Leishmania donovani Subverts Host Immune Response by Epigenetic Reprogramming of Macrophage M(Lipopolysaccharides + IFN-γ)/M(IL-10) Polarization.

Reciprocal changes in histone lysine methylation/demethylation of M(LPS + IFN-γ)/M(IL-10) genes is one of the factors that direct macrophage polarization and contribute to host defense/susceptibility toward infection. Although, histone lysine methyltransferases and lysine demethylases orchestrate these events, their role remains elusive in visceral leishmaniasis, a disease associated with macrophage M(IL-10) polarization. In this study, we observed that L. donovani induced the expression of histone lysine methyltransferases Ash1l, Smyd2, and Ezh2 and histone lysine demethylases Kdm5b and Kdm6b in J774 macrophages and BALB/c mice. Chromatin immunoprecipitation analysis revealed that L. donovani facilitated H3K36 dimethylation at TNF-α promoter by Smyd2 and H3K27 trimethylation at inducible NO synthase promoter by Ezh2 to suppress their expression in macrophages. Furthermore, infection-induced Kdm5b and Kdm6b modulated H3K4 and H3K27 trimethylation at IL-12, TNF-α, and arginase-1 promoters, respectively, whereas H3K4 trimethylation by Ash1l at IL-10 promoter induced its expression. Analysis of transductional events revealed that HIF-1α upregulated Kdm5b and Kdm6b expression, whereas Ash1l and Ezh2 expression were induced by transcription factor MeCP2. Additionally, Smyd2 was induced by c-Myc in infected macrophages. Knockdown of Ash1l, Ezh2, Kdm5b, and Kdm6b by specific small interfering RNA and Vivo-Morpholino, as well as inhibition of Smyd2 by its specific inhibitor, AZ505, led to increased protective proinflammatory response and inhibited amastigote multiplication in infected J774 macrophages and BALB/c mice, respectively. Collectively, our findings demonstrate that L. donovani exploits specific histone lysine methyltransferases/demethylases to redirect epigenetic programming of M(LPS + IFN-γ)/M(IL-10) genes for its successful establishment within the host.

A Semi-automated Organoid Screening Method Demonstrates Epigenetic Control of Intestinal Epithelial Differentiation.

Intestinal organoids are an excellent model to study epithelial biology. Yet, the selection of analytical tools to accurately quantify heterogeneous organoid cultures remains limited. Here, we developed a semi-automated organoid screening method, which we applied to a library of highly specific chemical probes to identify epigenetic regulators of intestinal epithelial biology. The role of epigenetic modifiers in adult stem cell systems, such as the intestinal epithelium, is still undefined. Based on this resource dataset, we identified several targets that affected epithelial cell differentiation, including HDACs, EP300/CREBBP, LSD1, and type I PRMTs, which were verified by complementary methods. For example, we show that inhibiting type I PRMTs, which leads enhanced epithelial differentiation, blocks the growth of adenoma but not normal organoid cultures. Thus, epigenetic probes are powerful tools to study intestinal epithelial biology and may have therapeutic potential.

Differential Induction of SOCS Isoforms by Leishmania donovani Impairs Macrophage-T Cell Cross-Talk and Host Defense.

Immune evasion strategies adopted by Leishmania donovani involve the exploitation of suppressor of cytokine signaling (SOCS) proteins that are well-known negative regulators of the JAK/STAT pathway. However, the cellular mechanism underpinning the induction of SOCS isoforms and their role in breaching the multilevel regulatory circuit connecting the innate and adaptive arms of immunity are still ambiguous during experimental visceral leishmaniasis. Using bone marrow-derived macrophages (BMMфs) and CD4+ T cells, we observed that L. donovani preferentially upregulates SOCS1 and SOCS3 expression in macrophages and T cells, respectively, whereas the SOCS1 level remains consistently high in BMMфs and SOCS3 expression is pronounced and long lasting in T cells. Consequently, this inhibits STAT1-mediated IL-12 induction in macrophages & STAT4-mediated IFN-γ synthesis in T cells. Mechanistically, PI3K/Akt-mediated SRF activation promotes nuclear translocation and binding of Egr2 to SOCS1 promoter for its early induction in infected BMMфs. Additionally, L. donovani activates IDO/kynurenine/AHR signaling in BMMфs to maintain prolonged SOCS1 expression. Later, PGE2, secreted from infected BMMфs induces cAMP-PKA pathway by binding to the EP2/EP4 receptor of CD4+ T cells, leading to SP1, CREB, and GATA1 activation and SOCS3 expression. Small interfering RNA-mediated silencing of SOCS1 and SOCS3 in macrophage and T cells, respectively, restored IL-12 and IFN-γ cytokine levels and BMMф-T cell interaction. Vivo morpholino-mediated silencing of SOCS1 and SOCS3 resulted in protective cytokine responses, thereby reducing organ parasite burden significantly in L. donovani-infected BALB/c mice. Collectively, our results imply that L. donovani orchestrates different SOCS isoforms to impair macrophage-T cell cross-talk and preserve its own niche.

Leishmania donovani Exploits Tollip, a Multitasking Protein, To Impair TLR/IL-1R Signaling for Its Survival in the Host.

IL-1R/TLR signaling plays a significant role in sensing harmful foreign pathogens and mounting effective innate and adaptive immune responses. However, the precise mechanism by which Leishmania donovani, an obligate intramacrophagic pathogen, breaches IL-1R/TLR signaling and host-protective immunity remains obscure. In this study, we report the novel biphasic role of Toll-interacting protein (Tollip), a negative regulator of the IL-1R/TLR pathway, in the disease progression of experimental visceral leishmaniasis. We observed that during early hours of infection, L. donovani induced phosphorylation of IRAK-1, resulting in the release of Tollip from the IL-1R-associated kinase (IRAK)-1 complex in J774 macrophages, which then acted as an endocytic adaptor on cell surface IL-1R1 and promoted its lysosomal degradation. In the later stage, Tollip shuttled back to IRAK-1, thereby inhibiting IRAK-1 phosphorylation in association with IRAK-M to neutralize downstream TLR signaling in infected macrophages. Moreover, during late infection, L. donovani enhanced nuclear translocation and recruitment of transcription factors early growth response protein 2, NF erythroid 2-related factor 2, and Ahr on Tollip promoter for its induction. Small interfering RNA-mediated silencing of Tollip in infected macrophages significantly enhanced NF-κB activation and induced host-defensive IL-12 and TNF-α synthesis, thereby reducing amastigote multiplication. Likewise, abrogation of Tollip in L. donovani-infected BALB/c mice resulted in STAT-1-, IRF-1-, and NF-κB-mediated upregulation of host-protective cytokines and reduced organ parasite burden, thereby implicating its role in disease aggravation. Taken together, we conclude that L. donovani exploited the multitasking function of Tollip for its own establishment through downregulating IL-1R1/TLR signaling in macrophages.

Ammonium trichloro [1,2-ethanediolato-O,O']-tellurate cures experimental visceral leishmaniasis by redox modulation of Leishmania donovani trypanothione reductase and inhibiting host integrin linked PI3K/Akt pathway.

In an endeavor to search for affordable and safer therapeutics against debilitating visceral leishmaniasis, we examined antileishmanial potential of ammonium trichloro [1,2-ethanediolato-O,O']-tellurate (AS101); a tellurium based non toxic immunomodulator. AS101 showed significant in vitro efficacy against both Leishmania donovani promastigotes and amastigotes at sub-micromolar concentrations. AS101 could also completely eliminate organ parasite load from L. donovani infected Balb/c mice along with significant efficacy against infected hamsters (˃93% inhibition). Analyzing mechanistic details revealed that the double edged AS101 could directly induce apoptosis in promastigotes along with indirectly activating host by reversing T-cell anergy to protective Th1 mode, increased ROS generation and anti-leishmanial IgG production. AS101 could inhibit IL-10/STAT3 pathway in L. donovani infected macrophages via blocking α4ß7 integrin dependent PI3K/Akt signaling and activate host MAPKs and NF-κB for Th1 response. In silico docking and biochemical assays revealed AS101's affinity to form thiol bond with cysteine residues of trypanothione reductase in Leishmania promastigotes leading to its inactivation and inducing ROS-mediated apoptosis of the parasite via increased Ca2+ level, loss of ATP and mitochondrial membrane potential along with metacaspase activation. Our findings provide the first evidence for the mechanism of action of AS101 with excellent safety profile and suggest its promising therapeutic potential against experimental visceral leishmaniasis.

15d-Prostaglandin J2 induced reactive oxygen species-mediated apoptosis during experimental visceral leishmaniasis.

UNLABELLED: 15-Deoxy-delta (12,14)-prostaglandin J2 (15d-PgJ2) is a potent bioactive lipid mediator, known to possess several roles in cell regulation and differentiation along with antimicrobial efficacy against different bacterial and viral infections. In the present study, we investigated the therapeutic efficacy and mechanism of action of 15d-PgJ2 in vitro in Leishmania donovani promastigotes and infected J774 macrophages, and in vivo in Balb/c mice/golden hamster model of experimental visceral leishmaniasis. 15d-PgJ2 effectively killed L. donovani promastigotes and amastigotes in vitro with IC50 of 104.6 and 80.09 nM, respectively. At 2 mg/kg (mice) and 4 mg/kg (hamster) doses, 15d-PgJ2 decreased >90 % spleen and liver parasite burden. It significantly reduced interleukin (IL)-10 and transforming growth factor (TGF)-ß synthesis in infected macrophages and splenocytes. 15d-PgJ2 induced reactive oxygen species (ROS)-dependent apoptosis of promastigotes by triggering phosphatidyl serine externalization, mitochondrial membrane damage and inducing caspase-like activity. In vitro drug interaction studies revealed an indifference to the synergistic association of 15d-PgJ2 with Miltefosine and Amphotericin-B (Amp-B). Moreover, when combined with sub-curative doses of Miltefosine and Amphotericin-B, 15d-PgJ2 resulted in >95 % parasite removal. Our results suggested that 15d-PgJ2 induces mitochondria-dependent apoptosis of L. donovani and is a good therapeutic candidate for adjunct therapy against experimental visceral leishmaniasis. KEY MESSAGE: 15d-PgJ2 effectively eliminated both promastigotes and amastigotes form of L. donovani. 15d-PgJ2 decreased parasite burden from infected mice and hamsters with reduced Th2 cytokines. 15d-PgJ2 induced ROS-mediated mitochondrial apoptosis of L. donovani promastigotes. 15d-PgJ2 is a good therapeutic candidate for adjunct therapy with Miltefosine and Amp-B.

Protection against filarial infection by 45-49 kDa molecules of Brugia malayi via IFN-γ-mediated iNOS induction.

Nitric oxide (NO) mediated mechanisms have been implicated in killing of some life-stages of Brugia malayi/Wuchereria bancrofti and protect the host through type 1 responses and IFN-γ stimulated toxic mediators' release. However, the identity of NO stimulating molecules of the parasites is not known. Three predominantly NO-stimulating SDS-PAGE resolved fractions F8 (45.24-48.64 kDa), F11 (33.44-38.44 kDa) and F12 (28.44-33.44 kDa) from B. malayi were identified and their proteins were analyzed by 2-DE and MALDI-TOF/TOF. Tropomyosin, calponin and de novo peptides were identified by 2-DE and MALDI-TOF/TOF in F8 and immunization with F8 conferred most significant protection against L3-initiated infection in Mastomys coucha. Immunized animals showed upregulated F8-induced NO, IFN-γ, TNF-α, IL-1ß, IL-10, TGF-ß release, cellular proliferative responses and specific IgG and IgG1. Anti-IFN-γ, anti-TNF-α, and anti-IL-1ß significantly reduced F8-mediated NO generation and iNOS induction at protein levels. Anti-IFN-γ treated cells showed maximum reduction (>74%) in NO generation suggesting a predominant role of IFN-γ in iNOS induction. In conclusion, the findings suggest that F8 which contains tropomyosin, calponin and de novo peptides protects the host via IFN-γ mediated iNOS induction and may hold promise as vaccine candidate(s). This is also the first report of identification of tropomyosin and calponin in B. malayi.