Niraj Shrestha

Eukaryotic Initiation Factor 2 (eIF2) Signaling Regulates Proinflammatory Cytokine Expression and Bacterial Invasion

Background: eIF2 is a critical point of stress-induced regulation of translation in eukaryotic cells. Results: eIF2 signaling is activated by bacterial pathogens and regulates two key infection-associated processes. Conclusion: Regulation of translation in eukaryotic cells is involved in innate immune responses. Significance: These findings enlarge the possible targets for therapeutic interventions against bacterial pathogens. In eukaryotic cells, there are two well characterized pathways that regulate translation initiation in response to stress, and each have been shown to be targeted by various viruses. We recently showed in a yeast-based model that the bacterial virulence factor YopJ disrupts one of these pathways, which is centered on the α-subunit of the translation factor eIF2. Here, we show in mammalian cells that induction of the eIF2 signaling pathway occurs following infection with bacterial pathogens and that, consistent with our yeast-based findings, YopJ reduces eIF2 signaling in response to endoplasmic reticulum stress, heavy metal toxicity, dsRNA, and bacterial infection. We demonstrate that the well documented activities of YopJ, inhibition of NF-κB activation and proinflammatory cytokine expression, are both dependent on an intact eIF2 signaling pathway. Unexpectedly, we found that cells with defective eIF2 signaling were more susceptible to bacterial invasion. This was true for pathogenic Yersinia, a facultative intracellular pathogen, as well as for the intracellular pathogens Listeria monocytogenes and Chlamydia trachomatis. Collectively, our data indicate that the highly conserved eIF2 signaling pathway, which is vitally important for antiviral responses, plays a variety of heretofore unrecognized roles in antibacterial responses.

Effective Treatment of Erythema Nodosum Leprosum with Thalidomide Is Associated with Immune Stimulation

The immunomodulatory drug thalidomide is the treatment of choice for erythema nodosum leprosum (ENL), an inflammatory cutaneous and systemic complication of multibacillary leprosy. To elucidate the mechanism of action of thalidomide in this syndrome, we prospectively investigated 20 patients with ENL who were treated with thalidomide for 21 days. All patients responded to treatment, with the majority of them having complete resolution of cutaneous lesions within 7 days. This response was associated with a marked but transient increase in ex vivo mitogen-induced expression of interleukin (IL)-2 and interferon- gamma by CD4(+) and CD8(+) T cells that was observed on treatment day 7, but these returned to pretreatment levels by day 21. Plasma tumor necrosis factor- alpha levels were not high at baseline, and they increased modestly during treatment. Plasma levels of IL-12 increased steadily during thalidomide treatment. Hence, the therapeutic effect of thalidomide in ENL appears to be associated with transient immune stimulation, which suggests that the drug may promote an active immunoregulatory response.

Selective Defect in Plasmacyoid Dendritic Cell Function in a Patient with AIDS-Associated Atypical Genital Herpes Simplex Vegetans Treated with Imiquimod

We report a case of acquired immunodeficiency syndrome (AIDS)-associated, acyclovir-refractory genital herpes infection treated with topical imidazoquinoline therapy. The patients plasmacytoid dendritic cells made a robust interferon- alpha response following in vitro stimulation with imidazoquinoline but not with herpes simplex virus. We hypothesize that disease resulting from defective herpes simplex virus-stimulated interferon- alpha may be overcome by stimulating intact alternative pathways.

Regulation of Acquired Immunity by γδ T‐Cell/Dendritic‐Cell Interactions

Abstract: In humans, innate immune recognition of mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae, involves toll‐like receptor‐2 (TLR‐2), expressed on immature dendritic cells (DCs), and the T‐cell γδ receptor expressed by a subpopulation of T cells that utilize Vδ2 (Vδ2 T cells). To investigate modulatory relationships between these host‐cell populations in a microbial context, in vitro experiments were performed with human DCs and Vδ2 T cells stimulated with model TLR‐2 ligands and phosphoantigens, respectively. We observed that TLR‐2‐stimulated DCs enhanced interferon‐γ (IFN‐γ) production by Vδ2 T cells; conversely, activated Vδ2 T cells enhanced TLR‐2‐induced DC maturation via soluble factors including IFN‐γ, which costimulated interleukin‐12 (IL‐12) p70 secretion by DCs. Exposure of DCs to activated Vδ2 T cells was critical for Th1 T‐cell priming when TLR‐2 stimulation was limiting. These results suggest that Vδ2 T cells may play an adjuvant role in priming protective antimycobacterial immunity when TLR‐2 stimulation is lacking, as may occur if the infectious inoculum is small, or if the pathogen is an intrinsically weak activator of DCs.

The Activities of the Yersinia Protein Kinase A (YpkA) and Outer Protein J (YopJ) Virulence Factors Converge on an eIF2α Kinase

The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-κB signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2α kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2α kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2α kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.

Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen

ABSTRACT The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes. Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2−/− mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2−/− cells that the vacuole-to-cytosol transitioning of L. monocytogenes is greatly accelerated. Unexpectedly, we found that in Perforin-2−/− macrophages, Listeria-containing vacuoles quickly (≤15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that a L. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2+/+ and Perforin-2−/− macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.

The Host-Encoded Heme Regulated Inhibitor (HRI) Facilitates Virulence-Associated Activities of Bacterial Pathogens

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria . For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking

ABSTRACT The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri−/− mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri−/− mice than in the liver of Hri+/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri+/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri−/− mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri−/− macrophages and fibroblasts was significantly higher than the rate seen with infected Hri+/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.