Deficiency in ST2 leads to pronounced microfilaremia and increased adult worm length.

<p>A, microfilariae count per 50 μl of peripheral blood of ST2-ko mice and wild type (WT) controls throughout <i>L. sigmodontis</i> infection. B, microfilarial burden in the thoracic cavity 60 days post <i>L. sigmodontis</i> infection. C, percentage of ST2-ko mice and WT controls that develop patent infections. D, embryogram of female worms 60dpi (6 mice per group and two female worms per mouse). E, adult worm burden in ST2-ko mice and WT controls 35, 60 and 100 dpi, (F and G) length of male and female <i>L. sigmodontis</i> worms in WT and ST2-ko mice during infection. A and C show pooled data from three independent experiments with a minimum of 8 mice per group. B shows pooled data from two independent experiments and E-G show representative data of two independent experiments with a minimum of 6 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05.</p

Anti-bacterial effector mechanisms are enhanced by <i>L. sigmodontis</i> infection in a TLR2 dependent manner.

<p>(<b>A</b>) Colony forming units (cfu) obtained by a gentamycin assay using peritoneal macrophages derived from chronic <i>L. sigmodontis</i> (<i>L.s.</i>)-infected wild type and TLR2^-/- mice and respective uninfected controls three hours after i.p. <i>E. coli</i> injection. (<b>B</b>) Nitrite concentrations of the same macrophages as in (<b>A</b>) after ex vivo cultivation for 48 hours. Frequency of peritoneal F4/80^-positive macrophages from <i>L.s.-</i>infected and uninfected wild type and TLR2^-/- mice (n = 5 per group) that phagocytosed pHrodo <i>E. coli</i>-BioParticles 90 minutes post injection (<b>C</b>) or from <i>L.s.</i>-infected and uninfected wild type mice (n = 5 per group) that phagocytosed pHrodo <i>S. aureus</i>-BioParticles three hours post injection (<b>D</b>). (<b>A</b>) Pooled data from two independent experiments with at least four mice per group. Data shown in (<b>A-C</b>) is illustrated as mean + SEM and was tested for statistical significance by 1-way ANOVA followed by Dunn’s multiple comparisons test; Data in (<b>D</b>) is also shown as mean + SEM and was tested for statistical significance by Mann-Whitney U test. *p< 0.05; **p< 0.01; ***p<0.001.</p

ST2-ko mice have a delayed splenic clearance of transferred microfilariae.

<p>Kinetics of blood microfilariae numbers in ST2-ko mice and wild type (WT) controls after i.v. inoculation of 50,000 microfilariae (A). Blood microfilariae counts in splenectomized and sham-treated ST2-ko mice and WT controls one hour after inoculation with 50,000 microfilariae per mouse (B). Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05. Representative data shown in (A) is from two independent experiments with at least 6 mice per group.</p

Reduced Th2 cytokine production after in vitro restimulation of thoracic cavity cells of acute, but not chronically <i>L. sigmodontis</i> infected ST2-ko mice.

<p>Isolated cells from the thoracic cavity of individual <i>L. sigmodontis</i> infected wild type (WT) or ST2-ko mice were cultured in vitro with either <i>L. sigmodontis</i> antigen (LsAg, left panel) or ConA (right panel). IL-4 (A,B), IL-5 (C,D), IL-10 (E,F), IFNγ (G,H), IL-33 (I,J) and IL-25 (K,L) within the culture supernatants were measured on days 35, 60 or 100 post infection. Data is representative for two independent experiments with at least 5 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05, **p<0.01.</p

ST2-ko mice have reduced thoracic cavity cell numbers throughout <i>L. sigmodontis</i> infection.

<p>Cell populations within the thoracic cavity were assessed by flow cytometry in wild type (WT) and ST2-ko mice prior to infection (day 0, only Fig. 6A) and after 35, 60 and 100 days post <i>L. sigmodontis</i> infection (Fig. 6B–F). Total number of cells within the thoracic cavity lavage (A), macrophages (B), CD4⁺ T-cells (C), B-cells (D), eosinophils (E) and neutrophils (F) are shown. Data shown is representative for two independent experiments with at least 5 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test, *p<0.05, **p<0.01.</p

Absence of the ST2 receptor does not change thoracic cavity IL-4, IL-5, IL-13, IL-25, IL-33 and IFNγ levels during <i>L. sigmodontis</i> infection.

<p>Local concentrations of IL-4 (A), IL-5 (B), IL-13 (C), IL-25 (D), IL-33 (E) and IFNγ (F) in the thoracic cavity lavage prior to infection (day 0) and 35, 60, and 100 days post <i>L. sigmodontis</i> infection of wild type (WT) and ST2-ko mice. Data is representative for two independent experiments with at least 5 mice per group. Differences were tested for statistical significance by Mann-Whitney-U-test.</p

Chronic Filarial Infection Provides Protection against Bacterial Sepsis by Functionally Reprogramming Macrophages

<div><p>Helminths immunomodulate their hosts and induce a regulatory, anti-inflammatory milieu that prevents allergies and autoimmune diseases. Helminth immunomodulation may benefit sepsis outcome by preventing exacerbated inflammation and severe pathology, but the influence on bacterial clearance remains unclear. To address this, mice were chronically infected with the filarial nematode <i>Litomosoides sigmodontis (L.s.)</i> and the outcome of acute systemic inflammation caused by i.p. <i>Escherichia coli</i> injection was determined. L.s. infection significantly improved <i>E. coli</i>-induced hypothermia, bacterial clearance and sepsis survival and correlated with reduced concentrations of associated pro-inflammatory cytokines/chemokines and a less pronounced pro-inflammatory macrophage gene expression profile. Improved sepsis outcome in <i>L.s.</i>-infected animals was mediated by macrophages, but independent of the alternatively activated macrophage subset. Endosymbiotic Wolbachia bacteria that are present in most human pathogenic filariae, as well as <i>L.s.</i>, signal via TLR2 and modulate macrophage function. Here, gene expression profiles of peritoneal macrophages from <i>L.s.</i>-infected mice revealed a downregulation of genes involved in TLR signaling, and pulsing of macrophages in vitro with <i>L.s.</i> extract reduced LPS-triggered activation. Subsequent transfer improved sepsis outcome in naïve mice in a <i>Wolbachia</i>- and TLR2-dependent manner. In vivo, phagocytosis was increased in macrophages from <i>L.s.</i>-infected wild type, but not TLR2-deficient animals. In association, <i>L.s.</i> infection neither improved bacterial clearance in TLR2-deficient animals nor ameliorated <i>E. coli</i>-induced hypothermia and sepsis survival. These results indicate that chronic <i>L.s.</i> infection has a dual beneficial effect on bacterial sepsis, reducing pro-inflammatory immune responses and improving bacterial control. Thus, helminths and their antigens may not only improve the outcome of autoimmune and allergic diseases, but may also present new therapeutic approaches for acute inflammatory diseases that do not impair bacterial control.</p></div

ELD mice display a strong type 2 immune response and eosinophilia in the lung.

(A) Experimental design (created with BioRender.com). Wild-type ELD and eosinophil-deficient dblGATA ELD mice were sensitized with dead MF and challenged with viable MF two weeks later. Controls received solely the MF challenge (MF) or remained naïve. Analyses were performed ten days after MF challenge. (B) Total cell count per g of lung tissue for naïve, MF-only challenged, as well as wild-type ELD and dblGATA ELD mice. Frequencies of (C) ILC2s (CD45+, linage-, TCRb-, CD90.2+, ST2+, GATA3+), (D) RELM-α positive macrophages (CD45+, CD206+, Siglec-F+, RELM-α+), (E) eosinophils (CD45+, CD11c-, Siglec-F+, CD11b+), (F) neutrophils (CD45+, Ly6G+), and (G) alveolar macrophages (CD45+, CD206+, Siglec-F+) in the CD45+ lung cell fraction. Data is pooled from 1–3 independent experiments with n = 6–18 animals per group. Data is shown as median with interquartile range. Statistical analysis was performed with Kruskal-Wallis followed by Dunn´s multiple comparison test. P values ≤ 0.05 are shown.</p

Lung eosinophils are highly activated during ELD.

Geometric mean of the fluorescence intensity (gMFI) of (A) CD11b, (B) Siglec-F, (C) CD86 and (D) ST2/IL-33R of lung eosinophils isolated from wild-type ELD mice, mice solely challenged with MF (MF) or naïve animals ten days after MF challenge. (E-H) Representative histograms comparing eosinophils from naïve (grey) and ELD mice (red). (A-D) Data is pooled from 1–3 independent experiments with n = 5–13 mice per group. Data is shown as median with interquartile range. Statistical analysis was performed with Kruskal-Wallis followed by Dunn´s multiple comparison test. p values ≤ 0.05 are shown.</p

Tissue remodeling and eosinophil-associated granular proteins and chemokines are reduced after HpARI2 treatment.

A) EPO, (B) MBP, (C) CCL5, (D) CCL11 and (E) amphiregulin levels in lung tissue homogenates ten days after MF challenge. (F) MMP activity assay from lung homogenates. Data shown is from one experiment with n = 4–9 animals per group and presented as median with interquartile range. Statistical analysis was performed with Kruskal-Wallis followed by Dunn´s multiple comparison test. p values ≤ 0.05 are shown.</p