MOSPD2 regulates the activation state of αLβ2 integrin to control monocyte migration: applicability for treatment of chronic inflammatory diseases

Abstract

Monocytes are innate immune cells that drive the chronicity of various inflammatory diseases. Monocyte migration to inflamed tissues involves multiple steps of interaction with the vascular endothelium and the extracellular matrix (ECM), a process mediated through conformational transitions in cell surface integrins. We previously described motile sperm domain-containing protein 2 (MOSPD2) as a surface protein expressed on myeloid cells that is essential for the migration of monocytes and a key regulator of inflammation. Investigating MOSPD2's mechanism of action, we assessed whether it plays a role in regulating integrin activation and monocyte adhesion. Data show that silencing of MOSPD2 expression in the THP-1 monocytic cell line significantly increased cell adhesion to various ECM molecules. Employing IW-601, a humanized anti-human MOSDP2 monoclonal antibody, on primary human monocytes increased adhesion to ECM molecules as well as to adhesion molecules. At the molecular level, silencing of MOSPD2 or blocking MOSPD2 using IW-601 led to a transition in integrin αLβ2 (CD11a/CD18, LFA-1) conformation into an active high-affinity binding form and to the induction of adhesion-associated signaling pathways. Co-immunoprecipitation experiments showed that MOSPD2 binds integrin-β2 (CD18), but not integrin-β1 (CD29). Our results reveal a novel mechanism controlling monocyte migration, in which MOSPD2 acts as an adhesion checkpoint that governs the balance between monocyte adhesion and release. By demonstrating the inhibitory effect of IW-601 on the migration of primary monocytes isolated from patients with chronic inflammatory diseases, we provide proof of concept for translating MOSPD2's mechanism into a potential treatment for inflammatory diseases, further supported by in vivo data in models of RA and IBD.

Keywords: Adhesion; Checkpoint; Inflammation; MOSPD2; Migration; αLβ2.

Fig. 1

MOSPD2 regulates monocyte adhesion. A Protein expression of MOSPD2 in THP- 1 cells transduced with CRISPR-control or CRISPR-MOSPD2 lentiviral particles. Two different isolated clones (M1, M2) are shown for the CRISPR-MOSPD2 cells. HSP- 90 was used as a loading control. B Trans-well migration of THP- 1 cells described in A toward SDF- 1 and MCP- 1 (100 ng/ml). Cell count is presented. Samples were run in triplicate. Mean ± S.D. is shown. C Adhesion of cells described in A on a plate coated with type IV collagen. Relative adhesion based on OD to CRISPR-control is presented. Samples were run in triplicate. Mean ± S.D. is shown. D Adhesion of THP- 1 cells transduced with CRISPR-control and the M1 MOSPD2-silenced clone seeded onto a plate coated with different ECM ligands. Relative adhesion based on OD to CRISPR-control is presented. Samples were run in triplicate. Mean ± S.D. is shown. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

IW- 601, a humanized anti-MOSPD2 antibody, enhances the adhesion of monocytes. A Dose-dependent effect of IW- 601 on the migration of human primary monocytes toward SDF- 1 and MCP- 1 (100 ng/ml). Cell count is presented. Samples were run in triplicate. Mean ± S.D. is shown. B Adhesion of human primary monocytes treated with human isotype control antibody or IW- 601 (10 µg/ml) to plates coated with different ECM ligands. Relative adhesion based on OD to human isotype control–treated cells is presented. Samples were run in triplicate. Mean ± S.D. is shown. C Adhesion of human primary monocytes treated with human isotype control antibody (10 µg/ml) or different doses of IW- 601 on a plate coated with fibronectin. Relative adhesion based on OD to isotype control–treated cells is presented. Samples were run in triplicate. Mean ± S.D. is shown. D Adhesion of human primary monocytes treated with human isotype control antibody or IW- 601 (10 µg/ml) to a plate coated with ICAM- 1 or VCAM- 1. Relative adhesion based on OD to isotype control–treated cells is presented. Samples were run in triplicate. Mean ± S.D. is shown

Fig. 3

MOSPD2 governs the conformational state of αLβ2. A THP- 1 CRISP-control or CRISPR-MOSPD2 clones (M1, M2) were stained with anti-αLβ2 clone m24. CRISPR-control cells were also stained with an isotype control antibody (IgG). B THP- 1 cells were treated with human isotype control antibody (10 µg/ml) or different doses of IW- 601 (0.1–10 µg/ml) and stained for 30 min with anti-αLβ2 clone m24. C THP- 1 cells were treated with human isotype control antibody or IW- 601 (10 µg/ml) for the indicated times and stained with anti-αLβ2 clone m24 for the last 30 min of treatment. D Human primary monocytes were treated with human isotype control antibody (10 µg/ml) or different doses of IW- 601 (1 and 10 µg/ml) and stained for 30 min with anti-αLβ2 clone m24. E Human primary T cells were treated with human isotype control antibody (10 µg/ml), IW- 601 (10 µg/ml) or PMA (5 ng/ml) as a positive control and stained for 30 min with anti-αLβ2 clone m24. F THP- 1 or G human primary monocytes were seeded onto a plate coated with fibronectin for 60 min, after which human isotype control antibody or IW- 601 (10 µg/ml) were added for the indicated times. Blots were stained to demonstrate the induction of signaling events. HSP- 90 and C-YES were used as loading controls for THP- 1 and human primary monocytes, respectively

Fig. 4

MOSPD2 binds β2-integrin. A–B HEK 293 cells stably expressing HA-tagged MOSPD2 were left untouched or transiently transfected with A FLAG-tagged β2-integrin or B FLAG-tagged β1-integrin for 48 h. Cell lysates were immunoprecipitated with either anti-FLAG mAb and blotted for HA or with anti-HA mAb and blotted for FLAG. C Comparison of trans-well migration between CRISPR-control, CRISPR-MOSPD2 clones (M1, M2), and CRISPR-β2-integrin clones (I1, I2) THP- 1 cells toward SDF- 1 and MCP- 1 (100 ng/ml). Cell count is presented. Samples were run in triplicate. Mean ± S.D. is shown. D Adhesion of CRISPR-control and β2-integrin-silenced clone I2 THP- 1 cells to a plate coated with different ECM ligands. Relative adhesion based on OD to CRISPR-control is presented. Samples were run in triplicate. Mean ± S.D. is shown. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

Inhibition of monocyte migration by IW- 601. Results are of mean + SEM normalized to IgG control antibody and shown in %. No statistically significant differences between disease types for IW- 601 were observed (one-way ANOVA)

Fig. 6

Treatment with Anti-MOSPD2 mAb curtails inflammatory disease progression. A–B Mice were injected with C-II to induce arthritis. On the first day of clinical manifestation (day 22) mice were apportioned (n = 9–10/group) to receive isotype control, anti-MOSPD2 mAb, or anti-TNF-α mAb every 3–4 days. Data is presented as mean ± SE. C–D Colitis was induced in mice (n = 15/group) by intra-rectal administration of TNBS. Isotype control or anti-MOSPD2 mAb was injected 2 days before disease induction, on the day of induction (day 0) and 3 days later. Colons for cytokine production were collected upon sacrifice (day 7). Data is presented as mean ± SE. p values were calculated by ANOVA with multiple comparisons test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Fig. 7

MOSPD2 is an adhesion checkpoint that regulates the activation state of integrin-β2 on monocytes. A MOSPD2 enables the normal β2 integrin conformational changes that are required for monocyte adhesion and migration. B Treatment with anti-MOSPD2 mAb IW- 601 keeps β2 integrin in its high-affinity conformation, blocking monocyte ability to detach from the extracellular matrix and migrate into the inflamed tissue