CHAPTER 12 PATHOGENIC PERSISTENCE AND

Pathogenic Persistence and Evasion mechanisms in Schistosomiasis Annapurna Nayak*,1,2 and Uday Kishore1

Centre for Infection, Immunity and Disease Mechanisms, Heinz Wolff Building, Brunel University, London, UK; Centre for Biotechnology and Bioinformatics, School of Life Sciences, Jawaharlal Nehru Institute for Advanced Studies, Secunderabad, Andhra Pradesh, India. *Corresponding Author: Annapurna Nayak—[email protected]; [email protected]

1 2

Abstract:

Schistosomiasis is caused by infection with parasitic flatworms of the genus Schistosoma. It affects 200 million people worldwide, especially in the developing countries. There are five known species of schistosomes that currently infect humans in various geographical locations. The infection leads to two forms of the disease; acute and chronic. Chronic infection can affect various organs within the human body including the brain, lungs, gut and the reproductive organs leading to neuroschistosomiasis, pulmonary schistosomiasis, hepatointerstinal schistosomiasis and urinary /genital schistosomiasis, respectively. All the Schistosoma spp have a common denominator and that is its ability to infect, invade and evade the host’s immune mechanism. The pathogen is a very complex organism, in that it requires two hosts to complete its life cycle and hence has developed specific immune evasion mechanisms for each host. The schistosome utilizes two hosts, mollusc and mammalian, in order for it to survive and propagate. Once the parasite has infected and established itself as an adult worm within its mammalian host, it escapes the immune mechanism of the host. However the antigens released by the eggs can elicit an immune response and lead to the formation of granulomas around the egg. Granuloma formation is the main characteristic lesion in schistosomias, which in the liver can cause hepatomegaly in hepatointerstinal schistosomiasis. This chapter endeavors to summarize various immune responses against the parasite as well as a range of strategies developed by the schistosomes to persist within the snail and human host.

Microbial Pathogenesis: Infection and Immunity, edited by Uday Kishore and Annapurna Nayak. ©2013 Landes Bioscience and Springer Science+Business Media. 255

©2013 Copyright Landes Bioscience and Springer. Not for Distribution

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Microbial Pathogenesis

APO: amoebocyte producing organ; BgMIF: B. glabrata macrophage migration inhibitory factor; BgTEP: Thioester‑containing protein from B. glabarata; CE: Cercarial Elastase; DC: Dendritic cells; ES: excretory/secretory; FREP: fibrinogen‑ related proteins; GAPDH: Glyceraldehyde 3‑phosphate dehydrogenase; HETE: Hydroxyeicosatetraenoic acid; IDS: Internal defense system; IFN: Interferon; IL: interleukin; MAC: Membrane attack complex; MASP: mannan binding lectin‑associated serine protease; NO: nitric oxide; PG: Prostaglandin; PGE2: prostaglandin E2; Pmy: Paramyosin; SEA: Soluble egg antigen; siRNA: short interference RNA; SMAF: S. mansoni apoptosis factor; SmPoMucs: S. mansoni mucin like antigen; SOD: superoxide dismutase; TNF: Tumour necrosis factor; TPX: thioredoxin peroxidase; VNTR: variable tandem repeat. Introduction Schistosomiasis is a chronic parasitic infection that has been prevalent in the world for over 5000 years and is responsible for nearly 280,000 deaths per year with 200 million people worldwide being infected.1,2 The prevalence of this disease from ancient times is proven by mummified Egyptian remains that show classical granulomatous lesions, characteristic of schistosomiasis. Schistosomiasis is also called bilharziasis, after Dr Theodor Bilharz, who discovered the causative parasite in humans in 1851. The disease is also termed as snail fever, after its intermediate host, the fresh water snail. Most people infected with S. mansoni are asymptomatic but a small number of infected subjects can develop life‑threatening clinical syndromes that are organ specific. Various organs that can get affected by chronic schistosomiasis are the brain, lungs, liver, bladder, gut and the reproductive organs. Hepatosplenic schistosomiasis, due to egg retention and consequent granuloma formation, involves liver fibrosis, enlargement of liver and spleen, and portal hypertension. In fact, 18.5% of patients with hepatosplenic schistosomiasis are diagnosed with pulmonary hypertension.3 This is because heavy egg infestations can cause severe periportal fibrosis leading to portal hypertension that, in turn, can lead to splenomegaly, esophageal variceal bleeding and development of portosystemic collaterals through which the egg travels into the pulmonary circulation. Once in the pulmonary circulation, the egg can elicit an inflammatory reaction leading to granuloma formation and, in turn, fibrosis thus leading to pulmonary hypertension. Granuloma formation in the urinary tract due to infection by S. haematobium can lead to haematuria, dysuria, bladder polyps and ulcers and even squamous cell bladder cancer.4 Eggs that travel through the blood brain barrier into the central nervous system can cause neuroschistosomiasis.5 The five species of schistosomes involved in the pathogenesis of schistosomiasis in humans are Schistosoma mansoni, S. japonicum, S. haematobium, S. mekongi and S. intercalatum. The species involved in the pathogenesis of schistosomiasis determines the clinical presentation of the disease. For example, S haematobium infection affects the urinary tract whereas all other species affect the hepatointestinal organs. S. mansoni occurs in most African countries, parts of Arabia, in northern and eastern parts of South America and in some Caribbean islands.1 S. haematobium is present in most African and in some middle‑eastern countries.1 S. japonicum occurs in the pacific side of the world and includes countries such as Japan, the Philippines, the Chinese mainland and Thailand.1


Abbreviations

Pathogenic Persistence and Evasion mechanisms in Schistosomiasis

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Schistosomes are trematodes that belong to the phylum Platyhelminthes (flatworms). Most trematodes usually require two intermediate hosts and one definitive host to complete their life cycle whereas schistosomes require only one intermediate host (the snail) and one definitive host (humans or other mammals). Unlike other trematodes, the schistosomes show distinct sexual dimorphism. Most trematodes localize within certain organs such as liver (Opisthorchis sinensis), lung (Paragonimus westermani) or intestine (Fasciolopsis buski). However, schistosomes are blood flukes i.e., they are intravascular obligates that reside in the mesenteric veins and affect many organs including the central nervous system. The life cycle of all schistosomes is similar with few modifications and consists of both asexual and sexual reproductive stages. The sexual stage of these dioecious parasites is where the interactions between male and female worms occur within their definitive hosts, whereas the asexual stage takes place within the aquatic intermediate mollusc host that leads to production of clonal larvae. This facilitates exposure and potential infection of any mammal that comes in contact with the water. Once infected, the transmission of the infection would occur through excretion of the schistosome eggs through stool (S. mansoni and S. japonicum), or via urine (S. haematobium) (Fig. 1).

Figure 1. Illustration of the life cycle of Schistosoma spp: the figure depicts the two stages in the life cycle of schistosomes in two different hosts. The eggs in water undergo clonal multiplication in the molluscan host. The cercariae infect the mammalian host by penetrating through the skin. Once the cercariae has penetrated the skin, it undergoes transformation and travels through the circulatory system and establishes as adult worms (male and female) in its preferred organ where they can reside for upto 30 years. The eggs are excreted either through feaces or urine and the cycle continues.


Life Cycle into Intermediate and Vector Host

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S. mansoni

S. japonicum

S. haematobium

Adult male • Length (mm) • Breadth (mm) • No of testes

6–12 2.00 4–13

12–20 0.50–0.55 6–9

10–14 0.75–1.00 4–5

Adult female • Length (mm) • Breadth (mm) • No of eggs in uterus

7–17 1.00 Usually 1

16–28 0.30 50 or more

16–20 0.25 10–100

Schistosomulum (All species – length x breadth) Size during penetration (mm) ~0.10–0.12 x 0.030 Size in lung (mm) ~0.12–0.18 x 0.029–0.037 Size on arrival in liver (mm) ~0.16–0.20 x 0.023–0.040

Schistosomes are dioecious in which the male schistosome possesses a groove or a gynaecophoric channel in which it clasps the longer and thinner female schistosome in a permanent embrace. The coupled schistosomes live within the perivesical or mesenteric venous plexus feeding on blood and globulins through anaerobic glycolysis with the waste being regurgitated into the host’s blood stream. Depending on the species, the eggs laid every day can range from 300 (S. mansoni) to 3000 (S. japonicum)6 (Table 1). Schistosome species are geographically separated, as mentioned previously, and are also host specific which helps to sustain species barriers. However, this barrier does not imply that crosses between species are not viable; the potential for cross‑breeding between species is restricted due to the host specificity and geographical distribution. Interestingly controlled laboratory experiments have demonstrated that heterospecific crosses can occur leading to either parthenogenesis or hybridization.7‑9 The outcome depends on the phylogenetic distance of the species involved and hybridization resulting from these crosses show enhanced phenotypic characteristics. These features involve higher fecundity, faster time of maturation, higher levels of infectivity and increased pathology. Moreover, these crosses can also infect both intermediate snail hosts of the parental species, thus increasing the spectrum of the intermediate host infection.9 Natural and anthropogenic environmental changes accompanied by migration of hosts can all serve to alter the distribution of Schistosoma species and thus lead to evolution of the parasite. Once infected, each egg harbouring a ciliated miracidium larva inside secretes several proteolytic enzymes that aid the egg to migrate to the lumen of the bladder (in S. haematobium) or the intestine (in other Schistosoma spp). The eggs are then excreted through faeces or urine (S. haematobium). Once the eggs are in contact with water, the miracidium is released, which then infects its molluscan intermediate host (Fig. 1). Different Schistosoma species tend to be transmitted by different fresh water snails. For example, S. mansoni is transmitted by Biomphalaria snails while S. haematobium and S. japonicum are transmitted by Bulinus and Oncomelama snails, respectively. The Biomphalaria species of snails are found throughout the world thus making S. mansoni the more common species that infects humans, from South America to Africa and the Arabian Peninsula. Once the schistosome occupies its preferred intermediate host, its asexual stage of the life cycle begins. The miracidium multiplies into multicellular sporocysts that develop into cercarial larva that have embryonic suckers and a bifurcated tail, a characteristic feature


Table 1. Information about physical characteristics that Schistosoma spp possess at different stages in its life cycle


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Skin Penetration by Cercariae: Breaking the First Line of Defense The cercariae, following contact with human skin, responds to chemical signals from the skin such as medium‑chain free fatty acids such as linoleic acid.10 The penetration through the primary defense layer i.e., the human skin is the first step of invasion by the cercariae (Fig. 2). The penetration is facilitated by serine proteases that are packaged in the acetabular glands of the cercariae.11 These proteases degrade epidermal and dermal protein components including keratin, elastin, collagens, fibronectins and laminin. It also disrupts cell‑cell contacts within the epidermis. One of the well‑studied serine protease that has been implicated in this process of penetration by S. mansoni is the Clan PA family S1 serine protease, also called cercarial elastase (CE). Several studies support the involvement of this family of proteases in the skin penetration and invasion by S. mansoni. Cercariae with intact acetabular gland contents, but whose tails were mechanically removed, were able to

Figure 2. Events during skin penetration by schistosome cercariae: Each stage of the schistosome life cycle within its mammalian host elicits an inflammatory response. The first line of defense is the skin and the cercariae would have to thwart attacks at every layer of the skin in order to gain entry into the circulation. The flowchart depicts the penetration of the cercariae through the different layers of the skin and the immune challenges that the cercariae faces during its journey through the skin. Once successfully penetrated, the cercariae then travel toward the lungs and other organs.


of the cercariae. The cercariae stay within the snail for 4–6 weeks after which it is shed by the snail. A single snail can shed thousands of cercariae for months, every single day. The shed cercariae, which are extremely motile organisms, are viable in the low level fresh water for up to 72 hours during which it seeks out suitable hosts, i.e., the human skin, which it penetrates to begin the rest of its life cycle. Cercariae are phototropic and hence align themselves on the surface of shallow waters where it can readily penetrate the host.


establish infection successfully when injected into animal models. However, in the same study, cercariae incubated in saline and thus lacking the acetabular gland contents, failed to establish infection.12 The immunolocalization studies have confirmed the release of CE by cercariae during penetration of skin.13 Once the cercariae have invaded the primary physical barrier, it progresses toward the next step i.e., evasion of the host immune system. After entering into the skin, the cercariae metamorphose into schistosomula which migrate to hepatic portal system via lungs. This is followed by their differentiation into male and female, pairing and relocation to mesenteric venous plexus where they can reside up to 10 years. Female schistosomes then start to deposit viable, active and highly antigenic eggs. These eggs can attach to the endothelium of mesenteric blood vessels, and cause inflammatory response in order to find their way into the intestine to be excreted in the faeces. These eggs can then hatch and the released miracidia can infect snails in a species‑dependent manner in order to complete the asexual life cycle. Few eggs may get trapped into liver, intestine or elsewhere and induce granuloma formation. This granulomatous inflammation is the cause of most pathological features and schistosomiasis and mortality due to S. mansoni. Modulation of the Host’s Immune Response The schistosome‑induced pathogenesis consists of evasion of the host immune system by the cercariae, the adult and the egg during different stages: penetration through the skin, migration through the circulation, incubation of the adult schistosomes, production of eggs and excretion of the eggs. Most immune responses are widely observed in chronic schistosomiasis when compared with acute schistosomiasis. During the earlier stages of the pathogenesis, the schistosome ES (excretory/secretory) products are involved in modulating the immune response while soluble egg antigens (SEA) are involved in the later stages of immune modulation. Schistosomal ES products are released or secreted from epithelial surfaces of the gut and/or tegument as well as other specialized ES organs throughout almost all life stages of the parasite (Table 2). The production and secretion of these products might be induced by factors present in the host fluid such as blood cells, phagocytic cells, hormones and complement proteins. Due to the complexity in collection and harvesting of ES products from host tissue and the inability to mimic in vivo environment in an in vitro environment, studies on the immune modulation by ES products is a daunting challenge for researchers. In the adult worms, ES products are mostly secreted by the excretory cells and co‑localized to the tegumental and subtegumental region along with the gut epithelium.14,15 Six of these ES products have been suggested as potential vaccine targets (Paramyosin, glutathione S‑transferase, IrV‑5, Triose phosphate isomerise, Sm23 and Sm14).16 Immune Response against Cercariae and Schistosomula The entry of the schistosomula through the skin does not go unnoticed by the host immune system. It elicits an inflammatory response due to infiltration of polymorphonuclear and mononuclear cells that is followed by the localized production of pro‑inflammatory cytokines (IL‑1b, IL‑12, TNF‑a, MIP‑1a and IL‑6) as well as immunoregulatory mediators such as IL‑10 and prostaglandins [PG] E2 and D2.17‑20 The invasion and consequent infection by the schistosomes leads to a predominantly Th2 immune response, in contrast


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Table 2. List of Excretory/Secretory products in different life stages References

Skin‑stage transforming cercariae • Paramyosin • SPO‑1 • Sm28GST • Leukocyte elastase inhibitor homolog • Heat shock proteins (‑70,‑86,‑90) • Triose phosphate isomerise • GAPDH • Glycolytic enzymes (aldolase, enolase) • Venom allergen‑like homologs • Sm16

14,15

Adult worms • FABP • Heat shock protein 70s • Glycolytic enzymes (enolase) • GAPDH • 14‑3‑3 protein • Detoxification molecules (TPX1, TPX2, SOD, GSTs, thioredoxin glutathione reductase) • Antigenic proteins (myosin, paramyosin, tropomyosin)

14,15

Egg

14,118

• Alpha‑1 • Omega‑1 • Glycolytic enzymes (aldolase, enolase) • Venom allergen‑like homologs • Sm‑p40 • 14–3‑3 protein1 • GAPDH • Heat shock protein 86 • Egg antigen SME16 • a‑tubulin • Actin‑1 • b ‑tubulin • Histones (H2A, H2Ab, H4)

to the expected pro‑inflammatory Th1 response. This skewing of the cytokine response is a rather complex and intriguing process by the virtue of which the parasite thrives and survives within the host immune system. One of the main immunomodulatory cytokine induced following exposure to cercariae is the anti‑inflammatory IL‑10.17,21‑22 The source of IL‑10 in the skin is not clear, although reports do suggest that keratinocytes might be the major source along with dendritic cells (DC), macrophages and B1 lymphocytes.17 In addition to IL‑10, other inhibitory molecules are also produced followed by stimulation by the cercarial ES products. These molecules include prostaglandins such as prostaglandin E2 (PGE2) and parasite‑derived prostaglandin D2 (PGD2) in all Schistosoma spp and IL‑1ra (IL‑1 receptor antagonist)


Excretory/Secretory Products


in S. mansoni and S. haematobium.21 The production of the prostaglandins leads to an increased production of IL‑10 in the skin. PGE2 aids the production of IL‑10 through a cyclooxygenase 2‑dependent pathway.23 Mouse studies have shown that PGE2 is the main immunoregulator in the skin in S. mansoni induced inflammation.17 Interestingly, parasite‑derived prostaglandins play a huge role in the immunomodulation within the host. The cercariae upon penetration into the skin turn themselves into schistosomula. This change of stage is also concomitant with the production of PGE1, PGE2, 5‑HETE, and 15‑HETE by the parasites.24,25 Studies have demonstrated the ability of schistosomula to induce PGE2 production by human keratinocytes.26 The schistosomula might be favoring the induction of increased production of PGE2 in the skin milieu for various reasons. First, schistosomula‑induced overexpression of PGE2 plays a role in the production of IL‑10 by host cells.17 Second, PGE2 acts as a potent vasodilator which might facilitate the easy passage of the parasite into the circulation.26 The S. mansoni schistosomula, following entry into the skin, remains in the skin for up to 3 days after which it gets into the circulation and progresses toward the other organs. Cytokine analysis shows a rapid increase in the levels of IL‑10 within a few hours of the parasite entry into the skin, along with significant reduction in the levels of IL‑1a and IL‑1b and increased levels of IL‑1ra.22,27 Yet another prostaglandin, PGD2, is produced as part of the ES components of the schistosomula. Parasite‑derived PGD2 has been reported to inhibit migration of epidermal Langerhans cells to the site of invasion.18 Physiologically, Langerhans cells are found anchored to neighboring keratinocytes and when the skin is penetrated by parasites, both keratinocytes and Langerhans cells produce pro‑inflammatory cytokines such as TNF‑a and IL‑1b. The expression of these cytokines, in turn, leads to the diminished expression of E‑cadherin and stimulates actin‑dependent movements of the Langerhans cells. However, during a schistosomal infection, the migration of Langerhans cells is inhibited due to the parasite‑induced production of PGE2 by the host cells and parasite‑derived PGD2 that both lead to an increased production of IL‑10. IL‑10 impedes migration of Langerhans cells by downregulating the production of IL‑1b and TNF‑a by epidermal cells.28 Thus, the purpose of the schistosome‑induced IL‑10 production is to create an anti‑inflammatory cytokine environment which can downregulate the host immune response against the invading parasite.17,29 The interruption of the migration of antigen presenting cells from site of exposure to the draining lymphoid tissue is another strategy adopted by the parasites to modulate the host’s immune response. The schistosomula also adopt additional strategies to evade the host immune response. The ES products from the schistosomula can induce in vitro mast cell degranulation, and hence, lead to production of IL‑4, release of histamine and 5‑hydroxytryptamine in an IgE‑independent manner.30 One of the components of the ES products, termed S. mansoni apoptosis factor (SMAF), has been shown to induce apoptosis specifically in the CD4+ lymphocyte population via a Fas‑FasL interaction. The CD4+ apoptosis allows the schistosomula to escape detection by the host immune system.31 Once the schistosomula have evaded the immune response, it gains entry into the portal veins and remains in the circulatory system. Within 1–3 weeks, it turns into a sexually active adult that adheres to the inner lining of the veins. The male and female adult schistosomes form a pair and can reside adhered to their chosen vein lining, escaping the host’s immune response for decades. S. mansoni and S. japonicum adhere to the inferior and superior mesenteric veins, respectively, while S. haematobium adheres to the venous plexus of the bladder. The adult pairs then produce 300–3000 eggs, depending on the species. The eggs are the second stage in the schistosomal life cycle that elicits an inflammatory response within the host body.


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The onset of egg production by the adult schistosomes is associated with the skewing of the CD4 response toward the Th2 polarization, characterized by production of IL‑4, IL‑5 and IL‑13. IL‑4 is one of the key cytokine that plays a role in the regulation of the development of the Th2 response. IL‑4 is produced in small amounts by naive CD4 cells. This IL‑4 in turn acts in an autocrine manner to induce GATA3 expression, and hence, establish the Th2 phenotype. The resultant IL‑4/IL‑4R/Stat6 signaling pathway plays an important role in stabilizing and expanding the Th2 cell populations32. In mouse models in which the egg antigens were injected, rapid induction of strong Th2 responses were observed.33 DCs, as the most potent antigen presenting cells and the sentinels of cell‑mediated adaptive immunity, are known to play a central role in initiation and polarization of T‑cell responses. S. mansoni eggs preparations have been shown to prime Th2 cells through the functional modulation of DCs.34‑37 Although the identity of the molecules responsible for this priming is still unclear, recent studies have reported Omega‑1 and S. mansoni glycoprotein w‑1 to be inducers of Th2 responses. One direct correlation of Th2 polarization is the presence of M2 macrophages in the granuloma, which undergo alternative activation by IL‑4 and IL‑13, important for the immune response to parasites as opposed to the classical macrophage activation induced by IFN‑g, which triggers a pro‑inflammatory response that is required to kill intracellular pathogens.38 Granulomas in Acute and Chronic Schistosomiasis Acute schistosomiasis, also called Katayama syndrome, is due to primary infection by the parasites and is observed in travelers visiting affected places and non‑ immune people.39 This phase of infection is usually asymptomatic but clinical manifestations can occur including fever, nausea, headache, irritating cough, blood‑and‑mucous‑ridden diarrhea for several months.40 Acute toxaemic schistosomiasis by S. mansoni and Katayama syndrome by S. japonicum are systemic reactions against the first cycle of eggs laid by the adult schistosomes, usually after 28–90 days of infection.39 Granulomas form around eggs that are trapped in the intestinal and liver wall leading to hepatosplenomegaly and leucocytosis with eosinophilia. Chronic schistosomiasis with complications occurs in affected individuals living in endemic areas. Intestinal schistosomiasis is the most frequently diagnosed form of chronic schistosomiasis. Schistosome eggs that have entered into circulation reach different organs including the intestinal wall. The eggs that get trapped in the intestinal wall provoke inflammation. Hepatointestinal schistotomiasis is due to embolisation of schistosome eggs in the liver and is the leading cause for hepatomegaly. In patients with severe longstanding infection, periportal collagen deposits lead to progressive obstruction of blood flow and portal hypertension (hepatosplenic form). S haematobium eggs, on the other hand, cause inflammation in the bladder and ureteral wall which can lead to haematuria and dysuria. With progressive involvement, fibrosis and calcification can occur, resulting in obstructive uropathy. Chronic disease caused by schistosome species is due to the immune response against entrapped eggs within tissues.41 Liver is the main organ that gets affected in S. mansoni and S. japonicum infections as the sinusoids of the liver are too small for the eggs to


Immune Responses Triggered by Schistosome Eggs


pass by. Contrastingly in S. haematobium infections, the bladder is affected as the eggs traverse across the bladder wall. Once trapped within the sinusoids, death of the eggs can cause the stimulation of the host response against the egg antigens. Granulomatous lesions, which comprise of collagen fibers and cells like macrophages, eosinophils and CD4+ T cells, form around the live eggs. Once the eggs die inside the granuloma, the resolution of the granuloma occurs leading to the formation of fibrotic plaques. The liver can become fibrotic, congested and harder to perfuse due to the granuloma‑induced fibrotic plaques and this can in turn lead to an increase in the portal blood pressure.42 Ascites and portal‑systemic venous shunts are also caused that can lead to excessive bleeding that could be life‑threatening. Infection with S. haematobium can lead to very serious diseases such as bladder cancer and genital schistosomiasis. Genital schistosomiasis is very common in women infected with S. haematobium and women affected can have some degree of inflammation in the genitalia. This inflammation is due to the egg migration through the urinary system that can lead to localization of the eggs in the womb or vagina. The most serious morbidity associated with genital schistosomiasis is infertility due to excessive tissue fibrosis.43 Thus, the causative factor for the hepatosplenomegaly and fibrosis is the immunopathology due to uncontrolled inflammatory response involving granulomatous formation induced by the trapped eggs in the tissues. The eggs generate a typical Th2 response that also includes infiltration of eosinophils, mast cells, and alternatively activated macrophages, followed by fibroblasts leading up to fibrosis.44,45 It is unclear whether granuloma formation is beneficial for the human host as the egg sequestration may reduce further tissue damage. For instance, mouse models that were tolerised against S. mansoni egg antigen did not develop granuloma but had severe hepatotoxic liver damage, which may be due to hepatotoxins secreted by the eggs.46 Granulomas along with egg‑antigen specific antibodies are likely to sequester these hepatotoxins away from the hepatocytes. However, what is evident via murine experiments is that the parasite uses host immune response for its proliferation, survival and excretion of eggs. The murine models using wild outbred strains‑ MOLF mice (as compared with inbred strains) appear to express more appropriate human immunopathology that coincides with the appearance of IL‑17 producing CD4+ T cells (Th17 cells).47 IRAK‑2 gene has been linked with severe immunopathology (than Th2 alone that induces only mild disease in murine models) since it appears to promote IL‑1b‑mediated Th17 cell development. Within 5 weeks of parasite infection, the immune response is characterized by a heightened Th1 response (IL‑12 and IFN‑g). As soon as female parasite starts to churn out eggs, there appears a shift Th polarization from a Th1 to Th2 phenotype.48 The egg antigen induces production of IL‑4, IL‑5 and IL‑13, and elevated IgE levels and eosinophilia.33 There appears to be a direct correlation between the intensity of the Th2 response against egg antigens and severity of granulomatous inflammation in murine models, which declines in the chronic phase (3 months). Thus, mice genetically deficient in IFN‑g or IL‑12p40 show no changes in granuloma formation following infection whereas IL‑4 deficient mice generate impaired granuloma and develop severe pathology.49 The modulation of T‑cell polarization from Th1 to Th2 response is due to secretory egg protein (SEA) that can suppress maturation of and subsequent cytokine production by DC (Dendritic cells co‑pulsed with microbial and helminth antigens undergo modified maturation, segregate the antigens to distinct intracellular compartments, and concurrently induce microbe‑specific Th1 and helminth‑specific Th2 responses.50,51


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Although granulomatous inflammation is principally triggered by CD4+ cells, cytotoxic CD8+ T lymphocytes, B cells, alternatively activated M2 macrophages, eosinophils and mast cells are also engaged in the development and maintenance of granuloma. Tissue eosinophil infiltration is aided by IL‑5 and IL‑13 in the granuloma.52‑54 However, transgenic mice deficient in eosinophils and infected with S. mansoni show no apparent defect in parasite load, granuloma formation and fibrosis.55 These infected mice, with no eosinophil detected in bone marrow and granuloma and high IL‑5 serum levels, were comparable to their wild type counterparts in terms of granuloma number, size, or fibrosis. The role of eosinophils as well as mast cells in S. mansoni induced immunopathology remains unclear. T‑cell deficient mice show impaired granuloma formation leading to mortality due to infection within 4–6 weeks.56 Without CD4+ T cells, the granuloma has preponderance of neutrophils rather than eosinophils, with extensive damage to liver. In the immunocompetent mice, the liver has normal functions, suggesting that granuloma formation may be helpful to the human host in order to sequester the eggs whose secretion can induce hepatotoxicity.46,56 CD4+ T cells have also been shown to be important for egg excretion in mice and human.56,57 Regulatory T cells (Tregs) with CD4+CD25+Fox3+ phenotype have been shown to suppress IL‑4 in the murine chronic stage that is reflected in the reduction in the size of granuloma.58 Thus, Tregs may play a role in limiting the pathogenesis in the chronic stage of the disease. Complement Evasion by Schistosome Paramyosin Schistosomula and adult worms also evade the immune system by developing resistance to complement attack. Complement system comprises of three different pathways; the classical pathway, the lectin pathway and the alternative pathway. All three pathways involve cascades of events that eventually attempt to the lysis of the target cell/pathogen or opsonisation and phagocytosis. The three complement activation pathways converge on the formation of C3 convertase (Fig. 3). The first component of the classical complement pathway is a macromolecular complex C1 that is composed of one C1q, two C1r and two C1s subunits, of which C1q is the ligand recognition subcomponent. C1q can recognize a diverse range of self, non‑self and altered self‑ligands in an antibody‑dependent or independent manner and this binding in turn activates the C1r.59,60 C1r cleaves and activates the C1s and this activation activates the other two proteins of the cascade, C4 and C2.61 The cleavage of C4 and C2 by C1s leads to the formation of a C4b2a complex collectively called the C3 convertase that cleaves C3. C3b, a cleavage product of C3, binds back the C3 convertase producing the C4b2a3b (C5 convertase) that cleaves the C5 component. This leads to the addition of several components such as C6, C7, C8 and several molecules of C9 forming C5b‑9, also termed as the membrane attack complex or MAC.62 The alternative pathway is initiated by low‑level activation of C3 by hydrolysed C3 and activated factor B. The activated C3b binds factor B that is cleaved by factor D to form C3 convertase.63 The alternative pathway amplifies the effects of the classical complement pathway through bound C3b. In mannose‑binding lectin (MBL) pathway, complement activation occurs following the binding of MBL to repetitive carbohydrate


Contribution of Various Immune Cells in the Immunopathology


Figure 3. Complement system: The complement system involves three pathways namely classical pathway, alternative pathway and lectin pathway. The funnel depicts the complement system and the common aim of the three pathways is to yield C3 convertase. The classical and lectin pathway, upon binding to their respective activation subcomponents, cleaves C4 and C2 to C4a/C4b and C2a/C2b respectively. C4b and C2b form a complex i.e., C3 convertase. This convertase facilitates the cleavage of C3 which in turn cleaves C5 to yield C5b. C5b hence forms a complex with C6, C7, C8 and C9 to form C5b‑9, also known as the Membrane Attack Complex (MAC) that leads to lysis of the target cells.

patterns on pathogen surfaces i.e., pathogen‑associated molecular patterns (PAMPs) through the MBL‑associated serine protease (MASP),64 designated as MASP‑2. MASP‑2 in turn leads to the activation of complement components C4, C2 and C3. The activation is analogous to the classical pathway where binding of C1q to target ligands leads to association and activation of the C1s–C1r–C1r–C1s serine protease complex. Of the three known MASPs (MASP‑1, MASP‑2 and MASP‑3), MASP‑2 resembles C1s in its ability to cleave C4 and C2, and thus generating a C3 convertase.65 The eventual assembly of the MAC and its insertion into the pathogens cell membrane leads to lysis of the pathogen. Schistosoma evades the complement attack and survives within the host system for years and the complement evasion mechanisms are yet to be fully understood. However, studies have been performed understand how the parasite escapes from or offers resistance to the complement‑mediated killing at every step of its life cycle within the mammalian host.


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Following host skin penetration by the schistosomal larva, it undergoes a change from being sensitive to complement attack to gaining resistance to the complement system. This is made possible by the shedding of the glycocalyx coat by the larva that otherwise contains strong complement activators.66,67 Once inside the host, the other life stages of the parasite employs several strategies to evade the hosts’ complement attack system. Parasitic proteins have been shown to bind to complement proteins such C1, C2, C8 and C9.68‑70 An important schistosomal protein that has been studied extensively as a complement pathway evader is a 97‑kDa protein named paramyosin (Pmy), a major core protein of thick filaments of invertebrate muscle. Immunolabelling studies in adult schistosomes have localized the detection of Pmy in regions just below the parasitic surface i.e., either the tegument or muscle layers of the male and female adult schistosome.71 Earlier studies identified a schistosome complement inhibitor, SCIP‑1, on the surface of S. mansoni larvae and adult worms70 which was later shown to be the exogenous form of Pmy.72 Pmy binds to C1q, the initial subcomponent of the classical complement pathway, in solution and this interaction fails to activate C4 and the MAC formation on sheep red blood cells.68 This suggests that the inhibition of the complement‑mediated killing of the parasite is modulated by paramyosin at the initiatial phase. Pmy has also been shown to bind to other complement proteins such as C8 and C9.72,73 Thus, Pmy appears to inhibit complement activation, and hence complement‑mediated killing of schistosomes, by binding to at least three complement proteins. Binding of C1q might inhibit the initial activation of the classical complement pathway and binding to C8 and C9 might ensure that the MAC is not generated. Pmy also shows binding ability to the Fc portion of IgG,74 which might possibly mask the surface of the parasite and block the binding of specific antibodies. Thus, Pmy is an attractive candidate for developing a potential vaccine against schistosomiasis. Trials in various animal models have demonstrated that immunization with native or recombinant paramyosin can substantially reduce the worm burden and liver/faecal egg counts in the infected animals.75‑77 Yet another protein, Sh‑TOR, from the surface of S. mansoni has been shown to be a functional receptor of human complement protein C2.69 Sh‑TOR is likely to inhibit the classical complement pathway by preventing C2 from binding to C4b.69 Immune Modulation of the Snail Several studies have examined the immune response of the mollusc to the entry of the parasite. Each Schistosoma spp has its own specific preferred host, i.e., S. mansoni infects Biomphalaria snails while S. haematobium and S. japonicum preferably infect Bulinus and Oncomelama snails, respectively. S. mansoni infection of its preferred intermediate host Biomphalaria occurs by penetrating through exposed parts of the snail, usually the base of the antennae and cephalopodal mass. The ciliated larvae of the schistosome involved in this process are called miracidia, which then undergoes several morphological and physiological changes to transform into primary sporocysts. These sporocysts remain in the fibro‑muscular tissue of the molluscs cephalopodal region and generate secondary sporocysts called daughter sporocysts. These in turn migrate to the digestive glands or hepatopancreas of the mollusc, the area in which they undergo many anatomical changes and generate cercariae that can infect hosts such as humans and animals78 (Fig. 1).



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In order to achieve the asexual maturation and reproduction within the snail, the parasite must evade the snail’s immune system. The immune system of the snail, also called the internal defense system which consists of hemocytes and soluble factors present in the hemolymph. B. glabarata and Bulinus species have a well‑defined area between the pericardium and posterior epithelium of the mantle cavity termed as the amebocyte producing organ (APO), which is the main site for production of hemocytes.79 Following inoculation of antigens from S. mansoni miracidia or cercariae, the mitoses of hemocytes were seen to increase in the APO thus suggesting that hemocytes are involved in the defense mechanism against invading parasites. This was further supported by the observations in resistant lineage of B. glabarata wherein the division of hemocytes was comparatively higher than the susceptible lineage of B. glabarata.80,81 The hemolymph of B. glabarata consists of two sub‑populations of circulating hemocytes called granulocytes and hyalinocytes. Granulocytes have extensive pseudopodia processes and primary and secondary lysosome‑like structures, required for phagocytosis of sporocysts and containment within phagolysosomes.82 On the other hand, hyalinocytes are small and roundish hemocytes probably lacking phagocytic properties.82,83 Hemocytes circulate throughout the tissue and hemolymph in order to recognize and phagocytise invading microorganisms including schistosome.84 Hemocytes also secrete soluble hemolymph factors that opsonize and aggregate potential pathogens and particulate antigens in order to facilitate the phagocytosis. Snail‑Schistosome Interaction and Compatibility Equilibrium Although Schistosoma can evade the snail’s immune system, a large diversity in the susceptibility of strains to infection has been reported. This diversity is contributed by genetic as well as physiological factors associated with the snail and schistosome. The lectin ligands on the surface of the hemocytes have been studied to be heterogeneous between Schistosoma species and snail strains.85,86 Susceptible snails mount a poor hemocyte reaction around the sporocysts that does not interfere with the parasite infection.78 By contrast, in resistant snails, the hemocytes are able to recognize, encapsulate and clear the parasites.87 The mollusc and the schistosomes have evolved together in a way that the parasitic virulence factors and the host defense are in equilibrium, a phenomenon called compatibility polymorphism.88 Compatibility is defined as a characteristic of a host–parasite interaction where the parasite is capable of establishing infection and achieving transmission using the host species.89 Susceptibility or resistance to parasite is dependent on the compatibility between snail and parasite genes and this compatibility influences the outcome of infection. To establish infection in a schistosome‑resistant phenotype, the schistosome larvae must prevent the snail from either detecting it or eliminating it. Two hypotheses exist to support the parasitic escape/evasion from the snail’s immune system: first that suggests that the snail IDS fails to detect the parasite and hence does not mount an immune response;90,91 second that suggests the parasite interferes/modifies the host immune response to facilitate establishment of infection.92,93 Humoral Factors That Contribute to Snail’s IDS In recent years, few humoral factors have been discovered that appear to contribute to snail’s IDS against schistosomes. One such factor includes snail‑derived lectins belonging


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to the fibrinogen‑related proteins (FREP) gene family.94,95 FREP lectins consist of one or two N‑terminal Immunoglobulin (Ig) Superfamily domains joined to a C‑terminal fibrinogen domain. Genes encoding the FREP molecules are capable of diversifying by undergoing somatic changes via gene conversions and point mutations.96 Thus, allelic polymorphism and somatic mutations can lead to the expression of nearly 36 variants of FREP3 per single snail.96 It appears that FREPs function as innate immune molecules in vertebrates and invertebrates.97,98 FREPs can precipitate soluble antigens derived from trematodes, bind to sporocysts of another trematode parasite, Echinostoma paraensei and a range of Gram‑positive and Gram‑negative bacteria.94,99 FREPs, especially FREP3, have also been observed to act as an opsonin thus favoring phagocytosis of S. mansoni by hemocytes.100 Knocking‑down FREP3 through siRNA‑mediated enhances snail’s susceptibility to the parasite by 30% in an otherwise resistant strain.100 FREPs have been shown to form complexes with certain Schistosomal mucins,101,102 which are known to be involved in immune evasion, host invasion and immune protection.103‑105 Studies have identified a set of highly glycosylated S. mansoni mucin‑like antigens (SmPoMucs), which are expressed only in snail‑specific larval stages. SmPoMucs are located in the apical gland of miracidia and sporocysts and are secreted as ES products.101,102 SmPoMucs have a ‘variable number of tandem repeats (VNTR) region’ that exhibit a high degree of polymorphic glycosylation. Thus, the inherent variability within FREPs (host) and SmPoMucs (parasite) may suggest co‑evolution of the genes as a part of host‑parasite stand‑off. A number of studies appear to suggest the interaction between FREPs and SmPoMucs as an excellent system to explore the molecular basis of the compatibility polymorphism between S. mansoni and B. glabrata. FREP2, a gene that is consistently upregulated following exposure of B. glabarata to S. mansoni, immunoprecipitates with SmPoMucs.106,107 It is likely that an incompatibility between the two variants will affect the outcome of the infection. SmPoMucs are released during larval transformation and might be creating an immunological “smoke‑screen” by generating antigen‑antibody complexes away from the schistosome as seen in other parasites.108 Thioester‑containing protein from B. glabrata (BgTEP) has been suggested to associate with parasite (SmPoMucs)‑bound FREPs, thus acting as an opsonic complex to recruit hemocytes at the site of infection.88 Thus, FREPs can be involved in playing a direct harmful role on the asexual development of the parasite within the snail. S. mansoni ES products also elicit intracellular nitric oxide (NO) production in both susceptible and resistant B. glabrata hemocytes, probably via the ERK signaling pathway.109 However parasite anti‑oxidant capacities appear to match closely to the host hemocyte oxidant responses in the case of sympatric B. glabrata vs. S. mansoni, which highlights the importance of oxidant production by resistant phenotype hemocytes.110 Migration, recognition and adhesion of hemocytes to the transforming miracidia and developing sporocysts are also likely important determinants of the resistance response. Integrin‑like cell surface receptors are known to regulate hemocyte adhesion and motility.111‑113 A tandem‑repeat galectin has been found to bind hemocytes and the tegument of S. mansoni sporocysts making it a candidate anti‑schistosome pattern recognition receptor.114 Finally, knock‑down of the recently‑characterized B. glabrata cytokine, Macrophage Migration Inhibitory Factor (BgMIF), was shown to reduce hemocyte encapsulation of S. mansoni sporocysts in vitro and increase mother sporocyst survival in vivo.115 Differences in gene expression, including those for immune/stress response, signal transduction and matrix/adhesion genes were identified between the resistant




and susceptible snail strains. Tests for asymmetric distributions of gene function also identified immune‑related gene expression in resistant snails, but not in susceptible.116 Recently, a putative mollusc immune effector cytolytic protein, called Biomphalysin in B. glabrata, which belongs to the b‑pore forming toxin (b‑PFT) superfamily, has been identified. Biomphalysin binds to the parasitic membranes and has been shown to be highly toxic toward S. mansoni sporocysts.117 Thus, the host‑parasite interaction mechanisms between snail and schistosome are highly complex and need to be elucidated further, especially with regards to resistance vs. susceptibility of certain species toward the Schistosome spp. Conclusion Schistsomes have co‑evolved with its two hosts over the years and population migration and traveling have further fuelled the evolution of this parasite. The schistosomes have developed three strategies to “cheat” its host’s immune system: utilization of parasitic proteins to gain entry, mimicry of the host proteins to establish itself within the host body and enable persistence by evasion of the host immune system. The survival of the host is dependent on the Th balance that in turn can affect the development of granuloma. The main characteristic lesion in schistosomiasis is the granuloma formation induced by the eggs laid by the adult worms that can persist in the host for up to 30 years. These granulomatous lesions can induce heavy inflammatory reactions that can cause fibrosis in the organs afflicted. Thus, by controlling the development of granuloma formation and consequent fibrosis, acute and chronic forms of the disease can be controlled. Renewed efforts to study the resistance vs. susceptibility phenotypes in intermediate and mammalian hosts can help control the infection by the parasite. Studies to understand the host‑pathogen interactions in both intermediate and mammalian hosts can pave way for better therapeutic approaches to control and cure subsequent infections, especially in the developing countries. By identifying target proteins, both host and parasitic, vaccines can be developed to destroy the parasite. References 1. Chitsulo, L, D Engels, A Montresor, and L Savioli. “The global status of schistosomiasis and its control.” Acta Trop. 2000 Oct 23;77(1):41-51. 77 (2000): 41-51. 2. Ross AG, Bartley PB, Sleigh AC, Olds GR, Li Y, Williams GM, et al. Schistosomiasis. N Engl J Med 2002; 346:1212-20; PMID:11961151; http://dx.doi.org/10.1056/NEJMra012396. 3. Lapa M, Dias B, Jardim C, Fernandes CJ, Dourado PM, Figueiredo M, et al. Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 2009; 119:1518-23; PMID:19273723; http://dx.doi.org/10.1161/ CIRCULATIONAHA.108.803221. 4. Mohammed AZ, Edino ST, Samaila AA. Surgical pathology of schistosomiasis. J Natl Med Assoc 2007; 99:570-4; PMID:17534016. 5. Ferrari TC, Moreira PR. Neuroschistosomiasis: clinical symptoms and pathogenesis. Lancet Neurol 2011; 10:853-64; PMID:21849166; http://dx.doi.org/10.1016/S1474-4422(11)70170-3. 6. Warren KS. The pathology, pathobiology and pathogenesis of schistosomiasis. Nature 1978; 273:609-12; PMID:351411; http://dx.doi.org/10.1038/273609a0. 7. Taylor MG. Hybridisation experiments on five species of African schistosomes. J Helminthol 1970; 44:253314; PMID:5505355. 8. Southgate VR, Rollinson D, Ross GC, Knowles RJ. Mating-behavior in mixed infections of Schistosoma haematobium and Schistosoma intercalatum. J Nat Hist 1982; 16:491-6; http://dx.doi. org/10.1080/00222938200770391.


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