Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Nov 19;8(1):17058.
doi: 10.1038/s41598-018-35189-7.

Genetic and Pharmacological Validation of TAK1 Inhibition in Macrophages as a Therapeutic Strategy to Effectively Inhibit TNF Secretion

Affiliations
Free PMC article

Genetic and Pharmacological Validation of TAK1 Inhibition in Macrophages as a Therapeutic Strategy to Effectively Inhibit TNF Secretion

Scott A Scarneo et al. Sci Rep. .
Free PMC article

Abstract

Immune challenge of invading macrophages at sites of infection is associated with release of TNF, which triggers a local cytokine storm as part of the normal inflammatory response. Whereas this response maybe beneficial in fighting off infections, similar responses triggered in autoimmune diseases contribute significantly to the underlying damaging pathology associated with these diseases. Here we show that Takinib, a highly discriminatory inhibitor of transforming growth factor Beta- activated kinase 1 (TAK1), selectively and potently reduces TNF production in pro-inflammatory THP-1 macrophages. A complete survey of 110 cytokines, showed robust loss of proinflammatory cytokine responsiveness to lipopolysaccharide (LPS) and interferon gamma (IFNγ) challenge in response to Takinib. The mechanisms of action of Takinib was recapitulated in TAK1 KO macrophages. TAK1 KO cells showed significant loss of TNF production as well as release of IL-6 in response to LPS challenge. Furthermore, Takinib blocked the ability of exogenously added LPS to promote phosphorylation of, c-Jun, p38 protein kinases as well as downstream transcription factors regulated by nuclear factor κ-light-chain-enhancer of activated B cells (NFκB). In a mouse LPS challenge model, Takinib significantly reduced TNF serum levels. Our findings demonstrate that Takinib has utility in the treatment inflammatory disease by locally suppressing TNF production from invading macrophages.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Takinib reduces the cytokine and chemokine response in pro inflammatory stimulated THP-1 human macrophages. THP-1 cells were differentiated in 100 nM PMA for 72 hours followed by a 48-hour rest period, cells were either left unstimulated (resting) or activated with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treated with 10 μM Takinib or DMSO. 110 cytokine and chemokine proteins were profiled. Takinib reduces the expression of 17 different cytokines and chemokines in response to LPS and IFNγ stimulation (a). GROα, IL-6, IL-23, TNF, MCP-3 and Thrombospondin-1 levels in pro-inflammatory treated DMSO (n = 3 ± SEM), 10 μM Takinib (n = 4 ± SEM), or resting cells (n = 4 ± SEM) (b–g). (ANOVA with Dunnett’s) RFU = Relative Fluorescent Units *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 2
Figure 2
TAK1 inhibition reduces TNF, IL-6, IL-1β and IL-8 secretion in a dose dependent manner. THP-1 human macrophage cell line was differentiated in 100 nM PMA, followed by a 48-hour rest period. Following the rest period, cells were pro-inflammatory stimulated with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treated with Takinib at varying doses or DMSO control. Takinib dose dependent effects of TNF (a), IL-6 (b), IL-1β (c), and IL-8 (d) secretion reported as a percent of vehicle control in THP-1 cells, (n = 3± SEM). Concentration of cytokines from DMSO treated or 21.8 μM Takinib are shown in the respective panel insets.
Figure 3
Figure 3
Takinib reduces the functional response of macrophages upon pro-inflammatory activation with LPS and IFNγ. Raw264.7 cells were plated at (0.25 × 106/well) in 24-well plates and activated with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treated with the indicated Takinib concentration or vehicle control for 24 hours (a). RAW 264.7 cell were plated at (3 × 106/plate) and serum starved for 4 hours. Cells were pretreated with either 10 μM Takinib or vehicle for 30 minutes followed by LPS (10 ng/mL) stimulation for the indicated times. mRNA levels compared to vehicle (n = 4± SEM) (b). Raw264.7 cells were plated at (0.75 × 106/well) in a 96-well Boyden migration chamber in serum free media. Cells migrated towards, 10% fetal bovine serum, media chambers for 18 hours and total cell migration determined (c). (One way ANOVA with Dunnett’s, n = 3± SEM). Reduced NO production following LPS + IFNγ stimulation and treatment with Takinib in RAW 264.7 murine macrophage cells (One-way ANOVA with Dunnett’s, n = 3± SEM) (d). RFU = Relative Fluorescent Units *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4
Figure 4
Takinib reduces phosphorylation of downstream p-38, c-Jun and NFκB in LPS stimulated Raw264.7 macrophages. Raw264.7 cells plated at (3 × 106/plate) and serum starved for 4 hours. Cells were pre-treated for 30 minutes with either 10 μM Takinib or DMSO followed by LPS (10 ng/mL) stimulation for the indicated times. Representative images of downstream phosphorylation of p-65, p-50, p-38 and p-c-Jun were detected via western blot and normalized to GAPDH (a). Images represent one biological replicate of 3, from the same biological blot stripped and re probed for the indicated proteins. Quantitative data shown as (b–e) (Student t-test, n = 3± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 5
Figure 5
Genetic KO of TAK1 in THP-1 cells recapitulate Takinib’s inhibition following stimulation. Immuno-blot representing relative TAK1, Cas9 and GAPDH protein expression in TAK1 KO, sgRNA control THP-1 cells, and wild type THP-1 cells (a,b). Western blot represents one biological replicate from the same gel. Following THP-1 cell differentiation as previously described, LPS (10 ng/mL) and IFNγ (50 ng/mL) treated THP-1 TAK1 KO cells release significantly less TNF and IL-6 than sgRNA control cells (c,d) (Students t-test, n = 3± SEM). TAK1 KO and sgRNA control THP-1 cells were differentiated as previously described and pro-inflammatory activated, simultaneously cells were treated in a dose dependent manner with Takinib. TNF and IL-6 secretion reported as percent of vehicle control treated cells (e,f). (2 way ANOVA with Sidak’s, n = 3± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 6
Figure 6
Takinib reduces TNF in a murine LPS challenge. FVB mice treated i.p. with varying concentrations of Takinib, once (qw) or twice weekly (qbw), caused no significant adverse events or weight change over 8 weeks of chronic treatment (a). Bone marrow derived macrophages (BMDM’s) were plated at (2 × 106/well) in a 24-well plate and treated with M-CSF (20 ng/mL) for 72 hours followed by a 24 hour rest period prior to activation with LPS (10 ng/mL) and IFNγ (50 ng/mL) and treatment with Takinib, at the indicated concentrations, or vehicle for 24 hours. Cytokine production was sampled from cell supernatant and examined by ELISA (b) (One-way ANOVA with Dunnett’s, n = 3± SEM). C57/bl6 male and female mice were sacrificed prior to LPS challenge and baseline TNF and IL-6 serum concentrations were examined by ELISA (c,d) (Student t-test, n = 3± SEM, 3 males, 3 females). C57/bl6 male and female mice were injected intraperitoneally (i.p.) with LPS (100 μg/kg in PBS) followed immediately by vehicle or Takinib (50 mg/kg in DMSO). 1 hour later, mice were sacrificed and blood samples clotted in centrifuge for 10 minutes at 3,000 rpm. Concentration of serum cytokines TNF and IL-6 determined by ELISA (c,d) (Student t-test, n = 5± SEM).

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Drutskaya MS, et al. Experimental models of arthritis in which pathogenesis is dependent on TNF expression. Biochemistry (Mosc) 2014;79:1349–1357. doi: 10.1134/S0006297914120086. - DOI - PubMed
    1. Komaki Y, et al. Efficacy, safety and pharmacokinetics of biosimilars of anti-tumor necrosis factor-alpha agents in rheumatic diseases; A systematic review and meta-analysis. J Autoimmun. 2017;79:4–16. doi: 10.1016/j.jaut.2017.02.003. - DOI - PubMed
    1. Spadaro A, et al. Remission in ankylosing spondylitis treated with anti-TNF-alpha drugs: a national multicentre study. Rheumatology (Oxford) 2013;52:1914–1919. doi: 10.1093/rheumatology/ket249. - DOI - PubMed
    1. Powrie F, et al. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity. 1994;1:553–562. doi: 10.1016/1074-7613(94)90045-0. - DOI - PubMed
    1. Roda G, Jharap B, Neeraj N, Colombel JF. Loss of Response to Anti-TNFs: Definition, Epidemiology, and Management. Clin Transl Gastroenterol. 2016;7:e135. doi: 10.1038/ctg.2015.63. - DOI - PMC - PubMed

Publication types

Substances

Feedback