IL-12 AND IFN-gamma mRNA EXPRESSION IS ENHANCED ON TICK-RESISTANT GUINEA PIGS

Carrapatos são artrópodes hematófagos de distribuição cosmopolita que parasitam vertebrados e transmitem uma grande variedade de agentes infecciosos para o homem e animais domésticos. Cobaias, diferentemente de cães e camundongos, são capazes de desenvolver resistência a carrapatos Rhipicephalus sanguineus após sucessivas infestações. Ao comparar o tipo de resposta imune desenvolvida por cobaias e cães frente a carrapatos observou-se que cobaias re-infestadas desenvolvem uma pequena reação de hipersensibilidade imediata e uma forte reação de hipersensibilidade tardia à inoculação cutânea com antígenos de carrapatos. Já em cães e camundongos, é notada somente uma forte reação de hipersensibilidade imediata. Também foi verificado que células dos linfonodos de cobaias infestadas três vezes com carrapatos (resistentes) proliferam intensamente na presença de saliva de carrapatos, diferentemente do que ocorre com células de cães e camundongos re-infestados (suscetíveis) que não proliferam. Esses achados sugerem o envolvimento de um padrão Th1 de resposta imune na aquisição de resistência, no entanto essa hipótese ainda não foi confirmada. Assim sendo, no atual trabalho procurou-se verificar a expressão de mRNA de citocinas na pele e linfonodos de cobaias infestadas e re-infestadas com carrapatos. Para tal foram delineados primers e padronizadas reações de PCR para detectar a expressão de mensagem das citocinas IL-12p40, IFN-gama e TNF-alfa, pertencentes a um padrão Th1, e IL-4, IL-5, IL-10 e TGF-beta, pertencentes a um padrão Th2 de resposta imune. Os resultados obtidos demonstraram que cobaias sucessivamente infestadas apresentaram um aumento significativo na intensidade de mensagem para IL-12p40 nos linfonodos, tanto comparado com animais uma vez infestados (aumento de 2,6 vezes), quanto comparado com os controles (aumento de 13 vezes). Embora a análise estatística não tenha apontado uma diferença significativa houve elevação consistente na intensidade de mRNA para IFN-gama nos linfonodos de cobaias re-infestadas comparadas às infestadas apenas uma vez (aumento de 2,4 vezes). Também foi observado um aumento significativo na intensidade da mensagem para IL-5 nos linfonodos de cobaias infestadas uma vez quando comparadas aos controles (aumento de 5 vezes). Não foi detectada expressão de mensagem para IL-4 e IL-10 nas amostras analisadas. Já a expressão de mensagem para TGF-beta foi observada em todos os animais (experimentais ou controles), sugerindo que essa citocina possa ter uma expressão constitutiva em cobaias. Tomados em conjunto, os resultados sugerem o envolvimento predominante de um perfil de citocinas de padrão Th1 na aquisição de resistência em cobaias a carrapatos. Nossos resultados poderão auxiliar o desenvolvimento de novas abordagens para o controle de carrapatos, como, por exemplo, sugerir adjuvantes mais adequados a serem utilizados em vacinas anti-carrapatos. O conhecimento gerado não se restringe à indução de proteção contra carrapatos como também a possibilidade de aumentar a resistência de hospedeiros a patógenos transmitidos por carrapatos que poderiam ser controlados por uma resposta tipo Th1.Ticks are hematophagous arthropods of cosmopolitan distribution and are significant vectors of several diseases for humans and animals. Guinea pigs, unlike dogs and mice, develop resistance to Rhipicephalus sanguineus ticks after successive infestations. When the immune reaction between tick-infested guinea pigs and dogs/mice are compared, guinea pigs develop both immediate and strong delayed type hypersensitivity reactions while dogs and mice develop only a strong immediate reaction. Additionally was shown that lymph node cells from tick-infested guinea pigs (resistant hosts) proliferate intensely when cultured with tick saliva, differently to what is observed with cells from tick-infested dogs and mice (susceptible hosts). These findings propose the contribution of a Th1 cytokine pattern on the acquired immune response to ticks; however this hypothesis still has to be tested. This being so, in this study we investigated the expression profile of genes coding for selected cytokines on R. sanguineus infested guinea-pigs. Messenger RNA for IL-12-p40, IFN-gamma and TNF-alfa (Th1 cytokine pattern) and IL-4, IL-5, IL-10 and TGF-beta (Th1 cytokine pattern) was measured in skin and lymph nodes biopsies from tick-infested guinea pigs. Our results demonstrated that repeatedly tick-infested guinea pigs presented a significant increase on the intensity of message for IL-12p40 in the lymph nodes compared to both, one time tick-infested guinea pigs (raise of 2.6 times) or controls (raise of 13 times). Although the statistical analysis did not point out differences, there was a consistent increase on the mRNA intensity for IFN-gamma on the lymph nodes from re-infested guinea pigs compared to one time tick-infested animals (raise of 2.4 times). In addition, a significant enhance on the intensity of message for IL-5 on the lymph nodes from one time tick-infested guinea pigs compared to the controls (raise of 5 times). No message for IL-4 and IL-10 was detected on the analyzed tissues. In contrast, TGF-beta was detected on tissues collected from all animals (experimental or controls), suggesting a spontaneous production of this cytokine in guinea pigs. Taken together, these data suggest that a T helper 1-type pattern of cytokine production might be associated with the resistance expressed by guinea-pigs to ticks. Moreover, our results can provide new approaches to control ticks, i.e. suggest adjuvants to be added to anti-tick vaccines that preferably induce a Th1-type of response

The immunobiology of infestations with the cattle tick Rhipiceplahus (Boophilus) microplus: comparative immunologic and molecular studies of the tick-host interface in susceptible and resistant bovine hosts.

A pele dos vertebrados é alvo da maioria das 15.000 espécies de artrópodes hematófagos existentes e pouco se sabe sobre as estratégias imunológicas utilizadas pelos hospedeiros para expulsar esse tipo de ectoparasitos. É fato que carrapatos, como artrópodes hematófagos, são capazes também de induzi-las. Entre a grande variedade de hospedeiros, os bovinos, que apresentam fenótipos variáveis de resistência ao Ripicephalus (Boophilus) microplus, constituem o único modelo no qual é possível correlacionar as respostas imunológicas entre os fenótipos contrastantes na mesma espécie hospedeira. Para tal, as populações celulares do infiltrado induzido na pele pelo carrapato foram quantificadas nos bovinos resistentes, Bos taurus indicus e nos suscetíveis, Bos taurus taurus. Como esperado, o carrapato induziu inflamação cutânea local nos bovinos estudados e a composição celular do infiltrado apresentou diferenças que variaram entre os fenótipos contrastantes de infestação. A pele resistente apresentou maior número de basófilos em comparação a pele suscetível infestada pelo carrapato adulto (P 0,05). The results suggest that mast cells are source of cytokines and inflammatory mediators that play effectors and modulator roles in immune responses, their reduction possibly due to degranulation by tick saliva. Amount of neutrophils in infested skin may reflect the fact that only nymphs express RGD-containing disintegrins, which are possibly neutrophils-specific. Eosinophils, as well as basophils, are important to ticks resistance, their skin kinetic suggesting a systemic effect of tick infestations. Basophils resistant host increasing suggesting that they are the pivotal cells that impair haematophagy. The observed increase of CD3+ T cells in nymph and adult-infested skin of resistants suggests their importance to regulate anti-tick immune responses. The diminished numbers of B cells in susceptible breed indicate that antibodies are important in acquired resistance to ticks. On the other hand, the reduction of gd/WC1+ T cells seen in the infested skin of susceptible bovines indicates that these cells may play a role in resistance to ticks. Natural Killers cells could help the development of efficacious immune responses to ticks-borne diseases. In conclusion, these results reflect the fact that local responses involve resident and infiltrating leukocytes and lymphocytes that are sources of immunoregulatory, effectors and memory responses elicited against ticks

Effect of Amblyomma cajennense Ticks on the Immune Response of BALB/c Mice and Horses

This work evaluated the effect of the Amblyomma cajennense tick on the immune response of BALB/c mice and on horse lymph node cell proliferation. We observed that mice do not develop resistance to nymphs of this tick species and that lymphocyte proliferation of this host is inhibited by tick saliva, nymphal extract, or infestations. Horse lymph node cell proliferation is inhibited by tick saliva as well. Mice lymphocytes under the effect of tick saliva, nymphal extract, or infestations display a predominantly. p Th-2 cytokine production pattern. Observed results partially explain this tick`s disease vectoring capacity and broad host range.National Council for Scientific and Technological Development (CNPq)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Effect of Amblyomma cajennense Ticks on the Immune Response of BALB/c Mice and Horses

This work evaluated the effect of the Amblyomma cajennense tick on the immune response of BALB/c mice and on horse lymph node cell proliferation. We observed that mice do not develop resistance to nymphs of this tick species and that lymphocyte proliferation of this host is inhibited by tick saliva, nymphal extract, or infestations. Horse lymph node cell proliferation is inhibited by tick saliva as well. Mice lymphocytes under the effect of tick saliva, nymphal extract, or infestations display a predominantly. p Th-2 cytokine production pattern. Observed results partially explain this tick's disease vectoring capacity and broad host range.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Modulation of cutaneous inflammation induced by ticks in contrasting phenotypes of infestation in bovines

Tick saliva contains molecules that are inoculated at the site of attachment on their hosts in order to modulate local immune responses and facilitate a successful blood meal. Bovines express heritable, contrasting phenotypes of infestations with the cattle tick, Rhipicephalus (Boophilus) microplus: breeds of Bos taurus indicus are significantly note resistant than those of Bos taurus taurus. Tick saliva may contain molecules that interfere with adhesion of leukocytes to endothelium and resistant hosts may mount an inflammatory profile that is more efficient to hamper the tick`s blood meal. We show in vitro that adhesion of peripheral blood mononuclear cells to monolayers of cytokine-activated bovine umbilical endothelial cells was significantly inhibited by tick saliva. The inflammatory response to bites of adults of R. microplus mounted by genetically resistant and susceptible bovine hosts managed in the same pasture was investigated in vivo. The inflammatory infiltrates and levels of message coding for adhesion molecules were measured in biopsies of tick-bitten and control skin taken when animals of both breeds were exposed to low and high tick infestations. Histological studies reveal that cutaneous reactions of resistant hosts to bites of adult ticks contained significantly more basophils and eosinophils compared with reactions of the susceptible breed. Expression of the adhesion molecules - intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin - was higher in adult-infested skin of susceptible hosts undergoing low infestations compared to resistant hosts; when host was exposed to high infestations expression of these adhesion molecules was down-regulated in both phenotypes of infestations. Expression of leukocyte adhesion glycoprotein-1 (LFA-1) was higher in skin from susceptible hosts undergoing low or high infestations compared to resistant hosts. Conversely, higher levels of E-selectin, which promotes adhesion of memory T cells, were expressed in skin of resistant animals. This finding may explain the resistant host`s ability to mount more rapid and efficient secondary responses that limit hematophagy and infestations. The expression profiles observed for adhesion molecules indicate that there are differences in the kinetics of the inflammatory reactions mounted by resistant and susceptible hosts and the balance between tick and host is affected by the number of tick bites a host receives. We show that the contrasting phenotypes of infestations seen in bovines infested with R. microplus are correlated with differences in the cellular and molecular composition of inflammatory infiltrates elicited by bites with adult ticks. (C) 2009 Published by Elsevier B.V.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico CNPq[420067/2005-1]Conselho Nacional de Desenvolvimento Cientifico e Tecnologico CNPq[505810/20042]Millennium Institute for Vaccine Development and Technology[CNPq-420067/2005-1]Fundacao de Amparo e Pesquisa do Estado de SAO Paulo - FAPESP[04/09992-7]Fundacao de Amparo e Pesquisa do Estado de SAO Paulo - FAPESP[06/55825-0

Immune and biochemical responses in skin differ between bovine hosts genetically susceptible and resistant to the cattle tick Rhipicephalus microplus

Abstract\ud \ud Background\ud Ticks attach to and penetrate their hosts’ skin and inactivate multiple components of host responses in order to acquire a blood meal. Infestation loads with the cattle tick, Rhipicephalus microplus, are heritable: some breeds carry high loads of reproductively successful ticks, whereas in others, few ticks feed and reproduce efficiently.\ud \ud \ud Methods\ud In order to elucidate the mechanisms that result in the different outcomes of infestations with cattle ticks, we examined global gene expression and inflammation induced by tick bites in skins from one resistant and one susceptible breed of cattle that underwent primary infestations with larvae and nymphs of R. microplus. We also examined the expression profiles of genes encoding secreted tick proteins that mediate parasitism in larvae and nymphs feeding on these breeds.\ud \ud \ud Results\ud Functional analyses of differentially expressed genes in the skin suggest that allergic contact-like dermatitis develops with ensuing production of IL-6, CXCL-8 and CCL-2 and is sustained by HMGB1, ISG15 and PKR, leading to expression of pro-inflammatory chemokines and cytokines that recruit granulocytes and T lymphocytes. Importantly, this response is delayed in susceptible hosts. Histopathological analyses of infested skins showed inflammatory reactions surrounding tick cement cones that enable attachment in both breeds, but in genetically tick-resistant bovines they destabilized the cone. The transcription data provided insights into tick-mediated activation of basophils, which have previously been shown to be a key to host resistance in model systems. Skin from tick-susceptible bovines expressed more transcripts encoding enzymes that detoxify tissues. Interestingly, these enzymes also produce volatile odoriferous compounds and, accordingly, skin rubbings from tick-susceptible bovines attracted significantly more tick larvae than rubbings from resistant hosts. Moreover, transcripts encoding secreted modulatory molecules by the tick were significantly more abundant in larval and in nymphal salivary glands from ticks feeding on susceptible bovines.\ud \ud \ud Conclusions\ud Compared with tick-susceptible hosts, genes encoding enzymes producing volatile compounds exhibit significantly lower expression in resistant hosts, which may render them less attractive to larvae; resistant hosts expose ticks to an earlier inflammatory response, which in ticks is associated with significantly lower expression of genes encoding salivary proteins that suppress host immunity, inflammation and coagulation.This work was supported by the Fundação de Amparo à Pesquisa do Estado\ud de São Paulo - FAPESP (grant number 2009/53645-3 to IKFMS), by the\ud Conselho Nacional de Desenvolvimento Científico e Tecnologico - CNPq\ud (grant numbers 490498/2007-8, 471946/2010-9, 300873/2010-7 and 559603/\ud 2009-6 to IKFMS) and by the Intramural Research Program of the Division of\ud Intramural Research, National Institute of Allergy and Infectious Diseases,\ud National Institutes of Health. AMF was supported by a scholarship from the\ud Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES;\ud SRM. and GRG were supported by scholarships from FAPESP (2012/04087-0\ud and 2009/51212-2, respectively). Because JMCR is a U.S. government employee\ud and this is a government work, the work is in the public domain in the United\ud States. Notwithstanding any other agreements, the NIH reserves the right to\ud provide the work to PubMedCentral for display and use by the public, and\ud PubMedCentral may tag or modify the work consistent with its customary\ud practices. You can establish rights outside of the U.S. subject to a government\ud use license

Rhipicephalus (Boophilus) microplus: Clotting time in tick-infested skin varies according to local inflammation and gene expression patterns in tick salivary glands

Ticks deposit saliva at the site of their attachment to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses. The anti-haemostatic properties of tick saliva have been described by many studies, but few show that tick infestations or its anti-haemostatic components exert systemic effects in vivo. In the present study, we extended these observations and show that, compared with normal skin, bovine hosts that are genetically susceptible to tick infestations present an increase in the clotting time of blood collected from the immediate vicinity of haemorrhagic feeding pools in skin infested with different developmental stages of Rhipicepahlus microplus; conversely, we determined that clotting time of tick-infested skin from genetically resistant bovines was shorter than that of normal skin. Coagulation and inflammation have many components in common and we determined that in resistant bovines, eosinophils and basophils, which are known to contain tissue factor, are recruited in greater numbers to the inflammatory site of tick bites than in susceptible hosts. Finally, we correlated the observed differences in clotting times with the expression profiles of transcripts for putative anti-haemostatic proteins in different developmental stages of R. microplus fed on genetically susceptible and resistant hosts: we determined that transcripts coding for proteins similar to these molecules are overrepresented in salivary glands from nymphs and males fed on susceptible bovines. Our data indicate that ticks are able to modulate their host`s local haemostatic reactions. In the resistant phenotype, larger amounts of inflammatory cells are recruited and expression of anti-coagulant molecules is decreased tick salivary glands, features that can hamper the tick`s blood meal. (C) 2010 Elsevier Inc. All rights reserved.Fundaco de Amparo A Pesquisa do Estado de SAO Paulo - FAPESPConselho Nacional de Pesquisa e Desenvolvimento Cientifico e Tecnologico - CNPqNational Institute of Allergy and Infectious Diseases (NIAID/NIH