The role of CuZn- and Mn-superoxide dismutases in earthworm Eisenia andrei kept in two distinct field-contaminated soils.

Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), together with polycyclic aromatic hydrocarbons (PAHs), represent highly toxic and persistent organic environmental pollutants, especially due to their capability for bioaccumulation in fatty tissues. To observe the environmentally relevant effect of these compounds on earthworms, two soils naturally contaminated with PCDD/Fs and PAHs were used in our experiments. We focused on the role of CuZn- and Mn-superoxide dismutases. We assembled a full-length sequences of these molecules from Eisenia andrei earthworm and confirmed their activity. We demonstrated the significant reduction of CuZn-SOD on both mRNA and enzyme activity levels and increased levels of reactive oxygen species in earthworms kept in PCDD/F-polluted soil, which corresponds to the observed histopathologies of the earthworm intestinal wall and adjacent chloragogenous tissue. The results show an important role of CuZn-SOD in earthworm tissue damage caused by PCDD/Fs present in soil. We did not detect any significant changes in the mRNA expression or activity of Mn-SOD in these earthworms. In earthworms maintained in PAH-polluted soil the activity of both CuZn-SOD and Mn-SOD significantly increased. No histopathological changes were detected in these worms, however significant decrease of coelomocyte viability was observed. This reduced viability was most likely independent of oxidative stress.

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms.

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.

Differential expression of immunity-related genes in larval Manduca sexta tissues in response to gut and systemic infection.

Introduction: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Methods: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. Results: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. Discussion: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

Current Aspects of the Role of Autoantibodies Directed Against Appetite-Regulating Hormones and the Gut Microbiome in Eating Disorders.

The equilibrium and reciprocal actions among appetite-stimulating (orexigenic) and appetite-suppressing (anorexigenic) signals synthesized in the gut, brain, microbiome and adipose tissue (AT), seems to play a pivotal role in the regulation of food intake and feeding behavior, anxiety, and depression. A dysregulation of mechanisms controlling the energy balance may result in eating disorders such as anorexia nervosa (AN) and bulimia nervosa (BN). AN is a psychiatric disease defined by chronic self-induced extreme dietary restriction leading to an extremely low body weight and adiposity. BN is defined as out-of-control binge eating, which is compensated by self-induced vomiting, fasting, or excessive exercise. Certain gut microbiota-related compounds, like bacterial chaperone protein Escherichia coli caseinolytic protease B (ClpB) and food-derived antigens were recently described to trigger the production of autoantibodies cross-reacting with appetite-regulating hormones and neurotransmitters. Gut microbiome may be a potential manipulator for AT and energy homeostasis. Thus, the regulation of appetite, emotion, mood, and nutritional status is also under the control of neuroimmunoendocrine mechanisms by secretion of autoantibodies directed against neuropeptides, neuroactive metabolites, and peptides. In AN and BN, altered cholinergic, dopaminergic, adrenergic, and serotonergic relays may lead to abnormal AT, gut, and brain hormone secretion. The present review summarizes updated knowledge regarding the gut dysbiosis, gut-barrier permeability, short-chain fatty acids (SCFA), fecal microbial transplantation (FMT), blood-brain barrier permeability, and autoantibodies within the ghrelin and melanocortin systems in eating disorders. We expect that the new knowledge may be used for the development of a novel preventive and therapeutic approach for treatment of AN and BN.

In Vitro Interactions of TiO2 Nanoparticles with Earthworm Coelomocytes: Immunotoxicity Assessment.

Titanium dioxide nanoparticles (TiO2 NPs) are manufactured worldwide. Once they arrive in the soil environment, they can endanger living organisms. Hence, monitoring and assessing the effects of these nanoparticles is required. We focus on the Eisenia andrei earthworm immune cells exposed to sublethal concentrations of TiO2 NPs (1, 10, and 100 µg/mL) for 2, 6, and 24 h. TiO2 NPs at all concentrations did not affect cell viability. Further, TiO2 NPs did not cause changes in reactive oxygen species (ROS) production, malondialdehyde (MDA) production, and phagocytic activity. Similarly, they did not elicit DNA damage. Overall, we did not detect any toxic effects of TiO2 NPs at the cellular level. At the gene expression level, slight changes were detected. Metallothionein, fetidin/lysenin, lumbricin and MEK kinase I were upregulated in coelomocytes after exposure to 10 µg/mL TiO2 NPs for 6 h. Antioxidant enzyme expression was similar in exposed and control cells. TiO2 NPs were detected on coelomocyte membranes. However, our results do not show any strong effects of these nanoparticles on coelomocytes at both the cellular and molecular levels.

Altered Serum Immunological and Biochemical Parameters and Microbiota Composition in Patients With AN During Realimentation.

Anorexia nervosa (AN) is a life-threatening psychiatric disorder with not well-described pathogenesis. Besides the genetic and sociological factors, autoimmunity is also considered to take part in AN pathogenesis. We evaluated general serological factors showing the physiological state of 59 patients with AN at hospital admission and their discharge. We detected the altered levels of some general biochemical and immunological parameters. We also detected decreased levels of appetite-regulating alpha-melanocyte stimulating hormone (α-MSH) in patients at hospital admission. Moreover, elevated anti-α-MSH IgM levels and decreased anti-α-MSH IgA levels were observed in patients with AN. Therefore, we analyzed the gut microbiota composition with special focus on α-MSH antigen-mimetic containing microbes from the Enterobacteriaceae family. We correlated gut bacterial composition with anti-α-MSH Ig levels and detected decreasing IgG levels with increasing alpha diversity. The upregulation of pro-inflammatory cytokines IL-6, IL-17, and TNF-α were detected in patients with AN both prior and after hospitalization. We also evaluated the treatment outcome and improvement was observed in the majority of patients with AN. We provide new data about various serum biochemical parameters and their changes during the patients' hospitalization, with emphasis on the immune system, and its possible participation in AN pathogenesis.

The intestinal microbiota and metabolites in patients with anorexia nervosa.

Brain-gut microbiota interactions are intensively studied in connection with various neurological and psychiatric diseases. While anorexia nervosa (AN) pathophysiology is not entirely clear, it is presumably linked to microbiome dysbiosis. We aimed to elucidate the gut microbiota contribution in AN disease pathophysiology. We analyzed the composition and diversity of the gut microbiome of patients with AN (bacteriome and mycobiome) from stool samples before and after renourishment, and compared them to healthy controls. Further, levels of assorted neurotransmitters and short-chain fatty acids (SCFA) were analyzed in stool samples by MS and NMR, respectively. Biochemical, anthropometric, and psychometric profiles were assessed. The bacterial alpha-diversity parameter analyses revealed only increased Chao 1 index in patients with AN before the realimentation, reflecting their interindividual variation. Subsequently, core microbiota depletion signs were observed in patients with AN. Overrepresented OTUs (operation taxonomic units) in patients with AN taxonomically belonged to Alistipes, Clostridiales, Christensenellaceae, and Ruminococcaceae. Underrepresented OTUs in patients with AN were Faecalibacterium, Agathobacter, Bacteroides, Blautia, and Lachnospira. Patients exhibited greater interindividual variation in the gut bacteriome, as well as in metagenome content compared to controls, suggesting altered bacteriome functions. Patients had decreased levels of serotonin, GABA, dopamine, butyrate, and acetate in their stool samples compared to controls. Mycobiome analysis did not reveal significant differences in alpha diversity and fungal profile composition between patients with AN and healthy controls, nor any correlation of the fungal composition with the bacterial profile. Our results show the changed profile of the gut microbiome and its metabolites in patients with severe AN. Although therapeutic partial renourishment led to increased body mass index and improved psychometric parameters, SCFA, and neurotransmitter profiles, as well as microbial community compositions, did not change substantially during the hospitalization period, which can be potentially caused by only partial weight recovery.

Earthworm immunity.

Earthworms belonging to oligochaete annelids became a model for comparative immunologists in the early sixties with the publication of results from transplantation experiments that proved the existence of self/nonself recognition in earthworms. This initiated extensive studies on the earthworm immune mechanisms that evolved to prevent the invasion of pathogens. In the last four decades important cellular and humoral pathways were described and numerous biologically active compounds were characterized and often cloned.

Mutual interactions of E. andrei earthworm and pathogens during the process of vermicomposting.

Vermicomposting is a process by which earthworms together with microorganisms degrade organic wastes into a humus-like material called vermicompost. This process does not include a thermophilic stage, and therefore, the possible presence of pathogens represents a potential health hazard. To elucidate the effect of earthworms in the selective reduction of pathogens, grape marc substrate was artificially inoculated with Escherichia coli, Enterococcus spp., thermotolerant coliform bacteria (TCB), and Salmonella spp., and their reduction during vermicomposting was monitored. Various defense mechanisms eliminating microorganisms in the earthworm gut were assumed to be involved in the process of pathogen reduction. Therefore, we followed the expression of three pattern recognition receptors (coelomic cytolytic factor (CCF), lipopolysaccharide-binding protein (LBP), and Toll-like receptor (v-TLR)), two antimicrobial molecules (fetidin/lysenins and lysozyme), and heat shock protein HSP70. We detected the significant decrease of some defense molecules (fetidin/lysenins and LBP) in all pathogen-inoculated substrates, and the increase of CCF and LBP in the Salmonella spp.-inoculated substrate. At the same time, the reduction of pathogens during vermicomposting was assessed. We observed the accelerated reduction of E. coli, Enterococcus spp., and TCB in pathogen-inoculated substrates with earthworms compared to that without earthworms. Moreover, the differences between the microbiome of grape marc substrate and earthworm intestines were determined by high throughput sequencing. This analysis revealed that the bacterial composition of grape marc substrate differed from the composition of the content of earthworm intestines, suggesting the elimination of specific bacterial species during food passage through the gut.

Anorexia nervosa: Gut microbiota-immune-brain interactions.

Anorexia nervosa is a psychiatric disorder defined by an extremely low body weight, a devastating fear of weight gain, and body image disturbance, however the etiopathogenesis remains unclear. The objective of the article is to provide a comprehensive review on the potential role of gut microbiota in pathogenesis of anorexia nervosa. Recent advances in sequencing techniques used for microbial detection revealed that this disease is associated with disruption of the composition of normal gut microbiota (dysbiosis), manifested by low microbial diversity and taxonomic differences as compared to healthy individuals. Microorganisms present in the gut represent a part of the so called "microbiota-gut-brain" axis that affect the central nervous system and thus human behavior via the production of various neuroactive compounds. In addition, cells of the immune system are equipped with receptors for these neuroactive substances. Microbiota of the intestinal system also represent a very important antigenic source. These antigens can mimic some host neuropeptides and neurohormones and thus trigger the production of autoantibodies which cross-react with these compounds. The levels and affinities of these antibodies are thought to be associated with neuropsychiatric conditions including anxiety, depression, and eating and sleep disorders. The study of microbiota function in diseases could bring new insights to the pathogenetic mechanisms.

Developmental and Immune Role of a Novel Multiple Cysteine Cluster TLR From Eisenia andrei Earthworms.

Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

Microbiota, Microbial Metabolites, and Barrier Function in A Patient with Anorexia Nervosa after Fecal Microbiota Transplantation.

The change in the gut microbiome and microbial metabolites in a patient suffering from severe and enduring anorexia nervosa (AN) and diagnosed with small intestinal bacterial overgrowth syndrome (SIBO) was investigated. Microbial gut dysbiosis is associated with both AN and SIBO, and therefore gut microbiome changes by serial fecal microbiota transplantation (FMT) is a possible therapeutic modality. This study assessed the effects of FMT on gut barrier function, microbiota composition, and the levels of bacterial metabolic products. The patient treatment with FMT led to the improvement of gut barrier function, which was altered prior to FMT. Very low bacterial alpha diversity, a lack of beneficial bacteria, together with a great abundance of fungal species were observed in the patient stool sample before FMT. After FMT, both bacterial species richness and gut microbiome evenness increased in the patient, while the fungal alpha diversity decreased. The total short-chain fatty acids (SCFAs) levels (molecules presenting an important source of energy for epithelial gut cells) gradually increased after FMT. Contrarily, one of the most abundant intestinal neurotransmitters, serotonin, tended to decrease throughout the observation period. Overall, gut microbial dysbiosis improvement after FMT was considered. However, there were no signs of patient clinical improvement. The need for an in-depth analysis of the donor´s stool and correct selection pre-FMT is evident.

Contribution of Eisenia andrei earthworms in pathogen reduction during vermicomposting.

Vermicomposting is a process of degradation of biowaste which involves complex interactions between earthworms and microorganisms. This process lacks a thermophilic stage and thus, the possible presence of pathogens poses a potential health hazard. To assess the contribution of earthworms during the selective reduction of various pathogens, apple pomace substrate was artificially inoculated with Escherichia coli, Salmonella spp., thermotolerant coliform bacteria, and Enterococci. The artificial bacterial load did not influence the weight, reproduction, or intestinal enzymatic activity of the earthworms, but it caused reversible histological changes to the epithelial layer and chloragogen tissue of their intestines. The reduction of pathogenic Enterococci and E. coli from the substrate was accelerated by earthworms (63-fold, 77-fold, and 840-fold for Enterococci and 6-fold, 36-fold, and 7-fold for E. coli inoculated substrates after 2, 4, and 6 weeks, respectively). Moreover, the rapid elimination of Salmonella spp. was supported by the upregulated expression of two pattern recognition receptors which bind lipopolysaccharide, coelomic cytolytic factor, and lipopolysaccharide-binding protein. Further, the microbiomes of the intestine and the composting substrate differed significantly. Graphical abstract.

Characterization, molecular cloning and localization of calreticulin in Eisenia fetida earthworms.

Calreticulin is a highly conserved calcium-binding protein affecting many cellular processes inside and outside of the endoplasmic reticulum (ER). It participates in the regulation of Ca(2+) homeostasis, acts as a chaperone and modulates gene transcription, integrin-mediated cell signalling as well as cell adhesion. Here we report on the sequence characterization of a calreticulin-coding cDNA of Eisenia fetida earthworms. The neighbor-joining phylogeny tree constructed based on the deduced amino acid sequence indicates a common origin of the E. fetida calreticulin molecule and that of mollusks. A polyclonal anti-calreticulin antibody used for immunocytochemistry and immunohistochemistry localized the protein in the mesenchymal lining of the coelomic cavity and in coelomocytes of E. fetida. In situ hybridization revealed high expression of E. fetida calreticulin in various cells and tissues, namely epidermis, neurons of the ventral nerve cord, intestine, sperms, body wall muscles and some coelomocytes. Real-time PCR confirmed the strong expression of calreticulin in the nervous system, particularly in cerebral ganglia, in body wall muscles and in seminal vesicles. Moreover, a high calreticulin expression was measured in the muscular pharynx.

Comparative study of the CCF-like pattern recognition protein in different Lumbricid species.

Coelomic fluid of the Lumbricid Eisenia fetida contains a 42-kDa pattern recognition protein named coelomic cytolytic factor (CCF) that binds microbial cell wall components and triggers the activation of the prophenoloxidase cascade, an important invertebrate defense pathway. Here we report on the sequence characterization of CCF-like molecules of other Lumbricids: Aporrectodea caliginosa, Aporrectodea icterica, Aporrectodea longa, Aporrectodea rosea, Dendrobaena veneta, Lumbricus rubellus and Lumbricus terrestris, and show that CCF from E. fetida has a broader saccharide-binding specificity, being the only one recognizing N,N'-diacetylchitobiose. We suggest that the broad recognition repertoire of E. fetida CCF reflects a particular microbial environment this species lives in.

Relationship between hemolytic molecules in Eisenia fetida earthworms.

The coelomic fluid of the earthworm Eisenia fetida has been reported to contain a variety of proteins causing the lysis of red blood cells-EFAF (Eisenia fetida andrei factor), fetidin, lysenin, eiseniapore, and hemolysins isolated either from coelomic fluid (H1, H2, H3) or from cell lysate (CL(39) and CL(41)). We document the presence of two distinct genes with a high level of homology. These genes encode fetidin and lysenin but their level of expression differs in individual E. fetida andrei animals.

LBP/BPI homologue in Eisenia andrei earthworms.

LBP/BPIs are pattern recognition receptors that are often present in vertebrates and in invertebrates, and they play a defense role against pathogens. We have identified 1698 bp cDNA sequence from the Eisenia andrei earthworm with predicted amino acid sequence that shares homology with the LBP/BPI family (EaLBP/BPI). Sequence analysis of EaLBP/BPI proved the existence of two conserved domains with the potential ability to bind LPS. The predicted molecular mass of the EaLBP/BPI protein is 53.5 kDa, and its high basicity (pI 9.8) is caused by its high arginine content. Constitutive transcription of the Ealbp/bpi gene was shown in all tested tissues, with the highest level in coelomocytes and seminal vesicles; the lowest level was detected in the intestine. On the contrary, another earthworm LPS-binding molecule CCF (coelomic cytolytic factor) was expressed only in the intestine and coelomocytes. In E. andrei coelomocytes, the transcription of Ealbp/bpi gene was up-regulated in response to bacterial stimulation, reaching a maximum at 8 and 16 h post stimulation with Bacillus subtilis and Escherichia coli, respectively.

Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms.

The tube-within-tube body plan of earthworms is appropriate for studying the interactions of microorganisms with the immune system of body cavities such as the digestive tract and coelom. This study aims to describe the immune response on the molecular and cellular level in the coelomic cavity and the gut of the earthworm Eisenia andrei after experimental microbial challenge by administering two bacterial strains (Escherichia coli and Bacillus subtilis) or yeast Saccharomyces cerevisiae to the environment. The changes in mRNA levels of defense molecules (pattern recognition receptor CCF, lysozyme, fetidin/lysenins) in the coelomocytes and gut tissue were determined by quantitative PCR. The immune response at a cellular level was captured in histological sections, and the expression of CCF was localized using in situ hybridization. Coelomocytes respond to the presence of bacteria in the coelomic cavity by increasing the mRNA levels of defense molecules, especially CCF. The immune response in gut tissue is less affected by microbial stimulation because the epithelial cells of gut exhibit basically strong mRNA synthesis of ccf as a defense against the continuous microbial load in the gut lumen. The cellular immune response is mediated by coelomocytes released from the mesenchymal lining of the coelomic cavity. These combined immune mechanisms are necessary for the survival of earthworms in the microbially rich environment of soil.

Effect of experimental microbial challenge on the expression of defense molecules in Eisenia foetida earthworm.

Earthworms are able to protect themselves against invading pathogens due to efficient innate defense mechanisms. Currently, two types of antimicrobial factors including lysozyme-like molecule and factors with hemolytic activity, as well as a pattern recognition protein named coelomic cytolytic factor (CCF) have been identified in Eisenia foetida earthworms. However, the modulations of these defense molecules during in vivo immune response have not been addressed. In this study, we investigated the effect of experimental challenge with live Gram-negative and Gram-positive bacteria and with beta-1,3-glucan on the expression of CCF and the hemolytic factor fetidin. In parallel, we followed levels of hemolytic activity and lysozyme-like activity in the coelomic fluid of challenged earthworms. We show that the biosynthesis of CCF, but not fetidin, is up-regulated upon microbial stimulation. Parenteral administration of bacteria or microbial polysaccharides in earthworms results, in the coelomic fluid, in augmented level of CCF, increased lysozyme-like activity and decreased hemolytic activity. The decreased hemolytic activity of the coelomic fluid reflects the increase of the whole protein content in the absence of synthesis of hemolytic proteins.