Electrostimulation suppresses allograft rejection via promoting lymphatic regulatory T cell migration mediated by lymphotoxin - lymphotoxin receptor ß signaling.

Conventional immunosuppressants that suppress allograft rejection cause various side effects. Although regulatory T cells (Tregs) are essential for allograft survival, the limited efficacy of Treg therapy demands improvement. Thus, it is imperative to seek new approaches to enhancing Treg suppression. Low-intensity electrostimulation (ES) has been shown to exert antiinflammatory effects without causing major adverse reactions. However, it remains unknown whether and how ES regulates alloimmunity. Here, we found that regional ES delayed murine skin allograft rejection and promoted long-term allograft survival induced by an mTOR inhibitor, rapamycin. ES also extended islet allograft survival. Mechanistically, ES enhanced the expression of lymphotoxin α (LTα) on Tregs after transplantation. Blockade of lymphotoxin ß receptor-mediated nonclassical NFκB signaling suppressed lymphatic Treg migration and largely reversed the effects of ES on allograft survival. Moreover, ES failed to extend allograft survival when recipients lacked LTα/lymph nodes or if transferred Tregs lacked LTα. Therefore, ES promoted the lymphatic migration of CD4+Foxp3+ Tregs by upregulating their surface expression of LTα. Finally, ES augmented expression of LTα on murine or human Tregs, but not conventional T cells, while promoting their calcium influx in vitro. This ES-mediated upregulation of LTα relied on calcium influx. Thus, our findings have unveiled novel mechanisms underlying ES-mediated immunoregulation.

A Novel Immunosuppressant, Luteolin, Modulates Alloimmunity and Suppresses Murine Allograft Rejection.

An allograft is rejected in the absence of any immunosuppressive treatment because of vigorous alloimmunity and thus requires extensive immunosuppression for its survival. Although there are many conventional immunosuppressants for clinical use, it is necessary to seek alternatives to existing drugs, especially in case of transplant patients with complicated conditions. Luteolin, a natural ingredient, exists in many plants. It exhibits multiple biological and pharmacological effects, including anti-inflammatory properties. In particular, luteolin has been shown to upregulate CD4+CD25+ regulatory T cells (Tregs) in the context of airway inflammation. However, it remains unknown whether luteolin regulates alloimmune responses. In this study, we demonstrated that luteolin significantly prolonged murine skin allograft survival, ameliorated cellular infiltration, and downregulated proinflammatory cytokine gene expression in skin allografts. Furthermore, luteolin increased the percentage of CD4+Foxp3+ Tregs while reducing frequency of mature dendritic cells and CD44highCD62Llow effector CD4+/CD8+ T cells posttransplantation. It also suppressed the proliferation of T cells and their production of cytokines IFN-γ and IL-17A in vitro while increasing IL-10 level in the supernatant. Moreover, luteolin promoted CD4+Foxp3+ Treg generation from CD4+CD25- T cells in vitro. Depleting Tregs largely, although not totally, reversed luteolin-mediated extension of allograft survival. More importantly, luteolin inhibited AKT/mTOR signaling in T cells. Thus, for the first time, to our knowledge, we found that luteolin is an emerging immunosuppressant as an mTOR inhibitor in allotransplantation. This finding could be important for the suppression of human allograft rejection, although it remains to be determined whether luteolin has an advantage over other conventional immunosuppressants in suppression of allograft rejection.

Mangiferin Attenuates Murine Lupus Nephritis by Inducing CD4+Foxp3+ Regulatory T Cells via Suppression of mTOR Signaling.

BACKGROUND/AIMS: Lupus nephritis (LN) is an autoimmune glomerulonephritis that frequently develops secondary to systemic lupus erythematosus. Patients with LN require extensive treatments with global immunosuppressive agents. However, long-term treatment with conventional immunosuppressants may cause various side effects. Therefore, it's important to seek alternative drugs for treating LN. Here we aimed to investigate the immunoregulatory effects of mangiferin (MG), an ingredient that was originally extracted from natural herbs, including Mangifera Indica Linn. and Rhizoma Anemarrhenae. METHODS: FasL-deficient B6/ gld mice were used as a spontaneous LN model. The serum anti-dsDNA Ab and creatinine levels were analyzed via ELISA. Renal histology and immunopathology were determined using H&E and PAS staining, immunofluorescence (IgG and C3), and IHC staining (CD3 and a-SMA). Cytokine gene expression was measured by RT-PCR assays while effector T cells and Tregs were enumerated by flow analysis. Finally, the proliferation and apoptosis of T cells were measured by CFSE staining and flow analysis while their mTOR signaling was detected through Western blotting. RESULTS: We found that administration of MG ameliorated LN in lupus-prone B6/gld mice by reducing the urinary protein and serum creatinine levels, diminishing T cell infiltration in kidneys and improving renal immunopathology. MG also significantly lowered the percentages of CD44highCD62Llow effector T cells in B6/gld mice. Importantly, treatments with MG augmented CD4+FoxP3+ Treg frequencies in spleens, lymph nodes and kidneys of B6/gld mice. It also induced CD4+FoxP3+ Tregs from CD3+ T cells in vitro and promoted Treg proliferation. Furthermore, it inhibited CD3+ T cell proliferation in vitro and suppressed their phosphorylation of mTOR and its downstream P70S6K. However, MG did not promote T cell apoptosis, implying that it is not cytotoxic. Depletion of CD4+CD25+FoxP3+ Tregs in B6/gld mice abrogated its therapeutic effects on LN. CONCLUSION: MG exerts a novel therapeutic effect on murine LN via upregulating CD4+FoxP3+ Tregs, downregulating mTOR/p70S6K pathway and improving renal immunopathology. It may be useful for treating LN in clinic.

Methotrexate and electrostimulation cooperate to alleviate the relapse of psoriasiform skin inflammation by suppressing memory T cells.

Methotrexate (MTX) is an immunosuppressant used to treat autoimmune diseases, including psoriasis. However, like other immunosuppressants, MTX alone does not prevent their recurrence. Electrostimulation (ES) has been utilized to treat some inflammatory disorders without any major side-effect. But it remains unknown if ES alone, or together with MTX, ameliorates autoimmune disease relapse: a sticky medical problem. In particular, the mechanisms underlying ES action remain unclear. The objective of this study was to determine an impact of ES and/or MTX on psoriasis relapse and their potential cooperation. We found that regional ES, but not MTX, ameliorated psoriasiform skin inflammation recurrence. Interestingly, treatment with both MTX and ES further prevented psoriasis recurrence compared to ES alone. Moreover, ES downregulated potassium channel Kv1.3 on T-cells and reduced CD4+/CD8+ effector memory (TEM) and CD8+ skin-resident memory T (TRM) cells, while ES plus MTX further decreased CD8+ TEM/TRM cells compared to ES alone. However, ES failed to further attenuate psoriasis recurrence or suppress T cell memory in Kv1.3-deficient mice, whereas lack of Kv1.3 itself ameliorated psoriasis relapse by shrinking T cell memory pool. Importantly, ES moderately inhibited T-cell proliferation in vitro. ES also reduced human CD8+ TRM cells and attenuated human skin lesions in humanized mice grafted with lesional skin from patients with recurrent psoriasis, with an enhanced efficacy in mice treated with both ES and MTX. Thus, ES and MTX cooperated to prevent psoriasis relapse by reducing T-cell memory via targeting potassium channel Kv1.3. Our studies may be implicated for treating human psoriasis.

The TOPK Inhibitor HI-TOPK-032 Enhances CAR T-cell Therapy of Hepatocellular Carcinoma by Upregulating Memory T Cells.

Chimeric antigen receptor (CAR) T cells are emerging as an effective antitumoral therapy. However, their therapeutic effects on solid tumors are limited because of their short survival time and the immunosuppressive tumor microenvironment. Memory T cells respond more vigorously and persist longer than their naïve/effector counterparts. Therefore, promoting CAR T-cell development into memory T cells could further enhance their antitumoral effects. HI-TOPK-032 is a T-LAK cell-originated protein kinase (TOPK)-specific inhibitor that moderately represses some types of tumors. However, it is unknown whether HI-TOPK-032 works on hepatocellular carcinoma (HCC) and whether it impacts antitumoral immunity. Using both subcutaneous and orthotopic xenograft tumor models of two human HCC cell lines, Huh-7 and HepG2, we found that HI-TOPK-032 significantly improved proliferation/persistence of CD8+ CAR T cells, as evidenced by an increase in CAR T-cell counts or frequency of Ki-67+CD8+ cells and a decrease in PD-1+LAG-3+TIM-3+CD8+ CAR T cells in vivo. Although HI-TOPK-032 did not significantly suppress HCC growth, it enhanced the capacity of CAR T cells to inhibit tumor growth. Moreover, HI-TOPK-032 augmented central memory CD8+ T cell (TCM) frequency while increasing eomesodermin expression in CD8+ CAR T cells in tumor-bearing mice. Moreover, it augmented CD8+ CAR TCM cells in vitro and reduced their expression of immune checkpoint molecules. Finally, HI-TOPK-032 inhibited mTOR activation in CAR T cells in vitro and in tumors, whereas overactivation of mTOR reversed the effects of HI-TOPK-032 on CD8+ TCM cells and tumor growth. Thus, our studies have revealed mechanisms underlying the antitumoral effects of HI-TOPK-032 while advancing CAR T-cell immunotherapy.

Astragalus polysaccharide enhances antitumoral effects of chimeric antigen receptor- engineered (CAR) T cells by increasing CD122+CXCR3+PD-1- memory T cells.

Chimeric antigen receptor-engineered T (CAR-T) cell therapy of cancer has been a hotspot and promising. However, due to rapid exhaustion, CAR-T cells are less effective in solid tumors than in hematological ones. CD122+CXCR3+ memory T cells are characterized with longevity, self-renewal and great antitumoral capacity. Thus, it's compelling to induce memory CAR-T cells to enhance their efficacy on solid tumors. Astragalus polysaccharide (APS) has reportedly exhibited antitumoral effects. However, it's unclear if APS has an impact on CD8+ memory T cell generation or persistence. Using two human cancer cell lines, here we found that APS significantly improved the persistence of GPC3-targeted CAR-T cells and enhanced their suppression of tumor growth in both Huh7 and HepG2 xenograft models of hepatocellular carcinoma. APS increased CD122+/CXCR3+ memory T cells, but decreased their PD-1+ subset within CD8+ CAR-T cells in tumor-bearing mice, while these effects of APS were also confirmed with in vitro experiments. Moreover, APS augmented the expression of chemokines CXCL9/CXCL10 by the tumor in vivo and in vitro. It also enhanced the proliferation and chemotaxis/migration of CAR-T cells in vitro. Finally, APS promoted the phosphorylation of STAT5 in CD8+ CAR-T cells, whereas inhibition of STAT5 activation reversed these in vitro effects of APS. Therefore, APS enhanced the antitumoral effects of CD8+ CAR-T cells by promoting formation/persistence of CD122+/CXCR3+/PD-1- memory T cells and their migration to the tumor.

Electroacupuncture and methotrexate cooperate to ameliorate psoriasiform skin inflammation by regulating the immune balance of Th17/Treg.

Psoriasis is an autoinflammatory dermatosis, while methotrexate (MTX) is an immunosuppressant used to treat psoriasis. However, conventional immunosuppressants may cause various side effects. Acupuncture has potential benefits in treating psoriasis based on its anti-inflammatory effects. However, the immune mechanisms underlying its effects remain unclear. In this study, imiquimod-induced psoriatic mice were used to investigate the effects and mechanisms of electroacupuncture (EA) and, in particular, its joint treatment with MTX. We found that treatment with either EA or MTX ameliorated psoriasiform skin lesions, improved skin pathology and reduced proinflammatory cytokines in the skin, while joint treatment with both EA and MTX further alleviated the skin lesions and inflammation compared to either one alone. Moreover, percentages of CD4+ IL-17A+ Th17 cells in the skin and lymph nodes were decreased by EA or MTX and further lowered by combined EA+MTX treatment. Similarly, EA or MTX also reduced their RORγt expression. On the contrary, CD4+ FoxP3+ Treg frequency in psoriatic mice was augmented by EA or MTX and further increased by the joint treatment. However, depleting Tregs mostly reversed the therapeutic effects of EA or EA plus MTX. Additionally, the phosphorylated NF-κB (p65) expression was suppressed by treatment with EA, MTX or better with EA+MTX. Meanwhile, the anti-inflammatory effects of EA plus MTX were offset by an NF-κB agonist. Thus, this study has revealed that EA cooperates with MTX to balance Th17/Treg responses and to ameliorate psoriasiform skin inflammation through suppressing NF-κB activation. Our findings may be implicated for treating human psoriasis.

Percutaneous Delivery of Hederacoside C-Loaded Nanoliposome Gel Alleviates Psoriasiform Skin Inflammation through the CCL17/Treg Axis.

Psoriasis is a chronic, recurrent, and inflammatory skin disease. Topical agents, which can avoid the adverse effects of systemic treatment, are the first-choice therapy for patients with mild-to-moderate psoriasis. Hederacoside C (HSC) with anti-inflammatory properties has been used to treat some inflammatory diseases. We speculated that HSC might also be effective for psoriasis treatment. However, topical application of HSC for psoriasis treatment is challenging because of its low water solubility and poor skin permeability. Therefore, it is important to effectively deliver HSC percutaneously using certain biomaterials. Here we constructed a hydroxypropyl-ß-cyclodextrin-coated liposome gel formulation for the loading and percutaneously delivering of HSC, referred to as HSC-Lipo@gel. The characterization, stability, release properties, and mechanical or transdermal features of the HSC-Lipo@gel were evaluated. Its therapeutic potential was also demonstrated using mouse models of IMQ-induced psoriasis. We found that HSC-Lipo@gel effectively improved the skin permeability of HSC with the property of good stability and sustained release. Importantly, HSC-Lipo@gel showed higher efficacy than HSC@gel without liposomes in alleviating psoriatic skin lesions. It attenuated epidermal hyperplasia and suppressed expression of IL-17A, TNF-α, IL-6, and IL-23 in lesional skin. Interestingly, HSC-Lipo@gel reduced the expression of CC chemokine ligand 17 (CCL17), but not CCL22, in the skin. Especially, HSC-Lipo@gel inhibited CCL17 expression by skin dendritic cells while increasing regulatory T cells (Tregs) in both skin and draining lymph nodes of psoriatic mice. Administration of CCL17 resulted in severe skin lesions and reduced CD4+FoxP3+ Tregs in psoriatic mice previously treated with HSC-Lipo@gel. Finally, HSC or HSC-Lipo also suppressed the CCL17 production by dendritic cells in vitro. Therefore, HSC-Lipo@gel alleviated psoriasiform skin inflammation by increasing cutaneous Tregs via downregulation of the expression of CCL17, but not CCL22. Thus, HSC-Lipo@gel may be a stable, highly permeable, and effective system for topical treatment of psoriasis.

Zhen-Wu-Tang ameliorates lupus nephritis by diminishing renal tissue-resident memory CD8+ T cells via suppressing IL-15/STAT3 pathway.

Zhen-Wu-Tang (ZWT), a conventional herbal mixture, has been recommended for treating lupus nephritis (LN) in clinic. However, its mechanisms of action remain unknown. Here we aimed to define the immunological mechanisms underlying the effects of ZWT on LN and to determine whether it affects renal tissue-resident memory T (TRM) cells. Murine LN was induced by a single injection of pristane, while in vitro TRM cells differentiated with IL-15/TGF-ß. We found that ZWT or mycophenolate mofetil treatment significantly ameliorated kidney injury in LN mice by decreasing 24-h urine protein, Scr and anti-dsDNA Ab. ZWT also improved renal pathology and decreased IgG and C3 depositions. In addition, ZWT down-regulated renal Desmin expression. Moreover, it lowered the numbers of CD8+ TRM cells in kidney of mice with LN while decreasing their expression of TNF-α and IFN-γ. Consistent with in vivo results, ZWT-containing serum inhibited TRM cell differentiation induced by IL-15/TGF-ß in vitro. Mechanistically, it suppressed phosphorylation of STAT3 and CD122 （IL2/IL-15Rß）expression in CD8+ TRM cells. Importantly, ZWT reduced the number of total F4/80+CD11b+ and CD86+, but not CD206+, macrophages in the kidney of LN mice. Interestingly, ZWT suppressed IL-15 protein expression in macrophages in vivo and in vitro. Thus, we have provided the first evidence that ZWT decoction can be used to improve the outcome of LN by reducing CD8+ TRM cells via inhibition of IL-15/IL-15R /STAT3 signaling.

MiR-124-3p mediates gastric cancer cell ferroptosis induced by an anti-cancer drug polyphyllin I.

Background: Ferroptosis is an emerging type of regulated cell death and associated with antitumoral therapy, while some microRNAs have been shown to regulate the tumorigenesis and cancer progression. Meanwhile, polyphyllin I (PPI) has exhibited antitumoral effects by promoting cancer cell apoptosis and ferroptosis. However, it is unclear whether PPI induces cancer cell ferroptosis by regulating microRNAs. Methods: We used two gastric cancer cell lines (AGS and MKN-45) to set up a tumor model of the nude mice, which were then treated daily with PPI to measure the cancer growth in vitro and in vivo. Ferroptosis was measured using immunofluorescence staining and flow cytometric analysis according to levels of intracellular ROS, lipid ROS and ferrous ions. Moreover, NRF2 expression was measured by Western blotting. In some experiments, the mimics or inhibitors of miR-124-3p were used to further confirm its involvement in PPI-induced cancer cell ferroptosis. Results: Here we found that miR-124-3p mediated cancer ferroptosis and tumor repression induced by PPI since PPI increased miR-124-3p expression in gastric cancer cells and promoted their ferroptosis, whereas inhibition of miR-124-3p mostly abolished the effects of PPI on tumor growth, ferroptosis and NRF2 expression. Moreover, miR-124-3p mimics promoted cancer cell ferroptosis by downregulating NRF2 through directly targeting 3'-UTR region of NRF2, confirming a role for miR-124-3p in regulating PPI-induced ferroptosis. Conclusion: PPI exerts its antitumoral effects on the gastric cancer by promoting cell ferroptosis via regulating miR-124-3p. Our findings have clinical implications for cancer chemotherapy.

Polyphyllin I suppresses the gastric cancer growth by promoting cancer cell ferroptosis.

Background: Ferroptosis is a new form of regulated cell death characterized by the accumulation of iron-dependent lipid peroxides and membrane damages. Recent studies have identified an important role for cancer cell ferroptosis in antitumor therapy. On the other hand, polyphyllin I (PPI) has been reported to exert antitumor effects on some types of cancers. However, it remains unknown whether or not PPI regulates cancer cell ferroptosis. Methods: Two types of human gastric cancer cells (AGS and MKN-45) were used to establish tumor xenograft models in nude mice that were treated with polyphyllin I (PPI) to observe tumor growth, while cells also were cultured for in vitro studies. Ferroptosis, based on the intracellular ROS/lipid ROS production and accumulation of ferrous ions, was detected using a fluorescence microscope and flow cytometer, while the expression of NRF2/FTH1 was measured using Western blotting assays. Results: Here we found that PPI inhibited the gastric cancer growth in vivo and in vitro while increasing the intracellular reactive oxygen species (ROS)/lipid peroxides and ferrous ions in the gastric cancer cells. PPI also decreased the levels of nuclear factor erythroid 2-related factor 2 (NRF2) and ferritin heavy chain 1 (FTH1) in gastric cancer cells in vitro. Moreover, liproxstain-1, an inhibitor of cell ferroptosis, mostly reversed the cell ferroptosis and tumor growth arrest induced by PPI. Finally, the effects of PPI on cancer cell ferroptosis were diminished by the overexpression of NRF2. Conclusion: For the first time, our results have demonstrated that PPI exerts its antitumor activity on the gastric cancer by, at least partially, inducing cancer cell ferroptosis via regulating NRF2/FTH1 pathway. These findings may be implicated for clinical replacement therapy of the gastric cancer.

Corrigendum: Polyphyllin I suppresses the gastric cancer growth by promoting cancer cell ferroptosis.

[This corrects the article DOI: 10.3389/fphar.2023.1145407.].

Total Glucosides of Paeony Ameliorate Pristane-Induced Lupus Nephritis by Inducing PD-1 ligands+ Macrophages via Activating IL-4/STAT6/PD-L2 Signaling.

Macrophages, a major subset of innate immune cells, are main infiltrating cells in the kidney in lupus nephritis. Macrophages with different phenotypes exert diverse or even opposite effects on the development of lupus nephritis. Substantial evidence has shown that macrophage M2 polarization is beneficial to individuals with chronic kidney disease. Further, it has been reported that PD-1 ligands (PD-Ls) contribute to M2 polarization of macrophages and their immunosuppressive effects. Total glucosides of paeony (TGP), originally extracted from Radix Paeoniae Alba, has been approved in China to treat some autoimmune diseases. Here, we investigated the potentially therapeutic effects of TGP on lupus nephritis in a pristane-induced murine model and explored the molecular mechanisms regulating macrophage phenotypes. We found that TGP treatment significantly improved renal function by decreasing the urinary protein and serum creatinine, reducing serum anti-ds-DNA level and ameliorating renal immunopathology. TGP increased the frequency of splenic and peritoneal F4/80+CD11b+CD206+ M2-like macrophages with no any significant effect on F4/80+CD11b+CD86+ M1-like macrophages. Immunofluorescence double-stainings of the renal tissue showed that TGP treatment increased the frequency of F4/80+Arg1+ subset while decreasing the percentage of F4/80+iNOS+ subset. Importantly, TGP treatment increased the percentage of both F4/80+CD11b+PD-L1+ and F4/80+CD11b+PD-L2+ subsets in spleen and peritoneal lavage fluid as well as the kidney. Furthermore, TGP augmented the expressions of CD206, PD-L2 and phosphorylated STAT6 in IL-4-treated Raw264.7 macrophages in vitro while its effects on PD-L2 were abolished by pretreatment of the cells with an inhibitor of STAT6, AS1517499. However, TGP treatment did not affect the expressions of STAT1 and PD-L1 in Raw264.7 macrophages treated with LPS/IFN-γ in vitro, indicating a possibly indirect effect of TGP on PD-L1 expression on macrophages in vivo. Thus, for the first time, we demonstrated that TGP may be a potent drug to treat lupus nephritis by inducing F4/80+CD11b+CD206+ and F4/80+CD11b+PD-L2+ macrophages through IL-4/STAT6/PD-L2 signaling pathway.

Berberine Promotes Induction of Immunological Tolerance to an Allograft via Downregulating Memory CD8+ T-Cells Through Altering the Gut Microbiota.

Emerging evidence has linked the gut microbiota dysbiosis to transplant rejection while memory T-cells pose a threat to long-term transplant survival. However, it's unclear if the gut microbiome alters the formation and function of alloreactive memory T-cells. Here we studied the effects of berberine, a narrow-spectrum antibiotic that is barely absorbed when orally administered, on the gut microbiota, memory T-cells, and allograft survival. In this study, C57BL/6 mice transplanted with islets or a heart from BALB/c mice were treated orally with berberine. Allograft survival was observed, while spleen, and lymph node T-cells from recipient mice were analyzed using a flow cytometer. High-throughput sequencing and qPCR were performed to analyze the gut microbiota. CD8+ T-cells from recipients were cultured with the bacteria to determine potential T-cell memory cross-reactivity to a specific pathogen. We found that berberine suppressed islet allograft rejection, reduced effector CD8+CD44highCD62Llow and central memory CD8+CD44highCD62Lhigh T-cells (TCM), altered the gut microbiota composition and specifically lowered Bacillus cereus abundance. Further, berberine promoted long-term islet allograft survival induced by conventional costimulatory blockade and induced cardiac allograft tolerance as well. Re-colonization of B. cereus upregulated CD8+ TCM cells and reversed long-term islet allograft survival induced by berberine plus the conventional costimulatory blockade. Finally, alloantigen-experienced memory CD8+ T-cells from transplanted recipients rapidly responded to B. cereus in vitro. Thus, berberine prolonged allograft survival by repressing CD8+ TCM through regulating the gut microbiota. We have provided the first evidence that donor-specific memory T-cell generation is linked to a specific microbe and uncovered a novel mechanism underlying the therapeutic effects of berberine. This study may be implicated for suppressing human transplant rejection since berberine is already used in clinic to treat intestinal infections.

Paeoniflorin ameliorates murine lupus nephritis by increasing CD4+Foxp3+ Treg cells via enhancing mTNFα-TNFR2 pathway.

Treg cells are essential for re-establishing self-tolerance in lupus. However, given that direct Treg therapies may be inadequate to control autoimmunity and inflammation, a strategy of inducing or expanding endogenous Treg cells in vivo may be a good option. Macrophages are main tissue-infiltrating cells and play a role in promoting Treg differentiation while paeoniflorin (PF), a monoterpene glycoside, exhibits anti-inflammatory and immunoregulatory effects. Here, we studied the effects of PF on CD4+FoxP3+ Treg frequency and the potential mechanisms involving M2 macrophages. We demonstrated that PF ameliorated lupus nephritis in lupus-prone B6/gld mice by reducing urinary protein, serum creatinine and anti-dsDNA levels, diminishing renal cellular infiltration, improving renal immunopathology and downregulating renal gene and protein expressions of key cytokines, including IFN-γ, IL-6, IL-12 and IL-23. PF also lowered the percentage of CD44highCD62Llow effector T cells while augmenting CD4+FoxP3+ Treg frequency in B6/gld mice. Importantly, PF increased TNFR2 expression on CD4+FoxP3+ Tregs, but not CD4+FoxP3- T cells, in vivo and in vitro. Furthermore, we found that CD206+ subset of F4/80+CD11b+ macrophages expressed a higher level of mTNF-α than their CD206- counterparts while PF increased mTNF-α expression on CD206+ macrophages in vitro and in vivo. In vitro studies showed that mTNF-α+ M2 macrophages were more potent in inducing Treg differentiation and proliferation than their mTNF-α- counterparts, whereas the effects of mTNF-α+ M2 macrophages were largely reversed by separation of M2 macrophages using a transwell or TNFR2-blocking Ab in the culture. Finally, PF also promoted in vitro Treg generation induced by M2 macrophages. Thus, we demonstrated that mTNFα-TNFR2 interaction is a new mechanism responsible for Treg differentiation mediated by M2 macrophages. We provided the first evidence that PF may be used to treat lupus nephritis.

Corrigendum: A New Immunosuppressive Molecule Emodin Induces both CD4+FoxP3+ and CD8+CD122+ Regulatory T Cells and Suppresses Murine Allograft Rejection.

[This corrects the article DOI: 10.3389/fimmu.2017.01519.].

Antitumor effects of immunity-enhancing traditional Chinese medicine.

Traditional Chinese Medicine (TCM) has been traditionally used to treat patients with cancers in China. It not only alleviates the symptoms of tumor patients and improves their quality of life, but also controls the size of tumors and prolongs the survival of tumor patients. While some herbs of TCM may exert therapeutic effects by directly targeting cancer cells or reducing side effects caused by antitumor drugs, others can control tumor growth and metastasis via enhancing antitumor immunity. In particular, TCM can exert antitumor effects by upregulating immune responses even in immunosuppressive tumor microenvironment. For instance, it reduces the number of M2-type macrophages and Treg cells in the tumor tissue. Although extensive reviews on directly killing cancer cells by TCM have been conducted, a review of anticancer activity of TCM solely based on its immunity-enhancing capacity is unusual. This review will summarize research progress of antitumor TCM that regulates the immune system, including both innate immunity, such as macrophages, dendritic cells, natural killer cells and MDSCs, and adaptive immunity, including CD4+/CD8+ T lymphocytes, regulatory T cells (Tregs) and B cells. As cancer immunotherapy has recently achieved certain success, it is expected that the clinical applications of immunity-enhancing TCM or traditional medicine for treating various cancer patients will be expanded. Further studies on the mechanisms by which TCM regulates immunity will provide new insights into how TCM controls tumor growth and metastasis, and may help improve its therapeutic effects on various cancers in clinic.

Resveratrol exerts antitumor effects by downregulating CD8+CD122+ Tregs in murine hepatocellular carcinoma.

CD4+Foxp3+ regulatory T cells (Tregs) in the tumor microenvironment restrain antitumor immunity, resulting in tumor aggression and poor survival in hepatocellular carcinoma (HCC). CD8+CD122+ Tregs have been previously shown to be more potent in immunosuppression than are CD4+Foxp3+ Tregs. Previous studies have demonstrated that resveratrol exerts its anti-cancer effects by downregulating CD4+Foxp3+ and M2-like macrophages, two key immunoregulatory cells that maintain the immunosuppressive tumor microenvironment. In this study, we found that resveratrol inhibited the tumor growth in a subcutaneous Hepa1-6 HCC model and decreased the frequency of CD8+CD122+ Tregs in the tumor as well as lymph nodes and spleen of the tumor-bearing mice. It also increased the percentage of IFN-γ-expressing CD8+ T cells in the tumor and peripheral lymphoid organs. The antitumor effects of resveratrol were partially reversed by the adoptive transfer of exogenous CD8+CD122+ Tregs into the tumor-bearing mice. Meanwhile, resveratrol treatment downregulated immunosuppressive cytokines, including TGF-ß1 and interleukin-10, in the tumor while elevating antitumor cytokines, TNF-α and IFN-γ. It also inhibited the activation of STAT3 signaling in the tumor. As expected, resveratrol reduced the percentage of M2-like macrophages in the mice. Importantly, resveratrol suppressed orthotopic H22 tumor growth and decreased the frequency of CD8+CD122+ Tregs and M2-like macrophages in the tumor-bearing mice. Furthermore, our studies showed that resveratrol, at non-cytotoxic concentrations, inhibited CD8+CD122+ Treg differentiation from CD8+CD122- T cells in vitro. Thus, our studies unveiled a new immune mechanism underlying the immunosuppressive tumor microenvironment and demonstrated that resveratrol could help reverse it by diminishing CD8+CD122+ Tregs.

Dihydroartemisinin ameliorates psoriatic skin inflammation and its relapse by diminishing CD8+ T-cell memory in wild-type and humanized mice.

Conventional immunosuppressants cause side effects and do not prevent the recurrence of autoimmune diseases. Moreover, they may not inhibit autoimmunity mediated by pathogenic memory T-cells. Dihydroartemisinin (DHA) has been shown to regulate autoimmunity. However, it remains unknown whether DHA impacts psoriasis and its recurrence. The objective of this study was to determine therapeutic effects of DHA on psoriasis and its relapse as well as its underlying mechanisms. Methods: We established animal models of imiquimod (IMQ)-induced psoriasis-like wild-type mice and humanized NSG mice receiving lesional human skin from patients with psoriasis. Many immunoassays, including immunohistochemistry, flow cytometry, quantitative RT-PCR and Western blotting, were performed. Results: We found that DHA not only ameliorated acute skin lesion of psoriatic mice, but also alleviated its recurrence by diminishing CD8+ central memory T (TCM) and CD8+ resident memory T (TRM) cells. It attenuated epidermal pathology and T-cell infiltration in the skin of IMQ-induced psoriatic mice while suppressing expression of IL-15, IL-17 and other proinflammatory cytokines in the skin. Surprisingly, DHA reduced the frequency and number of CD8+, but not CD4+, subset of CD44highCD62Lhigh TCM in psoriatic mice, whereas methotrexate (MTX) lowered CD4+, but not CD8+, TCM frequency and number. Indeed, DHA, but not MTX, downregulated eomesodermin (EOMES) and BCL-6 expression in CD8+ T-cells. Furthermore, DHA, but not MTX, reduced the presence of CD8+CLA+, CD8+CD69+ or CD8+CD103+ TRM cells in mouse skin. Interestingly, treatment with DHA, but not MTX, during the first onset of psoriasis largely prevented psoriasis relapse induced by low doses of IMQ two weeks later. Administration of recombinant IL-15 or CD8+, but not CD4+, TCM cells resulted in complete recurrence of psoriasis in mice previously treated with DHA. Finally, we demonstrated that DHA alleviated psoriatic human skin lesions in humanized NSG mice grafted with lesional skin from psoriatic patients while reducing human CD8+ TCM and CD103+ TRM cells in humanized mice. Conclusion: We have provided the first evidence that DHA is advantageous over MTX in preventing psoriasis relapse by reducing memory CD8+ T-cells.