Fungal infections claim an estimated 1.5 million lives each year. Mechanisms that protect from fungal infections are still elusive. Recognition of fungal pathogens relies on C-type lectin receptors (CLRs) and their downstream signaling kinase SYK. Here we report that the E3 ubiquitin ligase CBLB controls proximal CLR signaling in macrophages and dendritic cells. We show that CBLB associates with SYK and ubiquitinates SYK, dectin-1, and dectin-2 after fungal recognition. Functionally, CBLB deficiency results in increased inflammasome activation, enhanced reactive oxygen species production, and increased fungal killing. Genetic deletion of Cblb protects mice from morbidity caused by cutaneous infection and markedly improves survival after a lethal systemic infection with Candida albicans. On the basis of these findings, we engineered a cell-permeable CBLB inhibitory peptide that protects mice from lethal C. albicans infections. We thus describe a key role for Cblb in the regulation of innate antifungal immunity and establish a novel paradigm for the treatment of fungal sepsis.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Proteomics Identifications Database
Armstrong-James, D., Meintjes, G. & Brown, G.D. A neglected epidemic: fungal infections in HIV/AIDS. Trends Microbiol. 22, 120–127 (2014).
Ravikumar, S., Win, M.S. & Chai, L.Y. Optimizing outcomes in immunocompromised hosts: understanding the role of immunotherapy in invasive fungal diseases. Front. Microbiol. 6, 1322 (2015).
Lanternier, F. et al. Primary immunodeficiencies underlying fungal infections. Curr. Opin. Pediatr. 25, 736–747 (2013).
Brown, G.D. et al. Hidden killers: human fungal infections. Sci. Transl. Med. 4, 165rv13 (2012).
Marr, K.A. et al. Differential role of MyD88 in macrophage-mediated responses to opportunistic fungal pathogens. Infect. Immun. 71, 5280–5286 (2003).
Ramirez-Ortiz, Z.G. et al. Toll-like receptor 9–dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA. Infect. Immun. 76, 2123–2129 (2008).
Cambi, A. et al. The C-type lectin DC-SIGN (CD209) is an antigen-uptake receptor for Candida albicans on dendritic cells. Eur. J. Immunol. 33, 532–538 (2003).
Netea, M.G. et al. Immune sensing of Candida albicans requires cooperative recognition of mannans and glucans by lectin and toll-like receptors. J. Clin. Invest. 116, 1642–1650 (2006).
Hardison, S.E. & Brown, G.D. C-type lectin receptors orchestrate antifungal immunity. Nat. Immunol. 13, 817–822 (2012).
Brown, G.D. & Gordon, S. Immune recognition. A new receptor for β-glucans. Nature 413, 36–37 (2001).
Saijo, S. et al. Dectin-2 recognition of α-mannans and induction of TH17 cell differentiation is essential for host defense against Candida albicans. Immunity 32, 681–691 (2010).
Zhu, L.L. et al. C-type lectin receptors dectin-3 and dectin-2 form a heterodimeric pattern-recognition receptor for host defense against fungal infection. Immunity 39, 324–334 (2013).
Wells, C.A. et al. The macrophage-inducible C-type lectin, Mincle, is an essential component of the innate immune response to Candida albicans. J. Immunol. 180, 7404–7413 (2008).
van de Veerdonk, F.L. et al. The macrophage mannose receptor induces IL-17 in response to Candida albicans. Cell Host Microbe 5, 329–340 (2009).
Ishikawa, E. et al. Direct recognition of the mycobacterial glycolipid, trehalose dimycolate, by C-type lectin Mincle. J. Exp. Med. 206, 2879–2888 (2009).
Smeekens, S.P. et al. The classical CD14+CD16− monocytes, but not the patrolling CD14+CD16+ monocytes, promote TH17 responses to Candida albicans. Eur. J. Immunol. 41, 2915–2924 (2011).
Patel, D.D. & Kuchroo, V.K. TH17 cell pathway in human immunity: lessons from genetics and therapeutic interventions. Immunity 43, 1040–1051 (2015).
Whibley, N. & Gaffen, S.L. Brothers in arms: TH17 and Treg responses in Candida albicans immunity. PLoS Pathog. 10, e1004456 (2014).
Mócsai, A., Ruland, J. & Tybulewicz, V.L. The SYK tyrosine kinase: a crucial player in diverse biological functions. Nat. Rev. Immunol. 10, 387–402 (2010).
Deng, Z. et al. Tyrosine phosphatase SHP-2 mediates C-type-lectin-receptor-induced activation of the kinase Syk and antifungal TH17 responses. Nat. Immunol. 16, 642–652 (2015).
Zwolanek, F. et al. The nonreceptor tyrosine kinase Tec controls assembly and activity of the noncanonical caspase-8 inflammasome. PLoS Pathog. 10, e1004525 (2014).
Gross, O. et al. Syk kinase signaling couples to the Nlrp3 inflammasome for antifungal host defense. Nature 459, 433–436 (2009).
Ruland, J. CARD9 signaling in the innate immune response. Ann. NY Acad. Sci. 1143, 35–44 (2008).
Bachmaier, K. et al. Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b. Nature 403, 211–216 (2000).
Chiang, Y.J. et al. Cbl-b regulates the CD28 dependence of T cell activation. Nature 403, 216–220 (2000).
Loeser, S. & Penninger, J.M. The ubiquitin E3 ligase Cbl-b in T cell tolerance and tumor immunity. Cell Cycle 6, 2478–2485 (2007).
Paolino, M. et al. Essential role of E3 ubiquitin ligase activity in Cbl-b-regulated T cell functions. J. Immunol. 186, 2138–2147 (2011).
Paolino, M. et al. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature 507, 508–512 (2014).
Qu, X. et al. Negative regulation of FcɛRI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b. Blood 103, 1779–1786 (2004).
Han, C. et al. Integrin CD11b negatively regulates TLR-triggered inflammatory responses by activating Syk and promoting degradation of MyD88 and TRIF via Cbl-b. Nat. Immunol. 11, 734–742 (2010).
Wallner, S. et al. The role of the E3 ligase Cbl-b in murine dendritic cells. PLoS One 8, e65178 (2013).
Zhang, J., Chiang, Y.J., Hodes, R.J. & Siraganian, R.P. Inactivation of c-Cbl or Cbl-b differentially affects signaling from the high-affinity IgE receptor. J. Immunol. 173, 1811–1818 (2004).
Sohn, H.W., Gu, H. & Pierce, S.K. Cbl-b negatively regulates B cell antigen receptor signaling in mature B cells through ubiquitination of the tyrosine kinase Syk. J. Exp. Med. 197, 1511–1524 (2003).
Igyártó, B.Z. et al. Skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific T helper cell responses. Immunity 35, 260–272 (2011).
Drummond, R.A., Gaffen, S.L., Hise, A.G. & Brown, G.D. Innate defense against fungal pathogens. Cold Spring Harb. Perspect. Med. 5, a019620 (2015).
Aratani, Y. et al. Relative contributions of myeloperoxidase and NADPH oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus. Med. Mycol. 40, 557–563 (2002).
Gringhuis, S.I. et al. Dectin-1 is an extracellular pathogen sensor for the induction and processing of IL-1β via a noncanonical caspase-8 inflammasome. Nat. Immunol. 13, 246–254 (2012).
Qian, Q., Jutila, M.A., Van Rooijen, N. & Cutler, J.E. Elimination of mouse splenic macrophages correlates with increased susceptibility to experimental disseminated candidiasis. J. Immunol. 152, 5000–5008 (1994).
May, M.J. et al. Selective inhibition of NF-κB activation by a peptide that blocks the interaction of NEMO with the IκB kinase complex. Science 289, 1550–1554 (2000).
Zou, W., Reeve, J.L., Zhao, H., Ross, F.P. & Teitelbaum, S.L. Syk tyrosine 317 negatively regulates osteoclast function via the ubiquitin-protein isopeptide ligase activity of Cbl. J. Biol. Chem. 284, 18833–18839 (2009).
Yankee, T.M., Keshvara, L.M., Sawasdikosol, S., Harrison, M.L. & Geahlen, R.L. Inhibition of signaling through the B cell antigen receptor by the proto-oncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain. J. Immunol. 163, 5827–5835 (1999).
Glocker, E.O. et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N. Engl. J. Med. 361, 1727–1735 (2009).
Zheng, J. et al. Gain-of-function STAT1 mutations impair STAT3 activity in patients with chronic mucocutaneous candidiasis (CMC). Eur. J. Immunol. 45, 2834–2846 (2015).
van der Graaf, C.A. et al. Candida-specific interferon-γ deficiency and toll-like receptor polymorphisms in patients with chronic mucocutaneous candidiasis. Neth. J. Med. 61, 365–369 (2003).
Plantinga, T.S. et al. Human genetic susceptibility to Candida infections. Med. Mycol. 50, 785–794 (2012).
Plantinga, T.S. et al. Early stop polymorphism in human dectin-1 is associated with increased Candida colonization in hematopoietic stem cell transplant recipients. Clin. Infect. Dis. 49, 724–732 (2009).
Smeekens, S.P., van de Veerdonk, F.L., Kullberg, B.J. & Netea, M.G. Genetic susceptibility to Candida infections. EMBO Mol. Med. 5, 805–813 (2013).
Stevens, D.A. Advances in systemic antifungal therapy. Clin. Dermatol. 30, 657–661 (2012).
Bergholdt, R., Taxvig, C., Eising, S., Nerup, J. & Pociot, F. CBLB variants in type 1 diabetes and their genetic interaction with CTLA4. J. Leukoc. Biol. 77, 579–585 (2005).
Kosoy, R., Yokoi, N., Seino, S. & Concannon, P. Polymorphic variation in the CBLB gene in human type 1 diabetes. Genes Immun. 5, 232–235 (2004).
Nakao, R. et al. Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading. Mol. Cell. Biol. 29, 4798–4811 (2009).
Bechara, C. & Sagan, S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 587, 1693–1702 (2013).
Boxio, R., Bossenmeyer-Pourié, C., Steinckwich, N., Dournon, C. & Nüsse, O. Mouse bone marrow contains large numbers of functionally competent neutrophils. J. Leukoc. Biol. 75, 604–611 (2004).
Bourgeois, C., Majer, O., Frohner, I. & Kuchler, K. In vitro systems for studying the interaction of fungal pathogens with primary cells from the mammalian innate immune system. Methods Mol. Biol. 470, 125–139 (2009).
Wirnsberger, G. et al. Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense. Nat. Genet. 46, 1028–1033 (2014).
Dorfer, V. et al. MS Amanda, a universal identification algorithm optimized for high accuracy tandem mass spectra. J. Proteome Res. 13, 3679–3684 (2014).
Taus, T. et al. Universal and confident phosphorylation site localization using phosphoRS. J. Proteome Res. 10, 5354–5362 (2011).
We thank all members of the Penninger laboratory for helpful discussions and technical support. We thank all members of the IMP-IMBA Biooptics service facility, especially G. Schmauss and T. Lendl, for assistance in cell sorting and image quantification. We also thank A. Piszczek and M. Zeba from the Vienna Biocenter Core Facilities (VBCF) for excellent histopathology services, and K. Mechtler, R. Imre, and M. Madalinski from the Protein Biochemistry facility for providing excellent mass spectrometry and peptide synthesis services. This work was supported by a consolidator ERC grant (F.I.), the EC FP7 project 'FUNGITECT' (K.K.), the FWF project P-25333 (K.K.), a Marie-Curie 'Innovative Training Networks' ImResFun grant (contract MC-ITN-606786; K.K.), an Advanced ERC grant (J.M.P.), an 'Era of Hope/Innovator Award' (J.M.P.), a Helmsley foundation VEO–IBD network grant (J.M.P.), and the Austrian Academy of Sciences (J.M.P.).
J.M.P., G.W., F.Z., and K.K. are inventors on a patent application describing the use of a TKB binding peptide as a therapeutic for the modulation of CBLB-regulated immune responses.
About this article
Cite this article
Wirnsberger, G., Zwolanek, F., Asaoka, T. et al. Inhibition of CBLB protects from lethal Candida albicans sepsis. Nat Med 22, 915–923 (2016) doi:10.1038/nm.4134
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics (2020)
Clinical Significance of Potassium Channel and NLRP3 Expression in Platelets of Active Ulcerative Colitis
Inflammatory Bowel Diseases (2019)
Cellular Immunology (2019)
Nature Reviews Immunology (2019)
Dok3–protein phosphatase 1 interaction attenuates Card9 signaling and neutrophil-dependent antifungal immunity
Journal of Clinical Investigation (2019)