Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection
Parasites & Vectors volume 13, Article number: 51 (2020)
Different immune mechanisms are capable of killing developmental stages of filarial nematodes and these mechanisms are also likely to vary between the primary and a challenge infection. However, the lack of a detailed analysis of cytokine, chemokine and immunoglobulin levels in human loiasis is still evident. Therefore, detailed analysis of immune responses induced by the different developmental stages of Loa loa in immune-competent BALB/c mice will aid in the characterization of distinct immune responses that are important for the immunity against loiasis.
Different developmental stages of L. loa were obtained from human peripheral blood (microfilariae, MF), the transmitting vector, Chrysops (larval stage 3, L3) and infected immune-deficient BALB/cRAG2γc−/− mice (L4, L5, adult worms). Groups of wildtype BALB/c mice were then injected with the isolated stages and after 42 days post-infection (pi), systemic cytokine, chemokine and immunoglobulin levels were determined. These were then compared to L. loa-specific responses from in vitro re-stimulated splenocytes from individual mice. All parameters were determined using Luminex technology.
In a pilot study, BALB/c mice cleared the different life stages of L. loa within 42 days pi and systemic cytokine, chemokine and immunoglobulin levels were equal between infected and naive mice. Nevertheless, L. loa-specific re-stimulation of splenocytes from mice infected with L5, MF or adult worms led to induction of Th2, Th17 and chemokine secretion patterns.
This study shows that although host immunity remains comparable to naive mice, clearance of L. loa life-cycle development stages can induce immune cell memory leading to cytokine, chemokine and immunoglobulins secretion patterns which might contribute to immunity and protection against reinfection.
Loiasis is caused by the filarial nematode Loa loa that is endemic in equatorial rainforest regions of Central and West Africa. As with other filariae, humans are infected through a bite of an insect vector; here flies of the genus Chrysops which are transmitting infective third-stage larvae (L3) into the bite wound. L3 then migrate through subcutaneous tissues were they mature to adult worms that produce microfilariae (MF) which can be found in peripheral blood and other body fluids [1, 2]. Since adult worms migrate under the skin and the conjunctiva, L. loa is often referred to African eye worm . Although most infections remain asymptomatic, clinical symptoms can occur and are characterized by oedemas, Calabar swellings, purities and arthralgia [1, 3]. Moreover, high MF loads (> 30,000 MF/ml) are associated with severe encephalitis in individuals who were treated with ivermectin or diethylcarbamazine, drugs against onchocerciasis [4,5,6,7], another filarial disease. Thus, knowledge about the distribution of L. loa in regions were the African Programme for Onchocerciasis Control (APOC) is running is essential  and immune responses induced by the infection need to be taken into consideration to improve management of filarial-related adverse responses in man.
Interestingly, only a few human studies [9,10,11,12] or experiments with L. loa-infected mandrills or rhesus monkeys [13, 14], have analysed immunological responses during L. loa infection. In regard to immunoglobulins, it has been shown that IgG and IgE subclasses, especially non-specific polyclonal IgE and elevated levels of antigen-specific IgG4 are associated with L. loa infection [12, 13, 15, 16]. However, since all other immunoglobulin isotypes were shown to be important during other filarial infections [17,18,19], their role during loiasis remains uncertain. Knowledge of cytokine and chemokine responses based on a few studies using human peripheral blood mononuclear cells (PBMC), co-infection studies and experimental infections of mandrills or rhesus monkeys which suggest that Th1 and Th2 immune responses [9, 14] and eosinophil-associated cytokines  play a major role during L. loa infections. Nevertheless, a comprehensive analysis of cytokine, chemokine and immunoglobulin responses elicited throughout infection and the potential immunomodulatory capacity and mechanisms of L. loa remain unknown.
To decipher L. loa-specific immune responses in more detail, we performed an initial set of experiments that compared systemic and recall responses in groups of immune-competent BALB/c mice that had been exposed to different life stages of the filariae (Additional file 1: Figure S1). Whereas systemic cytokine, chemokine and immunoglobulin levels were comparable between L. loa-exposed and naive mice on 42 days post-infection (pi) (a time point where infection is cleared), we observed enhanced immunoglobulin, cytokine and chemokine levels in splenocytes from L. loa-exposed BALB/c mice upon re-stimulated with L. loa stage-specific antigen extracts. In summary, these findings show that although systemic host immunity was comparable to unexposed animals, developmental stages of L. loa trigger immune cells during an ongoing filarial infection leading to induction of recall immune responses that might be important for immunity against reinfections.
Collection, maintenance and dissection of Chrysops flies
Chrysops flies were captured in the Bakundu reserve forest in Meme Division (9°25′ E, 4°30′ N) in the South-West region of Cameroon. Flies were fed on a consenting L. loa positive individual with a microfilaria load above 10,000 MF/ml of blood. After a blood meal, flies were collected via aspiration in a dark net and then kept in separate 50-ml tubes which were filled a quarter with Plaster of Paris that formed a cement layer and helped to retain moisture. Engorged Chrysops were fed daily with 15% sucrose solution and kept at 23 °C for 2 weeks, the time required for the development of L3. After 14 days, flies were washed with distilled water containing 4% Tween20 (Sigma-Aldrich, St. Louis, USA) and then rinsed with distilled water in Petri dishes (Falcon, New York, USA). L3 were then isolated from the Chrysops in dissecting medium containing RPMI-1640 medium (Sigma-Aldrich) supplemented with a 2% antibiotic cocktail (pencillin-streptomycin-neomycin; Thermo Fisher Scientific, Schwerte, Germany) using a dissecting microscope (Motic, Wetzlar, Germany). The head, thorax and abdomen were separated and dissected in individual Petri dishes containing dissecting medium. L3 were allowed to migrate out of the various parts and washed 4 times in dissecting medium. L3 were then counted, pooled and either used for infection of mice or frozen at − 80 °C for antigen preparation.
Isolation of Loa loa microfilariae from human peripheral blood
Whole blood samples (4 ml) were collected from a donor infected with L. loa and MF were obtained using version of a previously described protocol [20, 21]. In brief, 2 ml of whole blood was layered onto 2 ml-modified Percoll gradient (Sigma-Aldrich) in a 15-ml tube (Falcon) and centrifuged at 2000× rpm for 20 min without brake using a bench-top centrifuge (Human Diagnostics, Wiesbaden, Germany). Using a Whatman® filter paper (pore size 5 µm) (Merck Millipore, Tullagreen, Ireland) placed in a filter paper holder, a dropper was used to discard the topmost part containing the serum. Then, the filter was mounted on another 15-ml tube and the whitish area containing the parasite was then filtered using a syringe (Terumo, Tokyo, Japan). The filter paper was then removed with a sterile pipette and placed in a Petri dish (Falcon) containing RPMI medium (Sigma-Aldrich) to aid migration of MF out of the paper into the medium. MF number and motility was determined using a dissecting microscope Leica M80 (Leica, Singapore, Republic of Singapore). MF were either frozen at − 80 °C for antigen preparation or used for infection of wildtype BALB/c mice.
Isolation of host-derived Loa loa worm stages from infected mice
Female and male BALB/c RAG2γc−/− mice were subcutaneously infected with 100 L3 in 100 µl RPMI-1640 (Sigma-Aldrich) that were isolated from Chrysops flies. To obtain L4, L5 and adult worms, mice were sacrificed and dissected 15 days, 1 and 3 months pi, respectively. To obtain parasite life stages, several organs (subcutaneous tissue, muscle tissue, peritoneal cavity, liver, lungs and heart) were placed into a Petri dish (Falcon) containing RPMI-1640 (Sigma-Aldrich) and numbers and motility was determined using a dissecting microscope Leica M80 (Leica). Obtained parasites were either frozen at − 80 °C for antigen preparation or used for infection of wildtype BALB/c mice.
Pre-clinical experimental studies with different Loa loa life-stages
Simultaneously, groups of female and male wildtype BALB/c mice were exposed to either: (i) a s.c. injection of 500 L3 in 100 µl RPMI-1640 medium isolated from Chrysops flies; (ii) 10,000 MF via the tail vein in 100 µl RPMI-1640 medium isolated from human peripheral blood , or (iii) 10 L4; (iv) 10 L5; and (v) 10 adult worms all isolated from infected-BALB/cRAG2γc−/− mice. Whereas L4 were injected s.c. in 100 µl of RPMI-medium, L5 and adult stages were implanted as previously described . In short, mice were anesthetized with ketamine (Ketaset, 70 mg/kg; Zoetis, Parsippany-Troy Hills Township, New Jersey, USA) and medetomidine (Domitor, 0.8 mg/kg; Zoetis), flanks were shaved, dosed with betadine and following a small incision, L5 or adult worms were implanted into the mice. The incised area was then sutured and a s.c. shot of penicillin (12.06 mg/ml) was administered based on mouse weight (i.e. 100 µl/20 g) at the back of the neck followed by 200 µl antiserdan to wake the mouse up.
Analysis of Loa loa-exposed wildtype BALB/c mice
Six weeks (42 days) pi, mice were sacrificed by exposing them to increased carbon dioxide (CO2) concentration. Then, the thoracic regions were opened, and cardiac blood was collected using a 1-ml insulin syringe (Terumo, Leuven, Belgium). Subsequently, blood was centrifuged at 2500× rpm for 10 min using a PRISMR centrifuge (Labnet, New Jersey, USA) and serum was collected and kept at − 80 °C for Luminex-based analysis of immunoglobulins, cytokines and chemokines. Additionally, spleens were isolated from individual mice for stage-specific re-stimulation assays.
Preparation of parasite antigen extract
Adult worms, L3, L4 and L5 were thawed and concentrated by centrifuging at 1500× rpm for 5 min using a bench-top centrifuge (Human Biochemica und Diagnostica GmbH, Wiesbaden, Germany). Parasites were then mechanically minced on ice in cold sterile endotoxin-free PBS (Sigma-Aldrich). Insoluble material was removed by centrifugation at 2000× rpm for 10 min at 4 °C. The resulting soluble parasite antigen extract was then filtered through a 0.22-µm pore size filter (Merck Millipore) and protein concentration was determined by a Bradford assay (Cytoskeleton, Denver, USA) according to manufacturer’s description. Aliquots were frozen at − 80 °C until required.
Re-stimulation of splenocytes
Isolated spleens from individual mice were crushed through a sieve (Corning, Durham, USA) and red blood cells were lysed for 5 min in the dark using lysing buffer (Sigma-Aldrich). Cell suspensions were then centrifuged for 5 min at 2000× rpm using a PRISMR centrifuge (Labnet) and cell numbers in the resulting pellet were counted using a cell counting chamber (VWR, Pennsylvania, USA). 1 × 106 splenocytes were plated/well into 48-well culture plates (Costar, Kennebunk, USA) and left either un-stimulated or re-stimulated with 100 µg/ml of stage-specific parasite extract in a total of 800 µl culture medium (RPMI-1640 containing 10% FCS and 0.4% beta-mecaptoethanol; Sigma-Aldrich) at 37 °C and 5% CO2 for 48 h (cytokines) and 72 h (immunoglobulins). Thereafter, culture supernatants were removed and frozen at − 20 °C until cytokine/chemokine/immunoglobulin levels were determined by Luminex technology.
Analysis of immunoglobulins, cytokines and chemokines levels using Luminex technology
Systemic immunoglobulin, cytokine and chemokine levels from individual mouse serum samples and supernatants of the splenocyte re-stimulation assays were determined using a ProcartaPlex Mouse Antibody Isotyping Panel 7 plex and a ProcartaPlex Mouse Cytokine/Chemokine Panel 1 26 plex (eBioscience, Frankfurt, Germany) according to the manufacturer’s instructions. The limits (upper limit of quantification/lower limit of quantification) of the cytokines and chemokines in pg/ml were as follows: eotaxin (2400/0.59), GM-CSF (10,400/2.54), CXCL1 (6400/1.56), IFN-γ (4250/1.04), TNF-α (12,800/3.13), IL-10 (9300/2.27), IL-12p70 (3900/3.81), IL-13 (11,000/2.69), IL-17A (5050/1.23), IL-18 (38,750/38), IL-1β (4800/1.17), IL-2 (6050/1.48), IL-22 (46,900/11), IL-23 (52,100/13), IL-27 (11,000/2.69), IL-4 (5350/1.31), IL-5 (9950/2.43), IL-6 (21,500/5.25), IL-9 (66,700/16), CXCL10 (1750/0.43), MCP-1 (31,200/7.62), MCP-3 (1300/1.27), MIP-1α (2000/0.49), MIP-1β (1338/1.31), MIP-2 (3100/3.03) and RANTES (3075/3.00). Results from the analysis were acquired using a MAGPIX Luminex system (Luminex Cooperation, Austin, USA) and analyzed with ProcartaPlex Analyst software 1.0 (eBioscience). Levels of immunoglobulins, cytokines and chemokines from the splenocytes which were re-stimulated with the different parasite antigen extracts were normalized by subtracting background levels of un-stimulated splenocytes. An overview of the study design is shown in Additional file 1: Figure S1.
Statistical analyses were performed using the software SPSS 22 (IBM, Armonk, NY, USA) and the PRISM 7 programme (GraphPad Software Inc., La Jolla, USA). Variables did not meet assumption to allow parametric analysis, therefore to compare more than two groups, a Kruskal-Wallis test (systemic immune responses) or Friedman test (cytokine, chemokine and immunoglobulin levels from re-stimulated splenocytes) was performed and, if significant, followed by a Dunn’s post-hoc multiple comparison test for a further comparison of the groups.
Comparable immunoglobulin levels in BALB/c upon parasite clearance
There is a growing body of evidence indicating that chronic helminth infections may influence the onset and outcome of further illnesses and disease such as allergies and tuberculosis. Due to the moderate pathologies associated with L. loa and Mansonella perstans infections, there have been few studies of the immune responses provoked by these filariae. To gain an impression as to whether L. loa imprint a long-lasting immune signature following clearance of infection, which might determine how the host responds to unrelated diseases at a later date, we designed an initial study using immunocompetent BALB/c mice (Additional file 1: Figure S1). In short, we exposed groups of mice to either MF, L3, L4, L5 or adult worms. Levels of immunoglobulins, cytokines and chemokines in the serum of individual mice were measured on day 42 pi. At this time point, no parasitic stages were observed in any group which supports our earlier in vivo studies using wildtype and cytokine-deficient BALB/c strains [24, 34]. Thus, we considered that sufficient time had passed so that retained infection profiles and memory responses could be evaluated. Interestingly, systemic total immunoglobulin patterns in the serum were not significantly regulated between naive mice and those exposed to the different L. loa life stages using a Kruskal-Wallis test followed by Dunn’s post-hoc multiple comparison test (Additional file 2: Figure S2). Nevertheless, exposure to adult stages appeared to reduce IgG1, IgG2b and IgM when compared to naive groups, albeit not significantly.
Levels of pro-inflammatory and Th1 cytokines (Additional file 3: Figure S3) as well as Th2 and Th17 cytokines (Additional file 4: Figure S4) were also comparable between naive and L. loa-exposed mice. Moreover, 10 chemokine parameters (Additional file 5: Figure S5) were also comparable between the groups using a Kruskal-Wallis test followed by Dunn’s post-hoc multiple comparison test. Albeit non-significant, some changes were seen between naive basal levels and adult stages, elevated IL-27 and IL-9 (Additional file 3: Figure S3h and Additional file 4: Figure S4d) and reduced CXCL-10, MIP-1β (Additional file 5: Figure S5b, g). Regarding L5, elevated IL-23, IL-6 and CXCL-1 (Additional file 3: Figure S3g, Additional file 4: Figure S4c, Additional file 5: Figure S5a) with reduced CXCL-10, MIP-1a, MIP-1b and RANTES (Additional file 5: Figure S5b, f, g, j) were detected.
Enhanced IgG2a, IgE and IgM secretion upon stage-specific re-stimulation with MF antigen extract
Next, we analysed stage-specific recall in splenocyte cultures from groups of BALB/c mice that had been exposed to different L. loa life stages. In short, splenocytes were isolated on day 42 pi and re-stimulated with the antigen extract from the parasitic stage that was originally used to infect the individual BALB/c mouse. After 72 h of re-stimulation, released immunoglobulin levels in the culture supernatants of spleen cells from naive and exposed BALB/c groups were determined using Luminex technology (bead-based multiplex assay) and levels were normalized by subtracting background levels from matched cultures without antigen. In contrast to the systemic responses, significant differences were detected between the different groups (Fig. 1). Whereas there were no significant differences in levels of IgA, IgG1, IgG2b and IgG3 (Fig. 1a–d) between the different infection scenarios, re-stimulation with MF lead to significantly enhanced IgG2a (Friedman test: F = 10.63, P = 0.0311; Dunn’s post-hoc test: P = 0.0466), IgE (Friedman test: F = 14.68, P = 0.0054; Dunn’s post-hoc test: P = 0.0026) and IgM (Friedman test: F = 19.73, P = 0.0006; Dunn’s post-hoc test: P = 0.0140 and P = 0.0009) in MF-exposed mice when compared to cultures from groups exposed to L4 and adult worms and responses to their specific antigens (Fig. 1e–g). Interestingly, no immunoglobulins were detected in cultures from L4-exposed mice when stimulated with L4 antigen extract. On an individual basis, spleen cultures from the L. loa-exposed groups showed enhanced levels of immunoglobulins upon specific antigen stimulation compared to matched cell cultures from naive mice (open symbols). For example, cultures from mice given adult worms produced elevated IgA and IgE levels (Fig. 1a, f). Moreover, there was strong IgG2b release from cultures stemming from L3- and MF-exposed mice (Fig. 1c). Indeed, apart from IgE, cultures from MF-exposed mice produced high levels of all Ig subtypes. These initial findings suggest that MF exposure induces a defined Ig profile that can be recalled when MF have been eliminated.
Reduced secretion of pro-inflammatory and Th1 cytokines in L3 and L4 antigen extract re-stimulated splenocytes
In addition to immunoglobulin levels, we also analysed cytokine release to parasitic extracts in cultures of splenocytes from L. loa-exposed BALB/c mice after 48 h. Interestingly, whereas secretion of IFN-γ, TNF-α, IL-1β and IL-23 were not significantly different between the groups there were differences between recall responses of naive and stage-specific cultures (Additional file 6: Figure S6). For example, MF elicited both IFN-γ and TNF-α (Additional file 7: Figure S7). This was also reflected in release of IL-27 and IL-18 (Fig. 2). Interestingly, L5 and adult stages also released higher amounts of IL-27 and IL-18 when compared to responses in cell cultures from naive mice (Fig. 2a, d). Moreover, we also observed that re-stimulation with L5 and adult worm antigen extracts lead to higher IL-18 (Fig. 2d) responses and significantly increased secretion of IL-27 (Friedman test: F = 17.41, P = 0.0016; Dunn’s post-hoc test: P = 0.0191, P = 0.0349 and P = 0.0466) (Fig. 2a) and IL-12p70 (Friedman test: F = 14.89, P = 0.0049; Dunn’s post-hoc test: P = 0.0466 and P = 0.0140) (Fig. 2b) when compared to recall responses by splenocyte cultures from L3- and L4-exposed BALB/c mice to their respective antigens. In addition, MF re-stimulation resulted in significantly enhanced IL-2 (Friedman test: F = 18.36, P = 0.0010; Dunn’s post-hoc test: P = 0.0009) (Fig. 2c) and IL-18 (Friedman test: F = 20.28, P = 0.0004; Dunn’s post-hoc test: P = 0.0037 and P = 0.0073) (Fig. 2d) secretion compared to L3 or L3 and L4 stage re-stimulation, respectively. Although the direct comparison of cytokine levels from the L. loa antigen extract re-stimulated splenocyte cultures is difficult due to limited sample size, these initial findings suggest that L3 and L4 stage larvae did not overtly provoke pro-inflammatory and/or Th1 immune responses, whereas infections with L5, adult worms and MF elicited recall immune responses.
Exposure to Loa loa adult worm antigen extracts leads to enhanced Th2 and Th17 recall responses to this life stage
Besides pro-inflammatory and Th1 secretion patterns, we analysed other T cell-based immune responses and noted that whereas cell cultures from naive or L4-exposed BALB/c groups failed to produce any of the eight measured cytokines, comparable cultures from other L. loa-exposed mice produced significant cytokine responses to their respective antigens (Fig. 3). Interestingly, only co-cultures of cells from mice exposed to adult worms released IL-5 (Fig. 3b) and IL-13 (Fig. 3e). This single instance shows an element of specific cell priming during exposure since overlapping structures, epitopes and molecules between adult- and L5-exposed animals should have also perhaps provoked IL-5 release in L5-specific co-cultures.
When comparing the groups, re-stimulated splenocytes with adult worm antigen extract secreted significantly more IL-5 (Friedman test: F = 1 7.6, P = 0.0015; Dunn’s post-hoc test: P = 0.0026 and P = 0.0102) (Fig. 3b), IL-6 (Friedman test: F = 12.37, P = 0.0148; Dunn’s post-hoc test: P = 0.0466) (Fig. 3c), IL-9 (Friedman test: F = 13.47, P = 0.0092; Dunn’s post-hoc test: P = 0.0349) (Fig. 3d) and IL-13 (Friedman test: F = 18.27, P = 0.0011; Dunn’s post-hoc test: P = 0.0140) (Fig. 3e) compared to L3- or L4-stimulated cells, respectively. In addition, L5 and MF antigen extract re-stimulation only led to significantly increased IL-9 secretion (Dunn’s post-hoc test: P = 0.0349 and P = 0.0191) (Fig. 3d). Interestingly, only cultures from L3-exposed mice and antigen extract significantly increased the release of IL-10 secretion (Friedman test: F = 13.34, P = 0.0097; Dunn’s post-hoc test: P = 0.0073) compared to L4 stage stimulation (Fig. 3f). Since L3 transition into L4, it will be interesting to determine in the future whether the absence of L4-specific responses is associated with the lack of proceeding L3-provoked responses. In regard to Th17 responses, re-stimulation with L5 and adult worm antigen extract also significantly increased the secretion of IL-17A (Friedman test: F = 16.53, P = 0.0024; Dunn’s post-hoc test: P = 0.0102 and P = 0.0026) (Fig. 3g) and IL-22 (Friedman test: F = 11.93, P = 0.0149; Dunn’s post-hoc test: P = 0.0191) (Fig. 3h) compared to the L4 stage group. In summary, as shown with the pro-inflammatory and Th1 immune responses, L5, MF and especially adult worms triggered Th2 and Th17 responses whereas L4 stage larvae did not induce remarkable Th immune responses.
Re-stimulation with antigen extracts from adult worms and MF induces distinct chemokine responses
Finally, we determined chemokine recall responses upon stage specific re-stimulation of splenocytes from L. loa-exposed BALB/c mice. Whereas, secretion of MIP-1α, MIP-1β, MCP-1, MCP-3, MIP-2, eotaxin and RANTES were comparable between the groups (Additional file 7: Figure S7), adult worm and MF antigen extracts triggered higher secretions of GM-CSF, CXCL1 and CXCL10 (Fig. 4). In detail, adult worm antigen extracts significantly induced GM-CSF secretion (Friedman test: F = 20.07, P = 0.0005; Dunn’s post-hoc test: P = 0.0026 and P = 0.0349) from adult worm-exposed lymphocytes when compared to cultures from L3- and L4-infected animals with their specific life stage (Fig. 4a). In association, cells from MF-exposed mice produced more GM-CSF (Dunn’s post-hoc test: P = 0.0191) (Fig. 4a) and CXCL10 (Friedman test: F = 14.18, P = 0.0067; Dunn’s post-hoc test: P=0.0349) (Fig. 4b) when compared to cultures from L3- and L4-exposed mice and respective antigens. Interestingly, similar to IL-10 secretion (Fig. 3f), re-stimulation with L3 antigen extract led to significant production of CXCL1 (Friedman test: F = 13.34, P = 0.0097; Dunn’s post-hoc test: P=0.0073) (Fig. 4c). Overall, these findings from the initial set of experiments show that upon clearance of infection, L. loa has provoked long-lasting host memory that can elicit distinct secretion patterns of cytokines, chemokines and immunoglobulins. These L. loa-specific responses might contribute to immunity and protection against reinfection or shape immune responses to other infections.
Although L. loa infects Mandrillus leucophaeus and can be maintained in splenectomised baboons [25, 26], research about L. loa-induced immunological mechanisms have been understudied and mainly concentrated on the analysis of unspecific antibody responses [15, 27, 28]. A major reason for the limited understanding of loiasis-associated immune responses and clinical picture [29, 30] is the lack of appropriate infection models. Besides the established rodent models of Brugia malayi and Onchocerca volvulus [23, 31,32,33], we recently showed that BALB/c with impaired IL-4, IL-5 and IL-13 signalling as well as a lymphopenic γc-deficient strain allow development of L. loa life stages [22, 34]. This was also reflected in our studies with the rodent Litomosoides sigmodontis model since in IL-4Rα/IL-5−/− BALB/c mice, worm burden and MF counts were significantly higher than in wildtype BALB/c control groups , suggesting that principally mice lacking Th2 responses provide a better environment for worm growth. However, these preclinical infection models have shown that host-adaptive immunity plays a crucial role for L. loa survival and fertility. Thus, to begin deciphering host immunity against L. loa, we analysed immune responses in immune-competent BALB/c mice upon exposure to different L. loa developmental stages. Previously, it was shown that L. loa cannot be maintained in wildtype BALB/c mice probably due to type-2 associated immunity [23, 36] that clears L. loa larvae after 1 week . Indeed, we did not isolate any L. loa developmental stages 42 days pi which was reflected in the relatively comparable levels of systemic cytokine, chemokine and immunoglobulins between the infected groups. In accordance with these findings, we recently showed that systemic regulatory immune cell frequencies (regulatory B cells) had returned to basal levels in humans who cleared Wuchereria bancrofti infection due to anti-filarial treatment compared to uninfected endemic normals . In association, some results did indicate a change in B cell activity since IgM and IgG levels in groups exposed to adult worms were lower than other groups which was accompanied by higher IL-27 (enhances germinal center B cell activity)  and IL-9 (IL-9R signalling in memory B cells regulates humoral recall responses) . Nevertheless, these findings confirm and support that L. loa infections cannot be established in immune-competent BALB/c mice . Interestingly, levels of permissiveness can be observed with other filarial, including the rodent-specific model L. sigmodontis. In that pre-clinical setting, mice infect all strains but are cleared in C57BL/6 mice after 40 days pi and in BALB/c mice, only a portion of infected mice become patent (MF release). The fact that adult worms can be produced in lymphopenic γc-deficient mice provides a much-needed platform for (i) obtaining the different life-stages to prepare antigen extracts; and (ii) elucidating cellular components involved in establishing infections per se. This model is equivalent to the RAG2IL-2Rγ−/− C57BL/6 mice that we have shown to allow fully infections of L. sigmodontis and moreover, these mice present higher worm burden and MF counts . All these upcoming models provide ways to show whether host immunity can establish filarial-specific memory to induce immune response upon L. loa reinfection or exposure to antigen leading to resistance or enhanced immune responses, respectively.
Interestingly, re-stimulation of splenocytes from MF-exposed BALB/c mice with MF antigen extract led to enhanced IgG2a, IgE and IgM secretion. Besides the association of IgE and active L. loa infection [12, 13, 15, 16], several studies about schistosomiasis revealed that antigen-specific IgE responses were associated with the duration of exposure  and resistance to reinfection [42,43,44]. Whereas re-stimulation with L4 antigen extract did not lead to significantly increased immunoglobulin, cytokine and chemokine secretion, antigen extracts from L5, adult worms and MF potently induced immune responses from splenocytes isolated from BALB/c mice that had cleared L. loa developmental stages, confirming previous studies suggesting an important role of Th1 and Th2 immune responses during L. loa infection [9, 14]. Since L5, adult worms and MF share an overwhelming majority of antigens also due to intrauterine MF in female worms, explains the overlapping induction of immune responses between these life stages. However, especially, adult worms induce distinct chemokines and especially Th2 and Th17 cytokines like IL-5, IL-9, IL-13 and IL-17A, which have been shown to play a crucial role in the host defence against filariae [17,18,19, 35, 45,46,47,48,49]. In addition, it has been shown that L. loa worm antigen extract strongly induced T cell proliferative responses in PBMC from individuals in villages with low L. loa transmission rates compared to PBMC from individuals in high transmission villages . These data suggest that distinct developmental stages modulate immune responses and contribute to memory recall responses that influence immunity against L. loa.
The reason for the insufficient immune responses of the L4 group might be due to the different amounts of worm material in terms of lower antigen load of the L4 larvae compared to the other stages, especially L5, adult worms and MF. In addition, L3 larvae might be coated in fly-host molecules and thus were seen as “non-self” leading to stronger immune responses compared to the L4 larvae. However, a general limitation of immunological studies is to equalise the parasite load and amount of antigen per se. For example, parasite load in humans vary strongly (e.g. microfilarial load) [17,18,19, 37, 47, 49] and results from the filarial mouse model L. sigmodontis are also based on different parasite loads when the natural infectious route via ticks is used [35, 40, 48]. Thus, comparisons of immune profiles in the mice groups following exposure to the different life stages are difficult to interpret and need cautious conclusions. Thus, further studies need to elucidate if L. loa L4 larvae can induce immune responses in the proposed mouse model.
Interestingly, re-stimulation of L3-exposed splenocytes with L3 antigen extract significantly induced IL-10 and CXCL1 secretion. However, IL-10 secretion was also induced in splenocytes upon L5, adult worm and MF antigen extract by tendency, confirming that chronic filarial infections are characterized by a dominate regulatory environment involving the secretion of IL-10 [17,18,19, 37]. In addition, several studies have shown that neutrophil activation play an important role for protective immunity against L3 [51,52,53,54,55], suggesting that the neutrophil-specific chemokine CXCL1 might be involved as well. Since L. loa developmental stages were difficult to obtain from mice or human volunteers, the resulting mouse infection studies performed here were restricted including the overall number of L. loa-exposed mice in each challenge group, recall responses in both infected and naive groups and measurements and thus comparisons of L. loa-specific immunoglobulin levels with total amounts. These initial data sets are also only based on one large comparative infection study but consider that these initial findings will become the precedent of such immune-modulatory research in loiasis. Furthermore, we could not perform cross-over stimulation studies like culturing splenocytes from one stage-specific exposed L. loa group with all the different L. loa developmental stage antigen extracts, nor were we able to analyse different time points upon parasite challenge to decipher how long the L. loa life stages can survive. Moreover, it was not possible to unravel if location of the parasite stage accompanied with antigen-drainage and immune cell migration influences splenocyte recall responses. Thus, further studies are required to optimize these initial L. loa in vitro cultures so that additional mouse infections with higher amounts of parasites and antigen extracts can be obtained. Such studies will evaluate in more detail the specificity, involvement and interactions of the studied immunoglobulins, chemokine and cytokines both systemically and at the site of infection.
Overall, this pilot study shows that infections with different developmental stages of L. loa were cleared within 42 days pi in immunocompetent BALB/c mice. Although no statistically significant findings were revealed in systemic immune parameters, re-stimulation of spleen lymphocytes with antigen extracts from each L. loa developmental life stage led to distinct immunoglobulin, cytokine and chemokine secretion patterns. Therefore, this pilot study highlights that the host immune system establish memory during an ongoing L. loa infection, leading to enhanced L. loa-specific recall responses in mice that cleared the infection. This mechanism might contribute to immunity and protection against reinfection.
Availability of data and materials
The data supporting the conclusions of this article are included within the article and its additional files. The raw datasets are available from the corresponding author upon reasonable request.
Padgett JJ, Jacobsen KH. Loiasis: African eye worm. Trans R Soc Trop Med Hyg. 2008;102:983–9.
CDC. 2015. Loiasis. US Centers for Disease Control and Prevention. https://www.cdc.gov/parasites/loiasis/. Accessed 23 Jan 2020.
Boussinesq M. Loiasis. Ann Trop Med Parasitol. 2006;100:715–31.
Chippaux JP, Boussinesq M, Gardon J, Gardon-Wendel N, Ernould JC. Severe adverse reaction risks during mass treatment with ivermectin in loiasis-endemic areas. Parasitol Today. 1996;12:448–50.
Gardon J, Gardon-Wendel N, Demanga-Ngangue, Kamgno K, Chippaux JP, Boussinesq M. Serious reactions after mass treatment of onchocerciasis with ivermectin in an area endemic for Loa loa infection. Lancet. 1997;350:18–22.
Boussinesq M, Gardon J, Gardon-Wendel N, Kamgno J, Ngoumou P, Chippaux JP. Three probable cases of Loa loa encephalopathy following ivermectin treatment for onchocerciasis. Am J Trop Med Hyg. 1998;58:461–9.
Lukiana T, Mandina M, Situakibanza NH, Mbula MM, Lepira BF, Odio WT, et al. A possible case of spontaneous Loa loa encephalopathy associated with a glomerulopathy. Filaria J. 2006;5:6.
Thomson MC, Obsomer V, Kamgno J, Gardon J, Wanji S, Takougang I, et al. Mapping the distribution of Loa loa in Cameroon in support of the African Programme for Onchocerciasis Control. Filaria J. 2004;3:7.
Baize S, Wahl G, Soboslay PT, Egwang TG, Georges AJ. T helper responsiveness in human Loa loa infection; defective specific proliferation and cytokine production by CD4+ T cells from microfilaraemic subjects compared with amicrofilaraemics. Clin Exp Immunol. 1997;108:272–8.
Herrick JA, Metenou S, Makiya SM, Taylar-Williams CA, Law MA, Klion AD, Nutman TB. Eosinophil-associated processes underlie differences in clinical presentation of loiasis between temporary residents and those indigenous to Loa-endemic areas. Clin Infect Dis. 2015;60:55–63.
Chatterjee S, Clark CE, Lugli E, Roederer M, Nutman TB. Filarial infection modulates the immune response to Mycobacterium tuberculosis through expansion of CD4+ IL-4 memory T cells. J Immunol. 2015;194:2706–14.
Njambe Priso GD, Lissom A, Ngu LN, Nji NN, Tchadji JC, Tchouangueu TF, et al. Filaria specific antibody response profiling in plasma from anti-retroviral naïve Loa loa microfilaraemic HIV-1 infected people. BMC Infect Dis. 2018;18:160.
Pinder M, Everaere S, Roelants GE. Loa loa: immunological responses during experimental infections in mandrills (Mandrillus sphinx). Exp Parasitol. 1994;79:126–36.
Leroy E, Baize S, Wahl G, Egwang TG, Georges AJ. Experimental infection of a nonhuman primate with Loa loa induces transient strong immune activation followed by peripheral unresponsiveness of helper T cells. Infect Immun. 1997;65:1876–82.
Akue JP, Egwang TG, Devaney E. High levels of parasite-specific IgG4 in the absence of microfilaremia in Loa loa infection. Trop Med Parasitol. 1994;45:246–8.
Akue JP, Hommel M, Devaney E. Markers of Loa loa infection in permanent residents of a loiasis endemic area of Gabon. Trans R Soc Trop Med Hyg. 1996;90:115–8.
Hoerauf A, Satoguina J, Saeftel M, Specht S. Immunomodulation by filarial nematodes. Parasite Immunol. 2005;27:417–29.
Adjobimey T, Hoerauf A. Induction of immunoglobulin G4 in human filariasis: an indicator of immunoregulation. Ann Trop Med Parasitol. 2010;104:455–64.
Babu S, Nutman TB. Immunology of lymphatic filariasis. Parasite Immunol. 2014;36:338–46.
Van Hoegaerden M, Ivanoff B. A rapid, simple method for isolation of viable microfilariae. Am J Trop Med Hyg. 1986;35:148–51.
Njouendou AJ, Fombad FF, O’Neill M, Zofou D, Nutting C, Ndongmo PC, et al. Heterogeneity in the in vitro susceptibility of Loa loa microfilariae to drugs commonly used in parasitological infections. Parasit Vectors. 2018;11:223.
Pionnier NP, Sjoberg H, Chunda VC, Fombad FF, Chounna PW, Njouendou AJ, et al. Mouse models of Loa loa. Nat Commun. 2019;10:1429.
Halliday A, Guimaraes AF, Tyrer HE, Metuge HM, Patrick CN, Arnaud KO. A murine macrofilaricide pre-clinical screening model for onchocerciasis and lymphatic filariasis. Parasit Vectors. 2014;7:472.
Wanji S, Tendongfor N, Vuong PN, Enyong P, Bain O. The migration and localization of Loa loa infective and fourth-stage larvae in normal and immunosuppressed rodents. Ann Trop Med Parasitol. 2002;96:823–30.
Duke BO. Studies on loiasis in monkeys. II. The population dynamics of the microfilariae of Loa in experimentally infected drills (Mandrillus leucophaeus). Ann Trop Med Parasitol. 1960;54:15–31.
Orihel TC, Moore PJ. Loa loa: experimental infection in two species of African primates. Am J Trop Med Hyg. 1975;24:606–9.
Churchill DR, Morris C, Fakoya A, Wright SG, Davidson RN. Clinical and laboratory features of patients with loiasis (Loa loa filariasis) in the U.K. J Infect. 1996;33:103–9.
Egwang TG, Dupont A, Leclerc A, Akué JP, Pinder M. Differential recognition of Loa loa antigens by sera of human subjects from a loiasis endemic zone. Am J Trop Med Hyg. 1989;41:664–73.
Nutman TB, Reese W, Poindexter RW, Ottesen EA. Immunologic correlates of the hyperresponsive syndrome of loiasis. J Infect Dis. 1988;157:544–50.
Klion AD, Massougbodji A, Sadeler BC, Ottesen EA, Nutman TB. Loiasis in endemic and nonendemic populations: immunologically mediated differences in clinical presentation. J Infect Dis. 1991;163:1318–25.
McGarry HF, Plant LD, Taylor MJ. Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway. Filaria J. 2005;4:4.
Sharma R, Al Jayoussi G, Tyrer HE, Gamble J, Hayward L, Guimaraes AF, et al. Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis. Sci Rep. 2016;6:23458.
Sjoberg HT, Pionnier N, Aljayyoussi G, Metuge HM, Njouendou AJ, Chunda VC, et al. Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models. PLoS Negl Trop Dis. 2019;13:e0006356.
Tendongfor N, Wanji S, Ngwa JC, Esum ME, Specht S, Enyong P, et al. The human parasite Loa loa in cytokine and cytokine receptor gene knock out BALB/c mice: survival, development and localization. Parasit Vectors. 2012;5:43.
Ritter M, Tamadaho RS, Feid J, Vogel W, Wiszniewsky K, Perner S, et al. IL-4/5 signalling plays an important role during Litomosoides sigmodontis infection, influencing both immune system regulation and tissue pathology in the thoracic cavity. Int J Parasitol. 2017;47:951–60.
Turner JD, Pionnier N, Furlong-Silva J, Sjoberg H, Cross S, Halliday A, et al. Interleukin-4 activated macrophages mediate immunity to filarial helminth infection by sustaining CCR3-dependent eosinophilia. PLoS Pathog. 2018;14:e1006949.
Ritter M, Osei-Mensah J, Debrah LB, Kwarteng A, Mubarik Y, Debrah AY, et al. Wuchereria bancrofti-infected individuals harbor distinct IL-10-producing regulatory B and T cell subsets which are affected by anti-filarial treatment. PLoS Negl Trop Dis. 2019;13:e0007436.
Vijayan D, Mohd Redzwan N, Avery DT, Wirasinha RC, Brink R, Walters G, et al. Directly enhances germinal center B cell activity and potentiates lupus in sanroque mice. J Immunol. 2016;197:3008–17.
Takatsuka S, Yamada H, Haniuda K, Saruwatari H, Ichihashi M, Renauld JC, et al. IL-9 receptor signaling in memory B cells regulates humoral recall responses. Nat Immunol. 2018;19:1025–34.
Layland LE, Ajendra J, Ritter M, Wiszniewsky A, Hoerauf A, Hübner MP. Development of patent Litomosoides sigmodontis infections in semi-susceptible C57BL/6 mice in the absence of adaptive immune responses. Parasit Vectors. 2015;8:396.
Vereecken K, Naus CW, Polman K, Scott JT, Diop M, Gryseels B, et al. Associations between specific antibody responses and resistance to reinfection in a Senegalese population recently exposed to Schistosoma mansoni. Trop Med Int Health. 2007;12:431–44.
Jiz M, Friedman JF, Leenstra T, Jarilla B, Pablo A, Langdon G, et al. Immunoglobulin E (IgE) responses to paramyosin predict resistance to reinfection with Schistosoma japonicum and are attenuated by IgG4. Infect Immun. 2009;77:2051–8.
Satti MZ, Lind P, Vennervald BJ, Sulaiman SM, Daffalla AA, Ghalib HW. Specific immunoglobulin measurements related to exposure and resistance to Schistosoma mansoni infection in Sudanese canal cleaners. Clin Exp Immunol. 1996;106:45–54.
Hagan P, Blumenthal UJ, Dunn D, Simpson AJ, Wilkins HA. Human IgE, IgG4 and resistance to reinfection with Schistosoma haematobium. Nature. 1991;349:243–5.
Martin C, Le Goff L, Ungeheuer MN, Vuong PN, Bain O. Drastic reduction of a filarial infection in eosinophilic interleukin-5 transgenic mice. Infect Immun. 2000;68:3651–6.
Anuradha R, George PJ, Hanna LE, Chandrasekaran V, Kumaran P, Nutman TB, et al. IL-4-, TGF-β-, and IL-1-dependent expansion of parasite antigen-specific Th9 cells is associated with clinical pathology in human lymphatic filariasis. J Immunol. 2013;191:2466–73.
Ritter M, Ndongmo WPC, Njouendou AJ, Nghochuzie NN, Nchang LC, Tayong DB, et al. Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses. PLoS Negl Trop Dis. 2018;12:e0006184.
Ritter M, Krupp V, Wiszniewsky K, Wiszniewsky A, Katawa G, Tamadaho RSE, et al. Absence of IL-17A in Litomosoides sigmodontis-infected mice influences worm development and drives elevated filarial-specific IFN-γ. Parasitol Res. 2018;117:2665–75.
Katawa G, Layland LE, Debrah AY, von Horn C, Batsa L, Kwarteng A, et al. Hyperreactive onchocerciasis is characterized by a combination of Th17-Th2 immune responses and reduced regulatory T cells. PLoS Negl Trop Dis. 2015;9:e3414.
Akue JP, Devaney E. Transmission intensity affects both antigen-specific and nonspecific T-cell proliferative responses in Loa loa infection. Infect Immun. 2002;70:1475–80.
Evans H, Killoran KE, Mitre BK, Morris CP, Kim SY, Mitre E. Ten weeks of infection with a tissue-invasive helminth protects against local immune complex-mediated inflammation, but not cutaneous type I hypersensitivity, in previously sensitized mice. J Immunol. 2015;195:2973–84.
Muhsin M, Ajendra J, Gentil K, Berbudi A, Neumann AL, Klaas L, et al. IL-6 is required for protective immune responses against early filarial infection. Int J Parasitol. 2018;48:925–35.
Karadjian G, Fercoq F, Pionnier N, Vallarino-Lhermitte N, Lefoulon E, Nieguitsila A, et al. Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung. PLoS Negl Trop Dis. 2017;11:e0005596.
Ajendra J, Specht S, Ziewer S, Schiefer A, Pfarr K, Parčina M, et al. NOD2 dependent neutrophil recruitment is required for early protective immune responses against infectious Litomosoides sigmodontis L3 larvae. Sci Rep. 2016;6:39648.
Pionnier N, Brotin E, Karadjian G, Hemon P, Gaudin-Nomé F, Vallarino-Lhermitte N, et al. Neutropenic mice provide insight into the role of skin-infiltrating neutrophils in the host protective immunity against filarial infective larvae. PLoS Negl Trop Dis. 2016;10:e0004605.
The authors would like to thank the Loa loa microfilaremic volunteers who took part in this study.
This work was funded by the German Research Foundation (DFG) within the “African-German Cooperation Projects in Infectiology” (RI 3036/1-1). In addition, SW and AH are financially supported by the Federal Ministry of Education and Research (BMBF; initiative Research Networks for Health Innovations in sub-Saharan Africa: TAKeOFF). AH and LEL are members of the Excellence Cluster Immunosensation (DFG, EXC 1023) and the German Center of Infectious Disease (DZIF).
Ethics approval and consent to participate
Animal husbandry and experimental investigations were carried out in strict accordance with international guidelines of rearing animals and using them in medical research under the official authorisation of the Ministry of Scientific Research in Cameroon (Research permit No. 028/MINRESI/B00/C00//C10/C12/2007). Enrolment into the study was done on a strictly voluntary basis and the objectives, risks and benefits of the study were explained in detail to all individuals. Verbal consent to perform the study in the villages was obtained from community leaders, i.e. chiefs and elders of the selected communities and written informed consent was obtained from all participants. The study was undertaken according to the principles of the Helsinki Declaration of 1975 (as revised 2008). Wildytpe BALB/c and BALB/c RAG2γc−/− mice originated from the Liverpool School of Tropical Medicine and bred at the Research Foundation in Tropical Disease and Environment (REFOTDE) in Buea, Cameroon under specific-pathogen-free (SPF) conditions in accordance with Cameroonian animal protection laws (028/MINRESI/B00/C00//C10/C12/2007).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Overview of the study design.
Comparable systemic immunoglobulin levels. Data show mean fluorescence intensity (MFI) of naive (n = 10) and BALB/c mice exposed to L3 (n = 5), L4 (n = 5), L5 (n = 5), adult worms (n = 5) or MF (n = 5).
Comparable systemic pro-inflammatory and Th1 cytokine levels. Data show concentration (pg/ml) of the different cytokines from groups of naive (n = 10) and BALB/c mice exposed to L3 (n = 5), L4 (n = 5), L5 (n = 5), adult worms (n = 5) or MF (n = 5).
Comparable systemic regulatory, Th2 and Th17 cytokine levels. Data show concentration (pg/ml) of the different cytokines from groups of naive (n = 10) and BALB/c mice exposed to L3 (n = 5), L4 (n = 5), L5 (n = 5), adult worms (n = 5) or MF (n = 5).
Comparable systemic chemokine levels. Data show concentration (pg/ml) of the different chemokines from groups of naive (n = 10) and BALB/c mice exposed to L3 (n = 5), L4 (n = 5), L5 (n = 5), adult worms (n = 5) or MF (n = 5).
Comparable IFN-γ, TNF-α, IL-1β and IL-23 levels of antigen extract re-stimulated splenocytes. Data show concentration (pg/ml) of the different cytokines from re-stimulated splenocytes of infected BALB/c mice (n = 6 per life stage/parasite antigen extract) and naive BALB/c mice (n = 3 per parasite antigen extract).
Comparable chemokine levels of antigen extract re-stimulated splenocytes. Data show concentration (pg/ml) of the different chemokines from re-stimulated splenocytes of infected BALB/c mice (n = 6 per life stage/parasite antigen extract) and naive BALB/c mice (n = 3 per parasite antigen extract).
About this article
Cite this article
Chunda, V.C., Ritter, M., Bate, A. et al. Comparison of immune responses to Loa loa stage-specific antigen extracts in Loa loa-exposed BALB/c mice upon clearance of infection. Parasites Vectors 13, 51 (2020). https://doi.org/10.1186/s13071-020-3921-x