Chemokine and cytokine profiles in preterm and term labor, in preterm prelabor rupture of the membranes, and in normal pregnancy.

The objective of this study was to investigate the immune mechanisms involved in preterm labor (PTL), preterm prelabor rupture of the membranes (PPROM), and normal pregnancies. The second objective was to explore immune profiles in PTL for association with early ( < 34 gestational weeks (gw)) or instant ( < 48 h) delivery. This prospective observational multi-center study included women with singleton pregnancies with PTL (n = 80) or PPROM (n = 40) before 34 gw, women with normal pregnancies scheduled for antenatal visits (n = 44), and women with normal pregnancies in active labor at term (n = 40). Plasma samples obtained at admission were analyzed for cytokine and chemokine quantification using a multiplex bead assay in order to compare the immune profiles between PTL, PPROM, and normal pregnancies. In PTL, CXCL1 and CCL17 were significantly higher compared to gestational age-matched women at antenatal visits, whereas for PPROM, CXCL1 and IL-6 were increased. Women in term labor had a more pronounced inflammatory pattern with higher levels of CXCL1, CXCL8, and IL-6 compared with PTL (p = 0.007, 0.003, and 0.013, respectively), as well as higher levels of CCL17, CXCL1 and IL-6 (all p < 0.001) compared with the women at antenatal visits. In PTL, CXCL8 was higher in women with delivery before 34 gw, whereas CXCL8, GM-CSF, and IL-6 were significantly higher in women with delivery within 48 h. To conclude, PTL and PPROM were associated with a complex pattern of inflammation, both involving Th17 (CXCL1) responses. Although further studies are needed, CXCL8, GM-CSF, and IL-6 may be potential candidates for predicting preterm birth in PTL.

Effects of extremely preterm birth on cytokine and chemokine responses induced by T-cell activation during infancy.

Objectives: Extremely preterm (EPT; gestational week < 28 + 0, < 1000 g) neonates are vulnerable to infections and necrotising enterocolitis, important contributors to mortality and morbidity. However, knowledge regarding their immune maturation remains limited. We here investigated the longitudinal development of functional T-cell capacity in EPT infants. Methods: Peripheral blood mononuclear cells were isolated at 14th and 28th day (D) and at gestational week 36 + 0 (Gw36) from EPT infants, participated in a randomised, double-blind, placebo-controlled study of Lactobacillus reuteri DSM 17938 probiotic supplementation. Blood collected from 25 full-term (FT) infants at D14 was used as control. The secretion of immune mediators was determined through comprehensive Luminex panels after stimulation with human T-cell activator CD3/CD28 beads. Results: The levels of many mediators were low in EPT infants at D14, whereas the secretion of several chemokines was higher in EPT than in FT infants. Furthermore, Th2:Th1 cytokine ratios were higher in EPT than in FT infants. Progressively elevated secretion of, for example, IFN-γ, TNF and IL-17A in EPT infants was observed from D14 to D28 and then at Gw36. Elevated levels were observed for many proinflammatory mediators at D28. Probiotic supplementation or perinatal factors (e.g. clinical chorioamnionitis, preeclampsia and delivery mode) did not influence the cytokine and chemokine responses. Conclusions: Immune mediators induced by T-cell activation in EPT infants were mainly reduced at D14 and Th2 skewed compared to those in FT infants, but mostly recovered at Gw36, indicating immune maturation. Increased proinflammatory responses at D28 may be related to the heightened risk of severe immune-associated complications seen in EPT infants.

Maternal prebiotic supplementation during pregnancy and lactation modifies the microbiome and short chain fatty acid profile of both mother and infant.

BACKGROUND & AIMS: Improving maternal gut health in pregnancy and lactation is a potential strategy to improve immune and metabolic health in offspring and curtail the rising rates of inflammatory diseases linked to alterations in gut microbiota. Here, we investigate the effects of a maternal prebiotic supplement (galacto-oligosaccharides and fructo-oligosaccharides), ingested daily from <21 weeks' gestation to six months' post-partum, in a double-blinded, randomised placebo-controlled trial. METHODS: Stool samples were collected at multiple timepoints from 74 mother-infant pairs as part of a larger, double-blinded, randomised controlled allergy intervention trial. The participants were randomised to one of two groups; with one group receiving 14.2 g per day of prebiotic powder (galacto-oligosaccharides GOS and fructo-oligosaccharides FOS in ratio 9:1), and the other receiving a placebo powder consisting of 8.7 g per day of maltodextrin. The faecal microbiota of both mother and infants were assessed based on the analysis of bacterial 16S rRNA gene (V4 region) sequences, and short chain fatty acid (SCFA) concentrations in stool. RESULTS: Significant differences in the maternal microbiota profiles between baseline and either 28-weeks' or 36-weeks' gestation were found in the prebiotic supplemented women. Infant microbial beta-diversity also significantly differed between prebiotic and placebo groups at 12-months of age. Supplementation was associated with increased abundance of commensal Bifidobacteria in the maternal microbiota, and a reduction in the abundance of Negativicutes in both maternal and infant microbiota. There were also changes in SCFA concentrations with maternal prebiotics supplementation, including significant differences in acetic acid concentration between intervention and control groups from 20 to 28-weeks' gestation. CONCLUSION: Maternal prebiotic supplementation of 14.2 g per day GOS/FOS was found to favourably modify both the maternal and the developing infant gut microbiome. These results build on our understanding of the importance of maternal diet during pregnancy, and indicate that it is possible to intervene and modify the development of the infant microbiome by dietary modulation of the maternal gut microbiome.

Proteomics reveal biomarkers for diagnosis, disease activity and long-term disability outcomes in multiple sclerosis.

Sensitive and reliable protein biomarkers are needed to predict disease trajectory and personalize treatment strategies for multiple sclerosis (MS). Here, we use the highly sensitive proximity-extension assay combined with next-generation sequencing (Olink Explore) to quantify 1463 proteins in cerebrospinal fluid (CSF) and plasma from 143 people with early-stage MS and 43 healthy controls. With longitudinally followed discovery and replication cohorts, we identify CSF proteins that consistently predicted both short- and long-term disease progression. Lower levels of neurofilament light chain (NfL) in CSF is superior in predicting the absence of disease activity two years after sampling (replication AUC = 0.77) compared to all other tested proteins. Importantly, we also identify a combination of 11 CSF proteins (CXCL13, LTA, FCN2, ICAM3, LY9, SLAMF7, TYMP, CHI3L1, FYB1, TNFRSF1B and NfL) that predict the severity of disability worsening according to the normalized age-related MS severity score (replication AUC = 0.90). The identification of these proteins may help elucidate pathogenetic processes and might aid decisions on treatment strategies for persons with MS.

Crohn's patients and healthy infants share immunodominant B cell response to commensal flagellin peptide epitopes.

About half of patients with Crohn's disease (CD) develop selective serum IgG response to flagellin proteins of the Lachnospiraceae family. Here, we identified a dominant B cell peptide epitope in CD, locating in the highly conserved "hinge region" between the D0 and D1 domains at the amino-terminus of Lachnospiraceae flagellins. Serum IgG reactive to this epitope is present at an elevated level in adult CD patients and in pediatric CD patients at diagnosis. Most importantly, high levels of serum IgG to the hinge epitope were found in most infants from 3 different geographic regions (Uganda, Sweden, and the USA) at one year of age. This vigorous homeostatic response decrements with age as it is not present in healthy adults. These data identify a distinct subset of CD patients, united by a shared reactivity to this dominant flagellin epitope that may represent failure of a homeostatic response beginning in infancy.

The role of chemokines in type 1 diabetes-associated neuropathy.

INTRODUCTION: To investigate whether circulating chemokines contribute to the development of diabetic peripheral neuropathy (DPN) in patients with type 1 diabetes (T1D). METHODS: Fifty-two patients with childhood-onset T1D (mean age 28 ± 4 yrs.; diabetes duration 19.5 ± 5.5 yrs.) and 19 control subjects (mean age 26.5 ± 4.5 yrs.) were included in a cross-sectional analysis of this long-term longitudinal cohort study. A subgroup of 24 patients was followed prospectively for a further 10 yrs. Plasma levels of Th1- (CXCL9, CXCL10 and CXCL11), Th2- (CCL17 and CCL22) and Th17-associated (CXCL8 and CCL20) chemokines were assessed in all subjects. Additionally, the TID patients underwent clinical examination and electroneurography. RESULTS: The frequency of neuropathy was 21% (11/52). Higher levels of CXCL9 levels were found in patients with DPN compared with control subjects (p = .019); by contrast, no difference between patients without DPN and control subjects was seen after adjustment for multiple comparisons. In patients with DPN, CXCL10 correlated negatively with suralis MCV and suralis SNAP (rho -0.966, p < .001 and rho -0.738, p < .001, respectively) and was positively correlated with the vibration perception threshold (rho 0.639, p = .034), while CXCL8 correlated negatively with the cold perception threshold (rho -0.645, p = .032). The frequency of neuropathy increased to 54% (13/24) in the subgroup of 23 TID patients, followed by an additional 10 yrs. CONCLUSIONS: Changes in Th1- and Th17-associated chemokines were associated with impaired peripheral sensory nerve function and nerve conduction after long disease duration in childhood-onset T1D.

Prominent epigenetic and transcriptomic changes in CD4+ and CD8+ T cells during and after pregnancy in women with multiple sclerosis and controls.

BACKGROUND: Multiple sclerosis (MS) is a neuroinflammatory disease in which pregnancy leads to a temporary amelioration in disease activity as indicated by the profound decrease in relapses rate during the 3rd trimester of pregnancy. CD4+ and CD8+ T cells are implicated in MS pathogenesis as being key regulators of inflammation and brain lesion formation. Although Tcells are prime candidates for the pregnancy-associated improvement of MS, the precise mechanisms are yet unclear, and in particular, a deep characterization of the epigenetic and transcriptomic events that occur in peripheral T cells during pregnancy in MS is lacking. METHODS: Women with MS and healthy controls were longitudinally sampled before, during (1st, 2nd and 3rd trimesters) and after pregnancy. DNA methylation array and RNA sequencing were performed on paired CD4+ and CD8+ T cells samples. Differential analysis and network-based approaches were used to analyze the global dynamics of epigenetic and transcriptomic changes. RESULTS: Both DNA methylation and RNA sequencing revealed a prominent regulation, mostly peaking in the 3rd trimester and reversing post-partum, thus mirroring the clinical course with improvement followed by a worsening in disease activity. This rebound pattern was found to represent a general adaptation of the maternal immune system, with only minor differences between MS and controls. By using a network-based approach, we highlighted several genes at the core of this pregnancy-induced regulation, which were found to be enriched for genes and pathways previously reported to be involved in MS. Moreover, these pathways were enriched for in vitro stimulated genes and pregnancy hormones targets. CONCLUSION: This study represents, to our knowledge, the first in-depth investigation of the methylation and expression changes in peripheral CD4+ and CD8+ T cells during pregnancy in MS. Our findings indicate that pregnancy induces profound changes in peripheral T cells, in both MS and healthy controls, which are associated with the modulation of inflammation and MS activity.

Immune-related microRNAs in breast milk and their relation to regulatory T cells in breastfed children.

BACKGROUND: The immunomodulatory capacity of breast milk may partially be mediated by microRNAs (miRNA), small RNA molecules that regulate gene expression on a post-transcriptional level and are hypothesized to be involved in modulation of immunological pathways. Here, we evaluate the expression of immune-related miRNAs in breast milk after pre- and postnatal supplementation with Limosilactobacillus reuteri and omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), and the association to infant regulatory T cell (Treg) frequencies. METHODS: One-hundred and twenty women included in a double-blind, randomized, placebo-controlled allergy intervention trial received L. reuteri and/or ω-3 PUFAs daily from gestational week 20. Using Taqman qPCR, 24 miRNAs were analyzed from breast milk obtained at birth (colostrum) and after 3 months (mature milk) of lactation. The proportion of activated and resting Treg cells were analyzed in infant blood using flow cytometry at 6, 12, and 24 months. RESULTS: Relative expression changed significantly over the lactation period for most of the miRNAs; however, the expression was not significantly influenced by any of the supplements. Colostrum miR-181a-3p correlated with resting Treg cell frequencies at 6 months. Colostrum miR-148a-3p and let-7d-3p correlated with the frequencies of activated Treg cells at 24 months, as did mature milk miR-181a-3p and miR-181c-3p. CONCLUSION: Maternal supplementation with L. reuteri and ω-3 PUFAs did not significantly affect the relative miRNA expression in breast milk. Interestingly, some of the miRNAs correlate with Treg subpopulations in the breastfed children, supporting the hypothesis that breast milk miRNAs could be important in infant immune regulation. TRIAL REGISTRATION: ClinicalTrials.gov-ID: NCT01542970.

Intralymphatic immunotherapy with birch and grass pollen extracts. A randomized double-blind placebo-controlled clinical trial.

INTRODUCTION: There is a need to evaluate the safety and efficacy of intralymphatic immunotherapy (ILIT) for inducing tolerance in patients with allergic rhinitis. METHODS: Thirty-seven patients with seasonal allergic symptoms to birch and grass pollen and skin prick test >3 mm and/or IgE to birch and timothy >0.35 kU/L were randomized to either ILIT, with three doses of 0.1 mL of birch pollen and 5-grass pollen allergen extracts on aluminium hydroxide (10,000 SQ-U/ml; ALK-Abelló) or placebo using ultrasound-guided intralymphatic injections at monthly intervals. Daily combined symptom medical score and rhinoconjunctivitis total symptom score were recorded during the peak pollen seasons the year before and after treatment. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were recorded annually starting 2 years after treatment. Circulating proportions of T helper cell subsets and allergen-induced cytokine and chemokine production were analysed using flow cytometry and ELISA. RESULTS: There were no differences between the groups related to daily combined symptom medical score the year before and after treatment. Two years after ILIT (after unblinding), the actively treated group reported significantly fewer symptoms, lower medication use and improved quality of life than did the placebo group. After the pollen seasons the year after ILIT, T regulatory cell frequencies and grass-induced IFN-γ levels increased only in the actively treated group. CONCLUSION: In this randomized controlled trial, ILIT with birch and grass pollen extract was safe and accompanied by immunological changes. Further studies are required to confirm or refute the efficacy of the treatment.

Functional SARS-CoV-2 cross-reactive CD4+ T cells established in early childhood decline with age.

Pre-existing SARS-CoV-2-reactive T cells have been identified in SARS-CoV-2-unexposed individuals, potentially modulating COVID-19 and vaccination outcomes. Here, we provide evidence that functional cross-reactive memory CD4+ T cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is established in early childhood, mirroring early seroconversion with seasonal human coronavirus OC43. Humoral and cellular immune responses against OC43 and SARS-CoV-2 were assessed in SARS-CoV-2-unexposed children (paired samples at age two and six) and adults (age 26 to 83). Pre-existing SARS-CoV-2-reactive CD4+ T cell responses targeting spike, nucleocapsid, and membrane were closely linked to the frequency of OC43-specific memory CD4+ T cells in childhood. The functional quality of the cross-reactive memory CD4+ T cell responses targeting SARS-CoV-2 spike, but not nucleocapsid, paralleled OC43-specific T cell responses. OC43-specific antibodies were prevalent already at age two. However, they did not increase further with age, contrasting with the antibody magnitudes against HKU1 (ß-coronavirus), 229E and NL63 (α-coronaviruses), rhinovirus, Epstein-Barr virus (EBV), and influenza virus, which increased after age two. The quality of the memory CD4+ T cell responses peaked at age six and subsequently declined with age, with diminished expression of interferon (IFN)-γ, interleukin (IL)-2, tumor necrosis factor (TNF), and CD38 in late adulthood. Age-dependent qualitative differences in the pre-existing SARS-CoV-2-reactive T cell responses may reflect the ability of the host to control coronavirus infections and respond to vaccination.

RNA-sequencing and mass-spectrometry proteomic time-series analysis of T-cell differentiation identified multiple splice variants models that predicted validated protein biomarkers in inflammatory diseases.

Profiling of mRNA expression is an important method to identify biomarkers but complicated by limited correlations between mRNA expression and protein abundance. We hypothesised that these correlations could be improved by mathematical models based on measuring splice variants and time delay in protein translation. We characterised time-series of primary human naïve CD4+ T cells during early T helper type 1 differentiation with RNA-sequencing and mass-spectrometry proteomics. We performed computational time-series analysis in this system and in two other key human and murine immune cell types. Linear mathematical mixed time delayed splice variant models were used to predict protein abundances, and the models were validated using out-of-sample predictions. Lastly, we re-analysed RNA-seq datasets to evaluate biomarker discovery in five T-cell associated diseases, further validating the findings for multiple sclerosis (MS) and asthma. The new models significantly out-performing models not including the usage of multiple splice variants and time delays, as shown in cross-validation tests. Our mathematical models provided more differentially expressed proteins between patients and controls in all five diseases. Moreover, analysis of these proteins in asthma and MS supported their relevance. One marker, sCD27, was validated in MS using two independent cohorts for evaluating response to treatment and disease prognosis. In summary, our splice variant and time delay models substantially improved the prediction of protein abundance from mRNA expression in three different immune cell types. The models provided valuable biomarker candidates, which were further validated in MS and asthma.

Plasma protein profiling reveals dynamic immunomodulatory changes in multiple sclerosis patients during pregnancy.

Multiple sclerosis (MS) is a chronic autoimmune neuroinflammatory and neurodegenerative disorder of the central nervous system. Pregnancy represents a natural modulation of the disease course, where the relapse rate decreases, especially in the 3rd trimester, followed by a transient exacerbation after delivery. Although the exact mechanisms behind the pregnancy-induced modulation are yet to be deciphered, it is likely that the immune tolerance established during pregnancy is involved. In this study, we used the highly sensitive and specific proximity extension assay technology to perform protein profiling analysis of 92 inflammation-related proteins in MS patients (n=15) and healthy controls (n=10), longitudinally sampled before, during, and after pregnancy. Differential expression analysis was performed using linear models and p-values were adjusted for false discovery rate due to multiple comparisons. Our findings reveal gradual dynamic changes in plasma proteins that are most prominent during the 3rd trimester while reverting post-partum. Thus, this pattern reflects the disease activity of MS during pregnancy. Among the differentially expressed proteins in pregnancy, several proteins with known immunoregulatory properties were upregulated, such as PD-L1, LIF-R, TGF-ß1, and CCL28. On the other hand, inflammatory chemokines such as CCL8, CCL13, and CXCL5, as well as members of the tumor necrosis factor family, TRANCE and TWEAK, were downregulated. Further in-depth studies will reveal if these proteins can serve as biomarkers in MS and whether they are mechanistically involved in the disease amelioration and worsening. A deeper understanding of the mechanisms involved may identify new treatment strategies mimicking the pregnancy milieu.

Study Protocol for a Randomised Controlled Trial Investigating the Effects of Maternal Prebiotic Fibre Dietary Supplementation from Mid-Pregnancy to Six Months' Post-Partum on Child Allergic Disease Outcomes.

Infant allergy is the most common early manifestation of an increasing propensity for inflammation and immune dysregulation in modern environments. Refined low-fibre diets are a major risk for inflammatory diseases through adverse effects on the composition and function of gut microbiota. This has focused attention on the potential of prebiotic dietary fibres to favourably change gut microbiota, for local and systemic anti-inflammatory effects. In pregnancy, the immunomodulatory effects of prebiotics may also have benefits for the developing fetal immune system, and provide a potential dietary strategy to reduce the risk of allergic disease. Here, we present the study protocol for a double-blinded, randomised controlled trial investigating the effects of maternal prebiotics supplementation on child allergic disease outcomes. Eligible pregnant women have infants with a first-degree relative with a history of medically diagnosed allergic disease. Consented women are randomised to consume either prebiotics (galacto-oligosaccharides and fructo-oligosaccharides) or placebo (maltodextrin) powder daily from 18-20 weeks' gestation to six months' post-partum. The target sample size is 652 women. The primary outcome is infant medically diagnosed eczema; secondary outcomes include allergen sensitisation, food allergies and recurrent wheeze. Breast milk, stool and blood samples are collected at multiple timepoints for further analysis.

Intralymphatic immunotherapy with one or two allergens renders similar clinical response in patients with allergic rhinitis due to birch and grass pollen.

INTRODUCTION: There is a need for a fast, efficient and safe way to induce tolerance in patients with severe allergic rhinitis. Intralymphatic immune therapy has been shown to be effective. METHODS: Patients with severe birch and timothy allergy were randomized and received three doses of 0.1 ml of birch and 5-grass allergen extracts (10,000 SQ units/ml, ALK-Abelló), or birch and placebo or 5-grass and placebo by ultrasound-guided injections into inguinal lymph nodes at monthly intervals. Rhinoconjunctivitis total symptom score, medication score and rhinoconjunctivitis quality of life questionnaire were evaluated before treatment and after each birch and grass pollen season during three subsequent years. Circulating proportions of T helper subsets and allergen-induced cytokine and chemokine production were analysed by flow cytometry and Luminex. RESULTS: The three groups reported fewer symptoms, lower use of medication and improved quality of life during the birch and grass pollen seasons each year after treatment at an almost similar rate independently of treatment with one or two allergens. Mild local pain was the most common adverse event. IgE levels to birch decreased, whereas birch-induced IL-10 secretion increased in all three groups. IgG4 levels to birch and timothy and skin prick test reactivity remained mainly unchanged. Conjunctival challenge tests with timothy extract showed a higher threshold for allergen. In all three groups, regulatory T cell frequencies were increased 3 years after treatment. CONCLUSIONS: Intralymphatic immunotherapy with one or two allergens in patients with grass and birch pollen allergy was safe, effective and may be associated with bystander immune modulatory responses. CLINICAL TRIAL REGISTRATION: EudraCT (2013-004726-28).

Progesterone Inhibits the Establishment of Activation-Associated Chromatin During TH1 Differentiation.

TH1-mediated diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) improve during pregnancy, coinciding with increasing levels of the pregnancy hormone progesterone (P4), highlighting P4 as a potential mediator of this immunomodulation. Here, we performed detailed characterization of how P4 affects the chromatin and transcriptomic landscape during early human TH1 differentiation, utilizing both ATAC-seq and RNA-seq. Time series analysis of the earlier events (0.5-24 hrs) during TH1 differentiation revealed that P4 counteracted many of the changes induced during normal differentiation, mainly by downregulating key regulatory genes and their upstream transcription factors (TFs) involved in the initial T-cell activation. Members of the AP-1 complex such as FOSL1, FOSL2, JUN and JUNB were particularly affected, in both in promoters and in distal regulatory elements. Moreover, the changes induced by P4 were significantly enriched for disease-associated changes related to both MS and RA, revealing several shared upstream TFs, where again JUN was highlighted to be of central importance. Our findings support an immune regulatory role for P4 during pregnancy by impeding T-cell activation, a crucial checkpoint during pregnancy and in T-cell mediated diseases, and a central event prior to T-cell lineage commitment. Indeed, P4 is emerging as a likely candidate involved in disease modulation during pregnancy and further studies evaluating P4 as a potential treatment option are needed.

CD4+ T-cell DNA methylation changes during pregnancy significantly correlate with disease-associated methylation changes in autoimmune diseases.

Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4+ T-cells in non-pregnant and pregnant women, during the 1st and 2nd trimester, using the Illumina Infinium Human Methylation 450K array, and explored how these changes were related to autoimmune diseases that are known to be affected during pregnancy. Pregnancy was associated with several hundreds of methylation differences, particularly during the 2nd trimester. A network-based modular approach identified several genes, e.g., CD28, FYN, VAV1 and pathways related to T-cell signalling and activation, highlighting T-cell regulation as a central component of the observed methylation alterations. The identified pregnancy module was significantly enriched for disease-associated methylation changes related to multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. A negative correlation between pregnancy-associated methylation changes and disease-associated changes was found for multiple sclerosis and rheumatoid arthritis, diseases that are known to improve during pregnancy whereas a positive correlation was found for systemic lupus erythematosus, a disease that instead worsens during pregnancy. Thus, the directionality of the observed changes is in line with the previously observed effect of pregnancy on disease activity. Our systems medicine approach supports the importance of the methylome in immune regulation of T-cells during pregnancy. Our findings highlight the relevance of using pregnancy as a model for understanding and identifying disease-related mechanisms involved in the modulation of autoimmune diseases.Abbreviations: BMIQ: beta-mixture quantile dilation; DMGs: differentially methylated genes; DMPs: differentially methylated probes; FE: fold enrichment; FDR: false discovery rate; GO: gene ontology; GWAS: genome-wide association studies; MDS: multidimensional scaling; MS: multiple sclerosis; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffered saline; PPI; protein-protein interaction; RA: rheumatoid arthritis; SD: standard deviation; SLE: systemic lupus erythematosus; SNP: single nucleotide polymorphism; TH: CD4+ T helper cell; VIStA: diVIsive Shuffling Approach.

Mucosa associated invariant T and natural killer cells in active and budesonide treated collagenous colitis patients.

Introduction: Collagenous colitis (CC) is an inflammatory bowel disease, which usually responds to budesonide treatment. Our aim was to study the immunological background of the disease. Methods: Analyses of peripheral and mucosal MAIT (mucosa associated invariant T cells) and NK (natural killer) cells were performed with flow cytometry. Numbers of mucosal cells were calculated using immunohistochemistry. We studied the same patients with active untreated CC (au-CC) and again while in remission on budesonide treatment. Budesonide refractory patients and healthy controls were also included. The memory marker CD45R0 and activation marker CD154 and CD69 were used to further study the cells. Finally B cells, CD4+ and CD8+ T cells were also analysed. Results: The percentages of circulating CD56dimCD16+ NK cells as well as MAIT cells (CD3+TCRVa7.2+CD161+) were decreased in au-CC compared to healthy controls. This difference was not seen in the mucosa; where we instead found increased numbers of mucosal CD4+ T cells and CD8+ T cells in au-CC. Mucosal immune cell numbers were not affected by budesonide treatment. In refractory CC we found increased mucosal numbers of MAIT cells, CD4+ and CD8+ T cells compared to au-CC. Discussion: Patients with active collagenous colitis have lower percentages of circulating MAIT and NK cells. However, there was no change of these cells in the colonic mucosa. Most mucosal cell populations were increased in budesonide refractory as compared to au-CC patients, particularly the number of MAIT cells. This may indicate that T cell targeting therapy could be an alternative in budesonide refractory CC.

Differential effects of estradiol and progesterone on human T cell activation in vitro.

Estradiol (E2) and progesterone (P4) are steroid hormones important for the regulation of immune responses during pregnancy. Their increasing levels coincide with an improvement of T cell-mediated diseases such as multiple sclerosis (MS). Although immune-endocrine interactions are involved in this phenomenon, the relative contribution of hormones is not known. We here report a direct comparison of E2- and P4-mediated effects on human CD4+ T cells, key cells in immune regulation. T cells were stimulated to obtain different activation levels and exposed to a broad range of hormone concentrations. Activation level was assessed by CD69/CD25 expression by flow cytometry, and secreted proteins (n = 196) were measured in culture supernatants using proximity extension assay and electrochemiluminescence immunoassay. We found that in low activated cells, pregnancy-relevant E2 concentrations increased activation and the secretion of several immune- and inflammation-related proteins. P4, on the other hand, showed a biphasic pattern, where serum-related concentrations upregulated activation and protein secretion while placenta-relevant concentrations induced a prominent dampening irrespective of the initial activation level. Our results demonstrate the importance of P4 as a major hormone in the immune modulation of T cells during pregnancy and emphasize the need to further evaluate its potency in the treatment of diseases like MS.

A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials.

16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials. For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).