Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63 ubiquitin chain formation

Abstract

Dimethyl fumarate (DMF) possesses anti-inflammatory properties and is approved for the treatment of psoriasis and multiple sclerosis. While clinically effective, its molecular target has remained elusive - although it is known to activate anti-oxidant pathways. We find that DMF inhibits pro-inflammatory cytokine production in response to TLR agonists independently of the Nrf2-Keap1 anti-oxidant pathway. Instead we show that DMF can inhibit the E2 conjugating enzymes involved in K63 and M1 polyubiquitin chain formation both in vitro and in cells. The formation of K63 and M1 chains is required to link TLR activation to downstream signaling, and consistent with the block in K63 and/or M1 chain formation, DMF inhibits NFκB and ERK1/2 activation, resulting in a loss of pro-inflammatory cytokine production. Together these results reveal a new molecular target for DMF and show that a clinically approved drug inhibits M1 and K63 chain formation in TLR induced signaling complexes. Selective targeting of E2s may therefore be a viable strategy for autoimmunity.

Figure 1. Inhibition of LPS stimulated cytokine induction by DMF.

(A) BMDMs were incubated with 0, 25, 50, 75 or 100 μM DMF for 4 h. Cells were then stimulated with 100 ng/ml LPS for 8 h and the levels of TNF, IL-6, IL-10, IL-13 and GM-CSF determined. (B) BMDMs were incubated with 50 μM DMF for the indicated times and cell viability was determined by staining with Zombie-Aqua. Results show average and standard deviation of 4 independent treatments. (C) BMDMs were incubated in 50 μM DMF where indicated. Cells were then stimulated with 100 ng/ml LPS for 5 h. Cells were then stained for intracellular TNF levels and viability (Zombie-Aqua) as described in the methods. Representative stimulations are shown in the FACS plots while the graphs show average and standard deviation from 3 independent stimulations. In (A,C) a p value (Students t-test) of less than 0.001 relative to the LPS alone condition is indicated by ***.

Figure 2. DMF inhibits LPS induced gene transcription.

BMDMs were incubated with 50 μM DMF for 4 h and then stimulated with LPS for 1 h as indicated. Total RNA was then isolated and the induction of TNF, IL-6, IL-10, GM-CSF, IL-12p40, IL-23p19, IκBα and IFNβ determined by qPCR as described in the methods. Error bars represent the standard deviation of 4 BMDM cultures. For a comparison of LPS stimulation with or without DMF treatment a p value (Students t-test) of <0.01 is indicated by ** and p < 0.001 by ***.

Figure 3. Effect of DMF on LPS stimulated mRNA induction in Nrf2 knockout BMDMs.

BMDMs were isolated from wild type and Nrf2 knockout mice. Cells were incubated with 50 μM DMF for 4 h and then stimulated with LPS for 1 h as indicated. Total RNA was then isolated and the induction of TNF, IL-6, IL-10, GM-CSF. IL-12p40, IL-23p19, IκBα and HO-1 determined by qPCR. Error bars represent the standard deviation of measurements from independent cultures from 4 mice per genotype. For a comparison of LPS stimulation in the presence and absence of DMF a p value (Students t-test) of <0.05 in indicated by *, p < 0.01 by ** and p < 0.001 by ***. For a comparison between wild type and Nrf2 knockout cells a p < 0.05 is indicated by ^# and p < 0.01 by ^##.

Figure 4. Effect of DMF on LPS stimulated mRNA induction in MSK1/2 knockout BMDCs.

BMDCs were isolated from wild type and MSK1/2 double knockout mice. Cells were incubated with 50 μM DMF for 4 h and then stimulated with LPS for 1 h or 16 h as indicated. Total RNA was then isolated and the induction of IL-10, IL-6, GM-CSF, IL-12p35, IL-12p40 and IκBα was determined by qPCR. Error bars represent the standard deviation of measurements from independent cultures from 4 (0 and 1 h) or 3 (16 h) mice per genotype. A p value (Students t-test) between the presence and absence of DMF of less that 0.01 is indicated by ** and a p < 0.001 by ***. For a comparison of wild type and MSK1/2 knockout cells in the absence of DMF, p < 0.05 and p < 0.01 are indicated by ^# and ^## respectively.

Figure 5. DMF inhibits multiple signals downstream of TLR4.

(A) TLR4 activates both NFκB and MAPK pathways via Tak1 and IKK in order to drive cytokine production. (B) BMDMs were treated with the indicated concentrations of DMF for 4 h. Cells were then stimulated with 100 ng/ml LPS for 30 min. Following cell lysis the levels of the indicated total and phospho proteins were determined by immunoblotting. (C) BMDMs were cultured in the presence or absence of 50 μM DMF for 4 h. Cells were then further stimulated for the indicated times with either 100 ng/ml LPS, 1 μg/ml Pam₃CSK₄ or 10 μg/ml poly(I:C). The levels of the indicated total and phospho proteins were determined by immunoblotting.

Figure 6. DMF can activate p38 MAPK and can covalently modify its targets in the cell.

BMDMs were incubated in 50 μM DMF for the indicated times. Alternatively BMDMs were stimulated with 100 ng/ml LPS for 30 min (L) or 60 μM sodium pervanadate for 10 min (P). (A) The levels of the indicated total and phospho proteins were determined by immunoblotting. (B) Lysates from A were immunoblotted for p-Tyr. Molecular weights (kDa) are indicated on the left and two exposures are shown. (C) Where indicated BMDMs were treated with 50 μM DMF for 4 h. Cells were then washed 3 times to remove the DMF and incubated for the indicated washout times. Finally cells were stimulated with 100 ng/ml LPS for 30 min. The levels of the indicated total and phospho proteins were determined by immunoblotting.

Figure 7. DMF can inhibit E2 conjugating enzymes in vitro.

(A) Reaction mechanism of DMF and cysteine. (B) Ubc13 or UbcH7 was incubated in the presence or absence of DMF for 30 minutes. E2 loading reactions for Ubc13 and UbcH7 in the presence of ubiquitin, Ube1 and Mg-ATP were carried out in the presence of increasing concentrations of DMF as described in the methods. Ubiquitin loading was resolved on 4-12% polyacrylamide gels. Example gels are shown on the left and the quantification of 3 separate reactions shown on the right. Error bars represent standard deviation. For DMF treated conditions, a p value of less than 0.001 (Students t-test) is indicated by ***. (C) Ubc13 or UbcH7 was incubated in the presence or absence of 50 μM DMF for 4 h. The molecular mass was then determined by MALDI-TOF mass spectrometry. Spectra show intensity vs. m/z and inserts show an expansion of the 2+ peak. (D) The indicated E2 conjugating enzymes were incubated with either 25 or 100 μM DMF for 30 minutes and E2 loading assays carried out as described in the methods. The % E2 loading relative to the 0 μM condition for each E2 tested is shown.

Figure 8. Effect of DMF on LPS stimulated polyubiquitin chain formation in RAW264.7 macrophages. RAW264.7 cells were incubated in the presence or absence of 50 μM DMF for 4 h.

Cells were then either left unstimulated or treated with 100 ng/ml LPS for a further 30 min. Cells were lysed and lysates were blotted for the phospho and total proteins indicated (A). Alternatively, Halo-NEMO beads were used to pulldown M1 polyubiquitin chains. Pull downs were then immunoblotted with antibodies specific for M1 chains, K63 chains, IRAK1 or IRAK2. Total ubiquitin was pulled down using Halo-TUBE beads and blotted for the presence of K48 chains (B).

Figure 9. Effect of DMF on LPS stimulated polyubiquitin chain formation in BMDMs.

(A) BMDMs were incubated in the presence or absence of 50 μM DMF for 4 h. Cells were then either left unstimulated or treated with 100 ng/ml LPS for a further 30 min. Halo-NEMO beads were used to pulldown M1 polyubiquitin chains. Pull downs were then immunoblotted with antibodies specific for M1 chains or IRAK2 (left hand panels). Alternatively the levels of IRAK1, phospho p105, phospho ERK1/2 and total ERK1/2 in the cell extracts were determined by immunoblotting (right hand panels). (B) As (A) but Halo-TUBE pull downs were used to isolate total ubiquitin chains and the pull downs blotted with antibodies against M1 and K48 chains.

Figure 10. Effect of DMF on IL-1 and TNF stimulated M1 chain formation.

(A) IL-1R HEK293 cells were incubated with 50 μM DMF for 4 h where indicated. Cells were stimulated with 5 ng/ml IL-1β for 30 min and the levels of phospho p105, phospho IKK, total IκBα, phospho ERK1/2 and total ERK1/2 determined by immunoblotting. (B) As (A) but Halo-NEMO beads were used to pull down M1 polyubiquitin chains. Pulldowns were blotted for M1 and K63 linked polyubiquitin chains. (C) HeLa cells were incubated with 50 μM DMF for 4 h where indicated. Cells were stimulated with 10 ng/ml TNF for 30 min and the levels of phospho p105, phospho IKK, total IκBα, phospho ERK1/2 and total ERK1/2 determined by immunoblotting. (D) As (C) but Halo-NEMO beads were used to pull down M1 polyubiquitin chains. Pull downs were blotted for M1 and K63 linked polyubiquitin chains.