RESUMO
Endotoxins, also known as lipopolysaccharides (LPS), are essential components of cell walls of diderm bacteria such as Escherichia coli. LPS are microbe-associated molecular patterns that can activate pattern recognition receptors. While trying to investigate the interactions between proteins and host innate immunity, some studies using recombinant proteins expressed in E. coli reported interaction and activation of immune cells. Here, we set out to provide information on endotoxins that are highly toxic to humans and bind to numerous molecules, including recombinant proteins. We begin by outlining the history of the discovery of endotoxins, their receptors and the associated signaling pathways that confer extreme sensitivity to immune cells, acting alone or in synergy with other microbe-associated molecular patterns. We list the various places where endotoxins have been found. Additionally, we warn against the risk of data misinterpretation due to endotoxin contamination in recombinant proteins, which is difficult to estimate with the Limulus amebocyte lysate assay, and cannot be completely neutralized (e.g., treatment with polymyxin B or heating). We further illustrate our point with examples of recombinant heat-shock proteins and viral proteins from severe acute respiratory syndrome coronavirus 2, dengue and HIV, for which endotoxin contamination has eventually been shown to be responsible for the inflammatory roles previously ascribed. We also critically appraised studies on recombinant Leptospira proteins regarding their putative inflammatory roles. Finally, to avoid these issues, we propose alternatives to express recombinant proteins in nonmicrobial systems. Microbiologists wishing to undertake innate immunity studies with their favorite pathogens should be aware of these difficulties.
Assuntos
Imunidade Inata , Leptospira , Lipopolissacarídeos , Proteínas Recombinantes , Humanos , Escherichia coli/genética , Lipopolissacarídeos/toxicidade , Proteínas Recombinantes/metabolismo , Leptospira/metabolismoRESUMO
Leptospira interrogans are bacteria that can infect all vertebrates and are responsible for leptospirosis, a neglected zoonosis. Some hosts, such as humans, are susceptible to the disease, whereas mice are resistant and get chronically colonized. Although leptospires escape recognition by some immune receptors, they activate the NOD-like receptor pyrin 3-inflammasome and trigger IL-1ß secretion. Classically, IL-1ß secretion is associated with lytic inflammatory cell death called pyroptosis, resulting from cytosolic LPS binding to inflammatory caspases, such as caspase 11. Interestingly, we showed that L. interrogans and Leptospira biflexa do not trigger cell death in either murine, human, hamster, or bovine macrophages, escaping both pyroptosis and apoptosis. We showed, in murine cells, that the mild IL-1ß secretion induced by leptospires occurred through nonlytic caspase 8-dependent gasdermin D pore formation and not through activation of caspase 11/noncanonical inflammasome. Strikingly, we demonstrated a potent antagonistic effect of pathogenic L. interrogans and their atypical LPS on spontaneous and Escherichia coli LPS-induced cell death. Indeed, LPS of L. interrogans efficiently prevents caspase 11 dimerization and subsequent massive gasdermin D cleavage. Finally, we showed that pyroptosis escape by leptospires prevents massive IL-1ß release, and we consistently found no major role of IL-1R in controlling experimental leptospirosis in vivo. Overall, to our knowledge, our findings described a novel mechanism by which leptospires dampen inflammation, thus potentially contributing to their stealthiness.
Assuntos
Leptospira interrogans , Leptospirose , Animais , Bovinos , Cricetinae , Humanos , Camundongos , Caspases/metabolismo , Gasderminas , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Leptospira interrogans/metabolismo , Leptospirose/metabolismo , Leptospirose/microbiologia , Lipopolissacarídeos , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Morte CelularRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1008639.].
RESUMO
Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccharide (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal microscopy and flow cytometry, we showed that the LPS of L. interrogans did not induce internalization of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicrobial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall, we describe here a novel leptospiral immune escape mechanism from mouse macrophages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Leptospira/imunologia , Leptospirose/imunologia , Lipopolissacarídeos/metabolismo , Lipoproteínas/metabolismo , Antígenos O/metabolismo , Receptor 4 Toll-Like/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Citocinas/metabolismo , Feminino , Leptospirose/metabolismo , Leptospirose/microbiologia , Leptospirose/patologia , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipoproteínas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/fisiologia , Antígenos O/genética , Transdução de Sinais , Receptor 2 Toll-Like/fisiologiaRESUMO
Leptospira interrogans are pathogenic spirochetes responsible for leptospirosis, a worldwide reemerging zoonosis. Many Leptospira serovars have been described, and prophylaxis using inactivated bacteria provides only short-term serovar-specific protection. Therefore, alternative approaches to limit severe leptospirosis in humans and morbidity in cattle would be welcome. Innate immune cells, including macrophages, play a key role in fighting infection and pathogen clearance. Recently, it has been shown that functional reprograming of innate immune cells through the activation of pattern recognition receptors leads to enhanced nonspecific antimicrobial responses upon a subsequent microbial encounter. This mechanism is known as trained immunity or innate immune memory. We have previously shown that oral treatment with Lactobacillus plantarum confers a beneficial effect against acute leptospirosis. Here, using a macrophage depletion protocol and live imaging in mice, we established the role of peritoneal macrophages in limiting the initial dissemination of leptospires. We further showed that intraperitoneal priming of mice with CL429, a TLR2 and NOD2 agonist known to mimic the modulatory effect of Lactobacillus, alleviated acute leptospiral infection. The CL429 treatment was characterized as a training effect since i.) it was linked to peritoneal macrophages that produced ex vivo more pro-inflammatory cytokines and chemokines against 3 different pathogenic serovars of Leptospira, independently of the presence of B and T cells, ii.) it had systemic effects on splenic cells and bone marrow derived macrophages, and iii.) it was sustained for 3 months. Importantly, trained macrophages produced more nitric oxide, a potent antimicrobial compound, which has not been previously linked to trained immunity. Accordingly, trained macrophages better restrict leptospiral survival. Finally, we could use CL429 to train ex vivo human monocytes that produced more cytokines upon leptospiral stimulation. In conclusion, host-directed treatment using a TLR2/NOD2 agonist could be envisioned as a novel prophylactic strategy against acute leptospirosis.
Assuntos
Memória Imunológica/imunologia , Leptospira interrogans/imunologia , Leptospirose/prevenção & controle , Macrófagos Peritoneais/imunologia , Proteína Adaptadora de Sinalização NOD2/agonistas , Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 2 Toll-Like/agonistas , Animais , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Memória Imunológica/efeitos dos fármacos , Leptospirose/imunologia , Leptospirose/metabolismo , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
Leptospirosis is a widespread zoonosis, potentially severe in humans, caused by spirochetal bacteria, Leptospira interrogans (L. interrogans). Host defense mechanisms involved in leptospirosis are poorly understood. Recognition of lipopolysaccharide (LPS) and lipoproteins by Toll-Like Receptors (TLR)4 and TLR2 is crucial for clearance of leptospires in mice, yet the role of Nucleotide Oligomerization Domain (NOD)-like receptors (NOD)1 and NOD2, recognizing peptidoglycan (PG) fragments has not previously been examined. Here, we show that pathogenic leptospires escape from NOD1 and NOD2 recognition both in vitro and in vivo, in mice. We found that leptospiral PG is resistant to digestion by certain hydrolases and that a conserved outer membrane lipoprotein of unknown function, LipL21, specific for pathogenic leptospires, is tightly bound to the PG. Leptospiral PG prepared from a mutant not expressing LipL21 (lipl21-) was more readily digested than the parental or complemented strains. Muropeptides released from the PG of the lipl21- mutant, or prepared using a procedure to eliminate the LipL21 protein from the PG of the parental strain, were recognized in vitro by the human NOD1 (hNOD1) and NOD2 (hNOD2) receptors, suggesting that LipL21 protects PG from degradation into muropeptides. LipL21 expressed in E. coli also resulted in impaired PG digestion and NOD signaling. We found that murine NOD1 (mNOD1) did not recognize PG of L. interrogans. This result was confirmed by mass spectrometry showing that leptospiral PG was primarily composed of MurTriDAP, the natural agonist of hNOD1, and contained only trace amounts of the tetra muropeptide, the mNOD1 agonist. Finally, in transgenic mice expressing human NOD1 and deficient for the murine NOD1, we showed enhanced clearance of a lipl21- mutant compared to the complemented strain, or to what was observed in NOD1KO mice, suggesting that LipL21 facilitates escape from immune surveillance in humans. These novel mechanisms allowing L. interrogans to escape recognition by the NOD receptors may be important in circumventing innate host responses.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Evasão da Resposta Imune , Leptospira interrogans/imunologia , Leptospira interrogans/patogenicidade , Lipoproteínas/metabolismo , Proteína Adaptadora de Sinalização NOD1/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Peptidoglicano/metabolismo , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Feminino , Humanos , Evasão da Resposta Imune/genética , Imunidade Inata , Leptospira/imunologia , Leptospira interrogans/genética , Leptospirose/genética , Leptospirose/imunologia , Leptospirose/microbiologia , Lipoproteínas/genética , Lipoproteínas/imunologia , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD2/deficiência , Proteína Adaptadora de Sinalização NOD2/genética , Peptidoglicano/química , Peptidoglicano/imunologia , Ligação Proteica , Transdução de Sinais , Especificidade da Espécie , Virulência/genética , Virulência/imunologiaRESUMO
Innate immunity encompasses immediate host responses that detect and respond to microbes. Besides recognition by the complement system (see the chapter by A. Barbosa, this volume), innate immunity concerns cellular responses. These are triggered through recognition of conserved microbial components (called MAMPs) by pattern recognition receptors (PRRs), leading, through secretion of cytokines, antimicrobial peptides, and immune mediators, to cellular recruitment and phagocytosis. Leptospira spp. are successful zoonotic pathogenic bacteria that obviously overcome the immune system of their hosts. The first part of this chapter summarizes what is known about leptospires recognition and interaction with phagocytes and other innate immune cells, and the second part describes specific interactions of leptospiral MAMPs with PRRs from the TLR and NLR families. On the one hand, pathogenic leptospires appear to escape macrophage and neutrophil phagocytosis. On the other hand, studies about PRR sensing of leptospires remain very limited, but suggest that pathogenic leptospires escape some of the PRRs in a host-specific manner, due to peculiar cell wall specificities or post-translational modifications that may impair their recognition. Further studies are necessary to clarify the mechanisms and consequences of leptospiral escape on phagocytic functions and hopefully give clues to potential therapeutic strategies aimed at restoring the defective activation of PRRs by pathogenic Leptospira spp.
Assuntos
Sistema Imunitário/imunologia , Imunidade Inata/imunologia , Leptospira/imunologia , Leptospirose/imunologia , Leptospirose/microbiologia , Receptores de Reconhecimento de Padrão/imunologia , Proteínas do Sistema Complemento , HumanosRESUMO
Respiratory infectious diseases are the third cause of worldwide death. The nasopharynx is the portal of entry and the ecological niche of many microorganisms, of which some are pathogenic to humans, such as Neisseria meningitidis and Moraxella catarrhalis. These microbes possess several surface structures that interact with the actors of the innate immune system. In our attempt to understand the past evolution of these bacteria and their adaption to the nasopharynx, we first studied differences in cell wall structure, one of the strongest immune-modulators. We were able to show that a modification of peptidoglycan (PG) composition (increased proportion of pentapeptides) and a cell shape change from rod to cocci had been selected for along the past evolution of N. meningitidis. Using genomic comparison across species, we correlated the emergence of the new cell shape (cocci) with the deletion, from the genome of N. meningitidis ancestor, of only one gene: yacF. Moreover, the reconstruction of this genetic deletion in a bacterium harboring the ancestral version of the locus together with the analysis of the PG structure, suggest that this gene is coordinating the transition from cell elongation to cell division. Accompanying the loss of yacF, the elongation machinery was also lost by several of the descendants leading to the change in the PG structure observed in N. meningitidis. Finally, the same evolution was observed for the ancestor of M. catarrhalis. This suggests a strong selection of these genetic events during the colonization of the nasopharynx. This selection may have been forced by the requirement of evolving permissive interaction with the immune system, the need to reduce the cellular surface exposed to immune attacks without reducing the intracellular storage capacity, or the necessity to better compete for adhesion to target cells.
Assuntos
Adaptação Fisiológica/genética , Estruturas da Membrana Celular/imunologia , Moraxella catarrhalis/genética , Neisseria meningitidis/genética , Mucosa Respiratória/microbiologia , Evolução Biológica , Proteínas de Ciclo Celular/genética , Humanos , Moraxella catarrhalis/imunologia , Moraxella catarrhalis/fisiologia , Nasofaringe/microbiologia , Neisseria meningitidis/imunologia , Neisseria meningitidis/fisiologia , Peptidoglicano/química , Peptidoglicano/imunologia , Mucosa Respiratória/imunologiaRESUMO
Muramyl dipeptide (MDP), a product of bacterial cell-wall peptidoglycan, activates innate immune cells by stimulating nucleotide-binding oligomerization domain containing 2 (NOD2) -dependent activation of the transcription factor NFkappaB and transcription of proinflammatory genes. A20 is a ubiquitin-modifying enzyme that restricts tumor necrosis factor (TNF) receptor and Toll-like receptor (TLR) -induced signals. We now show that MDP induces ubiquitylation of receptor- interacting protein 2 (RIP2) in primary macrophages. A20-deficient cells exhibit dramatically amplified responses to MDP, including increased RIP2 ubiquitylation, prolonged NFkappaB signaling, and increased production of proinflammatory cytokines. In addition, in vivo responses to MDP are exaggerated in A20-deficient mice and in chimeric mice bearing A20-deficient hematopoietic cells. These exaggerated responses occur independently of the TLR adaptors MyD88 and TRIF as well as TNF signals. These findings indicate that A20 directly restricts NOD2 induced signals in vitro and in vivo, and provide new insights into how these signals are physiologically restricted.
Assuntos
Cisteína Endopeptidases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Adaptadora de Sinalização NOD2/metabolismo , Transdução de Sinais/fisiologia , Ubiquitinação/fisiologia , Acetilmuramil-Alanil-Isoglutamina/farmacologia , Animais , Macrófagos/metabolismo , Camundongos , Camundongos Mutantes , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina/metabolismoRESUMO
CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance.
Assuntos
Anti-Infecciosos/metabolismo , Quimiocina CCL20/metabolismo , Criptosporidiose/imunologia , Cryptosporidium parvum/fisiologia , MicroRNAs/genética , Animais , Animais Recém-Nascidos , Linhagem Celular , Quimiocina CCL20/genética , Modelos Animais de Doenças , Células Epiteliais , Interferon gama/genética , Interferon gama/metabolismo , Intestinos/imunologia , Intestinos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes , Organismos Livres de Patógenos Específicos , EsporozoítosRESUMO
Cryptosporidium parvum is a zoonotic protozoan parasite found worldwide, that develops only in the gastrointestinal epithelium and causes profuse diarrhea. Using a mouse model of C. parvum infection, we demonstrated by conditional depletion of CD11c+ cells that these cells are essential for the control of the infection both in neonates and adults. Neonates are highly susceptible to C. parvum but the infection is self-limited, whereas adults are resistant unless immunocompromised. We investigated the contribution of DC to the age-dependent susceptibility to infection. We found that neonates presented a marked deficit in intestinal CD103+ DC during the first weeks of life, before weaning, due to weak production of chemokines by neonatal intestinal epithelial cells (IEC). Increasing the number of intestinal CD103+ DC in neonates by administering FLT3-L significantly reduced susceptibility to the infection. During infections in neonates, the clearance of the parasite was preceded by a rapid recruitment of CD103+ DC mediated by CXCR3-binding chemokines produced by IEC in response to IFNγ. In addition to this key role in CD103+ DC recruitment, IFNγ is known to inhibit intracellular parasite development. We demonstrated that during neonatal infection CD103+ DC produce IL-12 and IFNγ in the lamina propria and the draining lymph nodes. Thus, CD103+DC are key players in the innate immune control of C. parvum infection in the intestinal epithelium. The relative paucity of CD103+ DC in the neonatal intestine contributes to the high susceptibility to intestinal infection.
Assuntos
Antígenos CD/metabolismo , Criptosporidiose/imunologia , Cryptosporidium parvum/imunologia , Células Dendríticas/fisiologia , Imunidade Inata , Cadeias alfa de Integrinas/metabolismo , Intestinos/imunologia , Fatores Etários , Animais , Animais Recém-Nascidos , Bovinos , Criança , Células Dendríticas/metabolismo , Humanos , Intestinos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.
Assuntos
Quimiocinas/metabolismo , Ciclosporina/efeitos adversos , Imunossupressores/efeitos adversos , Transplante de Rim , Rim/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD1/metabolismo , Pielonefrite/induzido quimicamente , Doença Aguda , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Movimento Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Feminino , Inativação Gênica , Humanos , Imunidade Inata , Rim/metabolismo , Rim/patologia , Transplante de Rim/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Transcrição NFATC/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Técnicas de Cultura de Órgãos , Fagocitose/efeitos dos fármacosRESUMO
Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4(-/-) MCDs, but not in Tlr5(-/-) MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5(-/-) than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5(-/-) kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post-infected Tlr5(-/-) kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.
Assuntos
Infecções por Escherichia coli/imunologia , Flagelina/imunologia , Túbulos Renais Coletores/imunologia , Receptor 5 Toll-Like/imunologia , Escherichia coli Uropatogênica/patogenicidade , Animais , Aderência Bacteriana/fisiologia , Carga Bacteriana/imunologia , Quimiocina CXCL1/biossíntese , Quimiocina CXCL2/biossíntese , Infecções por Escherichia coli/microbiologia , Feminino , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/genética , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Pielonefrite/imunologia , Pielonefrite/microbiologia , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 5 Toll-Like/genética , Bexiga Urinária/imunologia , Bexiga Urinária/metabolismo , Infecções Urinárias/imunologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/imunologiaRESUMO
RATIONALE: Pattern recognition receptors are attractive targets for vaccine adjuvants, and polymorphisms of the innate receptor NOD1 have been associated with allergic asthma. OBJECTIVES: To elucidate whether NOD1 agonist may favor allergic asthma in humans through activation of dendritic cells, and to evaluate the mechanisms involved using an in vivo model. METHODS: NOD1-primed dendritic cells from allergic and nonallergic donors were characterized in vitro on their phenotype, cytokine secretion, and Th2 polarizing ability. The in vivo relevance was examined in experimental allergic asthma, and the mechanisms were assessed using transfer of NOD1-conditioned dendritic cells from wild-type or CCL17-deficient mice. MEASUREMENTS AND MAIN RESULTS: NOD1 priming of human dendritic cells promoted a Th2 polarization profile that involved the production of CCL17 and CCL22 in nonallergic subjects but only CCL17 in allergic patients, without requiring allergen costimulation. Moreover, NOD1-primed dendritic cells from allergic donors exhibited enhanced maturation that led to abnormal CCL22 and IL-10 secretion compared with nonallergic donors. In mice, systemic NOD1 ligation exacerbated allergen-induced experimental asthma by amplifying CCL17-mediated Th2 responses in the lung. NOD1-mediated sensitization of purified murine dendritic cells enhanced production of CCL17 and CCL22, but not of thymic stromal lymphopoietin and IL-33, in vitro. Consistently, adoptive transfer of NOD1-conditioned dendritic cells exacerbated the Th2 pulmonary response in a CCL17-dependent manner in vivo. CONCLUSIONS: Data from this study unveil a deleterious role of NOD1 in allergic asthma through direct induction of CCL17 by dendritic cells, arguing for a need to address vaccine formulation safety issues related to allergy.
Assuntos
Asma/imunologia , Quimiocina CCL17/imunologia , Quimiocina CCL22/imunologia , Células Dendríticas/imunologia , Proteína Adaptadora de Sinalização NOD1/imunologia , Alérgenos/imunologia , Animais , Asma/genética , Asma/prevenção & controle , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína Adaptadora de Sinalização NOD1/agonistas , Proteína Adaptadora de Sinalização NOD1/genética , Fenótipo , Polimorfismo Genético , Células Th2/imunologia , Regulação para Cima/imunologiaRESUMO
The neonatal intestinal immune system is still undergoing development at birth, leading to a higher susceptibility to mucosal infections. In this study, we investigated the effect of poly(I:C) on controlling enteric infection by the protozoan Cryptosporidium parvum in neonatal mice. After poly(I:C) administration, a rapid reduction in parasite burden was observed and proved to be dependent on CD11c(+) cells and TLR3/TRIF signaling. Protection against C. parvum required additional signals provided by the gut flora through TLR5 and MyD88 signaling. This cooperation gave rise to higher levels of expression of critical mutually dependent cytokines such as interleukin 12p40 and type 1 and type 2 interferons, the last 2 being known to play a key role in the elimination of infected enterocytes. Our findings demonstrate in neonatal mice how gut flora synergizes with poly(I:C) to elicit protective intestinal immunity against an intracellular pathogen.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Criptosporidiose/prevenção & controle , Cryptosporidium parvum/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/parasitologia , Poli I-C/administração & dosagem , Receptor 5 Toll-Like/imunologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C57BL , Microbiota/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de SinaisRESUMO
The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host's susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection.
Assuntos
Calcineurina/metabolismo , Imunidade Inata , Fatores de Transcrição NFATC/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Fagocitose , Transdução de Sinais , Animais , Infecções Bacterianas/imunologia , Calcineurina/genética , Humanos , Fatores de Transcrição NFATC/genética , Proteína Adaptadora de Sinalização NOD1/genética , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Leptospira interrogans is responsible for a zoonotic disease known to induce severe kidney dysfunction and inflammation. In this work, we demonstrate that L. interrogans induces NLRP3 inflammasome-dependent secretion of IL-1ß through the alteration of potassium transport in bone marrow-derived macrophages. Lysosome destabilization also contributed to the IL-1ß production upon stimulation with live, but not dead, bacteria. Using bone marrow-derived macrophages from various TLRs and nucleotide-binding oligomerization domain-deficient mice, we further determined that IL-1ß production was dependent on TLR2 and TLR4, suggesting a participation of the leptospiral LPS to this process. Hypokaliemia in leptospirosis has been linked to the presence of glycolipoprotein, a cell wall component of L. interrogans that is known to inhibit the expression and functions of the Na/K-ATPase pump. We show in this study that glycolipoprotein activates the inflammasome and synergizes with leptospiral LPS to produce IL-1ß, mimicking the effect of whole bacteria. These results were confirmed in vivo, as wild-type mice expressed more IL-1ß in the kidney than TLR2/4-deficient mice 3 d postinfection with L. interrogans. Collectively, these findings provide the first characterization, to our knowledge, of bacteria-induced activation of the NLRP3 inflammasome through the downregulation of a specific host potassium transporter.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Leptospirose/metabolismo , Macrófagos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Proteínas de Bactérias/imunologia , Western Blotting , Proteínas de Transporte/imunologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Inflamassomos/imunologia , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Leptospira/imunologia , Leptospira/metabolismo , Leptospirose/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Intestinal homeostasis is critical for efficient energy extraction from food and protection from pathogens. Its disruption can lead to an array of severe illnesses with major impacts on public health, such as inflammatory bowel disease characterized by self-destructive intestinal immunity. However, the mechanisms regulating the equilibrium between the large bacterial flora and the immune system remain unclear. Intestinal lymphoid tissues generate flora-reactive IgA-producing B cells, and include Peyer's patches and mesenteric lymph nodes, as well as numerous isolated lymphoid follicles (ILFs). Here we show that peptidoglycan from Gram-negative bacteria is necessary and sufficient to induce the genesis of ILFs in mice through recognition by the NOD1 (nucleotide-binding oligomerization domain containing 1) innate receptor in epithelial cells, and beta-defensin 3- and CCL20-mediated signalling through the chemokine receptor CCR6. Maturation of ILFs into large B-cell clusters requires subsequent detection of bacteria by toll-like receptors. In the absence of ILFs, the composition of the intestinal bacterial community is profoundly altered. Our results demonstrate that intestinal bacterial commensals and the immune system communicate through an innate detection system to generate adaptive lymphoid tissues and maintain intestinal homeostasis.
Assuntos
Bactérias Gram-Negativas/fisiologia , Homeostase , Intestinos/microbiologia , Intestinos/fisiologia , Tecido Linfoide/imunologia , Proteína Adaptadora de Sinalização NOD1/metabolismo , Animais , Quimiocina CCL20/metabolismo , Quimera , Feminino , Vida Livre de Germes , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/isolamento & purificação , Imunoglobulina A/imunologia , Intestinos/imunologia , Ligantes , Linfonodos/imunologia , Tecido Linfoide/citologia , Masculino , Camundongos , Proteína Adaptadora de Sinalização NOD1/deficiência , Proteína Adaptadora de Sinalização NOD1/genética , Proteína Adaptadora de Sinalização NOD1/imunologia , Peptidoglicano/imunologia , Nódulos Linfáticos Agregados/imunologia , Receptores CCR6/deficiência , Receptores CCR6/genética , Receptores CCR6/metabolismo , beta-Defensinas/metabolismoRESUMO
Leptospira interrogans are pathogenic bacteria responsible for leptospirosis, a worldwide zoonosis. All vertebrates can be infected, and some species like humans are susceptible to the disease whereas rodents such as mice are resistant and become asymptomatic renal carriers. Leptospires are stealth bacteria that are known to escape several immune recognition pathways and resist killing mechanisms. We recently published that leptospires may survive intracellularly in and exit macrophages, avoiding xenophagy, a pathogen-targeting form of autophagy. Interestingly, the latter is one of the antimicrobial mechanisms often highjacked by bacteria to evade the host immune response. In this study we explored whether leptospires subvert the key molecular players of autophagy to facilitate infection. We showed in macrophages that leptospires triggered a specific accumulation of autophagy-adaptor p62 in puncta-like structures, without altering autophagic flux. We demonstrated that Leptospira-induced p62 accumulation is a passive mechanism depending on the leptospiral virulence factor LPS signaling via TLR4/TLR2. p62 is a central pleiotropic protein, also mediating cell stress and death, via the translocation of transcription factors. We demonstrated that Leptospira-driven accumulation of p62 induced the translocation of transcription factor NRF2, a key player in the anti-oxidant response. However, NRF2 translocation upon Leptospira infection did not result as expected in antioxydant response, but dampened the production of inflammatory mediators such as iNOS/NO, TNF and IL6. Overall, these findings highlight a novel passive bacterial mechanism linked to LPS and p62/NRF2 signaling that decreases inflammation and contributes to the stealthiness of leptospires.
Assuntos
Leptospira , Leptospirose , Humanos , Camundongos , Animais , Lipopolissacarídeos , Fator 2 Relacionado a NF-E2/metabolismo , Regulação para Cima , Macrófagos/metabolismo , Inflamação , AutofagiaRESUMO
By providing innate immune modulatory stimuli, the early life immune system can be enhanced to increase resistance to infections. Activation of innate cell surface receptors called pattern recognition receptors (PRRs) by TLR ligands is one promising approach that can help to control infections as described for listeriosis and cryptosporidiosis. In this study, the effect of TLR2/TLR1 and TLR2/TLR6 agonists was compared when injected into neonatal mice. Surprisingly, the stimulation of TLR2/TLR6 led to the death of the neonatal mice which was not observed in adult mice. The TLR2/TLR6 agonist administration induced higher systemic and intestinal inflammation both in adult and neonatal mice when compared to TLR2/TLR1 agonist. The mortality of neonatal mice was IFN-γ dependent and involved the intestinal production of IL-22 and IL-17A. This study clearly demonstrates that targeting TLRs as new control strategy of neonatal infections has to be used with caution. Depending on its heterodimeric form, the TLR2 stimulation can induce adverse effects more or less severe relying on the age-related immune functions of the host.