1 Introduction

Host immune responses can influence the development of filarial parasites and their fecundity in human or experimental hosts (Kwarteng and Ahuno, 2017) Immune effector mechanisms against these large nematode parasites can be against larvae (anti-larval immunity) or microfilariae (anti-microfilarial immunity). Anti-larval immunity is directed towards incoming L3 larvae and reduces the possibility that the larvae develop into adult worms. In human filariasis, anti-microfilarial immunity has been demonstrated to be mediated by antibodies to the sheath of microfilariae, and there is an inverse association between circulating microfilaria and antibodies to the sheath (Ravindran et al., 1990). Although the immune response against larvae and microfilariae of filarial parasites has been understood with reasonable clarity (Ritter and Hubner, 2022), no mechanistic explanation is currently available explaining the mode of action of protective immune responses against large adult-stage filarial parasites.

The immune mechanism against large-stage adult nematode parasites is often mediated by host immune cells such as basophils and eosinophils (Yasuda and Kuroda, 2019; Peng and Siracusa, 2021), which act upon these parasites on their surface. The second possible effector mechanism is anti-fecundity immunity, which may operate inside the uterine cavity of adult parasites. This assumption was supported by the earlier studies, which indicated that sub-cutaneous infection of third-stage larvae of Brugia pahangi in IL-4-/- mice revealed that, at later time points of infection, the IL-4 −/- mice contained a large number of microfilariae in the peritoneal cavity than the wild type BALB/c mice and the differences in microfilariae levels appear to relate to differences in worm fecundity in the two strains of mice (Devaney et al., 2002). In another study in LPS-non-responsive C3H/HeJ and LPS-responsive C3H/HeN mouse strains, it was observed that the fertility of adult worms from C3H/HeJ mice after adult worm implantation in the peritoneal cavity was higher than those of C3H/HeN mice as indicated by the presence of higher numbers of microfilariae at the site of infection in C3H/HeJ mice suggesting a possible role of TLR-4 signaling in immune response that inhibits worm embryogenesis (Pfarr et al., 2003). In these two reports, the exact mechanism for difference in adult worms' fecundity was unknown. We assume this is due to the operation of anti-fecundity immunity inside filarial parasites. Using a flow cytometry-based assay system, we demonstrated the entry of macromolecules like Concanavalin-A and antibodies (Immunoglobulins) into the uterine cavity of live worms. We further propose that the entry of immunoglobulins into the worms may be through the cuticle through the process of endocytosis.

2 Methodology

3 Results

3.1

3.1 Concanavalin-A enters into the uterine cavity of adult s. Digitata and is bound to intrauterine stages

It is pertinent to note that, according to a report by Sahu et al., 2005, a flow cytometry-based technique was used to identify different embryonic stages of adult S. digitata. Although a flow cytometer is generally used to identify different cells (bacteria and eukaryotic cells), the author developed an assay by which different embryonic stages of adult S. digitata, such as microfilariae, early and late developmental stages, could be easily detected as three different clusters in the scatter plot. Using this novel method, we performed all experiments in the current research. The internalization of Con A into adult filarial worms was demonstrated by flow cytometry (Fig. 1). A significant proportion of microfilariae, early and late embryonic stages of adult worms was found to bind to Con A after incubating worms at different time points (1 h, 3hrs and 6hrs) (Fig. 1A) indicating that the molecule has entered into the uterine cavity of worms and then bound to IU stages. As shown in Fig. 1B, a gradual increase in the binding of embryonic stages to Con A was observed at different incubation time points, as noted from the mean intensity of fluorescence examined by flow cytometry.

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Fig. 1

Entry of Con-A into uterine cavity of adult S. digitata. % Reactivity of Concanavalin A to different intrauterine stages of adult S. digitata. 1, 5 and 9 – Control unpulsed worm; 2, 6 and 10 – Binding of Con A to IU stages after 1 hr of incubating adult worms; 3, 7 and 11 − Binding of Con A to IU stages after 3 hrs of incubating adult worms; 4, 8 and 12 − Binding of Con A to IU stages after 6 hrs of incubating adult worms. Data of mean of experiments performed with five different adult worms are shown. Mean % reactivity ± SEM. % reactivity − % of intrauterine stages binding to ConA-FITC as examined by flow cytometry.

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3.2

3.2 Human immunoglobulins enter into the uterine cavity of adult s. Digitata and bound to intrauterine

Internalization of Con A, a macromolecule, prompted us to show if immunoglobulins enter adult worms. We demonstrated this phenomenon by pulsing the female adult worms with media containing human serum (10 %). Live worms were pulsed for 6 hrs in vitro with human sera containing filarial-specific antibodies. Non-filarial human serum was used as a control. As shown in Fig. 2 (A and B), significant binding of IgG to different embryonic stages of filarial works was noted. This indicates that human immunoglobulins have entered the worms' uterine cavity and are subsequently bound to IU stages.

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Fig. 2

Immunoglobulins in human sera enter into live active worms. Binding of FITC labeled anti-human IgG to 3 distinct populations of IU stages was analyzed. (A) Data for experiment done with a single representative worm is shown. Scatter plot on left panels show background reactivity of anti-human-IgG – FITC conjugate to IU stages, Scatter plot on right panels show specific reactivity of antibodies in human filarial serum to R2 and R3 population of IU stages. Data of mean of experiments performed with five different adult worms are shown. Mean intensity of fluorescence ± SEM. (R1 Microfilariae, R2 early developmental stages of eggs and R3 late stages of eggs). Mean intensity of fluorescence – A value obtained in flow cytometer for overall fluorescence intensity of embryonic stages indicative of binding FITC conjugated antibody.

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3.3

3.3 Rabbit immunoglobulins enter into the uterine cavity of adult s. Digitata and are bound to intrauterine stages

We also demonstrated the entry of rabbit immunoglobulins into the uterine cavity of filaria worms. Female adult worms were pulsed with 10 % rabbit serum containing antibodies to embryonic stages for 6 hrs. Embryonic stages were harvested, and the suspension was probed anti-rabbit IgG FITC to detect bound immunoglobulins on the surface of embryonic stages. Worms were also pulsed with normal rabbit serum (without containing antibodies to embryonic stages). Normal rabbit serum was used as this does not contain embryonic stage-specific antibodies. Therefore, the idea was that if immunoglobulins enter the uterine cavity of worms, embryonic stages specific immunoglobulins in immunized rabbit sera will bind significantly to embryonic stages. In contrast, immunoglobulins in normal rabbit serum (without specific antibodies) would either not bind or bind less. Fig. 3 showed a significant increase in the binding of IgG to IU stages when worms were pulsed with immune rabbit sera.

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Fig. 3

Immunoglobulins in rabbit sera enter into live active worms. Binding of FITC labeled anti-rabbit IgG to 3 distinct populations of IU stages were analyzed Data of mean of experiments done with four worms for normal rabbit sera and seven worms for reactivity with immune rabbit sera are shown. Mean intensity of fluorescence ± SEM; t-test. R1 Mf, R2 early developmental stages of eggs and R3 late stages of eggs).

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3.4

3.4 Entry of rabbit immunoglobulins into filarial worms is inhibited by Latrunculin A

Latrunculin A, an endocytic inhibitor, was used to examine if the entry of immunoglobulins into worms occurs by the process of endocytosis. As shown in Fig. 4A, significant binding of rabbit immunoglobulins IU stages (R1, R2, and R3 stages combined) was noticed (Red line). On the other hand, a decreased binding (green line) was observed in IU stages to antibodies in the presence of Latrunculin A, the endocytic inhibitor. Here, we must mention that, as it was optional to show how inhibition of rabbit immunoglobulins occurred for individual embryonic stages, we showed the results by combining all three developmental stages of female adult S. digitata.

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Fig. 4

Inhibition of endocytosis of immunoglobulins in rabbit sera into uterine cavity of female adult S. digitata by Latrunculin A. Binding of FITC labeled anti-rabbit IgG to intra uterine egg population were analyzed. (I) Data for experiment done with a single representative worm is shown. Colour shaded area- background reactivity of anti rabbit IgG- FITC to eggs of an unpulsed worm. Red line- specific reactivity of filarial immune rabbit sera to embryogenic egg stages. Green line- specific reactivity of filarial immune rabbit sera to embryogenic egg stages in the presence of Latrunculin A. Numbers inside the histogram represent mean intensity of fluorescence. (II), Data of mean of experiments done with eight different worms for reactivity with immune rabbit sera and another eight worms for reactivity with filarial immune rabbit sera containing Latrunculin A are shown. Mean intensity of fluorescence ± SEM.

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3.5

3.5 Bovine IgG is present on the surface of IU stages in adult filarial worms

The natural habitat of S. digitata used in our study is cattle. Our observation on the entry of human immunoglobulins and rabbit immunoglobulins into the uterine cavity of adult S. digitata predicted a possible operation and existence of the host’s anti-fecundity immunity inside filarial parasites. As S. digitata dwells inside cattle host, we were interested to examine how bovine immunity can operate inside the filarial parasite. To demonstrate that, IU stages were directly harvested from adult worms and were probed with anti-bovine IgG (a polyclonal antisera raised in rabbits). As shown in Fig. 5A (right panel), all three embryonic stages were found to bind to rabbit anti-bovine IgG. Fig. 5B represents the results of the mean of 8 different worms showing a significant binding (mean intensity of fluorescence), particularly to the R2 and R3 stages. The results indicate the presence of bovine IgG on the surface of IU stages, which might have occurred due to entry of IgG and subsequent binding to IU stages.

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Fig. 5

Bovine immunoglobulins are present in situ on the surface of intrauterine embryogenic stages of Setaria digitata. Binding of bovine IgG to 3 distinct populations of IU stages were analyzed. (A), Data for experiment done with a single representative worm is shown. Scatter plot on the left panels show background reactivity of normal rabbit serum; Scatter plot on the right panels show binding of a polyclonal anti bovine IgG serum (raised in rabbit) to three intrauterine populations. Numbers inside plots represent percentage reactivity. (B), Data of eight independent experiments performed with eight adult worms are shown. Each dot represents reactivity for a single worm. Mean intensity of fluorescence, t-test.

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4 Discussion

We conducted the current study to demonstrate that anti-fecundity immunity exists and operates inside the uterine cavity of adult nematodes and proposed that it could be one of the host immune effector mechanisms against large adult parasites. Generally, during infection of hosts (human or experimental animals) with filarial parasites, the host’s immune effector mechanism to combat these parasites is poorly defined. As these parasites are multicellular in nature, it is believed that the host’s anti-filarial immunity against different developmental stages of parasites like L3 larvae, Microfilariae, and adult stage parasites could be operational from ‘outside’ of parasites, unlike unicellular pathogens where the host’s immunity act ‘inside’ of the parasites. However, the question arises if there is any mechanism in hosts that may block the production of embryos (microfilariae and eggs) in female adult filarial parasites, and parasites ultimately can not propagate inside hosts. One such possible mechanism is if the host’s molecules of effector immunity (chemokines, cytokines, interferons, and antibodies) can get access into the uterine cavity of female adult worms and act in embryonic stages. In other words, the anti-adult immunity may be influenced by the host’s anti-fecundity immunity during filariasis. However, there is no demonstrable evidence before examining if anti-fecundity immunity exists inside hosts during filariasis. In the current investigation, we examined whether host antibodies (immunoglobulins) can access adult worms. The use of flow cytometry to study the binding of antibodies to different embryonic stages of female adults becomes a tool for us to understand the entry of these molecules into the uterine cavity; however, internalization of immunoglobulins into adult worms was a surprising phenomenon for us as the mechanism of such entry into uterine cavity is not currently known. Previously, the uptake of several biomolecules into worms through cuticles has been well described. Initially, the cuticular surface was believed to be impermeable to high molecular weight polar solutes (Rogers et al., 1974; Pappas, 1975). However, the hypodermal membrane of adult worms of Brugia pahangi has several infoldings that increase the absorptive area of the worm (Vincent et al., 1975). There is adequate evidence to believe that radioactively labeled D-glucose, L-leucine, adenosine, and nucleic acid precursors are taken up in physiologically significant amounts by a transcuticular route of adult Brugia pahangi (Chen and Howells, 1981; Howells and Chen, 1981). According to study by Sasisekhar et al., (2005), in Setaria digitata, a hypodermally located protein named as SXP/RAL2 was identified which was of host origin.

To begin with, we wanted to examine if any large molecule such as Con-A, with a molecular weight of 108 kDa, can enter the uterine cavity of worms. Con-A, a lectin molecule, has a binding affinity for mannose residues, which are ubiquitously present in various components of filarial nematodes (van Die and Cummings, 2017) . Due to its binding specificity to embryonic stages of female adult worms, we selected this molecule to use in our experiment for pulsing female worms at different time points. The idea was that if Con-A entered the uterine cavity of female worms, it would bind the embryonic stages. We showed the binding of ConA to different IU stages of S. digitata, indicating that the molecule has entered the uterine cavity and then bound to embryonic stages. This observation revealed that not only small molecules could access nematode worms, but big molecules could also access worms.

We used filarial-specific antibodies to demonstrate the entry of these immunoglobulins into the uterine cavity. The assumption is that if immunoglobulins can enter the uterine cavity of worms, they should bind to epitopes present in the IU stages after their internalization. We pulsed female S. digitata in media containing filarial-specific antibodies for 6 hrs. This incubation time was selected based on data of ConA internalization, where we found a time-dependent entry of the molecule into worms with the highest amount of binding to embryonic stages at 6 hrs of incubation. We showed a significant binding of human filarial antibodies as well as rabbit filarial-specific antibodies to IU stages of S. digitata, demonstrating the phenomenon of operation of anti-fecundity immunity inside adult-stage filarial parasites. Our results were supported by previous findings that intravascular Schistosomes take up host immunoglobulins as part of enteric digestion and by surface Fc-receptor mediated mechanisms involving transport and processing within organelles as ‘elongate bodies.’(Thors et al., 2006).

To understand the mechanism of internalization of immunoglobulins, we used an endocytic inhibitor, Latrunculin A (Hessien et al., 2023). Pre-incubation of adult filaria worms with Latrunculin A followed by pulsing with rabbit serum containing filarial-specific antibodies suggested a decreased binding of antibodies to IU stages as compared to worms without incubation with the endocytic inhibitor. This experiment clearly showed that the immunoglobulins have entered into the uterine cavity by endocytosis, and the entry is inhibited by Latrunculin A. The most significant illustration of anti-fecundity immunity operating inside the uterine cavity of adult filarial worms was the presence of bound bovine immunoglobulins in situ on the surface of embryogenic stages of S. digitata. This was demonstrated by harvesting intrauterine stages of worms and then probing with polyclonal anti-bovine IgG serum (raised in rabbit) or normal rabbit serum (as negative control). It is reasonable to assume that the presence of bovine Immunoglobulin in situ on the surface of microfilariae (R1) and eggs (R2 and R3) is due to the entry of antibodies through the cuticle and subsequent binding to IU stages. Although microfilariae in human blood circulation are found to have bound host IgG on sheath (Shenoy et al., 1996), no report has been made in the past on the presence of immunoglobulins in the developing stages in utero.

Instead of using adult filarial worms of humans, we used S. digitata in our investigation. As mentioned earlier, S. digitata is a parasitic nematode found in the peritoneal cavity of cattle. We used this parasite in our study due to three important reasons. First, the adult worms of human filarial parasites, such as Wuchereria bancrofti and Brugia malayi, are difficult to access as they dwell in the lymphatics of humans. Second, worm structure has a morphological similarity between S. digitata and W. bancrofti. The third and most important reason is, the comparative genomic analysis among S. digitata, W. bancrofti and B. malayi genomes revealed similarity in several genome sequences (Senanayake et al., 2020).

It is important to note that the exact entry route of these macromolecules, such as ConA and immunoglobulins, into female adult S.digitata is still controversial. Although we argue here that the entry could be through the cuticular surface of the worms (Due to previous evidence of entry of small molecules like D-glucose, Fructose, etc..), we have no demonstrable evidence to examine this. There are oral and anal pores of adult worms, and these macromolecules could be entering into worms through these routes. We anticipate that, after entering through the oral cavity, these molecules could enter into the digestive tract of the worms and afterward could diffuse into the uterine cavity. Although we could sufficiently demonstrate the host’s anti-fecundity immunity inside adult parasites, the functional significance of entry of immunoglobulins into the uterine cavity of S. digitata is currently not known to us. To comprehend the functional aspect, we can align our findings with previous studies. According to Devaney and Gillan et al. (2002), wild-type Balb/C mice had significantly fewer microfilariae of B. pahangi in their peritoneal cavity compared to IL4 −/- animals after subcutaneous injection of third-stage larvae. This difference may be due to compromised viability of embryonic stages of B. pahangi caused by IL4, potentially occurring after IL4 entered the uterine cavity of adult B. pahangi. Another possible functional significance may be that host effector immunity inside filarial parasites is a part of host-parasite interaction (Jo, 2019). There are prior instances of the formation of extracellular vesicles from different helminth parasites, and these vesicles can access the host cells for communication purposes and may be helpful for establishing infection inside the host. Parasitic trematodes, like Echinostoma Caproni and Fasciola hepatica, produce exosome-like vesicles that contain several important parasite-specific proteins, and these vesicles are found to be internalized by rat intestinal epithelial cells, most probably by the process of endocytosis (Siles-Lucas et al., 2017; Marcilla et al., 2012). We, therefore, assume that, as a part of host-parasite interaction, the host immunoglobulins could be internalized into the uterine cavity and bound to Intra-uterine stages.

5 Conclusions

The movement of host macromolecules and immune molecules into nematode parasites has not been reported in the literature so far. Unlike another immune effector mechanism against larvae and microfilariae of filarial parasites, which normally act ‘outside’ these parasites, we, for the first time, demonstrate the existence and operation of the host’s anti-fecundity immunity that works ‘inside’ the adult stage filarial parasites and this type of immunity could be one effector mechanism against large stage adult nematode parasites. However, it is crucial to investigate further the actual biological consequence of the operation of anti-fecundity inside nematode parasites.

Funding

None.

CRediT authorship contribution statement

Bikash Ranjan Sahu: Writing – original draft, Software, Methodology, Formal analysis, Data curation, Conceptualization. Nityananda Mandal: Writing – review & editing, Methodology, Data curation, Conceptualization. Ahmad O. Babalghith: Visualization, Formal analysis, Conceptualization. Noha E. Abdel-razik: . Abdullah F. Aldairi: Writing – review & editing, Formal analysis, Data curation, Conceptualization. Farkad Bantun: Formal analysis, Conceptualization. Raju K. Mandal: Visualization, Formal analysis, Conceptualization. Shafiul Haque: Visualization, Software, Resources, Project administration, Conceptualization. Aditya K. Panda: .