Differentiated fibrocytes assume a functional mesenchymal phenotype with regenerative potential.

Fibrocytes (FCs) are hematopoietic lineage cells that migrate to sites of injury, transition to a mesenchymal phenotype, and help to mediate wound repair. Despite their relevance to human fibrotic disorders, there are few data characterizing basic FC biology. Herein, using proteomic, bioenergetic, and bioengineering techniques, we conducted deep phenotypic characterization of differentiating and mature FCs. Differentiation was associated with metabolic reprogramming that favored oxidative phosphorylation. Mature FCs had distinct proteomes compared to classic mesenchymal cells, formed functional stromae that supported epithelial maturation during in vitro organotypic culture, and exhibited in vivo survival and self-tolerance as connective tissue isografts. In an in vitro scratch assay, FCs promoted fibroblast migration and wound closure by paracrine signaling via the chemokine CXCL8 (interleukin-8). These findings characterize important aspects of FC differentiation and show that, in addition to their role in wound healing, FCs hold potential as an easily isolated autologous cell source for regenerative medicine.

Bioengineered vocal fold mucosa for voice restoration.

Patients with voice impairment caused by advanced vocal fold (VF) fibrosis or tissue loss have few treatment options. A transplantable, bioengineered VF mucosa would address the individual and societal costs of voice-related communication loss. Such a tissue must be biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration and physiologically capable of maintaining a barrier against the airway lumen. We isolated primary human VF fibroblasts and epithelial cells and cocultured them under organotypic conditions. The resulting engineered mucosae showed morphologic features of native tissue, proteome-level evidence of mucosal morphogenesis and emerging extracellular matrix complexity, and rudimentary barrier function in vitro. When grafted into canine larynges ex vivo, the mucosae generated vibratory behavior and acoustic output that were indistinguishable from those of native VF tissue. When grafted into humanized mice in vivo, the mucosae survived and were well tolerated by the human adaptive immune system. This tissue engineering approach has the potential to restore voice function in patients with otherwise untreatable VF mucosal disease.

Microarray-based characterization of differential gene expression during vocal fold wound healing in rats.

The vocal fold (VF) mucosa confers elegant biomechanical function for voice production but is susceptible to scar formation following injury. Current understanding of VF wound healing is hindered by a paucity of data and is therefore often generalized from research conducted in skin and other mucosal systems. Here, using a previously validated rat injury model, expression microarray technology and an empirical Bayes analysis approach, we generated a VF-specific transcriptome dataset to better capture the system-level complexity of wound healing in this specialized tissue. We measured differential gene expression at 3, 14 and 60 days post-injury compared to experimentally naïve controls, pursued functional enrichment analyses to refine and add greater biological definition to the previously proposed temporal phases of VF wound healing, and validated the expression and localization of a subset of previously unidentified repair- and regeneration-related genes at the protein level. Our microarray dataset is a resource for the wider research community and has the potential to stimulate new hypotheses and avenues of investigation, improve biological and mechanistic insight, and accelerate the identification of novel therapeutic targets.

T cell-derived interleukin (IL)-21 promotes brain injury following stroke in mice.

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell-derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21-deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4(+) T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4(+) T cells in the area surrounding acute stroke lesions, suggesting that IL-21-mediated brain injury may be relevant to human stroke.

Immune privilege of the CNS is not the consequence of limited antigen sampling.

Central nervous system (CNS) immune privilege is complex, and it is still not understood how CNS antigens are sampled by the peripheral immune system under steady state conditions. To compare antigen sampling from immune-privileged or nonprivileged tissues, we created transgenic mice with oligodendrocyte or gut epithelial cell expression of an EGFP-tagged fusion protein containing ovalbumin (OVA) antigenic peptides and tested peripheral anti-OVA peptide-specific sentinel OT-I and OT-II T cell activation. We report that oligodendrocyte or gut antigens are sampled similarly, as determined by comparable levels of OT-I T cell activation. However, activated T cells do not access the CNS under steady state conditions. These data show that afferent immunity is normally intact as there is no barrier at the antigen sampling level, but that efferent immunity is restricted. To understand how this one-sided surveillance contributes to CNS immune privilege will help us define mechanisms of CNS autoimmune disease initiation.

Morphology, classification, and distribution of the projection neurons in the dorsal lateral geniculate nucleus of the rat.

The morphology of confirmed projection neurons in the dorsal lateral geniculate nucleus (dLGN) of the rat was examined by filling these cells retrogradely with biotinylated dextran amine (BDA) injected into the visual cortex. BDA-labeled projection neurons varied widely in the shape and size of their cell somas, with mean cross-sectional areas ranging from 60-340 µm(2). Labeled projection neurons supported 7-55 dendrites that spanned up to 300 µm in length and formed dendritic arbors with cross-sectional areas of up to 7.0 × 10(4) µm(2). Primary dendrites emerged from cell somas in three broad patterns. In some dLGN projection neurons, primary dendrites arise from the cell soma at two poles spaced approximately 180° apart. In other projection neurons, dendrites emerge principally from one side of the cell soma, while in a third group of projection neurons primary dendrites emerge from the entire perimeter of the cell soma. Based on these three distinct patterns in the distribution of primary dendrites from cell somas, we have grouped dLGN projection neurons into three classes: bipolar cells, basket cells and radial cells, respectively. The appendages seen on dendrites also can be grouped into three classes according to differences in their structure. Short "tufted" appendages arise mainly from the distal branches of dendrites; "spine-like" appendages, fine stalks with ovoid heads, typically are seen along the middle segments of dendrites; and "grape-like" appendages, short stalks that terminate in a cluster of ovoid bulbs, appear most often along the proximal segments of secondary dendrites of neurons with medium or large cell somas. While morphologically diverse dLGN projection neurons are intermingled uniformly throughout the nucleus, the caudal pole of the dLGN contains more small projection neurons of all classes than the rostral pole.

Resolving the detailed structure of cortical and thalamic neurons in the adult rat brain with refined biotinylated dextran amine labeling.

Biotinylated dextran amine (BDA) has been used frequently for both anterograde and retrograde pathway tracing in the central nervous system. Typically, BDA labels axons and cell somas in sufficient detail to identify their topographical location accurately. However, BDA labeling often has proved to be inadequate to resolve the fine structural details of axon arbors or the dendrites of neurons at a distance from the site of BDA injection. To overcome this limitation, we varied several experimental parameters associated with the BDA labeling of neurons in the adult rat brain in order to improve the sensitivity of the method. Specifically, we compared the effect on labeling sensitivity of: (a) using 3,000 or 10,000 MW BDA; (b) injecting different volumes of BDA; (c) co-injecting BDA with NMDA; and (d) employing various post-injection survival times. Following the extracellular injection of BDA into the visual cortex, labeled cells and axons were observed in both cortical and thalamic areas of all animals studied. However, the detailed morphology of axon arbors and distal dendrites was evident only under optimal conditions for BDA labeling that take into account the: molecular weight of the BDA used, concentration and volume of BDA injected, post-injection survival time, and toning of the resolved BDA with gold and silver. In these instances, anterogradely labeled axons and retrogradely labeled dendrites were resolved in fine detail, approximating that which can be achieved with intracellularly injected compounds such as biocytin or fluorescent dyes.

Degeneration of axotomized projection neurons in the rat dLGN: temporal progression of events and their mitigation by a single administration of FGF2.

Removal of visual cortex in the rat axotomizes projection neurons in the dorsal lateral geniculate nucleus (dLGN), leading to cytological and structural changes and apoptosis. Biotinylated dextran amine was injected into the visual cortex to label dLGN projection neurons retrogradely prior to removing the cortex in order to quantify the changes in the dendritic morphology of these neurons that precede cell death. At 12 hours after axotomy we observed a loss of appendages and the formation of varicosities in the dendrites of projection neurons. During the next 7 days, the total number of dendrites and the cross-sectional areas of the dendritic arbors of projection neurons declined to about 40% and 20% of normal, respectively. The response of dLGN projection neurons to axotomy was asynchronous, but the sequence of structural changes in individual neurons was similar; namely, disruption of dendrites began within hours followed by cell soma atrophy and nuclear condensation that commenced after the loss of secondary dendrites had occurred. However, a single administration of fibroblast growth factor-2 (FGF2), which mitigates injury-induced neuronal cell death in the dLGN when given at the time of axotomy, markedly reduced the dendritic degeneration of projection neurons. At 3 and 7 days after axotomy the number of surviving dendrites of dLGN projection neurons in FGF-2 treated rats was approximately 50% greater than in untreated rats, and the cross-sectional areas of dendritic arbors were approximately 60% and 50% larger. Caspase-3 activity in axotomized dLGN projection neurons was determined by immunostaining for fractin (fractin-IR), an actin cleavage product produced exclusively by activated caspase-3. Fractin-IR was seen in some dLGN projection neurons at 36 hours survival, and it increased slightly by 3 days. A marked increase in reactivity was seen by 7 days, with the entire dLGN filled with dense fractin-IR in neuronal cell somas and dendrites.

Cross-sample validation provides enhanced proteome coverage in rat vocal fold mucosa.

The vocal fold mucosa is a biomechanically unique tissue comprised of a densely cellular epithelium, superficial to an extracellular matrix (ECM)-rich lamina propria. Such ECM-rich tissues are challenging to analyze using proteomic assays, primarily due to extensive crosslinking and glycosylation of the majority of high M(r) ECM proteins. In this study, we implemented an LC-MS/MS-based strategy to characterize the rat vocal fold mucosa proteome. Our sample preparation protocol successfully solubilized both proteins and certain high M(r) glycoconjugates and resulted in the identification of hundreds of mucosal proteins. A straightforward approach to the treatment of protein identifications attributed to single peptide hits allowed the retention of potentially important low abundance identifications (validated by a cross-sample match and de novo interpretation of relevant spectra) while still eliminating potentially spurious identifications (global single peptide hits with no cross-sample match). The resulting vocal fold mucosa proteome was characterized by a wide range of cellular and extracellular proteins spanning 12 functional categories.

E-cadherin and transglutaminase-1 epithelial barrier restoration precedes type IV collagen basement membrane reconstruction following vocal fold mucosal injury.

The vocal fold epithelium is critical to upper airway immunologic defense and water/ion transport; therefore, any form of physical trauma or insult increases the vulnerability of this structure to functional impairment and pathogen invasion/infection. In this study, we examined the reestablishment of epithelial and basement membrane barrier structures in a well-established rat model of vocal fold mucosal injury. We observed active cell recruitment culminating in peak hyperplasia at 3 days postinjury, the establishment of robust E-cadherin+ and transglutaminase-1+ biochemical barrier signals along the epithelial surface by 3 days postinjury, and the persistent absence of a type IV collagen+ basement membrane at 7 days postinjury. The distinct spatial and temporal immunoactivity of these molecules is consistent with a programmed repair process driving the restoration of vocal fold mucosal integrity and permeability. These data may inform future efforts to optimize functional mucosal recovery postinjury and avoid undesirable events such as barrier compromise or epithelial metaplasia.

Reactive response of fibrocytes to vocal fold mucosal injury in rat.

Fibrocytes hold a prominent role in inflammatory and tissue repair processes in various organ systems. In this study, we identified and quantified a reactive fibrocyte population in the vocal fold mucosa postinjury using immunohistochemistry and stereological analysis. These cells, which expressed CD11b on their surface and prolyl-4-hydroxylase ß (P4H-ß) intracellularly, were largely restricted to the lamina propria, and were morphologically and immunochemically distinguishable from newly recruited epithelial cells. We validated our immunohistochemistry findings using flow cytometry, and additionally characterized a reactive fibrocyte population in circulating peripheral blood using a novel detection panel (CD16(-) CD11b(+) P4H-ß(+) ). Fibrocyte recruitment peaked at 3 days postinjury in peripheral blood, and 5 days postinjury in the vocal fold mucosa. These findings suggest that circulating fibrocytes are recruited to sites of tissue injury in the vocal fold mucosa, and may play an important role in vocal fold tissue repair. The results of this study are consistent with published data from other organ systems and strongly suggest the importance of fibrocytes as therapeutic targets. Our newly reported antigen panel facilitating the direct characterization of fibrocytes via flow cytometry is a useful tool with the potential to facilitate improved study of this cell population.

Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury.

Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference genes in a well-established rat model of vocal fold mucosal injury. We selected and evaluated the expression stability of nine candidate reference genes. Ablim1, Sptbn1, and Wrnip1 were identified as stably expressed in a model-specific microarray dataset and were further validated as suitable reference genes in an independent qRT-PCR experiment using 2(-DeltaCT) and pairwise comparison-based (geNorm) analyses. Parallel analysis of six commonly used reference genes identified Sdha as the only stably expressed candidate in this group. Sdha, Sptbn1, and the geometric mean of Sdha and Sptbn1 each provided accurate normalization of target gene Tgfb1; Gapdh, the least stable candidate gene in our dataset, provided inaccurate normalization and an invalid experimental result. The stable reference genes identified here are suitable for accurate normalization of target gene expression in vocal fold mucosal injury experiments.

Alteration in cellular morphology, density and distribution in rat vocal fold mucosa following injury.

The vocal fold mucosa plays an important role in voice production. Its cellular composition and density frequently change under various pathological conditions, often contributing to altered extracellular matrix production, tissue viscoelasticity, and voice quality. In this study, cellular changes in the rat mucosa following a unilateral stripping injury were investigated and analyzed semi-quantitatively. Distinctive and sequential changes in cellular morphology, composition, and density were observed in the mucosa post-injury. Cellular recruitment was a major event during the early stage of injury and reached its peak level by day 5 post-injury. Several types of cells, including neutrophil-like cells, epithelial cells, and fibroblast-like cells, were sequentially recruited. The sequential emergence of reactive cell populations following injury and subsequent reconstruction of the mucosa suggests their involvement in vocal fold tissue repair and scar formation processes.

Pulsed dye laser-induced inflammatory response and extracellular matrix turnover in rat vocal folds and vocal fold fibroblasts.

BACKGROUND AND OBJECTIVES: Disruption of the vocal fold extracellular matrix (ECM) can induce a profound and refractory dysphonia. Pulsed dye laser (PDL) irradiation has shown early promise as a treatment modality for disordered ECM in patients with chronic vocal fold scar; however, there are limited data addressing the mechanism by which this laser energy might induce cellular and extracellular changes in vocal fold tissues. In this study, we examined the inflammatory and ECM modulating effects of PDL irradiation on normal vocal fold tissues and cultured vocal fold fibroblasts (VFFs). STUDY DESIGN/MATERIALS AND METHODS: We evaluated the effects of 585 nm PDL irradiation on inflammatory cytokine and collagen/collagenase gene transcription in normal rat vocal folds in vivo (3-168 hours following delivery of approximately 39.46 J/cm(2) fluence) and VFFs in vitro (3-72 hours following delivery of 4.82 or 9.64 J/cm(2) fluence). We also examined morphological vocal fold tissue changes 3 hours, 1 week, and 1 month post-irradiation. RESULTS: PDL irradiation altered inflammatory cytokine and procollagen/collagenase expression at the transcript level, both in vitro and in vivo. Additionally, PDL irradiation induced an inflammatory repair process in vivo that was completed by 1 month with preservation of normal tissue morphology. CONCLUSIONS: PDL irradiation can modulate ECM turnover in phenotypically normal vocal folds. Additional work is required to determine if these findings extend to disordered ECM, such as is seen in vocal fold scar. Lasers Surg. Med. 41:585-594, 2009. (c) 2009 Wiley-Liss, Inc.

Intracerebral Mycobacterium bovis bacilli Calmette-Guerin infection-induced immune responses in the CNS.

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-gamma(+)CD4(+) T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-gammaIL-17 double-positive CD4(+) T cells is unique to the CNS. The major CNS source of TNF-alpha is microglia, with modest production by CD4(+) T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3(+)CD4(+) T cells and PD-L2(+) dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection.

In situ activation of antigen-specific CD8+ T cells in the presence of antigen in organotypic brain slices.

The activation of Ag-specific T cells locally in the CNS could potentially contribute to the development of immune-mediated brain diseases. We addressed whether Ag-specific T cells could be stimulated in the CNS in the absence of peripheral lymphoid tissues by analyzing Ag-specific T cell responses in organotypic brain slice cultures. Organotypic brain slice cultures were established 1 h after intracerebral OVA Ag microinjection. We showed that when OVA-specific CD8(+) T cells were added to Ag-containing brain slices, these cells became activated and migrated into the brain to the sites of their specific Ags. This activation of OVA-specific T cells was abrogated by the deletion of CD11c(+) cells from the brain slices of the donor mice. These data suggest that brain-resident CD11c(+) cells stimulate Ag-specific naive CD8(+) T cells locally in the CNS and may contribute to immune responses in the brain.

Substance P receptor mediated maintenance of chronic inflammation in EAE.

Substance P (SP) is a modulatory, pro-inflammatory neuropeptide. We investigated the role of the SP receptor, neurokinin-1 (NK-1), in EAE. Our data show that in the chronic phase, mice lacking NK-1 have improved mobility and decreased numbers of LFA-1 high CD4+ T cells and MOG-specific, IFN-gamma producing CD4+ T cells. SR140333, an NK-1 antagonist, administered alone during the chronic phase of EAE was not sufficient to ameliorate symptoms. These results indicate that SP, through NK-1, contributes to maintenance of CNS inflammation, and combining NK-1 antagonists with conventional anti-inflammatory treatments may enhance the success of treatments for diseases like multiple sclerosis.

Traumatic injury and the presence of antigen differentially contribute to T-cell recruitment in the CNS.

T-cell recruitment into the brain is critical in inflammatory and autoimmune diseases of the CNS. We use intracerebral antigen microinjection and tetramer technology to track antigen-specific CD8+ T-cells in the CNS and to clarify the contribution of antigen deposition or traumatic injury to the accumulation of T-cells in the brain. We demonstrate that, after intracerebral microinjection of ovalbumin, ovalbumin-specific CD8+ T-cells expand systemically and then migrate into the brain where they complete additional proliferation cycles. T-cells in the brain are activated and respond to in vitro secondary antigen challenge. CD8+ T-cells accumulate and persist in sites of antigen in the brain without replenishment from the periphery. Persistent survival of CD8+ T-cells at sites of cognate antigen is significantly reduced by blocking CD154 molecules. A small traumatic injury itself does not lead to recruitment of CD8+ T-cells into the brain but attracts activated antigen-specific CD8+ T-cells from cognate antigen injection sites. This process is presumably antigen independent and cannot be inhibited by blocking CD154 molecules. These data show that activated antigen-specific CD8+ T-cells accumulate in the CNS at both cognate antigen-containing and traumatic injury sites after intracerebral antigen delivery. The accumulation of activated antigen-specific T-cells at traumatic injury sites, in addition to antigen-containing areas, could amplify local inflammatory processes in the CNS. Combination therapies in neuroinflammatory diseases to block both of these processes should be considered.

Initiation of immune responses in brain is promoted by local dendritic cells.

The contribution of dendritic cells (DCs) to initiating T cell-mediated immune response in and T cell homing into the CNS has not yet been clarified. In this study we show by confocal microscopy and flow cytometry that cells expressing CD11c, CD205, and MHC class II molecules and containing fluorescently labeled, processed Ag accumulate at the site of intracerebral Ag injection. These cells follow a specific pattern upon migrating out of the brain. To track their pathway out of the CNS, we differentiated DCs from bone marrow of GFP-transgenic mice and injected them directly into brains of naive C57BL/6 mice. We demonstrate that DCs migrate from brain to cervical lymph nodes, a process that can be blocked by fixation or pertussis toxin treatment of the DCs. Injection of OVA-loaded DCs into brain initiates a SIINFEKL (a dominant OVA epitope)-specific T cell response in lymph nodes and spleen, as measured by specific tetramer and LFA-1 activation marker staining. Additionally, a fraction of activated SIINFEKL-specific T cells home to the CNS. Specific T cell homing to the CNS, however, cannot be induced by i.v. injection of OVA-loaded DCs alone. These data suggest that brain-emigrant DCs are sufficient to support activated T cells to home to the tissue of DC origination. Thus, initiation of immune reactivity against CNS Ags involves the migration of APCs from nervous tissue to peripheral lymphoid tissues, similarly to that in other organs.

Dendritic cells in the initiation of immune responses against central nervous system-derived antigens.

Antigen presentation is essential for the activation and maintenance of antigen-specific T cell responses in the nervous tissue. Generally, it is becoming well accepted that the antigen presenting cell (APC) type responsible for the initiation of the primary immune response through the exclusive ability to activate naïve T cells is the dendritic cell (DC); however, the role of these cells in central nervous system (CNS) immunity is unclear at this time. The diverse phenotypes and origins of DCs make the characterization of their function in the CNS even more difficult. It is believed that DCs can influence the immune response in several ways: these cells are not only capable of initiating the immune response but they are also a major determinant of peripheral tolerance. DCs are characterized by the constitutive ability to express MHC class II molecules as well as high-level upregulation of these molecules in response to inflammatory stimuli. A pan DC marker that has proved to be useful in identifying them is CD11c (the alpha-chain of CR4); other markers include CD205 and MHC class II. DCs also actively participate in the humoral immune response. In this review, we would like to discuss how DCs appear in the CNS and their roles in initiation, maintenance and tolerance in the immune reactions in the CNS.