Experimental listeriosis: A study of purinergic and cholinergic inflammatory pathway

Journal Pre-proof Experimental listeriosis: a study of purinergic and cholinergic inflammatory pathway Antonise M. Jaguezeski, Aleksandro S. da Silva, Teane M.A. Gomes, Nathieli B. Bottari, Thalisson F. Lopes, Renan A. Cechin, Vera M. Morsch, Maria R.C. Schetinger, Janice L. Giongo, Rodrigo de A. Vaucher

PII:

S0378-1135(19)31033-8

DOI:

https://doi.org/10.1016/j.vetmic.2019.108528

Reference:

VETMIC 108528

To appear in:

Veterinary Microbiology

Received Date:

29 August 2019

Revised Date:

25 November 2019

Accepted Date:

25 November 2019

Please cite this article as: Jaguezeski AM, da Silva AS, Gomes TMA, Bottari NB, Lopes TF, Cechin RA, Morsch VM, Schetinger MRC, Giongo JL, de A. Vaucher R, Experimental listeriosis: a study of purinergic and cholinergic inflammatory pathway, Veterinary Microbiology (2019), doi: https://doi.org/10.1016/j.vetmic.2019.108528

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Experimental listeriosis: a study of purinergic and cholinergic inflammatory pathway Antonise M. Jaguezeskia,b, Aleksandro S. da Silvaa,b*, Teane M. A. Gomesc, Nathieli B. Bottaria, Thalisson F. Lopesa, Renan A. Cechinc, Vera M. Morscha, Maria R. C. Schetingera, Janice L. Giongod, Rodrigo de A. Vauchere a

Department of Molecular Biology and Toxicological Biochemistry, Universidade Federal de Santa

Maria, Santa Maria, RS, Brazil. b

Department of Animal Science, Universidade do Estado de Santa Catarina, Chapecó, Santa

Catarina, Brazil. Laboratory of Veterinary Pathology, Instituto Federal Catarinense – IFC, Concórdia, Santa

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c

Catarina, Brazil.

Pharmacy Laboratory, Faculdade Anhanguera, Pelotas, RS, Brazil.

e

Department of Biochemistry, Universidade Federal de Pelotas, RS, Brazil

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d

*Corresponding author: Rua Beloni Trombeta Zanin, 680E, Bairro Santo Antônio, Chapecó, SC.

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CEP: 89.815-630. Tel: (49) 2049 – 9524. [email protected] (Da Silva, A.S.). ORCID

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number: 0000-0002-9860-1933

Highlights

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Cerebral cholinergic and purinergic inflammatory markers. Infection by L. monocytogenes in adult gerbils did not cause clinical disease. ATP and ADP hydrolysis reduce in brain, which has a pro-inflammatory effect. ADA and AChE had neuroprotective behavior with the chronicity of the infection. Cerebral oxidative stress was observed in infected gerbils with subclinical listeriosis.

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ABSTRACT

The cholinergic, purinergic and oxidative stress systems were related to nervous system damage in some pathologies, as well as being involved in pro-inflammatory and anti-inflammatory pathways. The objective was to investigate changes in purinergic, cholinergic systems and oxidative stress related to the neuropathology of listeriosis. Gerbils were used as experimental models. The animals were divided in two groups: control and infected. The animals were orally infected with 5x108 CFU/animal of the pathogenic strain of Listeria monocytogenes. Collected of material was 6 and

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12th days post-infection (PI). Infected animals showed moderate mixed inflammatory infiltrates in the liver. The spleen and brain was used for PCR analyses, confirming infection by L. monocytogenes. Increase in number of total leukocytes because of an increase in lymphocytes in infected (P<0.001). ATP and ADP hydrolysis by NTPDase was lower at 6 and 12th days PI in infected animals than in the control group. ADA (adenosine deaminase) activity was higher on the 6th day PI (P<0.05) and decreased on the 12th day PI (P<0.05) in infected animals. AChE (acetylcholinesterase) activity did not differ between groups on the 6th day PI; however, activity decreased in infected group on the 12th day PI (P<0.05). On the 12th day PI, an increase of oxygenreactive species levels and lower catalase and superoxide dismutase activities in the infected group

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was observed, characterizing a situation of cerebral oxidative stress. The inflammatory and oxidative mechanisms are present in listeriosis in asymptomatic animals, and that ectonucleotidases and cholinesterase’s are involved in immunomodulation.

Keywords: Acetylcholine. Antioxidant/oxidants. Ectonucleotidases. Inflammatory marker.

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Neurobiology.

Introduction

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Infection by Listeria monocytogenes in humans has been among the first three causes of infectiouscontagious mortality in the world, after Salmonella spp. and Toxoplasma gondii infections (Scallan

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et al. 2011; Bundrant et al. 2011). L. monocytogenes is a food-borne pathogen that is potentially transmitted by contamined milk, drinking water and ready-to-eat food (Scallan et al. 2011; Soni et al. 2013; Chen et al. 2014) causing severe disease in humans (Almeida et al. 2017) and animals

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(Bundrant et al. 2011). Infection in humans causes bacteremia that can lead to meningoencephalitis or abortion when pregnant women are infected, progressing either to cure with sequelae or death (Charlier et al. 2017). In animals, similarly, abortion or neurological manifestations occur, including

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progressive ataxia, lateral head tilt, hyperthermia, horizontal nystagmus (Pritchard et al. 2016), walking in circles, depression, ear droop and ptosis of cheek tone (Scott 2013). Immunosuppression

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was reported to be a risk factor in both humans and animals (Charlier et al. 2017; Pritchard et al. 2017).

The mechanisms of symptomatology in humans and animals remain poorly understood. In

this context, it is known that ATP sequestration and the production of adenosine regulated by NTPDase and 5´-nucleotidase enzymes by immune cells are crucial for the regulation of various pathological processes (Dou et al. 2018). The release of extracellular ATP manifests a danger signal and activates pathways in the synthesis of pro-inflammatory cytokines (Jacob et al. 2013; Ferrari et al. 2016), which may aid in pathogen recognition and defense (Dou et al. 2018), but in intense

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response is damaging to cells. On the other hand, the neurotransmitter and immunomodulatory acetylcholine (ACh) has been shown to be beneficial in abnormal conditions for inflammatory homeostasis (Gilboa-Geffen et al. 2012), and its regulator, acetylcholinesterase (AChE), at low levels, has been associated with improved memory loss and cognitive disturbances (Kaizer et al. 2018). The generation of reactive oxygen species (ROS) may be related to occurs in degenerative brain pathologies (Toklu and Tümer 2015); however, antioxidant enzymes in the brain provide compensatory mechanisms against the increase of oxidants, because the brain consumes the O2 at the highest rate in the body, generating excess ROS (Solomon et al. 2002; Liu et al. 2012). In this way, both the purinergic system, the cholinergic system and the oxidant and inflammatory pathways

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are related to cognitive losses and neurological lesions. These alterations, already verified in other pathologies, may be related to and assist in the clarification of neurological changes and lesions observed in animals and humans with listeriosis. Therefore, we aimed to verify whether adult gerbils infected by L. monocytogenes have changes in activities of the purinergic and cholinergic enzymes, as well as whether there is cerebral oxidative

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stress in subclinical disease.

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Materials and methods Inoculum preparation

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L. monocytogenes was kept frozen in the Microbiology Laboratory of the Instituto Federal Catarinense – Concórdia (Brazil). For this experiment, L. monocytogenes, strain ATCC 7644, were plated in blood agar and incubated for 24 h at 37 ºC; and this replication process done three

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consecutive times for strain activation. Then, bacterial concentration was estimated by spectrophotometer using 1 OD600 = 1 x 109 colony forming unit (CFU) per mL. The bacterial suspension was centrifuged at 1000 x g for 5 min, and the pellet was resuspended in sterile

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phosphate buffer, resulting in the infecting dose of 5x109 CFU/ mL diluted in milk cream.

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Animals and experimental design Twenty-three gerbils (Meriones unguiculatus) were used between 6–8 months of age with mean weight of 75 g. Five to six animals were kept per cage (41 x 34 x 18), fed with food for rodents and water ad libitum. The animals underwent a period of adaptation with temperature (25 ºC), air circulation and photoperiod control (12 hours/light) controlled. Two groups were formed: negative control (C, n = 11), that were not infected but received 100 μL of milk cream (placebo effect); and infected group (I, n = 12), that received the infective dose of L. monocytogenes in 100 μL of milk cream, by gavage technique, i.e. 5x108 CFU/animal. The infectious dose used was based on a

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previous study by our research group (Jaguezeski et al., 2019). The moment of oral infection was set as day 0 (zero) of the experiment.

Sample collection At the 6th and 12th day post-infection, the animals were euthanized with isoflurane; 2 mL of blood were collected by intracardiac puncture and stored in Eppendorf tubes containing EDTA (ethylenediamine tetra acetic acid) until analysis. Then, euthanasia by cervical dislocation was performed, and we performed organ collection for histopathological examination, PCR and enzymatic and oxidative stress analyses. Importantly, the variable responses assessed in the gerbil

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brain may be affected by drugs (used as anesthetic or to euthanasia), which is why we opted for inhalation anesthesia (isofluorane), followed by euthanasia by cervical dislocation.

Blood tests

Blood samples containing anticoagulant EDTA was used to measure hematocrit (%) and perform

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leukogram analysis. The hematocrit was determined by microcapillary filling and centrifugation at 10000 rpm for 5 minutes. The leukocyte count was performed by on semi-automated CELM CC550

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equipment, and the relative differential count was performed manually on blood smears stained with rapid Panoptic. The remainder of the EDTA sample was centrifuged and the plasma were stored in

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Eppendorf tubes and frozen at -20 ºC for later enzymatic analysis.

Tissue collection

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Spleen, liver, small intestine, heart, and one right hemisphere (brain: cerebral cortex, brainstem and cerebellum) were collected for histopathology. Left hemispheres were collected and stored in Eppendorf tubes at -20 ºC for enzymatic analysis and measurement of levels of oxidants and

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antioxidants. A spleen fragment was collected and kept frozen for confirmation of L.

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monocytogenes infection by qPCR (quantitative polymerase chain reaction).

Ectonucleotidases and adenosine deaminase activities in brain The brain (left hemisphere) was placed in TRIS-HCl buffer, pH of 7.5 and then centrifuged at 1,800 g for 10 min. The supernatant was collected for biochemical analyzes described below, whereas the cerebral homogenate sediment was used to search for Listeria DNA in the brain (qPCR). The protein concentration was adjusted to 0.4–0.6 mg/ml in supernatant. The NTPDase (E.C. 3.6.1.5) and 5´-nucleotidase activities were determined in cerebral homogenates according to the method described by Schetinger et al. (2000). The samples were incubated at 37 °C for 10 min with ATP,

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ADP or AMP as the substrate (2.0 mM). The reaction was stopped with 10% trichloroacetic acid and colored by malachite green. The results were expressed as ɳmol Pi release/mg protein. The ADA activity in brain was measured according to the method of Giusti (1984), based on direct measurement of ammonia formation produced when the enzyme acts on adenosine. The results were expressed as ɳmol Pi release/mg protein.

Acetylcholinesterase (AChE) activity in brain AChE (E.C. 3.1.1.7) activity was measured in brain according to Ellman et al. (1961). Brain samples was adjusted between 1.4–1.8 mg/mL of protein. AChE activity is based on the formation

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of the yellow anion, 5,5′-dithio-bis-acid-nitrobenzoic, measured by absorbance at 412 nm during 2min incubation at 25 °C using acetylthiocholine iodide as substrate. All samples were assayed in triplicate. The results were expressed as µmol ACsCh/h/mg protein.

Oxidative status

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Determination in brain of ROS in cortex was evaluated by 2´-7´-dichlorofluorescein (DCFH) levels as an index of peroxide production by cellular components according to Halliwell and Gutteridge

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(2007). The results were expressed as U DCF/mg protein.

Catalase (CAT, E.C. 1.11.1.6) activity was determined in brain by the decomposition of

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H2O2 at 240 nm according to the method described by Aebi (1984). Superoxide dismutase (SOD, E.C. 1.15.1.1) activity was quantified spectrophotometrically by inhibition of auto-oxidation of epinephrine to adrenochrome at an alkaline pH at 480 nm in plasma, according to Misra and

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Fridovich (1972). For SOD and CAT assays, 10–40 μg of protein were used. The results were expressed as U CAT/mg protein or U SOD/mg protein.

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Protein determination

The protein levels were determined according to the methodology described by Bradford (1976)

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using albumin as standard. The results were expressed as mg/mL.

Histopathology

Spleen, liver, intestine, and brain were collected post-euthanasia and stored in 10% formaldehyde for 48 hours and then transferred to 70% alcohol for histopathology. The tissues were processed by routine methods, embedded in paraffin, followed by transversal sections of 4-µm thickness, and hematoxylin and eosin (HE) staining. Blinded readings were done by a pathologist using a light microscope.

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PCR The total DNA from the bacterium in spleen tissue and homogenate brain from infected and uninfected animal groups were extracted using a PureLinkTM Genomic DNA Mini Kit (Thermo Fisher Scientific, São Paulo, Brazil), according to the manufacturer's instructions. The detailed methodology used for qPCR analysis was published by Jaguezeski et al. (2018).

Statistical analysis We used the SAS statistical program (SAS Institute, Cary, NC, USA) for analysis of the data. These was subjected to Shapiro-Wilk’s normality test. The data were then subjected to one-way variance

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analysis (ANOVA), followed by the Student T-test (P ≤ 0.05) for comparison between groups. The results were presented as mean with standard deviation.

Results

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Course of infection and histopathology

The infected animals did not present clinical signs, characterizing subclinical listeriosis; there were

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no encephalic, enteric or splenic alterations. In the liver, four infected animals, primarily at day 6 PI, showed multifocal moderate inflammatory infiltrates of neutrophils and macrophages with

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hepatic necrosis. L. monocytogenes infection of the animals in the infected group was confirmed by positive spleen (n = 12/12) and brain (n = 12/12) qPCR. No histopathological lesions were observed

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in the control group, and qPCR was negative.

Hematocrit and Leukogram

The total leukocyte count revealed a statistical difference between groups, being higher in infected

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group on the 12th day PI due to the increase in the lymphocytes (P < 0.001). The other classes of leukocytes as well as the hematocrit showed no differences between groups (P > 0.05). The values

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are represented in Table 1.

L. monocytogenes infection modulate purines levels in brain The activities of NTPDase, 5´-NT and ADA are shown on Fig. 1. ATP and ADP hydrolysis levels at the 6th and 12th days PI were lower in infected animals than in control group. 5´- NT activity was lower at the 6th day PI (P = 0.045). By contrast, ADA activity was higher in the infected group on the 6th day PI (P = 0.023), but it decreased in this same group on the 12th day PI (P < 0.001) when compared with the control group.

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Down-regulation of the neuroimmune response by acetylcholinesterase enzyme during L. monocytogenes infection In order to confirm immunomodulation by the cholinergic system, AChE activity was investigated (Fig. 2). AChE activity did not differ between groups on the 6th day PI (P = 0.872); however, it was lower in the infected group on the 12th day PI (P < 0.001) when compared to the control group.

Oxidant and antioxidants status in brain infected by L. monocytogenes During L. monocytogenes infection, higher ROS levels was observed in brain at 12 days PI (p <

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0.05) in the infected group than in the control group (Fig. 3a), probably suggesting oxidative tissue damage. However, CAT (Fig. 3b) and SOD (Fig. 3c) activities at 12 days PI were lower in infected animals than in the control group (p < 0.05), suggesting absence of antioxidant repair by theses

enzymes. No significant differences of ROS, CAT, and SOD were observed at the 6th day PI (p >

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0.05).

Discussion

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Studies involving listeriosis lack information regarding cholinergic, purinergic and oxidative stress in brain; however, our group invested in this line of research, with recent publications in cattle

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(Jaguezeski et al. 2018a; Jaguezeski et al. 2018b). The gerbil is an appropriate model for experimental infection with Listeria spp. because it properly reproduces the mechanism and clinical signs of disease (Blanot et al. 1997). Understanding the immune mechanisms in diseases is

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important, because they can be used as indicators of damage and as markers of resolution of the disease via therapeutic pathways (Faas et al. 2017; Dou et al. 2018). In our study, we observed that adult gerbils did not develop clinical disease, but suffered cerebral oxidative stress and altered

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activities of regulatory and neuromodulatory enzymes. In general, the results demonstrated reduced ectonucleotidase activity in the infected animals

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during the course of infection, promoting increases of extracellular ATP and ADP. Our results are similar to those of a study conducted in cattle, in which ectonucleotidase activities decreased in the brains of infected animals (Jaguezeski et al. 2018b). In the context of stress or cell injury, immune cells release large amounts of extracellular ATP in addition to other nucleotides that bind the P2X7 receptor (Jacob et al. 2013), manifesting a danger sign or DAMP (damage associated to molecular pattern) (Zhou et al. 2010; Idzko et al. 2014). The probable increase of ATP activates caspase-1 via the NLRP (Soni et al. 2013) inflammatory pathway, resulting in the secretion of proinflammatory cytokines and apoptosis (Ferrari et al. 2016). When there is an infection, pathogens also signal their

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presence through PAMPs (pathogen-associated molecular patterns) (Janeway and Medzhitov 2002). Both mechanisms (DAMP and PAMPs) favor the activation of a signaling cascade useful in the recognition of intracellular pathogens (Dou et al. 2018), inducing a response by increasing ATP (Jacob et al. 2013; Faas et al. 2017), or chemotaxis of inflammatory cells (Kronlage et al. 2010), production of radical’s oxygen species (Gaikwad et al. 2017), and the synthesis of proinflammatory cytokines IL-6, IL-1β, TNF-α (Shieh et al. 2014), resulting in pathogen and inflammatory control. The increase of TNF-α is related to T lymphocyte proliferation (Veltkamp et al. 2011), and may be related to the increase in cell count found this study. ADA activity was elevated at the beginning of infection, but decreased at the end of the

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experiment, agreeing with results from Fass et al. (2017), who found that the end of the inflammatory process was associated with increasing levels of adenosine via ATP degradation. In elevated levels, adenosine activates receptors coupled to G-proteins that may increase adenylate cyclase activity and increase cyclic AMP levels (Sitkovsky et al. 2004). This, in turn, leads to PKA activation, inhibiting NF-κB activation with a reduction in ROS production, release of pro-

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inflammatory cytokines and increased release of IL-10 and monocyte anti-inflammatory cytokines (Koscsó et al. 2012). Furthermore, in the CNS, adenosine induces expression of the CX3CL1

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pathway that plays a role in the modulation of microglial activation and neuron survival (Lauro et al. 2010). Therefore, this may be a neuroprotection strategy present in asymptomatic listeriosis.

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In a report by our team (Jaguezeski et al. 2018a) of subclinical listeriosis bovine, cerebral acetylcholine levels were increased. Similarly, in the current experimental model, there was a decrease in AChE activity at the end of the experiment. AChE is the principal enzyme hydrolyzing

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acetylcholine in the cholinergic anti-inflammatory pathway in the nervous system, being associated with brain development, neuronal damage and control of immune responses (Jaguezeski et al. 2018a; Pick et al. 2006; Zimmerman et al. 2006). The nicotinic acetylcholine receptor α7 (α7

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nAChR) is widely distributed in the central and peripheral nervous system and immune cells (Wang et al. 2003; Borovikova et al. 2000); when activated by acetylcholine, this receptor mediates the

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negative regulation of NF-κB nuclear translocation by activation of the Jak2/STAT3 pathway, suppressing the transcription of proinflammatory cytokines TNF-α, IL-1 and IL-6 induced by lipopolysaccharides (Borovikova et al. 2000; Hamano et al. 2006). By the reverse mechanism, adhesion of L. monocytogenes to TLR2 receptors of immune cells or TLR4 in microglia results in activation of NF-kB and production of proinflammatory cytokines (Flo et al. 2000), potentiating neuroinflammation (increased blood lymphocytes) that may result in brain injury (Gaikwad et al. 2017). Therefore, the cholinergic system acts to contain infection and inflammation in cerebral listeriosis; the cholinergic system has a role in the immunomodulation of experimental bovine

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listeriosis, because the inhibition of AChE activity leads to an increase in ACh levels, contributing to neuroprotection and possibly avoiding the clinical manifestations of the disease (Jaguezeski et al. 2018a). Finally, CAT and SOD activities were lower in the infected animals, simultaneously with increased ROS during the infection period. The activities of SOD, CAT and glutathione peroxidase (GPX) constitute the first-line antioxidant defenses in biological systems (Ighodaro and Akinlove 2018), which in our study responded negatively to the infection. In response to oxidative stress conditions, the Nrf2 pathway (induced by α7 nAChR) is activated, inducing cytoprotective gene expression and anti-inflammatory and antioxidant responses45, such as glutathione S-transferase

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(GST), heme oxygenase-1 (HO-1) and superoxide dismutase 1 (SOD1), promoting microglia neuroprotection (Egea et al. 2015). It is known that ROS in low concentrations regulates

physiological processes such as signaling in cell proliferation (Kaspar et al. 2009; Dröge 2002), and containment of intracellular infection by L. monocytogenes (Noubade et al. 2014). However, at high

(Ighodaro and Akinlove 2018; Brentnall et al. 2013).

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concentrations, ROS were deleterious, triggering the apoptosis pathway based on caspases 9 and 6

Because any immune response is triggered under apoptosis, the sensitivity of the brain's

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immune system may be impaired by bacterial infection. This mechanism is also used by L. monocytogenes during brain infection in order to circumvent the immune system (Li et al. 2017).

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The increase in the activities of SOD and CAT may indicate an increase in antioxidant defenses (Yu et al. 2017); nevertheless, this mechanism was not observed in the present study. Therefore, the formation of ROS by immune cells (e.g., lymphocytes) may be promoting a response of infection,

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as reported by Noubade et al. (2014), because the purinergic and cholinergic pathways constitute a primary immune defense.

In conclusion, ectonucleotidases activity was reduced in brains of infected gerbils,

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characterizing pro-inflammatory enzymatic action, because less hydrolysis of ATP occurred, with consequently more ATP (a pro-inflammatory molecule) in the extracellular space; this response

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may be a consequence of bacterial infection in the CNS. The subsequent decrease in ADA and AChE activity suggests an anti-inflammatory response in formation, seeking to restore lesions and protect the tissue. Antioxidants and oxidants showed late responses in the infected group, without enzymatic antioxidant response (CAT and SOD showed reduced activity) during the experiment, characterizing cerebral oxidative stress in asymptomatic animals. We conclude that several mechanisms are involved in the process of cellular damage associated with listeriosis observed some in asymptomatic animals. These include inflammatory, excitatory or oxidative mechanisms, all of which need to be further clarified.

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Ethical Committee All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted, in Universidade do Estado de Santa Catarina (under protocol number 5265050617).

Conflict of interest The authors declared no conflict of interest.

Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível

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Superior – Brasil (CAPES). Capes/Proex process number: 88887.285983/2018-00 (Assistance number: 0737/2018). We also thank the CNPq for their financial support.

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Fig. 1 Ectonucleotidases activity in brain of gerbil experimentally infected with Listeria monocytogenes on the 6 and 12 th day post-infection. a) NTPDase (CD39) using ATP as substrate; b) NTPDase (CD39) using ADP as substrate; c) 5’-

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nucleotidase (CD73) using AMP as substrate; and d) adenosine deaminase (ADA) activity.

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Fig. 2 Acetylcholinesterase (AChE) in brain of gerbil experimentally infected with Listeria monocytogenes on the 6 and

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12 th day post-infection.

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Table 1 Hematocrit value and leukogram of control gerbils compared to experimentally infected with L. monocytogenes on the 6th and 12th days post-infection. Variable

Day

Control

Infected

P values

6

40 ± 7.0

44 ± 3.0

0.110

12

44 ± 3.0

46 ±1.0

0.340

6

3.0 ± 0.6

3.2 ± 0.5

0.670

12

2.4 ± 0.5

4.2 ± 0.6

0.001*

6

1.5 ± 0.3

0.91 ± 0.6

0.104

12

0.45 ± 0.12

0.87 ± 0.5

0.115

6

1.3 ± 0.5

2.8 ± 0.3

0.091

12

1.9 ± 0.3

3.2 ± 0.5

0.001*

6

0.18 ± 0.1

0.30 ± 0.13

0.260

12

0.84 ± 0.03

6

0.0 ± 0.0

12

0.0 ± 0.0

Hematocrit (%)

Total Leukocyte (x10³/µL)

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Neutrophil (x10³/µL)

0.06 ± 0.03

0.511

0.0 ± 0.00

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Monocyte (x10³/µL)

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Lymphocyte (x10³/µL)

Eosinophil (x10³/µL)

0.07 ± 0.2

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Mean values and standard deviation for the Student-t test (P < 0.05). *Variables with statistical difference between groups.

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