Sarah A. Wernimont

Delivery of antimicrobials into parasites

To eliminate apicomplexan parasites, inhibitory compounds must cross host cell, parasitophorous vacuole, and parasite membranes and cyst walls, making delivery challenging. Here, we show that short oligomers of arginine enter Toxoplasma gondii tachyzoites and encysted bradyzoites. Triclosan, which inhibits enoyl-ACP reductase (ENR), conjugated to arginine oligomers enters extracellular tachyzoites, host cells, tachyzoites inside parasitophorous vacuoles within host cells, extracellular bradyzoites, and bradyzoites within cysts. We identify, clone, and sequence T. gondii enr and produce and characterize enzymatically active, recombinant ENR. This enzyme has the requisite amino acids to bind triclosan. Triclosan released after conjugation to octaarginine via a readily hydrolyzable ester linkage inhibits ENR activity, tachyzoites in vitro, and tachyzoites in mice. Delivery of an inhibitor to a microorganism via conjugation to octaarginine provides an approach to transporting antimicrobials and other small molecules to sequestered parasites, a model system to characterize transport across multiple membrane barriers and structures, a widely applicable paradigm for treatment of active and encysted apicomplexan and other infections, and a generic proof of principle for a mechanism of medicine delivery.

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays

Improvements in neutrophil chemotaxis assays have advanced our understanding of the mechanisms of neutrophil recruitment; however, traditional methods limit biologic inquiry in important areas. We report a microfluidic technology that enables neutrophil purification and chemotaxis on-chip within minutes, using nanoliters of whole blood, and only requires a micropipette to operate. The low sample volume requirements and novel lid-based method for initiating the gradient of chemoattractant enabled the measurement of human neutrophil migration on a cell monolayer to probe the adherent and migratory states of neutrophils under inflammatory conditions; mouse neutrophil chemotaxis without sacrificing the animal; and both 2D and 3D neutrophil chemotaxis. First, the neutrophil chemotaxis on endothelial cells revealed 2 distinct neutrophil phenotypes, showing that endothelial cell-neutrophil interactions influence neutrophil chemotactic behavior. Second, we validated the mouse neutrophil chemotaxis assay by comparing the adhesion and chemotaxis of neutrophils from chronically inflamed and wild-type mice; we observed significantly higher neutrophil adhesion in blood obtained from chronically inflamed mice. Third, we show that 2D and 3D neutrophil chemotaxis can be directly compared using our technique. These methods allow for new avenues of research while reducing the complexity, time, and sample volume requirements to perform neutrophil chemotaxis assays.

Natural killer T-cell autoreactivity leads to a specialized activation state

Natural killer T (NKT) cells are innate-like T cells that recognize specific microbial antigens and also display autoreactivity to self-antigens. The nature of NKT-cell autoreactive activation remains poorly understood. We show here that the mitogen-activated protein kinase (MAPK) pathway is operative during human NKT-cell autoreactive activation, but calcium signaling is severely impaired. This results in a response that is biased toward granulocyte macrophage colony-stimulating factor (GM-CSF) secretion because this cytokine requires extracellular signal-regulated kinase (ERK) signaling but is not highly calcium dependent, whereas interferon-gamma (IFN-gamma), interleukin (IL)-4, and IL-2 production are minimal. Autoreactive activation was associated with reduced migration velocity but did not induce arrest; thus, NKT cells retained the ability to survey antigen presenting cells (APCs). IL-12 and IL-18 stimulated autoreactively activated NKT cells to secrete IFN-gamma, and this was mediated by Janus kinase-signal transducers and activators of transcription (JAK-STAT)-dependent signaling without induction of calcium flux. This pathway did not require concurrent contact with CD1d(+) APCs but was strictly dependent on preceding autoreactive stimulation that induced ERK activation. In contrast, NKT-cell responses to the glycolipid antigen alpha-galactosyl ceramide (alpha-GalCer) were dampened by prior autoreactive activation. These results show that NKT-cell autoreactivity induces restricted cytokine secretion and leads to altered basal activation that potentiates innate responsiveness to costimulatory cytokines while modulating sensitivity to foreign antigens.

Contact-Dependent T Cell Activation and T Cell Stopping Require Talin1

T cell–APC contact initiates T cell activation and is maintained by the integrin LFA-1. Talin1, an LFA-1 regulator, localizes to the immune synapse (IS) with unknown roles in T cell activation. In this study, we show that talin1-deficient T cells have defects in contact-dependent T cell stopping and proliferation. Although talin1-deficient T cells did not form stable interactions with APCs, transient contacts were sufficient to induce signaling. In contrast to prior models, LFA-1 polarized to T cell–APC contacts in talin1-deficient T cells, but vinculin and F-actin polarization at the IS was impaired. These results indicate that T cell proliferation requires sustained, talin1-mediated T cell–APC interactions and that talin1 is necessary for F-actin polarization and the stability of the IS.

Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome.

PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne) is an autosomal dominant, autoinflammatory disorder characterized by destructive inflammation of the skin and joints. Single amino acid substitutions in the gene encoding the Pombe Cdc15 homology (PCH) family member PSTPIP1 result in PAPA syndrome (1). PSTPIP1 is an adaptor protein expressed primarily in hematopoietic cells and is involved in cytoskeletal organization in part through its interactions with the phosphatase PTP-PEST, and Wiskott-Aldrich syndrome protein (WASp) (2, 3). Interestingly, PAPA syndrome causing mutations in PSTPIP1 disrupt binding to PTP-PEST (1) with unknown consequences on cytoskeletal organization and leukocyte motility. Highly motile leukocytes such as macrophages and dendritic cells form dynamic actin-containing adhesive and invasive structures known as podosomes, which facilitate migration and invasion (4). To determine if macrophages from PAPA patients display altered invasive motility and cytoskeletal organization, peripheral blood mononuclear cells were isolated from healthy volunteers and four patients with PAPA syndrome. Two of the PAPA patients were siblings and had PSTPIP1 A230T mutations. The other two PAPA patients were unrelated and had the PSTPIP1 A230T or the E250Q mutation, respectively. All mutations were confirmed by DNA sequence analysis (data not shown). Monocytes were differentiated into macrophages with 20 ng/ml human MCSF and used for experiments on day 7 using previously described methods (5). Macrophages were cultured as recommended by the American Type Culture Collection (ATCC). First we examined the directed migration of control and patient macrophages to the chemokine, MCSF. Macrophages isolated from two different patients with PAPA syndrome displayed significantly impaired chemotaxis to MCSF as compared to control macrophages (Figure 1A). In contrast to macrophage migration, T cell migration to SDF-1 was not altered in T cells isolated from PAPA patients (Figure 1B). These findings suggest that, although PSTPIP1 is expressed in both T cells and macrophages, patients with PAPA syndrome exhibit a specific defect in macrophage directed migration. To further characterize the function of macrophages from patients with PAPA syndrome, we examined the ability of PAPA patient macrophages to undergo invasive migration into gels and to degrade the extracellular matrix. We found that PAPA patient macrophages displayed significantly impaired invasion across matrigel invasion chambers (Figure 1C) as well as a decreased capacity of PAPA patient macrophages to degrade fluorescent conjugated gelatin-coated coverslips (Figure 1D). To determine if cytoskeletal regulation was altered in PAPA patient macrophages, we examined the organization of the actin cytoskeleton and podosome formation in macrophages isolated from PAPA patients. Control and PAPA patient macrophages were cultured on gelatin-coated coverslips and we examined podosome formation by staining for vinculin and actin (Figure 1E). Control macrophages showed robust polarized podosome formation with vinculin ring-like structures surrounding an actin core (Figure 1E). In contrast, PAPA patient macrophages showed a significant defect in podosome formation with many cells forming no podosomes and the cells that formed podosomes generally contained fewer than control (Figure 1E). This trend was observed in the three different PAPA patients tested (Figure 1E). In addition, PAPA patient macrophages that did form podosomes often showed abnormal architecture with increased numbers and size of vinculin-containing focal complexes (Figure 1E and data not shown). These findings indicate that PAPA patients display a specific defect in macrophage migration and invasion that may result from a switch from the formation of dynamic podosome adhesions, to more firm focal complexes. Figure 1 PAPA patient macrophages exhibit decreased invasive migration and podosome formation Defects in mononuclear cell podosome formation and migration have been reported in patients with WASp mutations and the primary immunodeficiency disorder Wiskott Aldrich Syndrome (WAS) (6-8). The similarities in macrophage morphology from patients with both PAPA syndrome and WAS, suggest that PSTPIP1 may regulate macrophage podosome formation through its interaction with WASp. Future studies will be needed to determine how PSTPIP1 regulates podosome formation and to examine if PAPA-associated mutations alter signaling through WASp. This is the first report to suggest that patients with a chronic inflammatory disease can also exhibit impaired macrophage migration and podosome formation. It is interesting, that unlike patients with WAS, PAPA patients show no evidence of immunodeficiency but instead exhibit chronic tissue inflammation and destruction. It is intriguing to speculate that abnormal macrophage trafficking and retention of macrophages within tissues because of impaired migration may contribute to the development of chronic inflammation through neutrophil recruitment, a hallmark of PAPA syndrome. Taken together, these findings suggest that defects in macrophage migration and podosome formation can contribute to the pathogenesis of both primary immunodeficiency disorders and chronic inflammatory disease.

PIPKI gamma 90 Negatively Regulates LFA-1-Mediated Adhesion and Activation in Antigen-Induced CD4(+) T Cells

T cell activation requires the formation and maintenance of stable interactions between T cells and APCs. The formation of stable T cell–APC contacts depends on the activation of the integrin LFA-1 (CD11aCD18). Several positive regulators of LFA-1 activation downstream of proximal TCR signaling have been identified, including talin; however, negative regulators of LFA-1 activity remain largely unexplored. Extended isoform of phosphatidylinositol phosphate kinase type I γ (PIPKIγ90) is a member of the type I phosphatidylinositol phosphate kinase family that has been shown previously to modulate talin activation of integrins through production of phosphatidylinositol 4,5-bisphosphate and direct binding to talin. In this study, we show that PIPKIγ90 negatively regulates LFA-1–mediated adhesion and activation of T cells. Using CD4+ T cells from PIPKIγ90-deficient mice, we show that CD4+ T cells exhibit increased LFA-1-dependent adhesion to ICAM-1 and increased rates of T cell–APC conjugate formation with enhanced LFA-1 polarization at the synapse. In addition to increased adhesiveness, PIPKIγ90-deficient T cells exhibit increased proliferation both in vitro and in vivo and increased production of IFN-γ and IL-2. Together, these results demonstrate that PIPKIγ90 is a negative regulator of Ag-induced T cell adhesion and activation.

Calpain inhibition impairs TNF-α-mediated neutrophil adhesion, arrest and oxidative burst

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration. Here, we show that calpain inhibition impairs TNF-alpha-induced neutrophil firm adhesion to fibrinogen-coated surfaces and the formation of vinculin-containing focal complexes. Calpain inhibition induces random migration in TNF-alpha-stimulated cells and prevents the generation of reactive oxygen species, but does not alter TNF-alpha-mediated activation of p38 MAPK and ERK MAPK. These findings suggest that the TNF-alpha-induced neutrophil arrest requires the activity of calpain independent of p38 MAPK and ERK signaling seen after TNF-alpha stimulation. Together, our data suggest that therapeutic inhibition of calpain may be beneficial for limiting TNF-alpha-induced inflammatory responses.

Adhesions ring: a structural comparison between podosomes and the immune synapse

Podosomes and the immune synapse are integrin-mediated adhesive structures that share a common ring-like morphology. Both podosomes and immune synapses have a central core surrounded by a peripheral ring containing talin, vinculin and paxillin. Recent progress suggests significant parallels between the regulatory mechanisms that contribute to the formation of these adhesive structures. In this review, we compare the structures, functions and regulation of podosomes and the immune synapse.

Gene related to anergy in lymphocytes (GRAIL) expression in CD4+ T cells impairs actin cytoskeletal organization during T cell/antigen-presenting cell interactions.

GRAIL (gene related to anergy in lymphocytes), is an E3 ubiquitin ligase with increased expression in anergic CD4+ T cells. The expression of GRAIL has been shown to be both necessary and sufficient for the induction of T cell (T) anergy. To date, several subsets of anergic T cells have demonstrated altered interactions with antigen-presenting cells (APC) and perturbed TCR-mediated signaling. The role of GRAIL in mediating these aspects of T cell anergy remains unclear. We used flow cytometry and confocal microscopy to examine T/APC interactions in GRAIL-expressing T cells. Increased GRAIL expression resulted in reduced T/APC conjugation efficiency as assessed by flow cytometry. Examination of single T/APC conjugates by confocal microscopy revealed altered polarization of polymerized actin and LFA-1 to the T/APC interface. When GRAIL expression was knocked down, actin polarization to the T/APC interface was restored, demonstrating that GRAIL is necessary for alteration of actin cytoskeletal rearrangement under anergizing conditions. Interestingly, proximal TCR signaling including calcium flux and phosphorylation of Vav were not disrupted by expression of GRAIL in CD4+ T cells. In contrast, interrogation of distal signaling events demonstrated significantly decreased JNK phosphorylation in GRAIL-expressing T cells. In sum, GRAIL expression in CD4+ T cells mediates alterations in the actin cytoskeleton during T/APC interactions. Moreover, in this model, our data dissociates proximal T cell signaling events from functional unresponsiveness. These data demonstrate a novel role for GRAIL in modulating T/APC interactions and provide further insight into the cell biology of anergic T cells.

The focal adhesion kinase inhibitor PF-562,271 impairs primary CD4+ T cell activation

The focal adhesion kinase inhibitor, PF-562,271, is currently in clinical development for cancer, however it is not known how PF-562,271 affects T cell function. Here, we demonstrate inhibitory effects of PF-562,271 on the activation of primary human and mouse T cells. PF-562,271 inhibits T cell receptor signaling-induced T cell adhesion to intercellular adhesion molecule-1 and T cell interactions with antigen-presenting cells. An additional focal adhesion kinase inhibitor, PF-573,228, and genetic depletion of focal adhesion kinase also impair T cell conjugation with antigen-presenting cells. PF-562,271 blocks phosphorylation of the signaling molecules zeta chain associate protein of 70 kDa, linker of activated T cells, and extracellular signal-regulated kinase, and impairs T cell proliferation. The effects observed on T cell proliferation cannot solely be attributed to focal adhesion kinase inhibition, as genetic depletion did not alter proliferation. The effect of PF-562,271 on T cell proliferation is not rescued when proximal T cell receptor signaling is bypassed by stimulation with phorbol-12-myristate-13-acetate and ionomycin. Taken together, our findings demonstrate that focal adhesion kinase regulates integrin-mediated T cell adhesion following T cell receptor activation. Moreover, our findings suggest that PF-562,271 may have immunomodulatory effects that could impact its therapeutic applications.