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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 RvD1

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 RvD1

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.

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.

Macrophages are a heterogeneous population of cells involved in tissue homeostasis, inflammation, and cancer. Although macrophages are densely distributed throughout the human intestine, our understanding of how gut macrophages maintain tissue homeostasis is limited. Here we show that colonic lamina propria macrophages (LpMs) and muscularis macrophages (MMs) consist of monocyte-like cells that differentiate into multiple transcriptionally distinct subsets. LpMs comprise subsets with proinflammatory properties and subsets with high antigen-presenting and phagocytic capacity. The latter are strategically positioned close to the surface epithelium. Most MMs differentiate along two trajectories: one that upregulates genes associated with immune activation and angiogenesis, and one that upregulates genes associated with neuronal homeostasis. Importantly, MMs are located adjacent to neurons and vessels. Cell–cell interaction and gene network analysis indicated that survival, migration, transcriptional reprogramming, and niche-specific localization of LpMs and MMs are controlled by an extensive interaction with tissue-resident cells and a few key transcription factors.

International audience; 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.

Macrophages are a heterogeneous population of cells involved in tissue homeostasis, inflammation and cancer. Although macrophages are densely distributed throughout the human intestine, our understanding of how gut macrophages maintain tissue homeostasis is limited. Here we show that colonic lamina propria (LpM) and muscularis macrophages (MM) consist of monocyte-like cells that differentiate into multiple transcriptionally distinct subsets. LpM comprise subsets with proinflammatory properties and subsets high antigen presenting and phagocytic capacity. The latter are strategically positioned close to the surface epithelium. Most MM differentiate along two trajectories; one that upregulates genes associated with immune activation and angiogenesis, whereas the other upregulates genes associated with neuronal homeostasis. Importantly, MM are located adjacent to neurons and vessels. Cell-cell interaction and gene network analysis indicated that survival, migration, transcriptional reprogramming, and niche-specific localization of LpM and MM are controlled by an extensive interaction with tissue-resident cells and a few key transcription factors.

Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality in hematopoietic stem cell transplantation (HSCT). Donor T cells are key mediators in pathogenesis, but a contribution from host T cells has not been explored, as conditioning regimens are believed to deplete host T cells. To evaluate a potential role for host T cells in GVHD, the origin of skin and blood T cells was assessed prospectively in patients after HSCT in the absence of GVHD. While blood contained primarily donor-derived T cells, most T cells in the skin were host derived. We next examined patient skin, colon, and blood during acute GVHD. Host T cells were present in all skin and colon acute GVHD specimens studied, yet were largely absent in blood. We observed acute skin GVHD in the presence of 100% host T cells. Analysis demonstrated that a subset of host T cells in peripheral tissues were proliferating (Ki67(+)) and producing the proinflammatory cytokines IFN-γ and IL-17 in situ. Comparatively, the majority of antigen-presenting cells (APCs) in tissue in acute GVHD were donor derived, and donor-derived APCs were observed directly adjacent to host T cells. A humanized mouse model demonstrated that host skin-resident T cells could be activated by donor monocytes to generate a GVHD-like dermatitis. Thus, host tissue-resident T cells may play a previously unappreciated pathogenic role in acute GVHD.

BackgroundRegulatory CD4 T cells (Tregs) in the mice 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 Tregs in the human gut is limited.ObjectiveHere, we performed a detailed characterization of Foxp3+ CD4 Tregs in the human small intestine.MethodsTregs and conventional CD4 T cells derived from normal intestine as well as from transplanted duodenum and celiac disease lesions were subjected to extensive immunophenotyping and their suppressive activity and ability to produce cytokines were assessed.ResultsSmall intestinal Foxp3+ CD4 T cells were CD45RA- CD127- CTLA4+ and suppressed proliferation of autologous T cells. Approximately 60% of the Tregs expressed the transcription factor Helios. When stimulated, Helios- Tregs produced IL-17, IFN-γ and IL-10, whereas Helios+ Tregs produced very low levels of these cytokines. By sampling mucosal tissue from transplanted human duodenum we demonstrated that donor Helios+ Tregs have a short life span whereas Helios- Tregs persisted for at least 1 year post-transplantation. In normal small intestine, Foxp3+ Tregs constituted only 2% of all CD4 T cells, while in active celiac disease both subsets expanded 5-10-fold.ConclusionThe small intestine contains two subsets of Tregs with different functional capacities and very different life span. Both subsets are scarce in the normal situation but increase dramatically in a chronic inflammatory setting.

Studies in mice and humans have shown that CD8+ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4+ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4+ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4+ T cells were still donor-derived one year after transplantation. In contrast to memory CD4+ T cells in peripheral blood, intestinal CD4+ TRM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4+ TRM cells were very potent cytokine producers; the vast majority being polyfunctional TH1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4+ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4+ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

Needle-free uptake across mucosal barriers is a preferred route for delivery of biologics, but the efficiency of unassisted transmucosal transport is poor. To make administration and therapy efficient and convenient, strategies for the delivery of biologics must enhance both transcellular delivery and plasma half-life. We found that human albumin was transcytosed efficiently across polarized human epithelial cells by a mechanism that depends on the neonatal Fc receptor (FcRn). FcRn also transported immunoglobulin G, but twofold less than albumin. We therefore designed a human albumin variant, E505Q/T527M/K573P (QMP), with improved FcRn binding, resulting in enhanced transcellular transport upon intranasal delivery and extended plasma half-life of albumin in transgenic mice expressing human FcRn. When QMP was fused to recombinant activated coagulation factor VII, the half-life of the fusion molecule increased 3.6-fold compared with the wild-type human albumin fusion, without compromising the therapeutic properties of activated factor VII. Our findings highlight QMP as a suitable carrier of protein-based biologics that may enhance plasma half-life and delivery across mucosal barriers.

Studies in mice and humans have shown that CD8

Studies in mice and humans have shown that CD8+ T cell immunosurveillance in non-lymphoid tissues is dominated by resident populations. Whether CD4+ T cells use the same strategies to survey peripheral tissues is less clear. Here, examining the turnover of CD4+ T cells in transplanted duodenum in humans, we demonstrate that the majority of CD4+ T cells were still donor-derived one year after transplantation. In contrast to memory CD4+ T cells in peripheral blood, intestinal CD4+ TRM cells expressed CD69 and CD161, but only a minor fraction expressed CD103. Functionally, intestinal CD4+ TRM cells were very potent cytokine producers; the vast majority being polyfunctional TH1 cells, whereas a minor fraction produced IL-17. Interestingly, a fraction of intestinal CD4+ T cells produced granzyme-B and perforin after activation. Together, we show that the intestinal CD4+ T-cell compartment is dominated by resident populations that survive for more than 1 year. This finding is of high relevance for the development of oral vaccines and therapies for diseases in the gut.

Objective— Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. Approach and Results— Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB–directed inflammatory enhancers. Conclusions— Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.

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.

Bujko et al. describe four distinct subsets of macrophages in human small intestine that are completely replaced in transplanted duodenum. These subsets show graduated changes in their phenotypes, function, and transcriptome profiles, suggesting that they represent stages of monocyte-derived macrophage maturation in tissue., Macrophages (Mfs) are instrumental in maintaining immune homeostasis in the intestine, yet studies on the origin and heterogeneity of human intestinal Mfs are scarce. Here, we identified four distinct Mf subpopulations in human small intestine (SI). Assessment of their turnover in duodenal transplants revealed that all Mf subsets were completely replaced over time; Mf1 and Mf2, phenotypically similar to peripheral blood monocytes (PBMos), were largely replaced within 3 wk, whereas two subsets with features of mature Mfs, Mf3 and Mf4, exhibited significantly slower replacement. Mf3 and Mf4 localized differently in SI; Mf3 formed a dense network in mucosal lamina propria, whereas Mf4 was enriched in submucosa. Transcriptional analysis showed that all Mf subsets were markedly distinct from PBMos and dendritic cells. Compared with PBMos, Mf subpopulations showed reduced responsiveness to proinflammatory stimuli but were proficient at endocytosis of particulate and soluble material. These data provide a comprehensive analysis of human SI Mf population and suggest a precursor-progeny relationship with PBMos.

This study provides a definite answer to the long-standing question concerning the longevity of the secretory antibody response. Landsverk et al. show that antigenic attrition affects a minor plasma cell subset and that distinct plasma cells are likely maintained for life in the human small intestine., Plasma cells (PCs) produce antibodies that mediate immunity after infection or vaccination. In contrast to PCs in the bone marrow, PCs in the gut have been considered short lived. In this study, we studied PC dynamics in the human small intestine by cell-turnover analysis in organ transplants and by retrospective cell birth dating measuring carbon-14 in genomic DNA. We identified three distinct PC subsets: a CD19+ PC subset was dynamically exchanged, whereas of two CD19− PC subsets, CD45+ PCs exhibited little and CD45− PCs no replacement and had a median age of 11 and 22 yr, respectively. Accumulation of CD45− PCs during ageing and the presence of rotavirus-specific clones entirely within the CD19− PC subsets support selection and maintenance of protective PCs for life in human intestine.

Background:Gastrointestinal tract (GIT) involvement is highly prevalent in systemic sclerosis (SSc) and associates with GIT symptoms that are present early and progress over time. Changes in gut microbiota are often reported in inflammatory disease settings but whether GIT symptoms associate with altered immune recognition of specific gut bacteria in early SSc is unknown.Objectives:Here, we profiled Ig coating patterns of gut bacteria in early disease from two well-characterized SSc cohorts to determine if the pattern and extent of bacterial immunoglobulin (Ig) coating differs in early SSc.Methods:We collected fecal material from early SSc patients (Results:We included 50 SSc patients (26 from Oslo, 24 from Lund) with early SSc and 9 gender and age matched HC. Mean age of SSc patients at time of inclusion was 53 years, mean time since diagnosis was 13 months; 82% were female, 61% had limited cutaneous SSc and 43% were anti-centromere antibody positive. In all, 82% were treatment naïve while 18% had received either cyclophosphomide or mycophenolate mofetil immunosuppressants. We found increased relative abundance of IgA coated Desulfovibrionaceae in both SSc cohorts compared to HC and increased IgM and IgG coating of Veillonellaceae and Streptococcaceae (Figure 1). All of these bacteria have previously been associated with other autoimmune diseases or pro-inflammatory status; Desulfovibrionaceae to immune activation in the gut, and Veillonellaceae and Streptococcaceae to other chronic inflammatory and fibrotic conditions. While abundance of IgA coated Desulfovibrionaceae was higher in cyclophosphomide or mycophenolate mofetil-treated SSc patients than untreated patients, Veillonellaceae and Streptococcaceae were not affected by treatment. A lower abundance of IgA and IgM coated Akkermansiaceae; and IgM and IgG coated Bifidobacteriaceae was detected in treated compared to treatment naïve early SSc patients (Figure 2).Conclusion:We find the pattern and extent of Ig coating to inflammatory-associated gut bacteria differs between treatment-naïve, early SSc patients treated with cyclophosphomide or mycophenolate mofetil and HC which suggests immunosuppressive treatments may modify gut microbiota in SSc. Overall these findings support the involvement of altered immune recognition of specific gut bacteria in early SSc.Disclosure of Interests:Anna-Maria Hoffmann-Vold Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Actelion, Bayer, GlaxoSmithKline, Speakers bureau: Boehringer Ingelheim, Actelion, Roche, Kristofer Andréasson: None declared, Simen Hyll Hansen: None declared, Simon Midtvedt: None declared, Håvard Fretheim: None declared, Henriette Didriksen Consultant of: Actelion, Torhild Garen: None declared, Espen Bækkevold: None declared, Øyvind Midtvedt: None declared, Roger Hesselstrand: None declared, Brian K Chung: None declared, Øyvind Molberg: None declared

Background: Lung fibrosis is characterized by excessive collagen deposition in the extracellular matrix that leads to destruction of lung architecture, and there is a need for novel therapeutic approaches for patients with lung fibrosis. Aim: We hypothesized that T cells in the lung may play a key part in the fibrotic process. We therefore investigated the cytokine secretion profile and clonal relationship of lung CD4 T cells in patients with lung fibrosis. Methods: BAL and peripheral blood were obtained from 25 patients (aged 59-75) diagnosed with idiopathic pulmonary fibrosis, non-classifiable lung fibrosis and hypersensitivity pneumonitis (non-CTD lung fibrosis) and 11 patients (aged 29-66) diagnosed with CTD (connective tissue disease) lung fibrosis. T cells from BAL and blood were stimulated in vitro (3.5h) for cytokine characterization (all samples) and T cell receptor (TCR) of single T cells was sequenced for clonality analysis (n=4). Results: The cytokine production capacity CD4 T cells from fibrosis patients revealed a unique expression of the profibrotic cytokine IL-13 in combination with the cytokine IFNγ in BAL but not in blood. In patients with non-CTD lung fibrosis these unique BAL cells constituted 13% of the CD4 T cells and only 3% in patients with CTD lung fibrosis (p Conclusion: Non-CTD lung fibrosis contains an unusual population of CD4+ T cells producing profibrotic IL-13 together with proinflammatory IFNγ in BAL but not in blood. Such cells are clonally expanded, thus indicating local proliferation by antigens present in the lung.

Celiac disease (CeD) is an autoimmune disorder in which ingestion of dietary gluten triggers an immune reaction in the small intestine1,2. The CeD lesion is characterized by crypt hyperplasia, villous atrophy and chronic inflammation with accumulation of leukocytes both in the lamina propria (LP) and in the epithelium3, which eventually leads to destruction of the intestinal epithelium1 and subsequent digestive complications and higher risk of non-hodgkin lymphoma4. A lifetime gluten-free diet is currently the only available treatment5. Gluten-specific LP CD4 T cells and cytotoxic intraepithelial CD8+ T cells are thought to be central in disease pathology1,6-8, however, CeD is a complex immune-mediated disorder and to date the findings are mostly based on analysis of heterogeneous cell populations and on animal models. Here, we comprehensively explore the cellular heterogeneity of CD45+ immune cells in human small intestine using index-sorting single-cell RNA-sequencing9,10. We find that myeloid and mast cell transcriptomes are reshaped in CeD. We observe extensive changes in the proportion and transcriptomes of CD4+ and CD8+ T cells and define a CD3zeta expressing NK-T-like cell population present in the control LP and epithelial layers that is absent and replaced in CeD. Our findings show that the immune landscape is dramatically changed in active CeD which provide new insights and considerably extend the current knowledge of CeD immunopathology.

Background Systemic sclerosis (SSc) is a progressive, multi organ, auto-immune disease marked by frequent and severe gastrointestinal (GI) afflictions and gut dysbiosis. Objectives Determine the safety and efficacy of fecal microbiota transplantation (FMT) using commercially-available anaerobic cultivated human intestinal microbiota (ACHIM) in patients with SSc. Methods The trial was a single-center, randomized, double-blind, placebo-controlled 16-week pilot of FMT by gastroduodenoscopy of ACHIM in SSc conducted at Oslo University Hospital. Primary endpoints were safety and clinical efficacy on GI symptoms assessed at weeks 4 and 16. Safety was assessed by observation, interviews and standardized safety form. Efficacy on GI symptoms was measured using the UCLA GIT 2.0 score questionnaire. Patients were defined as responders if reported symptom improvement was equivalent to the UCLA GIT definition of “minimally clinically important difference”. Secondary and explorative endpoints included changes in relative abundance of total, immunoglobulin (Ig)A- and IgM-coated fecal bacteria measured by 16s rRNA sequencing; changes in modified Rodnan skin score, lung function, CRP, ESR, and patient and physician global. Descriptive statistics were applied for clinical endpoints and linear mixed models for microbial analysis. Results Ten patients with limited cutaneous SSc randomized to ACHIM (n=5) or placebo (n=5) were included. All patients were female with clinical apparent GI symptoms, mean age of 62 years and mean time from diagnosis of 12 yrs. Two placebo controls experienced procedure-related serious adverse events; one developed laryngospasms at first gastroduodenoscopy necessitating study exclusion, one duodenal perforation at final gastroduodenoscopy. Improvement in total GIT score was reported by 3/5 FMT patients compared to 2/4 placebo controls at weeks 4 and 16 (Figure 1). FMT effects were most pronounced on lower GI symptoms, with improvement reported by 5/5 FMT patients with diarrhea, distention/bloating and/or fecal incontinence at baseline compared to 2/4 placebo controls (Figure). Clinical secondary endpoints showed no differences and other side effects (stomach discomfort, bloating and diarrhea) were mild and transient. Fecal microbiota diversity (observed number of operational taxonomic units) was increased after FMT compared to placebo treatment at week 16 (p Conclusion FMT of commercially-available ACHIM in patients with SSc appeared safe, effectively reduced lower GI symptoms, altered gut microbiota composition, richness and diversity and appeared to affect the mucosal immune system Disclosure of Interests Anna-Maria Hoffmann-Vold Grant/research support from: Received research funding or other remuneration from Boehringer Ingelheim, GSK, and Actelion, Consultant for: Received consulting fees or other remuneration from Boehringer Ingelheim, GSK, and Actelion, Speakers bureau: Actelion and Boehringer Ingelheim, Havard Fretheim Consultant for: Received consulting fees or other remuneration from GSK, and Actelion, Brian K Chung: None declared, Henriette Didriksen Consultant for: Received consulting fees or other remuneration from GSK, and Actelion, Espen S Baekkevold: None declared, Oyvind Midtvedt: None declared, Cathrine Brunborg: None declared, Torhild Garen: None declared, Tore Midtvedt Shareholder of: Owner of ACHIM, Johannes R Hov: None declared, Knut EA Lundin: None declared, Oyvind Molberg: None declared

Bartolomé-Casado et al. demonstrate that human gut contains large numbers of resident memory CD8 T cells that survive for years. Intestinal CD8 Trm cells have a clonally expanded immune repertoire that is stable over time and exhibit enhanced protective capabilities., Resident memory CD8 T (Trm) cells have been shown to provide effective protective responses in the small intestine (SI) in mice. A better understanding of the generation and persistence of SI CD8 Trm cells in humans may have implications for intestinal immune-mediated diseases and vaccine development. Analyzing normal and transplanted human SI, we demonstrated that the majority of SI CD8 T cells were bona fide CD8 Trm cells that survived for >1 yr in the graft. Intraepithelial and lamina propria CD8 Trm cells showed a high clonal overlap and a repertoire dominated by expanded clones, conserved both spatially in the intestine and over time. Functionally, lamina propria CD8 Trm cells were potent cytokine producers, exhibiting a polyfunctional (IFN-γ+ IL-2+ TNF-α+) profile, and efficiently expressed cytotoxic mediators after stimulation. These results suggest that SI CD8 Trm cells could be relevant targets for future oral vaccines and therapeutic strategies for gut disorders., Graphical Abstract

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.

This study identified an unusual phenotype of clonally expanded CD4 T-cells in BAL in lung fibrosis characterised by co-production of pro-fibrotic cytokine IL-13 and pro-inflammatory cytokine IFN-γ. These cells may be a promising target for therapy.https://bit.ly/2TgzWCX

Objectives Systemic sclerosis (SSc) is an auto-immune, multi organ disease marked by severe gastrointestinal (GI) involvement and gut dysbiosis. Here, we aimed to determine the safety and efficacy of fecal microbiota transplantation (FMT) using commercially-available anaerobic cultivated human intestinal microbiota (ACHIM) in SSc. Methods Ten patients with SSc were randomized to ACHIM (n = 5) or placebo (n = 5) in a double-blind, placebo-controlled 16-week pilot. All patients had mild to severe upper and lower GI symptoms including diarrhea, distention/bloating and/or fecal incontinence at baseline. Gastroduodenoscopy transfer of ACHIM or placebo was performed at weeks 0 and 2. Primary endpoints were safety and clinical efficacy on GI symptoms assessed at weeks 4 and 16. Secondary endpoints included changes in relative abundance of total, immunoglobulin (Ig) A- and IgM-coated fecal bacteria measured by 16s rRNA sequencing. Results ACHIM side effects were mild and transient. Two placebo controls experienced procedure-related serious adverse events; one developed laryngospasms at week 0 gastroduodenoscopy necessitating study exclusion whilst one encountered duodenal perforation during gastroduodenoscopy at the last study visit (week 16). Decreased bloating, diarrhea and/or fecal incontinence was observed in four of five patients in the FMT group (week 4 or/and 16) and in two of four in the placebo group (week 4 or 16). Relative abundance, richness and diversity of total and IgA-coated and IgM-coated bacteria fluctuated more after FMT, than after placebo. Conclusions FMT of commercially-available ACHIM is associated with gastroduodenoscopy complications but reduces lower GI symptoms by possibly altering the gut microbiota in patients with SSc.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

Thymic stromal lymphopoietin (TSLP) has multifaceted immunological functions ranging from maintenance of tolerance to induction of disease. Two human transcript variants of TSLP are described: a long form (variant 1; lfTSLP) consisting of four exons and an alternative, short form (variant 2; sfTSLP) that lacks two exons compared with variant 1. SfTSLP has not been described at the protein level or functionally studied. Here, we demonstrate that the human sfTSLP is the predominant form of TSLP, constitutively expressed at the mRNA and protein level in keratinocytes of oral mucosa and skin and in salivary glands, is released in saliva, and is not regulated in the same manner as the long form. Compared with lfTSLP, sfTSLP exhibits a markedly stronger antibacterial activity. Synthetic sfTSLP did not activate signal transducer and activator of transcription 5 (STAT5) signaling in CD1c(+) dendritic cells nor interfered with STAT5 activation by lfTSLP. SfTSLP may, therefore, act as an antimicrobial peptide in the oral cavity and on the skin to create a defense barrier that aids in the control of both commensal and pathogenic microbes. The results show that the two translational products of the TSLP gene have a different expression and different biological properties, and emphasize the importance of analyzing the two TSLP isoforms separately.

Streptococcus mitis colonizes all niches of the human oral cavity from early infancy and throughout life. Monocytes patrol blood vessels, lymphoid and non-lymphoid tissues and migrate into infected tissue where they participate in the inflammatory cascade and immune regulation. Here, we studied the effect of S. mitis on monocytes. Transcriptome analysis of monocytes exposed to S. mitis (SmMo) revealed increased transcription of chemotactic factors (CCL2, CCL3, CCL20, CXCL1, CXCL2) and cytokines (IL1A, IL1B, IL6, IL23, IL36G, TNF), indicating that S. mitis may trigger recruitment of leucocytes and initiate inflammation. Increased transcription in SmMo of IL1B, IL6 and IL23 indicated that S. mitis may participate in the induction of Th17 responses and agreed with our earlier findings of S. mitis-mediated memory Th17 reactivity. Furthermore, S. mitis inhibited tetanus toxoid-specific CD4 T cell proliferation. This can be due to the increased secretion of IL-10 and expression of PD-L1 that was observed in SmMo. PGE2 can modulate IL-10 and PD-L1 expression, concomitant with that of CCR7, IL-12 and IL-23 that also were changed. This, along with increased SmMo transcription of PTGS2 (COX2) and PTGER4 (EP4), pointed to a role of PGE2. Measurement of PGE2 secretion by SmMo showed indeed a marked increase, and chemical inhibition of PGE2 production lowered the PD-L1 expression on SmMo. In conclusion, our findings show that S. mitis may trigger immune modulation by recruiting immune cells to the site of infection, while at the same time dampening the severity of the response through expression of IL-10, PGE2 and PD-L1.

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.

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.

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

Expression of the chemokine receptor CCR4 is strongly associated with trafficking of specialized cutaneous memory T helper (Th) lymphocytes to the skin. However, it is unknown whether CCR4 itself participates in the development of cutaneous Th populations. We have addressed this issue via competitive bone marrow (BM) reconstitution assays; equal numbers of BM cells from CCR4(+/+) and CCR4(-/-) donors were allowed to develop side-by-side within RAG-1(-/-) hosts. Cells from both donor types developed equally well into B cells, naive CD8 T cells, naive CD4 T cells, interferon-gamma(+) Th1 cells, and interleukin-4(+) Th2 cells. In marked contrast, circulating cutaneous memory Th cells (i.e., E-selectin ligand(+) [E-lig(+)]) were more than fourfold more likely to be derived from CCR4(+/+) donors than from CCR4(-/-) donors. Most of this effect resides within the CD103(+) subset of the E-lig(+) Th population, in which donor CCR4(+/+) cells can outnumber CCR4(-/-) cells by12-fold. No similar effect was observed for alpha4beta7(+) intestinal memory Th cells or CD103(+)/E-lig(-) Th cells. We conclude that CCR4 expression provides a competitive advantage to cutaneous Th cells, either by participating in their development from naive Th cells, or by preferentially maintaining them within the memory population over time.

The homeostatic chemokine CXCL13 (also called B cell-attracting chemokine 1 [BCA-1] or B-lymphocyte chemoattractant [BLC]) is constitutively expressed in secondary lymphoid tissue and initiates lymphoid neogenesis when expressed aberrantly in mice. CXCL13 has also been detected in chronic inflammation associated with human lymphoid neogenesis, suggesting a pathogenic role. Follicular dendritic cells (FDCs) are generally considered to be the major source of CXCL13 both in normal and aberrant lymphoid tissue. We show here, instead, that most CXCL13-expressing cells in rheumatoid arthritis and ulcerative colitis are of monocyte/macrophage lineage. They are located in irregular lymphoid aggregates within an FDC network, but also within and near smaller collections of B cells in diseased tissue where no FDCs are detected. Some of these CXCL13-expressing cells are CD14+, suggesting derivation from recently extravasated monocytes. Interestingly, monocytes from healthy donors stimulated in vitro with lipopolysaccharide secrete CXCL13. This induced production is enhanced after in vitro maturation of the monocytes toward macrophages but markedly decreased after maturation toward dendritic cells. Together, our findings strongly suggest that newly recruited monocytes/macrophages play a role for lymphoid neogenesis in human inflammatory diseases. Circulating monocytes are therefore potential candidates for future targeted therapy of chronic inflammation.

Endothelial cells display remarkable heterogeneity in different organs and vascular beds. Although many studies suggest that tissues “speak” to endothelial cells, endothelial cell diversity remains poorly characterized at the molecular level. Here, we describe a novel strategy to characterize tissue-specific endothelial cell phenotypes and to identify endothelial cell genes that are under the control of the local microenvironment. By comparing post-capillary high endothelial venule endothelial cells (HEVECs), freshly isolated from human tonsils without any cell culture step, with HEVECs cultured for 2 days, we found that HEVECs rapidly lost their specialized characteristics when isolated from the lymphoid tissue microenvironment. Striking changes occurred as early as after 48 hours, with complete loss of the postcapillary venule–specific Duffy antigen receptor for chemokines (DARCs) and the HEV-specific fucosyltransferase Fuc-TVII. DNA microarray analysis identified several other candidate HEV genes that were rapidly down-regulated ex vivo, including type XV collagen, which we characterized as a novel, abundant HEV transcript in situ. Together, our results demonstrate that blood vessel type–specific and tissue-specific characteristics of endothelial cells are under the control of their microenvironment. Therefore, even short-term primary cultures of human endothelial cells may not adequately mimic the differentiated endothelial cell phenotypes existing in vivo.

Cerebrospinal fluid (CSF) from healthy individuals contains between 1,000 and 3,000 leukocytes per ml. Little is known about trafficking patterns of leukocytes between the systemic circulation and the noninflamed CNS. In the current study, we characterized the surface phenotype of CSF cells and defined the expression of selected adhesion molecules on vasculature in the choroid plexus, the subarachnoid space surrounding the cerebral cortex, and the cerebral parenchyma. Using multicolor flow cytometry, we found that CSF cells predominantly consisted of CD4+/CD45RA-/CD27+/CD69+-activated central memory T cells expressing high levels of CCR7 and L-selectin. CD3+T cells were present in the choroid plexus stroma in autopsy CNS tissue sections from individuals who died without known neurological disorders. P- and E-selectin immunoreactivity was detected in large venules in the choroid plexus and subarachnoid space, but not in parenchymal microvessels. CD4+T cells in the CSF expressed high levels of P-selectin glycoprotein ligand 1, and a subpopulation of circulating CD4+T cells displayed P-selectin binding activity. Intercellular adhesion molecule 1, but not vascular cell adhesion molecule 1 or mucosal addressin cell adhesion molecule 1, was expressed in choroid plexus and subarachnoid space vessels. Based on these findings, we propose that T cells are recruited to the CSF through interactions between P-selectin/P-selectin ligands and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 in choroid plexus and subarachnoid space venules. These results support the overall hypothesis that activated memory T cells enter CSF directly from the systemic circulation and monitor the subarachnoid space, retaining the capacity to either initiate local immune reactions or return to secondary lymphoid organs.

SummaryBackground Seasonal allergic rhinitis is a chronic inflammation in the nasal mucosa triggered by inhaled aeroallergens. The inflammatory reaction is controlled by allergen-specific T cells, but where and how these T cells become activated is not fully understood. Objectives We wanted to determine whether allergen-specific T-helper (Th) 2 cells are residing in the nasal mucosa under steady-state conditions outside of the pollen season and, if so, whether these cells are activated locally in response to allergen challenge. Methods Mucosal biopsies from the lower turbinate were obtained out of season from patients with either birch- or grass-pollen-allergic rhinitis and from healthy controls. Cultured explant samples were challenged with relevant pollen extract or with a mix of overlapping 20-mer peptides derived from the sequence of the major birch allergen, Betula verrucosa (Bet v) 1. After 24 h, culture medium was harvested for multiplex cytokine and tryptase analysis. Results Significant amounts of interleukin (IL)-5 were secreted from resident cells in response to ex vivo allergen challenge in the allergic group only. No increase was observed for the other cytokines measured. Production of IL-5 in response to both extract and the Bet v1-derived peptide mix strongly suggested that T cells were a major source of IL-5. Conclusion Our explant model indicated that local presentation of antigen to resident allergen-specific Th2 cells is the early event in the pathogenesis of allergic rhinitis. These findings identify possible cellular targets for anti-inflammatory treatment.

High endothelial venules (HEV) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. One of the major characteristics of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. Here, we show that HEVEC express two functional classes of sulfate transporters defined by their differential sensitivity to the anion-exchanger inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), and we report the molecular characterization of a DIDS-resistant sulfate transporter from human HEVEC, designated SUT-1. SUT-1 belongs to the family of Na + -coupled anion transporters and exhibits 40–50% amino acid identity with the rat renal Na + /sulfate cotransporter, NaSi-1, as well as with the human and rat Na + /dicarboxylate cotransporters, NaDC-1/SDCT1 and NaDC-3/SDCT2. Functional expression studies in cRNA-injected Xenopus laevis oocytes showed that SUT-1 mediates high levels of Na + -dependent sulfate transport, which is resistant to DIDS inhibition. The SUT-1 gene mapped to human chromosome 7q33. Northern blotting analysis revealed that SUT-1 exhibits a highly restricted tissue distribution, with abundant expression in placenta. Reverse transcription–PCR analysis indicated that SUT-1 and the diastrophic dysplasia sulfate transporter (DTD), one of the two known human DIDS-sensitive sulfate transporters, are coexpressed in HEVEC. SUT-1 and DTD could correspond, respectively, to the DIDS-resistant and DIDS-sensitive components of sulfate uptake in HEVEC. Together, these results demonstrate that SUT-1 is a distinct human Na + -coupled sulfate transporter, likely to play a major role in sulfate incorporation in HEV.

High endothelial venules (HEVs) are specialized postcapillary venules found in lymphoid organs and chronically inflamed tissues that support high levels of lymphocyte extravasation from the blood. ...

Lymphocyte recruitment to peripheral tissues is fundamental for immune surveillance and homeostasis, but the chemokines and chemokine receptors responsible for tissue-specific homing of T cells to the upper airway mucosa have not been determined. To address this, we analyzed the chemokines expressed in the normal human nasal mucosa and found that CCL28 is preferentially expressed at a high level on the lumenal face of vascular endothelial cells in the mucosa. Analysis of the cognate chemokine receptors revealed that close to 50% of the CD4(+) T cells in the human nasal mucosa expressed the CCL28 receptor CCR3, whereas CCR3 was hardly detectable on T cells in the small intestine and skin. In the circulation, CCR3(+) T cells comprised a small subset that did not express homing receptors to the intestine or skin. Moreover, depletion of CCR3(+)CD4(+) T cells abrogated the proliferative response of human blood CD4(+) T cells against the opportunistic nasopharyngeal pathogen Haemophilus influenzae, indicating that the CCR3(+)CD4(+) T-cell subset in the circulation contains antigen specificities relevant for the upper airways. Together, these findings indicate that CCL28-CCR3 interactions are involved in the homeostatic trafficking of CD4(+) T cells to the upper airways.

The name of the fifth author was given incorrectly. The correct name is: K. Elizabeth Tanner. The correct citation is: Hessle L, Stordalen GA, Wenglen C, Petzold C, Tanner KE, et al. (2013) The Skeletal Phenotype of Chondroadherin Deficient Mice. PLoS ONE 8(6): e63080. doi:10.1371/journal.pone.0063080. The correct abbreviation in Contributions is: KET.

Conventional dendritic cells (cDCs) comprise a heterogeneous population of cells that are important regulators of immunity and homeostasis. CD1c(+) cDCs are present in human blood and tissues, and found to efficiently activate naive CD4(+) T cells. While CD1c is thought to specifically identify this subset of human cDCs, we show here that also classical and intermediate monocytes express CD1c. Accordingly, the commercial CD1c (BDCA-1) Dendritic Cell Isolation Kit isolates two distinct cell populations from blood: CD1c(+) CD14 cDCs and CD1c(+) CD14(+) monocytes. CD1c(+) cDCs and CD1c(+) monocytes exhibited strikingly different properties, including their differential regulation of surface marker expression, their levels of cytokine production, and their ability to stimulate naive CD4(+) T cells. These results demonstrate that a commercial CD1c (BDCA-1) Dendritic Cell Isolation Kit isolates two functionally different cell populations, which has important implications for the interpretation of previously generated data using this kit to characterize CD1c(+) cDCs.

Background Activated T H 2 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 T H 2-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.