Enhanced Antibacterial Potential in UBP43-Deficient Mice against Salmonella typhimurium Infection by Up-Regulating Type I IFN Signaling1
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免疫学杂志 2005年第14期
Abstract
ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN- IFN regulatory factor 3 type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43–/– macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43–/– mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.
Introduction
Interferons are well-known cytokines that play pivotal roles in antiviral, antibacterial, cell growth, differentiation, and antitumor responses (1, 2). IFNs are also considered as signals for modulating innate and adaptive immunity (3). Type I IFNs induce several hundred IFN-stimulated genes (ISGs),4 including cytokine genes, through the Jak/Stat signaling pathway (4). It has been known that Jak/Stat signaling is tightly regulated by various means and dysregulation of this signaling is associated with a variety of immune disorders (5). There are three groups of negative regulators for type I IFN and other such cytokine signaling: several protein tyrosine phosphatases (Src homology proteins 1 and 2, CD45, protein tyrosine phosphatase 1B, and T cell protein tyrosine phosphatase), suppressor of cytokine signaling (SOCS) proteins, and protein inhibitor of activated STAT family members (5).
Recently, we have demonstrated that UBP43, a deconjugating protease of ISG15, is a new negative regulator of the type I IFN signaling pathway (6). ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are highly induced upon viral or bacterial infection (7, 8). The protein ISG15 conjugation process ISGylation follows an analogous mechanism to other ubiquitin-like protein modifications (8, 9, 10). However, the ISGylation system is unique among ubiquitin-like protein systems. The expression of ISG15 and currently known enzymes for ISGylation and deISGylation are all highly induced by type I IFN. Furthermore, the ISGylation system has been detected only in vertebrate genomes, whereas most ubiquitin-like protein systems are highly conserved throughout all eukaryotes from yeast to human. Thus, the ISGylation system may have specialized functions related to host defense against pathogens in higher eukaryotes. To date, three enzymes, UBE1L (E1), UBC8 (E2), and UBP43 (USP18, isopeptidase) have been identified for the ISGylation and deISGylation processes (11, 12, 13, 14). Genetic knockout studies demonstrated that UBP43 activity is not essential for the precursor processing of ISG15 in vivo because UBP43-deficient cells can generate ISGylated proteins upon IFN treatment (6). The most pronounced phenotype of UBP43 deficiency is hypersensitivity to type I IFN, with enhanced and prolonged activation of Jak/Stat signaling (6). Thus, UBP43 is another member of the negative regulators of type I IFN and the Jak/Stat signaling pathway.
Type I IFNs are produced in macrophages upon activation of TLR4 signaling by bacterial LPS, a pathogenic agent in endotoxin shock (15). Although TLRs have been shown to act as primary immune sensors for invading pathogens, over-activation of inflammatory signaling often causes a lethal shock to the host accompanied by multiple tissue damage and organ failure (16). TNF- has been known to be a major contributor to LPS-induced septic shock (17, 18, 19). In addition to TNF-, a recent report demonstrated that type I IFNs, especially IFN-, are central effecters in LPS-induced lethality (20). Several microarray studies showed that ISG15 is a highly up-regulated gene in human macrophages during various bacterial infections (21, 22) and both ISG15 and UBP43 were identified as LPS-induced genes in mouse macrophages (23). We questioned whether ISG15 and/or UBP43 are involved in antibacterial responses mediated by TLR4 signaling and addressed this question by using the UBP43 knockout mouse model, which is lacking an ISG15-specific deconjugating enzyme.
We report that LPS-induced UBP43 expression and protein ISGylation is mainly a TIR domain-containing adapter inducing IFN- (Trif) and type I IFN-dependent event in macrophages. We also show that UBP43-deficient mice have a higher sensitivity to endotoxin-induced septic shock than wild-type mice. However, TNF- production in UBP43-deficient macrophages was comparable to that of wild-type macrophages upon LPS treatment. Instead, ubp43–/– macrophages exhibited enhanced and prolonged activation of Jak/Stat signaling resulting in elevated expression of ISGs, as well as IFN-inducible cytokine and chemokine genes, which indicates a negative regulatory role of UBP43 in type I IFN signaling. ubp43–/– macrophages also showed enhanced phagocytic activity to IgG opsonized yeast particles. With these characteristics, ubp43–/– mice were more capable of restricting the growth of Salmonella typhimurium than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but detrimental to the body without strict control.
Results
Trif-dependent induction of protein ISGylation by TLR3 and TLR4 ligands in BMM
ISG15 has been reported as one of the most up-regulated genes in human macrophages during various bacterial infections (21, 22). Both ISG15 and UBP43 were identified as LPS-induced genes in mouse macrophages (23). To understand the signaling pathway that induces ISG15 expression and its conjugation upon bacterial infection, we first investigated which TLR signaling pathway is responsible for the induction of the ISGylation system in macrophages. BMM from C57BL/6 mice were treated with various TLR ligands and ISG15 conjugates were detected by Western blot analysis. IFN- treatment clearly induced protein ISGylation in macrophages (Fig. 1A). The TLR3 ligand (poly(I:C)) and TLR4 ligand (LPS) also induced ISG15 conjugates to a similar extent as IFN-. Other TLR ligands including peptidoglycan (TLR2), resiquimod (TLR7), and unmethylated CpG DNA (TLR9) did not induce protein ISGylation, although a basal level of ISGylated proteins were still detectable in those cells. Thus, TLR3 and TLR4 seem to be the major receptors that can induce protein ISGylation in macrophages.
The mRNA levels for cytokine IL-6 and chemokines including MCP-1, IP-10, and MIP-1 in wild-type BMM reached a maximum at 4 h after treatment with LPS and rapidly decreased with time to basal levels (Fig. 5A). Initial expression of these genes in UBP43-deficient BMM was comparable to or slightly higher (for MCP-1) than in wild-type BMM at 4 h post LPS challenge. However, a substantial amount of mRNA was still detected even after 24 h of LPS treatment (Fig. 5A). The expression of these genes was impaired in ifnar1–/– BMM treated with LPS (Fig. 5B). Together, these results suggest that type I IFN plays an important role in regulating the production of subsets of cytokines and chemokines, including IL-6, MCP-1, IP-10, and MIP-1 upon LPS treatment. In ubp43–/– macrophages, the production of type I IFN-inducible cytokines and chemokines is prolonged and/or enhanced in accordance with prolonged activation of Jak/Stat signaling.
Enhanced phagocytic activity in UBP43-deficient macrophage
One of the most important roles of macrophages upon bacterial infection along with cytokine and chemokine production is to engulf and degrade invading bacteria to clear the infected host body (33). We analyzed the phagocytic activity of macrophages from wild-type and ubp43–/– mice. Thioglycolate elicited peritoneal macrophages were incubated with a 50-fold excess amount of IgG opsonized zymosan particles, and particle uptake was visualized on the fluorescence microscope. As shown in Fig. 6, certain populations of ubp43–/– macrophages could take up a large number of fluorescent particles (up to 15–18 particles), whereas wild-type macrophages only contained a small number of particles. Average particle numbers for wild-type and ubp43–/– macrophages counted from a total of 200 cells were 3–4 and 7–9, respectively. This result indicates that a subpopulation of ubp43–/– macrophages were preactivated and had an enhanced ability to engulf particles compared with wild-type cells.
Virulent strains of Salmonella grow in the mouse and finally causes host death even with few numbers of initial infections (34). Surprisingly, the survival rate of ubp43–/– mice with a low dosage of Salmonella infection (40 PFU) was not significantly increased compared with wild-type mice. All wild-type mice died within 8 days postinfection and ubp43–/– mice survived slightly longer than wild-type hosts but all died at 11 days postinfection (Fig. 7D). This result indicates that although UBP43 deficiency provided an advantage in protecting the host from bacterial growth, it also caused detrimental effects to the host because of probable hypersensitivity to LPS.
Discussion
The innate immune system is the first line of the host defense against invading pathogens (35, 36). TLRs are now generally accepted as receptors, which recognize pathogen-associated molecular patterns. Among them, TLR4 has been known as a specific receptor of LPS, the cell wall component of Gram-negative bacteria (37). Binding of LPS to TLR4 initiates a signaling cascade to activate transcription factors including NF-B and IRF3, resulting in the production of immunoregulatory cytokines and chemokines (15). We previously found that LPS induces the expression of ISG15 and UBP43 as well as the conjugation of ISG15 to numerous target proteins in macrophages (32). Thus we questioned whether the protein ISGylation system and UBP43 are involved in the TLR4-mediated host defense mechanism. First we examined which TLR signaling other than TLR4 can activate protein ISGylation and how exactly TLR4 signaling induces protein ISGylation in macrophages. Our experiments revealed that TLR3 and TLR4 are major receptors inducing protein ISGylation in macrophages and that Trif IRF3 type I IFN is the main axis for inducing gene expression for the protein ISGylation system and for UBP43 in macrophages upon LPS induction. Interestingly, the ISG15 gene expression pattern showed a biphasic response upon LPS challenge. The expression at the late time period (8–12 h after LPS treatment) comes from the activation of Jak/Stat signaling by type I IFN because ifnar1–/– macrophages have a defect in expressing ISG15 gene at this point. The early expression of ISG15 (2–4 h) is probably induced by IRF3 because it is well known that IRF3 can directly activate ISG15 gene expression upon viral infection (38). In contrast, LPS induction of UBE1L, UBCm8, and UBP43 was mainly dependent on type I IFN signaling. Thus, type I IFN signaling plays a pivotal role in inducing protein ISGylation in macrophages upon LPS treatment. As reported previously, IFN production upon LPS treatment is due to the phosphorylation of IRF3 via a Trif pathway and the induction of NF-B via a MyD88 pathway (15).
We previously generated UBP43 knockout mice to study the biological function of the protein ISGylation system (6, 26). We used this mouse model to investigate the possible involvement of ISGylation or UBP43 in response to LPS-mediated TLR4 signaling. In our experiment, UBP43-deficient mice showed a hypersensitivity to LPS injection, resulting in over 50% death of ubp43–/– mice with a sublethal (for wild-type mice) dose of LPS injection. TNF- production was almost the same in wild-type and UBP43-deficient macrophages. Instead, we detected hyperactivation of the Jak/Stat signaling pathway in UBP43-deficient macrophages upon LPS treatment characterized by enhanced and prolonged phosphorylation of STAT1 and elevated expression of ISGs. One interesting finding is that several genes for cytokines (IL-6) and chemokines (MCP-1, IP-10, and MIP-1) were also highly expressed in ubp43–/– macrophages compared with wild-type cells by LPS treatment. Moreover, the expression of these genes were impaired or significantly reduced in ifnar1-deficient macrophages. Cytokine and chemokine production during the inflammatory response is regulated by complicated mechanisms. For example, IL-6 expression by LPS has been known to require at least two transcription factors: NF-B and the nuclear protein IB (25, 39). Now, our results clearly demonstrated that the type I IFN pathway plays a pivotal role in producing the maximum amount of inflammatory cytokines and chemokines synergistically with other signaling pathways and UBP43 is a crucial component for finishing type I IFN signaling upon LPS challenge. A defect in any one of these signaling molecules causes a significant reduction or overproduction of a subset of cytokines and chemokines. Recently, type I IFN has been identified as a major factor for LPS-induced mouse death (20). UBP43-deficient mice are not able to terminate inflammatory response efficiently due to its intrinsic hypersensitivity to type I IFN. Most probably, this is why UBP43 mice are more susceptible to LPS-induced septic shock compared with wild-type mice.
In contrast, the hypersensitivity to type I IFN helps mice to restrict bacterial growth in the body. Salmonella growth in ubp43–/– mice was substantially reduced compared with growth found in wild-type mice. UBP43-deficient macrophages exhibited elevated phagocytic activity, enhanced expression of antibacterial inducible NO synthase as well as cytokine and chemokine genes. All together, hypersensitivity to type I IFN was beneficial to the antibacterial response in ubp43–/– mice. However, survival of ubp43–/– mice was only slightly extended after peritoneal infection of S. typhimurium compared with the wild-type mice. Although ubp43–/– mice can delay bacterial growth, it cannot eradicate the infecting bacteria. Once the bacterial number has reached a certain point, it might cause systemic inflammation, resulting in rapid death of ubp43–/– mice with their intrinsic hypersensitivity to type I IFN, as was the case seen in the LPS-injection experiment. Thus, type I IFN signaling is beneficial to setup an antibacterial state against the Gram-negative bacteria S. typhimurium, though at the same time, it is detrimental to the body without proper termination of the signaling.
The mode of action of UBP43 as a negative regulator in type I IFN signaling is similar to that of SOCS-1 in IFN- signaling in that both proteins are induced by the signaling pathway and, in turn, function as negative regulators to terminate the signaling process (40). SOCS-1 has been identified as a negative regulator of LPS-induced TLR4 signaling as well (41, 42, 43). The socs-1–/– mouse itself, or with an IFN--deficient background, is hypersensitive to LPS-induced lethal effects as are the ubp43–/– mice. However, the mechanism whereby SOCS-1 deficiency causes LPS hypersensitivity is controversial. Two groups claimed that SOCS-1 directly suppresses LPS-induced NF-B activation (41, 42) but recently another group has found that type I IFN signaling but not NF-B signaling is a target of SOCS-1 upon LPS treatment (43). It is clear that UBP43 is a specific negative regulator for type I IFN signaling because we did not detect any changes in the activation of MyD88-dependent TLR4 signaling by LPS. More importantly, the proper termination of type I IFN-signaling by UBP43 is a critical requirement to prevent over-activation of TLR4 signaling by LPS. In the absence of UBP43, cells accumulate much higher levels of ISGylated proteins. To date, only five cellular targets for protein ISGylation have been reported, including Serpin 2a, Jak1, Stat1, phospholipase C1, and Erk1/2 (28, 44), among possibly hundreds of ISGylated proteins. We recently report that UBP43-deficient mice are resistant to certain virus infections (45). Currently, it is not clear how protein ISGylation contributes to innate immunity. Further identification of ISG15 targets and intensive studies for the consequence of ISGylation of these target proteins will eventually provide molecular explanation of how protein ISGylation is involved in innate immune responses against bacterial and viral infection.
In conclusion, UBP43, a deconjugating enzyme for the protein ISGylation system, is a crucial component in endotoxin-induced inflammatory responses by terminating type I IFN signaling. Deficiency of UBP43 causes hypersensitivity to LPS-induced sepsis. ubp43–/– mice could restrict Salmonella growth more efficiently than wild-type mice, though survival rates of ubp43–/– mice were only slightly extended after Salmonella infection as compared with the wild-type mice, the most likely reason being the intrinsic hypersensitivity to LPS in the ubp43–/– mice. These results clearly demonstrate a good model that the tight regulation of cytokine signaling is critical to protect the host from harmful overresponse to inflammatory signaling, which may have lethal effects.
Acknowledgments
We thank Drs. Michel Aguet (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) and Shizuo Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) for ifnar1–/– and myd88–/– mice, respectively, Drs. Li Li and Chang Hahn (Aventis Pharmaceuticals, Bridgewater, NJ) for technical assistance, and Dr. Herbert Virgin (Washington University School of Medicine, St. Louis, MO) and members of the Zhang laboratory for valuable discussions and critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work is supported by National Institutes of Health Grant CA079849 (to D.E.Z.) and GM060031 (to B.B.). The Stein Endowment Fund has partially supported the Molecular and Experimental Medicine Departmental Molecular Biology Service Laboratory for DNA Sequencing and Oligonucleotide Synthesis. This is manuscript no. 17087-MEM from The Scripps Research Institute.
2 Current address: Department of Biological Science, Sookmyung Women’s University, 53-12 Chungpa-dong 2 Ka, Yongsan-gu, Seoul 140-742, Korea.
3 Address correspondence and reprint requests to Dr. Dong-Er Zhang, MEM-L51, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: dzhang@scripps.edu
4 Abbreviations used in this paper: ISG, IFN-stimulated gene; BMM, bone marrow-derived macrophage; ISGylation, ISG15 protein conjugation; IRF, IFN regulatory factor; SOCS, suppressor of cytokine signaling; m, murine; IP-10, IFN--inducible protein-10, Trif, TIR domain-containing adapter inducing IFN-.
Received for publication March 21, 2005. Accepted for publication April 28, 2005.
References
Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264
Samuel, C. E.. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14: 778-809.
Pestka, S., J. A. Langer, K. C. Zoon, C. E. Samuel. 1987. Interferons and their actions. Annu. Rev. Biochem. 56: 727-777.
Der, S. D., A. Zhou, B. R. Williams, R. H. Silverman. 1998. Identification of genes differentially regulated by interferon , , or using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95: 15623-15628
Shuai, K., B. Liu. 2003. Regulation of JAK-STAT signalling in the immune system. Nat. Rev. Immunol. 3: 900-911.
Malakhova, O. A., M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, D. E. Zhang. 2003. Protein ISGylation modulates the JAK-STAT signaling pathway. Genes Dev. 17: 455-460.
Loeb, K. R., A. L. Haas. 1992. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J. Biol. Chem. 267: 7806-7813.
Kim, K. I., D. E. Zhang. 2003. ISG15, not just another ubiquitin-like protein. Biochem. Biophys. Res. Commun. 307: 431-434.
Hochstrasser, M.. 2000. Evolution and function of ubiquitin-like protein-conjugation systems. Nat. Cell Biol. 2: E153-E157.(Keun Il Kim2,*, Oxana A. )
ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN- IFN regulatory factor 3 type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43–/– macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43–/– mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.
Introduction
Interferons are well-known cytokines that play pivotal roles in antiviral, antibacterial, cell growth, differentiation, and antitumor responses (1, 2). IFNs are also considered as signals for modulating innate and adaptive immunity (3). Type I IFNs induce several hundred IFN-stimulated genes (ISGs),4 including cytokine genes, through the Jak/Stat signaling pathway (4). It has been known that Jak/Stat signaling is tightly regulated by various means and dysregulation of this signaling is associated with a variety of immune disorders (5). There are three groups of negative regulators for type I IFN and other such cytokine signaling: several protein tyrosine phosphatases (Src homology proteins 1 and 2, CD45, protein tyrosine phosphatase 1B, and T cell protein tyrosine phosphatase), suppressor of cytokine signaling (SOCS) proteins, and protein inhibitor of activated STAT family members (5).
Recently, we have demonstrated that UBP43, a deconjugating protease of ISG15, is a new negative regulator of the type I IFN signaling pathway (6). ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are highly induced upon viral or bacterial infection (7, 8). The protein ISG15 conjugation process ISGylation follows an analogous mechanism to other ubiquitin-like protein modifications (8, 9, 10). However, the ISGylation system is unique among ubiquitin-like protein systems. The expression of ISG15 and currently known enzymes for ISGylation and deISGylation are all highly induced by type I IFN. Furthermore, the ISGylation system has been detected only in vertebrate genomes, whereas most ubiquitin-like protein systems are highly conserved throughout all eukaryotes from yeast to human. Thus, the ISGylation system may have specialized functions related to host defense against pathogens in higher eukaryotes. To date, three enzymes, UBE1L (E1), UBC8 (E2), and UBP43 (USP18, isopeptidase) have been identified for the ISGylation and deISGylation processes (11, 12, 13, 14). Genetic knockout studies demonstrated that UBP43 activity is not essential for the precursor processing of ISG15 in vivo because UBP43-deficient cells can generate ISGylated proteins upon IFN treatment (6). The most pronounced phenotype of UBP43 deficiency is hypersensitivity to type I IFN, with enhanced and prolonged activation of Jak/Stat signaling (6). Thus, UBP43 is another member of the negative regulators of type I IFN and the Jak/Stat signaling pathway.
Type I IFNs are produced in macrophages upon activation of TLR4 signaling by bacterial LPS, a pathogenic agent in endotoxin shock (15). Although TLRs have been shown to act as primary immune sensors for invading pathogens, over-activation of inflammatory signaling often causes a lethal shock to the host accompanied by multiple tissue damage and organ failure (16). TNF- has been known to be a major contributor to LPS-induced septic shock (17, 18, 19). In addition to TNF-, a recent report demonstrated that type I IFNs, especially IFN-, are central effecters in LPS-induced lethality (20). Several microarray studies showed that ISG15 is a highly up-regulated gene in human macrophages during various bacterial infections (21, 22) and both ISG15 and UBP43 were identified as LPS-induced genes in mouse macrophages (23). We questioned whether ISG15 and/or UBP43 are involved in antibacterial responses mediated by TLR4 signaling and addressed this question by using the UBP43 knockout mouse model, which is lacking an ISG15-specific deconjugating enzyme.
We report that LPS-induced UBP43 expression and protein ISGylation is mainly a TIR domain-containing adapter inducing IFN- (Trif) and type I IFN-dependent event in macrophages. We also show that UBP43-deficient mice have a higher sensitivity to endotoxin-induced septic shock than wild-type mice. However, TNF- production in UBP43-deficient macrophages was comparable to that of wild-type macrophages upon LPS treatment. Instead, ubp43–/– macrophages exhibited enhanced and prolonged activation of Jak/Stat signaling resulting in elevated expression of ISGs, as well as IFN-inducible cytokine and chemokine genes, which indicates a negative regulatory role of UBP43 in type I IFN signaling. ubp43–/– macrophages also showed enhanced phagocytic activity to IgG opsonized yeast particles. With these characteristics, ubp43–/– mice were more capable of restricting the growth of Salmonella typhimurium than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but detrimental to the body without strict control.
Results
Trif-dependent induction of protein ISGylation by TLR3 and TLR4 ligands in BMM
ISG15 has been reported as one of the most up-regulated genes in human macrophages during various bacterial infections (21, 22). Both ISG15 and UBP43 were identified as LPS-induced genes in mouse macrophages (23). To understand the signaling pathway that induces ISG15 expression and its conjugation upon bacterial infection, we first investigated which TLR signaling pathway is responsible for the induction of the ISGylation system in macrophages. BMM from C57BL/6 mice were treated with various TLR ligands and ISG15 conjugates were detected by Western blot analysis. IFN- treatment clearly induced protein ISGylation in macrophages (Fig. 1A). The TLR3 ligand (poly(I:C)) and TLR4 ligand (LPS) also induced ISG15 conjugates to a similar extent as IFN-. Other TLR ligands including peptidoglycan (TLR2), resiquimod (TLR7), and unmethylated CpG DNA (TLR9) did not induce protein ISGylation, although a basal level of ISGylated proteins were still detectable in those cells. Thus, TLR3 and TLR4 seem to be the major receptors that can induce protein ISGylation in macrophages.
The mRNA levels for cytokine IL-6 and chemokines including MCP-1, IP-10, and MIP-1 in wild-type BMM reached a maximum at 4 h after treatment with LPS and rapidly decreased with time to basal levels (Fig. 5A). Initial expression of these genes in UBP43-deficient BMM was comparable to or slightly higher (for MCP-1) than in wild-type BMM at 4 h post LPS challenge. However, a substantial amount of mRNA was still detected even after 24 h of LPS treatment (Fig. 5A). The expression of these genes was impaired in ifnar1–/– BMM treated with LPS (Fig. 5B). Together, these results suggest that type I IFN plays an important role in regulating the production of subsets of cytokines and chemokines, including IL-6, MCP-1, IP-10, and MIP-1 upon LPS treatment. In ubp43–/– macrophages, the production of type I IFN-inducible cytokines and chemokines is prolonged and/or enhanced in accordance with prolonged activation of Jak/Stat signaling.
Enhanced phagocytic activity in UBP43-deficient macrophage
One of the most important roles of macrophages upon bacterial infection along with cytokine and chemokine production is to engulf and degrade invading bacteria to clear the infected host body (33). We analyzed the phagocytic activity of macrophages from wild-type and ubp43–/– mice. Thioglycolate elicited peritoneal macrophages were incubated with a 50-fold excess amount of IgG opsonized zymosan particles, and particle uptake was visualized on the fluorescence microscope. As shown in Fig. 6, certain populations of ubp43–/– macrophages could take up a large number of fluorescent particles (up to 15–18 particles), whereas wild-type macrophages only contained a small number of particles. Average particle numbers for wild-type and ubp43–/– macrophages counted from a total of 200 cells were 3–4 and 7–9, respectively. This result indicates that a subpopulation of ubp43–/– macrophages were preactivated and had an enhanced ability to engulf particles compared with wild-type cells.
Virulent strains of Salmonella grow in the mouse and finally causes host death even with few numbers of initial infections (34). Surprisingly, the survival rate of ubp43–/– mice with a low dosage of Salmonella infection (40 PFU) was not significantly increased compared with wild-type mice. All wild-type mice died within 8 days postinfection and ubp43–/– mice survived slightly longer than wild-type hosts but all died at 11 days postinfection (Fig. 7D). This result indicates that although UBP43 deficiency provided an advantage in protecting the host from bacterial growth, it also caused detrimental effects to the host because of probable hypersensitivity to LPS.
Discussion
The innate immune system is the first line of the host defense against invading pathogens (35, 36). TLRs are now generally accepted as receptors, which recognize pathogen-associated molecular patterns. Among them, TLR4 has been known as a specific receptor of LPS, the cell wall component of Gram-negative bacteria (37). Binding of LPS to TLR4 initiates a signaling cascade to activate transcription factors including NF-B and IRF3, resulting in the production of immunoregulatory cytokines and chemokines (15). We previously found that LPS induces the expression of ISG15 and UBP43 as well as the conjugation of ISG15 to numerous target proteins in macrophages (32). Thus we questioned whether the protein ISGylation system and UBP43 are involved in the TLR4-mediated host defense mechanism. First we examined which TLR signaling other than TLR4 can activate protein ISGylation and how exactly TLR4 signaling induces protein ISGylation in macrophages. Our experiments revealed that TLR3 and TLR4 are major receptors inducing protein ISGylation in macrophages and that Trif IRF3 type I IFN is the main axis for inducing gene expression for the protein ISGylation system and for UBP43 in macrophages upon LPS induction. Interestingly, the ISG15 gene expression pattern showed a biphasic response upon LPS challenge. The expression at the late time period (8–12 h after LPS treatment) comes from the activation of Jak/Stat signaling by type I IFN because ifnar1–/– macrophages have a defect in expressing ISG15 gene at this point. The early expression of ISG15 (2–4 h) is probably induced by IRF3 because it is well known that IRF3 can directly activate ISG15 gene expression upon viral infection (38). In contrast, LPS induction of UBE1L, UBCm8, and UBP43 was mainly dependent on type I IFN signaling. Thus, type I IFN signaling plays a pivotal role in inducing protein ISGylation in macrophages upon LPS treatment. As reported previously, IFN production upon LPS treatment is due to the phosphorylation of IRF3 via a Trif pathway and the induction of NF-B via a MyD88 pathway (15).
We previously generated UBP43 knockout mice to study the biological function of the protein ISGylation system (6, 26). We used this mouse model to investigate the possible involvement of ISGylation or UBP43 in response to LPS-mediated TLR4 signaling. In our experiment, UBP43-deficient mice showed a hypersensitivity to LPS injection, resulting in over 50% death of ubp43–/– mice with a sublethal (for wild-type mice) dose of LPS injection. TNF- production was almost the same in wild-type and UBP43-deficient macrophages. Instead, we detected hyperactivation of the Jak/Stat signaling pathway in UBP43-deficient macrophages upon LPS treatment characterized by enhanced and prolonged phosphorylation of STAT1 and elevated expression of ISGs. One interesting finding is that several genes for cytokines (IL-6) and chemokines (MCP-1, IP-10, and MIP-1) were also highly expressed in ubp43–/– macrophages compared with wild-type cells by LPS treatment. Moreover, the expression of these genes were impaired or significantly reduced in ifnar1-deficient macrophages. Cytokine and chemokine production during the inflammatory response is regulated by complicated mechanisms. For example, IL-6 expression by LPS has been known to require at least two transcription factors: NF-B and the nuclear protein IB (25, 39). Now, our results clearly demonstrated that the type I IFN pathway plays a pivotal role in producing the maximum amount of inflammatory cytokines and chemokines synergistically with other signaling pathways and UBP43 is a crucial component for finishing type I IFN signaling upon LPS challenge. A defect in any one of these signaling molecules causes a significant reduction or overproduction of a subset of cytokines and chemokines. Recently, type I IFN has been identified as a major factor for LPS-induced mouse death (20). UBP43-deficient mice are not able to terminate inflammatory response efficiently due to its intrinsic hypersensitivity to type I IFN. Most probably, this is why UBP43 mice are more susceptible to LPS-induced septic shock compared with wild-type mice.
In contrast, the hypersensitivity to type I IFN helps mice to restrict bacterial growth in the body. Salmonella growth in ubp43–/– mice was substantially reduced compared with growth found in wild-type mice. UBP43-deficient macrophages exhibited elevated phagocytic activity, enhanced expression of antibacterial inducible NO synthase as well as cytokine and chemokine genes. All together, hypersensitivity to type I IFN was beneficial to the antibacterial response in ubp43–/– mice. However, survival of ubp43–/– mice was only slightly extended after peritoneal infection of S. typhimurium compared with the wild-type mice. Although ubp43–/– mice can delay bacterial growth, it cannot eradicate the infecting bacteria. Once the bacterial number has reached a certain point, it might cause systemic inflammation, resulting in rapid death of ubp43–/– mice with their intrinsic hypersensitivity to type I IFN, as was the case seen in the LPS-injection experiment. Thus, type I IFN signaling is beneficial to setup an antibacterial state against the Gram-negative bacteria S. typhimurium, though at the same time, it is detrimental to the body without proper termination of the signaling.
The mode of action of UBP43 as a negative regulator in type I IFN signaling is similar to that of SOCS-1 in IFN- signaling in that both proteins are induced by the signaling pathway and, in turn, function as negative regulators to terminate the signaling process (40). SOCS-1 has been identified as a negative regulator of LPS-induced TLR4 signaling as well (41, 42, 43). The socs-1–/– mouse itself, or with an IFN--deficient background, is hypersensitive to LPS-induced lethal effects as are the ubp43–/– mice. However, the mechanism whereby SOCS-1 deficiency causes LPS hypersensitivity is controversial. Two groups claimed that SOCS-1 directly suppresses LPS-induced NF-B activation (41, 42) but recently another group has found that type I IFN signaling but not NF-B signaling is a target of SOCS-1 upon LPS treatment (43). It is clear that UBP43 is a specific negative regulator for type I IFN signaling because we did not detect any changes in the activation of MyD88-dependent TLR4 signaling by LPS. More importantly, the proper termination of type I IFN-signaling by UBP43 is a critical requirement to prevent over-activation of TLR4 signaling by LPS. In the absence of UBP43, cells accumulate much higher levels of ISGylated proteins. To date, only five cellular targets for protein ISGylation have been reported, including Serpin 2a, Jak1, Stat1, phospholipase C1, and Erk1/2 (28, 44), among possibly hundreds of ISGylated proteins. We recently report that UBP43-deficient mice are resistant to certain virus infections (45). Currently, it is not clear how protein ISGylation contributes to innate immunity. Further identification of ISG15 targets and intensive studies for the consequence of ISGylation of these target proteins will eventually provide molecular explanation of how protein ISGylation is involved in innate immune responses against bacterial and viral infection.
In conclusion, UBP43, a deconjugating enzyme for the protein ISGylation system, is a crucial component in endotoxin-induced inflammatory responses by terminating type I IFN signaling. Deficiency of UBP43 causes hypersensitivity to LPS-induced sepsis. ubp43–/– mice could restrict Salmonella growth more efficiently than wild-type mice, though survival rates of ubp43–/– mice were only slightly extended after Salmonella infection as compared with the wild-type mice, the most likely reason being the intrinsic hypersensitivity to LPS in the ubp43–/– mice. These results clearly demonstrate a good model that the tight regulation of cytokine signaling is critical to protect the host from harmful overresponse to inflammatory signaling, which may have lethal effects.
Acknowledgments
We thank Drs. Michel Aguet (Swiss Institute for Experimental Cancer Research, Lausanne, Switzerland) and Shizuo Akira (Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan) for ifnar1–/– and myd88–/– mice, respectively, Drs. Li Li and Chang Hahn (Aventis Pharmaceuticals, Bridgewater, NJ) for technical assistance, and Dr. Herbert Virgin (Washington University School of Medicine, St. Louis, MO) and members of the Zhang laboratory for valuable discussions and critical reading of the manuscript.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work is supported by National Institutes of Health Grant CA079849 (to D.E.Z.) and GM060031 (to B.B.). The Stein Endowment Fund has partially supported the Molecular and Experimental Medicine Departmental Molecular Biology Service Laboratory for DNA Sequencing and Oligonucleotide Synthesis. This is manuscript no. 17087-MEM from The Scripps Research Institute.
2 Current address: Department of Biological Science, Sookmyung Women’s University, 53-12 Chungpa-dong 2 Ka, Yongsan-gu, Seoul 140-742, Korea.
3 Address correspondence and reprint requests to Dr. Dong-Er Zhang, MEM-L51, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: dzhang@scripps.edu
4 Abbreviations used in this paper: ISG, IFN-stimulated gene; BMM, bone marrow-derived macrophage; ISGylation, ISG15 protein conjugation; IRF, IFN regulatory factor; SOCS, suppressor of cytokine signaling; m, murine; IP-10, IFN--inducible protein-10, Trif, TIR domain-containing adapter inducing IFN-.
Received for publication March 21, 2005. Accepted for publication April 28, 2005.
References
Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, R. D. Schreiber. 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227-264
Samuel, C. E.. 2001. Antiviral actions of interferons. Clin. Microbiol. Rev. 14: 778-809.
Pestka, S., J. A. Langer, K. C. Zoon, C. E. Samuel. 1987. Interferons and their actions. Annu. Rev. Biochem. 56: 727-777.
Der, S. D., A. Zhou, B. R. Williams, R. H. Silverman. 1998. Identification of genes differentially regulated by interferon , , or using oligonucleotide arrays. Proc. Natl. Acad. Sci. USA 95: 15623-15628
Shuai, K., B. Liu. 2003. Regulation of JAK-STAT signalling in the immune system. Nat. Rev. Immunol. 3: 900-911.
Malakhova, O. A., M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, D. E. Zhang. 2003. Protein ISGylation modulates the JAK-STAT signaling pathway. Genes Dev. 17: 455-460.
Loeb, K. R., A. L. Haas. 1992. The interferon-inducible 15-kDa ubiquitin homolog conjugates to intracellular proteins. J. Biol. Chem. 267: 7806-7813.
Kim, K. I., D. E. Zhang. 2003. ISG15, not just another ubiquitin-like protein. Biochem. Biophys. Res. Commun. 307: 431-434.
Hochstrasser, M.. 2000. Evolution and function of ubiquitin-like protein-conjugation systems. Nat. Cell Biol. 2: E153-E157.(Keun Il Kim2,*, Oxana A. )