RESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Criptosporidiosis/prevención & control , Cryptosporidium parvum/inmunología , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/parasitología , Poli I-C/administración & dosificación , Receptor Toll-Like 5/inmunología , Animales , Animales Recién Nacidos , Ratones , Ratones Endogámicos C57BL , Microbiota/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. METHODOLOGY/PRINCIPAL FINDINGS: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. CONCLUSIONS/SIGNIFICANCE: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases.
Asunto(s)
Inmunidad/efectos de los fármacos , Intestinos/efectos de los fármacos , Intestinos/inmunología , Oligodesoxirribonucleótidos/farmacología , Administración Oral , Envejecimiento/efectos de los fármacos , Animales , Animales Recién Nacidos , Movimiento Celular/efectos de los fármacos , Separación Celular , Quimiocinas/metabolismo , Enterocitos/citología , Enterocitos/efectos de los fármacos , Femenino , Inflamación/patología , Inyecciones Intraperitoneales , Interleucina-10/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Oligodesoxirribonucleótidos/administración & dosificación , Ratas , Células TH1/efectos de los fármacos , Células TH1/inmunologíaRESUMEN
Chemokines play a critical role in immune cell trafficking and the transition from an innate to an acquired immune response. We analyzed host response in neonatal mice deficient in chemokine receptor CCR5 following infection with the intracellular protozoan parasite Cryptosporidium parvum. CCR5 neonatal mice had a higher parasite burden at the early stage of infection but eliminated the parasite as efficiently as their wild-type counterparts. The higher sensitivity of neonates at the beginning of infection was not due to an altered IFNgamma response. An increased CCR2-attracting chemokine response associated with the recruitment of CCR2-positive cells in the infected mucosa may have compensated for the absence of CCR5. A lack of CCR5 thus has an impact in the early stage of C. parvum infection in neonates, but this receptor is dispensable for subsequent parasite elimination.
Asunto(s)
Criptosporidiosis/inmunología , Cryptosporidium parvum/fisiología , Receptores CCR5/inmunología , Animales , Cryptosporidium parvum/aislamiento & purificación , Interferón gamma/biosíntesis , Intestinos/parasitología , Ratones , Receptores CCR2/biosíntesis , Receptores CCR2/inmunología , Receptores CCR5/deficienciaRESUMEN
Dendritic cells (DCs) play a key role in activating and orientating immune responses. Little is currently known about DC recruitment during Cryptosporidium parvum infection. In the intestine, epithelial cells act as sensors, providing the first signals in response to infection by enteric pathogens. We analyzed the contribution of these cells to the recruitment of DCs during cryptosporidiosis. We found that intestinal epithelial cells produced a broad range of DC-attracting chemokines in vitro in response to C. parvum infection. The supernatant of the infected cells induced the migration of both bone marrow-derived DCs (BMDC) and the SRDC lymphoid dendritic cell line. Chemokine neutralization abolished DC migration in these assays. We next analyzed chemokine mRNA expression in the mucosa of C. parvum-infected neonatal mice and recruitment of the various subsets of DCs. Myeloid (CD11c+ CD11b+) and double-negative DCs (CD11c+ CD11b- CD8alpha-) were the main subsets recruited in the ileum during C. parvum infection, via a mechanism involving IFNgamma. DCs were also recruited and activated in the draining lymph nodes during C. parvum infection, as shown by the upregulation of expression of MHC II and of the costimulation molecules CD40 and CD86.
Asunto(s)
Quimiocinas/biosíntesis , Criptosporidiosis/patología , Cryptosporidium parvum/inmunología , Células Dendríticas/inmunología , Mucosa Intestinal/inmunología , Animales , Animales Recién Nacidos , Quimiocinas/genética , Criptosporidiosis/inmunología , Células Epiteliales/microbiología , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Neonates are particularly vulnerable to infections, in part because of the incomplete development of their immune system. Recent advances in immunostimulatory treatments based on conserved microbial components led us to assess the potential of oligodeoxynucleotides (ODNs) for decreasing the sensitivity of neonates to Cryptosporidium parvum infection. METHODS: Neonate mice were treated orally or intraperitoneally (ip) with CpG ODNs or non-CpG ODNs 24 h before C. parvum infection, and parasite load and cytokine up-regulation were evaluated. RESULTS: CpG ODN 1668 and non-CpG ODN 1668 administered orally, as well as CpG ODN 1668 administered ip, induced an 80%-95% decrease in intestinal parasite load 6 days after infection. Intraperitoneal and oral pretreatment with CpG ODN 1668 led to a strong initial up-regulation of cytokines and CD69 messenger RNA in the intestine and a decrease in parasite load by a Toll-like receptor 9 (TLR9)-dependent mechanism. By contrast, oral administration of non-CpG ODN 1668 decreased parasite load by a TLR9-independent mechanism. CONCLUSION: The control of neonatal C. parvum infection by ip or oral administration of ODNs is feasible by 2 different mechanisms: (1) the well-known interaction involving CpG/TLR9, leading to the production of cytokines and lymphocyte activation, and (2) a new unknown mechanism that is independent of TLR9 and effective orally.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Cryptosporidium parvum/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/uso terapéutico , Administración Oral , Animales , Animales Recién Nacidos , Criptosporidiosis/tratamiento farmacológico , Cryptosporidium parvum/patogenicidad , Modelos Animales de Enfermedad , Esquema de Medicación , Femenino , Humanos , Inyecciones Intraperitoneales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/uso terapéutico , Pruebas de Sensibilidad Parasitaria , Reacción en Cadena de la Polimerasa , ARN Protozoario/análisis , Organismos Libres de Patógenos EspecíficosRESUMEN
Numerous inflammatory cells are recruited in response to Cryptosporidium parvum infection. These cells include interferon gamma-producing T lymphocytes, which are of major importance for the resolution of infection. Here, we show that beta7 integrin is not essential for the control of infection in mice but that beta7-deficient neonatal mice are more susceptible during the early stages of infection.
Asunto(s)
Criptosporidiosis/inmunología , Cryptosporidium parvum/patogenicidad , Cadenas beta de Integrinas/genética , Cadenas beta de Integrinas/metabolismo , Animales , Animales Recién Nacidos , Bovinos , Criptosporidiosis/parasitología , Criptosporidiosis/fisiopatología , Susceptibilidad a Enfermedades , Humanos , Íleon/inmunología , Íleon/parasitología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Factores de TiempoRESUMEN
The effect of purine nucleosides on the in vitro growth of Cryptosporidium parvum was studied. Culturing the parasite in THP-1 cells for 72 h in growth medium supplemented with adenosine or inosine improved the parasite yields especially in the first 48 h. Similar results were obtained with parasites cultured in Madin-Darby bovine kidney cells and incubated for 24 h with inosine. The addition of inosine to 72-h cultures enhanced the growth of C. parvum in THP-1 cells, especially the trophic stages, whereas the analogue formycin B was toxic to the parasites and induced a marked decrease in the gamont stages. The monitoring of the added purine nucleosides by high performance liquid chromatography showed that at 37 degrees C in the presence of THP-1 cells, a rapid uptake of inosine occurred with hypoxanthine being the main purine present after 2 h in the medium.
Asunto(s)
Cryptosporidium parvum/crecimiento & desarrollo , Nucleósidos de Purina/metabolismo , Adenosina/metabolismo , Adenosina/farmacología , Animales , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Cryptosporidium parvum/citología , Cryptosporidium parvum/efectos de los fármacos , Cryptosporidium parvum/metabolismo , Medios de Cultivo/química , Formicinas/farmacología , Humanos , Hipoxantina/metabolismo , Hipoxantina/farmacología , Inosina/análogos & derivados , Inosina/metabolismo , Inosina/farmacología , Nucleósidos de Purina/farmacologíaRESUMEN
Detection of Cryptosporidium oocysts from goat kid faeces: comparison of a latex agglutination test with three other conventional techniques. A quantitative latex agglutination test (QLAT) with monoclonal antibodies for the detection of Cryptosporidium oocysts in faeces was compared with 3 other conventional techniques: Heine staining on faecal smears (HS) giving semi-quantitative results (scores from 1 to 5), sucrose flotation on diluted faeces (SF) with results expressed in oocysts/g of faeces (opg), direct ELISA (DE) giving qualitative results. Goat kid unconcentrated faecal samples (234) from 8 farms were processed according to the 4 techniques. Data were analyzed with Win Episcope 1.0 and Testview 1.1 softwares. The oocyst outputs ranged from 100 000 (detection limit for SF) to 200 millions opg (mean: 15.2 millions opg). A very good agreement was recorded between QLAT and HS, SF, DE: Kappa values ranged between 0.82 and 0.90. When considering the samples exhibiting oocysts (or not) as positive (or negative) using both HS and SF (n = 219), the sensitivity and specificity of QLAT were respectively 95.1 and 96.0%. The lack of sensitivity was observed in faeces harboring a few oocysts (< or = 200 000 opg, scores < or = 2) whereas the lack of specificity was only observed in 3 samples originating from the same farm. A significant correlation was calculated between the percentage of agglutination in QLAT and the number of oocysts in SF or scores in HS (Spearman correlation ranging from 0.45 to 0.48, p < 0.001). QLAT is a rapid, simple and reliable tool for routine detection of Cryptosporidium oocysts in faeces.
Asunto(s)
Criptosporidiosis/veterinaria , Cryptosporidium/aislamiento & purificación , Heces/parasitología , Enfermedades de las Cabras/parasitología , Pruebas de Fijación de Látex/veterinaria , Animales , Animales Recién Nacidos , Criptosporidiosis/diagnóstico , Enfermedades de las Cabras/diagnóstico , Cabras , Pruebas de Fijación de Látex/métodos , Oocitos , Sensibilidad y EspecificidadRESUMEN
Cryptosporidium parvum is a protozoan parasite that infects intestinal epithelial cells and induces inflammation of the intestine. To better understand the inflammatory process occurring during cryptosporidiosis, we investigated in this study the kinetics of chemokine expression in the mucosa of mice by quantitative reverse transcription-PCR. Our results demonstrate that among the chemokine mRNAs studied, gamma interferon (IFN-gamma)-inducible protein 10 (IP-10), monokine induced by IFN-gamma (MIG), i-TAC, lymphotactin, macrophage inflammatory protein 1 beta (MIP-1 beta), and RANTES mRNAs were strongly up-regulated in infected neonate mice, which correlated with the immunofluorescence staining results showing T-cell and macrophage infiltration in the mucosa. Our in vitro data showed that intestinal epithelial cells infected by C. parvum or stimulated by the proinflammatory cytokines (IFN-gamma, interleukin-1 beta, and tumor necrosis factor alpha) produce a pattern of chemokine secretion similar to that observed in vivo, suggesting that these cells may take part in the initial production of chemokines. In order to identify the chemokines responsible for the recruitment of the inflammatory cells leading to a protective immune response, we compared the patterns of chemokine expression in a healing neonate mouse model and a nonhealing IFN-gamma knockout (GKO) mouse model of cryptosporidiosis. In the absence of IFN-gamma, the chemokine response was altered for IP-10, MIG, i-TAC, RANTES, and MIP-1 beta mRNAs, while the three ELR C-X-C chemokine mRNAs studied (lipopolysaccharide-induced C-X-C chemokine, MIP-2 alpha, and KC mRNAs) were strongly overexpressed. These results are consistent with the neutrophil recruitment observed in the lamina propria of GKO mice at day 9 postinfection but are not consistent with the hypothesis that these cells play an important role in the resolution of the infection. On the contrary, the altered response of chemokines responsible for the recruitment of macrophages and T cells in GKO mice suggests that these two populations may be critical in the development of a protective immune response.