Morphometric Analysis of the Thymic Epithelial Cell (TEC) Network Using Integrated and Orthogonal Digital Pathology Approaches.

The thymus, a central primary lymphoid organ of the immune system, plays a key role in T cell development. Surprisingly, the thymus is quite neglected with regards to standardized pathology approaches and practices for assessing structure and function. Most studies use multispectral flow cytometry to define the dynamic composition of the thymus at the cell population level, but they are limited by lack of contextual insight. This knowledge gap hinders our understanding of various thymic conditions and pathologies, particularly how they affect thymic architecture, and subsequently, immune competence. Here, we introduce a digital pathology pipeline to address these challenges. Our approach can be coupled to analytical algorithms and utilizes rationalized morphometric assessments of thymic tissue, ranging from tissue-wide down to microanatomical and ultrastructural levels. This pipeline enables the quantitative assessment of putative changes and adaptations of thymic structure to stimuli, offering valuable insights into the pathophysiology of thymic disorders. This versatile pipeline can be applied to a wide range of conditions that may directly or indirectly affect thymic structure, ranging from various cytotoxic stimuli inducing acute thymic involution to autoimmune diseases, such as myasthenia gravis. Here, we demonstrate applicability of the method in a mouse model of age-dependent thymic involution, both by confirming established knowledge, and by providing novel insights on intrathymic remodeling in the aged thymus. Our orthogonal pipeline, with its high versatility and depth of analysis, promises to be a valuable and practical toolset for both basic and translational immunology laboratories investigating thymic function and disease.

Listeria delivers tetanus toxoid protein to pancreatic tumors and induces cancer cell death in mice.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

Deciphering structural bases of intestinal and hepatic selectivity in targeting pregnane X receptor with indole-based microbial mimics.

Microbial metabolite mimicry is a new concept that promises to deliver compounds that have minimal liabilities and enhanced therapeutic effects in a host. In a previous publication, we have shown that microbial metabolites of L-tryptophan, indoles, when chemically altered, yielded potent anti-inflammatory pregnane X Receptor (PXR)-targeting lead compounds, FKK5 and FKK6, targeting intestinal inflammation. Our aim in this study was to further define structure-activity relationships between indole analogs and PXR, we removed the phenyl-sulfonyl group or replaced the pyridyl residue with imidazolopyridyl of FKK6. Our results showed that while removal of the phenyl-sulfonyl group from FKK6 (now called CVK003) shifts agonist activity away from PXR towards the aryl hydrocarbon receptor (AhR), the imidazolopyridyl addition preserves PXR activity in vitro. However, when these compounds are administered to mice, that unlike the parent molecule, FKK6, they exhibit poor induction of PXR target genes in the intestines and the liver. These data suggest that modifications of FKK6 specifically in the pyridyl moiety can result in compounds with weak PXR activity in vivo. These observations are a significant step forward for understanding the structure-activity relationships (SAR) between indole mimics and receptors, PXR and AhR.

A Naturally Occurring Polymorphism in the HIV-1 Tat Basic Domain Inhibits Uptake by Bystander Cells and Leads to Reduced Neuroinflammation.

HIV-1 Tat protein contributes to HIV-neuropathogenesis in several ways including its ability to be taken up by uninfected bystander CNS cells and to activate inflammatory host genes causing synaptic injury. Here, we report that in the globally dominant HIV-1 clade C, Tat displays a naturally occurring polymorphism, R57S, in its basic domain, which mediates cellular uptake. We examined the effect of this polymorphism on Tat uptake and its consequences for cellular gene transactivation. In decapeptides corresponding to the basic domain, a R57S substitution caused up to a 70% reduction in uptake. We also used a transcellular Tat transactivation assay, where we expressed Tat proteins of HIV-1 clade B (Tat-B) or C (Tat-C) or their position 57 variants in HeLa cells. We quantified the secreted Tat proteins and measured their uptake by TZM-bl cells, which provide readout via an HIV-1 Tat-responsive luciferase gene. Transactivation by Tat-B was significantly reduced by R57S substitution, while that of Tat-C was enhanced by the reciprocal S57R substitution. Finally, we exposed microglia to Tat variants and found that R57 is required for maximal neuroinflammation. The R57S substitution dampened this response. Thus, genetic variations can modulate the ability of HIV-1 Tat to systemically disseminate neuroinflammation.

Optimizing leading edge F-actin labeling using multiple actin probes, fixation methods and imaging modalities.

We systematically evaluated the performance and reliability of several widely used, commercially available actin-filament probes in a highly motile breast adenocarcinoma cell line to optimize the visualization of F-actin-rich dynamic lamellipodia. We evaluated four Phalloidin-fluorophores, two anti-actin antibodies, and three live-cell actin probes in five fixation conditions across three imaging platforms as a basis for the design of optimized protocols. Of the fluorescent phalloidin-dye conjugates tested, Alexa Fluor-488 Phalloidin ranked best in overall labeling of the actin cytoskeleton and maintenance of the fluorescence signal over time. Use of actin monoclonal antibodies revealed significant limitations under a variety of fixation-permeabilization conditions. Evaluation of commonly used live-cell probes provides evidence for actin filament bias, with TagRFP-Lifeact excluded from lamellipodia, but not mEGFP-Lifeact or F-tractin-EGFP.

Targeting the PXR-TLR4 signaling pathway to reduce intestinal inflammation in an experimental model of necrotizing enterocolitis.

BackgroundThere is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC.MethodsWild-type and PXR-/- mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation.ResultsWe found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR-/- mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC.ConclusionThese findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.

Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation.

Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex-dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.

N-WASP and cortactin are involved in invadopodium-dependent chemotaxis to EGF in breast tumor cells.

Metastatic mammary carcinoma cells, which have previously been observed to form mature, matrix degrading invadopodia on a thick ECM matrix, are able to form invadopodia with similar characteristics on glass without previously applied matrix. They form in response to epidermal growth factor (EGF), and contain the usual invadopodium core proteins N-WASP, Arp2/3, cortactin, cofilin, and F-actin. The study of invadopodia on glass allows for higher resolution analysis including the use of total internal reflection microscopy and analysis of their relationship to other cell motility events, in particular, lamellipodium extension and chemotaxis toward an EGF gradient. Invadopodium formation on glass requires N-WASP and cortactin but not microtubules. In a gradient of EGF more invadopodia form on the side of the cells facing the source of EGF. In addition, depletion of N-WASP or cortactin, which blocks invadopodium fromation, inhibits chemotaxis of cells towards EGF. This appears to be a localized defect in chemotaxis since depletion of N-WASP or cortactin via siRNA had no effect on lamellipodium protrusion or barbed end generation at the lamellipodium's leading edge. Since chemotaxis to EGF by breast tumor cells is involved in metastasis, inhibiting N-WASP activity in breast tumor cells might prevent metastasis of tumor cells while not affecting chemotaxis-dependent innate immunity which depends on WASp function in macrophages.

Immunostaining evidence for PI(4,5)P2 localization at the leading edge of chemoattractant-stimulated HL-60 cells.

It is well known that in fMLP-stimulated neutrophils, phosphatidyl inositol 3,4,5-trisphosphate [PI(3,4,5)P3] localizes at the leading edge of the cells. However, no effort has been made to study the PI 4,5-bisphosphate [PI(4,5)P2] distribution in these cells. In fact, it has been suggested that PI(4,5)P2 is unlikely to localize, as its basal level is orders of magnitude higher than that of PI(3,4,5)P3. We developed an optimized immunostaining protocol for studying the endogenous distribution of PI(4,5)P2 in neutrophil-like HL-60 cells. We show that PI(4,5)P2 localizes sharply at the leading edge with an intensity gradient similar to that for PI(3,4,5)P3. The enzymes for the production of PI(4,5)P2, namely, PI5KIalpha and PI5KIgamma, were also found to localize at the leading edge, further supporting our finding that PI(4,5)P2 localizes at the leading edge. These results imply that complementary regulation of PI3K and phosphate and tensin homolog (PTEN) is not the sole or dominant mechanism of PI(3,4,5)P3 polarization in HL-60 cells.

WASP family members and formin proteins coordinate regulation of cell protrusions in carcinoma cells.

We examined the role of the actin nucleation promoters neural Wiskott-Aldrich syndrome protein (N-WASP) and WAVE2 in cell protrusion in response to epidermal growth factor (EGF), a key regulator in carcinoma cell invasion. We found that WAVE2 knockdown (KD) suppresses lamellipod formation and increases filopod formation, whereas N-WASP KD has no effect. However, simultaneous KD of both proteins results in the formation of large jagged protrusions with lamellar properties and increased filopod formation. This suggests that another actin nucleation activity is at work in carcinoma cells in response to EGF. A mammalian Diaphanous-related formin, mDia1, localizes at the jagged protrusions in double KD cells. Constitutively active mDia1 recapitulated the phenotype, whereas inhibition of mDia1 blocked the formation of these protrusions. Increased RhoA activity, which stimulates mDia1 nucleation, was observed in the N-WASP/WAVE2 KD cells and was shown to be required for the N-WASP/WAVE2 KD phenotype. These data show that coordinate regulation between the WASP family and mDia proteins controls the balance between lamellar and lamellipodial protrusion activity.

EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells.

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Transient early embryonic expression of Nkx2-5 mutations linked to congenital heart defects in human causes heart defects in Xenopus laevis.

Nkx2-5 is a homeobox containing transcription factor that is conserved and expressed in organisms that form hearts. Fruit flies lacking the gene (tinman) fail to form a dorsal vessel, mice that are homozygous null for Nkx2-5 form small, deformed hearts, and several human cardiac defects have been linked to dominant mutations in the Nkx2-5 gene. The Xenopus homologs (XNkx2-5) of two truncated forms of Nkx2-5 that have been identified in humans with congenital heart defects were used in the studies reported here. mRNAs encoding these mutations were injected into single cell Xenopus embryos, and heart development was monitored. Our results indicate that the introduction of truncated XNkx2-5 variants leads to three principle developmental defects. The atrial septum and the valve of the atrioventricular canal were both abnormal. In addition, video microscopic timing of heart contraction indicated that embryos injected with either mutant form of XNkx2-5 have conduction defects.

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis.

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.

Cofilin takes the lead.

Cofilin has emerged as a key regulator of actin dynamics at the leading edge of motile cells. Through its actin-severing activity, it creates new actin barbed ends for polymerization and also depolymerizes old actin filaments. Its function is tightly regulated in the cell. Spatially, its activity is restricted by other actin-binding proteins, such as tropomyosin, which compete for accessibility of actin filament populations in different regions of the cell. At the molecular level, it is regulated by phosphorylation, pH and phosphatidylinositol (4,5)-bisphosphate binding downstream of signaling cascades. In addition, it also appears to be regulated by interactions with 14-3-3zeta and cyclase-associated protein. In vivo, cofilin acts synergistically with the Arp2/3 complex to amplify local actin polymerization responses upon cell stimulation, which gives it a central role in setting the direction of motility in crawling cells.

A neural Wiskott-Aldrich Syndrome protein-mediated pathway for localized activation of actin polymerization that is regulated by cortactin.

Activation of the epidermal growth factor (EGF) receptor can stimulate actin polymerization via the Arp2/3 complex using a number of signaling pathways, and specific stimulation conditions may control which pathways are activated. We have previously shown that localized stimulation of EGF receptor with EGF bound to beads results in localized actin polymerization and protrusion. Here we show that the actin polymerization is dependent upon activation of the Arp2/3 complex by neural Wiskott-Aldrich Syndrome protein (N-WASP) via Grb2 and Nck2. Suppression of Grb2 or Nck2 results in loss of localization of N-WASP at the activation site and reduced actin polymerization. Although cortactin has been found to synergize with N-WASP for Arp2/3-dependent actin polymerization in vitro, we find that cortactin can restrict N-WASP localization around EGF-bead-induced protrusions. In addition, cortactin-deficient cells have increased lamellipod dynamics but show reduced net translocation, suggesting that cortactin can contribute to cell polarity by controlling the extent of Arp2/3 activation by WASP family members and the stability of the F-actin network.

Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation.

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.

Synergistic interaction between the Arp2/3 complex and cofilin drives stimulated lamellipod extension.

Both the Arp2/3 complex and cofilin are believed to be important for the generation of protrusive force at the leading edge; however, their relative contributions have not been explored in vivo. Our results with living cells show that cofilin enters the leading edge immediately before the start of lamellipod extension, slightly earlier than Arp2/3, which begins to be recruited slightly later as the lamellipod is extended. Blocking either the Arp2/3 complex or cofilin function in cells results in failure to extend broad lamellipods and inhibits free barbed ends, suggesting that neither factor on its own can support actin polymerization-mediated protrusion in response to growth factor stimulation. High-resolution analysis of the actin network at the leading edge supports the idea that both the severing activity of cofilin and the specific branching activity of the Arp2/3 complex are essential for lamellipod protrusion. These results are the first to document the relative contributions of cofilin and Arp2/3 complex in vivo and indicate that cofilin begins to initiate the generation of free barbed ends that act in synergy with the Arp2/3 complex to create a large burst in nucleation activity.

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation.

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.

Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge.

Rapid polymerization of a network of short, branched actin filaments takes place at the leading edge of migrating cells, a compartment enriched in activators of actin polymerization such as the Arp2/3 complex and cofilin. Actin filaments elsewhere in the cell are long and unbranched. Results reported here show that the presence or absence of tropomyosin in these different actin-containing regions helps establish functionally distinct actin-containing compartments in the cell. Tropomyosin, an inhibitor of the Arp2/3 complex and cofilin function, was localized in relation to actin filaments, the Arp2/3 complex, and free barbed ends of actin filaments in MTLn3 cells, which rapidly extend flat lamellipodia following EGF stimulation. All tropomyosin isoforms examined using indirect immunofluorescence were relatively absent from the dynamic leading edge compartment, but did colocalize with actin structures deeper in the lamellipodium and in stress fibers. An in vitro light microscopy assay revealed that tropomyosin protects actin filaments from cofilin severing. The results suggest that tropomyosin-free actin filaments under the membrane can participate in rapid, dynamic processes that depend on interactions between the activities of the Arp2/3 complex and ADF/cofilin that tropomyosin inhibits elsewhere in the cell.