Lactoferrin Potentiates Inducible Regulatory T Cell Differentiation through TGF-ß Receptor III Binding and Activation of Membrane-Bound TGF-ß.

Lactoferrin (LF) is known to possess anti-inflammatory activity, although its mechanisms of action are not well-understood. The present study asked whether LF affects the commitment of inducible regulatory T cells (Tregs). LF substantially promoted Foxp3 expression by mouse activated CD4+T cells, and this activity was further enhanced by TGF-ß1. Interestingly, blocking TGF-ß with anti-TGF-ß Ab completely abolished LF-induced Foxp3 expression. However, no significant amount of soluble TGF-ß was released by LF-stimulated T cells, suggesting that membrane TGF-ß (mTGF-ß) is associated. Subsequently, it was found that LF binds to TGF-ß receptor III, which induces reactive oxygen species production and diminishes the expression of mTGF-ß-bound latency-associated peptide, leading to the activation of mTGF-ß. It was followed by phosphorylation of Smad3 and enhanced Foxp3 expression. These results suggest that LF induces Foxp3+ Tregs through TGF-ß receptor III/reactive oxygen species-mediated mTGF-ß activation, triggering canonical Smad3-dependent signaling. Finally, we found that the suppressive activity of LF-induced Tregs is facilitated mainly by CD39/CD73-induced adenosine generation and that this suppressor activity alleviates inflammatory bowel disease.

Wnt3A Induces GSK-3ß Phosphorylation and ß-Catenin Accumulation Through RhoA/ROCK.

In canonical pathway, Wnt3A has been known to stabilize ß-catenin through the dissociation between ß-catenin and glycogen synthase kinase-3ß (GSK-3ß) that suppresses the phosphorylation and degradation of ß-catenin. In non-canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross-talk between canonical and non-canonical pathways by Wnt signaling; however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK-3ß and accumulation of ß-catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh-RhoA and Tat-C3 abolished both the phosphorylation of GSK-3ß and accumulation of ß-catenin. Y27632, an inhibitor of Rho-associated coiled coil kinase (ROCK) and si-ROCK inhibited both GSK-3ß phosphorylation and ß-catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK-3ß in vitro. In addition, Wnt3A-induced cell proliferation and migration, which were inhibited by Tat-C3 and Y27632. Taken together, we propose the cross-talk between canonical and non-canonical signaling pathways of Wnt3A, which induces GSK-3ß phosphorylation and ß-catenin accumulation through RhoA and ROCK activation. J. Cell. Physiol. 232: 1104-1113, 2017. © 2016 Wiley Periodicals, Inc.

Mechanism underlying the suppressor activity of retinoic acid on IL4-induced IgE synthesis and its physiological implication.

The present study extends an earlier report that retinoic acid (RA) down-regulates IgE Ab synthesis in vitro. Here, we show the suppressive activity of RA on IgE production in vivo and its underlying mechanisms. We found that RA down-regulated IgE class switching recombination (CSR) mainly through RA receptor α (RARα). Additionally, RA inhibited histone acetylation of germ-line ε (GL ε) promoter, leading to suppression of IgE CSR. Consistently, serum IgE levels were substantially elevated in vitamin A-deficient (VAD) mice and this was more dramatic in VAD-lecithin:retinol acyltransferase deficient (LRAT-/-) mice. Further, serum mouse mast cell protease-1 (mMCP-1) level was elevated while frequency of intestinal regulatory T cells (Tregs) were diminished in VAD LRAT-/- mice, reflecting that deprivation of RA leads to allergic immune response. Taken together, our results reveal that RA has an IgE-repressive activity in vivo, which may ameliorate IgE-mediated allergic disease.

Commensal bacteria protect against food allergen sensitization.

Environmentally induced alterations in the commensal microbiota have been implicated in the increasing prevalence of food allergy. We show here that sensitization to a food allergen is increased in mice that have been treated with antibiotics or are devoid of a commensal microbiota. By selectively colonizing gnotobiotic mice, we demonstrate that the allergy-protective capacity is conferred by a Clostridia-containing microbiota. Microarray analysis of intestinal epithelial cells from gnotobiotic mice revealed a previously unidentified mechanism by which Clostridia regulate innate lymphoid cell function and intestinal epithelial permeability to protect against allergen sensitization. Our findings will inform the development of novel approaches to prevent or treat food allergy based on modulating the composition of the intestinal microbiota.

APRIL stimulates NF-κB-mediated HoxC4 induction for AID expression in mouse B cells.

Activation-induced cytidine deaminase (AID) plays a key role in B cell immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM). We have previously reported that the highly conserved homeodomain HoxC4 transcription factor binds to the Aicda (AID gene) promoter to induce AID expression. Here, we investigated the regulation of HoxC4 transcription by a proliferation-inducing ligand (APRIL) and B cell-activating factor belonging to the TNF family (BAFF) in mouse B cells. APRIL substantially increased both HoxC4 and AID expression, whereas BAFF induced the expression of AID but not HoxC4. To elucidate the underlying mechanisms, we constructed a HoxC4 gene promoter reporter vector and analyzed the promoter induction after APRIL stimulation. APRIL enhanced the HoxC4 promoter activity by 2.3-fold, and this increase disappeared when the second putative NF-κB-binding promoter element (NBE2) was mutated. Based on ChIP assays, we found that NF-κB bound to the HoxC4 promoter NBE2 region. Furthermore, the overexpression of NF-κB augmented the APRIL-induced HoxC4 promoter activity, while the expression of dominant negative-IκBα suppressed it. Taken together, our findings suggest that NF-κB mediates APRIL-induced HoxC4 transcription.

Intravital Imaging of Intestinal Intraepithelial Lymphocytes.

Intestinal intraepithelial lymphocytes (IEL) are a numerous population of T cells located within the epithelium of the small and large intestines, being more numerous in the small intestine (SI). They surveil this tissue by interacting with epithelial cells. Intravital microscopy is an important tool for visualizing the patrolling activity of IEL in the SI of live mice. Most IEL express CD8α; therefore, here we describe an established protocol of intravital imaging that tracks lymphocytes labeled with a CD8α-specific monoclonal antibody in the SI epithelium of live mice. We also describe data acquisition and quantification of the movement metrics, including mean speed, track length, displacement length, and paths for each CD8α+ IEL using the available software. The intravital imaging technique for measuring IEL movement will provide a better understanding of the role of IEL in homeostasis and protection from injury or infection in vivo.

Transcriptomes and metabolism define mouse and human MAIT cell populations.

Mucosal-associated invariant T (MAIT) cells are a subset of T lymphocytes that respond to microbial metabolites. We defined MAIT cell populations in different organs and characterized the developmental pathway of mouse and human MAIT cells in the thymus using single-cell RNA sequencing and phenotypic and metabolic analyses. We showed that the predominant mouse subset, which produced IL-17 (MAIT17), and the subset that produced IFN-γ (MAIT1) had not only greatly different transcriptomes but also different metabolic states. MAIT17 cells in different organs exhibited increased lipid uptake, lipid storage, and mitochondrial potential compared with MAIT1 cells. All these properties were similar in the thymus and likely acquired there. Human MAIT cells in lung and blood were more homogeneous but still differed between tissues. Human MAIT cells had increased fatty acid uptake and lipid storage in blood and lung, similar to human CD8 T resident memory cells, but unlike mouse MAIT17 cells, they lacked increased mitochondrial potential. Although mouse and human MAIT cell transcriptomes showed similarities for immature cells in the thymus, they diverged more strikingly in the periphery. Analysis of pet store mice demonstrated decreased lung MAIT17 cells in these so-called "dirty" mice, indicative of an environmental influence on MAIT cell subsets and function.

Hypoxia induces adrenomedullin from lung epithelia, stimulating ILC2 inflammation and immunity.

Hypoxia contributes to airway inflammation and remodeling in several lung diseases; however, exactly how hypoxic pulmonary epithelium regulates allergic inflammation remains to be fully characterized. Here, we report that conditional deletion of the E3 ubiquitin ligase VHL in lung epithelial cells resulted in exacerbated type 2 responses accompanied by selective increase of group 2 innate lymphoid cells (ILC2s) at steady state and following inflammation or helminth infection. Ablation of expression of the hypoxia-inducible factor 2α (HIF2α) significantly reversed VHL-mediated ILC2 activation. VHL deficiency in lung epithelial cells caused increased expression of the peptide hormone adrenomedullin (ADM), and our data suggest that HIF2α controls Adm expression. ADM directly promoted ILC2 activation both in vitro and in vivo. Our findings indicate that the hypoxic response mediated by the VHL-HIF2α axis is critical for control of pulmonary type 2 responses by increasing ADM expression in lung epithelia, causing ILC2 activation.

Epithelial HVEM maintains intraepithelial T cell survival and contributes to host protection.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to ß1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on ß1 integrin binding to collagen IV and showed that ß1 integrin-collagen IV interactions supported IET survival in vitro. Moreover, the absence of ß1 integrin expression by T lymphocytes decreased TCR αß+ IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or ß1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged ß1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

iNOS potentiates mouse Ig isotype switching through AID expression.

The IgA antibody plays an important role in protecting mucosal surfaces against pathogens. It has recently been shown that nitric oxide (NO) plays a critical role in mouse IgA synthesis. In the present study, we further characterized inducible-nitric oxide synthase-deficient (iNOS(-/-)) mice in the context of Ig expression. The amount of IgA in fecal pellets was substantially diminished in iNOS(-/-) mice and was paralleled by a decrease in IgA production by Peyer's patch cells. Interestingly, the amount of all IgG subisotypes, as well as IgA, was substantially diminished in sera and in cultured spleen B cells from iNOS(-/-) mice. Moreover, the synthesis of TGF-ß1-inducible IgA and IgG2b in iNOS(-/-) mice was also lower than that in WT mice. However, levels of Ig germ-line transcripts, and expression of TGF-ß receptor type II (TßRII) and BAFF/APRIL, were comparable between iNOS(-/-) and WT mice. Expression of activation-induced cytidine deaminase (AID) was diminished in iNOS(-/-) B cells, but restored by a NO donor, SNAP. These results indicate that iNOS regulates Ig isotype switching events at the level of AID gene expression.

Transcriptome and chromatin landscape of iNKT cells are shaped by subset differentiation and antigen exposure.

Invariant natural killer T cells (iNKT cells) differentiate into thymic and peripheral NKT1, NKT2 and NKT17 subsets. Here we use RNA-seq and ATAC-seq analyses and show iNKT subsets are similar, regardless of tissue location. Lung iNKT cell subsets possess the most distinct location-specific features, shared with other innate lymphocytes in the lung, possibly consistent with increased activation. Following antigenic stimulation, iNKT cells undergo chromatin and transcriptional changes delineating two populations: one similar to follicular helper T cells and the other NK or effector like. Phenotypic analysis indicates these changes are observed long-term, suggesting that iNKT cells gene programs are not fixed, but they are capable of chromatin remodeling after antigen to give rise to additional subsets.

HVEM structures and mutants reveal distinct functions of binding to LIGHT and BTLA/CD160.

HVEM is a TNF (tumor necrosis factor) receptor contributing to a broad range of immune functions involving diverse cell types. It interacts with a TNF ligand, LIGHT, and immunoglobulin (Ig) superfamily members BTLA and CD160. Assessing the functional impact of HVEM binding to specific ligands in different settings has been complicated by the multiple interactions of HVEM and HVEM binding partners. To dissect the molecular basis for multiple functions, we determined crystal structures that reveal the distinct HVEM surfaces that engage LIGHT or BTLA/CD160, including the human HVEM-LIGHT-CD160 ternary complex, with HVEM interacting simultaneously with both binding partners. Based on these structures, we generated mouse HVEM mutants that selectively recognized either the TNF or Ig ligands in vitro. Knockin mice expressing these muteins maintain expression of all the proteins in the HVEM network, yet they demonstrate selective functions for LIGHT in the clearance of bacteria in the intestine and for the Ig ligands in the amelioration of liver inflammation.

Analyses of TGF-beta1-inducible Ig germ-line gamma2b promoter activity: involvement of Smads and NF-kappaB.

TGF-beta1 directs class switch recombination to IgG2b as well as IgA. We have shown that Smad3/4, Runx3, and p300 mediate TGF-beta1-induced germ-line (GL) gamma2b transcription and that there is a potential Smad-binding element (SBE, CAGAC, -38/-34) and Runx-binding element (TGTGGGT, +41/+47) in the promoter region. Here, we have characterized more putative transcription factor-binding elements in the promoter. Site-directed mutagenesis revealed that two more putative SBE (GTCTG, -67/-63 and +38/+42) are relevant to TGF-beta1-induced GLgamma2b promoter activity, a finding that was confirmed by EMSA. However, neither overexpression of Ets (i.e. Elf-1, Fli-1, or Pu.1) nor a mutation deleting a putative Ets-binding element (CAGGAA, -4/+2) affected basal or TGF-beta1-induced promoter activity. On the other hand, NF-kappaB repressed promoter activity without direct binding to two putative NF-kappaB-binding elements (GGACTCCCC, -63/-55; GGGCCTTTCC,+237/+246). Instead, NF-kappaB overexpression increased the expression of Smad7 transcripts. Moreover, p300 overexpression failed to rescue the inhibitory effect of NF-kappaB on GLgamma2b promoter activity. These results indicate that there are multiple SBE relevant to GLgamma2b promoter activity and that NF-kappaB acts in cooperation with p300 to downregulate promoter activity through increasing the gene expression of inhibitory Smad7.

Bacterial Infection Allows for Functional Examination of Adoptively Transferred Mouse Innate Lymphoid Cell Subsets.

Innate lymphoid cells (ILCs) are important regulators of the early responses to infection at mucosal barriers, including the intestine. Recently, we have shown that specific ILC3 subsets protect against enteric bacterial pathogens. Here, we describe a mouse model of oral infection by Yersinia enterocolitica (Y. enterocolitica) and several different methodologies to assess the severity of the infection. We also detail how ILC3 subsets can be isolated from the mouse small intestine and transferred into recipient immune deficient mice to study the function of these ILCs in the small intestine.

The role of innate lymphoid cells in response to microbes at mucosal surfaces.

Innate lymphoid cells (ILCs) are a lymphocyte population that is mostly resident at mucosal surfaces. They help to induce an appropriate immune response to the microbiome at homeostasis. In healthy people, the mucosal immune system works symbiotically with organisms that make up the microbiota. ILCs play a critical role in orchestrating this balance, as they can both influence and in turn be influenced by the microbiome. ILCs also are important regulators of the early response to infections by diverse types of pathogenic microbes at mucosal barriers. Their rapid responses initiate inflammatory programs, production of antimicrobial products and repair processes. This review will focus on the role of ILCs in response to the microbiota and to microbial infections of the lung and intestine.

TGF-beta1 and IFN-gamma stimulate mouse macrophages to express BAFF via different signaling pathways.

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF-beta1 and IFN-gamma induce BAFF expression to see if TGF-beta1 and IFN-gamma regulate B cell differentiation via macrophages. We found that TGF-beta1 stimulated mouse macrophages to express BAFF and that a typical TGF-beta signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad-binding elements. Importantly, TGF-beta1 enhanced the expression of membrane-bound and soluble forms of BAFF. IFN-gamma further augmented TGF-beta1-induced BAFF expression. IFN-gamma caused phosphorylation of CREB, and overexpression of CREB increased IFN-gamma-induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1alpha (a well-known transducing molecule of IFN-gamma) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN-gamma. Taken together, these results demonstrate that TGF-beta1 and IFN-gamma up-regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

The protein tyrosine kinase SYK regulates the alternative p38 activation in liver during acute liver inflammation.

Two distinct p38 signaling pathways, classical and alternative, have been identified to regulate inflammatory responses in host defense and disease development. The role of alternative p38 activation in liver inflammation is elusive, while classical p38 signaling in hepatocytes plays a role in regulating the induction of cell death in autoimmune-mediated acute liver injury. In this study, we found that a mutation of alternative p38 in mice augmented the severity of acute liver inflammation. Moreover, TNF-induced hepatocyte death was augmented by a mutation of alternative p38, suggesting that alternative p38 signaling in hepatocytes contributed more significantly to the pathology of acute liver injury. Furthermore, SYK-Vav-1 signaling regulates alternative p38 activation and the downregulation of cell death in hepatocytes. Therefore, it is suggested that alternative p38 signaling in the liver plays a critical role in the induction and subsequent pathological changes of acute liver injury. Collectively, our results imply that p38 signaling in hepatocytes plays a crucial role to prevent excessive liver injury by regulating the induction of cell death and inflammation.

Activin A stimulates IgA expression in mouse B cells.

In this study, a potential role for activin A in mouse immunoglobulin (Ig) regulation was investigated. We observed that activin A increased IgA secretion in B lymphoma cells. In contrast, little effect was observed on IgM and IgG2b secretion. Activin A also significantly increased surface IgA expression and Ig germ-line alpha transcript (GLT(alpha)) levels. In parallel, activin A increased GLT(alpha) and post-switch transcripts alpha (PST(alpha)) expression in normal B cells, which was augmented by IL-5. An increase in IgA production by surface IgA negative B cells by activin A was apparent. Finally, the increase of IgA secretion by activin A was blocked by an activin receptor inhibitor (SB431542). The increase of GLT(alpha) by activin A was augmented by Smad3/4 overexpression and abolished by Smad3 dominant negative overexpression. These results suggest that activin A induces IgA isotype switching via Smad3/4-mediated germ line alpha transcription.

Mechanisms underlying TGF-beta1-induced expression of VEGF and Flk-1 in mouse macrophages and their implications for angiogenesis.

TGF-beta induces vascular endothelial growth factor (VEGF), a potent angiogenic factor, at the transcriptional and protein levels in mouse macrophages. VEGF secretion in response to TGF-beta1 is enhanced by hypoxia and by overexpression of Smad3/4 and hypoxia-inducible factor-1alpha/beta (HIF-1alpha/beta). To examine the transcriptional regulation of VEGF by TGF-beta1, we constructed mouse reporters driven by the VEGF promoter. Overexpression of HIF-1alpha/beta or Smad3/4 caused a slight increase of VEGF promoter activity in the presence of TGF-beta1, whereas cotransfection of HIF-1alpha/beta and Smad3/4 had a marked effect. Smad2 was without effect on this promoter activity, whereas Smad7 markedly reduced it. Analysis of mutant promoters revealed that the one putative HIF-1 and two Smad-binding elements were critical for TGF-beta1-induced VEGF promoter activity. The relevance of these elements was confirmed by chromatin immunoprecipitation assay. p300, which has histone acetyltransferase activity, augmented transcriptional activity in response to HIF-1alpha/beta and Smad3/4, and E1A, an inhibitor of p300, inhibited it. TGF-beta1 also increased the expression of fetal liver kinase-1 (Flk-1), a major VEGF receptor, and TGF-beta1 and VEGF stimulated pro-matrix metalloproteinase 9 (MMP-9) and active-MMP-9 expression, respectively. The results from the present study indicate that TGF-beta1 can activate mouse macrophages to express angiogenic mediators such as VEGF, MMP-9, and Flk-1.

Murine γδ T Cells Render B Cells Refractory to Commitment of IgA Isotype Switching.

γδ T cells are abundant in the gut mucosa and play an important role in adaptive immunity as well as innate immunity. Although γδ T cells are supposed to be associated with the enhancement of Ab production, the status of γδ T cells, particularly in the synthesis of IgA isotype, remains unclear. We compared Ig expression in T cell receptor delta chain deficient (TCRδ-/-) mice with wild-type mice. The amount of IgA in fecal pellets was substantially elevated in TCRδ-/- mice. This was paralleled by an increase in surface IgA expression and total IgA production by Peyer's patches (PPs) and mesenteric lymph node (MLN) cells. Likewise, the TCRδ-/- mice produced much higher levels of serum IgA isotype. Here, surface IgA expression and number of IgA secreting cells were also elevated in the culture of spleen and bone marrow (BM) B cells. Germ-line α transcript, an indicator of IgA class switch recombination, higher in PP and MLN B cells from TCRδ-/- mice, while it was not seen in inactivated B cells. Nevertheless, the frequency of IgA+ B cells was much higher in the spleen from TCRδ-/- mice. These results suggest that γδ T cells control the early phase of B cells, in order to prevent unnecessary IgA isotype switching. Furthermore, this regulatory role of γδ T cells had lasting effects on the long-lived IgA-producing plasma cells in the BM.