Dynamic Interaction- and Phospho-Proteomics Reveal Lck as a Major Signaling Hub of CD147 in T Cells

Numerous publications have addressed CD147 as a tumor marker and regulator of cytoskeleton, cell growth, stress response, or immune cell function; however, the molecular functionality of CD147 remains incompletely understood. Using affinity purification, mass spectrometry, and phosphopeptide enrichment of isotope-labeled peptides, we examined the dynamic of the CD147 microenvironment and the CD147-dependent phosphoproteome in the Jurkat T cell line upon treatment with T cell stimulating agents. We identified novel dynamic interaction partners of CD147 such as CD45, CD47, GNAI2, Lck, RAP1B, and VAT1 and, furthermore, found 76 CD147dependent phosphorylation sites on 57 proteins. Using the STRING protein network database, a network between the CD147 microenvironment and the CD147-dependent phosphoproteins was generated and led to the identification of key signaling hubs around theG proteins RAP1B and GNB1, the kinases PKC beta, PAK2, Lck, and CDK1, and the chaperone HSPA5. Gene ontology biological process term analysis revealed that wound healing-, cytoskeleton-, immune system-, stress response-, phosphorylation-and protein modification-, defense response to virus-, and TNF production-associated terms are enriched within the microenvironment and the phosphoproteins of CD147. With the generated signaling network and gene ontology biological process term grouping, we identify potential signaling routes of CD147 affecting T cell growth and function

The mannose-6-phosphate analogue, PXS64, inhibits fibrosis via TGF-β1 pathway in human lung fibroblasts

© 2015 The Authors. Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a progressive decline in lung function which can be attributed to excessive scarring, inflammation and airway remodelling. Mannose-6-phosphate (M6P) is a strong inhibitor of fibrosis and its administration has been associated with beneficial effects in tendon repair surgery as well as nerve repair after injury. Given this promising therapeutic approach we developed an improved analogue of M6P, namely PXS64, and explored its anti-fibrotic effects in vitro. Normal human lung fibroblasts (NHLF) and human lung fibroblast 19 cells (HF19) were exposed to active recombinant human TGF-β1 to induce increases in fibrotic markers. rhTGF-β1 increased constitutive protein levels of fibronectin and collagen in the NHLF cells, whereas HF19 cells showed increased levels of fibronectin, collagen as well as αSMA (alpha smooth muscle actin). PXS64 demonstrated a robust inhibitory effect on all proteins analysed. IPF patient fibroblasts treated with PXS64 presented an improved phenotype in terms of their morphological appearance, as well as a decrease in fibrotic markers (collagen, CTGF, TGF-β3, tenascin C, αSMA and THBS1). To explore the cell signalling pathways involved in the anti-fibrotic effects of PXS64, proteomics analysis with iTRAQ labelling was performed and the data demonstrated a specific antagonistic effect on the TGF-β1 pathway. This study shows that PXS64 effectively inhibits the production of extracellular matrix, as well as myofibroblast differentiation during fibrosis. These results suggest that PXS64 influences tissue remodelling by inhibiting TGF-β1 signalling in NHLF and HF19 cell lines, as well as in IPF patient fibroblasts. Thus PXS64 is a potential candidate for preclinical application in pulmonary fibrosis

Association of CD147 and Calcium Exporter PMCA4 Uncouples IL-2 Expression from Early TCR Signaling

The Ig superfamily member CD147 is upregulated following T cell activation and was shown to serve as a negative regulator of T cell proliferation. Thus, Abs targeting CD147 are being tested as new treatment strategies for cancer and autoimmune diseases. How CD147 mediates immunosuppression and whether association with other coreceptor complexes is needed have remained unknown. In the current study, we show that silencing of CD147 in human T cells increases IL-2 production without affecting the TCR proximal signaling components. We mapped the immunosuppressive moieties of CD147 to its transmembrane domain and Ig-like domain II. Using affinity purification combined with mass spectrometry, we determined the domain specificity of CD147 interaction partners and identified the calcium exporter plasma membrane calcium ATPase isoform 4 (PMCA4) as the interaction partner of the immunosuppressive moieties of CD147. CD147 does not control the proper membrane localization of PMCA4, but PMCA4 is essential for the CD147-dependent inhibition of IL-2 expression via a calcium-independent mechanism. In summary, our data show that CD147 interacts via its immunomodulatory domains with PMCA4 to bypass TCR proximal signaling and inhibit IL-2 expression

The Late Endosomal Transporter CD222 Directs the Spatial Distribution and Activity of Lck

The spatial and temporal organization of T cell signaling molecules is increasingly accepted as a crucial step in controlling T cell activation. CD222, also known as the cation-independent mannose 6-phosphate/insulin-like growth factor 2 receptor, is the central component of endosomal transport pathways. In this study, we show that CD222 is a key regulator of the early T cell signaling cascade. Knockdown of CD222 hampers the effective progression of TCR-induced signaling and subsequent effector functions, which can be rescued via reconstitution of CD222 expression. We decipher that Lck is retained in the cytosol of CD222-deficient cells, which obstructs the recruitment of Lck to CD45 at the cell surface, resulting in an abundant inhibitory phosphorylation signature on Lck at the steady state. Hence, CD222 specifically controls the balance between active and inactive Lck in resting T cells, which guarantees operative T cell effector functions

Enhancing methotrexate tolerance with folate tagged liposomes in arthritic mice

Methotrexate is the first line of treatment of rheumatoid arthritis. Since many patients become unresponsive to methotrexate treatment, only very expensive biological therapies are effective and increased methotrexate tolerance strategies need to be identified. Here we propose the encapsulation of methotrexate in a new liposomal formulation using a hydrophobic fragment of surfactant protein conjugated to a linker and folate to enhance their tolerance and efficacy. In this study we aim to evaluate the efficiency of this system to treat rheumatoid arthritis, by targeting folate receptor β present at the surface of activated macrophages, key effector cells in this pathology. The specificity of our liposomal formulation to target folate receptor β was investigated both in vitro as in vivo using a mouse model of arthritis (collagen-induced arthritis in DBA/1J mice strain). In both systems, the liposomal constructs were shown to be highly specific and efficient in targeting folate receptor β. These liposomal formulations also significantly increase the clinical benefit of the encapsulated methotrexate in vivo in arthritic mice, together with reduced expression of CD39 and CD73 ectonucleotidases by joint-infiltrating macrophages. Thus, our formulation might be a promising cost effective way to treat rheumatoid arthritis and delay or reduce methotrexate intolerance

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis

<div><p>A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after <i>toll-like</i> receptor (TLR)-activation and glucose-deprivation or co-treatment with 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an <i>in-vivo</i> mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our <i>in vitro</i> observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone <i>Listeria monocytogenes</i> mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.</p></div

2-DG protects mice from Listeria monocytogenes <i>(L</i>.<i>m</i>.<i>)</i> infection.

<p>BALB/c mice (n = 5) were pretreated i.p. with 2-DG or PBS for 3 days and then challenged with 5 x 10⁴ <i>L</i>.<i>m</i>. On day 3 after infection the number of bacteria in the spleen and the liver were determined (a). Liver histomorphology was analyzed by H&E staining on day 3 after infection (b). Granulomatous lesions are indicated (by arrows). **p ≤ 0.005. (c) Livers (n = 3 per group) were analyzed by immunohistochemical staining of F4/80 on day 3 post infection. Representative images from each group of mice are shown (brown color, F4/80 staining; blue color, nuclear counterstaining with haematoxylin). Scale bar: 50 μm. Inserts: the high-power views. (d) Quantitative analysis of F4/80 staining in the liver specimens using HistoQuest software. The box-plot analyses represent the number of F4/80-positive cells (designated as “Count”) and also the total and mean areas of F4/80-positive cells (designated as “Total Area” and “Mean Area”, respectively); the total area values reflect both the magnitude and morphology/size of positive cells, while the mean area values reflect predominantly the morphology/size of positive cells. ***p ≤ 0.001; only significant p values are shown.</p

Impact of AMPK activators on IL-12p40/IL-10 induction.

<p>Human monocytes were preincubated for 90 minutes with different concentrations of A-769662 (a, b) or AICAR (c, d) or medium and then stimulated with LPS. Secretion of IL-10 (a, c) and IL-12p40 (b, d) was determined from 20 hr culture supernatants by ELISA. Data are representative of 3–5 independent experiments and presented as % response ± SD. A-769662 treatment alone induced no significant cytokine production: IL-10: 4 times below detection level, IL-12p40: 3 times below detection level 1x 1.8 pg/mL, Similarly, AICAR treatment alone induced no significant cytokine production: IL-10: 3x ≤ 3.1 pg/mL, IL-12p40: 3 times below detection level. *p ≤ 0.05, ***p ≤ 0.01.</p

Modulation of cytokine production by 2-DG in murine BMDMs.

<p>BMDMs from BALB/c mice were cultured in 6 well plates and treated with 1, 3, and 5 mM 2-DG and <i>L</i>.<i>m</i>. as indicated. IL-10 (a) and IL-12p40 (b) were determined from 20 hour culture supernatants by ELISA. *p ≤ 0.05, **p ≤ 0.01 <i>L</i>.<i>m</i>. <i>Listeria monocytogenes</i>. 2-DG treatment alone induced the following cytokine levels: IL-10: 4 times ≤ 84.5 pg/mL, IL12p40: 4 times ≤ 5.1 pg/mL.</p