TLR9-dependent induction of intestinal alpha-defensins by Toxoplasma gondii.

International audienceAlpha-defensins (or Cryptdins [Crps]) are a group of antimicrobial peptides produced as a component of Paneth cell (PC) secretory granules in the small intestine. In vivo ligation of TLR9 by synthetic agonists leads to PC degranulation, although the mechanism by which this occurs remains uncertain. In this report, we investigated TLR9-dependent mechanisms, triggered by the parasite Toxoplasma gondii, inducing Crp release in the lumen. Oral challenge of C57BL/6J (B6) wild-type (WT) mice with T. gondii induced TLR9 mRNA upregulation associated with a marked increase of type I IFN mRNA expression. PC secretory granules were released, and Crp-3/-5 mRNA expression by purified epithelial cells was increased following oral challenge of B6 WT mice. Although PCs failed to degranulate in infected B6 TLR9-/- mice, i.p. injection of mouse IFN-beta alone led to Crp-3/-5 mRNA upregulation in B6 WT and TLR9-/- mice. In addition, modulation of Crp mRNA expression in response to T. gondii infection was abrogated in B6 IFNAR-/- mice, which lack a functional type I IFN receptor. Taken together, these data demonstrate that T. gondii induces Crp-3/-5 production and release by PCs via a TLR9-dependent production of type I IFNs. Crps have a limited direct effect against T. gondii but may indirectly affect the early control of T. gondii invasiveness by promoting the initiation of a protective Th1 response against the parasite

A Novel Mechanism of Host-Pathogen Interaction through sRNA in Bacterial Outer Membrane Vesicles

Bacterial outer membrane vesicle (OMV)-mediated delivery of proteins to host cells is an important mechanism of host-pathogen communication. Emerging evidence suggests that OMVs contain differentially packaged short RNAs (sRNAs) with the potential to target host mRNA function and/or stability. In this study, we used RNA-Seq to characterize differentially packaged sRNAs in Pseudomonas aeruginosa OMVs, and to show transfer of OMV sRNAs to human airway cells. We selected one sRNA for further study based on its stable secondary structure and predicted mRNA targets. Our candidate sRNA (sRNA52320), a fragment of a P. aeruginosa methionine tRNA, was abundant in OMVs and reduced LPS-induced as well as OMV-induced IL-8 secretion by cultured primary human airway epithelial cells. We also showed that sRNA52320 attenuated OMV-induced KC cytokine secretion and neutrophil infiltration in mouse lung. Collectively, these findings are consistent with the hypothesis that sRNA52320 in OMVs is a novel mechanism of host-pathogen interaction whereby P. aeruginosa reduces the host immune response

Model of OMV sRNA mechanism of action.

<p>(1) <i>P</i>. <i>aeruginosa</i> (P.a.) resides in the airway mucus layer and produces OMVs. (2) OMVs traverse the mucus layer and reach the airway epithelial cells. (3) Pathogen-associated molecular patterns (PAMPs) on the outside of OMVs induce the host innate immune response by activating the Toll-like receptor and MAP-kinase (TLR/MAPK) signaling pathway. (4) Activation of transcription factors leads to up-regulation of IL-8 mRNA and IL-8 protein secretion. (5) IL-8 is a potent chemoattractant for neutrophils, which infiltrate the lungs and phagocytose <i>P</i>. <i>aeruginosa</i>. (6) OMVs also fuse with and deliver sRNA52320 into cells, which targets the mRNA of MAP-kinases upstream of IL-8, leading to reduced host IL-8 secretion and neutrophil recruitment. sRNA52320-mediated attenuation of the innate immune response to LPS is a novel mechanism of pathogen-host interaction that may facilitate chronic infection by <i>P</i>. <i>aeruginosa</i>.</p

sRNA52320 is inside of OMVs and protected from RNase digestion.

<p>(A) RNase A digests free RNA including RNA associated with the outside of OMVs, while RNA inside of intact OMVs is protected from degradation. (B) Agarose gel showing profiles of OMV-associated RNAs from untreated control OMVs (lane 1), RNase A treated OMVs (lane 2) and OMV RNA extracted from QIAzol lysed OMVs after digestion with RNase A (lane 3). RNA was visualized by staining with SYBR Safe. Samples were run on the same gel and were re-arranged for presentation. (C) qPCR for sRNA52320 using RNA isolated from control OMVs or RNase A-treated OMVs. RNase A treatment prior to RNA-Isolation (filled circles) increased the relative abundance of sRNA52320 compared to untreated OMVs (open circles). The difference in mean cycle threshold (Ct) of -2.5 ± 0.6 was statistically significant (95% CI = -4.1 to -0.9, N = 3, p = 0.013 indicated by an asterisk).</p

Transfection with sRNA52320 reduces LPS-stimulation of IL-8 mRNA abundance and IL-8 cytokine secretion in HBE cells.

<p>(A) HBE cells transfected with sRNA52320 (filled circles) had a lower induction of IL-8 mRNA (ΔCt = control Ct minus LPS-stimulated Ct) in response to LPS than control cells transfected with siNC (open circles). There was a statistically significant difference in the ΔCt means of -0.6 ± 0.2 (95% CI = -1.2 to -0.1, N = 5, p = 0.034 indicated by an asterisk). (B) sRNA52320 reduced IL-8 secretion by HBE cells in response to LPS. The difference in mean IL-8 secretion between cells transfected with siNC (open circles) and sRNA52320 (filled circles) of -149 ± 47 pg/ml was statistically significant (95% CI = -269 to -29, N = 6, p = 0.02 indicated by an asterisk).</p