Characteristics of the study population: Demographic details of the individuals included in the study.

<p>Note: Mf—Microfilaria; CFA—Circulating filarial antigen.</p><p>Characteristics of the study population: Demographic details of the individuals included in the study.</p

TLR4 signaling accounts for rWmhsp60-induced apoptosis.

<p>(a) Molecular docking of protein–receptor interface among TLR-2, TLR-4 and TLR-9 with rWmhsp60. (b) Surface expression of TLR-2, TLR-4 and TLR-9 in monocytes from EN with rWmhsp60 was evaluated by FCM analysis. (c) Monocytes were stimulated with rWmhsp60 and CHX and labeled with anti-TLR-4-APC antibody. Representative images showed TLR-4 surface expression by confocal microscopy (×1000). (d) Monocytes treated with rWmhsp60 and/or anti-TLR-4 blocking antibody was subjected to annexin-V-FITC/PI staining. Early apoptotic cells were annexin-V-FITC⁺/PI⁻, late apoptotic cells were annexin-V-FITC⁺/PI⁺ and dead cells were annexin-V-FITC⁻/PI⁺. (e) The statistical plots represent the percentage apoptotic and dead cells (n = 10; mean ± SD; * represents p<0.05).</p

ROS production is required for rWmhsp60-induced NF-κB activation.

<p>(a) Immunofluorescence of monocytes from EN treated with rWmhsp60 and LPS indicated the localization of NF-κB p65 (red fluorescence) at ×1000 magnification. DAPI (blue) was used as a nuclear counter stain. (b) The effect of ROS on rWmhsp60-induced NF-κβ activation was analyzed in monocytes of EN (n = 10) by Western blot and β-actin. IL-6 (c) and TNF-α (d) cytokine levels in the supernatant were quantified by ELISA using specific monoclonal primary antibodies and developed using chemiluminescence and expressed as fold change (mean ± SD; “*” represents p<0.05).</p

rWmhsp60-induced autophagy and senescence.

<p>(a) Visualization of autophagy activation with MDCin monocytes of EN. Cells treated with rWmhsp60 and rapamycin were incubated for 24 h. Cells were stained with MDC as described in Methods section and visualized under fluorescent microscope at 335 nm. (b) The percentage of MDC-stained cells was represented as % autophagic cells (n = 10; mean ±SD; “*” represents p<0.05). (c) rWmhsp60 stimulation potentiates senescence in monocytes of EN. Monocytes were treated as above and incubated overnight in the presence of X-Gal (1 mg/ml) at 37°C as described in the Methods section. SA-β-Gal activity was detected by a blue cell staining, visualized under Carl Zeiss inverted microscope, and analyzed using Axiovision software. (d). SA-b-Gal–positive cells were quantified by counting 10² cells on three separate fields for each condition. The bar graph represents average mean ± SD of fold changes (n = 10) and “*” denotes p<0.05.</p

rWmhsp60 induces ROS and mROS in monocytes of EN.

<p>(a) The generation of intracellular and mitochondrial ROS in monocytes of EN with CHX and rWmhsp60 was analyzed using DCF2-DA and MitoSOX staining. Representative images showed ROS and mROS production by confocal microscopy. (b) The rWmhsp60- and CHX-induced mitochondrial membrane dissipation was observed in confocal microscope using TMRE. (c) and (d) represents quantitative fluorimetric analysis of ROS and MMP using DCF2-DA and TMRE, respectively. The bar graph represents average mean ±SD of fold change in RFU (n = 10) and * denotes p<0.05. rWmhsp60 resulted in increased expression of pro-apoptotic markers. Monocytes of EN (n = 10) were stimulated with CHX and rWmhsp60 for 24 h and harvested, and the gene expression of pro- and anti-apoptotic markers (e) <i>Bax</i>, (f) <i>Bad</i>, (g) <i>Bid</i> and (h) <i>Bcl-2</i> determined by Real Time PCR is depicted as fold change over basal level (mean ± SD; “*” represents p<0.05).</p

Rapamycin protects monocytes of EN from rWmhsp60-induced apoptosis.

<p>(a) Monocytes were treated for 24 h with rWmhsp60 following 2 h incubation with or without rapamycin. Further, cells were stained with MDC as described in Methods section and visualized under fluorescent microscope at 335 nm. (b) The percentage of cells with MDC stained was represented as % autophagic cells. Rapamycin targets TLR4 to autophagosome. (c) Intracellular vesicle co-localization of TLR4 into autophagosome (LC3 positive: 3 h) following rapamycin and/or with rWmhsp60 treatment was assessed as described in the Methods section and visualized using confocal microscopy (×1000). (d) Immunoblot analysis was performed to assess the expression of LC3, mTOR, p16 and p53 in rapamycin pre-treated monocytes of EN following rWmhsp60 stimulation. β-actin was used as a loading control. (e) Representative distributions of the fluorescence intensity of annexin-V-FITC and PI binding of EN monocytes following rWmhsp60 and or rapamycin treatment. (f) The statistical plots represent the percentage apoptotic and dead cells (n = 10; mean ± SD; “*” represents p<0.05).</p

Rapamycin protects monocytes of EN from rWmhsp60 induced senescence and inflammation.

<p>(a) Monocytes treated with rWmhsp60 and/or rapamycin were subjected to SA-β-Gal activity staining as stated earlier and acquired in Carl Zeiss inverted microscope and analyzed using Axiovision software (Carl Zeiss, Jena). (b) SA-β-Gal–positive cells were quantified by counting 10² cells on three separate fields for each condition. IL-6 (c) and TNF-α (d) cytokine levels in the supernatant were quantified by ELISA using specific monoclonal primary antibodies and developed by chemiluminescence and expressed as fold (n = 10; mean ± SD; “*” represents p<0.05).</p

Activation of lymphocytes by rapamycin-treated monocytes from EN.

<p>Monocytes of EN were purified from PBMCs by percol gradient method and treated with rapamycin for 2 h. Rapamycin-treated monocytes were then co-cultured with monocyte depleted PBMCs (CC cells). (a) After 24 h, assessment of lymphocyte proliferation using [³H] thymidine was carried out following PHA treatment with or without rWmhsp60. Lymphocyte proliferation was expressed as stimulation index. (b) The concentration of IFN-γ in the cell culture medium was determined by enzyme-linked immunosorbent assay as described in Methods section (n = 10; mean ± S.D and * denotes p<0.05).</p

Quantitative analysis of apoptosis in PBMCs.

<p>Representative distributions of the fluorescence intensity of annexin-V-FITC and PI binding of (a) EN (n = 10) and (b) CP (n = 5) monocytes after 24-h stimulation with rWmhsp60 and CHX. Early apoptotic cells (E) were annexin-V-FITC⁺/PI⁻, late apoptotic cells (L) were annexin-V-FITC⁺/PI⁺ and dead cells (D) were annexin-V-FITC⁻/PI⁺. The scatter plot represents the percentage of early, late apoptotic and dead cells in (c) EN and (d) CP monocyte populations. Values are represented as mean ± S.D and “*” denotes p<0.05. rWmhsp60 induces apoptosis in monocytes of EN via caspase cascade activation. (e) rWmhsp60- and CHX-treated monocytes from EN were harvested, and the levels of caspase-3, caspase-9, p53 and PARP were determined by Western blot analysis as described in Methods section with the respective cleaved and total antibodies. (f) Bar graph represents the fold change of normalized IOD of the respective blots. (g) Monocytes of EN were cultured in the presence of CHX and rWmhsp60 for 24 h and determined the caspase-3 activity using the fluorogenic substrate Ac-DEVD-AMC as detailed in Methods section. The activity expressed as % fold increase in relative fluorescence units (RFU) (mean ±S.D, n = 10) and “*” represents p<0.05.</p

(a) Production of recombinant Wmhsp60.

<p>Western blot of recombinant and IMAC-purified Wmhsp60. Lane 1, molecular weight marker; lane 2, His-tagged rWmhsp60 probed with anti-rWmhsp60 antibody; lane 3, His-tagged rWmhsp60 probed with anti-histidine monoclonal antibody; lane 4, His-tagged rWmhsp60 probed with normal mouse serum. (b) rWmhsp60 inhibited the growth of PBMCs and monocytes in a dose-dependent manner. PBMCs and monocytes of EN were treated with different concentrations of rWmhsp60. After 48 h of incubation, cells were harvested, and proliferative effect was determined by MTT assay as described in Methods section. The optimum concentration of rWmhsp60 was found to be 5μg / 1 million cells / ml. To ensure endotoxin-free preparation of rWmhsp60, Polymyxin-B assay was performed as described in—Methods section with the optimum concentration. Values were represented as % inhibition (mean ± SD, n = 10; “*” and “^<b>a</b>” denote significance [p<0.05]).</p