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Abstract The respiratory system, including the lungs, is essential for terrestrial life. While recent research has advanced our understanding of lung development, much still relies on animal models and transcriptome analyses. In this study conducted within the Human Developmental Cell Atlas (HDCA) initiative, we describe the protein-level spatiotemporal organization of the lung during the first trimester of human gestation. Using high-parametric tissue imaging with a 30-plex antibody panel, we analyzed human lung samples from 6 to 13 post-conception weeks, generating data from over 2 million cells across five developmental timepoints. We present a resource detailing spatially resolved cell type composition of the developing human lung, including proliferative states, immune cell patterns, spatial arrangement traits, and their temporal evolution. This represents an extensive single-cell resolved protein-level examination of the developing human lung and provides a valuable resource for further research into the developmental roots of human respiratory health and disease.

Abstract Stromal cells support epithelial cell and immune cell homeostasis and play an important role in inflammatory bowel disease (IBD) pathogenesis. Here, we quantify the stromal response to inflammation in pediatric IBD and reveal subset-specific inflammatory responses across colon segments and intestinal layers. Using data from a murine dynamic gut injury model and human ex vivo transcriptomic, protein and spatial analyses, we report that PDGFRA+CD142− /low fibroblasts and monocytes/macrophages co-localize in the intestine. In primary human fibroblast-monocyte co-cultures, intestinal PDGFRA+CD142− /low fibroblasts foster monocyte transition to CCR2+CD206+ macrophages through granulocyte-macrophage colony-stimulating factor (GM-CSF). Monocyte-derived CCR2+CD206+ cells from co-cultures have a phenotype similar to intestinal CCR2+CD206+ macrophages from newly diagnosed pediatric IBD patients, with high levels of PD-L1 and low levels of GM-CSF receptor. The study describes subset-specific changes in stromal responses to inflammation and suggests that the intestinal stroma guides intestinal macrophage differentiation.

Abstract Effective humoral immune responses require well-orchestrated B and T follicular helper (Tfh) cell interactions. Whether these interactions are impaired and associated with COVID-19 disease severity is unclear. Here, longitudinal blood samples across COVID-19 disease severity are analysed. We find that during acute infection SARS-CoV-2-specific circulating Tfh (cTfh) cells expand with disease severity. SARS-CoV-2-specific cTfh cell frequencies correlate with plasmablast frequencies and SARS-CoV-2 antibody titers, avidity and neutralization. Furthermore, cTfh cells but not other memory CD4 T cells, from severe patients better induce plasmablast differentiation and antibody production compared to cTfh cells from mild patients. However, virus-specific cTfh cell development is delayed in patients that display or later develop severe disease compared to those with mild disease, which correlates with delayed induction of high-avidity neutralizing antibodies. Our study suggests that impaired generation of functional virus-specific cTfh cells delays high-quality antibody production at an early stage, potentially enabling progression to severe disease.

Abstract Background The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers. Results We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity. Conclusion We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria. Video Abstract

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) – a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis.

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.

Abstract Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co‐isolated entities, a combination of methods is needed. However, this is time‐consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size‐exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC‐MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non‐EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non‐EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.

Clinical and molecular heterogeneity of ulcerative colitis presents unresolved challenges to identify predictive biomarkers of response to therapies. Here, the authors combine mouse colitis time course with patient biopsy transcriptomes, achieving unsupervised clustering of UC patients correlating with therapeutic outcomes in independent data sets.

Tr1 cells are considered an immunosuppressive CD4 T cell population producing IL-10. Here the authors show that IL-10 is insufficient for Tr1 immunosuppression, define surface markers and transcriptional program of the immunosuppressive subset within Tr1, and reveal its deficiency in patients with IBD.

Toxoplasma gondii is known to cause congenital infection in humans and animals and severe disease in immunocompromised individuals; consequently development of vaccines against the parasite is highly necessary. Under stress conditions, T. gondii expresses the highly immunogenic heat shock protein 70 (TgHSP70). Here, we assessed the protective efficacy of rTgHSP70 immunization combined with Alum in oral ME-49 T. gondii infection and the mechanisms involved on it. It was observed that immunized mice with rTgHSP70 or rTgHSP70 adsorbed in Alum presented a significantly reduced number of cysts in the brain that was associated with increased iNOS+ cell numbers in the organ, irrespective the use of the adjuvant. Indeed, ex vivo experiments showed that peritoneal macrophages pre-stimulated with rTgHSP70 presented increased NO production and enhanced parasite killing, and the protein was able to directly stimulate B cells toward antibody producing profile. In addition, rTgHSP70 immunization leads to high specific antibody titters systemically and a mixed IgG1/IgG2a response, with predominance of IgG1 production. Nonetheless, it was observed that the pretreatment of the parasite with rTgHSP70 immune sera was not able to control T. gondii internalization and replication by NIH fibroblast neither peritoneal murine macrophages, nor anti-rTgHSP70 antibodies were able to kill T. gondii by complement-mediated lysis, suggesting that these mechanisms are not crucial to resistance. Interestingly, when in combination with Alum, rTgHSP70 immunization was able to reduce inflammation in the brain of infected mice and in parallel anti-rTgHSP70 immune complexes in the serum. In conclusion, immunization with rTgHSP70 induces massive amounts of iNOS expression and reduced brain parasitism, suggesting that iNOS expression and consequently NO production in the brain is a protective mechanism induced by TgHSP70 immunization, therefore rTgHSP70 can be a good candidate for vaccine development against toxoplasmosis.

The heat shock protein of Toxoplasma gondii (TgHSP70) is a parasite virulence factor that is expressed during T. gondii stage conversion. To verify the effect of dexamethasone (DXM)-induced infection reactivation in the TgHSP70-specific humoral immune response and the presence of the protein in the mouse brain, we produced recombinant TgHSP70 and anti-TgHSP70 IgY antibodies to detect the protein, the specific antibody and levels of immune complexes (ICs) systemically, as well as the protein in the brain of resistant (BALB/c) and susceptible (C57BL/6) mice. It was observed higher TgHSP70-specific antibody titers in serum samples of BALB/c compared with C57BL/6 mice. However, the susceptible mice presented the highest levels of TgHSP70 systemically and no detection of specific ICs. The DXM treatment induced increased parasitism and lower inflammatory changes in the brain of C57BL/6, but did not interfere with the cerebral parasitism in BALB/c mice. Additionally, DXM treatment decreased the serological TgHSP70 concentration in both mouse lineages. C57BL/6 mice presented high expression of TgHSP70 in the brain with the progression of infection and under DXM treatment. Taken together, these data indicate that the TgHSP70 release into the bloodstream depends on the death of the parasites mediated by the host immune response, whereas the increased TgHSP70 expression in the brain depends on the multiplication rate of the parasite.

Background and Aims Ulcerative colitis [UC] is associated with excessive neutrophil infiltration and collateral tissue damage, but the link is not yet completely understood. Since c-MET receptor tyrosine kinase [MET] is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor [HGF], we aimed to identify the function of HGF-MET signalling in neutrophils in UC patients and in mice during intestinal inflammation. Methods Serum and colonic biopsies from healthy controls and UC patients with active [Mayo endoscopic subscore 2–3] and inactive [Mayo endoscopic subscore 0–1] disease were collected to assess the level of serum and colonic HGF. Disease progression and immune cell infiltration were assessed during dextran sodium sulphate [DSS] colitis in wild-type and MRP8-Cre MET-LoxP mice. Results Increased mucosal HGF expression was detected in patients with active UC, and in mice during the inflammatory phase of DSS colitis. Similarly, serum HGF was significantly increased in active UC patients and positively correlated with C-reactive protein and blood neutrophil counts. Flow cytometric analysis also demonstrated an upregulation of colonic MET+ neutrophils during DSS colitis. Genetic ablation of MET in neutrophils reduced the severity of DSS-induced colitis. Concomitantly, there was a decreased number of TH17 cells, which could be due to a decreased production of IL-1β by MET-deficient neutrophils. Conclusions These data highlight the central role of neutrophilic HGF-MET signalling in exacerbating damage during intestinal inflammation. Hence, selective blockade of this pathway in neutrophils could be considered as a novel therapeutic approach in UC. [ABSTRACT FROM AUTHOR]

Background: Vitamin D deficiency is a risk factor for inflammatory bowel disease (IBD). The IL-23-driven tissueresident group 3 innate lymphoid cells (ILC3s) play essential roles in intestinal immunity, and targeting IL-23/12 is a promising approach in IBD therapy. Objective: We set out to define the role of 1α,25-dihydroxy vitamin D3 (1,25D) in regulating functional responses of human mucosal ILC3s to IL-23 plus IL-1β stimulation. Methods: Transcriptomes of sorted tonsillar ILC3s were assessed by using microarray analysis. ILC3 cytokine production, proliferation, and differentiation were determined by means of flow cytometry, ELISA, and multiplex immunoassay. Intestinal cell suspensions and ILC3s sorted from gut biopsy specimens of patients with IBD were also analyzed along with plasma 25-hydroxy vitamin D3 (25D) detection. Results: ILC3s stimulated with IL-23 plus IL-1β upregulated the vitamin D receptor and responded to 1,25D with downregulation of the IL-23 receptor pathway. Consequently, 1,25D suppressed IL-22, IL-17F, and GM-CSF production from tonsillar and gut ILC3s. In parallel, 1,25D upregulated genes linked to the IL-1β signaling pathway, as well as the IL-1β-inducible cytokines IL-6, IL-8, and macrophage inflammatory protein 1α/β. The 1,25D-triggered skewing in ILC3 function was not accompanied or caused by changes in viability, proliferation, or phenotype. Finally, we confirmed low 25D plasma levels in patients with IBD with active inflammation. Conclusion: In light of the beneficial targeting of IL-23/12 in patients with IBD, 1,25D appears as an interesting therapeutic agent that inhibits the IL-23 receptor pathway, providing a novel mechanism for how ILC3s could be manipulated to regulate intestinal inflammation. [ABSTRACT FROM AUTHOR]

Chronic systemic immune activation significantly influences human immunodeficiency virus (HIV) disease progression. Despite evidence of a pro-inflammatory environment in the genital tract of HIV-infected women, comprehensive investigations into cervical tissue from this region remain limited. Similarly, the consequences of chronic HIV infection on the integrity of the female genital epithelium are poorly understood, despite its importance in HIV transmission and replication. Ectocervical biopsies were obtained from HIV-seropositive (n = 14) and HIV-seronegative (n = 47) female Kenyan sex workers. RNA sequencing and bioimage analysis of epithelial junction proteins (E-cadherin, desmoglein-1, claudin-1, and zonula occludens-1) were conducted, along with CD4 staining. RNA sequencing revealed upregulation of immunoregulatory genes in HIV-seropositive women, primarily associated with heightened T cell activity and interferon signaling, which further correlated with plasma viral load. Transcription factor analysis confirmed the upregulation of pro-inflammatory transcription factors, such as RELA, NFKB1, and IKZF3, which facilitates HIV persistence in T cells. Conversely, genes and pathways associated with epithelial barrier function and structure were downregulated in the context of HIV. Digital bioimage analysis corroborated these findings, revealing significant disruption of various epithelial junction proteins in ectocervical tissues of the HIV-seropositive women. Thus, chronic HIV infection associated with ectocervical inflammation, characterized by induced T cell responses and interferon signaling, coupled with epithelial disruption. These alterations may influence HIV transmission and heighten susceptibility to other sexually transmitted infections. These findings prompt exploration of therapeutic interventions to address HIV-related complications and mitigate the risk of sexually transmitted infection transmission., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: P. Cz. is currently employed at DeepLife, located in 75013 Paris, France. None of the other authors declare any conflicts of interest., (Copyright: © 2024 Franzén Boger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Commensal-specific clusters of differentiation (CD)4 + T cells are expanded in patients with inflammatory bowel disease (IBD) compared to healthy individuals. How and where commensal-specific CD4 + T cells get activated is yet to be fully understood. We used CBir1 TCR-transgenic CD4 + T cells, specific to a commensal bacterial antigen, and different mouse models of IBD to characterize the dynamics of commensal-specific CD4 + T-cells activation. We found that CBir1 T cells proliferate following intestinal damage and cognate antigen presentation is mediated by CD11c + cells in the colon-draining mesenteric lymph nodes. Using assay for transposase-accessible chromatin sequencing and flow cytometry, we showed that activated CBir1 T cells preferentially acquire an effector rather than regulatory phenotype, which is plastic over time. Moreover, CBir1 T cells, while insufficient to initiate intestinal inflammation, contributed to worse disease outcomes in the presence of other CD4 + T cells. Our results suggest that the commensal-specific T-cell responses observed in IBD exacerbate rather than initiate disease., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: B cells are important in the pathogenesis of primary Sjögren's syndrome (pSS). Patients positive for Sjögren's syndrome antigen A/Sjögren syndrome antigen B (SSA/SSB) autoantibodies are more prone to systemic disease manifestations and adverse outcomes. We aimed to determine the role of B cell composition, gene expression, and B cell receptor usage in pSS subgroups stratified for SSA/SSB antibodies., Methods: Over 230,000 B cells were isolated from peripheral blood of patients with pSS (n = 6 SSA-, n = 8 SSA+ single positive and n = 10 SSA/SSB+ double positive) and four healthy controls and processed for single-cell RNA sequencing (scRNA-seq) and single-cell variable, diversity, and joining (VDJ) gene sequencing (scVDJ-seq)., Results: We show that SSA/SSB+ patients present the highest and lowest proportion of naïve and memory B cells, respectively, and the highest up-regulation of interferon-induced genes across all B cell subtypes. Differential usage of IGHV showed that IGHV1-69 and IGHV4-30-4 were more often used in all pSS subgroups compared with controls. Memory B cells from SSA/SSB+ patients displayed a higher proportion of cells with unmutated VDJ transcripts compared with other pSS patient groups and controls, indicating altered somatic hypermutation processes. Comparison with previous studies revealed heterogeneous clonotype pools, with little overlap in CDR3 sequences. Joint analysis using scRNA-seq and scVDJ-seq data allowed unsupervised stratification of patients with pSS and identified novel parameters that correlated to disease manifestations and antibody status., Conclusion: We describe heterogeneity and molecular characteristics in B cells from patients with pSS, providing clues to intrinsic differences in B cells that affect the phenotype and outcome and allowing stratification of patients with pSS at improved resolution., (© 2023 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Congenital toxoplasmosis may cause abortion, neonatal death, or foetal abnormalities. Despite little information from human studies, a genetic influence over congenital disease was demonstrated and, host genome have been implicated to resistance/susceptibility to Toxoplasma gondii infection in both human and mice. It was previously shown that BALB/c mice (H2 d ) were more resistant to congenital toxoplasmosis than C57BL/6 mice (H2 b ). However, it is unclear whether these differences are attributable to the MHC haplotype or to other components of the mouse's genetic background. Therefore, in this work, we intend to address this question by investigating the pregnancy outcome in H2 d -congenic C57BL/6 mice (C57BL/KsJ-H2 d ) and H2 b -congenic BALB/c mice (CB10-H2-H2 b ). For this, animals were infected by intragastric route on the first day of pregnancy and examined on days 8 (8dP/8dI) or 18 (18dP/18dI) of gestation and infection. The pregnancy outcome, parasite burden, systemic cytokine profile and antibody response to infection were evaluated. Infected mice showed adverse pregnancy outcomes, in parallel low parasite detection in the uterus/placenta, being that the C57BL/KsJ showed the worst results in relation to CB10-H2 mice. Both mouse lineages showed an increase in IFN-γ and TNF levels systemically on 8dP/8dI and on 18dP/18dI, and C57BL/KsJ showed an increase in IL-6 levels in both gestation/infection periods. Additionally, C57BL/KsJ showed 7- and 7-fold increase in IL-6, 4- and 2.5-fold increase in IFN-γ and, 6- and 4-fold increase in TNF production on 8dP/8dI and 18dP/18dI, respectively in association with 1.5-fold decrease in TGF-β levels on 8dP/8dI compared to CB10-H2 mice. In conclusion, the high IFN-γ and TNF serum levels observed in C57BL/KsJ (H2 d ) and CB10-H2 (H2 b ) mice were involved in the poor pregnancy outcomes in congenital toxoplasmosis. In addition, the higher IFN-γ, IL-6 and TNF levels detected in C57BL/KsJ in relation to CB10-H2 mice on 8dP/8dI seem to be related to the genetic background of C57BL/6J mice that may have contributed to the worse pregnancy outcome in this mouse lineage., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Venous thromboembolism (VTE) is a leading cause of preventable deaths in hospitals, and its incidence is not decreasing despite extensive efforts in clinical and laboratory research. Venous thrombi are primarily formed in the valve pockets of deep veins, where activated monocytes play a crucial role in bridging innate immune activation and hemostatic pathways through the production of inflammatory cytokines, chemokines, and tissue factor (TF) - a principal initiator of coagulation. In the valve pocket inflammation and hypoxia (sustained/intermittent) coexist, however their combined effects on immunothrombotic processes are poorly understood. Inflammation is strongly associated with VTE, while the additional contribution of hypoxia remains largely unexplored. To investigate this, we modelled the intricate conditions of the venous valve pocket using a state-of-the-art hypoxia chamber with software-controlled oxygen cycling. We comprehensively studied the effects of sustained and intermittent hypoxia alone, and in combination with VTE-associated inflammatory stimuli on primary monocytes. TF expression and activity was measured in monocytes subjected to sustained and intermittent hypoxia alone, or in combination with IL-1β. Monocyte responses were further analyzed in detailed by RNA sequencing and validated by ELISA. Stimulation with IL-1β alone promoted both transcription and activity of TF. Interestingly, the stimulatory effect of IL-1β on TF was attenuated by sustained hypoxia, but not by intermittent hypoxia. Our transcriptome analysis further confirmed that sustained hypoxia limited the pro-inflammatory response induced by IL-1β, and triggered a metabolic shift in monocytes. Intermittent hypoxia alone had a modest effect on monocyte transcript. However, in combination with IL-1β intermittent hypoxia significantly altered the expression of 2207 genes and enhanced the IL-1β-stimulatory effects on several chemokine and interleukin genes (e.g., IL-19, IL-24, IL-32, MIF), as well as genes involved in coagulation (thrombomodulin) and fibrinolysis (VEGFA, MMP9, MMP14 and PAI-1). Increased production of CCL2, IL-6 and TNF following stimulation with intermittent hypoxia and IL-1β was confirmed by ELISA. Our findings provide valuable insights into how the different hypoxic profiles shape the immunothrombotic response of monocytes and shed new light on the early events in the pathogenesis of venous thrombosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wahlund, Çaglayan, Czarnewski, Hansen and Snir.)

Therapeutic promotion of intestinal regeneration holds great promise, but defining the cellular mechanisms that influence tissue regeneration remains an unmet challenge. To gain insight into the process of mucosal healing, we longitudinally examined the immune cell composition during intestinal damage and regeneration. B cells were the dominant cell type in the healing colon, and single-cell RNA sequencing (scRNA-seq) revealed expansion of an IFN-induced B cell subset during experimental mucosal healing that predominantly located in damaged areas and associated with colitis severity. B cell depletion accelerated recovery upon injury, decreased epithelial ulceration, and enhanced gene expression programs associated with tissue remodeling. scRNA-seq from the epithelial and stromal compartments combined with spatial transcriptomics and multiplex immunostaining showed that B cells decreased interactions between stromal and epithelial cells during mucosal healing. Activated B cells disrupted the epithelial-stromal cross talk required for organoid survival. Thus, B cell expansion during injury impairs epithelial-stromal cell interactions required for mucosal healing, with implications for the treatment of IBD., Competing Interests: Declaration of interests E.J.V. has received research grants from F. Hoffmann-La Roche. C.E., L.L., and J.L. are scientific consultants for 10X Genomics Inc. J.S.-R. receives funding from GSK and Sanofi and consultant fees from Travere Therapeutics and Astex Pharmaceuticals., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa., Competing Interests: The authors have declared that no competing interests exist.

Background: Pulmonary sarcoidosis is an inflammatory disease characterised by granuloma formation and heterogeneous clinical outcome. Tumour necrosis factor (TNF) is a pro-inflammatory cytokine contributing to granuloma formation and high levels of TNF have been shown to associate with progressive disease. Mononuclear phagocytes (MNPs) are potent producers of TNF and highly responsive to inflammation. In sarcoidosis, alveolar macrophages have been well studied. However, MNPs also include monocytes/monocyte-derived cells and dendritic cells, which are poorly studied in sarcoidosis, despite their central role in inflammation., Objective: To determine the role of pulmonary monocyte-derived cells and dendritic cells during sarcoidosis., Methods: We performed in-depth phenotypic, functional and transcriptomic analysis of MNP subsets from blood and bronchoalveolar lavage (BAL) fluid from 108 sarcoidosis patients and 30 healthy controls. We followed the clinical development of patients and assessed how the repertoire and function of MNP subsets at diagnosis correlated with 2-year disease outcome., Results: Monocytes/monocyte-derived cells were increased in blood and BAL of sarcoidosis patients compared to healthy controls. Interestingly, high frequencies of blood intermediate monocytes at time of diagnosis associated with chronic disease development. RNA sequencing analysis showed highly inflammatory MNPs in BAL of sarcoidosis patients. Furthermore, frequencies of BAL monocytes/monocyte-derived cells producing TNF without exogenous stimulation at time of diagnosis increased in patients that were followed longitudinally. In contrast to alveolar macrophages, the frequency of TNF-producing BAL monocytes/monocyte-derived cells at time of diagnosis was highest in sarcoidosis patients that developed progressive disease., Conclusion: Our data show that pulmonary monocytes/monocyte-derived cells are highly inflammatory and can be used as a predictor of disease outcome in sarcoidosis patients., Competing Interests: Conflict of interest: R. Lepzien has nothing to disclose. Conflict of interest: S. Liu has nothing to disclose. Conflict of interest: P. Czarnewski has nothing to disclose. Conflict of interest: M. Nie has nothing to disclose. Conflict of interest: B. Österberg has nothing to disclose. Conflict of interest: F. Baharom has nothing to disclose. Conflict of interest: J. Pourazar has nothing to disclose. Conflict of interest: G. Rankin has nothing to disclose. Conflict of interest: A. Eklund has nothing to disclose. Conflict of interest: M. Bottai has nothing to disclose. Conflict of interest: S. Kullberg has nothing to disclose. Conflict of interest: A. Blomberg has nothing to disclose. Conflict of interest: J. Grunewald has nothing to disclose. Conflict of interest: A. Smed-Sörensen reports grants from Swedish Heart-Lung Foundation, Swedish Research Council and Karolinska Institutet, during the conduct of the study., (Copyright ©The authors 2021.)

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget., Competing Interests: Susanne Gabrielsson has a patent on B cell derived exosomes in immune therapy and is part of the Scientific Advisory Board of Anjarium Biosciences., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Clinical manifestations and response to therapies in ulcerative colitis (UC) are heterogeneous, yet patient classification criteria for tailored therapies are currently lacking. Here, we present an unsupervised molecular classification of UC patients, concordant with response to therapy in independent retrospective cohorts. We show that classical clustering of UC patient tissue transcriptomic data sets does not identify clinically relevant profiles, likely due to associated covariates. To overcome this, we compare cross-sectional human data sets with a newly generated longitudinal transcriptome profile of murine DSS-induced colitis. We show that the majority of colitis risk-associated gene expression peaks during the inflammatory rather than the recovery phase. Moreover, we achieve UC patient clustering into two distinct transcriptomic profiles, differing in neutrophil-related gene activation. Notably, 87% of patients in UC1 cluster are unresponsive to two most widely used biological therapies. These results demonstrate that cross-species comparison enables stratification of patients undistinguishable by other molecular approaches.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 + T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 neg CD4 + T cells that displays regulatory activity unlike other IL-10-producing CD4 + T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 + T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 neg CD4 + T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder of unclear etiology in women and is characterized by androgen excess, insulin resistance, and mood disorders. The gut microbiome is known to influence conditions closely related with PCOS, and several recent studies have observed changes in the stool microbiome of women with PCOS. The mechanism by which the gut microbiome interacts with PCOS is still unknown. We used a mouse model to investigate if diet-induced maternal obesity and maternal DHT exposure, mimicking the lean and obese PCOS women, cause lasting changes in the gut microbiome of offspring. Fecal microbiome profiles were assessed using Illumina paired-end sequencing of 16S rRNA gene V4 amplicons. We found sex-specific effects of maternal and offspring diet, and maternal DHT exposure on fecal bacterial richness and taxonomic composition. Female offspring exposed to maternal obesity and DHT displayed reproductive dysfunction and anxietylike behavior. Fecal microbiota transplantation from DHT and diet-induced obesity exposed female offspring to wild-type mice did not transfer reproductive dysfunction and did not cause the expected increase in anxietylike behavior in recipients. Maternal obesity and androgen exposure affect the gut microbiome of offspring, but the disrupted estrous cycles and anxietylike behavior are likely not microbiome-mediated.