Immune modulation by experimental filarial infection and its impact on <em>E. coli</em>-induced sepsis

Helminths cause so-called neglected tropical diseases in tropical and sub-tropical regions and are prevalent in almost one third of mankind. Thus, co-infections of helminths with other pathogens are common. However, the effects of helminths on outcomes of infections with unrelated pathogens like bacteria are rather poorly understood and underrepresented in biomedical research. In this thesis it was investigated, how chronic filarial infection influences acute bacterial challenge infections. To achieve this, mice chronically infected with the filarial nematode Litomosoides sigmodontis (L.s.) were intraperitoneally challenged with the gram-negative bacterium Escherichia coli. Sepsis severity was determined by survival, development of hypothermia, systemic pro-inflammatory cytokine and chemokine levels. Clearance of bacteria and recruitment of immune cells to the peritoneum were determined 6 hours after bacterial challenge. The role of nematode-induced immune cell populations as regulatory T cells, eosinophils and macrophages and their receptors (e.g. Toll-like receptor 2, IL-4 receptor) were investigated using various gene-deficient mouse strains. In order to further elucidate the protective mechanisms, in vitro studies and adoptive cell transfers were performed. This thesis demonstrates that chronic infection with the filarial nematode L. sigmodontis provides a significant survival benefit to E. coli-induced sepsis in mice. This was accompanied by attenuated hypothermia and reduced systemic cytokine/chemokine secretion. Chronically L.s.-infected mice displayed an improved bacterial control and increased recruitment of neutrophils and eosinophils, which was accompanied by a reduced activation and apoptosis of peritoneal macrophages. Depletion of macrophages by Clodronate liposomes indicated a protective role of macrophages in the L.s.-mediated protection against E. coli-induced sepsis. L.s. infection induced RELMα expression on peritoneal macrophages in wildtype BALB/c mice following E. coli challenge, indicating a possible switch to an alternatively activated macrophage (AAM) phenotype. However, L.s.-infected IL-4Rα/IL-5-/- and IL-4-/- mice that were devoid of AAM were still protected from E. coli sepsis. These experiments suggest that the presence of macrophages is necessary, but the induction of an AAM phenotype is not required to improve sepsis outcome by L.s. infection. Most filarial species have endosymbiotic Wolbachia bacteria that activate innate cells and reduce subsequent responses to innate stimuli via Toll-like receptor 2 (TLR2). In vitro stimulation with Wolbachia-containing preparations in wildtype but not TLR2-deficient macrophages reduced TNFα secretion following LPS-restimulation. These macrophages showed enhanced phagocytosis and uptake of bacteria and produced more bactericidal nitric oxide in a TLR2 dependent manner. Accordingly, the protective effect of chronic L.s. infection was lost in TLR2-deficient mice promoting a concept of Wolbachia- and TLR2-mediated immune modulation. Moreover, repeated injections of L.s. and Wolbachia-derived preparations improved sepsis outcome in a TLR2-dependent manner. Finally, macrophage transfer experiments demonstrated that macrophages from L.s.-infected mice improved sepsis outcome of recipient mice, whereas macrophages from L.s.-infected TLR2-/- mice and naïve BALB/c mice did not significantly improve sepsis outcome in recipient mice. This thesis provides immunologic insight to the complex interplay of filarial and bacterial co-infection and demonstrates a filariae- and Wolbachia-induced mechanism that protects mice via a dual beneficial effect on phagocytes, which permits improved containment of bacteria and reduced systemic inflammation. This may help to find new therapeutic interventions to prevent severe sepsis also in human patients

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

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

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

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

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

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

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

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