How Discovery of Toll-Mediated Innate Immunity in Impacted Our Understanding of TLR Signaling (and Vice Versa) This information is current as of September 24, 2021. Stefanie N. Vogel J Immunol 2012; 188:5207-5209; ; doi: 10.4049/jimmunol.1201050 http://www.jimmunol.org/content/188/11/5207 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. How Discovery of Toll-Mediated Innate Immunity in Drosophila Impacted Our Understanding of TLR Signaling (and Vice Versa) Stefanie N. Vogel

was delighted when The Journal of Immunology invited its body (Fig. 5 in Ref. 1) is undoubtedly the most widely me to write a Pillars of Immunology article on one of my reproduced figure shown in innate immunity lectures today! I favorite papers, the 1996 publication titled, “The Dor- The implications of this paper were far-reaching in that it soventral Regulatory Gene Cassette spa¨tzle/Toll/cactus Con- impacted the discovery of mammalian TLRs by providing trols the Potent Antifungal Response in Drosophila Adults” the first direct evidence that resistance to was the Downloaded from by Bruno Lemaitre et al. (1). In this seminal publication, the consequence of inducible expression of antimicrobial genes authors deftly combined genetic, biochemical, and molecular secondary to activation of the Toll NF-kB–like signaling approaches to reveal that the Toll signaling pathway, origi- pathway. The significance of this work was acknowledged nally recognized to regulate dorsoventral patterning in Dro- recently by naming the senior author, Jules Hoffmann, as sophila embryos by activating an NF-kB–like Rel protein corecipient of one half of the 2011 Nobel Prize in Physiology (recently reviewed in Ref. 2), was also responsible for tran- or Medicine. http://www.jimmunol.org/ scriptional activation of the gene that encodes Drosomysin, In a 2004 review, Lemaitre (3) provided a timeline of key an antifungal peptide. Using adult flies with gain-of-function observations in both insect and mammalian immunology or loss-of-function mutations in genes shown previously to that resulted in a convergence of ideas, leading first to their contribute to induction of both the antifungal peptide Droso- landmark publication and ultimately to the discovery of mycin and antibacterial peptides, Lemaitre et al. found the mammalian TLRs. Notably, the identification of inducible following: (1) The embryonic signaling pathway comprising antibacterial peptides in insects (4) and mammals (5), coupled gene products between spz and cact, and not upstream or with the finding that genes encoding antibacterial Attacin downstream genes—that is, genes constituting the Toll signal- peptides contained NF-kB–like binding sites in their pro- by guest on September 24, 2021 ing “cassette”—was necessary for the antifungal response in moters (6), pointed to a signaling pathway in insects that adult flies; (2) although this same pathway contributes par- was homologous to known immune signaling pathways lead- tially to the induction of antibacterial gene expression (includ- ing to NF-kB translocation in mammals. However, in 1991, ing genes encoding Cecropins, Attacin, and insect Defensin), Gay and Keith (7) and Schneider et al. (8) independently an additional pathway regulated by a distinct locus, imd, was reported a significant degree of homology between the also necessary for antibacterial peptide gene expression; (3) cytoplasmic domains of Drosophila Toll protein and the expression of other antibacterial peptide genes, Diptercin and human IL-1R. Gay and Keith found that this homology ex- Drosocin, is regulated by the imd signaling pathway, apparently tended for 135 aa with identical or conservative substitutions without cross-talk from the Toll signaling pathway; and (4) at 45% of positions (now known as the Toll/IL-1R/resistance finally, and most importantly, adult flies with mutations in the [TIR] domain). Consistent with this finding, Heguy et al. (9) Toll signaling pathway succumb to infection with the fungus described a 50-aa subregion within the TIR domain that was Aspergillus fumigatus, but not the bacterium Escherichia coli, essential for IL-1–mediated activity. When coupled with the and conversely, flies with defects in imd failed to survive E. coli 1988 observation by van der Meer et al. (10) that a low dose infection owing to uncontrolled bacterial replication, yet re- of IL-1 protected granulocytopenic mice from lethal Gram- sisted infection with A. fumigatus. Flies with mutations in both negative infection, it is clear that the idea of TIR domain- pathways failed to survive fungal or bacterial challenge. The mediated “nonspecific” host immunity was actually already image of the dead adult fly with fungal hyphae extending from established for mammals in the early 1990s. That NF-kB translocation to the nucleus was observed in IL-1–treated cells and paralleled the signal transduction events

Department of Microbiology and Immunology, University of Maryland School of mediated by Drosophila Toll upon activation by its endog- Medicine, Baltimore, MD 21201 enous ligand, spa¨tzle,provided further support for the idea S.N.V. is supported by National Institutes of Health Grant R01AI018797. that parallel signaling pathways leading to NF-kBtrans- Address correspondence and reprint requests to Dr. Stefanie N. Vogel, University location might regulate induction of innate immune response of Maryland, 685 West Baltimore Street, HSF1 Suite 380, Baltimore, MD 21201. genes in both insects and higher species. Between 1993 and E-mail address: [email protected] 1995, multiple studies in Drosophila provided evidence for an Abbreviation used in this article: TIR, Toll/IL-1R/resistance. additional, nondevelopmental role for Toll in insect immu- Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 nity (11–15), culminating in a series of papers by Lemaitre www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201050 5208 PILLARS OF IMMUNOLOGY et al. (16, 17), including their 1996 Cell paper showing that Although many laboratories attempted to clone the Lps gene mutations in Toll and imd in vivo resulted in increased sus- by various approaches (reviewed in Refs. 22 and 23), Bruce ceptibility to infection in Drosophila (1). Beutler and colleagues (29) were successful using a positional The idea of the existence of cellular receptors that could cloning approach to clone the murine TLR4 gene. This sense pathogens and deliver “danger” signals to cells was put achievement was followed by a landmark paper by Poltorak forward in 1989 by the late Charles Janeway (18). He pro- et al. (30), published in December 1998, showing that the posed that such receptors on mammalian APCs would not C3H/HeJ mouse strain carried a point mutation in the TIR only recognize conserved pathogenic structures but also signal domain of Tlr4 and that Tlr4 was largely deleted in the intracellularly, leading to the transcriptional upregulation of C57BL/10ScCr strain. For this discovery, Dr. Beutler was Dr. costimulatory molecules based, in part, on the observation Hoffmann’s corecipient of the 2011 Nobel Prize that was also that “a whole variety of microbial substances could stimulate shared with Dr. Ralph Steinman for his pioneering work on the antigen-presenting cells to express what we believed to dendritic cells. be costimulatory activity” (19). In 1997, Ruslan Medzhitov, An avalanche of studies in mammalian innate immunity while working with Dr. Janeway, cloned “hToll” (later identi- soon ensued. Within a few months after Poltorak et al. (30) fied as human TLR4) (20). Because these investigators did not published their findings, independent confirmation was pro- know the nature of the ligand for hToll, or if there existed vided by Malo and colleagues (31), followed in short order by a spa¨tzle homolog in mammals, they engineered a construct work from Shizuo Akira’s laboratory (32) showing that mice 2/2 that encoded the CD4 ectodomain with the TLR4 intracellular with a targeted mutation in TLR4 (i.e., TLR4 mice) both Downloaded from domain. This would have allowed them to cross-link the CD4 were unresponsive to LPS and exhibited increased suscepti- ectodomains with anti-CD4 Ab, if necessary. When this CD4– bility to infection. Kawai et al. (33) soon reported that 2 2 hToll fusion protein was overexpressed in mammalian cells, MyD88 / mice were refractory to lethal challenge with LPS 2 2 however, it constitutively activated NF-kB and resulted in the in vivo and that MyD88 / macrophages responded poorly upregulation of costimulatory molecules, as originally pre- to LPS to express NF-kB–dependent cytokine genes. This dicted by Janeway. This work, published just 1 y after Lemaitre observation was key because MyD88, the functional ho- http://www.jimmunol.org/ et al. (1), represents the first direct link between TLR signaling in molog of Drosophila MyD88 (dMyD88), served as a critical mammalian cells that leads to NF-kB activation, that in turn, adapter protein for both IL-1R and TLRs (reviewed in upregulates the costimulatory molecules, and thereby facilitates Ref. 34). However, although most assumed that the TLR4 the transition between innate and adaptive immune responses ligand was bacterial LPS, Shimazu et al. (35) showed that in mammals. The cloning of five human TLRs (named TLR1– MD-2, a protein that binds noncovalently to the extracellular 5) soon followed (21) and, importantly, these authors mapped domain of TLR4, serves as the actual “receptor” for LPS, the locations of each on the human genome. Still, the ligands whereas TLR4 serves as the signal-transducing component of

for these human TLRs were not known at the time. a larger “TLR4 signaling complex.” by guest on September 24, 2021 In contrast to the events that led to identification of human It is impossible in this venue to begin to detail the many TLRs, the cloning of murine TLR4 was preceded by more other studies constituting the unprecedented intellectual ex- than 30 y of publications showing that the LPS-unresponsive plosion in the field of innate immunity that followed the C3H/HeJ mouse strain, which arose by spontaneous mutation 1996 work of Lemaitre et al. Nonetheless, the first experi- sometime between 1960 and 1965, was highly susceptible to mental linkage of Toll signaling to innate immunity in Dro- infection by Gram-negative bacteria (reviewed in Refs. 22 and sophila by Lemaitre et al. is certainly deserving of recognition 23). With the use of standard genetic mapping techniques, as a Pillar of Immunology. the Lps gene (Lpsn, for the allele encoding normal LPS re- sponsiveness, and Lpsd, for the defective allele) was mapped to mouse Chromosome 4 and was inherited codominantly Acknowledgments (reviewed in Ref. 22). Macrophages from C3H/HeJ mice I thank Douglas Golenbock, Neal Silverman, Martin Flajnik, and Charles Dinarello for thoughtful discussions and insights during the preparation of failed to respond to highly purified LPS or lipid A to produce this article. cytokines or become tumoricidal in vitro, but responded normally to “endotoxin-associated proteins” that were present in less rigorously purified LPS preparations (24–27). Hence, a Disclosures relationship between sensitivity to a bacterial product (i.e., The author has no financial conflicts of interest. LPS or bacterial proteins) and resistance to infection in mam- mals was well established prior to 1980. In fact, in the mid- 1970s, Coutinho and colleagues prepared an antiserum that References 1. Lemaitre, B., E. Nicolas, L. Michaut, J.-M. Reichhart, and J. A. Hoffmann. 1996. not only distinguished between C3H/HeJ and LPS-responder The dorsoventral regulatory gene cassette spa¨tzle/Toll/cactus controls the potent lymphoid cells by immunofluorescent staining, but also was antifungal response in Drosophila adults. Cell 86: 973–983. used to identify the C57BL/10ScCr strain as LPS unrespon- 2. Ganesan, S., K. Aggarwal, N. Paquette, and N. Silverman. 2011. NF-kB/Rel proteins and the humoral immune responses of . Curr. sive (i.e., because this reagent also failed to stain cells from the Top. Microbiol. Immunol. 349: 25–60. C57BL/10ScCr strain, this group assessed its LPS sensitivity 3. Lemaitre, B. 2004. The road to Toll. Nat. Rev. Immunol. 4: 521–527. 4. Steiner, H., D. Hultmark, A. Engstro¨m, H. Bennich, and H. G. Boman. 1981. and showed that this strain was LPS unresponsive) (reviewed Sequence and specificity of two antibacterial proteins involved in insect immunity. in Ref. 23). These and other studies led to the hypothesis that Nature 292: 246–248. d 5. Lehrer, R. I., M. E. Selsted, D. Szklarek, and J. Fleischmann. 1983. Antibacterial Lps mice lacked a signaling receptor for LPS, even prior to activity of microbicidal cationic proteins 1 and 2, natural peptide antibiotics of the cloning of Drosophila Toll in 1988 (28). rabbit lung macrophages. Infect. Immun. 42: 10–14. The Journal of Immunology 5209

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