Human small intestine contains 2 functionally distinct regulatory T-cell subsets.

BACKGROUND: Regulatory T (Treg) CD4 cells in mouse gut are mainly specific for intestinal antigens and play an important role in the suppression of immune responses against harmless dietary antigens and members of the microbiota. However, information about the phenotype and function of Treg cells in the human gut is limited. OBJECTIVE: We performed a detailed characterization of Foxp3+ CD4 Treg cells in human normal small intestine (SI) as well as from transplanted duodenum and celiac disease lesions. METHODS: Treg cells and conventional CD4 T cells derived from SI were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines assessed. RESULTS: SI Foxp3+ CD4 T cells were CD45RA-CD127-CTLA-4+ and suppressed proliferation of autologous T cells. Approximately 60% of Treg cells expressed the transcription factor Helios. When stimulated, Helios-negative Treg cells produced IL-17, IFN-γ, and IL-10, whereas Helios-positive Treg cells produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum, we demonstrated that donor Helios-negative Treg cells persisted for at least 1 year after transplantation. In normal SI, Foxp3+ Treg cells constituted only 2% of all CD4 T cells, while in active celiac disease, both Helios-negative and Helios-positive subsets expanded 5- to 10-fold. CONCLUSION: The SI contains 2 subsets of Treg cells with different phenotypes and functional capacities. Both subsets are scarce in healthy gut but increase dramatically in active celiac disease.

Origin of langerin (CD207)-expressing antigen presenting cells in the normal oral mucosa and in oral lichen planus lesions.

The number of langerin-expressing antigen-presenting cells is higher in oral lichen planus than in normal oral mucosa. However, langerin may be expressed by several functionally different lineages of antigen presenting cells (APCs), and this has important implications for our understanding of the pathogenesis of oral lichen planus. The aim of this study was to determine the origin of the langerin-expressing APCs. To this end, we examined oral mucosal biopsies from healthy persons and patients with oral lichen planus using multicolor immunofluorescence. In normal oral mucosa, a substantial fraction of Langerhans cells expressed Ki-67, indicating that steady-state oral mucosal Langerhans cells are at least partially maintained by self-renewal. In oral lichen planus, the numbers of Langerhans cells were higher but proliferation was not altered, indicating that the higher cell numbers appeared to depend on recruited dendritic cell (DC)-precursors. Moreover, we found a markedly higher number of langerin+ APCs within the lamina propria of oral lichen planus lesions. Such cells did not display monocyte- or macrophage markers, but rather showed a phenotype compatible with tissue-elicited IRF4+ cDC2. Detailed understanding of how the oral mucosal APC network is regulated and the functional capacities of the different ontogenies may identify novel treatment targets for oral lichen planus.

Expression and function of resolvin RvD1n-3 DPA receptors in oral epithelial cells.

Chronic inflammatory responses can inflict permanent damage to host tissues. Specialized pro-resolving mediators downregulate inflammation but also can have other functions. The aim of this study was to examine whether oral epithelial cells express the receptors FPR2/ALX and DRV1/GPR32, which bind RvD1n-3 DPA , a recently described pro-resolving mediator derived from omega-3 docosapentaenoic acid (DPA), and whether RvD1n-3 DPA exposure induced significant responses in these cells. Gingival biopsies were stained using antibodies to FPR2/ALX and DRV1/GPR32. Expression of FPR2/ALX and DRV1/GPR32 was examined in primary oral epithelial cells by qRT-PCR, flow cytometry, and immunofluorescence. The effect of RvD1n-3 DPA on intracellular calcium mobilization and transcription of beta-defensins 1 and 2, and cathelicidin was evaluated by qRT-PCR. FPR2/ALX and DRV1/GPR32 were expressed by gingival keratinocytes in situ. In cultured oral epithelial cells, FPR2/ALX was detected on the cell surface, whereas FPR2/ALX and DRV1/GPR32 were detected intracellularly. Exposure to RvD1n-3 DPA induced intracellular calcium mobilization, FPR2/ALX internalization, DRV1/GPR32 translocation to the nucleus, and significantly increased expression of genes coding for beta-defensin 1, beta-defensin 2, and cathelicidin. This shows that the signal constituted by RvD1n-3 DPA is recognized by oral keratinocytes and that this can strengthen the antimicrobial and regulatory potential of the oral epithelium.

RvD1n-3 DPA Downregulates the Transcription of Pro-Inflammatory Genes in Oral Epithelial Cells and Reverses Nuclear Translocation of Transcription Factor p65 after TNF-α Stimulation.

Specialized pro-resolving mediators (SPMs) are multifunctional lipid mediators that participate in the resolution of inflammation. We have recently described that oral epithelial cells (OECs) express receptors of the SPM resolvin RvD1n-3 DPA and that cultured OECs respond to RvD1n-3 DPA addition by intracellular calcium release, nuclear receptor translocation and transcription of genes coding for antimicrobial peptides. The aim of the present study was to assess the functional outcome of RvD1n-3 DPA-signaling in OECs under inflammatory conditions. To this end, we performed transcriptomic analyses of TNF-α-stimulated cells that were subsequently treated with RvD1n-3 DPA and found significant downregulation of pro-inflammatory nuclear factor kappa B (NF-κB) target genes. Further bioinformatics analyses showed that RvD1n-3 DPA inhibited the expression of several genes involved in the NF-κB activation pathway. Confocal microscopy revealed that addition of RvD1n-3 DPA to OECs reversed TNF-α-induced nuclear translocation of NF-κB p65. Co-treatment of the cells with the exportin 1 inhibitor leptomycin B indicated that RvD1n-3 DPA increases nuclear export of p65. Taken together, our observations suggest that SPMs also have the potential to be used as a therapeutic aid when inflammation is established.

Do Long-Lived Plasma Cells Maintain a Healthy Microbiota in the Gut?

Disruptions to the gut microbiota have been associated with a variety of diseases. Understanding the underlying mechanisms that regulate the maintenance of a healthy microbiota may therefore have therapeutic implications. Secretory IgA play a unique role in immune-microbiota crosstalk by directly binding to bacteria in the gut lumen. Microbe-specific IgA responses co-develop with the assembly of the gut microbiota during infancy, and resemble those of adults by 2 years postnatally in the healthy host. We propose here that microbiota-specific IgA-producing gut plasma cells generated during infancy live for many decades and contribute to a stable microbiota community. We furthermore suggest that members of the microbiota that induce long-lasting IgA responses in the gut are putative targets for therapeutic interventions.

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates.

In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.

Targeting Influenza Virus Hemagglutinin to Xcr1+ Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

Targeting Ags to conventional dendritic cells can enhance Ag-specific immune responses. Although most studies have focused on the induction of T cell responses, the mechanisms by which targeting improves Ab responses are poorly understood. In this study we present data on the use of human XCL1 (hXCL1) and hXCL2 fusion vaccines in a murine model. We show that the human chemokines bound type 1 conventional dendritic cells (cDC1), and that immunization with influenza virus hemagglutinin fused to hXCL1 or hXCL2 induced full protection against influenza challenge. Surprisingly, the hXCL1- and hXCL2-fusion vaccines induced better long-term protection associated with stronger induction of neutralizing Abs, and more Ab-secreting cells in bone marrow. In contrast, murine Xcl1 fusion vaccines induced stronger CD8+ T cell responses compared with hXCL1. Further analysis revealed that although murine Xcl1 fusion vaccines induced chemotaxis and were rapidly endocytosed by cDC1, hXCL1 and hXCL2 fusion vaccines did not induce chemotaxis, were less efficiently endocytosed, and consequently, remained on the surface. This difference may explain the enhanced induction of Abs when targeting Ag to cDC1 using hXCL1 and hXCL2, and suggests that immune responses can be manipulated in directing Abs or T cells based on how efficiently the targeted Ag is endocytosed by the DC.

Monocytes accumulate in the airways of children with fatal asthma.

BACKGROUND: Activated T helper type 2 (Th2) cells are believed to play a pivotal role in allergic airway inflammation, but which cells attract and activate Th2 cells locally have not been fully determined. Recently, it was shown in an experimental human model of allergic rhinitis (AR) that activated monocytes rapidly accumulate in the nasal mucosa after local allergen challenge, where they promote recruitment of Th2 cells and eosinophils. OBJECTIVE: To investigate whether monocytes are recruited to the lungs in paediatric asthma. METHODS: Tissue samples obtained from children and adolescents with fatal asthma attack (n = 12), age-matched non-atopic controls (n = 9) and allergen-challenged AR patients (n = 8) were subjected to in situ immunostaining. RESULTS: Monocytes, identified as CD68+S100A8/A9+ cells, were significantly increased in the lower airway mucosa and in the alveoli of fatal asthma patients compared with control individuals. Interestingly, cellular aggregates containing CD68+S100A8/A9+ monocytes obstructing the lumen of bronchioles were found in asthmatics (8 out of 12) but not in controls. Analysing tissue specimens from challenged AR patients, we confirmed that co-staining with CD68 and S100A8/A9 was a valid method to identify recently recruited monocytes. We also showed that the vast majority of accumulating monocytes both in the lungs and in the nasal mucosa expressed matrix metalloproteinase 10, suggesting that this protein may be involved in their migration within the tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Monocytes accumulated in the lungs of children and adolescents with fatal asthma attack. This finding strongly suggests that monocytes are directly involved in the immunopathology of asthma and that these pro-inflammatory cells are potential targets for therapy.

Rapid recruitment of CD14(+) monocytes in experimentally induced allergic rhinitis in human subjects.

BACKGROUND: Activated TH2 cells and eosinophils are hallmarks of the allergic inflammation seen in patients with allergic rhinitis (AR). However, which cells activate and attract T cells and eosinophils to the inflammatory lesion has not been determined. OBJECTIVE: We wanted to assess the role of mucosal mononuclear phagocytes, consisting of monocytes, macrophages, and dendritic cells, in the local allergic inflammatory reaction. METHODS: Patients with AR and nonatopic control subjects were challenged with pollen extract, and nasal symptoms were recorded. Mucosal biopsy specimens obtained at different time points before and after challenge were used for immunostaining in situ and flow cytometric cell sorting. Sorted mononuclear phagocytes were subjected to RNA extraction and gene expression profiling. RESULTS: In an in vivo model of AR, we found that CD14(+) monocytes were recruited to the nasal mucosa within hours after local allergen challenge, whereas conventional dendritic cells accumulated after several days of continued provocation. Transcriptomic profiling of mucosal mononuclear phagocytes sorted after 1 week of continued allergen challenge showed an activated phenotype at least partially driven by IL-4 signaling, IL-13 signaling, or both. Importantly, gene expression of several TH2-related chemokines was significantly upregulated by the mononuclear phagocyte population concomitant with an increased recruitment of CD4(+) T cells and eosinophils. CONCLUSION: Our findings suggest that the mononuclear phagocyte population is directly involved in the production of proinflammatory chemokines that attract other immune cells. Rapid recruitment of CD14(+) monocytes to the challenged site indicates that these proinflammatory mononuclear phagocytes have a central role in orchestrating local allergic inflammation.

Long-term persistence of human donor alveolar macrophages in lung transplant recipients.

BACKGROUND: Alveolar macrophages (AMFs) are critical regulators of lung function, and may participate in graft rejection following lung transplantation. Recent studies in experimental animals suggest that most AMFs are self-maintaining cells of embryonic origin, but knowledge about the ontogeny and life span of human AMFs is scarce. METHODS: To follow the origin and longevity of AMFs in patients with lung transplantation for more than 100 weeks, we obtained transbronchial biopsies from 10 gender-mismatched patients with lung transplantation. These were subjected to combined in situ hybridisation for X/Y chromosomes and immunofluorescence staining for macrophage markers. Moreover, development of AMFs in humanised mice reconstituted with CD34+ umbilical cord-derived cells was assessed. RESULTS: The number of donor-derived AMFs was unchanged during the 2 year post-transplantation period. A fraction of the AMFs proliferated locally, demonstrating that at least a subset of human AMFs have the capacity to self-renew. Lungs of humanised mice were found to abundantly contain populations of human AMFs expressing markers compatible with a monocyte origin. Moreover, in patients with lung transplantation we found that recipient monocytes seeded the alveoli early after transplantation, and showed subsequent phenotypical changes consistent with differentiation into proliferating mature AMFs. This resulted in a stable mixed chimerism between donor and recipient AMFs throughout the 2-year period. CONCLUSIONS: The finding that human AMFs are maintained in the lung parenchyma for several years indicates that pulmonary macrophage transplantation can be a feasible therapeutic option for patients with diseases caused by dysfunctional AMFs. Moreover, in a lung transplantation setting, long-term persistence of donor AMFs may be important for the development of chronic graft rejection.