Maritza Jaramillo

Translational control of the innate immune response through IRF-7

Transcriptional activation of cytokines, such as type-I interferons (interferon (IFN)-α and IFN-β), constitutes the first line of antiviral defence. Here we show that translational control is critical for induction of type-I IFN production. In mouse embryonic fibroblasts lacking the translational repressors 4E-BP1 and 4E-BP2, the threshold for eliciting type-I IFN production is lowered. Consequently, replication of encephalomyocarditis virus, vesicular stomatitis virus, influenza virus and Sindbis virus is markedly suppressed. Furthermore, mice with both 4E- and 4E-BP2 genes (also known as Eif4ebp1 and Eif4ebp2, respectively) knocked out are resistant to vesicular stomatitis virus infection, and this correlates with an enhanced type-I IFN production in plasmacytoid dendritic cells and the expression of IFN-regulated genes in the lungs. The enhanced type-I IFN response in 4E-BP1-/- 4E-BP2-/- double knockout mouse embryonic fibroblasts is caused by upregulation of interferon regulatory factor 7 (Irf7) messenger RNA translation. These findings highlight the role of 4E-BPs as negative regulators of type-I IFN production, via translational repression of Irf7 mRNA.

Hemozoin induces macrophage chemokine expression through oxidative stress-dependent and -independent mechanisms.

Chemokine production has been associated with the immunopathology related to malaria. Previous findings indicated that hemozoin (HZ), a parasite metabolite released during schizogeny, might be an important source of these proinflammatory mediators. In this study we investigated the molecular mechanisms underlying HZ-inducible macrophage (Mφ) chemokine mRNA expression. We found that both Plasmodium falciparum HZ and synthetic HZ increase mRNA levels of various chemokine transcripts (MIP-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and MCP-1/CCL2) in murine B10R Mφ. The cellular response to HZ involved ERK1/2 phosphorylation, NF-κB activation, reactive oxygen species (ROS) generation, and ROS-dependent protein-tyrosine phosphatase down-regulation. Selective inhibition of either IκBα or the ERK1/2 pathway abolished both NF-κB activation and chemokine up-regulation. Similarly, blockage of HZ-inducible Mφ ROS with superoxide dismutase suppressed chemokine induction, strongly reduced NF-κB activation, and restored HZ-mediated Mφ protein-tyrosine phosphatase inactivation. In contrast, superoxide dismutase had no effect on EKR1/2 phosphorylation by HZ. Collectively, these data indicate that HZ triggers ROS-dependent and -independent signals, leading to increased chemokine mRNA expression in Mφ. Overall, our findings may help to better understand the molecular mechanisms through which parasite components, such as HZ, modulate the immune response during malaria infection.

Hemozoin-Inducible Proinflammatory Events In Vivo: Potential Role in Malaria Infection

During malaria infection, high levels of proinflammatory molecules (e.g., cytokines, chemokines) correlate with disease severity. Even if their role as activators of the host immune response has been studied, the direct contribution of hemozoin (HZ), a parasite metabolite, to such a strong induction is not fully understood. Previous in vitro studies demonstrated that both Plasmodium falciparum HZ and synthetic HZ (sHZ), β-hematin, induce macrophage/monocyte chemokine and proinflammatory cytokine secretion. In the present study, we investigated the proinflammatory properties of sHZ in vivo. To this end, increasing doses of sHZ were injected either i.v. or into an air pouch generated on the dorsum of BALB/c mice over a 24-h period. Our results showed that sHZ is a strong modulator of leukocyte recruitment and more specifically of neutrophil and monocyte populations. In addition, evaluation of chemokine and cytokine mRNA and protein expression revealed that sHZ induces the expression of chemokines, macrophage-inflammatory protein (MIP)-1α/CCL3, MIP-1β/CCL4, MIP-2/CXCL2, and monocyte chemoattractant protein-1/CCL2; chemokine receptors, CCR1, CCR2, CCR5, CXCR2, and CXCR4; cytokines, IL-1β and IL-6; and myeloid-related proteins, S100A8, S100A9, and S100A8/A9, in the air pouch exudates. Of interest, chemokine and cytokine mRNA up-regulation were also detected in the liver of i.v. sHZ-injected mice. In conclusion, our study demonstrates that sHZ is a potent proinflammatory agent in vivo, which could contribute to the immunopathology related to malaria.

Hemozoin Increases IFN-γ-Inducible Macrophage Nitric Oxide Generation Through Extracellular Signal-Regulated Kinase- and NF-κB-Dependent Pathways

NO overproduction has been suggested to contribute to the immunopathology related to malaria infection. Even though a role for some parasite molecules (e.g., GPI) in NO induction has been proposed, the direct contribution of hemozoin (HZ), another parasite metabolite, remains to be established. Therefore, we were interested to determine whether Plasmodium falciparum (Pf) HZ and synthetic HZ, β-hematin, alone or in combination with IFN-γ, were able to induce macrophage (Mφ) NO synthesis. We observed that neither Pf HZ nor synthetic HZ led to NO generation in B10R murine Mφ; however, they significantly increased IFN-γ-mediated inducible NO synthase (iNOS) mRNA and protein expression, and NO production. Next, by investigating the transductional mechanisms involved in this cellular regulation, we established that HZ induces extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase phosphorylation as well as NF-κB binding to the iNOS promoter, and enhances the IFN-γ-dependent activation of both second messengers. Of interest, cell pretreatment with specific inhibitors against either NF-κB or the ERK1/2 pathway blocked the HZ + IFN-γ-inducible NF-κB activity and significantly reduced the HZ-dependent increase on IFN-γ-mediated iNOS and NO induction. Even though selective inhibition of the Janus kinase 2/STAT1α pathway suppressed NO synthesis in response to HZ + IFN-γ, HZ alone did not activate this signaling pathway and did not have an up-regulating effect on the IFN-γ-induced Janus kinase 2/STAT1α phosphorylation and STAT1α binding to the iNOS promoter. In conclusion, our results suggest that HZ exerts a potent synergistic effect on the IFN-γ-inducible NO generation in Mφ via ERK- and NF-κB-dependent pathways.

Signalling events involved in interferon-γ-inducible macrophage nitric oxide generation

Nitric oxide (NO) produced by macrophages (Mφ) in response to interferon‐γ (IFN‐γ) plays a pivotal role in the control of intracellular pathogens. Current knowledge of the specific biochemical cascades involved in this IFN‐γ‐inducible Mφ function is still limited. In the present study, we evaluated the participation of various second messengers – Janus kinase 2 (JAK2), signal transducer and activator of transcription (STAT) 1α, MAP kinase kinase (MEK1/2), extracellular signal‐regulated kinases 1 and 2 (Erk1/Erk2) and nuclear factor kappa B (NF‐κB) – in the regulation of NO production by IFN‐γ‐stimulated J774 murine Mφ. The use of specific signalling inhibitors permitted us to establish that JAK2/STAT1α‐ and Erk1/Erk2‐dependent pathways are the main players in IFN‐γ‐inducible Mφ NO generation. To determine whether the inhibitory effect was taking place at the pre‐ and/or post‐transcriptional level, we evaluated the effect of each antagonist on inducible nitric oxide synthase (iNOS) gene and protein expression, and on the capacity of IFN‐γ to induce JAK2, Erk1/Erk2 and STAT1α phosphorylation. All downregulatory effects occurred at the pretranscriptional level, except for NF‐κB, which seems to exert its role in NO production through an iNOS‐independent event. In addition, electrophoretic mobility shift assay (EMSA) analysis revealed that STAT1α is essential for IFN‐γ‐inducible iNOS expression and NO production, whereas the contribution of NF‐κB to this cellular regulation seems to be minimal. Moreover, our data suggest that Erk1/Erk2 are responsible for STAT1α Ser727 residue phosphorylation in IFN‐γ‐stimulated Mφ, thus contributing to the full activation of STAT1α. Taken together, our results indicate that JAK2, MEK1/2, Erk1/Erk2 and STAT1α are key players in the IFN‐γ‐inducible generation of NO by Mφ.

Leishmania Repression of Host Translation through mTOR Cleavage Is Required for Parasite Survival and Infection

The protozoan parasite Leishmania alters the activity of its host cell, the macrophage. However, little is known about the effect of Leishmania infection on host protein synthesis. Here, we show that the Leishmania protease GP63 cleaves the mammalian/mechanistic target of rapamycin (mTOR), a serine/threonine kinase that regulates the translational repressor 4E-BP1. mTOR cleavage results in the inhibition of mTOR complex 1 (mTORC1) and concomitant activation of 4E-BP1 to promote Leishmania proliferation. Consistent with these results, pharmacological activation of 4E-BPs with rapamycin, results in a dramatic increase in parasite replication. In contrast, genetic deletion of 4E-BP1/2 reduces parasite load in macrophages ex vivo and decreases susceptibility to cutaneous leishmaniasis in vivo. The parasite resistant phenotype of 4E-BP1/2 double-knockout mice involves an enhanced type I IFN response. This study demonstrates that Leishmania evolved a survival mechanism by activating 4E-BPs, which serve as major targets for host translational control.

Hydrogen Peroxide Induces Murine Macrophage Chemokine Gene Transcription Via Extracellular Signal-Regulated Kinase- and Cyclic Adenosine 5′-Monophosphate (cAMP)-Dependent Pathways: Involvement of NF-κB, Activator Protein 1, and cAMP Response Element Binding Protein

Hydrogen peroxide (H2O2) has been shown to act as a second messenger that activates chemokine expression. In the present study, we investigated the mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that H2O2 increases mRNA expression of various chemokines, macrophage-inflammatory protein (MIP)-1α/CC chemokine ligand (CCL)3, MIP-1β/CCL4, MIP-2/CXC chemokine ligand 2, and monocyte chemoattractant protein-1/CCL2, by activating the extracellular signal-regulated kinase (ERK) pathway and the nuclear translocation of the transcription factors NF-κB, AP-1, and CREB. Blockage of the ERK pathway with specific inhibitors against mitogen-activated protein kinase kinase 1/2 and ERK1/ERK2 completely abolished both the H2O2-mediated chemokine up-regulation and the activation of all NF studied. Similarly, selective inhibition of cAMP and NF-κB strongly down-regulated the induction of all chemokine transcripts as well as CREB and NF-κB activation, respectively. Of interest, we detected a significant decrease of NF-κB, AP-1, and CREB DNA binding activities by reciprocal competition for these binding sites when either specific cold oligonucleotides (NF-κB, AP-1, and CREB) or Abs against various transcription factor subunits (p50, p65, c-Fos, Jun B, c-Jun, and CREB-1) were added. These findings indicate that cooperation between ERK- and cAMP-dependent pathways seems to be required to achieve the formation of an essential transcriptional factor complex for maximal H2O2-dependent chemokine modulation. Finally, experiments performed with actinomycin D suggest that H2O2-mediated MIP-1β mRNA up-regulation results from transcriptional control, whereas that of MIP-1α, MIP-2, and monocyte chemoattractant protein-1 is due to both gene transcription activation and mRNA posttranscriptional stabilization.

Translational control of the activation of transcription factor NF-κB and production of type I interferon by phosphorylation of the translation factor eIF4E

Type I interferon is an integral component of the antiviral response, and its production is tightly controlled at the levels of transcription and translation. The eukaryotic translation-initiation factor eIF4E is a rate-limiting factor whose activity is regulated by phosphorylation of Ser209. Here we found that mice and fibroblasts in which eIF4E cannot be phosphorylated were less susceptible to virus infection. More production of type I interferon, resulting from less translation of Nfkbia mRNA (which encodes the inhibitor IκBα), largely explained this phenotype. The lower abundance of IκBα resulted in enhanced activity of the transcription factor NF-κB, which promoted the production of interferon-β (IFN-β). Thus, regulated phosphorylation of eIF4E has a key role in antiviral host defense by selectively controlling the translation of an mRNA that encodes a critical suppressor of the innate antiviral response.

Synthetic Plasmodium-Like Hemozoin Activates the Immune Response: A Morphology - Function Study

Increasing evidence points to an important role for hemozoin (HZ), the malaria pigment, in the immunopathology related to this infection. However, there is no consensus as to whether HZ exerts its immunostimulatory activity in absence of other parasite or host components. Contamination of native HZ preparations and the lack of a unified protocol to produce crystals that mimic those of Plasmodium HZ (PHZ) are major technical limitants when performing functional studies with HZ. In fact, the most commonly used methods generate a heterogeneous nanocrystalline material. Thus, it is likely that such aggregates do not resemble to PHZ and differ in their inflammatory properties. To address this issue, the present study was designed to establish whether synthetic HZ (sHZ) crystals produced by different methods vary in their morphology and in their ability to activate immune responses. We report a new method of HZ synthesis (the precise aqueous acid-catalyzed method) that yields homogeneous sHZ crystals (Plasmodium-like HZ) which are very similar to PHZ in their size and physicochemical properties. Importantly, these crystals are devoid of protein and DNA contamination. Of interest, structure-function studies revealed that the size and shape of the synthetic crystals influences their ability to activate inflammatory responses (e.g. nitric oxide, chemokine and cytokine mRNA) in vitro and in vivo. In summary, our data confirm that sHZ possesses immunostimulatory properties and underline the importance of verifying by electron microscopy both the morphology and homogeneity of the synthetic crystals to ensure that they closely resemble those of the parasite. Periodic quality control experiments and unification of the method of HZ synthesis are key steps to unravel the role of HZ in malaria immunopathology.

Monosodium Urate Crystals Synergize with IFN-γ to Generate Macrophage Nitric Oxide: Involvement of Extracellular Signal-Regulated Kinase 1/2 and NF-κB

Elevated NO production has been detected in patients suffering from various arthropathies; however, its role and regulation during gouty arthritis remain largely unexplored. Monosodium urate (MSU) crystals, the causative agent of gout, have been shown to induce NO generation in vivo and inducible NO synthase (iNOS) expression in human monocytes. The present study was designed to evaluate the ability of MSU crystals to modulate macrophage (Mφ) iNOS expression and NO synthesis and to investigate the molecular mechanisms underlying these cellular responses. We found that MSU crystals did not induce NO production in murine J774 Mφ. However, a synergistic effect on the level of iNOS expression and NO generation was observed in cells exposed to MSU crystals in combination with IFN-γ. Characterization of the second messengers involved revealed the requirement of IFN-γ-mediated Janus kinase 2/STAT1α activation even though MSU crystals did not modulate this signaling cascade by themselves. MSU crystals exerted their up-regulating effect by increasing extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and NF-κB nuclear translocation in response to IFN-γ. The use of specific inhibitors against either NF-κB or the ERK1/2 pathway significantly reduced MSU + IFN-γ-inducible NF-κB activity, iNOS expression, and NO production. Altogether, these data indicate that MSU crystals exert a potent synergistic effect on the IFN-γ-inducible Mφ NO generation via ERK1/2- and NF-κB-dependent pathways. Understanding the molecular mechanisms through which MSU crystals amplify Mφ responses to proinflammatory cytokines such as IFN-γ will contribute to better define their role in NO regulation during gout, in particular, and inflammation, in general.