CFTR dictates monocyte adhesion by facilitating integrin clustering but not activation

Significance

This study changes our understanding of how integrin contributes to cell adhesion by underlining the importance of integrin clustering and the independence between integrin clustering and activation. Using superresolution microscopy, we provide evidence that G protein–coupled receptor–triggered inside-out signaling can induce integrin clustering. We showed that cystic fibrosis transmembrane conductance regulator is required for monocyte adhesion and integrin clustering but not integrin activation, which separates integrin activation and clustering, challenging the long-standing paradigm that attributes cell adhesion to integrin activation.

Abstract

Monocytes are critical in controlling tissue infections and inflammation. Monocyte dysfunction contributes to the inflammatory pathogenesis of cystic fibrosis (CF) caused by CF transmembrane conductance regulator (CFTR) mutations, making CF a clinically relevant disease model for studying the contribution of monocytes to inflammation. Although CF monocytes exhibited adhesion defects, the precise mechanism is unclear. Herein, superresolution microscopy showed that an integrin clustering but not an integrin activation defect determines the adhesion defect in CFTR-deficient monocytes, challenging the existing paradigm emphasizing an integrin activation defect in CF patient monocytes. We further found that the clustering defect is accompanied by defects in CORO1A membrane recruitment, actin cortex formation, and CORO1A engagement with integrins. Complementing canonical studies of leukocyte adhesion focusing on integrin activation, we highlight the importance of integrin clustering in cell adhesion and report that integrin clustering and activation are distinctly regulated, warranting further investigation for selective targeting in therapeutic strategy design involving leukocyte-dependent inflammation.

Cystic fibrosis (CF) is a progressive life-limiting genetic disease affecting >70,000 people worldwide, with major complications including lung infections and destructive inflammation of multiple organs (1). CF is caused by mutations in a CF transmembrane conductance regulator (CFTR) gene, which encodes a transmembrane chloride ion channel (1). Dysregulation of CFTR results in the dysfunction of anion transport and, in turn, airway dehydration, mucus retention, and chronic infection, eventually leading to airway inflammation and CF (1). The CFTR protein is also functional in monocytes and macrophages, compromising the innate immune response and bacterial phagocytosis in patients with CF (2, 3).

Monocytes are critical for host defense and the regulation of inflammation (4), wherein they directly secrete cytokines (5) and kill pathogens by phagocytosis (4). Monocytes can also differentiate into macrophages (6), which clear apoptotic debris, secrete cytokines for tissue homeostasis, and surveil the environment (7). On the other hand, monocyte dysregulation or hyperactivation is involved in multiple infectious and inflammatory diseases, such as CF (8), diabetes mellitus (9), dyslipidemia (9), atherosclerosis (10), heart failure (11), chronic kidney disease (12), multiple sclerosis (13), inflammatory bowel disease (14), and hepatitis (15). Monocyte-mediated immune protection begins with the recruitment of monocytes from the bloodstream, which proceeds with monocyte rolling along the endothelium and then transendothelial migration in a cascade-like fashion (15, 16). This process involves a key step of movement conversion from rolling to firm adhesion, or “arrest,” which is mediated by β₂ integrins (17, 18).

Integrins are heterodimeric adhesion molecules that regulate adhesiveness to ligands by conformational changes in their extracellular domain during a process called integrin “activation” (17–22), which is initiated by chemokine-induced integrin inside-out signaling (18, 23, 24). The fully activated β₂ integrins bind intercellular adhesion molecules (ICAMs) to support firm adhesion of monocytes. While the activation increases the affinity of integrins to ligands, “clustering” of integrins increases their avidity of binding interactions where cooperative strength supports larger forces compared to sporadic integrins (25–27). Both β₂ integrin activation (18–20) and clustering (25–27) are believed to be essential for monocyte arrest on vascular endothelium, wherein the two processes were speculated to be interlinked. However, integrin clustering upon inside-out signaling stimulation is extremely understudied. This is because most studies on integrin clustering are based on adhered and spreading cells (28–30), which involved integrin outside-in signaling triggered by integrin–ligand binding that only occurs after monocytes are already arrested on vascular endothelium (31). Whereas, during the physiological process of monocyte arrest, the β₂ integrins are primed only by inside-out signaling triggered by chemokine binding to G protein-coupled receptors (GPCRs). Integrin clustering induced by inside-out and outside-in signaling may have different molecular mechanisms. Thus, the regulatory mechanism of integrin clustering during inside-out signaling and its relationship with integrin activation are still unclear. In this study, we established a method based on superresolution stochastic optical reconstruction microscopy (STORM) imaging to overcome the technical challenge of assessing integrin clustering solely upon inside-out signaling and identified that the “activation” and “clustering” of β₂ integrins are discrete consequences of inside-out signaling, providing unique insights into the regulation of monocyte arrest and the treatment of CF targeting leukocyte recruitment.

Previous work showed that monocytes from CF patients exhibit deficiency in static adhesion with a partial defect in chemoattractant-induced β₁ and β₂ integrin activation (32). However, most CF patients have chronic infections and multiorgan inflammation. The systematic inflammation may cause monocyte tolerance in integrin activation. Thus, whether the integrin activation defect in patient samples is due to chronic infection-induced monocyte tolerance or a CFTR-dependent cell-intrinsic mechanism is unknown. Although the study tried to resolve this concern by showing that a CFTR-specific pharmacological inhibitor, CFTR inhibitor-172 (inh-172), weakened the static adhesion of healthy monocytes (32), whether this CFTR inh-172 impairs integrin activation in healthy monocytes was not tested. To fill this gap, we tested this and unexpectedly found that CFTR inhibition does not affect β₂ integrin activation. Instead, we observed a defect in β₂ integrin clustering on monocytes after CFTR inh-172 treatment. The unaffected integrin activation and deficient integrin clustering were also confirmed using monocytes isolated from CF mice. These results rewrite the canonical mechanism of how CF causes adhesion deficiency in monocytes. Our results also provided a set of evidence that integrin-clustering and activation are two parallel processes contributing to integrin-dependent monocyte adhesion, challenging the existing model that integrin-activation is the primary contributor in this context. Finally, we investigated the potential regulating mechanism and identified that CORO1A and the F-actin cytoskeleton might be critical for the β₂ integrin clustering-dependent adhesion defect in CF monocytes.

Results

CFTR Inhibition Reduces Monocyte Adhesion in Microfluidics.

To mimic monocyte adhesion on vascular endothelium, we used the microfluidic assay (20, 33) to determine the effect of CFTR inhibition on monocyte adhesion under shear flow. Purified blood monocytes from healthy donors were preincubated with CFTR inh-172, a specific inhibitor of CFTR ion channel activity (34), or dimethyl sulfoxide (DMSO) as vehicle control. Monocytes were then perfused over a microfluidic chamber cofunctionalized with P-selectin and ICAM-1 (Fig. 1A), which are both natively expressed on vascular endothelium, respectively mediating leukocyte rolling (via binding to P-selectin glycoprotein ligand-1) (35) and adhesion (via binding to β₂ integrins) (36). Under a shear stress of 6 dyn cm⁻², mimicking the shear stress in postcapillary venules during leukocyte recruitment into tissues during inflammatory processes (37), the monocytes rolled on P-selectin and ICAM-1 as expected (Fig. 1 B, Upper). Upon stimulation with human C–C motif ligand 2 (CCL2), a monocyte-specific chemokine that triggers β₂ integrin inside-out signaling, monocytes pretreated with vehicle control reduced their 60-s rolling distance from ~144 μm (Fig. 1 B, Upper Left) to ~17 μm (Fig. 1 B, Lower Left and Movie S1). In comparison, the 60-s rolling distance of monocytes pretreated with 10 μM CFTR inh-172 (CF10) was still considerably long (~77 μm) after CCL2 stimulation (Fig. 1 B, Lower Middle), whereas that of monocytes pretreated with 50 μM CFTR inh-172 (CF50) became indifferent to CCL2 stimulation (Fig. 1 B, Upper and Lower Right and Movie S2). Analyzing the rolling velocity of 60 individual monocytes showed that CCL2 slowed down monocyte rolling from ~2.6 to 1.8 μm s⁻¹ in vehicle control-treated monocytes but failed to do so in CF10- or CF50-treated monocytes (Fig. 1 C and D). The number of arrested monocytes after CCL2 stimulation in the vehicle control group (~200 per field-of-view) was also significantly higher than that in the CF10 or CF50 groups (~90 or ~50 per field-of-view, respectively) (Fig. 1E). By further categorizing the arrested monocytes into round and spreading shapes, it was found that CF10 or CF50 treatment significantly decreased the number of round cells from ~170 to ~80 or ~40 cells per field-of-view, respectively (Fig. 1F), and decreased the number of spreading cells from ~30 to ~10 cells per field-of-view (Fig. 1G). Considering the possible nonspecific effects of CFTR inh-172, we first assessed monocyte viability before and after CF10, CF50, or vehicle treatment and found that CFTR inh-172 treatment did not affect monocyte viability (SI Appendix, Fig. S1A). We also assessed whether CFTR inh-172 limited other ion channel functions, such as Ca²⁺ (38) or Na²⁺ (38) channels. We found that our CFTR inh-172 treatment did not affect monocyte intracellular Ca²⁺ or Na⁺ (SI Appendix, Fig. S1 B and C), regardless of CCL2 stimulation or not. These results show that CFTR inhibition suppresses β₂ integrin-dependent adhesion of human blood monocytes in a condition that mimics physiological flow.

Fig. 1.

CFTR inhibition causes an adhesion defect in human blood monocytes. Purified human blood monocytes incubated with 10 (CF10) or 50 μM CFTR Inhibitor-172 (CF50) or vehicle control were perfused over a P-selectin and ICAM-1 cofunctionalized surface without (CT) or with CCL2 stimulation under a shear stress of 6 dyn cm⁻². (A) Schematic diagram of the flow chamber. (B) The tracks of monocytes within 1-min records. n ≥ 23 cells from three individual experiments. (C and D) Cumulative frequency (C) and boxplots (D, median, 25 to 75% range box, and 0 to 100% range bars) of monocyte rolling velocity. n = 60 cells from three individual experiments. (E–G) Number of total (E), round (F), and spreading (G) arrested monocytes. Boxplots, n = 15 fields-of-view from three individual experiments. **P < 0.01; ****P < 0.0001, P-values were shown by the Mann–Whitney U test.

CFTR Is Dispensable for β₂ Integrin Activation on Monocytes.

To define the mechanism of the adhesion defect observed above, we first examined β₂ integrin activation on CFTR-deficient monocytes. The activation of β₂ integrins involves two major conformational changes in their extracellular domain—extension (E⁺), which allows ligand binding in trans, and headpiece opening (H⁺), which allows integrins to acquire a high affinity for ligand binding (39, 40). These conformational changes can be monitored by conformation-specific antibodies KIM127 (reporting E⁺) and mAb24 (reporting H⁺), respectively (41–43). Although a previous study (32) showed that monocytes from CF patients have defects in inducing H⁺ and E⁺ β₂ integrins, whether the defect is cell-intrinsic CFTR-dependent or a cell-extrinsic tolerance effect due to chronic infections in patients is unknown. Our study used CFTRΔF508 mice (44), which carry the most common CFTR mutation in CF patients (45) but are not susceptible to spontaneous lung infections. Since mAb24 and KIM127 are specific to human β₂ integrins, we introduced human β₂ integrin knock-in mice (46) and bred them with CFTRΔF508 mice to generate human β₂ integrin-expressing CF (hItgb2^KI/KICFTR^{ΔF508/ΔF508}) and control (CT, hItgb2^KI/KICFTR^WT/WT) mice. Peripheral blood samples were obtained from these mice, and β₂ integrin activation was assessed with or without mouse CCL2 stimulation. Different from the previous study using patient monocytes (32), we found that CCL2 increased the binding of mAb24 (Fig. 2A) and KIM127 (Fig. 2B) to both CF and CT blood monocytes, with no statistically significant difference between the two groups.

Fig. 2.

CFTR deficiency does not affect β₂ integrin activation in mouse and human blood monocytes. (A and B) Human β₂ integrin knock-in mice were crossed with CFTRΔF508 mice to generate cystic fibrosis (CF, hItgb2^KI/KICFTR^{ΔF508/ΔF508}) and control (CT, hItgb2^KI/KICFTR^WT/WT) mice. Peripheral blood samples were incubated with CCL2 (100 ng mL⁻¹) or vehicle control, and β₂ integrin activation on monocytes (CD115+) was quantified by flow cytometry using conformation-specific antibodies mAb24 (A) and KIM127 (B). Means ± SEM, n = 7 individual experiments. (C and D) Purified human blood monocytes incubated with 10 (CF10) or 50 μM CFTR Inhibitor-172 (CF50) or vehicle control. β₂ integrin activation on monocytes stimulated with CCL2 (100 ng mL⁻¹) or vehicle control was quantified by flow cytometry using conformation-specific antibodies mAb24 (C) and KIM127 (D). Means ± SEM, n = 3 individual experiments. (E and F) Purified human blood monocytes incubated with 50 μM CFTR Inhibitor-172 (CF50) or vehicle control. The number of mAb24 (E) and KIM127 (F) localizations per cell after CCL2 (100 ng mL⁻¹) stimulation were quantified by STORM imaging. Boxplots (median, 25 to 75% range box, and 0 to 100% range bars), n = 20 cells from three individual experiments. MFI, median fluorescence intensity. ns, not significant (P > 0.05) by the Mann–Whitney U test.

We also tested β₂ integrin activation on purified human blood monocytes incubated with CF10, CF50, or DMSO vehicle control (Fig. 2 C and D). Similarly, we observed comparable increases of mAb24 (Fig. 2C) and KIM127 (Fig. 2D) binding after human CCL2 stimulation in all groups, demonstrating that CF10 and CF50 treatment does not affect β₂ integrin activation in human monocytes. Consistent results were also seen in our STORM imaging of CCL2-stimulated human monocytes, where the localization numbers of mAb24 (Fig. 2E) and KIM127 (Fig. 2F) were comparable between CF50 and vehicle control group. These results suggested that CFTR deficiency does not affect β₂ integrin activation in monocytes, which challenges conventional wisdom (32).

β₂ Integrin Clustering Defect in CFTR-Deficient Monocytes.

The above results exclude the possibility that the β₂ integrin-dependent adhesion defects observed in CF mouse monocytes (44) and human monocytes treated with CFTR inh-172 (Fig. 1) under physiological flow were due to defects in β₂ integrin activation. Thus, we proceeded to test another mechanism that affects the ligand binding capacity of β₂ integrins—clustering (26, 27, 47). Superresolution STORM can achieve nanoscale single molecular resolution (48) and quantify molecular clustering (49–51), which is, therefore, a suitable tool to quantify β₂ integrin clustering on monocytes. Monocytes isolated from wildtype (WT) or CFTRΔF508 (CF) mice bone marrow were stained with pan-CD18 (the β subunit of β₂ integrins) antibody, stimulated with CCL2 or phosphate-buffered saline (PBS) vehicle control in suspension, fixed, and imaged by STORM (Fig. 3A). It is important to note that different from previous clustering studies using adhered and spreading cells (28–30), monocytes in our assay were kept in suspension and prevented from nonspecific surface adhesion before fixation, ensuring that they only underwent CCL2-initiated integrin inside-out signaling but not ligand binding-initiated outside-in signaling. After analyzing STORM images using the Voronoi tessellation algorithm (50, 52), we found that CCL2 stimulation facilitated β₂ integrin clustering in WT monocytes (Fig. 3A and SI Appendix, Fig. S2A), with significantly increased cluster number (Fig. 3B) and β₂ integrin localizations per cluster (Fig. 3D). In contrast, CF monocytes showed no sensitivity to CCL2 stimulation (Fig. 3 A and D). There was no significant change in cluster density (Fig. 3C). Similar results were shown in the cluster analysis using density-based spatial clustering of applications with noise (DBSCAN, SI Appendix, Fig. S3 A–C).

Fig. 3.

CFTR-deficient mouse and human blood monocytes have β₂ integrin clustering defects. (A) Representative STORM whole-cell (Upper) and zoomed-in (Lower) images of β₂ integrin (CD18) clusters on the mouse monocyte surface (the contact area with the coverslip). Monocytes were isolated from WT or CF mouse bone marrow and stimulated with CCL2 (100 ng mL⁻¹) or vehicle control. (B) The number of clusters, (C) the average cluster density, and (D) the average cluster localization number of each cell in STORM images. n = 15 cells from three individual experiments. (E) Representative STORM whole-cell (Upper) and zoomed-in (Lower) images of activated β₂ integrin clusters on the cell surface (the contact area with the coverslip) of CCL2-stimulated purified human blood monocytes preincubated with 50 μM CFTR Inh-172 (CF50) or vehicle control. (F–K) The number of activated β₂ integrin clusters (F and I), average cluster density (G and J), and average cluster localization number of each cell (H and K) in STORM images. n = 20 (F–H) and 15 (I–K) cells from three individual experiments. (F–H) Activated β₂ integrins were labeled with conformation-specific antibodies mAb24 or (I–K) KIM127. The clusters were labeled with different colors to distinguish adjacent clusters in (A) and (E). Boxplots (median, 25 to 75% range boxes, 0 to 100% range bars) in (B–D and F–K). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by the Mann–Whitney U test. (Scale bars are 1 μm.)

To extend our finding, the above β₂ integrin clustering assay was then performed using human monocytes incubated with CF50 or DMSO vehicle control, with mAb24 and KIM127 antibodies used to label activated β₂ integrins, which should better reflect the ligand binding avidity of the integrins than staining pan-β₂ integrins. To ensure adequate staining of mAb24 and KIM127, all groups of cells were stimulated with CCL2. The localization numbers of mAb24 and KIM127 remained comparable between CFTR-inhibited and control monocytes (Fig. 2 E and F). However, similar to mouse monocyte data (Fig. 3 A–D), after analyzing images using the Voronoi tessellation algorithm, the clustering of both mAb24-labeled H⁺ and KIM127-labeled E⁺ β₂ integrins in CFTR-inhibited human monocytes was dramatically weaker than in the vehicle control-treated monocytes (Fig. 3E and SI Appendix, Fig. S2B), with much lower cluster number (Fig. 3 F and I), similar cluster density (Fig. 3 G and J), and less localizations per cluster (Fig. 3 H and K). Similar results were shown in the cluster analysis using DBSCAN (SI Appendix, Fig. S3 D–I). We also used K-Ripley analysis (53) to identify global clustering patterns (SI Appendix, Fig. S3 J and K). Both control and CF50 monocytes showed a positive histogram in Ripley’s H function, indicating the existence of integrin clustering. A top-right shift was observed in the CF50 histogram, indicating an increase in the distance between clusters, which is consistent with the above Voronoi tessellation and DBSCAN data showing the decrease in cluster number. The x-axis position of the histogram peaks indicates the cluster size. We observed a decrease from ~1,000 to 1,500 nm in control monocytes to ~100 nm in CF50 monocytes, indicating a decreased size of integrin clusters after CFTR inhibition, which is consistent with our observation in the Voronoi and DBSCAN data that CF50 monocytes have similar cluster density but a smaller number of localizations in each cluster compared to controls.

Variations in cell surface topography and the moving of integrins to microvilli (20) may be interpreted as clustering. To avoid this misinterpretation, we analyzed our three-dimensional STORM images at the cell’s contact surface with the coverslip and found that the z-position distribution of integrin localizations has no difference between CF and WT monocytes, regardless of CCL2 stimulation (SI Appendix, Fig. S4A), suggesting there might be no change in the cell surface topography nor integrins moving to microvilli.

Taken together, our results demonstrate that CFTR-deficient monocytes have defects in β₂ integrin clustering, which is a main contributor to the adhesion defect of monocytes under shear flow.

Limited CORO1A Recruitment to the Cell Membrane and Actin Cortex Formation in CFTR-Deficient Monocytes.

To further determine the potential molecular mechanism that contributes to the integrin clustering defect in CFTR-deficient monocytes, we examined CORO1A, an actin-binding protein that interacts with β₂ integrins (54), hence directly contributing to neutrophil adhesion. Using deconvolution-boosted high-resolution epifluorescent imaging, we found that CCL2 stimulation induced the membrane recruitment of CORO1A in monocytes from WT mice (Fig. 4A), increasing membrane-located CORO1A from ~25 to ~33% (Fig. 4E). However, this effect was not observed in the monocytes of CF mice (Fig. 4 A and E). We also performed a radial analysis of the monocytes and confirmed the membrane recruitment of CORO1A in WT but not CF monocytes after CCL2 stimulation (Fig. 4I). Notably, the total expression of CORO1A was not altered in CF mouse monocytes as confirmed by both western blot (SI Appendix, Fig. S5 E and F) and flow cytometry (SI Appendix, Fig. S5G).

Fig. 4.

CFTR-deficient mouse and human blood monocytes have defects in CORO1A membrane recruitment and actin cortex formation. (A–D) Representative epifluorescence transverse images of mouse (A and B) or human (C and D) monocytes stimulated by CCL2 (200 ng mL⁻¹ for mouse and 100 ng mL⁻¹ for human) or vehicle control showing CORO1A (A and C) or F-actin (phalloidin, B and D) localization. Mouse monocytes were isolated from WT or CF mouse bone marrow. Purified human blood monocytes were preincubated with 50 μM CFTR Inhibitor-172 (CF50) or vehicle control. Deconvolution was used to improve the resolution of epifluorescence images. Three representative images are shown in each group. (Scale bars are 5 μm.) (E–H) Quantifications of the mouse (E and F) or human (G and H) monocyte epifluorescence transverse images showing the percentage of CORO1A (E and G) or F-actin (F and H) localized to the cell membrane. Boxplots (median, 25 to 75% range boxes, 0 to 100% range bars), n ≥ 28 cells from three individual experiments. ns, not significant (P > 0.05), *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by the Mann–Whitney U test. (I–L) Radial profile analysis of CORO1A (I and K) and phalloidin (J and L) in mouse (I and J) or human (K and L) monocytes. Mean ± SEM, n = 20 cells from three individual experiments.

The recruitment of CORO1A to the cell membrane might be due to its binding to the actin cortex (55), which is formed after leukocyte stimulation (56). Agreeing with this hypothesis, it was found that phalloidin-labeled F-actin molecules were recruited to the cell membrane and formed the actin cortex in WT monocytes after CCL2 stimulation, increasing membrane-located F-actin from ~20 to ~30%, which was not observed in CF monocytes (Fig. 4 B and F). This result was further demonstrated by a radial analysis (Fig. 4J).

Similar results were also seen in human monocytes—CCL2 stimulation induced the recruitment of CORO1A (Fig. 4 C, G, and K) and F-actin (Fig. 4 D, H, and L) to the cell membrane in vehicle-treated but not CF50-treated monocytes. These results determined that CFTR deficiency diminished CORO1A recruitment to the cell membrane and actin cortex formation in monocytes after chemokine stimulation,

Decreased Engagement of CORO1A and F-Actin to β₂ Integrins in CFTR-Deficient Monocytes.

We further tested the colocalization of CORO1A with β₂ integrins (CD18) to assess their engagement in monocytes using dual-color superresolution STORM, which provides ~20 nm spatial resolution (57) and thus can more accurately detect molecular engagement than other fluorescence imaging. Monocytes from WT mice showed an increase of CORO1A/CD18 colocalization after CCL2 stimulation (white in Fig. 5A, compare first and third columns). In comparison, monocytes from CF mice did not show this increase (Fig. 5A, compare second and fourth columns). Coordinate-based colocalization (CBC) analysis of STORM images showed an increase of CD18 colocalized with CORO1A from ~20 to ~35% (Fig. 5B) and an increase of CORO1A colocalized with CD18 from ~20 to ~45% (Fig. 5C) in WT monocytes after CCL2 stimulation. However, these CCL2-induced elevations of colocalization were not observed in CF monocytes (Fig. 5 B and C). Deconvolution-boosted high-resolution dual-color epifluorescence imaging was further used to reconfirm the above results (SI Appendix, Fig. S5A). Pearson’s correlation analysis showed the coefficient of CORO1A/CD18 increased from ~0.5 to ~0.75 in WT monocytes but remained at ~0.4 in CF monocytes after CCL2 stimulation (SI Appendix, Fig. S5C). Consistent results were shown in human monocytes–more CD18 and CORO1A colocalization area was formed (Fig. 5D), more CD18 was colocalized with CORO1A (Fig. 5E, increasing from ~24 to ~38%), and more CORO1A was colocalized with CD18 (Fig. 5F, increasing from ~25 to ~40%) in vehicle-treated but not CF50-treated monocytes after CCL2 stimulation, with deconvolution-boosted high-resolution epifluorescence imaging (SI Appendix, Fig. S5B) and Pearson’s correlation analysis (SI Appendix, Fig. S5D) also showing similar results. We also performed coimmunoprecipitation of CD18 and CORO1A (Fig. 5 G and H) and found that CCL2 stimulation doubled the binding of CORO1A to CD18 in vehicle-treated but not CF50-treated human monocytes. Overall, our results suggested that the CCL2-induced engagement of CORO1A with CD18 was impaired in CFTR-deficient monocytes.

Fig. 5.

CFTR-deficient mouse and human blood monocytes have CORO1A/β₂ integrin (CD18) colocalization defects. (A) Representative STORM images of CD18 (cyan) and CORO1A (magenta) on the mouse monocyte surface (the contact area with the coverslip). Monocytes were isolated from WT or CF mouse bone marrow and stimulated with CCL2 (200 ng mL⁻¹) or vehicle control. (Scale bars are 1 μm.) (B and C) Coordinate-based colocalization analysis of STORM images showing the percentage of CD18 that colocalizes with CORO1A (B) and CORO1A that colocalizes with CD18 (C). (D) Representative STORM images of CD18 (cyan) and CORO1A (magenta) on human monocyte surface. Purified blood monocytes were preincubated with 50 μM CFTR Inhibitor-172 (CF50) or vehicle control and stimulated with CCL2 (100 ng mL⁻¹) or vehicle control. (Scale bars are 1 μm.) (E and F) Coordinate-based colocalization analysis of STORM images showing the percentage of CD18 colocalizes with CORO1A (E) and CORO1A colocalizes with CD18 (F). Boxplots (median, 25 to 75% range boxes, 0 to 100% range bars). n = 20 cells from three individual experiments in (B, C, E, and F). ns, not significant (P > 0.05), **P < 0.01; ***P < 0.001; ****P < 0.0001, by the Mann–Whitney U test. (G and H) Purified human blood monocytes were preincubated with 50 μM CFTR Inhibitor-172 (CF50) or vehicle control, stimulated by CCL2 (100 ng mL⁻¹) or vehicle control. Cell lysates were used for immunoprecipitation of the CD18 binding components. Representative western blot images of CD18 and CORO1A (G) and quantifications of CORO1A (H) in the immunoprecipitated components. The CORO1A amount in (H) was normalized by the CD18 amount. Mean ± SEM, n = 3 individual experiments (six donors, two donors pooled together in each experiment). *P < 0.05; **P < 0.01; by paired Student’s t test.

Using STORM and deconvolution-boosted high-resolution epifluorescence imaging, we also found increased F-actin/CD18 colocalization in WT mouse monocytes (Fig. 6 A–C and SI Appendix, Fig. S6 A and C) and vehicle-treated human monocytes (Fig. 6 D–F and SI Appendix, Fig. S6 B and D) after CCL2 stimulation, which was diminished in CF mouse monocytes (Fig. 6 A–C and SI Appendix, Fig. S6 A and C) and CF50-treated human monocytes (Fig. 6 D–F and SI Appendix, Fig. S6 B and D).

Fig. 6.

CFTR-deficient mouse and human blood monocytes have a defect in actin/β₂ integrin (CD18) colocalization. (A) Representative STORM images of CD18 (cyan) and actin (magenta) on the mouse monocyte surface (the contact area with the coverslip). Monocytes were isolated from WT or CF mouse bone marrow and stimulated with CCL2 (200 ng mL⁻¹) or vehicle control. (Scale bars are 1 μm.) (B and C) Coordinate-based colocalization analysis of STORM images showing the percentage of CD18 colocalizes with actin (B) and actin colocalizes with CD18 (C). (D) Representative STORM images of CD18 (cyan) and actin (magenta) on human monocyte surface. Purified blood monocytes were preincubated with 50 μM CFTR Inhibitor-172 (CF50) or vehicle control and stimulated with CCL2 (100 ng mL⁻¹) or vehicle control. (Scale bars are 1 μm.) (E and F) Coordinate-based colocalization analysis of STORM images showing the percentage of CD18 colocalizes with actin (E) and actin colocalizes with CD18 (F). Boxplots (median, 25 to 75% range boxes, 0 to 100% range bars). n = 20 cells from three individual experiments in (B, C, E, and F). ns, not significant (P > 0.05), **P < 0.01; ****P < 0.0001 by the Mann–Whitney U test.

Finally, we examined the colocalization of CORO1A and F-actin. CORO1A maintained its association with F-actin in both mouse (SI Appendix, Figs. S7 A and C and S8 A–C) and human (SI Appendix, Figs. S7 B and D and S8 D–F) monocytes regardless of CCL2 treatment and regardless of whether CFTR function is normal or impaired. However, CCL2 stimulation induced more recruitment of colocalized CORO1A/F-actin to the cell membrane in control but not CFTR-deficient monocytes. These data collectively suggest a potential mechanism (SI Appendix, Fig. S9) as follows: After CCL2 stimulation, control monocytes will have F-actin reorganized to the actin cortex, allowing its colocalized CORO1A to be recruited to the cell membrane. CORO1A then can spatially bind to β₂ integrins to mediate β₂ integrin clustering. However, in CFTR-deficient monocytes, actin cortex formation is impaired, which impedes the recruitment of CORO1A to the cell membrane and its binding to β₂ integrins, thereby inhibiting the ensuing β₂ integrin clustering.

Discussion

Using a physiologically mimicked microfluidic chamber, we demonstrated the involvement of CFTR in shear flow–mediated monocyte adhesion following chemokine-induced β₂ integrin inside-out signaling. The results are consistent with our previous microfluidic adhesion assays using CF mouse monocytes (CFTRΔF508) (44) and previous static adhesion results using CF patient monocytes (32) and CFTR-inhibited healthy human monocytes (32). However, no defect in β₂ integrin activation was detected in CF mouse monocytes and CFTR-inhibited healthy human monocytes, suggesting that the β₂ integrin activation defect observed in the previous study using CF patient monocytes (32) might be a cell-extrinsic tolerance defect due to chronic infections and systemic inflammation. Further studies unraveled that the defective adhesion in CFTR-deficient monocytes was, in fact, due to the deficiency in integrin clustering—another necessary but understudied process of β₂ integrins. Notably, when leukocytes are arrested on vascular endothelium, the initiating step of leukocyte recruitment is dominated by chemokine/GPCR-triggered integrin inside-out signaling (40). Thus, unlike previous studies on integrin clustering (58–60) or cluster-like structures, e.g., focal adhesions (61–63) and podosomes (64), using adhered and spreading cells (28–30) where ligand binding and integrin outside-in signaling are known to regulate integrin clustering (31), our study focused on inside-out integrin signaling by stimulating and fixing cells in suspension. We showed that integrin clustering can be induced on monocytes solely by chemokine stimulation. Our finding that CFTR-deficient monocytes only have defects in integrin clustering but not activation also challenged the current paradigm that integrin activation and clustering are interlinked (65, 66). Overall, our results not only define a unique mechanism that may contribute to immune dysfunction in CF patients but also provide a unique model for studying inside-out signaling-induced integrin clustering.

Our study also reveals a central role of CORO1A in CFTR-dependent monocyte adhesion and β₂ integrin clustering. CFTR G551D mutation is a major mutation identified in 2 to 5% of CF patients (45). Restoring the function of G551D CFTR in CF patients using ivacaftor (67) resulted in an increased presence of CORO1A at the cell membrane of the patients’ monocytes (68). Also, CORO1A binding to the cytoplasmic tail of β₂ integrins was found to be critical to the integrin-dependent adhesion and soluble ligand binding of neutrophils (54). Our superresolution microscopy results on monocytes showed that inhibiting CFTR causes a deficiency of CORO1A in membrane recruitment and β₂ integrin engagement accompanied by defective β₂ integrin clustering but not activation, suggesting that CORO1A may be important for β₂ integrin clustering but not activation. Future studies using CORO1A knockout leukocytes and superresolution imaging will further define the unique role of CORO1A in integrin clustering.

CORO1A is an actin-binding protein (55). We showed both actin cortex formation and CORO1A membrane recruitment defects in CFTR-deficient monocytes, suggesting that CORO1A membrane recruitment is linked to actin cortex formation. A previous study using human lung microvascular endothelial cells showed that CFTR dysfunction leads to defects in actin rearrangement (69). Inconsistency, our study also showed defective actin cortex formation in monocytes with CFTR deficiency. However, the direct effect of how the CFTR defect alters actin rearrangement is unclear.

Besides adhesion, integrin clustering is also critically involved in phagocytosis (70), another essential function of monocytes when fighting infection. Monocytes and macrophages of CF patients were shown to both have defects in phagocytosis (71, 72), which may also be explained by the defective integrin clustering identified here. Meanwhile, rapid actin polymerization remodeling was observed during phagocytosis, especially for forming the phagocytic cup (73), which should also be impeded by the actin cortex deficiency we observed in CF monocytes. Ezrin is an F-actin binding protein that is critical for PI3K/Akt signaling activation downstream of the Toll-like receptor 4 pathway and thus induces an anti-inflammatory response in macrophages (74). Reducing ezrin results in impaired phagocytosis against Pseudomonas aeruginosa (74). Interestingly, CFTR is found to be crucial for the localization of ezrin to the filopodia of activated macrophages in response to lipopolysaccharides (74). It would be intriguing to inspect whether ezrin is involved in the CFTR-dependent β₂ integrin clustering and monocyte adhesion.

Even though we used both gene-edited mice and pharmacological inhibitor CFTR inh-172 in our study, the off-target effect of CFTR inh-172 is a concern because it has been reported that CFTR inh-172 may inhibit Ca²⁺ (38) or Na⁺ (38) channel in cancer cell lines. However, our results showed that 1-h incubation of CFTR inh-172 did not affect monocyte intracellular Ca²⁺ or Na⁺, regardless of cytokine stimulation. CFTR inh-172 also activates the nuclear factor-κB (NF-κB) pathway in Hela cells after 6 h of incubation (75). Although this activation may not happen in our experiments due to a much shorter treatment time, investigating the link between integrin clustering and the NF-κB pathway, such as whether NF-κB activation limits integrin clustering, will be an interesting future research direction.

In conclusion, our study explored the critical role of CORO1A in CFTR-dependent monocyte adhesion and β₂ integrin clustering. It should serve as a starting point for delineating the related signaling pathway(s) and identifying critical molecular targets, which should eventually pave the way to developing advanced therapeutic approaches for CF.

Materials and Methods

Mice.

C57BL/6J WT (000664) and CFTRΔF508 (002515) mice were obtained from The Jackson Laboratory. Human β₂ integrin knock-in mice were a gift from Klaus Ley from the La Jolla Institute for Immunology, La Jolla, CA (currently at the Immunology Center of Georgia, Augusta University, Augusta, GA) and are currently available from The Jackson Laboratory (037426). For the β₂ integrin activation study, human β₂ integrin knock-in mice were crossed with CFTRΔF508 mice to generate CF (hItgb2^KI/KICFTR^{ΔF508/ΔF508}) and control (hItgb2^KI/KICFTR^WT/WT) mice.

Monocyte Isolation.

Mouse monocytes were purified from bone marrow cells using the EasySep mouse monocyte isolation kit. Human monocytes were purified from fresh whole blood using the Ficoll density gradient centrifugation followed by the EasySep human monocyte enrichment kit without CD16 depletion. Heparinized whole blood samples were obtained from deidentified healthy donors after informed consent, as approved by the Institutional Review Board of UConn Health (#20-084-2) in accordance with the Declaration of Helsinki.

Microfluidic Perfusion Assay.

The assembly of the microfluidic devices and substrate coating were described before (20, 43) with some modifications to mimic the flow shear stress in postcapillary venules that commonly show leukocyte recruitment during inflammation. To study the adhesion, we perfused monocytes in the microfluidic chamber. Time-lapse images were taken by an iX83 Olympus inverted microscope with a 10× numerical aperture 0.4 air objective to calculate rolling distance and velocity. After washing, arrested monocytes were counted.

Flow Cytometry.

To monitor β₂ integrin activation, we labeled monocytes with AF488-conjugated mAb24 and DL550-conjugated KIM127 with or without CCL2 stimulation. After fixation and washes, the cell fluorescence was assessed with LSRII (BD Biosciences) and analyzed with FlowJo (version 10.6). For mouse whole blood assays, monocytes were gated as CD115+ cells. To assess monocyte viability, we use SYTOX AADvanced staining to indicate dead cells. To assess monocyte intracellular Ca²⁺ and Na⁺ changes, we used Fluo-4 AM and SBFI AM, respectively.

β₂ Integrin Clustering by STORM.

AF647-conjugated mAb24 and DL550-conjugated KIM127 were used to label activated β2 integrins on human monocytes. AF647-conjugated anti-mouse CD18 antibody was used to label β2 integrins on mouse monocytes. STORM images of the monocyte membrane surface contacting with the coverslip were captured using an iX83 Olympus inverted microscope equipped with the SAFe Light module (Abbelight, includes four color lasers, λ = 405 nm, 488 nm, 532 nm, and 640 nm), sCMOS fusion cameras (Hamamatsu), and a 100× NA 1.5 oil objective.

The raw images were analyzed to get the molecular localization spreadsheets using NEO Analysis (Abbelight) integrated with the redundant cross-correlation drift correction function (76). We merged consecutive localizations and multiple blinking events from the same fluorophore. To quantify integrin clustering, we identified clusters using the Voronoi tessellation algorithm (50, 52), DBSCAN (77), and K-Ripley analysis (53) incorporated in NEO Analysis.

CORO1A Membrane Recruitment and Actin Cortex Imaging.

Unconjugated CORO1A antibody and AF647-conjugated secondary antibody were used to label CORO1A. AF568-conjugated phalloidin was used to label F-actin. Epifluorescence images of monocyte transverse were acquired using the same microscope described above. In the images, CORO1A or F-actin fluorescence intensity was associated with their molecular amount, and their cytoplasmic and membrane-associated amounts were manually quantified by the “measure” function in FIJI-ImageJ v2.0. We also used a “radial profile” plugin in FIJI-ImageJ v2.0 to quantify the actin cortex formation and CORO1A membrane recruitment. To improve the resolution of representative epifluorescence images, we used a deconvolution algorithm in the Huygens Essential 23.04 software.

Coimmunoprecipitation and Western Blot.

Protein samples from isolated CF and WT mouse monocytes were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, stained by rabbit anti-CORO1A monoclonal antibody and HRP-conjugated horse anti-rabbit secondary antibody, and quantified by chemiluminescence imaging using ImageQuant LAS 4000 (GE).

To assess the interaction of CORO1A and CD18, we immunoprecipitated CD18 binding proteins from human monocytes incubated with different treatments and performed CORO1A western blot. A small portion of cell lysate samples before immunoprecipitation were also analyzed by western blots as input controls (SI Appendix, Fig. S5H).

Colocalization Assays.

To quantify the colocalization of CD18/CORO1A, CD18/F-actin, or CORO1A/F-actin, we performed dual-color immunofluorescence staining of monocytes. Dual-color epifluorescence images of monocyte transverse were acquired using the same microscope described above. Sequential dual-color STORM images of the monocyte membrane surface contacting with the coverslip were captured by the same microscope.

The colocalization analysis was performed using the CBC analysis (78) incorporated in NEO Analysis software. The colocalization of epifluorescence images was analyzed by JACoP FIJI-ImageJ v2.0 plugin (79) using Pearson’s coefficient algorithm. All representative epifluorescence images were deconvolved.

Statistical Analysis.

Statistical analyses were performed using PRISM software (version 9.4.1, GraphPad software). Group comparisons were performed using the Mann–Whitney U tests and paired or unpaired Student’s t tests, which are indicated in figure legends. All statistical tests were two sided, and P-values < 0.05 were considered statistically significant.

Supplementary Material

Appendix 01 (PDF)

Movie S1.

A representative movie showing purified human blood monocytes incubated with vehicle control rolling in the microfluidic chamber. The bottom surface of the chamber was co-functionalized with P-selectin and ICAM-1 without (CT) or with CCL2 stimulation under a shear stress of 6 dyn·cm⁻².

Movie S2.

A representative movie showing purified human blood monocytes incubated with 50 μM CFTR Inhibitor-172 (CF50) rolling in the microfluidic chamber. The bottom surface of the chamber was co-functionalized with P-selectin and ICAM-1 without (CT) or with CCL2 stimulation under a shear stress of 6 dyn·cm⁻².

Data, Materials, and Software Availability

Raw and analyzed quantification data have been deposited in the Harvard Dataverse (80) repository.