Requirements of transcription factor Smad-dependent and -independent TGF-β signaling to control discrete T-cell functions

TGF-β modulates immune response by suppressing non-regulatory T (Treg) function and promoting Treg function. The question of whether TGF-β achieves distinct effects on non-Treg and Treg cells through discrete signaling pathways remains outstanding. In this study, we investigated the requirements of Smad-dependent and -independent TGF-β signaling for T-cell function. Smad2 and Smad3 double deficiency in T cells led to lethal inflammatory disorder in mice. Non-Treg cells were spontaneously activated and produced effector cytokines in vivo on deletion of both Smad2 and Smad3. In addition, TGF-β failed to suppress T helper differentiation efficiently and to promote induced Treg generation of non-Treg cells lacking both Smad2 and Smad3, suggesting that Smad-dependent signaling is obligatory to mediate TGF-β function in non-Treg cells. Unexpectedly, however, the development, homeostasis, and function of Treg cells remained intact in the absence of Smad2 and Smad3, suggesting that the Smad-independent pathway is important for Treg function. Indeed, Treg-specific deletion of TGF-β-activated kinase 1 led to failed Treg homeostasis and lethal immune disorder in mice. Therefore, Smad-dependent and -independent TGF-β signaling discretely controls non-Treg and Treg function to modulate immune tolerance and immune homeostasis.     

TGF-β regulates T-cell neurokinin-1 receptor internalization and function

Substance P (SP) is a proinflammatory mediator implicated in inflammatory bowel disease (IBD) and other inflammatory states. SP acts by stimulating the neurokinin-1 receptor (NK-1R) on T lymphocytes and other cell types, and regulates these cells in a complex interplay with multiple cytokines. The mechanisms of interaction among these inflammatory mediators are not yet fully understood. Here, we demonstrate that function of the NK-1R, a member of the G protein-coupled receptor (GPCR) superfamily, is modulated by TGF-β. The latter acts not on a GPCR but via serine-threonine kinase-class receptors. By flow confocal image analysis, we demonstrate that TGF-β delays SP-induced NK-1R internalization on mucosal T cells isolated from a mouse model of IBD and on granuloma T cells in murine schistosomiasis. Furthermore, luciferase reporter-gene assays revealed that NK-1R stimulation activates the nuclear factor of activated T cell- and activator protein-1-dependent signaling pathways, which are known triggers of effector T-cell cytokine production. TGF-β markedly increases SP-induced activation of these signaling cascades, suggesting that delayed NK-1R internalization results in enhanced signaling. Providing a link to amplified immune function, SP and TGF-β, when applied in combination, trigger a strong release of the proinflammatory cytokines IFN-γ and IL17 from intestinal inflammatory T cells, whereas either agonist alone shows no effect. These observations establish precedent that members of two distinct receptor superfamilies can interact via a previously unrecognized mechanism, and reveal a paradigm of GPCR transregulation that is relevant to IBD and possibly other disease processes.     

Transforming growth factor-β signaling is constantly shaping memory T-cell population

The long-term maintenance of memory T cells is essential for successful vaccines. Both the quantity and the quality of the memory T-cell population must be maintained. The signals that control the maintenance of memory T cells remain incompletely identified. Here we used two genetic models to show that continuous transforming growth factor-β signaling to antigen-specific T cells is required for the differentiation and maintenance of memory CD8⁺ T cells. In addition, both infection-induced and microbiota-induced inflammation impact the phenotypic and functional identity of memory CD8⁺ T cells.Infectious diseases pose a significant public health burden, accounting for nearly one-fifth of annual deaths worldwide. Vaccines remain the most effective way to prevent infectious diseases. Functionally sustained memory T cells are the ideal cell population to be generated by T-cell–based vaccines. Considerable efforts have been made to elucidate the mechanisms that mediate the establishment of long-lived immunologic memory (1–6); however, the signals that control the differentiation and maintenance of memory T cells remain incompletely identified.During the early stages of an immune response, proinflammatory cytokines IL-12 and type I IFN promote the expansion of effector CD8⁺ T cells by sustaining the expression of the high-affinity IL-2 receptor CD25 (7, 8). In addition to its role in T-cell proliferation, IL-2 also functions as a differentiation factor for effector CD8⁺ T cells by promoting the differentiation of short-lived effector cells [SLECs; IL-7Rα⁻KLRG1⁺ (killer cell lectin-like receptor subfamily G, member 1)] and inhibiting the differentiation of memory precursor effector cells (MPECs; IL-7Rα⁺KLRG1⁻) (9–12). Furthermore, IL-10 and IL-21 signals promote MPEC differentiation through a STAT3-dependent mechanism (13, 14). During the late stages of an immune response, IL-15 and IL-7 are required to maintain the population of memory CD8⁺ T cells (15, 16); however, after the clearance of an infection, whether memory CD8⁺ T cells require any additional signals to maintain their phenotypic and functional identity remains unknown.Recent findings have revealed that effector and memory CD8⁺ T cells display nearly endless diversity based on the expression of surface and intracellular molecules that serve as the markers of antigen-experienced T cells (17). Thus, it is conceivable that memory CD8⁺ T cells might not be a fixed cell lineage, but instead represent an active differentiation state. Even in the absence of cognate antigens, memory CD8⁺ T cells may constantly receive diverse environmental signals; however, how memory T cells maintain their relatively stable characters under such circumstances remains unexplored.Here we show that TGF-β signaling to CD8⁺ T cells controls the differentiation of memory T cells at both early and late stages. By deleting TGF-β receptor in antigen-specific T cells at different time points following an acute infection, we demonstrate that during the effector phase of an immune response, TGF-β restrains the inflammatory signals associated with the infection. At the memory phase, both the TGF-β signal and the basal inflammation induced by microbiota cooperate to shape the memory T-cell population. Taken together, our findings show that continuous TGF-β signaling is required to maintain the identity of memory CD8⁺ T cells following acute infections.     

Cerebral small vessel disease-related protease HtrA1 processes latent TGF-β binding protein 1 and facilitates TGF-β signaling

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor β (TGF-β) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-β signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-β pathway activation. We identify latent TGF-β binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-β bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-β signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.Cerebral small vessel disease (SVD) accounts for roughly one fifth of all strokes worldwide and is recognized as a major cause of cognitive decline and dementia (1). Familial forms of SVD have successfully been used as model conditions for mechanistic studies on SVD (2–4). CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy), a recessive SVD, is characterized by juvenile and recurrent strokes, extensive brain white matter lesions, and premature dementia (5). The disease is caused by mutations in the HTRA1 (high temperature requirement protein A1) gene (6) encoding an evolutionarily conserved serine protease (7). CARASIL mutations typically result in loss of HtrA1 activity, suggesting impaired substrate processing as a disease mechanism (6, 8, 9).HtrA1 has been shown to degrade a number of substrates, most of which are situated in the extracellular compartment, thus suggesting the extracellular space as primary location of HtrA1 function. HtrA1-mediated proteolysis has been implicated in various disease processes such as neurodegeneration (10, 11), age-related macular degeneration (12, 13), carcinogenesis (14) and arthritis (15). Only recently, identification of HTRA1 as the CARASIL-causing gene has highlighted its role in the vascular system and in TGF-β signaling, a well-defined regulator of angiogenesis and vascular homeostasis (16, 17) known to be implicated in several vascular conditions including Marfan Syndrome, Loeys–Dietz Syndrome, and hereditary hemorrhagic telangiectasia (18).Initial studies on CARASIL reported increased levels of the TGF-β prodomain (also called latency-associated peptide, LAP) and of TGF-β target genes in the cerebral vasculature of affected patients. This finding led to the proposition that up-regulation of the TGF-β pathway drives CARASIL pathogenesis (6, 8). However, the initial results were mostly obtained using overexpressing cells and autopsy material from advanced cases. Also, there is some controversy as to how HtrA1 interferes with TGF-β signaling. Proposed mechanisms include HtrA1-mediated extracellular cleavage of mature TGF-β (19, 20), cleavage of TGF-β receptors (21), and intracellular degradation of LAP (8).To better define the mechanistic link between HtrA1 and TGF-β and to identify physiological HtrA1 substrates relevant for CARASIL we investigated the effects of HtrA1 deficiency on the TGF-β pathway in brain tissue and embryonic fibroblasts from HTRA1 knockout mice and in skin fibroblasts from a CARASIL patient. Unexpectedly, we observed a consistent reduction of TGF-β activity in both murine and human material suggesting a facilitating role of HtrA1 in TGF-β signaling. We further identified latent TGF-β binding protein-1 (LTBP-1), a matricellular factor with a major role in TGF-β bioactivation (16, 22) as a novel physiological HtrA1 substrate and report on the functional consequences of its processing. Our findings point to a down-regulation of the TGF-β pathway in CARASIL pathogenesis and suggest LTBP-1 as a key HtrA1 substrate.     

Connection between inflammation and carcinogenesis in gastrointestinal tract: Focus on TGF-β signaling

Inflammation is a primary defense process against various extracellular stimuli,such as viruses,pathogens,foods,and environmental pollutants.When cells respond to stimuli for short periods of time,it results in acute or physiological inflammation.However,if the stimulation is sustained for longer time or a pathological state occurs,it is known as chronic or pathological inflammation.Several studies have shown that tumorigenesis in the gastrointestinal (GI) tract is closely associated with chronic inflammati...     

Novel non-canonical TGF-β signaling networks: Emerging roles in airway smooth muscle phenotype and function

The airway smooth muscle (ASM) plays an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). ASM cells express a wide range of receptors involved in contraction, growth, matrix protein production and the secretion of cytokines and chemokines. Transforming growth factor beta (TGF-β) is one of the major players in determining the structural and functional abnormalities of the ASM in asthma and COPD. It is increasingly evident that TGF-β functions as a master switch, controlling a network of intracellular and autocrine signaling loops that effect ASM phenotype and function. In this review, the various elements that participate in non-canonical TGF-β signaling, including MAPK, PI3K, WNT/β-catenin, and Ca²⁺, are discussed, focusing on their effect on ASM phenotype and function. In addition, new aspects of ASM biology and their possible association with non-canonical TGF-β signaling will be discussed.     

TGF-β activation and lung fibrosis

Lung fibrosis can affect the parenchyma and the airways, classically giving rise to idiopathic pulmonary fibrosis (IPF) in the parenchyma or airway remodeling in asthma and chronic obstructive pulmonary disease. TGF-β activation has been implicated in the fibrosis of both IPF and airway remodeling. However, the mechanisms of TGF-β activation appear to differ depending on the cellular and anatomical compartments, with implications on disease pathogenesis. Although it appears that epithelial cell activation of TGF-β by the αvβ6 integrin is central in IPF, mesenchymal activation of TGF-β by the αvβ5 and αvβ8 integrins appears to predominate in airway remodeling. Interestingly, the mechanism of TGF-β by the integrins αvβ6 and αvβ5 is shared, relying on cytoskeletal changes, whereas activation of TGF-β by the αvβ8 integrin is distinct, relying on proteolytic cleavage of the latency-associated peptide of TGF-β by matrix metalloproteinase 14. This article describes the mechanisms through which epithelial cells activate TGF-β by the αvβ6 integrin and mesenchymal cells activate TGF-β by the αvβ5 integrin, and highlights their roles in lung fibrosis.     

Development and function of murine RORγt+ iNKT cells are under TGF-β signaling control

Invariant natural killer T (iNKT) cells have the ability to rapidly secret cytokines in response to diverse stimuli, and therefore influence numerous immune reactions. Although IFN-γ and IL-4 are thought to dominate iNKT cytokine production, a distinct subset of iNKT cells, expressing RORγt and producing IL-17, has now been identified in both mice and humans. Although a role in pathogen and allergic responses has been assigned to the RORγt(+) iNKT subset, factors controlling their development and function remain illusive. Here, we demonstrate that RORγt(+) iNKT-cell differentiation obeys transforming growth factor-β (TGF-β) signaling control, different from that described for conventional iNKT cells. We reveal that TGF-β signaling, and particularly its SMAD4-dependent pathway, is required for both the survival of RORγt(+) iNKT cells during their development and IL-17 production at the periphery. Moreover, constitutive TGF-β signaling in RORγt(+) iNKT cells drives higher peripheral numbers and increased tissue distribution. Finally, we found that SMAD4-dependent TGF-β signaling is mandatory for the peripheral expansion of the RORγt(+) iNKT cells responding to inflammatory signals. Thus, this work demonstrates that both the development and responsiveness of the newly described IL-17-producing iNKT cell subset is under the control of a dedicated TGF-β signaling pathway.