The larvae of N. brasiliensis parasites actively penetrate the skin and reach the lung after 18 to 32 hours, where they finish their development before migrating to the intestines, where they become sexually mature. Although the initial contact of the mammalian immune system with the invading nematode occurs in the skin, our current knowledge of these initial events is fragmented. Furthermore, as most studies hitherto focused on infection strategies using the subcutaneous route, less is known about the cutaneous immune responses following natural infection. The low efficiency and high variability of published “natural” N. brasiliensis infection models does not allow for a robust measure of late immune responses, and needle inoculation does not fully recapitulate skin immune responses to invading larvae. We, therefore, optimized an alternative model of “natural” infection using the percutaneous route, with worm migratory kinetics comparable to the more common subcutaneous inoculation. In accordance with previous findings, we observe an accumulation of immune cells early after larvae penetration in the skin. The use of high parameter flow cytometry allowed us to dissect with great detail parasite-induced changes in skin-resident and recruited immune cells, including less abundant populations. Moreover, we performed both a high-throughput analysis of inflammatory mediators within the tissue and analysed the kinetic changes in gene expression in the skin of infected mice. Thus, we generated the most complete roadmap to date describing the changes in skin immune system in response to nematode larvae. In order to test the contribution of skin-derived events in the regulation of distal immunity, we initially compared percutaneously-infected mice with intravenously-infected animals where the entirety of the skin immune response is bypassed. Interestingly, although intravenously-infected mice present similar numbers of larvae in the lungs as mice infected percutaneously, they display a distinct pulmonary immune response, both at the cellular and transcriptional level. Of note, these changes also translate in increased tissue damage in the lungs intravenously-infected mice, due to the effects of migrating larvae. By blocking lymphocyte migration and using a transgenic mouse models to track and manipulate lymphocytes from the skin, we were able to find that a subset of T lymphocytes migrates from the skin to the lungs, where they are responsible to kickstart the immune response observed in percutaneously infected mice. As such, when lymphocyte migration is blocked we observed the same immune hallmarks of intravenous infections, including increased tissue damage. Furthermore, using skin (but not pulmonary) allergic inflammation models we also observed the migration of lymphocytes from the skin to the lungs. Overall, our results confirm that we have a robust model to study the cutaneous (and distal) immune responses to N. brasiliensis, that strongly supports our initial hypothesis that early events in the skin regulate type-2 immunity at distal sites.
During the course of this project, these results have been presented in international conferences in Italy, Portugal and Greece. In addition, these findings were presented in local UK conferences. Finally, I was invited to speak about these results in institutional seminar series in Dundee, UK and Copenhagen, Denmark. Whereas the main body of work generated in the INTERCONNECTION project is still being prepared for publication in a peer-reviewed journal, it is important to note that results derived from the models and techniques developed for the present project figure in two peer-reviewed publications in the journals Cell Reports and Mucosal Immunology.
