The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice
Graphical abstract
Introduction
Mechanisms of the interaction between the liver (the main organ neutralizing foreign substances) and the brain (the primary regulator of all functions in the mammalian organism) are poorly studied, especially in the context of progressing pathological processes. Nonetheless, research interest in liver–brain interactions has been growing lately because diseases of the liver (e.g., steatosis, cirrhosis and hepatitis) may induce brain neuroinflammation (D'Mello and Swain, 2011, Montoliu et al., 2015, Azhari and Swain, 2018, Colognesi et al., 2020), which manifests itself behaviorally in the form of fatigue, increased anxiety, loss of appetite, sleep disturbances and loss of social interest, collectively termed “sickness behaviour” (D’Mello and Swain, 2014).
Pathological processes in the liver can be caused by helminths of the family Opisthorchiidae — Opisthorchis viverrini and Opisthorchis felineus — which enter the human (or animal) body after the individual consumes fishes from the family Cyprinidae without proper thermal cooking. The subsequent development of opisthorchiasis leads to severe complications in the helminth’s hosts (purulent cholangitis, cholecystitis, chronic hepatitis, constriction of biliary passages, abscesses of the liver, obstructive jaundice, or pancreatitis) including even cholangiocarcinoma (Sripa et al., 2007). Inflammation in the liver is accompanied by persistent monocyte infiltration, which differentiates into tissue macrophages. It is reported that after invasion by O. viverrini, there is polarisation of M1 macrophages (with their associated T helper 1 (Th1) immune response) into the M2 phenotype (and a Th2 immune response) together with the development of fibrosis in the liver of humans and animals (Bility and Sripa, 2014). Under such conditions, analysis of iNOS gene expression, which is characteristic for M1 macrophages (Martinez and Gordon, 2014, Shapouri-Moghaddam et al., 2018), shows downregulation in both blood- and liver-derived monocytes/macrophages. It is known that the excretory-secretory product (ESP) of the parasites plays a key role in various aspects of parasite–host relations in helminthiases (also in the polarisation of macrophages toward the M2 phenotype). It consists of multifunctional proteins actively secreted and excreted by helminths as well as metabolites, intestinal contents, and substances from the tegument surface. It has been demonstrated that all of these play a pivotal part in the penetration of host tissues by helminths, in the modulation of the host immune response, morphofunctional rearrangement of surrounding tissues, and in nutrient supply for the parasite (Berasain et al., 1997, Donnelly et al., 2005, Robinson et al., 2009, Smout et al., 2015), which, taken together, contribute to prolonged helminth parasitosis. This process is probably mediated by extracellular microvesicles (50–1000 nm in diameter) or exosomes (30–150 nm) participating in the secretion of a specific subset of components including proteins, mRNAs, microRNAs (miRNAs), and lipids in various organisms including parasites (Marcilla et al., 2014, Debs et al., 2019). For instance, there is evidence that in O. viverrini infection, most proteins of the helminth ESP are secreted into extracellular space via exosome-like vesicles, which then get into the host’s cells (Chaiyadet et al., 2015). The proof of the participation of the O. felineus helminth ESP in the polarisation of host blood macrophages can be obtained in vitro during co-cultivation of blood cells with adult maritae. Nonetheless, such data are currently unavailable.
Chronic inflammation in O. viverrini opisthorchiasis, just as in many diseases of the liver, is accompanied by upregulation of proinflammatory cytokine IL-6 in the blood (Sripa et al., 2009, Sripa et al., 2012). Because IL-6, together with two other proinflammatory cytokines, IL-1β and TNF, exerts not only a local but also distant action, at elevated concentrations in the blood, it can reach the brain and activate inflammatory processes there (D'Mello and Swain, 2011, Milewski and Oria, 2016). Consequently, it can be hypothesised that opisthorchiasis, especially a chronic case, is a potential contributing factor to severe anomalies in the CNS. This assumption is supported by numerous studies involving endotoxin lipopolysaccharide (LPS) as a classic proinflammatory agent, administration of which causes systemic inflammation with subsequent neuroinflammation, mostly reflected in the activation of the main resident immunocompetent cells of the brain i.e., microglia (Cazareth et al., 2014, Cheng et al., 2018, Brown, 2019).
Neuroinflammation in the brain is provoked not only by infectious but also by social factors, for example, repeated social defeat (RSD) stress (Wohleb et al., 2011, Kopschina Feltes et al., 2017, Niraula et al., 2018). In the latter case, stress promotes the release of bone marrow-derived monocytes into the circulation that are recruited to the brain, thereby enhancing neuroinflammation (D'Mello and Swain, 2011, Wohleb et al., 2011). It is reported that RSD acts bidirectionally. On the one hand, it stimulates the immune system, thus promoting increased formation of red bone marrow monocytes; on the other hand, RSD directly activates the sympathetic nervous system and hypothalamic–pituitary–adrenal axis (Reader et al., 2015). As a consequence, the blood level of corticosterone increases simultaneously with rising brain concentrations of IL-6, IL-1β and tumour necrosis factor (TNF), which all together augment neuroinflammation and cause prolonged anxiety-like behaviour (D'Mello and Swain, 2011, Wohleb et al., 2011, Wohleb et al., 2013). In RSD-exposed mice, by manganese-enhanced magnetic resonance imaging, researchers detected permanent activation of the brain regions known to regulate depressive- and anxiety-like behaviour, including the prefrontal cortex and ventral hippocampus (Laine et al., 2017).
It can be expected that a combination of two factors, i.e., chronic O. felineus infection and prolonged social stress, which are not uncommon among humans, will enhance each other’s pathological influence on the brain, thereby affecting behaviour, which is an indicator of the functional state of the brain in health and disease. This hypothesis is supported by studies on the combined action of LPS and RSD in mice. Some researchers have noted markedly stronger stress-induced ionised calcium-binding adapter protein 1 (Iba 1) immunoreactivity, especially in the hippocampus (Wohleb et al., 2012), which has a high concentration of microglia (Fonken et al., 2015) and expresses cytokine receptors at high density (Hopkins and Rothwell, 1995).
Previously, we have shown that a combination of RSD stress with 14-day infections in mice resulted in elevation of the IL-6 level in the blood and activation of cysteine proteases in the brain, cathepsins B and L (Avgustinovich et al., 2016), participating in many acute and chronic inflammatory processes. More prolonged infection of mice (6 months) concurrently with 30 day RSD stress induced leukocyte growth and activation of hemopoiesis in red bone marrow (Orlovskaya et al., 2018). In the present study, the aim was to evaluate the functional state of the liver and brain as well as the startle response (which reflects the ability of the brain to filtre sensory input) in mice during prolonged exposure to the two adverse factors.
Section snippets
Animals
In this work, males of inbred C57BL/6 and outbred CD-1 strains of mice were used, weighing 25–30 g and aged 2.5–3.0 months. During the experiments, the mice were maintained on a 12:12 h cycle (light:dark) at 24 °C, with ad libitum access to granulated feed and water. All the procedures were performed in compliance with international rules for experiments on animals (European Communities Council Directives of 24 November 1986, 86/609/EEC) and in accordance with the decision of the Bioethics
Histological examination of liver sections
Our semiquantitative analysis revealed that in OF and especially OF + SS mice, there was well-pronounced periductal fibrosis and cholangiofibrosis in the liver together with bile duct proliferation, hepatocyte dystrophy, and the presence of inflammation sites (Fig. 2A). Additionally, in the animals of the three experimental groups, there was noticeable enhancement of the hyperplasia of the bile duct epithelium in the absence of dysplasia and metaplasia. RSD stress caused weak dystrophy of
Discussion
In this study, we investigated a combined influence of two adverse factors of different nature (O. felineus infection and RSD stress) on the state of the liver and brain in mice from the standpoint of liver–brain interaction.
A comparison of histological samples of the liver indicated that the most pronounced negative effect is exerted by the helminth infection of the mice: periductal fibrosis, cholangiofibrosis, proliferation of bile ducts, hepatocyte dystrophy, and inflammatory infiltration.
Acknowledgements
This work was supported by the Russian Foundation for Basic Research (grant No. 20-04-00139), by the state program of the Federal Research Center ICG SB RAS, Russia (No. 0324-2019-0041-C-01), and a project within a state assignment (No. 0301-2019-0005) for the Institute of Solid State Chemistry and Mechanochemistry, SB RAS. The authors are grateful to the Multi-access Center for Microscopy of Biological Objects, Russia (http://www.bionet.nsc.ru/microscopy/) and to the Center for Genetic
References (59)
- et al.
Combined effects of social stress and liver fluke infection in a mouse model
Brain Behav. Immun.
(2016) - et al.
Role of peripheral inflammation in hepatic encephalopathy
J. Clin. Exp. Hepatol.
(2018) - et al.
Astrocyte-microglia cooperation in the expression of a pro-inflammatory phenotype
CNS Neurol. Disord.: Drug Targets
(2013) - et al.
Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components
J. Parasitol.
(1997) - et al.
Chronic Opisthorchis viverrini infection and associated hepatobiliary disease is associated with iron loaded M2-like macrophages
Korean J. Parasitol.
(2014) The endotoxin hypothesis of neurodegeneration
J. Neuroinflammation
(2019)- et al.
How microglia kill neurons
Brain Res.
(2015) - et al.
Molecular and cellular neuroinflammatory status of mouse brain after systemic lipopolysaccharide challenge: importance of CCR2/CCL2 signaling
J. Neuroinflammation
(2014) - et al.
Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production
Int. J. Parasitol.
(2015) - et al.
Brain–heart interaction: cardiac complications after stroke
Circ. Res.
(2017)
Kaempferol alleviates LPS-induced neuroinflammation and BBB dysfunction in mice via inhibiting HMGB1 release and down-regulating TLR4/MyD88 pathway
Int. Immunopharmacol.
Depression and cognitive impairment - extrahepatic manifestations of NAFLD and NASH
Biomedicines
Heterogeneity of Microglial Activation in the Innate Immune Response in the Brain
J. Neuroimmune Pharmacol.
Quantitative changes in hippocampal microvasculature of chronically stressed rats: no effect of fluoxetine treatment
Hippocampus.
Interplay of extracellular vesicles and other players in cerebral malaria pathogenesis
Biochim. Biophys. Acta Gen. Subj.
Cerebral microglia recruit monocytes into the brain in response to tumor necrosis factor alpha signaling during peripheral organ inflammation
J. Neurosci.
Liver-brain inflammation axis
Am. J. Physiol. Gastrointest. Liver Physiol.
Liver–brain interactions in inflammatory liver diseases: implications for fatigue and mood disorders
Brain Behav. Immun.
Thioredoxin peroxidase secreted by Fasciola hepatica induces the alternative activation of macrophages
IAI
Microglia inflammatory responses are controlled by an intrinsic circadian clock
Brain Behav. Immun.
Pharmacological studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review
Psychopharmacology
Arginase-1 is expressed exclusively by infiltrating myeloid cells in CNS injury and disease
Brain Behav. Immun.
Cytokines and the nervous system I: expression and recognition
Trends Neurosci.
Physiology of microglia
Physiol. Rev.
Repeated social defeat induced transient glial activation and brain hypometabolism: A positron emission tomography imaging study
J. Cereb. Blood Flow Metab.
The sensory contact model for the study of aggressive and submissive behaviors in male mice
Aggress Behav.
Brain activation induced by chronic psychosocial stress in mice
Sci. Rep.
Comparative histopathology of Opisthorchis felineus and Opisthorchis viverrini in a hamster model: An implication of high pathogenicity of the European liver fluke
Parasitol. Int.
Extracellular vesicles in parasitic diseases
J. Extracell. Vesicles
Cited by (3)
Prolonged liver fluke infection combined with alcoholization: An experimental mouse model
2022, Experimental ParasitologyCitation Excerpt :The prolonged alcoholization promoted fatty degeneration of hepatocytes in the mice, as noted by other researchers in such a mouse model (Szabo et al., 2012). Here, chronic opisthorchiasis was accompanied, as in humans and other animals (Sripa et al., 2009; Lvova et al., 2012; Dangtakot et al., 2017; Avgustinovich et al., 2021), by severe periductal fibrosis and cholangiofibrosis, massive infiltration of liver tissue by cells of predominantly lymphocytic-monocytic origin, and increased proliferation of bile ducts. Moreover, this proliferation was seen in the parenchyma areas distant from bile ducts, where the helminths were localized.
Behavioral effects and inflammatory markers in the brain and periphery after repeated social defeat stress burdened by Opisthorchis felineus infection in mice
2022, Physiology and BehaviorCitation Excerpt :It is reported that during O. viverrini infection, there is a positive correlation between increased plasma levels of IL-6 and higher risk of advanced fibrosis and bile duct cancer [23]. In our previous study, chronic O. felineus infection for 6 months in C57BL/6 mice provoked not only the development of a severe liver pathology but also higher mRNA expression of the Aif1 gene and greater density of Iba1-, IL-6-, and iNOS-positive cells in the hippocampus, thereby indicating microglial activation and a neuroinflammatory state of the brain [25]. Under these conditions, if RSDS was administered to the mice in the form of daily defeat in 30 intermale confrontations during the prolonged experimental opisthorchiasis, then the disturbances of the liver and brain became more pronounced.