TLR4 and NKT Cell Synergy in Immunotherapy against Visceral Leishmaniasis

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal β-(1–4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1–4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic

Complete Soluble Antigen from a UDP-Galactose:N-Acetylglucosamine β 1-4 Galactosyltransferase expressing Leishmania donovani promastigote induces complete protection in an experimental model of visceral leishmaniasis’

This disquisition is an attempt to formulate an effective immunoprophylactic and immunotherapeutic agent against protozoan Leishmania donovani, the causative pathogen for fatal visceral leishmaniasis (VL), commonly known as Kala-azar in India. Vaccination against this genetically diverse protozoan should be the most preferred approach for combating this disease, especially since chemotherapy is expensive, difficult to administer and is often ineffective due to the emerging drug resistance strains. Leishmania vaccine development has proven to be a difficult and challenging task, which is mostly hampered by inadequate knowledge of parasite pathogenesis and the complexity of immune responses needed for protection. At present most of the vaccines currently in market against tuberculosis, BCG, polio, measles and mumps are in the form of live attenuated vaccines. Though, several vaccination strategies against experimental leishmaniasis have been attempted, a successful vaccine against the disease has been elusive. Moreover, compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis has received limited attention. Our constant endeavor in search of an antileishmanial vaccine lead us to identify an UDP-Galactose:N-acetylglucosamine β 1–4 galactosyltransferase (GalT) expressing attenuated Leishmania parasite which can be used as live vaccine against visceral leishmaniasis. The live, attenuated vaccines are known to stimulate helper T-cell response and antibody immunity. The major problem of attenuation reversal could not be detected in these genetically defined stable clonal leishmanial parasites under long period of in vitro culture, making them risk-free

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28�C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42�C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-a-p-tosyl-Larginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca2? and Mn2?, but activated by Zn2?. The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar

TLR4 and MyD88 are required for GSPL induced anti-leishmanial response.

<p>(A) Splenic adherent cells were transfected with siRNAs specific to TLR2, 4 or MyD88 (specific). A control group was transfected with control siRNA (control). Twenty-four hours after transfection, cells were recovered and their TLR2, 4 and MyD88 levels assessed in Western blots. GAPDH in total proteins was used as loading controls. Blots are representative of three separate experiments. (B) Splenic adherent cells transfected with TLR2, TLR4, MyD88 or control siRNA (con), were infected with <i>LD</i> (APC/parasite 1∶20) for 12 h. Non-ingested promastigotes were removed by washing, and adherent cells were cultured for another 36 h. Infected APCs were then treated with GSPL (100 µg/mL) for 24 h. Intracellular parasite number was determined by Giemsa staining. Each experiment was conducted in triplicate and repeated at least three times each and one set of representative data is shown. Error bars represent mean ± SD. * p<0.0001; paired two-tailed Student's t-test. (C,D) Antileishmanial effect of GSPL on parasite growth in TLR4 deficient C3H/HeJ mice. Sixty days <i>LD</i> infected C3H/HeJ mice were given two subcutaneous injections of GSPL (100 µg each) at 15 days apart. The parasite burdens in liver (C), spleen (C), and bone marrow (D) of individual animals were then determined at 15 days after the last treatment. The results are representative of three independent experiments and data shown are means ± SD; n=5. *p<0.0001 versus corresponding infected control; paired two-tailed Student's t-test. (E,F) <i>In vivo</i> parasite load in liver, spleen and bone marrow of <i>LD</i> infected BALB/c mice treated with LPS. Sixty days <i>LD</i> infected BALB/c mice were given three intraperitoneal injections of LPS on alternate days (5 µg/injection or 10 µg/injection). The parasite burdens in liver (E), spleen (E), and bone marrow (F) of individual animals were then determined at days 1,3 and 12 after the last treatment. The results are representative of three independent experiments and data shown are means ± SD; n=3. *p<0.0001 versus corresponding infected control; paired two-tailed Student's t-test.</p

Induction of iNKT cell mediated IL-12 production by splenic adherent cells.

<p>Sixty days <i>LD</i> infected animals were treated with GSPL as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002646#ppat-1002646-g001" target="_blank">Figure 1</a>. (A) Animals were sacrificed 15 days after the last treatment and iNKT cells isolated from individual experimental animals (WT BALB/c and C3H/HeJ mice) were mixed with 1∶10 of autologous splenic adherent cells (1×10⁶ adherent cells) and were cultured in 24-well culture plates containing 100 µg/mL GSPL for 24 h. Culture supernatants were assayed for the concentration of IL-12p70 and IL-12p40 in ELISA. The results are representative of three independent experiments and data shown are means ± SD; *p<0.0001 versus corresponding infected control; Student's t-test. (B) mRNA expression of IL-12p40, IL-12p35, and IL-23p19 in the spleen of each GSPL treated <i>LD</i> infected animals was evaluated individually by real-time PCR. The fold up-regulation of mRNA post-GSPL treatment was calculated by normalizing the amount of cytokine mRNA with the housekeeping gene GAPDH, and comparing results from treated to infected; *p<0.0001; paired two-tailed Student's t-test. (means ± SD (n=3) of one from three independent experiments are shown).</p

GSPL mediated cure requires the dual activation of the IFN-γ and IL-17 signaling pathways.

<p>(A) Sixty days <i>LD</i>-infected BALB/c mice were treated with anti IL-17A Ab or isotype control Ab on days −1, 0, and +1 of GSPL treatment. The parasite burdens in (A) liver, spleen, and (C) bone marrow was then determined at 15 days after the last treatment. (B) Sixty days <i>LD</i>-infected WT C57BL/6 (WT) and IFN-γ KO mice were treated with GSPL and the parasite burdens in liver, spleen, and (D) bone marrow was then determined at 15 days after the last treatment. Experiments were repeated three times with three mice per group. The data are presented from individual mice of all three groups. *p<0.0001 versus corresponding infected control; paired two-tailed Student's t-test. T, GSPL treated; I, <i>LD</i> infected.</p

Kinetics of IL-17A production by iNKT cells of cured mice.

<p>Sixty days <i>LD</i> infected animals were treated with GSPL as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002646#ppat-1002646-g001" target="_blank">Figure 1</a>. Animals were sacrificed 15 days after the last treatment. NKT cells from spleens of individual experimental BALB/c (A) mice were identified as α-GC/CD1d tetramer⁺TCRβ⁺ cells and after enrichment by magnetic cell sorting (B) were further purified by FACS sorting (C). The NKT⁻ populations were identified as TCRβ⁺CD4⁺ cells (D). Isolated (E) iNKT cells or (F) iNKT depleted cell populations (R5, TCR-β⁺CD4⁺) were co-cultured with 1∶10 of autologous splenic adherent cells as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002646#ppat-1002646-g004" target="_blank">Figure 4</a>. Cells were stimulated with GSPL at 100 µg/mL, ± IL-6 Ab (E) or ± IL-6R Ab (F) for the time periods indicated and intracellular IL-17A was assessed using FACS analysis. Numbers above the horizontal bar indicates the mean fluorescence intensity and number below the bar indicates the percentage of IL-17 positive cells in the gated populations. Results show mean ± SD of three individual mice per group; paired two-tailed Student's t-test. Results of one from three independent experiments are shown.</p