Β1 Integrins Are Required to Mediate NK Cell Killing of Cryptococcus Neoformans Richard F

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 26, 2021. J Immunol 2018; 201:2369-2376; Prepublished online 10 September 2018; doi: 10.4049/jimmunol.1701805 http://www.jimmunol.org/content/201/8/2369 Downloaded from

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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. 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: 2369–2376.

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 26, 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 2370 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. The anti- pathways then converge into a central PI3K signaling pathway. cryptococcal activity of primary NK cells was determined by coculture with C. neoformans at an E:T ratio of 100–400:1 in flat-bottom 96-well plates. Plates were centrifuged at 400 3 g for 5 min to bring NK and Cryptococcus into contact. CFU were determined 24 h postinoculation. In Materials and Methods experiments in which CPD 22 was used, the inhibitor was added to the YT Chemicals and small molecule inhibitors or primary NK cells at the same time that Cryptococcus was added. Pri- FITC was purchased from Sigma-Aldrich (catalog no. 3326-32-7; St. Louis, mary NK cell and YT cell viability was determined by trypan blue staining. Percent viability was calculated as follows: (number of trypan MO), CPD 22 was purchased from EMD Millipore (catalog no. 407331; 3 Downloaded from Etobicoke, ON, Canada). SMIFH2 was purchased from Calbiochem (catalog blue positive cells) / (total number of cells) 100%. Concentrations of no. 344092). DMSO was obtained from Sigma-Aldrich (catalog no. 472301). CPD 22 had minimal impact on viability of YT and primary NK cells. Methyl-b-cyclodextran (MBCD) was obtained from Sigma-Aldrich (catalog Conjugate assay no. C4555). CFSE was obtained from Millipore (catalog no. 4500-0270). Trypan blue stain 0.4% was purchased from Life Technologies (catalog no. C. neoformans strain B3501 was labeled following the procedure for 15250; Burlington, ON, Canada). C. gattii as previously described by Huston et al. (21). Briefly, C. neoformans was cultured overnight to the exponential phase of proliferation and labeled Abs with 2.5 mg/ml FITC per 108 cells at 22˚C for 10 min. C. neoformans was http://www.jimmunol.org/ Proteins in immunoblots were revealed with specific Abs: rabbit anti– then washed three times with PBS. YT cells were labeled with 5 mlofanti- phospho-SFK (Y416) (2101S; Cell Signaling Technology, Whitby, ON, CD11a PE-Cy5 Ab for 30 min in a 37˚C CO2 incubator. For the conjugate Canada), rabbit anti–phospho-Erk1/2 (T202/Y204) (9101S; Cell Signaling assay involving MBCD, 2 mM MBCD or PBS control was added immedi- Technology), rabbit anti–p-ILK (AB1076; Millipore), mouse anti-Fyn ately before incubation. YT cells and Cryptococcus were then incubated (610163; BD Biosciences, San Jose, CA), mouse anti-Rac1 (1862341; together for 10 min at 37˚C in 200 ml of complete media. YT cells and Thermo Fisher Scientific, Waltham, MA), rabbit b1 integrin (4706S; Cell Cryptococcus were agitated by pipetting. Conjugates were detected by Signaling Technology), goat anti-rabbit IgG infrared dye 700DX (611-130-002; Guava easyCyte Flow Cytometer (Cytosoft version 5.3; Guava Technologies, Rockland Immunochemicals, Limerick, PA), and goat anti-mouse IgG infrared Millipore, Danvers, MA), and data were analyzed by FlowJo software (Tree Star, dye 800 (923-32210; LI-COR Biosciences, Lincoln, NE). Cells for flow Ashland, OR). The percentage of NK cells in conjugates with C. neoformans

was determined as follows: (number of green and red event) / (total number by guest on September 26, 2021 cytometry were labeled with specific Abs for mouse anti-CD11a PE-Cy5 3 (551131; BD Biosciences, San Jose, CA), rabbit anti-CD29 (4706; Cell of red events) 100%. Signaling Technology), and mouse anti-CD29 conjugated to Alexa Fluor 488 Rac-GTP precipitation (303015; BioLegend, San Diego, CA). YT cells were unstimulated or stimulated with C. neoformans for 4 min in a Cells and Cryptococcus 37˚C water bath. YT cells were centrifuged at 6000 3 g for 30 s, and the Cryptococcus YT cells are an NK-like cell line isolated from the pericardial fluid of a 15-y-old supernatant was decanted. YT cells were then lysed, and was boy with acute lymphocytic leukemia (20). YT cells possessed NK cytotoxic added to the unstimulated conditions to control for the additional volume Cryptococcus activity against a panel of target cells, including K562, T, and B cell lines of in the stimulated conditions. Rac-GTP was then extracted according to manufacturer instructions with two modifications (17-10394; (20). Our YT cells were a gift from C. Clayberger, Emeritus Faculty, Millipore). A protease inhibitor (Roche) was substituted for leupeptin, and Stanford University, Stanford, CA. YT cells were validated by their ex- m pression of NKp30 and NKp44 and lack of CD3. YT cells were maintained 5 l of magnetic beads conjugated to the p21 binding domain (PBD) of in complete medium containing RPMI 1640 supplemented with 10% FBS p21-activated kinase (PAK) was used. Aliquots of whole-cell lysate were (Invitrogen Life Technologies), 1% nonessential amino acids (catalog no. saved before the addition of PBD-coated beads so that the total levels of 11140; Invitrogen Life Technologies), 1% sodium pyruvate (catalog no. Rac1 could be determined. Active Rac, which had bound PBD-coated beads, was measured by immunoblot. 11360; Invitrogen Life Technologies), and 1% penicillin–streptomycin in a 37˚C 5% CO2 incubator. siRNA knockdown K562 cells were obtained from American Type Culture Collection (ATCC) (catalog no. CCL243; Manassas, VA). 721.221 cells were pur- siRNA specific against b1 integrin’s b-chain (CD29) (59-GGAACC- chased from ATCC (catalog no. CRL1855). Both K562 and 721.221 cells CUUGCACAAGUGA-39) were purchased from Invitrogen Life Technol- were maintained in complete media in a 37˚C 5% CO2 incubator. Primary ogies and Thermo Fisher Scientific. siRNA against b2 integrin was NK cells were isolated from healthy donors using an NK isolation kit purchased from Thermo Fisher Scientific. Nontargeting siRNA was pur- (catalog no. 130-092-657; Miltenyi Biotec, San Diego, CA) as per the chased from Cell Signaling Technology. YT cells were transfected with 2 mg manufacturer’s instructions. Isolated cells were routinely .92% CD56+ of siRNA specific against b1 integrin, b2 integrin, or control. A Nucleo- and ,0.5% CD3+. C. neoformans strain B3501 (catalog no. 34873; ATCC) fector Kit V (Amaxa, Walkersville, MD) and Nucleofector II (Amaxa) were was grown to log phase in Sabouraud dextrose broth (catalog no. 238230; used to perform the transfection using the Nucleofector program O-017. Becton Dickinson, Mississauga, ON, Canada) on a 32˚C shaker overnight. YT cells were transferred into 500 ml of prewarmed complete media after transfection and then placed in a 37˚C incubator for 10 min. Cells were then Immunoblotting transferred to six-well plates and allowed to recover for 24 h in a 37˚C CO2 YT cells (3 3 105–3 3 106) were preincubated with various inhibitors and incubator. The cells were then used in a killing assay as described above. their controls for 1 h in a 37˚C CO2 incubator. YT cells were then coin- Statistics cubated with C. neoformans strain B3501 at an E:T ratio of 1:100 for various time points in a 37˚C water bath. Cells were lysed in Nonidet P-40 GraphPad Prism (La Jolla, CA) was used to evaluate statistics. Error bars lysis buffer containing 50 mM Tris (pH 7.4), 250 mM NaCl, 5 mM EDTA, represent the SEM. Data were analyzed by one-way ANOVA with Bon- 50 mM NaF, 1 mM Na3VO4, 1% Nonidet P-40, and 0.02% NaN3. Lysis ferroni correction. A p value , 0.05 was considered to be a statistically buffer was supplemented with phosphatase (Roche, Mississauga, ON, Canada) significant difference. Percent reduction in conjugates compared with H2O and protease inhibitors (Roche). Lysates were separated on a 4–12% Bis-Tris or DMSO was analyzed by the column statistics program in GraphPad The Journal of Immunology 2371

Prism. Different experiments were performed on different days using cells was repeated, but Rac activation was assessed by precipitation with from different donors. PBD of PAK. Rac was activated in response to C. neoformans; Ethics however, unlike SFK, Cryptococcus-induced Rac1 activation was not inhibited by the addition of inhibitory anti-NKp30 (Fig. 1C). Experimental protocols were approved and performed following the This reveals that NKp30 does not stimulate the Rac1-mediated guidelines from the Conjoint Health Research Ethics Board of the Uni- versity of Calgary (protocol number REB15-0600). pathway and suggests that an unidentiﬁed NK receptor activates Rac1. Results ILK is required for Rac1-dependent NK cell Cryptococcus activates NKp30 and signals through SFK but anticryptococcal response not Rac1 NK cell tumor killing depends on activating receptors, such as Previous studies found that SFK and Rac1 were activated in NK NKp30, and integrins to optimize killing. Because integrins can cells independently and nonredundantly upstream of PI3K in re- activate Rac1 in NK cells, we investigated whether integrin sig- sponse to Cryptococcus (12). Additionally, NKp30 was required naling is involved in cryptococcal killing (17, 24). Because there for PI3K activation and cryptococcal killing (10). However, it was are a large number of integrins on NK cells, we examined ILK as unknown whether SFK or Rac1 contributes to NKp30-dependent an indication of integrin signaling. We stimulated YT cells with activation of PI3K. To determine whether SFK is activated Cryptococcus and investigated the activation of ILK by immu- downstream of NKp30, YT cells were cocultured with Crypto- noblot. Immunoblot for phosphorylated ILK showed that stimu- coccus in the presence of an inhibitory Ab to NKp30 (clone 1C01) lation with C. neoformans induced activation of ILK in YT cells Downloaded from (10), and SFK signaling was tested after cryptococcal stimulation (Fig. 2A, 2B). To determine the pathway downstream of ILK, the (Fig. 1A, 1B). An immunoblot showed that YT cells stimulated small molecule inhibitor CPD 22 was used to inhibit ILK activity. with C. neoformans had increased phosphorylation of SFK. CPD 22 inhibits ILK activation and the phosphorylation of ILK However, treatment with 1C01 caused a marked reduction in SFK targets. CPD 22 has been described as a speciﬁc inhibitor of ILK activation in response to Cryptococcus compared with isotype because the phosphorylation of the downstream targets was re-

control Ab, revealing that NKp30 recognizes Cryptococcus and stored with the introduction of constitutively active ILK, and when http://www.jimmunol.org/ activates the SFK pathway (Fig. 1A, 1B). Coincubation of NK tested at high concentrations against a panel of 20 kinases, CPD 22 cells with anti-CD56 did not inhibit SFK activation (Supplemental did not significantly impact the activity of 19 of those kinases, was Fig. 1), suggesting that the SFK activation pathway is specific to only 60% effective against P70S6K, and did not affect focal adhesion NKp30. CD56 was chosen as a control because it is a canonical NK kinase (FAK) (25). CPD 22 was also specifically chosen because it receptor that also leads to SFK activation (22, 23). To determine was shown to inhibit the ILK→ PI3K pathway (25, 26). YT cells whether Rac is activated downstream of NKp30, the experiment treated with CPD 22 and stimulated with Cryptococcus showed by guest on September 26, 2021

FIGURE 1. Cryptococcus activates SFK through NKp30. (A) YT cells were preincubated with various concentrations of anti-NKp30 or control isotype- matched Ab. YT cells were stimulated with Cryptococcus and lysates were separated by SDS-PAGE. Immunoblotting was performed for activated SFK and Fyn. Fyn was measured to ensure equal amounts of cell lysate were loaded into each well. Representative immunoblot shown. (B) Densitometry of three independent immunoblots performed in (A). (C) YT cells were treated with anti-NKp30 inhibitory Ab or isotype control and stimulated with Cryptococcus.YTcellswere lysed and active Rac-GTP was precipitated using the PBD of PAK. (C) Active Rac was measured by immunoblot. (D) Densitometry of data in (C). Data are the mean of three independent experiments. Bars are the mean 6 SEM. *p , 0.05. ISO, isotype control Ab; Stim YT, YT cells stimulated with Cryptococcus. 2372 b1 INTEGRINS AND NK CELL CRYPTOCOCCAL KILLING Downloaded from http://www.jimmunol.org/

FIGURE 2. Cryptococcus induces Rac1 activity through an ILK-dependent mechanism. (A) YT cells were stimulated with Cryptococcus for various time points and then lysed. Lysates were immunoblotted for p-ILK and b-actin. (B) Densitometry showing the mean of three independent experiments. (C) YT cells were preincubated with 2 mM CPD 22 or DMSO control for 1 h. YT cells were then lysed and levels of active Rac1 were measured by PBD pull- down and immunoblotting. (D) Densitometry of immunoblots testing Rac1 activation in the presence of CPD 22. Bars are mean of three independent experiments 6 SEM. *p , 0.05, **p , 0.01. C+YT, YT cells stimulated with Cryptococcus; n.s., not signiﬁcant. by guest on September 26, 2021 reduced Rac1 activation compared with control (Fig. 2C, 2D). assessing CFU. We found that YT cells and primary NK cells This reveals that ILK activity is required for activation of the treated with CPD 22 showed reduced anticryptococcal activity Rac1 pathway. (Fig. 3A, 3B). Taken together, these ﬁndings reveal that ILK and its signaling pathway play a crucial role in NK-mediated anti- ILK activity is required for NK cell–mediated cryptococcal killing. cryptococcal cytotoxicity b b Because Rac1 is involved in cryptococcal killing (12) and ILK 1 integrins but not 2 integrins are required for NK-mediated activates Rac1 (Fig. 2), we wished to examine if ILK was required cryptococcal killing for cryptococcal killing. We preincubated YT cells or primary NK NK cells express b1 and b2 integrins, although b2 integrins cells with CPD 22 and measured anticryptococcal activity by are the predominant integrins on NK cells that are involved in

FIGURE 3. ILK activity is required for NK-mediated cryptococcal killing. (A) YT cells were cocultured with Cryptococcus in the presence of CPD 22 or control. (B) Primary NK cells were cocultured with Cryptococcus in the presence of CPD 22 or control. Bars are means of quadruplicate wells in a single experiment 6 SEM. Data are representative of three independent experiments. **p , 0.01. CA, Cryptococcus alone; CNK, primary NK cells stimulated with Cryptococcus; CYT, YT cells stimulated with Cryptococcus. The Journal of Immunology 2373 Downloaded from http://www.jimmunol.org/

FIGURE 4. b1 but not b2 integrins are required for cryptococcal killing. (A) YT cells were transfected with siRNA to b1 integrin, nontargeting siRNA,

or were mock transfected. Cells were rested for 24 h before being lysed. Lysates were separated by SDS-PAGE, and an immunoblot was performed with a by guest on September 26, 2021 mAb for b1 integrin. Immunoblotting was chosen because the b1 integrin Ab was not recommended for flow cytometry. (B) YT cells were transfected with siRNA to b2 integrin or b1 integrin, nontargeting siRNA, or were mock transfected. Cells were labeled with anti–b2 integrin Ab conjugated to PE-Cy5. Surface expression of b2 integrin was assessed by flow cytometry (mean fluorescence intensity shown). (C) Transfected YT cells were coincubated with Cryptococcus for 48 h, and anticryptococcal activity was assessed by plating the Cryptococcus on Sabouraud dextrose agar plates and counting CFU. Data are representative of three independent experiments. Bars are means of quadruplicate wells in a single experiment 6 SEM. (D) YT cells were transfected with a different b1 integrin siRNA targeting a different sequence. YT cells were then rested for 24 h as described above. After 24 h, transfected YT cells were labeled with anti–b1 integrin Ab conjugated to PE-Cy5 that is appropriate for flow cytometry. Surface expression of b1 integrin was assessed by flow cytometry. (E) YT cells that had been transfected with the second siRNA sequence against b1 integrin were coincubated with Cryptococcus for 48 h, and anticryptococcal activity was assessed by plating the Cryptococcus on Sabouraud dextrose agar plates and counting CFU. Data are representative of three independent siRNA transfections. Bars are means of quadruplicate wells in a single experiment 6 SEM. (F) YT cells were cocultured with Cryptococcus in the presence of an inhibitory anti–b1 integrin (CD29) Ab or isotype control for 48 h. Cryptococcus was then plated on Sabouraud dextrose agar plates and CFU were counted. (G) Primary NK cells were cocultured with Cryptococcus in the presence of an inhibitory anti–b1 integrin Ab or isotype control. Data are representative of three independent experiments. Bars are means of quadruplicate wells in a single experiment 6 SEM. *p , 0.05, **p , 0.01. CA, Cryptococcus alone; CNK, primary NK cells stimulated with Cryptococcus; CYT, YT cells stimulated with Cryptococcus; NT, nontargetting siRNA. antitumor cytotoxic signaling (14, 27). b1 integrins are also 4E) that provided similar results. We also performed an anti- known to activate ILK signaling (28). Because b2 integrins are not cryptococcal cytotoxicity assay in the presence of a blocking Ab involved in anticryptococcal activity, we examined whether inhi- against b1 integrins (anti-CD29). We found that blocking b1 bition of b1 integrins inhibited anticryptococcal activity. To de- integrins also reduced the anticryptococcal activity of YT cells termine if b1 and not b2 integrins are involved in cytotoxicity, we and primary NK cells compared with control Ab (Fig. 4F, 4G). reduced the expression of b1 and b2 integrins by siRNA (Fig. 4A, These findings show that b1 integrins are necessary for NK cell– 4B). We found that YT cells with diminished expression of b1 mediated anticryptococcal killing, but b2 integrins are not. integrins had reduced anticryptococcal activity, whereas YT cells b with diminished b2 integrins experienced no change in anti- 1 integrins are required to activate ILK in response to cryptococcal activity (Fig. 4C). The lack of b2 integrin involve- cryptococcal stimulation ment agrees with previous literature that showed that blocking Because b1 integrin and ILK are involved in anticryptococcal Abs and siRNA knockdown of LFA-1 (CD11a/CD18) did not activity and b1 integrins are known to activate ILK (28), we ex- inhibit cryptococcal killing (16). To exclude potential off-target amined if b1 integrins activated ILK in response to cryptococcal effects of siRNA knockdown, we repeated the cryptococcal killing stimulation. Using siRNA knockdown of b1 integrin, we found assay with a different sequence of b1 integrin siRNA (Fig. 4D, that reduction in expression of CD29 caused a reduction in ILK 2374 b1 INTEGRINS AND NK CELL CRYPTOCOCCAL KILLING

FIGURE 5. b1 integrin is required for activation of ILK. YT cells were transfected with nontargeting siRNA or b1 integrin siRNA or were mock transfected. Treated YT cells were rested for 24 h. YT cells were then stimulated with Cryptococcus for 5 min and lysed. (A) Lysates were separated by SDS-PAGE and immunoblots performed for p-ILK and b-actin. (B) Densitometry is mean of three independent experiments 6 SEM. **p , 0.01.

activation in response to cryptococcal stimulation (Fig. 5A, 5B). NK cells and Cryptococcus. Therefore, the inhibition of ILK This reveals that Cryptococcus activates b1 integrins to initiate an signaling is likely the result of impaired b1 integrin signaling Downloaded from ILK → Rac1 signaling pathway. rather than reduced adherence. This is consistent with other studies that have shown that cross-linking b1 integrins on NK b 1 integrins not are required for conjugate formation cells enhances their cytotoxicity (17). b1 integrins in NK cells can function as both adhesion receptors and enhancers of cytotoxic signaling pathways (17, 29). Our Discussion ﬁndings that inhibiting b1 integrins inhibits ILK activation could In this paper, we showed that 1) the anticryptococcal receptor be due to b1 integrin activation of ILK and augmentation of the NKp30 activates the SFK pathway but not the Rac1 signaling http://www.jimmunol.org/ Rac1 cytotoxicity pathway, or b1 integrins could be enhancing pathway, 2) b1 integrins are required for NK cell–mediated adhesion between Cryptococcus and NK cells and assisting an- cryptococcal killing, 3) b1 integrins induce an ILK → Rac1 sig- other receptor binding to Cryptococcus, which is then responsible naling pathway that is required for anticryptococcal activity, for ILK activation. Adhesion mediated by b1 integrins is depen- and 4) b1 integrins are not required for conjugate formation be- dent on lipid rafts (30), and we found that disruption of lipid rafts tween NK cells and Cryptococcus. Together, these results describe with MBCD reduced NK cell–Cryptococcus conjugate formation a novel role of b1 integrins in activating NK cell–mediated anti- (Fig. 6A). We then proceeded to investigate if knockdown of b1 cryptococcal cytotoxicity.

integrins inhibited conjugate formation. Knockdown of b1 integ- We found that there are similarities but also important differ- by guest on September 26, 2021 rins reduced the ﬂuorescent intensity from anti–b1 integrin Ab by ences between fungal killing and tumor killing. Anticryptococcal 90%. We found that YT cells with reduced b1 integrin expression killing has been shown to require both SFK and Rac1 signaling did not have reduced conjugate formation compared with non- pathways (11, 12). We found that NKp30 is responsible for the targeting siRNA or mock transfected cells (Fig. 6B). This suggests activation of SFK (Fig. 1), and b1 integrins were found to activate that b1 integrins are not a major contributor to adherence between the ILK→ Rac1 signaling pathway (Figs. 2, 5). This observation

FIGURE 6. Knockdown of b1 integrins does not affect NK cryptococcal conjugate formation. YT cells and Cryptococcus were labeled with anti-CD11a and FITC, respectively. YT cells and Cryptococcus were then cocultured for 25 min at room temperature. (A) YT cells were preincubated with MBCD and then cocultured with Cryptococcus. Conjugates were analyzed by flow cytometry. Bars are mean conjugate formation of three independent experiments 6 SEM. (B) YT cells were transfected with nontargeting or b1 integrin–targeting siRNA and then cocultured with Cryptococcus. Mean conjugate formation of three independent experiments 6 SEM. *p , 0.05. n.s., not significant. The Journal of Immunology 2375 resembles tumor killing because both NK-activating receptors and interaction. In this case, knockdown of b1 integrins would not integrin signaling are required. However, activation of Rac1 by b1 affect NK cryptococcal conjugate formations because b1 integrins integrins represents a noncanonical pathway for integrin signaling. are not binding to a cryptococcal ligand. b1 integrins have shown Previous studies suggest a model of how signaling leads to Rac1 the capacity for forming cis interactions (41, 42), and NK cell activation. b1 integrin–mediated activation of ILK is likely through cytotoxicity can be regulated by cis interactions (43). Therefore, the cytoplasmic portions of their b-chains (reviewed in Ref. 31). future research will be needed to explore cryptococcal ligands or ILK then acts as a scaffold protein that contains ankyrin repeats, cis interactions that are required to activate b1 integrins. pleckstrein homology domains, and calponin homology domains In conclusion, b1 integrin activates a Rac-mediated cytotoxicity (28). Parvins are a family of proteins that interact with ILK by pathway required for NK cell–mediated killing of Cryptococcus. binding to the calponin homology domain of ILK and are involved The finding that b1 integrin provides an activation signal rather than in smooth muscle contraction, actin microtubule attachment, cell enhancing adherence highlights its novel role in fungal killing: it polarization, and cell survival (32, 33). Interestingly, b-parvin has functions as a pathogen-associated molecular pattern receptor rather been shown to interact with the guanine nucleotide exchange factor than an adhesion molecule. a-PIX, which is an activator of Rac1 (34). Further work will be required to determine whether this pathway or some other is in- Disclosures volved in NK cell killing of fungi. The authors have no financial conflicts of interest. Different classes of integrins signal differently in NK cell– mediated cryptococcal killing compared with tumor killing. In tumor killing, the b2 integrins, specifically LFA-1, serve as ad- References Downloaded from hesion molecules and signaling proteins that lead to granule po- 1. Rajasingham, R., R. M. Smith, B. J. Park, J. N. Jarvis, N. P. Govender, T. M. Chiller, D. W. Denning, A. Loyse, and D. R. Boulware. 2017. Global larization (15). The impact of LFA-1 on granule polarization is burden of disease of HIV-associated cryptococcal meningitis: an updated anal- independent of activation receptor signaling, such as from FcgRor ysis. Lancet Infect. Dis. 17: 873–881. 2. Butler, E. K., D. R. Boulware, P. R. Bohjanen, and D. B. Meya. 2012. Long term NKp30 (15). In cryptococcal killing, NKp30 activates SFK (Fig. 1), 5-year survival of persons with cryptococcal meningitis or asymptomatic sub- and b1 integrins activate Rac1 signaling (Figs. 2, 5). SFK and Rac1 clinical antigenemia in Uganda. PLoS One 7: e51291.

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Supplement figure 1: Anti-CD56 does not inhibit SFK activation by Cryptococcus. A) YT cells were pre-incubated with various concentrations of anti-CD56 for 30 minutes in a 37°C CO2 incubator. YT cells were then stimulated with Cryptococcus and lysed. Lysates were separated by SDS-PAGE and immunoblotted for activated SFK and Fyn. B) Densitometry of 2 independent immunoblots performed using the protocol in panel A.