Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector.

Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δmag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1ß (IL-1ß) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1ß release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1ß release) in Δmag1 parasites completely inhibited all IL-1ß release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection.IMPORTANCEToxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system.

Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5.

Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.

Toxoplasma GRA15 and GRA24 are important activators of the host innate immune response in the absence of TLR11.

The murine innate immune response against Toxoplasma gondii is predominated by the interaction of TLR11/12 with Toxoplasma profilin. However, mice lacking Tlr11 or humans, who do not have functional TLR11 or TLR12, still elicit a strong innate immune response upon Toxoplasma infection. The parasite factors that determine this immune response are largely unknown. Herein, we investigated two dense granule proteins (GRAs) secreted by Toxoplasma, GRA15 and GRA24, for their role in stimulating the innate immune response in Tlr11-/- mice and in human cells, which naturally lack TLR11/TLR12. Our results show that GRA15 and GRA24 synergistically shape the early immune response and parasite virulence in Tlr11-/- mice, with GRA15 as the predominant effector. Nevertheless, acute virulence in Tlr11-/- mice is still dominated by allelic combinations of ROP18 and ROP5, which are effectors that determine evasion of the immunity-related GTPases. In human macrophages, GRA15 and GRA24 play a major role in the induction of IL12, IL18 and IL1ß secretion. We further show that GRA15/GRA24-mediated IL12, IL18 and IL1ß secretion activates IFNγ secretion by peripheral blood mononuclear cells (PBMCs), which controls Toxoplasma proliferation. Taken together, our study demonstrates the important role of GRA15 and GRA24 in activating the innate immune response in hosts lacking TLR11.

Iron trafficking in patients with Indian Post kala-azar dermal leishmaniasis.

BACKGROUND: During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases. METHODOLOGY/PRINCIPAL FINDINGS: The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1. CONCLUSIONS/SIGNIFICANCE: In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted.

Assays to Evaluate Toxoplasma-Macrophage Interactions.

The obligate intracellular protozoan parasite Toxoplasma gondii can infect any nucleated cell from a warm-blooded host. However, its interaction with host macrophages plays a critical role in shaping the immune response during infection. Therefore, assessing Toxoplasma-macrophage interactions at a cellular level is important. In this chapter, we describe assays that can be used to characterize Toxoplasma-macrophage interactions. These assays can also be used to evaluate other host-pathogen interactions. We describe multiplex approaches for measuring arginase activity, indoleamine 2,3 dioxygenase activity, cell death, and parasite growth during Toxoplasma-macrophage interactions. These assays can be used to compare how different Toxoplasma strains differ in their interaction with macrophages, and we describe how to properly assess Toxoplasma strain differences in Toxoplasma-macrophage interactions.

Toxoplasma GRA15 Activates the NF-κB Pathway through Interactions with TNF Receptor-Associated Factors.

The protozoan parasite Toxoplasma gondii secretes proteins from specialized organelles, the rhoptries, and dense granules, which are involved in the modulation of host cell processes. Dense granule protein GRA15 activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. Exactly how GRA15 activates the NF-κB pathway is unknown. Here we show that GRA15 interacts with tumor necrosis factor receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. We identified several TRAF binding sites in the GRA15 amino acid sequence and showed that these are involved in NF-κB activation. Furthermore, a TRAF2 knockout cell line has impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.IMPORTANCE The parasite Toxoplasma can cause birth defects and severe disease in immunosuppressed patients. Strain differences in pathogenicity exist, and these differences are due to polymorphic effector proteins that Toxoplasma secretes into the host cell to coopt host cell functions. The effector protein GRA15 of some Toxoplasma strains activates the nuclear factor kappa B (NF-κB) pathway, which plays an important role in cell death, innate immunity, and inflammation. We show that GRA15 interacts with TNF receptor-associated factors (TRAFs), which are adaptor proteins functioning upstream of the NF-κB transcription factor. Deletion of TRAF-binding sites in GRA15 greatly reduces its ability to activate the NF-κB pathway, and TRAF2 knockout cells have impaired GRA15-mediated NF-κB activation. Thus, we determined the mechanism for GRA15-dependent NF-κB activation.

Impaired activation of lesional CD8+ T-cells is associated with enhanced expression of Programmed Death-1 in Indian Post Kala-azar Dermal Leishmaniasis.

Post Kala-azar dermal leishmaniasis (PKDL), caused by Leishmania donovani is the dermal sequel of Visceral Leishmaniasis and importantly, is the proposed disease reservoir. The survival of Leishmania parasites within monocytes/macrophages hinges on its ability to effectively nullify immune activation mechanisms. Thus, delineating the disease-promoting immune mechanisms can facilitate development of immunotherapeutic strategies. Accordingly, in the absence of an animal model, this study aimed to delineate the status of CD8+ T-cells in patients with PKDL. At disease presentation, the absence of CD4+ T-cells at lesional sites was concomitant with an overwhelming infiltration of CD8+ T-cells that demonstrated an absence of Perforin, Granzyme and Zap-70, along with an enhanced expression of Programmed Death-1 (PD-1) and the skin-homing CCL17. Additionally, the lesional CCR4+CD8+ population was associated with an enhanced expression of IL-10 and IL-5. In circulation, the enhanced CD8+CCR4+ T-cell population and raised levels of CCL17/22 was associated with an increased frequency of PD-1, while CD127 was decreased. Taken together, in PKDL, the enhanced plasma and lesional CCL17 accounted for the dermal homing of CD8+CCR4+ T-cells, that along with a concomitant upregulation of PD-1 and IL-10 mediated immune inactivation, emphasizing the need for designing immunotherapies capable of reinvigorating T-cell potency.

Decreased Frequency and Secretion of CD26 Promotes Disease Progression in Indian Post Kala-azar Dermal Leishmaniasis.

PURPOSE: Leishmania, the causative organisms for leishmaniasis, reside in host macrophages and survive by modulating the microbicidal pathways via attenuation of the oxidative burst and/or suppression of cell-mediated immunity. As post kala-azar dermal leishmaniasis (PKDL), the dermal sequela of visceral leishmaniasis, has no animal model, the underlying mechanism(s) that nullify the microbicidal effector mechanisms remain poorly understood. This study was aimed at assessing the status of dipeptidyl peptidase CD26, a co-stimulatory molecule that is essential for T-cell signal activation. METHODS: The frequency/expression of CD26 and CD45RO/RA was evaluated by flow cytometry, while levels of soluble CD26 (sCD26), CXCL-10, RANTES, IL-10 and TGF-ß along with adenosine deaminase (ADA) activity were measured using ELISA. RESULTS: In patients with PKDL vis-à-vis healthy individuals, there was a significant decrease in the frequency and expression of CD26 on CD3(+)CD8(+) T-cells, which was accompanied by a significant lowering of plasma levels of sCD26. Furthermore, these patients showed a significant decrease in the frequency of CD45RO(+)/CD8(+) T-cells, concomitant with a significant increase in the proportion of CD45RA(+)/CD8(+) T-cells. This could collectively translate into reduced formation of the immunological synapse of CD26, CD45RO, and ADA, and lead to an attenuation of the Th1 responses. The decreased levels of CD26 and sCD26 correlated negatively with raised levels of Th2 cytokines, IL-10, and TGF-ß along with the lesional parasite load, indicating disease specificity. CONCLUSIONS: Taken together, the decreased expression and secretion of CD26 in patients with PKDL resulted in impairment of the CD26-ADA interaction, and thereby possibly contributed to T-cell unresponsiveness, emphasizing the need to develop immunomodulatory therapies against PKDL and by extension, the leishmaniases.

M2 Polarization of Monocytes-Macrophages Is a Hallmark of Indian Post Kala-Azar Dermal Leishmaniasis.

The high level of functional diversity and plasticity in monocytes/macrophages has been defined within in vitro systems as M1 (classically activated), M2 (alternatively activated) and deactivated macrophages, of which the latter two subtypes are associated with suppression of cell mediated immunity, that confers susceptibility to intracellular infection. Although the Leishmania parasite modulates macrophage functions to ensure its survival, what remains an unanswered yet pertinent question is whether these macrophages are deactivated or alternatively activated. This study aimed to characterize the functional plasticity and polarization of monocytes/macrophages and delineate their importance in the immunopathogenesis of Post kala-azar dermal leishmaniasis (PKDL), a chronic dermatosis of human leishmaniasis. Monocytes from PKDL patients showed a decreased expression of TLR-2/4, along with an attenuated generation of reactive oxidative/nitrosative species. At disease presentation, an increased mRNA expression of classical M2 markers CD206, ARG1 and PPARG in monocytes and lesional macrophages indicated M2 polarization of macrophages which was corroborated by increased expression of CD206 and arginase-1. Furthermore, altered vitamin D signaling was a key feature in PKDL, as disease presentation was associated with raised plasma levels of monohydroxylated vitamin D3 and vitamin D3- associated genes, features of M2 polarization. Taken together, in PKDL, monocyte/macrophage subsets appear to be alternatively activated, a phenotype that might sustain disease chronicity. Importantly, repolarization of these monocytes to M1 by antileishmanial drugs suggests that switching from M2 to M1 phenotype might represent a therapeutic opportunity, worthy of future pharmacological consideration.

Decreased presence of Langerhans cells is a critical determinant for Indian Post kala-azar dermal leishmaniasis.

Post kala-azar dermal leishmaniasis (PKDL) is the dermal sequel of visceral leishmaniasis (VL) and occurs after apparent cure or alongside with VL. It is confined to South Asia (India, Nepal and Bangladesh) and East Africa (mainly Sudan), the incidence being 5-10% and 50-60% respectively. In South Asia, as the transmission of VL is anthroponotic, PKDL patients are the proposed disease reservoir, thus assuming epidemiological significance, its eradication being linked to the control of leishmaniasis. In the absence of an animal model and its low incidence, factors contributing towards the immunopathogenesis of PKDL remain an open-ended, yet pertinent question. This study delineated the lesional immunopathology in terms of granuloma formation, Langerhans cells, tissue macrophages along with mRNA expression of IL-12p40 and IL-10. Our study in Indian PKDL for the first time identified that the number of CD1a(+) /CD207(+) Langerhans cells are decreased and CD68(+) macrophages are increased along with the absence of an epitheloid granuloma. Importantly, this decrease in Langerhans cells was associated with decreased mRNA expression of IL-12p40 and increased IL-10. This was reverted with treatment allowing for elimination of parasites and disease resolution along with an increase in Langerhans cells and decrease in macrophages. Thus, in Indian PKDL, absence of a granuloma formation along with a decrease in Langerhans cells collectively caused immune inactivation essential for parasite persistence and disease sustenance.

Impact of iron deficiency anemia on cell-mediated and humoral immunity in children: A case control study.

OBJECTIVE: The precise role of iron in immune regulation especially in children vulnerable to iron deficiency is not fully known. Hence, this study was conducted to evaluate the effects of iron deficiency anemia (IDA) and its treatment with oral iron supplementation on cell-mediated immunity (CMI) and humoral immunity (HMI) in children. MATERIALS AND METHODS: A total of 40 children (<15 years) with IDA and 40 age-matched healthy children after satisfying the inclusion criteria were enrolled for this case-control study. Flow cytometric evaluation of absolute and relative numbers of cluster of differentiation 4 (CD4) and CD8 (cluster of differentiation 8) lymphocyte subgroups was carried out to assess the CMI and serum Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin M (IgM) were measured to assess the HMI at baseline and 3 months post oral iron supplementation. RESULTS: Significantly lower levels (P < 0.05) of CD4+ T-cells and decreased CD4:CD8 ratios were observed in the iron deficient children. Iron supplementation significantly improved the CD4+ cell counts and CD4:CD8 ratios. However, immunoglobulin levels weren't different between the two groups. CONCLUSIONS: Although IDA did not influence HMI, it significantly impaired CMI, which was improved following iron supplementation for 3 months.

Post kala-azar dermal leishmaniasis: an unresolved mystery.

Post kala-azar dermal leishmaniasis (PKDL), a cutaneous sequela of visceral leishmaniasis (VL), develops in some patients alongside but more commonly after apparent cure from VL. In view of the pivotal role of PKDL patients in the transmission of VL, here we review clinical, epidemiological, parasitological, and immunological perspectives of this disease, focusing on five hypotheses to explain the development of PKDL: (i) the role of antimonial drugs; (ii) UV-induced skin damage; (iii) reinfection; (iv) organ specific failure of memory T cell responses; and (v) genetic susceptibility of the host. This review will enable researchers and clinicians to explore the unresolved mystery of PKDL and provide a framework for future application of 'omic' approaches for the control and eventual elimination of VL.

Malabaricone-A induces a redox imbalance that mediates apoptosis in U937 cell line.

BACKGROUND: The 'two-faced' character of reactive oxygen species (ROS) plays an important role in cancer biology by acting both as secondary messengers in intracellular signaling cascades and sustaining the oncogenic phenotype of cancer cells, while on the other hand, it triggers an oxidative assault that causes a redox imbalance translating into an apoptotic cell death. PRINCIPAL FINDINGS: Using a tetrazolium [{3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium] based cell viability assay, we evaluated the cytotoxicity of a plant derived diarylnonanoid, malabaricone-A on leukemic cell lines U937 and MOLT-3. This cytotoxicity hinged on its ability to cause a redox imbalance via its ability to increase ROS, measured by flow cytometry using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate and by decreasing glutathione peroxidase activity. This redox imbalance mediated apoptosis was evident by an increase in cytosolic [Ca(2+)], externalization of phosphatidyl serine as also depolarization of the mitochondrial membrane potential as measured by flow cytometry. There was concomitant peroxidation of cardiolipin, release of free cytochrome c to cytosol along with activation of caspases 9, 8 and 3. This led to cleavage of the DNA repair enzyme, poly (ADP-ribose) polymerase that caused DNA damage as proved by labeling with 4',6-diamidino-2-phenylindole (DAPI); furthermore, terminal deoxy ribonucleotide transferase catalysed incorporation of deoxy uridine triphosphate confirmed DNA nicking and was accompanied by arrest of cell cycle progression. CONCLUSIONS: Taken together, compounds like MAL-A having pro-oxidant activity mediate their cytotoxicity in leukemic cells via induction of oxidative stress triggering a caspase dependent apoptosis.

Miltefosine effectively modulates the cytokine milieu in Indian post kala-azar dermal leishmaniasis.

BACKGROUND: The increasing incidence of unresponsiveness to antimonials in leishmaniasis prompted the use of newer drugs such as miltefosine. Miltefosine influences macrophage effector functions, but its effect on patients with post kala-azar dermal leishmaniasis (PKDL) has not been evaluated. METHODOLOGY: The immunomodulatory activity of miltefosine was evaluated in patients with PKDL by studying the expression of activation markers (CD14 and CD16) and costimulatory molecules (CD80 and CD86) on circulating monocytes, levels of pro-inflammatory cytokines (tumor necrosis factor α, interleukin 6, interleukin 1ß, and interleukin 8) and anti-inflammatory cytokines (interleukin 10, transforming growth factor ß, interleukin 4, and interleukin 13) in serum and peripheral blood mononuclear cell culture supernatants, and serum nitrite and arginase activity. RESULTS: Miltefosine on circulating monocytes upregulated expression of CD16 and CD86 and reduced that of CD14. Miltefosine also induced a significant increase in circulating levels of pro-inflammatory cytokines with a concomitant decrease in anti-inflammatory cytokines. Its macrophage activating potential was evidenced by its ability to decrease serum arginase activity and increase serum nitrite. CONCLUSIONS: Miltefosine increased the proportion of monocytes that have a pro-inflammatory phenotype, which was accompanied by an enhanced secretion of pro-inflammatory cytokines and increased levels of serum nitrite. The decrease in anti-inflammatory cytokine levels and serum arginase activity collectively indicated that miltefosine triggered a robust T-helper 1 response that facilitated parasite elimination.

Immunomodulation by chemotherapeutic agents against Leishmaniasis.

Leishmaniasis is caused by protozoan parasites of the genus Leishmania and causes a wide spectrum of clinical manifestations ranging from self-healing cutaneous lesions to the fatal visceral form. The use of pentavalent antimony, the mainstay of therapy of Leishmaniasis is now limited by its toxicity and alarming increase in unresponsiveness, especially in the Indian subcontinent. Furthermore, other anti-leishmanial drugs are unaffordable in many affected countries and as vaccination based approaches have not yet proved to be effective, chemotherapy remains the only alternative, emphasizing the need for identifying novel drug targets. In this review, we have described the different host immune signaling pathways that could be considered as potential drug targets for Leishmania chemotherapy.

Attenuation of oxidative stress by allylpyrocatechol in synovial cellular infiltrate of patients with Rheumatoid Arthritis.

Free radicals are involved in the pathogenesis of Rheumatoid arthritis, a systemic autoimmune disorder characterized by unchecked synovial inflammation. Allylpyrocatechol, a phytoconstituent of Piper betle leaves, has potent anti-inflammatory activity and this study evaluated its anti-oxidant effect on the synovial infiltrate of patients with Rheumatoid arthritis. The ex vivo effect of allylpyrocatechol upon generation of reactive oxygen species in neutrophils, macrophages and lymphocytes was measured by flow cytometry using dichlorodihydrofluorescein diacetate, wherein it significantly decreased basal levels as also scavenged phorbol myristate acetate generated reactive oxygen species. Furthermore, its effect on generation of superoxide and hydroxyl radicals produced within infiltrated neutrophils was measured by cytochrome c and deoxyribose assay, respectively. Allylpyrocatechol significantly scavenged superoxide and hydroxyl radicals in infiltrated neutrophils. The effect of allylpyrocatechol on nitric oxide was measured in macrophages using 4,5-diaminofluorescein diacetate by flow cytometry wherein it decreased production of nitric oxide in infiltrated macrophages, which correlated with its in vitro nitric oxide scavenging activity. Taken together, this ex vivo study has established that allylpyrocatechol has potent scavenging activity and could be considered as an add-on therapy in the treatment of inflammation-associated disorders like Rheumatoid Arthritis.

Enhanced lesional Foxp3 expression and peripheral anergic lymphocytes indicate a role for regulatory T cells in Indian post-kala-azar dermal leishmaniasis.

Indian post-kala-azar dermal leishmaniasis (PKDL) is a low-frequency (5-10%) dermal sequela of visceral leishmaniasis (VL) caused by Leishmania donovani; importantly, affected individuals are speculated to be parasite reservoirs. Insight into its immunopathogenesis could translate into rational immunomodulatory therapeutic approaches against leishmaniases. In patients with PKDL (n=21), peripheral lymphocytes were analyzed for surface markers, intracellular cytokines, and lymphoproliferative responses using flow cytometry. In lesional tissue biopsies (n=12), expression of counter-regulatory cytokines (IFN-gamma and IL-10) and the T-regulatory transcription factor forkhead box protein 3 (Foxp3) was analyzed using reverse transcriptase-PCR, along with immunohistochemical detection (n=8) of CD3 and Foxp3 positivity. In patients with PKDL, circulating CD8(+)CD28(-) and antigen-induced IL-10(+)CD3(+) lymphocytes were increased and receded with treatment. CD8(+) lymphocytes showed impaired proliferative responses to L. donovani antigen (LDA) and phytohemagglutinin, which were reinstated after treatment. At presentation, the upregulated lesional IFN-gamma and IL-10 messenger RNA (mRNA), Foxp3 mRNA, and protein were curtailed after treatment. In Indian patients with PKDL, increased frequency of the CD8(+)CD28(-) phenotype, enhanced antigen-specific IL-10 production, and accompanying anergy of circulating lymphocytes suggest their regulatory nature. Furthermore, the concomitantly elevated lesional expression of Foxp3 suggests their possible recruitment into the lesional site, which would sustain disease pathology.

A novel copper chelate modulates tumor associated macrophages to promote anti-tumor response of T cells.

BACKGROUND: At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis. METHODOLOGY/PRINCIPAL FINDINGS: The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-gamma but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-beta producing CD4(+)CD25(+) T cells toward IFN-gamma producing T cells. CONCLUSION/SIGNIFICANCE: Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth.