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. 2019 Jan 7;21(1):6.
doi: 10.1186/s13075-018-1783-9.

KCa1.1 and Kv1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis

Mark R Tanner  1   2 , Michael W Pennington  3   4 , Satendra S Chauhan  3 , Teresina Laragione  5 , Pércio S Gulko  5 , Christine Beeton  6   7
Affiliations

KCa1.1 and Kv1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis

Mark R Tanner et al. Arthritis Res Ther. .

Abstract

Background: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA.

Methods: We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies.

Results: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA.

Conclusions: Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

Keywords: Autoimmunity; Cell interactions; Dual therapy; Immunomodulation; Synovial fibroblast.

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Conflict of interest statement

Ethics approval

The experiments involving the use of rats were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine. De-identified human FLS were isolated by PSG’s group after patients provided written consent for their tissues to be studied, as approved by the Institutional Review Board at the Feinstein Institute for Medical Research. Baylor College of Medicine’s Institutional Review Board determined that the study of these cells did not constitute human research, because the samples were de-identified.

Consent for publication

Not applicable.

Competing interests

CB and MWP are inventors on the patent for ShK-186/dalazatide. CB and MWP are cofounders of Airmid, Inc., and sit on its board of directors. CB and MWP are investors in Kineta, Inc. The other authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Collagen-induced arthritis fibroblast-like synoviocytes (CIA-FLS) modulate T cell migration and serve as antigen-presenting cells in a KCa1.1-dependent manner. a Effector memory T (TEM) cell migration toward CIA-FLS that were cultured for 72 h in the presence or absence of interferon (IFN)-γ, paxilline, or both. Data are presented as mean ± SEM (n = 3). b Flow cytometric images of ovalbumin-loaded, IFN-γ-stimulated CIA-FLS (violet) and ovalbumin-specific TEM cell (green) cocultures that were stained for CD3 (red) after cells were allowed to interact for 30 min. c Flow cytometric plots of mixtures of ovalbumin-loaded, IFN-γ-stimulated CIA-FLS (CellTrace Violet) and ovalbumin-specific TEM cells (carboxyfluorescein succinimidyl ester [CFSE]) that were allowed to interact for 30 min in the presence or absence of paxilline (Pax) or ShK-186. Flow cytometric gating was completed to exclude single cells. d Quantifications of the proportion of CIA-FLS and TEM cells that form conjugates, as defined by the proportion of CellTrace Violet+CFSE+ multiplets or nonsingle cells detected by flow cytometry, from experiments in (c). Data are presented as mean ± SEM (n = 4 separate experiments). e Proliferation of cocultures of CIA-FLS with ovalbumin-specific CD4+ TEM cells in which CIA-FLS were pretreated with IFN-γ, paxilline (Pax), or both and loaded with either ovalbumin (OVA) or myelin basic protein (MBP). Data are presented as mean ± SEM (n = 6). *p < 0.05
Fig. 2
Fig. 2
Collagen-induced arthritis fibroblast-like synoviocytes (CIA-FLS) express major histocompatibility complex (MHC) class II molecules that are regulated through KCa1.1, as well as antigen-presenting proteins. a CIA-FLS plasma membrane expression of MHC class II protein following stimulation for 72 h with IFN-γ (gray circles), paxilline (Pax; black squares), or interferon (IFN)-γ and paxilline (open squares). Data are presented as mean ± SEM (n = 3–8 CIA-FLS donors). b Flow cytometric histogram of CIA-FLS stained for the plasma membrane expression of MHC class II protein in cells treated with paxilline for 72 h (dashed line), stimulated for 72 h with IFN-γ (solid line), or stimulated for 72 h with IFN-γ and paxilline (dotted line). The shaded histogram represents background staining. c MHC class II molecule expression of CIA-FLS that were permeabilized with saponin prior to staining for MHC class II in cells treated for 72 h with IFN-γ (gray circles), paxilline (black squares), or IFN-γ and paxilline (open squares). Data are presented as mean ± SEM (n = 3–6 CIA-FLS donors). d Flow cytometric histogram of CIA-FLS stained for MHC class II molecules following permeabilization with saponin in cells treated for 72 h with IFN-γ (solid line), paxilline (dashed line), or IFN-γ and paxilline (dotted line). The shaded histogram represents background staining. e Expression of intercellular adhesion molecule (ICAM)-1, B7-H3, and CD40 by CIA-FLS following treatment for 72 h with IFN-γ, paxilline, or IFN-γ and paxilline. Data are presented as mean ± SEM (n = 6 CIA-FLS donors). *p < 0.05, **p < 0.01
Fig. 3
Fig. 3
Effector memory T (TEM) cells and cytokines regulate the phenotype of fibroblast-like synoviocytes (FLS). KCa1.1α (a) and major histocompatibility complex (MHC) class II molecule (b) plasma membrane expression levels by collagen-induced arthritis (CIA)-FLS cultured for 72 h in the conditioned medium of TEM cells that were antigen-stimulated in the presence or absence of ShK-186. Data are presented as mean ± SEM (n = 6–9 CIA-FLS donors). c KCa1.1α protein expression by rheumatoid arthritis (RA)-FLS, as determined by flow cytometry, following 24 h of stimulation with recombinant cytokines. Data are presented as mean ± SEM (n = 7–9 RA-FLS donors). d CIA-FLS invasion through Matrigel-coated Transwells toward the conditioned medium of TEM cells that were antigen-stimulated in the presence or absence of ShK-186. Data are presented as mean ± SEM (n = 3 CIA-FLS donors). *p < 0.05, **p < 0.01
Fig. 4
Fig. 4
Kv1.3 and KCa1.1 blockers synergize to reduce disease severity in collagen-induced arthritis (CIA). a Clinical scores of rats with CIA treated with vehicle (open squares), iberiotoxin (IbTX; black triangles), ShK-186 (gray squares), or both IbTX and ShK-186 (open triangles) every other day (EOD) starting at disease onset. Data are presented as mean ± SEM (n = 11 or 12 rats per group). b Left: X-rays of hind paws from a healthy rat and from rats with CIA treated with vehicle, IbTX, ShK-186, or both IbTX and ShK-186 EOD for 14 days after disease onset. Center and right: Safranin O/Fast Green staining (center) and H&E staining (right) of tissue sections of hind paw joints of a healthy rat and of rats in each treatment group. Arrows indicate areas of cartilage erosions (Safranin O/Fast Green) or hyperplasia (H&E). Scale bar = 100 μm. c Scoring of disease parameters from tissue sections of paws from rats of each treatment group (n = 3 rats per group). *p < 0.05, ****p < 0.0001
Fig. 5
Fig. 5
Potassium channel blockers alter serum cytokines in collagen-induced arthritis (CIA). a–i Serum concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-12, IL-17A, monocyte chemoattractant protein (MCP)-1, IL-2, IL-1β, IL-6, and IL-10 from healthy rats or rats with CIA treated with vehicle, ShK-186, iberiotoxin (IbTX), or both ShK-186 and IbTX every other day for 14 days after disease onset. Data are presented as mean ± SEM (n = 5 or 6 rats per group). *p < 0.05
Fig. 6
Fig. 6
Fibroblast-like synoviocytes (FLS) from collagen-induced arthritis (CIA) rats treated with potassium channel blockers have reduced pathogenic phenotypes ex vivo. a Invasion through Matrigel-coated Transwell inserts of FLS isolated from healthy rats or from rats with CIA treated with either vehicle, ShK-186, iberiotoxin (IbTX), or ShK-186 and IbTX every other day (EOD) for 14 days after disease onset. Data are presented as mean ± SEM (n = 3 FLS donors per group). b KCa1.1α expression by FLS isolated from healthy rats and from rats with CIA treated with vehicle, ShK-186, IbTX, or ShK-186 and IbTX EOD for 14 days after disease onset. Data are presented as mean ± SEM (n = 5 FLS donors per group). c Data from (b) plotted against the clinical scores on the 14th day after disease onset of the rats with CIA from which the FLS were isolated, along with a linear regression plot. *p < 0.05, **p < 0.01
Fig. 7
Fig. 7
T cell populations are altered in rats with collagen-induced arthritis (CIA) treated with potassium channel blockers. a–o Expression of CD3, CD4, CD8, CD25, and CD45RC of inguinal lymph node cells of rats with CIA treated with vehicle, ShK-186, iberiotoxin (IbTX), or ShK-186 and IbTX every other day for 14 days after disease onset (n = 6 rats per group). *p < 0.05, **p < 0.01
Fig. 8
Fig. 8
KCa1.1 and Kv1.3 blockers work in tandem to reduce disease severity in pristane-induced arthritis (PIA). a Clinical scores of paw inflammation in rats with PIA treated with vehicle (open squares), paxilline (Pax; black triangles), ShK-186 (gray squares), or both paxilline and ShK-186 (open triangles) every other day for 21 days after disease onset. Data are presented as mean ± SEM (n = 18–27 rats per group). b Ex vivo invasiveness of fibroblast-like synoviocytes (FLS) isolated from healthy rats and from rats with PIA treated with vehicle, paxilline, ShK-186, or both paxilline and ShK-186. Data are presented as mean ± SEM, N = 3 FLS donors per group. c Left, example X-rays of hind paws of a healthy rat and from rats with PIA treated with vehicle, paxilline, ShK-186, or paxilline and ShK-186 every other day for 21 days after disease onset. Center and right: Safranin O/Fast Green staining (center) and H&E staining (right) of tissue sections of hind paw joints from a healthy rat and of rats from each treatment group. Arrows indicate areas of cartilage erosions (Safranin O/Fast Green) or hyperplasia (H&E). Scale bar = 100 μm. *p < 0.05, ****p < 0.0001
Fig. 9
Fig. 9
Schematic summarizing the model by which fibroblast-like synoviocytes (FLS) and T cells interact, within the context of previous studies showing the role of KCa1.1 in regulating integrin-mediated invasion in FLS and Kv1.3 in regulating calcium-mediated T cell activation [7, 16]
See this image and copyright information in PMC

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References

    1. Cope A. T cells in rheumatoid arthritis. Arthritis Res Ther. 2008;10(Suppl 1):S1. doi: 10.1186/ar2412. - DOI - PMC - PubMed
    1. Cope A, Schulze-Koops H, Aringer M. The central role of T cells in rheumatoid arthritis. Clin Exp Rheumatol. 2007;25((5 Suppl 46)):S4–11. - PubMed
    1. Smolen J, Aletaha D. Rheumatoid arthritis therapy reappraisal: strategies, opportunities, and challenges. Nat Rev Rheumatol. 2015;11:276–289. doi: 10.1038/nrrheum.2015.8. - DOI - PubMed
    1. Keyser F. Choice of biologic therapy for patients with rheumatoid arthritis: the infection perspective. Curr Rheumatol Rev. 2011;7:77–87. doi: 10.2174/157339711794474620. - DOI - PMC - PubMed
    1. Fasth A, Cao D, van Vollenhoven R, Trollmo C, Malmstrom V. CD28nullCD4+ T cells – characterization of an effector memory T-cell population in patients with rheumatoid arthritis. Scand J Immunol. 2004;60:199–208. doi: 10.1111/j.0300-9475.2004.01464.x. - DOI - PubMed

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