The pathogenic potential of the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice

Highlights

• Effects of infectious and social factors were investigated in C57BL/6 mice.

• Repeated social defeat (RSD) stress exacerbated Opisthorchis felineus-induced liver pathology.

• A combination of O. felineus and RSD increased neuroinflammation.

• The startle response was aberrant under the influence of both factors.

Abstract

Parasitic food-borne diseases and chronic social stress are frequent attributes of day-to-day human life. Therefore, our aim was to model the combined action of chronic Opisthorchis felineus infection and repeated social defeat stress in C57BL/6 mice. Histological examination of the liver revealed inflammation sites, pronounced periductal fibrosis, and cholangiofibrosis together with proliferation of bile ducts and hepatocyte dystrophy in the infected mice, especially in the stress-exposed ones. Simultaneously with liver pathology, we detected significant structural changes in the cerebral cortex. Immunohistochemical analysis of the hippocampus indicated the highest increase in numerical density of Iba 1-, IL-6-, iNOS-, and Arg1-positive cells in mice simultaneously subjected to the two adverse factors. The number of GFAP-positive cells rose during repeated social defeat stress, most strongly in the mice subjected to both infection and stress. Real-time PCR analysis showed that the expression of genes Aif1 and Il6 differed among the analysed brain regions (hippocampus, hypothalamus, and frontal cortex) and depended on the adverse factors applied. In addition, among the brain regions, there was no consistent increase or decrease in these parameters when the two adverse treatments were combined: (i) in the hippocampus, there was upregulation of Aif1 and no change in Il6 expression; (ii) in the hypothalamus, expression levels of Aif1 and Il6 were not different from controls; and (iii) in the frontal cortex, Aif1 expression did not change while Il6 expression increased. It can be concluded that a combination of two long-lasting adverse factors, O. felineus infection and repeated social defeat stress, worsens not only the hepatic but also brain state, as evidenced behaviorally by disturbances of the startle response in mice.

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.