Multi-omics analysis of innate and adaptive responses to BCG vaccination reveals epigenetic cell states that predict trained immunity.

Immune responses are tightly regulated yet highly variable between individuals. To investigate human population variation of trained immunity, we immunized healthy individuals with Bacillus Calmette-Guérin (BCG). This live-attenuated vaccine induces not only an adaptive immune response against tuberculosis but also triggers innate immune activation and memory that are indicative of trained immunity. We established personal immune profiles and chromatin accessibility maps over a 90-day time course of BCG vaccination in 323 individuals. Our analysis uncovered genetic and epigenetic predictors of baseline immunity and immune response. BCG vaccination enhanced the innate immune response specifically in individuals with a dormant immune state at baseline, rather than providing a general boost of innate immunity. This study advances our understanding of BCG's heterologous immune-stimulatory effects and trained immunity in humans. Furthermore, it highlights the value of epigenetic cell states for connecting immune function with genotype and the environment.

Seasonal and Nonseasonal Longitudinal Variation of Immune Function.

Different components of the immune response show large variability between individuals, but they also vary within the same individual because of host and environmental factors. In this study, we report an extensive analysis of the immune characteristics of 56 individuals over four timepoints in 1 single year as part of the Human Functional Genomics Project. We characterized 102 cell subsets using flow cytometry; quantified production of eight cytokines and two chemokines in response to 20 metabolic, bacterial, fungal, and viral stimuli; and measured circulating markers of inflammation. Taking advantage of the longitudinal sampling, both seasonal and nonseasonal sources of variability were studied. The circulating markers of inflammation IL-18, IL-18 binding protein, and resistin displayed clear seasonal variability, whereas the strongest effect was observed for α-1 antitrypsin. Cytokine production capacity also showed strong seasonal changes, especially after stimulation with the influenza virus, Borrelia burgdorferi, and Escherichia coli Furthermore, we observed moderate seasonality effects on immune cell counts, especially in several CD4+/CD8+ T cell subpopulations. Age of the volunteers was an important factor influencing IFN-γ and IL-22 production, which matched the strong impact of age on several T cell subsets. Finally, on average, genetics accounted for almost 50% of the interindividual variance not already explained by age, sex, and body mass index, although this varies strongly for different parameters. In conclusion, seasonality is an important environmental factor that influences immune responses, in addition to specific genetic and nongenetic host factors, and this may well explain the seasonal variation in the incidence and severity of immune-mediated diseases.

Immunomodulatory aged neutrophils are augmented in blood and skin of psoriasis patients.

BACKGROUND: Neutrophil accumulation in the skin is a hallmark of psoriasis. Novel insights on neutrophil phenotypic and functional heterogeneity raise the question to what extent these cells contribute to the sustained inflammatory skin reaction. OBJECTIVE: We sought to examine the phenotype and functional properties of neutrophils in blood and skin of patients with psoriasis, and the effect of TNF-α and p40(IL-12/IL-23) antibody therapy on circulating neutrophils. METHODS: Thirty-two patients with psoriasis were enrolled in an observational study performed in 2 university hospitals. We evaluated neutrophil phenotype and function using in vitro (co)culture stimulation assays, flow cytometry, multiplex immunohistochemistry, and multispectral imaging of patient-derived blood and skin samples. RESULTS: Cluster of differentiation (CD)10pos and CD10neg neutrophils were increased in peripheral blood of patients with psoriasis. In CD10neg neutrophils, different maturation stages were observed, including a subset resembling aged neutrophils that was 3 times more abundant than in healthy individuals. These aged neutrophils displayed suboptimal canonical neutrophil functions and induced IL-17 and IFN-γ production by T cells in vitro, mediated by neutrophil extracellular trap formation. Also, mature and aged neutrophils were present in psoriatic skin and were found in the vicinity of T cells. Upon antibody therapy, numbers of these cells in circulation decreased. CONCLUSIONS: Patients with psoriasis reveal a unique neutrophil profile in circulation, and 2 distinct neutrophil subsets are present in psoriatic skin. Targeted biological treatment may aid in the containment of sustained neutrophil-mediated inflammation.

Blood-Based Immune Profiling Combined with Machine Learning Discriminates Psoriatic Arthritis from Psoriasis Patients.

Psoriasis (Pso) is a chronic inflammatory skin disease, and up to 30% of Pso patients develop psoriatic arthritis (PsA), which can lead to irreversible joint damage. Early detection of PsA in Pso patients is crucial for timely treatment but difficult for dermatologists to implement. We, therefore, aimed to find disease-specific immune profiles, discriminating Pso from PsA patients, possibly facilitating the correct identification of Pso patients in need of referral to a rheumatology clinic. The phenotypes of peripheral blood immune cells of consecutive Pso and PsA patients were analyzed, and disease-specific immune profiles were identified via a machine learning approach. This approach resulted in a random forest classification model capable of distinguishing PsA from Pso (mean AUC = 0.95). Key PsA-classifying cell subsets selected included increased proportions of differentiated CD4+CD196+CD183-CD194+ and CD4+CD196-CD183-CD194+ T-cells and reduced proportions of CD196+ and CD197+ monocytes, memory CD4+ and CD8+ T-cell subsets and CD4+ regulatory T-cells. Within PsA, joint scores showed an association with memory CD8+CD45RA-CD197- effector T-cells and CD197+ monocytes. To conclude, through the integration of in-depth flow cytometry and machine learning, we identified an immune cell profile discriminating PsA from Pso. This immune profile may aid in timely diagnosing PsA in Pso.

Platelet microparticles inhibit IL-17 production by regulatory T cells through P-selectin.

Self-tolerance and immune homeostasis are orchestrated by FOXP3(+)regulatory T cells (Tregs). Recent data have revealed that upon stimulation, Tregs may exhibit plasticity toward a proinflammatory phenotype, producing interleukin 17 (IL-17) and/or interferon γ (IFN-γ). Such deregulation of Tregs may contribute to the perpetuation of inflammatory processes, including graft-versus-host disease. Thus, it is important to identify immunomodulatory factors influencing Treg stability. Platelet-derived microparticles (PMPs) are involved in hemostasis and vascular health and have recently been shown to be intimately involved in (pathogenic) immune responses. Therefore, we investigated whether PMPs have the ability to affect Treg plasticity. PMPs were cocultured with healthy donor peripheral blood-derived Tregs that were stimulated with anti-CD3/CD28 monoclonal antibodies in the presence of IL-2, IL-15, and IL-1ß. PMPs prevented the differentiation of peripheral blood-derived Tregs into IL-17- and IFN-γ-producing cells, even in the presence of the IL-17-driving proinflammatory cytokine IL-1ß. The mechanism of action by which PMPs prevent Treg plasticity consisted of rapid and selective P-selectin-dependent binding of PMPs to a CCR6(+)HLA-DR(+)memory-like Treg subset and their ability to inhibit Treg proliferation, in part through CXCR3 engagement. The findings that ~8% of Tregs in the circulation of healthy individuals are CD41(+)P-selectin(+)and that distinct binding of patient plasma PMPs to Tregs was observed support in vivo relevance. These findings open the exciting possibility that PMPs actively regulate the immune response at sites of (vascular) inflammation, where they are known to accumulate and interact with leukocytes, consolidating the (vascular) healing process.

Blood immune cell profiling in adults with longstanding type 1 diabetes is associated with macrovascular complications.

Aims/hypothesis: There is increasing evidence for heterogeneity in type 1 diabetes mellitus (T1D): not only the age of onset and disease progression rate differ, but also the risk of complications varies markedly. Consequently, the presence of different disease endotypes has been suggested. Impaired T and B cell responses have been established in newly diagnosed diabetes patients. We hypothesized that deciphering the immune cell profile in peripheral blood of adults with longstanding T1D may help to understand disease heterogeneity. Methods: Adult patients with longstanding T1D and healthy controls (HC) were recruited, and their blood immune cell profile was determined using multicolour flow cytometry followed by a machine-learning based elastic-net (EN) classification model. Hierarchical clustering was performed to identify patient-specific immune cell profiles. Results were compared to those obtained in matched healthy control subjects. Results: Hierarchical clustering analysis of flow cytometry data revealed three immune cell composition-based distinct subgroups of individuals: HCs, T1D-group-A and T1D-group-B. In general, T1D patients, as compared to healthy controls, showed a more active immune profile as demonstrated by a higher percentage and absolute number of neutrophils, monocytes, total B cells and activated CD4+CD25+ T cells, while the abundance of regulatory T cells (Treg) was reduced. Patients belonging to T1D-group-A, as compared to T1D-group-B, revealed a more proinflammatory phenotype characterized by a lower percentage of FOXP3+ Treg, higher proportions of CCR4 expressing CD4 and CD8 T cell subsets, monocyte subsets, a lower Treg/conventional Tcell (Tconv) ratio, an increased proinflammatory cytokine (TNFα, IFNγ) and a decreased anti-inflammatory (IL-10) producing potential. Clinically, patients in T1D-group-A had more frequent diabetes-related macrovascular complications. Conclusions: Machine-learning based classification of multiparameter flow cytometry data revealed two distinct immunological profiles in adults with longstanding type 1 diabetes; T1D-group-A and T1D-group-B. T1D-group-A is characterized by a stronger pro-inflammatory profile and is associated with a higher rate of diabetes-related (macro)vascular complications.

HIV immunological non-responders are characterized by extensive immunosenescence and impaired lymphocyte cytokine production capacity.

Introduction: Immunological non-responders (INR) are people living with HIV (PLHIV) who fail to fully restore CD4+ T-cell counts despite complete viral suppression with antiretroviral therapy (ART). INR are at higher risk for non-HIV related morbidity and mortality. Previous research suggest persistent qualitative defects. Methods: The 2000HIV study (clinical trials NTC03994835) enrolled 1895 PLHIV, divided in a discovery and validation cohort. PLHIV with CD4 T-cell count <350 cells/mm3 after ≥2 years of suppressive ART were defined as INR and were compared to immunological responders (IR) with CD4 T-cell count >500 cells/mm3. Logistic and rank based regression were used to analyze clinical data, extensive innate and adaptive immunophenotyping, and ex vivo monocyte and lymphocyte cytokine production after stimulation with various stimuli. Results: The discovery cohort consisted of 62 INR and 1224 IR, the validation cohort of 26 INR and 243 IR. INR were older, had more advanced HIV disease before starting ART and had more frequently a history of non-AIDS related malignancy. INR had lower absolute CD4+ T-cell numbers in all subsets. Activated (HLA-DR+, CD38+) and exhausted (PD1+) subpopulations were proportionally increased in CD4 T-cells. Monocyte and granulocyte immunophenotypes were comparable. INR lymphocytes produced less IL-22, IFN-γ, IL-10 and IL-17 to stimuli. In contrast, monocyte cytokine production did not differ. The proportions of CD4+CD38+HLA-DR+ and CD4+PD1+ subpopulations showed an inversed correlation to lymphocyte cytokine production. Conclusions: INR compared to IR have hyperactivated and exhausted CD4+ T-cells in combination with lymphocyte functional impairment, while innate immune responses were comparable. Our data provide a rationale to consider the use of anti-PD1 therapy in INR.

11-deoxycortisol positively correlates with T cell immune traits in physiological conditions.

BACKGROUND: Endogenous steroid hormones have significant effects on inflammatory and immune processes, but the immunological activities of steroidogenesis precursors remain largely unexplored. METHODS: We conducted a systematic approach to examine the association between steroid hormones profile and immune traits in a cohort of 534 healthy volunteers. Serum concentrations of steroid hormones and their precursors (cortisol, progesterone, testosterone, androstenedione, 11-deoxycortisol and 17-OH progesterone) were determined by liquid chromatography-tandem mass spectrometry. Immune traits were evaluated by quantifying cellular composition of the circulating immune system and ex vivo cytokine responses elicited by major human pathogens and microbial ligands. An independent cohort of 321 individuals was used for validation, followed by in vitro validation experiments. FINDINGS: We observed a positive association between 11-deoxycortisol and lymphoid cellular subsets numbers and function (especially IL-17 response). The association with lymphoid cellularity was validated in an independent validation cohort. In vitro experiments showed that, as compared to androstenedione and 17-OH progesterone, 11-deoxycortisol promoted T cell proliferation and Candida-induced Th17 polarization at physiologically relevant concentrations. Functionally, 11-deoxycortisol-treated T cells displayed a more activated phenotype (PD-L1high CD25high CD62Llow CD127low) in response to CD3/CD28 co-stimulation, and downregulated expression of T-bet nuclear transcription factor. INTERPRETATION: Our findings suggest a positive association between 11-deoxycortisol and T-cell function under physiological conditions. Further investigation is needed to explore the potential mechanisms and clinical implications. FUNDING: Found in acknowledgements.

Umbilical cord-mesenchymal stem cells induce a memory phenotype in CD4+ T cells.

Inflammation is a physiological state where immune cells evoke a response against detrimental insults. Finding a safe and effective treatment for inflammation associated diseases has been a challenge. In this regard, human mesenchymal stem cells (hMSC), exert immunomodulatory effects and have regenerative capacity making it a promising therapeutic option for resolution of acute and chronic inflammation. T cells play a critical role in inflammation and depending on their phenotype, they can stimulate or suppress inflammatory responses. However, the regulatory effects of hMSC on T cells and the underlying mechanisms are not fully elucidated. Most studies focused on activation, proliferation, and differentiation of T cells. Here, we further investigated memory formation and responsiveness of CD4+ T cells and their dynamics by immune-profiling and cytokine secretion analysis. Umbilical cord mesenchymal stem cells (UC-MSC) were co-cultured with either αCD3/CD28 beads, activated peripheral blood mononuclear cells (PBMC) or magnetically sorted CD4+ T cells. The mechanism of immune modulation of UC-MSC were investigated by comparing different modes of action; transwell, direct cell-cell contact, addition of UC-MSC conditioned medium or blockade of paracrine factor production by UC-MSC. We observed a differential effect of UC-MSC on CD4+ T cell activation and proliferation using PBMC or purified CD4+ T cell co-cultures. UC-MSC skewed the effector memory T cells into a central memory phenotype in both co-culture conditions. This effect on central memory formation was reversible, since UC-MSC primed central memory cells were still responsive after a second encounter with the same stimuli. The presence of both cell-cell contact and paracrine factors were necessary for the most pronounced immunomodulatory effect of UC-MSC on T cells. We found suggestive evidence for a partial role of IL-6 and TGFß in the UC-MSC derived immunomodulatory function. Collectively, our data show that UC-MSCs clearly affect T cell activation, proliferation and maturation, depending on co-culture conditions for which both cell-cell contact and paracrine factors are needed.

Gram-positive anaerobic cocci guard skin homeostasis by regulating host-defense mechanisms.

In atopic dermatitis (AD), chronic skin inflammation is associated with skin barrier defects and skin microbiome dysbiosis including a lower abundance of Gram-positive anaerobic cocci (GPACs). We here report that, through secreted soluble factors, GPAC rapidly and directly induced epidermal host-defense molecules in cultured human keratinocytes and indirectly via immune-cell activation and cytokines derived thereof. Host-derived antimicrobial peptides known to limit the growth of Staphylococcus aureus-a skin pathogen involved in AD pathology-were strongly upregulated by GPAC-induced signaling through aryl hydrocarbon receptor (AHR)-independent mechanisms, with a concomitant AHR-dependent induction of epidermal differentiation genes and control of pro-inflammatory gene expression in organotypic human epidermis. By these modes of operandi, GPAC may act as an "alarm signal" and protect the skin from pathogenic colonization and infection in the event of skin barrier disruption. Fostering growth or survival of GPAC may be starting point for microbiome-targeted therapeutics in AD.

Immunological Effects of Anti‒IL-17/12/23 Therapy in Patients with Psoriasis Complicated by Candida Infections.

Biologics that block the T helper (Th) 17 pathway are very effective in the treatment of psoriasis and other inflammatory diseases. However, IL-17 is also crucial for antifungal host defense, and clinical trial data suggest an increase in the incidence of Candida infections during IL-17 inhibitor (IL-17i) therapy. We investigated the innate and adaptive immune responses of patients with psoriasis with a history of skin and/or mucosal candidiasis during IL-17i or IL-12/23 inhibitor therapy, comparing those responses with those of healthy controls. Patients with psoriasis with IL-17i showed significantly lower CD4+Th1-like (CCR6‒CXCR3+CCR4‒) and Th1 Th17-like (CD4+CCR6+CXCR3+CCR4‒) cell percentages. Patient cells stimulated with Candida albicans produced significantly lower IL-6 in the IL-12/23 inhibitor group and IL-1ß in the IL-17i group, whereas the release of TNF-α and ROS was similar between patients and controls. IFN-γ and IL-10 production in response to several stimuli after 7 days was particularly decreased in patients receiving IL-17i therapy. Finally, after stimulation with the polarizing cytokines IL-1ß and IL-23, the Th17 cytokine response was significantly lower in the IL-17i patient group. These innate and adaptive immune response defects can diminish antifungal host immune response and thereby increase susceptibility to candidiasis in patients treated with IL-17i or IL-12/23 inhibitor.

Comprehensive phenotyping of circulating immune cell subsets in people living with HIV.

Systemic chronic inflammation and immune dysfunction are recognized as drivers of the development of non-AIDS related comorbidities (NARCs) in people living with HIV (PLHIV). In order to lower the risk of NARCs, it is critical to elucidate what is the contribution of alterations in the composition and function of circulating immune cells to NARCs-related pathogenesis. Findings from previous immunophenotyping studies in PLHIV are highly heterogeneous and it is not fully understood to what extent phenotypic changes on immune cells play a role in the dysregulated inflammatory response observed. In this study, three flow cytometry panels were designed and standardized to phenotypically and functionally identify the main circulating immune cell subsets in PLHIV. To reduce variability, up to 10 markers out of the approximately 20 markers in each panel were used in a custom dry format DURA Innovations (LUCID product line). Intra-assay precision tests performed for the selected cell subsets showed that the three panels had a %CV below 18% for percent of positive cells and the MFI (mean fluorescent intensity) of lineage markers. Our reported pipeline for immunophenotypic analysis facilitated the discrimination of 1153 cell populations, providing an integrated overview of circulating innate and adaptative immune cells as well as the cells' functional status in terms of activation, exhaustion, and maturation. When combined with unsupervised computational techniques, this standardized immunophenotyping approach may support the discovery of novel phenotypes with clinical relevance in NARCs and demonstrate future utility in other immune-mediated diseases.

Mature neutrophils and a NF-κB-to-IFN transition determine the unifying disease recovery dynamics in COVID-19.

Disease recovery dynamics are often difficult to assess, as patients display heterogeneous recovery courses. To model recovery dynamics, exemplified by severe COVID-19, we apply a computational scheme on longitudinally sampled blood transcriptomes, generating recovery states, which we then link to cellular and molecular mechanisms, presenting a framework for studying the kinetics of recovery compared with non-recovery over time and long-term effects of the disease. Specifically, a decrease in mature neutrophils is the strongest cellular effect during recovery, with direct implications on disease outcome. Furthermore, we present strong indications for global regulatory changes in gene programs, decoupled from cell compositional changes, including an early rise in T cell activation and differentiation, resulting in immune rebalancing between interferon and NF-κB activity and restoration of cell homeostasis. Overall, we present a clinically relevant computational framework for modeling disease recovery, paving the way for future studies of the recovery dynamics in other diseases and tissues.

Multi-Omics Integration Reveals Only Minor Long-Term Molecular and Functional Sequelae in Immune Cells of Individuals Recovered From COVID-19.

The majority of COVID-19 patients experience mild to moderate disease course and recover within a few weeks. An increasing number of studies characterized the long-term changes in the specific anti-SARS-CoV-2 immune responses, but how COVID-19 shapes the innate and heterologous adaptive immune system after recovery is less well known. To comprehensively investigate the post-SARS-CoV-2 infection sequelae on the immune system, we performed a multi-omics study by integrating single-cell RNA-sequencing, single-cell ATAC-sequencing, genome-wide DNA methylation profiling, and functional validation experiments in 14 convalescent COVID-19 and 15 healthy individuals. We showed that immune responses generally recover without major sequelae after COVID-19. However, subtle differences persist at the transcriptomic level in monocytes, with downregulation of the interferon pathway, while DNA methylation also displays minor changes in convalescent COVID-19 individuals. However, these differences did not affect the cytokine production capacity of PBMCs upon different bacterial, viral, and fungal stimuli, although baseline release of IL-1Ra and IFN-γ was higher in convalescent individuals. In conclusion, we propose that despite minor differences in epigenetic and transcriptional programs, the immune system of convalescent COVID-19 patients largely recovers to the homeostatic level of healthy individuals.

Baseline effector cells predict response and NKT cells predict pulmonary toxicity in advanced breast cancer patients treated with everolimus and exemestane.

BACKGROUND: The mTOR inhibitor everolimus used in cancer has immune-modulating effects, potentially contributing to an antitumor response but also leading to pulmonary toxicity. We studied the association of immunological cell subsets with antitumor response and pulmonary toxicity in breast cancer patients treated with everolimus plus exemestane. METHODS: In this exploratory analysis, peripheral blood mononuclear cells (PBMCs) were collected at baseline and 14, 35, 60, and 90 days after start of treatment, and at the moment of pulmonary toxicity. The percentage and absolute number of T-cells, B-cells, NK-cells, monocytes and numerous subtypes were measured in peripheral blood using flow cytometric analysis and were compared using a (paired) t-test. RESULTS: From 20 patients, a total of 89 samples were collected. At baseline, responders versus non-responders had 0.86% versus 0.32% CD4+ effector cells (CD45RA+CD27-) (p = 0.1266) and non-response could be predicted with 0.71 sensitivity and 0.82 specificity. Patients who developed pulmonary toxicity compared to patients without pulmonary toxicity had relatively more NKT-cells at baseline (6.0% versus 1.3%, p = 0.0068, 59 k versus 12 k * 109/l, p = 0.0081) and at the moment of toxicity (5.2% versus 1.2%, p = 0.0106 and 47 k versus 16 k * 109/l, p = 0.0466). Baseline percentage NKT cells predicted pulmonary toxicity with 0.78 sensitivity and 1.0 specificity. CONCLUSIONS: Our results suggest that baseline CD4+ effector cells may be predictive of antitumor responses and baseline NKT cells may be predictive of pulmonary toxicity. These results warrant further validation.

Maternal, Decidual, and Neonatal Lymphocyte Composition Is Affected in Pregnant Kidney Transplant Recipients.

Pregnancy after renal transplantation is associated with an increased risk of complications. While a delicately balanced uterine immune system is essential for a successful pregnancy, little is known about the uterine immune environment of pregnant kidney transplant recipients. Moreover, children born to kidney transplant recipients are exposed in utero to immunosuppressive drugs, with possible consequences for neonatal outcomes. Here, we defined the effects of kidney transplantation on the immune cell composition during pregnancy with a cohort of kidney transplant recipients as well as healthy controls with uncomplicated pregnancies. Maternal immune cells from peripheral blood were collected during pregnancy as well as from decidua and cord blood obtained after delivery. Multiparameter flow cytometry was used to identify and characterize populations of cells. While systemic immune cell frequencies were altered in kidney transplant patients, immune cell dynamics over the course of pregnancy were largely similar to healthy women. In the decidua of women with a kidney transplant, we observed a decreased frequency of HLA-DR+ Treg, particularly in those treated with tacrolimus versus those that were treated with azathioprine next to tacrolimus, or with azathioprine alone. In addition, both the innate and adaptive neonatal immune system of children born to kidney transplant recipients was significantly altered compared to neonates born from uncomplicated pregnancies. Overall, our findings indicate a significant and distinct impact on the maternal systemic, uterine, and neonatal immune cell composition in pregnant kidney transplant recipients, which could have important consequences for the incidence of pregnancy complications, treatment decisions, and the offspring's health.

The Architecture of Circulating Immune Cells Is Dysregulated in People Living With HIV on Long Term Antiretroviral Treatment and Relates With Markers of the HIV-1 Reservoir, Cytomegalovirus, and Microbial Translocation.

Long-term changes in the immune system of successfully treated people living with HIV (PLHIV) remain incompletely understood. In this study, we assessed 108 white blood cell (WBC) populations in a cohort of 211 PLHIV on stable antiretroviral therapy and in 56 HIV-uninfected controls using flow cytometry. We show that marked differences exist in T cell maturation and differentiation between PLHIV and HIV-uninfected controls: PLHIV had reduced percentages of CD4+ T cells and naïve T cells and increased percentages of CD8+ T cells, effector T cells, and T helper 17 (Th17) cells, together with increased Th17/regulatory T cell (Treg) ratios. PLHIV also exhibited altered B cell maturation with reduced percentages of memory B cells and increased numbers of plasmablasts. Determinants of the T and B cell composition in PLHIV included host factors (age, sex, and smoking), markers of the HIV reservoir, and CMV serostatus. Moreover, higher circulating Th17 percentages were associated with higher plasma concentrations of interleukin (IL) 6, soluble CD14, the gut homing chemokine CCL20, and intestinal fatty acid binding protein (IFABP). The changes in circulating lymphocytes translated into functional changes with reduced interferon (IFN)- γ responses of peripheral blood mononuclear cells to stimulation with Candida albicans and Mycobacterium tuberculosis. In conclusion, this comprehensive analysis confirms the importance of persistent abnormalities in the number and function of circulating immune cells in PLHIV on stable treatment.

Synthetic Extracellular Matrices as a Toolbox to Tune Stem Cell Secretome.

The application of stem cell-derived secretome in regenerative therapies offers the key advantage that instead of the stem cells, only their effective paracrine compounds are in vivo delivered. Ideally, the secretome can be steered by the culture conditions of the stem cells. So far, most studies use stem cells cultured on stiff plastic substrates, not representative of their native 3D environment. In this study, cells are cultured inside synthetic polyisocyanide (PIC)-based hydrogels, which are minimal, tailorable, and highly reproducible biomimetic matrices. Secretome analysis of human adipose-derived stem cells (multiplex, ELISA) displays that matrix manipulation is a powerful tool to direct the secretome composition. As an example, cells in nonadherent PIC gels secrete increased levels of IL-10 and the conditioned media from 3D culture accelerate wound closure. In all, our PIC-based approach opens the door to dedicated matrix design to engineer the secretome for custom applications.

Persistent Systemic Inflammation in Patients With Severe Burn Injury Is Accompanied by Influx of Immature Neutrophils and Shifts in T Cell Subsets and Cytokine Profiles.

Severe burn injury causes local and systemic immune responses that can persist up to months, and can lead to systemic inflammatory response syndrome, organ damage and long-term sequalae such as hypertrophic scarring. To prevent these pathological conditions, a better understanding of the underlying mechanisms is essential. In this longitudinal study, we analyzed the temporal peripheral blood immune profile of 20 burn wound patients admitted to the intensive care by flow cytometry and secretome profiling, and compared this to data from 20 healthy subjects. The patient cohort showed signs of systemic inflammation and persistently high levels of pro-inflammatory soluble mediators, such as IL-6, IL-8, MCP-1, MIP-1ß, and MIP-3α, were measured. Using both unsupervised and supervised flow cytometry techniques, we observed a continuous release of neutrophils and monocytes into the blood for at least 39 days. Increased numbers of immature neutrophils were present in peripheral blood in the first three weeks after injury (0.1-2.8 × 106/ml after burn vs. 5 × 103/ml in healthy controls). Total lymphocyte numbers did not increase, but numbers of effector T cells as well as regulatory T cells were increased from the second week onward. Within the CD4+ T cell population, elevated numbers of CCR4+CCR6- and CCR4+CCR6+ cells were found. Altogether, these data reveal that severe burn injury induced a persistent innate inflammatory response, including a release of immature neutrophils, and shifts in the T cell composition toward an overall more pro-inflammatory phenotype, thereby continuing systemic inflammation and increasing the risk of secondary complications.