Characterization of a novel microfilarial antigen for diagnosis of Wuchereria bancrofti infections.

BACKGROUND: Lymphatic filariasis (LF) is a neglected tropical disease caused by the filarial nematodes Wuchereria bancrofti, Brugia malayi and Brugia timori. The Global Program to Eliminate LF uses mass drug administration (MDA) of anti-filarial drugs that clear microfilariae (Mf) from blood to interrupt transmission by mosquitos. New diagnostic tools are needed to assess the impact of MDA on bancroftian filariasis, because available serologic tests can remain positive after successful treatment. METHODOLOGY/PRINCIPAL FINDINGS: We identified Wb-bhp-1, which encodes a W. bancrofti homologue of BmR1, the B. malayi protein used in the Brugia Rapid antibody test for brugian filariasis. Wb-bhp-1 has a single exon that encodes a 16.3 kD protein (Wb-Bhp-1) with 45% amino acid identity to BmR1. Immunohistology shows that anti-Wb-Bhp-1 antibodies primarily bind to Mf. Plasma from 124 of 224 (55%) microfilaremic individuals had IgG4 antibodies to Wb-Bhp-1 by ELISA. Serologic reactivity to Wb-Bhp-1 varied widely with samples from different regions (sensitivity range 32-92%), with 77% sensitivity for 116 samples collected from microfilaremic individuals outside of sub-Saharan Africa. This variable sensitivity highlights the importance of validating new diagnostic tests for parasitic diseases with samples from different geographical regions. Individuals with higher Mf counts were more likely to have anti-Wb-Bhp-1 antibodies. Cross-reactivity was observed with a minority of plasma samples from people with onchocerciasis (17%) or loiasis (10%). We also identified, cloned and characterized BmR1 homologues from O. volvulus and L. loa that have 41% and 38% identity to BmR1, respectively. However, antibody assays with these antigens were not sensitive for onchocerciasis or loiasis. CONCLUSIONS: Wb-Bhp-1 is a novel antigen that is useful for serologic diagnosis of bancroftian filariasis. Additional studies are needed to assess the value of this antigen for monitoring the success of filariasis elimination programs.

Genome editing as control tool for filarial infections.

Human filarial infections are vector-borne nematode infections, which include lymphatic filariasis, onchocerciasis, loiasis, and mansonella filariasis. With a high prevalence in developing countries, filarial infections are responsible for some of the most debilitating morbidities and a vicious cycle of poverty and disease. Global initiatives set to eradicate these infections include community mass treatments, vector control, provision of care for morbidity, and search for vaccines. However, there are growing challenges associated with mass treatments, vector control, and antifilarial vaccine development. With the emergence of genome editing tools and successful applications in other infectious diseases, the integration of genetic editing techniques in future control strategies for filarial infections would offer the best option for eliminating filarial infections. In this review, we briefly discuss the mechanisms of the three main genetic editing techniques and explore the potential applications of these powerful tools to control filarial infections.

Diethylcarbamazine activates TRP channels including TRP-2 in filaria, Brugia malayi.

Diethylcarbamazine is an important classic drug used for prevention and treatment of lymphatic filariasis and loiasis, diseases caused by filarial nematodes. Despite many studies, its site of action has not been established. Until now, the consensus has been that diethylcarbamazine works by activating host immune systems, not by a direct action on the parasites. Here we show that low concentrations of diethylcarbamazine have direct and rapid (<30 s) temporary spastic paralyzing effects on the parasites that lasts around 4 h, which is produced by diethylcarbamazine opening TRP channels in muscle of Brugia malayi involving TRP-2 (TRPC-like channel subunits). GON-2 and CED-11, TRPM-like channel subunits, also contributed to diethylcarbamazine responses. Opening of these TRP channels produces contraction and subsequent activation of calcium-dependent SLO-1K channels. Recovery from the temporary paralysis is consistent with inactivation of TRP channels. Our observations elucidate mechanisms for the rapid onset and short-lasting therapeutic actions of diethylcarbamazine.

Eosinophil-Mediated Immune Control of Adult Filarial Nematode Infection Can Proceed in the Absence of IL-4 Receptor Signaling.

Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R-dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Rα-deficient (IL-4Rα-/-) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Rα-/- mice. By treating IL-4Rα-/- mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Rα-/- mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL-13-dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Rα signaling and defined that IL-4Rα-/-/IL-5-/- double-knockout mice displayed significant eosinophil deficiency compared with IL-4Rα-/- mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R-dependent or IL-4R-independent/CCR3/IL-5-dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R-independent/IL-5- and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency.

Chronic infection with a tissue-invasive helminth attenuates sublethal anaphylaxis and reduces granularity and number of mast cells.

BACKGROUND: Immunoglobulin E (IgE)-mediated anaphylaxis is a potentially fatal condition in which allergy effector cells rapidly discharge pre-formed inflammatory mediators. Treatments that address the immune component of allergic anaphylaxis are inadequate. Helminths have been previously shown to suppress effector cell function; however, their ability to treat pre-existing allergy remains unclear. OBJECTIVE: To evaluate the ability of chronic helminth infection to protect against anaphylaxis in previously sensitized mice. METHODS: A sublethal model of anaphylaxis was used, in which BALB/c mice were sensitized by three intraperitoneal (i.p.) injections of OVA/alum. Temperature drop was then monitored after systemic OVA challenge in uninfected mice and in mice infected chronically with Litomosoides sigmodontis, a tissue-invasive filarial nematode. RESULTS: Litomosoides sigmodontis-infected mice exhibited significantly lower serum levels of mMCP-1 and were less hypothermic at 30-minute post-challenge compared to uninfected OVA-challenged controls. Characterization of anaphylaxis revealed that FcԑR1 and mast cells were required for hypothermia and elevated serum mMCP-1. OVA-IgE and OVA-IgG1 serum levels were not significantly altered by L sigmodontis infection, and experiments with IL-10-/- mice demonstrated that IL-10 was not required for protection against anaphylaxis. However, peritoneal mast cell numbers were significantly lower in infected mice, and those that were present exhibited decreased granularity by flow cytometry and marked depletion of intracytoplasmic granules by light microscopy. Mast cells from infected mice had lower expression of the activation markers CD200R and CD63 and contained significantly lower basal stores of histamine. CONCLUSIONS: Chronic L sigmodontis infection protects against anaphylaxis, likely due to reduction in mast cell numbers and depletion of pre-formed inflammatory mediators in remaining mast cells.

Comparative analysis of small RNAs released by the filarial nematode Litomosoides sigmodontis in vitro and in vivo.

BACKGROUND: The release of small non-coding RNAs (sRNAs) has been reported in parasitic nematodes, trematodes and cestodes of medical and veterinary importance. However, little is known regarding the diversity and composition of sRNAs released by different lifecycle stages and the portion of sRNAs that persist in host tissues during filarial infection. This information is relevant to understanding potential roles of sRNAs in parasite-to-host communication, as well as to inform on the location within the host and time point at which they can be detected. METHODOLOGY AND PRINCIPAL FINDINGS: We have used small RNA (sRNA) sequencing analysis to identify sRNAs in replicate samples of the excretory-secretory (ES) products of developmental stages of the filarial nematode Litomosoides sigmodontis in vitro and compare this to the parasite-derived sRNA detected in host tissues. We show that all L. sigmodontis developmental stages release RNAs in vitro, including ribosomal RNA fragments, 5'-derived tRNA fragments (5'-tRFs) and, to a lesser extent, microRNAs (miRNAs). The gravid adult females (gAF) produce the largest diversity and abundance of miRNAs in the ES compared to the adult males or microfilariae. Analysis of sRNAs detected in serum and macrophages from infected animals reveals that parasite miRNAs are preferentially detected in vivo, compared to their low levels in the ES products, and identifies miR-92-3p and miR-71-5p as L. sigmodontis miRNAs that are stably detected in host cells in vivo. CONCLUSIONS: Our results suggest that gravid adult female worms secrete the largest diversity of extracellular sRNAs compared to adult males or microfilariae. We further show differences in the parasite sRNA biotype distribution detected in vitro versus in vivo. We identify macrophages as one reservoir for parasite sRNA during infection, and confirm the presence of parasite miRNAs and tRNAs in host serum during patent infection.

Microfilariae infestation of goliath frogs (Conraua goliath) from Cameroon.

The goliath frog (Conraua goliath) is endemic to Equatorial Guinea and Cameroon. It is an endangered species but little information is known about its parasites. To understand the impact of blood parasites on this species, we microscopically examined blood smears from 78 goliath frogs in February and November 2016 (dry and wet seasons) from six localities in Littoral Region of Cameroon, and we sequenced mitochondrial DNA from positive samples. Microfilariae were found in 33/78 (42.3%) goliath frogs at six locations. No other haemoparasite species was detected. Morphological characteristics of microfilariae were also described, and specimens from each frog species were similar. DNA sequencing data from the mitochondrial Cytochrome Oxidases sub unit I (COI) gene revealed a close relationship with Icosiella neglecta, a microfilaria documented in other European, Asian, and African frogs. However, sequences were sufficiently genetically distant (0.118) that they may define a new species of Icosiella. The infection burden of microfilariae varied by site, with season (65% in dry season to 23% in rainy season), and by sex, (male frogs had significantly higher parasite burdens than females (p < 0.0001)). However, this may have been confounded by size as the microfilaria intensity increased with frog weight (p < 0.0001), and males were larger than females. Microfilaria infection intensity varied from 1 to 120 per 50 µl of blood. Microfilaria induced a significant increase (p < 0.05) in the number of white blood cells (WBC) counted compared to uninfected frogs, but there was no statistically significant variation in red blood cell (RBC) count, plasma cholesterol level (p = 0.210) or plasma glucose level (p = 0.100).

Exploring the homolog of a novel proinflammatory microfilarial sheath protein (MfP) of Wuchereria bancrofti in the adult-stage bovine filarial parasite Setaria cervi.

A novel microfilarial sheath protein (MfP) of the human filarial parasite Wuchereria bancrofti and its proinflammatory activity on host macrophages were identified recently. MfP is a homolog of the nematode bestrophin-9 superfamily that acts as a ligand of macrophage Toll-like receptor 4 (TLR4) to induce inflammation through NF-κB activation. Therefore, the presence and functional implication of this novel protein in adult-stage parasites were open questions to answer. In this study, the bovine filarial parasite Setaria cervi was used to simulate adult W. bancrofti. We detected the presence of MfP in adult-stage S. cervi through clear immunological cross-reactivity and immunolocalization employing an anti-MfP antibody developed in mice. Therefore, our findings put forward S. cervi as a cost-effective source of immunodominant filarial antigen MfP to simulate its future utilization in the immunotherapeutic intervention of lymphatic filariasis.

Exploring genetic variation in haplotypes of the filariasis vector Culex quinquefasciatus (Diptera: Culicidae) through DNA barcoding.

Culex quinquefasciatus (Diptera: Culicidae) is a vector of many pathogens and parasites of humans, as well as domestic and wild animals. In urban and semi-urban Asian countries, Cx. quinquefasciatus is a main vector of nematodes causing lymphatic filariasis. In the African region, it vectors the Rift Valley fever virus, while in the USA it transmits West Nile, St. Louis encephalitis and Western equine encephalitis virus. In this study, DNA barcoding was used to explore the genetic variation of Cx. quinquefasciatus populations from 88 geographical regions. We presented a comprehensive approach analyzing the effectiveness of two gene markers, i.e. CO1 and 16S rRNA. The high threshold genetic divergence of CO1 (0.47%) gene was reported as an ideal marker for molecular identification of this mosquito vector. Furthermore, null substitutions were lower in CO1 if compared to 16S rRNA, which influenced its differentiating potential among Indian haplotypes. NJ tree was well supported with high branch values for CO1 gene than 16S rRNA, indicating ideal genetic differentiation among haplotypes. TCS haplotype network revealed 14 distinct clusters. The intra- and inter-population polymorphism were calculated among the global and Indian Cx. quinquefasciatus lineages. The genetic diversity index Tajima' D showed negative values for all the 4 intra-population clusters (G2-4, G10). Fu's FS showed negative value for G10 cluster, which was significant and indicated recent population expansion. However, the G2-G4 (i.e. Indian lineages) had positive values, suggesting a bottleneck effect. Overall, our research firstly shed light on the genetic differences among the haplotypes of Cx. quinquefasciatus species complex, adding basic knowledge to the molecular ecology of this important mosquito vector.

Familial Aggregation and Heritability of Wuchereria bancrofti Infection.

BACKGROUND: The familial recurrence risk of lymphatic filariasis (LF) is unknown. This case study aimed to evaluate the familial susceptibility to infection with Wuchereria bancrofti and to microfilaremia in a village of the Republic of Congo. METHODS: The heritability and intrafamilial correlation coefficients were assessed for both W. bancrofti infection and microfilaremia by controlling for individual risk factors, environmental influence, and household effects. RESULTS: Pedigree charts were constructed for 829 individuals, including 143 individuals with a diagnosis of W. bancrofti circulating filarial antigens (CFAs) and 44 who also had microfilariae (MF). There was no intrafamilial correlation regarding CFA levels. However, the presence of MF (ρ = 0.45) and microfilarial density (ρ = 0.44) were significantly correlated among parent-offspring pairs. Heritability estimates for CFA positivity and intensity were 0.23 and 0.18, respectively. Heritability estimates were high for microfilarial positivity (h(2) = 0.74) and microfilarial density traits (h(2) = 0.81). CONCLUSIONS: Our study suggests that the acquisition of LF is mainly driven by environmental factors and habits and that genetic factors are moderately involved in the regulation of infection. By contrast, genetic factors play a major role in both the presence and intensity of microfilaremia.

Litomosoides sigmodontis induces TGF-ß receptor responsive, IL-10-producing T cells that suppress bystander T-cell proliferation in mice.

Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth-specific but also nonhelminth-specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T-cell suppression. When OT-II T cells specific for the third-party antigen ovalbumin are transferred into helminth-infected mice, these cells respond to antigen-specific stimulation with reduced proliferation compared to activation within non-infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two-staged process. Parasite products first engage the TGF-ß receptor on host-derived T cells that are central to suppression. In a second step, host-derived T cells produce IL-10 and subsequently suppress the adoptively transferred OT-II T cells. Terminal suppression was IL-10-dependant but independent of intrinsic TGF-ß receptor- or PD-1-mediated signaling in the suppressed OT-II T cells. Blockade of the same key suppression mediators, i.e. TGF-ß- and IL-10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis-infected mice.

Escherichia coli-induced immune paralysis is not exacerbated during chronic filarial infection.

Sepsis initially starts with a systemic inflammatory response (SIRS phase) and is followed by a compensatory anti-inflammatory response syndrome (CARS) that causes impaired adaptive T-cell immunity, immune paralysis and an increased susceptibility to secondary infections. In contrast, parasitic filariae release thousands of microfilariae into the peripheral blood without triggering inflammation, as they induce regulatory, anti-inflammatory host responses. Hence, we investigated the impact of chronic filarial infection on adaptive T-cell responses during the SIRS and CARS phases of a systemic bacterial infection and analysed the development of T-cell paralysis following a subsequent adenovirus challenge in BALB/c mice. Chronic filarial infection impaired adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ responses in the absence of a bacterial challenge and led to higher numbers of splenic CTLA-4(+)  CD4(+) T cells, whereas splenic T-cell expression of CD69 and CD62 ligand, serum cytokine levels and regulatory T-cell frequencies were comparable to naive controls. Irrespective of filarial infection, the SIRS phase dominated 6-24 hr after intravenous Escherichia coli challenge with increased T-cell activation and pro-inflammatory cytokine production, whereas the CARS phase occurred 6 days post E. coli challenge and correlated with high levels of transforming growth factor-ß and increased CD62 ligand T-cell expression. Escherichia coli-induced impairment of adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ production was not additionally impaired by chronic filarial infection. This suggests that filarial immunoregulation does not exacerbate E. coli-induced T-cell paralysis.

Human CD117 (cKit)+ innate lymphoid cells have a discrete transcriptional profile at homeostasis and are expanded during filarial infection.

Since innate lymphoid cells (ILCs) have been found to play a role in the immune response to helminth parasites in rodents, we sought to determine their role in human helminth infection. By developing multicolor flow cytometry-based methods to identify and enumerate circulating ILCs and their subsets, we were able to identify a subset of cKit+ ILCs defined as Lineage (Lin)-/CD45+/cKit+/CD127+ that were significantly expanded in the filarial-infected individuals (p=0.0473) as were those cKit+ ILCs that produced IL-13. Additionally, the frequency of these cKit+ ILCs correlated with the frequency of IL-17 producing CD4+ T cells (Th17 cells; p=0.025). To investigate the function of cKit+ ILCs, sorted, highly purified human ILCs were subjected to transcriptional profiling by RNAseq and compared to appropriate control cells. These cKit+ ILCs expressed TLRs, a broad range of cytokines/cytokine receptors and MHC Class II molecules suggesting that these ILCs sense pathogens independent of other cell types. Functional analysis revealed expanded cKit+ ILC-specific transcription and ILC-specific microRNA precursors.

ST2 deficiency does not impair type 2 immune responses during chronic filarial infection but leads to an increased microfilaremia due to an impaired splenic microfilarial clearance.

BACKGROUND: Interactions of the Th2 cytokine IL-33 with its receptor ST2 lead to amplified Type 2 immune responses. As Type 2 immune responses are known to mediate protection against helminth infections we hypothesized that the lack of ST2 would lead to an increased susceptibility to filarial infections. METHODOLOGY/PRINCIPAL FINDING: ST2 deficient and immunocompetent BALB/c mice were infected with the filarial nematode Litomosoides sigmodontis. At different time points after infection mice were analyzed for worm burden and their immune responses were examined within the thoracic cavity, the site of infection, and systemically using spleen cells and plasma. Absence of ST2 led to significantly increased levels of peripheral blood microfilariae, the filarial progeny, whereas L. sigmodontis adult worm burden was not affected. Development of local and systemic Type 2 immune responses were not impaired in ST2 deficient mice after the onset of microfilaremia, but L. sigmodontis infected ST2-ko mice had significantly reduced total numbers of cells within the thoracic cavity and spleen compared to infected immunocompetent controls. Pronounced microfilaremia in ST2-ko mice did not result from an increased microfilariae release by adult female worms, but an impaired splenic clearance of microfilariae. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the absence of ST2 does not impair the establishment of adult L. sigmodontis worms, but is important for the splenic clearance of microfilariae from peripheral blood. Thus, ST2 interactions may be important for therapies that intend to block the transmission of filarial disease.

Immunization of Mastomys coucha with Brugia malayi recombinant trehalose-6-phosphate phosphatase results in significant protection against homologous challenge infection.

Development of a vaccine to prevent or reduce parasite development in lymphatic filariasis would be a complementary approach to existing chemotherapeutic tools. Trehalose-6-phosphate phosphatase of Brugia malayi (Bm-TPP) represents an attractive vaccine target due to its absence in mammals, prevalence in the major life stages of the parasite and immunoreactivity with human bancroftian antibodies, especially from endemic normal subjects. We have recently reported on the cloning, expression, purification and biochemical characterization of this vital enzyme of B. malayi. In the present study, immunoprophylactic evaluation of Bm-TPP was carried out against B. malayi larval challenge in a susceptible host Mastomys coucha and the protective ability of the recombinant protein was evaluated by observing the adverse effects on microfilarial density and adult worm establishment. Immunization caused 78.4% decrease in microfilaremia and 71.04% reduction in the adult worm establishment along with sterilization of 70.06% of the recovered live females. The recombinant protein elicited a mixed Th1/Th2 type of protective immune response as evidenced by the generation of both pro- and anti-inflammatory cytokines IL-2, IFN-γ, TNF-α, IL-4 and an increased production of antibody isotypes IgG1, IgG2a, IgG2b and IgA. Thus immunization with Bm-TPP conferred considerable protection against B. malayi establishment by engendering a long-lasting effective immune response and therefore emerges as a potential vaccine candidate against lymphatic filariasis (LF).

Genomics of Loa loa, a Wolbachia-free filarial parasite of humans.

Loa loa, the African eyeworm, is a major filarial pathogen of humans. Unlike most filariae, L. loa does not contain the obligate intracellular Wolbachia endosymbiont. We describe the 91.4-Mb genome of L. loa and that of the related filarial parasite Wuchereria bancrofti and predict 14,907 L. loa genes on the basis of microfilarial RNA sequencing. By comparing these genomes to that of another filarial parasite, Brugia malayi, and to those of several other nematodes, we demonstrate synteny among filariae but not with nonparasitic nematodes. The L. loa genome encodes many immunologically relevant genes, as well as protein kinases targeted by drugs currently approved for use in humans. Despite lacking Wolbachia, L. loa shows no new metabolic synthesis or transport capabilities compared to other filariae. These results suggest that the role of Wolbachia in filarial biology is more subtle than previously thought and reveal marked differences between parasitic and nonparasitic nematodes.

Genome wide computational analysis of Brugia malayi helicases: a comparison with human host.

The availability of Brugia malayi genome sequence has paved ways for the search of homologues for a variety of genes. Helicases are ubiquitous enzymes involved in all the nucleic acid metabolic pathways and are essential for the development and growth. The genome wide analysis of B. malayi for different helicases showed the presence of a number of DEAD box helicases, 7 DEAH box helicases, RecQ helicases, repair helicases, super killer helicases, MCM2-7 complex, Rad54 and two subunits of Ku helicase. The comparison of protein sequence of each helicase with its human counterpart indicated characteristic differences in filarial helicases. There are noticeable differences in some of the filarial helicases such as DHX35, RecQL1 and Ku. Further characterization of these helicases will help in understanding physiological significance of these helicases in filarial parasites, which in future can be utilized for chemotherapy of parasitic infection.

Chronic helminth infection reduces basophil responsiveness in an IL-10-dependent manner.

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.

CCL17 controls mast cells for the defense against filarial larval entry.

Filarial parasites have to trespass many barriers to successfully settle within their mammalian host, which is equipped with mechanical borders and complex weaponry of an evolved immune system. However, little is known about mechanisms of early local events in filarial infections. In this study, bone marrow-derived dendritic cells not only upregulated activation markers CD40 and CD80 upon in vitro stimulation with filarial extracts, but also secreted CCL17, a chemokine known to be produced upon microbial challenge. Mice deficient for CCL17 had an up to 4-fold higher worm burden compared with controls by day 10 of infection with the murine filaria Litomosoides sigmodontis. Also, numbers of mast cells (MCs) invading the skin and degranulation were significantly increased, which was associated with enhanced vascular permeability and larval establishment. This phenotype was reverted by inhibition of MC degranulation with disodium cromoglycate or by blockade of histamine. In addition, we showed that CCL17-mediated vascular permeability was dependent on the presence of Wolbachia endosymbionts and TLR2. Our findings reveal that CCL17 controls filarial larval entry by limiting MC-dependent vascular permeability.

Characterization of the DMAE-modified juvenile excretory-secretory protein Juv-p120 of Litomosoides sigmodontis.

Juv-p120 is an excretory-secretory 160 kDa glycoprotein of juvenile female Litomosoides sigmodontis and exhibits features typical for mucins. 50% of its molecular mass is attributed to posttranslational modifications with the unusual substituent dimethylaminoethanol (DMAE). By that Juv-p120 corresponds to the surface proteins of the microfilarial sheath, Shp3 and Shp3a. The secreted protein consists of 697 amino acids, organized in two different domains of repeat elements separated by a stretch of polar residues. The N-terminal domain shows fourteen P/S/T/F-rich repeat elements highly modified with phospho-DMAE substituted O-glycans confering a negative charge to the protein. The C-terminal domain is extremely rich in glutamine (35%) and leucine (25%) in less organized repeats and may play a role in oligomerization of Juv-p120 monomers. A protein family with a similar Q/L-rich region and conserved core promoter region was identified in Brugia malayi by homology screening and in Wuchereria bancrofti and Loa loa by database similarity search. One of the Q/L-rich proteins in each genus has an extended S/T-rich region and due to this feature is supposed to be a putative Juv-p120 ortholog. The corresponding modification of Juv-p120 and the microfilarial sheath surface antigens Shp3/3a explains the appearance of anti-sheath antibodies before the release of microfilariae. The function of Juv-p120 is unknown.