David J. Gregory

Subversion Mechanisms by Which Leishmania Parasites Can Escape the Host Immune Response: a Signaling Point of View

SUMMARY The obligate intracellular parasite Leishmania must survive the antimicrobial activities of its host cell, the macrophage, and prevent activation of an effective immune response. In order to do this, it has developed numerous highly successful strategies for manipulating activities, including antigen presentation, nitric oxide and oxygen radical generation, and cytokine production. This is generally the result of interactions between Leishmania cell surface molecules, particularly gp63 and LPG, and less well identified macrophage surface receptors, causing the distortion of specific intracellular signaling cascades. We describe some of the signaling pathways and intermediates that are repressed in infected cells, including JAK/STAT, Ca2+-dependent protein kinase C (PKC) isoforms, and mitogen-activated protein kinases (especially ERK1/2), and proteasome-mediated transcription factor degradation. We also discuss protein tyrosine phosphatases (particularly SHP-1), intracellular Ca2+, Ca2+-independent PKC, ceramide, and the suppressors of cytokine signaling family of repressors, which are all reported to be activated following infection, and the role of parasite-secreted cysteine proteases.

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53‐dependent promoters and that endogenous p68 and p53 co‐immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53‐dependent apoptosis, but does not influence p53 stabilisation or expression of non‐p53‐responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53‐dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock‐down does not significantly affect NF‐κB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.

The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner

Backgroundp68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ERα). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation.ResultsIn this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein.ConclusionsIt is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

Role of Host Protein Tyrosine Phosphatase SHP-1 in Leishmania donovani-Induced Inhibition of Nitric Oxide Production

ABSTRACT In order to survive within the macrophages of its host organism, the protozoan parasite Leishmania inhibits a number of critical, gamma interferon (IFN-γ)-inducible, macrophage functions, including the generation of nitric oxide. We have previously shown that the protein tyrosine phosphatase SHP-1 (Src-homology 2 domain containing phosphatase-1) is activated during Leishmania infection and plays an important role in both the survival of Leishmania within cultured macrophages and disease progression in vivo by inhibiting nitric oxide production. Here we use a SHP-1−/− macrophage cell line derived from motheaten mice to address the mechanisms by which SHP-1 prevents IFN-γ-dependent nitric oxide production during Leishmania donovani infection. We show that Leishmania inhibits nitric oxide production in response to IFN-γ poorly in SHP-1-deficient macrophages. This correlates with the inability of Leishmania to alter JAK2 and mitogen-activated protein kinase extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation and to prevent nuclear translocation of transcription factors NF-κB and AP-1, although the latter two to a lesser extent. Surprisingly, Leishmania inactivated the transcription factor STAT1 to a similar extent in SHP-1-deficient and wild-type macrophages, so STAT1 is not necessary for nitric oxide production by infected macrophages. Overall, this study demonstrates that induction of SHP-1 by Leishmania is vital for inhibition of nitric oxide generation and that this inhibition occurs through the inactivation of JAK2 and ERK1/2, and transcription factors NF-κB and AP-1.

A novel form of NF-κB is induced by Leishmania infection : Involvement in macrophage gene expression

Leishmania 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‐κB 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‐κB p50. Importantly, induction of chemokine gene expression (MIP‐2/CXCL2, MCP‐1/CCL2, MIP‐1α/CCL3, MIP‐1β/CCL4) by Leishmania is NF‐κB dependent, which implies that p35RelA/p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF‐κB 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‐γ. These results indicate a novel mechanism by which a pathogen can subvert a macrophages regulatory pathways to alter NF‐κB activity.

Subversion of host cell signalling by the protozoan parasite Leishmania

The protozoa Leishmania spp. are obligate intracellular parasites that inhabit the macrophages of their host. Since macrophages are specialized for the identification and destruction of invading pathogens, both directly and by triggering an innate immune response, Leishmania have evolved a number of mechanisms for suppressing some critical macrophage activities. In this review, we discuss how various species of Leishmania distort the host macrophages own signalling pathways to repress the expression of various cytokines and microbicidal molecules (nitric oxide and reactive oxygen species), and antigen presentation. In particular, we describe how MAP Kinase and JAK/STAT cascades are repressed, and intracellular Ca2+ and the activities of protein tyrosine phosphatases, in particular SHP-1, are elevated.

Proteasome-mediated Degradation of STAT1α following Infection of Macrophages with Leishmania donovani

Activation of the Janus-activated kinase 2 (JAK2)/STAT1α signaling pathway is repressed in Leishmania-infected macrophages. This represents an important mechanism by which this parasite subverts the microbicidal functions of the cell to promote its own survival and propagation. We recently provided evidence that the protein tyrosine phosphatase (PTP) SHP-1 was responsible for JAK2 inactivation. However, STAT1 translocation to the nucleus was not restored in the absence of SHP-1. In the present study, we have used B10R macrophages to study the mechanism by which this Leishmania-induced STAT1 inactivation occurs. STAT1α nuclear localization was shown to be rapidly reduced by the infection. Western blot analysis revealed that cellular STAT1α, but not STAT3, was degraded. Using PTP inhibitors and an immortalized bone marrow-derived macrophage cell line from SHP-1-deficient mice, we showed that STAT1 inactivation was independent of PTP activity. However, inhibition of macrophage proteasome activity significantly rescued Leishmania-induced STAT1α degradation. We further demonstrated that degradation was receptor-mediated and involved protein kinase Cα. All Leishmania species tested (L. major, L. donovani, L. mexicana, L. braziliensis), but not the related parasite Trypanosoma cruzi, caused STAT1α degradation. Collectively, results from this study revealed a new mechanism for STAT1 regulation by a microbial pathogen, which favors its establishment and propagation within the host.

Comparison of the effects of Leishmania major or Leishmania donovani infection on macrophage gene expression.

ABSTRACT The intracellular parasite Leishmania causes a wide spectrum of human disease, ranging from self-resolving cutaneous lesions to fatal visceral disease, depending on the species of Leishmania involved. The mechanisms by which different Leishmania species cause different pathologies are largely unknown. We have addressed this question by comparing the gene expression profiles of bone marrow-derived macrophages infected with either Leishmania donovani or L. major promastigotes. We found that the two species had very similar effects on macrophage gene expression. Both species caused a small (<2.5-fold) but statistically significant repression of several hundred genes. In addition, both species strongly induced and repressed about 60 genes. Comparing the effects of the two species showed that only 26 genes were regulated differently by L. major as opposed to L. donovani, including those for metallothioneins 1 and 2, HSP70 and -72, CCL4, Gadd45β, Dsp1, matrix metalloprotease 13, T-cell death-associated gene 51 (Tdag51), RhoB, spermine/spermidine N1-acyl transferase 1 (SSAT), and Cox2. L. donovani-infected macrophages were also found to express higher levels of Cox2 protein and prostaglandin E synthase mRNA than L. major-infected macrophages. While both species have previously been shown to trigger prostaglandin E synthesis by bystander cells, this study suggests that infected macrophages themselves express prostaglandin E-synthesizing genes only in response to L. donovani.

MARCO regulates early inflammatory responses against influenza: a useful macrophage function with adverse outcome.

Lung macrophages use the scavenger receptor MARCO to bind and ingest bacteria, particulate matter, and post cellular debris. We investigated the role of MARCO in influenza A virus (IAV) pneumonia. In contrast to higher susceptibility to bacterial infection, MARCO(-/-) mice had lower morbidity and mortality from influenza pneumonia than wild-type (WT) mice. The early course of influenza in MARCO(-/-) lungs was marked by an enhanced but transient neutrophilic inflammatory response and significantly lower viral replication compared with the WT mice. At later time points, no significant differences in lung histopathology or absolute numbers of T lymphocyte influx were evident. Uptake of IAV by WT and MARCO(-/-) bronchoalveolar lavage macrophages in vitro was similar. By LPS coadministration, we demonstrated that rapid neutrophil and monocyte influx during the onset of influenza suppressed viral replication, indicating a protective role of early inflammation. We hypothesized that the presence of increased basal proinflammatory post cellular debris in the absence of scavenging function lowered the inflammatory response threshold to IAV in MARCO(-/-) mice. Indeed, MARCO(-/-) mice showed increased accumulation of proinflammatory oxidized lipoproteins in the bronchoalveolar lavage early in the infection process, which are the potential mediators of the observed enhanced inflammation. These results indicate that MARCO suppresses a protective early inflammatory response to influenza, which modulates viral clearance and delays recovery.

Induction of transcription through the p300 CRD1 motif by p21WAF1/CIP1 is core promoter specific and cyclin dependent kinase independent.

The tumor suppressors p300 and CREB-binding protein (CBP) are both multifunctional transcriptional coactivators. We have previously found that the cyclin dependent kinase (CDK) inhibitor p21WAF1/CIP1 can stimulate transactivation by p300 and CBP through inhibiting transcriptional repression by a discrete domain within these proteins termed CRD1. Given the large number of p300/CBP associated functions, it is unlikely that p21 regulates the expression of every gene under their control, however. Here we have investigated the factors that help determine this specificity. We have discovered that while CRD1 can repress the activity of p300 at multiple promoters, induction of transcription by p21 though this motif is highly variable. Analysis of this effect revealed that p21 inducibility is determined by sequences flanking the TATA box. Significantly, p21 regulation of CRD1 domain function is independent of Cyclin /CDK inhibition suggesting a novel function of this protein. p21 does not interact directly with the CRD1 motif, however. These results give further insight into how regulators of cell growth and tumorigenesis, such as p21, can specifically target and induce the expression of select groups of genes. Key Words: p21, p300, CBP, TATA box, Transcription