Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di ... more Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di hydro xy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.
American Journal of Physiology-lung Cellular and Molecular Physiology, Feb 15, 2012
The purpose of this study was to investigate the effects of chronically inhaled particulate matte... more The purpose of this study was to investigate the effects of chronically inhaled particulate matter <2.5 μm (PM2.5) on inflammatory cell populations in the lung and systemic circulation. A prominent component of air pollution exposure is a systemic inflammatory response that may exaggerate chronic diseases such as atherosclerosis and insulin resistance. T cell response was measured in wild-type C57B/L6, Foxp3-green fluorescent protein (GFP) “knockin,” and chemokine receptor 3 knockout (CXCR3−/−) mice following 24–28 wk of PM2.5 or filtered air. Chronic PM2.5 exposure resulted in increased CXCR3-expressing CD4+ and CD8+ T cells in the lungs, spleen, and blood with elevation in CD11c+ macrophages in the lung and oxidized derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine in wild-type mice. CXCR3 deficiency decreased T cells in the lung. GFP+ regulatory T cells increased with PM2.5 exposure in the spleen and blood of Foxp3-GFP mice but were present at very low levels in the lung irrespective of PM2.5 exposure. Mixed lymphocyte cultures using primary, PM2.5-treated macrophages demonstrated enhanced T cell proliferation. Our experiments indicate that PM2.5 potentiates a proinflammatory Th1 response involving increased homing of CXCR3+ T effector cells to the lung and modulation of systemic T cell populations.
STAT4 is critical for the production of IFN-γ during the generation of T h 1 immune responses. We... more STAT4 is critical for the production of IFN-γ during the generation of T h 1 immune responses. We investigated the role of STAT4 in mediating T h 1-inducing activity of a vaccine adjuvant monophosphoryl lipid A (MPL-A) using the standard antigen ovalbumin (OVA) in STAT4KO mice. Our results show that splenocytes from STAT4KO mice displayed lower OVA-specific T-cell proliferation and IL-2 production compared with wild-type (WT) mice. Further, IFN-γ production was diminished in STAT4KO-derived splenocytes but the levels of IL-12 and TNF-α were similar compared with WT mice. Interestingly, STAT4 deficiency also led to a decrease in IL-10 and T h 2 cytokines such as IL-4 and IL-13 upon MPL-A immunization, although IL-17 production was similar between WT-and STAT4KOderived splenocytes. Our observations for defective T h 1 and T h 2 responses in STAT4KO mice were further supported by the low levels of T h 1-associated IgG2a and T h 2-associated IgG1 in the sera of these mice. Taken together, our results show that STAT4 plays a critical role in mediating both T h 1 and T h 2 responses upon immunization with MPL-A. Our study provides a better understanding of how MPL-A mediates T-cell activation which will be critical for future vaccine development.
Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling wh... more Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1 ¡/¡ mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1 ¡/¡ mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1 ¡/¡ mice showed elevated Ly6G C CD11b C granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1 ¡/¡ mice suppressed accumulation of Ly6G C CD11b C cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.
Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no ... more Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen−/−) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen−/− parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen−/− amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (W...
Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malar... more Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1-/- mice we show that STAT1 is critical for mediating the recr...
Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling wh... more Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1 ¡/¡ mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1 ¡/¡ mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1 ¡/¡ mice showed elevated Ly6G C CD11b C granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1 ¡/¡ mice suppressed accumulation of Ly6G C CD11b C cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.
STAT4 is critical for the production of IFN-γ during the generation of Th1 immune responses. We i... more STAT4 is critical for the production of IFN-γ during the generation of Th1 immune responses. We investigated the role of STAT4 in mediating Th1-inducing activity of a vaccine adjuvant monophosphoryl lipid A (MPL-A) using the standard antigen ovalbumin (OVA) in STAT4KO mice. Our results show that splenocytes from STAT4KO mice displayed lower OVA-specific T-cell proliferation and IL-2 production compared with wild-type (WT) mice. Further, IFN-γ production was diminished in STAT4KO-derived splenocytes but the levels of IL-12 and TNF-α were similar compared with WT mice. Interestingly, STAT4 deficiency also led to a decrease in IL-10 and Th2 cytokines such as IL-4 and IL-13 upon MPL-A immunization, although IL-17 production was similar between WT- and STAT4KO-derived splenocytes. Our observations for defective Th1 and Th2 responses in STAT4KO mice were further supported by the low levels of Th1-associated IgG2a and Th2-associated IgG1 in the sera of these mice. Taken together, our resul...
Host-pathogen interaction is an area of considerable interest. Intracellular parasites such as Le... more Host-pathogen interaction is an area of considerable interest. Intracellular parasites such as Leishmania reside inside phagocytes such as macrophages, dendritic cells and neutrophils. Macrophages can be activated by cytokines such as IFN-γ and Toll like receptor (TLR) agonists resulting in enhanced microbicidal activity. Leishmania parasites hijack the microbicidal function of macrophages, mainly by interfering with intracellular signaling initiated by IFN-γ and TLR ligands. Here we used transgenic Leishmania donovani parasites expressing the red fluorescent protein DsRed2 and imaging-flow cytometry technology to evaluate parasitic loads inside the macrophage in vitro. Further, this methodology enables us to visualize impairment in NFκB translocation to the nucleus in L. donovani infected macrophages. Additionally we show that uninfected bystander macrophages have a similar impairment in NFκB translocation as in L. donovani infected macrophages in response to the TLR4 agonist LPS. This evidence suggests a possible immunosuppressive role for infected macrophages in regulating the activation of uninfected bystander macrophages.
The American Journal of Tropical Medicine and Hygiene, 2014
Cutaneous leishmaniasis (CL) is endemic in the Bikaner region situated in the Thar Desert of Raja... more Cutaneous leishmaniasis (CL) is endemic in the Bikaner region situated in the Thar Desert of Rajasthan, India. This study describes clinicoepidemiological data of pediatric CL in pre-school children (0-5 years of age) from this region during 2001-2012. In total, 151 patients with 217 lesions were reported during the study period. The mean age of the study group was 3.29 ± 1.43 years (0.25-5 years), with many (41.7%) cases being in the age group of 2-4 years. Face was the most common site involved, and morphologically, the lesions were either plaque type or papulonodular. Smear for parasitologic examination was positive in 84 (70%) of 120 cases, and histopathologic examination confirmed CL in 10 (55.55%) of 18 cases. Parasite species identification conducted for 13 randomly selected patients by polymerase chain reaction identified Leishmania tropica as the causative species. Intralesional sodium stibogluconate was the most commonly used treatment and found to be well-tolerated. Other therapies that were effective included oral rifampicin, oral dapsone, radiofrequency heat therapy (RFHT), and combinations of the three therapies.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 2, 2014
Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from co... more Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from conventional CD8(+) T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C-X-C motif) receptor 3 (CXCR3)-positive innate CD8(+) T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL-15 activated CXCR3(+) innate CD8(+) T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN-γ(-/-) mice compared with similarly activated CXCR3(-) subset. This was associated with enhanced proliferation and IFN-γ production in CXCR3(+) cells. Further, CXCR3(+) innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3(+) subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a,...
Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are... more Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are unable to achieve a sterile cure. Leishmania is able to persist in host cells by evading or exploiting host immune mechanisms. A thorough understanding of these mechanisms could lead to better strategies for effective management of Leishmania infections. Current research has focused on parasite modification of host cell signaling pathways, entry into phagocytic cells, and modulation of cytokine and chemokine profiles that alter immune cell activation and trafficking to sites of infection. Immuno-therapeutic approaches that target these mechanisms of immune evasion by Leishmania offer promising areas for preclinical and clinical research. Diseases caused by Leishmania are a major global health problem as over 12 million people currently suffer from leishmaniasis, with an incidence rate of approximately 2 million annually, according to recent estimates (www.who.int/tdr). It is transmitted by sandflies and presents a wide range of clinical manifestations that depend on the specie and strain of Leishmania, degree of virulence, and the immunological state of the host. Cutaneous leishmaniasis (CL) manifests as localized skin lesions that may resolve but can become chronic, leading to severe tissue destruction and disfigurement. Lesions could disseminate in immunocompromised patients giving rise to the diffuse cutaneous leishmaniasis (DCL) (Desjeux, 2004). This disease is caused by Leishmania major (the Middle East and Mediterranean Region), Leishmania mexicana (Central America), and Leishmania amazonensis (South America). Leishmania tropica and Leishmania aethiopica also cause CL in the Old World. Mucocutaneous leishmaniasis caused by Leishmania braziliensis is endemic in South America and is clinically characterized by the involvement of the nasal and oropharyngeal mucosa with extensive tissue destruction due to inflammation. Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani and Leishmania chagasi in the Old and New worlds, respectively. Clinical manifestations include hepatomegaly and splenomegaly due to parasite infiltration of the liver and spleen and if left untreated, it is almost always fatal (www.who.int/tdr) (Alexander,
Proceedings of the National Academy of Sciences, 2012
Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survi... more Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana . Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial ...
Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating... more Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (a-globin, b-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-c mRNA is highly increased by L. donovani infection. Expression of the IFN-c inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.
ObjectiveChemokine (C‐X‐C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regul... more ObjectiveChemokine (C‐X‐C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regulation of immune cell trafficking and activation. Increased CXCR3 expression in the visceral adipose of obese humans and mice was observed. A pathophysiologic role for CXCR3 in diet‐induced obesity (DIO) was hypothesized.MethodsWild‐type (WT) C57B/L6J and chemokine receptor 3 knockout (CXCR3−/−) mice were fed a high‐fat diet (HFD) for 20 weeks followed by assessment of glucose metabolism and visceral adipose tissue (VAT) inflammation.ResultsCXCR3−/− mice exhibited lower fasting glucose and improved glucose tolerance compared with WT‐HFD mice, despite similar body mass. HFD‐induced VAT innate and adaptive immune cell infiltration, including immature myeloid cells (CD11b+ F4/80lo Ly6C+), were markedly ameliorated in CXCR3−/− mice. In vitro IBIDI and in vivo migration assays demonstrated no CXCR3‐mediated effect on macrophage or monocyte migration, respectively. CXCR3−/− macrophages, however...
migrate through various tissues, to evade the host immune system, and to undergo intracellular re... more migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality. Leishmaniasis • American trypanosomiasis • Chagas disease • Toxoplasmosis • Malaria • Leishmania • Trypanosoma cruzi • Toxoplasma gondii • Plasmodium • Invasion Mechanisms of host-cell invasion in Leishmania B. S. McGwire, M. e. Drew and A. R. Satoskar share equal contribution in authorship.
The gamma isoform of PI3Kinase (PI3Kγ) controls leukocyte chemotaxis by participating in GPCR sig... more The gamma isoform of PI3Kinase (PI3Kγ) controls leukocyte chemotaxis by participating in GPCR signaling, and by regulating cellular polarization. Here we show that PI3Kγ is required for efficient induction of CXC chemokine receptor 3 (CXCR3) on T cells upon activation. T cells from PI3Kγ−/− mice up-regulated CXCR3 less efficiently than wild-type controls both upon activation in vitro as well as during Leishmania mexicana infection. Inhibition of PI3Kinases using wortmannin and LY294002 or blockade of PI3Kγ activity using a selective inhibitor or PI3Kγ siRNA suppressed induction of CXCR3 on T cells following activation. Levels of CXCR3 and T-bet mRNA were significantly lower in PI3Kγ inhibitor–treated T cells, indicating that PI3Kγ may control CXCR3 expression in part through induction of T-bet. These results reveal a novel role for PI3Kγ in the induction of CXCR3 on T cells and suggest that PI3Kγ may regulate leukocyte chemotaxis by controlling the expression of chemokine receptors.
Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di ... more Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di hydro xy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.
Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di ... more Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di hydro xy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.
American Journal of Physiology-lung Cellular and Molecular Physiology, Feb 15, 2012
The purpose of this study was to investigate the effects of chronically inhaled particulate matte... more The purpose of this study was to investigate the effects of chronically inhaled particulate matter <2.5 μm (PM2.5) on inflammatory cell populations in the lung and systemic circulation. A prominent component of air pollution exposure is a systemic inflammatory response that may exaggerate chronic diseases such as atherosclerosis and insulin resistance. T cell response was measured in wild-type C57B/L6, Foxp3-green fluorescent protein (GFP) “knockin,” and chemokine receptor 3 knockout (CXCR3−/−) mice following 24–28 wk of PM2.5 or filtered air. Chronic PM2.5 exposure resulted in increased CXCR3-expressing CD4+ and CD8+ T cells in the lungs, spleen, and blood with elevation in CD11c+ macrophages in the lung and oxidized derivatives of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine in wild-type mice. CXCR3 deficiency decreased T cells in the lung. GFP+ regulatory T cells increased with PM2.5 exposure in the spleen and blood of Foxp3-GFP mice but were present at very low levels in the lung irrespective of PM2.5 exposure. Mixed lymphocyte cultures using primary, PM2.5-treated macrophages demonstrated enhanced T cell proliferation. Our experiments indicate that PM2.5 potentiates a proinflammatory Th1 response involving increased homing of CXCR3+ T effector cells to the lung and modulation of systemic T cell populations.
STAT4 is critical for the production of IFN-γ during the generation of T h 1 immune responses. We... more STAT4 is critical for the production of IFN-γ during the generation of T h 1 immune responses. We investigated the role of STAT4 in mediating T h 1-inducing activity of a vaccine adjuvant monophosphoryl lipid A (MPL-A) using the standard antigen ovalbumin (OVA) in STAT4KO mice. Our results show that splenocytes from STAT4KO mice displayed lower OVA-specific T-cell proliferation and IL-2 production compared with wild-type (WT) mice. Further, IFN-γ production was diminished in STAT4KO-derived splenocytes but the levels of IL-12 and TNF-α were similar compared with WT mice. Interestingly, STAT4 deficiency also led to a decrease in IL-10 and T h 2 cytokines such as IL-4 and IL-13 upon MPL-A immunization, although IL-17 production was similar between WT-and STAT4KOderived splenocytes. Our observations for defective T h 1 and T h 2 responses in STAT4KO mice were further supported by the low levels of T h 1-associated IgG2a and T h 2-associated IgG1 in the sera of these mice. Taken together, our results show that STAT4 plays a critical role in mediating both T h 1 and T h 2 responses upon immunization with MPL-A. Our study provides a better understanding of how MPL-A mediates T-cell activation which will be critical for future vaccine development.
Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling wh... more Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1 ¡/¡ mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1 ¡/¡ mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1 ¡/¡ mice showed elevated Ly6G C CD11b C granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1 ¡/¡ mice suppressed accumulation of Ly6G C CD11b C cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.
Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no ... more Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen−/−) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen−/− parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen−/− amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (W...
Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malar... more Ly6Chi inflammatory monocytes (iMO) are critical for host defense against toxoplasmosis and malaria but their role in leishmaniasis is unclear. In this study, we report a detrimental role of Ly6Chi iMOs in visceral leishmaniasis (VL) caused by Leishmania donovani. We demonstrate that Ly6Chi iMOs are continuously recruited into the spleen and liver during L. donovani infection and they are preferential targets for the parasite. Using microarray-based gene expression profiling, we show that Ly6Chi iMOs isolated from the infected liver and spleen have distinct phenotypic and activation profiles. Furthermore, we demonstrate that blocking the recruitment of Ly6Chi iMOs into the liver and spleen during L. donovani infection using a CCR2 antagonist reduces the frequency of the pathogenic IFN-γ/IL10 dual producer CD4+ T cells in the spleen and leads to a significant reduction in parasite loads in the liver and spleen. Using STAT1-/- mice we show that STAT1 is critical for mediating the recr...
Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling wh... more Signal transducer and activator of transcription 1 (STAT1) mediates interferon gamma signaling which activates the expression of various genes related to apoptosis, inflammation, cell cycle and angiogenesis. Several experimental and clinical studies have investigated the role of STAT1 in primary tumor growth in breast cancer; however, its role in tumor metastasis remains to be determined. To determine the role of STAT1 in breast cancer metastasis, we analyzed growth and metastasis in WT or STAT1 ¡/¡ mice orthotopically implanted with metastatic 4T1.2 cells. Primary tumor development was faster in STAT1 ¡/¡ mice and these mice developed significantly bigger primary tumors and displayed more lung metastasis compared with WT counterparts. STAT1 ¡/¡ mice showed elevated Ly6G C CD11b C granulocytic MDSC infiltration in their primary tumors and spleens with concomitant upregulation of Mmp9 and Cxcl1 expression in tumors compared with WT counterparts. Blockade of IL-17A in primary tumor-bearing STAT1 ¡/¡ mice suppressed accumulation of Ly6G C CD11b C cells and markedly reduced lung metastasis. These data show that STAT1 is an important suppressor of primary breast tumor growth and metastasis. Importantly, we found anti-IL-17 treatment can rescue STAT1 deficient animals from developing exacerbated metastasis to the lungs which could be important for immunotherapies for immunocompromised breast cancer patients.
STAT4 is critical for the production of IFN-γ during the generation of Th1 immune responses. We i... more STAT4 is critical for the production of IFN-γ during the generation of Th1 immune responses. We investigated the role of STAT4 in mediating Th1-inducing activity of a vaccine adjuvant monophosphoryl lipid A (MPL-A) using the standard antigen ovalbumin (OVA) in STAT4KO mice. Our results show that splenocytes from STAT4KO mice displayed lower OVA-specific T-cell proliferation and IL-2 production compared with wild-type (WT) mice. Further, IFN-γ production was diminished in STAT4KO-derived splenocytes but the levels of IL-12 and TNF-α were similar compared with WT mice. Interestingly, STAT4 deficiency also led to a decrease in IL-10 and Th2 cytokines such as IL-4 and IL-13 upon MPL-A immunization, although IL-17 production was similar between WT- and STAT4KO-derived splenocytes. Our observations for defective Th1 and Th2 responses in STAT4KO mice were further supported by the low levels of Th1-associated IgG2a and Th2-associated IgG1 in the sera of these mice. Taken together, our resul...
Host-pathogen interaction is an area of considerable interest. Intracellular parasites such as Le... more Host-pathogen interaction is an area of considerable interest. Intracellular parasites such as Leishmania reside inside phagocytes such as macrophages, dendritic cells and neutrophils. Macrophages can be activated by cytokines such as IFN-γ and Toll like receptor (TLR) agonists resulting in enhanced microbicidal activity. Leishmania parasites hijack the microbicidal function of macrophages, mainly by interfering with intracellular signaling initiated by IFN-γ and TLR ligands. Here we used transgenic Leishmania donovani parasites expressing the red fluorescent protein DsRed2 and imaging-flow cytometry technology to evaluate parasitic loads inside the macrophage in vitro. Further, this methodology enables us to visualize impairment in NFκB translocation to the nucleus in L. donovani infected macrophages. Additionally we show that uninfected bystander macrophages have a similar impairment in NFκB translocation as in L. donovani infected macrophages in response to the TLR4 agonist LPS. This evidence suggests a possible immunosuppressive role for infected macrophages in regulating the activation of uninfected bystander macrophages.
The American Journal of Tropical Medicine and Hygiene, 2014
Cutaneous leishmaniasis (CL) is endemic in the Bikaner region situated in the Thar Desert of Raja... more Cutaneous leishmaniasis (CL) is endemic in the Bikaner region situated in the Thar Desert of Rajasthan, India. This study describes clinicoepidemiological data of pediatric CL in pre-school children (0-5 years of age) from this region during 2001-2012. In total, 151 patients with 217 lesions were reported during the study period. The mean age of the study group was 3.29 ± 1.43 years (0.25-5 years), with many (41.7%) cases being in the age group of 2-4 years. Face was the most common site involved, and morphologically, the lesions were either plaque type or papulonodular. Smear for parasitologic examination was positive in 84 (70%) of 120 cases, and histopathologic examination confirmed CL in 10 (55.55%) of 18 cases. Parasite species identification conducted for 13 randomly selected patients by polymerase chain reaction identified Leishmania tropica as the causative species. Intralesional sodium stibogluconate was the most commonly used treatment and found to be well-tolerated. Other therapies that were effective included oral rifampicin, oral dapsone, radiofrequency heat therapy (RFHT), and combinations of the three therapies.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 2, 2014
Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from co... more Innate CD8(+) T cells are a heterogeneous population with developmental pathways distinct from conventional CD8(+) T cells. However, their biology, classification, and functions remain incompletely understood. We recently demonstrated the existence of a novel population of chemokine (C-X-C motif) receptor 3 (CXCR3)-positive innate CD8(+) T cells. Here, we investigated the functional properties of this subset and identified effector molecules and pathways which mediate their function. Adoptive transfer of IL-15 activated CXCR3(+) innate CD8(+) T cells conferred increased protection against Listeria monocytogenes infection in susceptible IFN-γ(-/-) mice compared with similarly activated CXCR3(-) subset. This was associated with enhanced proliferation and IFN-γ production in CXCR3(+) cells. Further, CXCR3(+) innate cells showed enhanced cytotoxicity against a tumor cell line in vitro. In depth analysis of the CXCR3(+) subset showed increased gene expression of Ccl5, Klrc1, CtsW, GP49a,...
Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are... more Diseases caused by Leishmania present a worldwide problem, and current therapeutic approaches are unable to achieve a sterile cure. Leishmania is able to persist in host cells by evading or exploiting host immune mechanisms. A thorough understanding of these mechanisms could lead to better strategies for effective management of Leishmania infections. Current research has focused on parasite modification of host cell signaling pathways, entry into phagocytic cells, and modulation of cytokine and chemokine profiles that alter immune cell activation and trafficking to sites of infection. Immuno-therapeutic approaches that target these mechanisms of immune evasion by Leishmania offer promising areas for preclinical and clinical research. Diseases caused by Leishmania are a major global health problem as over 12 million people currently suffer from leishmaniasis, with an incidence rate of approximately 2 million annually, according to recent estimates (www.who.int/tdr). It is transmitted by sandflies and presents a wide range of clinical manifestations that depend on the specie and strain of Leishmania, degree of virulence, and the immunological state of the host. Cutaneous leishmaniasis (CL) manifests as localized skin lesions that may resolve but can become chronic, leading to severe tissue destruction and disfigurement. Lesions could disseminate in immunocompromised patients giving rise to the diffuse cutaneous leishmaniasis (DCL) (Desjeux, 2004). This disease is caused by Leishmania major (the Middle East and Mediterranean Region), Leishmania mexicana (Central America), and Leishmania amazonensis (South America). Leishmania tropica and Leishmania aethiopica also cause CL in the Old World. Mucocutaneous leishmaniasis caused by Leishmania braziliensis is endemic in South America and is clinically characterized by the involvement of the nasal and oropharyngeal mucosa with extensive tissue destruction due to inflammation. Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani and Leishmania chagasi in the Old and New worlds, respectively. Clinical manifestations include hepatomegaly and splenomegaly due to parasite infiltration of the liver and spleen and if left untreated, it is almost always fatal (www.who.int/tdr) (Alexander,
Proceedings of the National Academy of Sciences, 2012
Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survi... more Obligate intracellular pathogens such as Leishmania specifically target host phagocytes for survival and replication. Phosphoinositide 3-kinase γ (PI3Kγ), a member of the class I PI3Ks that is highly expressed by leukocytes, controls cell migration by initiating actin polymerization and cytoskeletal reorganization, which are processes also critical for phagocytosis. In this study, we demonstrate that class IB PI3K, PI3Kγ, plays a critical role in pathogenesis of chronic cutaneous leishmaniasis caused by L. mexicana . Using the isoform-selective PI3Kγ inhibitor, AS-605240 and PI3Kγ gene-deficient mice, we show that selective blockade or deficiency of PI3Kγ significantly enhances resistance against L. mexicana that is associated with a significant suppression of parasite entry into phagocytes and reduction in recruitment of host phagocytes as well as regulatory T cells to the site of infection. Furthermore, we demonstrate that AS-605240 is as effective as the standard antileishmanial ...
Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating... more Leishmania donovani is a parasite that causes visceral leishmaniasis by infecting and replicating in macrophages of the bone marrow, spleen, and liver. Severe anemia and leucopenia is associated with the disease. Although immune defense mechanisms against the parasite have been studied, we have a limited understanding of how L. donovani alters hematopoiesis. In this study, we used Syrian golden hamsters to investigate effects of L. donovani infection on erythropoiesis. Infection resulted in severe anemia and leucopenia by 8 weeks post-infection. Anemia was associated with increased levels of serum erythropoietin, which indicates the hamsters respond to the anemia by producing erythropoietin. We found that infection also increased numbers of BFU-E and CFU-E progenitor populations in the spleen and bone marrow and differentially altered erythroid gene expression in these organs. In the bone marrow, the mRNA expression of erythroid differentiation genes (a-globin, b-globin, ALAS2) were inhibited by 50%, but mRNA levels of erythroid receptor (c-kit, EpoR) and transcription factors (GATA1, GATA2, FOG1) were not affected by the infection. This suggests that infection has a negative effect on differentiation of erythroblasts. In the spleen, erythroid gene expression was enhanced by infection, indicating that the anemia activates a stress erythropoiesis response in the spleen. Analysis of cytokine mRNA levels in spleen and bone marrow found that IFN-c mRNA is highly increased by L. donovani infection. Expression of the IFN-c inducible cytokine, TNF-related apoptosis-inducing ligand (TRAIL), was also up-regulated. Since TRAIL induces erythroblasts apoptosis, apoptosis of bone marrow erythroblasts from infected hamsters was examined by flow cytometry. Percentage of erythroblasts that were apoptotic was significantly increased by L. donovani infection. Together, our results suggest that L. donovani infection inhibits erythropoiesis in the bone marrow by cytokine-mediated apoptosis of erythroblasts.
ObjectiveChemokine (C‐X‐C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regul... more ObjectiveChemokine (C‐X‐C motif) receptor 3 (CXCR3) is a chemokine receptor involved in the regulation of immune cell trafficking and activation. Increased CXCR3 expression in the visceral adipose of obese humans and mice was observed. A pathophysiologic role for CXCR3 in diet‐induced obesity (DIO) was hypothesized.MethodsWild‐type (WT) C57B/L6J and chemokine receptor 3 knockout (CXCR3−/−) mice were fed a high‐fat diet (HFD) for 20 weeks followed by assessment of glucose metabolism and visceral adipose tissue (VAT) inflammation.ResultsCXCR3−/− mice exhibited lower fasting glucose and improved glucose tolerance compared with WT‐HFD mice, despite similar body mass. HFD‐induced VAT innate and adaptive immune cell infiltration, including immature myeloid cells (CD11b+ F4/80lo Ly6C+), were markedly ameliorated in CXCR3−/− mice. In vitro IBIDI and in vivo migration assays demonstrated no CXCR3‐mediated effect on macrophage or monocyte migration, respectively. CXCR3−/− macrophages, however...
migrate through various tissues, to evade the host immune system, and to undergo intracellular re... more migrate through various tissues, to evade the host immune system, and to undergo intracellular replication. These cellular migration and invasion events are absolutely essential for the completion of the lifecycles of these parasites and lead to their for disease pathogenesis. This review is an overview of the molecular mechanisms of protozoan parasite invasion of host cells and discussion of therapeutic strategies, which could be developed by targeting these invasion pathways. Specifically, we focus on four species of protozoan parasites Leishmania, Trypanosoma cruzi, Plasmodium, and Toxoplasma, which are responsible for significant morbidity and mortality. Leishmaniasis • American trypanosomiasis • Chagas disease • Toxoplasmosis • Malaria • Leishmania • Trypanosoma cruzi • Toxoplasma gondii • Plasmodium • Invasion Mechanisms of host-cell invasion in Leishmania B. S. McGwire, M. e. Drew and A. R. Satoskar share equal contribution in authorship.
The gamma isoform of PI3Kinase (PI3Kγ) controls leukocyte chemotaxis by participating in GPCR sig... more The gamma isoform of PI3Kinase (PI3Kγ) controls leukocyte chemotaxis by participating in GPCR signaling, and by regulating cellular polarization. Here we show that PI3Kγ is required for efficient induction of CXC chemokine receptor 3 (CXCR3) on T cells upon activation. T cells from PI3Kγ−/− mice up-regulated CXCR3 less efficiently than wild-type controls both upon activation in vitro as well as during Leishmania mexicana infection. Inhibition of PI3Kinases using wortmannin and LY294002 or blockade of PI3Kγ activity using a selective inhibitor or PI3Kγ siRNA suppressed induction of CXCR3 on T cells following activation. Levels of CXCR3 and T-bet mRNA were significantly lower in PI3Kγ inhibitor–treated T cells, indicating that PI3Kγ may control CXCR3 expression in part through induction of T-bet. These results reveal a novel role for PI3Kγ in the induction of CXCR3 on T cells and suggest that PI3Kγ may regulate leukocyte chemotaxis by controlling the expression of chemokine receptors.
Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di ... more Here we studied ability of two naphthoquinones to inhibit Leishmania growth (2,3-dichloro-5,8-di hydro xy-1,4-naphthoquinone (TR 001) and 2,3-dibromo-1,4-naphthoquinone (TR 002). TR 001 was more efficient than TR 002 in inducing killing of promastigotes and intracellular amastigotes. These values compare well to those obtained with the standard first-line antileishmanial agent sodium stibogluconate (SSG). TR 001 also induced significantly more nitric oxide (NO) production than TR 002 or SSG. Taken together, these data show that TR 001 and TR 002 could be promising new drugs for treatment of visceral leishmaniasis.
Uploads
Papers by Abhay Satoskar