Vagotomy induces deregulation of the inflammatory response during the development of amoebic liver abscess in hamsters.

Original Paper Received: November 18, 2013 Accepted after revision: March 16, 2014 Published online: May 6, 2014

Neuroimmunomodulation DOI: 10.1159/000362240

Vagotomy Induces Deregulation of the Inflammatory Response during the Development of Amoebic Liver Abscess in Hamsters Esperanza Sánchez-Alemán a Andrés Quintanar-Stephano b Eustacio Galileo Escobedo-González c María del Rosario Campos-Esparza a Rafael Campos-Rodríguez d Javier Ventura-Juárez a a

Departamento de Morfología and b Departamento de Fisiología y Farmacología, Universidad Autónoma de Aguascalientes, Aguascalientes, and c Hospital General de México, and d Departamento de Bioquímica, Escuela Superior de Medicina, IPN, Mexico City, Mexico

Abstract Background: The parasympathetic nervous system modulates the immune response in the abdominal-pelvic gut through the vagus nerve, which releases acetylcholine. This endogenous ligand acts on α7 nicotinic receptors expressed on immune cells. Objective: To study the mechanism of the production and regulation of cytokines in parasympathectomized and control hamsters during the development of amoebic liver abscesses (ALA) caused by Entamoeba histolytica. Methodology: Six- to 8-week-old male hamsters with and without vagotomy were used in a model of ALA. The animals were infected with trophozoites (350,000; HM1:IMSS strain) via the intrahepatic route and sacrificed at 6, 12, and 24 h and at 2, 4, and 7 days postinfection. Immune parameters were recorded at each time point using morphometric techniques including immunofluorescence and immunohistochemistry assays. These parameters included signal transducer and activator of transcription 3 (STAT3) levels, pro- and

© 2014 S. Karger AG, Basel 1021–7401/14/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/nim

anti-inflammatory cytokine levels, and nuclear factor-κB (NFκB) activation in neutrophils and macrophages. Results: Compared to the control groups, the vagotomized (VAG) hamsters showed a significant increase in NFκB activation in neutrophils and macrophages, and higher levels of interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α. VAG hamsters showed an increase in the expression of IL-8 and phosphorylated STAT3 during the first 24 h postinfection as well as slightly increased levels of transforming growth factor-β on days 2–7 postinfection. No significant differences were demonstrated in the levels of IL-10. Conclusions: These results suggest that the vagus nerve plays an important role in the regulation of inflammation during ALA formation. © 2014 S. Karger AG, Basel

Introduction

The central nervous system, through humoral and neural pathways, regulates and integrates the immune response to pathogenic agents and damaged tissue. Throughout an infection, the nervous and immune systems maintain extensive communication [1, 2] via the Javier Ventura-Juárez, PhD, Departamento de Morfología Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes Avenida Universidad 940, Colonia Ciudad Universitaria, Edificio 202 Aguascalientes 20131 (Mexico) E-Mail jventur @ correo.uaa.mx

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Key Words Vagus nerve · Entamoeba histolytica · Inflammation · Amoebic hepatic abscess · Vagotomy · Parasympathetic nervous system

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own nerve fibers, thus, the liver can regulate the hepatic metabolisms, haemodynamic and regenerative response via neuroimmune connections [14, 15]. In the liver, the neuroimmune effects of the cholinergic system induce changes in the production of collagen, increases in the production of interferon (IFN)-γ, TNF-α, and IL-10, and modifications in the evolution of ALA (depending on the individual) [16]. Modulation of the neuroimmune response is carried out through various types of immune cells, the most abundant of these being the Kupffer cells, the resident macrophages of the liver. They are anchored in the space of Disse, protrude between endothelial cells into the lumen of sinusoids, and produce cytokines upon stimulation (e.g. TNF-α and IL1β) [17]. Other immune cells are also abundant, including natural killer cells and professional antigen-presenting dendritic cells [18, 19], T cells distributed throughout the liver lobes, and endothelial cells which express soluble adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and E-selectin. Adhesion molecules, regulated by TNF-α, increase the leukocyte migration from the bloodstream [20, 21]. Reports of in vitro assays have shown that human neutrophils (stimulated by IFN-γ or TNF-α) and human macrophages have amoebicidal activity against E. histolytica trophozoites [22–24]. In contrast, other evidence suggests that in vitro exposure of naive isolated gerbil macrophages (a susceptible species) to amoebic proteins results in the production of only a small quantity of TNF-α [22]. During the early stages of amoebic liver invasion, hepatic cellular destruction can be induced by interaction between the immune cells and parasites. Trophozoites have been found near undamaged hepatocytes, in both lysed and unlysed areas, with abundant or scant inflammatory infiltrate. In addition to macrophages and neutrophils, T CD8+ and T CD4+ cells are also present [23]. In the model of ALA in hamsters (a susceptible species), the early stages (the first 12 h) of the lesion are characterized by an acute cellular infiltrate composed principally of polymorphonuclear leukocytes that surround the trophozoites. Lysed histiocytes and leukocytes are located in the periphery of the lesions. Hepatocytes near the early lesions show degenerative changes leading to necrosis. In the later stages of the lesions, the degree of necrosis increases, epithelioid cells replace the majority of leukocytes, and well-organized granulomas develop. Extensive necrosis associated with the fusion of granulomas appears on approximately day 7. This pattern of damaged tissue Sánchez-Alemán et al.


hypothalamus-immune cells and organs, and they communicate with the autonomous nervous system via its parasympathetic and sympathetic branches [3]. These immune neuroendocrine interactions provide a context for understanding the causes and consequences of numerous phenomena at the molecular, cellular, and behavioral levels in both healthy individuals and those with hepatic disorders [4, 5]. The vagus nerve (VN) is the main pathway for parasympathetic innervation of the thorax and gut, including the pancreas and liver. It is composed of afferent (80%) and efferent nerve fibers. The latter are visceromotor preganglionic fibers that originate from the dorsal motor nucleus of the VN and the ambiguous nucleus of the medulla oblongata. These preganglionic fibers of the solar, gastric, and hepatic plexus synapse with postganglionic fibers in the parasympathetic ganglia in the target tissues [6]. Acetylcholine (Ach) is the principal neurotransmitter of the skeletal neuromuscular junction and the autonomous ganglia and in the parasympathetic postganglionic neurons and some sympathetic postganglionic neurons [7]. The efferent branch of the VN releases Ach, which in turn binds to α7 nicotinic receptors (α7nAChR) expressed on macrophages and other immune cells (T and B cells). This ligand-receptor binding inhibits the activation of nuclear factor-κB (NFκB) [8, 9], which in turn decreases the production of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)1β, and IL-6 without affecting the production of anti-inflammatory cytokines, including IL-10. On the other hand, IL-1 has been found to stimulate the VN at its terminal branches in the intestine, inducing modulation of the anti-inflammatory response [10]. Cholinergic receptors have been found in endothelial cells, where Ach inhibits the expression of adhesion molecules [11]. During natural Entamoeba histolytica infection, the intestine is initially colonized. After that, amoebas often pass from the intestine, via portal vessels, to the liver, where they are able to form abscesses. One percent of people infected with E. histolytica eventually develop potentially fatal pathologies, such as fulminant amoebic colitis or amoebic liver abscess (ALA) [12], which is 3 times more common in men than in women [13]. In the majority of cases, ALA involves the formation of only one abscess that is frequently located in the right hepatic lobe (which receives the greatest percentage of portal circulation). The liver is innervated by parasympathetic nerves, which release Ach in its efferent endings; whereas the sympathetic nerves release noradrenaline through their

Table 1. Primary antibodies for immunohistochemistry and immunofluorescence

Antibody

Host

Antibody type

Species reactivity

Catalog No.

Trademark

CD68a CD15a P-NFκB P65a

Mouse Mouse Rabbit

Monoclonal Monoclonal Monoclonal

AB955 M0733 3033L

Abcam 1:400 Dako 1:200 Cell Signaling 1:200

P-STAT3a IL-1β IL-6 IL-8

Mouse Mouse Mouse Rabbit

Monoclonal Monoclonal Monoclonal Polyclonal

MAB3705 MAB1001 CBL2117 AB7747

Chemicon Millipore Millipore Abcam

1:100 1:200 (5 μg/ml) 1:200 (5 μg/ml) 1:200 (1 μg/ml)

IFN-γ TNF-α IL-10 TGF-β

Rabbit Rabbit Rabbit Rabbit

Polyclonal Polyclonal Polyclonal Polyclonal

Mouse, human Human Human, mouse, rat, hamster, monkey, pig Mouse, human Goat, bovine Human Rat, human, cynomolgus monkey, rhesus monkey Human Human Human Mouse, rat, human

AB9657 AB1441 AB34843 AB66043

Abcam Millipore Abcam Abcam

1:1,000 (0.5 μg/ml) 1:100 (11 μg/ml) 1:400 (0.2 mg/ml) 1:200 (2.5 μg/ml)

The concentration is not shown; only the dilution suggested by the manufacturer is listed.

suggests that E. histolytica trophozoites do not produce hepatic abscesses in hamsters through the direct lysis of hepatocytes but these are instead due to the accumulation of toxic molecules produced by the lysis of leukocytes and macrophages surrounding the amoebas [24]. In this study, we provoked ALA in vagotomized (VAG) and control hamsters with E. histolytica and then analyzed the production of IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ, and transforming growth factor (TGF)-β in the liver by using immunohistochemistry and applying a morphometric method. We also assessed the activation of NFκB on macrophages and neutrophils using immunofluorescence. The aim of this analysis was to attain a broader understanding of the role of the parasympathetic nervous system as a modulator of the inflammatory response to infection.

Materials and Methods Amoebic Cultures Trophozoites of E. histolytica (HM1:IMSS strain) were cultivated in axenic conditions in a medium containing BI-S-33 + 15% (v/v) adult bovine serum (Microlab SU120) previously inactivated at 56 ° C for 30 min [25]. To maintain the virulent phase, amoebas were serially inoculated into hamster livers.

days by oral via) to eliminate intestinal parasites, the animals were divided into 6 groups (n=18): intact (IA), intact inoculated (IA+Eh), sham - operated (SHAM), sham-operated inoculated (SHAM+Eh), vagotomy (VAG) and vagotomy inoculated (VAG+Eh). After 24 h of fasting, VAG animals were anesthetized with pentobarbital sodium (25–40 mg/kg body weight). Vagotomy was performed by surgically removing the hepatic and intestinal branches of the VN. The sham surgery involved just surgical dissection followed by gentle manipulation of the hepatic and intestinal branches of the VN [26]. In order to corroborate the efficacy of each subdiaphragmatic vagotomy, during the autopsy the totality of the liver denervation was assessed; since gastric dilation is used as a sign of vagotomy, the stomach was removed and weighed [16]. ALA was induced 30 days after surgery via intrahepatic inoculation of E. histolytica trophozoites by injection of 350,000 trophozoites suspended in 100 μm of culture medium into the right liver lobe. Control IA, SHAM, and VAG hamsters were injected with culture medium only. The animals were sacrificed at 6 distinct time points after infection: 6, 12, and 24 h, and 2, 4, and 7 days. Samples of damaged and normal liver tissues were dissected and fixed in paraformaldehyde at 4% in phosphate-buffered saline (PBS) ×1. Animals were handled in accordance with the norms established by the Bioethics Commission of the Universidad Autónoma de Aguascalientes and in accordance with the NIH guidelines for research using animals (Guidelines for the Care and Use of Laboratory Animals, 2002).

Subdiaphragmatic Vagotomy, the ALA Model, and Collection of Tissue Samples One hundred eight golden male hamsters (Mesocricetus auratus) aged 6–8 weeks and weighing 100–150 g were used in this study. After pretreatment with metronidazole (7.5 mg/kg for 5

Immunolocalization and Quantity of Cytokines, STAT3, and Macrophages and Neutrophils Positive for Phosphorylated NFκB in Liver Abscesses Fixed liver tissues were immersed in paraffin and cut into 5-μm slices and placed on slides previously treated with 3-aminopropiltriethoxy-silane (Sigma A3648). After deparaffinization with xylene and rehydratation through different alcohol-water solutions

Vagotomy Induces Deregulation of the Inflammatory Response


3


a

Dilution (concentration)

Statistical Analysis The values for the control and experimental groups were compared using Student’s t test for means and ANOVA followed by a post hoc Tukey test for comparison of variances. The Windows version of GraphPad Prism 5.00 was used for all analysis. All experiments were performed in triplicate. Data are expressed as means ± SD.

Results

Liver Morphometric Analysis: Localization and Number of Macrophages and Neutrophils with P-NFκB The expression of NFκB was detected simultaneously in CD68+ macrophages and CD15+ neutrophils with the P-NFκB marker. Macrophages were observed in the cen4


ter of the inflammatory infiltrate at each measured time point during the evolution of ALA in control (fig. 1a, g) and VAG hamsters (fig. 1d, j). Although P-NFκB was detected in several inflammatory cells from both groups, it was observed mainly in the cytosol of macrophage cells (fig. 1b, e, h, k; arrowhead). Some positive CD68+ macrophages (fig. 1a, d; arrow) and NFκB were already present in the first stages of the infection (fig. 1b, e; arrowhead). CD68+ macrophages and NFκB were observed together mainly in the cytosol; however, quantitative differences in the number of labeled cells were evident in the VAG and control groups (fig. 1c, f, i, l; asterisks). There were also differences between VAG and control IA-SHAM hamsters regarding positive CD15+ neutrophils during the development of the ALA (fig. 2a, d, g, j; in red, marked with arrows). The presence of NFκB inside the neutrophils can also be seen in figure 2b, e, h, and k (in green, marked with arrowheads). It is worth mentioning that NFκB was found in the cytoplasm of some polymorphonuclear cells. Few CD15+ cells were found in coexistence with NFκB (fig. 2c, f, i, l; in yellow, marked with asterisks). The number of positive CD68+ (fig. 3a) and CD15+ cells (fig. 3b) counted per square millimeter at each of the evaluated time points is shown in figure 3. There was greater P-NFκB positivity (indicating the presence of NFκB) in CD68+ cells than in CD15+ cells at all time points. In VAG hamsters, at 7 days postinfection, there was a greater increase in CD68+ cells coexisting with NFκB (10.43 ± 0.93 cells/mm2) compared to the IA (3.97 ± 0.15 cells/mm2) and SHAM groups (4.97 ± 0.42 cells/ mm2) (p < 0.001). The same pattern exists with respect to CD15+ cells coexisting with NFκB (7.97 ± 0.09 cells/ mm2) compared to the IA (5.06 ± 0.52 cells/mm2) and SHAM animals (5.33 ± 0.38 cells/mm2) (p < 0.001). Analysis of STAT3+ Cells Utilizing immunohistochemistry with HRP, positive P-STAT3 were detected at several points in the inflammation process surrounding the granulomatose lesions (fig. 4a–d). Positive inflammatory cells were counted using morphometry. Infected VAG hamsters had the greatest quantity of cells positive for P-STAT3+ at 6, 12, and 24 h postinfection (fig. 4e); after that this parameter gradually decreased at days 2, 4, and 7 postinfection. In the short periods of interaction between parenchymal cells and parasites, P-STAT3 was seen in greater quantities in the inflammatory cells of infected VAG animals. Among infected animals, VAG hamsters at 24 h had 10.70 ± 2.50 positive cells/mm2, IA hamsters had 5.67 ± 0.76 positive Sánchez-Alemán et al.


(100, 96, 80 and 70%), finally with PBS, the slides were processed for histological staining with HE in order to verify the areas of injury, also, we performed immunohistochemical stains for detecting several cytokines and phosphorylated signal transducers and activators of transcription 3 (P-STAT3), whereas macrophages, neutrophils and NFκB were assessed by immunofluorescence. In order to unmask epitopes, the samples were subjected to treatment with citrate buffers (0.02 M at 120 ° C) for 2 min. For immunohistochemistry, endogenous peroxidase activity was blocked with methanol peroxide at 1% for 1 h at room temperature. Nonspecific reactions were blocked by treatment with fetal bovine serum at 10% in PBS for 1 h at room temperature. The slides were incubated overnight at 4 ° C with the appropriate antibody (table 1) and then incubated with the secondary antibody marked with peroxidase (Dako Envision Dual Link System-HRP K4061). The reaction was completed by adding 3,3′-diaminobenzidine (D-8001; Sigma) and H2O2 as a substrate for 1 min. The samples were counterstained with hematoxylin, dehydrated with alcohol, mounted with high-speed inclusion medium (Entellan; Merck), and examined using a light microscope (Axioskop 4.0) photograph system (CoolSnap-Pro Digital). Photographs were taken using Image-Pro Plus 4.5.0.19 (Media Cybernetics) [23, 27]. Double immunofluorescence was used to visualize the expression of phosphorylated p65 NFκB (P-NFκB) in macrophages and neutrophils. Tissue slides were incubated overnight at 4 ° C with the CD68 and CD15 antibodies, respectively (table 1). As a second antibody, Alexa Fluor 488 (A11008 and A21044 diluted 1:1,000) was used for CD68 and CD15. For the second immunofluorescence, slides were incubated overnight at 4 ° C with P-NFκB antibodies and, as a second antibody, Alexa Fluor 594 (diluted 1:1,000; A1105; Invitrogen). Autofluorescence was reduced with Sudan Black B (3645; Hycel) at 0.1% diluted with ethanol at 70%. Staining of the nuclei was performed with Hoechst dye (33258, 1:1,000 in PBS ×1). Samples were mounted using glycergel mounting medium (C0563; Dako) and examined under a microscope in the epifluorescent mode. Morphometric quantification of cells positive for the reaction was carried out by counting the cells located in the center, on the edge, and in the bordering areas of the abscess, and also at other foci of inflammation. The number of cells per area was expressed as cells per square millimeter.

CD68

P-NFǊB

CD68 + P-NFǊB + Hoechst stain

b

c

d

e

f

g

h

i

j

k

l

VAG + Eh 7 days

Control IA + Eh 7 days

VAG + Eh 24 h

Control IA + Eh 24 h

a

macrophages during the development of ALA. Figures are representative images of the double immunofluorescence analysis performed on liver tissue for 3 independent rounds of animal sacrifice for each of the times specified throughout the experiment. Fluorescent microscopy of macrophages is shown in red (arrows) and NFκB is shown in green (arrowheads), evaluated during the kinet-


ics of ALA development. a–c IA + E. histolytica at 24 h. d–f VAG + E. histolytica at 24 h. g–i IA + E. histolytica at 7 days. j–l VAG + E. histolytica at 7 days. Nuclear counterstaining with Hoechst dye. Colocalization of P-NFκB expression in CD68+ cells is shown in yellow (asterisks) at 24 h and at 7 days postinfection. Scale bar = 50 μm. Eh = E. histolytica.


5


Fig. 1. Absence of the VN induces a higher activation of NFκB in

CD15

P-NFǊB

CD15 + P-NFǊB + Hoechst stain

b

c

d

e

f

g

h

i

j

k

l

VAG + Eh 7 days

Control IA + Eh 7 days

VAG + Eh 24 h

Control IA + Eh 24 h

a

neutrophils during the development of ALA. Fluorescent microscopy of the hepatic areas damaged by E. histolytica in VAG and IA hamsters. Neutrophils are marked in red (arrows). The molecular complex P-NFκB is marked in green (arrowheads). Nuclei were stained with Hoechst dye. a–c IA + E. histolytica at 24 h. d–f VAG + E. histolytica at 24 h. g–i IA + E. histolytica at 7 days. j–l VAG + E. histolytica at 7 days. The evaluation times of 24 h and 7 days are

6


the most representative of changes during the evolution of ALA. In VAG hamsters, there was a greater quantity of neutrophils positive for P-NFκB in inflammatory areas at 24 h and at 7 days compared to the control groups. This colocalization is shown in yellow (asterisks). Figures are representative images of the double immunofluorescence analysis performed on liver tissue for 3 independent rounds of animal sacrifice for each of the times specified throughout the experiment. Scale bar = 50 μm. Eh = E. histolytica.

Sánchez-Alemán et al.


Fig. 2. Absence of the VN induces a higher activation of NFκB in

IA control + Eh

0

12 h

*** **

6h

***

***

24 h

2 days

*

12 h

4 days

7 days

10

*** 0

b

**

6h

12 h

**

***

24 h

2 days

4 days

Fig. 3. Activation of NFκB in macrophages and neutrophils during the development of ALA. Mean ± SD of the number of cells positive for P-NFκB: macrophages (a) and neutrophils (b) for IA, SHAM, and VAG hamsters from 6 h to 7 days postinfection with E. histolytica (n = 3). Significant differences between infected VAG hamsters and between infected IA or SHAM animals are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001. Eh = E. histolytica.

cells/mm2, and SHAM hamsters had 6.30 ± 2.02 positive cells/mm2.

b

c

d

10

e

* **

* ***

** ***

IA control + Eh SHAM + Eh VAG + Eh

*

* *

5 0

7 days

Time

a

15

*** Cells (n/mm2)

Neutrophils-NFǊB (n/mm2)

15

5

VAG + Eh

***

10

5

P-STAT3

6h

12 h

24 h

2 days Time

4 days

7 days

Fig. 4. Transcription factor P-STAT3 expressed in the cells of in-

flammatory areas in the livers of the hamsters. IA + E. histolytica (a, c) and VAG + E. histolytica hamsters (b, d). The number of cells positive for P-STAT3 was higher in VAG hamsters than in IA hamsters, with significant differences found at 6, 12, and 24 h and at 7 days. The photographs show the results at 12 h (a, b) and at 4 days (c, d) postinfection with E. histolytica. The arrows indicate inflammatory cells positive for P-STAT3. Scale bar = 50 μm. e Number of positive cells at all times evaluated. * p < 0.05; ** p < 0.01; *** p < 0.001. Representative images correspond to the immunohistochemical technique of HRP performed on hepatic tissue for 3 independent rounds of animal sacrifice for each of the times specified in the experiment. Eh = E. histolytica.

VAG Animals Exhibited the Greatest Number of Proinflammatory Cytokine-Producing Cells Compared to IA and SHAM Hamsters in Most Time Point Evaluations IL-1β was expressed by few cells at the foci of inflammation (fig. 5a–d). In infected VAG hamsters, the greatest number of positive cells was observed at 24 h and at 2 and 4 days postinfection (7.067 ± 1.59, 8.10 ± 1.0, and 5.88 ± 0.92 cells/mm2, respectively; fig. 6a), demonstrating a significant difference (p < 0.001) between IA (1.37 ± 0.19, 1.46 ± 0.15, and 0.99 ± 0.361 cell/mm2) and SHAM ani-

mals at the same points evaluated (2.32 ± 0.26, 1.43 ± 0.19, and 1.63 ± 0.26 cells/mm2). The expression of the main mediator of inflammation IL-8 [28] was greatest in infected VAG hamsters (8.15 ± 1.13 cells/mm2) during the first 24 h postinfection (fig. 5e, f; arrows), demonstrating a significant difference (p < 0.001) between IA (3.03 ± 0.53 cells/mm2) and SHAM (3.63 ± 0.78 cells/mm2) animals (fig. 6b). From day 2 to day 7, there was no significant difference in the numbers of these cells in the several infected groups.



7


a


4 days

Macrophages-NFǊB (n/mm2)

15

VAG + Eh

b

c

d

4 days

a

IL-8

VAG + Eh

12 h

IA control + Eh

f

g

h

4 days

e

Fig. 5. IL-1β (a–d) and IL-8 (e–h) expressed on cells in areas of

inflammation in hamster livers during the development of ALA. IA at 12 h (a, e) and at 4 days (c, g). VAG hamsters at 12 h (b, f) and at 4 days (d, h). With the immunohistochemical technique of HRP, a greater number of cells were positive for both of these cytokines in VAG hamsters compared to IA hamsters. Significant differences between the VAG and control groups were mainly found in the shorter time periods (p < 0.001). The arrows indicate inflammatory cells positive for IL-1β and IL-8. Scale bar = 50 μm. Eh = E. histolytica.

The TNF-α expression was highest in the inflammatory cells of VAG hamsters (17.02 ± 1.03 cells/mm2) at 24 h postinfection (fig. 7a–d; arrows) compared (p < 0.001) to IA (6.60 ± 0.61 cells/mm2) and SHAM (8.77 ± 0.66 cells/mm2) hamsters. However, this cytokine was observed to increase by an even lesser amount in VAG animals at other time points evaluated. The differences in TNF were highly significant (p < 0.001) on the first 5 studied times of the process of infection by E. histolytica and only decreased on the 7th day of the study (p < 0.05, 8


fig. 6c), indicating that vagotomy affects TNF-α producing cells through the process of infection by E. histolytica. The expression of IFN-γ was similar between the three groups during the first 24 h postinfection, with the greatest expression detected in IA and SHAM animals. A significant difference in IFN-γ cells was apparent between VAG and IA or SHAM animals starting at 2 days (p < 0.05), 4 days (p < 0.001), and 7 days (p < 0.05) postinfection (fig. 6d). The peak of IFN-γ expression occurred on day 4 postinfection in the VAG (13.3 ± 1.33 cells/mm2), IA (4.23 ± 0.69 cells/mm2), and SHAM (6.60 ± 0.81 cells/ mm2) groups (fig. 7e–h). At 12 h postinfection, VAG animals showed a higher expression of IL-6 compared to IA and SHAM animals (fig. 8a–d, 9a). This increase was also observed on day 4 postinfection in VAG (11.33 ± 0.38 cells/mm2), IA (2.93 ± 0.12 cells/mm2), and SHAM (2.31 ± 0.17 cells/mm2) animals. Except at 24 h postinfection, there was a similar expression of IL-10 in the remaining infected experimental groups. At 24 h postinfection, there was a higher IL-10 expression (p < 0.01) in VAG hamsters (6.63 ± 0.61 cells/ mm2) compared to IA (4.40 ± 0.42 cells/mm2) and SHAM (3.58 ± 0.46 cells/mm2) animals (fig. 8e–h; 9b). The expression of TGF-β (fig. 8i–l) was not different between groups during the first 24 h postinfection but did significantly increase (p < 0.01) at 2, 4, and 7 days to 6.87 ± 1.06, 6.90 ± 0.95, and 7.5 ± 0.5 cells/mm2, respectively, in VAG hamsters, to 4.80 ± 1.00, 4.57 ± 1.06, and 5.13 ± 1.11 cells/mm2, respectively, in IA hamsters, and to 3.53 ± 1.0, 3.92 ± 0.38, and 6.08 ± 0.63 cells/mm2, respectively, in SHAM animals (fig. 9c).

Discussion

Interaction between the immune and nervous systems is vital for modulation of the immune response to infection; the sympathetic and VN innervation of the thymus, liver, heart, gastrointestinal tract, lungs, pancreas, and kidneys represents hardwired pathways of the central nervous system regulation of innate immunity. Neural regulation is faster than endocrine processes; thereby the VN and the sympathetic division of the autonomic nervous system can mediate an initial and rapid immunomodulatory response. Slower neuroendocrine mechanisms, including the hypothalamus-pituitary-adrenal axis, regulate inflammation and protect against the deleterious effects of the proinflammatory mediators. Cytokine production is necessary to protect against pathogens Sánchez-Alemán et al.


IL-1Ǆ

12 h

IA control + Eh

10

***

5 0

TNF-į (cells/mm2)

***

*

6h

12 h

20

24 h

2 days

4 days

**

7 days

*** ***

*** *

5

6h

12 h

24 h

15 10

** **

5 0

**

6h

12 h

24 h

2 days

4 days

7 days

20

*** 10

b

***

15

0

c

***

IL-8 (cells/mm2)

15

2 days

4 days

Fig. 6. Immunohistochemical technique of HRP for IL-1β (a), IL-8 (b), TNF-α (c), and IFN-γ (d) expressed in the cells of inflamma-

tory areas in hamster livers during the development of ALA. Cell counts were made for all time periods evaluated and for all groups, including uninfected animals (data not shown). Note the greater variation in the acute phase of the infection (up to 24 h). IL-1β and TNF-α producer cells showed the greatest increase in VAG + E.

*

*** *

10 5 0

7 days

Time

15

6h

12 h

d

24 h

2 days

4 days

7 days

Time

histolytica hamsters; TNF-α had a higher increase in number in VAG + E. histolytica hamsters at 24 h. Cells producing IL-8 were observed to be increased during the first 24 h in this group of animals. IFN-γ did not show any significant differences between VAG and control animals until day 2. * p < 0.05; ** p < 0.01; *** p < 0.001. Eh = E. histolytica.

and promote tissue repair, but excessive cytokine release can lead to systemic inflammation, organ failure, and death. Inflammatory responses are finely regulated to effectively guard against noxious stimuli. The central nervous system interacts dynamically with the immune system to modulate inflammation through humoral and neural pathways [29, 30]. This study demonstrates, through an in situ analysis, NFκB and STAT3 activation in neutrophils and macrophages from the first 6 h to 7 days, and an increased production of proinflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) in the absence of the VN in the liver vagotomy hamster, thus highlighting the importance of the vagus nerve in regulating the mechanisms of inflammation during the development of amoebic hepatic abscesses.

The VN has traditionally been associated with the regulation of vital physiological functions, including the heart rate, bronchoconstriction, and gastrointestinal activity, through its principal neurotransmitter Ach [29]. Borovikova et al. [10] described the mechanisms by which the efferent branch of the VN can suppress the overproduction of proinflammatory cytokines, which they denominated the ‘cholinergic anti-inflammatory pathway’. These are the physiological mechanisms through which the nervous system interacts with the innate immune response to regulate inflammation [10]. Electric stimulation of the VN has been studied in laboratory animals to explore the modulatory function of the VN on the immune system, and significant reductions in the systemic levels of TNF-α and other proinflammatory cytokines during endotoxemia have been found [8, 10, 29,



9


a

20


IFN-ǅ (cells/mm2)

IL-1Ǆ (cells/mm2)

20

IA control + Eh

TNF-į

VAG + Eh

12 h

IA control + Eh

a

IL-6

VAG + Eh

4 days

12 h

b

c

a

b

c

d

IFN-ǅ

VAG + Eh

12 h

IA control + Eh

4 days

d

IA control + Eh

VAG + Eh

f

Fig. 8. Immunohistochemical technique of HRP performed on hepatic tissue for IL-6 (a–d), IL-10 (e–h), and TGF-β (i–l) expressed in the cells of inflammatory areas (arrows) during the development of ALA. IA at 12 h (a, e, i) and at 4 days (c, g, k). VAG hamsters at 12 h (b, f, j) and at 4 days (d, h, l). Note the greater number of cells positive for these cytokines in VAG vs. IA during the longer times of infection (2, 4, and 7 days) and the higher number of inflammatory cells positive for IL-6 and TGF-β (d, l). No significant differences were observed in the images shown for IL-10. k Amoeba surrounded by inflammatory cells, only some of which are marked positive for TGF-β. Representative images of the 3 independent rounds of animal sacrifice for each of the times tested. Scale bar = 50 μm.

10


g

h IA control + Eh

TGF-Ǆ

VAG + Eh

i

j

k

l



Fig. 7. Immunohistochemical technique of HRP for TNF-α (a–d) and IFN-γ (e–h) expressed in the cells of inflammatory areas (arrows) in hamster livers during the development of ALA. IA animals at 12 h (a, e) and at 4 days (c, g). VAG hamsters at 12 h (b, f) and at 4 days (d, h). Marked cells were counted for all time periods evaluated. b, d Amoebic forms positive for TNF-α. Inset Greater detail for a marked cell. h Highest number of labeled mononuclear cells of all time periods assessed. Scale bar = 50 μm. Eh = E. histolytica.

12 h

h

f

4 days

g

e

4 days

4 days

12 h

e

IL-10

20

15

15

*** 10

***

5 0

***

***

*** 6h

12 h

24 h

2 days

4 days

7 days

*

*

*

2 days

4 days

7 days

IL-10 (cells/mm2)

IL-6 (cells/mm2)

a

20

b

10

** 5 0

6h

12 h

TGF-Ǆ (cells/mm2)

15 10 5 0

c

6h

12 h

24 h

24 h 2 days Time

4 days

7 days


Time

31]. On the contrary, vagotomy has been shown to produce high levels of proinflammatory cytokines both in this model [10] and during intestinal inflammation [32], indicating a tonic inhibitory effect of the VN on the production of this kind of cytokine. Little work has been done in relation to NFκB in spite of its central role in E. histolytica-induced inflammatory processes. This protein complex controls the expression of genes that codify for proinflammatory cytokines (IL-6, TNF-α, IL-1, and IL2), chemokines (IL-8 and MCP-1), and adhesion molecules (ICAM-1, VCAM, and E-selectin) [8]. NFκB and STAT3 are transcription factors likely to play important roles in liver inflammatory responses. Signaling via α7nAChR induces recruitment to and phosphorylation of Jak2 by α7nAChR and the subsequent Jak2-induced activation of STAT3. In peritoneal macrophages, the nicotine-mediated suppression of TNF production via α7nAChR is dependent on P-STAT3 and its capacity to bind DNA [33]. Considering that STAT3 does not regulate TNF transcription directly [34] and that NFκB recruits and associates with STAT3, it is proposed that the

attenuation of cytokine production through α7nAChR may implicate the collaboration of the NFκB and Jak/ STAT pathways. It has recently been described that α7nAChR can control TNF production in macrophages via a mechanism that requires the expression of the STAT3 protein, in which α7nAChR prevents STAT3 tyrosine phosphorylation, and that unphosphorylated STAT3 controls the NFκB pathway to modulate the innate immune response to infection. Unphosphorylated STAT3 modulates α7 nicotinic receptor signaling and cytokine production in sepsis [35]. This coincides with our results which showed that vagotomy induces phosphorylation of STAT3 in the acute stages of ALA and therefore the activation of NFκB and the release of proinflammatory cytokines such as IL-1β, IL-8, TNF-α, and IL-6. A variety of cytokines have been shown to induce STAT3 activation in the liver, playing key roles in inducing the acute-phase response, protecting against hepatocellular damage, and promoting liver regeneration [36]. Inhibition of STAT3 protein expres-



11


Fig. 9. Immunohistochemical technique of HRP for IL-6 (a), IL-10 (b), and TGF-β (c) expressed in the inflammatory cells of hepatic tissue during the development of ALA. Note the higher significant differences between the VAG and control groups at all times tested for IL-6, at 24 h for IL-10, and at 2, 4, and 7 days postinfection for TGF-β. * p < 0.05; ** p < 0.01; *** p < 0.001.

12


NFκB and genes that codify for anti-inflammatory cytokines (like IL-10). Phosphorylation of the transcription factor STAT3, which is normally activated by α7nAChR, results in suppression of the production of proinflammatory cytokines in macrophages [33] and at the same time induces the production of anti-inflammatory cytokines as a result of being translocated to the cell nucleus [47]. By definition, liver vagotomy eliminates the stimulation to α7nAChR. However, immunohistochemistry techniques have shown the presence of STAT3 in VAG hamsters, and this suggests that an alternate pathway is responsible for the elevated levels of NFκB during the first few hours postinfection. It is possible that the IL-6 receptor is activated, which would in turn activate the tyrosine kinase Jak-2. Upon phosphorylation, Jak-2 would induce the phosphorylation of STAT3, which stimulates the production of IL-10 upon reaching the cell nucleus. This mechanism could explain the ability of an organism to control many inflammatory disorders [48]. The interaction of IL-6 and IL-10 with Jak-STAT also initiates JakSTAT phosphorylation [49]. During the pathogenesis of amoebiasis, in vitro and in vivo studies have shown an increase in the secretion of certain cytokines, including IL-1β, IL-8, and IFN-γ, by mononuclear cells in the bloodstream as well as intestinal epithelial cells [22, 50–52]. Tsutsumi et al. [24] and Ventura-Juarez et al. [53] showed that E. histolytica trophozoites are capable of inducing an important inflammatory response via polymorphonuclear and mononuclear leukocytes in hamsters. Pacheco-Yepez et al. [54] demonstrated, through immunohistochemistry and PCR techniques, that the immune response to E. histolytica invasion includes a gradual increase in TNF-α, IFN-γ, IL-8, and IL-1β during the early stages (the first 3 h) of ALA development. This response then slowly decreases, reaching its nadir at 48 h postinfection. The current results show that after vagotomy there is an increase in cells expressing IL-1β, TNF-α, IFN-γ, and IL-8 in the acute stage of the inflammatory process, and later there is a decrease in the number of these cells. Studies of intact hamsters have evaluated the presence of IL1α, IL-1β, and IL-8 and corroborated the presence of these cytokines during the acute response to infection by E. histolytica with both in vitro [50] and in vivo [54] models of hepatic amoebiasis. The presence of IL-1β is strongly associated with the VN and its regulation [55]. This study indicates an important role for IL-1β in ALA development and demonstrates that its quantity relies on cholinergic regulation, especially during the initial stages of infection. Sánchez-Alemán et al.


sion enhanced TNF-α responses proportionally to the concentration, suggesting that the STAT3 protein can inhibit TNF-α production. Furthermore, choline inhibited TNF-α production in control macrophages but not when STAT3 protein expression was inhibited using siRNA. Thus, α7nAChR can control TNF-α production in macrophages through a mechanism that requires STAT3 protein expression [35]. NFκB, which is also activated by TNF-α, was reported to be crucial for the stress response after LPS and open field stress in vivo [37]. In vitro studies of human intestinal epithelial tissue have shown that NFκB is activated in response to amoebic infection [38]. Additionally, Maldonado-Bernal et al. [39] demonstrated that NFκB is activated by Toll-like receptors (TLRs), including TLR2 and TLR4, in response to the recognition of E. histolytica-specific lipopeptidophosphoglycan by the innate immune system. This activation leads to the release of IL-10, IL-12 p40, TNF-α, and IL-8 from human monocytes [39]. The regulating effect of the cholinergic pathway on NFκB has been shown in smokers, acute respiratory distress syndrome, and epilepsy, focusing on the production of pro- and anti-inflammatory cytokines in monocytes during the inflammatory process [40–44]. In rats subjected to bilateral cervical vagotomy, the intemitent electrical stimulation at the caudal vagus ends in inhibited NFκB expression in the liver, thus, modulates the inflammatory response during acute hypovolemic hemorrhagic shock [45]. In the hamster model of ALA, subdiaphragmatic vagotomy has been shown to induce changes in the inflammatory response, specifically an increase in cells producing IL-10 and IFN-γ, while TNF-α does not change at 7 days postinfection [16]. When agonists to α7nAChR are utilized, proinflammatory cytokine release is inhibited and this has been found to protect animals in a variety of experimental inflammatory models through suppression of the activation of NFκB by the cholinergic pathway [46]. During ALA development in this study, VAG hamsters showed a greater number of macrophages and neutrophils with activated NFκB in lesions of the hepatic parenchyma compared to IA and SHAM animals. This activation of NFκB resulted in an increase in the number of cells expressing proinflammatory cytokines, suggesting that CD68+ macrophages and CD15+ neutrophils in the liver are more responsive to stimuli by E. histolytica in the absence of the VN compared to when it is intact. Triggering the anti-inflammatory pathway via the VN requires the activation of α7nAChR by Ach, mediated by the activation of phosphorylated Jak2-STAT3, which reaches the nucleus and induces an inhibitory effect on

During the first 48 h postinfection, there was an increase in IL-8 at the inflammatory foci of VAG animals. IL-8 can stimulate the production of IL-1β, TNF-α, and other cytokines [28]. It seems that IL-8 would then be a strong chemoattractor during this period in which the parasympathetic influence is absent. It is important to emphasize that in the IA and SHAM hamsters a greater expression of IL-8 was observed during the period of chronic inflammation (after 48 h), suggesting that in these two groups the ALA size was greater than in the VAG animals. TNF-α is a key proinflammatory cytokine in the inflammatory response produced by macrophages and T cells [56]. Mouse models have demonstrated that the release modulation of TNF-α by inflammatory cells requires cholinergic signaling of α7nAChR [57], as well as NFκB signaling of the transduction of this cytokine [58]. There were high levels of TNF-α in VAG hamsters during the 7 days postinfection. However, only after 2 days was there an increase in IFN-γ. Since the principal functions of IFN-γ are to activate macrophages, attract leukocytes, cause many cell types to mature, and regulate B cells in the innate and adaptive immune response [59, 60], our study indicates that the parasympathetic neuroimmune regulation of inflammation involves TNF-α in the early stages and IFN-γ in the later stages. Stimulation of the VN has been utilized as a treatment for patients with deep depression and uncontrolled epilepsy due to the communication that exists between the afferent vagal pathways and the nerve centers related to these functions. Upon measuring the serum levels of proand anti-inflammatory cytokines in these patients, a significant increase was found in IL-6, TNF-α, and TGF-β at the systemic level, without a significant change in IL-1β and IL-10. The result of this type of vagus stimulation was a generally suppressive effect on inflammation resulting from a decrease in circulating proinflammatory cytokines [44]. In this study a liver vagotomy induced the overproduction of cytokines at inflammatory foci and no effect of the VN on the production of IL-10. This evidence seems to indicate that a subdiaphragmatic vagotomy produces a different effect than that achieved via VN stimulation [44]. In vitro studies have shown that IL-10 exerts an anti-inflammatory effect by inhibiting the activation of macrophages and the production of IL-1β and TNF-α [61]. In cultures of macrophages and experimental models of hemorrhagic shock, arthritis, and pancreatitis there have been reports of Ach participation in attenuating the release of proinflammatory cytokines without producing any effect on the anti-inflammatory cytokine IL-10 [10,

62]. The slight increase in IL-10 at 24 h postinfection in VAG hamsters may indicate an innate immune response in which increased levels of proinflammatory cytokines activate the mechanism of STAT3 transcription independently of the cholinergic pathway [63]. A recent study on amoebiasis showed that E. histolytica induces the expression of TGF-β in hamster splenocytes, which could explain the inhibitory effects on the function of macrophages [64]. In parasitosis, this cytokine is an important regulator of inflammation, as well as macrophage repair and function. This is due to its proinflammatory effects at low concentrations and its anti-inflammatory effect at high concentrations (in the latter function, it inhibits the production of IFN-γ and TNF-α) [65]. TGF-β also contributes to the pathogenesis of fibrosis during the healing process in the majority of organs, including the liver [66]. When comparing these results with those of ALA histology (data not shown), a slight increase in fibrous tissue was observed along with cells positive for TGF-β at 2 days postinfection, indicating that there was no change in the response and transcription to TGF-β in the livers of VAG animals.



Conclusion

This study demonstrates for the first time the importance of the VN and the activation of transcription factors NFκB and STAT3 in modulating the inflammatory response induced during ALA development.

Acknowledgment This work was supported by the National Council of Science and Technology (CONACYT-49749), and by the Autonomous University of Aguascalientes, Mexico (PIBB11-3). CONACYT fellowship No. 371793 was granted to Esperanza Sánchez-Alemán. Dr. Blair Brown revised the English language of this report.

1 Webster JI, Tonelli L, Sternberg EM: Neuroendocrine regulation of immunity. Annu Rev Immunol 2002;20:125–163. 2 Otmishi P, Gordon J, El-Oshar S, Li H, Guardiola J, Saad M, Proctor M, Yu J: Neuroimmune interaction in inflammatory diseases. Clin Med Circ Respirat Pulm Med 2008; 2: 35–44. 3 Rosas-Ballina M, Tracey KJ: Cholinergic control of inflammation. J Intern Med 2009; 265: 663–679.

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