LPS-induced TNF-alpha factor (LITAF)-deficient mice express reduced LPS-induced cytokine: Evidence for LITAF-dependent LPS signaling pathways

Abstract

Previously we identified a transcription factor, LPS-Induced TNF-alpha Factor (LITAF), mediating inflammatory cytokine expression in LPS-induced processes. To characterize the role of LITAF in vivo, we generated a macrophage-specific LITAF-deficient mouse (macLITAF(-/-)). Our data demonstrate that in macrophages (i) several cytokines (such as TNF-alpha, IL-6, sTNF-RII, and CXCL16) are induced at lower levels in macLITAF(-/-) compared with LITAF(+/+) control macrophages; (ii) macLITAF(-/-) mice are more resistant to LPS-induced lethality. To further identify LITAF signaling pathways, we tested mouse TLR-2(-/-), -4(-/-), and -9(-/-) and WT peritoneal macrophages exposed to LPS. Using these cells, we now show that LITAF expression can be induced after challenge either with LPS from Porphyromonas gingivalis via agonism at TLR-2, or with LPS from Escherichia coli via agonism at TLR-4, both requiring functional MyD88. We also show that, in response to LPS, the MyD88-dependent LITAF pathway differs from the NF-kappaB pathway. Furthermore, using a kinase array, p38alpha was found to mediate LITAF phosphorylation and the inhibition of p38alpha with a p38-specific inhibitor (SB203580) blocked LITAF nuclear translocation and reduced LPS-induced TNF-alpha protein levels. Finally, macLITAF(-/-) macrophages rescued by LITAF cDNA transfection restored levels of TNF-alpha similar to those observed in WT cells. We conclude that LITAF is an important mediator of the LPS-induced inflammatory response that can be distinguished from NF-kappaB pathway and that p38alpha is the specific kinase involved in the pathway linking LPS/MyD88/LITAF to TNF.

Fig. 1.

Confirmation of LITAF conditional knockout mice by Western blot. For Western blot analysis, macrophages (macLITAF^−/− or LITAF^+/+ as control) were stimulated with 0.1 μg/ml E. coli LPS for 16 h, and their extracts were detected by Western blot with antibody directed against murine LITAF or actin as control.

Fig. 2.

Phenotype of MacLITAF mouse. (a) ELISA of LITAF rescue. MacLITAF^−/− macrophages were seeded in a six-well plate at 2 × 10⁶ cells per well, and transiently transfected with varying concentrations of pcDNA-musLITAF expression vector DNAs or mock DNA (pcDNA3) as control, for 3 h, then washed with PBS and maintained overnight. The supernatants from each treated culture were used in triplicate ELISAs (Abraxis, Warminster, PA). ELISA immunoreactivity was quantified by using a VerSaDoc Imaging System (Bio-Rad) and graphed. The protein extracts (30 μg) from each treated group were analyzed by Western blot with antibody directed against LITAF or actin as control. (b) Murine cytokine antibody array. Membranes containing 62 cytokine antibodies (Array III and 3.1; RayBiotech, Norcross, GA) were blotted with equal amounts of conditioned medium from macrophages (macLITAF^−/− or LITAF^+/+ as control) after treatment with 0.1 μg/ml E. coli LPS, and were assessed according to the array manufacturer's protocol. Cytokine array experiments were performed three times, and the intensities of the relative expression levels of cytokines were quantified by densitometry (VerSaDoc Imaging System; Bio-Rad). All cytokine expression levels were normalized with positive signals obtained with biotin-conjugated IgG. The density value of each test sample was calculated and graphed. (c). MacLITAF^−/− mice were more resistant to LPS-induce septic shock. Survival after LPS administration is shown. Age-matched male macLITAF^−/− (n = 17) and LITAF^+/+mice (n = 14) were injected i.p. with LPS (0.25 μg per mouse). Mortality was assessed every hour for 24 h. macLITAF^−/− mice showed improved survival compared with LITAF^+/+; P < 0.05. See Table 1.

Fig. 3.

LITAF signaling elements. (a) Detection of LITAF expression in mouse macrophages (TLR-2^−/−, -4^−/−, -9^−/−, MyD88^−/−, or WT as control) after LPS treatment. Proteins extracted from the LPS-stimulated mouse macrophages (from different genotypes) were analyzed by Western blot using antibody against LITAF or to actin as a control. (b and c) Analysis of the effect of BAY 11-7082 on the LPS-induced LITAF or NF-κB gene expression in macLITAF^−/− cells. Proteins extracted from macrophages (macLITAF^−/− or LITAF^+/+ as control) that had undergone no treatment or treatment of 0.1 μg/ml E. coli LPS were measured by Western blot with antibody against LITAF, NF-κB p50, NF-κB p52, c-Rel, or actin (b). The supernatant from macLITAF^−/− or LITAF^+/+ cells treated with 0.1 μg/ml E. coli LPS alone or 5 μM BAY 11-7082 alone, or cotreated with 0.1 μg/ml E. coli LPS plus 5 μM BAY 11-7082 or untreated as control were used in triplicate ELISAs at the same conditions (Abraxis). (c) The immunoreactivity of each test sample was quantified by using a VerSaDoc Imaging System (Bio-Rad) and graphed. ∗, P < 0.05.

Fig. 4.

Kinase array. (a and b) The human phospho-MAPK array was used to detect multiple phosphorylated kinases in elutriated human monocytes, either untreated (a) or treated with 0.1 μg/ml E. coli LPS (b). (b) The strong signals of p38α/δ, ERK1/2 or Hsp27 in response to LPS treatment are indicated by arrows. (c) Analysis of the effects of kinase inhibitors on the LPS-induced LITAF nuclear translocation in WT mouse macrophages. Treatment with inhibitors, e.g., BAY 11-7082 (inhibits NF-κB, 5 μM), PD98059 (inhibits ERK1/2, 30 μM), U0126 (inhibits MEK, 10 μM) or did not show any effects on LITAF nuclear translocation, but SB203580 (inhibits p38 MAP kinase, 20 μM) completely blocked LITAF nuclear translocation, whereas the total level of LPS-induced LITAF expression was unchanged. Anti-β-tubulin antibody was used as a cytoplasmic marker to ensure the purity of the proteins extracted from nuclei because β-tubulin is expressed only in cytoplasm.

Fig. 5.

Detection of p38α and phosphorylated p38α protein levels by Western blot in 0.1 μg/ml E. coli LPS-treated human monocytes (a) or mouse WT or MyD88^−/− macrophages (b) with antibody against LITAF, MyD88, p38, phospho-p38, or actin as control.

Fig. 6.

LITAF phosphorylation and translocation. (a) The phosphorylated kinases were detected by human phospho-MAPK array after cotreatment of human monocytes with 0.1 μg/ml E. coli LPS plus 20 μM SB203580 for 4–16 h. No signal of p38α/δ was detected, in contrast to the strong signals of ERK2 or Hsp27, as indicated by arrows. (b) Western blot was used to detect translocation of p38α-mediated LITAF in mouse macrophages as follows. Protein extracts were collected at various times from whole cells or nuclei after cotreatment with LPS (lanes 2–8) and SB203580 (lanes 2–4). Proteins were detected with the following antibodies: LITAF, p38α, p-p38α and actin as control. (c) Levels of p38α protein and phosphorylated p38 detected by Western blot above (b) and normalized to actin and graphed. (d) Mouse macrophages were seeded in a 96-well plate at 2 × 10⁴ cells per well, and transiently transfected with 0.5 μg of pcDNA-musLITAF expression vector DNAs or treated with 20 μM SB203580 alone or with 0.1 μg/ml E. coli LPS alone or cotreated with 0.5 μg of pcDNA-musLITAF expression vector DNAs and/or 0.1 μg/ml E. coli LPS and/or 20 μM SB203580, then maintained for 16 h. The supernatants from each treated culture were used in three separate ELISAs (Abraxis) to see effects of p38α inhibitor (SB203580) on TNF-α production in the treated macLITAF^−/− or LITAF^+/+ macrophages as control. ELISA immunoreactivity was quantified by using a VerSaDoc Imaging System (Bio-Rad) and graphed. ∗, P < 0.05; ∗∗, not significant.

Fig. 7.

Diagram of the proposed LITAF signaling pathway. LITAF and STAT6B (5) are induced by P. gingivalis LPS via TLR-2 or by E. coli LPS via TLR-4. Their production is MyD88-dependent. Subsequently, they are phosphorylated by p38α before protein–protein interactions aimed at forming a complex. This phosphorylation leads to the sequestration of the complex in the cytoplasm before translocation of the molecules to the nucleus. In the nucleus, the complex most likely separates to allow for LITAF alone to bind to the specific sequence (CTCCC) (4) of various cytokine genes and thus to activate their transcription.