<i>Leishmania donovani</i> induced Unfolded Protein Response delays host cell apoptosis in PERK dependent manner

<div><p>Background</p><p>Endoplasmic reticulum (ER) stress generated unfolded stress response (UPR) is a basic survival mechanism which protects cell under unfavourable conditions. Leishmania parasite modulates host macrophages in various ways to ensure its survival. Modulation of PI3K-Akt pathway in delayed apoptotic induction of host; enables parasite to stabilize the infection for further propagation.</p><p>Methodology</p><p>Infected RAW macrophages were exposed to campothecin or thagsigargin and phosphorylation status of PERK, Akt, BAD and Cyt-C was determined through western blotting using phospho specific antibody. Expression at transcriptional level for cIAP1 &2, ATF4, CHOP, ATF3, HO-1 and sXBP1 was determined using real time PCR. For inhibition studies, RAW macrophages were pre-treated with PERK inhibitor GSK2606414 before infection.</p><p>Findings</p><p>Our studies in RAW macrophages showed that induction of host UPR against <i>L</i>.<i>donovani</i> infection activates Akt mediated pathway which delays apoptotic induction of the host. Moreover, Leishmania infection results in phosphorylation and activation of host PERK enzyme and increased transcription of genes of inhibitor of apoptosis gene family (cIAP) mRNA. In our inhibition studies, we found that inhibition of infection induced PERK phosphorylation under apoptotic inducers reduces the Akt phosphorylation and fails to activate further downstream molecules involved in protection against apoptosis. Also, inhibition of PERK phosphorylation under oxidative exposure leads to increased Nitric Oxide production. Simultaneously, decreased transcription of cIAP mRNA upon PERK phosphorylation fates the host cell towards apoptosis hence decreased infection rate.</p><p>Conclusion</p><p>Overall the findings from the study suggests that Leishmania modulated host UPR and PERK phosphorylation delays apoptotic induction in host macrophage, hence supports parasite invasion at early stages of infection.</p></div

<i>L</i>. <i>donovani</i> infection induces transcription of host cIAP mRNA expression.

<p><b>A</b>) Expression level of cIAP1 mRNA in RAW 267.4 macrophages infected with <i>L</i>. <i>donovani</i> for 4h, 12 h and 36 h or exposed to thapsigargin (Thg) or campothecin (Cmpt). <b>B</b>) Expression level of cIAP2 mRNA in RAW 267.4 macrophages infected with <i>L</i>. <i>donovani</i> for 4h, 12 h and 36 h or exposed to thapsigargin (Thg) or campothecin (Cmpt). Uninfected RAW 267.4 macrophages (UNS) without any treatment were taken as control. Data are represented as the mean ± SEM of at least three experiments. (* p < 0.05).</p

<i>L</i>.<i>donovani</i> infection phosphorylates BAD protein in a PERK dependent manner.

<p><b>(A)</b> Western blot analysis to determine phosphorylated status of BAD protein using phosphos anti-BAD antibody in RAW 267.4 macrophages infected with <i>L</i>.<i>donovani</i> (LD) or exposed to campothecin (Cmpt). (<b>B</b>) Densitometry of western band for above experiment. <b>C</b>) Western analysis from lysates of infected RAW 267.4 macrophages pre-treated with GSK2606414 and exposed to campothecin (Cmpt+GSk) / thapsigargin (Thg+GSK) or untreated infected RAW cells subjected to campothecin (Cmpt) or thapsigargin (Thg) exposure. The band intensities were analysed densitometrically (<b>D</b>). For both the experiments, GAPDH was taken as control. Data are represented as the mean ± SEM of at least three experiments. (*p < 0.05).</p

Inhibition of host PERK decreases Akt phosphorylation and inhibits transcription of host cIAP mRNA expression under exposure to apoptotic inducers.

<p><b>(A</b>) RAW 267.4 macrophages pre-treated with GSK2606414 (2 hours) or untreated were infected with <i>L</i>.<i>donovani</i> promastigotes. After 4 h of infection, GSK pre-treated infected cells were exposed to campothecin (Cmpt+GSk) or thapsigargin (Thg+GSK). Similarly, untreated infected RAW macrophages were also exposed to campothecin (Cmpt) or thapsigargin (Thg). After treatment, cells were harvested and western analysis probed with phosphor-anti Akt, anti-Akt and GAPDH was performed from harvested cell lysates. (<b>B</b>) Band intensities of western blot bands after densitometry. (<b>C</b>) Relative expression of cIAP1 and cIAP2 mRNAs levels as analysed through real time in infected RAW 267.4 macrophages (4 h) either pre-treated with GSK2606414 or untreated, followed by campothecin (Cmpt+GSk) / (Cmpt) or thapsigargin (Thg+GSK) / (Thg) exposure. Infected RAW cell without any exposure were also taken in account. RAW 267.4 macrophages without any treatment or exposure were taken as control. Data are represented as the mean ± SEM of at least three experiments. (* p < 0.05).</p

Host PERK inhibition leads to increased Cyt-C release and induces caspase-3 activity in infected RAW macrophages.

<p>RAW267.4 macrophages either pre-treated with GSK2606414 or untreated were infected with <i>L</i>.<i>donovani</i> promastigotes. After 4hr post infection, the infected cells were exposed to camphothecin (Cmpt+GSK/ Cmpt) or thapsigargin (Thg+GSK/ Thg) and the level of released Cyt C in cytosolic fraction was determined through western blot analysis using anti-Cyt C antibody. (<b>A</b>) Western blot result showing released Cyt C level in camphothecin (Cmpt) or thapsigargin (Thg) exposure or in GSK2606414 pre-treated RAW cell exposed to camphothecin (Cmpt+GSK) or thapsigargin (Thg+GSK). Untreated or uninfected RAW macrophages (UNS) or RAW cells treated with GSK2606414 (RAW+GSK) were taken as control. (<b>B</b>) Densitometry analysis of blot bands against Cyt C for above mentioned experimental conditions. <b>(C</b>) Caspase-3 activity in <i>L</i>.<i>donovani</i> infected RAW 267.4 macrophages after campothecin or thapsigargin treatment: Prior to infection, RAW macrophages were incubated with GSK2606414, washed and allowed for infection with L.donovani promastigotes. Infected RAW (4 h) were further exposed to campothecin (LD+RAW+Cmpt+GSK) or thapsigargin (LD+RAW+Thg+GSK). In another set, RAW macrophages without GSK2606414 treatment were allowed to infect followed by campothecin (LD+RAW+Cmpt) or thapsigargin (LD+RAW+Cmpt) treatment. Untreated or uninfected RAW macrophages (RAW) were taken as control. (<b>D</b>) DNA analysis by agarose gel electrophoresis revealed DNA fragmentation into oligonucleosome-sized fragments in control RAW 267.4 macrophages (UNS), RAW cell infected with <i>L</i>.<i>donovani</i> (RAW+LD), GSK pre-treated; infected RAW (4 h) exposed to campothecin (LD+RAW+Cmpt+GSK) or thapsigargin (LD+RAW+Thg+GSK). Untreated or uninfected RAW macrophages (UNS) or RAW cells treated with GSK2606414 (RAW+GSK) were taken as control. Data are represented as the mean ± SEM of at least three experiments (*p < 0.05).</p

<i>L</i>. <i>donovani</i> infection reduces csapase-3 activity leading to decreased apoptotic induction in RAW macrophages.

<p><b>A)</b> Caspase-3 activity in infected host macrophages: RAW267.4 macrophages were infected with <i>L</i>.<i>donovani</i> promastigotes. After 4 h, 12 h or 36 h of infection, infected cells were subjected to camphothecin (Cmpt) or thapsigargin (Thg) treatment for apoptotic induction and caspase-3 activity was determined. <b>B</b>) Apoptotic induction in host macrophages was determined after PI staining and analysed with flow cytometry under above mentioned conditions. The obtained values were plotted in terms of percent apoptotic cells. Uninfected RAW (UNS) macrophages in both the conditions were taken as control. Thg and Cmpt represent thapsigargin and camphothecin treatment respectively Data are represented as the mean ± SEM of at least three experiments. (* p < 0.05).</p

PERK phosphorylation in <i>L</i>.<i>donovani</i> infected RAW macrophages is required for protection against oxidative stress.

<p><b>A)</b> Western blot analysis to determine host PERK phosphorylation status in <i>L</i>.<i>donovani</i> infected RAW 267.4 macrophages under oxidative exposure. GSK2606414 pre-treated or untreated RAW 267.4 macrophages, infected with <i>L</i>.<i>donovani</i> were exposed to H₂O_2. The cells were then harvested; lysed and equal amount of protein (50μg) was resolved on SDS-PAGE followed by probing with anti-phospho PERK antibody. Normal RAW macrophages (RAW) or <i>L</i>.<i>donovani</i> infected (LD+RAW) were considered as control. <b>B</b>) Densitometric analysis of western blot bands. <b>C</b>) Nitrite concentration was measured by Griess reaction in cell supernatant of infected RAW macrophages (LD+RAW) or in infected RAW cells exposed to H₂O₂ (LD+RAW+ H₂O₂) or pre-treated with GSK2606414 (LD+RAW+ H₂O₂+GSK) or in uninfected (H₂O₂+RAW) cells without any treatment. Untreated (UNS) or RAW macrophages exposed to H₂O₂ in presence of NAC (H₂O₂+ NAC) were taken as control. <b>D</b>) Real time plot to determine relative expression of HO-1 at mRNA level under above mentioned conditions.</p

Inhibition of host PERK induces Apoptosis in infected RAW macrophages and decreases infection rate.

<p>(<b>A</b>) RAW macrophages undergoing apoptosis was determined using PI dye for infected RAW macrophages either treated with GSK2606414 subjected to H₂O₂ exposure (H₂O₂+RAW) or campothecin (Cmpt+RAW) or thapsigargin (Thg+RAW) and for untreated RAW macrophages exposed to H₂O₂ (H₂O₂)_, campothecin (Cmpt) and thapsigargin (Thg). Normal uninfected RAW macrophages without any treatment (UNS) or infected with <i>L</i>.<i>donovani</i> (LD+RAW) or incubated along with wortmanin subjected to thapsigargin exposure (Thg+WRT) were considered as control. <b>B)</b> RAW macrophages either thapsigargin exposed (Thg+LD+RAW) or NAC incubated (LD+RAW+NAC) were infected with <i>L</i>. <i>donovani</i> in 10:1 MOI. Also, GSK2606414 pre-treated RAW macrophages (LD+RAW+GSK) or followed by thapsigargin exposure (LD+RAW+Thg+GSK) were infected. After 36 h of infection, the cells were fixed with methanol and stained with Giemsa, and the infection index was calculated (percent <i>L</i>. <i>donovani</i> infected cells X number of amastigotes per cell) Uninfected RAW macrophages (RAW) or normal RAW macrophages infected with <i>L</i>.<i>donovani</i> (LD+RAW) were taken as control (Significant difference * (<i>P</i> < 0.05).</p

<i>L</i>.<i>donovani</i> infection induces UPR in RAW macrophages.

<p><b>(A</b>) Gene expression profiling in RAW 267.4 macrophages 4 h, 12 h and 36 h after infection with <i>L</i>. <i>donovani</i> promastigotes. The graph shows the fold changes in comparison to the control (non-infected). (<b>B</b>) Gene expression in RAW 267.4 macrophages following thapsigargin (1.0μM/ 1hr) exposure. The graph shows the fold changes in comparison to the control (DMSO) values. (<b>C</b>) The induction of ER stress by thapsigargin (Thg) or tunicamycin (Tu) favours <i>L</i>. <i>donovani</i> infection. RAW264.7 cells pre-treated with thapsigargin for 1 h were infected with <i>L</i>. <i>donovani</i> promastigotes (10:1 MOI). After 36 h of infection, the cells were fixed with methanol and stained with Giemsa, and the infection index was calculated (% <i>L</i>. <i>donovani</i> infected cells X number of amastigotes per cell). The graph represents the infection index at 36 h. (<b>D</b>) Images showing thapsigargin (Thg) and tunicamycin (Tu) pre-treated RAW macrophages subjected to <i>L</i>.<i>donovani</i> infection. Untreated RAW macrophages (UNS) were taken as control. Data are represented as the mean ± SEM of at least three experiments. (* p < 0.05).</p

Primer sequences for real-time PCR.

<p>Primer sequences for real-time PCR.</p