Dynamic regulation of innate immune responses in Drosophila by Senju-mediated glycosylation Miki Yamamoto-Hinoa, Masatoshi Muraokab, Shu Kondoc, Ryu Uedac, Hideyuki Okanod, and Satoshi Gotoa,d,1 aDepartment of Life Science, Rikkyo University, Tokyo 171-8501, Japan; bStem Cell Project Group, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan; cInvertebrate Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima 411-8540, Japan; and dDepartment of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan Edited by Norbert Perrimon, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA, and approved March 30, 2015 (received for review December 22, 2014) The innate immune system is the first line of defense encountered by Most cell surface and secreted proteins are glycosylated in the invading pathogens. Delayed and/or inadequate innate immune endoplasmic reticulum (ER) and Golgi apparatus. Glycosylation responses can result in failure to combat pathogens, whereas ex- requires glycosyltransferases and nucleotide sugar substrates cessive and/or inappropriate responses cause runaway inflamma- that are synthesized in the cytosol and nucleus. The nucleotide tion. Therefore, immune responses are tightly regulated from sugars must be transported into the ER and Golgi lumens by initiation to resolution and are repressed during the steady state. It is nucleotide sugar transporters (NSTs) (6). On arrival, they are used well known that glycans presented on pathogens play important by glycosyltransferases. The Drosophila genome encodes at least roles in pathogen recognition and the interactions between host 10 NSTs, which transport different subsets of nucleotide sugars. molecules and microbes; however, the function of glycans of host Studies of mutated NST genes show that protein glycosylation organisms in innate immune responses is less well known. Here, we regulates intercellular signaling events. For example, Fringe-con- show that innate immune quiescence and strength of the immune nection (a UDP-sugar transporter) (7, 8) and the two GDP-fucose response are controlled by host glycosylation involving a novel UDP- transporters Gfr (9) and Efr (10) all affect Notch glycosylation and galactose transporter called Senju. In senju mutants, reduced expres- activity. Drosophila Gfr has a mammalian ortholog called SLC35C, sion of galactose-containing glycans resulted in hyperactivation of the which is responsible for the congenital disorder of glycosylation IIc Toll signaling pathway in the absence of immune challenges. Genetic Drosophila (11, 12). These reports suggest that mutant NSTs in INFLAMMATION epistasis and biochemical analyses revealed that Senju regulates the may be useful tools for investigating the roles of glycosylation in IMMUNOLOGY AND Toll signaling pathway at a step that converts Toll ligand Spatzle to its regulation of signaling and in human diseases. active form. Interestingly, Toll activation in immune-challenged wild Here, we report that flies harboring a mutation of a novel type (WT) flies reduced the expression of galactose-containing gly- UDP-galactose transporter named Senju showed hyperacti- cans. Suppression of the degalactosylation by senju overexpression vation of immune signal transduction via the Toll, JAK/STAT, resulted in reduced induction of Toll-dependent expression of an and JNK signaling pathways in the absence of immune chal- antimicrobial peptide, Drosomycin, and increased susceptibility to lenge. We focused on identifying the mechanism underlying Toll infection with Gram-positive bacteria. These data suggest that pathway activation and found that Spz was activated in the senju Senju-mediated galactosylation suppresses undesirable Toll signaling mutant. Moreover, we found that activation of the Toll pathway activation during the steady state; however, Toll activation in re- reduced the amount of galactose-containing glycan. Accordingly, sponse to infection leads to degalactosylation, which raises the im- senju overexpression increased galactose-containing glycan mune response to an adequate level and contributes to the prompt levels and repressed activation of the immune response against elimination of pathogens. pathogens. These results suggest that the immune response is dynamically regulated by the amount of galactose-containing innate immunity | glycosylation | Toll pathway | nucleotide sugar transporter | galactose Significance he innate immune system is the first line of defense against The innate immune system is the first line of defense against in- Tinvading pathogens. In the steady state, immune responses vading pathogens. Activation of the innate immune response by are restrained; however, the immune response is activated rap- infection and suppression during steady state are stringently idly on pathogen recognition. The Toll/TLR (Toll-like receptor) controlled to eliminate pathogens and prevent inflammation. We pathway plays a conserved role in the innate immune system of found that host glycosylation plays an important role in the ac- insects and vertebrates (1–3). In Drosophila, pathogens are not tivation of immune responses and in maintaining innate immune directly recognized by the Toll transmembrane receptor; rather, homeostasis. In the steady state, a high amount of galactose- they are recognized by several specific pattern recognition re- containing glycan suppresses undesirable activation of the im- ceptors. The binding of gram-positive bacteria and fungi by these mune response; however, activation of immune responses leads pattern recognition receptors triggers the activation of the serine to reduced levels of galactose-containing glycan, which is needed protease cascade. Finally, a precursor form of Spatzle (pro-Spz), an for raising immune responses to an adequate level. Our finding endogenous ligand for Toll, is converted to an active form, which suggests a novel mechanism for the regulation of innate immune binds Toll and triggers activation of the NF-κB−related transcrip- quiescence and activation via changes in glycosylation status. tion factor, Dorsal-related immunity factor (Dif), leading to tran- Author contributions: M.Y.-H. and S.G. designed research; M.Y.-H. and S.K. performed re- scription of genes encoding antimicrobial peptides (AMPs) in- search; M.Y.-H., M.M., S.K., R.U., and H.O. contributed new reagents/analytic tools; M.Y.-H. cluding Drosomycin (Drs). Many of the extracellular components and S.G. analyzed data; and M.Y.-H. and S.G. wrote the paper. required for Toll activation and suppression have been identi- The authors declare no conflict of interest. fied and are well understood; however, it is less known whether This article is a PNAS Direct Submission. the posttranslational modifications in host organisms, such as Freely available online through the PNAS open access option. glycosylation, regulate innate immune responses, despite the 1To whom correspondence should be addressed. Email: [email protected]. known significant roles of glycosylation in various other biological This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. processes (4, 5). 1073/pnas.1424514112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1424514112 PNAS | May 5, 2015 | vol. 112 | no. 18 | 5809–5814 Downloaded by guest on September 27, 2021 glycan, which is high during steady state to maintain immune were also observed in Toll gain-of-function (Tl10B) (17) and JAK homeostasis and low in infection to favor prompt elimination gain-of-function (hoptum-l)(18,19)mutantsofDrosophila.There- of pathogens. fore, we next investigated whether the Toll and JAK/STAT signaling pathways were activated in senju1. Because homo- Results zygous senju1 died at the late larval or early pupal stages, the Senju KO Flies Have Abnormal Immune Organs. To analyze the bi- majority of experiments (except for some that used adult flies) ological role of Drosophila NSTs, we generated a series of knock were performed using late-stage larvae. out (KO) flies by homologous recombination (SI Materials and Methods and Fig. S1). Of these, CG14040 KO flies, which are Signaling Pathways Involved in Innate Immunity Are Activated in called senju1 in the present study, showed various abnormalities in senju1 in the Absence of Immune Challenge. The Toll signaling their immune organs. These abnormalities included melanotic cells pathway is the most important pathway in innate immunity in the hemocoel and melanotic tumors in the lymph glands, which against gram-positive bacteria and fungi (2, 20). We found that the are a specialized larval hematopoietic organ in Drosophila (Fig. 1 Toll pathway was activated in senju1 by quantifying the expression A–C). The lymph glands were also enlarged (Fig. 1 B, E, G,andI). of Drs. In the absence of immune challenge, senju1 larvae expressed Therefore, we investigated whether normal hematopoiesis occurs in ∼50-fold higher Drs mRNA levels than unchallenged WT larvae. senju1. Normally, at the late third-instar larval stage, progenitor The Drs mRNA levels in senju1 were 50% of the Drs mRNA levels hemocytes mature into plasmatocytes, crystal cells, and lamellocytes of control larvae collected 12 h after infection with Micrococcus in the lymph gland (13); thus, lymph glands from WT and senju1 luteus (M. luteus), a gram-positive bacterium (Fig. 2A). The up- larvae were stained with anti-Nimrod C (a marker of plasmato- regulation of Drs in senju1 was significantly reduced by the