Macrophages play a crucial role in detecting, regulating, and resolving immune crises, requiring migration through complex extracellular matrices. Unwarranted macrophage inflammatory activity potentiates kidney disease, rheumatoid arthritis, and transplant rejection. Proper remodeling of the actin cytoskeleton, especially at adhesion structures, is essential to the translocation of macrophages. Macrophages form actin-rich adhesions termed “podosomes”, giving them the capacity to make contacts with the substratum for traction through interstitial tissues. Macrophages express multiple formins, including FMNL1, Dia1, and Fhod1, with potential to impact actin remodeling involved in migration. Formins are a family of proteins that are best known for modifying the actin cytoskeleton via nucleation, elongation, bundling, and/or severing actin filaments. In this study we demonstrate that the formin FMNL1 is a key regulator of podosomes and is required for normal macrophage migration. Additionally, this is the first study to demonstrate defects in primary human cell migration resulting from specific formin silencing. Pharmacologic inhibition of all formin activity results in a significant decrease in podosome formation and normal macrophage migration. Furthermore, targeted suppression of FMNL1 results in decreases in macrophage migration similar to inhibition of all expressed macrophage formins. These novel findings suggest FMNL1 as a possible chemotherapeutic target to hinder macrophage migration, which could offer an innovative method for limiting unnecessary macrophage-mediated inflammation. We hypothesize that formins are required in podosome actin dynamics to support macrophage migration.
Macrophages form actin-dense adhesion structures termed “podosomes”. These contain a dense pillar-like actin core that is tethered to the cortical actin cytoskeleton with radial actin filaments [
Formins are actin-binding proteins that have the capacity for nucleating, polymerizing, capping, severing and/ or bundling actin [
Previous studies in our lab have identified that the formin FMNL1 (formin-like 1) is associated with macrophage podosomes and is localized to the apex of the pillar-shaped dense actin core [
We have previously reported that FMNL1 is associated with macrophage podosomes [
Except were noted, all culture reagents were from Invitrogen (Carlsbad, CA, USA) and all plasticware from Corning Incorporated (Corning, NY, USA).
Peripheral blood was drawn into a 60 ml syringe containing 7.5 ml of 6% Dextran (Pharmacosmos, Holbaek, Denmark) in HBSS− for red blood cell sedimentation, and 5.625 ml of 96 mM sodium citrate (Sigma-Aldrich, St. Louis, MO, US) in HBSS− as an anti-coagulant. The blood was then transferred to 50 ml conical tubes for red blood cell sedimentation for 30 minutes at room temperature. Plasma and white blood cells were removed and placed on top of Ficoll-Paque PLUS (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in a 50 ml conical tube and centrifuged at 3000 g at 4˚C for 30 minutes. The buffy coat layer was removed and washed in HBSS− at 1000 g at 4˚C for 10 minutes. Monocyte pellets were resuspended in complete media (RPMI-1640 containing 20% FBS (Gemini Bio-Products, West Sacremento , CA , USA ), 2 mM glutamax, 50 μg/ml gentamicin (Sigma-Aldrich, St. Louis , MO , USA )), and induced to differentiate with 125 pg/ml GM-CSF (Berlex, Seattle , WA , USA ). Monocytes were cultured in complete media and allowed to differentiate into macrophages at 37˚C and 5% CO2. Macrophages were used between day 5 and day 10 of culture.
Adherent macrophages were washed quickly using ice cold 10 mM EDTA (Sigma-Aldrich, St. Louis, MO, USA) in HBSS−, and then 5 ml of ice cold 10 mM EDTA in HBSS− was added to the flask and placed at 4˚C for approximately 10 minutes. The bottom of the flask was smacked to help break adhesion of macrophages, and macrophages were gently detached from the flask using a disposable cell scraper. Macrophages were removed from flask and transferred to a 50 ml conical tube and washed twice with complete media. Macrophages were resuspended in 5 ml complete media and counted using a hemocytometer (Reichert, Buffalo , NY , USA ). Macrophages were plated in 24 well plates containing glass coverslips (Fisher Scientific, Pittsburg , PA , USA ) at a density of 120,000 cells/ well. Macrophages were allowed to adhere to the glass coverslips overnight. Small molecule inhibitor of the FH2 domain (SMIFH2) (Sigma-Aldrich, St. Louis, MO, USA) inhibitor was diluted into multiple concentrations in complete media, including 5 μM, 10 μM, 20 μM, 30 μM, and DMSO as a vehicle control (Sigma-Aldrich, St. Louis, MO, USA). Media was aspirated from wells containing macrophages and replaced with regular complete media and treatment medias in duplicate at 10 minute intervals to allow counting at each treatment condition. Adhered macrophages were counted at the center of each coverslip using a 20× objective at 0, 15, 30, 60, and 120 minutes for each treatment. Student’s t-test was performed for statistical analysis.
To determine viability, in parallel samples, media was aspirated from wells containing macrophages and replaced with regular complete media and treatment medias. Plates were placed back in incubator for 16 hours, the timespan used for migration assays. After the 16 hour time point, media was collected for each treatment and adhered macrophages were lifted and collected as previously described and added to collected media. Macrophage viability was performed for each treatment using trypan exclusion (Sigma-Aldrich, St. Louis , MO , USA ).
Macrophages were prepared as previously described. SMIFH2 inhibitor was diluted in complete media to a concentration of 30 μM with DMSO as a vehicle control. Media was aspirated from wells containing macrophages and replaced with regular complete media and treatment medias. Plates were placed back in incubator for two hours. Macrophages were then fixed using 3.7% ice cold formaldehyde (Fisher Scientific, Fair Lawn, NJ, USA) for 1 hour at 4˚C followed by permeabilization using ice cold 0.002% NP-40 (BMD Biosciences Inc, La Jolla, CA, USA) in PBS for 10 seconds. Macrophages were then treated with blocking buffer in PBS containing 0.01% Triton X-100 (Fisher Scientific, Pittsburg, PA, USA) and 3% goat serum (Invitrogen, Carlsbad, CA, USA) in PBS for 30 minutes at room temperature. Macrophages were stained with rhodamine phalloidin (Cytoskeleton, Denver, CO, USA) at [1:10,000] in PBS for 30 minutes, washed with PBS five times, and the coverslips were then mounted on glass slides (Globe Scientific, Paramus, NJ, USA) using an anti-fading solution. Using a Nikon Eclipse E800 fluorescent microscope (Nikon, Melville, NY, USA) equipped with a Hamamatsu ORCA-ER digital camera (Bridgewater, NJ, USA) with NIS-Elements software, macrophages were imaged, analyzed, and quantified for either exhibiting at least five podosomes per cell (actin-dense structures with a diameter of approximately 0.5 μm as described by Mersich et al. 2010, or having less than five podosomes per cell. Student’s t-test was performed for statistical analysis.
Macrophages were prepared as previously described. Macrophages were removed from flasks and transferred to a 50 ml conical tube and washed twice with RPMI with gentamicin. Macrophages were resuspended in 1 ml RPMI with gentamicin and counted using a hemocytometer. Transwell permeable polycarbonate support inserts with 5.0 μm pores were placed into wells of a 24 well plate. 600 μl of complete media was added into the lower chamber. The upper chamber was loaded with 175,000 macrophages resuspended in RPMI with gentamicin and either SMIFH2 inhibitor, DMSO, or nothing to a final volume of 100 μl. The migrations assays were placed in the incubator for 16 hours. After which, the media was removed from the upper well of the inserts and the membranes were fixed in 3.7% formaldehyde in PBS at 4˚C for 1 hour. The inserts were allowed to air dry for 1 hour. Polycarbonate membranes were stained using a Diff-Quick staining kit (IMEB Inc., San Marcos, CA, USA) following the manufacturer’s protocol. The inserts were then placed upside down and allowed to dry overnight. Membranes were cut from the transwell inserts and placed upside down on glass slides, covered with a drop of immersion oil (Cargille Laboratories, Cedar Grove, NJ, USA), and covered with a coverslip. Microscopy on the membranes was performed using light microscopy with a 40× objective and five different fields-of- view were counted for each membrane. Student’s t-test was performed for statistical analysis.
Macrophages were transduced with small interference RNA molecules targeting FMNL1 (Ambion, Inc, Austin, TX, USA) using the INTERFERin transduction reagent (PolyPlus Transfection, New York, NY, USA). Three unique target sequences were used for FMNL1 along with a scramble oligo for control. Macrophages prepared as above following 5 - 10 days of differentiation were seeded 1 day in advance of siRNA transduction in T25 flasks at ~2.0 × 106 macrophages in 5 ml complete media. On the day of transduction, complexes of siRNA and INTERFERin were prepared in 400 ul of serum-free media following the manufacturer’s protocol. A concentration of 600 nM siRNA in 4 ml of media was used to replace the media in the T25 flask. Macrophages were cultured with siRNA for 72 hours and harvested as described above for Western blot analysis or migration assays as previously described. For Western blot analysis, 150 μg of each macrophage lysate was prepared with sample buffer, loaded into a 10% SDS polyacrylamide gel, and resolved using electrophoresis. Separated proteins were transferred from the gel to a PVDF membrane (Millipore, Billerica, MA, USA), blocked with 3% BSA (Lampire Biological Laboratories, Pipersville, PA, USA) in TBS (Fisher Scientific, Fair Lawn, NJ, USA) buffer, and then probed for FMNL1 using mouse monoclonal 2369E4a (Santa Cruz, CA, USA) and transaldolase goat polyclonal T-20 (Santa Cruz, CA, USA) for a loading control. Student’s t-test was performed for statistical analysis.
We have previously reported that FMNL1 was required for normal podosome dynamics in macrophages [
Previous studies in our lab have shown that the formin FMNL1 plays an important role in macrophage adhesion and podosome stability [
following inhibition of all formins (
Human macrophages were plated on glass coverslips at identical densities and treated with 30 μM SMIFH2 or DMSO. After 2 hours of inhibitor treatment, macrophages were fixed, permeabilized, and stained with rhodamine phalloidin for actin visualization to quantitate podosomes. Podosomes were identified by staining for associated proteins including talin, vinculin, and paxillin as previously described [
To determine if inhibition of formin function using SMIFH2 affects the stabilization of macrophage adhesion, a dose and time dependent assay was performed. In this study, we replaced media in wells containing glass coverslip-adhered macrophages at identical density with media containing varying concentrations of SMIFH2. Ma
crophage de-adhesion was assessed after 15, 30, 60, and 120 minutes and in a dose dependent manner by SMIFH2. We determined that SMIFH2 at 30 μM reduced adherent macrophages by >50% within 120 minutes of treatment with no effect on cell viability, as shown in
Macrophages are crucial for appropriate immune responses to combat the invasion of pathogens and promoting wound healing. For macrophages to reach sites of inflammation and tissue damage, they must have the capacity to firmly adhere and migrate through complex extracellular matrices. This macrophage targeting mechanism is reliant on the proper formation and functioning of podosomes. Studies performed on Wiskott-Aldrich syndrome (WASP) models have reported that the lack of the protein WASp causes a loss of podosome formation [
Previous studies in our lab have indicated that FMNL1 is an indispensible protein for normal macrophage podosome dynamics. These findings were obtained via knockdown of FMNL1 protein expression and analysis of cell adhesion using light microscopy [
We demonstrated that SMIFH2 treatment of macrophages significantly reduces their macrophages capacity to migrate across a porous barrier. These data indicate that formins play a critical role in cellular changes during migration. However, this observation could result from an array of formin associated events including actin cytoskeleton remodeling, signaling, or even adapter protein functions. It remains unclear which actin regulating
events most formins may be performing in different cell types at any given time. Another interesting aspect of this data is that macrophage migration was not inhibited completely. This suggests some aspects of macrophage migration use podosomes, but additional mechanisms must exist, because all podosomes are lost by formin inhibition with 30 μM SMIFH2, but only 50% of macrophage migration is inhibited. This may mean that formins are important for optimal migration, or alternatively, formins may be important for a certain mode of migration such as mesenchymal or amoeboid [
We have previously reported that multiple formins, including Dia1, Dia3, FHOD1, FMNL2, and FMNL1, are expressed at high mRNA levels in human macrophages [
In our study we observed a significant decrease in macrophage migration across a porous barrier when FMNL1 protein levels were knocked down via siRNA. This observation ties together our previous observations that reduction of FMNL1 expression causes a loss in podosome stability and formation. Another interesting observation was that the reduction in macrophage migration using the formin inhibitor at 30 μM was 34.6%, and the average of the three siRNA targeting oligos was 42.1%. This suggests that FMNL1 is more than likely the largest contributing formin to macrophage migration. Since we have shown FMNL1 to localize to podosomes, and when it is knocked down podosomes are lost, we have further verified the necessity not only for FMNL1 in macrophage migration, but also for normal podosome dynamics. In accordance with our previous studies using siRNA knockdown of formins, we also observed that pharmacological manipulation of formins similarly effects podosome stability and adhesion in macrophages. It was interesting to observe that macrophage adhesion and podosome stability were affected by formin inhibition and siRNA-mediated knockdown of FMNL1 similarly. This is the first study to demonstrate changes in primary human cell migration caused by specific silencing of an individual formin. These exciting findings delineate the necessity of formins in macrophage migration and highlight FMNL1 as an important formin in this process.
As this research has shed some light on the requirement of formins in migration, many other questions remain to be investigated. It is still unclear how FMNL1 is recruited to macrophage podosomes. It has been suggested that spatial and temporal regulation of FMNL1 is controlled by small Rho GTPases, including Rac1, and CDC42 [
Our observations support FMNL1 as a target for chemotherapeutic intervention to inhibit macrophage migration. The work presented in this paper strongly suggests that the formin FMNL1 plays a prominent role in this immune process. Further studies on FMNL1 in this process may pave the way to understanding and attenuating macrophage mediated diseases such as atherosclerosis and rheumatoid arthritis.
This work was supported by NIH DK79884 to S.D.B.
The authors declare no conflict of interest.