Loss of Elp3 blocks intestinal tuft cell differentiation via an mTORC1-Atf4 axis.

peer reviewedIntestinal tuft cells are critical for anti-helminth parasite immunity because they produce IL-25, which triggers IL-13 secretion by activated group 2 innate lymphoid cells (ILC2s) to expand both goblet and tuft cells. We show that epithelial Elp3, a tRNA-modifying enzyme, promotes tuft cell differentiation and is consequently critical for IL-25 production, ILC2 activation, goblet cell expansion and control of Nippostrongylus brasiliensis helminth infection in mice. Elp3 is essential for the generation of intestinal immature tuft cells and for the IL-13-dependent induction of glycolytic enzymes such as Hexokinase 1 and Aldolase A. Importantly, loss of epithelial Elp3 in the intestine blocks the codon-dependent translation of the Gator1 subunit Nprl2, an mTORC1 inhibitor, which consequently enhances mTORC1 activation and stabilizes Atf4 in progenitor cells. Likewise, Atf4 overexpression in mouse intestinal epithelium blocks tuft cell differentiation in response to intestinal helminth infection. Collectively, our data define Atf4 as a negative regulator of tuft cells and provide insights into promotion of intestinal type 2 immune response to parasites through tRNA modifications

Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

peer reviewedMacrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization

NF-κB Signaling in Ex-Vivo Mouse Intestinal Organoids.

peer reviewedWe describe here a protocol to assess NF-κB activation in ex-vivo organoids generated from mouse intestinal crypts. These structures are maintained in culture as crypt-villus forming organoids. These ex-vivo organoids maintain both self-renewal and multilineage differentiation overtime. We also describe the generation of ex-vivo organoids from Apc-mutated mouse intestinal crypts. Both wild-type and Apc-mutated organoids respond very well to NF-κB-activating signals such as TNFα but not to LPS. The kinetic of NF-κB activation in response to these signals in ex-vivo intestinal organoids is very similar to what we see in 2D cell lines. This protocol provides investigators a powerful tool to assess NF-κB activation in both healthy and transformed intestinal epitheliums maintained in culture as 3D structures

IL-4 RECEPTOR SIGNALING REGULATES LUNG MACROPHAGES DURING HELMINTH COINFECTION RESULTING IN ENHANCED GAMMAHERPESVIRUS PERMISSIVENESS

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IL-4rα-dependent Lung Macrophage Responses To Helminths Increase Permissiveness To Gammaherpesvirus Infection

Helminth infection conditions lung macrophages in the long term, but little is known about how helminths affect the lung macrophage responses to respiratory viral coinfection. While comparing BALB/c and C57BL/6 mice, we found that helminth infection in C57BL/6 mice resulted in enhanced permissiveness to a subsequent infection with murid gammaherpesvirus 4 (MuHV-4), and that viral early tropism was mainly restricted to lung macrophages. Helminth infection resulted in enhanced type 2 airway inflammation and M(IL-4) polarization of interstitial macrophages (IntMs) in C57BL/6 mice, associated with an IL-4Rα-dependent disappearance reaction of alveolar macrophages (AlvMs) and enriched monocyte-derived IntMs proportions. Competent IL-4Rα responsiveness or intra-tracheal instillation of recombinant IL-4 or IL-13 significantly resulted in reduced numbers of AlvMs and enriched IntMs as well as increased permissiveness to MuHV-4 infection, which was restricted to AlvMs. Thus, direct IL-4Rα signaling during helminth infection affects macrophage permissiveness to gammaherpesvirus infection

Il-4rα-dependent Lung Macrophage Response To Helminth Increases Permissiveness To Gammaherpesvirus Infection

Helminth infection conditions lung macrophages in the long term, but little is known about how helminths affect the lung macrophage responses to respiratory viral coinfection. While comparing BALB/c and C57BL/6 mice, we found that helminth infection in C57BL/6 mice resulted in enhanced permissiveness to a subsequent infection with murid gammaherpesvirus 4 (MuHV-4), and that viral early tropism was mainly restricted to lung macrophages. Helminth infection resulted in enhanced type 2 airway inflammation and M(IL-4) polarization of interstitial macrophages (IntMs) in C57BL/6 mice, associated with an IL-4Rα-dependent disappearance reaction of alveolar macrophages (AlvMs) and enriched monocyte-derived IntMs proportions. Competent IL-4Rα responsiveness or intra-tracheal instillation of recombinant IL-4 or IL-13 significantly resulted in reduced numbers of AlvMs and enriched IntMs as well as increased permissiveness to MuHV-4 infection, which was restricted to AlvMs. Thus, direct IL-4Rα signaling during helminth infection affects macrophage permissiveness to gammaherpesvirus infection

IL-4 receptor signaling regulates lung macrophages during helminth coinfection resulting in enhanced gammaherpesvirus permissiveness

Helminth infection conditions lung macrophages in the long term, but little is known about how helminths affect the lung macrophage responses to respiratory viral coinfection. While comparing BALB/c and C57BL/6 mice, we found that helminth infection in C57BL/6 mice resulted in enhanced permissiveness to a subsequent infection with murid gammaherpesvirus 4 (MuHV-4), and that viral early tropism was mainly restricted to lung macrophages. Helminth infection resulted in enhanced type 2 airway inflammation and M(IL-4) polarization of interstitial macrophages (IntMs) in C57BL/6 mice, associated with an IL-4Rα-dependent disappearance reaction of alveolar macrophages (AlvMs) and enriched monocyte-derived IntMs proportions. Competent IL-4Rα responsiveness or intra-tracheal instillation of recombinant IL-4 or IL-13 significantly resulted in reduced numbers of AlvMs and enriched IntMs as well as increased permissiveness to MuHV-4 infection, which was restricted to AlvMs. Thus, direct IL-4Rα signaling during helminth infection affects macrophage permissiveness to gammaherpesvirus infection

Dusp3 deletion in mice promotes experimental lung tumour metastasis in a macrophage dependent manner

Vaccinia-H1 Related (VHR) dual-specificity phosphatase, or DUSP3, plays an important role in cell cycle regulation and its expression is altered in several human cancers. In mouse model, DUSP3 deletion prevents neo-angiogenesis and b-FGF-induced microvessel out- growth. Considering the importance of angiogenesis in metastasis formation, our study aimed to investigate the role of DUSP3 in tumour cell dissemination. Using a Lewis Lung carcinoma (LLC) experimental metastasis model, we observed that DUSP3-/- mice devel- oped larger lung metastases than littermate controls. DUSP3-/- bone marrow transfer to lethally irradiated DUSP3+/+ mice was sufficient to transfer the phenotype to DUSP3+/+ mice, indicating that hematopoietic cells compartment was involved in the increased tumour cell dissemination to lung tissues. Interestingly, we found a higher percentage of tumour- promoting Ly6Cint macrophages in DUSP3-/- LLC-bearing lung homogenates that was at least partially due to a better recruitment of these cells. This was confirmed by 1) the pres- ence of higher number of the Ly6Bhi macrophages in DUSP3-/- lung homogenates and by 2) the better migration of DUSP3-/- bone marrow sorted monocytes, peritoneal macrophages and bone marrow derived macrophages (BMDMs), compared to DUSP3+/+ monocytes, macrophages and BMDMs, in response to LLC-conditioned medium. Our study demon- strates that DUSP3 phosphatase plays a key role in metastatic growth through a mechanism involving the recruitment of macrophages towards LLC-bearing lungs