Recombinant Brugia malayi pepsin inhibitor (rBm33) induced monocyte function and absence of apoptotic cell death: An in vitro study

The effect of recombinant Brugia malayi pepsin inhibitor (rBm33) on human monocytes/macrophages has been examined using THP-1 cells. THP-1 cells stimulated with rBm33 showed enhanced levels of expression of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) and diminished levels of IL-12, iNOS and anti-inflammatory cytokine (IL-10) expression suggesting the predominant features of Th1 response. Phorbol-12-myristate-13-acetate (PMA) treated THP-1 cells stimulated with rBm33 and subsequent incubation with GFP expressing Escherichia coli (E. coli) for 2 h enhanced the uptake of E. coli. Nitric oxide (NO) levels measured in the supernatants of these cultures did not show significant changes. Apoptotic studies with Peripheral Blood Mononuclear Cells (PBMCs) from normal individuals stimulated with rBm33 did not induce apoptosis of monocytes or lymphocytes. These observations suggest that rBm33 stimulates macrophages to induce Th1 response and does not promote apoptosis

Autophagy Protects Monocytes from Wolbachia Heat Shock Protein 60–Induced Apoptosis and Senescence

Monocyte dysfunction by filarial antigens has been a major mechanism underlying immune evasion following hyporesponsiveness during patent lymphatic filariasis. Recent studies have initiated a paradigm shift to comprehend the immunological interactions of Wolbachia and its antigens in inflammation, apoptosis, lymphocyte anergy, etc. Here we showed that recombinant Wolbachia heat shock protein 60 (rWmhsp60) interacts with TLR-4 and induces apoptosis in monocytes of endemic normal but not in chronic patients. Higher levels of reactive oxygen species (ROS) induced after TLR-4 stimulation resulted in loss of mitochondrial membrane potential and caspase cascade activation, which are the plausible reason for apoptosis. Furthermore, release in ROS owing to TLR-4 signaling resulted in the activation of NF-κB p65 nuclear translocation which leads to inflammation and apoptosis via TNF receptor pathway following the increase in IL-6 and TNF-α level. Here for the first time, we report that in addition to apoptosis, rWmhsp60 antigen in filarial pathogenesis also induces molecular senescence in monocytes. Targeting TLR-4, therefore, presents a promising candidate for treating rWmhsp60-induced apoptosis and senescence. Strikingly, induction of autophagy by rapamycin detains TLR-4 in late endosomes and subverts TLR-4-rWmhsp60 interaction, thus protecting TLR-4-mediated apoptosis and senescence. Furthermore, rapamycin-induced monocytes were unresponsive to rWmhsp60, and activated lymphocytes following PHA stimulation. This study demonstrates that autophagy mediates the degradation of TLR-4 signaling and protects monocytes from rWmhsp60 induced apoptosis and senescence

Plasmodium Secretion Induces Hepatocyte Lysosome Exocytosis and Promotes Parasite Entry.

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosome-associated membrane protein 1 (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens

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

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

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