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.

TLR2, forming heterotypic associations with TLR1 or TLR6, recognizes triacetylated or diacetylated lipopeptides, respectively [15][16][17]. Ara-LAM, a cell wall glycolipid of Mycobacterium smegmatis, has been reported to confer protection against leishmanial pathogenesis via TLR2-dependent induction of the proinflammatory responses [18]. Ara-LAM-induced activation of p38 MAPK signalling in Leishmania infected macrophages shifts their Th2 phenotype towards Th1 via chromatin modification at various proinflammatory cytokine gene loci [19]. However, it was unclear if Ara-LAM would modulate TLR2 signalling in CD8 + T-cells, which might play a potential role in the regression of leishmanial pathogenesis.
In this study, we have demonstrated that Ara-LAM drives the activation of CD8 + T-cells, which are CD25 + CD28 + IL-12R + IFN-γR + and specifically destroys the L. donovani-infected macrophages. Ara-LAM enhances T-bet expression in CD8 + T-cells via upregulation of NF-κB and p38MAPK in a TLR2-dependent pathway. T-bet ultimately enhances the expression of IFN-γ, perforin, and granzyme-B in CD8 + T-cells via histone modifications at their respective promoter regions to restore host-protective CD8 + T-cell responses.
Preparation of TLR2 and T-bet-specific siRNA TLR2 and T-bet-specific siRNA were synthesized using the Silencer siRNA Construction kit (Ambion). Scrambled siRNA was synthesized with the similar GC content. Silencing primers are listed in the Table 1.

Isolation of RNA and Reverse Transcriptase polymerase chain reaction
Total RNA from purified CD8 + T-cells were extracted using TRI reagent using standard protocol [23]. The total RNA was reverse transcribed using Revert Aid M-MuLV reverse transcriptase (Fermentas). GAPDH was used as a loading control. Sequences of the PCR primers are given in the Table 1.

Chromatin immunoprecipitation (ChIP) assay
ChIP assays were conducted using the ChIP Assay kit following the manufacturers protocol. Purified CD8 + T-cells (1×10 6 ) from the indicated mice were co-cultured with autologous L. donovani-infected macrophages (1×10 5 ) for 45 min, paraformaldehyde(1%)-fixed for 10 min at 37°C and washed with ice-cold PBS containing 1mM PMSF, harvested and lysed in SDS lysis buffer. DNA was sheared by ultrasonication using a High Intensity Ultrasonic Processor (Hielscher, Teltow, Germany) for 3 × 10s pulses at 20% amplitude. Lysates were cleared by centrifugation and diluted in ChIP dilution buffer. Lysates were pre-cleared using protein A-agarose and a sample of "input DNA" was collected at this point. Protein-DNA complexes were immunoprecipitated with 5μg of antibodies (phospho H3, acetyl H3, T-bet) overnight at 4°C. Antibody-protein-DNA complexes were then captured using protein A-agarose for 1 h at 4°C. After washing beads with low and high salt, LiCl, and TE buffers, the protein/DNA complexes were eluted in buffer (1%SDS, 0.1M NaHCO 3 ). DNA was then extracted and precipitated. PCR was conducted using promoter specific primers ( Table 1).

Preparation of cell lysate and immunoblot analysis
CD8 + T-cells were lysed using lysis buffer for isolation of protein [26]. Equal amounts of protein (30μg) were subjected to 10% SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked overnight with 3%BSA in Tris-saline buffer (pH 7.5), and immunoblotting was performed to detect MyD88, IRAK 1, NF-κB, T-bet, β-Actin, GAPDH, and phosphorylated or total p38MAPK and ERK1/2, as described previously [27].

Statistical analysis
All experiments were performed in triplicate and a minimum of 4 mice was used per group. The data, represented as mean values ± SD, are from one experiment that was performed at least 3 times. One-way ANOVA test (using a statistical package, Instat3) was employed to assess the significance of the differences. p0.001 was considered significant.

Ara-LAM upregulates IL-12R and IFN-γR in CD8 + T-Cells in L. donovani infection
We studied the effect of Ara-LAM on BALB/c mice-derived CD8 + T-cells in indicated groups. Naïve CD8 + T cells proliferate in response to TCR and CD28 signals, but reqiure IFN-γ and IL-12 to develop effector functions [29][30]. We investigated the status of CD28 on CD8 + T cells expressing CD25, receptor for IL-12 (IL-12R) and IFN-γ (IFN-γR) [31][32]. 28 days after infection, compared to the splenic CD8 + T cells of untreated infected mice, Ara-LAM strongly induced the expression of IL-12R and a moderate induction of IFN-γR on splenic CD8 + T cells, co-expresseing CD25 (Fig 1A). Activation of TLR2 in CD8 + T-cells is associated with their enhanced effecter functions [18][19]. Therefore, we tested whether Ara-LAM, being a TLR2 ligand, could activate the CD8 + T-cells by upregulating the transcription of perforin and granzyme-B. We observed a significant enhancement in both perforin and granzyme-B expression in CD8 + T-cells isolated from Ara-LAM treated L. donovani infected mice compared to that of untreated infected mice (Fig 1B).

Ara-LAM-induced CD8 + T-cells activation in L. donovani infection is TLR2-dependent
We examined the effect of Ara-LAM treatment on TLR2 surface expression in CD8 + T-cells from different groups of BALB/c mice. Ara-LAM treatment significantly augmented the expression of TLR2 in splenic CD8 + T-cells on 14 and 28days post infection (Fig 2A). Because we observed significantly enhanced expressions of IFN-γ, perforin and granzyme-B in CD8 + T-cells isolated from Ara-LAM treated L. donovani infected mice compared to that of untreated infected mice (Fig 2A), we tested if TLR2 silencing could abrogate these effector functions. TLR2 silencing abrogated the Ara-LAM induced generation of IFN-γ, perforin, granzyme-B molecules in CD8 + T-cells isolated from the infected mice (Fig 2A and 2B). It has been noted earlier that Leishmania infection of the susceptible host results in apoptosis of T-cells, leading to impairment of cell-mediated immunity [33]. Therefore, we investigated whether Ara-LAM could restore the impaired CD8 + T-cell proliferation in Leishmaniainfected mice. Ara-LAM treatment resulted in significant enhancement of splenic CD8 + T-cell proliferation compared to the splenic CD8 + T-cells from untreated infected mice. The Ara-LAM-induced CD8 + T-cell proliferation was significantly attenuated during TLR2 silencing condition (Fig 2C). These results suggested that Ara-LAM stimulation leads to the enhanced effector function as well as the proliferation of CD8 + T-cells via TLR2 dependent pathway.
Ara-LAM triggers T-bet recruitment and histone modification at the IFNγ, Perforin and Granzyme-B promoter regions in CD8 + T-cells The expressions of IFN-γ, perforin and granzyme-B in CD8 + T cells is principally regulated at the level of transcription, which, in turn, is strictly dependent upon the favourable histone modifications at their respective promoter regions [19]. Therefore, we performed ChIP assays to investigate whether the Ara-LAM-mediated enhancement of IFN-γ, perforin and granzyme-B expression in CD8 + T-cells was due to chromatin modification at their respective promoter regions. We observed a significantly higher level of phosphorylated ( Fig 3A) and acetylated (Fig 3B) histones at the promoter regions of IFN-γ, perforin, granzyme-B in the splenic CD8 + T-cells of Ara-LAM treated L. donovani infected BALB/c mice relative to the splenic CD8 + Tcell from untreated infected mice. These Ara-LAM mediated histone modifications at the IFNγ, perforin and granzyme-B promoter regions in CD8 + T-cells were significantly attenuated in TLR2 silenced condition (Fig 3A and 3B). Therefore, Ara-LAM induced transcription favourable histone modifications at the IFN-γ, perforin, and granzyme-B loci of CD8 + T-cells in a TLR2 dependent pathway.
The effector functions of CD8 + T-cells are predominantly regulated by the transcription factor T-bet [34]. Therefore, we investigated the accumulaion of T-bet in the splenic CD8 + T-cells of different experimental mice groups. Immunoblot analysis showed that accmulation of T-bet was significantly higher in the splenic CD8 + T-cells of Ara-LAM treated L. donovani infected mice compared to that of the untreated infected mice (Fig 3C).
T-bet binding at the promoter regions of IFN-γ, perforin, granzyme-B is a crucial event for the optimal induction of these effectors molecules in CD8 + T-cells [13]. Therefore, we performed ChIP-on-ChIP assay to investigate the interaction of T-bet with the promoter regions of IFN-γ, perforin, and granzyme-B in CD8 + T-cells from Ara-LAM treated and untreated L. donovani infected BALB/c mice. Ara-LAM treatment induced a strong association of T-bet with the IFN-γ, perforin, and granzyme-B promoter regions in CD8 + T-cells compared with that of the untreated L. donovani infected mice; TLR2 silencing decreased Ara-LAM induced T-bet recruitment to the promoter regions of IFN-γ, perforin, granzyme-B in CD8 + T-cells ( Fig 3D). Collectively, Ara-LAM-induced TLR2 dependent activation of T-bet augmented the transcription of target effector genes in CD8 + T-cells of infected mice.

Ara-LAM promotes NF-κB activation and MAPK signaling in CD8 + Tcells
of TLR2 downstream signaling [18]. Therefore, we carried out co-immunoprecipitation studies to investigate the TLR2-MyD88 interaction in Ara-LAM-treated CD8 + T-cell. We observed a strong association between TLR2 and MyD88 in Ara-LAM-treated CD8 + T-cell compared with that of the untreated CD8 + T-cells. IRAK-1, crucial for activation of TLR2 downstream signaling [18], was found to be intricately associated with MyD88 in Ara-LAM-treated CD8 + T-cells compared to the untreated CD8 + T-cells (Fig 4A). Furthermore, nuclear translocation of NF-κB were significantly augmented in Ara-LAM treated CD8 + T-cell compared to the CD8 + T cells from differently treated mice groups were co-cultured with autologous infected macrophages for 45 min and chromatin immunoprecipitation (ChIP) assays were conducted. Immunoprecipitations were performed using Abs specific to phosphorylated H3 histone (IP: phospho-H3), acetylated H3 histone (IP: acetyl-H3) and conventional RT PCR was performed using primers specific to the IFN-γ, perforin and granzyme-B promoter region. (C) CD8 + T-cells were co-cultured with infected macrophages, lysed and the nuclear protein extracts were analyzed for the activation of T-bet by Western blot. (D) CD8 + T-cells from differently treated mice group were co-cultured with autologous L. donovani-infected macrophages, Immunoprecipitations were conducted using T-bet (IP: T-bet) specific Abs. Conventional RT-PCR was performed for amplifying the putative T-bet binding sites of the IFN-γ, perforin, granzyme-B promoter. Data represented were one of the three indepenedent experiments with similar results performed in the same way. untreated CD8 + T-cells. Ara-LAM induced TLR2 downstream signaling mediated NF-κB translocation was completely abrogated under TLR2 silenced condition (Fig 4B).
In addition to the NF-κB activation, TLR2 signaling can also modulate p38 and ERK-1/2 MAPK signaling cascades [19]. Therefore, we investigated whether Ara-LAM treatment could induce MAPK phosphorylation in CD8 + T-cells. Ara-LAM treatment resulted in significantly higher level of p38MAPK phosphorylation (Fig 4C), however; it failed to induce significant ERK1/2 phosphorylation in CD8 + T-cells (Fig 4C). TLR2 silencing abrogated the Ara-LAM induced p38 phosphorylation (Fig 4D). Taken together, Ara-LAM activated p38MAPK and NF-κB activation in infected CD8 + T-cells. Purified CD8 + T-cells (1×10 6 /mL) were stimulated with plate-bound anti-CD3ε mAbs (5μg/mL) and CD28 (1μg/mL) for 24 hrs and transfected with control siRNA or TLR2 siRNA, followed by Ara-LAM (3μg/mL) treatment for 24 hr. The cells were then lysed and subjected to immunoprecipitation with anti-TLR2 antibody, and the blots were probed with anti-MyD88 antibody. (B) Cells were lysed and immunoprecipitated with anti-MyD88 antibody; the blots were probed with anti-IRAK1 antibody. Cytosolic and nuclear protein extracts were analyzed for nuclear translocation of NF-κB. (C-D) In yet separate experiments, CD8 + T cells were treated by Ara-LAM for 5, 15, 30, and 60 min, and lysed. The lysate was subjected to Western blot analysis for the expression of p38MAPK, phospho-p38MAPK and ERK1/2, phospho-ERK1/2. Data represented were one of the three indepenedent experiments with similar results performed in the same way.

Ara-LAM enhances T-bet expression in CD8 + T-cells via upregulation of NF-κB and p38MAPK signaling
Since, Ara-LAM induced efficient activation of T-bet in CD8 + T-cells (Fig 3C) and enhanced the activation of NF-κB and p38MAPK in CD8 + T-cell (Fig 4), we hypothesized that T-bet activation in CD8 + T-cells might be regulated either by the NF-κB or MAPK signalling or by both of these signalling molecules. Ara-LAM mediated activation as well as mRNA expression of Tbet was attenuated in the presence of SB203580 and SN50, the pharmacological inhibitors of p38MAPK and NF-κB, respectively (Fig 5A and 5B), suggesting that the activation of T-bet in Ara-LAM treated CD8 + T-cells was regulated by TLR2 triggered NF-κB and p38MAPK signalling cascade. Besides, the enhanced IFN-γ, perforin and granzyme-B expression in Ara-LAM stimulated CD8 + T-cells were significantly decreased during T-bet silencing, as well as, NF-κB and the p38MAPK inhibition ( Fig 5B). Therefore, the upregulation of IFN-γ, perforin and granzyme-B expression in Ara-LAM stimulated CD8 + T-cells were due to the NF-κB and the p38MAPK dependent activation of T-bet.

Ara-LAM reduces hepatic and splenic parasitic burden in BALB/c mice
We have observed that Ara-LAM activates the CD8 + T cells via upregulation of IFN-γ and the cytotoxic molecules perforin and granzyme-B. These activated T cells have the ability to kill the infected macrophages to reduce the parasite burden [5][6]. In accordance with this phenomenon, we observed a significant reduction of the hepatic as well as splenic parasite burden in the Ara-LAM treated infected mice groups compared to the untreated infected mice groups (Fig 6) on 14 and 28days postinfection. However, Ara-LAM mediated clearance of parasites was significantly attenuated in TLR2 silenced condition. Collectively, the data clearly suggest that Ara-LAM plays a very important role to clear the L. donovani infection via a TLR2 dependent mechanism. We checked the effect of Ara-LAM on IFN-γ and IL-10 producing-CD8 + T-cells in both spleen and liver by flow cytometry. In both uninfected and infected mice, Ara-LAM up-regulated the IFN-γ secreting CD8 + T-cells in spleen as well as liver. IL-10 producing CD8 + T-cells were suppressed by Ara-LAM pretreatment in both spleen and liver of infected as well as uninfected mice. Although in case of hepatic CD8 + T-cells, the effect of Ara-LAM on both IFN-γ and IL-10 secretion was not very significant (Fig 7).

Discussion
Leishmanial pathogenesis is associated with abrogated pro-inflammatory responses, resulting in immune suppression of the host [9][10][11]. The persistence of the infection during VL was due to the impaired cell-mediated immunity which in turn was intricately associated with the TLR2 Mediated CD8 + T-Cell Activation severe dysfunction of cytotoxic CD8 + T-cells [7]. Therefore, activation of CD8 + T cells is very much important to eradicate L. donovani mediated infection. Recently, it has been reported that activation of TLR signalling could restore the impaired effector function of CD8 + T-cells in various models of infectious diseases [35]. Therefore, we intended to study whether Ara-LAM, so far known to be a TLR2 ligand [18], could restore the functional capacity of the effector CD8 + T-cells during VL.
We confirmed that Ara-LAM pre-treatment in L. donovani infected BALB/c mice significantly augmented the expression of TLR2 along with the concomitant increase in IFN-γ, perforin, and granzyme-B in the splenic CD8 + T-cells (Figs 1 and 2). Because knowing phenotype is important for its functional significance, we examined the phenotype of these T cells, which are IL-12R + IFN-γ + CD28 + co-expressing CD25 (Fig 1A). Moreover, Ara-LAM pre-treatment was found to be associated with significant enhancement of CD8 + T-cell proliferation (Fig 2C), a prerequisite for its effector function. TLR2 silencing significantly attenuated such Ara-LAM mediated enhanced expression of these effector molecules in CD8 + T-cells (Fig 2).
The optimal transcriptional induction of the IFN-γ, perforin, granzyme-B genes in CD8 + Tcells requires histone modification at their respective promoter regions [19]. Our study revealed that Ara-LAM pretreatment led to transcription favourable histone H3 phosphorylation and acetylation specifically at the promoter region of IFN-γ, perforin, granzyme-B in the splenic CD8 + T-cells of L. donovani-infected BALB/c mice (Fig 3A and 3B). In line with the fact that the activation of the transcription factor T-bet is a crucial event for the expression of IFN-γ, perforin, granzyme-B in CD8 + T-cells [13,34], we observed an increased T-bet accumulaion ( Fig 3C) along with enhanced T-bet binding to the promoter regions of IFN-γ, perforin, granzyme-B in the Ara-LAM treated splenic CD8 + T-cells of L. donovani infected BALB/c mice ( Fig 3D). Hence, our study revealed that Ara-LAM induces T-bet dependent activation of effector molecules in CD8 + T-cells to hinder leishmanial pathogenesis. Further, we intended to investigate the underlying molecular mechanism of Ara-LAM induced CD8 + T-cells activation during VL. Since, Ara-LAM confers its protective functions against VL via activation of the TLR2 downstream signaling in host macrophages [18][19], we investigated Ara-LAM mediated modulation of TLR2 signaling in CD8 + T-cells. Ara-LAM treatment led to successful initiation of TLR2 signalling in naive CD8 + T-cells via the TLR2-MyD88 association (Fig 4A), resulting in the selective activation of intermediate signalling molecules, IRAK1 and ultimately leading to nuclear translocation of NF-κB (Fig 4B). Ara-LAM treatment also led to increased p38MAPK phosphorylation along with concomitant attenuation of ERK1/2 phosphorylation in splenic CD8 + T-cells (Fig 4C and 4D). These observations indicate that Ara-LAM induced activation of TLR2 downstream signalling molecules leads to enhanced effector function of CD8 + T-cells during VL.
We observed that inhibition of NF-κB and p38MAPK, by their respective pharmacological inhibitors, significantly abrogated the Ara-LAM-induced T-bet expression and activation in CD8 + T-cells (Fig 5A and 5B). Our results indicated the active involvement of NF-κB and p38MAPK signalling in the regulation of Ara-LAM driven T-bet activation. Further, Ara-LAM treatment significantly reduced the parasite burden in the liver and spleen 14 and 28 days after the infection (Fig 6) accompanied with the expansion of IFN-γ + CD8 + T-cells (Fig 7). It is therefore feasible to conclude that Ara-LAM mediated reduced parasitic burden may be due to the CD8 + T-cell driven killing of the infected macrophages. It is noteworthy to mention that Ara-LAM works well when utilized as an immunotherapiutic agent administered 2days prior to L. donovani infection [18][19][20]28].
In summary, Ara-LAM confers significant protection through activation of CD8 + T-cells in L. donovani infected BALB/c mice. The novelty of our work lies in the fact that Ara-LAMinduced TLR2 signaling leads to the activation of the transcription factor T-bet which plays a pivotal role in restoring the effector functions of CD8 + T-cells in L. donovani infected susceptible host. Thus, we are one the way to devise a strategy in near future so that Ara-LAM can be used as a suitable vaccine during L. donovani infection.