Fig 1.
Expression of host defense genes in L. donovani infected THP-1 cells.
THP-1 cells (105 cells/ml) were treated with PMA (50 ng/ml for 48 h), as explained in the methods section. Uninfected and infected cells were harvested at A: 6 h and B: 24 h post-infection. RNA was extracted from infected and uninfected THP-1 cells, and expression of defense genes was quantitated by qRT-PCR. Gene expression changes are depicted as fold change in transcription compared to that of uninfected cells (UI). Numbers >1 indicates up-regulation, and <1 indicates the down-regulation of host genes in infected cells as compared to the uninfected cells. RNU6A was used as the housekeeping gene. Data analysis was performed using a 2^-ΔΔCT method. The basal level of transcript expression of uninfected cells was used for data normalization and to assess relative abundance. The results represent the mean ± SD (n = 3).
Fig 2.
Analysis of histone promoter acetylation and HDAC1 occupancy pattern of host defense genes in response to Leishmania donovani infection.
THP-1 cells (105 cells/ml) were infected with L. donovani. At 6 and 24 h post-infection, the histone acetylation pattern on the defense genes promoter was analysed by ChIP using antibodies specific for H3 acetylation A: 6 h post infection B: 24 h post-infection. HDAC1 occupancy at the defense genes promoters was also determined by ChIP using antibodies specific for HDAC1 C: 6 h post-infection D: 24 h post-infection. The immunoprecipitated DNA fragments were quantitated by qRT-PCR using primers specific for the promoter region of each of the host genes studied. The changes in the level of H3-acetylation and HDAC1 occupancy are depicted as fold change over uninfected cells (UI). Basal levels of acetylation and HDAC1 occupancy of uninfected cells were used for data normalization and assessed relative abundance. Numbers >1 indicates up-regulation, and <1 indicates down regulation of host genes in infected cells as compared to uninfected cells. The results are mean ± SD (n = 3).
Fig 3.
Histone deacetylase 1 (HDAC1) expression and enzyme activity in host cells post L. donovani infection.
THP-1 cells (105cells/ml) were infected with L. donovani (20:1, MOI). The cells were harvested at different time points. A: 6 and 24 h post-infection HDAC1 was immunoprecipitated from infected and unifected host cells, and lysates were analysed for HDAC1 enzyme activity by fluorescent assay kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was calculated, as mentioned in the methods section. B: RNA was extracted from uninfected (UI) and infected cells (IN) at 6 h, 12 h and 24 h post-infection. HDAC1 expression was quantitated by qRT-PCR. Fold change values of HDAC1 mRNA is represented relative to uninfected condition (UI). Basal levels of HDAC1 expression in uninfected cells was used for data normalization and was taken as 1.0. C: Nuclear extracts of uninfected and infected THP-1 cells were harvested at 6, 12 and 24 h post-infection and lysed. Western blotting was performed using HDAC1 specific antibodies to observe HDAC1 protein expression. The symbols (-) and (+) represent uninfected and infected conditions respectively. H3 was used as housekeeping control to confirm equal loading of protein. Molecular weight markers are mentioned at left hand side of the blot and ‘ns’ refers to non-specific protein bands—D: Densitometric analysis of western blot using ImageJ software and normalized to H3 levels. The results represent the mean ± SD (n = 3). ANOVA was used to determine statistical significance. P-value for significance: * (P≤0.01 to 0.05), **** (P≤0.00001).
Fig 4.
Effect of HDAC1 silencing on the expression of host defense genes and intracellular parasite load in L. donovani infected THP-1 cells.
THP-1 cells (105 cells/ml) were transfected with HDAC1-siRNA (600 pmol) using lipofectamine. Scrambled-siRNA transfected THP1 cells were used as a control. After 24 h of transfection, the THP-1 cells were infected with L. Donovani (20:1 MOI). The cells were then harvested at 6 h post-infection, and RNA was extracted, followed by qRT-PCR to determine the impact of HDAC1 silencing on host defense genes and intracellular parasite load. A: Expressions of HDAC1were quantified by qRT-PCR. Fold change values of HDAC1 mRNA is represented relative to uninfected-Sc-siRNA transfected condition. The basal level of HDAC1 in uninfected-Sc-siRNA transfected cells was used for data normalization and was taken as 1.0. B: THP1 cells were transfected with HDAC1-siRNA or Sc-siRNA for 24 h and were then incubated with L. donovani (IN) for 3 h. After 6 h post-infection, cells were stained with Giemsa and the number of infected cells and the number of amastigotes were counted visually. C: Expression of defense genes in infected THP-1 cells transfected with HDAC1-siRNA or Sc-siRNA was determined by qRT-PCR.Results represent mean ± SD (n = 3). Significance was calculated by ANOVA, ** (P≤.01), *** (P≤0.001) and **** (P≤0.00001).
Fig 5.
Effect of inhibition of HDACI activity by pharmacological inhibitor NaB on defense gene expression and parasite load in L. donovani infected THP-1 cells.
THP-1 cells (105cells/ml) were incubated with L. donovani (20:1 MOI) for 3 h. Subsequently, the infected (IN) cells were incubated with increasing amounts of sodium butyrate (NaB) and were harvested at 6 h. A: Expression of HDAC1 was measured by qRT-PCR and transcription levels were plotted. The significance of the difference in expression levels was measured in infected (IN) and untreated samples (NaB concentration– 0 mM). B: Total HDAC activity in nuclear extracts of infected cells in the presence of increasing amounts of NaB was detected by fluorescent assay kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was calculated as mentioned in the methods section. The significance of the difference in activity was measured in infected (IN) and untreated samples (NaB concentration– 0 mM). C: Dose dependent impact of HDAC1 inhibitor on the parasitaemia count. Infected THP-1 cells (IN) were incubated in increasing concentrations of NaB. After 6 h, cells were stained and amastigotes enumerated visually. D: Expression levels of the host defense genes, post-infection in the presence and absence of NaB (5 mM) were quantified by qRT-PCR. Results represent the mean ± SD (n = 3). The statistical significance was determined using ANOVA, ns (P>0.05),* (P≤0.01 to 0.05), ** (P≤0.01), *** (P≤0.001), *** (P≤0.0001) and **** (P≤0.00001).
Fig 6.
HDAC1 inhibiting drug SAHA impairs parasite survival.
THP-1 cells (105 cells/ml) were infected with L. donovani (20:1 MOI) for 3 h.The infected (IN) cells were then incubated with increasing amounts of SAHA and were then harvested at 6 h. A: Expression of HDAC1 infected (IN) THP-1 cells after treatment with different concentrations of SAHA. Significance was measured concerning infected and untreated samples (SAHA concentration– 0 uM). B: Dose-dependent impact of HDAC1 inhibitor, SAHA, on the parasitemia count. Infected THP1 cells (IN) were incubated with increasing concentrations of SAHA for 6 h. Cells were stained after 6 h and amastigotes enumerated visually. C: The expression levels of the host defense genes, 6 h post-infection in the absence and presence of SAHA (5 μM) were quantified by qRT-PCR. Results represent the mean ± SD (n = 3). The statistical significance was determined using ANOVA, ns (P>0.05), * (P≤0.05), ** (P≤0.01), *** (P≤0.001), *** (P≤0.0001) and **** (P≤0.00001).
Fig 7.
Mechanism of action of siRNA mediated HDAC1 down-regulation and its impact on intracellular L. donovani A: Infection with L. donovani (LD), increases histone deacetylase (HDAC) levels which further leads to decrease in expression of the antimicrobial factors. Thereby facilitating the establishment of parasite infection within the host cells. B: Transfection of host cells with HDAC1-siRNA, leads to reduced HDAC1 and reduced deacetylation of histones, facilitating increased transcription of host defense genes and decreased intracellular load of L. donovani.