Gram-Positive Bacteria Cell Wall Peptidoglycan Polymers Activate Human Dendritic Cells to Produce IL-23 and IL-1ß and Promote TH17 Cell Differentiation.

Gram-positive bacterial infections are a major cause of organ failure and mortality in sepsis. Cell wall peptidoglycan (PGN) is shed during bacterial replication, and Bacillus anthracis PGN promotes a sepsis-like pathology in baboons. Herein, we determined the ability of polymeric Bacillus anthracis PGN free from TLR ligands to shape human dendritic cell (DC) responses that are important for the initiation of T cell immunity. Monocyte-derived DCs from healthy donors were incubated with PGN polymers isolated from Bacillus anthracis and Staphylococcus aureus. PGN activated the human DCs, as judged by the increased expression of surface HLA-DR, CD83, the T cell costimulatory molecules CD40 and CD86, and the chemokine receptor CCR7. PGN elicited the DC production of IL-23, IL-6, and IL-1ß but not IL-12p70. The PGN-stimulated DCs induced the differentiation of naïve allogeneic CD4+ T cells into T helper (TH) cells producing IL-17 and IL-21. Notably, the DCs from a subset of donors did not produce significant levels of IL-23 and IL-1ß upon PGN stimulation, suggesting that common polymorphisms in immune response genes regulate the PGN response. In sum, purified PGN is a highly stimulatory cell wall component that activates human DCs to secrete proinflammatory cytokines and promote the differentiation of TH17 cells that are important for neutrophil recruitment in extracellular bacterial infections.

ILC Differentiation in the Thymus.

The thymus provides a microenvironment conducive to the differentiation of innate lymphoid cells (ILCs), supplying IL-7 as well as Notch ligands. Early T cell precursors also express a number of obligatory transcription factors essential for ILC differentiation. Therefore, the thymus could be a powerhouse for ILC production. However, coordinated regulation by transcription factors and T cell receptor signaling events ensure that T cell production is the dominating output of the thymus. One group of the key regulators are the basic helix-loop-helix E protein transcription factors and their inhibitors, Id proteins. When E protein activities are downregulated, T cell development is blocked and massive ILC2 production occurs in the thymus. Normally, the thymus indeed generates a small number of ILCs, mostly group 2 ILCs (ILC2s). It has been shown in vitro that ILC2s can be differentiated from multipotent early T cell progenitors (ETPs) as well as committed T cell precursors. Moreover, thymus-derived ILC precursors have been found in the blood of adult mice. They then home to peripheral tissues and undergo differentiation into distinct ILC subsets. These ILC precursors may replenish tissue ILC pools in steady state or on demand in pathophysiological conditions. Collectively, emerging evidence suggests that the thymus plays an underappreciated role in ILC homeostasis.

Correlation between circulating innate lymphoid cell precursors and thymic function.

The thymus has a high capacity to support the differentiation of ILCs, especially when E protein transcription factors are ablated. Whether it contributes to the homeostasis of ILC pools in tissues is not clear. Single-cell RNA sequencing analysis shows a substantial amount of ILC precursors in wild type but not athymic nude blood. The precursors express CD3 intracellularly (ic) but not on the surface. The abundance of Lin-CD127+CD62L+icCD3ε+ precursors varies with age, peaking at 2-3 months. These cells can differentiate into various ILC subsets on OP9-DL1 stroma in vitro. In the lung, small intestine, and epidermis, icCD3ε+ cells differentiate into diverse ILC subsets in different tissue environments in steady state. Helminth infection promotes their differentiation toward functional ILC2s. Thus, the thymus appears to play a role in replenishing ILC pools in different peripheral tissues. Because thymic activity is age-dependent, this finding may help explain age-related differences in immune responses.

Type 2 innate lymphoid cells from Id1 transgenic mice alleviate skin manifestations of graft-versus-host disease.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is one of the most common causes of morbidity for patients undergoing allogeneic stem cell transplantation. There is preliminary evidence that activated Group 2 innate lymphoid cells (ILC2s) from wild type (WT) mice reduces the lethality of aGVHD and is effective in treating lower gastrointestinal (GI) tract manifestations of aGVHD. This raises the prospect that ILC2s may be used for cell-based therapy of aGVHD but vigorous investigation is necessary to assess their impacts on different aspects of aGVHD. Genetically engineered mice which either express Id1 protein (Id1tg/tg), an inhibitor of E protein transcription factors or have E protein genes knocked out (dKO) in the thymus produce massive numbers of ILC2s, thus allowing extensive evaluation of ILC2s. We investigated whether these ILC2s have protective effects in aGVHD as WT ILC2s do using an established mouse model of aGVHD. RESULTS: bone marrow transplant was performed by irradiating BALB/c strain of recipient mice and transplanting with bone marrow and T cells from the MHC-disparate C57BL/6 strain. We isolated ILC2s from Id1tg/tg and dKO mice and co-transplanted them to study their effects. Our results confirm that activated ILC2s have a protective role in aGVHD, but the effects varied depending on the origin of ILC2s. Co-transplantation of ILC2s from Id1tg/tg mice were beneficial in aGVHD and are especially helpful in ameliorating the skin manifestations of aGVHD. However, ILC2s from dKO mice were less effective at the protection and behaved differently depending on if the cells were isolated from dKO mice were pre-treated with IL-25 in vivo. CONCLUSION: These findings support the notion that thymus-derived ILC2s from Id1tg/tg mice are protective against aGVHD, with a significant improvement of skin lesions and they behave differently from dKO mice in the setting of aGVHD.

Augmenting E Protein Activity Impairs cDC2 Differentiation at the Pre-cDC Stage.

Dendritic cell (DC) specification and differentiation are controlled by a circuit of transcription factors, which regulate the expression of DC effector genes as well as the transcription factors themselves. E proteins are a widely expressed basic helix-loop-helix family of transcription factors whose activity is suppressed by their inhibitors, ID proteins. Loss-of-function studies have demonstrated the essential role of both E and ID proteins in different aspects of DC development. In this study, we employed a gain-of-function approach to illustrate the importance of the temporal control of E protein function in maintaining balanced differentiation of conventional DC (cDC) subsets, cDC1 and cDC2. We expressed an E protein mutant, ET2, which dimerizes with endogenous E proteins to overcome inhibition by ID proteins and activate the transcription of E protein targets. Induction of ET2 expression at the hematopoietic progenitor stage led to a dramatic reduction in cDC2 precursors (pre-cDC2s) with little impact on pre-cDC1s. Consequently, we observed decreased numbers of cDC2s in the spleen and lung, as well as in FLT3L-driven bone marrow-derived DC cultures. Furthermore, in mice bearing ET2, we detected increased expression of the IRF8 transcription factor in cDC2s, in which IRF8 is normally down-regulated and IRF4 up-regulated. This aberrant expression of IRF8 induced by ET2 may contribute to the impairment of cDC2 differentiation. In addition, analyses of the transcriptomes of splenic cDC1s and cDC2s revealed that ET2 expression led to a shift, at least in part, of the transcriptional profile characteristic of cDC2s to that of cDC1. Together, these results suggest that a precise control of E protein activity is crucial for balanced DC differentiation.

Suppression of ILC2 differentiation from committed T cell precursors by E protein transcription factors.

Current models propose that group 2 innate lymphoid cells (ILC2s) are generated in the bone marrow. Here, we demonstrate that subsets of these cells can differentiate from multipotent progenitors and committed T cell precursors in the thymus, both in vivo and in vitro. These thymic ILC2s exit the thymus, circulate in the blood, and home to peripheral tissues. Ablation of E protein transcription factors greatly promotes the ILC fate while impairing B and T cell development. Consistently, a transcriptional network centered on the ZBTB16 transcription factor and IL-4 signaling pathway is highly up-regulated due to E protein deficiency. Our results show that ILC2 can still arise from what are normally considered to be committed T cell precursors, and that this alternative cell fate is restrained by high levels of E protein activity in these cells. Thymus-derived lung ILC2s of E protein-deficient mice show different transcriptomes, proliferative properties, and cytokine responses from wild-type counterparts, suggesting potentially distinct functions.

The effect of soluble E-selectin on tumor progression and metastasis.

BACKGROUND: Distant metastasis resulting from vascular dissemination of cancer cells is the primary cause of mortality from breast cancer. We have previously reported that E-selectin expression on the endothelial cell surface mediates shear-resistant adhesion and migration of circulating cancer cells via interaction with CD44. As a result of shedding, soluble E-selectin (sE-selectin) from the activated endothelium is present in the serum. In this study, we aimed to understand the role of sE-selectin in tumor progression and metastasis. METHODS: We investigated the effect of sE-selectin on shear-resistant adhesion and migration of metastatic breast cancer cells and leukocytes in vitro and in vivo. RESULTS: We found that sE-selectin promoted migration and shear-resistant adhesion of CD44(+) (/high) breast cancer cell lines (MDA-MB-231 and MDA-MB-468) to non-activated human microvessel endothelial cells (ES-HMVECs), but not of CD44(-/low) breast cancer cell lines (MCF-7 and T-47D). This endothelial E-selectin independent, sE-selectin-mediated shear-resistant adhesion was also observed in a leukocyte cell line (HL-60) as well as human peripheral blood mononuclear cells (PBMCs). Additionally, the incubation of MDA-MB-231 cells with sE-selectin triggered FAK phosphorylation and shear-resistant adhesion of sE-selectin-treated cells resulted in increased endothelial permeabilization. However, CD44 knockdown in MDA-MB-231 and HL-60 cells resulted in a significant reduction of sE-selectin-mediated shear-resistant adhesion to non-activated HMVECs, suggesting the involvement of CD44/FAK. Moreover, functional blockade of ICAM-1 in non-activated HMVECs resulted in a marked reduction of sE-selectin-mediated shear-resistant adhesion. Finally, the pre-incubation of CD44(+) 4 T1 murine breast cancer cells with sE-selectin augmented infiltration into the lung in E-selectin K/O mice and infusion of human PBMCs pre-incubated with sE-selectin stimulated MDA-MB-231 xenografted breast tumor growth in NSG mice. CONCLUSIONS: Our data suggest that circulating sE-selectin stimulates a broad range of circulating cells via CD44 and mediates pleiotropic effects that promote migration and shear-resistant adhesion in an endothelial E-selectin independent fashion, in turn accelerating tissue infiltration of leukocytes and cancer cells.

IRF4 and IRF8 Act in CD11c+ Cells To Regulate Terminal Differentiation of Lung Tissue Dendritic Cells.

Dendritic cells (DCs) initiate immune responses in barrier tissues including lung and skin. Conventional DC (cDC) subsets, CD11b(-) (cDC1s) or CD11b(+) (cDC2s), arise via distinct networks of transcription factors involving IFN regulatory factor 4 (IRF4) and IRF8, and are specialized for unique functional responses. Using mice in which a conditional Irf4 or Irf8 allele is deleted in CD11c(+) cells, we determined whether IRF4 or IRF8 deficiency beginning in CD11c(+) cDC precursors (pre-cDCs) changed the homeostasis of mature DCs or pre-DCs in the lung, dermis, and spleen. CD11c-cre-Irf4(-/-) mice selectively lacked a lung-resident CD11c(hi)CD11b(+)SIRPα(+)CD24(+) DC subset, but not other lung CD11b(+) DCs or alveolar macrophages. Numbers of CD11b(+)CD4(+) splenic DCs, but not CD11b(+) dermal DCs, were reduced, indicating cDC2s in the lung and dermis develop via different pathways. Irf4 deficiency did not alter numbers of cDC1s. CD11c-cre-Irf8(-/-) mice lacked lung-resident CD103(+) DCs and splenic CD8α(+) DCs, yet harbored increased IRF4-dependent DCs. This correlated with a reduced number of Irf8(-/-) pre-cDCs, which contained elevated IRF4, suggesting that Irf8 deficiency diverts pre-cDC fate. Analyses of Irf4 and Irf8 haploinsufficient mice showed that, although one Irf4 allele was sufficient for lung cDC2 development, two functional Irf8 alleles were required for differentiation of lung cDC1s. Thus, IRF8 and IRF4 act in pre-cDCs to direct the terminal differentiation of cDC1 and cDC2 subsets in the lung and spleen. These data suggest that variation in IRF4 or IRF8 levels resulting from genetic polymorphisms or environmental cues will govern tissue DC numbers and, therefore, regulate the magnitude of DC functional responses.

In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells.

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro. In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al., 2015).

Increased level of E protein activity during invariant NKT development promotes differentiation of invariant NKT2 and invariant NKT17 subsets.

E protein transcription factors and their natural inhibitors, Id proteins, play critical and complex roles during lymphoid development. In this article, we report that partial maintenance of E protein activity during positive selection results in a change in the cell fate determination of developing iNKT cells, with a block in the development of iNKT1 cells and a parallel increase in the iNKT2 and iNKT17 subsets. Because the expression levels of the transcription factors that drive these alternative functional fates (GATA-3, RORγT, T-bet, and Runx-3) are not altered, our results suggest that E protein activity controls a novel checkpoint that regulates the number of iNKT precursors that choose each fate.

IRF4 promotes cutaneous dendritic cell migration to lymph nodes during homeostasis and inflammation.

Migration of resident dendritic cells (DC) from the skin to local lymph nodes (LN) triggers T cell-mediated immune responses during cutaneous infection, autoimmune disease, and vaccination. In this study, we investigated whether the development and migration of skin-resident DC were regulated by IFN regulatory factor 4 (IRF4), a transcription factor that is required for the development of CD11b(+) splenic DC. We found that the skin of naive IRF4(-/-) mice contained normal numbers of epidermal Langerhans cells (eLC) and increased numbers of CD11b(+) and CD103(+) dermal DC (dDC) populations, indicating that tissue DC development and skin residency is not disrupted by IRF4 deficiency. In contrast, numbers of migratory eLC and CD11b(+) dDC were significantly reduced in the cutaneous LN of IRF4(-/-) mice, suggesting a defect in constitutive migration from the dermis during homeostasis. Upon induction of skin inflammation, CD11b(+) dDC in IRF4(-/-) mice did not express the chemokine receptor CCR7 and failed to migrate to cutaneous LN, whereas the migration of eLC was only mildly impaired. Thus, although dispensable for their development, IRF4 is crucial for the CCR7-mediated migration of CD11b(+) dDC, a predominant population in murine and human skin that plays a vital role in normal and pathogenic cutaneous immunity.

Tolerogenic dendritic cells generated with different immunosuppressive cytokines induce antigen-specific anergy and regulatory properties in memory CD4+ T cells.

Dendritic cells (DCs) are professional APCs involved in the initiation of both immunity and immunological tolerance. In autoimmune diseases or graft rejections, most reactive lymphocytes are effector/memory cells. It is believed that memory T cells are more resistant to tolerance induction than naive lymphocytes; however, studies on mechanisms for their efficient tolerization are still scarce. In this study, we generated human monocyte-derived DCs by culture with GM-CSF and IL-4 (control DCs), as well as tolerogenic DCs (tDCs) by adding IL-10, IL-10/TGF-beta1, or IL-10/IL-6. Cells were maturated with TNF-alpha/PGE(2). Compared with control DCs, tDCs had similar expression of HLA-DR, CD80, and CD86, lower expression of CD40, higher levels of macrophage markers, enhanced endocytic ability, increased secretion of IL-6, IL-10 (only tDCs generated with IL-10 and tDCs generated with IL-10/IL-6), and PGE(2), and lower secretion of IL-12 and IL-23. In vitro, tDCs had the capacity to induce anergy in tetanus toxoid-specific memory CD4(+) T cells, whereas the proliferative response to an unrelated Ag was intact. Anergy could be reverted upon exposure to IL-2. tDC-primed T cells have low suppressive ability. Nevertheless, the generation of both anergic and regulatory T cells was more efficient with tDCs generated with IL-10/TGF-beta1. Microarray-based gene expression profiling reflected modulated expression of several transcripts in tDCs. Surface CLIP-HLA-DR complexes and intracellular thrombospondin-1 were increased in the three tDCs. CD39 was highly expressed only in tDC-TGF, which correlated with increased adenosine production. We propose that these molecules, together with IL-10 and prostanoids, are key factors to induce Ag-specific tolerance in memory T cells.

Deficiency of type I IFN receptor in lupus-prone New Zealand mixed 2328 mice decreases dendritic cell numbers and activation and protects from disease.

Type I IFNs are potent regulators of innate and adaptive immunity and are implicated in the pathogenesis of systemic lupus erythematosus. Here we report that clinical and pathological lupus nephritis and serum anti-nuclear Ab levels are greatly attenuated in New Zealand Mixed (NZM) 2328 mice deficient in type I IFN receptors (IFNAR). To determine whether the inflammatory environment in NZM 2328 mice leads to IFNAR-regulated changes in dendritic cells (DC), the number, activation, and function of DC subsets were compared in 2- and 5-mo-old (clinically healthy) female NZM and NZM-IFNAR(-/-) mice. Numbers of activated CD40(high) plasmacytoid DC (pDC) were significantly increased in renal lymph nodes of 2-mo-old NZM but not NZM-IFNAR(-/-) mice, suggesting an early IFNAR-dependent expansion and activation of pDC at disease sites. Relative to NZM spleens, NZM-IFNAR(-/-) spleens in 5-mo-old mice were significantly decreased in size and contained reduced numbers of conventional DC subsets, but not pDC. Splenic and renal lymph node NZM-IFNAR(-/-) DC analyzed directly ex vivo expressed significantly less CD40, CD86, and PDL1 than did NZM DC. Upon activation with synthetic TLR9 ligands in vitro, splenic NZM-IFNAR(-/-) DC produced less IL-12p40/70 and TNF-alpha than did NZM DC. The limited IFNAR(-/-) DC response to endogenous activating stimuli correlated with reduced numbers of splenic activated memory CD4(+) T cells and CD19(+) B cells in older mice. Thus, IFNAR signaling significantly increases DC numbers, acquisition of Ag presentation competence, and proinflammatory function before onset of clinically apparent lupus disease.

Differential CD4(+) T-cell memory responses induced by two subsets of human monocyte-derived dendritic cells.

Dendritic cells (DC) are powerful inducers of primary T-cell responses, but their role in secondary responses has not been extensively analysed. Here, we address the role of two DC subsets derived from human CD16(+) (16(+) mDC) or CD16(-) (16(-) mDC) monocytes on the reactivation of memory responses. CD4(+) CD45RA(-) memory T cells were obtained from adult blood donors, and central (T(CM)) and effector (T(EM)) memory T cells were isolated by fluorescence-activated cell sorting with anti-CCR7 antibodies. The 16(+) mDC and 16(-) mDC were cocultured with autologous lymphocytes, either unpulsed or loaded with purified protein derivatives of Mycobacterium tuberculosis (PPD) or tetanus toxoid (TT), and were analysed for up to 8 days. Over a range of doses, 16(+) mDC drove stronger T-cell proliferative responses against both antigens. Overall, antigen-specific memory cells tended to acquire a phenotype of T(EM) at later time-points in the culture, whereas cells that had completed fewer cycles of division were similar to T(CM). The 16(+) mDC induced higher rates of proliferation on both T(CM) and T(EM) lymphocytes than 16(-) mDC. This phenomenon was not related to the ability of both DC to induce CD25 expression on T cells, to lower secretion of interleukin-2, or to raise production of interleukin-10 during T-cell/16(-) mDC cocultures. The induction of T(CM) effector capacity in terms of interferon-gamma production was faster and more pronounced with 16(+) mDC, whereas both DC had similar abilities with T(EM). In conclusion, these data might reveal new potentials in vaccination protocols with 16(+) mDC aimed at inducing strong responses on central memory T cells.

Lung squamous cell carcinoma and adenocarcinoma cell lines use different mediators to induce comparable phenotypic and functional changes in human monocyte-derived dendritic cells.

Tumor-derived immunosuppressive factors contribute to the evasion of malignant cells from the immune response, partially by hampering dendritic cell (DC) differentiation. Here, we analyze whether soluble mediators released by the most frequent histological types of non-small cell lung carcinoma, squamous cell carcinoma (SCC), and adenocarcinoma (AD) cells, affect the development and functionality of DC. Monocytes from healthy donors were differentiated in vitro into DC with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, in the absence or presence of soluble factors (SF) from SCC or AD cell lines. Monocytes were differentiated in parallel into macrophages (MPhi s) with macrophage colony-stimulating factor (M-CSF). SF-treated DC were phenotypically and functionally more similar to MPhi s than to untreated DC [control DC (Ctrl-DC)]. Both tumors increased myelomonocytic markers (CD14, CD16, CD32, and CD163) and impaired CD1a expression on DC. SF-treated DC increased their endocytic capacity, and released higher levels of IL-6, IL-10, and lower levels of IL-12, compared to Ctrl-DC. SF-treated DC were poor stimulators in mixed lymphocyte reactions, and naïve CD4(+) T lymphocytes stimulated by SF-treated DC secreted lower levels of interferon (IFN)-gamma and higher amounts of IL-10 than controls. In contrast to AD, the effects caused by SCC were mostly abolished by IL-6 neutralization during monocyte differentiation. However, tumor-derived prostanoid blockade recovered the IFN-gamma levels secreted by lymphocytes stimulated with SF-treated DC, whereas prostanoid/IL-6 or prostanoid/IL-10 blockade decreased IL-10 production only by SCC-DC-stimulated lymphocytes. Thus, we provide evidence that lung SCC and AD cause comparable deficiencies on DC in vitro, skewing monocyte differentiation from DC to MPhi -like cells, but most of these changes occurred via different mediators.

CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote similar allogeneic Th2 responses: regulation by pro- and anti-inflammatory cytokines.

We previously demonstrated that tumor necrosis factor (TNF)-alpha-matured CD16- and CD16+ human monocyte-derived dendritic cells (16-mDC and 16+mDC) differentially stimulate naive CD4+ lymphocytes by inducing Th1- and Th2-like responses, respectively. Here, we further characterized the role of different DC maturation factors on Th polarization. Immature 16+mDC and 16-mDC (iDC) obtained by culture of purified monocytes with GM-CSF and IL-4 were maturated with (i) Toll-like receptor (TLR) ligands [lipopolysaccharide (LPS)], (ii) lymphocyte-derived (soluble CD40 ligand, IFN-gamma) and (iii) endogenous inflammatory stimuli [TNF-alpha, prostaglandin (PG)E2]. After activation with these stimuli, DC secrete IL-12 only in presence of LPS, and 16+mDC produced lower amounts of IL-12 and IL-10 than 16-mDC. Allogeneic CD4+CD45RO- lymphocytes co-cultured with 16+mDC secreted higher levels of IL-4 and IL-10 than those co-cultured with 16-mDC, regardless of the maturation stimuli. Results were similar when DC were activated with TLR-2 or TLR-3 ligands. The higher induction of IL-4 by 16+mDC was primarily dependent on IL-12, IL-4 and IL-10. IFN-gamma production by CD4+ T cells was similar with all the conditions except with LPS-16+mDC, which induced reduced amounts of this cytokine. Those differences were totally eliminated by neutralization of IL-12, IL-4 or IL-10. Finally, 16-mDC could reverse the Th2 phenotype of already committed lymphocytes toward a Th1 pattern in short-term cultures, whereas 16+mDC had less ability to skew this phenotype. These results indicate that 16+mDC elicit superior Th2 responses independently of the maturation factors that they received, and suggest that they could represent an important population of regulatory DC.