Listeria monocytogenes infection in macrophages induces vacuolar-dependent host miRNA response.

Listeria monocytogenes is a gram-positive facultative intracellular pathogen, causing serious illness in immunocompromised individuals and pregnant women. Upon detection by macrophages, which are key players of the innate immune response against infection, L. monocytogenes induces specific host cell responses which need to be tightly controlled at transcriptional and post-transcriptional levels. Here, we ask whether and how host miRNAs, which represent an important mechanism of post-transcriptional regulation in a wide array of biological processes, are altered by a model pathogen upon live infection of murine bone marrow derived macrophages. We first report that L. monocytogenes subverts the host genome-wide miRNA profile of macrophages in vitro. Specifically, we show that miR-155, miR-146a, miR-125a-3p/5p and miR-149 were amongst the most significantly regulated miRNAs in infected macrophages. Strikingly, these miRNAs were highly upregulated upon infection with the Listeriolysin-deficient L. monocytogenes mutant Δhly, that cannot escape from the phagosome thus representing a vacuolar-contained infection. The vacuolar miRNA response was significantly reduced in macrophages deficient for MyD88. In addition, miR-146a and miR-125a-3p/5p were regulated at transcriptional levels upon infection, and miR-125a-3p/5p were found to be TLR2 responsive. Furthermore, miR-155 transactivation in infection was regulated by NF-κB p65, while miR-146a and miR-125a-3p/5p expression was unaffected in p65-deficient primary macrophages upon L. monocytogenes infection. Our results demonstrate that L. monocytogenes promotes significant changes in the miRNA expression profile in macrophages, and reveal a vacuolar-dependent miRNA signature, listeriolysin-independent and MyD88-dependent. These miRNAs are predicted to target immune genes and are therefore most likely involved in regulation of the macrophage innate immune response against infection at post-transcriptional levels.

Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria.

C-type lectins (CTLs) are a family of proteins that share a common structural motif, the carbohydrate recognition domain, and may act as receptors in pathogen recognition. Indeed, some vertebrate CTLs, particularly the collectins, are unequivocally implicated in the innate immune response to certain microbes. Although studies in insects and other invertebrates have described CTL activation of effector immune responses in vitro, the contribution of these CTLs to immune defenses in vivo is still poorly understood. Here we report that two CTLs, CTL4 and CTLMA2, which were shown previously to inhibit Plasmodium berghei ookinete melanization in the malaria vector Anopheles gambiae, are transcriptionally induced by bacterial challenge. Using in vivo reverse genetic analysis, we show that both CTLs are required for the clearance of Escherichia coli, but not Staphylococcus aureus, from adult female mosquitoes. Silencing either CTL dramatically reduces mosquito survival to Gram-negative but not to Gram-positive bacterial infections, suggesting a role in defense against Gram-negative bacteria. Furthermore, molecular characterization reveals that both CTLs are secreted into the mosquito hemolymph mainly in the form of a disulfide-linked heterodimer. This association explains the similar roles of these CTLs in bacterial defense as well as in the melanization response to P. berghei ookinetes. Apparently, CTL4 and CTLMA2 serve pleiotropic functions in the innate immune response of A. gambiae.

The melanization reaction is not required for survival of Anopheles gambiae mosquitoes after bacterial infections.

The melanization reaction of insects requires activation of pro-phenoloxidase by a proteolytic cascade leading to melanin production. Studies in adult mosquitoes have shown that bacteria are efficiently melanized in the hemocoel, but the contribution of melanization to survival after bacterial infections has not been established. Here we show that the Anopheles gambiae noncatalytic serine protease CLIPA8, an essential factor for Plasmodium ookinete melanization, is also required for melanization of bacteria in adult mosquitoes. CLIPA8 silencing by RNA interference inhibits pro-phenoloxidase activation and melanization of bacteria in the hemolymph following microbial challenge. However, CLIPA8 is not required for wound melanization nor for melanotic pseudotumor formation in serpin2 knockdown mosquitoes, suggesting a specific role for pathogen melanization. Surprisingly, CLIPA8 knockdown mosquitoes are as resistant to bacterial challenge as controls, indicating that melanization is not essential for defense against bacteria and questions its precise role in mosquito immunity.

Combined application of saponin and chimeric toxins drastically enhances the targeted cytotoxicity on tumor cells.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

13-cis retinoic acid inhibits development and progression of chronic allograft nephropathy.

Chronic allograft nephropathy is characterized by chronic inflammation and fibrosis. Because retinoids exhibit anti-proliferative, anti-inflammatory, and anti-fibrotic functions, the effects of low and high doses of 13-cis-retinoic acid (13cRA) were studied in a chronic Fisher344-->Lewis transplantation model. In 13cRA animals, independent of dose (2 or 20 mg/kg body weight/day) and start (0 or 14 days after transplantation) of 13cRA administration, serum creatinine was significantly lower and chronic rejection damage was dramatically reduced, including subendothelial fibrosis of preglomerular vessels and chronic tubulointerstitial damage. The number of infiltrating mononuclear cells and their proliferative activity were significantly diminished. The mRNA expression of chemokines (MCP-1/CCL2, MIP-1alpha/CCL3, IP-10/CXCL10, RANTES/CCL5) and proteins associated with fibrosis (plasminogen activator inhibitor-1, transforming growth factor-beta1, and collagens I and III) were strikingly lower in treated allografts. In vitro, activated peritoneal macrophages of 13cRA-treated rats showed a pronounced decrease in protein secretion of inflammatory cytokines (eg, tumor necrosis factor-alpha, interleukin-6). The suppression of the proinflammatory chemokine RANTES/CCL5 x 13cRA in fibroblasts could be mapped to a promoter module comprising IRF-1 and nuclear factor-kappaB binding elements, but direct binding of retinoid receptors to promoter elements could be excluded. In summary, 13cRA acted as a potent immunosuppressive and anti-fibrotic agent able to prevent and inhibit progression of chronic allograft nephropathy.

The renal retinoid system: time-dependent activation in experimental glomerulonephritis.

Retinoids reduce renal damage in rat experimental glomerulonephritis. It is unknown, however, how local and systemic retinoid pathways respond to renal injury. We used a rat model of artificially induced acute anti-Thy1.1-nephritis (THY-GN). We examined the extrarenal and glomerular expression of the retinol (RoDH) and retinal (RalDH) dehydrogenases 1 and 2 as well as the expression of the retinoic acid (RAR) and retinoid X (RXR) receptor subtypes alpha, beta, and gamma. Furthermore, we investigated serum and glomerular retinoid concentration patterns. On days 3, 7, and 14, we compared nonnephritic rats (control group; CON) to THY-GN rats with respect to systolic blood pressure and glomerular cell count per cross section. Systolic blood pressure and glomerular cell count were significantly higher in THY-GN rats on days 7 and 14 (P < 0.001). We found a 60% reduction in expression levels for retinoid receptors and dehydrogenases in nephritic glomeruli on day 3, but a threefold increase on day 7 (P < 0.001 vs. CON). The same applies to RAR alpha protein. Hepatic expression of retinoid receptors was not influenced. On day 14, glomerular expression levels for retinoid receptors and retinoid-metabolizing enzymes had returned to a normal level, glomerular cell count being still increased. Administering 13-cis retinoic acid (isotretinoin) lowered blood pressure and glomerular cell count in nephritic rats but failed to influence the glomerular expression of retinoid receptors or retinoid-metabolizing enzymes. Our data document a stimulation of glomerular retinoid-synthesizing enzymes and expression of retinoid receptors in the early repair phase of THY-GN, suggesting activation of this system in acute renal disease.