Syk inhibitor R406 down-regulates inflammation in an in vitro model of Pseudomonasaeruginosa infection

Alaa Alhazmi1, Joshua Choi2, Marina Ulanova1,2*1Department of Biology, Lakehead University, 2Northern Ontario School of Medicine, Thunder Bay, Ontario, CanadaCorresponding Author


As Pseudomonas aeruginosa infections are characterized by strong inflammation of infected

tissues anti-inflammatory therapies in combination with antibiotics have been considered for the

treatment of associated diseases. Syk tyrosine kinase is an important regulator of inflammatory

responses, and its specific inhibition was explored as a therapeutic option in several inflammatory

conditions; however, this has not been studied in bacterial infections. We used a model of in vitro

infection of human monocytic cell line THP-1 and lung epithelial cell line H292 with both wild

type and flagella-deficient mutant of P. aeruginosa strain K, as well as with clinical isolates from

cystic fibrosis patients, to study the effect of a small molecule Syk inhibitor R406 on

inflammatory responses induced by this pathogen. One-hour long pretreatment of THP-1 cells

with 10 µM R406 resulted in a significant down-regulation of the expression of the adhesion

molecule ICAM-1, pro-inflammatory cytokines TNFα and IL-1β, and phosphorylated signaling

proteins ERK2, JNK, p-38, and IκBα, as well as significantly decreased TNF-α release by

infected H292 cells. The results suggest that Syk is involved in the regulation of inflammatory
responses to P. aeruginosa, and R406 may potentially be useful in dampening the damage caused
by severe inflammation associated with this infection.
Key Words: Pseudomonas aeruginosa, cystic fibrosis, Syk, small molecule inhibitor, R406,


inflammation, cytokine




43Pseudomonas aeruginosa is the major cause of chronic pulmonary infection in cystic fibrosis

(CF) patients as well as of other serious conditions in immunocompromised individuals (Saiman

and Siegel 2004; Hakki et al. 2007; Crouch Brewer et al. 1996; Lieberman and Lieberman 2003).

P. aeruginosa is a Gram-negative opportunistic pathogen armed with potent virulence factors

including the type III secretion and quorum sensing systems, lipopolysaccharide, several powerful

exotoxins, and various enzymes that contribute to disease pathogenesis via severe tissue damage

and inflammation as well as immune evasion (Kipnis 2006). As P. aeruginosa infection is

44characterized by exaggerated inflammatory responses, anti-inflammatory therapy is considered

important for treatment of P. aeruginosa-associated conditions (Cheng et al. 2013). In particular,

45intracellular protein kinases involved in the regulation of pro-inflammatory signaling pathways

46may represent potential therapeutic targets. We have recently found that an inhibitor of Syk

tyrosine kinase piceatannol is able to down-regulate inflammatory responses in P. aeruginosa-

47infected lung epithelial cells (Aval et al. 2013). However, the effect of piceatannol in this model

extended beyond inhibition of Syk, i.e. via potential modulation of Syk-independent signaling

pathways (Aval et al. 2013). A small molecule inhibitor, N4-(2,2-dimethyl-3-oxo-4H-

48pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406) was

49demonstrated to selectively inhibit Syk kinase activity in an ATP-competitive manner both in

50vitro and in vivo (Cha et al. 2006; Braselmann et al. 2006; Spalton et al. 2009; McAdoo and Tam

512011). R406 is the active metabolite of an orally available drug Fostamatinib, which had

52undergone several clinical trials for treatment of some autoimmune and allergic diseases and

53hematological malignancies (Riccaboni et al. 2010). However, it is unknown whether R406 can

54modulate inflammatory responses in infections. In this study, we sought to assess the effect of

55R406 on inflammatory markers associated with P. aeruginosa infection of human monocytic and

lung epithelial cells.







Materials and Methods
70Cell culture conditions

71The THP-1 human acute monocytic leukemia cell line (ATCC, Manassas, VA) was used at the

72passage numbers of 6-20. These cells were maintained in RPMI 1640 medium (Sigma-Aldrich,

73Oakville, ON, Canada) supplemented with 10% heat inactivated fetal bovine serum (FBS) (SAFC

74Biosciences, Lenexa, KS) and 1% antibiotic-antimycotic (Invitrogen, Burlington, ON, Canada).

75Cells were grown at 37°C with 5% CO2 and seeded every 3-4 days when cell counts neared 1×106

76cells/mL. In preparation for experiments, the cells were centrifuged at 400 × g for 5 minutes,

77washed with sterile PBS (pH 7.4), and suspended in culture medium without antibiotics. To

78induce differentiation, THP-1 cells were plated at 1×106 cells/mL/well in 24-well plates (Costar,

79Corning Incorporated, Corning NY), in serum- and antibiotic-RPMI 1640 medium. Cells were

80then treated with 20 ng/mL phorbol myristate acetate (PMA; Sigma-Aldrich) at 37°C in 5%

81CO2 for 12 hours, then washed and re-suspended in the same medium. After 48 hours of further

82incubation, the cells were washed twice with serum- and antibiotic-free medium and used for


84The H292 human muco-epidermoid bronchiolar carcinoma cell line (ATCC) was used at the

85passage numbers of 10-25. These cells were maintained in RPMI 1640 medium supplemented

86with 10% heat inactivated FBS without antibiotics. Cells were grown at 37°C with 5% CO2 and

87seeded every 3-4 days when confluency approached 80%. For viability testing, the cells were

88detached using 0.5% Trypsin-EDTA (Gibco, Eugene, OR), centrifuged at 400 × g for 5 minutes,

89washed with sterile PBS (pH 7.4), and suspended in culture medium. Cell viability was

90determined by the trypan blue exclusion method using a ViCell XR Cell Viability Analyzer

(Beckman Coulter, Brea, CA, USA).
93Pseudomonas aeruginosa strains and in vitro infectious model



94Pseudomonas aeruginosa strain K wild type (PAK WT, provided by Dr. RJ Irvin, University of

95Alberta, Edmonton, AB) and the isogenic P. aeruginosa mutant PAK fliC (flagella deficient,

96provided by Dr. AS Prince, Columbia University, New York), as well as P. aeruginosa clinical

97isolates from sputum samples of CF patients were used (Table). One clinical isolate from an

98intermittently colonized and another from a chronically infected patient (the latter obtained during

99longitudinal observation at the Danish CF Center) were kindly provided by Dr. N Høiby

100(University Hospital Rigshospitalet, Copenhagen, Denmark). The characteristics of the isolates

101are described in our previous study (Hawdon et al. 2010).

102The bacteria were maintained on Luria Burtani (LB) medium (Fischer Scientific, Fair Lawn, NJ)

103with 1% agar (LBA). A single colony of P. aeruginosa was grown overnight in sterile LB

104medium on a shaking platform at 150 rpm and diluted by a factor of 20 into fresh sterile LB

105medium. Cultures were allowed to grow for approximately 1 hour, until mid-log phase when

106optical density at 600 nm (OD600) reached 0.30. The culture was then centrifuged at 3,500 × g for

10720 minutes at 4°C and washed twice in PBS. Following the final re-suspension, bacteria were

108diluted to an OD600 of 0.30 in RPMI 1640 that corresponded to approximately 2×108 CFU/mL, as

109determined by serial dilutions and drop plating on LBA. From this stock, bacteria were added to

110either H292 cells to obtain a multiplicity of infection (MOI) of 50, as was optimized in our

111previous experiments (Aval et al. 2013), or THP-1 cells at a MOI of 5. The latter conditions were

112optimized using THP-1 cells infected with PAK during 1, 2, 6, 12, or 18 hours at MOI of 1, 5, or


115Stimulation of THP-1 cells via Fcγ-receptor cross-linking

116The 96-well plates (Falcon, Corning Incorporated) were coated with human IgG (Sigma-Aldrich)

117at concentrations of 10 and 100 µg/mL and incubated for 3 hours at 37°C, followed by overnight

118incubation at 4°C, then the plates were washed twice with sterile PBS. THP-1 cells at



119concentration of 0.4×106 cells/mL in 200 µL were added to the coated wells and incubated for 18

hours at 37°C with 5% CO2.
122Pretreatment with R406

123THP-1 or H292 cells were grown for 24 hours to 0.4×106 cells/mL, or until they reached

124approximately 80% confluence, respectively, and R406 (AstraZeneca) dissolved in DMSO was

125added to the medium to achieve a final concentration of 10 µM. The cells were incubated in the

126presence of R406 for 1 hour, then washed once with PBS and used for experiments. These

127conditions were developed based on published literature describing R406 pretreatments

128(Braselmann et al. 2006; Spalton et al. 2009; Chen et al. 2008; Quiroga et al. 2009) and our

129cellular viability testing using R406 concentrations of 1, 5, 10, 15, and 20 µM. No noticeable

130effect of R406 concentrations up to 10 µM on cell viability tested during one hour was detected

131(97-99% viable cells), nor significant decrease in either THP-1 or H292 cell viability following

13218-hour-long incubation with 10 µM of R406 occurred. Viability of THP-1 following 18 hour-

133long incubation with R406 or without R406 was 82% and 75% (P>0.05), for H292 cells, it was

84% and 81%, correspondingly (P>0.05).
136Flow cytometry analysis of ICAM-1 expression

137THP-1 cells (0.4×106) were infected with PAK WT for 6 hours at 37°C, 5% CO2, then washed

138and re-suspended in 100 µL of 0.1% BSA-PBS containing PE-conjugated mAb against ICAM-1

139(Mouse anti-human CD54, BD Pharmigen, Mississauga, ON) at a dilution of 1:50 and incubated

140for 1 hour at 4°C. Following incubation, cells were washed twice with PBS and analyzed by flow

141cytometry on the FACSCailbur (BD Bioscience, Mississauga, ON, Canada). The data were

analyzed using CellQuest Pro software and expressed as mean fluorescence intensity (MFI).
144ELISA for cytokine detection



145To measure the release of cytokines, PMA-differentiated THP-1 or H292 cells were infected with

146P. aeruginosa (MOI of 10, or 50, correspondingly), for 1 hour at 37˚C, with 5% CO2, and then

147100 µg/mL gentamicin was added, followed by incubation for a further 17 hours at 37°C with 5%

148CO2. Following stimulation, cell culture supernatants were collected and stored at -80°C until

149analysis. The levels of TNFα and IL-1β were measured using eBioscience Ready-Set-Go ELISA

150kits (San Diego, CA) according to the manufacturer’s protocol. The lower detection limits of the

151assays were 2 pg/mL for IL-1β and 4 pg/mL for TNFα. Samples from three independent

experiments were run in triplicate.
154Immunoprecipitation and Western blot

155THP-1 cells (2×106) were infected with PAK WT at an MOI of 5 for 2 hours at 37°C, 5% CO2.

156Following stimulation, the cells were centrifuged, washed, re-suspended in 100 µL of ice-cold

157RIPA lysis buffer, and incubated for 30 minutes at 4 °C. Following incubation, the cells were

158centrifuged at 8,000 × g for 10 minutes and protein lysate was collected. Isolated proteins were

159immunoprecipitated with polyclonal anti-Syk antibody (N-19) (Santa Cruz Biotehnology, CA)

160using magnetic Protein A beads (Bio-Rad, Hercules CA), according to the manufacturer’s

161protocol. Samples were resolved by 12% SDS-PAGE and transferred to a nitrocellulose

162membrane. Blots were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.1%

163Tween 20, probed with primary antibody, i.e. anti-phospho-tyrosine (P-Tyr-100) (Cell Signaling

164Technology), or monoclonal anti-syk antibodies (4D10) (Santa Cruz Biotechnology, CA)

165followed by HRP-conjugated secondary antibody (7074S) (Cell Signaling Technology), and

166developed using enhanced chemiluminescence. Bands were scanned and images analyzed using

167ChemiDoc XRS (Bio-Rad). For analysis of total and phosphorylated intracellular signaling

168proteins, THP-1 cells were stimulated with PAK (MOI of 5), for 15, 30, or 60 minutes at 37°C.

169Protein lysates were collected and stored at -80 °C. For analysis of protein expression by Western

170blot we used: monoclonal anti-JNK (D-2), anti-phospho JNK (G-7), anti-ERK 2 (12A4), anti-



171phospho ERK 2 (E-4), anti-p38α (9F12), anti-phospho p38α (E-1), anti-IκB-α (H-4), anti-

172phospho IκB-α (B-9), anti- β-actin (C4) and mouse IgGκ BP-HRP (Santa Cruz Biotechnology). In

some cases, the blots were stripped and re-probed with other antibodies.
175Statistical analysis

176All the experiments were repeated at least 3 times. Data were expressed as mean +/- SEM for n

177independent experiments. For comparison of two sample means, Student’s t test was applied.

178GraphPad Prism 7.0 (La Jolla, CA, USA) was used for the analysis. P-values <0.05 were

179considered significant.




R406 down-regulates ICAM-1 expression induced by P. aeruginosa infection
183As the intercellular adhesion molecule 1 (ICAM-1) typically becomes up-regulated during

184inflammatory responses, particularly in cells infected with P. aeruginosa (Roebuck and Finnegan

1851999; Sadikot et al. 2005) we tested the effect of R406 on the cell-surface expression of ICAM-1

186in THP-1 cells exposed to a virulent P. aeruginosa strain K (PAK) at an MOI of 5. As shown on

187Fig.1A, 6-hour long infection resulted in >15-fold increase in ICAM-1 mean fluorescence

188intensity (MFI) compared to uninfected cells. One hour-long pre-incubation of THP-1 cells with

189R406 used in concentrations between 0.1 and 20 µM down-regulated ICAM-1 expression in a

190dose-dependent manner, with a statistically significant effect for all the R406 concentrations >0.5

191µM (Fig. 1A). There was no noticeable effect of R406 concentrations up to 10 µM on cell

192viability; the percentage of viable cells after one hour of incubation with R406 was 97-99% (data

193not shown). Likewise, one hour-long incubation of THP-1 cells with 1, 5, or 10 µM of R406 did

not have any visible effect on the baseline ICAM-1 expression (data not shown).
196To confirm the effect of R406 on Syk in our model, we stimulated THP-1 cells via Fcγ-receptor

197(FcγR) cross-linking, which is known to induce Syk-dependent signaling (Darby et al. 1994).

198While stimulation of THP-1 cells with immobilized human IgG resulted in a significant increase

199in ICAM-1 surface expression in a dose-dependent manner, pre-treatment with 10 µM R406

200caused an attenuation of ICAM-1 expression at both 10 and 100 µg/mL IgG concentrations

201(P<0.05) (Fig. 1B). Moreover, tyrosine phosphorylation of Syk induced by two hour-long

202exposure of THP-1 cells to live bacteria was significantly down-regulated in cells, pre-treated

with 10 µM R406 (Fig. 1C).



205 These experiments imply that down-regulation of ICAM-1 in P. aeruginosa infected cells by

R406 could be mediated by inhibition of Syk-mediated signaling.
208 R406 down-regulates the release of pro-inflammatory cytokines TNFα and IL-1β induced by P.

aeruginosa infection
211To further test the effect of R406 in our model, we studied the release of cytokines TNFα and IL-

2121β, which are the hallmarks of inflammatory responses caused by P. aeruginosa infection, by

213using a virulent P. aeruginosa strain K (PAK WT), the isogenic P. aeruginosa mutant PAK fliC

214(flagella-deficient), and clinical isolates from two CF patients (intermittently colonized and

215chronically infected). One hour-long infection of differentiated THP-1 cells, or H292 cells with

216PAK WT, followed by adding gentamicin with further 17 hours of incubation resulted in a large

217TNFα release by both cell types, with THP-1 cells producing over 35-fold greater amount of this

218cytokine as compared to H292 cells (Fig. 2B-C).

219When flagella-deficient mutant (PAK fliC) was used for stimulation, TNFα release by both cell

220lines was lower compared to stimulation with PAK WT (P<0.01) Interestingly, although

221stimulation of THP-1 cells with either clinical P. aeruginosa isolate resulted in lower TNFα

222release (P<0.0001), in case of H292 cells, an isolate from a chronically infected CF patient

223induced higher TNFα release compared to PAK WT (P<0.001), PAK fliC (P<0.0001), and isolate

224from a CF patient with intermittent P. aeruginosa infection (P<0.001). Nevertheless, TNFα

225release was significantly decreased in all infected cell cultures pretreated with R406, except for

226THP-1 cells stimulated with an isolate from a chronically infected CF patient (Fig. 2B-C).

227While unstimulated differentiated THP-1 cells only produced a low amount of IL-1β (78 ±9

228pg/mL), infection with PAK WT resulted in a large increase in IL-1β release (3993 ±245 pg/mL,

229P<0.0001). Stimulation with PAK fliC, or either isolate from a CF patient with intermittent or

230chronically P. aeruginosa infected also significantly upregulated IL-1β release, although to a



231lesser degree compared to PAK WT (P<0.0001, P<0.01, and P<0.05 respectively). The lowest

232amount of IL-1β (1310 ±281 pg/mL) was released by THP-1 cells stimulated with an isolate from

233a chronically infected CF patient (significant lower than following stimulation with PAK WT,

234P<0.0001). Despite of different degrees of IL-1β release induced by P. aeruginosa strains,

pretreatment of differentiated THP-1 cells with R406 down-regulated this response (Fig. 2A).
237 R406 down-regulates the expression of phosphorylated ERK2, JNK, p-38, and IκBα in P.

aeruginosa infected THP-1 cells
240As intracellular signaling molecules ERK2, JNK, p-38, and IκBα have been recognized as

241important regulators of inflammatory responses induced by P. aeruginosa (Li et al. 1998; Ratner

242et al. 2001; Esen et al. 2001), we investigated the effect of R406 on the expression of these total

243and phosphorylated proteins in our model. Stimulation of THP-1 cells with PAK induced

244significant up-regulation of phosphorylated ERK2 at 30 min (P<0.01) and 60 min (P<0.01), JNK

245at 15 min (P<0.001), 30 min (P<0.001), and 60 min (P<0.001), p38 at 15 min (P<0.001), 30 min

246(P<0.0001), and 60 min (P<0.0001), and IκBα at 30 min (P<0.01) and 60 min (P<0.001).

247Pretreatment of infected cells with R406 led to a decreased expression of all phosphorylated

248signaling molecules that was statistically significant for JNK and p-38 at 15, 30, and 60 minutes


of stimulation, and for IκBα and ERK2 at 30 and 60 minutes of stimulation (Fig. 3A-D).







254This study shows that a small molecule inhibitor of Syk down-regulates inflammatory responses

255of human cells infected with P. aeruginosa. Specifically, in monocytic cell line THP-1, R406

256caused a significant decrease in cell surface expression of ICAM-1, an adhesion molecule, which

257mediates leukocyte migration to inflammatory sites, in a dose-dependent manner, as well as

258down-regulated the release of pro-inflammatory cytokines TNFα and IL-1β. The transcriptional

259regulation of all these three molecules is largely dependent on the activation of transcription

260factor NF-κB, which is known to be a downstream target of Syk-mediated signaling along with

261the MAPK cascade (Costello et al. 1996; Darby et al. 1994). Indeed, R406 caused a decrease in

262the expression of phosphorylated ERK2, JNK and p-38, as well as of IκBα; the latter, when

263phosphorylated, facilitates nuclear translocation of NF-κB, which is required for its activation and

264resulting production of inflammatory mediators (Akira and Kishimoto 1997). In our previous

265study, inhibition of Syk using small interfering RNA caused down-regulation of the MAPK

266cascade phosphorylation and nuclear translocation of p65 NF-κB induced by TNFα stimulation of

267lung epithelial cells (Ulanova et al. 2006). The data of the present study extend our earlier

268observations to monocytic cells and indicate that Syk is involved in the regulation of pro-

269inflammatory responses to P. aeruginosa infection via activation of downstream signalling

270pathways, including MAPK-mediated one. In support of this idea, we found an increase in the

271expression of tyrosine-phosphorylated Syk, an indicator of Syk activation, following two hour-

272long P. aeruginosa infection, and a decrease in the expression of phospho-Syk following pre-

273treatment with R406 (Fig. 1C). As release of mature IL-1β requires inflammasome activation, in

274addition to IL-1β gene transcription, the effect of R406 on IL-1β release suggests Syk

275involvement in the regulation of inflammasome activation in our model (Dinarello 2009). This is

276not surprising as previous studies identified Syk as a key mediator of NLRP3 inflammasome

277activation and IL-1β secretion in innate immune cells stimulated with fungi and crystals (Gross et

278al. 2009; del Fresno et al. 2013; Mao et al. 2014; Lin et al. 2015; Lima-Junior et al. 2017).




280There are potentially multiple pathways of Syk activation during P. aeruginosa infection of

281monocytic cells. This non-receptor protein tyrosine kinase is best known as a critical component

282of immunoreceptor tyrosine-based activation motifs (ITAM)-dependent signaling in

283hematopoietic cells involving Fc receptors, T-, B-, and NK cell receptors (Turner et al. 2000).

284Congruently, in our experiments, we observed a strong inhibitory effect of R406 on ICAM-1

285expression induced by a classical mechanism of Syk activation, i.e. via Fcγ receptor cross-linking,

286with ICAM-1 expression level decreased to the baseline while using a 10 µg/mL concentration of

287human IgG for receptor activation (Fig. 1B). However, none of the cellular responses to P.

288aeruginosa have been completely inhibited by R406, although we could achieve their significant

289down-regulation using a concentration of 10 µM, which was commonly used in studies by others

290(Braselmann et al. 2006); in case of ICAM-1, lower concentrations of 0.5 to 5 µM were also

291effective (Fig 1A). The data suggest that although Syk is certainly involved in the regulation of

292inflammatory responses to P. aeruginosa infection, it does not represent the major pathway

293among multiple mechanisms operating in cellular responses to this highly virulent

294microorganism, which is capable to interact with many pathogen-recognition receptors, including

295Toll-like receptors, Nod-like receptors, integrins, C-type lectins, asialoGM1, etc (Sadikot et al.

2962005; DiMango et al. 1995; Skerrett et al. 2007). Ability of P. aeruginosa to stimulate TNFα and

297IL-1β synthesis and release from human monocytes, and activation of transcription factors NF-κB

298and AP in infected cells have been established by previous studies (Li et al. 1998; Kube et al.

2992001; Lagoumintzis et al. 2003; Wehkamp et al. 2006). Syk involvement in the regulation of

300signals generated by the engagement of TLR-4 complex by its ligand LPS in human neutrophils

301and macrophages has also been previously demonstrated (Arndt et al. 2004; Ulanova et al. 2007;

302Miller et al. 2012), and this mechanism likely operates in our model. Recent studies expanded our

303understanding of the role of Syk in fine-tuning of cellular responses stimulated by the

304engagement of innate immune receptors (Aouar et al. 2016; Yin et al. 2016). For example, it was



305demonstrated that in macrophages and dendritic cells, Syk regulates TNFα exocytosis induced by

306stimulation of TLR9 by bacterial CpG DNA (Rao et al. 2013); such mechanism may potentially

307be involved in responses of differentiated THP-1 cells to P. aeruginosa. In addition, innate

308immune responses activated by P. aeruginosa result in the amplification of inflammatory

309responses, as for example, TNFα further activates the inflammatory cascade via its own receptor

310associated signaling (Newton and Dixit 2012). The complexity of cellular responses to P.

311aeruginosa is further augmented by cross talk among multiple signalling pathways, including

both pro- and anti-inflammatory (Lee and Kim 2007).
314Syk may become activated following P. aeruginosa infection via several potential mechanisms. It

315is well recognized that Syk is significantly involved in several ITAM-independent signalling

316pathways, which are mediated by its interaction with G-protein coupled receptors, pattern

317recognition, and cytokine receptors (Ulanova et al. 2005a; Mocsai et al. 2010). In particular, Syk

318can be activated via interaction with integrin receptor cytoplasmic domains that is especially

319significant in lung epithelial cells, which do not express the plethora of innate immune receptors

320typical for leukocytes (Ulanova et al. 2005b). Our previous research demonstrated the

321involvement of integrin receptors in P. aeruginosa internalization and recognition by A549

322alveolar epithelial cells; moreover, the data suggested an important role of integrin-mediated

323signaling in inflammation induced by this infection (Gravelle et al. 2010). In the present study,

324the release of TNFα by infected bronchiolar epithelial cells was significantly down-regulated by

325R406 implicating the involvement of Syk-dependent signaling in inflammatory responses to P.

326aeruginosa by lung epithelial cells, in addition to monocytes (Fig 1C). Indeed, we have

327previously demonstrated that H292 cells express Syk (Aval et al. 2013); however, it is uncertain

328whether or not Syk is exclusively engaged via integrin receptors in this particular cell line, or

329some other mechanisms, for example, those mediated by TNF-receptor signaling are involved

330(Takada and Aggarwal 2004).




332Because Syk combines both kinase and adaptor protein properties, this molecule is capable to

333interact with multiple protein targets, and this explains why its inhibition leads to numerous

334biological effects. Indeed, Syk has been considered as a target for therapy of such diverse

335conditions as allergic diseases, rheumatoid arthritis, systemic lupus erythematosus, idiopathic

336thrombocytopenic purpura, and B-cell lymphoma, with several pharmacological compounds

337undergoing clinical trials (Riccaboni et al. 2010). One potential application could be the use of

338Syk inhibitors to dampen severe pro-inflammatory responses associated with pulmonary P.

339aeruginosa infection, which affects CF patients, as well as occurs in ventilator-associated

340pneumonia, aggravates the course of chronic obstructive pulmonary disease (COPD), and causes

341severe complications in cancer patients with neutropenia, caused by chemotherapy that

342predisposes to P. aeruginosa pneumonia (Saiman and Siegel 2004; Hakki et al. 2007; Crouch

343Brewer et al. 1996; Lieberman and Lieberman 2003). In our previous study, we found that a

344natural Syk inhibitor piceatannol significantly suppressed inflammation, oxidative stress,

345apoptosis, and bacterial internalization in a model of P. aeruginosa infected pulmonary epithelial

346cells, although not all of these outcomes could be attributed to Syk-specific effect (Aval et al.

3472013). Results of the current study corroborate our previous observations using this time both a

348model of infected THP-1 cells, which represent innate immune cells, and a bronchiolar epithelial

cell line H292 (Carney et al. 1985)
351As bronchiolar epithelial cells represent the major component of the airway lining, have receptors

352for P. aeruginosa, are the site of infection, generate inflammatory responses to this infectious

353agent, and express Syk, they could be the major targets for potential therapeutic intervention

354using Syk inhibitors. Importantly, the response of H292 cells to stimulation with various strains

355of P. aeruginosa was noticeably different from the response by differentiated monocytic THP-1

356cells (Fig. 2B-C). Although the release of TNFα by H292 cells infected with PAK WT or PAK



357fliC was approximately 50-times lower than the one by THP-1 cells, infection with clinical P.

358aeruginosa isolates caused relatively higher TNFα production in H292 cells. In particular, H292

359cells infected with P. aeruginosa of a CF patient with long-term chronic infection released the

360largest amount of TNFα in comparison to other P. aeruginosa strains, i.e. 202 ±5 pg/mL,

361although THP-1 cells produced significantly less TNFα when stimulated with either clinical

362isolate (1545 ±237 pg/mL and 714 ±102 pg/mL) as compared to both wild-type (4795 ±463

363pg/mL) and flagella-deficient (3691 ±255 pg/mL) laboratory strains PAK. These data corroborate

364our previous observations that P. aeruginosa isolates from chronically infected CF patients have

365increased abilities of causing inflammatory responses of A549 alveolar epithelial cells in

366comparison to bacteria from patients with intermittent P. aeruginosa colonization, owing to the

367adaptation process in the CF long during long-term infectious process (Hawdon et al. 2010). The

368isolate #19731A/92 was obtained from a CF patient with 18-year long chronic P. aeruginosa

369infection (Hawdon et al. 2010). As a flagella-deficient strain (PAK fliC) induced significantly

370lower release of cytokines TNFα and IL-1β compared to the wild-type bacteria (Fig 2A-C) these

371data emphasize importance of flagella in stimulating potent pro-inflammatory responses to P.

372aeruginosa infection via the activation of pattern-recognition receptors such as TLR5 and NLRC4

373inflammasome (Blohmke et al. 2010; Zhao et al. 2011). Importantly, in bronchiolar epithelial

374cells, R406 was able to significantly down-regulate TNFα release caused by P. aeruginosa

375isolates from both chronically infected and intermittently colonized CF patients, although to a

376lesser degree than when the inhibitor was applied to cells, stimulated with PAK WT or PAK fliC

377(Fig 2C) suggesting potential clinical application of this inhibitor. However, as recent studies

378found that Syk is essential for flagellin-specific T cell responses, it is important to consider

379complexity of the regulatory role of this signaling molecule in immune responses (Atif et al.




382Compared to an early used inhibitor piceatannol, R406 has been demonstrated to be much more

383selective for Syk. However, R406 is not entirely specific to Syk, and able to inhibit JAK2 in

384addition to Syk of similar potency (Rolf et al. 2015). Although the present findings suggest Syk

385involvement in the regulation of P. aeruginosa triggered inflammatory responses in both human

386monocytic and bronchiolar epithelial cells, it will be highly desired to test more specific Syk

387inhibitors. However, creating a truly selective Syk inhibitor apparently represents a challenge;

388indeed, all of the existing compounds with Syk-inhibitory capacities, including the most recent

389ones express certain off-target specificity (Ferguson et al. 2016; Yaron et al. 2016). When

390Fostamatinib, of which R406 is the active metabolite, was tested in phase II-III clinical trials for

391rheumatoid arthritis, adverse events related to its off-target effect have been noticed (Kunwar et

392al. 2016). As it was demonstrated that inhibition of JAK2 down-regulated inflammatory

393responses in an animal model of polymicrobial sepsis, certain off-target effects of R406 may

394potentially be beneficial in case of P. aeruginosa infection (Pena et al. 2010). Conducting

395clinical trials to ascertain capacity of this Syk inhibitor to alleviate exaggerated inflammatory

396responses, which significantly contribute to the pathogenesis of P. aeruginosa pulmonary

infections may represent a sensible approach.

400This work was supported by a National Science and Engineering Research Council Discovery

401Grant and Ontario Lung Association Grant to M.U. We thank Dr. Niels Høiby (University

402Hospital Rigshospitalet, Copenhagen, Denmark), Dr. Randal Irvin (University of Alberta,

403Edmonton, Canada), and Dr. Alice Prince, Columbia University, New York) for kindly providing

404P. aeruginosa clinical isolates and strains, AstraZeneka AB (Södertälje, Sweden) for providing

405R406), and Jessica Rosengren (Lakehead University, Thunder Bay, Canada) for help with

optimizing experimental conditions.




409Akira, S. and Kishimoto, T. 1997. NF-IL6 and NF-kappa B in cytokine gene regulation. Adv

410 Immunol, 65, 1-46.

411 Aouar, B., Kovarova, D., Letard, S., Font-Haro, A., Florentin, J., Weber, J., Durantel, D.,

412 Chaperot, L., Plumas, J., Trejbalova, K., Hejnar, J., Nunes, J. A., Olive, D., Dubreuil, P.,

413 Hirsch, I. and Stranska, R. 2016. Dual Role of the Tyrosine Kinase Syk in Regulation of

414 Toll-Like Receptor Signaling in Plasmacytoid Dendritic Cells. PLoS One, 11(6), e0156063.

415 doi: 10.1371/journal.pone.0156063. PMID: 27258042.

416 Arndt, P. G., Suzuki, N., Avdi, N. J., Malcolm, K. C. and Worthen, G. S. 2004.

417 Lipopolysaccharide-induced c-Jun NH2-terminal kinase activation in human neutrophils:

418 role of phosphatidylinositol 3-Kinase and Syk-mediated pathways. J Biol Chem, 279(12),

419 10883-91. doi: 10.1074/jbc.M309901200. PMID: 14699155.

420 Atif, S. M., Lee, S. J., Li, L. X., Uematsu, S., Akira, S., Gorjestani, S., Lin, X., Schweighoffer, E.,

421 Tybulewicz, V. L. and McSorley, S. J. 2015. Rapid CD4+ T-cell responses to bacterial

422 flagellin require dendritic cell expression of Syk and CARD9. Eur J Immunol, 45(2), 513-

423 24. doi: 10.1002/eji.201444744. PMID: 25430631.

424 Aval, P. S., Werner, J., Cerqueira, A., Balfour-Boehm, J. and Ulanova, M. 2013. Piceatannol

425 modulates lung epithelial cellular responses to Pseudomonas aeruginosa. Inflamm Allergy

426 Drug Targets, 12(5), 297-307.

427 Blohmke, C. J., Park, J., Hirschfeld, A. F., Victor, R. E., Schneiderman, J., Stefanowicz, D.,

428 Chilvers, M. A., Durie, P. R., Corey, M., Zielenski, J., Dorfman, R., Sandford, A. J., Daley,

429 D. and Turvey, S. E. 2010. TLR5 as an anti-inflammatory target and modifier gene in cystic

430 fibrosis. J Immunol, 185(12), 7731-8. doi: 10.4049/jimmunol.1001513. PMID: 21068401.

431 Braselmann, S., Taylor, V., Zhao, H., Wang, S., Sylvain, C., Baluom, M., Qu, K., Herlaar, E.,

432 Lau, A., Young, C., Wong, B. R., Lovell, S., Sun, T., Park, G., Argade, A., Jurcevic, S.,

433 Pine, P., Singh, R., Grossbard, E. B., Payan, D. G. and Masuda, E. S. 2006. R406, an orally



434 available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune

435 complex-mediated inflammation. J Pharmacol Exp Ther, 319(3), 998-1008. doi:

436 10.1124/jpet.106.109058. PMID: 16946104.

437 Carney, D. N., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, T. W., Zweig,

438 M. H. and Minna, J. D. 1985. Establishment and identification of small cell lung cancer cell

439 lines having classic and variant features. Cancer Res, 45(6), 2913-23. 2985257.

440 Cha, H. S., Boyle, D. L., Inoue, T., Schoot, R., Tak, P. P., Pine, P. and Firestein, G. S. 2006. A

441 novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene

442 expression in synoviocytes. J Pharmacol Exp Ther, 317(2), 571-8. doi:

443 10.1124/jpet.105.097436. PMID: 16452391.

444 Chen, L., Monti, S., Juszczynski, P., Daley, J., Chen, W., Witzig, T. E., Habermann, T. M.,

445 Kutok, J. L. and Shipp, M. A. 2008. SYK-dependent tonic B-cell receptor signaling is a

446 rational treatment target in diffuse large B-cell lymphoma. Blood, 111(4), 2230-7. doi:

447 10.1182/blood-2007-07-100115. PMID: 18006696.

448 Cheng, K., Ashby, D. and Smyth, R. L. 2013. Oral steroids for long-term use in cystic fibrosis.

449 Cochrane Database Syst Rev(6), CD000407. doi: 10.1002/14651858.CD000407.pub3.

450 PMID: 23794322.

451 Costello, P. S., Turner, M., Walters, A. E., Cunningham, C. N., Bauer, P. H., Downward, J. and

452 Tybulewicz, V. L. 1996. Critical role for the tyrosine kinase Syk in signalling through the

453 high affinity IgE receptor of mast cells. Oncogene, 13(12), 2595-605.

454 Crouch Brewer, S., Wunderink, R. G., Jones, C. B. and Leeper, K. V., Jr. 1996. Ventilator-

455 associated pneumonia due to Pseudomonas aeruginosa. Chest, 109(4), 1019-29.

456 Darby, C., Geahlen, R. L. and Schreiber, A. D. 1994. Stimulation of macrophage Fc gamma

457 RIIIA activates the receptor-associated protein tyrosine kinase Syk and induces

458 phosphorylation of multiple proteins including p95Vav and p62/GAP-associated protein. J

459 Immunol, 152(11), 5429-37.



460 del Fresno, C., Soulat, D., Roth, S., Blazek, K., Udalova, I., Sancho, D., Ruland, J. and Ardavin,

461 C. 2013. Interferon-beta production via Dectin-1-Syk-IRF5 signaling in dendritic cells is

462 crucial for immunity to C. albicans. Immunity, 38(6), 1176-86. doi:

463 10.1016/j.immuni.2013.05.010. PMID: 23770228.

464 DiMango, E., Zar, H. J., Bryan, R. and Prince, A. 1995. Diverse Pseudomonas aeruginosa gene

465 products stimulate respiratory epithelial cells to produce interleukin-8. J Clin Invest, 96(5),

466 2204-10. doi: 10.1172/JCI118275. PMID: 7593606.

467 Dinarello, C. A. 2009. Immunological and inflammatory functions of the interleukin-1 family.

468 Annu Rev Immunol, 27, 519-50. doi: 10.1146/annurev.immunol.021908.132612. PMID:

469 19302047.

470 Esen, M., Grassme, H., Riethmuller, J., Riehle, A., Fassbender, K. and Gulbins, E. 2001. Invasion

471 of human epithelial cells by Pseudomonas aeruginosa involves src-like tyrosine kinases

472 p60Src and p59Fyn. Infect Immun, 69(1), 281-7. doi: 10.1128/IAI.69.1.281-287.2001.

473 PMID: 11119516.

474 Feldman, M., Bryan, R., Rajan, S., Scheffler, L., Brunnert, S., Tang, H. and Prince, A. 1998. Role

475 of flagella in pathogenesis of Pseudomonas aeruginosa pulmonary infection. Infect Immun,

476 66(1), 43-51. 9423837.

477 Ferguson, G. D., Delgado, M., Plantevin-Krenitsky, V., Jensen-Pergakes, K., Bates, R. J., Torres,

478 S., Celeridad, M., Brown, H., Burnett, K., Nadolny, L., Tehrani, L., Packard, G., Pagarigan,

479 B., Haelewyn, J., Nguyen, T., Xu, L., Tang, Y., Hickman, M., Baculi, F., Pierce, S.,

480 Miyazawa, K., Jackson, P., Chamberlain, P., LeBrun, L., Xie, W., Bennett, B. and Blease, K.

481 2016. A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint

482 Inflammation. PLoS One, 11(1), e0145705. doi: 10.1371/journal.pone.0145705. PMID:

483 26756335.

484 Gravelle, S., Barnes, R., Hawdon, N., Shewchuk, L., Eibl, J., Lam, J. S. and Ulanova, M. 2010.

485 Up-regulation of integrin expression in lung adenocarcinoma cells caused by bacterial



486 infection: in vitro study. Innate Immun, 16(1), 14-26. doi: 10.1177/1753425909106170.

487 PMID: 19710103.

488 Gross, O., Poeck, H., Bscheider, M., Dostert, C., Hannesschlager, N., Endres, S., Hartmann, G.,

489 Tardivel, A., Schweighoffer, E., Tybulewicz, V., Mocsai, A., Tschopp, J. and Ruland, J.

490 2009. Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence.

491 Nature, 459(7245), 433-6. doi: 10.1038/nature07965. PMID: 19339971.

492 Hakki, M., Limaye, A. P., Kim, H. W., Kirby, K. A., Corey, L. and Boeckh, M. 2007. Invasive

493 Pseudomonas aeruginosa infections: high rate of recurrence and mortality after

494 hematopoietic cell transplantation. Bone Marrow Transplant, 39(11), 687-93. doi:

495 10.1038/sj.bmt.1705653. PMID: 17401395.

496 Hawdon, N. A., Aval, P. S., Barnes, R. J., Gravelle, S. K., Rosengren, J., Khan, S., Ciofu, O.,

497 Johansen, H. K., Hoiby, N. and Ulanova, M. 2010. Cellular responses of A549 alveolar

498 epithelial cells to serially collected Pseudomonas aeruginosa from cystic fibrosis patients at

499 different stages of pulmonary infection. FEMS Immunol Med Microbiol, 59(2), 207-20. doi:

500 10.1111/j.1574-695X.2010.00693.x. PMID: 20528926.

501 Kipnis, E., Sawa, T. and Wiener-Kronish, J. 2006. Targeting mechanisms of Pseudomonas

502 aeruginosa pathogenesis. Med Mal Infect, 36(2), 78-91. doi: 10.1016/j.medmal.2005.10.007.

503 PMID: 16427231.

504 Kube, D., Sontich, U., Fletcher, D. and Davis, P. B. 2001. Proinflammatory cytokine responses to

505 P. aeruginosa infection in human airway epithelial cell lines. Am J Physiol Lung Cell Mol

506 Physiol, 280(3), L493-502.

507 Kunwar, S., Devkota, A. R. and Ghimire, D. K. 2016. Fostamatinib, an oral spleen tyrosine

508 kinase inhibitor, in the treatment of rheumatoid arthritis: a meta-analysis of randomized

509 controlled trials. Rheumatol Int, 36(8), 1077-87. doi: 10.1007/s00296-016-3482-7. PMID:

510 27113955.

511 Lagoumintzis, G., Christofidou, M., Dimitracopoulos, G. and Paliogianni, F. 2003. Pseudomonas



512 aeruginosa slime glycolipoprotein is a potent stimulant of tumor necrosis factor alpha gene

513 expression and activation of transcription activators nuclear factor kappa B and activator

514 protein 1 in human monocytes. Infect Immun, 71(8), 4614-22.

515 Lee, M. S. and Kim, Y. J. 2007. Signaling pathways downstream of pattern-recognition receptors

516 and their cross talk. Annu Rev Biochem, 76, 447-80. doi:

517 10.1146/annurev.biochem.76.060605.122847. PMID: 17328678.

518 Li, J. D., Feng, W., Gallup, M., Kim, J. H., Gum, J., Kim, Y. and Basbaum, C. 1998. Activation

519 of NF-kappaB via a Src-dependent Ras-MAPK-pp90rsk pathway is required for

520 Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells. Proc Natl Acad

521 Sci U S A, 95(10), 5718-23.

522 Lieberman, D. and Lieberman, D. 2003. Pseudomonal infections in patients with COPD:

523 epidemiology and management. Am J Respir Med, 2(6), 459-68.

524 Lima-Junior, D. S., Mineo, T. W. P., Calich, V. L. G. and Zamboni, D. S. 2017. Dectin-1

525 Activation during Leishmania amazonensis Phagocytosis Prompts Syk-Dependent Reactive

526 Oxygen Species Production To Trigger Inflammasome Assembly and Restriction of Parasite

527 Replication. J Immunol, 199(6), 2055-2068. doi: 10.4049/jimmunol.1700258. PMID:

528 28784846.

529 Lin, Y. C., Huang, D. Y., Wang, J. S., Lin, Y. L., Hsieh, S. L., Huang, K. C. and Lin, W. W.

530 2015. Syk is involved in NLRP3 inflammasome-mediated caspase-1 activation through

531 adaptor ASC phosphorylation and enhanced oligomerization. J Leukoc Biol, 97(5), 825-835.

532 doi: 10.1189/jlb.3HI0814-371RR. PMID: 25605870.

533 Mao, L., Zhang, L., Li, H., Chen, W., Wang, H., Wu, S., Guo, C., Lu, A., Yang, G., An, L., Abliz,

534 P. and Meng, G. 2014. Pathogenic fungus Microsporum canis activates the NLRP3

535 inflammasome. Infect Immun, 82(2), 882-92. doi: 10.1128/IAI.01097-13. PMID: 24478101.

536McAdoo, S. P. and Tam, F. W. 2011. Fostamatinib Disodium. Drugs Future, 36(4), 273.

537Miller, Y. I., Choi, S. H., Wiesner, P. and Bae, Y. S. 2012. The SYK side of TLR4: signalling



538 mechanisms in response to LPS and minimally oxidized LDL. Br J Pharmacol, 167(5), 990-

539 9. doi: 10.1111/j.1476-5381.2012.02097.x. PMID: 22776094.

540 Mocsai, A., Ruland, J. and Tybulewicz, V. L. 2010. The SYK tyrosine kinase: a crucial player in

541 diverse biological functions. Nat Rev Immunol, 10(6), 387-402. doi: 10.1038/nri2765.

542 PMID: 20467426.

543 Newton, K. and Dixit, V. M. 2012. Signaling in innate immunity and inflammation. Cold Spring

544 Harb Perspect Biol, 4(3). doi: 10.1101/cshperspect.a006049. PMID: 22296764.

545 Pasloske, B. L., Finlay, B. B. and Paranchych, W. 1985. Cloning and sequencing of the

546 Pseudomonas aeruginosa PAK pilin gene. FEBS Lett, 183(2), 408-12. 2985436.

547 Pena, G., Cai, B., Deitch, E. A. and Ulloa, L. 2010. JAK2 inhibition prevents innate immune

548 responses and rescues animals from sepsis. J Mol Med (Berl), 88(8), 851-9. doi:

549 10.1007/s00109-010-0628-z. PMID: 20393690.

550 Quiroga, M. P., Balakrishnan, K., Kurtova, A. V., Sivina, M., Keating, M. J., Wierda, W. G.,

551 Gandhi, V. and Burger, J. A. 2009. B-cell antigen receptor signaling enhances chronic

552 lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen

553 tyrosine kinase inhibitor, R406. Blood, 114(5), 1029-37. doi: 10.1182/blood-2009-03-

554 212837. PMID: 19491390.

555 Rao, S., Liu, X., Freedman, B. D. and Behrens, E. M. 2013. Spleen tyrosine kinase (Syk)-

556 dependent calcium signals mediate efficient CpG-induced exocytosis of tumor necrosis

557 factor alpha (TNFalpha) in innate immune cells. J Biol Chem, 288(18), 12448-58. doi:

558 10.1074/jbc.M113.454405. PMID: 23515313.

559 Ratner, A. J., Bryan, R., Weber, A., Nguyen, S., Barnes, D., Pitt, A., Gelber, S., Cheung, A. and

560 Prince, A. 2001. Cystic fibrosis pathogens activate Ca2+-dependent mitogen-activated

561 protein kinase signaling pathways in airway epithelial cells. J Biol Chem, 276(22), 19267-

562 75. doi: 10.1074/jbc.M007703200. PMID: 11278360.

563 Riccaboni, M., Bianchi, I. and Petrillo, P. 2010. Spleen tyrosine kinases: biology, therapeutic



564 targets and drugs. Drug Discov Today, 15(13-14), 517-30. doi:

565 10.1016/j.drudis.2010.05.001. PMID: 20553955.

566 Roebuck, K. A. and Finnegan, A. 1999. Regulation of intercellular adhesion molecule-1 (CD54)

567 gene expression. J Leukoc Biol, 66(6), 876-88.

568 Rolf, M. G., Curwen, J. O., Veldman-Jones, M., Eberlein, C., Wang, J., Harmer, A., Hellawell, C.

569 J. and Braddock, M. 2015. In vitro pharmacological profiling of R406 identifies molecular

570 targets underlying the clinical effects of fostamatinib. Pharmacol Res Perspect, 3(5), e00175.

571 doi: 10.1002/prp2.175. PMID: 26516587.

572 Sadikot, R. T., Blackwell, T. S., Christman, J. W. and Prince, A. S. 2005. Pathogen-host

573 interactions in Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med, 171(11),

574 1209-23. doi: 10.1164/rccm.200408-1044SO. PMID: 15695491.

575 Saiman, L. and Siegel, J. 2004. Infection control in cystic fibrosis. Clin Microbiol Rev, 17(1), 57-

576 71.

577 Skerrett, S. J., Wilson, C. B., Liggitt, H. D. and Hajjar, A. M. 2007. Redundant Toll-like receptor

578 signaling in the pulmonary host response to Pseudomonas aeruginosa. Am J Physiol Lung

579 Cell Mol Physiol, 292(1), L312-22. doi: 10.1152/ajplung.00250.2006. PMID: 16936244.

580 Spalton, J. C., Mori, J., Pollitt, A. Y., Hughes, C. E., Eble, J. A. and Watson, S. P. 2009. The

581 novel Syk inhibitor R406 reveals mechanistic differences in the initiation of GPVI and

582 CLEC-2 signaling in platelets. J Thromb Haemost, 7(7), 1192-9. doi: 10.1111/j.1538-

583 7836.2009.03451.x. PMID: 19422460.

584 Takada, Y. and Aggarwal, B. B. 2004. TNF activates Syk protein tyrosine kinase leading to TNF-

585 induced MAPK activation, NF-kappaB activation, and apoptosis. J Immunol, 173(2), 1066-

586 77. 15240695.

587 Turner, M., Schweighoffer, E., Colucci, F., Di Santo, J. P. and Tybulewicz, V. L. 2000. Tyrosine

588 kinase SYK: essential functions for immunoreceptor signalling. Immunol Today, 21(3), 148-

589 54.



590 Ulanova, M., Asfaha, S., Stenton, G., Lint, A., Gilbertson, D., Schreiber, A. and Befus, D. 2007.

591 Involvement of Syk protein tyrosine kinase in LPS-induced responses in macrophages. J

592 Endotoxin Res, 13(2), 117-25. doi: 10.1177/0968051907079125. PMID: 17621553.

593 Ulanova, M., Duta, F., Puttagunta, L., Schreiber, A. D. and Befus, A. D. 2005a. Spleen tyrosine

594 kinase (Syk) as a novel target for allergic asthma and rhinitis. Expert Opin Ther Targets,

595 9(5), 901-21. doi: 10.1517/14728222.9.5.901. PMID: 16185147.

596 Ulanova, M., Marcet-Palacios, M., Munoz, S., Asfaha, S., Kim, M. K., Schreiber, A. D. and

597 Befus, A. D. 2006. Involvement of Syk kinase in TNF-induced nitric oxide production by

598 airway epithelial cells. Biochem Biophys Res Commun, 351(2), 431-7. doi:

599 10.1016/j.bbrc.2006.10.073. PMID: 17070777.

600 Ulanova, M., Puttagunta, L., Marcet-Palacios, M., Duszyk, M., Steinhoff, U., Duta, F., Kim, M.

601 K., Indik, Z. K., Schreiber, A. D. and Befus, A. D. 2005b. Syk tyrosine kinase participates in

602 beta1-integrin signaling and inflammatory responses in airway epithelial cells. Am J Physiol

603 Lung Cell Mol Physiol, 288(3), L497-507. doi: 10.1152/ajplung.00246.2004. PMID:

604 15557085.

605 Wehkamp, K., Schwichtenberg, L., Schroder, J. M. and Harder, J. 2006. Pseudomonas

606 aeruginosa- and IL-1beta-mediated induction of human beta-defensin-2 in keratinocytes is

607 controlled by NF-kappaB and AP-1. J Invest Dermatol, 126(1), 121-7. doi:

608 10.1038/sj.jid.5700020. PMID: 16417227.

609 Yaron, J. R., Rao, M. Y., Gangaraju, S., Zhang, L., Kong, X., Su, F., Tian, Y., Glenn, H. L. and

610 Meldrum, D. R. 2016. The oxindole Syk inhibitor OXSI-2 blocks nigericin-induced

611 inflammasome signaling and pyroptosis independent of potassium efflux. Biochem Biophys

612 Res Commun, 472(3), 545-50. doi: 10.1016/j.bbrc.2016.03.021. PMID: 26970308.

613 Yin, H., Zhou, H., Kang, Y., Zhang, X., Duan, X., Alnabhan, R., Liang, S., Scott, D. A., Lamont,

614 R. J., Shang, J. and Wang, H. 2016. Syk negatively regulates TLR4-mediated IFNbeta and

615 IL-10 production and promotes inflammatory responses in dendritic cells. Biochim Biophys



616 Acta, 1860(3), 588-98. doi: 10.1016/j.bbagen.2015.12.012.PubMed PMID: 26708990.

617 Zhao, Y., Yang, J., Shi, J., Gong, Y. N., Lu, Q., Xu, H., Liu, L. and Shao, F. 2011. The NLRC4

618 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature,

477(7366), 596-600. doi: 10.1038/nature10510. PMID: 21918512.







Figure Legends
623Fig. 1 The effect of R406 on ICAM-1 expression induced by Pseudomonas aeruginosa strain K

624(PAK WT) infection, or Fcγ receptor (FcγR) cross-linking. a) One hour-long pre-treatment of

625THP-1 cells with various concentrations of R406 decreased ICAM-1 expression in THP-1 cells

626infected with PAK at an MOI of 5 for 6 hours in a dose-dependent manner. b) Pre-treatment of

627THP-1 cells with 10µM R406 for 1 hour prior to their stimulation with immobilized human IgG

628at concentrations of 10 and 100 µg/mL decreased up-regulation of ICAM-1. THP-1 cells were

629infected with PAK or stimulated via FcγR cross-linking as described in Materials & Methods, and

630ICAM-1 surface expression determined using immunostaining and flow cytometry analysis. Data

631are expressed as mean fluorescence intensity (MFI). Results represent the mean ± SEM of 3

632independent experiments; ###P<0.001, difference between un-stimulated and stimulated cells; *P

633< 0.05, **P<0.01, ***P<0.001, difference between stimulated R406 treated vs. un-treated cells. c)

634Ratios of Western blotting band intensity of Syk phosphorylated on tyrosine to total Syk. The

635lanes from left to right: un-stimulated THP-1 cells, THP-1 cells infected with PAK for 2 hours,

636THP-1 cells pretreated with 10µM R406 followed by infection with PAK WT. The bands of Syk

637and phosphotyrosine on immunoprecipitated Syk were detected at 72 kDa. Results represent 3

638independent experiments. *** P < 0.001, difference between R406-treated and un-treated infected

THP-1 cells.
641Fig. 2 The effect of R406 pre-treatment on cytokine expression induced by Pseudomonas


643Differentiated THP-1 cells were infected with P. aeruginosa for 1 hours at an MOI of 10 per 1 ×

644106 cells, and cultured for another 17 h in the presence of 100 µg/mL gentamicin. Unstimulated

645differentiated THP-1 cells (1 × 106) in complete culture medium served as a negative control. The

646supernatant was collected and IL-1β (A) and TNFα (B) concentrations (pg/mL) in culture



647supernatants were examined using ELISA. For TNFα expression by H292 cells (C), the cells

648were infected at an MOI of 50 for 1 h and further cultured as described above. In samples

649involving R406, cells were pre-treated with 10 µM R406 for 1 hour prior to infection. Results

650represent the mean ± SEM of 3 independent experiments; * P < 0.05, **P <0.01, *** P < 0.001,

****P <0.0001, difference between R406-treated and un-treated infected cells.
653Fig. 3 The effect of R406 on expression of phosphorylated and total intracellular signaling


655THP-1 cells were stimulated with P. aeruginosa strain K (PAK WT) at an MOI of 5 for 15, 30, or

65660 minutes. Following stimulation, the levels of total and phosphorylated ERK2 (42 kDa), JNK

657(46 kDa), IκBα (41 kDa), and p-38 (38 kDa) were determined in cellular lysates by Western blot.

658Results are expressed as ratios of phosphorylated/total protein band intensity. In samples

659involving R406, cells were pre-treated with 10µM R406 for 1 hour prior to infection. β-actin

660served as a loading control. Results represent the mean ± SEM of 2 independent experiments; * P

661< 0.05, **P <0.01, *** P < 0.001, ****P <0.0001, difference between R406-treated and un-

treated infected cells.






Table 1: Strains and Clinical Isolates of P. aeruginosa used in this study.

Strain/Isolate Source/Reference

Wild-type PAK (PAK WT) R.J. Irvin/Pasloske et al. (1985)
Flagella-deficient PAK (PAK fliC) Isolate from intermittently colonized CF
A.S. Prince/Feldman et al. (1998)

Danish CF Centre/Hawdon et al. (2010)
patient (9793/92)

Isolate from chronically infected CF
Danish CF Centre/Hawdon et al. (2010)
patient (19731A/92)

















































Fig. 1 The effect of R406 on ICAM-1 expression induced by Pseudomonas aeruginosa strain K (PAK WT) infection, or Fcγ receptor (FcγR) cross-linking. a) One hour-long pre-treatment of THP-1 cells with various concentrations of R406 decreased ICAM-1 expression in THP-1 cells infected with PAK at an MOI of 5 for 6
hours in a dose-dependent manner. b) Pre-treatment of THP-1 cells with 10µM R406 for 1 hour prior to their stimulation with immobilized human IgG at concentrations of 10 and 100 µg/mL decreased up-regulation of ICAM-1. THP-1 cells were infected with PAK or stimulated via FcγR cross-linking as described in Materials &
Methods, and ICAM-1 surface expression determined using immunostaining and flow cytometry analysis.
Data are expressed as mean fluorescence intensity (MFI). Results represent the mean ± SEM of 3 independent experiments; ###P<0.001, difference between un-stimulated and stimulated cells; *P < 0.05,
**P<0.01, ***P<0.001, difference between stimulated R406 treated vs. un-treated cells. c) Ratios of Western blotting band intensity of Syk phosphorylated on tyrosine to total Syk. The lanes from left to right: un-stimulated THP-1 cells, THP-1 cells infected with PAK for 2 hours, THP-1 cells pretreated with 10µM R406 followed by infection with PAK WT. The bands of Syk and phosphotyrosine on immunoprecipitated Syk were



detected at 72 kDa. Results represent 3 independent experiments. *** P < 0.001, difference between R406-
treated and un-treated infected THP-1 cells. 232x305mm (300 x 300 DPI)



























Fig. 2 The effect of R406 pre-treatment on cytokine expression induced by Pseudomonas aeruginosa.%”Differentiated THP-1 cells were infected with P. aeruginosa for 1 hours at an MOI of 10 per 1
× 106 cells, and cultured for another 17 h in the presence of 100 µg/mL gentamicin. Unstimulated differentiated THP-1 cells (1 × 106) in complete culture medium served as a negative control. The
supernatant was collected and IL-1β (A) and TNFα (B) concentrations (pg/mL) in culture supernatants were examined using ELISA. For TNFα expression by H292 cells (C), the cells were infected at an MOI of 50 for 1
h and further cultured as described above. In samples involving R406, cells were pre-treated with 10 µM R406 for 1 hour prior to infection. Results represent the mean ± SEM of 3 independent experiments; * P < 0.05, **P <0.01, *** P < 0.001, ****P <0.0001, difference between R406-treated and un-treated infected

231x351mm (300 x 300 DPI)



Leave a Reply

Your email address will not be published. Required fields are marked *


You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>