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In Small Intestine Inflammation, CD103 + CD11b + DCs and CD103 ? CD11b + DCs Expand Inflammatory Th17 Cells (A) Representative flow plot of SI-LP DCs in cohoused , littermate A20 cko -DTA ? mice and A20 cko - DTA + mice at 9?12 weeks of age. (B) Absolute number of each SI-LP DC subset in A20 wt or A20 cko mice (either DTA ? or DTA + ). (C?F) Absolute number of SI-LP CD4 T cells (IL-17 + in C, IFNg + in D, IL-17 + IFNg + in E, or Foxp3 + CD4 T cells in F) in co-housed A20 wt or A20 cko mice (additionally either DTA ? or DTA + ). Results were combined from three independent experiments including at least one mouse of each genotype. Each dot represents one mouse. Error bars represent mean ? SEM. *p < 0.05, **p < 0.01, ****p < 0.0001 (unpaired Student's t test).
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Normal dynamics between microbiota and dendritic cells (DCs) support modest numbers of T cells, yet these do not cause inflammation. The DCs that induce inflammatory T cells and the signals that drive this process remain unclear. Here, we demonstrate that small intestine DCs lacking the signaling attenuator A20 induce inflammatory T cells and that...
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... upregulation of IL-23a and IL-1b mRNA was exclusive to CD103 + CD11b À DCs, and both cytokines were upregulated in a MyD88-independent fashion in this DC subset (Figures 6D-6I). IL-12a(p35) and IL-12b(p40) mRNA were not upregulated, indicating that gene expression was not generally ''promiscuous'' in CD103 + CD11b À DCs from A20 cko or A20/Myd88 cko mice ( Figure S7). From these data, we conclude that physiologic signals that are independent of MyD88 can induce IL-23a, IL-1b, and IL-6 mRNA in CD103 + CD11b À DCs. ...
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... A20 cko -DTA + mice, CD103 + CD11b + DCs were numerically reduced to just 7% that of co-housed littermate A20 cko -DTA À mice ( Figures 7A and 7B). CD103 + CD11b + DCs in A20 wt -DTA + mice were ablated with even higher efficiency (98%). ...
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... + CD11b + DCs in A20 wt -DTA + mice were ablated with even higher efficiency (98%). Although previous studies reported the Langerin-DTA transgene did not ablate other SI-LP DCs ( Welty et al., 2013;Panea et al., 2015), we found numbers of CD103 À CD11b + DCs in both A20 wt -DTA + mice and A20 cko -DTA + mice were markedly reduced ( Figures 7A and 7B). CD103 À CD11b + DCs were on average reduced 2-fold in A20 cko -DTA + mice and as much as 8-fold in A20 wt - DTA + mice. ...
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... the two DC subsets with potent IL-17-inducing APC functions in DC/T cell co-cultures were depleted in A20 cko - DTA + mice. These mice were not deficient in CD103 + CD11b À DCs or macrophages ( Figure 7B). ...
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... to A20 wt -DTA À mice, littermate A20 wt -DTA + mice had fewer SI-LP Th17 cells ( Figure 7C). This is consistent with previous reports and supports a specialized role for CD103 + CD11b + DCs and also CD103 -CD11b + DCs in expansion of mucosal Th17 cells during health. ...
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... is consistent with previous reports and supports a specialized role for CD103 + CD11b + DCs and also CD103 -CD11b + DCs in expansion of mucosal Th17 cells during health. In inflamed small intestine of A20 cko -DTA À mice numbers of mucosal Th17 cells were mark- edly expanded ( Figure 7C). By contrast, in co-housed, littermate A20 cko -DTA + mice, Th17 expansion was significantly attenuated. ...
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... contrast, in co-housed, littermate A20 cko -DTA + mice, Th17 expansion was significantly attenuated. On average, Th17 cells in A20 cko -DTA + mice were just one- third that of A20 cko -DTA À mice, suggesting that inflammatory Th17 cells in A20 cko mice required CD103 + CD11b + DCs and CD103 À CD11b + DCs ( Figure 7C). Unlike Th17 cells, Th1 cells were expanded >55-fold in both A20 cko -DTA À and DTA + mice ( Figure 7D). ...
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... average, Th17 cells in A20 cko -DTA + mice were just one- third that of A20 cko -DTA À mice, suggesting that inflammatory Th17 cells in A20 cko mice required CD103 + CD11b + DCs and CD103 À CD11b + DCs ( Figure 7C). Unlike Th17 cells, Th1 cells were expanded >55-fold in both A20 cko -DTA À and DTA + mice ( Figure 7D). Additionally, both mice had expanded numbers of IL-17 + IFNg + and Foxp3 + T cells (Figures 7E and 7F). ...
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... Th17 cells, Th1 cells were expanded >55-fold in both A20 cko -DTA À and DTA + mice ( Figure 7D). Additionally, both mice had expanded numbers of IL-17 + IFNg + and Foxp3 + T cells (Figures 7E and 7F). The persistence of these SI-LP T cells and the marked reduction of Th17 cells in A20 cko -DTA + mice indicates that APC functions of CD103 + CD11b + DCs, and also CD103 -CD11b + DCs, are required to instruct and expand inflammatory Th17 cells in the pathogenesis of small intestine inflammation, sharing histologic features with Crohn's disease. ...
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Th17 cells drive autoimmune disease but also control commensal microbes. A common link among antigens from self-proteins or commensal microbiota is relatively low activation of T cell receptor (TCR) and costimulation signaling. Indeed, strong TCR/CD28 stimulation suppressed Th17 cell differentiation from human naive T cells, but not effector/memory...
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... The expression of CD80 and CD86 on colonic mDCs in DKO recipients was significantly lower than that in Rag1 −/− recipients (Fig. 5E). Flow cytometric analysis of mDCs recovered from the inflamed colonic LP identified four populations of CD11b-and CD103-expressing cells in Rag1 -/recipients (12). The CD103 -CD11b + phenotype was the most prominent (90%) (Fig. 5F). ...
... the LP-DC population, which has been identified as a potent inducer of Th1 and Th17 differentiation (12). Moreover, Tl1a −/− LP-DCs exhibited reduced maturity and MHC II expression, as well as costimulatory molecules CD80 and CD86. ...
... Moreover, Tl1a −/− LP-DCs exhibited reduced maturity and MHC II expression, as well as costimulatory molecules CD80 and CD86. In fact, IL-12 and IL-23 produced by DCs are required for intestinal Th1 and Th17 differentiation, respectively (12,22). ...
The binding of tumor necrosis factor-like cytokine 1A (TL1A) to death receptor 3 (DR3) plays an important role in the interaction between dendritic cells (DCs) and T cells and contributes to intestinal inflammation development. However, the mechanism by which DCs expressing TL1A mediate helper T (Th) cell differentiation in the intestinal lamina propria (LP) during the pathogenesis of inflammatory bowel disease remains unclear. In this study, we found that TL1A/DR3 promoted Th1 and Th17 cell differentiation in T-T and DC-T cell interaction-dependent manners. TL1A-deficient CD4+ T cells failed to polarize into Th1/Th17 cells and did not cause colonic inflammation in a T cell transfer colitis model. Notably, TL1A was located in the cytoplasm and nuclei of DCs, positively regulated the DC-specific ICAM-grabbing nonintegrin/RAF1/nuclear factor κB signaling pathway, enhanced the antigen uptake ability of DCs, and promoted TLR4-mediated DC activation, inducing naive CD4+ T cell differentiation into Th1 and Th17 cells. Our work reveals that TL1A plays a regulatory role in inflammatory bowel disease pathogenesis.
... BSA/1mM DTT for 10 min at 37°C. 60 Supernatant was collected from these washes to isolate intraepithelial lymphocytes. Tissue was then washed for 10 min at 37°C with HBSS/10mM HEPES/0.625% ...
... BSA followed by incubation in C-tubes (Miltenyi) with digestion cocktail containing Liberase (57.6 μg/mL) and DNase I (8 U/mL). 60 Tissue was digested for 37°C for 30 min with shaking, followed by GentleMACS dissociation (Miltenyi). Proximal and distal small intestine samples were each ~10 cm long and defined as the first (stomach adjacent) or last (cecum adjacent) quarter of the small intestinal organ. ...
Little is known about how microbiota regulate innate-like γδ T cells or how these restrict their effector functions within mucosal barriers, where microbiota provide chronic stimulation. Here, we show that microbiota-mediated regulation of γδ17 cells is binary, where microbiota instruct in situ interleukin-17 (IL-17) production and concomitant expression of the inhibitory receptor programmed cell death protein 1 (PD-1). Microbiota-driven expression of PD-1 and IL-17 and preferential adoption of a PD-1high phenotype are conserved for γδ17 cells across multiple mucosal barriers. Importantly, microbiota-driven PD-1 inhibits in situ IL-17 production by mucosa-resident γδ17 effectors, linking microbiota to their simultaneous activation and suppression. We further show the dynamic nature of this microbiota-driven module and define an inflammation-associated activation state for γδ17 cells marked by augmented PD-1, IL-17, and lipid uptake, thus linking the microbiota to dynamic subset-specific activation and metabolic remodeling to support γδ17 effector functions in a microbiota-dense tissue environment.
... In the PP, the genera unclassified Bacteroidales, Lactobacillus, Akkermansia, Faecalibaculum, unclassified Erysipelotrichales, Odoribacter and Alloprevotella correlated negatively with this dendritic cell population. This dendritic cell population is unique to the intestinal immune system [10] and drives Th17 differentiation in the intestinal immune response [28]. The Th17 cytokines support the integrity of the gut barrier [29]. ...
The gut microbiota are involved in adaptations of the maternal immune response to pregnancy. We therefore hypothesized that inducing gut dysbiosis during pregnancy alters the maternal immune response. Thus, pregnant mice received antibiotics from day 9 to day 16 to disturb the maternal gut microbiome. Feces were collected before, during and after antibiotic treatment, and microbiota were measured using 16S RNA sequencing. Mice were sacrificed at day 18 of pregnancy and intestinal (Peyer's patches (PP) and mesenteric lymph nodes (MLN)) and peripheral immune responses (blood and spleen) were measured using flow cytometry. Antibiotic treatment decreased fetal and placental weight. The bacterial count and the Shannon index were significantly decreased (Friedman, followed by Dunn's test, p < 0.05) and the bacterial genera abundance was significantly changed (Permanova, p < 0.05) following antibiotics treatment as compared with before treatment. Splenic Th1 cells and activated blood monocytes were increased, while Th2, Th17 and FoxP3/RoRgT double-positive cells in the PP and MLNs were decreased in pregnant antibiotics-treated mice as compared with untreated pregnant mice. In addition, intestinal dendritic cell subsets were affected by antibiotics. Correlation of immune cells with bacterial genera showed various correlations between immune cells in the PP, MLN and peripheral circulation (blood and spleen). We conclude the disturbed gut microbiota after antibiotics treatment disturbed the maternal immune response. This disturbed maternal immune response may affect fetal and placental weight.
... Also, in Batf3 -/mice which lack intestinal CD103 + CD11b -DCs, there was no evidence of spontaneous gastrointestinal inflammation (22). So, CD103 + CD11b + DCs have been postulated to play a role for immune tolerance in the intestine; however, paradoxically, it has been reported that those cells induce a Th1 and Th17 response under inflammatory conditions (23,24). Likewise, a minor population of intestinal CD103 − CD11b + DCs prime naïve CD4 + T cells and induce differentiation to IL-17-or IFN-γ-producing effector CD4 + T cells (25). ...
The gastrointestinal tract is the first organ directly affected by fasting. However, little is known about how fasting influences the intestinal immune system. Intestinal dendritic cells (DCs) capture antigens, migrate to secondary lymphoid organs, and provoke adaptive immune responses. We evaluated the changes of intestinal DCs in mice with short-term fasting and their effects on protective immunity against Listeria monocytogenes (LM). Fasting induced an increased number of CD103+CD11b- DCs in both small intestinal lamina propria (SILP) and mesenteric lymph nodes (mLN). The SILP CD103+CD11b- DCs showed proliferation and migration, coincident with increased levels of GM-CSF and C-C chemokine receptor type 7, respectively. At 24 h post-infection with LM, there was a significant reduction in the bacterial burden in the spleen, liver, and mLN of the short-term-fasted mice compared to those fed ad libitum. Also, short-term-fasted mice showed increased survival after LM infection compared with ad libitum-fed mice. It could be that significantly high TGF-β2 and Aldh1a2 expression in CD103+CD11b- DCs in mice infected with LM might affect to increase of Foxp3+ regulatory T cells. Changes of major subset of DCs from CD103+ to CD103- may induce the increase of IFN-γ-producing cells with forming Th1-biased environment. Therefore, the short-term fasting affects protection against LM infection by changing major subset of intestinal DCs from tolerogenic to Th1 immunogenic.
... Intestinal leukocyte preparation Leukocytes from the colon and SI were prepared as previously described (Liang et al., 2016). Briefly, colon and SI were removed, opened longitudinally, and gently washed with ice-cold PBS. ...
Osteopontin (OPN) has been considered a potential biomarker of graft-versus-host disease (GVHD). However, the function of OPN in GVHD is still elusive. Using a mouse model of acute GVHD (aGVHD), we report that OPN generated by CD4⁺ T cells is sufficient to exert a beneficial effect in controlling aGVHD through limiting gastrointestinal pathology, a major target organ of aGVHD. CD4⁺ T cell-derived OPN works on CD44 expressed in intestinal epithelial cells (IECs) and abates cell death of IECs. OPN also modulates gut microbiota with enhanced health-associated commensal bacteria Akkermansia. Importantly, we use our in vivo mouse mutant model to specifically express OPN isoforms and demonstrate that secreted OPN (sOPN), not intracellular OPN (iOPN), is solely responsible for the protective role of OPN. This study demonstrates that sOPN generated by CD4⁺ T cells is potent enough to limit aGVHD.
... An additional layer of functional specialization is embedded within intestinal DC and macrophage populations since both cell types are composed of discrete subsets, some of which are specific to the intestine and do not exist in other tissues. Evidence suggests that each DC and macrophage subset is functionally distinct and therefore, accurate and complete identification of these subsets is a major priority, since these mononuclear phagocytes are key for pathogen defense, intestinal homeostasis, and also, the aberrant activity of intestinal DCs and macrophages is linked to pathogenesis of inflammatory bowel disease and colorectal cancer (5)(6)(7)(8)(9)(10). ...
... Accurate identification of mononuclear phagocyte subsets is fundamental to understanding intestinal immunity and based on several landmark studies (10)(11)(12)(13)(14)(15), murine macrophages and DCs (within MHC-II + CD11b + /CD11c + populations) are commonly distinguished on the basis of CD64 or CD14. Both markers are highly expressed on intestinal macrophages, whereas DCs must meet two criteria: 1) Negative for CD14 and CD64, and 2) Positive for CD24 or CD26 (10)(11)(12)(13)(14)(15). ...
... Accurate identification of mononuclear phagocyte subsets is fundamental to understanding intestinal immunity and based on several landmark studies (10)(11)(12)(13)(14)(15), murine macrophages and DCs (within MHC-II + CD11b + /CD11c + populations) are commonly distinguished on the basis of CD64 or CD14. Both markers are highly expressed on intestinal macrophages, whereas DCs must meet two criteria: 1) Negative for CD14 and CD64, and 2) Positive for CD24 or CD26 (10)(11)(12)(13)(14)(15). Although these subsetting strategies are widely used, previous studies validating these approaches largely focus on small intestine and it remains to be thoroughly investigated whether these strategies are equally as effective in the colon (10)(11)(12)(13)(14)(15). ...
Intestinal immunity is coordinated by specialized mononuclear phagocyte populations, constituted by a diversity of cell subsets. Although the cell subsets constituting the mononuclear phagocyte network are thought to be similar in both small and large intestine, these organs have distinct anatomy, microbial composition, and immunological demands. Whether these distinctions demand organ-specific mononuclear phagocyte populations with dedicated organ-specific roles in immunity are unknown. Here we implement a new strategy to subset murine intestinal mononuclear phagocytes and identify two novel subsets which are colon-specific: a macrophage subset and a Th17-inducing dendritic cell (DC) subset. Colon-specific DCs and macrophages co-expressed CD24 and CD14, and surprisingly, both were dependent on the transcription factor IRF4. Novel IRF4-dependent CD14⁺CD24⁺ macrophages were markedly distinct from conventional macrophages and failed to express classical markers including CX3CR1, CD64 and CD88, and surprisingly expressed little IL-10, which was otherwise robustly expressed by all other intestinal macrophages. We further found that colon-specific CD14⁺CD24⁺ mononuclear phagocytes were essential for Th17 immunity in the colon, and provide definitive evidence that colon and small intestine have distinct antigen presenting cell requirements for Th17 immunity. Our findings reveal unappreciated organ-specific diversity of intestine-resident mononuclear phagocytes and organ-specific requirements for Th17 immunity.
... Nevertheless, CD11b − CD103 + DCs are involved in antigen cross-presentation and priming of CD8T cells (Ruane and Lavelle, 2011); CD11b − CD103 + DCs are also a crucial mediator of Treg cells that works in immune tolerance (Coombes et al., 2007). In contrast, CD11b + CD103 − DCs are involved in priming of Th1 and Th17 CD4T cells (Liang et al., 2016). Consistently, the IFNγ and IL17-producing CD8T and Treg cells increased, but Th1 and Th17 cells decreased in AKR1B8 -/-mice. ...
Aldo-keto reductase 1B10 (AKR1B10) is downregulated in human ulcerative colitis (UC) and colorectal cancer, being a potential pathogenic factor of these diseases. Aldo-keto reductase 1B8 (AKR1B8) is the ortholog in mice of human AKR1B10. Targeted AKR1B8 deficiency disrupts homeostasis of epithelial self-renewal and leads to susceptibility to colitis and carcinogenesis. In this study, we found that in AKR1B8 deficient mice, Muc2 expression in colon was diminished, and permeability of colonic epithelium increased. Within 24 h, orally administered FITC-dextran penetrated into mesenteric lymph nodes (MLN) and liver in AKR1B8 deficient mice, but not in wild type controls. In the colon of AKR1B8 deficient mice, neutrophils and mast cells were markedly infiltrated, γδT cells were numerically and functionally impaired, and dendritic cell development was altered. Furthermore, Th1, Th2, and Th17 cells decreased, but Treg and CD8T cells increased in the colon and MLN of AKR1B8 deficient mice. In colonic epithelial cells of AKR1B8 deficient mice, p-AKT (T308 and S473), p-ERK1/2, p-IKBα, p-p65 (S536), and IKKα expression decreased, accompanied with downregulation of IL18 and CCL20 and upregulation of IL1β and CCL8. These data suggest AKR1B8 deficiency leads to abnormalities of intestinal epithelial barrier and immunity in colon.
... Bacterial eATP in the gut elicits a variety of inflammatory responses and interacts with the host's mucosal immune system [3]. For example, intestinal ATP increases the number of Th17 cells in the gut [6], which is associated with the pathogenesis of inflammatory bowel disease (IBD) [7]. Similarly, ATP release has been observed in eukaryotic cells including neuronal and immune cells, as well as in injured or damaged cells [4]. ...
Adenosine triphosphate (ATP) is one of the most important indicators of cell viability. Extracellular ATP (eATP) is commonly detected in cultures of both eukaryotic and prokaryotic cells but is not the focus of current scientific research. Although ATP release has traditionally been considered to mainly occur as a consequence of cell destruction, current evidence indicates that ATP leakage also occurs during the growth phase of diverse bacterial species and may play an important role in bacterial physiology. ATP can be conveniently measured with high sensitivity in luciferase-based bioluminescence assays. However, wild-type luciferases suffer from low stability, which limit their use. Here we demonstrate that an engineered, thermostable luciferase is suitable for real-time monitoring of ATP release by bacteria, both in broth culture and on agar surfaces. Different bacterial species show distinct patterns of eATP accumulation and decline. Real-time monitoring of eATP allows for the estimation of viable cell number by relating luminescence onset time to initial cell concentration. Furthermore, the method is able to rapidly detect the effect of antibiotics on bacterial cultures as Ampicillin sensitive strains challenged with beta lactam antibiotics showed strongly increased accumulation of eATP even in the absence of growth, as determined by optical density. Patterns of eATP determined by real-time luminescence measurement could be used to infer the minimal inhibitory concentration of Ampicillin. Compared to conventional antibiotic susceptibility testing, the method presented here is faster and more sensitive, which is essential for better treatment outcomes and reducing the risk of inducing antibiotic resistance. Real-time eATP bioluminescence assays are suitable for different cell types, either prokaryotic or eukaryotic, thus, permitting their application in diverse fields of research. It can be used for example in the study of the role of eATP in physiology and pathophysiology, for monitoring microbial contamination or for antimicrobial susceptibility testing in clinical diagnostics.
... CD103 + CD11b − CD8α + DCs are known to be specialized for the cross-presentation of cell-associated antigens and priming of CD8 + T cells 18 . By contrast, CD103 + CD11b + DCs appear to induce immune tolerance at the steady-state, particularly in intestine; however, paradoxically, these cells were prone to induce Th1 response if exposed to in ammatory condition 19 . Likewise, a minor population of intestinal CD103 − CD11b + DCs appear to e ciently prime naïve CD4 + T cells and induce differentiation to either IL-17 or IFN-γ producing effector CD4 + T cells 20 . ...
Gastrointestinal tract is the first organ to be directly affected upon fasting. However, little is known about how the fasting influences intestinal immune system. In the present study, we focused on the changes of intestinal dendritic cells (DCs) in mice upon short-term fasting and how the changes influence protective immunity against Listeria monocytogenes (LM) infection. We found that the fasting induces an increased number of CD103 ⁺ CD11b ⁻ DCs in both small intestinal lamina propria (SI LP) and mesenteric lymph nodes (mLN) and the SI LP CD103 ⁺ CD11b ⁻ DCs undergo an active proliferation and migration by increased levels of GM-CSF and CCR7, respectively. At 24 hours post-infection (hpi) of LM, there was a significant reduction of bacterial burden from the spleen, liver, and mLN of the short-term fasting mice compared to those of ad libitum mice. Accordingly, short-term fasting mice showed enhanced survival against LM infection when compared with ad libitum mice. Furthermore, significantly high amount of TGF-β2 and Aldh1a2 expression from CD103 ⁺ CD11b ⁻ DCs in mouse infected with LM sequentially caused the following events: the increase of Foxp3 ⁺ Tregs, preferential change in the composition of CD103 ⁺ to CD103 ⁻ DCs, and the induction of IFN-γ producing cells. Collectively, increase of intestinal CD103 ⁺ DCs by short-term fasting is a key player for protection against LM infection through the changes of functional features from tolerogenic to Th1 immunogenic.
... The DC-specific deletion of A20 promotes Th17 cell production both in vivo and in vitro through secreting high amounts of IL-23 (30). Intestinal DCs from DC-conditional A20 knockout mice have enhanced and biased abilities to promote inflammatory Th1 and Th17 cell differentiation, which is associated with lymphocyte-dependent colitis (68). In addition, Rhbdd3 recruits A20 to inhibit IL-6 production in DCs, and thus suppresses the generation of Th17 cells. ...
Dendritic cells (DCs) are specialized antigen-presenting cells that play a key role in immune homeostasis and the adaptive immune response. DC-induced immune tolerance or activation is strictly dependent on the distinct maturation stages and migration ability of DCs. Ubiquitination is a reversible protein post-translational modification process that has emerged as a crucial mechanism that regulates DC maturation and function. Recent studies have shown that ubiquitin enzymes, including E3 ubiquitin ligases and deubiquitinases (DUBs), are pivotal regulators of DC-mediated immune function and serve as potential targets for DC-based immunotherapy of immune-related disorders (e.g., autoimmune disease, infections, and tumors). In this review, we summarize the recent progress regarding the molecular mechanisms and function of ubiquitination in DC-mediated immune homeostasis and immune response.
