Autoimmunity in human CE: Correlative with the fertility status of the CE cyst

Echinococcosis is one of the 17 neglected tropical and zoonotic diseases (NTDs) documented by the World Health Organization (WHO). In endemic regions, prevalence rates in humans exceed 50 per 100,000 people/ year. In some regions in Central Asia, China, Argentina, Peru, and East Africa incidence rate may increase up to 5 – 10 %. In hyperendemic areas of South America prevalence of echinococcosis in the livestock in slaughterhouses, diverges from 20 % up to 95 %. This parasitic infection ensues also extensively in Australia, particularly in eastern regions, where it cycles chiefly in a wildlife pattern (Craig et al., 2007; Brunetti et al., 2010; Barnes et al., 2012; Thompson et al., 2014; WHO, 2020). According to WHO in 2018, increased numbers of infected people reported in a multicenter study revealed the real burden of this neglected parasitic disease in some European Regions and Turkey. In Egypt, a recent study determined the wide diversity of this parasitic disease in slaughterhouses (El Dakhly et al., 2019). Therefore, it becomes necessary for the World Health Organization (WHO) to prioritize efforts for human cystic echinococcosis (CE) (WHO, 2018; WHO, 2020).

It worthy to mention that the establishment of CE disease conserves infection of the intermediate host (herbivorous animal) or the end-host (man) by ingesting the infective stage of the parasite (eggs) that disperse in the dogs’ feces; consequently, oncospheres would release and approach the portal blood circulation to lodge in the hepatic parenchyma in about 60 % of the cases. Later, each oncospheres undergoes multiple insidious morphological alterations that involve disintegration of its hooklets, vesiculation, and multiplication of its cellular territories, to be recreated into a cyst surrounded by a germinal layer that frequently produces the characteristic CE cyst fluid. Accordingly, in many years it can attain a larger size. The inner germinal layer reproduces asexually forming inside buds of protoscoleces (PSC) and brood capsules (asexual-reproduction) while depositing to the outside the laminated layer for its protection (Bortoletti et al., 1973; Galindo et al., 2002; Atmaca et al., 2019).

Probably in the intermediate host cystic echinococcosis exists in two variable forms: fertile CE cyst, in which PSC is attached to the germinative layer and liberated into the CE cyst fluid. Also, the viability of the fertile CE cyst, that is, the proportion of live PSC, fluctuates between 100 % and 2,8 % (Dueger et al., 2001; Lahmar et al., 2009; Salem et al., 2011; Lahmar et al., 2013; Zait et al., 2013). Contrarily, non-fertile CE cysts have no PSC neither connected to the germinal layer nor detached free in the CE cyst fluid, and thus are incapable of continuing the parasite life cycle; and the reason beyond CE cysts infertility remains unclear (Rogan et al., 2006; Irshadullah et al., 2011).

However, the innate immunity against the developing oncospheres tempts to defeat the infection via recruitments of eosinophils and macrophages. Consequently, in few weeks, lymphocytic T cells become stimulated and incorporated in the inflammatory response that starts preliminary with the activation of T helper (Th)-1 cells to eliminate the parasite; however, dynamic alterations in the T cells retain the predominance of the Th-2 and T regulatory (Treg) cells (Gottstein et al., 1991; Vatankhah et al., 2018; Abo Aziza et al., 2020; Jiménez et al., 2020). Interestingly, the immunomodulatory mechanisms supervened by the host-derived IL-10 and TGF-β would be invested by the parasite to house itself silently at the expense of the surrounding liver microenvironment (Hanilou et al., 2008; Siracusano et al., 2008). Therefore, the clinical presentation of CE disease remains asymptomatic or presents with non-specific symptoms to be undetectable for a long time until complications ensue (Sahin et al., 2006; Saidi, 2006; Frider et al., 2010; Amit et al., 2015).

Recently, IL-17, a signature interleukin produced chiefly by Th17 cells, has been defined in the progression of liver inflammation (Hammerich et al., 2011) and gastric cancers (Zhong et al., 2015). Besides, in a human study, the expression level of IL-17 appeared to affect the progression of cystic echinococcosis (Tursun et al., 2010). However, the factual role of IL-17 in CE remains not well understood especially if this interleukin had been implicated in several autoimmune (Furue & Kadono, 2019; Nalbandian et al., 2019) and infectious diseases (Iwakura et al., 2008).

In this framework and on the account of previous studies, CE in humans seemed to be related to autoimmune diseases (Girelli et al., 1993; De Rosa et al., 2001; Tekaya et al., 2009). Review articles published by Zhang et al. (2003), Zhang et al. (2012), and Grubor et al. (2017) proposed several mechanisms exerted by the parasite to avoid the host-protective immunity that involves antigen mimicry.

In the current study, serum samples for CE were used to define autoimmune populations implied in the diagnosis of autoimmune and rheumatic diseases; anti-nuclear antibody (ANA) and anti-cyclic citrullinated peptide (anti-CCP) antibodies.

ANAs are a subset of autoantibodies that attack the nuclear macromolecules including DNA, histones, centromeres, cyclin A, and small nuclear ribonucleoproteins; that share chiefly in the pathogenesis of SLE (Wu et al., 2006). ANAs were found to be elicited as a part of the anti-parasite immune response including cystic echinococcosis (CE), plasmodium infections, and schistosomiasis (Hsieh et al., 2008; Romani, 2008; Aslan et al., 2009; Brahimi et al., 2011; Wang et al., 2018; Rivera-Correa & Rodriguez, 2019). However, the real function of ANAs in echinococcosis still not clear. Anti-CCP, citrullination is a physiologic process that occurs during cellular death by the diminution of arginine amino acid that exists in certain human proteins; a process that requires the presence of the peptidyl arginine-deiminase enzyme (PAD). Expression of PAD occurs normally in the leucocytes during the inflammatory processes. However, the release of PAD in the bloodstream may citrullinate extracellular proteins. Production of anti-cyclic citrullinated peptide antibodies appeared to be critical for the development of rheumatoid arthritis (RA) (Schellekens et al., 2000; Lee & Schur, 2003; Zendman et al., 2006; Ortancıl, 2011).

In the present work, based on the fertility rate of the metacestode we determined the serum levels of IL-17A and auto-immune populations (ANA and anti-CCP antibodies) in CE patients comparing with healthy controls. Localizations of the inflammatory cytokine IL-17A and ANA in the metacestode and adventitial layer were also investigated.

The specimens were obtained from the National Hepatology and Tropical Medicine Research Institute and Nasser Institute for Research and Treatment in the period from January 2018 up to May 2020. The 63 patients were confirmed for CE in respect to clinical, radiological, and serological findings; and underwent either surgical removal or PAIR technique in association with medical treatment. All these patients were with no known disease at the time of sample recruitment or preceding anti-CE therapy in earlier times. Small CE cysts were considered still developing and have not initiated producing PSC. Control subjects were selected on account of being age and gender harmonized to the assessed sera and of no known illness meanwhile obtaining the blood samples. Sample processing regarding the standardized procedures was performed in the Pathology Department, Faculty of Medicine, Cairo University, Egypt. Readings and titrations of the target autoimmune immunoglobulins were performed in Theodor Bilharz Research Institute (TBRI), Immunology and Therapeutic Evaluation Department, Cairo, Egypt.

In a descriptive-analytic cross-sectional study, the influence of CE infection on autoimmune antibody populations in the serum of the patients and in situ in surgical CE cysts specimens was evaluated besides the possible role of IL-17A. The study involved 63 patients diagnosed with CE and 63 age and sex-matched healthy subjects were used as controls in the current study.

Quantification of autoimmune antibodies-sera levels (ELISA): Using pre-operative serum samples the autoimmune populations; Antinuclear antibody (ANA) and Cyclic citrullinated peptides (Anti-CCP) were quantified using enzyme-linked immunosorbent assays (ELISA) as recommended in the manufacturers’ instructions (Biocompare, Inc. company, Biorbyt, # orb339622, South San Francisco, United States for ANA, MyBioSource, # MBS703683, Creative Diagnostics, # DEIA1227, the USA for Anti-CCP, and RayBiotech #ELH-IL17-1, Norcross, Georgia for IL17A). Optical density was measured by switching the plate reader at 450nm for ANAs, anti-CCP, and anti-IL-17A. IL-17A detection level was 80 pg/ml. In further absolute quantification of the ANAs (using lab-to-lab service), titer less than 1:80 was considered negative, ≤1:160 was a cut-off, and ≥1:320 was positive meanwhile in anti-CCP titers more than 20 EU/mL were regarded as being positive.

Characterization and classification of Cystic Echinococcosis HCF obtained by PAIR and cystectomy was evacuated into sterilized test tubes such that the shreds of the germinative layers, brood capsules, and protoscoleces were allowed to precipitate. The parasite’s products were then washed three times in NaCl sterile solution at 0.9 %. Thereafter, the liquid was filtered using sterilized gauze (with wide porous mesh) to eliminate large fragments. The obtained filtrate was poured into sterile test tubes left to precipitate at room temperature for 5 min (Khammari, 2019). Obtained samples were classified based on their fertility rate, a process that involved estimation of the average number of protoscoleces and brood capsules/ml, and the viability rates in percentage where viability below 85 % were to be considered low.

The supernatant was removed and 100 μL of the protoscoleces’ sediment was obtained for counting; where 20 μL was smeared and the parasites (protoscoleces and brood capsules) were enumerated and logged as the parasite number per each 100 μL sediment. The process was repeated 2 – 3 times to assure the average number (Shahnazi et al., 2013; Hajihossein et al., 2015). After the average protoscoleces counting, one drop from the sediments of the CE sand (protoscoleces; brood capsules, and shreds of the germinal layer) was inspected microscopically as wet unstained mounts to determine the existence of protoscoleces or hooks. Protoscoleces were examined for amoeboid-like movements, the activity of the flame cells, and integral morphology by light microscopy. Dye- exclusion test was performed using eosin 0.1 % solution as a vital stain where eosin was added to the same volume of cyst fluid holding CE sand. Due to loss of membrane integrity, the dye is absorbed by dead protoscoleces while viable ones remained unstained (Hanks and Wallace, 1958; Ducci et al., 2002). Thereafter viability was calculated in percentage. Moreover, viability was assured using the method of the urine strip test, where the highest glucose and the lowest protein measurements signified high viability and vice versa with those specimens of low viability (El Saftawy et al., 2017).

Processing of the metacestode: Cyst wall specimens from both fertile and non-fertile cysts were formalin-fixed, paraffin-embedded, and subjected to the ensuing staining processes:

Hematoxylin and Eosin stain (H&E) (Kiernan, 2001): H&E stain was used to demonstrate the considerable details of the metacestodes. All slides were examined for the germinal and laminated layers, attached protoscoleces, and the adventitial layer where photomicrographs were captured using Olympus Microscopy, CX-23.

(1) Immunohistochemical Staining for IL-17A (El Aal et al., 2015): tissue cut sections were dewaxed and the intrinsic peroxidases were blocked with Hydrogen-Peroxide Block stock for 10 – 15 min. to hinder the nonspecific background staining. Tissue sections were then washed 2 – 3 times with PBS and incubated with ScyTek Pro-Block solution for 20 – 30 min to block proteins. Tissue sections of both fertile and non-fertile cysts were then incubated with Anti-IL-17A antibody, polyclonal antibody (Abcam, catalog number ab214588) (1:200) for 30 minutes at 4°C. After washing in PBS, tissue sections were incubated with Ultra-Vision Detection System, a labeled streptavidin-biotin immune-enzymatic antigen detection kit, (TP-015-HD, Thermo-Fisher Scientific, USA) for 10 min. at 25°C. Finally, tissue cut sections were embedded in DAB solution for 15 – 25 min. to be finally counter-stained with hematoxylin. Control slides were performed where the step of immune staining with the ani-IL-17A antibody was omitted.

Estimation of IL-17A immune-reactive cells: the intensity of the immunohistochemical stain was performed by Image J software Images by being transformed to the format, 8-bit gray-scale. Two definite assessments were made in the current study: a) the digitized immune-stained tissue images were segmented into IL-17A immune reactive areas versus the non-immune reactive areas and

b) the quantification of the staining intensity within IL-17A immune positive areas where the threshold of positive pixels was set above the background intensity (Smorodinova et al., 2015; Smorodinova et al., 2017). Positive signals were automatically selected to designate counts of the positive signals per mm².

Tissue sections from both fertile and non-fertile CE cysts were subjected to the staining procedure. Besides, the study held a process to convert hydatid sand into cut sections that involved embedding the samples of informal aldehyde in a test tube, then the sediment was obtained and allowed to be settled and wrapped in gauze. Thereafter it was applied in tissue cassettes to be embedded in melted paraffin wax. Cut sections were consistently prepared using microtome to be ready for staining.

Satisfactory CE treatment entails an acquaintance of certain features related to the viability of the parasite (Odev et al., 2000; Pauluzzi et al., 1966). Protoscoleces viability was relatively high in the viable solitary and multivesicular cysts, while almost absent viability was detected in solitary cysts. There are few studies concerning protoscoleces viability and CE cyst fertility, however, the results obtained in the current study were similar to a prior study published by Lahmar et al. (2012) in Tunisia. Manterola et al. (2006) employed the association between fertility and some variables including type, location, and the diameter of CE cysts as well as the incidence of biliary communications.

High fertility and viability of the cysts evidences the high metabolic activity of the parasite and thus the production of antigenic secretory/excretory by-products being available to host immunity to be phagocytosed and introduced to T helper cells by antigen-presenting cells (Virginio et al., 2012).

Histological analysis revealed the infiltration of mixed inflammatory cells in the adventitial layer similar to a prior study published by Barnes et al. (2011). However, Moudgil et al. (2019) conveyed the existence of mononuclear infiltrations in the lung tissues adjacent to cystic echinococcosis. Meng et al. (2012) assumed the recruitment of the inflammatory cells to be related to apoptosis in the hepatocytes adjacent to the metacestode.

The higher percentage of IL-17A in the inflammatory milieu observed in CE patients with fertile highly viable cysts than in patients with non-fertile cysts suggests the implication of the cytokine in host immune responses against the helminthic infection, following prior-studies (Mezioug & Touil-Boukoffa, 2012; Pan et al. 2017). These data indicate a possible relationship between CE cyst fertility and cytokine production in patients’ serum and the possible use of IL-17A in the immunosurveillance of CE disease. Labsi et al. (2018) reported that in vivo treatment with IL-17A attenuated the development of cystic echinococcosis and the associated liver fibrogenesis in an experimental model. Such findings drive the attention if IL-17A may have a possible role in the progression of cystic echinococcosis towards non-fertility?

However, the somatic antigens of the protoscoleces were suggested to be related to the regulation of IL-17A production in B cells and CD-4⁺T cells. Pan et al. (2017) evidenced that in vitro cultivation of B cells with the excretory/ secretory products released by the protoscoleces exhibited lower percentages of Th-17 and B-17 cells and higher percentages of B-10 cells.

Interestingly, it has been assumed that the balance between B-17 cells and B-10 is similar to that between Th-17 and T-reg cells; however, during chronic infection, there are higher percentages of T-reg cells and B-10 contributing to the immune evasion and pathogenesis of the metacestode (Tuxun et al., 2012; Pan et al., 2013; Tan et al., 2013; Pang et al., 2014; Pan et al., 2017).

Similarly, using animal models Wang et al. (2014) and Kim et al. (2019) postulated the possible therapeutic effects of CE cystic fluid on airway inflammation due to allergy by different strategies including down-regulation of IL-17A.

ANA is the easiest and most frequently assessed biomarkers for autoimmune diseases at a population level (Wandstrat et al., 2006). Estimating the prevalence of ANA in the CE seemed to be critical to understanding the possible parasitic relationship.

Our findings of high ANA seropositivity in fertile CE cysts with high viability is puzzling; and similar to our results Aslan et al. (2010) detected significantly high levels of ANAs and other autoantibodies in the sera of patients with CE. One prospect is that ANA binds to nuclear antigens that are charged molecules, chiefly with histones and DNA (Tan et al., 1989). As a consequence, seropositivity shows a robust response and their interpretation necessitates caution, especially in clinical settings of this parasitic disease (Copple et al., 2007; Meroni & Schur, 2010).

Another explanation for the wide frequency of ANA expression (wide standard deviation) among patients with fertile and highly viable CE cysts might be related to intrinsic immunological disorders. Perhaps as a species-specific criterion, humans are more liable to autoimmunity, with ANA response a shot in the arm in several autoimmune diseases (Pisetsky, 2011; Rai & Wakeland, 2011). In animal-based experiments, the production of ANA can occur in absence of other systemic lupus erythematosus-related manifestations, reflecting that enhancement of some immune cells’ activities seemed to be dependable on the actions of specific genes (Richard & Gilkeson, 2018).

Rai and Wakeland (2011) postulated that the human genome may contain several polymorphisms to increase the species qualification to defeat infections or to help in wound healing. Also, Oates et al. (2003) suggested that the selective and deep pressure created by certain infections, that may involve for instance nitric oxide production on a molecular level, that associates diseases such as rheumatoid arthritis and systemic lupus erythematosus as well as resistance against malaria.

However, several studies illustrated that ANA can be existing in healthy individuals (Grygiel-Górniak et al., 2018) as well as in patients with non-rheumatic diseases such as hepatitis-C (Hsieh et al., 2008), cancers (Mohammed & Abdelhafiz, 2015), and thyroid diseases (Inamo & Harada, 1997). In prior studies ANA had been postulated to be of low diagnostic value and that ANA-seropositive individuals in the general population shouldn’t necessarily associate autoimmune disease or even develop the disease in the future. In this regard, monitoring of ANA sera-levels is speculated to be only mandatory in cases of clinical symptoms for further rheumatologic diagnosis (Grygiel-Górniak et al., 2018; Karamehic et al., 2007; Pisetsky & Lipsky, 2019).

The finding of being higher in females simulates several earlier studies (Watanabe et al., 2004; Hayashi et al., 2008; Satoh et al., 2012). However, Li et al., (2011) postulated the increased risk for autoimmunity in ANA seropositivity, especially in females.

Another notable finding was the minor changes in ANA levels in individuals with fertile CE and low viability and those with non-fer tile cysts that were of insignificant differences compared with disease-free subjects that seemed to be related to the low expression of the parasite antigens.

We investigated serum samples from all patients and their controls to test for the presence of any disease-specific effects as anti-CCP antibodies are recently overlooked as a serologic marker more sensitive and specific than rheumatoid factor (El Sawi et al., 2011). Among the 63 serum samples assayed, positive titers were of insignificant differences with healthy subjects. These findings propose that there is no definite relationship between CE and the level of autoantibodies. In agreement with our results; Colebrook and Lightowlers (1995), concluded the lack of association between CE and autoantibodies to a wide-scale of self-antigens.

Interestingly, several case reports documented reactive-arthritis secondary to the infestation of cystic echinococcosis at a distant site in absence of a broad-spectrum of autoantibodies including ANA and anti-CCP antibodies or evidence of other auto-immune disorders, a matter that seems to be related to the potential intense anti-helminthic immunological response (Buskila et al., 1992; Tekaya et al., 2009; Alım et al., 2017). Sánchez et al. (1983) reported the existence of special anti-CE antibodies, complement deposits, and vasculitic changes in synovial vessels in patients with a prior diagnosis for Reiter’s syndrome. However, Al-Sakee (2011) postulated the significant increase of anti-phospholipid and anti-ardiolipin levels in CE patients and suggested a substantial association of these autoreactive antibodies to cystic echinococcosi T cells are assumed to be the key mononuclear inflammatory cells in CE that to be differentiated into T-helper cells encompassing the proinflammatory Th1 cells, the anti-inflammatory Th2 cells, immunomodulatory Treg cells, and recently Th-17 cells (Gottstein et al., 1991; Vatankhah et al., 2018; Jiménez et al., 2020; Abo Aziza et al., 2020). In concern to our work, the immuno-reactive expression of IL-17A was mainly of the mononuclear inflammatory infiltrates that seemed to mirror Th-17 cells possess a definite share in the lymphocytic compartment of the adventitial layer. However, Tan et al. (2013) and Ma et al (2014) described neutrophils and Kupffer cells to be involved in the production of IL-17A. Prior studies assumed that the protective role of IL-17A against CE involves the attraction of the macrophages and neutrophils to the site of metacestode to act consecutively with their panel of cytokines (interferon-γ (INF-γ) and IL-6) (Hetta & ElKady, 2017).

Similar to our results in the non-fertile cysts, Monin and Gaffen (2017) reported IL-17A in the laminated membrane within the extracellular traps of the neutrophils along with the enzymes released from their cytoplasmic granules and nuclear histones. Nakajima et al. (2014) and Veríssimo et al. (2019) assumed IL-17A expression to be directly related to the inflammation intensity via inducing Th-2 cells. According to Diaz-Godinez and Carrero (2019), this immunological setting contributes to pathology development rather than being curative.

The dynamics of IL-17A were reported to occur in a time-dependent manner (Hattori et al., 2015). Hai et al. (2019) hypothesized that IL-17A alters the immune response milieu towards an effective pro-fibrotic process that encompasses the release of TGF-β1 from Kupffer cells and HSCs to share in vascular and architectural changes in the liver cells.

IL-17A production was affected significantly by the parasite viability and fertility; in accordance to Das and Khader (2017). Amusingly, it has been suggested that TGF‑β1 released by the IL-17A primed-kupffer cells not only contribute to the parasite immune evasion and chronicity but also facilitates the expansion and differentiation of IL-17A producing cells (Tian et al., 2020).

The current study introduces the first representative estimates of the incidence of ANA in the parasite cells in human CE. In dead, the definite burden of the cytotoxic ANA auto-immune reactions in different populations was mainly allocated in the highly cellular structures of the parasite and the adventitial layer in a viability-dependent manner.

In the metacestode, the homogeneous nuclear pattern of ANAs seemed to signify the metabolically active parasite cells encompassing protoscoleces, germinal layer, and brood capsule (Galindo et al., 2008). Castedo et al. (2004) speculated that DNA proliferation can be typified by ANA homogenous pattern where ANAs in the metaphase stage bind to the dense chromatin plate and the interphase stage is found in the outer rim of the nuclei.

In the germinal cells rod and/or ring patterns of ANAs, according to previous descriptions of the ANA fluorescent pattern, are mostly supposed to attack the peri-nuclear cytoplasm of the parasite cells that to our knowledge, had no apparent explanation. In prior studies, it was reported in the perinuclear cytoplasm, against myeloperoxidase enzyme, and considered as a distinctive trait to viral hepatitis-C therapy (Feldrihan et al., 2019).

Infiltration of IL-17A significantly related to ANAs-immune-complex production similar to prior results published by Hetta and ElKady, 2017, Hetta et al. (2017), and Zhao et al. (2011) who supposed that IL-17A multi-facets involve priming for autoimmune diseases. In this accordance, Nalbandian et al. (2009) presumed the direct influence of IL-17A to stimulate the production of auto-antibodies against self-antigens including DNA. Moreover, Miletić et al. (2012) defined IL-17A in the inflammatory reactions due to autoimmunity.

Our study examined the serum levels of IL-17A and autoimmune populations (ANA and anti-CCP antibodies) and evaluated and compared their relevance following CE cyst fertility and simultaneously, for the first time, we investigated the localization of IL-17A and ANA in the metacestode and adventitial layer. The present work demonstrated that IL-17A serum levels were significantly increased in CE patients following the fertility of CE. ANA despite being of a four-fold increase in fertile cysts with high viability there was no correlation between IL-17A cytokine and ANA and anti-CCP antibodies serum levels along with the study. There is a lack of association between CE and anti-CCP antibodies. Relative IL-17A expressions in the adventitial layer and among parasites’ cells were significantly increased and were associated with elevated ANA intensities being higher in fertile cysts with highly viable protoscoleces compared with non-fertile cysts. The present work suggests that, in addition to IL-17A, ANA also seems to participate in immune responses against CE infection in a fertility and viability-dependent manner.