Reducing the activities of the pro‐inflammatory cytokine IL‐17 is an effective treatment strategy for several chronic autoimmune disorders. Rho‐associated coiled‐coil containing kinase 2 (ROCK2) is a ...member of the serine‐threonine protein kinase family that regulates IL‐17 secretion in T cells via signal transducer and activator of transcription 3 (STAT3)‐dependent mechanism. We reported here that the selective ROCK2 inhibitor KD025 significantly reduced in vitro production of IL‐17 in unfractionated human peripheral blood mononuclear cells (PBMCs) stimulated with the dectin‐1 agonist Candida albicans. C. albicans induced IL‐17 was reduced by 70% (p < 0.0001); a similar reduction (80%) was observed in PBMC stimulated with the Toll‐like receptor 2 agonist Staphylococcus epidermidis (p < 0.0001). Treatment of PBMC with KD025 was not associated with a reduction in IL‐1β, IL‐6 or IL‐1α levels; in contrast, a 1.5 fold increase in the level of IL‐1 receptor antagonist (IL‐1Ra) was observed (p < 0.001). KD025 down‐regulated C. albicans‐induced Myosin Light Chain and STAT3, whereas STAT5 phosphorylation increased. Using anti‐CD3/CD28 activation of the TCR, KD025 similarly suppressed IL‐17 independent of a reduction in IL‐1β. Thus, ROCK2 directly regulates IL‐17 secretion independent of endogenous IL‐1 and IL‐6 supporting development of selective ROCK2 inhibitors for treatment of IL‐17‐driven inflammatory diseases.
ROCK2 targeting bypasses IL‐1 signaling and regulates C. albicans –induced IL‐17 secretion by modulating STAT3/STAT5 phosphorylation in human PBMCs. In contrast, IL‐1Ra inhibits IL‐17 secretion by blocking intracellular IL‐1 signaling via STAT3‐independent manner, underlying different molecular mechanisms involved in regulation of IL‐17 production induced by C. albicans.
Targeted inhibition of Rho-associated kinase (ROCK)2 downregulates the proinflammatory T cell response while increasing the regulatory arm of the immune response in animals models of autoimmunity and ...Th17-skewing human cell culture in vitro. In this study, we report that oral administration of a selective ROCK2 inhibitor, KD025, reduces psoriasis area and severity index scores by 50% from baseline in 46% of patients with psoriasis vulgaris, and it decreases epidermal thickness as well as T cell infiltration in the skin. We observed significant reductions of IL-17 and IL-23, but not IL-6 and TNF-α, whereas IL-10 levels were increased in peripheral blood of clinical responders after 12 wk of treatment with KD025. Collectively, these data demonstrate that an orally available selective ROCK2 inhibitor downregulates the Th17-driven autoimmune response and improved clinical symptoms in psoriatic patients via a defined molecular mechanism that involves concurrent modulation of cytokines without deleterious impact on the rest of the immune system.
The growth factor progranulin (PGRN) has been implicated in embryonic development, tissue repair, tumorigenesis, and inflammation, but its receptors remain unidentified. We report that PGRN bound ...directly to tumor necrosis factor receptors (TNFRs) and disturbed the TNFα-TNFR interaction. PGRN-deficient mice were susceptible to collagen-induced arthritis, and administration of PGRN reversed inflammatory arthritis. Atsttrin, an engineered protein composed of three PGRN fragments, exhibited selective TNFR binding. PGRN and Atsttrin prevented inflammation in multiple arthritis mouse models and inhibited TNFα-activated intracellular signaling. Collectively, these findings demonstrate that PGRN is a ligand of TNFR, an antagonist of TNFα signaling, and plays a critical role in the pathogenesis of inflammatory arthritis in mice. They also suggest new potential therapeutic interventions for various TNFα-mediated pathologies and conditions, including rheumatoid arthritis.
CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both ...CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25- or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-gamma and TNF-alpha secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-beta and IL-10. In addition, the expression of ERK, NF-kappaB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.
Rho-associated coiled-coil kinase (ROCK)2 targeting down-regulates autoimmune responses in animal models and patients, however the underlying molecular mechanism is still an enigma. We report that ...ROCK2 binds phosphorylated-STAT3 and its kinase activity controls the formation of ROCK2/STAT3/JAK2 complex and optimal STAT3 phosphorylation in human CD4
T cells during T helper 17 (TH17)-skewing. Moreover, chromatin-immunoprecipitation sequencing (ChIP-seq) analysis revealed that, genome-wide, about 70% of ROCK2 and STAT3 peaks overlapped and co-localized to several key genes controlling TH17 and T follicular helper (TFH) cell functions. Specifically, the co-occupancy of ROCK2 and STAT3 on the Irf4 and Bcl6 genes was validated by ChIP-qPCR analysis. Furthermore, the binding of ROCK2 to both the Irf4 and Bcl6 promoters was attenuated by STAT3 silencing as well as by selective ROCK2 inhibitor. Thus, the present study demonstrated previously unidentified evidence that ROCK2-mediated signaling in the cytosol provides a positive feed-forward signal for nuclear ROCK2 to be recruited to the chromatin by STAT3 and potentially regulates TH17/TFH gene transcription.
Significance Rho-associated kinase 2 (ROCK2) is implicated in the regulation of proinflammatory cytokines, such as IL-17 and IL-21, and the development of autoimmunity in mice. However, the role of ...ROCK2 signaling pathway in regulation of immune responses in humans is still an enigma. Here we show that targeted ROCK2 inhibition down-regulates proinflammatory responses via concurrent regulation of STAT3/STAT5 phosphorylation and shifting Th17/Treg balance in human T cells with a minimal effect on the rest of the immune response. This work provides previously unidentified insights into the molecular mechanism of ROCK2-mediated modulation of the immune response in man and has profound implications for development of a selective ROCK2 inhibitor as a new therapeutic target for autoimmunity treatment.
Rho-associated kinase 2 (ROCK2) regulates the secretion of proinflammatory cytokines and the development of autoimmunity in mice. Data from a phase 1 clinical trial demonstrate that oral administration of KD025, a selective ROCK2 inhibitor, to healthy human subjects down-regulates the ability of T cells to secrete IL-21 and IL-17 by 90% and 60%, respectively, but not IFN-γ in response to T-cell receptor stimulation in vitro. Pharmacological inhibition with KD025 or siRNA-mediated inhibition of ROCK2, but not ROCK1, significantly diminished STAT3 phosphorylation and binding to IL-17 and IL-21 promoters and reduced IFN regulatory factor 4 and nuclear hormone RAR-related orphan receptor γt protein levels in T cells derived from healthy subjects or rheumatoid arthritis patients. Simultaneously, treatment with KD025 also promotes the suppressive function of regulatory T cells through up-regulation of STAT5 phosphorylation and positive regulation of forkhead box p3 expression. The administration of KD025 in vivo down-regulates the progression of collagen-induced arthritis in mice via targeting of the Th17-mediated pathway. Thus, ROCK2 signaling appears to be instrumental in regulating the balance between proinflammatory and regulatory T-cell subsets. Targeting of ROCK2 in man may therefore restore disrupted immune homeostasis and have a role in the treatment of autoimmunity.
Abstract
In vitro
induced human regulatory T cells (iTregs) have demonstrated
in vivo
therapeutic utility, but pathways regulating their function have not been elucidated. Here, we report that human ...iTregs generated
in vitro
from naïve cord blood cells preferentially recruit Disc large homolog 1 (Dlgh1) and exclude protein kinase C (PKC)-θ from immunological synapses formed on supported lipid bilayers with laterally mobile ICAM-1 and anti-CD3 mAb. Also, iTregs display elevated Dlgh1 overall and Dlgh1-dependent p38 phosphorylation, higher levels of phosphatase and tensin homolog (PTEN), and diminished Akt phosphorylation. Pharmacological interruption of PKC-θ increases and Dlgh1 silencing decreases the ability of iTregs to suppress interferon-γ production by CD4
+
CD25
−
effector T cells (Teff). Comparison with expanded cord blood-derived CD4
+
CD25
hi
tTreg and expanded Teffs from the same donors indicate that iTreg are intermediate between expanded CD4
+
CD25
hi
tTregs and Teffs, whereas modulation of suppressive activities by PKC-θ and Dlgh1 signaling pathways are shared.
Rho-associated kinase 2 (ROCK2) determines the balance between human T helper 17 (TH17) cells and regulatory T (Treg) cells. We investigated its role in the generation of T follicular helper (TFH) ...cells, which help to generate antibody-producing B cells under normal and autoimmune conditions. Inhibiting ROCK2 in normal human T cells or peripheral blood mononuclear cells from patients with active systemic lupus erythematosus (SLE) decreased the number and function of TFH cells induced by activation ex vivo. Moreover, inhibition of ROCK2 activity decreased the abundance of the transcriptional regulator Bcl6 (B cell lymphoma 6) and increased that of Blimp1 by reducing the binding of signal transducer and activator of transcription 3 (STAT3) and increasing that of STAT5 to the promoters of the genes Bcl6 and PRDM1, respectively. In the MRL/lpr murine model of SLE, oral administration of the selective ROCK2 inhibitor KD025 resulted in a twofold reduction in the numbers of TFH cells and antibody-producing plasma cells in the spleen, as well as a decrease in the size of splenic germinal centers, which are the sites of interaction between TFH cells and B cells. KD025-treated mice showed a substantial improvement in both histological and clinical scores compared to those of untreated mice and had reduced amounts of Bcl6 and phosphorylated STAT3, as well as increased STAT5 phosphorylation. Together, these data suggest that ROCK2 signaling plays a critical role in controlling the development of TFH cells induced by autoimmune conditions through reciprocal regulation of STAT3 and STAT5 activation.
Toll-like receptors (TLRs) are widely expressed and play an essential role in the activation of innate immune cells. However, certain TLRs are also expressed on T cells, and TLR ligands can directly ...modulate T cell functions. Here, we discuss findings indicating that T cells directly respond to Heat Shock Protein (HSP) 60, a self molecule, or to the HSP60-derived peptide, p277, via a TLR2-dependent mechanism. HSP60 has been considered to be a "danger signal" for the immune system because of its ability to induce pro-inflammatory phenotypes in innate immune cells - in this case via TLR4 activation; nevertheless, TLR2 engagement by HSP60 on T cells can lead to resolution of inflammation by up-regulating the suppression function of regulatory T cells and shifting the resulting cytokine secretion balance toward a Th2 phenotype. Moreover, T cell TLR4 engagement by LPS leads to up-regulation of suppressor of cytokine signaling 3 expression and consequently down-regulates T cell chemotaxis. Thus, TLR2 and TLR4 activation can contribute to both induction and termination of effector immune responses by controlling the activities of both innate and adaptive immune cells.
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