature,
902! and may be both adaptive and maladaptive. In order to understand individual differences in
903! pain, human studies of long-term effects of inflammatory stressors are needed.
904!
905!
906! Acknowledgements: Zouikr I is an overseas researcher under Postdoctoral Fellowship of
907! Japan Society for the Promotion of Science (JSPS). Karshikoff B is supported by AFA

l
908! Insurance and the Swedish Society of Medicine.
909!
910!
911! References:

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