Eosinophils Are Important for Protection, Immunoregulation and Pathology during Infection with Nematode Microfilariae

Eosinophil responses typify both allergic and parasitic helminth disease. In helminthic disease, the role of eosinophils can be both protective in immune responses and destructive in pathological responses. To investigate whether eosinophils are involved in both protection and pathology during filarial nematode infection, we explored the role of eosinophils and their granule proteins, eosinophil peroxidase (EPO) and major basic protein-1 (MBP-1), during infection with Brugia malayi microfilariae. Using eosinophil-deficient mice (PHIL), we further clarify the role of eosinophils in clearance of microfilariae during primary, but not challenge infection in vivo. Deletion of EPO or MBP-1 alone was insufficient to abrogate parasite clearance suggesting that either these molecules are redundant or eosinophils act indirectly in parasite clearance via augmentation of other protective responses. Absence of eosinophils increased mast cell recruitment, but not other cell types, into the broncho-alveolar lavage fluid during challenge infection. In addition absence of eosinophils or EPO alone, augmented parasite-induced IgE responses, as measured by ELISA, demonstrating that eosinophils are involved in regulation of IgE. Whole body plethysmography indicated that nematode-induced changes in airway physiology were reduced in challenge infection in the absence of eosinophils and also during primary infection in the absence of EPO alone. However lack of eosinophils or MBP-1 actually increased goblet cell mucus production. We did not find any major differences in cytokine responses in the absence of eosinophils, EPO or MBP-1. These results reveal that eosinophils actively participate in regulation of IgE and goblet cell mucus production via granule secretion during nematode-induced pathology and highlight their importance both as effector cells, as damage-inducing cells and as supervisory cells that shape both innate and adaptive immunity

HeLa cells retard growth of <i>Toxoplasma</i> by an IFNγ and ubiquitin-dependent but acidification-independent mechanism.

<p>(A) Ubiquitin is recruited to type II <i>Toxoplasma</i> vacuoles in IFNγ-stimulated HeLa cells. Inhibiting ubiquitination with the E1 inhibitor UBEI-41 reduces recruitment of ubiquitin to type II vacuoles at 6h p.i.. Counts are expressed as % positive vacuoles and the mean of 3 experiments is shown. Significance was determined using 2way ANOVA, ***, p ≤ 0.001, ****, p ≤ 0.0001 and ns, not significant. (B) Inhibiting ubiquitination increased the ability of type II <i>Toxoplasma</i> to replicate in IFNγ-stimulated HeLa at 24h and 18h p.i., but no difference in replication was observed at 6h. HeLa were pre-treated with 50μm UBEI-41 to inhibit ubiquitination prior to infecting with type I or type II <i>Toxoplasma</i> for 24, 18 and 6h. Replicating <i>Toxoplasma</i> counts with and without inhibition of ubiquitination are shown, mean of ≥4 experiments are shown Significance was determined using 2way ANOVA where * indicates p ≤ 0.05, **** indicates p ≤ 0.0001 and ns not significant. (C) LC3B is recruited to the type II vacuole. HeLa cells stimulated or not with IFNγ for 18h were infected with type I or type II parasites for the indicated times. Recruitment of LC3B was monitored by antibody staining. Graphs indicating the percentage of positive vacuoles are shown for 3 experiments. Significance was determined using 2way ANOVA, *, p ≤ 0.05, ***, p ≤ 0.001, ****, p ≤ 0.0001 and ns, not significant. (D) The type II <i>Toxoplasma</i> vacuole in IFNγ-stimulated HeLa cells does not acquire lysosomal markers and does not acidify. HeLa cells were infected with type I or type II <i>Toxoplasma</i> for the indicated times. LAMP1 staining was recorded and expressed as % positive vacuoles for 3 experiments. Significance was determined using 2way ANOVA, **, p ≤ 0.01 and ns, not significant. (E) Inhibiting acidification has no effect on the ability of type II <i>Toxoplasma</i> to replicate in IFNγ-stimulated HeLa, 6h p.i.. HeLa cells were treated with 10mM NH₄Cl 1h after infection to prevent acidification and infection continued until 6h. Replicating <i>Toxoplasma</i> counts with and without NH₄Cl are shown, mean of ≥3 experiments are shown. Significance was determined using 2way ANOVA, ns = not significant.</p

Model for K63 ubiquitin-dependent endolysosomal destruction of type II <i>Toxoplasma</i> in IFNγ-stimulated HUVEC.

<p>Upon entry into IFNγ-stimulated HUVEC, type II <i>Toxoplasma</i> (green) enveloped within their PV (blue line) are recognised by host immune effectors (K63Ub, p62, NDP52, LAMP1, Rab7) leading to lysosomal fusion and subsequent destruction of the parasite.</p

Autophagy does not control <i>Toxoplasma</i> replication in HUVEC.

<p>(A), (B) and (C) The autophagy proteins LC3B, GABARAP and Atg16L1 localise minimally to both type I and type II <i>Toxoplasma</i> in IFNγ-stimulated HUVEC. Confocal images were taken of HUVEC infected with type II <i>Toxoplasma</i> for 2.5, 4 and 6h, fixed and stained with LC3B, GABARAP or Atg16L1 antibodies. Representative images are shown. Scale bar 10μm. Quantitation of autophagy protein-positive PVs under the indicated conditions is shown. The mean of 3 experiments is shown. Significance was calculated using 2way ANOVA, *, p ≤ 0.05, **, p ≤ 0.01, **** p ≤ 0.0001 and ns, not significant. (D) Electron micrographs of type I and type II <i>Toxoplasma</i> in IFNγ-stimulated HUVEC exhibit no vacuolar disruption or obvious autophagosomal membranes. Ultrastructural analysis was performed in IFNγ-stimulated HUVEC 4h p.i.. Scale bar = 2μm. (E) Knock down of Atg16L1 does not impact IFNγ-mediated restriction of type I and type II <i>Toxoplasma</i> in HUVEC. HUVEC were knocked down for Atg16L1 by nucleofection, incubated for 24h, then stimulated or not with IFNγ for 24h before infecting with type II <i>Toxoplasma</i> for 18h. Results of ≥3 experiments are shown. Parasite numbers/vacuole were counted and significance determined using 2way ANOVA, *, p≤0.05 and ns, not significant. (F) HUVEC were knocked down for Atg16L1 by nucleofection of siRNA, incubated for 24h, then stimulated or not with IFNγ for 24h before infecting with type II <i>Toxoplasma</i> for 18h. Results of 5 experiments are shown. Percentage infected cells were determined in >100 cells by fluorescence microscopy and significance calculated by 2way ANOVA, **, p≤0.001, *, p≤0.05 and ns, not significant. (G) HUVEC were incubated for 24h with 50units/ml IFNγ and 100nM rapamycin, before washing and infecting with type I or type II <i>Toxoplasma</i> for 18h, when the cultures were fixed for microscopy. The number of parasites/vacuole was scored. The mean of 3–4 experiments is shown. Significance was calculated using 2way ANOVA, *, p≤0.05, **, p ≤ 0.01, ****, p ≤ 0.0001 and ns, not significant.</p

p62 and NDP52 are recruited to microdomains on the type II <i>Toxoplasma</i> PV in dependence of ubiquitination.

<p>(A) p62 accumulates specifically at the type II <i>Toxoplasma</i> PV in IFNγ-stimulated HUVEC. Confocal images were taken of IFNγ-stimulated HUVEC infected with type II <i>Toxoplasma</i> for 2.5h, fixed and stained with p62 antibody and a representative image is shown. Scale bar 10μm. Quantitation of p62-positive PVs under the indicated conditions is shown, 2.5h p.i. The mean of 3 experiments is shown. Significance was calculated using 2way ANOVA, ***, p ≤ 0.001. (B) NDP52 accumulates specifically at the type II <i>Toxoplasma</i> PV in IFNγ-stimulated HUVEC. Confocal images were taken of HUVEC infected with type II <i>Toxoplasma</i> for 2.5h, fixed and stained with NDP52 antibody and a representative image is shown. Scale bar 10μm. Quantitation of NDP52-positive PVs under the indicated conditions is shown, 2.5h p.i.. The mean of 3 experiments is shown. Significance was calculated using 2way ANOVA, ****, p≤ 0.0001. (C) p62 and NDP52 occupy overlapping microdomains at the type II <i>Toxoplasma</i> PV in IFNγ-stimulated HUVEC. Superresolution Structured Illumination Microscopy images of 3 representative vacuoles demonstrated mostly a patchy and distinct localisation of p62 and NDP52 with some small overlapping microdomains. Scale bar 2μm. (D) Host defence proteins p62 and NDP52 are recruited to the PV subsequent to ubiquitin binding to the PV. Ubiquitin accumulates on the type II PV within 30min-1h p.i., reaching a maximum already at 1h. p62 sequentially follows the ubiquitin recruitment, with a maximum at 2–4h p.i. and NDP52, appearing last to maximum levels at 2h p.i and is maintained until at least 4h p.i. Quantitation of recruitment-positive type II PVs, at the indicated time-points p.i. is shown. The mean of ≥3 experiments is shown. (E) p62 and NDP52 recruitment to the PV depends upon the ubiquitination of the PV. Inhibition of ubiquitination in HUVEC with the E1 inhibitor UBEI-41 leads to a reduction in ubiquitin as well as p62 and NDP52 at the vacuole of type II <i>Toxoplasma</i>. IFNγ-stimulated HUVEC were pre-incubated with 50μM UBEI-41 for 2h before washing and infecting with type II <i>Toxoplasma</i> for 2.5h. Cells were stained for ubiquitin, p62, NDP52 and positive staining recorded. The mean of ≥3 experiments is shown. Significance was calculated using 2way ANOVA, *, p ≤ 0.05, ****, p ≤ 0.0001 and ns, not significant. (F) Knockdown of p62 by siRNA leads to the loss of IFNγ-restriction of type II <i>Toxoplasma</i>. siRNA nucleofection of p62 was used to knock down the protein in HUVEC, comparing to a control siRNA. 24h after siRNA nucleofection, the cells were stimulated with 50units/ml IFNγ for a further 24h. Infection with type II <i>Toxoplasma</i> was then allowed to proceed for 18h and the effect on replication recorded in fixed cells by scoring the numbers of parasites/vacuole in >100 vacuoles. Significance was determined by 2way ANOVA, ***, p ≤ 0.001 and ns, not significant.</p

IFNγ-mediated K63-linked ubiquitination of type II <i>Toxoplasma</i> vacuoles.

<p>(A) Replication of both type I and type II <i>Toxoplasma</i> is diminished in IFNγ-stimulated HUVEC at 6h and more substantially at 24h post invasion. HUVEC were infected (-/+ stimulation with 50 units/ml IFNγ for 18h) with type I or type II parasites for 6h and 24h. Results from ≥3 experiments are shown. Significance was calculated using 2way ANOVA, *, p≤0.05, **, p≤0.01, ***, p≤0.001 and ns, not significant. (B) Ubiquitin is recruited to the PV of type II <i>Toxoplasma</i> in HUVEC. HUVEC were stimulated with 50 units/ml IFNγ for 18h before infecting with type II <i>Toxoplasma</i> for 2.5h. Confocal image of a representative vacuole is shown. Scale bar 10μm. The ubiquitin staining is mainly at the PV and not substantially on the parasite. 3D surface profile (Image J) of the pixel intensity over a ubiquitin-stained vacuole containing type II <i>Toxoplasma</i>, on a confocal section. Ubiquitin intensity (red) and parasite (green) are shown. (C) Ubiquitin around the type II <i>Toxoplasma</i> vacuole is mostly of the K63 linkage. Antibodies specific for ubiquitin linkages K63, K48 and M1 linear were used alongside an antibody staining total ubiquitin. Quantitation of the indicated linkage-specific ubiquitin-positive PVs, at 2.5h p.i., is shown. The mean of 3 experiments is shown. Significance was calculated using 2way ANOVA, **, p≤0.01, ****, p≤ 0.0001 and ns, not significant. (D) K63-linked and M1-linear ubiquitin accumulated at the type II <i>Toxoplasma</i> PV. Confocal images were taken of HUVEC stimulated with 50 units/ml IFNγ for 18h, before infecting with type II <i>Toxoplasma</i> for 2.5h. Infected cells were then fixed and stained with linkage-specific ubiquitin antibodies. Representative images are shown. Scale bar 10μm.</p

K63-ubiquitin-dependent endo-lysosomal acidification of <i>Toxoplasma</i> PV inhibits parasite replication.

<p>(A) Acidification of the PV is dependent on IFNγ and <i>Toxoplasma</i> virulence type. Type II <i>Toxoplasma</i> PV acidifies in IFNγ-stimulated HUVEC. Representative confocal image of infected cell stained with LAMP-1 is shown 4h p.i.. Scale bar = 10 μm. Quantitation of LAMP1 positive PVs under the indicated conditions is shown. The mean of ≥3 experiments is shown. Significance was determined using 2way ANOVA, **, p≤ 0.01, ****, p≤ 0.0001 and ns, not significant. (B) Representative confocal image of an infected cell stained with LysoTracker is shown 3h p.i.. Scale bar 10 μm. (C) Ubiquitinated type II <i>Toxoplasma</i> are destined for acidification in IFNγ-stimulated HUVEC. Representative confocal image showing K63-ubiquitin antibody co-staining with LAMP-1 antibody or LysoTracker, 4h p.i. Scale bar 10μm. (D) Inhibiting ubiquitination and acidification restores type II <i>Toxoplasma’s</i> ability to replicate in IFNγ-stimulated HUVEC. HUVEC were stimulated or not with IFNγ for 18h before being treated with 50μM UBEI-41 for 2h to inhibit host E1. Cells were then washed prior to infection with type II <i>Toxoplasma</i> for 6h. Replicating <i>Toxoplasma</i> counts with and without E1 inhibitor are shown, mean of ≥3 experiments. Significance was determined using 2way ANOVA, where *** indicates p ≤ 0.001. HUVECs were treated with 10mM NH₄Cl 1h after infection with type II <i>Toxoplasma</i> to prevent acidification and infection continued in the presence of NH₄Cl until 24h. Replicating <i>Toxoplasma</i> counts with and without NH₄Cl are shown as parasites/vacuole, mean of 3 experimental replicates. Significance was determined using 2way ANOVA, ****, p ≤ 0.0001. (E) HUVEC were stimulated or not with IFNγ for 18h before being treated with 50μM UBEI-41 for 2h to inhibit host E1. Cells were then washed prior to infection with type II <i>Toxoplasma</i> for 18h. Percentage infected cells were counted in >100 cells. The mean of 4 experiments is shown. Significance was determined using 2way ANOVA, **, p ≤ 0.01 and ns, not significant. (F) HUVEC were stimulated or not with IFNγ for 18h before being treated with 50μM UBEI-41 for 2h to inhibit host E1. Cells were then washed prior to infection with type II <i>Toxoplasma</i> for 18h. Infected cells were prepared and fixed for FACS and cells containing fluorescent parasites scored as a measure parasite survival. Results are expressed as percentage infected cells. A representative of 3 experiments is shown, each performed in triplicate. Significance was determined using unpaired student t test, **, p ≤ 0.01, *, p ≤ 0.05 and ns, not significant. (G) Inhibiting ubiquitination decreases the presence of LysoTracker-positive structures in IFNγ-stimulated HUVEC infected with type II <i>Toxoplasma</i> 6h p.i.. Confocal image of infected cells stained with LysoTracker in the presence or absence of the E1 inhibitor UBEI-41. Scale bar 10 μm.</p

Eosinophils are required for clearance of <i>B. malayi</i> Mf during primary (1°), but not challenge (2°) infection.

<p><b>A.</b> Mf survival in groups of PHIL (black squares) and C57Bl/6 (open circles) mice, following primary (dotted line) and secondary (solid line) infection. Mf were counted in blood from the tail vein of mice on days 6 and 12 p.i. or in cardiac blood on day 21 p.i. (d21 PHIL mice 1° versus C57Bl/6 1° p = 0.0018) <b>B–D.</b> Cell recruitment into BALF of naïve PHIL and C57Bl/6 mice and both mouse strains mice given primary or challenge Mf infections on day 21 p.i. <b>B.</b> Mean total and differential cell recruitment into BALF. <b>C.</b> Numbers of mast cells recruited into BALF (Mean ± S.E) of naïve and infected PHIL and C57Bl/6 mice. <b>D.</b> Numbers of eosinophils recruited into BALF (Mean ± S.E) of naïve and infected PHIL and C57Bl/6 mice. <b>E.</b> Mf-specific IgG1 antibody in serum during infection of PHIL and C57Bl/6 mice (Mean ± S.E.) (dotted line represents naïve levels in both PHIL and C57Bl/6). <b>F.</b> Total IgE antibody in serum during infection of PHIL and C57Bl/6 mice (Mean ± S.E.). <b>G–I.</b> Cytokines produced by splenocytes upon stimulation with Mf antigen and measured in cell culture supernatants by ELISA 72 h later. Graphs show mean ± S.E. cytokine concentration of naïve PHIL and C57Bl/6 mice and mice 21 days post primary (1°) and challenge (2°) infections of live Mf. <b>G.</b> IFN-γ responses <b>H.</b> IL-5 responses. <b>I.</b> IL-13 responses. This figure represents data from two independent experiments with 6 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 ****p<0.0001 between groups of PHIL mice and C57Bl/6 mice given the same infection regimen.</p

MBP-1 is not required for Mf survival but contributes to pulmonary eosinophil recruitment and goblet cell mucus production following Mf infection.

<p><b>A.</b> Mf survival in MBP-1^−/− (black squares) and C57Bl/6 (open circles) mice following primary (dotted line) and challenge (solid line) infection. <b>B.</b> Mean total and differential cell recruitment to BALF in MBP-1^−/− and C57Bl/6 mice given primary or challenge Mf infections. <b>C.</b> Lung function (Penh) on day 12 post live Mf challenge showing mean ± S.E. Penh. <b>D.</b> Mucus-secreting goblet cells in 6 µm sections of lung, stained with Periodic Acid Schiff (magnification ×40). The graph shows mean ± S.E. percentage of positively staining cells per airway at day 20 post live Mf infection. <b>A–D.</b> These graphs represent data from two independent experiments with 4 individual mice per group. *represents a significant difference at p<0.05, ** p<0.01 between groups of gene-targeted mice and C57Bl/6 mice given the same infection regimen.</p