Interdependencies between Toll-like receptors in Leishmania infection

Abstract

Multiple pathogen-associated molecular patterns (PAMPs) on a pathogen's surface imply their simultaneous recognition by the host cell membrane-located multiple PAMP-specific Toll-like receptors (TLRs). The TLRs on endosomes recognize internalized pathogen-derived nucleic acids and trigger anti-pathogen immune responses aimed at eliminating the intracellular pathogen. Whether the TLRs influence each other's expression and effector responses-termed TLR interdependency-remains unknown. Herein, we first probed the existence of TLR interdependencies and next determined how targeting TLR interdependencies might determine the outcome of Leishmania infection. We observed that TLRs selectively altered expression of their own and of other TLRs revealing novel TLR interdependencies. Leishmania major-an intra-macrophage parasite inflicting the disease cutaneous leishmaniasis in 88 countries-altered this TLR interdependency unfolding a unique immune evasion mechanism. We targeted this TLR interdependency by selective silencing of rationally chosen TLRs and by stimulation with selective TLR ligands working out a novel phase-specific treatment regimen. Targeting the TLR interdependency elicited a host-protective anti-leishmanial immune response and reduced parasite burden. To test whether this observation could be used as a scientific rationale for treating a potentially fatal L. donovani infection, which causes visceral leishmaniasis, we targeted the inter-TLR dependency adopting the same treatment regimen. We observed reduced splenic Leishman-Donovan units accompanied by host-protective immune response in susceptible BALB/c mice. The TLR interdependency optimizes TLR-induced immune response by a novel immunoregulatory framework and scientifically rationalizes targeting TLRs in tandem and in sequence for redirecting immune responses against an intracellular pathogen.

Keywords: Leishmania major; Toll-like receptors; cytokines; macrophages.

FIGURE 1

Thioglycolate‐elicited BALB/c‐derived macrophages were infected with L. major promastigotes at a ratio of 1:10 for 6 h followed by washing of the extracellular parasites. After incubation for further 63, 60 or 54 h, cells were treated with TLR1/2 ligand (Pam₃CSK₄—10, 50 and 100 ng) or left untreated till 72 h of total incubation. Cells were lysed, and subjected to RNA isolation and cDNA preparation for qPCR analyses. GAPDH was used as internal control to normalize the value of gene of interest. Data shown represent the relative fold change of TLRs by using ΔΔC_T method, compared with untreated controls. Filled and open circles show the expression of TLRs in uninfected and infected macrophages, respectively. Experiments were performed three times, and the data are shown as mean ± SE. *P < 0·05, **P < 0·01 and ***P < 0·001

FIGURE 2

BALB/c‐derived peritoneal macrophages were infected with L. major promastigotes at a ratio of 1:10 for 6 h followed by washing of the extracellular parasites. After incubation for further 63, 60 and 54 h, cells were treated with different doses of TLR2 ligand PGN (1 μg, 5 μg and 10 μg) or left untreated for indicated time duration. RNA was isolated for the synthesis of cDNA. Real‐time PCR was performed from cDNA templates for the amplification of TLRs. GAPDH was used as a reference control to normalize the value of gene of interest. Data shown represent the relative fold change of TLRs by using ΔΔCT method, compared with untreated controls. Filled and open circles show the expression of TLRs in uninfected and infected macrophages, respectively. Experiments were performed three times, and the data are shown as mean ± SE. *P < 0·05, **P < 0·01 and ***P < 0·001

FIGURE 3

The elicited BALB/c‐derived macrophages were infected with L. major promastigotes at a ratio of 1:10 for 6 h followed by washing of the extracellular parasites. After incubation for further 63, 60 and 54 h, cells were treated with different doses of TLR2/6 ligand (FSL—10, 50 and 100 ng) or left untreated. RNA was isolated for the synthesis of cDNA. Real‐time PCR was performed from cDNA templates for the amplification of TLRs. GAPDH was used as a reference control to normalize the value of gene of interest. Data shown represent the relative fold change of TLRs by using ΔΔCT method, compared with untreated controls. Filled and open circles show the expression of TLRs in uninfected and infected macrophages, respectively. Experiments were performed three times, and the data are shown as mean ± SE. *P < 0·05, **P < 0·01 and ***P < 0·001

FIGURE 4

L. major‐infected macrophages were treated with different doses of CpG‐ODN (0·0625, 0·125 and 0·25 μm) for indicated time periods or left untreated. RNA was isolated for the synthesis of cDNA. Real‐time PCR was performed from cDNA templates for the amplification of TLRs. GAPDH was used as a reference control to normalize the value of gene of interest. Data shown represent the relative fold change of TLRs by using ΔΔCT method, compared with untreated controls. Filled and open circles show the expression of TLRs in uninfected and infected macrophages, respectively. Experiments were performed three times, and the data are shown as mean ± SE. *P < 0·05, **P < 0·01 and ***P < 0·001

FIGURE 5

BALB/c‐derived, thioglycolate‐elicited, peritoneal macrophages were plated and incubated for 24‐h resting. The cells were infected with stationary phase L. major promastigotes at a ratio of 1:10 for 6 h, and extracellular parasites were washed. Cells were further incubated for 48 h and treated with TLR11 ligand (profilin—125, 250 and 500 ng) or left untreated for indicated time duration. Cells were lysed for total RNA isolation, and cDNA was prepared for real‐time PCR analysis for TLR expression. Filled and open circles show the expression of TLRs in uninfected and infected macrophages, respectively. Experiments were performed three times, and the data are shown as mean ± SE. *P < 0·05, **P < .01 and ***P < .001

FIGURE 6

Alteration of inter‐TLR networks in uninfected and L. major‐infected macrophages. For simplicity of visualization, TLR fold change values from (a–d) uninfected macrophages and (e–h) L. major‐infected macrophages were reassigned with +5 or −5 log scales and plotted using Cytoscape. The negative regulations are shown with red colour and positive regulations with blue colour. Regulations at 3, 6 and 12 h are shown with dotted, dashed and solid lines, respectively

FIGURE 7

TLRs control the Leishmania infection by enhancing iNOS expression. (a) Mouse macrophages were isolated from BALB/c mice and 60 h L. major‐infected macrophages or uninfected macrophages treated with different doses of TLR ligands, as previously described, for another 8 h. The line graphs show the relative fold changes in iNOS expression. GAPDH was used as internal control for normalization, and fold changes were calculated by ΔΔCT method. (b) After collecting the cell‐free supernatants, cells were washed with PBS, lysed and subjected to Western blotting analysis of the expression of inducible nitric oxide synthase (iNOS). (c) In another experiment, infected and uninfected cells were treated with the selected dose of TLR ligands, as described earlier. The cell‐free supernatants were collected after 24 h of TLR treatment, and nitrite generation (μM) was measured using Griess reagent and shown as bar graphs. (d) Similar experiment was conducted by plating macrophages in chamber slides for checking the parasite count. Macrophages were infected with L. major promastigotes at a ratio of 1:10 for 6 h. Extracellular parasites were washed out and after another 12 h of infection; cells were treated with indicated doses of TLR ligands (stimulation of TLR ligands was used in per ml culture media) for more 60 h. Cells were washed with 1xPBS, fixed with chilled methanol for 4 min and stained with Giemsa for amastigote enumeration

FIGURE 8

TLR2, TLR6, TLR9 and TLR11 are key modules of inter‐TLR dependency. (a) BALB/c macrophages were transduced with TLR1 shRNA‐Lv (left), TLR2 shRNA‐Lv (middle) and TLR6 shRNA‐Lv (right) (2 lentiviral particles/cells) or with control lentiviral particles for 48 h. TLR1 shRNA‐Lv and TLR2 shRNA‐Lv‐transduced or untransduced macrophages were stimulated with Pam₃CSK₄ (50 ng/ml), and TLR6 shRNA‐Lv‐transduced or untransduced macrophages were stimulated with FSL (50 ng/ml) for another 6 h and assessed for TLR9 and TLR11 expression. (b) Macrophages were transduced with TLR11 shRNA‐Lv (two lentiviral particles/cell) or with control lentiviral particles for 48 h. Lentivirally transduced or untransduced macrophages were stimulated with profilin (250 ng/ml) for another 6 h and examined for TLR2 and TLR9 expression. (c) BALB/c macrophages were transduced with TLR1 shRNA‐Lv and TLR6 shRNA‐Lv alone and with combination (two lentiviral particles/cells) or with control lentiviral particles for 48 h. Lentivirally transduced or untransduced macrophages were stimulated with PGN (5 μg/ml) for another 6 h and assessed for TLR9 expression. (d) Mouse macrophages were treated with the indicated doses of Pam₃CSK₄ (1 ng), PGN (0·2 μg), LPS (5 ng), FSL (2·5 ng) and profilin (20 ng) for 6 h and assessed for TLR9 expression. (e) Macrophages were transduced with TLR1 shRNA‐Lv (left) and TLR2 shRNA‐Lv (right) (two lentiviral particles per cells) or with control lentiviral particles for 48 h. Transduced or untransduced macrophages were stimulated with CpG (0·125 μm) for another 6 h and examined for IL‐12 expression. (f) Macrophages were transduced with TLR6 shRNA‐Lv for 48 h. Transduced or untransduced macrophages were treated with FSL (50 ng/ml) for another 6 h and assessed IL‐10 production. RNA was isolated for the synthesis of cDNA. Real‐time PCR was performed from cDNA templates for the amplification of TLRs, IL‐10 and IL‐12 by using their specific forward and reverse primers. GAPDH was used as a control reference to normalize the value of gene of interest. Data shown represent the relative fold change in TLRs by using ΔΔCT method compared with untreated controls. Experiments were performed three times, and the data are shown as mean ± SEM

FIGURE 9

TLR1, TLR2, TLR6, TLR9 and TLR11 rewiring reduces the parasite load in vivo. TLR11 was silenced on 3rd day after 2 × 10⁶ L. major infection of BALB/c mice. The mice were subcutaneously treated with TLR2/6 ligand alone on 7th and 9th day after the infection, followed by TLR2/6 ligand BPPcysMPEG (1 μg/mouse) and TLR9 ligand CpG (10 μg/mouse) on 11th day of infection. TLR2 was silenced on 25th day after infection. (a) Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 weeks. (b) 5 weeks after infection, indicated group of mice were killed, and parasites were enumerated from the popliteal lymph node cells. Silencing of TLR2 and TLR11 in footpad was confirmed by performing RT‐PCR (Inset). (c) BALB/c‐derived peritoneal macrophages were infected with L. major for 72 h. After washing out the extracellular parasites, macrophages were co‐cultured with the CD4⁺ T cells isolated from the lymph nodes of indicated group of BALB/c mice at 1:3 ratio (MФ:T cells). After 72‐h infection, macrophages were washed, fixed, stained with Giemsa stain, and evaluated for the amastigotes/ 100 macrophages. (d) BPPcysMPEG or CpG or TLR11 shRNA or TLR2 shRNA ‐treated or untreated L. major‐infected BALB/c mice were killed on 35th day. Lymph node T cells analysed for the Treg cell expansion by gating FoxP3⁺ IL‐10⁺ cells on CD3⁺ CD4⁺ CD25⁺ CD127^low cells by FACS. (e) Uninfected and L. major‐infected mice treated with different combination of TLRs were killed, their spleens were crushed, RBCs were lysed with Gey's solution, and the lymph node cells were lysed to extract RNA for real‐time PCR. Real‐time PCR was performed from cDNA templates for the amplification of IFN‐γ and IL‐4 by using their specific forward and reverse primers. GAPDH was used as a reference control to normalize the value of gene of interest. Data shown represent the relative fold change in IFN‐γ and IL‐4 by using ΔΔCT method compared with uninfected controls. (f) TLR1 and TLR11 were silenced on day 3 after 2 × 10⁶ L. major infection of BALB/c mice. The mice were treated with FSL (10 μg/mouse), a TLR2/6 ligand, and CpG (10 μg/mouse), a TLR9 ligand on 7th, 9th and 11th day after the infection. Disease progression was scored weekly by evaluating the net footpad swelling (the thickness of the uninfected left footpad subtracted from the infected right footpad) by digital micrometer for 5 weeks. (g) Mice were killed, and parasites were enumerated from the popliteal lymph node cells, as indicated. (h) TLR1 silencing in footpad was validated by RT‐PCR. (i) Lymph nodes were isolated from the above groups. Stamp smears of the lymph nodes from the group A (left) and the group F (right) are shown here as representative of the effects of targeting TLR interdependencies

FIGURE 10

(a) Phase‐specific TLR treatment in L. donovani‐infected BALB/c mice. BALB/c mice were infected with 2 × 10⁷ L. donovani 2 days prior to lentivirally expressed TLR11shRNA or control shRNA treatment followed by treatment with BPPcysMPEG (1 μg/mouse) on 7th and 9th day or CpG (10 μg/mouse) on 11th day; these treatments were followed by TLR2shRNA on 21st day or control shRNA in different combinations or were left untreated. Mice were euthanized on the 28th day. (b) Spleens were isolated from above groups. Giemsa‐stained stamp smears were used for splenic parasite load assessment. The graph represents the parasite load as Leishman–Donovan unit (LDU) in spleens of the control and treated mice. (c) Naive and infected mice treated with different combinations of TLRs were killed, their spleens were crushed and RBCs were lysed with Gey's solution, and the splenocytes were lysed to extract RNA for real‐time PCR. Real‐time PCR was performed from cDNA templates for the amplification of IL‐12, IFN‐γ, IL‐10 and IL‐4 by using their specific forward and reverse primers. GAPDH was used as a reference control to normalize the value of gene of interest. Data shown represent the relative fold change in TLRs by using ΔΔCT method compared with uninfected controls. Inhibition of TLR2‐ and TLR11‐specific shRNA was checked by RT‐PCR by transducing the mouse macrophages with their respective TLR‐Lv or control lentiviral particles for 48 h, as shown in Figure 8 and Figure 9. (d) The collected splenocytes were stained for T‐regulatory cell‐specific fluorochrome‐conjugated antibodies and analysed by flow cytometry. T cells were analysed for the FoxP3⁺ IL10⁺ cells by serial gating of CD3⁺ CD4⁺ CD25⁺ CD127^low cells. The data shown are representative of one of the triplicate experiment. Experiments were performed three times, and the data are shown as mean ± SEM