Study population, samples collection and ethics

Patients and endemic controls’ samples were collected between 2008 and 2010 in villages of the Ahanta West and Nzema East Districts in the western region of Ghana endemic for LF (S1 Table). No other human filarial species were endemic in the region. The donors were recruited as part of a diagnostic and a clinical trial in LF (Clinical Trials Registration: ISRCTN15216778 and ISRCTN14757) [9,50,51]. Written informed consent was obtained from all participants. Persons eligible for participation were male adults in good health, 18–60 years of age, with a minimum body weight of more than 40 kg and without any clinical condition requiring chronic medication. Exclusion criteria included abnormal hepatic and renal enzyme levels (γ-glutamyltransferase > 28 U/L, glutamyl pyruvic transaminase > 30 U/L, creatinine > 1.2 mg/100 mL) assessed by dipstick chemistry, alcohol, drug abuse or antifilarial therapy in the past 10 months. Study participants were examined by a clinician using physical methods and a portable ultrasound machine (180 Plus; SonoSite, Bothell, WA) as described previously [51]. In addition, the presence of infections with other helminths (Ascaris lumbricoides, Trichuris trichiura, Schistosoma spp.) and protozoa (Plasmodium) was investigated using respectively Kato-Katz and finger prick tests. All samples included in the present study were free of such infections as previously described [9]. Ethical clearance was given by the Committee on Human Research Publication and Ethics at the University of Science and Technology in Kumasi, and the Ethics Committee at the University Hospital Bonn. Microfilarial load was determined by microscopic examination of fingerprick night blood samples as published [51]. Subsequently, 10 mL of venous blood was collected from each eligible volunteer and plasma was taken, aliquoted, and frozen at −80°C until used.

Samples included EN, residing in the endemic region but free of infection (CFA-, Mf-, n = 14), clinically asymptomatic microfilaraemic (CFA+, Mf+, n = 14) and amicrofilaraemic (CFA+, Mf-, n = 14) subjects, positive for circulating filarial antigen and a group of chronic pathological individuals with lymphedema and/or elephantiasis termed “CP” (n = 14), negative for filarial antigen. Also, plasma from European non-endemic blood donors (NEC, n = 14) was used as controls. Serum samples were obtained in an anonymized and de-identified form. All samples and controls used in the present study were randomly picked from the initial batches using a computer-based simple random algorithm as previously described [52]. Each sample in the initial batch was assigned a unique number and the samples corresponding to computer generated list were picked in each group and used in the study.

Preparation of Brugia malayi antigen extracts

Brugia malayi worms recovered from the peritoneal cavity of jirds (Meriones unguiculatus) were obtained from NAID Filariasis Research Reagent Resource, FR3 (University of Georgia, Athens, GA). To prepare B. malayi antigen extract (BmAg), 100–300 frozen adult worms were thawed and transferred to a Petri dish pre-filled with sterile PBS (PAA, Pasching, Austria). Following several washes in PBS, worms were placed inside a glass mortar (VWR, Langenfeld, Germany). 3–5 ml of medium (RPMI without supplements) were added and worms were crushed until the solution was homogenous. The extract was then centrifuged for 10 minutes at 300 x g at 4°C to remove insoluble material. The supernatant was carefully transferred to a new tube. Protein concentration was measured using Bradford Assay. Aliquots were stored at -80°C until use. The extract was titrated to determine the optimal concentration for cell stimulation and the level of endotoxin was defined using the Pierce Limulus amoebocyte lysate (LAL) Chromogenic quantification kit (Thermo Fisher Scientific, Schwerte, Germany). The endotoxin level was below the detection limit of 0.1 EU/ml.

Granulocyte purification from healthy blood donors

Granulocytes used in this study were purified from buffy coats of healthy European donors provided by the Institute for Experimental Haematology and Transfusion Medicine, University Clinic Bonn, Germany. Ethical clearance was given by the Ethics Committee of the University of Bonn (“Ethikkommission der Medizinischen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn”). Granulocytes were isolated using Ficoll-Hypaque (Pancoll, PAN Biotech, Aidenbach Germany) method. The density gradient was performed according to the manufacturer's instructions. Briefly, 15 mL heparinized venous blood samples were diluted with an equal volume of cold phosphate-buffered saline (PBS) in a 50 mL conical centrifuge tube, layered over Ficoll and centrifuged at 900 x g for 30 min at 4°C in a swinging bucket centrifuge (Thermo Scientific, Germany) with brake off. The opaque layer below the Ficoll/plasma interface containing granulocytes was transferred to another tube. After that, red cells were lysed by 10 min incubation at room temperature in 1x red blood cell lysis solution (Miltenyi Biotech, Bergisch Gladbach, Germany). Granulocytes were then centrifuged at 200 x g for 8 min at 4°C to remove contaminating red blood cells. Cell pellets were washed twice at 200 x g for 8 min in RPMI 1640 (Life Technologies, NY, USA). Supernatants were discarded and the purity of isolated granulocytes was assessed by flow cytometry. The purity was routinely ≥ 96%.

Affinity chromatography for the purification of total IgG and IgG4

Total IgG was isolated from the plasma of EN, Mf+, Mf- and CP using prepacked HiTrap Protein G columns (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. Briefly, 100 μl of plasma samples were diluted with 1400 μl PBS and passed through a pre-equilibrated protein G-Sepharose column (GE Healthcare, Freiburg, Germany). Since Protein G binds to all human IgG subclasses, non-IgG plasma components were washed out from the column. Bound IgG was eluted in 1 ml fractions using IgG Elution Buffer (0.2 M Glycine/HCl, pH 3.0) and neutralized with saturated Tris-HCl (pH 9.0). The antibody concentration was then assessed at 280 nm using a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific, Wilmington, USA).

IgG4 antibodies were purified from IgG-enriched fractions using the CaptureSelect Human IgG4 affinity matrix (Life Technologies, Paisley, UK) according to the manufacturer’s instructions. Briefly, CaptureSelect affinity matrix was gently loaded and equilibrated in 10 ml affinity chromatography column with 1x PBS (pH 7.3). Diluted IgG-enriched fractions (1:1 volume PBS) were loaded onto the column and the linear flow rate was set at 15 cm/hour. After washing with 1x PBS, the column was eluted with 0.1 M Glycine (pH 3.0) and the fractions were immediately neutralized with Tris-HCl (pH 9.0). IgG4 fractions were collected and the purity of fractions assessed by determining the level of IgG subclasses, IgA, IgE and IgM antibodies by Luminex assay. In addition, the total protein concentration in plasma, IgG and IgG4 fractions was determined using a Bradford Protein Assay Kit (Thermo Fischer Scientific, Wilmington, USA).

Affinity chromatography for the purification of human IgA

IgA purification was done using immobilized Peptide M/Agarose (InvivoGen, San Diego, USA) according to the manufacturer’s instructions. 1 ml of peptide M/Agarose gel was loaded into an appropriate microcentrifuge spin column (Thermo Scientific, Rockford, USA). 1 ml of IgG-depleted plasma from EN were then added to the column and incubated at room temperature for 30 min. After incubation, the column was washed and the flow-through was collected and labeled as IgA negative fractions (IgA-). The bound antibodies were then eluted with IgG elution buffer as described above. The resulting IgA positive (IgA+) fractions were neutralized immediately with neutralization buffer (1 M Tris–HCl, pH 9.0) and store at 4°C until use. Purity was assessed by Luminex analysis as described above. The purity was routinely > 90%

Quantification of total protein in plasma and purified IgA, IgG and IgG4 fractions

To determine the protein concentration of plasma and purified IgA, IgG and IgG4 fractions, a Bradford protein assay kit (Thermo Scientific, Rockford, USA) was used according to the manufacturer’s instructions. In brief, serial dilutions of bovine serum albumin (BSA) was performed and used as standards against the samples. Another serial dilution of the samples was done in PBS. 300 μl per well of Coomassie blue G-250 (Cytoscelecton, Denver, USA) reagent was distributed in duplicate in a 96 well plate (Greiner Bio-One, Frickenhausen, Germany) and 3 μl of diluted samples and standard were added. The protein concentration in plasma, IgA, IgG and IgG4 fractions was then measured at 595 nm using a SpectraMAX 190 microplate reader (Molecular Devices, California, USA).

Assessment of immunoglobulin isotypes expression in plasma and IgG/IgG4 positive and negative fractions

To analyze the isotype composition in IgA, IgG/IgG4 positive and negative fractions and in the plasma of EN and LF patients, ProcartaPlex Human Antibody Isotyping Panels (eBioscience, Vienna, Austria) were used according to manufacturer’s instructions. Briefly, antibody coated magnetic bead mixtures were incubated with 25 μl of assay buffer, kit standards or diluted plasma samples in a ProcartaPlex 96-wells plates at room temperature for 1 hour. 25 μl of detection antibodies mixture was then added and the plates were incubated on an orbital shaker at 500 rpm for 30 min. After that, each well was incubated with 50 μl of diluted Streptavidin-Phycoerythrin for 30 min. Plates were then washed using a hand-held magnetic plate washer. All incubations were performed at room temperature in the dark. Afterwards, samples were suspended in 120 μl reading buffer. Data were acquired using a MAGPIX Luminex system (Luminex Cooperation) and analyzed with ProcartaPlex Analyst software 1.0.

Western blot analysis of eluted IgG fractions

The purity of eluted IgG fractions was analyzed by western blot. Samples were treated with 50 mM 2-mercaptoethanol for 5 min, and equal quantities (2.5 μg) of the purified proteins and controls were loaded onto separate lanes of a polyacrylamide gel (10–12%) and resolved by SDS-PAGE (100 v, 45–60 min). The resolved proteins were transferred onto nitrocellulose membranes (GE Healthcare, Freiburg, Germany) using a Bio-Rad Trans-Blot Turbo Transfer system (Bio-Rad, Germany). The membranes were then blocked with gelatin blocking buffer (3% gelatin in Tris Buffered Saline (TBS)) (Bio-Rad, Germany) for 1 hour prior incubation with the primary antibody (polyclonal mouse anti-human IgG (H+L)) (Thermo Scientific, Rockford, USA) for 1.5 hours at room temperature. The nitrocellulose membranes were then washed with TBS/0.05% Tween 20 before incubation for 1 hour with alkaline phosphatase-conjugated goat anti-mouse IgG (Bio-Rad Laboratories, USA). Immune complexes were finally detected with NBT (nitro blue tetrazolium) and BCIP (5-bromo-4-chloro-3-indolyl-phosphate, Bio-Rad Laboratories, USA). Experiments were repeated at least three times.

In vitro cell culture and granulocyte suppression assay

After establishing the optimal concentrations of IgE, anti-IgE and rIL-3 to induce granulocyte activation and degranulation (S1 Fig), the cells were purified as described above and 2 x 10⁵ cells/well were plated and pre-incubated with 40 ng/ml of natural human IgE antibody (Abcam, Cambridge, UK) for 30 min at 37°C/5% CO₂ as previously described [53,54]. The cells were first preactivated at 37°C/5% CO₂ for 10 min in the presence of 2 ng/ml rIL-3 (Miltenyi Biotech, Bergisch Gladbach, Germany), before being stimulated with 25 ng/ml anti-IgE mAb (Clone BE5) (Abnova, Taipei, Taiwan) and 10 μg/ml Brugia malayi Ag. Thereafter, the granulocytes were further incubated for 18 hours at 37°C/5% CO₂ either alone (with culture medium), or in the presence of appropriately diluted plasma samples (5% v:v, containing 5 μg/ml of total proteins), IgG and corresponding IgG depleted fractions (5 μg/ml of total proteins) or 2.5 μg/ml IgG4 antibodies purified from the IgG positive fractions from the different clinical groups.

Assessment of granule content release

Granulocyte culture supernatants were collected after 30 min and 18 hours to assess the level of histamine, and after 18 hours to investigate the release of ECP and NE using ELISA-Kits respectively from Abnova (Taipei, Taiwan), Abbexa (Cambridge, UK) and eBioscience (Vienna, Austria) according to the manufacturer’s recommendations. For histamine detection, samples and standards were first acylated by reacting 50 μl of samples, 25 μl of standards or control with 25 μl of acylation reagent and 25 μl of acylation buffer supplied in the test kit for 45 minutes. 25 μl aliquots of acylated standards, controls and samples were pipetted into wells of the antibody-coated microplate provided with the kit. Then the wells received 100 μl of histamine antiserum and the mixture was allowed to incubate for 3 hours at room temperature. The plates were then washed with the provided washing buffer to remove unbound materials. After that, the bound antibodies were detected using 100 μl of anti-rabbit IgG-peroxidase conjugate using TMB as a substrate. The color was allowed to develop for 20 minutes at room temperature in the dark. The reaction was stopped and the resulting OD values were measured at 450 nm. The histamine concentration, inversely proportional to the OD, was calculated using the SoftMax Pro Data Acquisition and Analysis Software.

For the assessment of ECP and NE, pre-coated ELISA plates were incubated with supernatants and standards for 1 hour at room temperature. The plates were then washed and incubated for 1 hour with HRP-conjugated anti-ECP and anti-NE polyclonal antibodies. After a final wash, the plates were developed using the provided TMB substrate and analyzed at 450 nm.

Flow cytometry

To assess granulocyte activation and degranulation, the cells were harvested and washed with FACS buffer (PBS/2% FCS) at 1300 rpm for 8 min. 1x10⁵ cells were then resuspended in 100 μl of FACS buffer and blocked with 1 μl of FC- block (Affymetrix eBioscience, San Diego, CA, USA) for 15 min. 5μg/1x10⁵ cells of either anti-human CD66b-FITC (clone: G10F5) or CD63-PE (clone: H5C6) and HLADR-FITC (clone: LN3) (all from Affymetrix eBioscience) were then added and the cell suspension was incubated for 30 min at 4°C. Cells were then washed two times with FACS buffer and fixed in 200 μl PFA (4%). To correct spectral overlaps, fluorescence compensation was done using UltraComp ebeads (Affymetrix eBioscience). Data were acquired and analyzed using a FACS Canto flow cytometer and the BD-FACS-DIVA analysis software (BD Biosciences). Before the analysis, granulocyte viability was assessed by FACS using propidium iodide (PI-PE) and annexin-V (Annexin-FITC) (all from BD Biosciences, Heidelberg, Germany). All samples presented less than 1 and 10% necrotic cells respectively before and after 18 hours incubation.

Cytospin and immunofluorescent staining for IgG4

Granulocytes were cultured as previously described and 2 x 10⁵ cells were harvested and washed with PBS. Then 100 μl of diluted cells were aliquoted into cytospin funnels and spun at 500 x g for 5 min onto glass slides (Engelbrecht, Edermünde, Germany) in a Hettich Cytospin centrifuge (Hettich, Tuttlingen, Germany) and immediately fixed in 4% PFA for 15 min. The slides were then blocked with PBS/1% BSA for 30 min and incubated with the primary antibody (mouse anti-human monoclonal IgG4) (Thermo Fischer Scientific, Rockford, USA) for 1 hour. After washing 3 times, the slides were incubated with the Alexa Fluor 488 coupled secondary antibody (goat anti-mouse polyclonal IgG antibody) (Thermo Fischer Scientific, Rockford, USA) for 1 hour at room temperature in a humidifying chamber. For the investigation of the Fc-receptors associated with IgG4-mediated granulocyte suppression, blocking antibodies against human FcγRI (2 μg/ml) (Biolegend, San Diego, CA, USA, clone:10.1), FcγRII (1 μg/ml) (Biolegend, San Diego, CA, USA, clone: FUN-2) and FcγRIII (4 μg/ml) (Biolegend, San Diego, CA, USA, clone: 3G8) were added before the incubation with purified IgG4 antibodies. Nuclear DNA was labeled with 0.25 μg/ml DAPI (Thermo Fischer Scientific, Rockford, USA) in PBS for 5 min. Cells were then mounted in VECTASHIELD-Antifade mounting medium (Vector Laboratories, CA, USA) and the slides were analyzed using a Zeiss LM-Set Axiocam MRm microscope (Carl Zeiss, Thornwood, NY, USA).

Antigen-specific ELISA

To evaluate the capacity of purified IgG4 from EN, Mf+, Mf- and CP individuals to interact with Brugia antigen, 50 μl Brugia antigen (10 μg/ml) were coated on high binding ELISA plates (Greiner Bio-One, Frickenhausen, Germany) overnight at 4°C. The plates were then washed 5 times with PBS/0.05% Tween 20 and blocked with PBS/1% BSA for 1 hour at room temperature. The wash step was repeated and 50 μl/well of purified IgG4 from EN, Mf+, Mf- and CP (2.5 μg/ml) were added and the plates were incubated again at 4°C overnight. The wells were washed again as described above and diluted biotin-conjugated mouse anti-human IgG4 (clone JDC-14) (1:1000) (from BD Biosciences, Heidelberg, Germany) was added, followed by incubation at room temperature for 2 hours. After an additional washing step, the plates were incubated with 50 μl/well of Streptavidin-HRP for 45 min in the dark. After a final washing step, 50 μl/well TMB substrate solution were added and the reaction was stopped with 25 μl/well 2N H2SO4 (Merck KGAA, Darmstadt, Germany). Optical density was measured at 450 nm using the SpectraMAX ELISA reader and the results were expressed as arbitrary units (AU) using as a standard a plasma sample arbitrarily set at 5 AU.

Statistical analysis

All statistical analyzes were performed using Prism 5.03 software (GraphPad Software, Inc., La Jolla, USA). Comparative analyzes among groups were conducted using the Kruskal-Wallis test with a Dunn’s nonparametric post-hoc test (> 2 groups). Significance was accepted when p < 0.05. Correlation between the levels of antibodies in IgG negative fractions and inhibition capacity was analyzed using Spearman’s rank correlation.

IgG4 and IgA are characteristic for Mf+ and EN respectively, whereas IgE is prominent in Mf- and CP

To define the initial antibody profile of EN, Mf+, Mf- and CP, we compared the plasma levels of IgG1, IgG2, IgG3, IgG4, IgE, IgM and IgA in different groups using a Luminex-based immunoassay. We found that the IgG1 expressions observed in EN and CP were similarly high. In contrast, Mf+ and Mf- individuals presented reduced IgG1 levels (Fig 1A). However, while the highest levels of IgG2 were detected in the plasma of CP individuals, plasmatic IgG2 in EN and Mf- were significantly lower compared to Mf+ and CP (Fig 1B). The differences observed in the expression of IgG3 between the four groups were not statistically significant (Fig 1C). Interestingly, the expression of IgG4 was relatively low in EN, Mf- and CP but significantly elevated in Mf+ (Fig 1D). This contrasts with lower levels of IgE in those patients in comparison to Mf- and patients with chronic pathological manifestations (Fig 1E). In addition, plasma of EN expressed higher IgA levels compared to Mf+, Mf- and CP (Fig 1F), whereas no significant differences were seen in the expression of IgM (Fig 1G).

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Fig 1. Preferential expression of IgG4 in Mf+.

Plasma samples from EN (n = 14) and LF infected Mf+ (n = 14), Mf-(n = 14) and CP (n = 14) patients were diluted and analyzed for the expression of IgG1 (A), IgG2 (B), IgG3 (C), IgG4 (D), IgE (E), IgA (F) and IgM (G) using Luminex-based immunoassay. Bars depict the plasmatic antibody expressions as mean ± SEM. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g001

Plasma from EN and Mf+ but not those of Mf- and CP inhibits granulocyte activation and degranulation

Because plasma samples from Mf+ individuals presented high levels of IgG4 antibody and since IgG4 antibodies are known to exhibit anti-inflammatory properties, we hypothesized that plasma from Mf carriers, and specifically IgG4 molecules, would preferentially down-modulate granulocyte activation and degranulation. We then next investigated how crude plasma of NEC, EN, Mf+, Mf- or CP modulates the function of IL-3/anti-IgE/BmAg activated granulocytes by monitoring the expression levels of CD63/HLADR and analyzing the release of granule components (histamine, ECP and NE). While plasma from NEC, CP and Mf- had no effect on granulocytes (Fig 2 and S2 Fig), those from EN and Mf+ significantly inhibited activation of granulocytes as indicated by the lower percentages of CD63+HLADR- cells (Fig 2A). Interestingly, the plasma of EN presented a higher inhibitory potential on granulocyte activation when compared to those of Mf+. In line with the activation data, plasma from both EN and Mf+ significantly suppressed the release of histamine (Fig 2B and S2 Fig) and NE (Fig 2C). In addition, histamine levels were higher after 30 min and were lower, but detectable, after 18 hours. However, while the plasma of Mf+ individuals significantly inhibited the release of ECP in granulocyte cultures, those of EN failed to suppress the release of ECP (Fig 2D). These results indicate that, in lymphatic filariasis, active factors in EN and Mf+ infected patients’ plasma environment but not present in Mf- and CP patients impaired granulocyte activation.

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Fig 2. Plasma from EN and Mf+ patients suppressed granulocyte activation and degranulation.

Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) as control (dark bars) and then cultured in presence of 5% (v/v) of plasma (with a final concentration of 5 μg/ml of total proteins) of either non-endemic controls (NEC), endemic normal (EN), microfilaria positive (Mf+), microfilaria negative individuals (Mf-) or plasma of patients with chronic pathology manifestations (CP) (grey bars). Cells were then stained for CD63 and HLADR antigens expression. The proportion of activated granulocyte cells (CD63+/HLADR- cells) was determined after 18 hours of incubation (A). The release of histamine after 30 min (B), neutrophil elastase (C) and eosinophil cationic protein (D) after 18 hours was measured in culture supernatants. Bars represent means ± SEM. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g002

Depletion of IgG reduces the suppressive capacity of plasma from Mf+ but not of EN

To define the role of IgGs in the suppression of granulocytes by plasma of EN and Mf, we depleted IgG antibodies per affinity chromatography. The purity was analyzed (S3 Fig), and the ability of IgG positive and negative fractions to modulate granulocyte activation was tested. Interestingly, while IgG negative (IgG-) fractions of EN significantly suppressed granulocyte activation, IgG positive (IgG+) fractions showed no effect (Fig 3A). Both IgG+ and IgG- fractions from Mf+ significantly inhibited granulocyte activation (Fig 3B) whereas neither IgG+ nor IgG- fractions from NEC, Mf- and CP affected granulocyte activation (Fig 3C–3E). Moreover, in Mf+, the IgG-related inhibition was significantly higher than that observed with negative fractions. These trends were also reflected in the release of histamine (Fig 3F) and NE (Fig 3G). Surprisingly both fractions from EN did not impair ECP release (Fig 3H) when compared with histamine and NE. These data suggest that whereas total IgG from Mf+ individuals inhibited granulocyte activation, IgG-independent factors, seem to be involved in the suppression by plasma of both EN and Mf+ individuals.

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Fig 3. Suppression of granulocyte activation and degranulation is IgG-independent in EN but IgG-dependent in Mf+.

Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 5 μg/ml of IgG negative fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) and NEC (E) or the corresponding IgG positive fractions (grey bars). After that, cells were stained for CD63 and HLADR expression. Activated granulocytes were characterized as CD63+/HLADR- cells. Bars represent means ± SEM of the percentage of activated granulocyte CD63⁺/HLADR^- cells. The release of histamine after 30 min (F), neutrophil elastase (G) and eosinophil cationic protein (H) after 18 hours in culture supernatants was assessed. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g003

IgA expression is associated with the suppressive capacity of IgG-negative fractions from EN

To define the IgG-independent factors responsible for granulocyte suppression in EN, we correlated the expression of the remaining antibodies in the IgG-negative fractions (IgA, IgE and IgM) with the ability of these plasma to inhibit granulocyte functions. Interestingly, while IgE and IgM presented no correlation with the inhibitory capacity of IgG-negative fractions of EN (Fig 4A and 4B), IgA expression significantly correlated with the inhibition capacity on activated granulocytes (Fig 4C). In addition, while IgA-depleted fractions [(EN) IgA-] lose their ability to suppress granulocyte activation as shown by similar expression of CD63+ cells when compared to the control, peptide M purified IgA+ fractions significantly reduced granulocyte activation (Fig 4D). These data strongly suggest that IgA expression in EN is associated with the inhibition effect observed when their plasma were incubated with activated granulocytes.

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Fig 4. IgA expression correlated with the suppressive capacity in the IgG-negative fractions of EN.

Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone or in the presence of 5 μg/ml of IgG negative fractions of EN. Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells and the percentage of inhibition was calculated using the control without plasma as 100% of activation (0% of inhibition). The percentage of inhibition was then correlated with the expression of IgE, IgM and IgA determined by Luminex assay. Each dot represents the plot of the expression of IgE (A), IgM (B) or IgA (C) to the corresponding value of the inhibition. Thereafter, the percentage of activated granulocytes (CD63+/HLADR- cells) was determined after 18 hours of incubation (D) in the presence of IgE/IL-3 alone (dark bar) or in combination with either bulk plasma from EN (EN), IgA negative fractions (EN) IgA- or IgA positive fractions (EN) IgA+. Bars represent means ± SEM of the percentage of activated granulocyte CD63⁺/HLADR^- cells. Statistical comparison was based on Spearman’s rank correlation (A-C) or Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test (D). The indicated p-values refer to the significance level among all groups and asterisks indicate the level of significance, determined by Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01. Graphs are representative of 3 independent experiments.

https://doi.org/10.1371/journal.pntd.0005777.g004

IgG4 antibodies purified from EN, Mf+, Mf- but not CP dampen granulocyte functions in a dose-dependent manner

We next investigated whether the modulation of granulocyte activation and degranulation by Mf+ IgG fractions is associated with the presence of the anti-inflammatory isotype IgG4. Highly pure fractions of IgG4 antibodies were prepared and the purity validated (S4 Fig). Thereafter the purified fractions were tested on activated granulocytes. Strikingly, while IgG4 antibodies from EN, Mf+ and Mf- significantly suppressed granulocyte activation (Fig 5A–5C and S4 Fig), those from CP have failed to suppress granulocyte activation (Fig 5D) compared to the control. In addition, the suppressive effect was completely abrogated after IgG4 removal from IgG fractions (Fig 5A–5C), suggesting that the suppressive effect of IgG fractions stems from IgG4 molecules and not from other IgG antibodies. Furthermore, we investigated whether these effects were dose-dependent. Whereas increasing concentrations of IgG4 from EN, Mf+ and Mf- proportionally reduced the percentage of activated cells (CD63+/HLADR-) in a dose-dependent manner, no dose effect was seen when IgG4 from CP patients were used (Fig 5E). Interestingly, the suppressive effect of IgG4 affected mostly neutrophils and basophils but not eosinophils (S5 Fig). Consistent with the granulocyte activation data, we detected lower levels of histamine and NE in supernatants of granulocyte cultures treated with IgG4 antibodies from EN, Mf+ and Mf- compared to those treated with CP-IgG4 (Fig 5F and 5G). However, no significant reduction in the release of ECP was observed after incubation with IgG4 from EN or Mf- (Fig 5H). Table 1 summarizes the effects of plasma, IgG and IgG4 fractions on granulocyte activation.

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Fig 5. Depletion of IgG4 abrogates the suppressive capacity of IgG positive fractions from plasma of LF infected individuals.

Freshly isolated granulocytes from healthy blood donors (n = 9) were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml) and Brugia antigen extracts (10 μg/ml) alone (dark bars) or in presence of 2.5 μg/ml of IgG4 negative fractions obtained from IgG enriched fractions (n = 8) (light bars) from EN (A), Mf+ (B), Mf- (C), CP (D) or the corresponding IgG4 positive fractions (n = 8) (grey bars) and increasing concentration (1.25 μg/ml, 2.5 μg/ml and 5 μg/ml) of IgG4 positive fractions from different groups (E). Cells were stained with anti-CD63 and HLADR antibodies. Activated granulocytes were characterized as CD63+/HLADR- cells. The release of histamine (F), neutrophil elastase (G) and eosinophil cationic protein (H) in supernatants from cultures with IgG4 positive fractions was assessed after 18 hours. Bars represent means ± SEM. Graphs are representative of 3 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test;*: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g005

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Table 1. Effect of plasma and purified IgG and IgG4 fractions from EN, Mf+, Mf- and CP on granulocyte activation.

https://doi.org/10.1371/journal.pntd.0005777.t001

IgG4 antibodies modulate granulocyte activities via FcγRI and II

To further explore the mechanisms by which IgG4 interfere with granulocyte activities, we examined the ability of purified IgG4 antibodies from each group to bind to granulocytes and Brugia antigen. While IgG4 molecules from EN, Mf+ and Mf- were able to interact with effector cells, those from CP had no affinity to granulocytes (Fig 6). However, IgG4 from Mf+ presented a much higher affinity for the cells in comparison to those from Mf- and EN (Fig 6A–6C and 6E). Since differences in the affinity of IgG4 from EN, Mf+, Mf- and CP to form complex with Brugia antigen can also affect granulocyte modulation, we analyzed the capacity of IgG4 from the different groups to interact with Brugia antigen. We detected no significant differences in the capacity of IgG4 from EN, Mf+, Mf- and CP to form a complex with Brugia antigen (Fig 6F).

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Fig 6. IgG4 antibodies from Mf+ patients present a higher affinity for granulocytes compared to IgG4 from EN and Mf-.

Purified granulocytes from healthy blood donors were stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and cultured in presence of 2.5 μg/ml of IgG4 molecules from EN (A), Mf+ (B), Mf- (C) and CP (D) for 18 hours. The cells were then stained with DAPI (blue) and IgG4 binding on activated cells was revealed with Alexa fluor 488 labelled antibody (green). Original magnification x100. The median green fluorescence intensities are illustrated in E. A representative experiment of 3 is shown. Thereafter, the relative affinity of IgG4 from EN, Mf+, Mf- and CP with Brugia antigen was analyzed by ELISA (F). Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-value refers to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g006

Because IgG4 from Mf+, Mf- and EN bound to granulocytes, we next investigated which FcγRs are involved in their fixation by using blocking antibodies against FCγRI, FCγRII, and FCγRIII. We observed that the blockade of FcγRI (Fig 7B and 7H) and FcγRII (Fig 7C and 7H) but not FcγRIII (Fig 7D and 7H) significantly reduced IgG4 binding to granulocytes. Interestingly, the capacity of IgG4 to bind to granulocytes was completely abrogated when FcγRI and FcγRII were simultaneously blocked (Fig 7E and 7H). Corresponding results were also observed when the activation of granulocytes in the presence of IgG4 and anti-FCγRs was measured (Fig 7I). These findings suggest that IgG4-mediated granulocyte suppression in Mf+ patients involves FcγRI and FcγRII but not FcγRIII.

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Fig 7. Anti-inflammatory IgG4 antibodies modulate granulocyte functions via FcγRI and FcγRII.

Granulocytes from healthy blood donors were purified and stimulated with IL-3 (2 ng/ml), anti-IgE (25 ng/ml), and Brugia antigen extracts (10 μg/ml) and incubated with either medium or anti-FcγRI (2μg/ml), FcγRII (1 μg/ml) or FcγRIII (4 μg/ml) antibodies alone or in combinations. Thereafter granulocytes were incubated with 2.5 μg/ml of affinity purified IgG4 for 18 hours. The cells were then stained with DAPI (blue) and the presence of IgG4 was detected with anti-IgG Alexa fluor 488 antibody (green). A representative experiment out of 6 is shown: A-G are representative fluorescence microscopy images of granulocytes incubated with culture medium (A) or in the presence of blocking antibodies against FcγRI (B), FcγRII (C), FcγRIII (D) or a combination of antibodies against FcγRI and II (E), FcγRI and III (F), or FcγRII and III (G). Bars represent mean fluorescence intensities ± SEM (H) or the percentages of CD63+/HLADR- activated granulocytes (I) in 6 independent experiments. Statistical comparison was based on Kruskal-Wallis one-way ANOVA followed by Dunn post-hoc test. The indicated p-values refer to the significance level among all groups according to Kruskal-Wallis test. Asterisks indicate the level of differences after Dunn’s multiple comparisons test; *: p<0.05; **: p<0.01; ***: p<0.001.

https://doi.org/10.1371/journal.pntd.0005777.g007