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Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease., Inflammasome activation plays a role in malaria pathogenesis, but details aren’t well understood. Here, the authors show that caspase-8 is a central mediator of systemic inflammation in rodent malaria and that monocytes from malaria patients express active caspases-1, -4 and -8.

Heterogeneity and high plasticity are common features of cells from the mononuclear phagocyte system: monocytes (MOs), macrophages, and dendritic cells (DCs). Upon activation by microbial agents, MO can differentiate into MO-derived DCs (MODCs). In previous work, we have shown that during acute infection with Plasmodium berghei ANKA (PbA), MODCs become, transiently, the main CD11b+ myeloid population in the spleen (SP) and once recruited to the brain play an important role in the development of experimental cerebral malaria (ECM). Here, we isolated 4 cell populations: bone marrow (BM) MOs (BM-MOs) and SP-MOs from uninfected mice; BM inflammatory MOs (BM-iMOs) and SP-MODCs from PbA-infected mice and used a system biology approach to a holistic transcriptomic comparison and provide an interactome analysis by integrating differentially expressed miRNAs (DEMs) and their differentially expressed gene targets (DEGs) data. The Jaccard index (JI) was used for gauging the similarity and diversity among these cell populations. Whereas BM-MOs, BM-iMOs, and SP-MOs presented high similarity of DEGs, SP-MODCs distinguished by showing a greater number of DEGs. Moreover, functional analysis identified an enrichment in canonical pathways, such as DC maturation, neuroinflammation, and IFN signaling. Upstream regulator analysis identified IFNγ as the potential upstream molecule that can explain the observed DEMs–Target DEGs intersections in SP-MODCs. Finally, directed target analysis and in vivo/ex vivo assays indicate that SP-MODCs differentiate in the SP and IFNγ is a main driver of this process.

Biological rhythms appear to be an elegant solution to the challenge of coordinating activities with the consequences of the Earth's daily and seasonal rotation. The genes and molecular mechanisms underpinning circadian clocks in multicellular organisms are well understood. In contrast, the regulatory mechanisms and fitness consequences of biological rhythms exhibited by parasites remain mysterious. Here, we explore how periodicity in parasite traits is generated and why daily rhythms matter for parasite fitness. We focus on malaria (Plasmodium) parasites which exhibit developmental rhythms during replication in the mammalian host's blood and in transmission to vectors. Rhythmic in-host parasite replication is responsible for eliciting inflammatory responses, the severity of disease symptoms, and fueling transmission, as well as conferring tolerance to anti-parasite drugs. Thus, understanding both how and why the timing and synchrony of parasites are connected to the daily rhythms of hosts and vectors may make treatment more effective and less toxic to hosts.

The pathogenesis of malaria is a multifactorial syndrome associated with a deleterious inflammatory response that is responsible for many of the clinical manifestations. While dendritic cells (DCs) play a critical role in initiating acquired immunity and host resistance to infection, they also play a pathogenic role in inflammatory diseases. In our recent studies, we found in different rodent malaria models that the monocyte-derived DCs (MO-DCs) become, transiently, a main DC population in spleens and inflamed non-lymphoid organs. These studies suggest that acute infection with Plasmodium berghei promotes the differentiation of splenic monocytes into inflammatory monocytes (iMOs) and thereafter into MO-DCs that play a pathogenic role by promoting inflammation and tissue damage. The recruitment of MO-DCs to the lungs and brain are dependent on expression of CCR4 and CCR5, respectively, and expression of respective chemokine ligands in each organ. Once they reach the target organ the MO-DCs produce the CXCR3 ligands (CXCL9 and CXCL10), recruit CD8+ T cells, and produce toxic metabolites that play an important role in the development of experimental cerebral malaria (ECM) and acute respiratory distress syndrome (ARDS).

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1β and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.

Cellular nucleic acid–binding protein (Cnbp) regulates IL-12β transcription and IL-12–driven, Th1-mediated immune responses., In this issue of JEM, the study by Chen et al. (https://doi.org/10.1084/jem.20181031) reveals a previously unrecognized role of cellular nucleic acid–binding protein (Cnbp) as a novel transcriptional regulator of interleukin-12β (IL-12β) transcription and IL-12–driven, Th1-mediated immune responses, which has important implications for both host defense and inflammatory disease.

SummaryAn inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Herein, we identified Cellular nucleic acid binding protein (Cnbp) as a specific regulator of interleukin-12β gene transcription and Th1 immunity. Cnbp resides in the cytosol of macrophages and translocates to the nucleus in response to a broad range of microbial ligands. Cnbp-deficient macrophages had a selective impairment in their ability to induce IL12β gene transcription. Cnbp interacted with c-Rel, an NFκB/Rel family member that controls IL12β gene transcription. c-Rel nuclear translocation and DNA binding activity were dependent on Cnbp. Furthermore, Cnbp itself bound the IL12β promoter. Lastly, Cnbp-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL12β, as well as a reduced Th1 cell IFNγ response essential to control parasite replication. Collectively, these findings identify Cnbp as a key regulator of c-Rel dependent IL12β gene transcription and Th1 immunity.

Prostaglandin E2 (PGE2) suppresses macrophage effector mechanisms; however, little is known about the function of PGD2 in infected alveolar macrophages (AMs). Using serum-opsonized Histoplasma capsulatum (Ops-H. capsulatum) in vitro, we demonstrated that AMs produced PGE2 and PGD2 in a time-dependent manner, with PGE2 levels exceeding those of PGD2 by 48 h postinfection. Comparison of the effects of both exogenous PGs on AMs revealed that PGD2 increased phagocytosis and killing through the chemoattractant receptor-homologous molecule expressed on Th2 lymphocytes receptor, whereas PGE2 had opposite effects, through E prostanoid (EP) receptor 2 (EP2)/EP4-dependent mechanisms. Moreover, PGD2 inhibited phospholipase C-γ (PLC-γ) phosphorylation, reduced IL-10 production, and increased leukotriene B4 receptor expression. In contrast, exogenous PGE2 treatment reduced PLC-γ phosphorylation, p38 and nuclear factor κB activation, TNF-α, H2O2, and leukotriene B4, but increased IL-1β production. Using specific compounds to inhibit the synthesis of each PG in vitro and in vivo, we found that endogenous PGD2 contributed to fungicidal mechanisms and controlled inflammation, whereas endogenous PGE2 decreased phagocytosis and killing of the fungus and induced inflammation. These findings demonstrate that, although PGD2 acts as an immunostimulatory mediator to control H. capsulatum infection, PGE2 has immunosuppressive effects, and the balance between these two PGs may limit collateral immune damage at the expense of microbial containment.

Summary The Plasmodium cell cycle, wherein millions of parasites differentiate and proliferate, occurs in synchrony with the vertebrate host's circadian cycle. The underlying mechanisms are unknown. Here we addressed this question in a mouse model of Plasmodium chabaudi infection. Inflammatory gene expression and carbohydrate metabolism are both enhanced in interferon-γ (IFNγ)-primed leukocytes and liver cells from P. chabaudi-infected mice. Tumor necrosis factor α (TNFα) expression oscillates across the host circadian cycle, and increased TNFα correlates with hypoglycemia and a higher frequency of non-replicative ring forms of trophozoites. Conversely, parasites proliferate and acquire biomass during food intake by the host. Importantly, cyclic hypoglycemia is attenuated and synchronization of P. chabaudi stages is disrupted in IFNγ−/−, TNF receptor−/−, or diabetic mice. Hence, the daily rhythm of systemic TNFα production and host food intake set the pace for Plasmodium synchronization with the host's circadian cycle. This mechanism indicates that Plasmodium parasites take advantage of the host's feeding habits.

Background Halofuginone (HF) is a low-molecular-weight alkaloid that has been demonstrated to interfere with Metalloproteinase-2 (MMP-2) and Tumor Growth Factor-β (TGF-β) function and, to present antiangiogenic, antiproliferative and proapoptotic properties in several solid tumor models. Based on the fact that high levels of Vascular Endothelial Growth Factor (VEGF) and increased angiogenesis have been described in acute myeloid leukemia and associated with disease progression, we studied the in vivo effects of HF using an Acute Promyelocytic Leukemia (APL) mouse model. Methods NOD/SCID mice were transplanted with leukemic cells from hCG-PML/RARA transgenic mice (TM) and treated with HF 150 μg/kg/day for 21 days. The leukemic infiltration and the percentage of VEGF+ cells were evaluated by morphology and flow cytometry. The effect of HF on the gene expression of several pro- and antiangiogenic factors, phosphorylation of SMAD2 and VEGF secretion was assessed in vitro using NB4 and HUVEC cells. Results HF treatment resulted in hematological remission with decreased accumulation of immature cell and lower amounts of VEGF in BM of leukemic mice. In vitro, HF modulated gene expression of several pro- and antiangiogenic factors, reduced VEGF secretion and phosphorylation of SMAD2, blocking TGF-β-signaling. Conclusion Taken together, our results demonstrate that HF inhibits SMAD2 signaling and reduces leukemia growth and angiogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s13046-015-0181-2) contains supplementary material, which is available to authorized users.

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB(4). However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-α and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-γ. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NFκB but was not dependent on peroxisome proliferator-activated receptor-γ (PPAR-γ) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-IκBα formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.

Promyelocytic leukemia-retinoic acid receptor alpha (PML-RARα) expression in acute promyelocytic leukemia (APL) impairs transforming growth factor beta (TGFβ) signaling, leading to cell growth advantage. Halofuginone (HF), a low-molecular-weight alkaloid that modulates TGFβ signaling, was used to treat APL cell lines and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice subjected to transplantation with leukemic cells from human chorionic gonadotrophin-PML-RARα transgenic mice (TG). Cell cycle analysis using incorporated bromodeoxyuridine and 7-amino-actinomycin D showed that, in NB4 and NB4-R2 APL cell lines, HF inhibited cellular proliferation (P

Made available in DSpace on 2014-12-03T13:09:12Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-01-21Bitstream added on 2014-12-03T13:23:14Z : No. of bitstreams: 1 WOS000330244500034.pdf: 682376 bytes, checksum: 8a807f26b964e0bd71f9341cd7902561 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) National Institutes of Health (NIH) The bioactive lipid mediator leukotriene B-4 (LTB4) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B-4 receptor 1 (BLT1) and decreased LTB4 production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB4 than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB4 production and BLT1 signaling are required for a histoplasmosis-resistant phenotype. Univ Sao Paulo, Dept Anal Clin Toxicol & Bromatol, Fac Ciencias Farmaceut Ribeirao Preto, Sao Paulo, Brazil Univ Sao Paulo, Dept Odontopediat, Fac Odontol Ribeirao Preto, Sao Paulo, Brazil Indiana Univ Sch Med, Dept Microbiol & Immunol, Indianapolis, IN 46202 USA Univ Estadual Paulista, Dept Ciencias Biol, Fac Ciencias Farmaceut, Sao Paulo, Brazil Univ Estadual Paulista, Dept Ciencias Biol, Fac Ciencias Farmaceut, Sao Paulo, Brazil CNPq: 302097/2010-4 National Institutes of Health (NIH)HL103777-04

Differentiation of Th17 cells requires the combination of IL-6 and TGF-β and is further maintained by IL-23. This T cell subtype is important for both host defense against extracellular pathogens and the pathogenesis of autoimmune diseases. The main cytokines produced by Th17 cells are IL-17, which induces strong proinflammatory effects, and IL-22, an enhancer of epithelial proliferation and healing responses. Previous research has demonstrated that the expression of the transcription factor c-Maf is important for inhibition of IL-22 production in Th17 cells. The transcription factor AHR (aryl hydrocarbon receptor) has also been shown crucial for proper Th17 differentiation. The adapter protein PTIP (Pax transactivation domain-interacting protein) has recently been shown to have a role in the maturation of thymocytes. PTIP stably associates with complexes containing the chromatin methyltransferases MLL3 and MLL4, which modify chromatin in order to activate gene transcription. Here we demonstrate that PTIP contributes to Th17 differentiation and function via regulation of both c-Maf and AHR. Using CD4+ T cells from Ptipf/fCD4 Cre+ (PTIP−/−) mice and Cre- (WT) controls in a T cell transfer model of chronic colitis, we determined that RAG2−/− mice receiving WT cells had aggressive colitis with high expression of IL-17 in the intestine. However, in mice receiving PTIP−/− cells there was a reduction in IL-17 and an increase in IL-22 expression. The change in IL-17 and IL-22 expression in PTIP−/− mice was confirmed in vitro. Further analysis by chromatin immunoprecipitation (ChIP) assay showed that MLL3 binding and its dependent chromatin modifications at the loci for IL-17, c-Maf and AHR were enhanced in WT Th17 cells when compared to Th0 conditions and reduced in PTIP−/− Th17 cells. These data highlight, for the first time, that PTIP is essential for proper differentiation of Th17 cells.

Abstract 1845 Acute Promyelocytic Leukemia (APL) patients present increased bone marrow microvascular density (MVD) compared to normal bone marrow, which has been associated with the aberrant secretion of the proangiogenic factor VEGF by leukemic cells. The APL associated fusion protein PML-RARα is thought to deregulate the TGF-β pathway, through its dominant negative action on cytoplasmic PML, thus down regulating SMAD2/3 signaling, and VEGF transcription. However, PML-RARα expression was associated with increased TGF-β gene transcription and secretion. We used the low molecular weight quinazolinone alkaloid Halofuginone (HF), which has been shown to be a potent TGF-β inhibitor, to test the association between TGF-β/VEGF/angiogenesis. HF inhibited the VEGF secretion by NB4 (an APL cell lineage) and cell proliferation. To determine the effects of HF in vivo, irradiated NOD/SCID mice were transplanted with leukemic cell from hCG-PML-RARα transgenic mice. Twenty-four hours after transplantation, mice were treated with 150μg/kg of HF by intraperitoneal injections for 21 days. All recipients developed leukemia, however the leukemic infiltration of bone marrow and WBC were significantly lower in animals treated HF (4.2 ± 3.89 vs. 20.6 ± 21.9, p Disclosures: Assis: FAPESP: Research Funding; CNPq: Research Funding.

Background: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. Results: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. Conclusions: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.