A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

The Transcription Factor Ets1 Suppresses T Follicular Helper Type 2 Cell Differentiation to Halt the Onset of Systemic Lupus Erythematosus.

Single-nucleotide polymorphisms in ETS1 are associated with systemic lupus erythematosus (SLE). Ets1-/- mice develop SLE-like symptoms, suggesting that dysregulation of this transcription factor is important to the onset or progression of SLE. We used conditional deletion approaches to examine the impact of Ets1 expression in different immune cell types. Ets1 deletion on CD4+ T cells, but not B cells or dendritic cells, resulted in the SLE autoimmunity, and this was associated with the spontaneous expansion of T follicular helper type 2 (Tfh2) cells. Ets1-/- Tfh2 cells exhibited increased expression of GATA-3 and interleukin-4 (IL-4), which induced IgE isotype switching in B cells. Neutralization of IL-4 reduced Tfh2 cell frequencies and ameliorated disease parameters. Mechanistically, Ets1 suppressed signature Tfh and Th2 cell genes, including Cxcr5, Bcl6, and Il4ra, thus curbing the terminal Tfh2 cell differentiation process. Tfh2 cell frequencies in SLE patients correlated with disease parameters, providing evidence for the relevance of these findings to human disease.

ETV5 promotes lupus pathogenesis and follicular helper T cell differentiation by inducing osteopontin expression.

Follicular helper T (TFH) cells mediate germinal center reactions to generate high affinity antibodies against specific pathogens, and their excessive production is associated with the pathogenesis of systemic autoimmune diseases such as systemic lupus erythematosus (SLE). ETV5, a member of the ETS transcription factor family, promotes TFH cell differentiation in mice. In this study, we examined the role of ETV5 in the pathogenesis of lupus in mice and humans. T cell-specific deletion of Etv5 alleles ameliorated TFH cell differentiation and autoimmune phenotypes in lupus mouse models. Further, we identified SPP1 as an ETV5 target that promotes TFH cell differentiation in both mice and humans. Notably, extracellular osteopontin (OPN) encoded by SPP1 enhances TFH cell differentiation by activating the CD44-AKT signaling pathway. Furthermore, ETV5 and SPP1 levels were increased in CD4+ T cells from patients with SLE and were positively correlated with disease activity. Taken together, our findings demonstrate that ETV5 is a lupus-promoting transcription factor, and secreted OPN promotes TFH cell differentiation.

Forkhead box protein D2 suppresses colorectal cancer by reprogramming enhancer interactions.

Somatic stem cells contribute to normal tissue homeostasis, and their epigenomic features play an important role in regulating tissue identities or developing disease states. Enhancers are one of the key players controlling chromatin context-specific gene expression in a spatial and temporal manner while maintaining tissue homeostasis, and their dysregulation leads to tumorigenesis. Here, epigenomic and transcriptomic analyses reveal that forkhead box protein D2 (FOXD2) is a hub for the gene regulatory network exclusive to large intestinal stem cells, and its overexpression plays a significant role in colon cancer regression. FOXD2 is positioned at the closed chromatin and facilitates mixed-lineage leukemia protein-4 (MLL4/KMT2D) binding to deposit H3K4 monomethylation. De novo FOXD2-mediated chromatin interactions rewire the regulation of p53-responsive genes and induction of apoptosis. Taken together, our findings illustrate the novel mechanistic details of FOXD2 in suppressing colorectal cancer growth and suggest its function as a chromatin-tuning factor and a potential therapeutic target for colorectal cancer.

NFAT1 and NFκB regulates expression of the common γ-chain cytokine receptor in activated T cells.

INTRODUCTION: Cytokines of the common γ chain (γc) family are critical for the development, differentiation, and survival of T lineage cells. Cytokines play key roles in immunodeficiencies, autoimmune diseases, allergies, and cancer. Although γc is considered an assistant receptor to transmit cytokine signals and is an indispensable receptor in the immune system, its regulatory mechanism is not yet well understood. OBJECTIVE: This study focused on the molecular mechanisms that γc expression in T cells is regulated under T cell receptor (TCR) stimulation. METHODS: The γc expression in TCR-stimulated T cells was determined by flow cytometry, western blot and quantitative RT-PCR. The regulatory mechanism of γc expression in activated T cells was examined by promoter-luciferase assay and chromatin immunoprecipitation assays. NFAT1 and NFκB deficient cells generated using CRISPR-Cas9 and specific inhibitors were used to examine their role in regulation of γc expression. Specific binding motif was confirmed by γc promotor mutant cells generated using CRISPR-Cas9. IL-7TgγcTg mice were used to examine regulatory role of γc in cytokine signaling. RESULTS: We found that activated T cells significantly upregulated γc expression, wherein NFAT1 and NFκB were key in transcriptional upregulation via T cell receptor stimulation. Also, we identified the functional binding site of the γc promoter and the synergistic effect of NFAT1 and NFκB in the regulation of γc expression. Increased γc expression inhibited IL-7 signaling and rescued lymphoproliferative disorder in an IL-7Tg animal model, providing novel insights into T cell homeostasis. CONCLUSION: Our results indicate functional cooperation between NFAT1 and NFκB in upregulating γc expression in activated T cells. As γc expression also regulates γc cytokine responsiveness, our study suggests that γc expression should be considered as one of the regulators in γc cytokine signaling and the development of T cell immunotherapies. Video Abstract.

hnRNP K supports the maintenance of RORγ circadian rhythm through ERK signaling.

Retinoic acid-related orphan receptor γ (RORγ) maintains the circadian rhythms of its downstream genes. However, the mechanism behind the transcriptional activation of RORγ itself remains unclear. Here, we demonstrate that transcription of RORγ is activated by heterogeneous nuclear ribonucleoprotein K (hnRNP K) via the poly(C) motif within its proximal promoter. Interestingly, we confirmed the binding of endogenous hnRNP K within RORγ1 and RORγ2 promoter along with the recruitment of RNA polymerase 2 through chromatin immunoprecipitation (ChIP). Furthermore, an assay for transposase accessible chromatin (ATAC)-qPCR showed that hnRNP K induced higher chromatin accessibility within the RORγ1 and RORγ2 promoter. Then we found that the knockdown of hnRNP K lowers RORγ mRNA oscillation amplitude in both RORγ and RORγ-dependent metabolic genes. Moreover, we demonstrated that time-dependent extracellular signal-regulated kinase (ERK) activation controls mRNA oscillation of RORγ and RORγ-dependent metabolic genes through hnRNP K. Taken together, our results provide new insight into the regulation of RORγ by hnRNP K as a transcriptional activator, along with its physiological significance in metabolism.

Bone marrow CX3CR1+ mononuclear cells relay a systemic microbiota signal to control hematopoietic progenitors in mice.

The microbiota regulate hematopoiesis in the bone marrow (BM); however, the detailed mechanisms remain largely unknown. In this study, we explored how microbiota-derived molecules (MDMs) were transferred to the BM and sensed by the local immune cells to control hematopoiesis under steady-state conditions. We reveal that MDMs, including bacterial DNA (bDNA), reach the BM via systemic blood circulation and are captured by CX3CR1+ mononuclear cells (MNCs). CX3CR1+ MNCs sense MDMs via endolysosomal Toll-like receptors (TLRs) to produce inflammatory cytokines, which control the basal expansion of hematopoietic progenitors, but not hematopoietic stem cells, and their differentiation potential toward myeloid lineages. CX3CR1+ MNCs colocate with hematopoietic progenitors at the perivascular region, and the depletion of CX3CR1+ MNCs impedes bDNA influx into the BM. Moreover, the abrogation of TLR pathways in CX3CR1+ MNCs abolished the microbiota effect on hematopoiesis. These studies demonstrate that systemic MDMs control BM hematopoiesis by producing CX3CR1+ MNC-mediated cytokines in the steady-state.

Longitudinal Label-Free Two-Photon Microscopy of Cellular Healing Processes in Non-Ablative Fractional Laser Wounds.

BACKGROUND AND OBJECTIVES: Wound healing is an important biomedical problem with various associated complications. Although cutaneous wound healing has been studied in vivo extensively using various optical imaging methods, early-stage cellular healing processes were difficult to study due to scab formation. The objective of this study is to demonstrate that minimal laser wounds and optical microscopy can access the detailed cellular healing processes of cutaneous wounds from the early stage. STUDY DESIGN/MATERIALS AND METHODS: A non-ablative fractional laser (NAFL) and label-free two-photon microscopy (TPM) were used to induce minimal cutaneous wounds and to image the wounds in three-dimension. Sixteen hairless mice and a single human volunteer were used. NAFL wounds were induced in the hindlimb skin of the mice and in the forearm skin of the human subject. The NAFL wounds were longitudinally imaged during the healing period, starting from an hour post wound induction in the earliest and until 21 days. Cells in the wound and surrounding normal skin were visualized based on two-photon excited auto-fluorescence (TPAF), and cellular changes were tracked by analyzing longitudinal TPM images both qualitatively and quantitatively. Damage and recovery in the skin dermis were tracked by using the second harmonic generation (SHG) signal of collagen. Immunofluorescence and hematoxylin and eosin histology analysis were conducted to validate the TPM results of the murine skin. RESULTS: Cellular healing processes in NAFL wounds and surroundings could be observed by longitudinal TPM. In the case of murine skin, various healing phases including inflammation, re-epithelization, granulation tissue formation, and late remodeling phase including collagen regeneration were observed in the same wounds owing to minimal or no scab formation. The re-epithelization process was analyzed quantitatively by measuring cell density and thickness of the epithelium in the wound surroundings. In the case of the human skin, the access inside the wound was blocked for a few days post wound induction due to scabs but the cellular changes in the wound surroundings were observed from the early stage. Cellular healing processes in the NAFL wound of the human skin were similar to those in murine skin. CONCLUSIONS: The minimal NAFL wound model and label-free TPM demonstrated the cell level assessment of wound healing processes with applicability to human subjects. © 2021 Wiley Periodicals LLC.

Intestinal microbiota control acute kidney injury severity by immune modulation.

Intestinal microbiota impacts the host immune system and influences the outcomes of chronic diseases. However, it remains uncertain whether acute kidney injury (AKI) impacts intestinal microbiota or vice versa. To determine this, we investigated the mechanistic link between AKI, microbiota, and immune response in ischemia/reperfusion injury. Microbiota alteration and its biological consequences after ischemia/reperfusion injury were examined and the effect of dysbiotic microbiota on the outcome of AKI was also assessed by colonizing germ-free mice with post-AKI microbiota. The role of Th17, Th1, Tregs cells and macrophage polarization in mediating the renoprotective effect of antibiotic induced microbiota depletion in ischemia/reperfusion injury was also determined. Increase of Enterobacteriacea, decrease of Lactobacilli, and Ruminococacceae were found to be the hallmarks of ischemia/reperfusion injury induced dysbiosis and were associated with a decreased levels of short-chain fatty acids, intestinal inflammation and leaky gut. Colonizing germ-free mice with post-AKI microbiota worsened ischemia/reperfusion injury severity with exaggerated inflammation in recipient mice compared to colonizing with microbiota from sham operated mice. Microbiota depletion by oral antibiotics protected against ischemia/reperfusion injury. This renoprotective effect was associated with reduced Th 17, Th 1 response along with expansion of regulatory T cells, and M2 macrophages. Our study demonstrated a unique bidirectional relationship between the kidney and the intestine during AKI. Intestinal dysbiosis, inflammation and leaky gut are consequences of AKI but they also represent an important modifier determining post-AKI severity. Thus, targeting the intestinal microbiota might provide a novel therapeutic strategy in AKI.

Locus-Specific Reversible DNA Methylation Regulates Transient IL-10 Expression in Th1 Cells.

IL-10 is a pleiotropic cytokine with multifaceted functions in establishing immune homeostasis. Although expressed by Th1 and Th2 cells, conventional Th1 cells produce marginal levels of IL-10 compared with their Th2 counterparts. In this study, we investigated the epigenetic mechanisms of Il-10 gene expression in Th1 cells. Bioinformatics EMBOSS CpG plot analysis and bisulfite pyrosequencing revealed three CpG DNA methylation sites in the Il-10 gene locus. Progressive DNA methylation at all of the CpG regions of interest (ROIs) established a repressive program of Il-10 gene expression in Th1 cells. Interestingly, Th1 cells treated with IL-12 and IL-27 cytokines, thereby mimicking a chronic inflammatory condition in vivo, displayed a significant increase in IL-10 production that was accompanied by selective DNA demethylation at ROI 3 located in intron 3. IL-10-producing T cells isolated from lymphocytic choriomeningitis virus-infected mice also showed enhanced DNA demethylation at ROI 3. Binding of STAT1 and STAT3 to demethylated ROI 3 enhanced IL-10 expression in an IL-12/IL-27-dependent manner. Accordingly, CD4+ T cells isolated from STAT1- or STAT3-knockout mice were significantly defective in IL-10 production. Our data suggest that, although stably maintained DNA methylation at the promoter may repress IL-10 expression in Th1 cells, locus-specific reversible DNA demethylation may serve as a threshold platform to control transient Il-10 gene expression.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

NFAT1 Regulates Systemic Autoimmunity through the Modulation of a Dendritic Cell Property.

The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.

Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice.

BACKGROUND & AIMS: Obesity and metabolic syndrome have been associated with alterations to the intestinal microbiota. However, few studies examined the effects of obesity on the intestinal immune system. We investigated changes in subsets of intestinal CD4+ T-helper (TH) cells with obesity and the effects of gut-tropic TH17 cells in mice on a high-fat diet (HFD). METHODS: We isolated immune cells from small intestine and adipose tissue of C57BL/6 mice fed a normal chow diet or a HFD for 10 weeks and analyzed the cells by flow cytometry. Mice fed a vitamin A-deficient HFD were compared with mice fed a vitamin A-sufficient HFD. Obese RAG1-deficient mice were given injections of only regulatory T cells or a combination of regulatory T cells and TH17 cells (wild type or deficient in integrin ß7 subunit or interleukin 17 [IL17]). Mice were examined for weight gain, fat mass, fatty liver, glucose tolerance, and insulin resistance. Fecal samples were collected before and after T cell transfer and analyzed for microbiota composition by metagenomic DNA sequencing and quantitative polymerase chain reaction. RESULTS: Mice placed on a HFD became obese, which affected the distribution of small intestinal CD4+ TH cells. Intestinal tissues from obese mice had significant reductions in the proportion of TH17 cells but increased proportion of TH1 cells, compared with intestinal tissues from nonobese mice. Depletion of vitamin A in obese mice further reduced the proportion of TH17 cells in small intestine; this reduction correlated with more weight gain and worsening of glucose intolerance and insulin resistance. Adoptive transfer of in vitro-differentiated gut-tropic TH17 cells to obese mice reduced these metabolic defects, which required the integrin ß7 subunit and IL17. Delivery of TH17 cells to intestines of mice led to expansion of commensal microbes associated with leanness. CONCLUSIONS: In mice, intestinal TH17 cells contribute to development of a microbiota that maintains metabolic homeostasis, via IL17. Gut-homing TH17 cells might be used to reduce metabolic disorders in obese individuals.

Nuclear Speckle-related Protein 70 Binds to Serine/Arginine-rich Splicing Factors 1 and 2 via an Arginine/Serine-like Region and Counteracts Their Alternative Splicing Activity.

Nuclear speckles are subnuclear storage sites containing pre-mRNA splicing machinery. Proteins assembled in nuclear speckles are known to modulate transcription and pre-mRNA processing. We have previously identified nuclear speckle-related protein 70 (NSrp70) as a novel serine/arginine (SR)-related protein that co-localizes with classical SR proteins such as serine/arginine-rich splicing factor 1 (SRSF1 or ASF/SF2) and SRSF2 (SC35). NSrp70 mediates alternative splice site selection, targeting several pre-mRNAs, including CD44 exon v5. Here we demonstrated that NSrp70 interacts physically with two SR proteins, SRSF1 and SRSF2, and reverses their splicing activity in terms of CD44 exon v5 as exon exclusion. The NSrp70 RS-like region was subdivided into three areas. Deletion of the first arginine/serine-rich-like region (RS1) completely abrogated binding to the SR proteins and to target mRNA and also failed to induce splicing of CD44 exon v5, suggesting that RS1 is critical for NSrp70 functioning. Interestingly, RS1 deletion also resulted in the loss of NSrp70 and SR protein speckle positioning, implying a potential scaffolding role for NSrp70 in nuclear speckles. NSrp70 contains an N-terminal coiled-coil domain that is critical not only for self-oligomerization but also for splicing activity. Consistently, deletion of the coiled-coil domain resulted in indefinite formation of nuclear speckles. Collectively, these results demonstrate that NSrp70 acts as a new molecular counterpart for alternative splicing of target RNA, counteracting SRSF1 and SRSF2 splicing activity.

Targeted Inhibition of the NCOA1/STAT6 Protein-Protein Interaction.

The complex formation between transcription factors (TFs) and coactivator proteins is required for transcriptional activity, and thus disruption of aberrantly activated TF/coactivator interactions could be an attractive therapeutic strategy. However, modulation of such protein-protein interactions (PPIs) has proven challenging. Here we report a cell-permeable, proteolytically stable, stapled helical peptide directly targeting nuclear receptor coactivator 1 (NCOA1), a coactivator required for the transcriptional activity of signal transducer and activator of transcription 6 (STAT6). We demonstrate that this stapled peptide disrupts the NCOA1/STAT6 complex, thereby repressing STAT6-mediated transcription. Furthermore, we solved the first crystal structure of a stapled peptide in complex with NCOA1. The stapled peptide therefore represents an invaluable chemical probe for understanding the precise role of the NCOA1/STAT6 interaction and an excellent starting point for the development of a novel class of therapeutic agents.

Hypoxia-inducible factor-2α is an essential catabolic regulator of inflammatory rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that manifests as chronic inflammation and joint tissue destruction. However, the etiology and pathogenesis of RA have not been fully elucidated. Here, we explored the role of the hypoxia-inducible factors (HIFs), HIF-1α (encoded by HIF1A) and HIF-2α (encoded by EPAS1). HIF-2α was markedly up-regulated in the intimal lining of RA synovium, whereas HIF-1α was detected in a few cells in the sublining and deep layer of RA synovium. Overexpression of HIF-2α in joint tissues caused an RA-like phenotype, whereas HIF-1α did not affect joint architecture. Moreover, a HIF-2α deficiency in mice blunted the development of experimental RA. HIF-2α was expressed mainly in fibroblast-like synoviocytes (FLS) of RA synovium and regulated their proliferation, expression of RANKL (receptor activator of nuclear factor-κB ligand) and various catabolic factors, and osteoclastogenic potential. Moreover, HIF-2α-dependent up-regulation of interleukin (IL)-6 in FLS stimulated differentiation of TH17 cells-crucial effectors of RA pathogenesis. Additionally, in the absence of IL-6 (Il6-/- mice), overexpression of HIF-2α in joint tissues did not cause an RA phenotype. Thus, our results collectively suggest that HIF-2α plays a pivotal role in the pathogenesis of RA by regulating FLS functions, independent of HIF-1α.

NFAT1 and JunB cooperatively regulate IL-31 gene expression in CD4+ T cells in health and disease.

IL-31 is a key mediator of itching in atopic dermatitis (AD) and is preferentially produced by activated CD4(+) T cells and Th2 cells. Although pathophysiological functions of IL-31 have been suggested in diverse immune disorders, the molecular events underlying IL-31 gene regulation are still unclear. In this study we identified the transcription start site and functional promoter involved in IL-31 gene regulation in mouse CD4(+) T cells. TCR stimulation-dependent IL-31 expression was found to be closely linked with in vivo binding of NFAT1 and JunB to the IL-31 promoter. Although NFAT1 alone enhanced IL-31 promoter activity, it was further enhanced in the presence of JunB. Conversely, knockdown of either NFAT1 or JunB resulted in reduced IL-31 expression. NFAT1-deficient CD4(+) T cells showed a significant defect in IL-31 expression compared with wild-type CD4(+) T cells. In agreement with these findings, mice subjected to atopic conditions showed much higher levels of IL-31, which were closely correlated with a significant increase in the number of infiltrated NFAT1(+)CD4(+) T cells into the AD ears. Amelioration of AD progression by cyclosporin A treatment was well correlated with downregulation of IL-31 expressions in CD4(+) T cells and total ear residual cells. In summary, our results suggest a functional cooperation between NFAT1 and JunB in mediating IL-31 gene expression in CD4(+) T cells and indicate that interference with this interaction or their activity has the potential of reducing IL-31-mediated AD symptoms.

Flagellin suppresses experimental asthma by generating regulatory dendritic cells and T cells.

BACKGROUND: Although the hygiene hypothesis suggests that microbial infections could subvert asthma and thus a microbial product might serve as a therapeutic adjuvant for asthma, the relationship between bacterial components and asthma is complex. Recently, low levels of flagellin, the Toll-like receptor (TLR) 5 ligand, have been reported to promote asthma. OBJECTIVE: We show that a therapeutic dose of flagellin suppresses asthma and that the effect occurs through generating regulatory dendritic cells (rDCs) and regulatory T (Treg) cells. METHODS: Ovalbumin (OVA)-induced wild-type and TLR5 knockout asthmatic mice were treated intranasally with a mixture of OVA and 10 µg of a flagellin B (FlaB; of Vibrio vulnificus). OVA/FlaB-treated rDCs were adoptively transferred to mice with OVA-induced asthma. Anti-CD25 mAb was used to deplete Treg cells. A mixture of house dust mite (HDM) and FlaB was used to treat mice with HDM-induced asthma. Blood CD14(+) monocyte-derived dendritic cells from HDM-sensitive asthmatic patients were treated with FlaB and incubated with autologous CD4(+) T cells. RESULTS: An OVA/FlaB mixture ameliorated OVA-induced asthma by inhibiting TH1/TH2/TH17 responses in a TLR5-dependent manner through generating rDCs and Treg cells. The adoptive transfer of OVA/FlaB-treated dendritic cells inhibited OVA-induced asthma, whereas the depletion of CD25(+) cells eliminated the inhibitory effect. A similar effect of FlaB was observed in mice with HDM-induced asthma. In patients with HDM-sensitive asthma, FlaB-treated rDCs inhibited HDM-stimulated TH1/TH2 responses while enhancing Treg cells in an IL-10-dependent manner. CONCLUSION: These findings collectively suggest that flagellin could be used as a tolerogenic adjuvant to treat allergic asthma.