In 1989, Charles Janeway gave the introduction to the Cold Spring Harbor Symposium on Quantitative Biology, in which he proposed a revolutionary framework for immune recognition whereby pathogen-associated molecular patterns (PAMPs) are recognized by pattern recognition receptors (1). This classic paradigm remains a tenet of innate immunology today (2). Following Janeway’s “Approaching the Asymptote (1),” also featured in the Pillars of Immunology series, there was a rising interest in linking PAMPs with their cognate receptors, and LPS was at the top of the list. A prototypic PAMP, LPS is an outer membrane component produced by Gram-negative bacteria and was well known for its ability to increase humoral- and cell-mediated immune responses (3), the latter of which were readily measured by TNF-α production (4). In late 1998, using genetic evidence, Bruce Beutler’s group (5) proposed that TLR4 was the receptor for LPS; however, their publication lacked definitive functional evidence. Within a matter of weeks, Shizuo Akira’s group (6) confirmed that TLR4 was indeed the receptor for LPS and demonstrated this conclusively using functional and genetic studies. This paper is the focus of this installment of the Pillars of Immunology series.

The first clues about the identity of the LPS receptor came from reports published between 1960 and 1980 that described two mouse strains that were uniquely nonresponsive to LPS. The C3H/HeJ strain was highly resistant to sublethal doses of heat-killed Salmonella typhosa, with a calculated LD50 > 1000 μg of LPS (7). It was subsequently shown, by using bone marrow chimeras, that the LPS resistance observed in C3H/HeJ mice was conferred through hematopoietic cells (8). Another strain, C57BL/10ScCr, was also identified to be resistant to LPS (9). When these mice were crossed with C3H/HeJ mice, the F1 generation lacked complementation of the LPS response and exhibited the parental resistance to LPS, suggesting a single lps locus that coded for a nonredundant LPS receptor (10).

Meanwhile, other investigators worked to determine the mediators involved in LPS signaling. CD14 was indicated to play a role in LPS signaling early on, because anti-CD14 Ab abrogated LPS-driven TNF-α production (11); however, CD14 was a cell surface protein that could not potentiate the LPS signal across the cellular membrane. Additionally, the LPS-binding protein (LBP) present in serum was thought to control the LPS response by binding to LPS and creating LBP-LPS complexes that would bind to immune cells and signal through the unknown receptor for LPS (12). After TLR4 was discovered as the receptor for LPS, another cell surface protein, MD-2, was shown to also interact with TLR4 during LPS signaling (13). Crystal structures further demonstrated, using LPS (14) and Eritoran, an analog of LPS (15), that TLR4 and MD-2 dimerized with the Lipid A component of LPS and then, like other members of the IL-1/IL-18/Toll receptor superfamily, induced NF-κB through the signal adaptor protein MyD88, which was featured previously in Pillars of Immunology (16).

Another important clue to the identity of the receptor for LPS came from an unexpected place, the Drosophila Toll protein. Toll is a Drosophila type 1 transmembrane protein initially ascribed to establishing dorsal–ventral polarity (17), but it was later discovered to also be critical for antifungal responses and Drosophila survival after fungal infection (18). Drosophila Toll was also known to be remarkably similar to IL-1R (19), which, when stimulated by IL-1, induced activation of NF-κB (20), a human homolog of Drosophila dorsal protein. The localization of a gene encoding a human analog of Toll on chromosome 4p14 (21) increased the likelihood that human TLR played an important role in the activation of inflammation (22). Furthermore, a constitutively active human homolog of the Drosophila Toll receptor, hToll, was found to activate NF-κB and increase expression of proinflammatory cytokines IL-1, IL-6, and IL-8 (23).

Several reports using a molecular biology approach were published between 1998 and 1999 that supported TLR2 as the receptor for LPS. Yang et al. (24) reported that HEK 293 cells expressing an epitope-tagged version of TLR2 had increased reporter gene activity when stimulated with LPS relative to unmodified HEK 293 cells. Additionally, Kirschning et al. (25) observed that overexpression of TLR2 in an NF-κB luciferase-transfection system resulted in increased luciferase activity after stimulation with LPS. In contrast, Heine et al. (26) found that Chinese hamster ovary fibroblasts transfected with CD14 were highly responsive to LPS yet lacked the transmembrane and intracellular domains of TLR2, suggesting that TLR2 is not required for the LPS response.

Simultaneously, other groups were pursuing the question using genetic mapping of LPS-resistant mice. A single backcross of the C3H/HeJ mice determined that the Lps locus was located on chromosome 4, in between the loci for major urinary protein and polysyndactyl (27). Positional cloning done by Poltorak et al. (28) used two separate backcrosses of the LPS-resistant mouse strain C3H/HeJ and localized the Lps locus to a 0.9-cM genetic interval. They identified gene candidates by subsequent sequencing runs that yielded only one intact gene, TLR4. Poltorak et al. (5) and Hoshino et al. (6) identified a single point mutation in the TLR4 gene in C3H/HeJ mice that resulted in replacement of a proline with a histidine. This point mutation changed a highly conserved Toll family residue; these results were published initially by Beutler’s group and then a few weeks later by Akira’s group. However, in addition to identifying a point mutation in C3H/HeJ mice and showing that mutant TLR4 was functionally impaired, Hoshino et al. (6) generated TLR4−/− mice and used them in functional studies. TLR4−/− mice were unresponsive to LPS and synthetic Lipid A, conclusively demonstrating that TLR4 was the receptor for LPS (6). This conclusion was supported by other studies (29, 30).

In the wake of the controversy surrounding whether TLR2 or TLR4 was the receptor for LPS, the preparation of LPS was discovered to be key in elucidating immune responses (31). LPS preparations are typically contaminated with other microbial products, including lipopeptides (TLR2 agonists) (32, 33) and peptidoglycan fragments (nucleotide-binding oligomerization domain 1 and 2 agonists) (34, 35), which are found in bacterial cell walls and are known to stimulate responses via their cognate innate immune receptors. Repurification of LPS and the use of transfected human cell lines deficient in either TLR2 or TLR4 confirmed that TLR4 was indeed the receptor for LPS (36) and suggested that low levels of lipopeptides in LPS preparations were responsible for the positive results seen with TLR2. This was further demonstrated through the generation of TLR2−/− mice [by Takeuchi et al. (37)], which responded well to LPS but poorly to components of Gram-positive bacterial cell walls.

In summary, Akira’s group was the first to link genetic evidence for TLR4 as the receptor for LPS with functional studies using TLR4-deficient mice, and for this reason it was chosen to be featured in Pillars of Immunology. This characterization of the first mammalian TLR was pivotal for our understanding of innate immunology signaling and influenced future studies of TLRs, earning its place as one of the 100 most highly cited articles published in The Journal of Immunology.

Abbreviations used in this article:

LBP

LPS-binding protein

PAMP

pathogen-associated molecular pattern.

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The authors have no financial conflicts of interest.