Podoplanin Drives Motility of Active Macrophage via Regulating Filamin C During Helicobacter pylori Infection

Podoplanin (Pdpn) is a mucin-type transmembrane protein that has been implicated in multiple physiological settings including lymphangiogenesis, platelet aggregation, and cancer metastasis. Here, we reported an absence of Pdpn transcript expression in the resting mouse monocytic macrophages, RAW264.7 cells; intriguingly, a substantial upregulation of Pdpn was observed in activated macrophages following Helicobacter pylori or lipopolysaccharide stimulation. Pdpn-knockout macrophages demonstrated intact phagocytic and intracellular bactericidal activities comparable to wild type but exhibited impaired migration due to attenuated filopodia formation. In contrast, an ectopic expression of Pdpn augmented filopodia protrusion in activated macrophages. NanoString analysis uncovered a close dependency of Filamin C gene on the presence of Pdpn, highlighting an involvement of Filamin C in modulation of actin polymerization activity, which controls cell filopodia formation and migration. In addition, interleukin-1β production was significantly declined in the absence of Pdpn, suggesting a role of Pdpn in orchestrating inflammation during H. pylori infection besides cellular migration. Together, our findings unravel the Pdpn network that modulates movement of active macrophages.

Current knowledge of Pdpn functions relies heavily on the extracellular domain interaction with its ligand platelet C-type lectin-like receptor 2 (Clec2). Interaction of Pdpn to Clec2 stimulates deep vein thrombosis (12) while point mutations of threonine residues at the highly conserved platelet-activating (PLAG) domain of Pdpn obliterate platelet aggregation (13). By activating platelets, Pdpn is able to promote pulmonary cancer growth, malignant progression, and metastasis through cancer embolization that protects cancer cells from immunological assault (14,15). In addition, Pdpn-Clec2 axis controls the development and maintains the integrity and contractibility of high endothelial venules (HEVs) (4,5,11), permitting the travel of lymphocytes and dendritic cells across the lymphatic system (16,17). While the extracellular domain of Pdpn binds to Clec2, the intracellular domain of Pdpn interacts with the ezrin-radixin-moesin (ERM) complex to modulate Rho-GTPases and actin cytoskeletal rearrangement (10,18). High expression of Pdpn has been associated with cancer metastasis such as advanced-stage gastric carcinoma (15,19). On the contrary, Pdpn inhibition by neutralizing antibody depresses the metastasis of breast cancer cells xenografted into immunodeficient NOD-SCID mouse model (20). As such, Pdpn has hitherto been considered a potential marker as well as therapeutic target for aggressive cancer (21,22). In the immune system, Pdpn expression has been reported in T helper 17 (T H 17) cells and macrophages (23,24). Pdpn-expressing T H 17 cell promotes ectopic lymphoid follicles formation in mouse experimental autoimmune encephalomyelitis (EAE) model (23), while its deletion exacerbates EAE in a genetically susceptible mouse model (25). In macrophages, Pdpn expression can be induced by thioglycollate or lipopolysaccharide (LPS) (24). A subset of Pdpn-expressing macrophage is highly phagocytic (26), associates with pro-inflammatory response (27), and promotes platelet aggregation through Pdpn-Clec2 crosstalk (24). Presence of Pdpn-expressing CD68 + macrophage-like cells and synovial fibroblasts at the joint of rheumatoid arthritis patients exaggerate joint cartilage inflammation and destruction (28,29). Inflammatory response can be relieved with Vav1-promoter driven Pdpn deletion in myeloid cells as reported in mouse peritonitis model (30).
Chronic inflammation triggered by long-term Helicobacter pylori infection is the major mechanism underlying the development of gastric cancer in human. Macrophage is a key player in orchestrating inflammatory response to H. pylori infection since increased M1 cells and related proinflammatory markers were detected in the stomach of H. pylori-infected humans (31)(32)(33)(34), while transient macrophage depletion reduces the H. pylori-associated inflammation and gastric pathology in a mouse model (35). While T H 17 cells also play an important role during the chronic inflammation induced by H. pylori, the current study focuses on the role of Pdpn in macrophages only. This study investigates the expression and potential role of Pdpn in macrophage during inflammation by applying the H. pylori infection model. Subsequent generation of Pdpn-knockout and overexpressing macrophages enabled us to clarify the function and signaling pathway of Pdpn. Here, we demonstrated that, during H. pylori infection, Pdpn promotes Filamin C expression to enhance filopodia formation and migratory activity of macrophages. Nonetheless, there are no differences in bacterial uptake and killing in the absence of Pdpn. High Pdpn expression is also associated with a greater IL-1b secretion, which implies intervening Pdpn as a therapeutic opportunity in limiting inflammation.

Phagocytosis Assay
H. pylori (10 8 cells/ml) were labeled with FITC in the dark for 30 min and washed 4× with sterile PBS. Cells were infected with FITC-labeled bacteria at MOI 1 for 1 and 4 h before analyzed. Cells harvested at 1 and 4 h were washed and analyzed with FACS Canto II (BD Bioscience). For immunofluorescent staining, cells were seeded on a coverslip and infected with H. pylori at MOI 10. At 24 h, cells were gently washed and mounted with ProLong Gold Antifade reagent with DAPI (Thermo Fisher Scientific) before visualization under an Eclipse TE2000-E fluorescence microscope (Nikon, Tokyo, Japan).

Electron Microscopy
Cells were pelleted, fixed in 4% glutaraldehyde overnight, and post-fixed in osmium tetroxide. Cell pellet was dehydrated with an ascending series of ethanol and embedded in the epon mixture prepared with Agar 100, dodecyl succinic anhydride, methyl nadic anhydride, and tri-dimethylaminomethyl phenol. Samples were sectioned using an ultramicrotome and stained with uranyl acetate and lead citrate before observation with transmission electron microscope LEO LIBRA120 (Carl Zeiss, Oberkochen, Germany).

Macrophage Intracellular Killing Assay
Culture medium of H. pylori-infected cells at 24 h post-infection (h.p.i.) was replaced with DMEM with 100 mg/ml of gentamycin for extracellular bacteria killing. After 1-h incubation, cells were washed and lysed with 0.1% saponin at 37°C for 15 min. Viable intracellular bacteria count was determined by calculating colony-forming units (CFU) of the serially diluted bacteria that were plated and cultured on chocolate agar plate.

Real-Time Cell Analysis Assay and Transwell Migration Assay
Real-time cell migration was monitored using electronically integrated Boyden chamber CIM-16 plate in a xCELLigence RTCA DP instrument (ACEA Bioscience, San Diego, CA). Briefly, the lower chamber was detached and loaded with DMEM with 20% FBS, 100 ng/ml CCL2, and 1 μg/ml LPS from E. coli (O111:B4, Sigma Aldrich, USA). The upper chamber was reassembled and 50 μl of serum-free DMEM was added. CIM-16 plate was placed on RTCA instrument in a 37°C incubator for 1 h and background signal was measured for 5 min. Then, cells were seeded at 5 × 10 5 cells/ml in DMEM media supplemented with 10% FBS in the upper chamber and allowed to equilibrate and attached at room temperature for 30 min before initiating measurement. Data were analyzed in a xCELLigence RTCA Software Pro V2.0 (ACEA Bioscience).
Conventional migration assay was performed using 8-mm 24well transwell inserts. Cells were seeded in the upper chamber at 3 × 10 5 cells/ml. Lower chamber was filled with DMEM with 20% FBS, 100 ng/ml CCL2, and 1 μg/ml LPS. After 24 h, nonmigrated cells inside the insert were removed using a cotton tip while migrated cells were fixed with methanol for 10 min, stained with crystal violet staining, and visualized under a light microscope. Ten images from different fields were captured randomly for cell calculation. For migration assay using H. pylori, bacteria suspension was adjusted to 10 8 cells/ml and heat-inactivated at 56°C for 30 min. Subsequently, inactivated bacteria were added at MOI 50:1 in the lower chamber and incubated for 24 h.

Wound Healing Assay
Cells were seeded onto a 96-well plate at a concentration of 5 × 10 5 cells/ml to achieve around 90% confluency. Media was changed into fresh DMEM supplemented with 10% FBS and a 10-μl pipette tip was used to scratch a wound in the middle of the well. Wound healing was recorded every 5 min with JuLi BRD04 live cell imager (NanoEntek, Pleasanton, CA) for 24 h. Area of wound was measured every 4 h using ImageJ (version 1.50b). Percentage of wound healed was calculated with the following equation, where T 0 indicates 0 h; T n indicates selected time point.

Immunofluorescence Microscopy
Cells were seeded at 2 × 10 5 cells/ml in a four-well chamber slide and incubated overnight. After 24-h treatment with 100 ng/ml CCL2 and 10 ng/ml LPS, cells were fixed with ice-cold methanol for 10 min, washed with PBS/0.01% Triton-X, and blocked with PBS/3% BSA. Cells were incubated with rabbit anti-mouse-bactin antibody (Cell Signaling Technology, Beverly, MA) for 1 h followed by incubation with secondary antibodies Alexa Fluor 555-conjugated anti-rabbit IgG (H+L) F(ab') 2 (Cell Signaling Technology). Cells were washed with PBS/0.01% Triton-X again before counterstained with Prolong gold antifade DAPI reagent. Slide was viewed in a Leica TCS SP5 II confocal microscope (Leica Microsystems, Mannheim, Germany).

Cell Proliferation Assay
Cells were seeded at 2 × 10 5 cells/ml in different wells of four 96well plate and incubated for 4 days. Each day, a plate was removed and processed. A total of 50 μl of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) substrate was added and incubated for 1 h. Cell supernatant was removed carefully and replaced with 100 μl of DMSO. Absorbance reading at 570 nm was measured using a Synergy HTX Multi-Mode microplate reader (BioTek Instruments, Winooski, VT).

NanoString Gene Expression Analysis
A total of 100 ng of RNA (50 ng/μl) was added to hybridization buffer, reporter, and capture probe sets (Cat: NNS_NAA-AKIT-012, Lot: CP6643X1), and hybridized at 65°C for 20 h. Subsequent sample preparation and scanning were performed using an automated nCounter Prep station for hybridization onto the sample cartridge (Cat: NNS_XT-CSO-MPATH1-1, Lot: RC7091X1) followed by scanning with Digital analyzer (NanoString Technologies, Seattle, WA). Data were analyzed using the nSolver Analysis Software V4.0. Two-step data normalization was performed using an internally developed Pipeline Pilot Tool (NAPPA) to account for the background using the spike in positive and negative controls, followed by input correction using housekeeping genes. Genes that had a mean value fewer than 10 counts across all samples were removed from analysis. NanoString gene expression data have been deposited to the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) repository available at https://www.ncbi.nlm.nih.gov/geo/query/ acc.cgi?acc=GSE151269.

ELISA
Enzyme-linked immunosorbent assay (ELISA) was carried out using ELISA MAX ™ Deluxe Set Mouse (Biolegend) according to the manufacturer's protocol. Cell lysate was used for pro-IL-1b detection since RAW264.7 cell lacks apoptosis-associated specklike protein containing a Card (Asc), an inflammasome adaptor indispensable for IL-1b maturation (39). Supernatant from cell culture was used for the detection of TNFa.

Statistics
Data were shown as mean ± standard deviation (SD). Data were analyzed with unpaired two-tailed Student's t-test when comparing between two groups, in GraphPad Prism (GraphPad software, La Jolla, CA, www.graphpad.com). Statistical significance among three groups were analyzed using two-way ANOVA test. p-values of <0.05 were considered as statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical analysis for NanoString data was performed by R-based statistic program by either loglinear regression or simplified negative binomial model and adjusted with the Benjamini-Yekutieli method in nCounter Advanced Analysis 2.0 (NanoString Technologies). Samples were considered significant if p < 0.01 (−log 10 > 2).

H. pylori Infection Upregulates Pdpn Expression in Macrophages
Our previous microarray analysis revealed an altered transcriptional profile of multiple genes in H. pylori-infected macrophages (37). Out of the genes attained, we selected Pdpn gene for further investigation given its uncharacterized function in macrophage and intense upregulation following H. pylori infection ( Figure 1A). To validate the microarray data, we infected RAW264.7 macrophages with H. pylori SS1 for examination at both transcriptional and translational levels. H. pylori SS1 was used in the current study as this strain is the mouse adapted strain widely employed in studies of H. pylori  increased in a time-dependent fashion ( Figure 1C, arrows). We then hypothesized that the upregulation of Pdpn in response to H. pylori infection could also occur with other bacteria. Two gram-positive (S. aureus and S. epidermidis) and two gramnegative (H. pylori and N. gonorrhoeae) bacteria were selected for the test. Interestingly, we noted that Pdpn expression was strongly induced following infection with gram-negative but not gram-positive bacteria ( Figure 1D). Induction of Pdpn was likely triggered by bacteria surface lipopolysaccharide (LPS), as shown in Figure 1E and a previous report (24). Additional stimulation with C-C chemokine ligand 2 (CCL2) chemoattractant showed no additive or synergistic effect on Pdpn expression ( Figure 1E). Further confirmation of PDPN expression in human cells was also carried out in human THP-1 monocytic macrophages using H. pylori J99 strain, a highly pathogenic strain in human, where upregulation of expression was observed at both mRNA and protein levels ( Figures 1F, G).

Pdpn Deletion Does Not Affect Macrophage Phagocytic or Bactericidal Activities
To further investigate the functional role of Pdpn in macrophages, we disrupted Pdpn gene in the parental RAW264.7 cells using CRISPR/Cas9 gene editing technology. Two stable clones with early non-sense mutation were selected (Supplementary Figures S1A, B). Three-dimensional protein structure prediction suggested that the mutations resulted in disrupted alpha helix folding at the N terminal extracellular domain of Pdpn protein, indicating loss of protein function (Supplementary Figure S1C). The first clone exhibited a partial (~70%) reduction in expression with a lower molecular weight indicative of protein truncation and was labeled as podoplanin knockdown with truncation (Pdpn-KD/TC), whereas the second clone showed no detectable protein and thus was labeled as podoplanin knockout (Pdpn-KO) (Supplementary Figure S1D). Most of the following analyses were conducted using Pdpn-KO cells.
A previous study reported high phagocytic activity of podoplanin-expressing inflammatory macrophages (24). Hence, we first assessed the ability of Pdpn-KO cells in phagocytosing H. pylori. In transmission electron microscopy images, internalized bacteria in Pdpn-KO cells can be clearly detected as densely stained, curved rod structure in the phagocytic vacuoles (Supplementary Figure S2A). By using FITC-labeled H. pylori, immunofluorescence images revealed that Pdpn-KO cells effectively engulfed bacteria comparable to the wild-type parental cells (Supplementary Figure S2B). Flow cytometric analysis at 1 h.p.i. suggested an effective albeit minimal delay in the phagocytic ability of Pdpn-KO; data at 4 h.p.i. demonstrated that >98% of Pdpn-KO successfully phagocytosed the bacteria (Supplementary Figure S2C). In intracellular bactericidal assay, no significant difference was observed in the CFU of the viable H. pylori that remained within the Pdpn-KO and wild-type macrophages following infection (Supplementary Figure S2D), suggesting that Pdpndeletion caused no direct defect in bacterial uptake and killing.

Pdpn-Knockout Cells Exhibit Impaired Migration Activity
Given that fewer CD11b + F4/80 + cells are recruited to the site of inflamed peritonitis in Vav-Cre Pdpn-floxed mice (30), we postulated that Pdpn mediates macrophage migration. Using wound healing assay, we observed that Pdpn-KO cells were inefficient in closing the scratched area compared to wild-type cells (Figure 2A). We exclude the possibility that the inefficient wound healing ability was a result of defective cell proliferation capability, as shown by parallel cellular proliferation rates over a period of 4 days between wild-type and knockout cells ( Figure 2B). Strikingly, live recording demonstrated static or limited movement of Pdpn-KO cells compared to highly motile wild-type cells (Supplementary Video S1).
We further compared cellular migration of parental and Pdpn-KO cells towards LPS and CCL2 chemoattractant. The number of the migrated cells in the Boyden chamber assay was remarkably reduced by approximately 50% and >90%, in Pdpn-KD/TC and Pdpn-KO cells, respectively, compared to wild-type control ( Figures 2C, D). Notably, the morphology of the mutant cells appeared smaller and rounder compared to that of the migrated parental cells ( Figure 2C). Next, we repeated the migration assay by using H. pylori induction. The heatinactivated form of H. pylori was used to avoid the migrated cell count from being affected by live bacterial infectionmediated cell death. We noted that wild-type cells migrated efficiently towards lower chamber containing the heatinactivated form of H. pylori. Consistent with the above induction using LPS and CCL2, a significantly lower number of migrated Pdpn-KD/TC and Pdpn-KO cells were induced by H. pylori, suggesting the defective migratory activity (Supplementary Figure S3). Results from an impedance-based real-time cell analyzer analysis demonstrated that the cellular migration rate towards CCL2 chemoattractant increased steadily over time in the wild-type parental cells, but it was substantially declined in Pdpn-KO or Pdpn-KD/TC cells ( Figure 2E). These observations collectively suggest that Pdpn modulates cell migration of activated macrophages.

Filopodia Formation Is Diminished by Pdpn Deletion and Intensified by Pdpn Overexpression
When cells were stained and visualized under confocal microscopy, there was no overt differences observed in the WT, Pdpn-KD/TC, and Pdpn-KO cells prior to activation ( Figure 3A, upper panel). Upon treatment with LPS (and CCL2), wild-type cells displayed increased cellular size with extended filopodia protrusion and long cytoplasmic tail formation, suggesting active actin polymerization and directional cell migration. In contrast, LPS (and CCL2) treatment failed to induce the formation of filopodia and long cytoplasmic tail in the mutant clones ( Figure 3A, as indicated by arrows, medium, and lower panels). Although Pdpn-KD/TC and Pdpn-KO cells lacked long filopodia, the formation of lamellipodia and podosome structures ( Figure 3A, as indicated by broken arrow and arrowhead) can still be detected.
To further confirm the involvement of Pdpn in filopodia formation, a stable clone ectopically expressing Pdpn was generated using a retrovirus transduction system. The level of Pdpn expression in the Pdpn-TG cells was strongly evident at the protein level under unstimulated condition, at a greater level compared to that of H. pylori-stimulated WT cells ( Figure 3B). Intriguingly, an explosive amount of filopodia protrusion was detected in the Pdpn-TG cells after LPS stimulation ( Figure 3C). Absence of filopodia in the knockout cells and its excessive presence in the transgenic cells underscore an essential role of  Pdpn in controlling filopodia formation. It should be noted that this phenomenon was not observed in unstimulated Pdpn-TG cells, suggesting that additional factors derived from cell signaling are indispensable for initiating actin polymerization process compulsory for filopodia formation.

Pdpn Deletion Abrogates the Production of Interleukin-1b From the Activated Macrophages
To further investigate the changes of Pdpn-KO and Pdpn-TG cells at the molecular level, mRNA transcripts were analyzed  using a NanoString nCounter ® PanCancer Pathways panel that included genes in immune-oncology, innate, and adaptive immune responses. Prior to the assay, the expression levels of Pdpn among the parental WT, Pdpn-KO, and Pdpn-TG cells were verified before and 24 h after H. pylori infection using RT-PCR ( Figure 4A). For WT cells, Pdpn expression was low in resting cells, and increased at approximately 600-fold after H. pylori infection. As expected, no Pdpn expression was observed in KO cells, while strong expression of >50,000-fold was detected in Pdpn-TG cells regardless of infection status.
In general, wild-type, Pdpn-KO, and Pdpn-TG cells displayed a highly homogeneous gene expression profile that altered comparably upon H. pylori infection, as depicted by the heatmap (Figure 4B), suggesting minimal influence of Pdpn disruption on the transcriptional programming of the macrophages. Among the top 10 upregulated genes following H. pylori infection in wild-type cells were cytokines for myeloid differentiation Colony stimulating factor 2 and 3 (Csf2, Csf3), proinflammatory cytokines interleukin-1b (Il1b) and Il6, chemokine C-X-C ligand 2 (Cxcl2), as well as signaling molecules Suppressor of cytokine signaling 3 (Socs3) and Linker of T cell activation (Lat) (Supplementary Figure S4A, red dot cluster), indicating a robust immune response in the macrophages following H. pylori infection. A similar pattern is observed in KO and TG groups following infection (Supplementary Figures S4B, C). Venn diagrams also depict most of the differentially expressed genes (log 2 fold change > 2 or <−2) following infection for KO and TG groups, which were in common to that of WT ( Figure 4C).
Despite similar expression changes across all cells following infection, we noted that Il1b was missing from the list of top 10 upregulated genes of the activated Pdpn-KO cells (Supplementary Figure S4B, red dot cluster), but retained in Pdpn-TG cells (Supplementary Figure S4C, red dot cluster). Normalized log 2 fold changes highlighted expression of Il1b and Il1a in sharp "V" trendlines, reflecting lower expressions in activated Pdpn-KO compared to both WT and Pdpn-TG cells ( Figure 4D, red and blue lines). Other genes, for example, Tumor necrosis factor (Tnf) exhibited rather equivalent level of fold changes across three comparison groups ( Figure 4D, green line).
The mRNA transcripts of Il1b were undetectable (<10 counts) in resting macrophages but were drastically increased to 684.8 ± 110.7 and 616.6 ± 182.3 counts in the activated wild-type and Pdpn-TG cells, respectively ( Figure 4E). However, in Pdpn-KO cells, H. pylori-mediated expression was merely 93.4 ± 27.8 counts, approximately sevenfold lower than that of wild-type and Pdpn-TG cells. Similarly, transcription of Il1a was greatly augmented to 66.5 ± 11.0 and 119.1 ± 11.4 counts in wild-type and Pdpn-TG cells post-infection ( Figure 4F), but not in Pdpn-KO cells. In contrast, no drastic changes were observed in the transcript levels of tumor necrosis factor (Tnf) across all cells post-infection (Supplementary Figure S5A). Production of pro-IL-1b and TNFa was verified by using ELISA (Figure 4G,  Supplementary Figure S5B). Concentration of pro-IL-1b in cell lysate increased at two-to threefold in the activated WT and Pdpn-TG cells, while no such increase was detected in Pdpn-KO cells, in accordance with the mRNA transcript level ( Figure 4G).

Pdpn Regulates the Transcriptional Expression of Filamin C Actin-Binding Protein
We then compared, in various pairwise combinations, the transcriptional outputs of knockout, transgenic, and wild-type cells in uninfected and H. pylori-infected contexts (Figures 5A-D). We were intrigued by the expression of a particular gene, Filamin C (Flnc), which was consistently greater in the high Pdpn-expressing cells compared to low/no Pdpn-expressing cells before (Figures 5A, B) or after H. pylori infection (Figures 5C, D). Filamin C encodes for an actin-binding protein essential for muscle movement (41) and is involved in actin cytoskeleton modulation (42). Given its actin regulation function, it is plausible that Filamin C is involved in macrophage migration via a Pdpn-dependent pathway. Comparison of normalized transcript counts showed a low expression of Filamin C in the absence of Pdpn ( Figure 5E). Prior to infection, Ubiquitously transcribed tetratricopeptide repeat containing, Y-linked (Uty) was also consistently greater in the high Pdpnexpressing cells compared to low/no Pdpn-expressing cells before ( Figures 5A, B) or after H. pylori infection. Uty encodes for a histone demethylase enzyme although its significance in macrophage migration remains to be identified (43).

DISCUSSION
The present study reports that the Pdpn gene that encodes for a transmembrane molecule was significantly upregulated in macrophages ensuing H. pylori infections. In addition to H. pylori, stimulation by LPS or other gram-negative bacteria infection were found sufficient to trigger Pdpn expression in macrophages, as reported in the current ( Figure 1) and previous studies (24). Pdpn can also be induced by TLR1/2 and TLR2/6 agonists in addition to LPS (24); however, it is still unclear if other H. pylori virulent factors such as CagA can trigger its expression. Using the Pdpn-knockout cell model generated in the present study, we discovered that phagocytosis and bactericidal activities were not perturbed. Although a previous study reports that a subset of Pdpn-expressing macrophages are highly phagocytic (26), our data using knockout cell model rectify that there is no direct role of Pdpn in phagocytosis activity. (G) Bar chart shows ELISA analysis of pro-IL-1b production in cell lysate. y-axis represents cytokine concentrations in pg/ ml. Data were shown as mean ± SD of duplicates obtained from representative of two independent experiments. Statistical significance by unpaired Student's t-test (**p < 0.01). Rather, we anticipate that high phagocytic activity in Pdpnexpressing cells is a consequence of aggressive cell movement directed towards the bacteria, as cell migratory activity was significantly attenuated in Pdpn-disrupted cells. Furthermore, we also report impeded filopodia protrusion following Pdpn modes of actin polymerization assembly. For instance, depletion of capping protein results in loss of lamellipodia but extensive filopodia protrusion, whereas Ena/VASP deficiency resulted in membrane ruffling without filopodia (45). Mechanistically, our findings highlight that the defective cellular migration in the absence of Pdpn is attributable to a consequence of altered Filamin C expression. We anticipate that Pdpn may promote Filamin C transactivation through activating RhoGTPase signaling pathway, but further investigation is required to confirm this notion (10). This study adds novel knowledge to the mechanism and function of Pdpn, on top of the previous reports that credited its role predominantly to its extracellular interaction with Clec2 (13).
As implicated in the migration and would healing assays, motility was significantly impaired in Pdpn-KO and Pdpn-KD/ TC cells as a result of diminished filopodia elongation. Filopodia formation is important to control directional cell movement. The involvement of Pdpn in migration has been established using non-immune cells such as mouse embryonic fibroblasts and cancer cells (8,(46)(47)(48). Our findings extend its role in regulating migration of immune cells. Interestingly, a previous study that relates Pdpn to dendritic cells motility suggests a model that is close to cancer metastasis, in which binding of Clec2 on dendritic cells to Pdpn on the stroma network activates actin rearrangement and promotes movement of dendritic cell in the lymphatic system (17). Since in vitro knockout setting was utilized in the current study, we suggest that Pdpn regulating macrophage migration is independent of external interaction with Clec2. Rather, our findings highlight an association between Pdpn with Filamin C, a member of actin-crosslinking protein family that stabilizes and links actin web to the cell membrane (49). Patients with mutated Filamin C gene demonstrate high incidence of cardiomyopathy (50). Filamin C maintains the structural integrity of skeletal and cardiac muscles by interacting with the sarcomeric Z-disc (41,51). Given that Pdpn is indispensable in heart development, we anticipate that Filamin C activity in cardiomyocytes is potentially dependent on Pdpn signaling, but further analysis is required to validate this notion. Notably, H. pylori infection promotes aggressive metastasis of gastric epithelial cells by downregulating Filamin C level (52). Filamin A, another family member of filamin, has been implicated in Rac1/Cdc42-mediated monocyte migration, but the role of Filamin C remains understudied (53). Our findings warrant further study to clarify its link with Pdpn during immune cell migration.
H. pylori is a class I carcinogen that causes gastric adenocarcinoma through prolonged colonization, triggering chronic inflammation and destruction of gastric epithelial layer, which can eventually lead to chronic gastritis and gastric adenocarcinoma in humans. The current study suggests a correlation of Pdpn expression with the degree of H. pylori infection-mediated inflammation, through regulation of IL-1b proinflammatory cytokine. Production of IL-1b was evidently impaired in Pdpn-KO while enhanced in Pdpn-TG cells, as outlined in Figure 4. Increased IL-1b production aggravates development of gastric cancer by repressing secretion of gastric acid and orchestrating inflammatory response (54). Hence, we anticipate that a Pdpn inhibitor could be applied to alleviate inflammation and cancer in H. pylori-infected patients. On the other hand, Pdpn-Clec2 axis between macrophages and platelets is responsible for the local inflammatory response in either the peritoneum or liver during E. coli infection (30,55) as well as in acetaminophen induced acute liver injury (56). Pdpn is also expressed by primary microglial cells during inflammation in traumatic brain injury (57). Our results acquired through examination of Pdpn-knockout and Pdpn-transgenic models provide evidence of Pdpn in controlling inflammatory diseases as its presence is essential for IL-1b cytokine expression and secretion.
Collectively, our findings suggest that Pdpn is crucial in regulating actin filament reorganization to enhance cell motility and filopodia extension, by altering Filamin C protein level. This study is limited by the employment of an in vitro infection model that may not accurately reflect the true complexity of host-pathogen interaction in an in vivo infection scenario. Nevertheless, this study opens up avenues for further research into clarifying the downstream mechanisms by which Pdpn modulates macrophage biology and suggests that Pdpn blockade may provide a new approach to relieve H. pyloriassociated chronic inflammation.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.