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,
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.
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
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.
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
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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
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.
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)
Isolate from chronically infected CF
Danish CF Centre/Hawdon et al. (2010)
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)