Abrupt Expression of TLR4 in TLR4-Deficient Macrophages Imposes a Selective Disadvantage: Genetic Evidence for TLR4-Dependent Responses to Endogenous, Nonmicrobial Stimuli

Abstract

TLR4 is crucial for macrophage responses to LPS. It is less clear whether TLR4 may also transduce signals from host factors, and if so, with what consequences. Immortalized bone marrow-derived macrophage cell lines, termed T4Cr and T4ko, were established from TLR4null strains, C57BL/10ScNCr and TLR4 knockout mice, respectively. Multiple transfections and selections were conducted to stably introduce TLR4 into these cell lines. Among 196 individual clones isolated, 48 expressed TLR4 on the cell surface but did not respond to LPS due to a deletion in the MyD88 gene. The remaining clones integrated TLR4 DNA into the genome but expressed neither detectable TLR4 mRNA nor TLR4 protein. To test the possibility that TLR4null cells lack modulating factors to protect against a harmful effect of TLR4, 15 stably transfected clones were generated in the presence of conditioned media from wild-type macrophages. Some of these cells expressed a small amount of TLR4 and regained responsiveness to LPS. Because no microbial ligands were available to the cell lines during their generation, signaling via endogenous ligands is likely to have occurred in TLR4-expressing, signal-competent macrophages and imposed a proliferative or other selective disadvantage. These studies support the existence of constitutive signaling via TLR4 during in vitro culture of macrophages without microbial products, and help account for the lack of reports of restoration of TLR4 expression in normally TLR4-expressing types of cells in vitro whose TLR4 genes are deleted or disrupted.

Transcriptional regulation of bone sialoprotein gene by Porphyromonas gingivalis lipopolysaccharide

“…In this study, we could not conclude which TLR was activated by P. gingivalis LPS, despite of 0.01 µg/ml P. gingivalis LPS induced both TLR4 and TLR2 mRNA expression in ROS17/2.8 (data not shown). TLR4 as a transmembrane protein contains a conserved intracellular domain and occurs in a series of intracellular adaptors including mitogen activated protein kinase (MAPK) family (p38, ERK1/2, JUNK) signaling pathways, which play a critical role in the LPS‐induced inflammation and periodontal bone loss [Darveau et al, 2002; Wang and Ohura, 2002; David et al, 2005; Cohn et al, 2006; Kirkwood et al, 2007]. Since BSP transcription is regulated mainly by tyrosine kinase, cAMP, and MAPK [Shimizu‐Sasaki et al, 2001; Nakayama et al, 2006; Ogata, 2008], we investigated the effects of several kinase inhibitors on P. gingivalis LPS regulated BSP gene transcription.…”