2Leishmania spp. are obligate intracellular parasites that inhabit the phagolysosomes of macrophages. Manipulation of host cell signaling pathways and gene expression by Leishmania is critical for Leishmania's survival and resultant pathology. Here, we show that infection of macrophages with Leishmania promastigotes in vitro causes specific cleavage of the NF-jB p65RelA subunit. Cleavage occurs in the cytoplasm and is dependent on the Leishmania protease gp63. The resulting fragment, p35RelA , migrates to the nucleus, where it binds DNA as a heterodimer with NF-jB p50. Importantly, induction of chemokine gene expression (MIP-2/CXCL2, MCP-1/CCL2, MIP-1a/CCL3, MIP-1b/CCL4) by Leishmania is NF-jB dependent, which implies that p35 RelA /p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF-jB cleavage was observed following infection with a range of pathogenic species, including L. donovani, L. major, L. mexicana, and L. (Viannia) braziliensis, but not the non-pathogenic L. tarentolae or treatment with IFN-c. These results indicate a novel mechanism by which a pathogen can subvert a macrophage's regulatory pathways to alter NF-jB activity.

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.

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.

Chemokine production has been associated with leukocyte infiltration into the joint during gouty arthritis, and monosodium urate (MSU) crystals, the causative agent of this arthropathy, have been shown to modulate their expression. In the present study, we investigated the transductional mechanisms underlying this cellular regulation in the murine macrophage cell line B10R. We report that MSU crystals rapidly and transiently increase mRNA levels of various chemokines in a concentration-dependent manner. Examination of second messenger activation revealed that macrophage exposure to MSU crystals led to MEK1/2, ERK1/2, and inhibitory protein B␣ phosphorylation as well as to NF-B and AP-1 nuclear translocation. Of interest, specific blockage of the ERK1/2 pathway drastically reduced up-modulation of MSU crystal-mediated chemokine production and activation of nuclear factors. Similarly, selective inhibition of NF-B suppressed NF-B DNA binding activity and the induction of all chemokine transcripts. These findings indicate that ERK1/2-dependent signals seem to be required for AP-1 and NF-B activation and subsequent mRNA expression of the various macrophage chemokines. In addition, transcription and stability assays performed in presence of actinomycin D showed that MSU crystal-mediated MIP-1␤ mRNA up-regulation resulted solely from transcriptional control, whereas that of MIP-1␣, MIP-2, and MCP-1 was due to both gene transcription activation and mRNA posttranscriptional stabilization. Overall, the results of this study help to define the molecular events that govern macrophage chemokine regulation in response to MSU crystals, which is of paramount importance to better understand, and eventually to tame, the inflammatory response during acute gout.

We have characterized cDNA clones of 1G5, an mRNA highly enriched in the mammalian forebrain that encodes a 504-residue protein found in association with perikaryal membranes and neurites. The protein, which accumulates predominantly postnatally, is associated with vesicles in both axons and dendrites. The sequence of the 1G5 protein highly resembles those of protein kinases with serine/threonine specificity; however, although most residues universally conserved among protein kinases are present, a few signature residues are absent from the 1G5 protein. Furthermore, although recombinant 1G5 protein binds calmodulin in the presence of calcium, it lacks kinase activity with a sample substrate.