Background

Pathogens of the genus Leishmania cause vector born zoonotic diseases with a variety of clinical manifestations and a variability in disease severity (Mansueto et al., 2007). The cutaneous form of leishmaniasis is caused by several Leishmania species including Leishmania amazonensis (McMahon-Pratt and Alexander, 2004; Christensen et al., 2019).

The existence of mouse models, with high, intermediate or no susceptibility to the cutaneous form of leishmaniasis have contributed to the development of experimental protocols for parasite dissemination (de Oliveira Cardoso et al., 2010). The parasites are naturally transmitted to the mammalian hosts by the bite of infected phlebotomine sandflies of the genus Lutzomyia (Bates, 2007). Thus transmission protocols use the ear dermis, the dorsal skin or the hind footpad (Schuster et al., 2014). In our protocol the Leishmania amazonensis parasites were inoculated through the ear dermis of the mouse, a method that closely imitates the natural transmission of the parasites by the sandfly (Giraud et al., 2019b). In addition, the ear pinna is appropriate for phenotypic evaluation of the lesion as well as for the isolation of nucleic acids from the inoculation sites, for two reasons: 1) parasites, in their infectious path, induce two distinct phases: the first, is a clinically silent phase characterized by the absence of lesions and the increase of parasite load and the second phase, during which visible lesions develop, and are associated with immune cell infiltration at the site of inoculation (Liu and Uzonna 2012; Giraud et al., 2019a). 2) The immune cells expand in the draining lymph nodes, which are also used for nucleic acids preparation (Cortes et al., 2010). Therefore the immune response of the host to the parasites can be studied on the site of inoculation and on the secondary immune adjacent tissue, the ear draining lymph nodes.

Leishmania amazonensis infection triggers an inflammatory response characterized by the presence of macrophages on the lesion sites. The parasites develop in the resident dermal macrophages (MF) and in Dendritic cells (DC) (Liu and Uzonna, 2012). The mechanisms of the parasite adaptation in the mammalian phagocytes and tissues remain elusive. Macrophages do not prevent the proliferation of the infection but on the contrary are the resident cells for these parasites.

We have studied the immune response of the host to Leishmania amazonensis infection in mice (Giraud et al., 2019b). Parasites transmitted through the ear dermis induce the immune response of the adjacent lymph nodes. RNAs prepared from the ear lesions and the lymph nodes were used to address the immune-related gene expression prior and after infection with the parasites. Several methods exist for preparation of ribonucleic acids. The classical protocols use guanidium isothiocyanate, a chaotropic agent that disintegrates the cellular structures and dissociates rapidly the nucleoproteins from the nucleic acids (Reid, 1991). RNases are also inactive in the presence of 4 M guanidium thiocyanate. Beta-mercaptoethanol (β-ME) is added to reduce RNA degradation by RNases. These protocols require disposable sterile glassware and plastic ware for the preparation of non-degraded RNA. Often pretreatment with diethylpyrocarbonate (DEPC) (0.1%) is used to treat glassware for 12 h at 37 °C and then heating at 100 °C for 15 min to remove any traces of diethylpyrocarbonate. The cellular proteins are removed from the nucleic acids preparations by extractions with phenol/chloroform. Ethanol precipitation of the isolated RNAs is then required and the nucleic acids are obtained by precipitation in the presence of 0.3 M sodium acetate. Our method benefits from the existence of a simplified protocol provided by the Qiagen kits. The methodological advantages of this protocol are as follows: 1) the method is simplified and quick, 2) there is no need to use DEPC, 3) phenol extraction of the nucleic acids from the proteins is not required, 4) the silica columns for separation of the nucleic acids from the cells or tissues constituents are very efficient, 5) RNAs are at a correct concentration and ethanol precipitation is not necessary and finally this method yields a high quality of RNAs. Whilst accompanied protocols are provided by the Qiagen kits, the additional details in the description of our method, applied to the mouse ear and lymph nodes, completes the methodology of the corresponding published article (Giraud et al., 2019b), presents with an interest for the isolation of pure non-degraded RNAs and may be useful to other investigators.