FOXP3+ regulatory T cell perturbation mediated by the IFNγ-STAT1-IFITM3 feedback loop is essential for anti-tumor immunity.

Targeting tumor-infiltrating regulatory T cells (Tregs) is an efficient way to evoke an anti-tumor immune response. However, how Tregs maintain their fragility and stability remains largely unknown. IFITM3 and STAT1 are interferon-induced genes that play a positive role in the progression of tumors. Here, we showed that IFITM3-deficient Tregs blunted tumor growth by strengthening the tumor-killing response and displayed the Th1-like Treg phenotype with higher secretion of IFNγ. Mechanistically, depletion of IFITM3 enhances the translation and phosphorylation of STAT1. On the contrary, the decreased IFITM3 expression in STAT1-deficient Tregs indicates that STAT1 conversely regulates the expression of IFITM3 to form a feedback loop. Blocking the inflammatory cytokine IFNγ or directly depleting STAT1-IFITM3 axis phenocopies the restored suppressive function of tumor-infiltrating Tregs in the tumor model. Overall, our study demonstrates that the perturbation of tumor-infiltrating Tregs through the IFNγ-IFITM3-STAT1 feedback loop is essential for anti-tumor immunity and constitutes a targetable vulnerability of cancer immunotherapy.

Therapeutic effects of tea polyphenol-loaded nanoparticles coated with platelet membranes on LPS-induced lung injury.

Patients with ALI (acute lung injury)/ARDS (acute respiratory distress syndrome) are often septic and with poor prognosis, which leads to a high mortality rate of 25-40%. Despite the advances in medicine, there are no effective pharmacological therapies for ALI/ARDS due to the short systemic circulation and poor specificity in the lungs. To address this problem, we prepared TP-loaded nanoparticles (TP-NPs) through the emulsification-and-evaporation method, and then the platelet membrane vesicles were extracted and coated onto the surface of the NPs to constitute the biomimetic PM@TP-NPs. In a LPS-induced ALI mouse model, PM@TP-NPs showed good biocompatibility and biosafety, which was evidenced by no significant toxic effect on cell viability and no hemolysis of red blood cells. In ALI mice, the PM@TP-NPs showed favorable anti-inflammation and enhanced therapeutic activity of TPs compared to the free drug. Administration of PM@TP-NPs effectively inhibited lung vascular injury, evidenced by the decreased lung vascular permeability, reduced pro-inflammatory cytokine burden, evidenced by decreased inflammatory cell (macrophages, neutrophils, etc.) infiltration in the bronchoalveolar lavage fluid (BALF) and lung tissues, and inhibited the secretion of pro-inflammatory cytokines and NLRP3 inflammasome activation. ALI/ARDS is defined by damage to the alveolar epithelium and endothelium; thus, effective intervention targeting pulmonary vascular endothelial cells (VECs) is crucial for the treatment of respiratory diseases. For further determination of the targeting of PM cloaked NPs, healthy mice were also administered with the same NPs. Interestingly, the PM cloaked NPs only showed highly efficient targeting to the inflamed lungs and VECs, but no accumulation in healthy lungs and VECs. The data demonstrated that this biomimetic nanoplatform could be used as a potential strategy for personalized therapies in the treatment of inflammatory diseases, such as ALI/ARDS, and even COVID-19-associated pneumonia.

A novel role of Fas in delaying cellular senescence.

Fas-mediated apoptosis is a major player of many physiological and pathological cellular processes. Fas-regulated immune regulation exhibits either the beneficial or the harmful effects which is associated with the onset or development of immune disorders. Alterations in apoptosis may contribute to age-associated changes. However, the role of apoptosis in the ageing process remains ambiguous. Here we demonstrated Fas signaling-mediated premature senescence in young mouse embryonic fibroblast (MEF) cells. Activated Fas signaling by agonist Jo-2 resulted in declined senescence in young and aged MEFs. Premature senescence induced the early activation of senescence markers, including the increase in the percentage of SA-ß-galactosidase (SA-ß-gal) cells, the induction of p53 phosphorylation, and the enhanced expression of p16 and p21 protein and elevated IL-6 pro-inflammatory cytokine in the absence of Fas. The elevated production of reactive oxygen species (ROS) in Fas-deficient MEFs was associated with dysfunctional mitochondria. Further, we determined that the known ROS scavenger NAC (N-acetyl-l-cysteine) could reverse the process of premature senescence in absence of Fas. Therefore, this study signifies a novel role of Fas in the control of cellular senescence.

Protein kinase p38α signaling in dendritic cells regulates colon inflammation and tumorigenesis.

Dendritic cells (DCs) play pivotal roles in maintaining intestinal homeostasis, but how the DCs regulate diverse immune networks on homeostasis breakdown remains largely unknown. Here, we report that, in response to epithelial barrier disruption, colonic DCs regulate the differentiation of type 1 regulatory T (Tr1) cells through p38α-dependent IL-27 production to initiate an effective immune response. Deletion of p38α in DCs, but not in T cells, led to increased Tr1 and protected mice from dextran sodium sulfate-induced acute colitis and chronic colitis-associated colorectal cancer. We show that higher levels of IL-27 in p38α-deficient colonic cDC1s, but not cDC2s, were responsible for the increase of Tr1 cells. Moreover, p38α-dependent IL-27 enhanced IL-22 secretion from intestinal group 3 innate lymphoid cells and protected epithelial barrier function. In p38α-deficient DCs, the TAK1-MKK4/7-JNK-c-Jun axis was hyperactivated, leading to high IL-27 levels, and inhibition of the JNK-c-Jun axis suppressed IL-27 expression. ChIP assay revealed direct binding of c-Jun to the promoter of Il27p28, which was further enhanced in p38α-deficient DCs. In summary, here we identify a key role for p38α signaling in DCs in regulating intestinal inflammatory response and tumorigenesis, and our finding may provide targets for the treatment of inflammatory intestinal diseases.

MAPK Phosphatase-1 Deficiency Exacerbates the Severity of Imiquimod-Induced Psoriasiform Skin Disease.

Persistent activation of mitogen-activated protein kinase (MAPK) is believed to be involved in psoriasis pathogenesis. MAPK phosphatase-1 (MKP-1) is an important negative regulator of MAPK activity, but the cellular and molecular mechanisms of MKP-1 in psoriasis development are largely unknown. In this study, we found that the expression of MKP-1 was decreased in the imiquimod (IMQ)-induced psoriasiform mouse skin. MKP-1-deficient (MKP-1-/-) mice were highly susceptible to IMQ-induced skin inflammation, which was associated with increased production of inflammatory cytokines and chemokines. MKP-1 acted on both hematopoietic and non-hematopoietic cells to regulate psoriasis pathogenesis. MKP-1 deficiency in macrophages led to enhanced p38 activation and higher expression of interleukin (IL)-1ß, CXCL2, and S100a8 upon R848 stimulation. Moreover, MKP-1 deficiency in the non-hematopoietic compartments led to an enhanced IL-22 receptor signaling and higher expression of CXCL1 and CXCL2 upon IMQ treatment. Collectively, our data suggest a critical role for MKP-1 in the regulation of skin inflammation.

p38α signaling in Langerhans cells promotes the development of IL-17-producing T cells and psoriasiform skin inflammation.

Dendritic cells (DCs) contribute to psoriasis pathogenesis. In a mouse model of imiquimod-induced psoriasiform skin inflammation, we found that p38α activity in Langerhans cells (LCs), a skin-resident subset of DCs, promoted the generation of T cells that produce IL-17, a proinflammatory cytokine that is implicated in autoimmune disease. Deletion of p38α in LCs, but not in other skin or circulating DC subsets or T cells, decreased T cell-mediated psoriasiform skin inflammation in mice. The activity of p38α in LCs specifically promoted IL-17 production from γδ and CD4+ T cells by increasing the abundance of IL-23 and IL-6, two cytokines that stimulate IL-17 secretion. Inhibition of p38 activity through either pharmacological inhibition or genetic deletion also reduced the severity of established psoriasiform skin inflammation. Together, our findings indicate a critical role for p38α signaling in LCs in promoting inflammatory responses in the skin and suggest that targeting p38α signaling in LCs may offer an effective therapeutic approach to treat psoriasis.

Fas Signaling in Dendritic Cells Mediates Th2 Polarization in HDM-Induced Allergic Pulmonary Inflammation.

Fas-Fas ligand (FasL) signaling plays an important role in the development of allergic inflammation, but the cellular and molecular mechanisms are still not well known. By using the bone marrow-derived dendritic cell (BMDC) transfer-induced pulmonary inflammation model, we found that house dust mite (HDM)-stimulated FAS-deficient BMDCs induced higher Th2-mediated allergic inflammation, associated with increased mucus production and eosinophilic inflammation. Moreover, FAS-deficient BMDCs promoted Th2 cell differentiation upon HDM stimulation in vitro. Compared to wild-type BMDCs, the Fas-deficient BMDCs had increased ERK activity and decreased IL-12 production upon HDM stimulation. Inhibition of ERK activity could largely increase IL-12 production, consequently restored the increased Th2 cytokine expression of OT-II CD4+ T cells activated by Fas-deficient BMDCs. Thus, our results uncover an important role of DC-specific Fas signaling in Th2 differentiation and allergic inflammation, and modulation of Fas signaling in DCs may offer a useful strategy for the treatment of allergic inflammatory diseases.

Tuberous sclerosis 1 (Tsc1)-dependent metabolic checkpoint controls development of dendritic cells.

Coordination of cell metabolism and immune signals is crucial for lymphocyte priming. Emerging evidence also highlights the importance of cell metabolism for the activation of innate immunity upon pathogen challenge, but there is little evidence of how this process contributes to immune cell development. Here we show that differentiation of dendritic cells (DCs) from bone marrow precursors is associated with dynamic regulation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling and cell metabolism. Unexpectedly, enhancing mTORC1 activity via ablation of its negative regulator tuberous sclerosis 1 (Tsc1) impaired DC development in vivo and in vitro, associated with defective cell survival and proliferation. Moreover, Tsc1 deficiency caused DC spontaneous maturation but a propensity to differentiate into other lineages, and attenuated DC-mediated effector TH1 responses. Mechanistically, Tsc1-deficient DCs exhibited increased glycolysis, mitochondrial respiration, and lipid synthesis that were partly mediated by the transcription factor Myc, highlighting a key role of Tsc1 in modulating metabolic programming of DC differentiation. Further, Tsc1 signaled through Rheb to down-regulate mTORC1 for proper DC development, whereas its effect at modulating mTOR complex 2 (mTORC2) activity was largely dispensable. Our results demonstrate that the interplay between Tsc1-Rheb-mTORC1 signaling and Myc-dependent bioenergetic and biosynthetic activities constitutes a key metabolic checkpoint to orchestrate DC development.

Control of T cell fates and immune tolerance by p38α signaling in mucosal CD103+ dendritic cells.

Dendritic cells (DCs) play a crucial role in launching protective adaptive immunity against pathogens while maintaining immune tolerance to self-Ags. However, how intracellular signaling pathways program DCs to mediate tolerogenic responses remains largely unexplored. In this study, we describe that p38α signaling in CD103(+) mesenteric lymph node DCs reciprocally regulates the differentiation of anti-inflammatory induced regulatory T cells and proinflammatory Th1 cells from naive precursors and promotes mucosal tolerance. Deficiency of p38α in CD103(+) DCs inhibited the generation of induced regulatory T cells while promoting Th1 cell development in a TGF-ß2-dependent manner. Consequently, loss of p38α in DCs prevented induction of oral tolerance in vivo. Moreover, p38α in CD103(+) DCs was required for optimal expression of retinaldehyde dehydrogenase, a key enzyme for retinoic acid synthesis, which in turn imprinted gut-homing receptors on responding T cells. Consistent with a crucial role of p38α to program the tolerogenic activity of CD103(+) DCs, such DC subset contained constitutive activity of p38α and abundant expression of TGF-ß2 and retinaldehyde dehydrogenase. Our studies identify a key mechanism of DC-mediated coupling of T cell differentiation and trafficking that orchestrates mucosal immune tolerance.

Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasis and function.

Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c(+) cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8(+) and CD103(+) DC subsets in lymphoid and nonlymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Prosurvival signals from Toll-like receptors, CD40 and receptor activator of nuclear factor-κB (RANK) are integrated by TAK1 in DCs, which in turn mediated activation of downstream NF-κB and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T-cell homeostasis, and prevented effective T-cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a prosurvival checkpoint in DCs that affects the homeostasis and function of the immune system.

Signaling via the kinase p38α programs dendritic cells to drive TH17 differentiation and autoimmune inflammation.

Dendritic cells (DCs) bridge innate and adaptive immunity, but how DC-derived signals regulate T cell lineage choices remains unclear. We report here that the mitogen-activated protein kinase p38α programmed DCs to drive the differentiation of the T(H)17 subset of helper T cells. Deletion of p38α in DCs protected mice from T(H)17 cell-mediated autoimmune neuroinflammation, but deletion of p38α in macrophages or T cells did not. We also found that p38α orchestrated the expression of cytokines and costimulatory molecules in DCs and further 'imprinted' signaling via the receptor for interleukin 23 (IL-23R) in responding T cells to promote T(H)17 differentiation. Moreover, p38α was required for tissue-infiltrating DCs to sustain T(H)17 responses. This activity of p38α was conserved in mouse and human DCs and was dynamically regulated by pattern recognition and fungal infection. Our results identify p38α signaling as a central pathway for the integration of instructive signals in DCs for T(H)17 differentiation and inflammation.

The primitive immune system of amphioxus provides insights into the ancestral structure of the vertebrate immune system.

Amphioxus is considered to be the basal chordate. However, the structural and anatomical features of the amphioxus immune system are still elusive. Here we report a profile of structural studies of the amphioxus gill and gut, the first line of defending against microbes, through optical and electron microscopy. The amphioxus gut and gill are characterized by the following morphological criteria compared with vertebrates: primary and secondary lymphoid-like tissue clustered in the gill, a thicker basement membrane with a large villus channel and lack of muscular layer in the gut, along with blood vessels that fill with phagocytes following microbial challenge. The phenomena of tissue repair after microbial invasion was observed, though no phagocytes were observed in the region of tissue necrosis. The epithelium cells of amphioxus gut showed active phagocytosis after the microbial challenge. A small number of free and fixed macrophage-like cells were also found in the amphioxus gut. The current results described the structure of the immune system and cellular defense against infection in a protochordate, which may help us in understanding the structural origin of the vertebrate immune system.

The receptor S1P1 overrides regulatory T cell-mediated immune suppression through Akt-mTOR.

Regulatory T cells (T(reg) cells) are critically involved in maintaining immunological tolerance, but this potent suppression must be 'quenched' to allow the generation of adaptive immune responses. Here we report that sphingosine 1-phosphate (S1P) receptor type 1 (S1P1) delivers an intrinsic negative signal to restrain the thymic generation, peripheral maintenance and suppressive activity of T(reg) cells. Combining loss- and gain-of-function genetic approaches, we found that S1P1 blocked the differentiation of thymic T(reg) precursors and function of mature T(reg) cells and affected T(reg) cell-mediated immune tolerance. S1P1 induced selective activation of the Akt-mTOR kinase pathway to impede the development and function of T(reg) cells. Dynamic regulation of S1P1 contributed to lymphocyte priming and immune homeostasis. Thus, by antagonizing T(reg) cell-mediated immune suppression, the lipid-activated S1P1-Akt-mTOR pathway orchestrates adaptive immune responses.

[Screening ubiquitin/ribosomal protein S27a by yeast two-hybridization system].

BACKGROUND & OBJECTIVE: Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession. Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth. In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library. METHODS: The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR). Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I. The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments. Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcript method was used to construct a cDNA library in yeast cells. Library plasmid was named AD plasmid. BD plasmid and AD plasmid were co-transformed into competent yeast AH109. Yeast two-hybridization was used to screen positive clones. RESULTS: Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened. DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%). CONCLUSION: Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells.