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

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

Diminished mitogen stimulated levels of common γ-chain cytokines in PTB individuals and restoration following anti-TB treatment.

<p>(A) Mitogen stimulated levels of common γ-chain cytokines IL-2, IL-7, IL-15 and IL-2 were measured in PTB (n = 30), LTB (n = 30) and NTB (n = 30) individuals. The data are represented as bar graphs showing geometric means and 95% confidence intervals. P values were calculated using the Kruskal-Wallis test with Dunn's post hoc comparison. (B) Mitogen stimulated levels of common γ-chain cytokines IL-2, IL-7, IL-15 and IL-2 were measured in PTB individuals before (pre-T) and after (post-T) standard anti-TB chemotherapy. The data are represented as line graphs with each line representing a single individual. P values were calculated using the Wilcoxon signed rank test.</p

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

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

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

Demographics of study groups.

<p>Demographics of study groups.</p