Dias-Teixeira et al. group has investigated the importance of the PERK/p-eIF2α signaling branch in Leishmania amazonensisinfection. The western blotting results of the total extracts of the infected cells showed an increased level of PERK, 8 hours post-infection. The observed levels were similar to that of thapsigangin-treated cells which are used as positive controls. To determine the importance of the PERK/p-eIF2α/ATF4 signaling branch, mouse macrophages transduced with lentiviral short hairpin RNA expression vectors that target PERK (shPERK), were used as test samples and, mouse macrophages transduced with scrambled shRNA (shSCR) were used as controls. Significant down-regulation of p-eIF2α, which is the downstream effector of PERK was detected in PERK knockout cells compared to that of cells transduced with shSCR. In addition to that, a decrease in the infection index was also observed in PERK knockdown cells. These results were further confirmed using Leishmania amazonensisinfected cells treated with PERK inhibitors. The cells treated with the inhibitor were reported to have a negative effect on infection. However, no effect of PERK was observed on the parasite load. Collectively with these results, they concluded that Leishmania amazonensisinfection indeed activates the PERK/eIF2α signaling cascade and that PERK/eIF2α signaling favors parasite infection[48]. ATF4 is the immediate downstream effector of the PERK/eIF2α signaling axis. To determine whether Leishmania amazonensis infection induced the expression of ATF4 through the PERK/eIF2α branch, whole cell lysates were subjected to western blotting at specific time intervals and an increase in ATF4 was observed in infected cells compared to uninfected cells. Using shATF4 transduced infected cells, the importance of ATF4 inLeishmania amazonensis infection was determined. The ATF4 knockout infected cells showed a reduced parasite burden compared to shSCR transinfected cells which are the controls. These observations decipher that upon infection with Leishmania amazonensis , PERK/eIF2α/ATF4 axis is induced and plays a critical role in shaping theLeishmania amazonensis infection[48].

PERK protein sensor triggered branch can phosphorylate and activate the NRF2 transcription factor which is responsible for inducing the expressions of genes involved in anti-oxidative response[8]. In a study done to evaluate the effect of ATF4 on oxidative stress defense inLeishmania amazonensis infection, researchers observed an increase in (NO) production and nitric oxide synthase (iNOS) in shATF4 transduced Leishmania amazonensis infected cells. Heme oxygenase-1 (HO-1) is a key enzyme triggered by cellular stress and is a target of NRF2[48], [50]. Heme Oxygenase-1 can diminish the reactive oxygen species, thereby ensuring parasite survival[50]. The luciferase assay data of infected cells transduced with shATF4 showed a down-regulation in NRF2, concluding that the activation PERK/eIF2α/ATF4 signaling protects Leishmania amazonensis infected macrophages from oxidative stress[48].

Galluzzi et al. demonstrated that Leishmania infantuminfection induces a mild UPR. The main aim of their study was to investigate the ER stress responses in macrophages infected withLeishmania infantum and uncover underlying molecular mechanisms which lead to anti-apoptotic properties in infected cells, using tunicamycin-treated cells as positive controls. Tunicamycin triggers apoptosis via the induction of ER stress. The gene expression analysis studies showed significant induction of several ER stress markers, namely, DDIT3/CHOP, ATF3, ATF4, and CEBPB at 6-hour and 24-hour post-infection in infected cells, but the expressions were much lower compared to positive controls. The western blot analyses of infected U937-derived macrophages showed the induction of sXBP1 and GRP78/HSPA5 proteins. However, Leishmania infantum infection did not appear to induce the ER stress markers phospho-eIF2α and DDIT3/CHOP. These results concluded that there is a mild but significant induction in ER stress markers tested in infected cells, but the magnitude of induction is significantly higher in positive controls compared to the infected cells. Furthermore, they have pointed out that different kinetic properties and activation of main UPR branches or the cells not being infected simultaneously might be the reason for uneven induction between the tested UPR markers. (GRP78/HSPA5 and sXBP1 being induced and lack of induction of phospho-eIF2α and DDIT3/CHOP)[7] Additionally, they assessed the ability of Leishmania infantum to modulate the UPR to shape the infection. After 4 hours of treatment with tunicamycin, a significant reduction in both phospho-eIF2α and DDIT3/CHOP protein levels was detected in infected cells compared to non-infected cells. These results suggested that Leishmania infantum infection delays or attenuates the effects of the host UPR[7]. Prolonged ER stress is reported to cause cell death, but mild ER stress is reported as a possible adaptive mechanism for the cell to build up resistance to subsequent ER stress. This is known as hormesis. Mild ER stress has been shown to have a protective role in neurodegenerative diseases, diabetes, cancer, and in certain cell lines[51]–[53]. The mechanism underlying hormesis is shown in figure 6.

Additionally, their findings may suggest the induction of ER stress as a mechanism by which Leishmania triggers host antioxidant enzymes that act as scavengers of superoxide anions generated during infection. The qPCR results showed a significant induction in XBP1 both in human and mouse cells confirming the observations of Dias-Teixeira et al . 2016, which suggest a common pathogenic mechanism with minor alterations between cutaneous and visceral species[7]. Their study also shows an increase in ATF3 and CHOP expression, indicating potential involvement of the PERK-ATF4 arm but this needs further investigation[7], [8].

The RT-qPCR assays performed on RNA samples obtained from skin lesions of Leishmania braziliensis infected human CL patients showed that samples from patients displayed elevated ATF4 expression compared to healthy individuals, indicating that patients with CL have high ATF4 expression in infected tissue. With their previous findings[47], these data suggest that Leishmania parasites activate at least two branches of the ER stress response during human infection; namely, the IRE1/XBP1 branch and PERK/ eIF2α/ATF4 branch[48]. Overall, data demonstrates that Leishmania parasites activate the PERK/eIF2α/ATF4 pathway and that this pathway is important for parasite survival and favors pathogenesis[48].

A study done on Leishmania donovani in the Indian subcontinent by Abhishek et al. 2018 showed that Leishmania donovaniinfection induces the UPR in PERK dependent manner and it has an important role in delaying apoptosis of infected macrophages. They demonstrated that Leishmania donovani infection induces UPR and ER stress inducers that enhance parasite infection. Western blotting data showed that host PERK phosphorylation gets induced byLeishmania donovani . Moreover, real-time PCR data shows an up-regulation in mammalian inhibitor of apoptosis (IAP) proteins cIAP1 and cIAP2 in infected cells, whereas no such induction is detected in normal cells.