RESUMO
The most common congenital viral infection is CMV, which leads to numerous neurologic disabilities. Using a mouse model of congenital CMV, we previously determined that Ag-specific CD8+ T cells traffic to the brain in a CCR9-dependent manner. The mechanism by which these CD8+ T cells acquire a CCR9-dependent "brain-tropic" phenotype remains unclear. In this study, we identify the key factor that imprints brain homing specificity on CD8+ T cells, the source of production, and the location where CCR9 expression is induced. Specifically, we discovered that CCR9 is induced on CD8+ T cells by retinoic acid-producing CD8α+ dendritic cells in the cervical lymph node postinfection. We found that retinoic acid is important for CD8+ T cells to establish tissue residency in the brain. Collectively, our data expand the role of retinoic acid during infection and mechanistically demonstrate how CD8+ T cells are primed to protect the brain during congenital viral infection.
RESUMO
CD8+ T lymphocytes infiltrate the brain during congenital CMV infection and promote viral clearance. However, the mechanisms by which CD8+ T cells are recruited to the brain remain unclear. Using a mouse model of congenital CMV, we found a gut-homing chemokine receptor (CCR9) was preferentially expressed in CD8+ T cells localized in the brain postinfection. In the absence of CCR9 or CCL25 (CCR9's ligand) expression, CD8+ T cells failed to migrate to key sites of infection in the brain and protect the host from severe forms of disease. Interestingly, we found that expression of CCR9 on CD8+ T cells was also responsible for spatial temporal positioning of T cells in the brain. Collectively, our data demonstrate that the CMV-infected brain uses a similar mechanism for CD8+ T cell homing as the small intestine.
Assuntos
Infecções por Citomegalovirus , Receptores CCR , Humanos , Receptores CCR/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Intestino Delgado/metabolismo , Infecções por Citomegalovirus/metabolismo , Encéfalo/metabolismoRESUMO
RATIONALE: Circulating monocytes can have proinflammatory or proreparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet-derived ß2M (ß-2 microglobulin) and TGF-ß (transforming growth factor ß) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes, respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. OBJECTIVE: To determine the molecular mechanisms and signal transduction pathways by which ß2M and TGF-ß regulate monocyte responses both in vitro and in vivo. METHODS AND RESULTS: Wild-type- (WT) and platelet-specific ß2M knockout mice were treated intravenously with either ß2M or TGF-ß to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma ß2M increased proinflammatory monocytes, while increased plasma TGFß increased proreparative monocytes. TGF-ßR (TGF-ß receptor) inhibition blunted monocyte responses to both ß2M and TGF-ß in vivo. Using imaging flow cytometry, we found that ß2M decreased monocyte SMAD2/3 nuclear localization, while TGF-ß promoted SMAD nuclear translocation but decreased noncanonical/inflammatory (JNK [jun kinase] and NF-κB [nuclear factor-κB] nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. ß2M, but not TGF-ß, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked noncanonical SMAD-independent monocyte signaling and skewed monocytes towards a proreparative monocyte response. CONCLUSIONS: Our findings indicate that elevated plasma ß2M and TGF-ß dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor but induce SMAD-dependent canonical signaling (TGF-ß) versus noncanonical SMAD-independent signaling (ß2M) in a ubiquitin ligase dependent manner. This work has broad implications as ß2M is increased in several inflammatory conditions, while TGF-ß is increased in fibrotic diseases. Graphic Abstract: A graphic abstract is available for this article.
Assuntos
Monócitos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Microglobulina beta-2/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Proteínas Smad/metabolismo , Células THP-1 , Microglobulina beta-2/farmacologiaRESUMO
Although platelets are the cellular mediators of thrombosis, they are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered bone marrow-resident (BM-resident) cells. However, platelet-producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared with BM Mks. We therefore sought to define the immune functions of lung Mks. Using single-cell RNA sequencing of BM and lung myeloid-enriched cells, we found that lung Mks, which we term MkL, had gene expression patterns that are similar to antigen-presenting cells. This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment, as evidenced by BM Mks having an MkL-like phenotype under the influence of pathogen receptor challenge and lung-associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4+ T cell activation in an MHC II-dependent manner both in vitro and in vivo. These data indicated that MkL had key immune regulatory roles dictated in part by the tissue environment.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Pulmão/imunologia , Megacariócitos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , RNA-Seq , Análise de Célula ÚnicaRESUMO
Platelets have central roles in both immune responses and development. Stimulated platelets express leukocyte adhesion molecules and release numerous immune modulatory factors that recruit and activate leukocytes, both at the sites of activation and distantly. Monocytes are innate immune cells with dynamic immune modulatory functions that change during the aging process, a phenomenon termed "inflammaging". We have previously shown that platelets are a major source of plasma beta-2 microglobulin (ß2M) and that ß2M induced a monocyte pro-inflammatory phenotype. Plasma ß2M increases with age and is a pro-aging factor. We hypothesized that platelet derived ß2M regulates monocyte phenotypes in the context of aging. Using wild-type (WT) and platelet specific ß2M knockout mice (Plt-ß2M-/-) mice, we found that plasma ß2M increased with age and correlated with increased circulating Ly6CHi monocytes. However, aged Plt-ß2M-/- mice had significantly fewer Ly6CHi monocytes compared to WT mice. Quantitative real-time PCR of circulating monocytes showed that WT mouse monocytes were more "pro-inflammatory" with age, while Plt-ß2M-/- derived monocytes adopted a "pro-reparative" phenotype. Older Plt-ß2M-/- mice had a significant decline in heart function compared to age matched WT mice, as well as increased cardiac fibrosis and pro-fibrotic markers. These data suggest that platelet-derived ß2M regulates age associated monocyte polarization, and a loss of platelet derived ß2M shifted monocytes and macrophages to a pro-reparative phenotype and increased pro-fibrotic cardiac responses. Platelet regulation of monocyte phenotypes via ß2M may maintain a balance between inflammatory and reparative signals that affects age related physiologic outcomes.
Assuntos
Envelhecimento/imunologia , Envelhecimento/metabolismo , Plaquetas/metabolismo , Macrófagos/metabolismo , Microglobulina beta-2/metabolismo , Envelhecimento/patologia , Animais , Plaquetas/imunologia , Fibrose/imunologia , Fibrose/patologia , Macrófagos/imunologia , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Miocárdio/patologia , FenótipoRESUMO
ß-2 Microglobulin (ß2M) is a molecular chaperone for the major histocompatibility class I (MHC I) complex, hemochromatosis factor protein (HFE), and the neonatal Fc receptor (FcRn), but ß2M may also have less understood chaperone-independent functions. Elevated plasma ß2M has a direct role in neurocognitive decline and is a risk factor for adverse cardiovascular events. ß2M mRNA is present in platelets at very high levels, and ß2M is part of the activated platelet releasate. In addition to their more well-studied thrombotic functions, platelets are important immune regulatory cells that release inflammatory molecules and contribute to leukocyte trafficking, activation, and differentiation. We have now found that platelet-derived ß2M is a mediator of monocyte proinflammatory differentiation through noncanonical TGFß receptor signaling. Circulating monocytes from mice lacking ß2M only in platelets (Plt-ß2M-/-) had a more proreparative monocyte phenotype, in part dependent on increased platelet-derived TGFß signaling in the absence of ß2M. Using a mouse myocardial infarction (MI) model, Plt-ß2M-/- mice had limited post-MI proinflammatory monocyte responses and, instead, demonstrated early proreparative monocyte differentiation, profibrotic myofibroblast responses, and a rapid decline in heart function compared with WT mice. These data demonstrate a potentially novel chaperone-independent, monocyte phenotype-regulatory function for platelet ß2M and that platelet-derived 2M and TGFß have opposing roles in monocyte differentiation that may be important in tissue injury responses.
Assuntos
Plaquetas/metabolismo , Monócitos/metabolismo , Microglobulina beta-2/metabolismo , Animais , Diferenciação Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Chaperonas Moleculares , Ativação Plaquetária , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Células THP-1 , Microglobulina beta-2/genéticaRESUMO
Platelets have dual physiologic roles as both cellular mediators of thrombosis and immune modulatory cells. Historically, the thrombotic function of platelets has received significant research and clinical attention, but emerging research indicates that the immune regulatory roles of platelets may be just as important. We now know that in addition to their role in the acute thrombotic event at the time of myocardial infarction, platelets initiate and accelerate inflammatory processes that are part of the pathogenesis of atherosclerosis and myocardial infarction expansion. Furthermore, it is increasingly apparent from recent studies that platelets impact the pathogenesis of many vascular inflammatory processes such as autoimmune diseases, sepsis, viral infections, and growth and metastasis of many types of tumors. Therefore, we must consider platelets as immune cells that affect all phases of immune responses.