TNF-α gene expression is elevated in FA-C lymphoblasts. (A) Mutant FA-C lymphoblasts (HSC536N and PD149) produce more TNF-α than isogenic complemented FA-C/C cells. Secreted TNF-α was measured in culture media by the use of ELISA assays. Shown are results from 1 of 3 independent experiments. All error bars in this figure represent mean (± SD). (B) TNF-α RNA is elevated in FA-C lymphoblasts. TNF-α mRNA is increased in FA-C cells. TNF-α mRNA was analyzed by real-time RT-PCR. Results shown are from 1 of 3 identical experiments. (C) TNF-α mRNA decay rates are equivalent in mutant and complemented cells. To rule out differential TNF-α mRNA decay as an explanation for the increase in mutant cells, mRNA stability was measured by real-time RT-PCR after treatment of FA-C lymphoblasts (HSC536) with the transcription inhibitor actinomycin-D. The mRNA decay rates were identical, demonstrating that the differences in mRNA levels between FA-C and FA-C/C cells are attributable to differences in mRNA production rather than differences in mRNA stability. (D) TLR8 siRNA decreases TLR8 and TNF-α mRNA in FA-C cells. By using real-time RT-PCR for both TLR8 and TNF-α mRNA, we found that both were reduced in FA-C cells treated with siRNA targeting TLR8, demonstrating that TNF-α production in these cells is TLR8-dependent. (E) Ground-state activation of NF-κB was demonstrated by use of the electromobility shift assay in 2 different FA-C lymphoblastoid cell lines in the 2 lanes to the far left, HSC536N and HSC536N/FANCC (complemented with FANCC cDNA) and in the 6 lanes on the right, PD149 and PD149/FANCC. The top right panel is a short exposure of the same gel exposed longer shown in the bottom right panel. NF-κB signal is reduced in the both complemented cells. Supershift experiments were performed by the use of nuclear lysates of PD149 and PD149/FANCC cells and revealed that both p50 and p65 (best seen on the longer exposure) were contained within the NF-κB band. Shown are results of 1 representative of 4 identical experiments. Autoradiographs of both full gels are shown in supplemental Figure 7. This figure also shows that in mutant FA-C lymphoblasts, NF-κB activation is high in the ground state and does not require exposure to interferon-γ or TNF-α. Again, this is distinctly different from primary mononuclear phagocytes and the THP-1 shFANCC cells, which require exposure to a TLR-agonist to reveal overexpression of TNF-α.

Inhibition of the TLR8 pathway suppresses R848 activation of NF-κB in FANCC-deficient cells. THP-1 Blue cells expressing either control (nontargeted shRNA) or FANCC shRNA (target and shRNA sequences are shown in supplemental Table 4) were pretreated for 2 hours with 25μM IKK inhibitor (A) or 25μM IRAK-1/4 inhibitor (B) and then incubated for 24 hours with 30μM R-848. Both inhibitors reduced NF-κB activation in THP-1 Blue cells exposed to R-848. (C) Stable expression of TLR8 shRNA (target and shRNA sequences are shown in supplemental Table 4) in THP-1 Blue cells reduced TLR8 mRNA levels by 73%. Nontargeted shRNA had no effect on TNF-α mRNA. (D) Stable coexpression of TLR8 shRNA with either control (nontarget) or FANCC shRNAs lowered R-848–induced NF-κB activation. Specificity of TLR8 shRNA was confirmed by in experiments that showed that this shRNA did not suppress LPS-induced NF-κB activity (not shown). Error bars represent mean (± SD). (E) NF-κB activation (assessed by quantification of SEAP, y-axis) was quantified in the ground state and in cells treated with R848 in the presence and absence of SB203580, a p38 MAPK inhibitor. The inhibitor suppressed the R848 response in both control and shFANCC cells. (F) TNF-α production was likewise suppressed by SB203580 in THP1 Blue shFANCC cells treated with R848. Control cells not treated with R848 did not produce TNF-α. (G) Likewise, high-level endogenous TNF-α production in FANCC-deficient HSC536 lymphoblasts (control) was also suppressed by SB203580. HSC536/FANCC cells are isogenic FA-C lymphoblasts expressing wild-type FANCC cDNA.

TLR8-ubiquitin coimmunoprecipitation. Immunoprecipitations were performed by the use of antiubiquitin antibodies (lanes 1-2) or nonspecific control immunoglobulin (lanes 3-4), and immunoblots of the precipitated material were performed by the use of anti-TLR8 antibodies (left). A greater amount of ubiquitinylated TLR8 was detected in immunoprecipitates of mutant cells, confirming the proteomics result. The distinct upper band (lane 1 vs lanes 3-4) is consistent with monoubiquitinylation (8.5 kDa). Total TLR8 protein levels in whole cell lysates were identical in mutant and complemented cells (right). The data shown are representative of 3 identical experiments. A second experiment with loading controls and nonspecific immunoglobulin controls is shown in supplemental Figure 1.

FANCC suppresses TNF production and NF-κB activation induced by TLR8 ligands. Results in THP-1 cells are from 1 of 4 identical experiments. THP-1 Blue cells transduced with control (not targeted) shRNA or with shRNA targeting FANCC were incubated 24 hours with various concentrations of R-848. Expression of the NF-κB reporter was quantified colorimetrically (A), and TNF-α production was quantified by the use of ELISA (B). Dose-response curves shown are representative of 4 (A) and 2 (B) assays. SEAP and TNF-α responses of untransduced cells matched precisely those of cells transduced with the control shRNA (not shown). The specific lentiviral shRNA targeting FANCC (shFANCC) was selected based upon its capacity to induce MMC hypersensitivity and suppress monoubiqutinylation of FANCD2 in THP1 Blue cells (data are shown in supplemental Figure 5). (C) TNF-α release from primary murine splenic cd11b⁺ macrophages exposed for 24 hours to multiple doses of R848 showed a 4-fold increase in TNF-α released in cultures of Fancc^−/− macrophages. Macrophage-depleted mononuclear cells produced no detectable TNF-α either before or after exposure to R848. Error bars represent mean (± SD).

TNF-α overproduction in FA-C cells is not a secondary effect of hypersensitivity to cross-linking agents. (A) FANCC alanine substitutions are located in 3 highly conserved domains.² M55 is a naturally occurring truncation mutant. All of the alanine mutants are known to fully complement FA-C cells in the MMC sensitivity test.² (B) HSC536 cells were transduced with wild-type FANCC cDNA (bar 2), 2 naturally occurring FANCC mutant constructs (bar 1, L554P and bar 9, M55) and 6 engineered point mutant constructs. With the exception of the partially effective S249A mutant and the completely effective normal cDNA (“wild-type”), which normalized TNF production in FA-C cells, TNF-α levels remain high in all of the isogenic cells expressing the other alanine mutants and M55.