Interleukin-17 as a molecular target in immune-mediated arthritis: Immunoregulatory properties of genetically modified murine dendritic cells that secrete interleukin-4

Objective Our previous studies have shown that murine dendritic cells (DCs) genetically modified to express interleukin-4 (IL-4) reduce the incidence and severity of murine collagen-induced arthritis. The present studies were performed to assess the immunoregulatory mechanisms underlying this response, by assessing the effects of IL-4 DCs on cytokine production by subsets of T helper cells. Methods Male DBA mice ages 6–8 weeks old were immunized with type II collagen. Splenic T cells obtained during the initiation phase and the end stage of arthritis were cultured with IL-4 DCs or untransduced DCs in the presence of collagen rechallenge. Interferon-Γ (IFNΓ) and IL-17 responses were measured. Antibodies to IL-4, IL-12, and IL-23, and recombinant IL-4, IL-12, and IL-23 were used to further study the regulation of T cell cytokine production by IL-4 DCs. Results Splenic T cells obtained during the initiation phase of arthritis produced less IL-17 when cultured in the presence of IL-4 DCs, despite their production of increased quantities of other proinflammatory cytokines (IFNΓ and tumor necrosis factor). T cell IL-17 production after collagen rechallenge was not inhibited by a lack of IL-23, since IL-4–mediated suppression of IL-17 was not reconstituted by IL-23, an otherwise potent inducer of IL-17 production by T cells. Although IL-4 DCs can produce increased quantities of IL-12 and IFNΓ, suppression of IL-17 production by IL-4 DCs was independent of both. While IL-17 production by T cells obtained during the initiation phase of arthritis was regulated by IL-4 DCs, IL-17 production by T cells obtained during end-stage arthritis was not altered. Conclusion Our data suggest that IL-4 DCs exert a therapeutic effect on collagen-induced arthritis by targeting IL-17. IL-17 suppression by IL-4 DCs is robust and is not reversed by IL-23. Timing might be important in IL-17–targeted therapy, since IL-17 production by T cells obtained during end-stage arthritis did not respond to suppression by IL-4 DCs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55925/1/22311_ftp.pd

A polymorphism in the interleukin-4 receptor affects the ability of interleukin-4 to regulate Th17 cells: a possible immunoregulatory mechanism for genetic control of the severity of rheumatoid arthritis

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94408/1/Wallis_2011_Polymorphism_interleukin-4_receptor.pdf165

Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes.

Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA

Molecular Interactions between T Cells and Fibroblast-Like Synoviocytes Role of Membrane Tumor Necrosis Factor-α on Cytokine-Activated T Cells

The mechanism of fibroblast-like synoviocyte (FLS) transformation into an inflammatory phenotype in rheumatoid arthritis (RA) is not fully understood. FLS interactions with invading leukocytes, particularly T cells, are thought to be a critical component of this pathological process. Resting T cells and T cells activated through the T-cell receptor have previously been shown to induce inflammatory cytokine production by FLS. More recently, a distinct population of T cells has been identified in RA synovium that phenotypically resembles cytokine-activated T (Tck) cells. Using time lapse microscopy, the interactions of resting, superantigen-activated, and cytokine-activated T cells with FLS were visualized. Rapid and robust adhesion of Tck and superantigen-activated T cells to FLS was observed that resulted in flattening of the T cells and a crawling movement on the FLS surface. Tck also readily activated FLS to produce interleukin IL-6 and IL-8 in a cell contact-dependent manner that was enhanced by exogenous IL-17. Although LFA-1 and ICAM-1 co-localized at the Tck-FLS synapse, blocking the LFA-1/ICAM-1 interaction did not substantially inhibit Tck effector function. However, antibody blocking of membrane tumor necrosis factor (TNF)-α on the Tck surface did inhibit FLS cytokine production, thus illustrating a novel mechanism for involvement of TNF-α in cell-cell interactions in RA synovium and for the effectiveness of TNF-α blockade in the treatment of RA

CD13 is upregulated in FLS at the mRNA level but the effects on CD13 protein levels are varied.

<p>FLS were stimulated over a time course of 0–72 hours with IFNγ (1000U/ml), TNFα (10ng/ml), or IL-17 (10ng/ml). Cells were harvested and processed for either mRNA (A), surface expression (B), total cell lysate CD13 (C), or total CD13 in supernatant (D). mRNA was measured by qRT-PCR. CD13 was measured on the surface with anti-CD13 (1D7) and flow cytometry. Cell lysate and supernatant CD13 concentrations were measured by CD13 ELISA. Gating was done to isolate the major cell population and exclude debris and dead cells. Data is expressed as a ratio to unstimulated FLS at the same time point in either ΔΔCt normalized to GAPD (mRNA), mean fluorescent intensity corrected for background florescence with MsIg staiming (surface), or CD13 concentration in lysate or supernatant. A n = 3 B-D n = 1 *p≤0.05</p

Metalloproteinases cleave CD13 from the surface of FLS.

<p>Five different protease inhibitors were added to FLS cultures covering all classes of proteases. <b>(A)</b> The only inhibitor to decrease shedding of CD13 into the supernatant of the cultures was GM6001. <b>(B)</b> No significant decreases were seen in cell lysate CD13 concentrations. Cultures were incubated with serum free media containing protease inhibitors for 48 hours: Pepstatin A (aspartic) 10μM, Aprotinin (serine) 100nM, Leupeptin (serine/cysteine) 10μM, GM6001 (metalloproteinase) 25μM, and E-64 (cysteine) 10μM. <b>(C)</b> TIMP-2 (0.6μg/ml) inhibited the secretion of CD13 from RA FLS while TIMP-1(0.6μg/ml) did not. We used FLS from 3 different RA patients. Secretion was measured by ELISA and optical density (OD) of the samples was measured. (n = 3) mean of % change ± SEM *p≤0.05 ***p≤0.0001</p

CD13 is found as a soluble protein and on extracellular vesicles.

<p><b>(A)</b> Supernatants from 3 flasks of RA fibroblasts were concentrated through a 30K centrifugal filter. RA synovial fluid was diluted with PBS (4:1), and 10mls of plasma was obtained from a healthy individual. Vesicle fractions were isolated from the samples by serial centrifugation. Supernatants from the final centrifugation were collected as the soluble protein fraction. The vesicle pellet was resuspended in 1ml PBS. CD13 was measured by ELISA and aminopeptidase activity was analyzed through cleavage of L-leu-AMC. Data is normalized to concentration in original fluid. mean±SEM n≥3 <b>(B)</b> Exosomes were lysed and purity was confirmed by western blot for flotillin-1 and CD9. Single bands appeared at expected sizes with a weak band for CD9 and a strong band for flotillin-1 confirming exosomes from FLS. <b>(C)</b> A discontinuous optiprep gradient was created in seven fractions from 1.268g/ml to 1.031g/ml. 500ul of the resuspended vesicles was layered onto the top of the gradient. The loaded gradients were centrifuged at 100kg for one hour. Fractions were collected in reverse. Fractions were washed in PBS at 110kg for 2hr and the pellets were resuspended in 500ul PBS. Soluble CD13 is present in the first supernatant separation, exosomes are in fractions 3–5, and other extracellular vesicles are shown from the other fractions. CD13 was observed in all three fractions. Data was converted to percentage of total fluid CD13. % total n≥3</p