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NBR1 is a new PB1 signalling adapter in Th2 differentiation and allergic airway inflammation in vivo

2010; Springer Nature; Volume: 29; Issue: 19 Linguagem: Inglês

10.1038/emboj.2010.214

1460-2075

Jun‐Qi Yang, Hongzhu Liu, Marı́a T. Diaz-Meco, Jorge Moscat,

Pediatric health and respiratory diseases

Article31 August 2010free access NBR1 is a new PB1 signalling adapter in Th2 differentiation and allergic airway inflammation in vivo Jun-Qi Yang Jun-Qi Yang Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Hongzhu Liu Hongzhu Liu Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Maria T Diaz-Meco Maria T Diaz-Meco Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jun-Qi Yang Jun-Qi Yang Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Hongzhu Liu Hongzhu Liu Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Maria T Diaz-Meco Maria T Diaz-Meco Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Author Information Jun-Qi Yang1, Hongzhu Liu1, Maria T Diaz-Meco1 and Jorge Moscat 1 1Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA *Corresponding author. Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267, USA. Tel.: +1 513 558 8419; Fax: +1 513 558 5061; E-mail: [email protected] The EMBO Journal (2010)29:3421-3433https://doi.org/10.1038/emboj.2010.214 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Allergic airway inflammation is a disease in which T helper 2 (Th2) cells have a critical function. The molecular mechanisms controlling Th2 differentiation and function are of paramount importance in biology and immunology. Recently, a network of PB1-containing adapters and kinases has been shown to be essential in this process owing to its function in regulating cell polarity and the activation of critical transcription factors. Here, we show in vivo data showing that T-cell-specific NBR1-deficient mice show impaired lung inflammation and have defective Th2 differentiation ex vivo with alterations in T-cell polarity and the selective inhibition of Gata3 and nuclear factor of activated T c1 activation. These results establish NBR1 as a novel PB1 adapter in Th2 differentiation and asthma. Introduction PB1-domain-containing signalling regulators include kinases such as MEK5α, MEKK3 and the atypical PKCs (aPKCs) PKCζ and PKCλ/ι, as well as the signalling adapters p62 and Par-6 (Moscat et al, 2006). These latter two molecules serve to locate the aPKCs into the NF-κB and cell polarity signalling cascades, respectively (Moscat et al, 2009). The analysis of gene-knockout (KO) mice deficient in the different PB1 molecules is shedding light on their actual functions in vivo and at the cellular level. That is, PKCζ-deficient mice show impaired T-cell differentiation towards the T helper 2 (Th2) lineage because of the critical function that PKCζ has in IL-4 signalling ex vivo and in vivo (Martin et al, 2005). Extensive evidence shows a critical function of Th2 cells in the genesis of asthma and other allergic diseases (Paul, 1997; Luster and Tager, 2004). Naive Th cells can differentiate in response to antigen stimulation into different effector lineages, including T helper 1 (Th1) and Th2; these are characterized by the secretion of different sets of cytokines as well as by performing different regulatory functions in the immune system (Mosmann and Coffman, 1989; Shuai and Liu, 2003). Th1 cells mainly produce IFN-γ and IL-2 and have an essential function in cell-mediated immune responses against intracellular pathogens. On the other hand, Th2 cells produce IL-4, IL-5, IL-10 and IL-13 and are important in the control of humoural immunity and allergy. The differentiation of CD4+ T cells along the Th2 lineage is modulated by signals emanating from the T-cell receptor (TCR), in combination with pathways triggered by cytokines generated during polarization, particularly IL-4, which is extensively used for the in vitro differentiation of CD4+ T cells towards Th2 (Ho and Glimcher, 2002; Murphy and Reiner, 2002). As IL-4 is important for induction and maintenance of differentiated Th2 cells, our data showed that PKCζ impinges on Th2 differentiation because it is a critical target of IL-4 signalling (Martin et al, 2005). More recent findings showed that the other aPKC, PKCλ/ι, is likewise important for Th2 differentiation but, in contrast to PKCζ, is not involved in IL-4 signalling. Instead, it has a more general function in the control of T-cell polarity (Yang et al, 2009), a critical mechanism whereby essential regulators are located at the immunological synapse (IS) and the opposite pole during TCR activation (Ludford-Menting et al, 2005; Krummel and Macara, 2006; Chang et al, 2007; Yeh et al, 2008). Consistent with this, PKCλ/ι-deficient mice show impaired responses to allergic airway inflammation, a typical Th2 response, and show diminished induction of Th2 differentiation in ex vivo experiments (Yang et al, 2009). These observations establish that at least two PB1-containing kinases perform similar cellular functions through different signalling mechanisms. Interestingly, the genetic inactivation of the aPKC-interacting, PB1-containing, signalling adapter p62 also reveals its function in Th2 differentiation in ex vivo studies, as well as in vivo in the above-mentioned lung inflammatory model (Martin et al, 2006). Recently, another PB1-domain protein has been identified that has a remarkably similar domain organization to that of p62, including zinc-finger and UBA domains (see below). From overexpression and transfection studies, it has been suggested that NBR1 is involved in growth factor trafficking (Mardakheh et al, 2009), and/or p62-mediated processes (Lange et al, 2005; Kirkin et al, 2009). However, its precise in vivo function has not been elucidated yet owing to a lack of studies involving gene inactivation at an organismal level. As at least three PB1-containing proteins are involved in Th2 differentiation, PKCζ, PKCλ/ι and p62, we tested whether NBR1 was part of a novel PB1-organized network of signalling molecules controlling T-cell differentiation and allergic airway inflammation in vivo. Here, we report the generation and characterization of mice in which NBR1 has been specifically knocked out in activated T cells, and show for the first time, through ex vivo and in vivo studies, that it is a critical mediator of T-cell activation in the control of Th2 differentiation and allergic airway inflammation ex vivo and in vivo. Results Generation of conditional NBR1 KO mice in activated T cells We generated a conditional NBR1-KO (NBR1fl/flCreOX40) mouse line, in which NBR1 is specifically deleted in activated T cells (Supplementary Figure S1). Thus, we bred NBR1fl/fl mice with CreOX40 mice in which the expression of Cre is under the control of the Tnfrsf4 locus (Zhu et al, 2004; Klinger et al, 2009). OX40 is expressed almost exclusively in activated T cells, especially CD4+ cells, upon stimulation (Zhu et al, 2004; Klinger et al, 2009). By using this strategy, we prevent potential embryonic lethality and possible confounding effects resulting from the deletion of NBR1 during development or in resting cells. This very same approach has been used previously to specifically delete the GATA3 and PKCλ/ι genes in activated T cells during Th2 differentiation experiments (Zhu et al, 2004; Yang et al, 2009). PCR genotyping was used to screen for homozygous conditional NBR1-deficient (NBR1fl/flCreOX40) and wild-type (NBR1fl/fl) mice. No Cre-mediated effects were detected when NBR1wt/wtCreOX40 and NBR1fl/fl mice were compared (data not shown). The deletion of NBR1 in activated CD4+ T cells was confirmed by western blot of extracts from T cells activated with anti-CD3 plus anti-CD28. Interestingly, we observed a significant upregulation of NBR1 in T cells from wild-type mice, but not in those from conditional NBR1-KO mice (Figure 1A and B). Therefore, NBR1 is normally induced during sustained T-cell stimulation and is effectively deleted in the mutant cells. This implies that NBR1 levels are very low in resting cells, but that it is induced in T cells activated for a relatively long period (Figure 1A). Of note, the NBR1-interacting partner p62 is also induced upon T-cell activation, as is PKCλ/ι, which is a partner of p62 (Figure 1A). This is consistent with the previously published data (Martin et al, 2006; Yang et al, 2009). The loss of NBR1 in the mutant mice does not affect p62 or PKCλ/ι induction (Figure 1B). On the basis of these observations, we hypothesized that NBR1 might be required for the sustained signalling leading to T-cell differentiation. Figure 1.NBR1 is upregulated upon T-cell activation and is required for Th2 cytokine secretion. (A, B) Purified splenic CD4+ T cells from WT (A) or conditional NBR1-deficient mice (B) were stimulated with anti-CD3/CD28 for 0–48 h. Cells lysates were analysed by western blot for NBR1, p62, PKCλ/ι and actin. The results shown are representative of three independent experiments. (C–F) Purified splenic CD4+ T cells from mice of different genotypes were stimulated with anti-CD3/CD28 for 1 and 3 days; supernatants were collected for ELISA assays to detect cytokines. Data (mean±s.e.) are from three experiments with measurements for each sample performed in triplicate. (G–I) Cells were stimulated with anti-CD3/CD28 for 1 day with BD GolgiPlug™ in the last 5 h, and collected for intracellular cytokine staining. Percentages of cytokine-positive CD4+ T cells are shown. The results are representative of two independent experiments. (J–M) Purified CD4+ T cells were also differentiated under Th0, Th1 or Th2 conditions for 4 days and then re-stimulated with plate-bound anti-CD3/CD28 for 1 day. Cells were harvested for NBR1 expression by immunoblotting (J). Supernatants were collected for ELISA assays to detect IL-4 (K), IL-5 (L) or IFN-γ (M). Data (mean±s.e.) are from three experiments with measurements for each sample performed in triplicate. **P 300 cells were performed on cytospins stained with Kwik-Diff. The numbers of eosinophils (Eo), macrophages (Mϕ), neutrophils (Neu) and lymphocytes (Lym) in BAL are shown. (C) Representative H&E staining of lung tissue sections. (D–G) Cytokine levels in BAL fluid were determined by ELISA. The results are expressed as mean±s.e. from two independent experiments (n=10 per group). *P<0.05; **P<0.01. Download figure Download PowerPoint Figure 3.Cytokine and mucin glycoprotein mRNA levels. Total RNA from the right lower lobe of the lungs used in Figure 2 was extracted for real-time PCR analysis. The mRNA levels of IL-4 (A), IL-5 (B), IL-13 (C), RANTES (D), Eotaxin (E), IFN-γ (F), Muc-5AC (G) and Gob-5 (H) were determined by RT–PCR and expressed as arbitrary units. All samples were determined in triplicate; the data are normalized to an 18S reference. The results are expressed as mean±s.e. from two independent experiments (n=10 per group). *P 300 cells for each staining, and are representative of three independent experiments. (B) Cells with nuclear translocation were quantified by cell counting (n>400) under a microscope. Nuclear (C), cytoplasmic (D) and total (E) extracts of T cells treated as above were analysed by immunoblotting with antibodies for the respective transcription factors and signalling kinases. T cells were pooled from 5 to 10 mice per genotype. (F) Gata3 mRNA levels are shown (arbitrary units). Total RNA was extracted from splenic CD4+ T cells differentiated into Th0 and Th2 cells and re-stimulated with anti-CD3/CD28 for 1 day for real-time PCR analysis. All samples were determined by triplicate, the data are normalized to an 18S reference. *P<0.05; **P 300 cells (B). Cells with nuclear translocation were quantified by cell counting (n>400) under a microscope (C). T cells were pooled from 3 to 5 mice per genotype. The results shown are representative of two independent experiments. (D) Cells were transfected with the luciferase reporter pGL3-NFAT along with different doses of HA-NBR1 (50, 100, 500 ng) and a Renilla control plasmid. Luciferase activity was determined 2 days after transfection and normalized for Renilla. NBR1 expression levels were analysed by immunoblotting with anti-HA antibody. The results are the means±s.d. for triplicates. Download figure Download PowerPoint On the other hand, NBR1 has been shown to regulate autophagy (Kirkin et al, 2009; Moscat and Diaz-Meco, 2009). Our results in T-cell activation, however, show that the loss of NBR1 has no effect on autophagy as determined by LC3 immunofluorescence and immunoblot analysis (Supplementary Figure S6). Consistent with this, we found no changes in the surface expression of CD3/CD28 between T cells of the two different genotypes (Supplementary Figure S6). Function of NBR1 in T-cell polarity and recruitment to the immunological synapse The aPKCs have been implicated in the control of cell polarity in several mammalian in vitro cell culture experiments (Goldstein and Macara, 2007). More recently, it has been suggested that cell polarity has a function in T-cell activation (Krummel and Macara, 2006; Chang et al, 2007). In agreement with this, our previous data showed that PKCλ/ι is required for proper polarization of T cells during chronic activation through the TCR (Yang et al, 2009), which is characterized by the recruitment of PKCλ/ι and other polarity proteins, such as talin and scribble, to the IS (Yang et al, 2009). To determine whether NBR1, like PKCλ/ι, is recruited to the IS, we incubated CD4+ T cells, from either wild-type or NBR1-mutant mice, with latex beads coated with anti-CD3 plus anti-CD28. The results of these experiments show that NBR1 is clearly translocated to the IS (see Figure 6A as an example) in at least 80% of T cells upon activation with anti-CD3/CD28-coated latex beads. We then used confocal immunofluorescence to determine whether the loss of NBR1 impaired the localization of PKCλ/ι. Figure 6B shows that the loss of NBR1 had no effect on the recruitment of PKCλ/ι to the IS. Interestingly, the genetic inactivation of PKCλ/ι, by using the OX40-CRE system as previously reported (Yang et al, 2009), did not affect the IS translocation of NBR1 (Figure 6C). Together, these results indicate that the translocation of NBR1 and PKCλ/ι are mutually independent. As p62 binds PKCλ/ι and NBR1 (Lange et al, 2005), our next experiments were to determine whether p62 is translocated to the IS upon CD3/CD28 stimulation. Figure 6A shows that this is, in fact, the case in >80% of activated cells. Interestingly, this depends on the presence of NBR1, but is independent of PKCλ/ι (Figure 6B and C). Surprisingly, the translocation of PKCλ/ι is independent of p62, but that of NBR1 is not (Figure 6D). Together, these results show that the likely interaction between p62 and NBR1 is required for their translocation to the IS, whereas the translocation of PKCλ/ι is independent of both adapters, which, likewise, translocates independently of PKCλ/ι. Figure 6.NBR1 recruitment to the immunological synapse. Purified splenic CD4+ T cells from mice with different genotypes were incubated for 14 h