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1.
Mucosal Immunol ; 15(6): 1243-1256, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35288645

RESUMO

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMß, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-ß, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection.


Assuntos
Fatores Inibidores da Migração de Macrófagos , Infecções por Strongylida , Camundongos , Animais , Fatores Inibidores da Migração de Macrófagos/genética , Imunidade Inata , Linfócitos , Nippostrongylus
2.
Int J Parasitol ; 51(1): 39-48, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32931780

RESUMO

Apyrases are a recurrent feature of secretomes from numerous species of parasitic nematodes. Here we characterise the five apyrases secreted by Heligmosomoides polygyrus, a natural parasite of mice and a widely used laboratory model for intestinal nematode infection. All five enzymes are closely related to soluble calcium-activated nucleotidases described in a variety of organisms, and distinct from the CD39 family of ecto-nucleotidases. Expression is maximal in adult worms and restricted to adults and L4s. Recombinant apyrases were produced and purified from Pichia pastoris. The five enzymes showed very similar biochemical properties, with strict calcium dependence and a broad substrate specificity, catalysing the hydrolysis of all nucleoside tri- and diphosphates, with no activity against nucleoside monophosphates. Natural infection of mice provoked very low antibodies to any enzyme, but immunisation with an apyrase cocktail showed partial protection against reinfection, with reduced egg output and parasite recovery. The most likely role for nematode secreted apyrases is hydrolysis of extracellular ATP, which acts as an alarmin for cellular release of IL-33 and initiation of type 2 immunity.


Assuntos
Enteropatias Parasitárias , Nematospiroides dubius , Animais , Apirase/genética , Camundongos , Saccharomycetales
3.
Front Immunol ; 10: 2375, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708913

RESUMO

Immunity to intestinal helminths is known to require both innate and adaptive components of the immune system activated along the Type 2 IL-4R/STAT6-dependent pathway. We have found that macrophage migration inhibitory factor (MIF) is essential for the development of effective immunity to the intestinal helminth Heligmosomoides polygyrus, even following vaccination which induces sterile immunity in wild-type mice. A chemical inhibitor of MIF, 4-IPP, was similarly found to compromise anti-parasite immunity. Cellular analyses found that the adaptive arm of the immune response, including IgG1 antibody responses and Th2-derived cytokines, was intact and that Foxp3+ T regulatory cell responses were unaltered in the absence of MIF. However, MIF was found to be an essential cytokine for innate cells, with ablated eosinophilia and ILC2 responses, and delayed recruitment and activation of macrophages to the M2 phenotype (expressing Arginase 1, Chil3, and RELM-α) upon infection of MIF-deficient mice; a macrophage deficit was also seen in wild-type BALB/c mice exposed to 4-IPP. Gene expression analysis of intestinal and lymph node tissues from MIF-deficient and -sufficient infected mice indicated significantly reduced levels of Arl2bp, encoding a factor involved in nuclear localization of STAT3. We further found that STAT3-deficient macrophages expressed less Arginase-1, and that mice lacking STAT3 in the myeloid compartment (LysMCrexSTAT3fl/fl) were unable to reject a secondary infection with H. polygyrus. We thus conclude that in the context of a Type 2 infection, MIF plays a critical role in polarizing macrophages into the protective alternatively-activated phenotype, and that STAT3 signaling may make a previously unrecognized contribution to immunity to helminths.


Assuntos
Imunidade Celular , Oxirredutases Intramoleculares/imunologia , Ativação de Macrófagos , Fatores Inibidores da Migração de Macrófagos/imunologia , Macrófagos/imunologia , Nematospiroides dubius/imunologia , Infecções por Strongylida/imunologia , Linfócitos T Reguladores/imunologia , Animais , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Macrófagos/patologia , Camundongos Endogâmicos BALB C , Camundongos Mutantes , Infecções por Strongylida/genética , Infecções por Strongylida/patologia , Linfócitos T Reguladores/patologia
4.
Elife ; 72018 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-30238872

RESUMO

Interleukin 25 (IL-25) is a major 'alarmin' cytokine, capable of initiating and amplifying the type immune response to helminth parasites. However, its role in the later effector phase of clearing chronic infection remains unclear. The helminth Heligmosomoides polygyrus establishes long-term infections in susceptible C57BL/6 mice, but is slowly expelled in BALB/c mice from day 14 onwards. We noted that IL-25R (Il17rb)-deficient BALB/c mice were unable to expel parasites despite type 2 immune activation comparable to the wild-type. We then established that in C57BL/6 mice, IL-25 adminstered late in infection (days 14-17) drove immunity. Moreover, when IL-25 and IL-4 were delivered to Rag1-deficient mice, the combination resulted in near complete expulsion of the parasite, even following administration of an anti-CD90 antibody to deplete innate lymphoid cells (ILCs). Hence, effective anti-helminth immunity during chronic infection requires an innate effector cell population that is synergistically activated by the combination of IL-4Rα and IL-25R signaling.


Assuntos
Imunidade Inata/imunologia , Nematospiroides dubius/imunologia , Receptores de Superfície Celular/imunologia , Receptores de Interleucina-17/imunologia , Infecções por Strongylida/imunologia , Células Th2/imunologia , Animais , Interações Hospedeiro-Parasita/imunologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Interleucina-17/imunologia , Interleucina-17/farmacologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nematospiroides dubius/fisiologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Células Th2/metabolismo
5.
J Parasitol ; 2018 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-30085900

RESUMO

In the course of a structure based drug discovery program the known anticancer candidate marimastat was uncovered as a potent inhibitor of an enzyme in nematode cuticle biogenesis. It was shown to kill Caenorhabditis elegans, and the sheep parasites Haemonchus contortus and Teladorsagia circumcinta via an entirely novel nematode-specific pathway, specifically by inhibiting cuticle-remodelling enzymes that the parasites require for the developmentally essential moulting process. This discovery prompted an investigation of the compound's effect on Heligmosomoides polygyrus parasites in a mouse model of helminth infection. Mice were administered the drug via oral gavage daily from day of infection for a period of 2 wk. A second group received the drug via intra-peritoneal implantation of an osmotic minipump for 4 wk. Control groups were administered identical volumes of water by oral gavage in both cases. Counts of H. polygyrus faecal egg and larval load showed that marimastat effected a consistent and significant reduction in egg laying, and a consistent but minor reduction in adult worm load when administered every day, starting on the first day of infection. However, the drug failed to have any significant effect on egg counts or worm burdens when administered to mice with established infections. Therefore, marimastat does not appear to show promise as an anthelmintic in gastrointestinal nematode infections, although other metalloproteases such as batimastat may prove more effective.

6.
Int J Parasitol ; 48(5): 379-385, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29510118

RESUMO

We recently reported the discovery of a new parasite-derived protein that functionally mimics the immunosuppressive cytokine transforming growth factor (TGF)-ß. The Heligmosomoides polygyrus TGF-ß Mimic (Hp-TGM) shares no homology to any TGF-ß family member, however it binds the mammalian TGF-ß receptor and induces expression of Foxp3, the canonical transcription factor of both mouse and human regulatory T cells. Hp-TGM consists of five atypical Complement Control Protein (CCP, Pfam 00084) domains, each lacking certain conserved residues and 12-15 amino acids longer than the 60-70 amino acids consensus domain, but with a recognizable 3-cysteine, tryptophan, cysteine motif. We now report on the identification of a family of nine related Hp-TGM homologues represented in the secreted proteome and transcriptome of H. polygyrus. Recombinant proteins from five of the nine new TGM members were tested for TGF-ß activity, but only two were functionally active in an MFB-F11 reporter assay, and by the induction of T cell Foxp3 expression. Sequence comparisons reveal that proteins with functional activity are similar or identical to Hp-TGM across the first three CCP domains, but more variable in domains 4 and 5. Inactive proteins diverged in all domains, or lacked some domains entirely. Testing truncated versions of Hp-TGM confirmed that domains 1-3 are essential for full activity in vitro, while domains 4 and 5 are not required. Further studies will elucidate whether these latter domains fulfill other functions in promoting host immune regulation during infection and if the more divergent family members play other roles in immunomodulation.


Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Nematospiroides dubius/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Clonagem Molecular , Fatores de Transcrição Forkhead/metabolismo , Proteínas de Helminto/genética , Camundongos , Baço/citologia , Fator de Crescimento Transformador beta/genética
7.
Nat Commun ; 8(1): 1741, 2017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29170498

RESUMO

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-ß signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-ß mimic (Hp-TGM) that replicates the biological and functional properties of TGF-ß, including binding to mammalian TGF-ß receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-ß or other members of the TGF-ß family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.


Assuntos
Mimetismo Molecular/imunologia , Nematospiroides dubius/imunologia , Nematospiroides dubius/patogenicidade , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/química , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mimetismo Molecular/genética , Nematospiroides dubius/genética , Ligação Proteica , Domínios Proteicos , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia
8.
Cell Rep ; 19(8): 1545-1557, 2017 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-28538175

RESUMO

Recent studies have demonstrated that many parasites release extracellular vesicles (EVs), yet little is known about the specific interactions of EVs with immune cells or their functions during infection. We show that EVs secreted by the gastrointestinal nematode Heligmosomoides polygyrus are internalized by macrophages and modulate their activation. EV internalization causes downregulation of type 1 and type 2 immune-response-associated molecules (IL-6 and TNF, and Ym1 and RELMα) and inhibits expression of the IL-33 receptor subunit ST2. Co-incubation with EV antibodies abrogated suppression of alternative activation and was associated with increased co-localization of the EVs with lysosomes. Furthermore, mice vaccinated with EV-alum generated protective immunity against larval challenge, highlighting an important role in vivo. In contrast, ST2-deficient mice are highly susceptible to infection, and they are unable to clear parasites following EV vaccination. Hence, macrophage activation and the IL-33 pathway are targeted by H. polygyrus EVs, while neutralization of EV function facilitates parasite expulsion.


Assuntos
Vesículas Extracelulares/metabolismo , Imunidade , Ativação de Macrófagos , Nematospiroides dubius/metabolismo , Parasitos/metabolismo , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/imunologia , Formação de Anticorpos/efeitos dos fármacos , Células da Medula Óssea/citologia , Citocalasina D/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Imunidade/efeitos dos fármacos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Parasitos/efeitos dos fármacos , Receptores de Interleucina/metabolismo , Vacinação
9.
Nature ; 529(7585): 226-30, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26762460

RESUMO

Helminth parasitic infections are a major global health and social burden. The host defence against helminths such as Nippostrongylus brasiliensis is orchestrated by type 2 cell-mediated immunity. Induction of type 2 cytokines, including interleukins (IL) IL-4 and IL-13, induce goblet cell hyperplasia with mucus production, ultimately resulting in worm expulsion. However, the mechanisms underlying the initiation of type 2 responses remain incompletely understood. Here we show that tuft cells, a rare epithelial cell type in the steady-state intestinal epithelium, are responsible for initiating type 2 responses to parasites by a cytokine-mediated cellular relay. Tuft cells have a Th2-related gene expression signature and we demonstrate that they undergo a rapid and extensive IL-4Rα-dependent amplification following infection with helminth parasites, owing to direct differentiation of epithelial crypt progenitor cells. We find that the Pou2f3 gene is essential for tuft cell specification. Pou2f3(-/-) mice lack intestinal tuft cells and have defective mucosal type 2 responses to helminth infection; goblet cell hyperplasia is abrogated and worm expulsion is compromised. Notably, IL-4Rα signalling is sufficient to induce expansion of the tuft cell lineage, and ectopic stimulation of this signalling cascade obviates the need for tuft cells in the epithelial cell remodelling of the intestine. Moreover, tuft cells secrete IL-25, thereby regulating type 2 immune responses. Our data reveal a novel function of intestinal epithelial tuft cells and demonstrate a cellular relay required for initiating mucosal type 2 immunity to helminth infection.


Assuntos
Imunidade nas Mucosas/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Nippostrongylus/imunologia , Parasitos/imunologia , Animais , Linhagem da Célula , Proliferação de Células , Retroalimentação Fisiológica , Feminino , Células Caliciformes/citologia , Células Caliciformes/imunologia , Interleucina-13/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Fatores de Transcrição de Octâmero/deficiência , Receptores de Interleucina-4/imunologia , Transdução de Sinais/imunologia , Células-Tronco/citologia , Células-Tronco/imunologia , Infecções por Strongylida/imunologia , Células Th2/citologia , Células Th2/imunologia
11.
J Vis Exp ; (98): e52412, 2015 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-25867600

RESUMO

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).


Assuntos
Nematospiroides dubius/crescimento & desenvolvimento , Nematospiroides dubius/metabolismo , Animais , Estágios do Ciclo de Vida , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA
12.
PLoS Pathog ; 11(3): e1004676, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25816012

RESUMO

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.


Assuntos
Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Imunoglobulina G/imunologia , Interleucina-4/imunologia , Interleucinas/imunologia , Nematospiroides dubius/imunologia , Infecções por Strongylida/imunologia , Vacinação , Animais , Anticorpos Anti-Helmínticos/genética , Humanos , Imunoglobulina G/genética , Interleucina-4/genética , Interleucinas/genética , Larva/imunologia , Camundongos , Camundongos Knockout , Infecções por Strongylida/genética , Infecções por Strongylida/prevenção & controle
13.
Nat Commun ; 5: 5488, 2014 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-25421927

RESUMO

In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.


Assuntos
Exossomos/metabolismo , Imunidade Inata , MicroRNAs/metabolismo , Nematoides/imunologia , Infecções por Nematoides/imunologia , RNA de Helmintos/imunologia , Alternaria/imunologia , Alternaria/fisiologia , Alternariose/genética , Alternariose/imunologia , Alternariose/microbiologia , Animais , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/imunologia , Exossomos/genética , Exossomos/imunologia , Humanos , Proteína 1 Semelhante a Receptor de Interleucina-1 , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/química , MicroRNAs/genética , MicroRNAs/imunologia , Nematoides/genética , Nematoides/metabolismo , Infecções por Nematoides/genética , Infecções por Nematoides/parasitologia , RNA de Helmintos/química , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/imunologia
14.
Gut Microbes ; 5(4): 522-32, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25144609

RESUMO

The intestinal microbiota are pivotal in determining the developmental, metabolic and immunological status of the mammalian host. However, the intestinal tract may also accommodate pathogenic organisms, including helminth parasites which are highly prevalent in most tropical countries. Both microbes and helminths must evade or manipulate the host immune system to reside in the intestinal environment, yet whether they influence each other's persistence in the host remains unknown. We now show that abundance of Lactobacillus bacteria correlates positively with infection with the mouse intestinal nematode parasite, Heligmosomoides polygyrus, as well as with heightened regulatory T cell (Treg) and Th17 responses. Moreover, H. polygyrus raises Lactobacillus species abundance in the duodenum of C57BL/6 mice, which are highly susceptible to H. polygyrus infection, but not in BALB/c mice, which are relatively resistant. Sequencing of samples at the bacterial gyrB locus identified the principal Lactobacillus species as L. taiwanensis, a previously characterized rodent commensal. Experimental administration of L. taiwanensis to BALB/c mice elevates regulatory T cell frequencies and results in greater helminth establishment, demonstrating a causal relationship in which commensal bacteria promote infection with an intestinal parasite and implicating a bacterially-induced expansion of Tregs as a mechanism of greater helminth susceptibility. The discovery of this tripartite interaction between host, bacteria and parasite has important implications for both antibiotic and anthelmintic use in endemic human populations.


Assuntos
Trato Gastrointestinal/microbiologia , Lactobacillus/fisiologia , Interações Microbianas , Nematospiroides dubius/fisiologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Trato Gastrointestinal/imunologia , Interações Hospedeiro-Patógeno , Lactobacillus/crescimento & desenvolvimento , Lactobacillus/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nematospiroides dubius/crescimento & desenvolvimento , Nematospiroides dubius/imunologia
15.
J Immunol ; 193(6): 2984-93, 2014 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-25114104

RESUMO

Helminth parasites remain one of the most common causes of infections worldwide, yet little is still known about the immune signaling pathways that control their expulsion. C57BL/6 mice are chronically susceptible to infection with the gastrointestinal helminth parasite Heligmosomoides polygyrus. In this article, we report that C57BL/6 mice lacking the adapter protein MyD88, which mediates signaling by TLRs and IL-1 family members, showed enhanced immunity to H. polygyrus infection. Alongside increased parasite expulsion, MyD88-deficient mice showed heightened IL-4 and IL-17A production from mesenteric lymph node CD4(+) cells. In addition, MyD88(-/-) mice developed substantial numbers of intestinal granulomas around the site of infection, which were not seen in MyD88-sufficient C57BL/6 mice, nor when signaling through the adapter protein TRIF (TIR domain-containing adapter-inducing IFN-ß adapter protein) was also ablated. Mice deficient solely in TLR2, TLR4, TLR5, or TLR9 did not show enhanced parasite expulsion, suggesting that these TLRs signal redundantly to maintain H. polygyrus susceptibility in wild-type mice. To further investigate signaling pathways that are MyD88 dependent, we infected IL-1R1(-/-) mice with H. polygyrus. This genotype displayed heightened granuloma numbers compared with wild-type mice, but without increased parasite expulsion. Thus, the IL-1R-MyD88 pathway is implicated in inhibiting granuloma formation; however, protective immunity in MyD88-deficient mice appears to be granuloma independent. Like IL-1R1(-/-) and MyD88(-/-) mice, animals lacking signaling through the type 1 IFN receptor (i.e., IFNAR1(-/-)) also developed intestinal granulomas. Hence, IL-1R1, MyD88, and type 1 IFN receptor signaling may provide pathways to impede granuloma formation in vivo, but additional MyD88-mediated signals are associated with inhibition of protective immunity in susceptible C57BL/6 mice.


Assuntos
Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Nematospiroides dubius/imunologia , Infecções por Strongylida/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Granuloma/genética , Granuloma/imunologia , Interleucina-17/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/genética , Receptores Tipo I de Interleucina-1/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Infecções por Strongylida/parasitologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 5 Toll-Like/genética , Receptor Toll-Like 9/genética
16.
Mol Cell Proteomics ; 13(10): 2736-51, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24994561

RESUMO

Hookworms infect more than 700 million people worldwide and cause more morbidity than most other human parasitic infections. Nippostrongylus brasiliensis (the rat hookworm) has been used as an experimental model for human hookworm because of its similar life cycle and ease of maintenance in laboratory rodents. Adult N. brasiliensis, like the human hookworm, lives in the intestine of the host and releases excretory/secretory products (ESP), which represent the major host-parasite interface. We performed a comparative proteomic analysis of infective larval (L3) and adult worm stages of N. brasiliensis to gain insights into the molecular bases of host-parasite relationships and determine whether N. brasiliensis could indeed serve as an appropriate model for studying human hookworm infections. Proteomic data were matched to a transcriptomic database assembled from 245,874,892 Illumina reads from different developmental stages (eggs, L3, L4, and adult) of N. brasiliensis yielding∼18,426 unigenes with 39,063 possible isoform transcripts. From this analysis, 313 proteins were identified from ESPs by LC-MS/MS-52 in the L3 and 261 in the adult worm. Most of the proteins identified in the study were stage-specific (only 13 proteins were shared by both stages); in particular, two families of proteins-astacin metalloproteases and CAP-domain containing SCP/TAPS-were highly represented in both L3 and adult ESP. These protein families are present in most nematode groups, and where studied, appear to play roles in larval migration and evasion of the host's immune response. Phylogenetic analyses of defined protein families and global gene similarity analyses showed that N. brasiliensis has a greater degree of conservation with human hookworm than other model nematodes examined. These findings validate the use of N. brasiliensis as a suitable parasite for the study of human hookworm infections in a tractable animal model.


Assuntos
Ancylostomatoidea/crescimento & desenvolvimento , Trato Gastrointestinal/parasitologia , Proteínas de Helminto/metabolismo , Estágios do Ciclo de Vida , Proteoma/análise , Ancylostomatoidea/metabolismo , Animais , Sequência de Bases , Sequência Conservada , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Filogenia , Proteoma/metabolismo , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Análise de Sequência de RNA
17.
PLoS Pathog ; 10(2): e1003930, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24586152

RESUMO

Human lymphatic filariasis is a major tropical disease transmitted through mosquito vectors which take up microfilarial larvae from the blood of infected subjects. Microfilariae are produced by long-lived adult parasites, which also release a suite of excretory-secretory products that have recently been subject to in-depth proteomic analysis. Surprisingly, the most abundant secreted protein of adult Brugia malayi is triose phosphate isomerase (TPI), a glycolytic enzyme usually associated with the cytosol. We now show that while TPI is a prominent target of the antibody response to infection, there is little antibody-mediated inhibition of catalytic activity by polyclonal sera. We generated a panel of twenty-three anti-TPI monoclonal antibodies and found only two were able to block TPI enzymatic activity. Immunisation of jirds with B. malayi TPI, or mice with the homologous protein from the rodent filaria Litomosoides sigmodontis, failed to induce neutralising antibodies or protective immunity. In contrast, passive transfer of neutralising monoclonal antibody to mice prior to implantation with adult B. malayi resulted in 60-70% reductions in microfilarial levels in vivo and both oocyte and microfilarial production by individual adult females. The loss of fecundity was accompanied by reduced IFNγ expression by CD4⁺ T cells and a higher proportion of macrophages at the site of infection. Thus, enzymatically active TPI plays an important role in the transmission cycle of B. malayi filarial parasites and is identified as a potential target for immunological and pharmacological intervention against filarial infections.


Assuntos
Brugia Malayi/patogenicidade , Filariose Linfática/enzimologia , Microfilárias , Triose-Fosfato Isomerase/metabolismo , Animais , Anticorpos Anti-Helmínticos/imunologia , Anticorpos Neutralizantes/imunologia , Western Blotting , Brugia Malayi/enzimologia , Brugia Malayi/imunologia , Filariose Linfática/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Gerbillinae , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C
18.
Immunol Cell Biol ; 92(5): 436-48, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24492801

RESUMO

The nematode Heligmosomoides polygyrus is an excellent model for intestinal helminth parasitism. Infection in mice persists for varying lengths of time in different inbred strains, with CBA and C57BL/6 mice being fully susceptible, BALB/c partially so and SJL able to expel worms within 2-3 weeks of infection. We find that resistance correlates not only with the adaptive Th2 response, including IL-10 but with activation of innate lymphoid cell and macrophage populations. In addition, the titer and specificity range of the serum antibody response is maximal in resistant mice. In susceptible strains, Th2 responses were found to be counterbalanced by IFN-γ-producing CD4(+) and CD8(+) cells, but these are not solely responsible for susceptibility as mice deficient in either CD8(+) T cells or IFN-γ remain unable to expel the parasites. Foxp3(+) Treg numbers were comparable in all strains, but in the most resistant SJL strain, this population does not upregulate CD103 in infection, and in the lamina propria the frequency of Foxp3(+)CD103(+) T cells is significantly lower than in susceptible mice. The more resistant SJL and BALB/c mice develop macrophage-rich IL-4Rα-dependent Type 2 granulomas around intestinal sites of larval invasion, and expression of alternative activation markers Arginase-1, Ch3L3 (Ym1) and RELM-α within the intestine and the peritoneal lavage was also strongly correlated with helminth elimination in these strains. Clodronate depletion of phagocytic cells compromises resistance of BALB/c mice and slows expulsion in the SJL strain. Thus, Type 2 immunity involves IL-4Rα-dependent innate cells including but not limited to a phagocyte population, the latter likely involving the action of specific antibodies.


Assuntos
Imunidade Adaptativa , Resistência à Doença/genética , Resistência à Doença/imunologia , Helmintíase/genética , Helmintíase/imunologia , Helmintos/imunologia , Imunidade Inata , Enteropatias/genética , Enteropatias/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Anticorpos Anti-Helmínticos/imunologia , Ácido Clodrônico/farmacologia , Feminino , Granuloma , Helmintíase/tratamento farmacológico , Helmintíase/metabolismo , Helmintíase/patologia , Helmintíase Animal , Imunidade Celular , Imunidade Inata/efeitos dos fármacos , Interferon gama/metabolismo , Enteropatias/tratamento farmacológico , Enteropatias/metabolismo , Enteropatias/patologia , Enteropatias Parasitárias , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Camundongos , Receptores de Interleucina-4/metabolismo , Transdução de Sinais
19.
PLoS Pathog ; 9(8): e1003492, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23966853

RESUMO

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.


Assuntos
Antígenos de Helmintos/metabolismo , Proteínas de Helminto/metabolismo , Larva/metabolismo , Infecções por Nematoides/imunologia , Nematospiroides dubius/imunologia , Proteômica , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Western Blotting , Cromatografia Líquida , Biologia Computacional , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Feminino , Perfilação da Expressão Gênica , Proteínas de Helminto/imunologia , Interações Hospedeiro-Parasita , Imunização , Imunoprecipitação , Larva/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Infecções por Nematoides/parasitologia , Nematospiroides dubius/crescimento & desenvolvimento , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vacinação
20.
Infect Immun ; 80(10): 3481-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22851746

RESUMO

Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11c(high) dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11c(high) cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11c(high) cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-α, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-ß in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid ("nuocyte") populations all proceeded irrespective of depletion of CD11c(high) cells or basophils. Thus, while CD11c(high) DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection.


Assuntos
Antígeno CD11c/metabolismo , Células Dendríticas/imunologia , Imunidade Inata/fisiologia , Nematospiroides dubius/imunologia , Nippostrongylus/imunologia , Infecções por Strongylida/imunologia , Animais , Basófilos/imunologia , Antígeno CD11c/genética , Citocinas/genética , Citocinas/metabolismo , Citometria de Fluxo , Macrófagos/metabolismo , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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