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.