Intracellular TLR4/MD-2 in macrophages senses Gram-negative bacteria and induces a unique set of LPS-dependent genes

Int Immunol. 2011 Aug;23(8):503-10. doi: 10.1093/intimm/dxr044. Epub 2011 Jun 28.


Toll-like receptor (TLR)4/MD-2, a sensor for LPS, delivers the MyD88-dependent signal from the cell surface, then traffics to endolysosomes and delivers the TRIF/TICAM-1-dependent signal. Both signals are thought to be dependent on cell surface TLR4/MD-2. Although TLR4/MD-2 is located also in recycling endosomes, the Golgi apparatus or the endoplasmic reticulum, little is known about a role for intracellular TLR4/MD-2 in LPS responses. We here studied intracellular LPS sensing in macrophages. PRAT4A (protein associated with TLR4 A) is a cochaperone for a general chaperone gp96 and required for cell surface expression of TLR4/MD-2. Cell surface TLR4/MD-2 was undetectable on PRAT4A(-/-) thioglycollate-elicited peritoneal macrophages (P-Macs) and bone marrow-derived macrophages (BM-Macs). LPS responses were all abolished in PRAT4A(-/-) P-Macs, whereas a part of LPS responses remained detectable in PRAT4A(-/-) BM-Macs. Of note, LPS responses in PRAT4A(-/-) BM-Macs were not necessarily dependent on TRIF/TICAM-1 signaling. PRAT4A(-/-) BM-Macs showed unimpaired production of both TRIF/TICAM-1-dependent chemokine RANTES (CCL5) and MyD88-dependent chemokine MCP-1 (CCL2). Moreover, up-regulation of co-stimulatory molecules, CD40 and CD86 was not altered. In contrast, TRIF/TICAM-1-dependent production of type I IFN was profoundly impaired. In response to heat-killed bacteria Escherichia coli, BM-Macs also required PRAT4A-independent TLR4/MD-2 for production of MCP-1 (CCL2) and RANTES (CCL5) and for up-regulation of CD40 and CD86, indicating that intracellular TLR4/MD-2 is able to sense phagocytosed bacteria and activate immune responses. These results demonstrate that intracellular TLR4/MD-2 is responsible for unique set of LPS responses.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blood Cells / immunology
  • Blood Cells / metabolism
  • Bone Marrow Cells / immunology
  • Bone Marrow Cells / metabolism
  • Cytokines / biosynthesis
  • Gene Expression Regulation* / drug effects
  • Gene Expression Regulation* / immunology
  • Gram-Negative Bacteria / immunology*
  • Intracellular Space / immunology
  • Intracellular Space / metabolism
  • Lipopolysaccharides / immunology*
  • Lipopolysaccharides / pharmacology
  • Macrophages / immunology*
  • Macrophages / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*
  • Up-Regulation / genetics
  • Up-Regulation / immunology


  • Cytokines
  • Lipopolysaccharides
  • Toll-Like Receptor 4