RESUMO
The stratum corneum is the outermost skin layer with a vital role in skin barrier function. It is comprised of dead keratinocytes (corneocytes) and is known to maintain its thickness by shedding cells, although, the precise mechanisms that safeguard stratum corneum maturation and homeostasis remain unclear. Previous ex vivo studies have suggested a neutral-to-acidic pH gradient in the stratum corneum. Here, we use intravital pH imaging at single-corneocyte resolution to demonstrate that corneocytes actually undergo differentiation to develop three distinct zones in the stratum corneum, each with a distinct pH value. We identified a moderately acidic lower, an acidic middle, and a pH-neutral upper layer in the stratum corneum, with tight junctions playing a key role in their development. The upper pH neutral zone can adjust its pH according to the external environment and has a neutral pH under steady-state conditions owing to the influence of skin microbiota. The middle acidic pH zone provides a defensive barrier against pathogens. With mathematical modeling, we demonstrate the controlled protease activation of kallikrein-related peptidases on the stratum corneum surface that results in proper corneocyte shedding in desquamation. This work adds crucial information to our understanding of how stratum corneum homeostasis is maintained.
Assuntos
Epiderme , Homeostase , Queratinócitos , Concentração de Íons de Hidrogênio , Animais , Queratinócitos/metabolismo , Epiderme/metabolismo , Pele/metabolismo , Camundongos , Humanos , Diferenciação Celular , Junções Íntimas/metabolismo , Masculino , Feminino , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: Itch is the most troublesome symptom of atopic dermatitis, and it is important to assess it appropriately for optimal treatment. We discussed issues regarding itch and the most appropriate methods of assessment at the Atopic Itch Consensus Meeting (AICOM), attended by physicians and researchers with expertise in itch treatment and research. METHODS: The AICOM participants prepared a draft consensus statement that addressed the most appropriate itch assessment methods for age groups <2 years, 2-6 years, 7-14 years, and ≥15 years. Consensus was defined as agreement by ≥80% of the participants. RESULTS: Votes were cast by 20 participants (8 dermatologists, 7 pediatricians, and 5 researchers), and a consensus on the best current methods of itch assessment was reached with 95% agreement. For infants and preschool children, because subjective evaluation is difficult, a checklist for itch assessment was developed for caregivers. CONCLUSION: For itch assessment, we recommend subjective evaluation by the patient using a rating scale. For infants and preschoolers, evaluation should be done by the caregiver using a checklist, combined with objective evaluation (of skin lesions, for example) by a physician. We anticipate that more objective itch assessment indices will be established in the future.
Assuntos
Dermatite Atópica , Prurido , Lactente , Pré-Escolar , Humanos , Índice de Gravidade de Doença , Prurido/diagnóstico , Prurido/etiologia , Dermatite Atópica/complicações , Dermatite Atópica/diagnóstico , Dermatite Atópica/terapiaRESUMO
BACKGROUND: The integumentary system of the skin serves as an exceptional protective barrier, with the stratum corneum situated at the forefront. This outermost layer is composed of keratinocytes that biosynthesize filaggrin (encoded by the gene Flg), a pivotal constituent in maintaining skin health. Nevertheless, the precise role of sensory nerves in restoration of the skin barrier after tape stripping-induced epidermal disruption, in contrast to the wound-healing process, remains a tantalizing enigma. OBJECTIVE: This study aimed to elucidate the cryptic role of sensory nerves in repair of the epidermal barrier following tape stripping-induced disruption. METHODS: Through the implementation of resiniferatoxin (RTX)-treated denervation mouse model, we investigated the kinetics of barrier repair after tape stripping and performed immunophenotyping and gene expression analysis in the skin or dorsal root ganglia (DRG) to identify potential neuropeptides. Furthermore, we assessed the functional impact of candidates on the recovery of murine keratinocytes and RTX-treated mice. RESULTS: Ablation of TRPV1-positive sensory nerve attenuated skin barrier recovery and sustained subcutaneous inflammation, coupled with elevated IL-6 level in ear homogenates after tape stripping. Expression of the keratinocyte differentiation marker Flg in the ear skin of RTX-treated mice was decreased compared with that in control mice. Through neuropeptide screening, we found that the downregulation of Flg by IL-6 was counteracted by somatostatin or octreotide (a chemically stable somatostatin analog). Furthermore, RTX-treated mice given octreotide exhibited a partial improvement in barrier recovery after tape stripping. CONCLUSION: Sensory neurons expressing TRPV1 play an indispensable role in restoring barrier function following epidermal injury. Our findings suggest the potential involvement of somatostatin in restoring epidermal repair after skin injury.
Assuntos
Interleucina-6 , Neuropeptídeos , Camundongos , Animais , Interleucina-6/metabolismo , Octreotida/metabolismo , Epiderme/metabolismo , Somatostatina/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismoRESUMO
IL-31 receptor blockade suppresses pruritus of atopic dermatitis. However, cell-type-specific contributions of IL-31 receptor to itch, its expression mechanism, and the downstream signaling pathway to induce itch remain unknown. Here, using conditional knockout mice, we demonstrate that IL-31-induced itch requires sensory neuronal IL-31 receptor and STAT3. We find that IL-31 receptor expression is dependent on STAT3 in sensory neurons. In addition, pharmacological experiments suggest that STAT3 activation is important for the itch-inducing signaling downstream of the IL-31 receptor. A cutaneous IL-31 injection induces the nuclear accumulation of activated STAT3 first in sensory neurons that abundantly express IL-31 receptor and then in other itch-transmitting neurons. IL-31 enhances itch induced by various pruritogens including even chloroquine. Finally, pruritus associated with dermatitis is partially dependent on sensory neuronal IL-31 receptor and strongly on sensory neuronal STAT3. Thus, sensory neuronal STAT3 is essential for IL-31-induced itch and further contributes to IL-31-independent inflammatory itch.
Assuntos
Dermatite Atópica , Prurido , Animais , Camundongos , Dermatite Atópica/metabolismo , Expressão Gênica , Camundongos Knockout , Prurido/induzido quimicamente , Prurido/genética , Prurido/metabolismo , Células Receptoras Sensoriais/metabolismo , Pele/metabolismoRESUMO
RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.
Assuntos
Nanopartículas , Linfócitos T Citotóxicos , Animais , Camundongos , Linfócitos T CD8-Positivos , Vitamina E/farmacologia , Vacinas Sintéticas , Vacinas de mRNA , Antígenos , Ovalbumina , RNA Mensageiro/genética , Lipídeos/farmacologia , Camundongos Endogâmicos C57BL , Células DendríticasRESUMO
Unique molecular vulnerabilities have been identified in the aggressive MCD/C5 genetic subclass of diffuse large B-cell lymphoma (DLBCL). However, the premalignant cell-of-origin exhibiting MCD-like dependencies remains elusive. In this study, we examined animals carrying up to 4 hallmark genetic lesions found in MCD consisting of gain-of-function mutations in Myd88 and Cd79b, loss of Prdm1, and overexpression of BCL2. We discovered that expression of combinations of these alleles in vivo promoted a cell-intrinsic accumulation of B cells in spontaneous splenic germinal centers (GCs). As with MCD, these premalignant B cells were enriched for B-cell receptors (BCRs) with evidence of self-reactivity, displayed a de novo dependence on Tlr9, and were more sensitive to inhibition of Bruton's tyrosine kinase. Mutant spontaneous splenic GC B cells (GCB) showed increased proliferation and IRF4 expression. Mice carrying all 4 genetic lesions showed a >50-fold expansion of spontaneous splenic GCs exhibiting aberrant histologic features with a dark zone immunophenotype and went on to develop DLBCL in the spleen with age. Thus, by combining multiple hallmark genetic alterations associated with MCD, our study identifies aberrant spontaneous splenic GCBs as a likely cell-of-origin for this aggressive genetic subtype of lymphoma.
Assuntos
Linfoma Difuso de Grandes Células B , Baço , Animais , Linfócitos B/patologia , Centro Germinativo/patologia , Linfoma Difuso de Grandes Células B/patologia , Camundongos , Mutação , Baço/patologiaRESUMO
T follicular helper (Tfh) cells are defined by a Bcl6+CXCR5hiPD-1hi phenotype, but only a minor fraction of these reside in germinal centers (GCs). Here, we examined whether GC-resident and -nonresident Tfh cells share a common physiology and function. Fluorescently labeled, GC-resident Tfh cells in different mouse models were distinguished by low expression of CD90. CD90neg/lo GCTfh cells required antigen-specific, MHCII+ B cells to develop and stopped proliferating soon after differentiation. In contrast, nonresident, CD90hi Tfh (GCTfh-like) cells developed normally in the absence of MHCII+ B cells and proliferated continuously during primary responses. The TCR repertoires of both Tfh subsets overlapped initially but later diverged in association with dendritic cell-dependent proliferation of CD90hi GCTfh-like cells, suggestive of TCR-dependency seen also in TCR-transgenic adoptive transfer experiments. Furthermore, the transcriptomes of CD90neg/lo and CD90hi GCTfh-like cells were enriched in different functional pathways. Thus, GC-resident and nonresident Tfh cells have distinct developmental requirements and activities, implying distinct functions.
Assuntos
Centro Germinativo/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores CXCR5/metabolismo , Células T Auxiliares Foliculares/metabolismo , Subpopulações de Linfócitos T/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Comunicação Celular/imunologia , Diferenciação Celular , Proliferação de Células , Células Dendríticas/imunologia , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Células T Auxiliares Foliculares/imunologia , Subpopulações de Linfócitos T/imunologia , Antígenos Thy-1/metabolismoRESUMO
The stratum corneum (SC), the outermost epidermal layer, consists of nonviable anuclear keratinocytes, called corneocytes, which function as a protective barrier. The exact modes of cell death executed by keratinocytes of the upper stratum granulosum (SG1 cells) remain largely unknown. Here, using intravital imaging combined with intracellular Ca2+- and pH-responsive fluorescent probes, we aimed to dissect the SG1 death process in vivo. We found that SG1 cell death was preceded by prolonged (â¼60 min) Ca2+ elevation and rapid induction of intracellular acidification. Once such intracellular ionic changes were initiated, they became sustained, irreversibly committing the SG1 cells to corneocyte conversion. Time-lapse imaging of isolated murine SG1 cells revealed that intracellular acidification was essential for the degradation of keratohyalin granules and nuclear DNA, phenomena specific to SC corneocyte formation. Furthermore, intravital imaging showed that the number of SG1 cells exhibiting Ca2+ elevation and the timing of intracellular acidification were both tightly regulated by the transient receptor potential cation channel V3. The functional activity of this protein was confirmed in isolated SG1 cells using whole-cell patch-clamp analysis. These findings provide a theoretical framework for improved understanding of the unique molecular mechanisms underlying keratinocyte-specific death mode, namely corneoptosis.
Assuntos
Morte Celular/fisiologia , Células Epidérmicas/metabolismo , Queratinócitos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Diferenciação Celular , Epiderme/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Queratinócitos/fisiologia , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp/métodos , PeleRESUMO
The sentinel lymph node (LN) is the first LN to which lymph fluid flows from tumor tissue. We identified the key parameters of liposomes (LPs) that affect their accumulation in regional (primary) LNs with minimum leakage to its connecting (secondary) LNs by a comprehensive analysis of the LN-to-LN trafficking of LPs with various surface charges and various sizes. We used a lymphatic flow-modified (LFM) mouse that allows for the chronological analysis of inguinal (primary) LN-to-axillary (secondary) LN at the body surface. As a result, the anionic medium-sized LPs (130 nm on average) exhibited the highest accumulation in the primary LNs. A mechanism-based analysis revealed that CD169-positive macrophages in LNs were the dominant cell population that captures anionic LPs. Sentinel LN imaging was also performed by the intratumoral injection of fluorescent medium-sized anionic LPs using a breast cancer orthotopic model. In comparison with the typically used contrast agent indocyanine green, the anionic LPs were detected in sentinel LNs with a high sensitivity. Additionally, the co-injection of hyaluronidase significantly improved the sensitivity of detection of the fluorescent LPs in sentinel LNs. In conclusion, medium-sized anionic LPs combined with hyaluronidase represents a potent strategy for investigating sentinel LNs.
Assuntos
Biomarcadores , Lipossomos , Metástase Linfática/diagnóstico por imagem , Metástase Linfática/patologia , Linfonodo Sentinela/diagnóstico por imagem , Meios de Contraste , Humanos , Cinética , Lipossomos/administração & dosagem , Lipossomos/metabolismo , Macrófagos/metabolismo , Estadiamento de Neoplasias , Imagem Óptica/métodos , Linfonodo Sentinela/patologiaRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disorder with a high prevalence, especially in industrialized countries. Dysbiosis of the intestinal microbiota has been observed in RA patients. For instance, new-onset untreated RA (NORA) is associated with the underrepresentation of the Clostridium cluster XIVa, including Lachnospiraceae, which are major butyrate producers, although the pathological relevance has remained obscure. Follicular regulatory T (TFR) cells play critical regulatory roles in the pathogenesis of autoimmune diseases, including RA. Reduced number of circulating TFR cells has been associated with the elevation of autoantibodies and disease severity in RA. However, the contribution of commensal microbe-derived butyrate in controlling TFR cell differentiation remains unknown. METHODS: We examined the contribution of microbe-derived butyrate in controlling autoimmune arthritis using collagen-induced arthritis (CIA) and SKG arthritis models. We phenotyped autoimmune responses in the gut-associated lymphoid tissues (GALT) in the colon and joint-draining lymph nodes in the CIA model. We developed an in vitro CXCR5+Bcl-6+Foxp3+ TFR (iTFR) cell culture system and examined whether butyrate promotes the differentiation of iTFR cells. FINDINGS: Microbe-derived butyrate suppressed the development of autoimmune arthritis. The immunization of type II collagen (CII) caused hypertrophy of the GALT in the colon by amplifying the GC reaction prior to the onset of the CIA. Butyrate mitigated these pathological events by promoting TFR cell differentiation. Butyrate directly induced the differentiation of functional TFR cells in vitro by enhancing histone acetylation in TFR cell marker genes. This effect was attributed to histone deacetylase (HDAC) inhibition by butyrate, leading to histone hyperacetylation in the promoter region of the TFR-cell marker genes. The adoptive transfer of the butyrate-treated iTFR cells reduced CII-specific autoantibody production and thus ameliorated the symptoms of arthritis. INTERPRETATION: Accordingly, microbiota-derived butyrate serves as an environmental cue to enhance TFR cells, which suppress autoantibody production in the systemic lymphoid tissue, eventually ameliorating RA. Our findings provide mechanistic insights into the link between the gut environment and RA risk. FUNDING: This work was supported by AMED-Crest (16gm1010004h0101, 17gm1010004h0102, 18gm1010004h0103, and 19gm1010004s0104 to KH), the Japan Society for the Promotion of Science (JP17KT0055, JP16H01369, and JP18H04680 to KH; JP17K15734 to DT), Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects (KH), Keio Gijuku Fukuzawa Memorial Fund for the Advancement of Education and Research (DT), the SECOM Science and Technology Foundation (KH), the Cell Science Research Foundation (KH), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (DT), the Suzuken Memorial Foundation (KH and DT), the Takeda Science Foundation (KH and DT), The Science Research Promotion Fund, and The Promotion and Mutual Aid Corporation for Private Schools of Japan (KH).
Assuntos
Artrite Experimental/terapia , Artrite Reumatoide/terapia , Bactérias/metabolismo , Butiratos/farmacologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/transplante , Acetilação , Transferência Adotiva , Idoso , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/imunologia , Autoimunidade , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Microbioma Gastrointestinal , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Tecido Linfoide/citologia , Tecido Linfoide/efeitos dos fármacos , Tecido Linfoide/imunologia , Pessoa de Meia-Idade , Linfócitos T Reguladores/efeitos dos fármacosRESUMO
Recent clinical and experimental evidence has evoked the concept of the gut-brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1-4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver-brain-gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver-brain-gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut.
Assuntos
Encéfalo/citologia , Intestinos/citologia , Intestinos/inervação , Fígado/citologia , Fígado/inervação , Neurônios/fisiologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Vias Aferentes , Animais , Células Apresentadoras de Antígenos/imunologia , Colite/imunologia , Colite/metabolismo , Colite/patologia , Homeostase , Humanos , Intestinos/imunologia , Masculino , Camundongos , Ratos , Receptores Muscarínicos/metabolismo , Baço/citologia , Baço/imunologia , Nervo Vago/fisiologiaRESUMO
Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Memória Imunológica/imunologia , Imunidade Adaptativa/imunologia , Animais , Formação de Anticorpos/imunologia , Formação de Anticorpos/fisiologia , Antígenos/imunologia , Linfócitos B/metabolismo , Células CHO , Linhagem Celular , Cricetulus , Feminino , Centro Germinativo/metabolismo , Humanos , Memória Imunológica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos AnimaisRESUMO
The epidermal barrier is thought to protect sensory nerves from overexposure to environmental stimuli, and barrier impairment leads to pathological conditions associated with itch, such as atopic dermatitis (AD). However, it is not known how the epidermal barrier continuously protects nerves for the sensory homeostasis during turnover of the epidermis. Here we show that epidermal nerves are contained underneath keratinocyte tight junctions (TJs) in normal human and mouse skin, but not in human AD samples or mouse models of chronic itch caused by epidermal barrier impairment. By intravital imaging of the mouse skin, we found that epidermal nerve endings were frequently extended and retracted, and occasionally underwent local pruning. Importantly, the epidermal nerve pruning took place rapidly at intersections with newly forming TJs in the normal skin, whereas this process was disturbed during chronic itch development. Furthermore, aberrant Ca2+ increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca2+ increases in epidermal nerves and itch. These results suggest that epidermal nerve endings are pruned through interactions with keratinocytes to stay below the TJ barrier, and that disruption of this mechanism may lead to aberrant activation of epidermal nerves and pathological itch.
Assuntos
Epiderme/inervação , Epiderme/patologia , Homeostase , Tecido Nervoso/patologia , Prurido/patologia , Animais , Cálcio/metabolismo , Doença Crônica , Dermatite Atópica/patologia , Humanos , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Terminações Nervosas/patologia , Células Receptoras Sensoriais/patologia , Canal de Cátion TRPA1/metabolismo , Junções Íntimas/patologiaRESUMO
The tumor necrosis factor receptor superfamily member HVEM is one of the most frequently mutated surface proteins in germinal center (GC)-derived B cell lymphomas. We found that HVEM deficiency increased B cell competitiveness during pre-GC and GC responses. The immunoglobulin (Ig) superfamily protein BTLA regulated HVEM-expressing B cell responses independently of B-cell-intrinsic signaling via HVEM or BTLA. BTLA signaling into T cells through the phosphatase SHP1 reduced T cell receptor (TCR) signaling and preformed CD40 ligand mobilization to the immunological synapse, thus diminishing the help delivered to B cells. Moreover, T cell deficiency in BTLA cooperated with B cell Bcl-2 overexpression, leading to GC B cell outgrowth. These results establish that HVEM restrains the T helper signals delivered to B cells to influence GC selection outcomes, and they suggest that BTLA functions as a cell-extrinsic suppressor of GC B cell lymphomagenesis.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Proliferação de Células , Sinapses Imunológicas , Ativação Linfocitária , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Comunicação Parácrina , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Imunológicos/genética , Transdução de SinaisRESUMO
PURPOSE: The purpose of this study was to elucidate the specific regional cerebral blood flow (rCBF) alterations for idiopathic normal pressure hydrocephalus (iNPH) by comparing the proportional rCBF and gray matter change from those of a normal database at the same point of SPECT and MRI examinations. METHODS: Thirty subjects with iNPH underwent both CBF SPECT and MRI. After normalization, voxel-wise two-sample t tests between patients and 11 normal controls were conducted to compare the regional alteration in the gray matter density and rCBF. RESULTS: The rCBF reduction and the gray matter decrease were seen in almost similar regions surrounding Sylvian fissure, the left parietotemporal region and frontal lobes, whereas we did not find rCBF increase at the top of the high convexity, where the increase of the gray matter density was the highest (p < 0.05). CONCLUSION: Our study showed regional associations and dissociations between the relative gray matter density and rCBF in patients with iNPH.
Assuntos
Circulação Cerebrovascular , Substância Cinzenta/diagnóstico por imagem , Hidrocefalia de Pressão Normal/diagnóstico por imagem , Imageamento por Ressonância Magnética , Fluxo Sanguíneo Regional , Tomografia Computadorizada de Emissão de Fóton Único , Idoso , Idoso de 80 Anos ou mais , Feminino , Substância Cinzenta/patologia , Humanos , Hidrocefalia de Pressão Normal/patologia , Masculino , Pessoa de Meia-IdadeRESUMO
Higher- or lower-affinity germinal center (GC) B cells are directed either to plasma cell or GC recycling, respectively; however, how commitment to the plasma cell fate takes place is unclear. We found that a population of light zone (LZ) GC cells, Bcl6loCD69hi expressing a transcription factor IRF4 and higher-affinity B cell receptors (BCRs) or Bcl6hiCD69hi with lower-affinity BCRs, favored the plasma cell or recycling GC cell fate, respectively. Mechanistically, CD40 acted as a dose-dependent regulator for Bcl6loCD69hi cell formation. Furthermore, we found that expression of intercellular adhesion molecule 1 (ICAM-1) and signaling lymphocytic activation molecule (SLAM) in Bcl6loCD69hi cells was higher than in Bcl6hiCD69hi cells, thereby affording more stable T follicular helper (Tfh)-GC B cell contacts. These data support a model whereby commitment to the plasma cell begins in the GC and suggest that stability of Tfh-GC B cell contacts is key for plasma cell-prone GC cell formation.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linfócitos B/citologia , Antígenos CD40/metabolismo , Centro Germinativo/imunologia , Lectinas Tipo C/metabolismo , Plasmócitos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Animais , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Molécula 1 de Adesão Intercelular/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família de Moléculas de Sinalização da Ativação Linfocitária/biossíntese , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Protective immunity against pathogens depends on the efficient generation of functionally diverse effector and memory T lymphocytes. However, whether plasticity during effector-to-memory CD8+ T cell differentiation affects memory lineage specification and functional versatility remains unclear. Using genetic fate mapping analysis of highly cytotoxic KLRG1+ effector CD8+ T cells, we demonstrated that KLRG1+ cells receiving intermediate amounts of activating and inflammatory signals downregulated KLRG1 during the contraction phase in a Bach2-dependent manner and differentiated into all memory T cell linages, including CX3CR1int peripheral memory cells and tissue-resident memory cells. "ExKLRG1" memory cells retained high cytotoxic and proliferative capacity distinct from other populations, which contributed to effective anti-influenza and anti-tumor immunity. Our work demonstrates that developmental plasticity of KLRG1+ effector CD8+ T cells is important in promoting functionally versatile memory cells and long-term protective immunity.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Receptores Imunológicos/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Linhagem da Célula/imunologia , Vírus da Influenza A/imunologia , Subunidade p35 da Interleucina-12/imunologia , Lectinas Tipo C , Listeria monocytogenes/imunologia , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Imunológicos/genética , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
A hallmark of humoral immune responses is the production of antibodies. This process involves a complex cascade of molecular and cellular interactions, including recognition of specific antigen by the B cell receptor (BCR), which triggers activation of B cells and differentiation into plasma cells (PCs). Although activation of the small GTPase Rac has been implicated in BCR-mediated antigen recognition, its precise role in humoral immunity and the upstream regulator remain elusive. DOCK2 is a Rac-specific guanine nucleotide exchange factor predominantly expressed in hematopoietic cells. We found that BCR-mediated Rac activation was almost completely lost in DOCK2-deficient B cells, resulting in defects in B cell spreading over the target cell-membrane and sustained growth of BCR microclusters at the interface. When wild-type B cells were stimulated in vitro with anti-IgM F(ab')2 antibody in the presence of IL-4 and IL-5, they differentiated efficiently into PCs. However, BCR-mediated PC differentiation was severely impaired in the case of DOCK2-deficient B cells. Similar results were obtained in vivo when DOCK2-deficient B cells expressing a defined BCR specificity were adoptively transferred into mice and challenged with the cognate antigen. In addition, by generating the conditional knockout mice, we found that DOCK2 expression in B-cell lineage is required to mount antigen-specific IgG antibody. These results highlight important role of the DOCK2-Rac axis in PC differentiation and IgG antibody responses.
Assuntos
Diferenciação Celular/imunologia , Proteínas Ativadoras de GTPase/fisiologia , Imunidade Humoral , Imunoglobulina G/metabolismo , Plasmócitos/fisiologia , Transferência Adotiva , Animais , Membrana Celular/imunologia , Feminino , Fatores de Troca do Nucleotídeo Guanina , Imunoglobulina G/imunologia , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Células-Tronco Embrionárias Murinas/transplante , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Quimeras de Transplante , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Identification of germinal center (GC) B cells is typically reliant on the use of surface activation markers that exhibit a wide range of expression. Here, we identify Ephrin-B1, a ligand for Eph-related receptor tyrosine kinases, as a specific marker of mature GC B cells. The number of Ephrin-B1+ GC B cells increases during the course of an immune response with Ephrin-B1+ GC B cells displaying elevated levels of Bcl6, S1pr2, and Aicda relative to their Ephrin-B1- counterparts. We further identified a small proportion of recently dividing, somatically mutated Ephrin-B1+ GC B cells that have begun to down-regulate Bcl6 and S1pr2 and express markers associated with memory B cells, such as CD38 and EBI2. Transcriptional analysis indicates that these cells are developmentally related to memory B cells, and likely represent a population of GC memory precursor (PreMem) B cells. GC PreMem cells display enhanced survival relative to bulk GC B cells, localize near the edge of the GC, and are predominantly found within the light zone. These findings offer insight into the significant heterogeneity that exists within the GC B cell population and provide tools to further dissect signals regulating the differentiation of GC B cells.
Assuntos
Linfócitos B/imunologia , Efrina-B1/fisiologia , Centro Germinativo/imunologia , Memória Imunológica , Animais , Biomarcadores , Efrina-B1/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Lisoesfingolipídeo/análise , Receptores de Esfingosina-1-Fosfato , Sindecana-1/análiseRESUMO
Surface coatings delivering BMP are a promising approach to render biomaterials osteoinductive. In contrast to soluble BMPs which can interact with their receptors at the dorsal side of the cell, BMPs presented as an insoluble cue physically bound to a biomimetic matrix, called here matrix-bound (bBMP-2), are presented to cells by their ventral side. To date, BMP-2 internalization and signaling studies in cell biology have always been performed by adding soluble (sBMP-2) to cells adhered on cell culture plates or glass slides, which will be considered here as a "reference" condition. However, whether and how matrix-bound BMP-2 can be internalized by cells and its relation to canonical (SMAD) and non-canonical signaling (ALP) remain open questions. In this study, we investigated the uptake and processing of BMP-2 by C2C12 myoblasts. This BMP-2 was presented either embedded in polyelectrolyte multilayer films (matrix-bound presentation) or as soluble form. Using fluorescently labeled BMP-2, we showed that the amount of matrix-bound BMP-2 internalized is dependent on the level of crosslinking of the polyelectrolyte films. Cav-1-mediated internalization is related to both SMAD and ALP signaling, while clathrin-mediated is only related to ALP signaling. BMP-2 internalization was independent of the presentation mode (sBMP-2 versus bBMP-2) for low crosslinked films (soft, EDC10) in striking contrast with high crosslinked (stiff, EDC70) films where internalization was much lower and slower for bBMP-2. As anticipated, internalization of sBMP-2 barely depended on the underlying matrix. Taken together, these results indicate that BMP-2 internalization can be tuned by the underlying matrix and activates downstream BMP-2 signaling, which is key for the effective formation of bone tissue. STATEMENT OF SIGNIFICANCE: The presentation of growth factors from material surfaces currently presents significant challenges in academic research, clinics and industry. Being able to deliver efficiently these growth factors by a biomaterial will open new perspectives for regenerative medicine. However, to date, very little is known about how matrix-bound growth factors are delivered to cells, especially whether they are internalized and how they are signaling to drive key differentiation events. These initial steps are crucial as they will guide the subsequent processes leading to tissue regeneration. In this work, we investigate the uptake and processing by cells of BMP-2 ligands embedded in polyelectrolyte multilayer films in comparison to soluble BMP-2. We show that BMP-2 responsive cells can internalize matrix-bound BMP-2 and that internalization is dependent on the cross-linking level of the polyelectrolyte films. In addition, we show that internalization is mediated by both clathrin- and caveolin-dependent pathways. While inhibiting clathrin-dependent endocytosis affects only non-canonical signaling, blocking caveolin-1-dependent endocytosis reduces both canonical and non-canonical BMP signaling. The signaling pathways found for matrix-bound BMP-2 are similar to those found for soluble BMP-2. These results highlight that BMP-2 presented by a biomaterial at the ventral side of the cell can trigger major endocytic and associated signaling pathways leading to bone regeneration.