Superimposed visceral leishmanial infection aggravates response to Heligmosomoides polygyrus

Background Polyparasitism is the rule in all animal species, including humans, and has an important role in pathogenicity, diagnosis and control measures. Among them, co-infections by gastrointestinal helminths and protists are very prevalent under natural conditions but experimental infections are relatively scarce. Thus, despite the frequent association of visceral Leishmania infections and intestinal helminth parasitism the experimental co-infection has not been addressed. Heligmosomoides polygyrus, an intestinal nematode of mice, is related to other helminths causing important pathologies and is a model species for immunological studies. Mice are valuable experimental model for visceral leishmaniasis. Methods BALB/c mice infected with H. polygyrus (200 third-stage larvae, L3) were subsequently infected seven days later with Leishmania infantum (107 promastigotes) with the aim of determining the effect of the overinfection on the host response to the primary infection with the helminth. Results Overinfection with the protist did not affect the establishment rate of the nematode but induced a higher fecal egg output. Helminth burdens in co-infected animals were significant at the end of the experiment. Early unspecific immune suppression induced by the nematode in mesenteric lymph nodes was not switched by L. infantum infection. Co-infection elicited a higher serum antibody (IgG1) response against the helminth. Conclusions Visceral leishmanial overinfection aggravated the early host response against primary infections with the intestinal helminth. This effect was evidenced by an increased longevity and higher production of non-protective antibodies.


Background
Gastrointestinal nematodiases are a major problem worldwide, both for human and animal health. Helminth infections are present in all species of wild and domestic animals, with different management practices and climates. Soil-transmitted helminthiases (STHs) are among the most common and persistent parasitic infections worldwide and it has been estimated that over three billion people are infected with one or more helminth species [1]. In the veterinary arena these diseases have a strong impact on health status, productivity and therefore on the sustainability of animal production. Visceral leishmaniasis is the second most lethal parasitic disease for humans [2]. Leishmania infantum zoonotic infection is found in the Mediterranean and Brazil where millions of dogs are naturally infected.
Multiparasite infections are the rule and the coexistence of more than one species in a host has significant effects on their pathogenicity, clinical course and design of control systems [3,4]. Information is relatively scarce and only recently has a growing interest on the study of multiparasitism been observed [5,6].
Heligmosomoides polygyrus (Nematoda: Strongylida), a natural parasite of mice intestines, is a species taxonomically related to those causing processes of relevance in humans (e.g. Necator, Ancylostoma) and in animal health and production (e.g. Ostertagia, Haemonchus). Primary exposure to H. polygyrus provokes a predominant Th2 response [7] accompanied by regulatory T cell (Treg) activation, thus failing to achieve an effective expulsion of the worms and causing chronic infections in most mouse strains [8][9][10]. The lack of protective response in primary infections is related to the immunosuppressive abilities of the nematode since an effective response appears when the primary infection is cleared (i.e. with drug treatment) [11]. Immune interference between helminths and protozoans has been described and experimentally addressed in some combinations [12][13][14][15][16]. Cross-sectional studies have been carried out in human patients with cutaneous (caused by Leishmania braziliensis) [17] and visceral leishmaniasis [18] and helminth infections with conflicting results. However, the potential effect of a visceral Leishmania infection on previously infected hosts with intestinal helminths has apparently not been experimentally addressed. This lack of information is surprising, given the widespread distribution of helminths all over the world and the high prevalence of visceral leishmanial infections. Since H. polygyrus infections are a well established model of Th2 response in mice and L. infantum infection in mice elicits a mixed Th1/Th2 response [19][20][21], we administered an overinfection with L. infantum to BALB/c mice previously infected with H. polygyrus. Results obtained in this surrogate model showed that visceral leishmanial infection aggravated the primary response against the intestinal helminth.

Parasites
Heligmosomoides polygyrus larvae were provided by M. Grueiro (Faculty of Pharmacy, UCM, Madrid, Spain) and the isolate was maintained in our laboratory by passage in susceptible mice every 6 months. The infective thirdstage larvae (L3) were obtained by incubation of fecal material on filter paper disks placed on a Petri dish with distilled water at 22°C for 7 days. The isolate of L. infantum (M/CAN/ES/97/10.445 zymodeme MON-1) was supplied by M. Domínguez (ISCIII, Madrid, Spain) and has been maintained as promastigotes by passage in RPMI 1640 medium (Lonza Group, Basel, Switzerland) at 26°C supplemented with heat inactivated fetal bovine serum (30 min, 56°C) (Sera Laboratories International, Horsted Keynes, UK) and 100 U/ml penicillin + 100 μg/ml streptomycin (BioWhittaker, Verviers, Belgium).

Mice, experimental design and follow-up
Female BALB/c mice (Harlan Laboratories Models SL, Barcelona, Spain) were housed in our facilities (No. ES280790000155) in polystyrene cages (4 animals per cage) at a controlled temperature of 22-25°C with a 12 h light 12 h darkness cycle and received water and commercial rodent feed ad libitum. Mice were randomly allocated to four experimental groups of eight animals per group. G1 and G2 animals were infected with 200 L3 of H. polygyrus in 0.2 ml water, using a bucoesophagic catheter, on day 0 of the experiment. G1 and G3 animals were infected by intraperitoneal injection, on day 7 post-infection (pi), with 10 7 stationary promastigotes of L. infantum in 0.1 ml PBS. G4 was the uninfected control group.
Individual blood samples were obtained on day -1, 6, 14, 21, 28 and 35 pi with H. polygyrus by puncture of submandibular vein. The blood volume obtained was 50 μl/ mouse/sample day except for the days when animals were euthanatized (14 and 35 pi) when 150 μl were taken. Blood was allowed to clot and the sera preserved at -20°C until used. Samples taken at the end point of the experiment were employed for ELISA determinations. Coproscopical analyses were performed every 3 days from the 9th day pi onwards. Mice were individually isolated for 30 min, their feces collected and egg counts performed by a modified flotation technique. The results were expressed as eggs per gram of feces (epg). Cumulative epg output was estimated using the trapezoidal method to determine areas under the curve (AUC) of the animals and groups. Mice were euthanatized (CO 2 inhalation -isoflurane) on days 14 and 35 pi, 4 animals per group at each time point. Intestines, spleens and mesenteric lymph nodes were removed and employed for further determinations. Individual intestines were opened and placed in physiological saline solution, kept overnight at 37°C and the worms were recovered and counted. Spleens were extracted under sterile conditions and weighed individually. To assess L. infantum infection, a spleen sample was employed to prepare smears, stained with May Grünwald Giemsa and microscopically examined.

Antigens
Heligmosomoides polygyrus soluble extract (ASE) was obtained from adult helminths. Nematodes were cleaned with cold PBS containing protease inhibitors (Roche, Mannheim, Germany), subjected to 8 freeze-thaw cycles (-80°C to room temperature), homogenized in a glassin-glass homogenizer and centrifuged at 3000× g for 30 min at 4°C. Soluble leishmanial antigen (SLA) was obtained by 10 freeze-thaw cycles of L. infantum promastigotes followed by centrifugation and recovery of the supernatant. The concentration of protein was measured [22] and the antigen extracts (SLA, ASE) stored at -80°C until use.

Lymphoproliferation
Lymphocyte proliferation was determined using the tetrazolium salt (MTT) method [23] with some modifications.

Results
Overinfection with L. infantum provokes higher fecal egg output of H. polygyrus-infected mice Animal groups infected with H. polygyrus (G1 and G2) started eggs excretion on day 10 pi; however, the epg pattern greatly varied depending on the infections received ( Fig. 1). There was a high intragroup individual variability within a sample day and along the experiment. Thus, cumulative egg excretion, estimated by AUC from day 15 to the end of the experiment, showed that mice infected only with H. polygyrus shed c.25% of the AUC value of co-infected mice (G1: H. polygyrus + L. infantum) although the difference was not significant. There was a different pattern of epg values between animal groups and egg excretion (AUC) of co-infected mice was significantly higher between days 15 and 21 pi (t (6) = 2.342, P = 0.0288). No eggs were found in the mice infected only with L. infantum (G3) and the uninfected control group.

Leishmania infantum overinfection does not affect establishment of H. polygyrus although it increases helminth survival
On day 14 pi, estimated total worm burden was comparable in both mice groups (G1: 86.25 and G2: 88.25 helminths) and no differences were found in sex ratio of helminths (Fig. 2). Conversely, 35 days pi parasite burdens were significantly different depending on the presence of concurrent L. infantum infection. In a similar way to that found in epg patterns, mice co-infected (G1) hosted higher worm burdens than those observed in animals subjected only to  (Fig. 5b), in the presence of H. polygyrus soluble antigen was low in all groups. Despite the helminth-induced unspecific suppression, the most reactive animals in MLN were those co-infected with L. infantum (F (2, 18) = 8.762, P = 0.002] with significant differences on day 14 (2.181 ± 0.251) to both the control group (1.032 ± 0.376) and the animals with monospecific helminth infection (0.904 ± 0.104). This pattern was also observed after 35 days although differences were not significant. In the spleen (Fig. 5b) co-infected mice (G1) also displayed a higher proliferative response in the last sampling (1.474 ± 0.365). Lymphoproliferative response against leishmanial antigen was low and not related to the infection schedule of the animal groups (not shown).
Overinfection with L. infantum increased the antibody response to H. polygyrus Antibody response against H. polygyrus was slow to develop and on day 35 pi showed a significant IgG 1 serum response against Heligmosomoides ASE in the groups infected with the helminth (Fig. 6a) (F (3,24) = 37.939, P < 0.0001]. The response was higher in the group co-infected (G1: 0.362 ± 0.098) than in the mice with monospecific infection (G2: 0.311 ± 0.054) although differences were not significant. No specific IgG 2a response to Heligmosomoides ASE was observed except for an outlier value (Fig. 6b). Monospecific Leishmania infection (G3) elicited a significant IgG apparently related to the IgG 2a response (35 days pi) against homologous antigen extract (SLA) whereas no IgG 1 specific response was observed (not shown).

Discussion
Under natural conditions, co-infections are the rule in all host species, including humans, [3] and they have a significant impact on disease course outcome, diagnosis and preventive measures [6]. Helminth-protist interactions are very complex ranging from a lack of interference [14] to strong impairment of protective immunity [13]. For the most part, some type of modulation/switching of immune response has been found in the co-infections so far explored. Direct comparison of the results is extremely difficult since the mechanisms involved and final outcome depend on the parasite species and also the schedule of infections (e.g. timing and doses administered). In our experiments, overinfection with L. infantum of previously infected mice with H. polygyrus strongly impaired the immune response against helminth infection. This interference was evidenced by a worsening of major parameters such as the apparent higher fertility of female worms in early patency and higher helminth burdens at the end of the experiment.
The number of helminths found at the beginning of the patency evidenced a high establishment rate (c.40%), in-line with previous observations in primary infections by H. polygyrus [24,25]. It has been reported that a previous infection with Toxoplasma gondii led to a higher fecundity of H. polygyrus female worms 14 days pi [26]. These results were comparable to those obtained by us with an unrelated protist species, L. infantum. Thus, a notable increase in fecal egg output was found in co-infected mice from day 15 to 21 pi without helminth number differences compared to the animals subjected Moreover, we found at the end of the experiment (35 days pi) a significantly higher number of worms in co-infected mice. These findings are consistent with the partial abrogation of the Th2 response in H. polygyrus infections [10] and it has also been described in other co-infections with this helminth species [16,26] irrespective of the experimental design. In H. polygyrus/ Trypanosoma congolense co-infections, no differences were found in female helminth fertility [24,27]. However, this experiment was not designed to determine the response in primary helminth infections but the acquired resistance after challenge. Our results suggest that the timing, order and protist species involved is apparently less relevant and in both cases immune response polarizes towards a Th1 type this resulting in both higher fecundity and longevity of helminths. The actual mechanistic basis of this interference is poorly known and deserves attention since it is much relevant within the framework of naturally infected hosts where the polyparasitism is the rule [5].
Single infection with L. infantum did not elicit significant changes in spleen weight in any of the experimental time points (1 week pi, 4 weeks pi). Intravenous infection of susceptible mice (e.g. BALB/c) with visceral Leishmania leads to self-limiting liver invasion and a progressive spleen parasitism. Thus, our results could be possibly related to the pi time elapsed [28]. Mice infected with H. polygyrus alone or co-infected with L. infantum showed a notable increase in spleen weight, this being consistent with the immune response mounted against the nematode infection. It is noteworthy to indicate that co-infection with L. infantum actually caused an early reduction of spleen weights (14 days pi) compared to those seen in the mice with the helminth infection alone. This result supports the interference of the co-infection and the impairment of the characteristic Th2 protective response against the nematode [8,29]. Poor anti-H. polygyrus specific lymphoproliferation in both lymph nodes and spleen was consistent with the unspecific immune suppression (ConA) elicited by nematode infection, particularly in the first parasitic stages (< 14 days pi). Conversely, the immune suppression in L. infantum-infected mice (G3) was seen in later stages (day 35 pi), perhaps related to the course of the infection [28]. Visceral Leishmania infections cause a mixed Th1/Th2 response [19][20][21] and H. polygyrus is highly effective in blocking the protective Th2 immunity [10]. In our experiment with primary helminth infections, high IgG 1 anti-H. polygyrus levels were found after 35 days whereas no IgG 2a was detected. This suggests a Th2 polarized response [29] and, in our experiment, L. infantum overinfection actually increased the antibody response against the helminth. A lack of correlation between IgG 1 levels and protection (e.g. reduced parasite burden or fecal egg output) points towards the limited protective role of the antibodies detected by ELISA [30]. Although not strictly comparable to our experimental approach, it has been observed that human patients co-infected with visceral Leishmania and helminths displayed no alteration of the course of leishmaniasis [19] whereas the clinical outcome of cutaneous infection by L. braziliensis was significantly influenced by helminth co-infection [17]. It is possible that the initial clinical condition of patients, chemotherapeutic regimen, differential immune response against visceral and cutaneous Leishmania and the timing of infections (protist, helminth) could account for the variable response observed.

Conclusions
Considering all results together, our experiment confirmed the unspecific and specific immune suppression elicited by primary infection by H. polygyrus in mice. More importantly, overinfection with L. infantum of previously infected animals with the helminth aggravated the suppression, leading to higher parasite burdens, fecal egg output and longevity. Co-infection apparently polarized the immune response towards a non-protective Th1 type. However, no complete Th1/Th2 switch was found since co-infected mice developed a notable specific IgG 1 response against Heligmosomoides. A lack of correlation between protection and IgG 1 levels suggests the limited role played by antibodies in coping with helminth infection.