Uterine WNTS modulates fibronectin binding activity required for blastocyst attachment through the WNT/CA2+ signaling pathway in mice

Abstract Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5β1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5β1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium

Bacterial Cyclic Diguanylate Signaling Networks Sense Temperature

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria

A balanced IL-1β activity is required for host response to Citrobacter rodentium infection.

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1β. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1β to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3(-/-) and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1β (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3(-/-) mice developed severe colitis; IL-1β treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3(-/-) mice. In contrast, IL-1β treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3(-/-) mice, and increased severity of disease in WT mice with IL-1β treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3(-/-) compared to WT mice. IL-1β treatments elevated macrophage infiltration into infected crypts in Nlrp3(-/-) mice, suggesting that IL-1β may improve macrophage function, as exogenous administration of IL-1β increased phagocytosis of C. rodentium by peritoneal Nlrp3(-/-) macrophages in vitro. As well, the exogenous administration of IL-1β to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3(-/-) mice with IL-1β seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1β appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1β improved bacterial clearance in Nlrp3(-/-) mice but increased tissue damage when given to WT mice

IL-1β treatments augment macrophage colonic infiltration.

<p>Macrophage infiltration was assessed by staining cryosections of distal colons. An increase in the number of macrophages (red; indicated by arrowhead in higher magnification panels on the left) in close proximity of <i>C. rodentium</i> (green) at 10 DPI in <i>Nlrp3^−/−</i> mice is noted. With IL-1β treatments, the macrophages in crypts and mucosal lining appeared increased and bacterial infiltration into the crypts reduced. Bar 50 µm.</p

IL-1β treatments reduce intestinal colonization and dissemination of <i>C. rodentium</i> in <i>Nlrp3^−/−</i> mice.

<p>Infected mice were given repeated IL-1β treatments on 0, 2, and 4 DPI. <i>C. rodentium</i> colonization of the (A) cecum, (B) colon, and (C) dissemination to MLN were assessed on 6 and 10 DPI by plating homogenates on MacConkey agar plates in serial dilutions. IL-1β treatments of <i>Nlrp3^−/−</i> mice significantly lowered cecum colonization on 6 DPI, and colon colonization and dissemination to MLN on 10 DPI. Values were Log₁₀ transformed and data presented as mean ± SE. One asterisk: <i>P</i><0.05, two asterisks: <i>P</i><0.01.</p

IL-1β treatments modulate the systemic cytokine response.

<p>IL-6 (A), IL-12p70 (B), TNF-α (C), IL-1β (D), KC/gro (E), and IFN-γ (F) were measured using ELISA-based assays. Generally, secretion of pro-inflammatory cytokines was dissipated in <i>Nlrp3^−/−</i> mice at 10 DPI compared to WT mice. One asterisk: <i>P</i><0.05, two asterisks: <i>P</i><0.01, three asterisks: <i>P</i><0.001; ND, not detected.</p

IL-1β treatments modulate <i>C. rodentium</i> penetration and epithelial integrity.

<p>Biotin was administered into the lumen of the distal colon at 6 or 10 DPI. Cryosections were stained and viewed under fluorescent microscopy for <i>C. rodentium</i> infiltration (in red; indicated by arrowheads in the enlarged image of the inset highlighted by boxes in the left panels) and biotin penetration (green; indicated by asterisks). Uninfected IL-1β-treated mice retained biotin to the apical surface. In non-treated mice at 6 DPI, the bacterial infiltration and biotin penetration was increased. IL-1β treatments at 6 DPI did not seem to have a major effect on infiltration or epithelial integrity. In non-treated mice at 10 DPI, the bacterial infiltration and epithelial barrier disruption were lowered in WT and elevated in <i>Nlrp3^−/−</i> mice, relative to 6 DPI. In IL-1β-treated mice at 10 DPI, the bacterial infiltration and barrier disruption were elevated in WT and lowered in <i>Nlrp3^−/−</i> mice. Bacterial colonization followed the same trend as biotin penetration. Magnification X400, bar 50 µm.</p

IL-1β enhances <i>C. rodentium</i> phagocytosis, while overcompensation of IL-1β augments epithelial barrier damage <i>in vitro</i>.

<p>(A) Exogenous IL-1β induced <i>C. rodentium</i> phagocytosis (arrowhead) by peritoneal macrophages <i>in vitro</i>, especially in <i>Nlrp3^−/−</i> macrophages. Magnification X630, bar 50 µm; (B) Quantification showed an increase of intracellular <i>C. rodentium</i> in IL-1β treated <i>Nlrp3^−/−</i> macrophages but no effect on WT macrophages. Data represents mean ± SE. One asterisk <i>P</i><0.05; (C) Western blot analysis of mature IL-1β in the supernatant of peritoneal macrophages and pro-IL-1β in the cellular component; (D) The epithelial integrity of <i>C. rodentium</i>-infected CMT-93 cells assessed by ECIS, deteriorated in presence WT macrophages and even more with the addition of IL-1β. Data represents the mean of two independent experiments.</p