β1 Integrins Are Required To Mediate NK Cell Killing of Cryptococcus neoformans Richard F. Xiang, ShuShun Li, Henry Ogbomo, Danuta Stack and Christopher H. Mody This information is current as of September 30, 2021. J Immunol published online 10 September 2018 http://www.jimmunol.org/content/early/2018/09/07/jimmun ol.1701805 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/09/07/jimmunol.170180 Material 5.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 30, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 10, 2018, doi:10.4049/jimmunol.1701805 The Journal of Immunology b1 Integrins Are Required To Mediate NK Cell Killing of Cryptococcus neoformans Richard F. Xiang,*,† ShuShun Li,*,† Henry Ogbomo,*,† Danuta Stack,*,† and Christopher H. Mody*,†,‡ Cryptococcus neoformans is a fungal pathogen that causes fatal meningitis and pneumonia. During host defense to Cryptococcus, NK cells directly recognize and kill C. neoformans using cytolytic degranulation analogous to killing of tumor cells. This fungal killing requires independent activation of Src family kinase (SFK) and Rac1-mediated pathways. Recognition of C. neoformans requires the natural cytotoxicity receptor, NKp30; however, it is not known whether NKp30 activates both signal transduction pathways or whether a second receptor is involved in activation of one of the pathways. We used primary human NK cells and a human NK cell line and found that NKp30 activates SFK → PI3K but not Rac1 cytotoxic signaling, which led to a search for the receptor leading to Rac1 activation. We found that NK cells require integrin-linked kinase (ILK) to activate Rac1 for effective Downloaded from fungal killing. This observation led to our identification of b1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including b1 integrins and NKp30 and their proximal signaling pathways, are required for recognition of Cryptococcus, which activates a central cytolytic antimicrobial pathway leading to fungal killing. The Journal of Immunology, 2018, 201: 000–000. ryptococcus neoformans is a ubiquitous fungal pathogen the target cell. However, it is not known how nontumor ligands, http://www.jimmunol.org/ that causes over 220,000 cases of meningitis per year and such as those on fungal pathogens, trigger NK cell intracellular C over 180,000 deaths annually (1). C. neoformans pri- cytotoxic pathways. marily infects AIDS patients. Current antifungal therapies have Human and murine NK cells have been shown to directly kill limited efficacy and are cost prohibitive in this population, and C. neoformans (5–7). NK cells are recruited to the lung in re- unfortunately, symptomatic individuals have a 10-wk mortality sponse to pulmonary Cryptococcus (8), and mice lacking NK cells rate of 57% (2). This unacceptable morbidity and mortality has led are more susceptible to cryptococcal infections (9). Once in to a search of mechanisms of host defense to Cryptococcus, with a contact, C. neoformans binds to the natural cytotoxicity receptor view to enhanced therapies. (NCR) NKp30 and initiates a cytotoxic PI3K → Erk1/2 signaling NK cells are innate immune cells that are best known for their cascade (10). However, NKp30 does not directly interact with by guest on September 30, 2021 role in killing of malignant and virus-infected cells. However, NK PI3K. Instead, cryptococcal stimulation activates both Rac1 and cells are also capable of killing microbes such as fungi (3). NK Src family kinase (SFK) upstream of PI3K. Activation of Rac1 cell–mediated tumor killing depends on ligation of multiple re- and SFK are independent of one another, but both classes of ceptors that trigger release of cytolytic proteins. When the signals proteins are necessary for the activation of the downstream cyto- from activating receptors and integrins dominate, the NK cell and toxic PI3K → Erk1/2 pathway (11, 12). These observations trig- the tumor target cell form a conjugate that creates an NK immune gered important unanswered questions. Does NKp30 activate two synapse (reviewed in Ref. 4). The synapse provides a platform that separate pathways (SFK and Rac1), and do these two separate leads to intracellular signaling and trafficking of secretory lyso- pathways converge to activate PI3K, or does NKp30 activate one somes, resulting in directional release of cytolytic proteins toward pathway and cooperate with other receptors to activate that other pathway? *Department of Microbiology, Immunology and Infectious Diseases, Cumming School of During NK cell–mediated tumor killing, more than one receptor Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada; †Calvin, Phoebe is involved. Both activating receptors and integrin signaling syn- and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta ‡ ergize to achieve cytotoxicity (13–15). Signaling through activa- T2N 4N1, Canada; and Department of Internal Medicine, Cumming School of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada tion receptors leads to fusion of cytolytic granules with the plasma ORCIDs: 0000-0001-7448-5125 (H.O.); 0000-0001-7264-3679 (C.H.M.). membrane (degranulation), whereas signaling through b2 integ- Received for publication January 2, 2018. Accepted for publication August 8, 2018. rins facilitates the movement of granules toward the immune synapse (granule polarization) (15). Both integrin-mediated This work was supported by a studentship from The Lung Association, Alberta & NWT (to R.F.X.). C.H.M. was supported by the Jessie Bowden Lloyd Professorship. granule polarization and activating receptor–mediated degranula- This work was also supported by Canadian Institute for Health Research Grant tion are essential to effective tumor lysis. 365812 (to C.H.M.). Although b2 integrins play a crucial role in tumor killing, they Address correspondence and reprint requests to Dr. Christopher H. Mody, University do not play a role in anticryptococcal activity (16). Although b2 of Calgary, Room 273 Heritage Medical Research Building, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada. E-mail address: [email protected] integrins play the major role, b1 integrins have been implicated in The online version of this article contains supplemental material. tumor killing because cross-linking b1 integrins enhanced NK cell Abbreviations used in this article: ATCC, American Type Culture Collection; ILK, antitumor activity (17, 18). Interestingly, b1 integrins on neutro- integrin-linked kinase; MBCD, methyl-b-cyclodextran; NCR, natural cytotoxicity phils bind to and are activated by b-glucans, which are a con- receptor; PAK, p21-activated kinase; PBD, p21 binding domain; SFK, Src family served molecular pattern on fungal pathogens (19). Because kinase; siRNA, small interfering RNA. Cryptococcus also expresses similar b-glucans, it is plausible that Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 b1 integrins could be an anticryptococcal adhesion molecule. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701805 2 b1 INTEGRINS AND NK CELL CRYPTOCOCCAL KILLING To determine which signaling pathway NKp30 activated, we NuPAGE gradient gel (catalog no. NP0335BOX; Invitrogen). After separa- used blocking Abs to NKp30 and phospho-immunoblots to de- tion, samples were transferred to a nitrocellulose membrane and revealed termine whether SFK or Rac is activated by this NCR. Having with indicated Abs. Bands were recorded using an Odyssey infrared imaging system (LI-COR Biosciences). Densitometry was performed by measuring demonstrated that Rac is activated independently of NKp30, we the area under the intensity plot using ImageJ (version 1.48; National In- used immunoblots following small interfering RNA (siRNA) stitutes of Health). The fold increase in signaling compared with unstimu- knockdown or pharmacologic inhibitors to explore the role of lated cells was calculated as (intensity of stimulated condition normalized to integrin-linked kinase (ILK) upstream of Rac. Blocking Abs and loading control) / (intensity of unstimulated condition normalized to loading control) 2 1. siRNA knockdown were then used to investigate the roles of b2 and b1 integrins in activation of ILK → Rac and cryptococcal NK anticryptococcal killing assay killing. Our results demonstrated that NK cells require two inde- YT cells were cocultured with C. neoformans at an E:T ratio of 200:1 in pendent signaling pathways from NKp30 and b1 integrins. NKp30 round-bottom 96-well plates (catalog no. 163320; Thermo Fisher Scien- activates SFK, and b1 integrins activate ILK. The SFK and ILK tific). CFU were determined at 24 and 48 h postinoculation.