c mice: Contribution of endogenous spinal cord TNFα and NFκB activation

Accepted Manuscript Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation Sergio M. Borghi, Victor Fattori, Kenji W. Ruiz-Miyazawa, Milena M. Miranda-Sapla, Rúbia Casagrande, Phileno Pinge-Filho, Wander R. Pavanelli, Waldiceu A. Verri PII:

S0009-2797(16)30685-8

DOI:

10.1016/j.cbi.2017.02.009

Reference:

CBI 7931

To appear in:

Chemico-Biological Interactions

Received Date: 7 December 2016 Revised Date:

27 January 2017

Accepted Date: 14 February 2017

Please cite this article as: S.M. Borghi, V. Fattori, K.W. Ruiz-Miyazawa, M.M. Miranda-Sapla, R. Casagrande, P. Pinge-Filho, W.R. Pavanelli, W.A. Verri, Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα and NFκB activation, Chemico-Biological Interactions (2017), doi: 10.1016/j.cbi.2017.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Leishmania (L). amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous

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spinal cord TNFα and NFκB activation

3 Sergio M. Borghia, Victor Fattoria, Kenji W. Ruiz-Miyazawaa, Milena M. Miranda-Saplaa, Rúbia

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Casagrandeb, Phileno Pinge-Filhoa, Wander R. Pavanellia, Waldiceu A. Verri Jra,*

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Londrina, Rod. Celso Garcia Cid PR445 KM380, 86057-970 Londrina, Paraná, Brasil

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Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Hospital Universitário,

Universidade Estadual de Londrina, Av. Robert Koch, 60, 86038-350 Londrina, Paraná, Brasil

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Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de

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*Corresponding author. Waldiceu A. Verri Jr., Department of Pathology, Biological Sciences Center,

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Londrina State University. Rod. Celso Garcia Cid, KM380, PR445, Londrina, Paraná, Brazil, Cx.

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Postal 10.011, CEP 86057-970. Fax: + 55 43 33714979, Tel: + 55 43 33714979, Email address:

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[email protected]; [email protected].

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Abstract

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Cutaneous leishmaniasis (CL) is the most common form of the leishmaniasis in humans. Ulcerative

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painless skin lesions are predominant clinical features of CL. Wider data indicate pain accompanies

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human leishmaniasis, out with areas of painless ulcerative lesions per se. In rodents, Leishmania

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(L.) major infection induces nociceptive behaviors that correlate with peripheral cytokine levels.

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However, the role of the spinal cord in pain processing after Leishmania infection has not been

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investigated. Balb/c mice received intraplantar (i.pl.) injection of Leishmania (L). amazonensis and

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hyperalgesia, edema, parasitism, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression, and

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NFκB activation were evaluated. The effects of intrathecal (i.t.) injection of morphine, TNFα, TNFα

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inhibitors (etanercept and adalimumab) and NFκB inhibitor (PDTC) were investigated. The present

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study demonstrates that Leishmania (L.) amazonensis infection in balb/c mice induces chronic

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mechanical and thermal hyperalgesia in an opioid-sensitive manner. Spinal cord TNFα mRNA

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expression increased in a time-dependent manner, peaking between 30-40 days after infection. At

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the peak of TNFα mRNA expression (day 30), there was a concomitant increase in TNFR1 and

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TNFR2 mRNA expression. TNFα i.t. injection enhanced L. (L.) amazonensis-induced hyperalgesia.

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Corroborating a role for TNFα in L. (L.) amazonensis-induced hyperalgesia, i.t. treatment with the

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TNFα inhibitors, etanercept and adalimumab inhibited the hyperalgesia. L. (L.) amazonensis also

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induced spinal cord activation of NFκB, and PDTC (given i.t.), also inhibited L. (L.) amazonensis-

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induced hyperalgesia, and spinal cord TNFα, TNFR1 and TNFR2 mRNA expression. Moreover, L.

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(L.) amazonensis-induced spinal cord activation of NFκB was also inhibited by etanercept and

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adalimumab as well as PDTC i.t. treatment. These results demonstrate that endogenous spinal cord

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TNFα and NFκB activation contribute to L. (L.) amazonensis-induced hyperalgesia in mice. Thus,

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spinal cord TNFα and NFκB are potential therapeutic targets for Leishmania infection-induced pain.

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Keywords: L. (L.) amazonensis; TNFα; NFκB; spinal cord; pain; hyperalgesia.

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1. Introduction 2

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American tegumentary leishmaniasis or cutaneous leishmaniasis (CL) is a public health and social problem neglected in many countries across the world. The infected female phlebotomine

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sandflies are vectors that transmit the parasite to humans. CL is the most common form of

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leishmaniasis affecting 0,7 to 1,3 million people worldwide. CL endemic regions include the

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Americas and the Mediterranean. CL is characterized by skin lesions distributed on exposed areas

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of the body that form ulcers, which result in life-long scars and severe disability. Although there are

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considerable variations in humans, cutaneous lesions are described as a painless papule or ulcer,

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covered by an adherent crust or dried exudate that can range from 0,5 to 3 cm in diameter in parts of

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the body such as face, arms and legs [1,2]. The finding that CL lesions are not painful is intriguing,

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with evidence suggesting that Leishmania infection may drive hypoalgesic processes at some time-

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points in the course of the disease [3]. However, an increasing number of clinical studies report

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painful areas in CL patients [4-11]. Clarification as to the pathophysiology of CL pain processing is

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clearly required.

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Evidence shows Leishmania infection induces hyperalgesia in domestic animals such as dogs

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[12,13], as well as when CL is experimentally induced in rats and mice [3,14-18]. The studies

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evaluating CL-induced pain in rodents assessed thermal hyperalgesia using the hot plate and tail

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flick tests. These studies are restricted to Leishmania (L.) major spp. and show up-regulation of

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hyperalgesic factors and cytokines, including nerve growth factor (NGF), interleukin (IL)-1β, tumor

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necrosis factor (TNF) α, IL-6 and keratinocyte-derived cytokine (KC)/chemokine (C-X-C motif)

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ligand 1 (CXCL1) [3,14-18]. IL-13 possesses analgesic effects in models of inflammatory and

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neuropathic pain [19-21]. However, IL-13 treatment intensifies L. (L.) major-induced hyperalgesia

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in rats [16], whilst in mice, IL-13 reduces L. (L.) major-induced hyperalgesia and IL-1β production

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[17]. These previous studies only investigated peripheral cytokines. As such, no previous studies

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explored any changes in leishmaniasis-induced spinal cord nociceptive signaling.

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TNFα is an important cytokine regulating inflammatory, neuropathic and cancer pain with 3

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indirect and direct nociceptive neuron actions [22-28]. TNFα, via TNFR1 and TNFR2, triggers

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nuclear factor kappa B (NFκB) activation in resident and migrating cells, peripherally and centrally,

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thereby inducing additional nociceptive molecules, including cytokines and lipid mediators [29].

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Consequently, inhibiting NFκB activation reduces inflammatory, neuropathic and cancer pain

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[28,30-33]. TNFα also has receptor-mediated direct neuronal effects, leading to neuronal

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depolarization and mechanical hyperalgesia [34]. Despite the importance of TNFα in hyperalgesia

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[22,24] and the pathophysiology of leishmaniasis as well as in macrophage killing of Leishmania

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[35-38], it requires investigation as to the role of spinal cord TNFα in leishmaniasis-induced pain.

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The current study evaluated the role of spinal cord TNFα and NFκB in L. (L.) amazonensis-induced

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hyperalgesia in a balb/c mouse CL model.

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2. Methods 4

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2.1. Ethics statement

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All animals were used according to the protocols approved by the Ethics Committee of the State University of Londrina, registered under the number 1067.2015.64. Animals’ care and handling

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procedures were carried following the Brazilian Council on Animal Experimentation (CONCEA)

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and in accordance with the International Association for Study of Pain (IASP) guidelines. All efforts

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were made to minimize the number of animals used and their suffering.

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113 2.2. L. (L.) amazonensis promastigotes

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Promastigotes forms of L. (L.) amazonensis (MHOM/BR/1989/166MJO) in the stationary

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growth phase were obtained from homogenate of popliteal lymph nodes of infected balb/c mice.

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The division of promastigote forms were cultured in 199 medium (Invitrogen-GIBCO)

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supplemented with 10% fetal bovine serum, 1 M Hepes, 0.1% L-glutamine, 1% penicillin-

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streptomycin solution, 10% sodium bicarbonate and 1% human urine [39]. Cultures were incubated

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in a BOD-type incubator at 25°C in 25-cm2 flasks [40]. In our laboratory, the use of 199 medium is

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well established as promastigote culture medium [40, 41] and follows the formulation media and

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chemical composition defined previously [42].

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2.3. Animals

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The experiments were conducted on male balb/c mice obtained from Fundação Oswaldo Cruz

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(FIOCRUZ), PR, Brazil, weighing between 20-25g, 4-6 weeks old. Mice were carefully kept under

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pathogen-free conditions. Mice were housed in standard clear plastic cages with free access to water

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and food, light/dark cycle of 12/12 h and controlled temperature, and were maintained in the 5

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vivarium of the Department of Pathology of State University of Londrina for at least one week

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before the experiments. Mice were used only once and were acclimatized to the testing room at

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least 1 hour before the experiments, which were conducted during the light cycle. At the end of

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experiments, mice were anesthetized with isoflurane 3% (Abbott Park, IL, USA) and terminally

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killed by cervical dislocation followed by decapitation.

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2.4. Experimental infection and general procedures

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Mice were infected subcutaneously in the plantar region of the right hind paw with L. (L.)

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amazonensis promastigote forms (1 x 105/20 µL) and were divided initially into control non-

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infected and infected groups for evaluation of mechanical and thermal hyperalgesia, edema,

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parasitism and spinal cord TNFα mRNA expression to determine the peak of each response in the

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following 40 days. Subsequently, TNFR1 and TNFR2 mRNA expression were determined at the

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30th day post-infection given this is the peak of TNFα mRNA expression. In the next set of

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experiments, mice were divided into a total of seven groups, namely: control non-infected, infected

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+ vehicle (saline), infected + morphine, infected + recombinant mouse TNFα, infected + etanercept,

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infected + adalimumab and, infected + PDTC. At the 30th day post-infection, mice were treated by

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intrathecal (i.t.) route with their respective drugs, with measurement of mechanical and thermal

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hyperalgesia and edema taken 1, 3, 5 and 7 h after the treatment. After the last measurement at the

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7th h, mice were euthanized, and spinal cord samples (L4-L6, the segment responsible for paw

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innervation) were collected for molecular assays (TNFα, TNFR1 and TNFR2 mRNA expression by

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qPCR; and NFκB activation by ELISA). The maximum period of behavioral tests was 40 days

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given that no ulcerated nodules or ulcerative skin lesions at the site of parasite inoculation were

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observed up to this time point, following recommended procedures [14]. A lower dose of the

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parasite was chosen since a dose of 1.5 x 107 L. (L.) major could induce non-ulcerated nodules after

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3 weeks and wide ulcerative lesions after 5 weeks of inoculation [4]. Preliminary studies using

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variable doses of L. (L.) amazonensis (1 x 107 and 1 x 108), in comparison to the dose used in the

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present investigation (1 x 105), confirmed that high doses of L. (L.) amazonensis induce the

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development of ulcerative lesions in the paw skin at the 40th day post-infection (data not shown).

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The following drugs were obtained from the sources indicated: saline solution 0.9% (Gaspar Viana S/A., Fortaleza, CE, Brazil, 5µL); morphine sulphate (Cristalia, São Paulo, SP, Brazil, 3

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nmol/5µL in saline); recombinant mouse TNFα (eBioscience, San Diego, CA, USA, 1 ng/5µL in

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saline); etanercept (Enbrel®, Wyeth, São Paulo, SP, Brazil, 10 ng/5µL in saline); adalimumab

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(Humira®, Abbott Laboratórios do Brasil LTDA, São Paulo, SP, Brazil, 30 ng/5µL in saline); and

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pyrrolidine dithiocarbamate (PDTC, Sigma Chemical Co., St. Louis, MO, USA, 300 µg/5µL in

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saline). The doses described above were based on previous studies [28,43,44], except for

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adalimumab, which was selected according to the dose of etanercept), to make the doses of these

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TNFα inhibitors equivalent.

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2.6. Mechanical hyperalgesia

Mechanical hyperalgesia was evaluated in mice according to the detailed methodology described

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previously [45]. In the first set of experiments, mechanical hyperalgesia was evaluated before and

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during 40 days after experimental infection, and subsequently, in the next phase, it was evaluated

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only at day 30, before and after (1-7 h) i.t. treatment with vehicle, morphine, TNFα, etanercept,

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adalimumab, and PDTC. Briefly, in a quiet room, mice were placed in acrylic cages (12 x 10 x 17

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cm3) with wire grid floors, 15-30 min before the start of testing. The test consisted of evoking a

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hind paw flexion reflex with a hand-held force transducer (electronic anaesthesiometer; Insight,

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Ribeirão Preto, SP, Brazil) adapted with a 0.5 mm2 polypropylene tip. The results are expressed by 7

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delta (∆) withdrawal threshold (in g), calculated by subtracting the zero-time mean measurements

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from the mean measurements (indicated time points) after experimental infection. The basal

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mechanical withdrawal threshold was 9.5 ± 0.1 g (mean ± S.E.M. of 18 groups, 6 mice per group)

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before experimental infection in both control non-infected and infected groups treated with vehicle

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or with the respective inhibitors described above. There was no difference of basal mechanical

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withdrawal thresholds between groups in the same experiment.

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Thermal hyperalgesia was evaluated in mice as described previously [46]. In the first set of

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experiments, thermal hyperalgesia was evaluated before and during 40 days after experimental

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infection, and subsequently, in the next phase, it was evaluated only at day 30, before and after (1-7

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h) i.t. treatment with vehicle, morphine, TNFα, etanercept, adalimumab, and PDTC. In brief, mice

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were placed in a hot plate apparatus (EFF 361, Insight, Ribeirão Preto, SP, Brazil) maintained at

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55±1 °C. The reaction times were registered when the animal jump as well as lick or flinch the paw.

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A maximum latency (cut-off) was set at 15 s to avoid tissue damage.

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2.8. Edema

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The paw edema was measured in mice as described previously [46]. In the first set of

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experiments, paw edema was evaluated before and during 40 days after experimental infection, and

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subsequently, in the next phase, it was evaluated only at day 30, before and after (1-7 h) i.t.

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treatment with vehicle, morphine, TNFα, etanercept, adalimumab, and PDTC. The measurements

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were made using a calliper (Digmatic Calliper, Mitutoyu Corporation, Kanagawa, Japan). Paw

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thickness was expressed as the difference (∆ mm) between the values obtained just before (basal)

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and after the experimental infection. 8

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2.9. DNA extraction and parasite quantification

211 Quantitative PCR (qPCR) for parasite quantification was performed to determine tissue parasite

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load in infected group at days 5, 10, 20, 30 and 40 after infection. A hind paw plantar fragment was

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collected and DNA was extracted using trizol reagent following manufacturer’s instructions (Life

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Technologies). The DNA purity was measured with a spectrophotometer (Multiskan GO Microplate

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Spectrophotometer, ThermoScientific, Vantaa, Finland) and the wavelength absorption ratio

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(260/280 nm) was between 1.6 and 1.8 for all preparations. Quantitative PCR (qPCR) was

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performed by using Platinum SYBR Green qPCR SuperMix UDG with ROX reagent (Invitrogen

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Corporation, New York, NY) with 100 ng total genomic DNA (gDNA). Parasite quantification was

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performed using JW11 (forward, 5´-CCTATTTTACACCAACCCCCAGT-3´) and JW12 (reverse,

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5´-GGGTAGGGGCGTTCTGCGAAA-3´) Leishmania specific primers. The results were presented

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as parasite DNA expression, using β-actin as a reference gene to normalize data.

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2.10. Reverse Transcription and quantitative polymerase chain reaction (RT-qPCR)

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RT-qPCR was performed following the protocol as described previously [47]. Spinal cord samples were collected at day 30 after the infection with L. amazonensis and homogenized in trizol

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reagent for total RNA isolation according to the manufacturer’s direction. The purity of total RNA

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was measured with a spectrophotometer and the wavelength absorption ratio (260/280 nm) was

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between 1.8 and 2.0 for all preparations. Reverse transcription of total RNA to cDNA and qPCR

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were carried out using Go Taq® 2-Step RT-qPCR system (Promega) following the manufacturer’s

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instructions. The relative gene expression was measured using the comparative 2-(∆∆Cq) method. The

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primers used were TNFα, sense: 5´-TCTCATCAGTTCTATGGCCC-3´, antisense: 5´-

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GGGAGTAGACAAGGTACAAC-3´; TNFR1, sense: 5´-TCCGCTTGCAAATGTCACA-3′,

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antisense: 5′-GGCAACAGCACCGCAGTAC-3′; TNFR2, sense: 5′-

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GGAACCTGGGTACGAGTGCCA-3′, antisense: 5′-GCGGATCTCCACCTGGTCAGT-3′ and β-

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actin, sense: 5´- AGCTGCGTTTTACACCCT TT-3´, antisense: 5´-

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AAGCCATGCCAATGTTGTCT-3´. The expression of β-actin RNA was used as a reference gene to

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normalize data.

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2.11. NFκB activation

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The determination of NFκB activation in spinal cord samples was performed following the protocol as described previously [47]. Spinal cord samples were collected at day 30 after the

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infection with L. amazonensis and homogenized in ice-cold lysis buffer (Cell Signaling Technology,

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Beverly, MA, USA). The homogenates were centrifuged (200 g x 10 min x 4°C) and the

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supernatants used to assess the levels of total and phosphorylated NFκB p65 subunit by ELISA

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using PathScan kits (Cell Signaling Technology, Beverly, MA, USA) according to the

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manufacturer’s directions. The results represent the total p65/phospho-p65 ratio measured at 450

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nm.

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2.12. Statistical analysis

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Results are presented as means ± SEM of measurements made on six mice in each group per

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experiment and are representative of two separate experiments. Two-way analysis of variance

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(ANOVA) was used to compare the groups and doses at all times when responses were measured at

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different times after the stimulus injection. Analyzed factors were treatments, time and time versus

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treatment interaction, and when interaction was significant, a one-way ANOVA followed by

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Tukey’s post hoc was performed for each time point. Differences between responses were evaluated

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by one-way ANOVA followed by Tukey’s post hoc for data of single time point. Statistical 10

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differences were considered significant when P < 0.05.

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3. Results 11

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3.1. L. (L.) amazonensis intraplantar (i.pl.) administration induces chronic mechanical and thermal

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hyperalgesia, and paw edema

290 Mechanical hyperalgesia, thermal hyperalgesia and paw edema were measured before and 2 to

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40 days after i.pl. infection with L. (L.) amazonensis (Fig. 1A-C). Samples of paw skin tissue were

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collected at days 5, 10, 20, 30 and 40 after infection to evaluate L. (L.) amazonensis parasitism by

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qPCR (Fig. 1D). L. (L.) amazonensis induced significant long-lasting mechanical hyperalgesia

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compared to control non-infected animals starting at day 10, reaching maximal mechanical

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hyperalgesia at the 26th day, which remained significant until the 40th day (Fig. 1A). Thermal

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hyperalgesia presented a delayed profile compared to mechanical hyperalgesia, with a significant

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reduction of the thermal threshold of infected mice, compared to non-infected mice, starting at the

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18th day after the infection and remaining significant until the 40th day post-infection (Fig. 1B).

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Although the inflammatory response in the paw tissue showed a tendency to be more evident in

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infected animals from the 2nd to 32th days, when compared to the control non-infected mice,

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significant differences in paw edema were observed only from the 32th to 40th days after the

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infection (Fig. 1C). Representative images of days 8, 16, 24, 32 and 40 of control and infected paws

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are presented at the bottom of Fig. 1C. Parasitism analysis in paw skin tissue samples showed a

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time-dependent gradual increase from day 5 peaking at day 40 (Fig. 1D). Therefore, L. (L.)

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amazonensis i.pl. injection induces chronic inflammatory hyperalgesia associated with delayed paw

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edema and parasitism increase over time.

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3.2. Morphine i.t. administration inhibits L. (L.) amazonensis-induced mechanical and thermal

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hyperalgesia without affecting paw edema

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Thirty days after L. (L.) amazonensis i.pl. injection, mice received i.t. treatment with vehicle or 12

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morphine (3 nmol) (Fig. 2). Morphine treatment significantly inhibited L. (L.) amazonensis-induced

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mechanical and thermal hyperalgesia between 1 to 7 h after treatment compared to the vehicle-

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treated group (Fig. 2A and B, respectively). Morphine treatment did not alter L. (L.) amazonensis-

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induced paw edema (Fig. 2C). This result indicates that in experimental CL, at least in the case of L.

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(L.) amazonensis, the altered mechanical and thermal threshold responses have a nociceptive

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characteristic, given such inhibition by a known analgesic.

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3.3. L. (L.) amazonensis induces TNFα, TNFR1 and TNFR2 mRNA expression in the spinal cord

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Samples of L4-L6 segment of spinal cord were collected 5-40 days after L. (L.) amazonensis i.pl.

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injection and processed for RT-qPCR (Fig. 3). Although no statistically significant difference was

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observed between infected and non-infected mice, TNFα mRNA expression started to increase (up

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to 5 fold) at day 5 and remained stable until day 20. Importantly, spinal cord TNFα mRNA levels

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significantly increased in L. (L.) amazonensis-infected mice at the 30th and 40th days compared to

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non-infected mice (Fig. 3A). Considering the time points of TNFα mRNA expression, TNFR1 and

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TNFR2 mRNA expression were evaluated at 30th day post-infection (Fig. 3B and C, respectively).

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L. (L.) amazonensis induced significant increase of spinal cord TNFR1 (Fig. 3B) and TNFR2 (Fig.

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3C) mRNA expression, compared to non-infected mice. Therefore, L. (L.) amazonensis paw

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infection induces significant up regulation of TNFα, TNFR1 and TNFR2 mRNA expression in the

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spinal cord.

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3.4. Exogenous TNFα i.t. injection enhances L. (L.) amazonensis-induced mechanical and thermal

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hyperalgesia, without affecting paw edema

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Recombinant mouse TNFα (1 ng) was injected by i.t. route in order to assess whether spinal cord TNFα could contribute to L. (L.) amazonensis-induced hyperalgesia and edema (Fig. 4). TNFα i.t. 13

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injection induced mechanical (Fig. 4A) and thermal (Fig. 4B) hyperalgesia (1-7 h) with a slight

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increase in paw volume at 1 h (Fig. 4C) in non-infected animals. In L. (L.) amazonensis infected

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mice, TNFα i.t. injection induced a slight, but significant, enhancement of mechanical hyperalgesia

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at 5 and 7 h (Fig. 4A) and of thermal hyperalgesia at 7 h (Fig. 4B) without affecting paw edema

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(Fig. 4C). These results, together with the L. (L.) amazonensis-induced spinal cord TNFα, TNFR1

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and TNFR2 mRNA expression, suggest that TNFα can contribute to enhanced L. (L.) amazonensis-

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induced hyperalgesia. Of note, it is likely that the TNFα enhancement of the L. (L.) amazonensis-

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induced hyperalgesia was limited, given that hyperalgesia was already significantly elevated by the

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infection.

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3.5. Soluble TNFR2 (etanercept) i.t. treatment inhibits L. (L.) amazonensis-induced mechanical and

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thermal hyperalgesia without affecting paw edema

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The effect of etanercept (10 ng) i.t. treatment in L. (L.) amazonensis-induced hyperalgesia and edema was also investigated. Etanercept i.t. significantly inhibited L. (L.) amazonensis-induced

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mechanical hyperalgesia between 3 to 7 h after the treatment (Fig. 5A), and thermal hyperalgesia

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between 1-7 h after the treatment (Fig. 5B), compared to the vehicle-treated group, with no

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etanercept effect on L. (L.) amazonensis-induced paw edema (Fig. 5C). These results demonstrate

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that inhibiting endogenous spinal cord TNFα reduces L. (L.) amazonensis-induced hyperalgesia.

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3.6. Anti-TNFα antibody, adalimumab i.t., inhibits L. (L.) amazonensis-induced mechanical and

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thermal hyperalgesia without affecting paw edema

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Corroborating the effect of etanercept, the i.t. treatment with the anti-TNFα antibody

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adalimumab (30 ng) inhibited L. (L.) amazonensis-induced mechanical and thermal hyperalgesia

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from 3 to 7 h after the treatment compared to the vehicle-treated group (Fig. 6A and 6B, 14

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respectively). I.t. treatment with adalimumab did not affect L. (L.) amazonensis-induced paw edema

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(Fig. 6C). Therefore, two different approaches targeting TNFα (a soluble TNFR2 and anti-TNFα

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antibody) demonstrated that spinal cord TNFα contributes to L. (L.) amazonensis-induced

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hyperalgesia.

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3.7. Inhibition of spinal cord NFκB with PDTC i.t. treatment diminishes L. (L.) amazonensis-

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induced mechanical hyperalgesia, thermal hyperalgesia and paw edema

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The increase of spinal cord expression of the hyperalgesic cytokine TNFα suggests possible activation of the major pro-inflammatory transcription factor NFκB. To address this point, at day 30

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of L. (L.) amazonensis infection mice received one i.t. treatment with PDTC (300 µg), an inhibitor

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of NFκB. Mechanical hyperalgesia, thermal hyperalgesia and paw edema were evaluated 1, 3, 5 and

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7 h after treatment (Fig. 7). PDTC inhibited L. (L.) amazonensis-induced mechanical hyperalgesia

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(Fig. 7A), thermal hyperalgesia (Fig. 7B) and paw edema (Fig. 7C) between 1-7 h, 3-7 h, and 3-7 h,

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respectively. These data indicate a prominent role of endogenous spinal cord NFκB in the

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pathophysiology of L. (L.) amazonensis-induced pain as well as the regulation of peripheral

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inflammation.

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3.8. Reciprocal interaction of spinal cord TNFα and NFκB in L. (L.) amazonensis infection

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PDTC i.t. treatment at day 30 post-infection inhibited L. (L.) amazonensis-induced TNFα,

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TNFR1 and TNFR2 mRNA expression (Fig. 8A). Fig. 8B shows that the i.t. injection of exogenous

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TNFα induced spinal cord NFκB activation, and L. (L.) amazonensis also induced NFκB activation

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at day 30, which was inhibited by i.t. treatments with analgesic doses of etanercept, adalimumab

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and PDTC. TNFα was not able to increase L. (L.) amazonensis-induced NFκB activation, possibly

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because the infection already induced a maximal effect (Fig. 8B). This suggests that there is a 15

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reciprocal stimulation between NFκB and TNFα in the spinal cord of L. (L.) amazonensis-infected

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mice that contributes to hyperalgesia.

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4. Discussion 16

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The clinical characteristics of CL have been widely portrayed as involving the painless evolution of non-ulcerative and/or ulcerative skin lesions [1,2]. However, increasing number of cases of

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patients reporting pain independently of the region of the body infected during the course of

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leishmaniasis have been described around the world [4-11]. Therefore, investigating leishmaniasis-

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induced pain is a growing field. The present study demonstrates that L. (L.) amazonensis induces

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mechanical and thermal hyperalgesia in balb/c mice by a mechanism dependent on endogenous

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spinal cord TNFα and NFκB.

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In experimental models using mice and rats, nociceptive behaviors related to Leishmania infection can be easily observed [3,14-18]. Evidence shows that peripheral production of cytokines

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associates with thermal hyperalgesia in CL infected rodents. All these studies used L. (L.) major

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infection model [3,14-18], and whether other species of Leishmania induce hyperalgesia has

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remained to be investigated. Other important species of Leishmania include L. (L.) amazonensis,

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which is classified as a New World component of the genus Leishmania, subgenus Leishmania

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(Euleishmania phylogenetic lineage) belonging to the Leishmania (Leishmania) mexicana complex.

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L. (L.) amazonensis is one of the main causes of human CL in countries such as Brazil, Bolivia and

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Venezuela, where it is responsible for localized and diffuse clinical forms of CL, as well as human

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visceral leishmaniasis [39,48,49]. The present study is the first to demonstrate that i.pl. inoculation

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of L. (L.) amazonensis induces chronic mechanical and thermal hyperalgesia in balb/c mice. A

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parasite load of 1x105 was used, given that higher doses induce paw tissue ulceration and increase

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paw volume (data not show). Also low doses of L. (L.) major induce short-lived thermal

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hyperalgesia while high doses induce long-lasting thermal hyperalgesia [15]. A possible explanation

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is that a high parasite load causes a loss of tissue ability to respond to a nociceptive stimulus, with

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data here showing that paw edema increased with higher parasite load. On the other hand,

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mechanical hyperalgesia and thermal hyperalgesia increase before the peak of parasite load,

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suggesting that pain is a clinical sign that depends on the course of the infection. This might also be

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an explanation for the painless ulcers, which are present in tissue with extensive parasitic

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destruction that drives an inability to respond to a nociceptive stimulus. The present data confirm

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that the nature of L. (L.) amazonensis-induced changes is nociceptive, given that treatment with the

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classical opioid morphine reduced both mechanical and thermal hyperalgesia. Not all types of pain

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are alleviated by opioids, but the inhibition by this class of analgesics is indicative of changes in

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nociceptive processes [50].

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L. (L.) amazonensis induced a time-dependent increase in TNFα mRNA expression levels in the

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L4-L6 segment of the spinal cord, which is the site for spinal innervation of the paw. TNFα mRNA

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expression peaked at day 30 post parasite inoculation. At this time point, L. (L.) amazonensis also

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increased the mRNA of the TNFα receptors, TNFR1 and TNFR2. In agreement with these qPCR

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data, TNFα i.t. injection enhanced L. (L.) amazonensis-induced mechanical and thermal

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hyperalgesia without affecting paw edema at day 30. The enhancement of hyperalgesia was

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significant, but not dramatic likely due to the inflammatory processes driven by the infection per se.

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The role of spinal TNFα is further supported by the striking inhibition of L. (L.) amazonensis-

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induced mechanical and thermal hyperalgesia following etanercept i.t., a soluble TNFR2, and

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adalimumab i.t., an anti-TNFα antibody. Such data indicate clinical utility from the targeting of

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endogenous TNFα in regard to the inhibition of ongoing L. (L.) amazonensis-induced hyperalgesia.

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In agreement with the present data, the systemic treatment with atenolol inhibited L. (L.) major-

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induced thermal hyperalgesia and TNFα production in the infection foci, indicating a role for TNFα

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inhibition in the analgesic effects of atenolol [18].

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TNFα has a role in pain regulation more widely, including the enhancement of mechanical

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hyperalgesia, thermal hyperalgesia and overt pain-like behaviors (paw flinching, paw licking and

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abdominal contortions) in models of inflammation, neuropathy and cancer. Accordingly, data also

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supports the participation of both TNFR1 and TNFR2, depending on the experimental model

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[22,25-29,51-54]. TNFα can directly and indirectly activate nociceptive neurons [22,24,34]. TNFα

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can induce mechanical and thermal hyperalgesia via the mitogen activated protein kinase (MAPK) 18

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pathways, including the p38 MAPK pathway that modulates Nav1.8 and Nav1.9 channels

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[34,55,56] and the activation of transient receptors potential cation channels subfamily V member 1

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(TRPV1) and subfamily A member 1 (TRPA1) [34,57]. TNFα may therefore impact on nociceptive

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neuron ionic regulation. TNFα, via TNFR1, is a major inducer of the canonical NFκB pathway [58,59]. This interaction is

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reciprocal, as NFκB activation induces TNFα gene transcription, with TNFα being a crucial

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downstream NFκB effector during inflammation [60,61]. The present data indicate that endogenous

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TNFα is a relevant effector of NFκB in Leishmania infection given that i.t. treatment with the NFκB

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inhibitor PDTC abolished L. (L.) amazonensis-induced TNFα, TNFR1 and TNFR2 mRNA

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expression in the spinal cord. The inhibition of TNFα with etanercept and adalimumab significantly

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inhibited L. (L.) amazonensis-induced hyperalgesia and NFκB activation as well. However, the fact

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that anti-TNF therapies did not abolish the hyperalgesia and did not affect paw edema suggests that

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other inflammatory hyperalgesic molecules that activate NFκB or are produced upon NFκB

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activation may participate in the regulation of L. (L.) amazonensis-induced paw edema in addition

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to TNFα. In fact, TNFα i.t. injection in naïve mice induced only a small edema in the paw that was

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detectable solely at 1h after stimulus. Therefore, although peripheral TNFα contributes to paw

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edema [62], spinal cord TNFα at a hyperalgesic dose is not a major contributor to peripheral

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neurogenic paw edema. Furthermore, spinal cord NFκB is also responsive to other signaling

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pathways and molecules such as oxidative stress, toll-like receptors and other cytokines including

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IL-1β and IL-33 [28,59,63-68], which suggests that as varied inflammatory and hyperalgesic

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molecules converge to the activation of NFκB, the contribution of this transcription factor in

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regulating inflammation and hyperalgesia is wider than the role of one cytokine. Lining up with this

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rationale, the i.t. treatment with PDTC diminished L. (L.) amazonensis-induced mechanical

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hyperalgesia and thermal hyperalgesia, but also presented a greater effect than targeting TNF in

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reducing paw edema. Notably, the inhibition of peripheral inflammatory paw and articular edema as

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well as associated hyperalgesia by PDTC were previously observed in a models of superoxide

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anion-induced paw inflammation in mice and adjuvant-induced arthritis in rats [46,69]. It is not

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unlikely that spinal cord NFκB activation induces an important retrograde nociceptive neuron

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mechanism that accounts for peripheral edema. For instance, nociceptive neurons release

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neuropeptides such as calcitonin gene-related peptide (CGRP) and substance P that induce edema

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[68]. Thus, further studies are necessary to clarify the anti-edematogenic effect of i.t. PDTC

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treatment [46] and whether additional hyperalgesic molecules contribute to the spinal cord

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processing of nociceptive information in L. (L.) amazonensis infection.

The present data also shows that L. (L.) amazonensis infection and TNFα i.t. injection per se

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induce spinal cord activation of NFκB, as observed by a decreased ratio of total

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NFκB/phosphorylated NFκB. Targeting TNFα with etanercept and adalimumab therefore inhibited

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L. (L.) amazonensis-induced NFκB activation in the spinal cord. TNFα i.t. injection could not

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dramatically enhance L. (L.) amazonensis-induced spinal cord NFκB activation, given the near

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maximal effect induced by infection per se including an increase of TNFR1 and TNFR2 mRNA

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expression, as well as TNFα mRNA expression.

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5. Conclusions 20

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In summary, this is the first report demonstrating that L. (L.) amazonensis infection induces pain in balb/c mice. L. (L.) amazonensis-induced pain depends on endogenous spinal cord TNFα and

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NFκB activation, which can be targeted by currently available anti-TNF therapies, including

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etanercept and adalimumab. Given that a single i.t. administration of anti-TNF or NFκB inhibitor

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attenuated ongoing chronic L. (L.) amazonensis-induced pain, these data indicate a clinically

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relevant therapeutic utility of inhibitors of these pathways. Fig. 9 summarizes the schematic

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proposed mechanism for reciprocal interaction of spinal cord endogenous TNFα and NFκB in L.

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(L.) amazonensis-induced hyperalgesia in balb/c mice. These preclinical findings contribute to

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address significant questions related to biological events observed in human leishmaniasis and may

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represent great advances in the complex understanding about the pathophysiology of pain during

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the evolution of lesions in CL.

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Acknowledgements 21

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This work was supported by grants from Coordenadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secretaria da

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Ciência, Tecnologia e Ensino Superior (SETI), Departamento de Ciência e Tecnologia / Ministério

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da Saúde (DECIT/MS), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq),

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Fundação Araucária, Secretaria de Saúde do Estado do Paraná (SESA) and Governo do Estado do

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Paraná (Brazil). We thank Fundação Osvaldo Cruz (FIOCRUZ) from Curitiba, Paraná, Brazil, for

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providing the balb/c mice for the study. The authors appreciate the helpful technical assistance of

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Pedro S. R. Dionísio Filho during the process of infection of the animals.

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The authors declare no competing financial interests.

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Authors’ contributions

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All authors listed have substantially contributed to the manuscript as follows: S.M.B., R.C. and

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W.A.V.J. designed the study, planned experiments and analyze the data. S.M.B., V.F., K.W.R.M. and

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M.M.M.S. performed the experiments. W.R.P. and P.P.F. provided essential materials and

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contributed to the process of culture and infection with L. (L.) amazonensis. S.M.B. and W.A.V.J.

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wrote the manuscript. W.A.V.J. supervised the study. All authors read and approved the manuscript.

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[69] J.G. Luo, X.L. Zhao, W.C. Xu, X.J. Zhao, J.N. Wang, X.W. Lin, T. Sun, Z.J. Fu, Activation of spinal NF-κB/p65 contributes to peripheral inflammation and hyperalgesia in rat adjuvant-induced arthritis, Arthritis Rheumatol. 66 (2014) 896-906. DOI: 10.1002/art.38328. [70] F.A. Pinho-Ribeiro, W.A. Verri Jr., I.M. Chiu, Nociceptor sensory neuron-immune interactions in pain and inflammation, Trends Immunol. 38 (2017) 5-19. DOI: 10.1016/j.iy.2016.10.001.

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Figure captions 27

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Fig. 1. L. (L.) amazonensis infection induces pain in balb/c mice. Mechanical hyperalgesia (A),

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thermal hyperalgesia (B) and paw edema (C) were measured in non-infected and infected (1x105

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parasites) mice over 40 days. Representative images of the evolution of paw edema in infected in

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comparison to control (non-infected) animals are presented on days 8, 16, 24, 32 and 40 (C;

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bottom). Parasitism in infected mice was evaluated by qPCR from day 5 to day 40 post-infection

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(D). Results are presented as mean ± SEM of six mice per group per experiment and are

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representative of two separate experiments. [*p < 0.05 compared to non-infected mice (one-way

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ANOVA followed by Tukey test)].

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Fig. 2. Morphine i.t. treatment inhibits L. (L.) amazonensis-induced hyperalgesia, but not paw

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edema. Mechanical (A) and thermal (B) hyperalgesia and paw edema (C) were measured in non-

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infected and infected mice on day 30 after the infection, and subsequently, infected mice received

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i.t. injection of morphine (opioid agonist, 3 nmol) or vehicle for new measurements of mechanical

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and thermal hyperalgesia and paw edema 1, 3, 5 and 7 h after the treatment. Results are presented as

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mean ± SEM of six mice per group per experiment and are representative of two separate

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experiments. [*p < 0.05 compared to non-infected mice; #p < 0.05 compared to vehicle treated-

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infected mice (one-way ANOVA followed by Tukey test)].

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Fig. 3. L. (L.) amazonensis induces spinal cord TNFα, TNFR1 and TNFR2 mRNA expression.

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Time-course expression of TNFα mRNA was measured 5, 10, 20, 30 and 40 days after the infection

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and compared to non-infected animals (A). TNFR1 (B) and TNFR2 (C) mRNA expression were

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measured in non-infected and infected mice at day 30 post-infection (peak of TNFα mRNA

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expression). Results are presented as mean ± SEM of six mice per group per experiment and are

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representative of two separate experiments. [*p < 0.05 compared to non-infected mice (one-way

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ANOVA followed by Tukey test)]. 28

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Fig. 4. TNFα i.t. injection enhances L. (L.) amazonensis-induced mechanical and thermal

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hyperalgesia, but not paw edema. Mechanical (A) and thermal (B) hyperalgesia and paw edema (C)

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were measured in non-infected and infected mice on day 30 post-infection, and subsequently, mice

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received i.t. injection of recombinant mouse TNFα (1 ng) or vehicle for measurement of mechanical

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and thermal hyperalgesia and paw edema at 1, 3, 5 and 7 h after the treatment. Results are presented

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as mean ± SEM of six mice per group per experiment and are representative of two separate

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experiments. [*p < 0.05 compared to non-infected mice; #p < 0.05 compared to vehicle treated-

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infected mice (one-way ANOVA followed by Tukey test)].

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Fig. 5. Etanercept i.t. treatment inhibits L. (L.) amazonensis-induced hyperalgesia, but not paw

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edema. Mechanical (A) and thermal (B) hyperalgesia and paw edema (C) were measured in non-

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infected and infected mice on day 30 after the infection, and subsequently, infected mice received

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i.t. injection of etanercept (TNFα soluble receptor, 10 ng) or vehicle for measurement of mechanical

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and thermal hyperalgesia and paw edema at 1, 3, 5 and 7 h after the treatment. Results are presented

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as mean ± SEM of six mice per group per experiment and are representative of two separate

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experiments. [*p < 0.05 compared to non-infected mice; #p < 0.05 compared to vehicle treated-

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infected mice (one-way ANOVA followed by Tukey test)].

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Fig. 6. Adalimumab i.t. treatment inhibits L. (L.) amazonensis-induced hyperalgesia, but not paw

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edema. Mechanical (A) and thermal (B) hyperalgesia and paw edema (C) were measured in non-

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infected and infected mice on day 30 after the infection, and subsequently, infected mice received

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i.t. injection of adalimumab (antibody anti-TNFα, 30 ng) or vehicle for measurement of mechanical

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and thermal hyperalgesia and paw edema at 1, 3, 5 and 7 h after the treatment. Results are presented

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as mean ± SEM of six mice per group per experiment and are representative of two separate

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experiments. [*p < 0.05 compared to non-infected mice; #p < 0.05 compared to vehicle treated29

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infected mice (one-way ANOVA followed by Tukey test)].

864 Fig. 7. PDTC i.t. treatment inhibits L. (L.) amazonensis-induced hyperalgesia and paw edema.

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Mechanical (A) and thermal (B) hyperalgesia and paw edema (C) were measured in non-infected

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and infected mice on day 30 after the infection, and subsequently, infected mice received i.t.

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injection of PDTC (NFκB inhibitor, 300 µg) or vehicle for measurement of mechanical and thermal

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hyperalgesia and paw edema at 1, 3, 5 and 7 h after the treatment. Results are presented as mean ±

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SEM of six mice per group per experiment and are representative of two separate experiments. [*p

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< 0.05 compared to non-infected mice; #p < 0.05 compared to vehicle treated-infected mice (one-

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way ANOVA followed by Tukey test)].

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Fig. 8. L. (L.) amazonensis induces spinal cord NFκB activation in a TNFα reciprocal manner. L.

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(L.) amazonensis-induced spinal cord TNFα, TNFR1 and TNFR2 mRNA expression were inhibited

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by PDTC (NFκB inhibitor, 300 µg, after 7 h of i.t. treatment) at day 30 post-infection (A). TNFα

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and L. (L.) amazonensis induced spinal cord NFκB activation, as well as L. (L.) amazonensis-

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induced spinal cord NFκB activation, were inhibited by etanercept (TNFα soluble receptor, 10 ng),

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adalimumab (antibody anti-TNFα, 30 ng) and PDTC (NFκB inhibitor, 300 µg) after 7 h of i.t.

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treatment. TNFα (1 ng) i.t. injection did not enhance L. (L.) amazonensis-induced spinal cord NFκB

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activation at day 30 post-infection after 7 h of i.t. treatment. Results are presented as mean ± SEM

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of six mice per group per experiment and are representative of two separate experiments. [*p < 0.05

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compared to non-infected mice; #p < 0.05 compared to vehicle treated-infected mice (one-way

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ANOVA followed by Tukey test)].

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Fig. 9. Schematic proposed mechanism by which Leishmania (L.) amazonensis induces chronic

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hyperalgesia in balb/c mice. The peripheral infection with L. (L.) amazonensis generates an immune

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response in dermal tissue leading to the activation and/or recruitment of leukocytes, which release 30

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hyperalgesic mediators that sensitize primary nociceptor sensory neurons culminating in spinal cord

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activation of NFκB as well as TNFα, TNFR1 and TNFR2 mRNA expression. Spinal cord TNFα

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induces hyperalgesia. Therapies targeting spinal cord NFκB (PDTC) and/or TNFα (adalimumab and

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etanercept) are effective and promising approaches to control L. (L.) amazonensis infection-induced

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pain.

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ACCEPTED MANUSCRIPT Leishmania (L.) amazonensis induces hyperalgesia in balb/c mice: Contribution of endogenous spinal cord TNFα α and NFκ κB activation

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Sergio M. Borghia, Victor Fattoria, Kenji W. Ruiz-Miyazawaa, Milena M. Miranda-Sapla a, Rúbia

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Casagrandeb, Phileno Pinge-Filhoa, Wander R. Pavanellia, Waldiceu A. Verri Jra,*

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Highlights

L. (L.) amazonensis intraplantar injection induces chronic hyperalgesia

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Spinal cord TNFα mRNA increased gradually over the infection course

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Endogenous spinal cord TNFα and NFκB contribute to L. (L.) amazonensis-induced pain

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Targeting spinal cord TNFα and NFκB inhibited L. (L.) amazonensis-induced pain

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TNFα and NFκB act reciprocally in the spinal cord during Leishmania-induced pain

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