IL-27 induces an IFN-like signature in murine macrophages which in turn modulate colonic epithelium

Mucosal delivery of IL-27 has been shown to have a therapeutic benefit in murine models of inflammatory bowel disease (IBD). The IL-27 effect was associated with phosphorylated STAT1 (pSTAT1), a product of IL27 receptor signaling, in bowel tissue. To determine whether IL-27 acted directly on colonic epithelium, murine colonoids and primary intact colonic crypts were shown to be unresponsive to IL-27 in vitro and to lack detectable IL-27 receptors. On the other hand, macrophages, which are present in inflamed colon tissue, were responsive to IL-27 in vitro. IL-27 induced pSTAT1 in macrophages, the transcriptome indicated an IFN-like signature, and supernatants induced pSTAT1 in colonoids. IL-27 induced anti-viral activity in macrophages and MHC Class II induction. We conclude that the effects of mucosal delivery of IL-27 in murine IBD are in part based on the known effects of IL27 inducing immunosuppression of T cells mediated by IL-10. We also conclude that IL-27 has potent effects on macrophages in inflamed colon tissue, generating mediators that in turn act on colonic epithelium

The phorbol 12-myristate-13-acetate differentiation protocol is critical to the interaction of THP-1 macrophages with Salmonella Typhimurium.

THP-1 cells differentiated with phorbol 12-myristate 13-acetate (PMA) are widely used as a model for function and biology of human macrophages. However, the conditions used for differentiation, particularly the concentration of PMA and the duration of treatment, vary widely. Here we compare several differentiation conditions and compare the ability of THP-1 macrophages to interact with the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The results show that THP-1 macrophages differentiated in high concentrations of PMA rapidly died following infection whereas those differentiated in low concentrations of PMA survived and were able to control the intracellular bacteria similar to primary human macrophages

Predictable, Tunable Protein Production in Salmonella for Studying Host-Pathogen Interactions

Here we describe the use of synthetic genetic elements to improve the predictability and tunability of episomal protein production in Salmonella. We used a multi-pronged approach, in which a series of variable-strength synthetic promoters were combined with a synthetic transcriptional terminator, and plasmid copy number variation. This yielded a series of plasmids that drive uniform production of fluorescent and endogenous proteins, over a wide dynamic range. We describe several examples where this system is used to fine-tune constitutive expression in Salmonella, providing an efficient means to titrate out toxic effects of protein production

Surface receptor expression and cell morphology are dependent on differentiation conditions.

<p>(A) Representative bright field images of THP-1 cells differentiated for 1, 2, or 5 days in the presence of PMA (200 ng/mL). Scale bar is 100 μm. (B) Representative flow cytometric analysis of THP-1 cells stained using anti- CD11b or -CD14. (C) Quantification of mean fluorescence intensity (MFI) for CD11b (black) and CD14 (white). Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p

Replication of <i>Salmonella</i> in differentiated THP-1 cells.

<p>(A) THP-1 cells differentiated in PMA (200 ng/mL) for 2 days or 5 days and infected with mCherry-<i>Salmonella</i> (red) were stained with DAPI (blue) at 1 and 24 h pi. Scale bars are 25 and 5 μm (inset). (B, C) Intracellular growth of <i>Salmonella</i> in THP-1 cells differentiated 2 days (open circles) or 5 days (closed circles) at 1, 6, 12, and 24 h pi. (C) Growth normalized to 1 h pi. Data are means ± SD from three independent experiments. Statistical significance was determined using 2-way ANOVA with Sidak post hoc test.</p

LPS induced cell death in differentiated THP-1 cells.

<p>(A) Quantification of percentage of propidium iodide positive THP-1 cells differentiated in the presence of PMA (200 ng/mL) for 2 days (white circles) or 5 days (black circles) following internalization of <i>Salmonella</i> or <i>E</i>. <i>coli</i> DH5α or a 30 min treatment with LPS (10 ng/mL). Data shown as individual experimental means. Statistical significance was determined as compared to uninfected controls using 2-way ANOVA with Tukey’s post hoc test. (B) Representative bright field images of THP-1 cells stained with propidium iodide (red) 120 min after internalization of <i>Salmonella</i> or <i>E</i>. <i>coli</i> or treatment with LPS. Scale bar 15 μm. (C) THP-1 cells were treated with DMSO or staurosporine (1 μM) for 3 h, infected with <i>Salmonella</i> or <i>E</i>.<i>Coli</i> for 30 min, or treated with LPS (10 ng/mL) for 30 min. At 2 h post-treatment cells were incubated with poly-caspase FAM-VAD-FMK before fixation, staining, and analysis. Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p

<i>Salmonella</i> are killed by THP1 cells and primary human macrophage cells.

<p>(A) Intracellular <i>Salmonella</i> in THP-1 cells differentiated for 3 days in the presence of 20 ng/mL PMA (open circles) or primary human monocyte derived macrophages (MDMs, closed circles) differentiated for 7 days in the presence of 100 ng/mL MCSF. Growth normalized to 1 h pi. Data are means ± SD from three independent experiments. (B) Cytokine expression as measured by ELISA for TNF-α, IL-8, and IL-12p40 in THP-1 cells (white bar) and human macrophages (black bars) in uninfected and <i>Salmonella</i>-infected cells at 6 and 24 h pi. Representative plots from three independent experiments. Statistical significance was determined as compared to the uninfected control for each strain using an unpaired <i>t</i>-test. (C) Representative maximum intensity projection images of <i>Salmonella</i> (red) infected MDMs and THP-1 cells differentiated in the presence of either 200 ng/mL PMA (2 day and 5 day) or 20 ng/mL PMA (3 day) at 1 h pi and stained for LAMP1 (green). Scale bars are 15μm and 5uM (insert). (D) Quantification of LAMP1 positive <i>Salmonella</i> in MDMs (red circles) and THP1 cells differentiated for 2 days (open circles), 3 days (grey squares), and 5 days (closed circles). Data are means ± SD from three independent experiments.</p

THP-1 surface receptor expression in cells differentiated with 20 ng/mL PMA.

<p>(A) Representative flow cytometric analysis of THP-1 cells stained using anti- CD11b or -CD14 antibodies. THP-1 cells were differentiated for 2 days (red), 3 days (blue), and 4 days (green) in the presence of PMA (20 ng/mL). (B) Quantification of mean fluorescence intensity (MFI) for CD11b (black) and CD14 (white). Data are means ± SD from three independent experiments. Statistical significance was determined using 1-way ANOVA with Dunnett post hoc test.</p

Cell death of <i>Salmonella</i>-infected THP1 cells is determined by PMA concentration.

<p>(A) Representative bright field images of THP-1 cells differentiated for 3 days in the presence of 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA, as described in Material and Methods. Scale bar is 100 μm. (B) Quantification of percentage of propidium iodide positive THP-1 cells differentiated for 2 days, 3 days, and 4 days at either 20 ng/mL, 50 ng/mL, 100 ng/mL, or 200 ng/mL PMA following infection with <i>Salmonella</i> (filled circle) for 2 h as compared to uninfected (open circles). Data are means ± SD from three independent experiments. Statistical significance was determined using 2-way ANOVA with Sidak post hoc test. (C) Representative images of differentiated THP-1 cells stained with propidium iodide (red) 2 h pi with <i>Salmonella</i> (bottom panel) or uninfected (top panel). Scale bar 25μm.</p

A second wave of <i>Salmonella</i> T3SS1 activity prolongs the lifespan of infected epithelial cells

<div><p>Type III secretion system 1 (T3SS1) is used by the enteropathogen <i>Salmonella enterica</i> serovar Typhimurium to establish infection in the gut. Effector proteins translocated by this system across the plasma membrane facilitate invasion of intestinal epithelial cells. One such effector, the inositol phosphatase SopB, contributes to invasion and mediates activation of the pro-survival kinase Akt. Following internalization, some bacteria escape from the <i>Salmonella</i>-containing vacuole into the cytosol and there is evidence suggesting that T3SS1 is expressed in this subpopulation. Here, we investigated the post-invasion role of T3SS1, using SopB as a model effector. In cultured epithelial cells, SopB-dependent Akt phosphorylation was observed at two distinct stages of infection: during and immediately after invasion, and later during peak cytosolic replication. Single cell analysis revealed that cytosolic <i>Salmonella</i> deliver SopB via T3SS1. Although intracellular replication was unaffected in a SopB deletion mutant, cells infected with Δ<i>sopB</i> demonstrated a lack of Akt phosphorylation, earlier time to death, and increased lysis. When SopB expression was induced specifically in cytosolic <i>Salmonella</i>, these effects were restored to levels observed in WT infected cells, indicating that the second wave of SopB protects this infected population against cell death via Akt activation. Thus, T3SS1 has two, temporally distinct roles during epithelial cell colonization. Additionally, we found that delivery of SopB by cytosolic bacteria was translocon-independent, in contrast to canonical effector translocation across eukaryotic membranes, which requires formation of a translocon pore. This mechanism was also observed for another T3SS1 effector, SipA. These findings reveal the functional and mechanistic adaptability of a T3SS that can be harnessed in different microenvironments.</p></div