Leishmania, a unicellular kinetoplastid protozoan flagellate that infects people by the bite of phlebotomine sand flies, causes leishmaniasis, a parasitic disease which accounts for 1.5 to 2 million new cases worldwide each year, 350 million people are at risk of contracting it, and it results in 70,000 annual fatalities (Arenas et al., 2017). WHO 2010 expert committee study categorizes many clinical symptoms that occurred in the Old World into three basic forms: 1) Visceral leishmaniasis (VL), often known as kala-azar, caused by L. donovani and L. infantum; 2) Cutaneous leishmaniasis (CL), most commonly caused by L. tropica, L. major, and L. aethiopica; and 3) Mucocutaneous leishmaniasis (MCL), which can be caused by any species. 4) Diffused cutaneous leishmaniasis (induced by L. aethiopica) and 5) post-kala-azar dermal leishmaniasis (present in all areas due to L. donovani). While CL is the most prevalent type of the disease, VL is the most severe and nearly invariably fatal if ignored (https://www.who.int/news-room/fact-sheets/detail/leishmaniasis). Leishmaniasis is widespread in Africa, Latin America, Asia, the Mediterranean Basin, and the Middle East. Although the cutaneous form (CL) of the illness accounts for more than half of all new cases of leishmaniasis, 90% of CL cases occur in South America, the Middle East, and Afghanistan. The bulk of VL cases are reported in five countries: India, Bangladesh, Ethiopia, Sudan, and Brazil (Guhe et al., 2023).

The life cycle of Leishmania is digenetic, i.e., it rotates between two hosts. During infection, blood feeding parasites are engulfed by macrophages in which parasite promastigotes transform into amastigotes within phagolysosomal compartments, where they are able to survive and multiply. Thus, in order to establish successful infection and maintain parasite survival inside the mammalian host, macrophages are crucial. Mammalian cells have been observed to induce autophagy in response to microbe infections, which can either result in parasite elimination or pathogen persistence (Sampaio et al., 2019). For Leishmanial parasites, the role played by autophagy in the context to their infection is not fully elucidated.

Macroautophagy henceforth referred as autophagy is an adaptive cellular response to intra- or extracellular stress and signals (including starvation-induced nutrition restriction, ER-stress, and pathogenic encounter) and this evolutionary conserved process was first described by Christian de Duve in mammalian systems in 1963 (Feng et al., 2014) (Singh et al., 2016). Similar to apoptotic programmed cell death; it is a crucial process for the control of development and the preservation of homeostasis in multicellular organisms. There are predominantly three major forms of autophagy described in mammalian cells, which are microautophagy, macroautophagy (mainly known as autophagy or canonical autophagy) and chaperone-mediated autophagy (CMA) with different mode of cargo delivery to the lysosome and different physiological roles (Kelekar, 2005).

The initiation, elongation, and maturation phases—also known as the autophagosome formation process—are each characterized by distinct morphological and metabolic processes. A protein complexes having ATG gene products successively coordinate the formation of autophagosome. There are 31 ATG (autophagy-related) proteins found in yeast, and many of them aggregate in a pre-autophagosomal site (PAS) that may be seen as a punctate area by fluorescent microscopy (Mizushima, 2007). A key positive regulator of the development of autophagosomes is the Atg1/ULK1 complex (Atg1 in yeast and ULK1 (Unc-51-like autophagy activating kinase 1) in mammals). When nutrients are in ample supply, autophagy is inhibited by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) binding to the ULK1 complex. When there is starvation, mTORC1 separates from the ULK1 complex, freeing it to cause the formation and elongation of autophagosomes. The autophagic pathway is then triggered by the activation of the ULK1-ATG13-FIP200-ATG101 complex. Phosphorylaion of FIP200 (Focal adhesion kinase interacting protein of 200 kD) and ATG13 initiate phagophore formation at phagophore nucleation site, where it phosphorylates the PI3K (phosphatidylinositol-3-kinase) complex (Vps34 (vacuolar sorting protein 34), Vps15, Vps30/Atg6 and ATG14) that generates phosphatidylinositol 3- phosphate (PI3P). ATG9 is a transmembrane protein that transits between the PAS and peripheral sites; this represents sites from which membrane is delivered to the formation of phagophore. Return of ATG9 to peripheral site requires ATG2 and ATG18. ER-accumulated PtdIns3P recruits WIPI proteins which later decode the PtdIns3P signal to permit the formation of an autophagosomal precursor membrane. ATG14 is a component of the ATG14 complex, which is regulated by the binding of BECN1 to AMBRA1 (activating molecule in Beclin1-regulated autophagy 1) mainly because to stimulate the complex and binding of BECN1 to BCL2 (B-cell lymphoma 2) to inhibit the complex (Parzych and Klionsky, 2014).

After nucleation step, elongation and expansion of autophagosome is started with the help of ubiquitin like conjugation system. ATG4 proteases break the LC3 (microtubule associated protein light chain 3) protein off, revealing its glycine residue at the c-terminus. The E1-like enzyme ATG7 eventually activates the LC3 proteins. ATG7 transfers LC3 proteins to the E2 like enzyme ATG3 and further to the phosphatidylethanolamine (PE). This step is massively promoted by ATG12-ATG5-ATG16 complex, Thus LC3 keeps getting added on PE and expansion of autophagosome happens. In the final step, autophagosomes fuses with lysosomes to form autolysosomes. Following fusion cargo gets digested by lysosomal acid hydrolases.

For a long time, autophagy has been considered to be an essential cell recycling mechanism. Recent studies have discovered that it also performs roles in innate immunity and antimicrobial defence, among other things. The survival of many pathogens, including Mycobacterium TB, Shigella flexneri, Listeria monocytogenes, and Toxoplasma gondii, is significantly reduced in macrophages due to autophagy (Thomas et al., 2018).

Bioactive molecules called lipid mediators are produced by the metabolism of polyunsaturated fatty acids (PUFA) (Jordan and Werz, 2022). The three acids arachidonic (AA), eicosapentanoic (EPA), and docosahexaenoic (DHA) are the main sources of the lipid mediator precursors. Leishmania and other trypanosomatids can convert AA to eicosanoids via specialised enzymes such as cyclooxygenase (COX) and prostaglandin synthases (PG synthases), (Díaz-gandarilla et al., 2018; Tavares et al., 2021; Kubata et al., 2007). Lipid mediators are produced in the cytoplasm and in organelles termed lipid droplets (LD, lipid bodies) (Bozza et al., 2011; Almeida et al., 2018). Triacylglycerols (TAGs), cholesteryl-esters, and retinyl-esters, which all play a part in energy homeostasis, membrane formation, and cell signalling, make up the neutral hydrophobic core of lipid droplets (Bouazizi-Ben Messaoud et al., 2017). They play significant functions in controlling how lipids are stored and used by various cells. Potentially to defend the body from the negative effects of various stimuli, LD are altered during infection and inflammation (Saka and Valdivia, 2012) suggesting its role in host defence (Ganguly et al., 2022).

On the other hand, interactions between these LDs and pathogen-containing phagosomes may offer the pathogen a rich supply of energy, which may have a substantial effect on the survival of the microbes infecting the hosts (Melo and Dvorak, 2012). Since Leishmania has been shown to have inefficient de novo lipid synthesis and therefore must scavenge lipids from the host environment, this is very pertinent to this parasite (Cicco et al., 2012). In the context of the function of LDs in inflammation, LDs are characterised as abundant deposits of esterified arachidonic acid (AA), which serve as precursors for the synthesis of eicosanoids. It has been reported that Enzymes necessary for this synthesis, including cyclooxygenases (COX) and prostaglandin E2 synthase (PGE2) are localized within LD (Bozza et al., 2007). Prostaglandins (PG), an eicosanoid, are biologically active substances produced from the metabolism of arachidonic acid (AA) by certain enzymes such cyclooxygenase (COX) and prostaglandin synthases. Prostaglandins may play a role in the regulation of immunological response (Araújo-santos et al., 2014). Prostaglandins have a various roles including regulation of immunological responses and suppressing macrophage activity. Notably, LDs are the primary site for increased PGE2 synthesis after T. cruzi infection because COX-2 and PGE-synthase accumulation inside them (Vallochi et al., 2018). Higher plasma levels of PGE2 in patients with localised or diffuse cutaneous leishmaniasis than in patient controls serve as an excellent illustration of the pivotal function that PGE2 plays in the modulation of the host immune response (França-costa et al., 2015). More than that, as shown in Leishmania spp., PGE2 synthesis helps parasite survival (Avila et al., 2009; Lonardoni and Barbieri, 1994; Content, 2014).

A class of small non-coding RNAs known as micro-RNAs are important post-transcriptional regulators of gene expression. They target mRNA which leads to either translational repression or degradation. After infection with L. major or L. donovani, it was discovered that many miRNAs were modulated in human macrophages as a result of inflammation and toll-like receptor activation brought on by either a pathogenic challenge or bacterial lipopolysaccharides (Laouini et al., 2013; Geraci et al., 2015). A significant alteration in the expression pattern of many miRNAs was seen in human macrophages infected with L. major, suggesting that these miRNAs may have a function in modifying the gene expression profile of the infected cells. According to recent studies, a number of microRNAs (miRNAs) may directly target autophagy-related genes such as BECN1 and ATG4 in order to modulate autophagy (Singh et al., 2016; Su et al., 2015; Xu et al., 2012).

Key miRNAs that were differentially modulated following L. major infection were identified during our earlier study (Nimsarkar et al., 2020). First, we describe important data addressing the molecular mechanism underlying the autophagy response in host cells infected with L. major. The potential influence of miR-146a-3p, a member of the miR146 family, on autophagy and L. major infection is then covered in detail. Subsequently, we report miR-146a-3p helps in control of lipid metabolism by targeting HPGD. As far as we know, we, for the first time demonstrate the regulatory role of miR-146a-3p in autophagy and lipid metabolism together post L.major infection.