The proprotein convertase PC1/3 regulates TLR9 trafficking and the associated signaling pathways.

Endosomal TLR9 is considered as a potent anti-tumoral therapeutic target. Therefore, it is crucial to decipher the mechanisms controlling its trafficking since it determines TLR9 activation and signalling. At present, the scarcity of molecular information regarding the control of this trafficking and signalling is noticeable. We have recently demonstrated that in macrophages, proprotein convertase 1/3 (PC1/3) is a key regulator of TLR4 Myd88-dependent signalling. In the present study, we established that PC1/3 also regulates the endosomal TLR9. Under CpG-ODN challenge, we found that PC1/3 traffics rapidly to co-localize with TLR9 in CpG-ODN-containing endosomes with acidic pH. In PC1/3 knockdown macrophages, compartmentalization of TLR9 was altered and TLR9 clustered in multivesicular bodies (MVB) as demonstrated by co-localization with Rab7. This demonstrates that PC1/3 controls TLR9 trafficking. This clustering of TLR9 in MVB dampened the anti-inflammatory STAT3 signalling pathway while it promoted the pro-inflammatory NF-kB pathway. As a result, macrophages from PC1/3 KO mice and rat PC1/3-KD NR8383 macrophages secreted more pro-inflammatory cytokines such as TNF-α, IL6, IL1α and CXCL2. This is indicative of a M1 pro-inflammatory phenotype. Therefore, PC1/3 KD macrophages represent a relevant mean for cell therapy as "Trojan" macrophages.

Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.

Cg-TGF-beta, a TGF-beta/activin homologue in the Pacific Oyster Crassostrea gigas, is involved in immunity against Gram-negative microbial infection.

Transforming growth factor-beta (TGF-beta) members represent a widespread protein superfamily in the animal kingdom, but few members have been characterised in lophotrochozoans, a major clade of invertebrates. Here, we report the identification of Crassostrea gigas-TGF-beta (Cg-TGF-beta), a homologue of vertebrate TGF-beta and activin, from the bivalve mollusc C. gigas. Phylogenetic analysis suggests an early ancestral origin of this subgroup of TGF-beta superfamily member. Investigation of the spatio-temporal expression of Cg-TGF-beta gene by real-time quantitative RT-PCR showed a ubiquitous pattern in all adult tissues. These findings imply that Cg-TGF-beta has multiple functions as described for its vertebrate counterparts. Moreover, Cg-TGF-beta was upregulated in haemocytes during infection by a Gram-negative bacterium, suggesting that it could act as a cytokine involved in immunity in molluscs.

Expression and activity of a multixenobiotic resistance system in the Pacific oyster Crassostrea gigas.

The multidrug resistance (MDR) mechanism corresponds to a defence system relying on the expression of high molecular membrane proteins that can actively lower the intracellular concentration of a wide variety of toxins, thus maintaining them below their toxic level. Using RT-PCR, expression levels of a gene belonging to the class I of mammalian mdr genes, has been assessed in different developmental stages of the oyster Crassostrea gigas. While no expression was found in the oocyte or the trocophore stage, a rise of mRNA content was observed from the veliger stage to the juvenile stage, thus indicating the induction of the system as the animal is developing in the environment. The incubation of gill fragments in the dye rhodamine B and subsequent measurements of intracellular fluorescence using a microplate reader indicates that the system can effectively decrease the accumulation of the test compound in a competitive manner with known inhibitors or environmental contaminants as observed in vertebrate cells. The oyster MXR system is thus becoming active in adult oyster and could be of importance in environmentally contaminated areas.