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Digital archive of theses discussed at the University of Pisa


Thesis etd-02132011-235358

Thesis type
Tesi di dottorato di ricerca
Thesis title
“Impiego di un adiuvante Th1 di origine batterica per la modulazione della risposta Th2 in vivo: studio in un modello di trichinellosi sperimentale”
Academic discipline
Course of study
tutor Prof. Bruschi, Fabrizio
  • Helicobacter pylori
  • HP-NAP
  • Th1 adiuvante
  • trichinellosi sperimentale
Graduation session start date
During Trichinella spiralis infection host defense is based on eosinophilia and increased IgE levels, as observed in Th2 mediated diseases like asthma and allergic rhinitis. Nowadays allergic diseases are treated with an alternative approach to the usual immunesuppressive method, using the mutual antagonism between Th1 and Th2 cytokines. For this reason the research has investigated new Th1 adjuvant molecules able to contrast the exacerbated Th2 responses to allergens. In particular among a large panel of pro-Th1 adjuvants, the protein of bacterial origin Helicobacter pylori neutrophil activating protein (HP-NAP) can induce immunity cells to activate towards a Th1 phenotype, also acting in vitro on Th2 polarized cells.
The aim of the present PhD thesis was to verify the immunological efficacy of HP-NAP in the experimental mouse model of T. spiralis infection, characterized by a Th2 immune response, which represents an alternative to the classical approach of ovalbumin-induced asthma. This experimental study offers not only the possibility to evaluate the immunological effects of the bacterial adjuvant, but also to assess parasitological issue in the immunomodulated host-parasite relationship.
After a preliminary study a dosage of 10 μg of HP-NAP was chosen to be administered intraperitoneally at 10th and 28th day after experimental infection in correspondence with eosinophilia peak and plasma IgE level plateau in our mouse model of T. spiralis infection, respectively. HP-NAP used was cloned and espressed in the Gram positive Bacillus subtilis to avoid endotoxin contamination. Peripheral blood collection from different experimental groups (infected animals treated with HP-NAP or with PBS) allowed us to study the immune response along the infection using leukocyte count, total and parasite specific IgE level and cytokine profile determination. In addition, splenocytes from treated and control animals were used in an ex vivo system to confirm HP-NAP immunomodulation and identify cellular target and the respective receptor involved in HP-NAP signaling pathway on immunity cells. Further, in vivo experiments were performed using our bacterial adjuvant in addition to the antibody blocking the toll-like receptor 2 (TLR2) which is responsible for HP-NAP signal transmission.
For the evaluation of parasitological aspects, animals were examined by histological, histochemical and immunohistochemistry analysis at muscle level and their carcasses were digested in a chloride pepsin solution for parasitological burden assessment.
Results obtained from our experimental work showed HP-NAP ability to immunomodulate the Th2 response, reducing significantly eosinophils peak and total and parasite IgE levels during the helminthic infection. HP-NAP action resulted strongly active in increasing IFN-γ and IL-12 levels and diminishing IL-4 and IL-5 levels both in animals treated with HP-NAP and in ex-vivo system derived from treated infected mouse spleens compared to cells from infected animal controls. Besides, the ex-vivo experiments confirmed HP-NAP action on innate immunity cells through HP-NAP/TLR2 binding, able to stimulate a cytokine profile expression different from that of another TLR2 agonist such as PAM3. All these data show that HP-NAP, acting through the TLR2, is capable also in vivo to shift the immune response to a Th1 phenotype.
After having studied the effects of HP-NAP in immunological response, we aimed to understand the host-parasite relation focusing on Trichinella muscle phase, during which the parasite enters the striated skeletal muscle cell inducing the nurse cell formation, devoted to nourishing and to protection of the helminth.
Histological analysis evidenced by means of an appropriate software an increased inflammation infiltrate surrounding nurse cell-parasite complex in HP-NAP treated animals, in addition to elevated active matrix metalloproteinase 9 (MMP-9) plasma levels, considered a leukocyte transendothelial migration marker. Histochemical staining revealed a reduction in eosinophil number in muscular pericapsular inflammation in HP-NAP treated animals, confirming the decrease of these polymorphonucleate cells at blood level. This suggests that the decrease in the blood induced by HP-NAP is not due to an increased extravasation of eosinophils but rather to the reduced IL-5 plasma levels observed in HP-NAP treated animals.
Immunohistochemistry with an anti-arginase 1 antibody, an enzyme which is considered a marker of alternative macrophage activation, showed a decreased staining of this enzyme in muscle inflammatory infiltrate of treated infected animals. Surprisingly, parasite burden of HP-NAP treated animals is thrice times that of control animal. It is important underline that the first HP-NAP administration when intestinal parasite phase is not yet ended in the mouse strains used (BALB/c) shifted host immunity to a Th1 response, which might have delayed worm expulsion, increasing the number of migrant larvae produced. Besides inverse correlation between parasite burden and Th2 parameters (eosinophils, total and specific IgE) suggests the importance of this type of host immune response against parasite infection.
Our experimental design offers a dualistic point of view. In experimental helminthic infection the molecule of bacterial origin confirms its immunological potential, shifting host immune response to Trichinella infection. Immunomodulation of parasite infection provides new information on host- parasite relationship: in our model the Th2 immune response acquires an host protective value in favor of host defense versus massive Trichinella infection in muscle.