<i>L. monocytogenes</i> causes calcium-independent LLO-mediated mast cell degranulation with delayed kinetics.

<p>(A and B) BMMC were incubated for the times indicated with (A) media alone (shaded) or WT <i>L. monocytogenes</i> at the MOI indicated (black line); IgE-loaded cells were unstimulated (shaded) or stimulated with 10 ng/ml antigen (black line); (B) WT or <i>Δhly L. monocytogenes</i> at the MOI indicated. (C) BMMC were incubated for 2 h alone (shaded), or with WT or <i>Δhly L. monocytogenes</i> at MOI 100:1 (black line) in media or calcium-free buffer. IgE-loaded cells were stimulated with 10 ng/ml antigen or PMA/I. CD107a expression levels were determined by flow cytometry. Data are (A and C) representative of 3 independent experiments; (B) mean +/- SEM for 3 independent experiments shown relative to media alone.</p

<i>L. monocytogenes</i> infects mast cells at low levels and survives intracellularly for up to 24 h.

<p>(A) BMMC were incubated with WT or <i>Δhly</i> bacteria for 2 h at MOI 10∶1, washed then transferred to fresh media containing 50 µg/ml gentamicin for 24 h. Cells were stained with anti-<i>Listeria</i> (green), wheat germ agglutinin (red) and DAPI (blue) and analysed by confocal microscopy. Intracellular whole bacteria (unbroken arrow) and bacterial fragments (broken arrow) are indicated. (B) BMMC were incubated with WT, <i>Δhly</i> or EGDe bacteria for 2 h at MOI 1∶1 and 100∶1, washed then transferred to fresh media containing 50 µg/ml gentamicin for the times indicated. At each time point BMMC were washed, lysed and viable intracellular bacteria determined. nd, non detected. Data are (A) representative images of mast cells showing intracellular bacteria from a single experiment; (B) the mean +/- SD from 2 independent experiments.</p

<i>L. monocytogenes</i> causes LLO-mediated downregulation of CD117 but not FcεRI on BMMC and decreases chemotaxis.

<p>(A, B and C) BMMC were incubated with WT or <i>Δhly</i> bacteria at the MOI indicated for (A) the times indicated; (B and C) 2 h, then washed and transferred to media containing 50 µg/ml gentamicin for an additional 24 h. Cells were analysed at the time points indicated. CD117 and FcεRI levels were determined by flow cytometry and are shown relative to untreated cells. (D and E) BMMC were incubated with or without WT bacteria at the MOI 10∶1 for 2 h prior transfer to the top section of a transwell. Cells were incubated in the transwells for 4 h in the presence or absence of 10 ng/ml SCF in the bottom compartment. Migration was assessed by counting number of cells per 1000 beads by flow cytometry (D). (E) CD117 levels were determined by flow cytometry in cells incubated in media alone (shaded) or with 10:1 MOI WT bacteria (Black line) for cells incubated with media or in the presence of 10 ng/ml SCF. Data are the mean +/- SEM for 3 (A, B and C); 7 (D); 4 (E) independent experiments. Statistical significance p<0.05 compared to media treatment is indicated (*).</p

<i>L. monocytogenes</i> causes the release of various cytokines and chemokines from BMMC.

<p>BMMC were incubated with media alone or WT <i>L. monocytogenes</i> at MOI 100:1 for 2 h, washed and transferred to media containing 50 µg/ml gentamicin for an additional 24 h. Cell supernatants were harvested after the initial 2 h and the additional 24 h and mediator release determined by Bio-plex. Concentrations of (A) TNF-α; (B) IL-6 (light grey bars), CCL2 (open bars), CCL3 (dark grey bars) and CCL4 (black bars); (C) IL-2 (light grey bars), GM-CSF (dark grey bars) and CCL5 (open bars); (D) the mediators indicated at 2 h and 24 h in media alone (light grey bars and dark grey bars, respectively) or with <i>Listeria</i> (open bars and black bars, respectively). Undetectable levels are indicated (ND). Data are mean +/- SEM for 4 independent experiments.</p

BMMC show mediator release in response to bacterial PAMPs and <i>L. monocytogenes</i> in an LLO-dependent manner.

<p>(A, B, C, D and F) BMMC were incubated with media alone, WT or <i>Δhly L. monocytogenes</i> at the MOI indicated for 2 h, washed and transferred to media containing 50 µg/ml gentamicin for an additional 24 h. Cell supernatants were harvested after the initial 2 h (open bars) and the additional 24 h (grey bars). (E) BMMC were incubated with media alone, 100 ng/ml LPS or 10 µg/ml PGN for 24 h. (B) IL-6, (C) CCL2, (D) TNF-α, and (E and F) OPN concentrations were determined by ELISA, with values below the limit of detection indicated (ND). Data are mean +/- SEM for (B) 5, (C) 4, (D) 4, (E) 8, and (F) 3 independent experiments. Statistical significance p<0.05 compared to media treatment is indicated (*).</p

<i>L. monocytogenes</i> strain with a modified InlA causes the release of higher concentration of IL-6 and CCL2.

<p>(A and B) BMMC were incubated with media alone, WT or EGDe <i>L. monocytogenes</i> at the MOI indicated for 2 h, washed and transferred to media containing 50 µg/ml gentamicin for an additional 24 h. Cell supernatants were harvested after the initial 2 h (open bars) and the additional 24 h (grey bars). (A) IL-6 and (B) CCL2 concentrations were determined by ELISA. Data are mean +/- SEM. Statistical significance between WT and EGDe treated at p<0.05 (*) and p<0.01 (**) is indicated.</p

ILC2s mediate systemic innate protection by priming mucus production at distal mucosal sites

Host immunity to parasitic nematodes requires the generation of a robust type 2 cytokine response, characterized by the production of interleukin 13 (IL-13), which drives expulsion. Here, we show that infection with helminths in the intestine also induces an ILC2-driven, IL-13-dependent goblet cell hyperplasia and increased production of mucins (Muc5b and Muc5ac) at distal sites, including the lungs and other mucosal barrier sites. Critically, we show that type 2 priming of lung tissue through increased mucin production inhibits the progression of a subsequent lung migratory helminth infection and limits its transit through the airways. These data show that infection by gastrointestinal-dwelling helminths induces a systemic innate mucin response that primes peripheral barrier sites for protection against subsequent secondary helminth infections. These data suggest that innate-driven priming of mucus barriers may have evolved to protect from subsequent infections with multiple helminth species, which occur naturally in endemic areas

Listeria monocytogenes alters mast cell phenotype, mediator and osteopontin secretion in a listeriolysin-dependent manner

Whilst mast cells participate in the immune defence against the intracellular bacterium Listeria monocytogenes, there is conflicting evidence regarding the ability of L. monocytogenes to infect mast cells. It is known that the pore-forming toxin listeriolysin (LLO) is important for mast cell activation, degranulation and the release of pro-inflammatory cytokines. Mast cells, however, are a potential source of a wide range of cytokines, chemokines and other mediators including osteopontin, which contributes to the clearing of L. monocytogenes infections in vivo, although its source is unknown. We therefore aimed to resolve the controversy of mast cell infection by L. monocytogenes and investigated the extent of mediator release in response to the bacterium. In this paper we show that the infection of bone marrow-derived mast cells by L. monocytogenes is inefficient and LLO-independent. LLO, however, is required for calcium-independent mast cell degranulation as well as for the transient and selective downregulation of cell surface CD117 (c-kit) on mast cells. We demonstrate that in addition to the key pro-inflammatory cytokines TNF-α and IL-6, mast cells release a wide range of other mediators in response to L. monocytogenes. Osteopontin, IL-2, IL-4, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF), and chemokines including CCL2, CCL3, CCL4 and CCL5 are released in a MyD88-dependent manner. The wide range of mediators released by mast cells in response to L. monocytogenes may play an important role in the recruitment and activation of a variety of immune cells in vivo. The cocktail of mediators, however, is unlikely to skew the immune response to a particular effector response. We propose that mast cells provide a hitherto unreported source of osteopontin, and may provide an important role in co-ordinating the immune response during Listeria infection