Contribution à la compréhension de l'immunopathogénie de l'aspergillose nasosinusale chez le chien

Sinonasal aspergillosis is a sever fungal rhinosinusitis mainly affecting large breed dogs in their mid ages. Its most common causal agent is A. fumigatus, a fungus that is largely spread in the atmosphere. As of today, the diagnostic and treatment for this disease remain a challenge for the practicing veterinary doctor. Very little data is available to explain why such a ubiquitous fungus induces a sever rhinosinusitis in otherwise healthy dogs, while other dogs do not present any sign of fungal infection. The authors of a study analysing the expression of mRNA encoding for certain cytokines and chimiokines in the nasal mucosa of SNA affected dogs, propose the assumption that dogs develop a protective immunity (Th1) against A. fumigatus, but that it could be blocked by an excessively intense regulating immunity (massive production of IL-10). Indeed, it is commonly described that in humans affected by invasive aspergillosis, as well as in mice-based models, the production of immunoregulating cytokines (IL-10) should be considered as a sign of the escalation of the sickness and an absence of its remission. The objective of this thesis was to investigate the adaptive immunity reaction of SNA affected dogs based on the assumption that sick subjects develop a protective immunity that is antagonised by a disproportional regulating immunity. Three axes of analysis have been considered to answer this objective. The first looked into the difference of expression and/or production of cytokines and transcriptor factors prototypics of the different adaptative and regulatory immunological paths: Th1 (IFN-γ and Tbet) – Th2 (IL-4 and GATA3) – Th17 (IL-17A and RORc) and Treg (IL-10 and FoxP3) in PBMC of affected and healthy dogs after A. fumigatus stimulation. Secondly, an analysis of genes by microarray has been carried on nasal mucosa biopsies of affected and healthy dogs. Thirdly, the promotor zone of the gene encoding IL-10 in dogs has been analysed by sequencing. This study has been done within three cohortes of dogs: Rottweiler-Labrador and Golden containing affected and healthy dogs. The objective was to investigate, as it is the case in human medicine, the possibility of a genetic modification as a factor susceptible of leading to SNA development. The results of the first study revealed that: (1) the PBMC of half the controls dogs and every affected dogs expressed a relevant overexpression of IFN-γ. This increase was significantly more important within PBMC of affected dogs. The analysis of IFN-γ production in culture supernatants was in accordance with these last observations. A significant increase in the expression of mRNA coding for Tbet was also observed in half of the PBMC of affected dogs. (2) a significant increase in expression of mRNA encoding IL-4 was observed in the PBMC of most of the affected and healthy dogs. This increase was significantly higher in the PBMC of affected dogs than in the healthy dogs. (3) the PBMC of most control and affected dogs also revealed an increase in expression of mRNA encoding IL-17A. This increase was statistically more important in the PBMC of affected dogs than in the healthy dogs. (4) a relevant decrease in mRNA expression encoding IL-10 was observed in the PBMC of more than half of the affected dogs. The expression of the mRNA encoding IL-10 was significantly smaller in the PBMC of affected dogs than in the healthy dogs. The microarray analysis showed that: (1) amongst the 49 overexpressed biological groups, 13 were associated to the immunological or inflammatory process; (2) the nasal mucosa of affected dogs presented an increase in the expression of genes encoding for molecules involved directly (IFN-γ and STAT4) and indirectly (IL-16, CCL3, CCL4, and CXCL10) in the development of protective Th1 immunity, as well as molecules involved in the regulatory branch of the immune response (IL-16 and Ikaros). The sequencing analysis of the promotor region of the gene encoding IL-10 revealed the presence of polymorphisms. Three polymorphisms were observed more frequently in clones belonging to the three studied cohorts, excepted for the clones belonging to SNA affected Rottweiler. The polymorphisms observed in dogs were not similar to those described in humans. The first study showed an increase in the expression of mRNa encoding IFN-γ - Tbet – IL-4 and IL-17A in most of the PBMC of the affected dogs, and a decrease in the expression of IL-10 in comparison with the PBMC of healthy dogs. Similar results were observed in mice repeatedly affected by A. fumigatus. The suggested hypothesis was that an intense Th17 immunity resulted in a massive inflammatory reaction leading to a favourable environment were A. fumigatus was able to proliferate as hyphae. In return, hyphae would lead to the development of a non-protective Th2 immunity. It is tempting to suggest that the same hypothesis could be made for dogs affected by SNA. In order to reinforce this hypothesis, we should compare the expression of the different molecules involved in the Th17 immunity inside the nasal mucosa of affected and healthy dogs. Additionally, we should be running a kinetic study based on the expression of prototypical cytokines in parallel with the analysis of the production of these cytokines in culture supernatants. Ideally these studies should use DC and lymphocytes isolated from the nasal mucosa of affected and healthy dogs. In conclusion, an new hypothesis could be formulated: the possibility that not the overstimulation of the regulatory branch of the immunity response but an overstimulation of the Th17 branch of the immune response could be the cornerstone of the incapacity of dogs to clear from their SNA. The results of the microarray study were partially in accordance to the starting hypothesis. Indeed, the results showed an overexpression of genes involved in in the development of the protective Th1 (IFN-γ, STAT4, IL-16, CCL3, CCL4, and CXCL10) as well as genes involved in the regulatory path of the adaptive immunity (IL-16 and Ikaros). But the results of this study did not show an increase in IL-10. No conclusion could be drawn from these results; indeed, they were only the reflection of a fixed image at a given moment and we cannot consider qPCR results as the exact replica of the production of cytokines in the microenvironment. Nevertheless this study pointed out new possible areas of research. The results obtained after the sequencing of the promotor zone of the gene encoding IL-10 did not show any clear difference between affected and healthy dogs. However, this study was undertaken with a very limited number of dogs. In order to further assess the possibility of a genetic modification as the cornerstone of the development of SNA, more dogs should be analysed and the sequencing analysis should be run in parallel with an ELISA analysis.

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