Fluorescein isothiocyanate (FITC), Fluo-3-AM, Fura-2-AM, pluronic F, bovine serum albumin (BSA), 4′,6-diamidino-2-phenylindole (DAPI), trypan blue, paraformaldehyde, ionomycin, and cytochalasin-D were obtained from Sigma (St. Louis, MO). Arthrobacter ureafaciens sialidase was from Roche Applied Science (Mannheim, Germany); Mounting medium was from Amersham Biosciences (Uppsala, Sweden); 2′7′- dichlorodihydro fluorescein diacetate chloromethyl ester (CM-H₂DCFDA), 4-Amino-5-Methylamino-2′,7′-Difluorofluorescein Diacetate (DAF-FM Diacetate), Lysotracker Red DND-99, carboxyfluorescein succinimidyl ester (CFSE) dye, Alexafluor-647 conjugated anti-Rabbit secondary antibody was from Molecular Probes, Thermo Fisher Scientific (OR, USA); siglec-E siRNA and anti-siglec-E antibody was from Santacruz Biotechnology (Texas, USA). All cytokine ELISA kits, CD16/32, CD11b, SHP-2, and SHP-1 antibodies were obtained from BD pharmingen and BD Biosciences (San Jose, CA, USA). Anti-Siglec antibodies (siglec-1, 3, 5, 7, 9, and siglec-1 and E) were from R&D systems (MN, USA). All other antibodies were from Cell Signaling Technologies (MA, USA) unless indicated otherwise. All cell culture medium, fetal calf serum (FCS), lipofectamine, and other reagents were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). West-pico enhanced chemiluminescent substrate (ECL) and BCA assay kit was purchased from Pierce (Thermo Scientific, Waltham, MA, USA).

Pseudomonas aeruginosa strain PA14 is a virulent burn-wound isolate, gifted by Prof. Richard D. Cummings from the Emory University School of Medicine (Atlanta, GA, USA). Bacteria were overnight cultured in tryptic soy broth in shaking condition at 37°C to obtain non-sialylated PA (PA^−Sia).

Additionally, bacteria cultured in tryptic soy broth supplemented with 10% heat-inactivated FCS (Gibco, Thermo Fisher Scientific) were designated as sialylated PA (PA^+Sia). For infection, overnight grown bacteria were pelleted, washed with phosphate buffered saline (PBS, 0.01 M) and bacterial counts estimated by measuring optical density (OD_600nm) by spectrophotometry.

Murine macrophage (J774A.1) and human monocytic (THP-1) cell lines were obtained from Cell Repository of National Centre for Cell Science, Pune, India and was routinely cultured at 37°C with 5% CO₂ in IMDM (Gibco, Invitrogen) and RPMI (Sigma) mediums, respectively, supplemented with 10% FCS (15). THP-1 derived macrophages were generated by treating the cells with PMA (100 ng/mL) for 48 h and differentiation was monitored by cellular morphology as well as expression of macrophage-specific cellular markers.

In brief, overnight cultured PA^−Sia/PA^+Sia (1 × 10⁷) were washed, enumerated and then incubated with FITC labeled Sambucus nigra (SNA) lectin or Maackia amurensis II (MAA) lectin (0.02 μg, Vectorlabs, California, USA) at 4°C for 30 min. Additionally, PA^+Sia (1 × 10⁷/100 μL) was desialylated by incubating them with A. ureafaciens sialidase (10 mU/100 μL, 10269611001, Roche) for 30 min at 37°C (7) and then used in FITC tagged lectin binding assay. Bacteria were then extensively washed to remove non-specifically bound lectins and analyzed by flow cytometry.

As before, equal numbers of bacteria (5 × 10⁹) were suspended in PBS and lysed by sonication in ice. Protein estimation was done and lysates (500 μg) were used to detect content of sialic acid by the method described by Shukla and Schauer (16). Mild oxidation of lysates by treatment with periodate solution (2.5 mM) at 4°C in 15 min dark was followed by treatment with sodium arsenite (2% in 0.5 M HCl) and acetyl acetone (750 ml glacial acetic acid, 3.75 g ammonium acetate and 500 μl of acetyl acetone in 250 mL water) at 60°C for 10 min then addition of 2.5 mL of water. Sialic acids present in lysates form a fluorogenic product after incubation and this product was quantified using a fluorimeter with excitation at 410 nm and collecting emission at a wavelength of 510 nm.

Overnight cultured PA^−Sia or PA^+Sia was labeled with FITC as stated previously (7). Bacteria were pelleted, washed with PBS and its OD₆₀₀ value was determined. Equal numbers of PA^−Sia or PA^+Sia (1 × 10⁹) were then incubated with 0.1% FITC in a minimal volume of bicarbonate buffer (pH 8.0) for 45 min at 37°C. Unbound FITC was removed by repeated washing with PBS and extent of labeling with FITC was verified by flow cytometry.

Additionally, FITC-labeled bacteria were incubated for 0, 2, or 4 h in PBS at 37°C followed by fixation with 2% para-formaldehyde to preserve FITC signal. Bacteria were analyzed by flow cytometry to detect stability of FITC tag.

J774A.1 cells (5 × 10⁴) were seeded in glass coverslips and incubated overnight in IMDM medium at 37°C with 5% CO₂. FITC-PA was added to the coverslips at a ratio of 10:1 and incubated at 4°C for 30 min. Unbound bacteria were removed by washing with PBS. These infected macrophages were finally fixed using 2% paraformaldehyde and mounted onto slides using DAPI containing mounting medium to visualize the binding of PA with macrophages by confocal microscopy. All images were acquired by Andor Spinning Disc Confocal microscope (Belfast, U.K.) with a 60 × /1.42 NA oil immersion objective.

In order to check the internalization of bacteria by macrophages, the above experiment was performed by incubating for 30 min at 37°C followed by fixation and staining. Macrophages were also briefly treated with trypan blue solution (0.02%) to quench fluorescence coming from extracellularly adherent bacteria (17). Images were analyzed and bacterial fluorescence associated with individual cells was quantified by Fiji software (18). We have also calculated phagocytic index, which is the mean number of bacteria adhered or internalized per phagocytosing macrophage.

Macrophages were similarly incubated with FITC-PA^−Sia or PA^+Sia at 10 multiplicity of infection (MOI) and incubated for 15 min at 4°C to synchronize bacterial binding. Cells were then incubated at 37°C for 30 min followed by removal of unbound bacteria by PBS wash. Infected macrophages were further incubated for 60 min. Lysotracker Red DND-99 (100 nM, L7528, Molecular Probes, Invitrogen) was then added and incubation continued for another 30 min at 37°C to label lysosomes. Cells were fixed, mounted on slides, and analyzed by confocal microscopy as mentioned before.

As previously described, coverslip-adherent macrophages were incubated with 1:10 MOI of FITC-PA^−Sia or PA^+Sia for 15 min at 4°C. Plates were transferred to 37°C for 10 min to allow synchronous uptake of bacteria and incubated further for 20 min, 40 and 60 min. Cells were fixed, permeabilized using PBS containing 0.3% Triton-X. Cells infected for 20 min were then overnight incubated at 4°C with primary antibodies against Rab5 (1:100, #12666, Cell Signaling Technology) in blocking buffer (PBS containing 10% goat serum and 0.3% Triton-X). Cells infected with PA for 40 min were incubated with anti-Rab7 antibodies (1:100, #12666, Cell Signaling Technology) and those infected for 60 min were incubated with anti-LAMP1 antibodies (1:100, #3243, Cell Signaling Technology). Coverslips were washed with PBS containing 0.3% Triton-X and then counterstained with Alexa Fluor-647 tagged secondary antibodies (1:2000, A-21245, Invitrogen) in blocking buffer for 2 h at 4°C. Coverslips were once again washed, mount and sealed. Images were randomly acquired from multiple fields in Fluoview FV10i (Olympus Life science). In order to confirm co-localization, multiple z stacks of 1 μm distance were also acquired. Images were processed and co-localization analyzed using JaCOP plugin (19) from Fiji software by calculating both Pearson's correlation coefficient (R) and Mander's overlap coefficient. For publication, images were brightened and background subtracted. On an average 100 cells from different fields were analyzed to determine colocalization.

Macrophages were also stained with Fluo-3-AM (5.0 μM, Sigma-Aldrich) for 30 min at 37°C to allow the dye to permeate. They were then infected with PA^−Sia or PA^+Sia at 1:10 ratio, incubated for 15 min at 37°C before fixation and processing for microscopy. Images were acquired in Fluoview FV10i (Olympus Life science). A similar experimental set up was repeated using siglec-E silenced macrophages for infection with PA^+Sia. Images were analyzed as discussed above.

J774A.1 (1 × 10⁶) cells were incubated with 10 MOI of FITC-PA^−Sia or PA^+Sia at 4°C for 30 min to detect their binding. Additionally, PA^+Sia (1 × 10⁶) were desialylated as mentioned earlier and used for binding.

To prevent sialic acid-siglec binding, macrophages (1 × 10⁶) were additionally incubated with anti-murine siglec-1 and siglec-E antibodies (1:1,000, R&D systems) for 1 h at 4°C. In the case of THP-1 derived macrophages, anti-Siglec-1, 3, 5, 7, and 9 antibodies (1:1,000, R&D systems) were used. Macrophages were also treated with blocking antibodies against CD11b (complement receptor-3, 550282, BD Biosciences) or CD16/32 (FcγR III/II, 553142, BD Biosciences). Bacteria and macrophages were then allowed to bind at 4°C for 30 min and assessed by flow cytometry in FACS LSR Fortessa (BD Biosciences) and analyzed using FACSDiva 8.0.2 software.

Macrophages were incubated with 10 MOI of FITC-PA^−Sia/PA^+Sia/sialidase-treated PA^+Sia at 37°C for 30 min. As before, anti-siglec antibodies were used to block siglecs present on murine/human macrophages. Incubation was followed by washing to remove unbound bacteria. In order to distinguish between surface bound and internalized FITC-PA, cells were briefly treated with Trypan blue (0.02%) to quench FITC fluorescence coming from bacteria which were adherent on the surface. Thus, fluorescence associated with infected macrophages represents phagocytosed PA and was measured by flow cytometry as before.

Macrophages (1 × 10⁶) were infected with 10 MOI of PA^−Sia/PA^+Sia/sialidase-treated PA^+Sia and incubated for 30 min at 37°C. Non-adherent bacteria were removed by washing and cells were incubated with a ROS sensitive dye-CM-H₂DCFDA (10 μM, C6827, Molecular Probes, Invitrogen) for an additional 30 min at 37°C. Cells were washed and analyzed by flow cytometry to estimate ROS generation.

Reactive nitrogen species (RNS) represented by NO, is another component of respiratory burst generated by macrophages in response to foreign particles. Intracellular nitric oxide was estimated using DAF-FM diacetate dye. As before, macrophages (1 × 10⁶) infected with PA^−Sia/PA^+Sia/sialidase-treated PA^+Sia were incubated with DAF-FM (100 nM, D23844, Invitrogen) for 30 min at 37°C. Cells were washed and analyzed by flow cytometry.

J774A.1 cells were activated using PMA (100 ng/mL) (20). These cells (1 × 10⁶) were infected with bacteria (10 MOI) for 45 min at 37°C to allow internalization. Unbound bacteria were washed off. Infected macrophages were further incubated for 3 h at 37°C at 5% CO₂. Culture supernatants were used to estimate levels of secretory cytokines-IL-10 (555252, BD Biosciences), IL-4 (555232, BD Biosciences), TGF-β (BMS608-4, Invitrogen), and IFN-γ (551866, BD Biosciences) using ELISA kits.

Intracellular survival and persistence of PA were assessed by gentamicin assay. Macrophages before and after treatment with anti-siglec antibodies were incubated with PA^−Sia/PA^+Sia/sialidase-treated PA^+Sia at 10 MOI for 30 min at 37°C. Unbound bacteria were removed by washing. Infected macrophages were further incubated for 3 h with gentamicin (200 μg/mL, G1397, Sigma-Aldrich) in IMDM medium to kill extracellular or surface adherent bacteria. To check the viability of internalized PA, macrophages were lysed in 1% w/v saponin solution. Lysate was serially diluted and spread on tryptic soy agar plates followed by overnight incubation at 37°C. Viable and persistent PA gave rise to colonies and colony forming units (cfu) were used to calculate the number of viable bacteria inside macrophages.

Overnight cultured PA^−Sia and PA^+Sia were pelleted, washed with PBS and enumerated by spectrophotometry. Equal numbers of bacteria were incubated with CFSE dye (50 μM, C34554, Invitrogen) at 37°C for 45 min to ensure uniform staining. Bacteria were repeatedly washed with PBS and then incubated with J774A.1 macrophages at 10:1 ratio at 37°C for 30 min. Unbound bacteria were removed by PBS wash. Remaining uninternalized bacteria were killed by incubation with 200 μg/mL gentamicin. Following 8 h after incubation, cells were visualized by confocal microscopy to detect fluorescence from internalized CFSE stained bacteria.

J774A.1 (1 × 10⁶) were incubated with 1:10 MOI of FITC-PA^−Sia or PA^+Sia for 15 min at 4°C. Plates were then transferred to 37°C to synchronize bacteria uptake and incubated for 20, 40, and 60 min. These cells were scraped off into a phagosome isolation buffer (20 mM HEPES, 0.5 mM EGTA, 0.25 M sucrose, 0.1% gelatin, protease inhibitor cocktail from Calbiochem, Merck-Millipore) (21). Cells were disrupted by passaging (15–20 times) through a 23-gauge needle fitted syringe. The suspension was centrifuged at 200 g for 10 min to separate most of the nucleus and intact cells. Phagosomes were collected by centrifugation of the supernatant at 13,000 g for 5 min and incubated for 5 min with 0.2% saponin, 10% normal goat serum in RPMI. Different endosomal compartments were stained by incubating with primary antibodies against Rab5 (1:100, #12666, Cell Signaling Technology), Rab7 (1:100, #12666, Cell Signaling Technology), and LAMP1 (1:100, #3243, Cell Signaling Technology) in the same buffer for 2 h at 4°C. Phagosomes were counterstained with Alexa Fluor-647 tagged secondary antibodies (A-21245, Invitrogen). The localization of PA in various endosomal compartments was detected by flow cytometry (22).

Additionally, macrophages were infected for 45, 90, and 120 min, labeled with Lysotracker red dye and processed similarly as described above. The colocalization of FITC-PA into Lysotracker red positive lysosomal compartment was detected by flow cytometry.

J774A.1 (1 × 10⁴) in microwell plates were incubated with Fura-2-AM (10 μM, Sigma-Aldrich) in presence of 0.04% pluronic-F in Hank's balanced salt solution (HBSS, pH 7.4) containing calcium (CaCl₂ 0.14 g/L) at 37°C for 30 min. Additionally, few cells were treated with ionomycin (10 μM) to estimate Rmax (high calcium). Similarly, cells were also incubated in calcium-free HBSS and treated with EGTA (10 mM) to estimate Rmin (low calcium). Fura-2-AM stained cells were infected with PA^−Sia or PA^+Sia at MOI 10 at 37°C and intracellular calcium ions were detected by microplate fluorescence readers detecting fluorescence at 510 nm upon excitation both at 340 and 380 nm. Intracellular calcium concentrations in infected cells were estimated using reference wells as per formula developed by Grynkyewicz (23). Fluorescence readings were taken in Synergy™ 2 Multi-Mode Microplate Reader (Biotek Instruments Inc.).

J774A.1 macrophages were incubated in calcium-free Hank's balanced salt solution (HBSS) buffer or with additional 2.5, 5, 10 mM Ca²⁺ (as CaCl₂). These cells were infected with 10 MOI of PA^−Sia/PA^+Sia at 4°C for 30 min for bacterial binding followed by transfer to 37°C. Cells were incubated for 30 min to allow phagocytosis in presence of extracellular calcium and then gentamicin (200 μg/mL) was added. Cells were incubated till 3 h followed by quantification of viable internalized bacteria by serial dilution and plating.

Additionally, J774A.1 cells incubated in calcium-free Hank's balanced salt solution (HBSS) buffer were infected with PA^+Sia at 10 MOI for 30 min at 4°C. Plates were transferred to 37°C followed by addition of 5 mM Ca²⁺ (as CaCl₂), ionomycin (10 μM) and were incubated further for 90 min to allow phagocytosis. Macrophages were also stained with Lysotracker red (100 nM) and processed for phagosome purification and flow cytometry as mentioned earlier (22). Macrophages were also prepared for confocal microscopy to detect phagosome lysosome fusion as noted before.

J774A.1 macrophages (1 × 10⁶) were infected with PA^−Sia or PA^+Sia at 10 MOI at 37°C for 15, 30, and 45 min, respectively. Infected cells were scraped into PBS, sonicated in presence of protease and phosphatase inhibitor cocktail (Calbiochem, Merck -Millipore) and protein was estimated by BCA assay (Pierce, Thermo Scientific). Equal amounts of protein were resolved in SDS-PAGE (10%) gel and transferred on to a PVDF membrane via wet transfer method (24). Membranes were blocked and incubated with antibodies (1:1,000) against different signaling proteins for overnight. Membranes were next washed using Tris-buffered Saline containing 0.1% Tween-20 (TBS-T) then incubated with appropriate HRP-conjugated secondary antibodies and visualized using chemiluminescence substrate (Pierce, Thermo Scientific) in a Chemidoc imaging system (BioRad Laboratories, CA, USA). The primary antibodies used includes—calmodulin (#4830), calcineurin A (#2614), PI3K-p85 (#4257), PI3K-p110γ (#4252), p-p38 MAPK (#4511), p38 MAPK (#9212), p-Akt Ser473 (#4060), pan-Akt (#4691), p-ERK1/2 (#4377), ERK1/2 (#4695), p-JNK/SAPK (#4668), JNK/SAPK (#9252), β-Actin (#4970), Lamin B (#13435), NF-κB Pathway Sampler Kit (#9936) from Cell Signaling Technology; biotinylated-anti phosphotyrosine (309304) from Biolegend (San Diego, CA, USA); calmodulin-dependent kinase type II (611293), SHP-1 (610126), SHP-2 (610622) from BD Biosciences; siglec-E (sc-377477) from Santacruz Biotechnology.

Additionally, cell lysates were overnight incubated with antibodies against phospho-tyrosine proteins, SHP-1 or SHP-2 phosphatases. Proteins were immunoprecipitated from the lysate using avidin-agarose beads (Invitrogen) or protein-A–sepharose 4B beads (Sigma-Aldrich). Beads were washed with chilled PBS to remove unbound proteins. Protein complexes bound to the beads were solubilized in sample buffer, resolved by SDS-PAGE, transferred to membrane and probed by western blotting process. Membranes were stripped off using stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.8 for 30 min at 50°C) and reprobed using the same antibody used for pulldown to check for protein loading.

Furthermore, cells were suspended into cytosol isolation buffer (10 mM Tris-Cl, 10 mM NaCl, 1.5 mM MgCl₂, 1 mM PMSF, 0.05% NP-40, pH 6.8), vortexed to allow partial lysis of membranes, centrifuged at 1,000 g for 5 min to precipitate nuclear fractions and cytosol was collected as supernatant. The pellet was washed with chilled PBS, incubated in nuclear isolation buffer (20 mM Tris-Cl, 137 mM NaCl, 1 mM CalCl₂, 1 mM MgCl₂, 1 mM PMSF, 1% NP-40, pH 8.0) for 30 min. Nuclear fraction was collected after centrifuging the suspension at 1,000 g for 5 min at 4°C. Both cytosolic and nuclear fractions were similarly resolved via SDS-PAGE and probed by western blotting.

Densitometric analysis of bands was performed using ImageJ software and values normalized with respect to β-actin band intensity have been represented as bar diagrams.

J774A.1 cells were transiently transfected with short interfering RNA (siRNA) designed against murine siglec-E (sc-153462, Santacruz Biotechnology) using Lipofectamine and Plus reagent (Invitrogen) as per manufacturer's instructions (15). Silencing of siglec-E was verified by western blotting. Siglec-E silenced J774A.1 cells were similarly infected with PA^+Sia and macrophage response to infection was analyzed.

The data represented are mean values derived from at least three independent experiments. Statistical analysis was performed using the two-tailed Student's t-test for two groups of samples and one-way analysis of variance (ANOVA) for more than two groups followed by pair wise multiple comparison procedures using Tukey test with p < 0.05 deemed statistically significant. Error-bars represent mean ± standard error of the mean (SEM) from three independent experiments. Significant differences were set at ^nsp > 0.05, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, and ^****p ≤ 0.0001 and analyzed by GraphPad Prism version 6.01.

We routinely confirmed the presence of sialic acid in PA before each experiment by sialic acid specific SNA, MAA lectin binding, and acetyl acetone based fluorimetric estimation of sialic acids.

Lectin binding was always higher with the sialylated (PA^+Sia) than non-sialylated bacteria (PA^−Sia) indicating presence of sialic acids in PA^+Sia. This was further corroborated by sialidase treatment which abrogates this binding with PA^+Sia. The MFI values of lectin binding have been presented in Figure 1A.

Additionally, sialic acid content from PA^−Sia/PA^+Sia/PA^+Sia-sialidase was measured by fluorimetric acetyl acetone method (16). The estimated sialic acid content was always higher in case of PA^+Sia as shown in Figure 1A.

Bacteria (PA^−Sia/PA^+Sia) were labeled using FITC and analyzed by flow cytometry to confirm extent of labeling. No significant difference in labeling was observed between PA^−Sia and PA^+Sia despite presence of additional surface sialic acids (Figure 1B). FITC tag was also found to remain stable till 4 h post-labeling (Figure 1C).

Phagocytosis of FITC-PA^+Sia and FITC-PA^−Sia bacteria by human monocytic (THP-1), as well as murine (J774A.1) macrophage cell lines, were investigated. Phagocytosis involves the recognition and binding of the bacteria to macrophage surface through interactions between bacterial ligands and cellular receptors. This is followed by actual physical internalization by the phagocytic cell (9).

Association between J774A.1 macrophage and FITC-PA^−Sia/PA^+Sia at 4°C was visualized by confocal microscopy (Figure 1D). Higher numbers of FITC-PA^+Sia (with phagocytic index = 8.875 ± 0.37) were found to be adherent on macrophage surfaces compared to PA^−Sia (4.855 ± 0.14, p ≤ 0.01). Images were analyzed to quantitate average FITC fluorescence associated with each macrophage. On an average, macrophages incubated with PA^−Sia showed lower fluorescence signal (3.504 ± 0.35 a.u.) as compared to those incubated with PA^+Sia (6.067 ± 0.54 a.u., p ≤ 0.05).

To check for the internalization of PA, macrophages were incubated with FITC-PA^−Sia/PA^+Sia at 37°C for 30 min and subsequently visualized by confocal microscopy (Figure 1E). A greater number of FITC-PA^+Sia, represented by green dots (phagocytic index = 7.775 ± 1.02) were visualized inside macrophages, compared to PA^−Sia (4.480 ± 0.23). Fluorescence quantitation reveals similar results (macrophages with PA^−Sia = 4.178 ± 0.54 a.u., macrophages with PA^+Sia = 7.263 ± 0.53 a.u., p ≤ 0.01). Thus, macrophages phagocytose more of PA^+Sia compared to PA^−Sia.

Bacterial binding and internalization by macrophages were also analyzed by flow cytometry (Figure 2). As observed in microscopy, binding of FITC-PA^+Sia to J774A.1 was significantly higher (~1.5-fold) with mean fluorescence intensity (MFI) values of 90.67 ± 0.88, p ≤ 0.05 compared to PA^−Sia (59.67 ± 0.88; Figure 2A).

Additionally, PA^+Sia were treated with sialidase to remove sialic acids. Desialylation of PA^+Sia significantly abrogates (MFI = 63.67 ± 1.45, p ≤ 0.01) the higher binding seen in interactions of untreated PA^+Sia with J774A.1 (MFI = 90.67 ± 0.88; Figure 2A). Thus, sialic acids on bacteria can be considered as ligands responsible for the differential binding between PA^+Sia and PA^−Sia to macrophages.

A similar trend was observed when THP-1 derived macrophages were used for binding (Figure 2C). Association of PA^+Sia with these macrophages (MFI = 322.7 ± 5.20, p ≤ 0.01) was significantly higher compared to PA^−Sia (MFI = 85.67 ± 2.60). Also, removal of sialic acids significantly reduces the binding of PA^+Sia (MFI = 97.33 ± 6.17, p ≤ 0.01).

Sialic acids are recognized and bound by siglecs on host cells. In order to identify particular siglecs responsible for the increased association of PA^+Sia with macrophages, cells were pretreated with blocking antibodies against siglecs. Flow cytometry data revealed that blocking of siglecs on J774A.1 macrophages with anti-siglec-E antibodies results in significant reduction in PA^+Sia binding (MFI = 63.00 ± 0.57, p ≤ 0.05) as compared to untreated macrophages (MFI = 90.67 ± 0.88; Figure 2A).

Binding of PA^+Sia to THP-1 derived macrophages (MFI = 322.7 ± 5.20) was similarly reduced when siglecs were blocked by anti-siglec 9 antibodies (MFI = 148.7 ± 4.05, p ≤ 0.05; Figure 2C). Treatment of macrophages with anti-siglec-5 antibody also slightly reduces the binding of PA^+Sia (MFI = 272.7 ± 3.18, p ≤ 0.05). Representative histograms for the binding assays with J774A.1 cells are also shown in Figure 2B and with THP-1 is shown in Figure 2D. Blocking of other siglecs, namely murine siglec-1 and human siglecs-1, 3, and 7 show minute changes in PA binding (Supplementary Figure S1A).

Few other macrophage receptors are also known to mediate non-opsonic phagocytosis of PA (11). Contribution of such receptors in binding of PA^+Sia was also checked by using anti-CD11b (CR-3 alpha chain) and anti-CD16/32 (FcγRII and FcγRIII) antibodies. Although these macrophage receptors are also involved in binding, siglecs also play an important role in PA^+Sia binding with macrophages (Supplementary Figure S1A).

In similar experimental set up, macrophages were incubated with FITC-PA at 37°C for 30 min to allow for bacterial uptake, which was measured by flow cytometry (Figures 2E,G). PA^+Sia was internalized more (MFI = 54.33 ± 0.33, p ≤ 0.0001) by J774A.1 compared to PA^−Sia (MFI = 42.00 ± 0.57). This higher internalization was abrogated when sialic acid was removed from PA^+Sia (MFI = 42.33 ± 0.33, p ≤ 0.05; Figure 2E).

Furthermore, blocking of siglecs on J774A.1 by anti-siglec-E antibodies also reduced PA^+Sia phagocytosis (MFI = 41.00 ± 1.000, p ≤ 0.01) suggesting sialic acid and siglecs interactions are responsible for this higher internalization (Figure 2E). Representative histogram for the internalization assay with J774A.1 is shown as Figure 2F.

THP-1 derived macrophages also show similar pattern of interaction with PA (Figure 2G). Macrophages phagocytose more of PA^+Sia (MFI = 352.7 ± 5.78, p ≤ 0.01) compared to PA^−Sia (MFI = 107.3 ± 3.52). Also, desialylation of PA^+Sia abolishes this higher internalization (MFI = 116.3 ± 4.66, p ≤ 0.05; Figure 2G). Moreover, anti-siglec-9 antibodies treatment of THP-1 derived macrophages exhibited reduced bacterial internalization (MFI = 147.7 ± 4.05, p ≤ 0.001) indicating sialic acid-siglec-9 interaction to be majorly responsible for higher phagocytosis of PA^+Sia compared to siglec-5 (Figure 2G). As before, representative histogram for the internalization assay with THP-1 cells is shown as Figure 2H.

In order to further verify that PA entry into macrophages is due to its uptake by macrophages, cells were treated with cytochalasin D, which inhibits actin polymerization. Cytochalasin D nearly abolished bacterial uptake in both J774A.1 (MFI = 16.50 ± 0.50, p ≤ 0.05) and THP-1 derived macrophages (MFI = 23.33 ± 2.40, p ≤ 0.01; Figures 2E,G) confirming that macrophages are actively internalizing bacteria.

Taken together, these results demonstrate that PA^+Sia binding to macrophages is mediated through sialic acid-siglec interactions mainly through siglec-E and siglec-9 subsequently leading to higher phagocytosis.

J774A.1 were infected with PA^−Sia or PA^+Sia for 30 min at 37°C followed by staining with CM-H₂DCFDA, an indicator dye for cellular ROS (Figure 3A). PA^+Sia infected macrophages were found to exhibit significantly lowered ROS levels (MFI = 314.5 ± 7.81, p ≤ 0.0001) compared to PA^−Sia infected macrophages (MFI = 513.7 ± 8.24). Interestingly, removal of sialic acids from PA^+Sia resulted in enhancement of ROS production in macrophages (MFI = 442.7 ± 1.20, p ≤ 0.0001).

Similarly, intracellular levels of nitric oxide generated were measured by staining infected macrophages with DAF-FM (Figure 3B). It was observed that macrophages infected with PA^+Sia show lower levels of intracellular nitric oxide (MFI = 255.2 ± 2.85, p ≤ 0.01) compared to PA^−Sia infected macrophages (MFI = 401.6 ± 10.10). Removal of sialic acids from PA^+Sia also resulted in enhanced generation of nitric oxide (MFI = 304.4 ± 18.10, p ≤ 0.05).

Furthermore, we compared the modulation of cytokine response in PA^−Sia or PA^+Sia infected macrophages (Figure 3C). Macrophages were stimulated with PMA and culture supernatant from stimulated uninfected cells were used to establish baseline of cytokine secretion. Culture supernatants from stimulated macrophages infected with PA^+Sia show further elevation in levels of TH2 type cytokines (IL-10 = 154.0 ± 9.00 pg/mL, TGF-β = 177.0 ± 1.00 pg/mL, IL-4 = 100.5 ± 4.50 pg/mL) and reduced secretion of IFN-γ (77.50 ± 2.50 pg/mL). In contrast, macrophages infected with PA^−Sia showed comparatively reduced levels of TH2 cytokines (IL-10 = 92.50 ± 0.50 pg/mL, TGF-β= 98.00 ± 1.00 pg/mL, IL-4 = 56.00 ± 4.00 pg/mL,) and slightly higher secretion of pro-inflammatory cytokine IFN-γ (122.5 ± 7.50 pg/mL).

Removal of sialic acids from PA^+Sia reversed the cytokine profile leading to lowered secretion of TH2 type cytokines (IL-10= 81.00 ± 2.00 pg/mL, TGF-β= 84.50 ± 0.50 pg/mL, IL-4= 77.50 ± 2.50 pg/mL) and increased IFN-γ secretion (90.50 ± 0.50 pg/mL) suggesting an immunosuppressive role of sialic acid.

Thus, it may be noted that reduced respiratory burst and anti-inflammatory cytokine secretion in PA^+Sia-infected macrophages, can be reversed by desialylation of PA^+Sia, suggesting that sialic acids-siglec interactions may play a role in this altered immune response.

So far, we have observed that higher numbers of PA^+Sia are internalized by macrophages relative to PA^−Sia. However, attenuation of respiratory burst and anti-inflammatory cytokine responses by macrophages will have a negative impact on bacterial killing. Thus, the clearance of phagocytosed PA^+Sia depends on the endocytic pathway, specifically phagosome maturation and phagolysosome formation.

Accordingly, we checked the final fate of PA^+Sia phagosomes inside the macrophages (Figure 4A). FITC-PA^−Sia or PA^+Sia infected J774A.1 macrophages were stained with Lysotracker red. Confocal microscopy revealed that across multiple fields, more numbers of green dots (intracellular PA^−Sia) colocalized with the red lysosomes giving a composite yellow color in the merged fields in contrast to PA^+Sia-infected macrophages. Quantification of colocalization revealed that 98.7% of PA^−Sia containing phagosomes colocalize with the lysosomal compartment while only 31.7% of PA^+Sia containing phagosomes were found to do so.

We used flow cytometry to further quantify phagosome-lysosome fusion. Lysotracker red stained macrophages infected with FITC-PA^−Sia or PA^+Sia for 45, 90 and 120 min were gently lysed and released phagosomes were analyzed in a flow cytometer. Out of all phagosomes, lysosomes and phagolysosomes were identified by incorporated lysotracker stain. The presence of FITC-PA in lysosomes, signifying phagosome-lysosome fusion, was quantified by dual color (FL1-FL2 channel) positivity (Figure 4B).

In infected J774A.1, we observed that PA^+Sia colocalized with lysosomes was lower being only 8.800 ± 0.30% (p ≤ 0.01) of all analyzed events compared to PA^−Sia (17.90 ± 0.60%) after 45 min of infection (Figure 4C). A similar trend was observed after 90 min of infection. The percentage of dual positive phagosomes diminished at 120 min post infection. We confirmed our observations in THP-1 derived macrophages, which showed a similar kind of colocalization at 45, 90 and 120 min upon infection with FITC-PA^−Sia/PA^+Sia (Figure 4D). Thus, the flow cytometry data corroborates with our observations from confocal microscopy, that fusion of PA^+Sia-containing phagosomes with lysosomes was reduced.

Earlier, we demonstrated a comparatively lowered oxidative burst and reduced pro-inflammatory cytokines secretion (Figure 3) in PA^+Sia-infected macrophages compared to PA^−Sia. Furthermore, phagosome-lysosome fusion, which eliminates phagocytosed bacteria, is also reduced in PA^+Sia-infected macrophages (Figure 4). Thus, we checked if PA^+Sia bacteria remain viable following internalization by macrophages through gentamicin protection assay (Figure 4E). Results indicate that approximately 3.4-fold higher number of PA^+Sia survived inside macrophages compared to PA^−Sia; colony forming units being 8.33 ± 0.43 × 10⁵ vs. 2.90 ± 0.55 × 10⁵ for PA^+Sia vs. PA^−Sia respectively (p ≤ 0.05) at 3 h post-infection.

Removal of sialic acids from PA^+Sia resulted in ~ >2.6-fold reduction in the number of viable bacteria isolated from infected macrophages. These observations clearly indicate that bacterial sialic acid provide internalized PA^+Sia with additional advantage to survive inside macrophages.

So far we have observed higher numbers of viable PA^+Sia inside macrophages after 3 h post infection by gentamicin protection assay. Staining of bacteria with CFSE is one of the methods to check bacterial multiplication inside phagocytic cells (25–27). Therefore, to check if internalized PA^+Sia can replicate inside macrophages, J774A.1 macrophages were infected with CFSE stained PA^−Sia/PA^+Sia.

Macrophages infected for 8 h were visualized by confocal microscopy to check for the presence of PA (Figure 4F). While only a few PA^−Sia bacteria were observed as bright green dots inside the cells, multiple dots of faintly glowing PA^+Sia were visible. This suggests that CFSE stained PA^+Sia undergoes division inside macrophages which resulted in dilution of CFSE dye.

Phagosome maturation consists of early, mid and late stage phagosomes. Each stage is characterized by sequential association and dissociation of specific effector Rab proteins (28). The final step always is the fusion of the phagosome with lysosomes subsequently resulting in degradation of its cargo. Since phagosome-lysosome fusion was reduced in PA^+Sia-infected macrophages, we wanted to identify if any intermediate step in phagosome maturation was hindered by checking for changes in the acquisition of stage-specific Rab proteins.

Previous experiments have demonstrated that internalized PA is trafficked into the lysosomal compartment within 45 min of infection. Accordingly, J774A.1 infected with PA^−Sia/PA^+Sia were fixed at 20, 40, and 60 min after infection, permeabilized and stained for early endosomal (Rab5), late endosomal (Rab7) and lysosome specific (LAMP1) markers, respectively. Cells were counterstained with secondary antibody and visualized by confocal microscopy. Nearly 100 cells from different randomly chosen fields as well as z stacks based visualizations were analyzed for colocalization of internalized bacteria with the endosomal compartments.

At the initial phase of infection (20 min), 79.67 ± 3.53% of internalized PA^−Sia and 80.82 ± 6.15% of internalized PA^+Sia were located in Rab5-stained compartment (Figure 5A). Although PA^−Sia and PA^+Sia were equally localized in the Rab5 labeled early phagosomes, the number of PA^+Sia taken up by the macrophages remained higher.

Maturation of early phagosomes into late phagosomes is marked by the appearance of Rab7 protein which is necessary for the final step of phagosome fusion to lysosomes (29). Accordingly, 40 min post-infected macrophages were stained with anti-Rab7 antibody. It was observed that 74.67 ± 9.61% of phagocytosed PA^−Sia colocalized within the Rab7 stained compartment after 40 min of incubation (Figure 5B). Interestingly, reduced numbers of PA^+Sia-containing phagosomes (54.70 ± 11.31%, p ≤ 0.001) were found to acquire Rab7. Additionally, we compared colocalization between macrophages that had internalized higher numbers of PA^−Sia and PA^+Sia. Even at comparable numbers, PA^−Sia always showed higher colocalization with Rab7 (Supplementary Figure S1B) while PA^+Sia evaded fusion with the Rab7 compartment.

Next we wanted to observe successful fusion of PA-containing phagosomes with lysosomes. Accordingly, lysosomes were labeled with anti-LAMP1 antibody in 60 min post-infected macrophages. Higher percentage of PA^−Sia-containing phagosomes (81.72 ± 5.33%) was found to have reached the lysosomal compartments (Figure 5C). However, at the same time, only 53.46 ± 14.23% (p ≤ 0.05) of PA^+Sia-containing phagosomes were LAMP1 positive.

These observations were validated using flow cytometry based analysis of PA containing phagosomes. FITC-PA^−Sia/PA^+Sia infected J774A.1 were incubated for 20, 40, and 60 min and prepared phagosomal fractions were stained for early endosomal (Rab5), late endosomal (Rab7) and lysosome specific (LAMP1) markers. Flow cytometry revealed that at an early time point of infection (20 min), higher numbers of PA^+Sia colocalized with the Rab5 labeled compartment (10.30 ± 1.30%, p ≤ 0.05) compared to PA^−Sia (3.750 ± 0.05%; Figure 5D). This trend remains even up to 40 min post-infection (PA^+Sia = 3.300 ± 0.20%, PA^−Sia = 1.900 ± 0.0%, p ≤ 0.05). Possibly, the greater presence of PA^+Sia in Rab5 positive early phagosomes at early time points is due to its higher phagocytosis by J774A.1. By 60 min post-infection, only a few PA^+Sia/PA^−Sia remain in the Rab5 labeled compartment.

Interestingly, we observed that PA^+Sia harboring phagosomes acquire approximately 0.8-fold less Rab7 (Figure 5E). The percentage of PA^+Sia phagosomes that were positive for Rab7 were 3.950 ± 0.05 % out of all events at 20 min post infection which was significantly lower compared to Rab7 positive PA^−Sia phagosomes (5.350 ± 0.15 %, p ≤ 0.05). At 40 min post infection, Rab7-positive phagosomes containing PA^+Sia were even less (1.900 ± 0.10%) as compared to PA^−Sia (3.950 ± 0.05%, p ≤ 0.01).

We also checked for colocalization of bacteria with the LAMP1 labeled compartment, which represents lysosomes and phagolysosomes (Figure 5F). At 20 min, the difference between PA^−Sia and PA^+Sia phagosomes was low mostly because very few phagosomes had matured at this early time point. We observed that PA^−Sia phagosomes associated with LAMP1 at 40 min was higher (3.050 ± 0.05%) than PA^+Sia (2.350 ± 0.15%, p ≤ 0.05). At 60 min post-infection, a similar trend was observed. Colocalization of PA^−Sia to lysosomes was 3.550 ± 0.05% while in case of PA^+Sia this value was lower (2.450 ± 0.05, p ≤ 0.01).

Taken together, our observations indicate that 20 min after infection, significantly higher numbers of PA^+Sia was localized in Rab5 positive early phagosomes than PA^−Sia. However, at the same time point, more PA^−Sia had colocalized with the Rab7 compartment compared to PA^+Sia. At 40 min after infection, higher numbers of PA^+Sia continue to remain associated with early phagosomes while comparatively more PA^−Sia-phagosomes mature and become Rab7 positive. Simultaneously, the PA^−Sia colocalization with LAMP1 was higher than PA^+Sia, signifying successful fusion to lysosomes. This observation was also confirmed by flow cytometry in FITC-PA^−Sia and PA^+Sia infected THP-1 derived macrophages (Supplementary Figure S1C).

Thus, our observations indicate that reduced acquisition of Rab7 by phagosomes harboring PA^+Sia may be responsible for the subsequent prevention of phagosome-lysosome fusion as demonstrated by the reduced acquisition of LAMP1.

Modulation of intracellular calcium ions during phagocytosis has been linked to evasion of phagosome-lysosome fusion in Mycobacterium tuberculosis infections (30). Thus, we next evaluated intracellular calcium levels in PA^−Sia or PA^+Sia-infected macrophages (Figure 6A). Microscopy revealed that intracellular calcium levels, [Ca²⁺]_c were lower in Fluo-3-AM loaded PA^+Sia infected macrophages (7.075 ± 0.34 a.u., p ≤ 0.001) compared to PA^−Sia (12.50 ± 0.70 a.u.) at 15 min post infection.

Additionally, intracellular calcium concentrations in Fura-2-AM stained infected macrophages were also analyzed by fluorescence plate reader and concentration of calcium ions (nM) was calculated using the Grynkewicz equation (Figure 6B). Following infection, intracellular calcium concentrations remained lower in PA^+Sia-infected macrophages as compared to PA^−Sia.

Alteration of intracellular calcium levels results in modulating a cascade of signaling events in the cytosol. Calmodulin is an important cytosolic calcium-binding protein that is known to regulate phagosome-lysosome fusion in Mycobacterium (30, 31). Accordingly, we analyzed the levels of a few calcium-related proteins in infected macrophages by western blotting (Figure 6C). Statistically significant differences were observed in levels of calmodulin, calcineurin, and calmodulin-dependent kinase type II expression only after 45 min post infection in PA^+Sia infected macrophages.

So far we have observed that PA^+Sia infection of J774A.1 macrophages are associated with lowered intracellular calcium concentrations and calcium-related proteins. As mentioned before, lowered calcium concentrations are correlated with phagosome lysosome fusion inhibition in Mycobacterium (30, 31). Normally, infection was carried out in IMDM medium containing 1.5 mM calcium concentration (as CaCl₂). Gentamicin protection assay was used to determine if addition of extracellular calcium (final concentration −2.5, 5, 10 mM) affects the viability of PA^+Sia at 3 h post infection. It was observed that bacterial viability was not affected in Ca²⁺ free condition or at lower concentrations of extracellular calcium. However, viability of PA^−Sia and PA^+Sia was inhibited onwards from 5 mM extracellular Ca²⁺ concentration (Figure 6D).

We next checked if loss of viability of PA^+Sia-infected macrophages in 5 mM extracellular Ca²⁺ was due to enhanced phagosome-lysosome fusion. Macrophages in calcium free HBSS were infected with PA^+Sia at 4°C for binding. Plates were transferred to 37°C and 5mM extracellular Ca²⁺ ionomycin (10 μM) was added. Phagocytosis was continued in macrophages incubated in calcium-free HBSS or containing Ca²⁺ (5 mM) or both Ca²⁺ (5 mM) and ionomycin for 90 min. Cells were finally labeled with lysotracker red, phagosomes were prepared and analyzed by flow cytometry. Surprisingly, compared to infected macrophages in absence of calcium, addition of only extracellular Ca²⁺ did not greatly increase phagolysosome fusion events (Figure 6E). However, introduction of ionomycin along with extracellular Ca²⁺ (5 mM) resulted in increase in the percentage of phagosome-lysosome fusion events. Ionomycin is known to increase intracellular calcium levels. Representative scatter plots indicating colocalization between lysotracker stained lysosome or phagolysosomes with FITC-PA^+Sia are shown (Figure 6F).

Confocal microscopy also confirmed these observations. Phagosome-lysosome fusion remained unchanged at high extracellular Ca²⁺ concentrations (Figure 6G). But, addition of ionomycin increased the intracellular calcium concentration and enhanced phagolysosome fusion in PA^+Sia infected macrophages (Figure 6G).

These observations indicate that loss of viability of bacteria in presence of extracellular calcium was not related to phagolysosome fusion. However, in presence of extracellular calcium, ionomycin-mediated rapid increase in intracellular calcium enhanced phagosome-lysosome fusion in PA^+Sia infected macrophages. This further confirms that intracellular Ca²⁺ plays a regulatory role in bacterial killing through phagolysosome formation in PA^+Sia-infected macrophages.

Inflammatory cytokine response by macrophages incubated with PA^−Sia or PA^+Sia in presence of increased extracellular calcium was also checked by ELISA based estimation of IFN-γ (pro-inflammatory) and IL-10 (anti-inflammatory). No significant change in cytokine secretion was observed with increase in calcium (Figure 6H). Interestingly, the differences in cytokine response between PA^−Sia/PA^+Sia-infected macrophages were diminished with increasing Ca²⁺.

Binding of sialic acids by siglecs result in phosphorylation of tyrosine residues present in the ITIM motifs of inhibitory siglec-E leading to the recruitment of SHP-1 and SHP-2 phosphatases. We have already demonstrated that PA^+Sia binds to J774A.1 through siglec-E (Figure 2). Immunoprecipitation revealed 2.75-fold higher phosphorylation of tyrosine residues of siglec-E in PA^+Sia-infected macrophages compared to PA^−Sia, indicating such engagement of siglec-E leads to its activation (Figure 7A). A representative Ponceau blot is provided to account for equal loading across lanes. Next, we analyzed the association between siglec-E and SHP1/2 phosphatases (Figures 7B,C). Higher levels of siglec-E were found to be associated with SHP-2 in macrophages infected with PA^+Sia compared to PA^−Sia at each time point (20, 40, and 60 min). At 40 min, association between siglec-E and SHP-2 was significantly ~1.5-fold times higher (Figure 7C). These observations suggest that siglec-mediated inhibitory signaling is active in PA^+Sia-infected macrophages. A similar trend was also observed with SHP-1 (Figure 7B). Association between siglec-E and SHP-1/2 was also significantly higher at 40 min post infection in PA^+Sia infected macrophages than PA^−Sia infection.

The role of sialic acid-siglec-E interaction on macrophage responses against PA^+Sia was further established by silencing expression of siglec-E in J774A.1. Reduction in siglec-E expression due to transient siRNA transfection was confirmed by western blotting (Figure 8A). Consequently, confocal microscopy revealed 3-fold less PA^+Sia were internalized by siglec-E knockdown macrophages (Figure 8B). Phagocytic index of PA^+Sia infected macrophages was 11.62 ± 0.19 while it decreased to 6.467 ± 0.72 upon siglec-E knockdown.

Subsequently, we have observed enhanced ROS generation in siglec-E silenced macrophages in response to PA^+Sia (MFI = 640.5 ± 31.50, p ≤ 0.05) as compared to mock-transfected PA^+Sia-infected macrophages (MFI = 349.8 ± 32.20; Figure 8C). Similarly, reactive nitrogen species generation in these PA^+Sia -infected macrophages was higher (MFI = 2,569 ± 609.0) in absence of siglec-E rather than in its presence (MFI = 1,436 ± 195.0, p ≤ 0.05; Figure 8D). Siglec-E silenced PA^+Sia-infected macrophages also showed increased IFN-γ levels (203.0 ± 3.00, p ≤ 0.01) and reduced IL-10 secretion (153.2 ± 6.76 pg/mL, p ≤ 0.01; Figure 8E). Cytokine levels of siglec-E expressing macrophages upon PA^+Sia infection were characterized by higher levels of IL-10 (328.7 ± 4.33 pg/mL) and lower levels of IFN-γ (105.0 ± 5.05 pg/mL). Thus, siglec-E knockdown alters macrophage cytokine response against PA^+Sia from a TH2 anti-inflammatory type to a pro-inflammatory TH1 type, as characterized by these two cytokines. Survival of PA^+Sia in siglec-E blocked macrophages was also checked by gentamicin protection assay. In absence of interacting siglec-E, the number of PA^+Sia that remains viable inside macrophages also decreases after 3 h of infection (7.73 ± 0.29 × 10⁵ vs. 3.31 ± 0.34 × 10⁵, p ≤ 0.01; Figure 8F). These observations suggest that absence of siglec-E results in stronger anti-bacterial response by macrophages leading to PA^+Sia elimination.

We have observed that phagosome maturation is altered in PA^+Sia-infected macrophages (Figures 4, 5). We have also observed modulation of intracellular calcium levels upon PA^+Sia infection, which is known to play a significant role in such phagolysosome fusion inhibition (Figure 6). By microscopy, we identified that siglec-E has a role in such modulation, where we found higher intracellular calcium concentrations in siglec-E silenced macrophages (9.267 ± 0.29 a.u., p ≤ 0.01) compared to mock-transfected macrophages (6.833 ± 0.33 a.u.; Figure 9A) upon PA^+Sia infection. This observation was corroborated by flow cytometry, where we observed that PA^+Sia-infected siglec-E silenced macrophages show higher calcium levels (MFI = 1,994 ± 17.50, p ≤ 0.05) as compared to infected mock-transfected macrophages (MFI = 1,583 ± 92.00; Figure 9B). Additionally, we also demonstrated that calcium monitoring proteins such as calmodulin and calmodulin-dependent kinase II were also elevated upon siglec-E knockdown in macrophages (Figure 9C).

We have also confirmed that siglec-E knockdown resulted in reduction in expression of SHP-2 and SHP-1 in these PA^+Sia infected transfected cells (Figure 9C).

Furthermore, we have observed elevated phosphoproteins of p-P38, p-AKT (Ser473), p-ERK1/2, and p-JNK/SAPK in PA^+Sia-infected siglec-E silenced macrophages compared to mock transfectants (Figure 9C). Moreover, knockdown of siglec-E resulted in an increase in IκB-α phosphorylation and translocation of NF-κB p65 subunit from the cytosol into the nucleus of PA^+Sia-infected macrophages (Figure 9D).

Statistical analysis of densitometry values of each band from three independent western blots were performed in all cases to evaluate the significance of observed differences in band intensities.

All these observations support that siglec-E plays an important role in regulating antimicrobial responses, intracellular calcium levels and key cellular signaling molecules in PA^+Sia-infected macrophages.

So far we have demonstrated the important role of siglec-E in sialic acid-siglec-E interactions in PA^+Sia infected macrophages (Figure 9). Now, the status of a few key signaling molecules, previously identified to be affected by sialic acid-siglec-E interactions were also checked in PA^−Sia/PA^+Sia infected macrophages at different time points post-infection (Figures 9C,D). We have observed that expression of PI3K p85 and phosphorylated forms of ERK1/2, JNK/SAPK are consistently lower in case of PA^+Sia-infected macrophages than PA^−Sia infection (Figure 10A). Average of densitometric values of observed protein bands normalized with respect to β-actin bands are reported as bar diagrams and statistical significance is also reported.

We also observed that the p65 subunit of NF-κB pathway remains in the cytosol in PA^+Sia infected macrophages, resulting in lower activation of NF-κB pathway. However, p65 translocates into the nucleus of macrophages post infection with PA^−Sia (Figure 10B) indicating activation of NF-κB pathway.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.