Despite the increasing number of studies concerning insect immunity, Lutzomyia longipalpis immune responses in the presence of Leishmania infantum chagasi infection has not been widely investigated. The few available studies analyzed the role of the Toll and IMD pathways involved in response against Leishmania and microbial infections. Nevertheless, effector molecules responsible for controlling sand fly infections have not been identified. In the present study we investigated the role a signal transduction pathway, the Transforming Growth Factor-beta (TGF-β) pathway, on the interrelation between L. longipalpis and L. i. chagasi. We identified an L. longipalpis homolog belonging to the multifunctional cytokine TGF-β gene family (LlTGF-β), which is closely related to the activin/inhibin subfamily and potentially involved in responses to infections. We investigated this gene expression through the insect development and in adult flies infected with L. i. chagasi. Our results showed that LlTGF-β was expressed in all L. longipalpis developmental stages and was upregulated at the third day post L. i. chagasi infection, when protein levels were also higher as compared to uninfected insects. At this point blood digestion is finished and parasites are in close contact with the insect gut. In addition, we investigated the role of LlTGF-β on L. longipalpis infection by L. i. chagasi using either gene silencing by RNAi or pathway inactivation by addition of the TGF-β receptor inhibitor SB431542. The blockage of the LlTGF-β pathway increased significantly antimicrobial peptides expression and nitric oxide levels in the insect gut, as expected. Both methods led to a decreased L. i. chagasi infection. Our results show that inactivation of the L. longipalpis TGF-β signal transduction pathway reduce L. i. chagasi survival, therefore suggesting that under natural conditions the parasite benefits from the insect LlTGF-β pathway, as already seen in Plamodium infection of mosquitoes.

Laboratório de Biologia Molecular de Parasitas e Vetores Instituto Oswaldo Cruz Rio de Janeiro Brazil

Parasitology Department Faculty of Science Charles University Prague Czechia

Zobrazit více v PubMed

Aleman-Muench G. R., Soldevila G. (2012). When versatility matters: activins/inhibins as key regulators of immunity. Immunol. Cell. Biol. 90, 137–148. 10.1038/icb.2011.32 PubMed DOI

Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. (1990). Basic local alignment search tool. J. Mol. Biol. 215, 403–410. 10.1016/S0022-2836(05)80360-2 PubMed DOI

Barral A., Teixeira M., Reis P., Vinhas V., Costa J., Lessa H., et al. . (1995). Transforming growth factor-beta in human cutaneous leishmaniasis. Am. J. Pathol. 147, 947–954. PubMed PMC

Berg D. T., Gupta A., Richardson M. A., O'Brien L. A., Calnek D., Grinnell B. W. (2007). Negative regulation of inducible nitric-oxide synthase expression mediated through transforming growth factor-beta-dependent modulation of transcription factor TCF11. J. Biol. Chem. 282, 36837–36844. 10.1074/jbc.M706909200 PubMed DOI

Bosco-Drayon V., Poidevin M., Boneca I. G., Narbonne-Reveau K., Royet J., Charroux B. (2012). Peptidoglycan sensing by the receptor PGRP-LE in the Drosophila gut induces immune responses to infectious bacteria and tolerance to microbiota. Cell Host Microbe 12, 153–165. 10.1016/j.chom.2012.06.002 PubMed DOI

Boulanger N., Lowenberger C., Volf P., Ursic R., Sigutova L., Sabatier L., et al. (2004). Characterization of a defensin from the sand fly Phlebotomus duboscqi induced by challenge with bacteria or the protozoan parasite Leishmania major. Infect. Immun. 72, 7140–7146. 10.1128/IAI.72.12.7140-7146.2004 PubMed DOI PMC

Chen W., Ten Dijke P. (2016). Immunoregulation by members of the TGFbeta superfamily. Nat. Rev. Immunol. 16, 723–740. 10.1038/nri.2016.112 PubMed DOI

Chng W. A., Sleiman M. S. B., Schüpfer F., Lemaitre B. (2014). Transforming growth factor beta/activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression. Cell Rep. 9, 336–348. 10.1016/j.celrep.2014.08.064 PubMed DOI

Coggins S. A., Estévez-Lao T. Y., Hillyer J. F. (2012). Increased survivorship following bacterial infection by the mosquito Aedes aegypti as compared to Anopheles gambiae correlates with increased transcriptional induction of antimicrobial peptides. Dev. Comp. Immunol. 37, 390–401. 10.1016/j.dci.2012.01.005 PubMed DOI

Covarrubias L., Hernandez-Garcia D., Schnabel D., Salas-Vidal E., Castro-Obregon S. (2008). Function of reactive oxygen species during animal development: passive or active? Dev. Biol. 320, 1–11. 10.1016/j.ydbio.2008.04.041 PubMed DOI

Crampton A., Luckhart S. (2001). The role of As60A, a TGF-beta homolog, in Anopheles stephensi innate immunity and defense against Plasmodium infection. Infect. Genet. Evol. 1, 131–141. 10.1016/S1567-1348(01)00017-X PubMed DOI

Diaz-Albiter H., Sant' Anna M. R., Genta F. A., Dillon R. J. (2012). Reactive oxygen species-mediated immunity against Leishmania mexicana and Serratia marcescens in the phlebotomine sand fly Lutzomyia longipalpis. J. Biol. Chem. 287, 23995–24003. 10.1074/jbc.M112.376095 PubMed DOI PMC

Foley E., O'Farrell P. H. (2003). Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila. Genes Dev. 17, 115–125. 10.1101/gad.1018503 PubMed DOI PMC

Freitas V. C., Parreiras K. P., Duarte A. P., Secundino N. F., Pimenta P. F. (2012). Development of Leishmania (Leishmania) infantum chagasi in its natural sandfly vector Lutzomyia longipalpis. Am. J. Trop. Med. Hyg. 86, 606–612. 10.4269/ajtmh.2012.11-0386 PubMed DOI PMC

Graça-Souza A. V., Maya-Monteiro C., Paiva-Silva G. O., Braz G. R., Paes M. C., Sorgine M. H., et al. . (2006). Adaptations against heme toxicity in blood-feeding arthropods. Insect Biochem. Mol. Biol. 36, 322–335. 10.1016/j.ibmb.2006.01.009 PubMed DOI

Ha E. M., Oh C. T., Ryu J. H., Bae Y. S., Kang S. W., Jang I. H., et al. . (2005). An antioxidant system required for host protection against gut infection in Drosophila. Dev. Cell. 8, 125–132. 10.1016/j.devcel.2004.11.007 PubMed DOI

Heerman M., Weng J. L., Hurwitz I., Durvasula R., Ramalho-Ortigao M. (2015). Bacterial infection and immune responses in lutzomyia longipalpis sand fly larvae midgut. PLoS Negl. Trop. Dis. 9:e0003923. 10.1371/journal.pntd.0003923 PubMed DOI PMC

Hickner P. V., Mori A., Zeng E., Tan J. C., Severson D. W. (2015). Whole transcriptome responses among females of the filariasis and arbovirus vector mosquito Culex pipiens implicate TGF-beta signaling and chromatin modification as key drivers of diapause induction. Funct. Integr. Genomics 15, 439–447. 10.1007/s10142-015-0432-5 PubMed DOI PMC

Hinck A. P., Mueller T. D., Springer T. A. (2016). Structural biology and evolution of the TGF-beta family. Cold Spring Harb Perspect. Biol. 8:12. 10.1101/cshperspect.a022103 PubMed DOI PMC

Hu X., Jiang Y., Gong Y., Zhu M., Zhu L., Chen F., et al. . (2016). Important roles played by TGF-beta member of Bmdpp and Bmdaw in BmNPV infection. Mol. Immunol. 73, 122–129. 10.1016/j.molimm.2016.04.004 PubMed DOI

Huminiecki L., Goldovsky L., Freilich S., Moustakas A., Ouzounis C., Heldin C., et al. . (2009). Emergence, development and diversification of the TGF-beta signalling pathway within the animal kingdom. BMC Evol. Biol. 9:28. 10.1186/1471-2148-9-28 PubMed DOI PMC

Jones K. L., de Kretser D. M., Patella S., Phillips D. J. (2004). Activin A and follistatin in systemic inflammation. Mol. Cell. Endocrinol. 225, 119–125. 10.1016/j.mce.2004.07.010 PubMed DOI

Kaneko T., Goldman W. E., Mellroth P., Steiner H., Fukase K., Kusumoto S., et al. (2004). Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20, 637–649. 10.1016/S1074-7613(04)00104-9 PubMed DOI

Kelly P. H., Bahr S. M., Serafim T. D., Ajami N. J., Petrosino J. F., Meneses C., et al. . (2017). The gut microbiome of the vector lutzomyia longipalpis is essential for survival of leishmania infantum. MBio 8:1. 10.1128/mBio.01121-16 PubMed DOI PMC

Kleino A., Silverman N. (2014). The Drosophila IMD pathway in the activation of the humoral immune response. Dev. Comp. Immunol. 42, 25–35. 10.1016/j.dci.2013.05.014 PubMed DOI PMC

Kodrík D., Bednárová A., Zemanová M., Krishnan N. (2015). Hormonal regulation of response to oxidative stress in insects-an update. Int. J. Mol. Sci. 16, 25788–25816. 10.3390/ijms161025788 PubMed DOI PMC

Lainson R., Rangel E. F. (2005). Lutzomyia longipalpis and the eco-epidemiology of American visceral leishmaniasis, with particular reference to Brazil: a review. Mem. Inst. Oswaldo Cruz 100, 811–827. 10.1590/S0074-02762005000800001 PubMed DOI

Lee K. B., Taghavi C. E., Murray S. S., Song K. J., Keorochana G., Wang J. C. (2012). BMP induced inflammation: a comparison of rhBMP-7 and rhBMP-2. J. Orthop. Res. 30, 1985–1994. 10.1002/jor.22160 PubMed DOI

Lemaitre B., Hoffmann J. (2007). The host defense of Drosophila melanogaster. Annu. Rev. Immunol. 25, 697–743. 10.1146/annurev.immunol.25.022106.141615 PubMed DOI

Leulier F., Parquet C., Pili-Floury S., Ryu J. H., Caroff M., Lee W. J., et al. . (2003). The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat. Immunol. 4, 478–484. 10.1038/ni922 PubMed DOI

Li H. Y., Lin X. W., Geng S. L., Xu W. H. (2018). TGF-beta and BMP signals regulate insect diapause through Smad1-POU-TFAM pathway. Biochim. Biophys. Acta Mol. Cell. Res. 1865, 1239–1249. 10.1016/j.bbamcr.2018.06.002 PubMed DOI

Lindsay S. A., Wasserman S. A. (2014). Conventional and non-conventional Drosophila Toll signaling. Dev. Comp. Immunol. 42, 16–24. 10.1016/j.dci.2013.04.011 PubMed DOI PMC

Luckhart S., Crampton A. L., Zamora R., Lieber M. J., Dos Santos P. C., Peterson T. M., et al. . (2003). Mammalian transforming growth factor beta1 activated after ingestion by Anopheles stephensi modulates mosquito immunity. Infect. Immun. 71, 3000–3009. 10.1128/IAI.71.6.3000-3009.2003 PubMed DOI PMC

Luckhart S., Vodovotz Y., Cui L., Rosenberg R. (1998). The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc. Natl. Acad. Sci. U.S.A. 95, 5700–5705. 10.1073/pnas.95.10.5700 PubMed DOI PMC

Massague J. (1990). The transforming growth factor-beta family. Annu. Rev. Cell. Biol. 6, 597–641. 10.1146/annurev.cb.06.110190.003121 PubMed DOI

Massague J. (2012). TGFbeta signalling in context. Nat. Rev. Mol. Cell. Biol. 13, 616–630. 10.1038/nrm3434 PubMed DOI PMC

Morikawa M., Derynck R., Miyazono K. (2016). TGF-beta and the TGF-beta family: context-dependent roles in cell and tissue physiology. Cold Spring Harb. Perspect. Biol. 8:5. 10.1101/cshperspect.a021873 PubMed DOI PMC

Mullen A. C., Wrana J. L. (2017). TGF-beta family signaling in embryonic and somatic stem-cell renewal and differentiation. Cold Spring Harb. Perspect. Biol. 9:7. 10.1101/cshperspect.a022186 PubMed DOI PMC

Murphy M. P., Holmgren A., Larsson N. G., Halliwell B., Chang C. J., Kalyanaraman B., et al. . (2011). Unraveling the biological roles of reactive oxygen species. Cell. Metab. 13, 361–366. 10.1016/j.cmet.2011.03.010 PubMed DOI PMC

Myllymäki H., Rämet M M. (2014). JAK/STAT pathway in Drosophila immunity. Scand. J. Immunol. 79, 377–385. 10.1111/sji.12170 PubMed DOI

O'Connor M. B., Umulis D., Othmer H. G., Blair S. S. (2006). Shaping BMP morphogen gradients in the Drosophila embryo and pupal wing. Development 133, 183–193. 10.1242/dev.02214 PubMed DOI PMC

Ogawa K., Funaba M. (2011). Activin in humoral immune responses. Vitam. Horm. 85, 235–253. 10.1016/B978-0-12-385961-7.00012-3 PubMed DOI

Pruzinova K., Sadlova J., Myskova J., Lestinova T., Janda J., Volf P. (2018). Leishmania mortality in sand fly blood meal is not species-specific and does not result from direct effect of proteinases. Parasit. Vectors 11:37 10.1186/s13071-018-2613-2 PubMed DOI PMC

Ramalho-Ortigao J. M., Temporal P., de Oliveira S. M. P., Barbosa A. F., Vilela M. L., Rangel E., et al. . (2001). Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis. Mem. Inst. Oswaldo Cruz. 96, 105–111. 10.1590/S0074-02762001000100012 PubMed DOI

Rogers M. E., Hajmová M., Joshi M. B., Sadlova J., Dwyer D. M., Volf P., et al. . (2008). Leishmania chitinase facilitates colonization of sand fly vectors and enhances transmission to mice. Cell Microbiol. 10, 1363–1372. 10.1111/j.1462-5822.2008.01132.x PubMed DOI PMC

Sadlova J., Myskova J., Lestinova T., Votypka J., Yeo M., Volf P. (2017). Leishmania donovani development in Phlebotomus argentipes: comparison of promastigote- and amastigote-initiated infections. Parasitology 144, 403–410. 10.1017/S0031182016002067 PubMed DOI PMC

Sant'Anna M. R., Alexander B., Bates P. A., Dillon R. J. (2008). Gene silencing in phlebotomine sand flies: xanthine dehydrogenase knock down by dsRNA microinjections. Insect Biochem. Mol. Biol. 38, 652–660. 10.1016/j.ibmb.2008.03.012 PubMed DOI PMC

Sant'Anna M. R., Diaz-Albiter H., Aguiar-Martins K., Al Salem W. S., Cavalcante R. R., Dillon V., et al. . (2014). Colonisation resistance in the sand fly gut: Leishmania protects Lutzomyia longipalpis from bacterial infection. Parasit. Vectors 7:329. 10.1186/1756-3305-7-329 PubMed DOI PMC

Schefe J. H., Lehmann K. E., Buschmann I. R., Unger T., Funke-Kaiser H. (2006). Quantitative real-time RT-PCR data analysis: current concepts and the novel gene expression's CT difference formula. J. Mol. Med. 84, 901–910. 10.1007/s00109-006-0097-6 PubMed DOI

Schneider C. A., Rasband W. S., Eliceiri K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. PubMed PMC

Shrinet J., Nandal U. K., Adak T., Bhatnagar R. K., Sunil S. (2014). Inference of the oxidative stress network in Anopheles stephensi upon Plasmodium infection. PLoS ONE 9:e114461. 10.1371/journal.pone.0114461 PubMed DOI PMC

Sideras P., Apostolou E., Stavropoulos A., Sountoulidis A., Gavriil A., Apostolidou A., et al. . (2013). Activin, neutrophils, and inflammation: just coincidence? Semin. Immunopathol. 35, 481–499. 10.1007/s00281-013-0365-9 PubMed DOI PMC

Souza A. V., Petretski J. H., Demasi M., Bechara E. J., Oliveira P. L. (1997). Urate protects a blood-sucking insect against hemin-induced oxidative stress. Free Radic. Biol. Med. 22, 209–214. 10.1016/S0891-5849(96)00293-6 PubMed DOI

Spottiswoode N., Armitage A. E., Williams A. R., Fyfe A. J., Biswas S., Hodgson S. H., et al. . (2017). Role of activins in hepcidin regulation during malaria. Infect. Immun. 85:12. 10.1128/IAI.00191-17 PubMed DOI PMC

Surachetpong W., Singh N., Cheung K. W., Luckhart S. (2009). MAPK ERK signaling regulates the TGF-beta1-dependent mosquito response to Plasmodium falciparum. PLoS Pathog. 5:e1000366. 10.1371/journal.ppat.1000366 PubMed DOI PMC

Telleria E. L., Martins-da-Silva A., Tempone A. J., Traub-Cseko Y. M. (2018). Leishmania, microbiota and sand fly immunity. Parasitology 2018, 1–18. 10.1017/S0031182018001014 PubMed DOI PMC

Telleria E. L., Pitaluga A., O Ortigão-Farias N. J., de Araújo R. A. P., Ramalho-Ortigão J. M., Traub-Cseko Y. M. (2007). Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis. Arch. Insect. Biochem. Physiol. 66, 53–63. 10.1002/arch.20198 PubMed DOI

Telleria E. L., Sant'Anna M. R., Alkurbi M. O., Pitaluga A. N., Dillon R. J., Traub-Cseko Y. M. (2013). Bacterial feeding, Leishmania infection and distinct infection routes induce differential defensin expression in Lutzomyia longipalpis. Parasit. Vectors 6:12. 10.1186/1756-3305-6-12 PubMed DOI PMC

Telleria E. L., Sant'Anna M. R. V., Ortigao-Farias J. R., Pitaluga A. N., Dillon V. M., Bates P. A., et al. . (2012). Caspar-like gene depletion reduces leishmania infection in sand fly host lutzomyia longipalpis. J. Biol. Chem. 287, 12985–12993. 10.1074/jbc.M111.331561 PubMed DOI PMC

Tinoco-Nunes B., Telleria E. L., Silva-Neves M., Marques C., Azevedo-Brito D. A., Pitaluga A. N. (2016). The sandfly Lutzomyia longipalpis LL5 embryonic cell line has active Toll and Imd pathways and shows immune responses to bacteria, yeast and Leishmania. Parasit Vectors 9:4. 10.1186/s13071-016-1507-4 PubMed DOI PMC

Vodovotz Y., Zamora R., Lieber M. J., Luckhart S. (2004). Cross-talk between nitric oxide and transforming growth factor-beta1 in malaria. Curr. Mol. Med. 4, 787–797. 10.2174/1566524043359999 PubMed DOI PMC

Wilson R., Bates M. D., Dostalova A., Jecna L., Dillon R. J., Volf P., et al. . (2010). Stage-specific adhesion of Leishmania promastigotes to sand fly midguts assessed using an improved comparative binding assay. PLoS Negl. Trop. Dis. 4:9. 10.1371/journal.pntd.0000816 PubMed DOI PMC

Evidence of a conserved mammalian immunosuppression mechanism in Lutzomyia longipalpis upon infection with Leishmania

RNAi-mediated gene silencing of Phlebotomus papatasi defensins favors Leishmania major infection

Phenotypical Differences between Leishmania (Leishmania) amazonensis PH8 and LV79 Strains May Impact Survival in Mammal Host and in Phlebotomine Sand Flies

The gene expression of Leishmania infantum chagasi inside Lutzomyia longipalpis, the main vector of visceral leishmaniasis in Brazil