Toll-Like Receptor 2 Targeted Rectification of Impaired CD8⁺ T Cell Functions in Experimental Leishmania donovani Infection Reinstates Host Protection.

Leishmania donovani, a protozoan parasite, causes the disease visceral leishmanisis (VL), characterized by inappropriate CD8+ T-cell activation. Therefore, we examined whether the Toll-like Receptor 2 (TLR2) ligand Ara-LAM, a cell wall glycolipid from non-pathogenic Mycobacterium smegmatis, would restore CD8+ T-cell function during VL. We observed that by efficient upregulation of TLR2 signaling-mediated NF-κB translocation and MAPK signaling in CD8+ T-cells (CD25+CD28+IL-12R+IFN-γR+), Ara-LAM triggered signaling resulted in the activation of T-bet, which in turn, induced transcription favourable histone modification at the IFN-γ, perforin, granzyme-B promoter regions in CD8+ T-cells. Thus, we conclude that Ara-LAM induced efficient activation of effector CD8+ T-cells by upregulating the expression of IFN-γ, perforin and granzyme-B in an NF-κB and MAPK induced T-bet dependent manner in VL

Effect of Ara-LAM and IFN-γ signaling against leishmanial pathogenesis in BALB/c mice.

<p>(A) Mice were injected with respective shRNAs (for 2 days) followed by treatment with either phosphate-buffered saline (PBS) (control) or Ara-LAM (30 μg intraperitoneally) for 2 days, after which mice were infected. After 28 days mice were sacrificed, splenocytes (2x10⁶) was collected in Trizol for mRNA extraction and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117247#sec002" target="_blank">Methods</a>). Data are from one of three representative experiment. (B-C) A separate set of splenocytes (2x10⁵cells/well) was stimulated with soluble leishmanial antigen (SLA) at 5 μg/ml for 48 h. Release of Interferon-γ (IFN-γ) and nitric oxide in culture supernatants were determined by enzyme-linked immunosorbent assay and the Nitric Oxide Colorimetric Assay kit, respectively. Data represent means ± SD for four animals per group. ***<i>P</i> <.001 and **<i>P</i> <. 01 for the comparison with infected mice. (D-E) Differently treated infected mice were sacrificed 28 days after infection. Levels of parasite burden in liver and spleen are expressed in Leishman-Donovan units (LDUs). Data represent means ± SD for 4 animals per group. ***<i>P</i> <.001 and **<i>P</i> < .01 for the comparison with infected mice. (F) Proliferative responses to soluble leishmanial antigen (SLA) (5 μg/ml) of splenocytes from above mentioned group of mice were examined by measuring [³H]thymidine incorporation. At 5 μg/ml SLA, optimal proliferation was obtained. Data represent means ± SD for 4 animals per group. ***<i>P</i> <.001 and **<i>P</i> < .01 for the comparison with infected mice.</p

Moderation of IFN-γ induced JAK-STAT signaling by Ara-LAM in <i>Leishmania</i> infected macrophages.

<p>(A) BALB/c derived peritoneal macrophages were pre-treated with Ara-LAM for 3 hrs, followed by <i>L</i>. <i>donovani</i> infection for 24hrs. Macrophages were treated with rIFN-γ (20ng/ml) for 45min, followed by cell lysate preparation and Western blot for JAK1, p-JAK1, JAK2, p-JAK2, STAT-1 and p-STAT-1. The blots shown are representative of triplicate experiments. (B) Both the nuclear extracts and the cytosolic extracts of differently treated peritoneal macrophages were prepared followed by Western blot to analyze the nuclear translocation of p-STAT-1 in <i>L</i>. <i>donovani</i> infected macrophages. Blots shown here are from one of three representative experiments.</p

Ara-LAM induced the MHC-II expression in the membrane of <i>L</i>. <i>donovani</i> infected cells and helped in parasite killing.

<p>(A) Peritoneal macrophages (2x10⁶cells) isolated from BALB/c mice were pre-treated with Ara-LAM (3μg/ml) for 3hrs, followed by <i>Leishmania donovani</i> challenge for 24hrs. Both rIFN-γ (20ng/ml) stimulated (for 24hrs) and non-stimulated cells were then stained with anti-MHCII-FITC antibody and analyzed by Flow cytometry for MHC-II. Data are from one of three representative experiments. (B) In a similar set of experiment, the macrophages were cultured in cover slips, treated with Ara-LAM for 3 hrs followed by <i>Leishmania</i> infection and rIFN-γ stimulation (20ng/ml). After 24hrs of incubation intracellular parasite number were assessed as described in methods. Data represent means ± SD for three sets of experiments. ***<i>P</i> <.001 for the comparison with infected macrophages.</p

Induction of IFN-γ and IL-10 secreting CD4⁺ T cells by Ara-LAM in <i>Leishmania</i>-infected mice.

<p>Twenty eight days after infection, different groups of mice were sacrificed, splenocytes and hepatocytes were isolated and stimulated with soluble leishmanial antigen (SLA) for 48hrs. Before harvesting, cells were incubated with brefeldin A (10 mg/mL) for 4 hrs. CD4⁺ T cells were purified by Magnetic Associated Cell Sorter (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117247#sec002" target="_blank">Materials and Methods</a>), permeabilized (0.1% saponin) and stained with anti-mouse IFN-γ-FITC (A) and anti-mouse IL-10-PE (B) antibodies and were analyzed by flow cytometry. Data are from one of three experiments conducted in the same way with similar results.</p

The Host-Protective Effect of Arabinosylated Lipoarabinomannan against <i>Leishmania donovani</i> Infection Is Associated with Restoration of IFN-γ Responsiveness

<div><p>Visceral leishmaniasis (VL), which is endemic as a major infectious disease in the tropical and subtropical countries, is caused by a protozoan parasite <i>Leishmania donovani</i>. At present, restricted treatment options and lack of vaccines intensify the problem of controlling VL. Therefore, finding a novel immunoprophylactic or therapeutic principle is a pressing need. Here, we report that arabinosylated lipoarabinomannan (Ara-LAM), a TLR2-ligand isolated from <i>Mycobacterium smegmatis</i>, exhibits a strong immunomodulatory property that conferred protection against <i>L. donovani</i> infection. Although, Ara-LAM modulates TLR2 and MAPK signaling, it is not known whether Ara-LAM involves IFN-γ signaling for effective parasite clearance. Because, it is reported that IFN-γ signaling, a principle mediator of NO generation and macrophage and Tcell activation, is hampered during leishmanial pathogenesis. Ara-LAM increases IFN-γ receptor expression and potentiates IFN-γ receptor signaling through JAK-STAT pathway. Moreover, Ara-LAM reciprocally modulates IRF4 and IRF8 expression and reinstates anti-leishmanial Th1 response that eventuates in significantly reduced parasite load in spleen and liver of <i>L. donovani</i>-infected BALB/c mice. IFN-γRα silencing resulted in the suppression of these host-protective mechanisms affected by Ara-LAM. Thus, Ara-LAM-mediated restoration of IFN-γ responsiveness is a novel immuno-modulatory principle for protection against <i>L. donovani</i> susceptible host.</p></div

Ara-LAM reciprocally regulated IRF4 and IRF8 expression and corresponding immune response during <i>L</i>. <i>donovani</i> infection both in vitro and in vivo.

<p>(A) <i>L</i>. <i>donovani</i> infected and uninfected control peritoneal macrophages were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. Data represent means ± SD for three experiments. <b>Inset 1</b>: Comparison between IRF4 and IRF8 expression in <i>L</i>. <i>donovani</i> infected and uninfected control macrophages. (B) Peritoneal macrophages (2x10⁶cells) from BALB/c mice were cultured and subjected to Ara-LAM pre-treatment (3μg/ml) for 3hrs, followed by <i>L</i>. <i>donovani</i> challenge for 6hrs. Both rIFN-γ stimulated (20ng/ml, 45mins) and unstimulated cells were collected in Trizol for RNA extraction and semi-quantitative RT-PCR was performed. Data are from one representative experiment, which was performed at least thrice. <b>Inset 2:</b> Reciprocal expression of IRF4 and IRF8 by Ara-LAM in <i>L</i>. <i>donovani</i> infected macrophages. (C-D) BALB/c derived peritoneal macrophages (2x10⁶cells) were treated with Ara-LAM (3μg/ml) for 3hrs, followed by <i>L</i>. <i>donovani</i> challenge for 6hrs. After 45 min of rIFN-γ stimulation immuno-precipitations were conducted using IRF8 (IP:IRF8) and IRF4 (IP: IRF4) specific Abs. Semi quantitative RT-PCR was performed for amplifying the putative IRF8 binding sites of the IL-12p40 promoter and IRF4 binding sites of the IL-10 promoter. Data represent means ± SD for three sets of experiments. <b>Inset 3:</b> Ara-LAM mediated up-regulation of IRF8 binding to the IL-12 promoter and down-regulation of IRF4 binding to the IL-10 promoter in infected macrophages. (E-F) BALB/c mice were injected with respective shRNAs (for 2 days) followed by treatment with either phosphate-buffered saline (PBS) (control) or Ara-LAM (30 μg intraperitoneally) for 2 days, after which mice were infected. 28 days later, mice were sacrificed and splenocytes (2x10⁶) were collected in Trizol for mRNA extraction and semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117247#sec002" target="_blank">Methods</a>). Data are from one of three representative experiments. <b>Inset 4:</b> Correlations (R-values) between IRF4 and IL-10 or IL-12 expression in Ara-LAM treated and untreated infected splenocytes. <b>Inset 5:</b> Correlations (R-values) between IRF8 and IL-10 or IL-12 expression in same set of mice. (G-H) A separate set of splenocytes (2x10⁶) were stimulated with soluble leishmanial antigen (SLA) at 5 μg/ml for 48 h. Release of Interleukin 12 (IL-12) p70 and Interleukin 10 (IL-10) culture supernatants were determined by enzyme-linked immunosorbent assay. Data represent means ± SD for 4 animals per group. ***<i>P</i> <.001 and **<i>P</i> < .01 for the comparison with infected mice.</p

Ara-LAM promptly regulates effector functions in CD8⁺ T-cells through NF-κB and p38MAPK mediated T-bet signalling.

<p>(A) CD8⁺ T-cells were isolated by MACS from the spleen BALB/c mice. Purified CD8⁺ T cells were stimulated as described previously and allowed to transfect with control siRNA or T-bet siRNA, or treated with SB203580 (SB) (5μg/ml), or SN50 (SN) (20μg/ml), subsequently followed by Ara-LAM (3μg/mL) treatment for 24 hr. The cells were then lysed and nuclear protein extracts were prepared, followed by subjected to Western blot with anti-T-bet. (B) The blot shown is representative of triplicate experiments that yield similar type of results. In a separate set of experiments, after the treatment schedule, the cells were collected in Trizol for RNA extraction, and conventional RT PCR analysis was performed to determine the expression of T-bet, IFN-γ, perforin, granzyme-B. Data represented were one of the three indepenedent experiments with similar results performed in the same way.</p

Ara-LAM facilitates TLR2 dependent activation and expansion of CD8⁺ T-cells in <i>Leishmania donovani</i> infected BALB/c mice.

<p>(A) Purified CD8⁺ T-cells were subjected to FACS analysis for TLR2 expression. Separately, purified CD8⁺ T-cells from differently treated mice were co-cultured with autologous infected macrophages (10:1) for 48hrs and IFN-γ, perforin, granzyme-B expression were determined by intracellular FACS. (B) CD8⁺ T-cells from differently treated mice groups were stimulated as described previously and conventional RT PCR was done after RNA extraction. (C) Purified CD8⁺ T-cells from differently treated mice and autologous <i>L</i>. <i>donovani</i>–infected macrophages were co-cultured for 72 hours. Proliferation was determined by an 18 h [³H] thymidine incorporation assay. Data were presented as count/million (×10³). Results were mean value ±SD. from triplicate wells. The asterisk indicated a statistically significant induction (**<i>P</i><0.001) of T-cell proliferation, compared with that in infected mice.</p