Systemic Staphylococcus aureus infection in restraint stressed mice modulates impaired immune response resulting in improved behavioral activities

    Systemic Staphylococcus aureus infection in restraint stressed mice modulates impaired immune response resulting in improved behavioural activities Sayantika Mahanti, Arnab Majhi, Kiran Kundu, Anirban Basu, Biswadev Bishayi PII: DOI: Reference:

S0165-5728(15)30038-2 doi: 10.1016/j.jneuroim.2015.09.003 JNI 476203

To appear in:

Journal of Neuroimmunology

Received date: Revised date: Accepted date:

10 March 2015 7 September 2015 9 September 2015

Please cite this article as: Mahanti, Sayantika, Majhi, Arnab, Kundu, Kiran, Basu, Anirban, Bishayi, Biswadev, Systemic Staphylococcus aureus infection in restraint stressed mice modulates impaired immune response resulting in improved behavioural activities, Journal of Neuroimmunology (2015), doi: 10.1016/j.jneuroim.2015.09.003

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ACCEPTED MANUSCRIPT Systemic Staphylococcus aureus infection in restraint stressed mice modulates impaired immune response resulting in improved behavioural activities

Department of Physiology, Immunology laboratory. University of Calcutta, University Colleges

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Sayantika Mahantia, Arnab Majhia, Kiran Kundub, Anirban Basub, and Biswadev Bishayia*

of Science and Technology, 92 APC Road, Calcutta-700009, West Bengal, India.

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National Brain Research Centre, Manesar, Haryana 122051, India.

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*Corresponding Author:

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Prof. Biswadev Bishayi, Ph.D.

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Fax: 91-33-2351-9755

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Telephone No: 91-33-2350-8386 Extn: 225

E mail: [email protected]

Abbreviations:

HPA, hypothalamus-pituitary-adrenal; COX, Cyclooxygenase; iNOS,

inducible nitric oxide

synthase; HSP, heat shock proteins; SOD, super oxide dismutase; GC, glucocorticoids; LPS, lipopolysaccharides; IAEC, Institutional Animal Ethics Committee; CFU, colony forming units; TSST, toxic shock syndrome toxin; PBS, phosphate buffered saline; AChE, esterase; MPO, myeloperoxidase; GSH, reduced glutathione.

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acetylcholine

ACCEPTED MANUSCRIPT Abstract Stress leads to immune malfunction and increased susceptibility to infection resulting in

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impaired cognitive behavior and depression. Working with an animal model of S. aureus

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infection and restraint stress we demonstrated impaired immune response and altered behavior

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against the S. aureus infection after exposure to acute or chronic restraint stress. This enhanced the resistance of mice to S. aureus infection via inhibiting the production of pro-inflammatory

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cytokines, free radicals, and up regulating corticosterone and anti-inflammatory cytokines production, resulting in altered exploratory behavior, compared to non-stressed infected group

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(p<0.05), thereby helping the animals to recover from depressive-like symptoms due to stress.

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Staphylococcus aureus.

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Keywords: Behavior, Corticosterone, Cytokines, Restraint stress, Immunity, Nitric oxide,

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ACCEPTED MANUSCRIPT 1. Introduction Rodent restraint stress has been used in modeling human disease and is regularly

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employed in studying symptoms associated with human depressive disorders (Dantzer et al.,

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2008; Maccari and Morley-Fletcher, 2007). Although a great deal of work has been done to characterize the biological and immunological events following acute psychological stress

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exposure, the vast majority of research utilizing restraint stress is conducted over short

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experimental windows and conveys results obtained at single time points following completion of stress exposure (Buynitski and Mostofsky, 2009; Houshyar et al., 2001; Jankord and Herman,

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2008).

Glucocorticoids (GC) are the main endogenous anti-inflammatory agents in vivo,

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interfering with virtually every aspect of immune and inflammatory response. Changes in physiological regulation of GC response to inflammation could, therefore have very important

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consequences for inflammatory diseases (Da Silva, 1995). It has been reported that restraint stress resulted in depression-like symptoms in adult rats; but surprisingly those rats that suffered a bacterial infection early in life, had blunted corticosterone responses to the stressor and were remarkably protected from depression-like symptoms compared to controls (Bilbo et al., 2008). Moreover, prolonged periods of stress were found to be detrimental through excess production of neuroendocrine mediators that could dampen the immune responses to invasive pathogens (Radek, 2010). Thus stress may have significant impacts on immune responses via GCs that may alter the susceptibility of hosts to various diseases. Much of the research addressing the effects of infection and inflammation on behavior has used lipopolysaccharide (LPS) to induce immune system activity. However there are few reports of other agents like viable S. aureus or toxic shock syndrome toxin (TSST) superantigen,

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ACCEPTED MANUSCRIPT producing behavioral changes in comparison to those seen with LPS or E. coli (Nicolson and Haier, 2009; Mahanti et al., 2013).

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Acetylcholinesterase (AChE) is an enzyme that terminates the neurotransmission at

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cholinergic synapses by splitting the neurotransmitter acetylcholine (ACh) to choline and acetate (Tripathi and Srivastava, 2008). It has been reported that inhibition of AChE activity protects

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animals from neurodegeneration and brain damage initiated due to infection of the CNS or

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immune activation (Habila et al., 2012). Thus, the activity of this enzyme following stress and live S. aureus infection would help us to understand the underlying mechanisms of the

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involvement of these neurotransmitter molecules to mediate their anti-inflammatory role during stress and infection.

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In a broader perspective, since the immune system is intricately associated with learning, memory and neural plasticity (Yirmiya and Goshen, 2011), stress induced disruption could be

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critical for such processes. Changes in behavior produced by infections and cytokines are also dependent on the existing physiological state of the infected organism. However, differences in the sensitivity and reactions of the immune system stimulated by viable S. aureus in response to restraint stress were never studied. It is unclear, therefore, if the previously reported observations would hold ground if organisms are challenged with living, infectious bacteria. The aim of the current investigation was to test the effect of restraint stress on an in vivo model of infection. Viable Staphylococcus aureus (S. aureus), a ubiquitous bacteria, was chosen because earlier studies have indicated that Staphylococcus aureus, stimulated a more consistent and robust inflammatory response (Rossi-George et al., 2004). 2. Materials and Methods 2.1 Animals

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ACCEPTED MANUSCRIPT All experiments involving animals were conducted according to the protocols that had been approved by the Institutional Animal Ethics Committee (IAEC), Department of Physiology,

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University of Calcutta, under the guidance of Committee for the purpose of control and

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supervision of experiments on animal (CPCSEA) [Approval Number: 820/04/ac/CPCSEA.2010 dated: 16.11.2011], Ministry of Environment and Forest, Govt. of India. This study did not

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involve any invasive study using human subjects. Male BALB/c (6-8 weeks) mice were obtained

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from a registered breeder in our department and were used for all studies. All animals were maintained and utilized in accordance with recommendations from the IAEC and were provided

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with food and water ad libitum. Mice were housed 4-5 per cage and maintained on a 12 hr light: 12 hr dark cycle (lights on at 08.00 am) in a temperature controlled room (22 ± 2oC).

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2.2 Restraint Stress

Due to the inherent correlation of the circadian rhythm with corticosterone production,

15.00 hrs.

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mice were exposed to restraint stress each day during a fixed time period, from 09.00 hrs to Restraint was applied in a separate room to eliminate the possible effects of

vocalizations or pheromones on the control mice. Non restrained mice were left in their home cages in a noise-free environment, with food and water during the restraint period. Mice (n=15) per group were restrained each day according to procedures described in previous reports (Delgado-Morales et al., 2012). Briefly, following the acclimatization period, individual animals were randomly assigned to six groups. Group I: Acute stress alone; was subjected to restraint stress only once for 6 h (acute stress); after 6 h of restraint stress, mice were returned to their home cages and allowed free access to food and water for the duration of the experiment. Group II: non-stressed S. aureus infected only; was infected with viable S. aureus cells once intravenously through the tail vein. Group III: Acute stress + S. aureus infection; after subjecting

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ACCEPTED MANUSCRIPT the mice to acute restraint stress for the same period, they were allowed to acclimatize for 5 min and then infected with viable S. aureus cells, intravenously, into the tail vein. After recording

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and analyzing the behavioral changes 4 hours post infection, mice from both the groups

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(n=15/group) were sacrificed at 24 h post infection. Group IV: chronic stress only. In this group, mice were exposed to restraint stress every day, on same time 09.00 h to 15.00 h, for a

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period of 21 days. Group V: Chronic stress + S. aureus infection; animals in this group were

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subjected to restraint stress every day, at the same time, for a period of 21 days and after completion of the stress period, mice were infected once with viable S. aureus through the tail

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vein. After observing the behavioral changes 4 hours post infection, mice from both the groups (n=15 per group), were also sacrificed at 24 h post infection. Group VI: Control mice; was not

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subjected to restraint stress and received sterile saline 24 h prior to sacrifice. Restraint stress was performed in well ventilated 50 mL polystyrene tubes and food and water were not provided

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during the restraint period. Restraint animals were also allowed to move freely in their cages until the next restraint cycle. 2.3 Bacterial culture

S. aureus (strain # AG-789) were obtained from Apollo Gleneagles Hospital, Calcutta, and was maintained in our laboratory. We have extensively used few clinical isolates of S. aureus in a mouse model of arthritis with short term but non-lethal infection (Mal et al., 2012). Bacteria were cultured on blood agar (5% human erythrocytes) for 24 h and then re-incubated on blood agar for another 24 h. Before experimentation bacteria were grown overnight at 37 oC in 5 ml of Luria Bertani (LB) broth, diluted in fresh broth and cultured until mid-logarithmic phase of growth. Bacteria were harvested, washed in sterile phosphate buffered saline (PBS) and adjusted to the desired inoculums (Yao et al., 1997) spectrophotometrically before infection (OD620 = 0.2

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ACCEPTED MANUSCRIPT = 5 x 107 cells/ml for S. aureus) and the colony forming unit (CFU) were confirmed by serial dilution and culture on blood agar.

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2.4 S. aureus challenge

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Live S. aureus (5 x 107 CFU/mouse) in a total volume of 200 µl was injected intravenously once into the tail vein of acute and chronic stress + S. aureus challenged group

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immediately after releasing the animal from the restraint condition. Both acute and chronic

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stressed mice, used as stress control to observe the effect of S. aureus infection on restraint stress, were injected equal volume of sterile saline once immediately after completion of the

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stress period. Non-stressed control mice also received equal volume of sterile saline once (Mahanti et al., 2013; Wang et al., 2008).

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2.5 Recovery of bacteria from blood

Working under a sterile laminar flow hood, we performed bacterial CFU count in blood

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24 hours post-infection. Blood samples from mice of different groups were obtained by cardiac puncture under ether anesthesia, and collected in tubes containing an anticoagulant solution of 4% sodium citrate at selected intervals. A serial 10-fold dilution of blood samples in PBS (pH 7.2) was then spread on mannitol agar plates and viability of S. aureus was determined by method described earlier (Mal et al., 2013). 2.6 Determination of level of corticosterone in serum Blood samples were collected at 24 hours post infection, in anticoagulant free tubes and centrifuged at 1,000 x g for 10 min. Serum obtained was distributed into separate tubes. Serum for assessment of cytokine levels were stored at -70°C until use and that for determining corticosterone concentration, using a corticosterone EIA kit from Cayman Chemical as per the manufacturer’s instructions, was done on the same day of blood collection. For each study,

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ACCEPTED MANUSCRIPT corticosterone levels were determined, in duplicate, in a single run to avoid inter-assay variability, and intra-assay variability was less than 10%.

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2.7 Acetyl cholinesterase (AChE) assay in brain

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A decrease in AChE activity leading to an increase in cholinergic activity of the brain may help to enhance cognitive functions. In order to estimate this cholinergic activity among

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different groups, the whole intact brain was removed carefully and placed in the petri dish, over

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ice. The brain was washed with ice-chilled normal saline repeatedly to clean. A 10% (w/v) homogenate of brain samples was prepared first by homogenizing in a polytron homogenizer at

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a speed of 9500 rpm with three intervals of a few seconds in between the runs, using a sodium phosphate buffer (30 mM, pH 7.0). Sodium phosphate buffer was taken in a volume half that of

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the final volume required for the 10% homogenate. The assay of AChE in the above mentioned

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supernatant was performed by the modified Ellman’s method (Ellman et al., 1961) using

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acetylthiocholine iodide as substrate at a final concentration of 1 mmol/L. A kinetic profile of the enzyme activity was studied spectrophotometrically at 412 nm at an interval of 15 s. The assay for each sample was run in duplicate and each experiment was performed three times. Protein was estimated in the range 0.01– 0.1 mg/ml in the brain samples by the Folin–Lowry method, using bovine serum albumin (BSA) as standard at a concentration of 1 mg /ml. 2.8 Estimation of hydroxy steroid dehydrogenase (HSD) enzyme activity 3βHSD and 17βHSD enzyme activities reflect the intracellular levels of biologically active steroid hormones in the adrenal glands. Thus, adrenal glands were homogenized separately in 20% spectroscopic glycerol containing 5 mM potassium phosphate and 1 mM ethylene-diamine-tetra-acetic acid (EDTA) at a tissue concentration of 100 mg/ml of

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ACCEPTED MANUSCRIPT homogenizing mixture followed by centrifugation at 4oC at 10,000g for 30 min and enzyme activities were estimated by methods described in earlier studies (Mukhopadhyay et al., 2010).

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2.9 Cytokine ELISA

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Serum TNF-α, IFN-γ, IL-6 and IL-10 concentrations were done by Sandwich ELISA and calculated based on the standard curve. Serum cytokine levels were expressed in pg/ml,

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following manufacturer’s protocol. (Ray Biotech, Inc.) For each study, cytokine levels were

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determined, in duplicate, in a single run to avoid inter-assay variability, and intra-assay variability was less than 10-12%. The minimum detectable dose of the cytokines TNF-α, IFN-γ,

2.10 Estimation of NO Production

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IL-6 and IL-10 was 60 pg/ml, 5 pg/ml 2 pg/ml and 45 pg/ml respectively.

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NO plays a complex role in free radical mediated injury in the brain during exposure to stressful condition and/or during bacterial infection. Thus, the concentration of nitrite in the brain

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tissues was measured as an index for NO production. Equal weights of the brains of infected mice were homogenized in sterile PBS (1ml), supernatants were collected, and analyzed for NO production by modified Greiss method as described earlier (Nandi et al., 2010). Briefly, nitrate was converted to nitrites with β-nicotinamide adenine dinucleotide phosphate (NADPH; 1.25 mg/ml) and nitrate reductase followed by addition of Griess reagent. The reaction mixture was incubated at room temperature for 20 minutes followed by addition of TCA. Samples were centrifuged, clear supernatants were collected, and optical density was recorded at 550 nm. The amounts of NO produced were determined by calibrating a standard curve using sodium nitrite. 2.11 Determination of blood brain barrier (BBB) leakage Disruption of the BBB as a result of free radicals induced peroxidation generated during stress and infection, results in increased vascular permeability, brain edema and secondary brain

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ACCEPTED MANUSCRIPT damage.

Thus the permeability of the BBB was quantitatively evaluated by detection of

extravasated Evans blue dye. Briefly, 2% Evans blue dye in saline was injected intraperitoneally,

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and after 4 hours mice were deeply anesthetized with Nembutal and transcardially perfused until

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colorless perfusion fluid was obtained from the right atrium. After decapitation, brain tissue was removed, weighed, and homogenized. The supernatant was obtained by centrifugation, and

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protein concentration was determined. Evans blue intensity was determined by a microplate

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reader at 550 nm. Calculations were based on the external standards dissolved in the same solvent. The amount of extravasated Evans blue dye was quantified as micrograms per milligram

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protein (Mittal et al., 2010).

2.12 Neutrophil accumulation in spleen

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Myeloperoxidase (MPO) enzyme activity was analyzed as index of neutrophil infiltration in the spleen tissue, because it is closely related with the number of neutrophil present in the

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tissue. Spleen tissues were separated from mouse and were first homogenized in 10 volumes of a buffer containing 20 mM Tris–HCl, (pH 7.0), EDTA, sucrose and protease inhibitor cocktail and then centrifuged at 2,000×g for 10 min at 4°C. The supernatants were sterilized by passing through a Milipore filter (0.45 μm pore size) and stored at −80°C until analysis. Protein levels in the tissue homogenates were determined by Bradford method. An aliquot of the supernatant was allowed to react with a solution of O-dianisidine dihydrochloride (0.167 mg/ml) and 0.005% H2O2. The rate of change in absorbance was measured spectrophotometrically at 405 nm. MPO enzyme activity has been defined as the concentration of enzyme degrading 1 μM of peroxide/min at 37°C and was expressed as change in absorbance/min.mg of protein (Majumdar et al., 2011).

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ACCEPTED MANUSCRIPT 2.13 Assay of antioxidant enzymes from liver, brain and adrenal To determine the activity of antioxidant enzymes in neutralizing the reactive oxygen

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species (ROS) molecules produced due to infiltration of leukocytes at the site of infection, we

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estimated the antioxidant activity in liver, brain and adrenal tissues which were key sites to understand the modulation of HPA axis due to stress and infection. In order to do so, the liver,

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brain and adrenal tissues were separately homogenized in 10 volumes of 50 mM phosphate

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buffer (pH 7.4) on ice for 30 s using a power driven polytron homogenizer. The homogenate was transferred into centrifuge tubes and centrifuged at 9000 x g at 4°C for 20 min. The supernatant

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was used to measure activity of antioxidant enzymes and the amount of protein present. 2.13.1 Measurement of reduced glutathione level (GSH)

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Reduced glutathione content (as acid soluble sulfhydryl) was estimated by its reaction

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with DTNB (Ellman’s reagent) following the method of Sedlak and Lindsay with some

1968).

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modifications. Values were expressed as nmoles of GSH per mg protein (Sedlak and Lindsay,

2.13.2 Superoxide dismutase (SOD) activity Tissue homogenate (100μl) was mixed with 1.5 ml of a Tris-EDTA-HCl buffer (pH 8.5), then 100μl of 7.2 mM pyrogallol was added and the reaction mixture was incubated at 25 oC for 10 min. The reaction was terminated by the addition of 50 μl of 1M HCl and measured at 420 nm. One unit was determined as the amount of enzyme that inhibited the oxidation of pyrogallol by 50%. The activity was expressed as U/mg protein (Dutta et al., 2009). 2.13.3 Catalase activity Decomposition of H2O2 due to catalase activity was assayed by the decrease in absorbance of H2O2 at 240 nm. Catalase activity in the cell free homogenate was determined

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ACCEPTED MANUSCRIPT spectrophotometrically by measuring the decrease in H2O2 concentration at 240 nm. At time zero, 1.8 ml of each homogenate was mixed with 0.2 ml of a phosphate buffer containing 10

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mmol H2O2. One ml of the mixture was immediately added to a cuvette and placed into a

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spectrophotometer. Catalase activity was observed via degradation of H2O2 as determined by a decrease in UV light absorbance over time. Measurement of absorbance was taken at 15 sec

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interval after addition of the homogenate to hydrogen peroxide buffer. Units of catalase activity

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present in 1 ml of homogenate were calculated (Dutta et al., 2009).

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2.14 Expression of Cox-2, iNOS, SOD-1, HSP 70, HSP90 in hypothalamic tissue Expression of cyclooxegenase-2 (cox-2), inducible nitric oxide synthase (iNOS),

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superoxide dismutase (SOD-1), heat shock protein (HSP) 70 and 90 in hypothalamic tissues was

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determined by immunoblotting. Production of NO correlates with the level of expression of the

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iNOS gene. Moreover induction of iNOS has been directly correlated with production of cox-2 during stress and infection which may be partly regulated by the NO pathway. HSPs are also synthesized in significant quantity to protect eukaryotic cells from various insults during periods of stress caused by infection, inflammation or similar events. Thus estimation of these protein levels in the tissue was determined by immunoblotting after estimating the protein level in the tissue homogenates by the Bradford method. Twenty micrograms of each sample was electrophoresed on polyacrylamide gel and transferred onto a nitrocellulose membrane. After blocking with 7% skimmed milk, the blots were incubated overnight at 4°C with primary antibodies against cox-2 (1:1000; Chemicon, USA), HSP 90 (a kind gift from Dr. N. R. Jana, NBRC), HSP 70, SOD1 (1:1000; Santa Cruz Biotechnology, USA). After extensive washes in PBS–Tween, blots were incubated with appropriate secondary antibodies conjugated with peroxidase (Vector Laboratories, USA). The blots were again washed in PBS–Tween and 12

ACCEPTED MANUSCRIPT processed for development using chemiluminescence reagent (Millipore, USA). The images were captured and analyzed using Chemigenius, Bioimaging System (Syngene, Cambridge, UK).

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The blots were then stripped (30 min at 50°C in 62.5mmol/l Tris–HCl pH 6.8, 2% sodium

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dodecyl sulfate, and 100 mM β-mercaptoethanol) and reprobed with anti-β-tubulin (Santa Cruz Biotechnology,, USA) to determine equivalent loading of samples (Dutta et al., 2010).

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2.15 Activity and exploratory behavior in open-field test

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Activity and exploratory behavior of mice were observed in open field for a 5-min period, and the following behavioral activities were scored:

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TNSCP- total number of squares crossed in periphery, DTP- distance traveled in the periphery, LRC- latency to reach centre, DI - duration of immobilization, TNR – total number of rearing,

freezing.

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ULC – unable to locate central region, TNG – total number of grooming, TNF – total number of

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The open field was constructed from plywood (painted white) and measured 72 x 72 cm, with 36 cm high walls. One of the walls was made of clear Plexiglas so that mice were visible within the maze. Blue lines were drawn on the floor, and the lines were visible through the clear Plexiglas floor. The lines divided the floor into 16 x 18 x 18 cm squares, and these lines were used to measure motor activity. A central square was drawn in the middle of the open field and was used to measure exploration (Brown et al., 1999). The central square is used because some mouse strains have high locomotor activity and cross the lines of the test chamber many times during a test session. The central square has sufficient space surrounding it to give meaning to the central location as being distinct from the outer locations (Carrey et al., 2000). A digital camera was fixed to the ceiling 2.1 m above the apparatus and was used to measure line crosses. 2.16 Anxiety Test-Elevated plus maze (EPM)

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ACCEPTED MANUSCRIPT One of the most popular tests of anxiety-like behavior in mice is the EPM, in which the reduced number of entries or time spent in the open arms of the EPM suggests the operation of anxiety-

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like processes. The elevated plus maze was built as described previously elsewhere (Lister,

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1987). This maze was made of Plexiglas, and consisted of a central square (5 x 5 cm), from which radiated four arms (5 x 45 cm). Two of the arms had clear Plexiglas walls (15 cm high)

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around the edge (closed arms); whereas the other two arms did not have walls (open arms). A

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0.25 cm high edge was placed around the open arms to prevent mice from falling from the maze. A white line was drawn half way (22.5 cm) along each of the four arms to measure motor

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activity. The maze was elevated 45 cm above the floor on a plus-shape plywood stand. A camera- was fixed to the ceiling 2.1 m above the apparatus to measure entries and duration in the

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center, open and closed zones. We repeated this test three times due to our different results on EPM from other investigations and the results represent an average of the three test sessions.

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The observer measured: (a) TSC-total time spent at centre, b) TSCA-the time spent in the closed arms, (c) TSOA-time spent in open arm, (d) NRCA- number of rearing in closed arms, during the 5-min test period. An entry was defined as all four paws in the arm. The elevated plus-maze was thoroughly cleaned with distilled water following the testing of each animal to avoid possible biasing effects due to odor clues left by previous mice. The total number of open arms entered, as well as the total number of closed arms entered was used as indexes of general locomotor activity 2.17 Effect of acute and chronic restraint stress on humoral immune response In a separate set of experiment to determine any alteration in the humoral immune responses, acute and chronic restraint stressed mice were challenged with heat killed S .aureus (i. p.) as described elsewhere (Bishayi and Ghosh, 2007).

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ACCEPTED MANUSCRIPT 2.18 Statistical analysis The data are expressed as mean ± SD and statistical significance was assessed with an

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one-way ANOVA. Analysis of variance was done using the statistical software Origin, version 8.

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Post hoc comparison of means was done by Scheffe’s test. Significance for all tests was set at P

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< 0.05.

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3. Results

3.1 Restraint stressed mice were partially resistant to S. aureus infection

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To explore the effects of bacterial infection on acute and chronic restraint stress, we stressed mice using the physical restraint stress model as described in Materials and Methods

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before the mice were infected with S. aureus. Interestingly, the acutely stressed mice showed increased resistance to post infection with a single dose of pathogenic S. aureus as compared

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with the S. aureus infected group (Table 1). However, the chronic stressed mice was also able to reduce the bacterial burden in blood compared to S. aureus infected group, which indicates that the chronic stressed mice were also resistant to S. aureus infection (Table 1). 3.2 Effect of acute and chronic restraint stress on the alteration in humoral immune response

To assess possible mechanisms of this bacterial translocation, levels of one of the main defense mechanisms, antibody (Ab) titer, were assessed 24 hours post-immunization. Circulating Ab represents a first-line defense mechanism against pathogens in the mucosal surfaces by means of, among other mechanisms, agglutinating bacteria and preventing them from binding to epithelial cells. Chronic stress before S. aureus immunization induced a significant decrease in serum antibody level when compared with other experimental groups (Table 2). In acute restraint

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ACCEPTED MANUSCRIPT stress and heat killed S. aureus immunized serum; agglutination titer was obtained at lowest antibody concentration (1:1024) whereas serum from pre-exposure of chronic restraint stress

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followed by S. aureus immunization showed early agglutination (1:256). (Table 2)

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3.3 Effect of S. aureus infection after acute and chronic restraint stress on serum corticosterone concentration

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The concentrations of corticosterone in mice that were not exposed to stress were low in

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the serum. However, exposure to 6 h of restraint stress (acute stress) or to repeated restraint for longer period of time (chronic stress) significantly increased the serum corticosterone

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concentrations (P<0.05). When acute restraint mice were infected with live S. aureus (single dose) the corticosterone concentration was increased significantly in serum than the only stressed

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mice. However, when chronic stressed mice were infected with viable S. aureus, these mice were able to increase the serum corticosterone level when compared to acute stressed plus S. aureus

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infected group or to S. aureus infected unstressed mice (P<0.05) (Fig. 1A). 3.4 Effect of S. aureus infection after acute and chronic restraint stress on the brain AChE activity

AChE-specific activity in (µM/min /mg tissue protein) in the chronic stressed group was significantly low as compared to the acute stressed group. AChE activity was also observed to be significantly low in the control animals compared to all other experimental groups. (P<0.05) However, when acute restraint stressed mice were infected with viable S. aureus the mice showed significantly elevated brain AChE activity when compared with the acute stressed alone group or with the unstressed S. aureus infected group. Brain AChE activity was also increased in the chronic stressed plus S. aureus infected mice with respect to chronic stressed only group or with the unstressed S. aureus infected group (Fig. 1B)

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ACCEPTED MANUSCRIPT 3.5 Effects of S. aureus infection after acute and chronic restraint stress on steroidogenic enzyme (3βHSD and 17βHSD) activities in adrenal gland

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Mice undergone chronic restraint stress showed elevated 3βHSD activity in the adrenal

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gland when compared with the acute stressed mice as well as the control. However, when the mice belonging to either acute or chronic restraint stress group was infected with S. aureus, the

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3βHSD activity in the adrenal gland increased significantly compared to the stressed group or

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with the unstressed S. aureus infected group and the most significant change was found in the chronic stressed mice receiving S. aureus infection (Fig.1C). Mice underwent restraint for longer

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period showed elevated 17βHSD when compared with acutely stressed mice and the control group. After inoculation of the stressed mice with viable S. aureus, chronic stressed mice showed

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significant increase in 17βHSD activity in the adrenal gland compared to acute stress + S. aureus infected group or with the unstressed S. aureus infected group. (P<0.05) (Fig. 1D).

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3.6 Effect of S. aureus infection after acute and chronic restraint stress on serum IL-6, IL10, TNF-α and IFN-γ level

All the estimated level of cytokines showed significant difference with respect to control animals (P<0.05).

Serum IL-6 level in the chronic restraint stressed mice was increased significantly with respect to acute stressed mice. However, when the acute restraint stressed mice were given S. aureus infection it showed significant elevation in serum IL-6 level when compared with the only acute stressed group of mice or with the unstressed S. aureus infected group. (Fig. 2A). Serum IL-10 level in the chronic restraint stressed mice was increased significantly with respect to acute stressed mice. However, when the acute restraint stressed mice were given S. aureus infection it showed significant elevation in serum IL-10 level when compared with the

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ACCEPTED MANUSCRIPT only acute stressed group of mice. IL-10 concentration also increased significantly in both S. aureus treated acute and chronic stressed group compared to unstressed S. aureus infected group

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(P<0.05). (Fig. 2B)

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Both serum TNF-α and IFN-γ levels in the chronic restraint stressed mice were increased with respect to acute stressed mice. However, when the acute or chronic restraint stressed mice

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were given S. aureus infection it showed significant reduction in serum TNF-α level in acute

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condition but change in chronic condition was not significant. Moreover serum TNF-α levels decreased significantly in acute and chronic stress + S. aureus infected group compared to

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unstressed S. aureus infected group (P<0.05) (Fig. 2C). Contrastingly IFN-γ (Fig. 2D) levels when compared with the only stressed group of mice decreased significantly (P<0.05) in both

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acute and chronic stress group infected with S. aureus. Decrease in level was more remarkable in chronic stress + S. aureus infected group compared to acute stress + S. aureus infected group.

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Moreover serum IFN-γ levels decreased significantly in acute and chronic stress + S. aureus infected group compared to unstressed S. aureus infected group (P<0.05). 3.7 Elevated NO production in the brain of mice Significant differences in NO levels were observed in all the experimental groups compared to the control group. Significantly higher NO production in chronic stressed and S. aureus infected brains was observed in comparison with the brains of acute stressed mice inoculated with S. aureus or with the unstressed S. aureus infected group, as assessed by Griess reagent (Fig. 3A ; P <0.05). NO levels increased from 31.44 μg/g brain tissue (control) to 59.36 μg/g brain tissue (chronic restraint stress plus S. aureus infected group). To examine whether there was an increase in iNOS expression during the stress followed by S. aureus infection, protein was extracted from brain tissues and subjected to Western Blot using iNOS specific

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ACCEPTED MANUSCRIPT antibody. The constitutive expression of iNOS in stressed and infected animals was also observed by immunoblot. (Fig 5A or C)

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3.8 Blood brain barrier (BBB) leakage

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Because the BBB leakage is hallmark of brain inflammation, we next examined the BBB leakage by using Evans blue extravasation method. The dye was injected IP at 72 hours post

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infection, and after two hours the brains were removed and Evans blue concentration was

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determined. Significant increase in BBB permeability was observed in other experimental group compared to the control. A marked increase in the permeability of the BBB was observed in

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chronic stressed animals compare to the acute stressed mice, which was significantly increased in chronic stressed and S. aureus infected mice with respect to acute stressed plus S. aureus

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infected animals or with the unstressed S. aureus infected group. (P<0.05)

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(Evans blue concentration, 26.59 μg/mg protein for acute stressed + S. aureus infected mice

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versus 45.27 μg/mg protein for chronic stressed and S. aureus infected mice (Fig. 3B). 3.9 Effect of restraint stress and S. aureus infection on splenic MPO activity Splenic MPO activity was found to be significantly different in rest of the test groups compared to the control. (P<0.05) MPO activity in the chronic restraint stressed mice was decreased significantly in respect to acute stressed mice. However, in this study, the long term restraint stress and S. aureus infection showed decreased MPO activity than the acute stressed and S. aureus infected group in spleen or when compared with the unstressed S. aureus infected group. (Fig. 3C) 3.10 Effect of S. aureus infection after acute and chronic restraint stress on the hepatic, brain and adrenal GSH, SOD and catalase activity

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ACCEPTED MANUSCRIPT Mice undergone chronic stress showed reduced hepatic and brain GSH level but significant increase in SOD and catalase activity when compared with acutely stressed mice and

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control group (P<0.05). However, when acute or chronic restraint stressed mice were infected

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with S. aureus, both the groups showed increased hepatic, brain and adrenal GSH and SOD activity although there was significant variation in catalase activity in between liver, brain and

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adrenal gland in chronic stress plus S. aureus infected group (P<0.05), when compared with the

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stressed only group or with the non-stressed S. aureus infected group. (Fig. 4A to C) 3.11 Expression of Cox-2, iNOS, SOD-1, HSP 70, HSP90 in hypothalamic tissue

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Densitometric analysis of immunoblots, normalized to equal protein content, showed a significant increase in the expression of heat shock proteins (HSP-70 and HSP-90) in the

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hypothalamus, post infection with S. aureus, in acute or chronic stressed animals. However, there was also identical observation in acute or chronic stressed animals, where the levels of SOD-1,

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iNOS and COX-2 showed moderate increases after S. aureus infection, as compared to respective non-infected stressed alone group. (Figure 5) 3.12 Locomotor activity and exploratory behavior As reduced locomotion is a common sickness behavior and an adaptive response to illness and infection, locomotor activity and exploration in the open field test provides an excellent index for the assessment of both the effects of and responses to viable bacterial infection in acute and chronic stressed animal. In the open field test it was observed that the total number of squares crossed by the mice in the periphery, along with distance traveled in periphery, number of rearing behavior was increased, whereas the total time of restraint in periphery was decreased, in the group of mice subjected to acute or chronic stress and subsequently infected with S. aureus when compared with the acute and chronic stressed only

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ACCEPTED MANUSCRIPT group or with the unstressed S. aureus infected group, indicating improvement of exploratory

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behavior when stressed mice were infected with low bolus of bacteria (Table 3).

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3.13 Anxiety Test-Elevated plus maze (EPM)

EPM provide reliable measures of anxiety- like behavior. When tested in EPM, it was

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observed that the time spent in the central area of the maze decreased, time spent in closed arm

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increased, rearing behavior in closed arm was increased in the group of mice who were stressed for short time and subsequently infected with viable S. aureus than the only stressed mice (acute)

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indicating presence of anxiety in these group of mice and improvement of behavior when stressed mice were infected with bacteria. Non-stressed S. aureus infected group showed altered

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levels of anxiety-like behavior like decreased exploration, spending more time in the closed arm of the maze and increased non-exploratory behavior like grooming, which were typical to those

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that are elicited during infection. Thus the effect of S. aureus infection on stress reflected improvement of behavior. In case of mice subjected to restraint stress for longer duration (chronic) showed opposite behavioral activities like the time spent in the central area of the maze increased, time spent in closed arm decreased, indicating less anxiety in those group of mice and improvement of behavior when repeatedly stressed mice were infected with live bacteria. Moreover none of the group of mice entered the unsafe open arms of the maze, except the control and unstressed S. aureus infected group, indicating persistence of anxiety-like behavior. (Table 4) 4. Discussion Stress is capable of altering the physiological homeostasis of the body, and hence coping up with such stress is crucial for maintaining normal health. The question of whether restraint

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ACCEPTED MANUSCRIPT stress and infection induced biochemical changes in the oxidant status, cytokine levels and cholinergic system were enough to cause alteration in the behavioral responses, has not been

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well elucidated by previous studies. In this current investigation, we have attempted to answer

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this question by exploring the effect of restraint stress and infection induced changes in immunological and anti-oxidant activities along with cognitive behavior. Restraint stress and

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subsequent infection was able to activate the HPA axis leading to significant increase in the level

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of serum corticosterone and also the activities of the associated steroidogenic enzymes 3β and 17β HSD, that was supported by findings from other studies (Bishayi and Ghosh, 2007;

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Silverstein et al., 2000).

Increased corticosterone in systemic circulation due to repeated restraint stress prior to S.

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aureus infection, were also able to clear the bacteria from blood as evident from the bacterial density, suggesting the role of restraint stress in resistance to S. aureus infection. The possible

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mechanism might be due to acute stress induced activation of sympathetic nervous system that primes the immune system by mobilizing the leukocytes from the spleen into the blood. Circulating proinflammatory cytokines such as TNF-α, IL-6 induces a release of CRH which in turn induce the release of ACTH and corticosterone. Acute stress also stimulated the release of TNF-α in the brain and initiated direct activation of the HPA axis, which culminated in the secretion of GC thereby playing its anti-inflammatory role. However, despite higher circulating levels of GC under conditions of chronic stress, the anti-inflammatory effects of GCs appeared to be substantially lessened. This might be due to the formation of GC insensitivity by many immune cells and may be mediated by MIF production (Escher et al., 1998). The major cytokines such as IL-6, TNF-α and IFN-γ are potent activators of HPA axis and are known to affect the development and proliferation of pituitary gland and the release of anterior pituitary

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ACCEPTED MANUSCRIPT hormones under the control of hypothalamus. The end result was that the ability to clear bacterial

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infection and improvement of behavior was found to be greater in restrained stressed mice.

In addition, in a previous study, we demonstrated a marked decrease in the antibody level

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after exposure to chronic restraint stress (Mahanti et al., 2013). Circulating antibody (particularly

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IgG) represents a first line of defense against pathogens and by means of, agglutinating bacteria. Prolonged stress exposure may be responsible in regulation of adaptive immune responses

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through HPA mediated corticosterone activity. These changes may depend on GC-induced

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selective alteration in the circulation pattern of certain subpopulation of lymphocytes. Therefore, during prior exposure of mice to acute stress, due to less corticosterone secretion, the GC

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induced immunosuppressive effect is minimal, leading to immune-potentiation and thus we

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obtained a higher antibody titer. However, exposure to chronic stress for 3 weeks, when GC level

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is restored towards normal, steroids may dampen the expression of genes encoding cytokine or co-stimulatory factors thereby decreasing their availability to induce an immune response. Restraint stress prior to S. aureus infection might regulate cognitive function by increasing brain AChE activity. Most of the tests used to study cognitive responses in animals involve locomotor activation that appears to be associated with an increase in ACh release. It is therefore important to define whether changes in AChE activity from the brain are always associated with, in some way related to motor activity or conditions exist in which increase in AChE activity unrelated to motor activity takes place. Further evidence that ACh release and motor activity are not necessarily related has been reported in earlier studies. (Sorrells and Sapolsky, 2007). Currently the predominant hypothesis is that cytokines are mediators of immune signaling to the CNS that affects behavior. Serum concentration of IL-6 was greater among high 23

ACCEPTED MANUSCRIPT than low corticosterone responders. In addition IL-6 is essential for activation of HPA axis during immunological challenge in the absence of hypothalamic input from CRH (Ragozzino et

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al., 1996) and can also act directly on the pituitary to elicit ACTH release, but it may also act on

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the hypothalamus and even on the adrenal gland to elicit corticosterone secretion (Dunn, 2006). Although corticosterone released during acute stress inhibits serum IL-6 levels, an inverse

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relationship was observed between circulating corticosterone and the IL-6 levels when the

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chronic stressed mice were infected with S. aureus. Similarly IL-6 is associated with greater viability of cortisol via inhibition of cortisol binding globulin synthesis (Bethin et al., 2000).

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Therefore, it may be possible that the modulatory effect of IL-6 on the adrenal secretion of corticosterone could be additive, so as to promote an anti-inflammatory response. Furthermore, it

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was observed that GC levels can remain elevated over several days due to IL-6 mediated induction of ACTH and GC levels, again supporting the role of circulating IL-6 in the

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corticosterone response to intravenous S. aureus infection. In an earlier study, we had established protective effects of IL-6 in S. aureus infection induced experimental arthritis and its significance in alteration of hepatic anti-oxidant enzymes and endogenous cytokines (Mal et al., 2013; Zhou et al., 1993). Here, our data suggests that IL-6 acts as an anti-inflammatory molecule whose functions can also be compensated for by another anti- inflammatory cytokine, IL-10, particularly during acute stress and low dose infection. IL-10 enhances CRF and ACTH production in hypothalamus and pituitary tissue. Further downstream in the HPA axis, endogenous IL-10 has the potential to contribute to regulation of GC production both tonically and following stressors in accordance with earlier reports indicating that IL-10 might be an important endogenous regulator in HPA axis activity and in CNS pathologies (Mal et al., 2012). Mice that have been genetically engineered to over express IL-10 display less

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ACCEPTED MANUSCRIPT anxiety while IL-10 knockouts display greater level of anxiety and depressive behavior on a force swim test. Both IL-10 and IL-6 inhibit proinflammatory responses, enhance the release of

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anti-inflammatory molecule, and induces the generation of specific immune responses that might also be contributing to clear the S. aureus in our case. Exaggerated expression of IL-6 but not

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TNF-α was reported in mice lacking IL-10 (Haddad et al., 2002). Previous results also indicated

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that the stress-induced elevation of serum IL-6 negatively regulates the serum TNF-α level and

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may thus contribute to the maintenance of homeostasis (Huey et al., 2008). NO has been shown to exert beneficial, detrimental, or no effects on disease severity and

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brain injury, depending on the model and the bacteria studied. Since the neuroprotective and neurodestructive effects of NO in different models depend on the redox status of the host, the

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sustained brain NO content may be due to restraint stress as well as S. aureus infection. These results might suggest that endogenous NO is an important modulator of BBB

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permeability, an important hallmark of brain inflammation. Neurological manifestations caused by neuroinvading pathogens are typically attributed to penetration of the BBB and invasion of the CNS. We observed that infection with S. aureus after chronic restraint stress lead to significant BBB disruption as indicated by the leakage of Evans blue into brain tissues of mice. MPO is an enzyme found in neutrophils, and its activity in the spleen is related to neutrophil infiltration (Majumdar et al., 2011). Acute restraint stress before S. aureus infection exhibited an increase in splenic MPO activity when compared with the rest of the treatment groups indicating enhanced recruitment of neutrophil into the spleen. However, long term restraint stress prior to S. aureus infection led to reduced neutrophil infiltration into the spleen. In contrast chronic stress seems to depress the migration of immune cells from blood, an effect that correlates with the alterations of responsiveness to corticosterone. Previous studies also

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ACCEPTED MANUSCRIPT suggested that the recruitment of absolute number of specific blood leukocytes may be significantly affected by the elevated level of corticosterone (Nukina et al., 2001).

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Glutathione dysregulation is associated with the etiology and progression of several

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diseases including neurotoxic and neurodegenerative diseases. Corticosterone secreted during stress reduces GSH levels in liver and disrupt anti-oxidant capacity in hepatic tissue (Black,

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2002). In our study, elevated hepatic and brain GSH levels suggest that infection post chronic

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stress may restore the GSH levels. When we compared SOD activities in liver, brain, and adrenal tissues, the highest percentage of SOD activity, indicating high level of H2O2, was

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observed in chronic restraint stress plus S. aureus infected mice than in only stressed animals. Therefore, it may be suggested that in order to escape the elevated H2O2 level in the liver, brain

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and adrenal those tissues have more SOD activity. However, paradoxically the brain and adrenal catalase activity was significantly reduced in the chronic restraint stressed mice after S. aureus

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infection except in liver. A combination of cytokines (IFN-γ and TNF-α) and elevated NO level was found to inhibit catalase activity, not only in normal cells but also in catalase-over expressing cells and in rat and human islets of Langerhans, (Beck and Levander, 1998), therefore, reduction of brain catalase activity in our case may also be mediated by elevated brain NO and serum TNF-α and IFN-γ. These data suggest that suppression of catalase activity was sufficient to cause an accumulation of H2O2 in brain and may support that catalase is a key factor in oxidative damage. However, activity, or protein or mRNA of catalase are decreased (Sigfrid et al., 2003; Clerch et al., 1996; Singh et al., 1993); increased (Cvetkovic et al., 1998) or unchanged during oxidative stress (Clerch et al., 1991). Up regulation of the heat shock proteins in the hypothalamus of acute or chronic restraint stressed group post infection with S. aureus indicated sustained stress in the brain. Since we

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ACCEPTED MANUSCRIPT obtained an elevated level of corticosterone in infected and repeatedly stressed groups, both HSP-70 and HSP-90 could not bind with cytosolic GC receptor, implicated for sustained stress in

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brain. We also observed that the expression of SOD-1 in hypothalamus increased in both post-

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infected stressed groups and also corroborated with the SOD enzyme activity in the brain tissue. COX-2 expression is important in regulating prostaglandin signaling in brain and both basal and

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induced expression of brain COX-2 were observed in infected and non-infected groups in both

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acute and chronic stressed animals even though their levels show a trend similar to that of brain iNOS.

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In the present study, observed behavioral changes might be due to alteration in the brain regions controlling motor activity and anxiety-like behavior, while impaired motor activity The presumed functions of the CNS-mediated

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could be due to stress-induced depression.

responses during infection are to enhance immunity, conserve energy and prevent hyper

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inflammation. The viewpoint of the integrated physiology, therefore, is that cytokines which are induced during immune responses modulate the function of the CNS to recruit physiological, behavioral and endocrine mechanism to combat infection. Role of TNF-α and IFN-γ in induction of depressive like behavior in response to Bacillus Calmette–Guerin in mice has been demonstrated (Dieterich et al., 2000). Changes in the open field behavior have also been documented in animals administered cytokines in isolation, including TNF-α and in animals with relevant cytokine genes deleted or over expressed (O’Connor et al., 2009). The level of cortisol hormone is often elevated in depressed individuals. But in depressed individuals the negative feedback system for dampening a response does not work well. The problem with CRH not being suppressible in depressed individuals may be related to the inflammatory state in chronic stress (Silverman et al., 2007). It is reported that stressors and cytokines may synergistically

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ACCEPTED MANUSCRIPT influences biological and behavioral responses and that these treatments may have long term ramifications through the sensitization of processes associated with stress responses (Raison et

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al., 2010). One of the mechanisms proposed for this neurodegeneration due to glucocorticoid

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hyper-secretion is the inhibition of glucose transport in brain neurons and glia, which could be the result of the redirection of the energy towards the muscles in order to facilitate the escape

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from the stressor leading to increased locomotor activity.

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In addition to the open field test, behavioral study such as EPM provides reliable measures of anxiety-related behavior. When tested in EPM, animals with a more anxiogenic

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phenotype will spend a greater percentage of their time in the closed arm of the maze relative to the open, unsafe arms. Moreover, the latency to exit the closed arm of the EPM will be

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significantly greater than that of animals that is not anxious. Surprisingly, all groups of mice did not entered into the open arm except the control animals. As behavior is controlled by neuronal

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function, behavioral changes associated with infection are as a result of proinflammatory cytokine direct interaction with neurons. A systemic inflammatory response results in the production of TNF-α which circulates in the blood and communicates with neurons within brain. There are a number of different routes by which systemic inflammatory response may communicate with the CNS. There may be active transport of cytokines across the BBB and the evidence suggests that in doing so they may impact on cognitive process. A very recent study used extremely low doses of TNF-α administered intravenously to show that TNF-α within the brain causes depressive like behavior (Anisman, 2009). IFN-γ action may be necessary to maintain the expression of other cytokines or elicit a separate but parallel signal that is insufficient alone but is needed to drive depressive like behavior (Kaster et al., 2012). Indeed a low level of proinflammatory cytokine activity within the brain is necessary for normal cognition

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ACCEPTED MANUSCRIPT via maintenance of proper neurotransmitter levels (Sayre et al., 2008). It is only when the neurotransmitter response and input on neurons is at an imbalance that behavior shifts to a

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sickness or depressive like behavior, and unable to explore the open arm (Litteljohn et al., 2010). Recipients of IFN-γ therapy typically report reduced alertness, with sustained slowing of reaction

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time at higher doses (McCusker and Kelley, 2013) also supported the role of IFN-γ in alteration

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of cognitive function in our case. Symptoms of depression appear after pro-inflammatory

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cytokines are produced by the body or administered exogenously. There is a strong correlation between infection-related TNF-α expression in the periphery and the degree of sickness

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behavior, as blocking cytokine expression during inflammation attenuates sickness behavior (Dieterich et al., 2000).

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Although acute or chronic restraint stress prior to S .aureus infection was able to elevate the systemic level of IL-6 and IL-10, these anti-inflammatory cytokines could not completely

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inhibit the production of pro-inflammatory cytokines like TNF-α and IFN-γ in the brain. Taken together with our results, these findings support the notion that cytokines particularly TNF-α and IFN-γ

and brain NO produced

during immune activation and under the influence of

corticosterone can modulate the open field behavior both in terms of locomotor activity as well as exploration. When acute stressed mice were infected with low bolus of bacteria, they showed improvement of exploratory behavior in OFT than the acute stressed alone group, might be due to less TNF-α and IFN-γ under the control of elevated brain AChE activity and systemic IL-6 and IL-10. One of the features observed was presence of anxiety in the acute stress plus S. aureus infected mice compared to non-infected stressed group, whereas in case of mice subjected to restraint stress for longer duration (chronic) showed identical behavioral activities like the time

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ACCEPTED MANUSCRIPT spent in central area of the maze decreased, time spent in closed arm increased, indicating persistent anxiety in those group of mice and very little improvement of exploratory behavior

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when repeatedly stressed mice were infected with a single bolus of live bacteria.

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Role of the Funding sources

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The funding agency had no involvement with the design, execution or data analysis of this study, manuscript preparation, or in the decision to submit the paper for publication.

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Conflict of interest None declared.

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Contributors

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Authors AB and BB designed the study and designed protocol. SM and AM performed all the

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experiments. KK performed the immunoblots. SM and AM managed the literature searches and analyses. SM and AM undertook the statistical analysis; BB and AM wrote the manuscript. All authors contributed to and have approved the final manuscript.

Acknowledgments

This study was financially supported by Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi under the DBT-Neuroscience scheme. A.B. is recipient of Tata Innovation Fellowship from the Department of Biotechnology, Government of India (BT/HRD/35/01/02/2014).

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restraint-stressed mice to sepsis. J. Immunol. 181, 3441- 3448. Yao, L., Berman, J.W., Factor, S.M., Lowy, F.D., 1997. Correlation of histopathologic and

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bacteriologic changes with cytokine expression in an experimental murine model of bacteremic Staphylococcus aureus infection. Infect. Immun. 65, 3889-3895.

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Yirmiya, R., Goshen, I., 2011. Immune modulation of learning, memory, neural plasticity and neurogenesis. Brain Behav. Immun. 25, 181-213. Zhou, D., Kusnecov, A.W., Shurin, M.R., DePaoli, M., Rabin, B.S., 1993. Exposure to physical and psychological stressors elevates plasma interleukin 6: relationship to the activation of hypothalamic-pituitary-adrenal axis. Endocrinol. 133, 2523-2530. Legends to the Figures Fig. 1A Effect of S. aureus infection post acute and chronic restraint stress on the serum corticosterone concentration in mice Effect of S. aureus infection post exposure to acute and chronic restraint stress on serum corticosterone level were expressed as mean ± standard deviation from the serum of mice from each group (n=15).

*

Significant difference from control group.

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#

Significant difference from

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$

Significant difference from chronic stress group.

from acute stress + S. aureus infected group.

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^

Significant difference

Significant difference from S. aureus infected

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group. (P<0.05)

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Fig. 1B Effect of S. aureus infection post acute and chronic restraint stress on the brain AChE activity in mice

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The results in this figure showing the effect of S. aureus challenge on restraint stressed mice on

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the specific activity of AChE in mice brain. The AChE activity for each group was denoted by the mean ± SD values of triplicate observations.

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Significant difference from control group.

#

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Fig. 1C Effects of S. aureus infection on steroidogenic enzyme (3βHSD) activity during

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restraint stress in mouse adrenal gland The results in this figure represent the enzyme activity (Units/min.mg) in the adrenal gland homogenate isolated from acute and chronic restraint stressed mice (in presence of S. aureus infection). DHEA was the substrate for 3β HSD activity. One unit of enzyme activity is equivalent to a change in absorbance 0.001/min at 340 nm. Data are expressed as the mean ± SD of triplicate experiments.

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Significant difference from control group.

#

Significant difference

from acute stress group. $Significant difference from chronic stress group.

^

Significant

difference from acute stress + S. aureus infected group. ξ Significant difference from S. aureus infected group. (P<0.05) Fig. 1D Effects of S. aureus infection

on steroidogenic enzyme (17βHSD) activity during

restraint stress in mouse adrenal gland

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ACCEPTED MANUSCRIPT The results in this figure represent the enzyme activity (Units/min.mg) in the adrenal gland homogenate isolated from acute and chronic restraint stressed mice (in presence of live S. aureus

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infection). Testosterone was the substrate for 17β HSD activity. One unit of enzyme activity is

from acute stress group.

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of triplicate experiments.

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equivalent to a change in absorbance 0.001/min at 340 nm. Data are expressed as the mean ± SD

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difference from acute stress + S. aureus infected group. ξ Significant difference from S. aureus infected group. (P<0.05) C- Control, AS- acute stress, CS- chronic stress, SA- S. aureus

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infected, ASSA – acute stress + S. aureus infection and CSSA- chronic stress + S. aureus infection.

restraint stressed mice

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Fig. 2 Effect of S. aureus infection on the serum IL-6, IL-10, TNF-α and IFN-γ level in

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Levels of IL-6 (A), IL-10 (B) TNF-α (C), and IFN-γ (D),

in serum from different groups of

animal were determined by utilizing ELISA according to the manufacturer’s recommendations and were expressed from triplicate experiments. A = IL-6, B = IL-10, C = TNF-α and D = IFN-γ. Data were expressed as mean ± standard deviation from the serum of mice from each group *

(n=15). group.

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Significant difference from chronic stress group.

stress + S. aureus infected group.

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Significant difference from S. aureus infected group.

(P<0.05) C- Control, AS- acute stress, CS- chronic stress, SA- S. aureus infected, ASSA – acute stress + S. aureus infection and CSSA- chronic stress + S. aureus infection.

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ACCEPTED MANUSCRIPT Fig. 3A. Effects of S. aureus infection on nitric oxide (NO) production during restraint stress in mouse brain

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NO production was determined by Griess reagent, in the brain tissues of different groups of

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mice. Results are given as the mean ± SD and are representative of three independent experiments. The increase in the production of NO was significantly greater in infected mice. #

Significant difference from acute stress group. ^

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Significant difference from chronic stress group.

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Fig. 3B. Effects of S. aureus infection on blood brain barrier leakage (BBB) during restraint stress in mouse brain

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The blood–brain barrier permeability was assessed using the Evans blue extravasation technique Data represent the mean ± SD and are representative of three independent experiments. The

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increase in the BBB permeability was significantly greater in infected mice. difference from control group.

#

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difference from chronic stress group.

^

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infected group. ξ Significant difference from S. aureus infected group. (P<0.05) Fig. 3C. Effects of S. aureus infection

on splenic MPO activity during restraint stress in

mouse spleen MPO activity was analyzed as index of neutrophil infiltration in the spleen tissue. The rate of change in absorbance was measured spectrophotometrically at 405 nm. MPO activity has been defined as the concentration of enzyme degrading 1 μ mole of peroxide/min at 37°C and was expressed as change in absorbance/min.mg of protein. Data were expressed as mean ± standard deviation. * Significant difference from control group. # Significant difference from acute stress

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stress + S. aureus infected group.

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Significant difference from S. aureus infected group.

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(P<0.05) C- Control, AS- acute stress, CS- chronic stress, SA- S. aureus infected, ASSA – acute

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stress + S. aureus infection and CSSA- chronic stress + S. aureus infection. Fig. 4. Alteration in mouse hepatic anti-oxidant status like reduced glutathione level and

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the activities of the anti-oxidant enzymes like SOD and catalase

Significant difference from control group.

#

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aureus infected group.

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*

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aureus infection and CSSA- chronic stress + S. aureus infection.

Fig. 5 Expression of Cox-2, iNOS, SOD-1, HSP 70, HSP90 in hypothalamic tissue Expression of Cox-2, iNOS, SOD-1, HSP 70, HSP90 were measured in terms of fold change among the different groups.

Panel A: western blot for the proteins tested Panel B = Fold change for Cox-2, Panel C: Fold change for iNOS, Panel D: Fold change for SOD-1, Panel E: Fold change for HSP-70, Panel F: Fold change for HSP-90

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ACCEPTED MANUSCRIPT C= Control; AS= Acute stress; CS = Chronic stress; SA = S. aureus infected only; AS + SA = Acute Stress + S. aureus infection; CS + SA: = Chronic Stress + S. aureus infection. * Significant #

Significant difference compared to acute stress group.

$

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difference from control group.

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infected group at P<0.05 level of significance.

Legends to the tables

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Table-1: Recovery of bacteria from blood after intravenous injection of S. aureus to the acute and chronic restraint stressed mice

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Blood was collected from different groups of mice 24 hours post-infection and processed as detailed in materials and methods. Numbers of bacterial cells in blood were determined by CFU

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assay. Each result is the representative of triplicate experiments. a = Number of bacterial CFU in blood,

S. aureus infection Vs Acute stress + S. aureus

infection, significant decrease at P<0.05. b = S. aureus infection P<0.05.

Vs Chronic stress + S. aureus infection, significant decrease at

Table-2 Effect of acute and chronic restraint stress on the alteration of humoral immune response Stress induced changes in serum agglutination titer was measured at 24 hours postimmunization. Table-3 Effects of S. aureus infection during restraint stress on the motor activity of mice in the open field test

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ACCEPTED MANUSCRIPT Activity and exploratory behavior of mice were observed in open field for a 5-min period, and the following behavioral activities were scored:

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TNSCP- Total number of squares crossed in periphery, DTP- distance traveled in the periphery, LRC- latency to reach centre, DI - duration of immobilization, TNR – total number of rearing,

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ULC – unable to locate central region, TNG – total number of grooming, TNF – total number of

#

significant difference from acute stress group.

*

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Significant difference from control group.

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freezing. Each value represent the mean ± SD (n=15) of counts in 5 min open field test.

Significant difference from chronic stress group.

^

Significant difference from acute stress + S.

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aureus infected group. ξ Significant difference from S. aureus infected group. (P<0.05) Table-4 Effects of S. aureus infection during restraint stress on the anxiety test of mice in

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the elevated plus maze test

To test the anxiety-like behavior in mice is the EPM, in which the reduced number of entries or

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time spent in the open arms of the EPM suggests the operation of anxiety-like processes. Mouse from different groups was placed individually in the center portion of the plus-maze, facing one of the open arms. The observer measured: (a) TSC-total time spent at centre, b) TSCA-the time spent in the closed arms, (c) TSOA-time spent in open arm, (d) NRCA- number of rearing in closed arms, during the 5-min test period. An entry was defined as all four paws in the arm. Significant difference from control group.

#

significant difference from acute stress group.

Significant difference from chronic stress group.

^

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Significant difference from acute stress + S.

aureus infected group. ξ Significant difference from S. aureus infected group. (P<0.05)

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ACCEPTED MANUSCRIPT Table 1: Bacterial CFU count Group

Bacterial CFU count/ml of blood Mean ± SD Not detectable Not detectable Not detectable 116.67 ± 8.54 58.33 ± 7.51a 81.67 ± 9.16b

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Control Acute stress Chronic stress S. aureus infected Acute stress + S. aureus Chronic stress + S. aureus

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Number of bacterial CFU in blood, S. aureus infection vs acute stress + S. aureus infection, significant decrease at P< 0.05. b S. aureus infection vs chronic stress + S. aureus infection, significant decrease at P < 0.05.

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ACCEPTED MANUSCRIPT Table 2: Antibody titer to determine alteration in humoral immune response Antibody titer of serum

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Experimental group

Control (no immunization)

No visible bacterial agglutination 1:512

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Heat killed S. aureus immunized Acute IS + Heat killed S. aureus immunized

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Chronic IS + Heat killed S. aureus immunized

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1:1024 1:256

ACCEPTED MANUSCRIPT Table 3: Open field test

117.66 ± 8.50*# 207 ± 12.08 297.66 ± 16.04#ξ 158 ± 10.81*$^ξ

941.33 ± 68.03*# 1656 ± 106.64 2381.33 ± 120.32#ξ 1264 ± 86.53*$^ξ

ULC 112 ± 24.56 182.66 ± 17.24ξ ULC

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48.66 ± 17.01 107.33 ± 20.50* 48.66 ± 7.50# 88.32 ± 15.29 7.66 ± 3.21*#ξ 17.66 ± 4.72*$^ξ

TNR Mean ± SD 32 ± 5

3 ± 1*

9 ± 1*

7 ± 1*#

9±3

21 ± 4.26

12 ± 2.66

12.33 ± 2.53*# 5.66 ± 1.57*^

2.33 ± 1.53*#ξ 6.33 ± 0.57*$^ξ

1.66 ± 0.57*#ξ 4 ± 1*$^ξ

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Chronic stress S. aureus infected Acute stress + S. aureus Chronic stress + S. aureus

Behavioral activity TNG TNF Mean ± SD Mean ± SD 17.66 ± 13.67 ± 4.72 2.51 8 ± 1* 12 ± 1 12 ± 1

DI (s) Mean ± SD

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Acute stress

Locomotor activity DTP (cm) LRC (s) Mean ± SD Mean ± SD 2296 ± 178 ± 552.69 61.48 32 ± 8* ULC

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Control

TNSCP Mean ± SD 287 ± 69.08 4 ± 1*

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Group

TNSCP- total number of squares crossed in periphery, DTP- distance travelled in the periphery,

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LRC-latency to reach centre, DI- duration of immobilization, TNR- total number of rearing,

group.

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freezing.

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ULC- unable to locate central region, TNG- total number of grooming, TNF- total number of #

Significant difference from acute stress

Significant difference from chronic stress group.

stress + S. aureus infected group.

ξ

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Significant difference from acute

Significant difference with respect to S. aureus infected

group. (P<0.05)

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ACCEPTED MANUSCRIPT Table 4: Elevated plus maze TSOA(s) Mean ± SD 48.33 ± 7.09 DEOA

283.75 ± 12.63* 257.18 ± 15.46 269.25 ± 22.75

DEOA 27.82 ± 6.81 DEOA

253 ± 19.54

DEOA

NRCA Mean ± SD 16.66 ± 2.08 6 ± 0.81* 3.75 ± 0.5* 8±3 10.5 ± 1.29*# 7.33 ± 1.57*

NGCA Mean ± SD 12 ± 1 7.75 ± 0.95* 5.5 ± 0.57* 18 ± 2.36 2 ± 0.81*#ξ 3.5 ± 0.57*ξ

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Chronic stress S. aureus infected Acute stress + S. aureus Chronic stress + S. aureus

TSCA(s) Mean ± SD 242.67 ± 18.50 175.75 ± 29.13*

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Control Acute stress

TSC(s) Mean ± SD 9±2 124.25 ± 29.13* 16.25 ± 2.62*# 15 ± 4 30.75 ± 2.75*#ξ 47 ± 9.53*$^ξ

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Group

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TSC- time spent at the centre, TSCA- time spent in the closed arm, TSOA- time spent in the open arm, DEOA- did not entered the open arm, NRCA- number of rearing in closed arm, *

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NGCA- number of grooming in the closed arm.

Significant difference from control group.

#

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from S. aureus infected unstressed group. (P<0.05)

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Significant difference

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Graphical abstract

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ACCEPTED MANUSCRIPT

Research Highlights: Effect of S. aureus infection during acute and chronic restraint stress.



Acute and chronic restraint stress enhances the resistance of mice to S. aureus infection.



Pro-inflammatory cytokines and free radicals production down regulated.



iNOS, COX-2, HSP 70 and 90 expressions and AChE activity in hypothalamus along

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

Cytokines and nitric oxide in presence of corticosterone modulate locomotion and

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exploration.

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

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with the behavioral changes support our observation.

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