Peptidase inhibitor 16 identifies a human regulatory T-cell subset with reduced FOXP3 expression over the first year of recent onset type 1 diabetes.

CD4+ T-cell subsets play a major role in the host response to infection, and a healthy immune system requires a fine balance between reactivity and tolerance. This balance is in part maintained by regulatory T cells (Treg), which promote tolerance, and loss of immune tolerance contributes to autoimmunity. As the T cells which drive immunity are diverse, identifying and understanding how these subsets function requires specific biomarkers. From a human CD4 Tconv/Treg cell genome wide analysis we identified peptidase inhibitor 16 (PI16) as a CD4 subset biomarker and we now show detailed analysis of its distribution, phenotype and links to Treg function in type 1 diabetes. To determine the clinical relevance of Pi16 Treg, we analysed PI16+ Treg cells from type 1 diabetes patient samples. We observed that FOXP3 expression levels declined with disease progression, suggesting loss of functional fitness in these Treg cells in Type 1 diabetes, and in particular the rate of loss of FOXP3 expression was greatest in the PI16+ve Treg. We propose that PI16 has utility as a biomarker of functional human Treg subsets and may be useful for tracking loss of immune function in vivo. The ability to stratify at risk patients so that tailored interventions can be applied would open the door to personalised medicine for Type 1 diabetes.

Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients.

Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit colo-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The ß-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). ß-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived ß-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in µ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for ß-endorphin secretion in humans. IBS patients have lower monocyte derived ß-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.

CD4(+) and CD8(+) regulatory T cells in chronic rhinosinusitis mucosa.

BACKGROUND: Chronic rhinosinusitis (CRS) mucosal inflammation is characterized by an accumulation of effector-memory T cells, but their immune regulatory potential has not been adequately examined. Coexpression of transcription factor, forkhead box P3 (Foxp3), and interleukin-2 receptor, CD25, in CD4(+) and CD8(+) T cells is linked with regulatory function in humans. The aim of this study was to investigate the regulatory T cell (Treg) phenotype of CD4(+) (CD4Treg) and CD8(+) (CD8Treg) T cells in peripheral blood (PB) and sinus mucosa of CRS patients. METHODS: Prospective study was performed involving 32 CRS with nasal polyp (CRSwNP), 14 CRS without nasal polyp (CRSsNP), and 8 control patients. Sinus and PB T lymphocytes were stained with CD3, CD4, CD8, CD25, and Foxp3 and analyzed using flow cytometry. Relevant clinical characteristics, sinus bacterial culture results, and eosinophilic mucus were examined. RESULTS: Sinus mucosa had a higher percentage of CD4Treg (CD3(+)CD4(+)CD25(+)Foxp3(+)) population compared with PB in all patients. The percentage of PB CD4Treg and CD8Treg (CD3(+)CD8(+)CD25(+)Foxp3(+)) was not significantly different between the study groups. CRS mucosal tissue had a higher percentage of CD4Treg and activated T-helper cells than controls. There was no significant difference in PB and mucosal CD4Treg populations in CRS patients based on the presence of allergy, sinus culture results, or eosinophilic mucus. In controls, increased mucosal CD4Treg correlated with coexisting allergy. Although overall CD4Treg numbers were higher, the regulatory potential of activated CD4(+) T cells (CD4Treg/activated T-helper cell ratio) was significantly lower in CRS mucosa compared with controls. The CD8Treg subset was also significantly reduced in CRSwNP mucosa compared with controls. CONCLUSION: A higher percentage of CD4Treg and activated T-helper cells in CRS mucosa suggests increased inflammation in CRS, independent of the presence of allergy, microbial culture results, or eosinophilic mucus. However, the decreased ratio of CD4Treg versus activated T-helper cells in CRS and reduced CD8Treg population in CRSwNPs indicates an inflammatory bias and the inability to control mucosal disease.

CD4+ and CD8+ regulatory T cells in chronic rhinosinusitis mucosa.

BACKGROUND: Chronic rhinosinusitis (CRS) mucosal inflammation is characterized by an accumulation of effector-memory T cells, but their immune regulatory potential has not been adequately examined. Coexpression of transcription factor, forkhead box P3 (Foxp3), and interleukin-2 receptor, CD25, in CD4+ and CD8+ T cells is linked with regulatory function in humans. The aim of this study was to investigate the regulatory T cell (Treg) phenotype of CD4+ (CD4Treg) and CD8+ (CD8Treg) T cells in peripheral blood (PB) and sinus mucosa of CRS patients. METHODS: Prospective study was performed involving 32 CRS with nasal polyp (CRSwNP), 14 CRS without nasal polyp (CRSsNP), and 8 control patients. Sinus and PB T lymphocytes were stained with CD3, CD4, CD8, CD25, and Foxp3 and analyzed using flow cytometry. Relevant clinical characteristics, sinus bacterial culture results, and eosinophilic mucus were examined. RESULTS: Sinus mucosa had a higher percentage of CD4Treg (CD3+CD4+CD25+Foxp3+) population compared with PB in all patients. The percentage of PB CD4Treg and CD8Treg (CD3+CD8+CD25+Foxp3+) was not significantly different between the study groups. CRS mucosal tissue had a higher percentage of CD4Treg and activated T-helper cells than controls. There was no significant difference in PB and mucosal CD4Treg populations in CRS patients based on the presence of allergy, sinus culture results, or eosinophilic mucus. In controls, increased mucosal CD4Treg correlated with coexisting allergy. Although overall CD4Treg numbers were higher, the regulatory potential of activated CD4+ T cells (CD4Treg/activated T-helper cell ratio) was significantly lower in CRS mucosa compared with controls. The CD8Treg subset was also significantly reduced in CRSwNP mucosa compared with controls. CONCLUSION: A higher percentage of CD4Treg and activated T-helper cells in CRS mucosa suggests increased inflammation in CRS, independent of the presence of allergy, microbial culture results, or eosinophilic mucus. However, the decreased ratio of CD4Treg versus activated T-helper cells in CRS and reduced CD8Treg population in CRSwNPs indicates an inflammatory bias and the inability to control mucosal disease.

Accumulation of effector memory CD8+ T cells in nasal polyps.

BACKGROUND: T lymphocytes are prevalent in sinus mucosa and are implicated in chronic rhinosinusitis (CRS) pathogenesis. However, the major T-cell subpopulations, helper (CD4+) and cytotoxic (CD8+), have not been adequately examined in CRS. This study was designed to characterize human sinus mucosa and peripheral blood (PB) CD4+ and CD8+ T cells and their level of differentiation in CRS with nasal polyps (NPs), CRS without NPs, and control patients. METHODS: A prospective study was performed. Percentages of CD4+ and CD8+ T cells and their levels of differentiation were analyzed in sinus mucosa and PB by flow cytometry. Cell populations were defined as naive, central memory, effector memory, and effector T cells using cell surface markers CD45RA, CD62L, and CD27. The influence of coexisting allergy, sinus eosinophilic mucus (EM), and culture results were examined. RESULTS: In all patients, sinus mucosa had a lower percentage of CD4+ and a higher percentage of CD8+ T cells compared with PB. However, CRS with NPs (n = 86) had a significantly higher percentage of mucosal CD8+ T cells compared with CRS without NPs (n = 40) in control (n = 13) patients (p < 0.0001). Effector memory T cells were increased in sinuses compared with PB in all patients; however, the percentage of effector memory CD8+ T cells was greatest in CRS with NP mucosa (p = 0.002). Surprisingly coexisting allergy or culture results did not influence the mucosal T-cell phenotype. CRS with NP patients with sinus EM had a significantly higher percentage of mucosal CD8+ T cells. CONCLUSION: Sinus mucosa in CRS with NPs is characterized by a significant enrichment of CD8+ T cells and a relative deficiency of CD4+ T cells. The majority of NP CD8+ T cells had a terminally differentiated, mature, effector memory phenotype, which raises the question, whether these cells are pathogenic or appear as a consequence of inflammation, independent of the presence of allergy or positive microbial culture.

Immune activation in irritable bowel syndrome: can neuroimmune interactions explain symptoms?

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.

Cytoskeletal protein Flightless (Flii) is elevated in chronic and acute human wounds and wound fluid: neutralizing its activity in chronic but not acute wound fluid improves cellular proliferation.

Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.

Comparison of blood and synovial fluid th17 and novel peptidase inhibitor 16 Treg cell subsets in juvenile idiopathic arthritis.

OBJECTIVE: Early recognition and treatment of juvenile idiopathic arthritis (JIA) can prevent joint damage and minimize side effects of medication. The balance between proinflammatory and antiinflammatory mechanisms is known to be important in JIA, and we therefore investigated T cell subsets including Th cells, autoaggressive Th17 cells, and regulatory T cells (Treg), including a novel Treg subset in peripheral blood (PB) and synovial fluid (SF) of patients with JIA. METHODS: Fifty children with JIA were enrolled in our study. Frequency, phenotype, and function of T lymphocytes in PB and SF were characterized using flow cytometry. Migration capabilities of PB and SF cells were compared. RESULTS: Synovial T cells showed different phenotype and function compared with PB T cells, with an increased proportion of memory T cells, expression of CCR4, CCR5, CXCR3, interleukin 23R, and an increased ratio of Th17 to Treg. Although Treg were increased in SF compared with the PB, we found a significant decrease in the numbers of peptidase inhibitor 16 (PI16)+ Treg in active joints compared with peripheral blood. Coexpression of CCR4 and CCR6 was reduced on PI16+ Treg in PB and SF of patients with JIA compared with healthy children, however the ability of these cells to migrate toward their ligands was unaffected. CONCLUSION: This is a comprehensive characterization of novel PI16+ Treg and Th17 cells in matched blood and synovial fluid samples of patients with JIA. Despite an increased number of Treg within the inflamed joint, lower numbers of PI16+ Treg but high numbers of Th17 cells might contribute to the inability to control disease.

Inhibition of activation induced CD154 on CD4+ CD25- cells: a valid surrogate for human Treg suppressor function.

Natural Regulatory T cells (Tregs) are defined by stable expression of the cell surface proteins CD4 and CD25, low surface expression of CD127 and expression of the transcription factor FOXP3. The contribution of Treg to the prevention of autoimmunity and the maintenance of immune homoestasis is the subject of ongoing interest, as alterations in Treg numbers and function are implicated in a wide range of diseases. The in vitro benchmark for determining Treg function is suppression of proliferation of unmatched effector T cells in a mixed lymphocyte reaction (MLR) over a 3-6-day time period. As an alternative to this assay, we show that a 7-h CD154 expression assay is rapid, simple and provides a reliable readout of suppressor function. Using multiple Treg-like cell types including natural (n)Treg, inducible (i)Treg and Treg cell lines, we show that suppression of CD154 expression is a surrogate for suppression of proliferation. We propose this as a suitable alternative to the MLR assay, as it is rapid and may be more amenable to high-throughput screening, analysing large cohorts of clinical samples or assaying transiently suppressive populations.

PI16 is expressed by a subset of human memory Treg with enhanced migration to CCL17 and CCL20.

The peptidase inhibitor PI16 was shown previously by microarray analysis to be over-expressed by CD4-positive/CD25-positive Treg compared with CD4-positive/CD25-negative Th cells. Using a monoclonal antibody to the human PI16 protein, we found that PI16-positive Treg have a memory (CD45RO-positive) phenotype and express higher levels of FOXP3 than PI16-negative Treg. PI16-positive Treg are functional in suppressor assays in vitro with potency similar to PI16-negative Treg. Further phenotyping of the PI16-positive Treg revealed that the chemokine receptors CCR4 and CCR6 are expressed by more of the PI16-positive/CD45RO-positive Treg compared with PI16-negative/CD45RO-positive Treg or Th cells. PI16-positive Treg showed enhanced in vitro migration towards the inflammatory chemokines CCL17 and CCL20, suggesting they can migrate to sites of inflammation. We conclude that PI16 identifies a novel distinct subset of functional memory Treg which can migrate to sites of inflammation and regulate the pro-inflammatory response at those sites.

Isolation, expansion and characterisation of alloreactive human Th17 and Th1 cells.

Interleukin 17 producing T helper cells (Th17) and IFNγ producing Th1 cells are distinct subsets of effector memory CD4(+) T cells that are crucial to host immunity and have been linked to the pathology of certain inflammatory autoimmune diseases. We have developed a method for the isolation and long term culture of human Th17 and Th1 cells. Using allogeneic stimulation we have cultured homogeneous populations of Th17 and Th1 cells to large cell numbers. These alloreactive cell lines were established from CD4(+)CD45RO(+) memory T cells expressing, or lacking, CCR6 and CCR4. The Th17 cells were derived only from cells expressing both CCR6 and CCR4 whereas the Th1 cells, secreting IFNγ, were derived from cells lacking CCR6 and CCR4. The CCR6(+) and CCR4(+) memory T cells also gave rise to a third population of polyfunctional cells expressing both IL-17 and IFNγ. All cell populations expressed the TCR αß and the Th17 cells characteristically expressed CCR6, CCR4 and CD161. The use of this protocol will ultimately allow for the comparative analysis of the Th17 and Th1 cells.

Cordycepin is an immunoregulatory active ingredient of Cordyceps sinensis.

We have reported that cordycepin, an adenosine derivative from the fungus Cordyceps, increased interleukin (IL)-10 expression, decreased IL-2 expression and suppressed T lymphocyte activity. In the present study, we further characterized the regulatory effects of cordycepin on human immune cells. Moreover, a traditional Chinese drug, Cordyceps sinensis (CS) that contains cordycepin, was also investigated. Cytometric Bead Array (CBA) was used to determine the concentrations of IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, TNF-alpha and IFN-gamma in culture of peripheral blood mononuclear cells (PBMCs). The results showed that both cordycepin and CS up-regulated IL-10, IL-1beta, IL-6, IL-8 and TNF-alpha; at the same time, they suppressed phytohemagglutinin (PHA)-induced production of IL-2, IL-4, IL-5, IFN-gamma and IL-12. As compared to cordycepin, CS displayed its regulatory effects on IL-2 and IL-10 in a similar dose-dependent manner even with higher efficiency. The binding activity of transcription factors in a human monocytic cell line THP-1 was tested by the trans-AM method, and a higher binding activity of SP1 and SP3 was observed in cordycepin or CS treated cells compared to the control. These results led to the opinion that cordycepin and CS pleiotropically affected the actions of immune cells and cytokine network in a similar fashion. Cordycepin could be an important immunoregulatory active ingredient in Cordyceps sinensis. In addition, CS may contain substances which possess synergism with cordycepin, as CS showed a higher efficiency in the production of IL-10 and IL-2 than cordycepin. However, merits of these effects in pharmacology and clinical medicine have yet to be proven and the precise mechanism of these immune regulatory actions should be researched.

Enhanced expression of IL-27 mRNA in human newborns.

Interleukin (IL)-27, a heterodimer composed of Epstein-Barr virus-induced gene 3 (EBI3) and p28, is an early product of activated dendritic cells (DC). Binding of IL-27 to the WSX-1 receptor initiates Th1 (Thelper 1) responses in naïve T cells. In order to assess the Th1 responses in human neonates with high susceptibility to infectious diseases, expression of EBI3-, p28- and WSX-1-mRNA in response to Toll-like receptor ligands was compared in neonate and adult monocyte-derived (m)DC. Only the combined addition of ligands induced expression of IL-27p28 mRNA. Surprisingly, neonatal mDC produced significantly more IL-27p28 mRNA than that obtained from adults. Furthermore, there was enhanced expression of EBI3 mRNA in cord blood as compared with adult blood. In addition, the secretion of WSX-1 mRNA in neonatal mDC and T cells was also significantly increased. Taken together, these findings indicate that the restricted Th1 responses in human newborns owing to deficient IL-12 production may be compensated for, in part, by enhanced IL-27 secretion.

Combined Toll-like receptor agonists synergistically increase production of inflammatory cytokines in human neonatal dendritic cells.

Dendritic cells (DC) are thought to be responsible for the reduced ability of human newborns to induce protective T-helper 1 (Th1) immune responses. The key player in Th1 differentiation, interleukin-12 (IL-12), is primarily produced in response to Toll-like receptor (TLR) binding by adult DC but not by neonatal DC. The potential use of various TLR agonist combinations for initiating neonatal monocyte-derived DC to prime Th1 responses was investigated. Single TLR ligands induced maturation only in adult DC; neonatal DC matured with combined targeting of TLR3/TLR8 or TLR4/TLR8, based on the expression of maturation markers. Similarly, the synergistic effects of combined TLR ligands could also be shown with adult and neonatal cytokine production, but different expression patterns were noted. In particular, IL-12p70 was produced by neonatal DC exclusively after combined TLR stimulation. Surprisingly, it was found that supernatants of combined stimulated neonatal DC could induce interferon-gamma production in autologous naïve T cells. Moreover, this interferon-gamma secretion was blocked by anti-IL-12p70 antibodies and increased after addition of recombined IL-12. In conclusion, these findings underline the differences between adult and neonatal DC and might suggest new strategies for promoting newborn Th1 immunity in response to pathogens and vaccine antigens.

Efficient maturation and cytokine production of neonatal DCs requires combined proinflammatory signals.

Specific functional properties of dendritic cells (DCs) have been suspected as being responsible for the impaired specific immune responses observed in human neonates. To analyze stimulatory requirements for the critical transition from immature, antigen-processing DCs to mature, antigen-presenting DCs, we investigated the effect of different proinflammatory mediators and antigens on phenotype and cytokine secretion of human neonatal DCs derived from hematopoietic progenitor cells (HPCs). Whereas single proinflammatory mediators were unable to induce the maturation of neonatal DCs, various combinations of IFNgamma, CD40L, TNFalpha, LPS and antigens, induced the maturation of neonatal DCs documented by up-regulation of HLA-DR, CD83 and CD86. Combinations of proinflammatory mediators also increased cytokine secretion by neonatal DCs. Especially combined stimulation with LPS and IFNgamma proved to be very efficient in inducing maturation and cytokine synthesis of neonatal DCs. In conclusion, neonatal DCs can be stimulated to express maturation as well as costimulatory surface molecules. However, induction of maturation requires combined stimulation with multiple proinflammatory signals.