A Listeria monocytogenes-based vaccine that secretes sand fly salivary protein LJM11 confers long-term protection against vector-transmitted Leishmania major.

Cutaneous leishmaniasis is a sand fly-transmitted disease characterized by skin ulcers that carry significant scarring and social stigmatization. Over the past years, there has been cumulative evidence that immunity to specific sand fly salivary proteins confers a significant level of protection against leishmaniasis. In this study, we used an attenuated strain of Listeria monocytogenes as a vaccine expression system for LJM11, a sand fly salivary protein identified as a good vaccine candidate. We observed that mice were best protected against an intradermal needle challenge with Leishmania major and sand fly saliva when vaccinated intravenously. However, this protection was short-lived. Importantly, groups of vaccinated mice were protected long term when challenged with infected sand flies. Protection correlated with smaller lesion size, fewer scars, and better parasite control between 2 and 6 weeks postchallenge compared to the control group of mice vaccinated with the parent L. monocytogenes strain not expressing LJM11. Moreover, protection correlated with high numbers of CD4(+), gamma interferon-positive (IFN-γ(+)), tumor necrosis factor alpha-positive/negative (TNF-α(+/-)), interleukin-10-negative (IL-10(-)) cells and low numbers of CD4(+) IFN-γ(+/-) TNF-α(-) IL-10(+) T cells at 2 weeks postchallenge. Overall, our data indicate that delivery of LJM11 by Listeria is a promising vaccination strategy against cutaneous leishmaniasis inducing long-term protection against ulcer formation following a natural challenge with infected sand flies.

Misregulation of the broad-range phospholipase C activity increases the susceptibility of Listeria monocytogenes to intracellular killing by neutrophils.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that tightly regulates the activities of various virulence factors during infection. A mutant strain (the plcBDpro mutant) that has lost the ability to control the activity of a phospholipase C (PC-PLC) is attenuated a hundred fold in mice. This attenuation is not due to a lack of bacterial fitness, but appears to result from a modified host response to infection. The transcriptomic pattern of immune-related genes indicated that PC-PLC did not enhance the innate immune response in infected macrophages. However, it partially protected the cells from bacteria-mediated mitochondrial fragmentation. In mice, the plcBDpro mutant transiently caused an increase in liver pathology, as judged by the size of neutrophil-filled micro-abscesses. Moreover, the plcBDpro mutant was more susceptible to intracellular killing by neutrophils than wild-type L. monocytogenes. Together, these data indicate that in vivo attenuation of the plcBDpro mutant results from its reduced ability to disrupt mitochondrial homeostasis and to resist intracellular killing by neutrophils.

Toxoplasma gondii inhibits mast cell degranulation by suppressing phospholipase Cγ-mediated Ca(2+) mobilization.

Toxoplasma gondii is well-known to subvert normal immune responses, however, mechanisms are incompletely understood. In particular, its capacity to alter receptor-activated Ca(2+)-mediated signaling processes has not been well-characterized. In initial experiments, we found evidence that T. gondii infection inhibits Ca(2+) responses to fMetLeuPhe in murine macrophages. To further characterize the mechanism of inhibition of Ca(2+) mobilization by T. gondii, we used the well-studied RBL mast cell model to probe the capacity of T. gondii to modulate IgE receptor-activated signaling within the first hour of infection. Ca(2+) mobilization that occurs via IgE/FcεRI signaling leads to granule exocytosis in mast cells. We found that T. gondii inhibits antigen-stimulated degranulation in infected cells in a strain-independent manner. Under these conditions, we found that cytoplasmic Ca(2+) mobilization, particularly antigen-mediated Ca(2+) release from intracellular stores, is significantly reduced. Furthermore, stimulation-dependent activation of Syk kinase leading to tyrosine phosphorylation and activation of phospholipase Cγ is inhibited by infection. Therefore, we conclude that inhibitory effects of infection are likely due to parasite-mediated inhibition of the tyrosine kinase signaling cascade that results in reduced hydrolysis of phosphatidylinositol 4,5-bisphosphate. Interestingly, inhibition of IgE/FcεRI signaling persists when tachyzoite invasion is arrested via cytochalasin D treatment, suggesting inhibition is mediated by a parasite-derived factor secreted into the cells during the invasion process. Our study provides direct evidence that immune subversion by T. gondii is initiated concurrently with invasion.

CCR2 and CD44 promote inflammatory cell recruitment during fatty liver formation in a lithogenic diet fed mouse model.

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a spectrum of presentations. The current study utilized a lithogenic diet model of NAFLD. The diet was fed to mice that are either resistant (AKR) or susceptible (BALB/c and C57BL/6) to hepatitis followed by molecular and flow cytometric analysis. Following this, a similar approach was taken in congenic mice with specific mutations in immunological genes. The initial study identified a significant and profound increase in multiple ligands for the chemokine receptor CCR2 and an increase in CD44 expression in susceptible C57BL/6 (B6) but not resistant AKR mice. Ccr2(-/-) mice were completely protected from hepatitis and Cd44(-/-) mice were partially protected. Despite protection from inflammation, both strains displayed similar histological steatosis scores and significant increases in serum liver enzymes. CD45(+)CD44(+) cells bound to hyaluronic acid (HA) in diet fed B6 mice but not Cd44(-/-) or Ccr2(-/-) mice. Ccr2(-/-) mice displayed a diminished HA binding phenotype most notably in monocytes, and CD8(+) T-cells. In conclusion, this study demonstrates that absence of CCR2 completely and CD44 partially reduces hepatic leukocyte recruitment. These data also provide evidence that there are multiple redundant CCR2 ligands produced during hepatic lipid accumulation and describes the induction of a strong HA binding phenotype in response to LD feeding in some subsets of leukocytes from susceptible strains.

Toxoplasma gondii triggers phosphorylation and nuclear translocation of dendritic cell STAT1 while simultaneously blocking IFNγ-induced STAT1 transcriptional activity.

The protozoan Toxoplasma gondii actively modulates cytokine-induced JAK/STAT signaling pathways to facilitate survival within the host, including blocking IFNγ-mediated STAT1-dependent proinflammatory gene expression. We sought to further characterize inhibition of STAT1 signaling in infected murine dendritic cells (DC) because this cell type has not previously been examined, yet is known to serve as an early target of in vivo infection. Unexpectedly, we discovered that T. gondii infection alone induced sustained STAT1 phosphorylation and nuclear translocation in DC in a parasite strain-independent manner. Maintenance of STAT1 phosphorylation required active invasion but intracellular parasite replication was dispensable. The parasite rhoptry protein ROP16, recently shown to mediate STAT3 and STAT6 phosphorylation, was not required for STAT1 phosphorylation. In combination with IFNγ, T. gondii induced synergistic STAT1 phosphorylation and binding of aberrant STAT1-containing complexes to IFNγ consensus sequence oligonucleotides. Despite these findings, parasite infection blocked STAT1 binding to the native promoters of the IFNγ-inducible genes Irf-1 and Lrg47, along with subsequent gene expression. These results reinforce the importance of parasite-mediated blockade of IFNγ responses in dendritic cells, while simultaneously showing that T. gondii alone induces STAT1 phosphorylation.

Neutrophils cast extracellular traps in response to protozoan parasites.

Release of extracellular traps by neutrophils is a now well-established phenomenon that contributes to the innate response to extracellular bacterial and fungal pathogens. The importance of NETs during protozoan infection has been less explored, but recent findings suggest an emerging role for release of neutrophil-derived extracellular DNA in response to this class of microbial pathogens. The present review summarizes findings to date regarding elicitation of NETs by Toxoplasma gondii, Plasmodium falciparum, Eimeria bovis, and Leishmania spp.

Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps.

Neutrophils have recently been shown to release DNA-based extracellular traps that contribute to microbicidal killing and have also been implicated in autoimmunity. The role of neutrophil extracellular trap (NET) formation in the host response to nonbacterial pathogens has received much less attention. Here, we show that the protozoan pathogen Toxoplasma gondii elicits the production of NETs from human and mouse neutrophils. Tachyzoites of each of the three major parasite strain types were efficiently entrapped within NETs, resulting in decreased parasite viability. We also show that Toxoplasma activates a MEK-extracellular signal-regulated kinase (ERK) pathway in neutrophils and that the inhibition of this pathway leads to decreased NET formation. To determine if Toxoplasma induced NET formation in vivo, we employed a mouse intranasal infection model. We found that the administration of tachyzoites by this route induced a rapid tissue recruitment of neutrophils with evidence of extracellular DNA release. Taken together, these data indicate a role for NETs in the host innate response to protozoan infection. We propose that NET formation limits infection by direct microbicidal effects on Toxoplasma as well as by interfering with the ability of the parasite to invade target host cells.

Mouse neutrophils are professional antigen-presenting cells programmed to instruct Th1 and Th17 T-cell differentiation.

Neutrophils play a major role in the innate immune system and are normally considered to be short-lived effector cells that exert anti-microbial activity and sometimes immunopathology. Here, we show that these cells possess an additional function as professional antigen-presenting cells capable of priming a T(h)1- and T(h)17-acquired immune response. Using flow cytometry, fluorescence microscopy and western blotting, we show that mouse neutrophils express MHC class II and co-stimulatory molecules CD80 and CD86 after T-cell co-incubation. Neutrophils pulsed with ovalbumin (OVA) process and present peptide antigen to OVA-specific T cells in an MHC class II-dependent manner. Importantly, we demonstrate that neutrophils can prime antigen-specific T(h)1 and T(h)17 immune responses even without the addition of exogenous cytokines to cell cultures.

Toxoplasma gondii prevents chromatin remodeling initiated by TLR-triggered macrophage activation.

Macrophages infected with the opportunistic protozoan Toxoplasma gondii are unable to up-regulate many proinflammatory cytokine genes, including TNF (TNF-alpha), upon stimulation with LPS and other TLR ligands. In this study, we examined the influence of T. gondii on transcription factors associated with TNF-alpha transcription, as well as phosphorylation and acetylation of histone H3 at distal and proximal regions of the TNF-alpha promoter. During LPS stimulation, we found that Toxoplasma blocks nuclear accumulation of transcription factor c-Jun, but not that of cAMP response element-binding protein or NF-kappaB. However, chromatin immunoprecipitation studies revealed that binding of all of these transcription factors to the TNF promoter was decreased by T. gondii infection. Furthermore, the parasite blocked LPS-induced Ser(10) phosphorylation and Lys(9)/Lys(14) acetylation of histone H3 molecules associated with distal and proximal regions of the TNF-alpha promoter. Our results show that Toxoplasma inhibits TNF-alpha transcription by interfering with chromatin remodeling events required for transcriptional activation at the TNF promoter, revealing a new mechanism by which a eukaryotic pathogen incapacitates proinflammatory cytokine production during infection.