Eko Raharjo

An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients

Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LTB4) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants. p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.

LSP1 is an endothelial gatekeeper of leukocyte transendothelial migration

Leukocyte-specific protein 1 (LSP1), an F-actin binding protein and a major downstream substrate of p38 mitogen-activated protein kinase as well as protein kinase C, has been reported to be important in leukocyte chemotaxis. Although its distribution has been thought to be restricted to leukocytes, herein we report that LSP1 is expressed in endothelium and is essential to permit neutrophil emigration. Using intravital microscopy to directly visualize leukocyte rolling, adhesion, and emigration in postcapillary venules in LSP1-deficient (Lsp1 −/−) mice, we found that LSP1 deficiency inhibits neutrophil extravasation in response to various cytokines (tumor necrosis factor-α and interleukin-1β) and to neutrophil chemokine keratinocyte-derived chemokine in vivo. LSP1 deficiency did not affect leukocyte rolling or adhesion. Generation of Lsp1 −/− chimeric mice using bone marrow transplantation revealed that in mice with Lsp1 −/− endothelial cells and wild-type leukocytes, neutrophil transendothelial migration out of postcapillary venules is markedly restricted. In contrast, Lsp1 −/− neutrophils in wild-type mice were able to extravasate normally. Consistent with altered endothelial function was a reduction in vascular permeability to histamine in Lsp1 −/− animals. Western blot analysis and immunofluorescence microscopy examination confirmed the presence of LSP1 in wild-type but not in Lsp1 −/− mouse microvascular endothelial cells. Cultured human endothelial cells also stained positive for LSP1. Our results suggest that LSP1 expressed in endothelium regulates neutrophil transendothelial migration.

Complexity of Inducible Nitric Oxide Synthase Cellular Source Determines Benefit Versus Toxicity

Background—Inducible nitric oxide synthase (iNOS) has been shown to have both beneficial and detrimental effects in sepsis. We focused on a single organ, the heart, and used 2 distinct cell types that express iNOS—the cardiac myocyte and the infiltrating neutrophil—to study the distinct functional effects of iNOS derived from heterogeneous cellular sources. Methods and Results—In the first series of experiments, extravascular neutrophils were exposed to isolated single endotoxemic cardiac myocytes. Adhesion of wild-type neutrophils caused a rapid decrease in myocyte shortening and a concomitant increase in neutrophil-derived intracellular oxidative stress within the myocytes that was not observed with neutrophils from iNOS-deficient animals. We previously demonstrated that neutrophil-derived superoxide was essential for myocyte dysfunction; however, superoxide production was not compromised in the iNOS-deficient neutrophils. Because both superoxide and NO were essential for the neutrophil dysfunction, we probed for but could not detect any peroxynitrite assessed by detection of nitrotyrosine. There was a significant increase in length shortening in response to &bgr;-adrenergic stimulation of wild-type myocytes. Surprisingly, myocyte iNOS activity was essential rather than detrimental for the development of &bgr;-adrenergic receptor–mediated increases in shortening in endotoxemic iNOS-deficient myocytes. Conclusions—These results demonstrate that iNOS, when expressed in isolated cardiac myocytes, can regulate the response to &bgr;-adrenergic stimulation during sepsis. However, as the neutrophils migrate in proximity to myocytes, iNOS now becomes essential for the ability of neutrophils to damage myocytes. These findings demonstrate that cellular source strongly modulates the beneficial and detrimental effect of iNOS.

Hair Follicle Dermal Stem Cells Regenerate the Dermal Sheath, Repopulate the Dermal Papilla, and Modulate Hair Type

The dermal papilla (DP) provide instructive signals required to activate epithelial progenitors and initiate hair follicle regeneration. DP cell numbers fluctuate over the hair cycle, and hair loss is associated with gradual depletion/atrophy of DP cells. How DP cell numbers are maintained in healthy follicles remains unclear. We performed in vivo fate mapping of adult hair follicle dermal sheath (DS) cells to determine their lineage relationship with DP and found that a subset of DS cells are retained following each hair cycle, exhibit self-renewal, and repopulate the DS and the DP with new cells. Ablating these hair follicle dermal stem cells and their progeny retarded hair regrowth and altered hair type specification, suggesting that they function to modulate normal DP function. This work identifies a bipotent stem cell within the adult hair follicle mesenchyme and has important implications toward restoration of hair growth after injury, disease, and aging.

Neuronal nitric oxide synthase (NOS) regulates leukocyte-endothelial cell interactions in endothelial NOS deficient mice

The present study was designed to examine the possible role of neuronal nitric oxide synthase (nNOS) in regulation of leukocyte – endothelial cell interactions in the absence of endothelial nitric oxide synthase (eNOS), using intravital microscopy of the cremasteric microcirculation of eNOS−/− mice. Baseline leukocyte rolling and adhesion revealed no differences between wild‐type and eNOS−/− mice in either the cremasteric or intestinal microcirculations. Superfusion with L‐NAME (100 μM) caused a progressive and significant increase in leukocyte adhesion in both wild‐type and eNOS−/− mice, without detecting differences between the two strains of mice. Superfusion with 7‐nitroindazole (100 μM), a selective inhibitor of nNOS, had no effect on leukocyte adhesion in wild‐type animals. However, it increased leukocyte adhesion significantly in eNOS−/− mice, which was reversed by systemic L‐arginine pre‐administration. Stimulation of the microvasculature with H2O2 (100 μM) induced a transient elevation in leukocyte rolling in wild‐type mice. Conversely, the effect persisted during the entire 60 min of experimental protocol in eNOS−/− mice either with or without 7‐nitroindazole. Semi‐quantitative analysis by RT – PCR of the mRNA for nNOS levels in eNOS−/− and wild‐type animals, showed increased expression of nNOS in both brain and skeletal muscle of eNOS−/− mice. In conclusion, we have demonstrated that leukocyte‐endothelial cell interactions are predominantly modulated by eNOS isoform in postcapillary venules of normal mice, whereas nNOS appears to assume the same role in eNOS−/− mice. Interestingly, unlike eNOS there was insufficient NO produced by nNOS to overcome leukocyte recruitment elicited by oxidative stress, suggesting that nNOS cannot completely compensate for eNOS.

Exclusive Neutrophil Recruitment with Oncostatin M in a Human System

Oncostatin M (OSM), a member of the IL-6 family has been postulated to be a potent recruiter of leukocytes, however information regarding the molecular mechanism(s) underlying this event is extremely limited. Therefore, the aim of this study was to investigate the role of OSM-mediated leukocyte recruitment in a human system in vitro under flow conditions. A parallel-plate flow chamber assay was used to examine leukocyte recruitment from whole blood by human umbilical vein endothelium treated for 24 hours with OSM. OSM in a dose-response manner revealed very significant leukocyte rolling and adhesion reaching optimal levels at a very low concentration of OSM (10 ng/ml). The OSM-induced leukocyte rolling and adhesion was comparable to levels seen with tumor necrosis factor. OSM was extremely selective for neutrophil recruitment (96%) with <3% lymphocyte recruitment. By contrast, tumor necrosis factor-alpha revealed no such selectivity, recruiting 70% neutrophils and at least 25% lymphocytes and detectable levels of eosinophils at 24 hours. The molecular mechanism underlying the leukocyte recruitment seemed to be entirely dependent on P-selectin as leukocyte recruitment could be completely blocked by the addition of a P-selectin-blocking antibody. An elevation in both P-selectin message and protein was observed with 24 hours of OSM stimulation of endothelium. By contrast, E-selectin and VCAM-1 were not detectable after OSM stimulation. Similar results were seen with passaged dermal microvascular endothelium that does not have a prestored pool of P-selectin. Based on these results, we conclude that OSM may be a very selective potent recruiter of neutrophils in more prolonged inflammatory conditions, an event exclusively dependent on P-selectin.

Levels of the ubiquitin ligase substrate adaptor MEL-26 are inversely correlated with MEI-1/katanin microtubule-severing activity during both meiosis and mitosis

The MEI-1/MEI-2 microtubule-severing complex, katanin, is required for oocyte meiotic spindle formation and function in C. elegans, but the microtubule-severing activity must be quickly downregulated so that it does not interfere with formation of the first mitotic spindle. Post-meiotic MEI-1 inactivation is accomplished by two parallel protein degradation pathways, one of which requires MEL-26, the substrate-specific adaptor that recruits MEI-1 to a CUL-3 based ubiquitin ligase. Here we address the question of how MEL-26 mediated MEI-1 degradation is triggered only after the completion of MEI-1s meiotic function. We find that MEL-26 is present only at low levels until the completion of meiosis, after which protein levels increase substantially, likely increasing the post-meiotic degradation of MEI-1. During meiosis, MEL-26 levels are kept low by the action of another type of ubiquitin ligase, which contains CUL-2. However, we find that the low levels of meiotic MEL-26 have a subtle function, acting to moderate MEI-1 activity during meiosis. We also show that MEI-1 is the only essential target for MEL-26, and possibly for the E3 ubiquitin ligase CUL-3, but the upstream ubiquitin ligase activating enzyme RFL-1 has additional essential targets.

Adult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells

Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

Enhanced Expansion and Sustained Inductive Function of Skin-Derived Precursor Cells in Computer-Controlled Stirred Suspension Bioreactors

Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443

Improved method to track and precisely count Schwann cells post-transplantation in a peripheral nerve injury model

BACKGROUND To optimize survival evaluation of Schwann cells (SCs) in vivo, we tested fluorescent labeling of the nucleus as an improved method of tracking and counting the transplanted SCs at sciatic nerve injury sites in rodents. We also investigated if co-administering cells with the glial growth factor Neuregulin-1 β (NRG1β) improves in vivo survival. NEW METHOD We transduced SCs using a Lentiviral vector with a nuclear localization signal (NLS) fused with mCherry and transplanted them in the sciatic nerve of rat post-crush injury (bilateral) either in the presence or absence of NRG1β in the injectate media. For comparison, in a separate group of similar injury, GFP-labeled cells were transplanted. After 10 days, nerves were harvested and sections (14μm) were counterstained with Hoechst and imaged. Cells showing co-localization with Hoechst and GFP or mCherry were exhaustively counted and data analyzed. RESULTS Percentage cells counted in with- and without-NRG condition in both the groups were 0.83±0.13% and 0.06±0.04% (Group 1) & 2.83*±1.95% and 0.23*±0.29% (Group 2). COMPARISON TO EXISTING METHOD We are introducing fluorescent labeling of the nucleus as a reliable and efficient technique to perform survival assessments in Schwann cell based treatment studies in animal model. This method can overcome the challenges and limitations of the existing method that could result in underestimation of the therapeutic outcome. CONCLUSIONS Nucleus-restricted fluorescent labeling technique offer improved method of tracking as well as accurately counting transplanted SCs in vivo while NRG1β in the injectate media can improve survival.