Role of Brain Transmigrating Neutrophils in Depression-Like Behavior During Systemic Infection
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Molecular Psychiatry (2014) 19, 599–606 & 2014 Macmillan Publishers Limited All rights reserved 1359-4184/14 www.nature.com/mp ORIGINAL ARTICLE Role of brain transmigrating neutrophils in depression-like behavior during systemic infection A Aguliar-Valles1,3, J Kim1, S Jung1, B Woodside2 and GN Luheshi1 Peripheral inflammation induces transmigration of interleukin (IL)-1b-expressing neutrophils to the brain. We investigated the possibility that this presents a new route of immune-to-brain communication by assessing their role in sickness behaviors relevant for mood disorders. Mice treated with lipopolysaccharide (LPS) developed despair-like behavior, and administration of an anti- polymorphonuclear antibody abolished LPS-induced despair-like and asocial behaviors, which correlated with the levels of IL-1b expression in the brain. These behavioral changes were directly mediated by the energy-regulating hormone, leptin. Increasing the concentration of endogenous leptin during obesity exacerbated, whereas its neutralization using a specific antiserum attenuated sickness behaviors and importantly the neutrophil transmigrating process. Our results indicate a role for peripheral neutrophils in conveying inflammatory signals to the brain, which appears to be dependent on the energy status of the organism. This constitutes a novel mechanism of immune-to-brain communication relevant to mood disorders. Molecular Psychiatry (2014) 19, 599–606; doi:10.1038/mp.2013.137; published online 15 October 2013 Keywords: depression; IL-1b; infection; inflammation; leptin; neutrophil; obesity; sickness behavior INTRODUCTION to-brain communication through these routes depends on the Chronic inflammation and the associated display of brain-regulated transient increase in circulating cytokine levels that is usually sickness behaviors have been implicated in the etiology of a associated with acute responses. It is unlikely that these routes of number of psychiatric illnesses.1–3 It is of critical importance then to communication explain the sustained inflammatory conditions in identify those processes that contribute to the maintenance of the the brain that maintain sickness behaviors, suggesting the existence inflammatory response in the brain. Here we present data to of alternate mechanisms. suggest that leptin-regulated neutrophil transmigration to the brain, Recently, we have demonstrated that severe systemic, LPS- following an acute inflammatory challenge,4 represents a novel induced inflammation triggers neutrophil recruitment into the brain mode of immune-to-brain communication. This may contribute to a parenchyma, through a process modulated by the energy-regulating 4 sustained neuro-inflammatory response and the ensuing prolonged hormone leptin. Given that leukocyte recruitment is normally display of chronic depression-like sickness behaviors. associated with targeted invasion of the area of infection or Several lines of evidence support a link between inflammation inflammation, the physiological significance of these cells entering and the etiology of mood disorders, including similarities between the intact brain remains unknown. We sought to determine whether sickness-type behavioral responses and symptoms of depression neutrophils entering the brain, following peripheral infection, act as (that is, weakness, malaise, listlessness, inability to concentrate, vehicles for delivering inflammatory mediators to propagate the lethargy, decreased interest in immediate environment and reduced acute inflammatory mechanisms in the brain. appetite).1,2 Indeed, sickness-type behaviors are recapitulated by acute administration of the Gram-negative bacterial cell wall product 5 MATERIALS AND METHODS lipopolysaccharide (LPS) and reversed by chronic antidepressant Animals treatment before the administration of the inflammatory challenge.6 There are also similarities in the underlying mechanisms leading to Adult (8-week) male mice (C57BL/6J; The Jackson Laboratories, Bar Harbor, ME, USA) were group housed (four per cage) with ad libitum access to water both, namely, the involvement of cytokines such as interleukin (IL)- and standard laboratory chow (Teklad Global 18% protein rodent diet, 1b, IL-6 and tumor necrosis factor (TNF)-a. The circulating levels of Harlan Laboratories, Madison, WI, USA), with an average body weight of 20 g these mediators increase significantly following acute infection and (unless otherwise specified). They were kept on a light:dark cycle of 12:12 h have a critical role in relaying the inflammatory signal from the (lights on at 0800 hours) and at an ambient temperature of 21±2.1 1C. The periphery to the brain and the subsequent activation of brain institutional Animal Care Committee pursuant to the Canadian Council of mechanisms regulating sickness-type behavioral responses.3,7,8 Animal Care guidelines approved all experimental procedures. Cytokines gain access to the brain via a number of pathways including, active transport across the blood–brain barrier (BBB), Treatment and experimental procedures activation of endothelial cells lining the BBB or through areas devoid Mice were treated with LPS (intraperitoneal, 2.5 mg kg À 1 Escherichia coli of barrier protection such as the circumventricular organs.7 Immune- 0111:B4; cat: L-2630; Sigma-Aldrich, Oakville, ON, Canada) or pyrogen-free 1Department of Psychiatry, Douglas Mental Health University Institute, McGill University, Montreal, QC, Canada and 2Groupe de recherche en neurobiologie comportementale/ Center for Studies in Behavioral Neurobiology, Concordia University, Montreal, QC, Canada. Correspondence: Professor GN Luheshi, Department of Psychiatry, Douglas Mental Health University Institute, McGill University, 6875 LaSalle Boulevard, Montreal, H4H 1R3 QC, Canada. E-mail: [email protected] 3Current address: Department of Physiology, Universite´ de Montre´al and Department of Biochemistry and Goodman Cancer Research Centre, McGill University, Montreal, QC, Canada. Received 3 July 2013; revised 20 August 2013; accepted 6 September 2013; published online 15 October 2013 Neutrophils and depression-like sickness behavior A Aguliar-Valles et al 600 saline (IP, 4 ml kg À 1).4 For the time course experiment, mice underwent APC-conjugated anti-pro-IL-1b or anti-TNF-a (both at 1:333, eBiosciences) behavioral testing at either 24, 48, 72, 96 or 120 h after treatment and were were used. Staining was analyzed using a FACScalibur apparatus (BD killed immediately after each time point (n ¼ 4–11/group). Saline-treated Biosciences, Mississauga, ON, Canada) and FlowJo software (Tree Star Inc., mice were tested and killed at the same time points and pooled into one Ashland, OR, USA). group (n ¼ 2–3 per time point, final n ¼ 13). Neutropenia was induced with a rabbit anti-polymorphonuclear (aPMN) antibody (Accurate Chemical and Scientific Corporation, Westbury, NY, Neutrophil isolation USA) at 2 mg kg À 1 and injected 0 and 24 h after saline or LPS treatment; From naı¨ve anesthetized mice (pentobarbital, IP, 70 mg kg À 1), both femurs normal rabbit serum (NRS) was used as control (Accurate Chemical and were removed and bone marrow was flushed out with 3 ml of RPMI-1640 Scientific Corporation, 2 mg kg À 1). Leptin was neutralized using sheep medium using a 21-G needle. The collected cells were centrifuged and anti-mouse leptin antiserum (LAS; S4159, NIBSC, Potters Bar, UK) incubated in red blood cells lysis buffer (eBiosciences) at room temperature and normal sheep serum (NSS; NIBSC) was used as control (both IP at for 5 min. Lysis reaction was stopped with Hank’s balanced salt solution, 25 ml kg–1, 0 and 24 h after LPS or saline treatment4,9). Mice in diet-induced and cells were run through a 70-mm cell strainer. For brain neutrophil obesity (DIO) were fed with a high-fat diet (HFD) for 5–6 weeks (TD.06414 isolation, saline-perfused brains from LPS-treated mice were dissociated as Adjusted Calories Diet 60/Fat, Harlan Laboratories). Control mice were fed before by passage through a 70-mm pore-size cell strainer and then with a low-fat diet (LFD; TD.06416 Adjusted Calories Diet 10/Fat, Harlan centrifuged in 37% Percoll. Ly6G þ neutrophils from either bone marrow or Laboratories). After reaching criterion weight (15% difference between brain preparations were immunomagnetically separated following the HFD and LFD), mice were treated with LPS or saline as described above. In manufacturer’s protocol (Miltenyi Biotec, Cambridge, MA, USA). all cases, behavioral testing was conducted 48 h after treatment and mice were killed immediately afterwards (15–25 min after the 48 h time point). A cohort of HFD- and LFD-fed mice was used to test for leptin resistance, Immunocytochemistry –1 for which they received an injection of either NSS or LAS (25 ml kg ) and Bone marrow and brain transmigrated neutrophils (isolated as for FACS) food intake was monitored for 24 h. To measure food intake, animals were were plated on 15 mm diameter poly-L-lysine-coated coverslips in 24 well habituated to eat powdered food from a jar for 7 days before the test for plates. After, cells were fixed in 4% paraformaldehyde for 20 min at room leptin resistance. Food jars were weighed before and 24 h after serum temperature. Fixed cells were washed in 0.1 M phosphate-buffered saline injection and intake was calculated by subtracting the 24 h jar weight from (PBS), and permeabilized in 0.2% Triton X-100 in PBS. To minimize the one before antiserum injection and correcting for caloric content of the background and nonspecific binding, slides were