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TREML4 adds fuel to the TLR7 fire Mihai G Netea & Frank L van de Veerdonk The cell-surface receptor TREML4 amplifies cellular responses to single-stranded RNA by regulating recruitment of the adaptor MyD88 to the receptor TLR7. Mice lacking TREML4 show impaired antiviral immunity but also reduced severity of lupus-like disease. he Toll-like receptor TLR7 recognizes nucleic acids and is thought to have a key role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). In this issue of Nature Immunology, RamirezOrtiz et al. demonstrate that the receptor TREML4 amplifies TLR7-induced signaling and type I interferon responses by recruiting TLR7 and the adaptor Myd88 to the endolysosomal compartment, which subsequently leads to activation of the mitogen-activated protein kinase p38 and phosphorylation of the transcription factor STAT1 (ref. 1). Furthermore, the authors show that TREML4 deficiency results in amelioration of the development of SLE-like symptoms in lupus-prone MRL/lpr mice. SLE is a systemic autoimmune disorder that can affect the skin, joints, lungs and kidneys and the nervous system. Defects in the clearance of necrotic and apoptotic cells are thought to lead to the accumulation of self DNA and RNA that trigger the inflammation associated with this disease2. Single-stranded RNA is recognized by TLR7 in both humans and mice, whereas double-stranded DNA is recognized by TLR9. This process activates dendritic cells and, subsequently, T cells and B cells, which results in the production of autoantibodies that then form complexes with the self DNA and RNA. These complexes can be ingested by phagocytosis by dendritic cells via the receptor FcγRIIa and, in turn, trigger the production of type I interferon, which is thought to have a central role in the pathogenesis of SLE3. The importance of TLRs that recognize nucleic acids in the pathogenesis of SLE is underscored by the observation that lupus-prone MRL/lpr mice on a TLR7-deficient background have less severe disease than that of their TLR7-expressing counterparts4 and by the early onset of lupus in BXSB mice, which have a duplication of the
gene encoding TLR7 (ref. 5). Strikingly, TLR9 deficiency in MRL/lpr mice actually increases disease severity, an observation that has not been explained so far4. Intracellular recognition of RNA most probably evolved as a mechanism of protection against RNA viruses. However, when this process runs out of hand during auto immune processes such as SLE, it becomes very deleterious to the host. Understanding how this pathway is regulated, and especially which amplifiers of autoimmunity are induced by TLR7, is crucial for the identification of novel therapeutic targets. Ramirez-Ortez et al. elucidate a key step in that direction1. Using a lentivirus-based short-hairpin RNA screen targeting 8,000 mouse genes in RAW264.7 mouse macrophages, the authors search for genes encoding products involved in the TLR7-induced expression of tumor-necrosis factor and of the transcription factor NF-κB. In addition to genes encoding products already known to be involved in this, such as Myd88, they identify several genes encoding products previously not known to be involved, including TREML4, a member of the TREM (‘triggering receptor expressed on myeloid cells’) family of receptors. Although not much is known about TREML4, several functions have been reported that link it with the etiology of SLE, such as binding to dead cells6, involvement in antigen presentation7 and modulation of dendritic cell function8.
The authors then systematically elucidate the molecular mechanisms through which TREML4 amplifies TLR7 signaling (Fig. 1). They show that knockdown of TREML4 in RAW264.7 macrophages results in specific defects in cytokine induction by the TLR7 agonist gardiquimod (GRD) and the TLR9 agonist CpG, whereas cytokine production induced by binding to TLR2, TLR3 or TLR4 is not affected. Human embryonic kidney (HEK) cells transfected to express TREML4 alone do not transcribe the gene encoding interleukin 8 (IL-8) in response to stimulation with GRD, but HEK-293 cells transfected to express TLR7 (HEK-TLR7 cells) do transcribe this gene. When HEK-TLR7 cells are transfected to express TREML4 lacking its intracytoplasmic domain, GRD still induces transcription of the gene encoding IL-8, but transfection of HEK-TLR7 cells to express a TREML4 variant with substitution of the charged lysine residue in the transmembrane region results in defective induction of IL-8 expression. These data suggest that TREML4 does not contribute to TLR7 responses by inducing signaling but instead acts as a chaperone that modulates TLR7-induced responses. Human TREML4 has insertion of a stop codon just downstream of sequence encoding the transmembrane lysine residue, which suggests that in humans, TREML4 probably also does not modulate immune responses via direct signaling.
Immune Viral RNA complexes p38 TREML4
Nucleus
Type I interferons STAT1
MyD88 TLR7
Antiviral response
Autoimmunity in SLE
ER ssRNA Endolysosome
Mihai G. Netea and Frank L. van de Veerdonk are in the Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands. e-mail: [email protected] or [email protected]
Figure 1 Amplification of TLR7 signaling by TREML4. TLR7 recognizes internalized single-stranded RNA derived from viruses or from immunocomplexes containing self RNA. TLR7 activity requires interaction with MyD88; recognition of RNA by this complex leads to the activation of p38 and STAT1, which in turn leads to the production of type 1 interferons that mediate both antiviral responses and the deleterious autoimmune responses of SLE. Ramirez-Ortiz et al. show that TLR7-induced responses are potentiated by TREML4, which regulates trafficking and co-localization of TLR7 and MyD88 to endolysosomes1.
nature immunology volume 16 number 5 MAy 2015
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Katie Vicari/Nature Publishing Group
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Ramirez-Ortiz et al. also demonstrate that TREML4 is needed for the binding and recruitment of MyD88 to TLR7 in response to GRD and that the absence of TREML4 results in impaired trafficking and co-localization of TLR7 and MyD88 to endosomes and lysosomes1 (Fig. 1). The consequence of these defects is an inability to induce accumulation of phosphorylated p38 and phosphorylation of STAT1 at Ser727 in response to GRD. Moreover, TREML4-deficient splenic macrophages do not produce type I interferons in response to stimulation of TLR7 with GRD, although their interferon responses to the stimulation of TLR9 are normal. However, the production of other cytokines, such as tumornecrosis factor, is impaired in TREML4deficient cells in response to stimulation of either TLR7 or TLR9. The authors then assess the effects of TREML4 in two in vivo models: the lupusprone MRL/lpr mice described above, and infection with influenza virus, an RNA virus. In MRL/lpr mice, TREML4 deficiency ameliorates several aspects of their disease, including diminishing the loss of body weight and the severity of nephritis and prolonging survival; TREML4-deficient MRL/lpr mice also show lower autoantibody production. In contrast, mice lacking TREML4 are more susceptible to influenza virus than are wild-type mice, and they produce suboptimal amounts of protective type I interferons during infection.
These findings show that TREML4 is a nonredundant amplifier of TLR7-dependent immunological activation in vivo and that the absence of TREML4 results in a phenotype similar to that of TLR7 deficiency. Some members of the TREM family, such as TREM1, amplify TLR signaling, whereas others, such as TREML1, suppress inflammation9; the findings reported here suggest that TREML4 belongs to the former category. Ramirez-Ortiz et al. also identify TREML4 as a key component of the pathway by which the recognition by TLR7 of RNA viruses or RNAcontaining immune complexes leads to strong induction of type I interferon responses1. In this context, the potential for therapeutic targeting of TREML4 will depend on the ability to specifically target pathological autoimmune processes while sparing the capacity to respond to viral infection. Several avenues remain for follow-up investigations. For example, several issues remain unresolved, including what the ligand of TREML4 is, and how TREML4 regulates the co-localization of TLR7 and MyD88 or whether it also interacts with other receptors, such as those that recognize apoptotic cells, such as TIM4. In addition to its role in responding to the recognition of nucleic acids, TLR7 is involved in the induction of other cellular processes, including autophagy10 and the formation of neutrophil extracellular traps11; whether TREML4 also amplifies these
processes remains an open question. TLR7 has also been linked to adult-onset Still’s disease12, and TLR7 activity shows the surprising effect of inducing anergy in CD4+ T cells during infection with human immunodeficiency virus13; future studies should determine the involvement of TREML4 in these diseases as well. Such studies will increase the understanding of the entire potential of TREML4-TLR7 interactions. Competing financial interests The authors declare no competing financial interests. 1. Ramirez-Ortiz, Z.G. et al. Nat. Immunol. 16, 495–504 (2015). 2. Means, T.K. et al. J. Clin. Invest. 115, 407–417 (2005). 3. Baccala, R., Hoebe, K., Kono, D.H., Beutler, B. & Theofilopoulos, A.N. Nat. Med. 13, 543–551 (2007). 4. Christensen, S.R. et al. Immunity 25, 417–428 (2006). 5. Pisitkun, P. et al. Autoreactive Science 312, 1669–1672 (2006). 6. Hemmi, H. et al. J. Immunol. 182, 1278–1286 (2009). 7. Hemmi H. et al. J. Immunol. 188, 1147–1155 (2012). 8. Idoyaga, J. et al. J. Clin. Invest. 123, 844–854 (2013). 9. Washington, A.V. et al. J. Clin. Invest. 119, 1489–1501 (2009). 10. Delgado, M.A., Elmaoued, R.A., Davis, A.S., Kyei, G. & Deretic, V. EMBO J. 27, 1110–1121 (2008). 11. Guiducci, C. et al. J. Exp. Med. 207, 2931–2942 (2010). 12. Chen, D.Y. et al. Arthritis Res. Ther. 15, R39 (2013). 13. Dominguez-Villar, M., Gautron, A.S., de Marcken, M., Keller, M.J. & Hafler, D.A. Nat. Immunol. 16, 118–128 (2015).
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TREML4 adds fuel to the TLR7 fire Mihai G Netea & Frank L van de Veerdonk The cell-surface receptor TREML4 amplifies cellular responses to single-stranded RNA by regulating recruitment of the adaptor MyD88 to the receptor TLR7. Mice lacking TREML4 show impaired antiviral immunity but also reduced severity of lupus-like disease. he Toll-like receptor TLR7 recognizes nucleic acids and is thought to have a key role in the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). In this issue of Nature Immunology, RamirezOrtiz et al. demonstrate that the receptor TREML4 amplifies TLR7-induced signaling and type I interferon responses by recruiting TLR7 and the adaptor Myd88 to the endolysosomal compartment, which subsequently leads to activation of the mitogen-activated protein kinase p38 and phosphorylation of the transcription factor STAT1 (ref. 1). Furthermore, the authors show that TREML4 deficiency results in amelioration of the development of SLE-like symptoms in lupus-prone MRL/lpr mice. SLE is a systemic autoimmune disorder that can affect the skin, joints, lungs and kidneys and the nervous system. Defects in the clearance of necrotic and apoptotic cells are thought to lead to the accumulation of self DNA and RNA that trigger the inflammation associated with this disease2. Single-stranded RNA is recognized by TLR7 in both humans and mice, whereas double-stranded DNA is recognized by TLR9. This process activates dendritic cells and, subsequently, T cells and B cells, which results in the production of autoantibodies that then form complexes with the self DNA and RNA. These complexes can be ingested by phagocytosis by dendritic cells via the receptor FcγRIIa and, in turn, trigger the production of type I interferon, which is thought to have a central role in the pathogenesis of SLE3. The importance of TLRs that recognize nucleic acids in the pathogenesis of SLE is underscored by the observation that lupus-prone MRL/lpr mice on a TLR7-deficient background have less severe disease than that of their TLR7-expressing counterparts4 and by the early onset of lupus in BXSB mice, which have a duplication of the
gene encoding TLR7 (ref. 5). Strikingly, TLR9 deficiency in MRL/lpr mice actually increases disease severity, an observation that has not been explained so far4. Intracellular recognition of RNA most probably evolved as a mechanism of protection against RNA viruses. However, when this process runs out of hand during auto immune processes such as SLE, it becomes very deleterious to the host. Understanding how this pathway is regulated, and especially which amplifiers of autoimmunity are induced by TLR7, is crucial for the identification of novel therapeutic targets. Ramirez-Ortez et al. elucidate a key step in that direction1. Using a lentivirus-based short-hairpin RNA screen targeting 8,000 mouse genes in RAW264.7 mouse macrophages, the authors search for genes encoding products involved in the TLR7-induced expression of tumor-necrosis factor and of the transcription factor NF-κB. In addition to genes encoding products already known to be involved in this, such as Myd88, they identify several genes encoding products previously not known to be involved, including TREML4, a member of the TREM (‘triggering receptor expressed on myeloid cells’) family of receptors. Although not much is known about TREML4, several functions have been reported that link it with the etiology of SLE, such as binding to dead cells6, involvement in antigen presentation7 and modulation of dendritic cell function8.
The authors then systematically elucidate the molecular mechanisms through which TREML4 amplifies TLR7 signaling (Fig. 1). They show that knockdown of TREML4 in RAW264.7 macrophages results in specific defects in cytokine induction by the TLR7 agonist gardiquimod (GRD) and the TLR9 agonist CpG, whereas cytokine production induced by binding to TLR2, TLR3 or TLR4 is not affected. Human embryonic kidney (HEK) cells transfected to express TREML4 alone do not transcribe the gene encoding interleukin 8 (IL-8) in response to stimulation with GRD, but HEK-293 cells transfected to express TLR7 (HEK-TLR7 cells) do transcribe this gene. When HEK-TLR7 cells are transfected to express TREML4 lacking its intracytoplasmic domain, GRD still induces transcription of the gene encoding IL-8, but transfection of HEK-TLR7 cells to express a TREML4 variant with substitution of the charged lysine residue in the transmembrane region results in defective induction of IL-8 expression. These data suggest that TREML4 does not contribute to TLR7 responses by inducing signaling but instead acts as a chaperone that modulates TLR7-induced responses. Human TREML4 has insertion of a stop codon just downstream of sequence encoding the transmembrane lysine residue, which suggests that in humans, TREML4 probably also does not modulate immune responses via direct signaling.
Immune Viral RNA complexes p38 TREML4
Nucleus
Type I interferons STAT1
MyD88 TLR7
Antiviral response
Autoimmunity in SLE
ER ssRNA Endolysosome
Mihai G. Netea and Frank L. van de Veerdonk are in the Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands. e-mail: [email protected] or [email protected]
Figure 1 Amplification of TLR7 signaling by TREML4. TLR7 recognizes internalized single-stranded RNA derived from viruses or from immunocomplexes containing self RNA. TLR7 activity requires interaction with MyD88; recognition of RNA by this complex leads to the activation of p38 and STAT1, which in turn leads to the production of type 1 interferons that mediate both antiviral responses and the deleterious autoimmune responses of SLE. Ramirez-Ortiz et al. show that TLR7-induced responses are potentiated by TREML4, which regulates trafficking and co-localization of TLR7 and MyD88 to endolysosomes1.
nature immunology volume 16 number 5 MAy 2015
445
Katie Vicari/Nature Publishing Group
npg
© 2015 Nature America, Inc. All rights reserved.
T
Ramirez-Ortiz et al. also demonstrate that TREML4 is needed for the binding and recruitment of MyD88 to TLR7 in response to GRD and that the absence of TREML4 results in impaired trafficking and co-localization of TLR7 and MyD88 to endosomes and lysosomes1 (Fig. 1). The consequence of these defects is an inability to induce accumulation of phosphorylated p38 and phosphorylation of STAT1 at Ser727 in response to GRD. Moreover, TREML4-deficient splenic macrophages do not produce type I interferons in response to stimulation of TLR7 with GRD, although their interferon responses to the stimulation of TLR9 are normal. However, the production of other cytokines, such as tumornecrosis factor, is impaired in TREML4deficient cells in response to stimulation of either TLR7 or TLR9. The authors then assess the effects of TREML4 in two in vivo models: the lupusprone MRL/lpr mice described above, and infection with influenza virus, an RNA virus. In MRL/lpr mice, TREML4 deficiency ameliorates several aspects of their disease, including diminishing the loss of body weight and the severity of nephritis and prolonging survival; TREML4-deficient MRL/lpr mice also show lower autoantibody production. In contrast, mice lacking TREML4 are more susceptible to influenza virus than are wild-type mice, and they produce suboptimal amounts of protective type I interferons during infection.
These findings show that TREML4 is a nonredundant amplifier of TLR7-dependent immunological activation in vivo and that the absence of TREML4 results in a phenotype similar to that of TLR7 deficiency. Some members of the TREM family, such as TREM1, amplify TLR signaling, whereas others, such as TREML1, suppress inflammation9; the findings reported here suggest that TREML4 belongs to the former category. Ramirez-Ortiz et al. also identify TREML4 as a key component of the pathway by which the recognition by TLR7 of RNA viruses or RNAcontaining immune complexes leads to strong induction of type I interferon responses1. In this context, the potential for therapeutic targeting of TREML4 will depend on the ability to specifically target pathological autoimmune processes while sparing the capacity to respond to viral infection. Several avenues remain for follow-up investigations. For example, several issues remain unresolved, including what the ligand of TREML4 is, and how TREML4 regulates the co-localization of TLR7 and MyD88 or whether it also interacts with other receptors, such as those that recognize apoptotic cells, such as TIM4. In addition to its role in responding to the recognition of nucleic acids, TLR7 is involved in the induction of other cellular processes, including autophagy10 and the formation of neutrophil extracellular traps11; whether TREML4 also amplifies these
processes remains an open question. TLR7 has also been linked to adult-onset Still’s disease12, and TLR7 activity shows the surprising effect of inducing anergy in CD4+ T cells during infection with human immunodeficiency virus13; future studies should determine the involvement of TREML4 in these diseases as well. Such studies will increase the understanding of the entire potential of TREML4-TLR7 interactions. Competing financial interests The authors declare no competing financial interests. 1. Ramirez-Ortiz, Z.G. et al. Nat. Immunol. 16, 495–504 (2015). 2. Means, T.K. et al. J. Clin. Invest. 115, 407–417 (2005). 3. Baccala, R., Hoebe, K., Kono, D.H., Beutler, B. & Theofilopoulos, A.N. Nat. Med. 13, 543–551 (2007). 4. Christensen, S.R. et al. Immunity 25, 417–428 (2006). 5. Pisitkun, P. et al. Autoreactive Science 312, 1669–1672 (2006). 6. Hemmi, H. et al. J. Immunol. 182, 1278–1286 (2009). 7. Hemmi H. et al. J. Immunol. 188, 1147–1155 (2012). 8. Idoyaga, J. et al. J. Clin. Invest. 123, 844–854 (2013). 9. Washington, A.V. et al. J. Clin. Invest. 119, 1489–1501 (2009). 10. Delgado, M.A., Elmaoued, R.A., Davis, A.S., Kyei, G. & Deretic, V. EMBO J. 27, 1110–1121 (2008). 11. Guiducci, C. et al. J. Exp. Med. 207, 2931–2942 (2010). 12. Chen, D.Y. et al. Arthritis Res. Ther. 15, R39 (2013). 13. Dominguez-Villar, M., Gautron, A.S., de Marcken, M., Keller, M.J. & Hafler, D.A. Nat. Immunol. 16, 118–128 (2015).
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