Immunopathology of Dirofilaria immitis Infection - Springer Link

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Dec 23, 2006 - both D. immitis and Wolbachia, in the immunology and immunopathology of dirofilariosis. Keywords: canine, feline and human infections; ...
Veterinary Research Communications, 31 (2007) 161–171 DOI: 10.1007/s11259-006-3387-0

 C Springer 2007

Immunopathology of Dirofilaria immitis Infection F. Sim´on1,∗ , L.H. Kramer3 , A. Rom´an1,2 , W. Blasini1,2 , R. Morch´on1 , C. Marcos-Atxutegi1 , G. Grandi3 and C. Genchi4 1 Laboratorio de Parasitolog´ıa, Universidad de Salamanca, Salamanca, Spain; 2 University of Puerto Rico, Medicine School, Puerto Rico; 3 Dipt. Di Produzione Animale, Universit`a di Parma, Parma; 4 DIPAV–Sezione di Patologia Generale e Parassitologia Universit`a degli Studi di Milano, Milan Italy ∗ Correspondence: E-mail: [email protected]

ABSTRACT Heartworm disease caused by Dirofilaria immitis affects canine and feline hosts, with infections occasionally being reported in humans. Studies have shown that both dirofilarial antigens and those derived from its bacterial endosymbiont Wolbachia, interact with the host organism during canine, feline and human infections and participate in the development of the pathology and in the regulation of the host’s immune response. Both innate and acquired immune responses are observed and the development of the acquired response may depend on the host and, or on its parasitological status. This review aims at illustrating current research on the role of both D. immitis and Wolbachia, in the immunology and immunopathology of dirofilariosis. Keywords: canine, feline and human infections; D. immitis, inflammatory reactions, pro- and antiinflammatory cytokine expression, TH 1 and TH 2-type response; Wolbachia endosymbionts Abbreviations: Dipp, Dirofilaria immitis derived peptides; GroEl, heat-shock protein of D. immitis (hsp60); iNOs, inducible nitric oxide synthase; IL, interleukin; LPS, lipopolysacharide; L3 , third-stage larvae; TGF-β, transforming growth factor β; TNF-α, tumour necrosis factor α; WSP, Wolbachia surface protein

INTRODUCTION Heartworm disease is a parasitic infection caused by Dirofilaria immitis in canine and feline populations worldwide. The adult worm lives in the pulmonary arteries where mature females release first-stage larvae (microfilariae) into the bloodstream. These are taken up by an arthropod vector (several species of mosquitoes are competent vectors of D. immitis, including Culex pipiens, Aedes albopictus, Anopheles maculipenis and Coquilletidia richiardii), where they develop to infective third-stage larvae (L3 ). These are then inoculated into the dermis of the final host where, after several months of migration and maturation, they reach the pulmonary arteries. Heartworm infection in dogs usually presents as a chronic disease. Initially the pulmonary vasculature is affected, and later the lung itself and, finally, the right chambers of the heart. The initial inflammatory reaction that occurs in the walls of the pulmonary vasculature is critical in the development of the entire disease process (Furlanello et al., 1998). Feline infection is diagnosed with increasing frequency in areas where the disease is endemic in canines. However, the development of the parasite and the clinical findings in cats are different from those that occur in dogs. The development of the parasite takes longer compared to dogs and most infections are amicrofilaraemic. Additionally, the parasite burden

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Figure 1. Main clinical/parasitological characteristics Dirofilaria immitis infection in each of its hosts

is low and the infection is generally asymptomatic, although some cats present with severe disease or even sudden death in the presence of a small number of adult worms (1–3) (Genchi et al., 1992; McCall et al., 1994; Atkins et al., 2000). Humans are incidental hosts for D. immitis since the larvae do not normally develop into adult worms and microfilaraemia is absent. Infections with immature worms have been identified in humans. In these cases, pulmonary nodules develop when the parasite lodges in the small branches of the pulmonary artery, dies and causes a granulomatous reaction. This can be seen radiologically as a well-circumscribed, non-calcified or calcified nodule (Cordero et al., 1992; Orihel and Eberhard, 1998; Muro et al., 1999) that may be confused with a neoplastic lesion. Figure 1 summarizes the main differences between D. immitis infection in canine, feline and human hosts. The immunopathology of filarial disease is extremely complex and clinical manifestations depend on the type of immune response that is stimulated by the parasite. It is currently accepted that the inflammatory changes associated with the disease appear when parasites die, either naturally or as a consequence of specific drug treatment. (Dreyer et al., 2000; Taylor et al., 2001). The recent ‘rediscovery’ of the presence of bacterial endosymbionts belonging to the genus Wolbachia in many filarial species, including Wuchereria bancrofti, Brugia malayi, Onchocerca volvulus and D. immitis (Sironi et al., 1995; Taylor et al., 2000), has led to a revolutionary change in the interpretation of studies related to filarial worms and, in particular, of the pathogenesis of infection. Recent studies have focused on understanding

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the role these bacteria play in the immunological response and the immunopathology of filariasis. There are, however, very few data available regarding Dirofilaria infections. The aim of this review is to present the current data on the role of worm antigens, and in particular Wolbachia-derived molecules, during D. immitis infection in its various hosts.

Wolbachia INTERACTS WITH D. immitis- INFECTED HOSTS There is substantial evidence that Wolbachia comes into contact with the D. immitis-infected host organism. Wolbachia has been identified in many organs and cells of D. immitis-infected dogs such as renal tubular cells, glomeruli or inflammatory pulmonary cells by the use of immunohistochemistry techniques employing a polyclonal antibody against the Wolbachia surface protein (WSP) (Figure 2; Kramer et al., 2003, 2005). Moreover, IgG antibodies against WSP have been detected in felids with experimental and natural D. immitis infections (Bazzochi et al., 2000; Morchon et al., 2004), in humans who have been diagnosed with pulmonary dirofilariosis, and in healthy, seropositive humans living in areas where canine dirofilariosis is endemic (Sim´on et al., 2003). High titres of specific IgG antibodies have also been detected in dogs with different clinical presentations of dirofilariosis (Kramer et al., 2005). Similar findings have also been found in human filarioses. Individuals with lymphatic filariosis and onchocercosis mount an antibody response against

Figure 2. Immunohistochemical localization (arrows) of Wolbachia surface protrein (WSP) in the lung from a dog naturally infected with D. immitis

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WSP and other molecules such as the heat-shock protein (hsp60) of Wolbachia (Punkosdy et al., 2001, 2003; Taylor et al., 2001), while Keiser and colleagues (2002) reported Wolbachia DNA in the blood of patients following diethylcarbamazine (DEC) treatment for O. volvulus.

THE INTENSITY OF THE ANTIBODY RESPONSE DURING INFECTION IS RELATED TO BOTH THE PRESENCE OF MICROFILARIAE AND THE CLINICAL STATUS OF THE HOST Several past studies, reviewed by Sim´on and colleagues (1998, 2001), have shown that there is a relationship between the intensity of the humoral response to D. immitis and the parasitological status of dogs with heartworm disease. Recent studies carried out in naturally infected dogs (Marcos-Atxutegi et al., 2004), using molecules derived from D. immitis (synthetic peptides derived from molecules of 22 and 30 kDa from adult worms, (D. immitis peptide pool, Dipp) and from Wolbachia (WSP), have demonstrated that there is a stronger IgG antibody response towards both D. immitis and Wolbachia antigens in microfilaraemic dogs than in canine infections without microfilariae (Table I). This is in accordance with the observations of Grieve and colleagues (1979), who showed that microfilaraemic dogs exhibit high levels of IgG antibodies, which then decrease when microfilariae disappear. However, in an amicrofilaraemic dog with massive pulmonary thromboembolism examined in our study, the IgG response to both Wolbachia and D. immitis antigens was much higher than in other amicrofilaraemic dogs that were asympomatic. This may suggest that the level of antibody response is also related to the host’s clinical status. In fact, Punkosdy and colleagues (2003) found high levels of IgG antibodies against WSP in half of the individuals with lymphoedema or hydrocele, in a region of Haiti where W. bancrofti is endemic, suggesting that the antibody response against WSP correlates more with chronic disease than with the parasitic status of infection. The differences in the infection in cats have led to investigation of the particular immunological responses in feline heartworm disease. During early infection, a strong IgG antibody response against D. immitis has been demonstrated using antigenic fractions containing the 22 and 33 kDa polypeptides (Prieto et al., 2002). However, a low and transient antibody response against L3 somatic antigen has also been observed (Prieto et al., 2001), suggesting that this parasite stage can evade the host’s humoral immune response and continue development until adulthood. The antigenic role of Wolbachia endosymbionts in feline heartworm disease has also been a matter of interest for researchers. Bazzocchi and colleagues (2000), using a recombinant WSP in western blot analysis, reported the presence of IgG antibodies against Wolbachia in D. immitis-infected cats. In a further study by Morchon and colleagues (2004), the antibody response against specific molecules of D. immitis and Wolbachia endosymbionts in both naturally and experimentally infected cats with and without larvicidal (ivermectin) treatment, was evaluated. Increased IgG antibody production against filarial antigens and WSP was observed in experimentally infected cats without treatment. However, in experimentally infected cats (infection with 30 L3 ) treated with a larvicidal drug one month post -infection, there was a transient increase in anti-D. immitis IgG that decreased dramatically in correspondence with the death of the larvae,

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while the anti-WSP IgG response increased continuously until the end of the experiment (6 months). The immune response to Wolbachia antigens was detected as early as 2 months after infection, before detection of specific antibodies against D. immitis antigens. These results demonstrate that the humoral response in cats with heartworm disease is not only directed against the parasite but also against the Wolbachia endosymbionts, which may be massively shed following the death of larvae and/or preadult worms. Indeed, according to Fenn and Blaxter (2004), there is a sharp increase in Wolbachia load in L4 larvae of Brugia malayi compared to the other developmental stages. According to these findings, one may suggest that Wolbachia also plays an important role in the inflammatory reactions in heartworm infection in cats. Specific immunological responses have been observed in patients with human pulmonary dirofilariosis and in healthy seropositive donors living in areas where the infection is endemic in dogs. IgG or IgM antibodies against somatic and excretory/secretory antigenic complexes from D. immitis adult worms are consistently present in cases of pulmonary dirofilariosis, while IgE predominates in healthy seropositive donors without symptoms (Sato et al., 1985; Sim´on et al., 1991., Espinoza et al., 1993). As in canids and felids, the role of the filarial Wolbachia endosymbionts in human dirofilariosis is currently being investigated. Sim´on and colleagues (2003) demonstrated that IgG against recombinant WSP of Wolbachia was consistently detected in all patients studied with pulmonary nodules, but only in some of the healthy seropositive asymptomatic donors living in endemic areas. These results have led to the hypothesis that the surface protein of Wolbachia (and probably other molecules) stimulates the host immune system mainly after the death of the larvae and/or the worms in pulmonary nodules, and have provided further insight of the role of these endosymbionts in the course of the disease.

INNATE IMMUNITY AND ACQUIRED TH 1/TH 2-TYPE RESPONSES ARE PRESENT IN DIROFILARIOSIS Recent data from our laboratory on the mRNA expression of the different cytokines produced in infected dogs have shown that both innate and acquired immune responses are characteristic of natural infection. Induced nitric oxide synthase (iNOs) and tumour necrosis factor (TNF-α) important pro-inflammatory mediators, are consistently present. This type of response, which plays a key role in the possible subsequent development of the disease, has also been described in lymphatic filariosis (Taylor et al., 2000) and in onchocercosis (Brattig et al., 2004). The expression of cytokines (interleukins (IL)) related to both the TH 1-and TH 2type acquired responses (IL-2, IL-4, IL-5, IL-10) has also been detected in naturally infected dogs. Preliminary results indicate that IL-10 is present in dogs with patent infections, while it is not expressed at detectable levels in amicrofilariaemic dogs. This cytokine has been implicated in the hyporesponsiveness observed in filarioses. IL-10 has been detected in phagocyte cultures stimulated by antigens of O. volvulus (Brattig et al., 2000) and during lymphatic filarial infections (Mahanty et al., 1996; Ravichandran et al., 1997; Osborne and Devaney, 1999). In a recent study, the lack of cell-mediated immune responses against helminths was attributed to the existence of cells of the type TH 3/TR 1, a subgroup characterized by producing IL-10 and TGF-β, which are inhibitors of inflammation (Doetze et al., 2000).

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There are several studies showing the participation of both T helper 1 (TH 1) and T helper 2 (TH 2) responses in various models of filariosis (Winkler et al., 1996; Mar´echal et al., 1997; Babu et al., 2000; Goff et al., 2000; Tauber and Zanher, 2001). Nevertheless, these studies have not taken into account the presence of Wolbachia in filarial parasites. An analysis of the type of immune response (TH 1/TH 2) in canine dirofilariosis (Marcos-Atxutegi et al., 2004) indicated that microfilaraemic dogs mount a strong IgG1 response against both Dipp and WSP antigens, while amicrofilaraemic dogs showed no significative responses when compared to healthy control dogs. On the other hand, an IgG2 response was detected in both microfilaraemic and amicrofilaraemic dogs and was directed exclusively against the WSP of Wolbachia (Figure 3, Table I), a situation similar to that observed by Dimock and colleagues (1996) in a human population living in an area endemic for lymphatic filariosis in Haiti and in humans infected with O. volvulus (see Brattig, 2004). It should be noted that, in contrast to humans, IgG1 production in dogs is thought to be indicative of a predominantly TH 2-type response while IgG2 is considered of type TH 1. These results would suggest a TH 2-type response in the presence of active infections, while occult infections appear to feature a TH 1-type response. In humans, Dirofilaria infections are always amicrofilaraemic and very frequently asymptomatic. Interestingly, only TH 1-type response against WSP have been observed (Marcos-Atxutegi et al., 2004) and IgG1 (TH 1) production against WSP was the only response consistently detected in all individuals with pulmonary dirofilariosis studied. Some of the healthy seropositive donors developed IgG1 against WSP or D. immitis antigens, but no IgG2 response was observed in these individuals.

THE CONTRIBUTION OF EXPERIMENTAL MODELS AND ‘IN VITRO’ STUDIES There are a very limited number of experimental models or ‘in vitro’ studies related to the immune response and immunopathology of dirofilariosis. The first evidence that filarial antigens induce a preferential TH 2 response while those of Wolbachia stimulate only the TH 1-type response was reported by our group using a murine model of immunization (Marcos-Atxutegui et al., 2003). Moreover, in this work we have shown that populations of eosinophils and neutrophils increase when the immune system of mice is stimulated by somatic L3 or adult antigens of D. immitis. Neutrophils accumulate in the kidneys and in the walls of the pulmonary arteries during canine dirofilariosis infection, suggesting that neutrophils participate in the development of the inflammatory reactions in heartworm disease. In an ‘in vitro’ study, Bazzocchi and colleagues (2003) demonstrated that WSP stimulates chemokinesis and IL-8 expression in canine neutrophils, thus implicating Wolbachia in the development of inflammatory reactions in a lipopolysaccharide (LPS)-independent manner in canine dirofilariosis. A recent study carried out in our laboratory in BALB/c mice inoculated with a combination of WSP and Wolbachia-hsp60 (GroEL) has shown that these molecules stimulate the intense expression of pro-inflammatory innate mediators, especially iNOs and TNF-α, confirming the hypothesis of Bazzocchi and colleagues (2003). Indeed, there is strong evidence that Wolbachia do not possess LPS (Wu et al., 2004) and our findings provide evidence that Wolbachia can participate in the inflammatory reactions of heartworm disease through other molecules than LPS, as has been reported in onchocercosis and lymphatic filariosis (Brattig et al., 2001; Taylor et al., 2001; Brattig et al., 2004).

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TABLE I Summary of the currently known data on the immunology and immunopathology of dirofilariosis Host

Antibody response to Wolbachia antigens

Dogs

Cats

Humans

Stronger IgG response against WSPa in mf+ than in mf− dogsb

IgG response against WSP has been detected

IgG response against WSP has been identified in all individuals with pulmonary dirofilariosis, but only in some healthy seropositive donors

Adulticide treatment induces an increase in the IgG anti-WSP response Type of antibody response

IgG1 antibodies against both D. immitis and Wolbachia antigens have been detected in mf+ infections, with different intensity

IgG2 antibodies exclusively directed against Wolbachia antigens have been detected both in mf+ and mf− infections Cytokine mRNA TNF-α , iNOs, IL-2, IL-4 expression and IL-5 have been identified in both mf+ and mf− infections IL-10 is expressed much more intensely in patent than in occult infections Cells involved WSP stimulates canine during the course neutrophils to produce of dirofilariosis IL-8 a Wolbachia surface protein b mf+ , microfilaria-positive;

mf− , microfilaria-negative

IgG1 antibody response predominates in human dirofilariosis. In cases of pulmonary dirofilariosis, only IgG1 against WSP has been detected consistently Only some healthy seropositive donors develop IgG1 against WSP and/or D.immitis antigens

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Figure 3. Antibody response (IgG1, IgG2) against D. immitis peptide pool (Dipp) and Wolbachia surface protein (WSP) in dogs naturally infected by Dirofilaria immitis. Black bars, microfilaria+ dogs; light gray bars, microfilaria− dogs; dark gray, bars, healthy control dogs.

DO Dirofilaria ANTIGENS PLAY A ROLE IN THE REGULATION OF INFLAMMATORY REACTIONS? Despite the fact that most recent research on the molecular mechanisms of inflammatory response during filarial infections has focused almost exclusively on the elucidation of the role of Wolbachia, there does exist some evidence suggesting that molecules derived from the filariae themselves can also participate in instigation of pathology and in the regulation of the host’s immune response. Molecular and immunohistochemical studies on Loa loa (McLaren et al., 1975; McGarry et al., 2003) have suggested that this species lacks endosymbiont bacteria, but, in spite of this, inflammatory reactions occur in infected individuals. Moreover, the observation of cutaneous inflammatory reactions after microfilaricide treatment in patients with onchocercosis, in which Wolbachia was previously eliminated by the administration of antibiotics, suggest that these reactions depend only partially, not exclusively, on the presence of bacteria (N.W. Brattig, personal communication). It has been proposed that some molecules from O. volvulus that bind fatty acids and retinols may participate in the development of the cutaneous and ocular pathology (Bradley et al., 2001). Finally, some molecules secreted by D. immitis that mimic host molecules seem to stimulate the production of IL-10, a cytokine that is associated with immunosuppression. These parasite-derived molecules could contribute to the long-term survival of

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the parasite and benefit the host by inhibition of immunomediated pathology (Tezuka et al., 2003).

CONCLUSION In conclusion, there is compelling evidence that molecules derived from Wolbachia are important protagonists in the inflammatory pathology in dirofilariosis. Indeed, a recent report by Higazi and colleagues (2005) has shown that the Wolbachia load in severe strains of O. volvulus is significantly higher than in to mild strains, thus supporting the idea that Wolbachia is responsible for inflamation during infection. Even though Wolbachia lacks LPS, it is likely that other molecules such as WSP, hsp60 or DNA, are able to stimulate the inflammatory response. For this reason, Wolbachia bacteria have become the target of antibiotic treatment, which not only affects worm fecundity and survival but may decrease inflammatory pathology. Moreover, it is likely, and worthy of further study, that molecules from filariae contribute to inflammatory and anti-inflammatory reactions during filarial diseases.

ACKNOWLEDGMENTS This work was supported by the Spanish Ministerio de Ciencia y Tecnolog´ıa (SAF200305829) and the MIRT Program (NIH), USA.

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