Pathological manifestations in lymphatic filariasis correlate with lack of inhibitory properties of IgG4 antibodies on IgE-activated granulocytes

<div><p>Helminth parasites are known to be efficient modulators of their host’s immune system. To guarantee their own survival, they induce alongside the classical Th2 a strong regulatory response with high levels of anti-inflammatory cytokines and elevated plasma levels of IgG4. This particular antibody was shown in different models to exhibit immunosuppressive properties. How IgG4 affects the etiopathology of lymphatic filariasis (LF) is however not well characterized. Here we investigate the impact of plasma and affinity-purified IgG/IgG4 fractions from endemic normals (EN) and LF infected pathology patients (CP), asymptomatic microfilaraemic (Mf+) and amicrofilaraemic (Mf-) individuals on IgE/IL3 activated granulocytes. The activation and degranulation states were investigated by monitoring the expression of CD63/HLADR and the release of granule contents (neutrophil elastase (NE), eosinophil cationic protein (ECP) and histamine) respectively by flow cytometry and ELISA. We could show that the activation of granulocytes was inhibited in the presence of plasma from EN and Mf+ individuals whereas those of Mf- and CP presented no effect. This inhibitory capacity was impaired upon depletion of IgG in Mf+ individuals but persisted in IgG-depleted plasma from EN, where it strongly correlated with the expression of IgA. In addition, IgA-depleted fractions failed to suppress granulocyte activation. Strikingly, affinity-purified IgG4 antibodies from EN, Mf+ and Mf- individuals bound granulocytes and inhibited activation and the release of ECP, NE and histamine. In contrast, IgG4 from CP could not bind granulocytes and presented no suppressive capacity. Reduction of both the affinity to, and the suppressive properties of anti-inflammatory IgG4 on granulocytes was reached only when FcγRI and II were blocked simultaneously. These data indicate that IgG4 antibodies from Mf+, Mf- and EN, in contrast to those of CP, natively exhibit FcγRI/II-dependent suppressive properties on granulocytes. Our findings suggest that quantitative and qualitative alterations in IgG4 molecules are associated with the different clinical phenotypes in LF endemic regions.</p></div

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

<p>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.</p

Preferential expression of IgG4 in Mf+.

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p