Modulation of the immune response and infection pattern to Leishmania donovani in visceral leishmaniasis due to arsenic exposure: An in vitro study.

The arsenic contamination of ground water in visceral leishmaniasis (VL) endemic areas in Bihar, India leads to human exposure through drinking water. Possibly, the consumed arsenic (As) accumulates in the tissues of VL patients, who subsequently internalize intracellular amastigotes to confer resistance against chemotherapy to the parasite, leading to modulation in the host's immune response. This hypothesis appears to be consistent with the in vitro findings that in arsenic-exposed parasites, the mitochondrial membrane potential became depolarized, whereas the reduced thiol and lactate production was overexpressed with enhanced glucose consumption; therefore, the reduced thiol possibly supports an immunosuppressive state in the host cells. This observation was well supported by the down-regulated expression of pro-inflammatory cytokines (IL-2, IL-12, IFN-γ, and TNF-α) with a suppressed anti-leishmanial function of macrophage (NO, ROS). In contrast, the pathophysiological mechanism of VL has received ample support by the promotion of Th2 cytokines (IL-4 and IL-10) in the presence of arsenic-exposed Leishmania parasites (LdAS). Dysfunction of mitochondria and the overexpression of lactate production raise the possibility of the Warburg effect being operative through the up-regulation of glucose consumption by parasites to enhance the energy production, possibly augmenting virulence. Therefore, we surmise from our data that arsenic exposure to Leishmania donovani modulates the immune response and infection pattern by impairing parasite function, which may affect the anti-leishmanial effect in VL

Identification of <i>Leishmania donovani</i> antigen in circulating immune complexes of visceral leishmaniasis subjects for diagnosis

<div><p>The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the <i>Leishmania</i> antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of <i>L</i>. <i>donovani</i> proteins in circulating antigens were further validated using anti-<i>Ld</i> actin and anti-α tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.</p></div

Figure showing accumulation of CICs in human VL subjects during <i>L</i>. <i>donovani</i> infection in comparison to others.

<p>For this, one ml of peripheral blood serum was taken from the different study groups. It was incubated with precipitation buffer (5% polyethylene glycol and 0.1 M sodium borate) for overnight. The precipitated CICs was washed with wash buffer containing 2.5% PEG 6000 and re-dissolved in 100 μl PBS. Protein content (in μg/μl) was evaluated using Lowry method. 16 μg/μl was evaluated as cut-off concentration analyzed using graph pad prism 6.0.</p

SDS-PAGE showing the banding pattern of electrophoresed CICs antigens in VL-BT subjects compared to healthy, treated VL, relapse, reinfection and other diseases subjects.

<p>Briefly, 50μg of proteins was electrophoresed (10% SDS-PAGE) and stained with Coomassie Brilliant Blue R-250 or immunoblotted. <b>Fig</b> 3.a.i. SDS-PAGE comparing VL-BT subjects to healthy and treated VL cases. Molecular weight marker in lane 1; CICs antigen of VL-AT samples in lane-2 & 6,CICs antigen of VL-BT samples in lane-3 and healthy endemic and non-endemic in lane 4 and 5. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑). VL-AT (lane 2 & 6) is also showing comparatively down-regulated expression of 55kDa and 23kDaband. Fig 3.a.ii. SDS-PAGE showing the banding pattern of electrophoresed CICs antigens in VL-BT (relapse/reinfection) subjects compared to healthy and treated VL cases and SLA. Molecular weight marker in lane 1; CICs antigen of VL-BT samples in lane-2,3 (Reinfection/relapse) & 5; healthy endemic and non-endemic in lane 4 and 6; SLA in lane 7 and VL-AT in lane 8. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑). VL-AT (lane 8) and healthy endemic (lane 4) is also showing comparatively downregulated expression of 55 and 23kDaband.Fig 3.b. Immunoblotting data of SDS-PAGE (the gel of Fig 3.a.) showing immunoreactive bands in different study groups. Electrophoresed gel was transferred to NCP membrane and exposed to anti-leishmanial antibody HRP conjugated followed by substrate (DAB) exposure. Molecular weight marker in lane 1;CICs antigen of VL-BT samples in lane-2,3 and 8–10;VL-AT in lane 5; SLA in lane 7 and healthy in lane 4 and 6. 55 and 23 kDa can be recognized in all VL-BT samples (indicated by ↑). VL-AT (lane 5) is also showing comparatively downregulated expression of 55 kDa band. 23 kDa band is not recognizable in this sample. Fig 3.c. SDS-PAGE showing the banding pattern of electrophoresed CICs antigens of VL-BT subjects in comparison to healthy and treated VL cases and other diseases. Molecular weight marker in lane 1;CICs antigen of VL-AT samples in lane-2;VL-BT in lane 3; healthy endemic and non-endemic in lane 4 and 5; SLA in lane 6; Filaria in lane 7 and TB in lane 8. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑) in comparison to others. Fig 3.d. Immunoblotting data of SDS-PAGE (the gel of Fig 3.c.) showing immunoreactive bands in different study groups. Molecular weight marker in lane 1;CICs antigen of VL-AT samples in lane-2;VL-BT in lane 3; healthy in lane 4 and 5; SLA in lane 6; Filaria in lane 7, TB in lane 8, Asthma in lane 9 and viral in lane 10. VL-BT samples are expressing up-regulated 55 and 23 kDa antigen (indicated by ↑) in comparison to others except low intensity band at 55kDa band appeared in VL-AT. Fig 3.e. Histogram showing comparative band intensity of 55 and 23 kDa SDS-PAGE gel and after immunoblotting. Relative band intensity was evaluated using Quantity one software. Band intensity of SLA in stained gel and after blotting was revealed as 10.5, 7.9 and 5.4, 11 respectively, confirmed the presence of the protein of similar molecular weight in <i>L</i>. <i>donovani</i>.</p

Pie chart representing the percentage and biological functional categories of ESI-LC-MS/MS identified antigen isolated from CICs (based upon their putative functions assigned using protein function database).

<p>Hypothetical protein was abundant with maximum coverage (50%). Motor proteins are second abundant protein. Protein of phosphorylation ant protein synthesis was third most abundant protein. Proteins of cytoskeleton organization, carbohydrate metabolism, transcription, DNA replication, Cell adhesion, signal transduction and nucleosome assembly are present as minor proteins.</p

Validatory evaluation of circulating antigen.

<p>Figure showing presence of <i>L</i>. <i>donovani</i> antigen in CICs of VL-BT samples using (a) anti-<i>Ld</i> actin antibody and (b) anti-<i>Ld</i> tubulin antibody. (c) Figure showing immuno-reactivity of MS derived synthetic peptide with VL patient sera.</p

Flow diagram showing procurement and HRP labeling of anti-leishmanial primary antibody.

<p>Blood serum was isolated from parasitologically confirmed VL patient sera. Anti-leishmanial antibodies present in VL-BT was immunoprecipitated with protein A agarose. MgCl₂ was used to dissociate VL specific antibody. Eluted antibody was tagged with and HRP using HRP labeling kit to prepare Kala-azar antigen detection antibody.</p

Figure showing 2D gel electrophoresis data.

<p>40μg of proteins was subjected to IPG strips (3–10 pH, 4–7 cm, non-linear) in the first dimension followed by SDS-PAGE (12%) in the second dimension. (a). Silver stained 2D spots are visualized in the sample from VL-BT subject (A) and healthy subject (B). The number of 2D spots was more in healthy subjects whereas in VL-BT intense and specific 2D spots were observed. (b). Figure showing differences in different regions of the gel and exclusive expression of spots. (c). Figure showing immunoblot of the gel of Fig a.(A) and (B). The sample of VL-BT subject showed 100 kDa, 93.3 kDa, 87 kDa, 67.6 kDa, 63 kDa, 55 kDa, 45.7 kDa, 41.6 kDa, 37 kDa all having pI 8.5 as well as 24.5 kDa (pI 8.5), 23 kDa (pI 6.8, 6.2) immunoreactive spots. Besides, 55 kDa (pI7.7) 26.3 kDa (pI 8) and 25.7 kDa (pI 7.7) dots were upregulated in the immunoblot of the sample from VL-BT subjects in comparison to the sample from healthy subjects. (d). Figure showing comparative densitometry analysis of immunoreactive spots. Densitometry data of immunoreactive 2D spots of VL-BT and healthy subject after western immune-blotting with anti-<i>Leishmania</i> antibody isolated from patients are given in term of volume (INT*mm2), and area (mm²). Immuno-reactive 2D spots present in both VL-BT and healthy are indicated in <b>↑</b>_<b>1,</b> ↑_<b>2</b> indicates 2D spots only present in the healthy subject and 2D spots only present in VL-BT subject indicated by <b>↑</b>_<b>3.</b></p