RESUMO
Lactate is a key metabolite for the normal occurrence of spermatogenesis. In the testis, lactate is produced by the Sertoli cells and transported to germline cells. Monocarboxylate transporters (MCTs) are key players in that process. Among the family of MCTs, MCT1 is at least partly responsible for lactate uptake by the germ cells. We aimed to perform a first assessment of the role of MCT1 in male reproductive potential. Mct1 conditional knockout (cKO) mice were used for morphometric evaluation, testicular morphology, and sperm parameter assessment. Serum steroid hormones levels were also measured. cKO animals showed a decrease in gonadosomatic index, testis weight, and seminiferous tubular diameters. Deletion of MCT1 also causes morphological changes in the organization of the seminiferous tubules and on Sertoli cell morphology. These changes resulted in failure of spermatogenesis with depletion of germ cells and total absence of spermatozoa. MCT1 cKO animals presented also hormonal dysregulation, with a decrease in serum 17ß-estradiol levels. In conclusion, MCT1 is pivotal for male reproductive potential. Absence of MCT1 results in maintenance of undifferentiated spermatogonia pool and compromised sperm production.
Assuntos
Fertilidade/fisiologia , Transportadores de Ácidos Monocarboxílicos/fisiologia , Túbulos Seminíferos/metabolismo , Células de Sertoli/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Simportadores/fisiologia , Animais , Estradiol/sangue , Ácido Láctico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos/genética , Células de Sertoli/citologia , Espermatozoides/citologia , Simportadores/genéticaRESUMO
Effective immune responses depend upon appropriate T cell differentiation in accord with the nature of an infectious agent, and the contingency of differentiation depends minimally on TCR, coreceptor, and cytokine signals. In this reverse genetic study, we show that the MAPK Erk2 is not essential for T cell proliferation in the presence of optimum costimulation. Instead, it has opposite effects on T-bet and Gata3 expression and, hence, on Th1 and Th2 differentiation. Alternatively, in the presence of TGF-ß, the Erk pathway suppresses a large program of gene expression, effectively limiting the differentiation of Foxp3(+) regulatory T cells. In the latter case, the mechanisms involved include suppression of Gata3 and Foxp3, induction of Tbx21, phosphorylation of Smad2,3, and possibly suppression of Socs2, a positive inducer of Stat5 signaling. Consequently, loss of Erk2 severely impeded Th1 differentiation while enhancing the development of Foxp3(+)-induced T regulatory cells. Selected profiles of gene expression under multiple conditions of T cell activation illustrate the opposing consequences of Erk pathway signaling.
Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Polaridade Celular/imunologia , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Polaridade Celular/genética , Proliferação de Células , Células Cultivadas , Fatores de Transcrição Forkhead/biossíntese , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/deficiência , Proteína Quinase 1 Ativada por Mitógeno/genética , Subpopulações de Linfócitos T/virologia , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Células Th1/enzimologia , Células Th1/imunologia , Células Th1/virologia , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
The magnitude of T cell responses is determined by proliferation and survival decisions made by the responding cells. We now demonstrate that the Erk MAPK pathway plays a critical role in these cell fate decisions within CD8 T cells. While Erk1 is dispensable for all aspects of CD8 T cell activation, Erk2 is required for the proliferation of CD8 T cells activated in the absence of costimulation. Surprisingly, Erk2 is not required for proliferation following the addition of a costimulatory signal in vitro, or upon viral infection in vivo, but regulates the size of the responding population by enhancing cell survival. An important component of this Erk2-derived signal is the transcriptional regulation of Bcl-2 family members Bcl-x(L) and Bim, and impaired Erk2-deficient CD8 T cell survival can be rescued by genetic ablation of Bim. These studies ascribe multifaceted functions specific to Erk2 in CD8 T cell activation, proliferation, and survival.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD8-Positivos/enzimologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Ativação Linfocitária/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/imunologia , Proteína bcl-X/genética , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismoRESUMO
Designing drugs to treat diseases associated with articular joints, particularly those targeting chondrocytes, is challenging due to unique local environmental constraints including the avascular nature of cartilage, the absence of a closed joint compartment, and a highly cross-linked extracellular matrix. In an effort to address these challenges, we developed a novel strategy to prolong residence time of intra-articularly administered protein therapeutics. Avimer domains are naturally found in membrane polypeptides and mediate diverse protein-protein interactions. Screening of a phage Avimer domain library led to identification of several low affinity type II collagen-binding Avimers. Following several rounds of mutagenesis and reselection, these initial hits were transformed to high affinity, selective type II collagen-binding Avimers. One such Avimer (M26) persisted in rat knees for at least 1 month following intra-articular administration. Fusion of this Avimer to a candidate therapeutic payload, IL-1Ra, yielded a protein construct which simultaneously bound to type II collagen and to IL-1 receptor. In vitro, IL-1Ra_M26 bound selectively to cartilage explants and remained associated even after extensive washing. Binding appeared to occur preferentially to pericellular regions surrounding chondrocytes. An acute intra-articular IL-1-induced IL-6 challenge rat model was employed to assess in vivo pharmacodynamics. Whereas both IL-1Ra_M26 and native IL-1Ra inhibited IL-6 output when co-administered with the IL-1 challenge, only IL-1Ra_M26 inhibited when administered 1 week prior to IL-1 challenge. Collagen-binding Avimers thus represent a promising strategy for enhancing cartilage residence time of protein therapeutics. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1238-1247, 2018.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Artropatias/tratamento farmacológico , Proteínas/administração & dosagem , Animais , Colágeno Tipo II/metabolismo , Feminino , Humanos , Injeções Intra-Articulares , Masculino , Domínios Proteicos , Engenharia de Proteínas , Ratos Endogâmicos Lew , Ratos Sprague-DawleyRESUMO
Inflammatory bowel disease (IBD) is associated with a loss of intestinal barrier function and dysregulated immune responses. It has been shown that short chain fatty acids (SCFAs) are protective in IBD and that GPR43 mediates the protective effects of SCFAs. In this study, we investigated the effects of SCFAs in comparison to highly specific GPR43 agonists on human intestinal epithelial and immune cells. Our results confirm that SCFAs are enhancers of barrier function in intestinal epithelial cells. Additionally, SCFAs also displayed potent immunoregulatory properties based upon the ability to inhibit LPS-induced cytokine production in PBMC, and human T cell proliferation and cytokine production. Unexpectedly, and in contrast to the current belief, specific GPR43 agonists failed to exhibit similar barrier enhancing and anti-inflammatory properties. These findings demonstrate that SCFA possess broad protective functions in IBD and agonizing GPR43 alone is unlikely to be beneficial in patients.
Assuntos
Células Epiteliais/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Receptores de Superfície Celular/agonistas , Animais , Células CACO-2 , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Ácidos Graxos Voláteis , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , CamundongosRESUMO
Osteoarthritis (OA) is a complex disease plagued by a significant unmet need for treatment. To date, no disease- modifying OA drugs (DMOADs) exist and the available symptom-modifying OA drugs (SMOADs) have limitations. Although a complete understanding of the mechanisms of OA pain in humans is lacking, animal models have helped provide insight into the multifaceted origin and manifestation of OA pain. Success in discovering new therapeutics will likely require reliance on good animal models. This review summarizes the animal models available for studying pain associated with OA.
Assuntos
Modelos Animais de Doenças , Osteoartrite/fisiopatologia , Dor/fisiopatologia , Animais , HumanosRESUMO
Factors that influence T cell responses, such as Ag load, APCs, costimulatory molecules, and cytokines, dramatically change during the course of an immune response. We observed that antiviral CD8 T cells were not recruited from circulation simultaneously, but over a period of 3-4 days. Consequently, locally resident T cells and those that entered secondary lymphoid tissue later were primed in very different environments. The cells recruited later in the response were imprinted with a unique differentiation program, such that their magnitude of proliferation was reduced and their kinetics of expansion was delayed. In addition, we found that the "latecomer" CD8 T cells displayed a unique surface phenotype indicative of reduced stimulation but were not preferentially recruited into the surviving pool of memory cells. This finding demonstrates that the timing of recruitment of individual T cell clones determines the population dynamics of the subsequent immune response.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/transplante , Linfócitos T CD8-Positivos/virologia , Movimento Celular/imunologia , Sobrevivência Celular/imunologia , Memória Imunológica , Imunofenotipagem , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
Based primarily on in vitro data, IL-2 is believed to be the key cytokine for initiation of the cell cycle of activated T cells. However, the role of IL-2 remains unresolved for T cell responses in vivo. We examined whether the absence of IL-2-mediated signaling in CD8 T cells affected initiation of proliferation. Our results conclusively demonstrated that initial division of Ag-specific CD8 T cells following priming was IL-2 independent, regardless of the context in which Ag was presented. In contrast, the latter stage of the proliferative phase was IL-2-dependent, particularly in nonlymphoid tissues. Thus, activated CD8 T cells initially undergo IL-2-independent proliferation, but reach a critical juncture where the requirement for IL-2 as a growth factor gains prominence.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Ciclo Celular/imunologia , Interleucina-2/fisiologia , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Divisão Celular/imunologia , Linhagem Celular Tumoral , Humanos , Injeções Intraperitoneais , Interleucina-2/administração & dosagem , Interleucina-2/biossíntese , Interleucina-2/deficiência , Subunidade alfa de Receptor de Interleucina-2 , Linfonodos/imunologia , Linfonodos/patologia , Linfonodos/virologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Receptores de Interleucina/biossíntese , Receptores de Interleucina/fisiologia , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/administração & dosagem , Estomatite/imunologia , Estomatite/patologia , Vírus da Estomatite Vesicular Indiana/imunologiaRESUMO
IL-2 is an important cytokine that is capable of inducing both proliferation and apoptosis of activated T cells. CD4 T cells are thought to be the major producers of IL-2, but CD8 T cells also produce copious amounts of this cytokine. However, our current understanding regarding the kinetics of IL-2 production by antigen-specific CD8 T cells, and the proportion of these cells that produce IL-2 in vivo, is extremely limited. We now demonstrate that virus-specific CD8 T cells initiate IL-2 production by 6 h post-infection and prior to cell division in vivo. Interestingly, peak levels of IL-2 production were achieved very early during the response and prior to the proliferative peak. We also show -- using transgenic mice expressing herpes simplex virus-1 thymidine kinase under the control of the IL-2 promoter -- that, unlike what has been reported for antigen-specific CD4 T cells, the majority of antigen-specific CD8 T cells produce IL-2 during primary as well as secondary immune responses in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/biossíntese , Infecções por Rhabdoviridae/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Transferência Adotiva , Animais , Antivirais/farmacologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/virologia , Proliferação de Células , Epitopos de Linfócito T/imunologia , Ganciclovir/farmacologia , Herpes Simples/imunologia , Memória Imunológica/imunologia , Interleucina-2/imunologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Simplexvirus/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
IL-2 is a cytokine produced primarily by activated T cells and is thought to be the quintessential T cell growth factor. The precise role of IL-2 in the regulation of CD8 T cell responses to foreign Ag in vivo however remains enigmatic. Using an adoptive transfer system with IL-2- or IL-2R-deficient TCR transgenic CD8 T cells and MHC class I tetramers, we demonstrated that the expansion of antiviral CD8 T cells in secondary lymphoid tissues was IL-2 independent, whereas IL-2 played a more significant role in supporting the continued expansion of these cells within nonlymphoid tissues. Paradoxically, autocrine IL-2 negatively regulated the overall magnitude of the CD8 T cell response in nonlymphoid tissues via a Fas-independent mechanism. Furthermore, autocrine IL-2 did not regulate the contraction or memory phase of the response. These experiments identified a novel role for IL-2 in regulation of antiviral CD8 T cell responses and homeostasis in nonlymphoid tissues.