Cellular Targeting in Autoimmunity

Many biologic agents that were first approved for the treatment of malignancies are now being actively investigated and used in a variety of autoimmune diseases such as rheumatoid arthritis (RA), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, systemic lupus erythematosus (SLE), and Sjogren’s syndrome. The relatively recent advance of selective immune targeting has significantly changed the management of autoimmune disorders, and in part, can be attributed to the progress made in understanding effector cell function and their signaling pathways. In this review, we will discuss the recent FDA approved biologic therapies that directly target immune cells as well as the most promising investigational drugs affecting immune cell function and signaling for the treatment of autoimmune disease

CX3CR1 deficient mice have decreased Th17 and antigen-specific humoral responses in the collagen induced arthritis (CIA) model

CX3CR1 is a chemokine receptor that uniquely binds to its ligand fractalkine (FKN or CX3CL1) and has been shown to be important in inflammatory arthritis responses largely due to effects on cellular migration. In this study, we tested the hypothesis that genetic deficiency of CX3CR1 would be protective in the chronic inflammatory arthritis model, collagen induced arthritis (CIA). Because CX3CR1 is expressed on T cells and antigen-presenting cells, we additionally examined adaptive immune functions in this model

G-protein signaling modulator-3, a gene linked to autoimmune diseases, regulates monocyte function and its deficiency protects from inflammatory arthritis.

Polymorphism at the GPSM3 gene locus is inversely associated with four systemic autoimmune diseases, including rheumatoid arthritis and ankylosing spondylitis. G-protein signaling modulator-3 (GPSM3) expression is most pronounced in myeloid cells, in which it targets heterotrimeric G-protein Gαi subunits of chemokine receptors, critical to immune function. To begin to explore the regulatory role of GPSM3 in monocytes, human THP-1 and primary mouse myeloid cells were cultured under stimulus conditions; GPSM3 was found by immunoblotting to be expressed at highest levels in the mature monocyte. To evaluate the effects of GPSM3 deficiency on a myeloid-dependent autoimmune disease, collagen antibody-induced arthritis (CAIA) was induced in Gpsm3-/- and control mice, which were then analyzed for clinical score, paw swelling, intra-articular proinflammatory markers, and histopathology. Mice lacking GPSM3 were protected from CAIA, and expression of monocyte-representative pro-inflammatory chemokine receptors and cytokines in paws of Gpsm3-/- mice were decreased. Flow cytometry, apoptosis, and transwell chemotaxis experiments were conducted to further characterize the effect of GPSM3 deficiency on survival and chemokine responsiveness of monocytes. GPSM3-deficient myeloid cells had reduced migration ex vivo to CCL2, CX3CL1, and chemerin and enhanced apoptosis in vitro. Our results suggest that GPSM3 is an important regulator of monocyte function involving mechanisms of differentiation, survival, and chemotaxis, and deficiency in GPSM3 expression is protective in acute inflammatory arthritis. Mol Immunol 2013 Jun; 54(2):193-8

G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

<div><p>Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent <i>in vivo</i> mouse model, we further demonstrate that alterations in <i>GRK3</i> expression levels in tumor cells directly affect migration and invasion <i>in vitro</i> and the establishment of distant metastasis <i>in vivo</i>. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.</p></div

GRK3 regulates CXCL12-specific CXCR4 internalization and β-arrestin recruitment.

<p>(A) Control cells (empty plasmid) and GRK3-overexpressed MDA-MB-231 cells were treated for the indicated times with 100 nM CXCL12 at 37°C. Surface expression of CXCR4 on the surface of cells was determined by flow cytometry. Data shown are the mean of three experiments normalized to the zero time point. Error bars represent SEM. Statistical analysis was performed using a two-tailed t-test. **p < 0.01. (B) Using a TANGO arrestin-recruitment assay, HTLA cells were transfected with either CXCR4 alone or CXCR4 plus GRK3 as detailed in the Materials and Methods. Cells were plated in a 384 well plate and stimulated with CXCL12 at the indicated Molar concentrations. Luminescence was measured 24 hours post-stimulation. Error bars represent +/- SEM (n = 3). (C) TANGO results testing the CXCR4 antagonist AMD-3100. As in (B), HTLA cells were transfected with CXCR4 and GRK3 plasmids and stimulated with 10⁻⁷ M CXCL12 (one concentration point above EC₅₀) following pre-treatment with AMD3100 at the indicated Molar concentrations. (D) TANGO results testing the CXCR4 antagonist MSX-122. HTLA cells were transfected with CXCR4 and GRK3 plasmids and stimulated with 10⁻⁷ M CXCL12 following pre-treatment with MSX-122 at the indicated Molar concentrations.</p

Summary model of GRK3 regulation of CXCR4-driven metastasis.

<p>Extracellular ligand stimulation of CXCL12 on G protein-coupled receptor (GPCR) CXCR4 elicits conformational changes of receptor, activation and dissociation of guanine nucleotide binding proteins, and downstream signaling for tumor cell migration (A). Negative regulation of surface receptor expression is mediated by G protein-coupled receptor kinase 3 (GRK3), which phosphorylates the carboxyl terminus of CXCR4 for desensitization, thus prompting β-arrestin recruitment for receptor internalization. The presence of GRK3 limits ligand/receptor signaling by contributing to desensitization and by reducing CXCR4 surface expression (A, inset). Upon GRK3 deficiency, CXCR4 receptor expression is enhanced thus allowing increased opportunities for CXCL12 extracellular ligand/receptor stimulation, signaling, and migration (B). The absence of GRK3 enhances ligand/receptor signaling by prolonging CXCR4 surface expression (B, inset).</p

GRK3-deficient 66cl4-luc mammary tumors disseminate distant metastasis.

<p>GRK3-deficient 66cl4-luc mammary tumors disseminate distant metastasis.</p

Alterations in GRK3 affect migratory responses of human breast cancer lines to CXCL12.

<p><i>GRK3</i> expression was altered by overexpression or shRNA silencing in human breast cancer lines. (A) Chemotaxis toward media or 50 nM CXCL12 of MDA-MB-231 transiently transfected with GRK3 or control plasmid was assessed by a real-time modified Transwell assay. Transfection efficiency was routinely 40%. Results shown are the mean of 5 independent experiments. (B) MDA-MB-231 invasion through Matrigel was analyzed after 24 hours and staining with Calcien-AM. The chemoinvasion index is defined as the ratio of relative fluorescence of cells migrated toward CXCL12 over media control. Results shown are the mean of 3 independent experiments. (C) Chemotaxis toward media or 500nM CXCL12 of stably transduced MDA-MB-468 cells was assessed by a real-time modified Transwell assay. Results shown are the mean of 4 independent experiments. (D) MDA-MB-468 invasion through Matrigel was analyzed using a 96-well invasion assay. Results shown are the mean of 3 independent experiments. Error bars for all data represent the SEM. Statistical significance determined by analysis of covariance (ANCOVA) linear regression model (A and C) or by a two-tailed t-test (B and D): * p < 0.05; ** p < 0.01; *** p < 0.001; N.S. not significant.</p

<i>CXCR4</i>:<i>GRK3</i> ratio correlates with the invasiveness of human breast cancer lines.

<p>Human breast cancer lines were analyzed by quantitative real time PCR to determine the mRNA expression levels of <i>GRK3</i> and <i>CXCR4</i>. Transcript copy number was determined using the standard curve method. Data shown are the average of two to four independent experiments. Error bars represent the SEM.</p