Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation

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

Sustained and intermittent hypoxia differentially modulate primary monocyte immunothrombotic responses to IL-1β stimulation

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

Tissue-specific transcriptional imprinting and heterogeneity in human innate lymphoid cells revealed by full-length single-cell RNA-sequencing

The impact of the microenvironment on innate lymphoid cell (ILC)-mediated immunity in humans remains largely unknown. Here we used full-length Smart-seq2 single-cell RNA-sequencing to unravel tissue-specific transcriptional profiles and heterogeneity of CD127+ ILCs across four human tissues. Correlation analysis identified gene modules characterizing the migratory properties of tonsil and blood ILCs, and signatures of tissue-residency, activation and modified metabolism in colon and lung ILCs. Trajectory analysis revealed potential differentiation pathways from circulating and tissue-resident na\uefve ILCs to a spectrum of mature ILC subsets. In the lung we identified both CRTH2+ and CRTH2− ILC2 with lung-specific signatures, which could be recapitulated by alarmin-exposure of circulating ILC2. Finally, we describe unique TCR-V(D)J-rearrangement patterns of blood ILC1-like cells, revealing a subset of potentially immature ILCs with TCR-δ rearrangement. Our study provides a useful resource for in-depth understanding of ILC-mediated immunity in humans, with implications for disease

Single-cell BCR and transcriptome analysis after influenza infection reveals spatiotemporal dynamics of antigen-specific B cells

B cell responses are critical for antiviral immunity. However, a comprehensive picture of antigen-specific B cell differentiation, clonal proliferation, and dynamics in different organs after infection is lacking. Here, by combining single-cell RNA and B cell receptor (BCR) sequencing of antigen-specific cells in lymph nodes, spleen, and lungs after influenza infection in mice, we identify several germinal center (GC) B cell subpopulations and organ-specific differences that persist over the course of the response. We discover transcriptional differences between memory cells in lungs and lymphoid organs and organ-restricted clonal expansion. Remarkably, we find significant clonal overlap between GC-derived memory and plasma cells. By combining BCR-mutational analyses with monoclonal antibody (mAb) expression and affinity measurements, we find that memory B cells are highly diverse and can be selected from both low- and high-affinity precursors. By linking antigen recognition with transcriptional programming, clonal proliferation, and differentiation, these finding provide important advances in our understanding of antiviral immunity

Community-driven ELIXIR activities in single-cell omics

Single-cell omics (SCO) has revolutionized the way and the level of resolution by which life science research is conducted, not only impacting our understanding of fundamental cell biology but also providing novel solutions in cutting-edge medical research. The rapid development of single-cell technologies has been accompanied by the active development of data analysis methods, resulting in a plethora of new analysis tools and strategies every year. Such a rapid development of SCO methods and tools poses several challenges in standardization, benchmarking, computational resources and training. These challenges are in line with the activities of ELIXIR, the European coordinated infrastructure for life science data. Here, we describe the current landscape of and the main challenges in SCO data, and propose the creation of the ELIXIR SCO Community, to coordinate the efforts in order to best serve SCO researchers in Europe and beyond. The Community will build on top of national experiences and pave the way towards integrated long-term solutions for SCO research. Keywor

Papel de HSP70 de Toxoplasma gondii como antígeno vacinal e como ferramenta diagnóstica de toxoplasmose em camundongos BALB/c e C57BL/6

Toxoplasma gondii is an obligate intracellular protozoan parasite of warm-blooded animals that can express the heat shock protein of 70 kDa (TgHSP70) during stage conversion. TgHSP70 is a highly antigenic protein and its presence indicates a danger signal to the host. The aim of this work was to verify the role of TgHSP70 as an alternative tool for active toxoplasmosis diagnosis, detecting the protein, specific antibodies and immune complexes (IC) in the experimental infection with T. gondii. Also, we observed the localization of this protein in T. gondii tachyzoites and its role in immunization procedures. Recombinant TgHSP70 was purified from E. coli and used to immunize chickens and Calomys callosus in order to produce specific antibodies anti-TgHSP70. It was observed by immunofluorescence that TgHSP70 is present in the cytoplasm of tachyzoites and could be associated to the inner layer of plasma membrane. It was also demonstrated by immunoblotting and ELISA that this protein is not secreted by T. gondii. Immunization of BALB/c and C57BL/6 mice only with TgHSP70 did not induce protection to challenge with ME-49 and RH strains of T. gondii after 30 of the last immunization. However, it was observed that spleen cells of non-immunized mice proliferate when stimulated in vitro with TgHSP70, indicating unspecific stimulation effect. Moreover, BALB/c and C57BL/6 mice were infected with T. gondii ME-49 strain and treated with dexamethasone (DXM) for toxoplasmosis reactivation. C57BL/6 mice treated with DXM presented higher parasite load in the brain than treated BALB/c mice. In addition, the treatment reduced the inflammatory changes in the brain of both lineages, although, these lesions were higher in C57BL/6 mice. It was verified by qPCR that infection induces TgHSP70 expression in the brain mainly in C57BL/6 mice, and also increases the specific protein concentration in sera from both lineages, but only leads IC formation in BALB/c mice. Finally, these data suggest that circulating TgHSP70 could be used as diagnostic tool for active infection in susceptible C57BL/6 mice and also that TgHSP70, in the other hand, could be used as adjuvant in immunization procedures.Fundação de Amparo a Pesquisa do Estado de Minas GeraisMestre em Imunologia e Parasitologia AplicadasToxoplasma gondii é um parasita intracelular obrigatório de animais homeotérmicos que expressa a proteína de choque térmico de 70 kDa (TgHSP70) durante a conversão de estágios. TgHSP70 é uma proteína com elevado potencial antigênico e sua presença no curso da infecção é um sinal de perigo para o hospedeiro. O objetivo deste trabalho foi avaliar o papel da TgHSP70 como ferramenta alternativa para o diagnóstico de toxoplasmose ativa, detectando a proteína, anticorpos específicos e complexos imunes (CI) circulantes em infecção experimental pelo parasita. Além disso, avaliamos a localização da proteína em taquizoítas de T. gondii e o papel dessa como antígeno vacinal. Deste modo, TgHSP70 recombinante foi purificada de E. coli e foi utilizada na imunização de galinhas e Calomys callosus para produção de anticorpos específicos anti-TgHSP70. Foi observado por imunofluorescência que TgHSP70 esta presente no citoplasma de taquizoítas e que esta pode estar associada com a membrana plasmática interna, sendo demonstrado por immunoblotting e ELISA que TgHSP70 não é secretada por T. gondii. A imunização de camundongos BALB/c e C57BL/6 com TgHSP70 apenas, não induziu proteção quando esses animais foram desafiados 30 dias após com as cepas RH ou ME-49 de T. gondii. Foi observado também que células de baço de animais não imunizados proliferam in vitro quando estimuladas com TgHSP70. Além disso, para verificar a presença de reativação da toxoplasmose, camundongos BALB/c e C57BL/6 foram infectados com a cepa ME-49 de T. gondii e tratados com dexametazona (DXM). Foi observado que animais C57BL/6 tratados com DXM apresentaram carga parasitária e alterações histológicas no cérebro aumentadas em relação aos camundongos BALB/c tratados, embora a inflamação tenha sido menor em ambas as linhagens quando comparado aos animais infectados e não tratados. Detectamos também por qPCR que a infecção induziu a expressão de TgHSP70 no cérebro dos animais, sendo maior no órgão dos camundongos C57BL/6, e também aumentou a concentração de TgHSP70 circulante no soro de ambas as linhagens e maior indução de anticorpos específicos e formação de CI em camundongos BALB/c. Por fim, estes dados sugerem que TgHSP70 circulante pode ser utilizada como ferramenta diagnostica de infecção ativa em camundongos C57BL/6, sendo que essa infecção é persistente nesses animais e que a TgHSP70, por outro lado, poderia ser utilizada como adjuvante em experimentos de imunização

Mecanismos de proteção induzidos pela imunização com TgHSP70 e de controle da inflamação pelo tratamento com STAg na infecção por Toxoplasma gondii

Toxoplasma gondii is an obligatory protozoan parasite that present several antigens capable of activating immune responses. In this work, we aimed to study two immunomodulatory antigens in pre-treatment for acute and immunization for chronic infection. The first was to evaluate the role of soluble tachyzoite antigen (STAg) on epithelial cells to control intestinal inflammation during acute infection. It was demonstrated STAg treatment was able to preserve Paneth cell numbers during infection, and to maintain epithelial integrity and function. We also observed that STAg treated and infected C57BL/6 mice produce secretory IgA against pathogenic bacteria, controlling microbiota and diminishing intestinal inflammation. The second objective was to evaluate the role of T. gondii heat shock protein of 70 kDa (TgHSP70) as a vaccinal antigen in murine chronic infection. It was observed that immunization with alum-adsorbed TgHSP70 reduced cyst numbers and diminished inflammation in the brain, and also induced high titers of TgHSP70-specific antibodies. We demonstrated that these antibodies do not act directly on the parasite, but seem to neutralize free TgHSP70 that induce nitric oxide production in RAW264.7 cells. In summary, this work demonstrated action mechanisms of two T. gondii immunomodulatory antigens, STAg and TgHSP70, and strengthens the use of these antigens as tools for vaccination or pre-treatment in order to control infection and intestinal inflammation.Fundação de Amparo a Pesquisa do Estado de Minas GeraisDoutor em Imunologia e Parasitologia AplicadasToxoplasma gondii é um parasita intracelular obrigatório que apresenta diversos antígenos capazes de modular a resposta imune. Neste trabalho, visamos estudar dois antígenos imunomoduladores através do pré-tratamento para fase aguda e de imunização para fase crônica da infecção. O primeiro objetivo foi estudar o papel do antígeno solúvel de taquizoítas (STAg) nas células epiteliais para controle da inflamação intestinal durante a infecção aguda por T. gondii. Foi demonstrado que o tratamento com STAg preserva o número de células de Paneth durante a infecção, além de manter a integridade e a função do epitélio intestinal. Observamos também que camundongos tratados com STAg produzem IgA secretória contra bactérias patogênicas, controlando a microbiota e diminuindo a inflamação intestinal. Em outra vertente, avaliamos o papel da proteína de choque térmico de 70 kDa de T. gondii (TgHSP70) como um antígeno vacinal contra a infecção crônica. Observamos que a imunização com TgHSP70 adsorvida em alúmen reduziu o número de cistos e a inflamação no cérebro de animais infectados, bem como induziu a produção de elevados títulos de anticorpos específicos e imunocomplexos anti-TgHSP70. Mostramos que estes anticorpos não exercem sua função diretamente no parasita, mas parece desenvolver seu papel neutralizando a TgHSP70 livre, a qual induz a produção de óxido nítrico pelas células RAW264.7. Em suma, este trabalho demonstrou os mecanismos de ação de dois antígenos imunomoduladores de T. gondii, TgHSP70 e STAg, e reforça o uso destes antígenos como ferramentas para vacinação ou pré-tratamento no controle da infecção e da inflamação intestinal

Retinoic Acid and Its Role in Modulating Intestinal Innate Immunity

Vitamin A (VA) is amongst the most well characterized food-derived nutrients with diverse immune modulatory roles. Deficiency in dietary VA has not only been associated with immune dysfunctions in the gut, but also with several systemic immune disorders. In particular, VA metabolite all-trans retinoic acid (atRA) has been shown to be crucial in inducing gut tropism in lymphocytes and modulating T helper differentiation. In addition to the widely recognized role in adaptive immunity, increasing evidence identifies atRA as an important modulator of innate immune cells, such as tolerogenic dendritic cells (DCs) and innate lymphoid cells (ILCs). Here, we focus on the role of retinoic acid in differentiation, trafficking and the functions of innate immune cells in health and inflammation associated disorders. Lastly, we discuss the potential involvement of atRA during the plausible crosstalk between DCs and ILCs

Recombinant TgHSP70 Immunization Protects against Toxoplasma gondii Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression

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

Clonotypic analysis of protective influenza M2e-specific lung resident Th17 memory cells reveals extensive functional diversity

The fate of tissue-resident memory CD4 T cells (Trm) has been incompletely investigated. Here we show that intranasal, but not parenteral, immunization with CTA1-3M2e-DD stimulated M2e-specific Th17 Trm cells, which conferred strong protection against influenza virus infection in the lung. These cells rapidly expanded upon infection and effectively restricted virus replication as determined by CD4 T cell depletion studies. Single-cell RNAseq transcriptomic and TCR VDJ-analysis of M2e-tetramer-sorted CD4 T cells on day 3 and 8 post infection revealed complete Th17-lineage dominance (no Th1 or Tregs) with extensive functional diversity and expression of gene markers signifying mature resident Trm cells (Cd69, Nfkbid, Brd2, FosB). Unexpectedly, the same TCR clonotype hosted cells with different Th17 subcluster functions (IL-17, IL-22), regulatory and cytotoxic cells, suggesting a tissue and context-dependent differentiation of reactivated Th17 Trm cells. A gene set enrichment analysis demonstrated up-regulation of regulatory genes (Lag3, Tigit, Ctla4, Pdcd1) in M2e-specific Trm cells on day 8, indicating a tissue damage preventing function. Thus, contrary to current thinking, lung M2e-specific Th17 Trm cells are sufficient for controlling infection and for protecting against tissue injury. These findings will have strong implications for vaccine development against respiratory virus infections and influenza virus infections, in particular