A conserved population of MHC II-restricted, innate-like, commensal-reactive T cells in the gut of humans and mice.

Interactions with commensal microbes shape host immunity on multiple levels and play a pivotal role in human health and disease. Tissue-dwelling, antigen-specific T cells are poised to respond to local insults, making their phenotype important in the relationship between host and microbes. Here we show that MHC-II restricted, commensal-reactive T cells in the colon of both humans and mice acquire transcriptional and functional characteristics associated with innate-like T cells. This cell population is abundant and conserved in the human and murine colon and endowed with polyfunctional effector properties spanning classic Th1- and Th17-cytokines, cytotoxic molecules, and regulators of epithelial homeostasis. T cells with this phenotype are increased in ulcerative colitis patients, and their presence aggravates pathology in dextran sodium sulphate-treated mice, pointing towards a pathogenic role in colitis. Our findings add to the expanding spectrum of innate-like immune cells positioned at the frontline of intestinal immune surveillance, capable of acting as sentinels of microbes and the local cytokine milieu.

IL-1-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies.

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.

Host-microbiota maladaptation in colorectal cancer.

Colorectal cancer (CRC) is a heterogeneous disease of the intestinal epithelium that is characterized by the accumulation of mutations and a dysregulated immune response. Up to 90% of disease risk is thought to be due to environmental factors such as diet, which is consistent with a growing body of literature that describes an 'oncogenic' CRC-associated microbiota. Whether this dysbiosis contributes to disease or merely represents a bystander effect remains unclear. To prove causation, it will be necessary to decipher which specific taxa or metabolites drive CRC biology and to fully characterize the underlying mechanisms. Here we discuss the host-microbiota interactions in CRC that have been reported so far, with particular focus on mechanisms that are linked to intestinal barrier disruption, genotoxicity and deleterious inflammation. We further comment on unknowns and on the outstanding challenges in the field, and how cutting-edge technological advances might help to overcome these. More detailed mechanistic insights into the complex CRC-associated microbiota would potentially reveal avenues that can be exploited for clinical benefit.

The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer.

PURPOSE: The cytokine IL22 promotes tumor progression in murine models of colorectal cancer. However, the clinical significance of IL22 in human colorectal cancer remains unclear. We sought to determine whether the IL22 pathway is associated with prognosis in human colorectal cancer, and to identify mechanisms by which IL22 can influence disease progression. EXPERIMENTAL DESIGN: Transcriptomic data from stage II/III colon cancers in independent discovery (GSE39582 population-based cohort, N = 566) and verification (PETACC3 clinical trial, N = 752) datasets were used to investigate the association between IL22 receptor expression (encoded by the genes IL22RA1 and IL10RB), tumor mutation status, and clinical outcome using Cox proportional hazard models. Functional interactions between IL22 and mutant KRAS were elucidated using human colorectal cancer cell lines and primary tumor organoids. RESULTS: Transcriptomic analysis revealed a poor-prognosis subset of tumors characterized by high expression of IL22RA1, the alpha subunit of the heterodimeric IL22 receptor, and KRAS mutation [relapse-free survival (RFS): HR = 2.93, P = 0.0006; overall survival (OS): HR = 2.45, P = 0.0023]. KRAS mutations showed a similar interaction with IL10RB and conferred the worst prognosis in tumors with high expression of both IL22RA1 and IL10RB (RFS: HR = 3.81, P = 0.0036; OS: HR = 3.90, P = 0.0050). Analysis of human colorectal cancer cell lines and primary tumor organoids, including an isogenic cell line pair that differed only in KRAS mutation status, showed that IL22 and mutant KRAS cooperatively enhance cancer cell proliferation, in part through augmentation of the Myc pathway. CONCLUSIONS: Interactions between KRAS and IL22 signaling may underlie a previously unrecognized subset of clinically aggressive colorectal cancer that could benefit from therapeutic modulation of the IL22 pathway.

High-Dose IL-2 Skews a Glucocorticoid-Driven IL-17+IL-10+ Memory CD4+ T Cell Response towards a Single IL-10-Producing Phenotype.

Glucocorticoids are known to increase production of the anti-inflammatory cytokine IL-10, and this action is associated with their clinical efficacy in asthmatics. However, glucocorticoids also enhance the synthesis of IL-17A by PBMCs, which, in excess, is associated with increased asthma severity and glucocorticoid-refractory disease. In this study, we show that the glucocorticoid dexamethasone significantly increased IL-10 production by human memory CD4+ T cells from healthy donors, as assessed by intracellular cytokine staining. In addition, dexamethasone increased production of IL-17A, IL-17F, and IL-22, with the most striking enhancement in cells coproducing Th17-associated cytokines together with IL-10. Of note, an increase in IFN-γ+IL-10+ cells was also observed despite overall downregulation of IFN-γ production. These dexamethasone-driven IL-10+ cells, and predominantly the IL-17+IL-10+ double-producing cells, were markedly refractory to the inhibitory effect of dexamethasone on proliferation and IL-2Rα expression, which facilitated their preferential IL-2-dependent expansion. Although lower concentrations of exogenous IL-2 promoted IL-10+ cells coproducing proinflammatory cytokines, higher IL-2 doses, both alone and in combination with dexamethasone, increased the proportion of single IL-10+ T cells. Thus, glucocorticoid-induced IL-10 is only accompanied by an increase of IL-17 in a low IL-2 setting, which is, nevertheless, likely to be protective owing to the induction of regulatory IL-17+IL-10+-coproducing cells. These findings open new avenues of investigation with respect to the role of IL-2 in glucocorticoid responsiveness that have potential implications for optimizing the benefit/risk ratio of glucocorticoids in the clinic.

Effects of vitamin D on inflammatory and oxidative stress responses of human bronchial epithelial cells exposed to particulate matter.

BACKGROUND: Particulate matter (PM) pollutant exposure, which induces oxidative stress and inflammation, and vitamin D insufficiency, which compromises immune regulation, are detrimental in asthma. OBJECTIVES: Mechanistic cell culture experiments were undertaken to ascertain whether vitamin D abrogates PM-induced inflammatory responses of human bronchial epithelial cells (HBECs) through enhancement of antioxidant pathways. METHODS: Transcriptome analysis, PCR and ELISA were undertaken to delineate markers of inflammation and oxidative stress; with comparison of expression in primary HBECs from healthy and asthmatic donors cultured with reference urban PM in the presence/absence of vitamin D. RESULTS: Transcriptome analysis identified over 500 genes significantly perturbed by PM-stimulation, including multiple pro-inflammatory cytokines. Vitamin D altered expression of a subset of these PM-induced genes, including suppressing IL6. Addition of vitamin D suppressed PM-stimulated IL-6 production, although to significantly greater extent in healthy versus asthmatic donor cultures. Vitamin D also differentially affected PM-stimulated GM-CSF, with suppression in healthy HBECs and enhancement in asthmatic cultures. Vitamin D increased HBEC expression of the antioxidant pathway gene G6PD, increased the ratio of reduced to oxidised glutathione, and in PM-stimulated cultures decreased the formation of 8-isoprostane. Pre-treatment with vitamin D decreased CXCL8 and further decreased IL-6 production in PM-stimulated cultures, an effect abrogated by inhibition of G6PD with DHEA, supporting a role for this pathway in the anti-inflammatory actions of vitamin D. CONCLUSIONS: In a study using HBECs from 18 donors, vitamin D enhanced HBEC antioxidant responses and modulated the immune response to PM, suggesting that vitamin D may protect the airways from pathological pollution-induced inflammation.

miR-29b directly targets activation-induced cytidine deaminase in human B cells and can limit its inappropriate expression in naïve B cells.

Class-switch recombination (CSR) is an essential B cell process that alters the isotype of antibody produced by the B cell, tailoring the immune response to the nature of the invading pathogen. CSR requires the activity of the mutagenic enzyme AID (encoded by AICDA) to generate chromosomal lesions within the immunoglobulin genes that initiate the class switching recombination event. These AID-mediated mutations also participate in somatic-hypermutation of the immunoglobulin variable region, driving affinity maturation. As such, AID poses a significant oncogenic threat if it functions outside of the immunoglobulin locus. We found that expression of the microRNA, miR-29b, was repressed in B cells isolated from tonsil tissue, relative to circulating naïve B cells. Further investigation revealed that miR-29b was able to directly initiate the degradation of AID mRNA. Enforced overexpression of miR-29b in human B cells precipitated a reduction in overall AID protein and a corresponding diminution in CSR to IgE. Given miR-29b's ability to potently target AID, a mutagenic molecule that can initiate chromosomal translocations and "off-target" mutations, we propose that miR-29b acts to silence premature AID expression in naïve B cells, thus reducing the likelihood of inappropriate and potentially dangerous deamination activity.

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes.

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) -stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T-lymphocyte responses despite their importance in anti-viral immunity. To address this, we examined the effects of UPM on DC-stimulated naive CD8 T-cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte-macrophage colony-stimulating factor (GM-CSF). The capacity of these mDCs to stimulate naive CD8 T-lymphocyte responses in allogeneic co-culture was then assessed by measuring T-cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon-γ (IFN-γ)-producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co-cultures, UPM treatment of mDCs enhanced CD8 T-cell proliferation and the frequency of IFN-γ+ cells. The secretion of tumour necrosis factor-α, interleukin-13, Granzyme A and Granzyme B were also increased. GM-CSF alone, and in concert with UPM, enhanced many of these T-cell functions. The PM-induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro-inflammatory cytokines. Such UPM-induced stimulation of CD8 cells may potentiate T-lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.

Vitamin D Counteracts an IL-23-Dependent IL-17A+IFN-γ+ Response Driven by Urban Particulate Matter.

Urban particulate matter (UPM) air pollution and vitamin D deficiency are detrimentally associated with respiratory health. This is hypothesized to be due in part to regulation of IL-17A, which UPM is reported to promote. Here, we used a myeloid dendritic cell (DC)-memory CD4+ T cell co-culture system to characterize UPM-driven IL-17A+ cells, investigate the mechanism by which UPM-primed DCs promote this phenotype, and address evidence for cross-regulation by vitamin D. CD1c+ myeloid DCs were cultured overnight with or without a reference source of UPM and/or active vitamin D (1,25[OH]2D3) before they were co-cultured with autologous memory CD4+ T cells. Supernatants were harvested for cytokine analysis on Day 5 of co-culture, and intracellular cytokine staining was performed on Day 7. UPM-primed DCs increased the proportion of memory CD4+ T cells expressing the T helper 17 cell (Th17)-associated cytokines IL-17A, IL-17F, and IL-22, as well as IFN-γ, granulocyte-macrophage colony-stimulating factor, and granzyme B. Notably, a large proportion of the UPM-driven IL-17A+ cells co-expressed these cytokines, but not IL-10, indicative of a proinflammatory Th17 profile. UPM-treated DCs expressed elevated levels of il23 mRNA and increased secretion of IL-23p40. Neutralization of IL-23 in culture reduced the frequency of IL-17A+IFN-γ+ cells without affecting cell proliferation. 1,25(OH)2D3 counteracted the UPM-driven DC maturation and inhibited the frequency of IL-17A+IFN-γ+ cells, most prominently when DCs were co-treated with the corticosteroid dexamethasone, while maintaining antiinflammatory IL-10 synthesis. These data indicate that UPM might promote an inflammatory milieu in part by inducing an IL-23-driven proinflammatory Th17 response. Restoring vitamin D sufficiency may counteract these UPM-driven effects without obliterating important homeostatic immune functions.

ASTHMA - comparing the impact of vitamin D versus UVR on clinical and immune parameters.

The incidence of asthma has increased markedly since the 1960s and is currently estimated to affect more than 300 million individuals worldwide. A number of environmental factors are implicated in asthma pathogenesis, one of which is vitamin D. Vitamin D deficiency is a global health concern and has increased in parallel with asthma incidence. Epidemiological studies report associations between low vitamin D status, assessed as circulating levels of 25-hydroxyvitamin D, with asthma incidence, severity, exacerbations and responses to treatment. This has led to clinical studies to test whether increasing the levels of vitamin D improves asthma management. Despite being highly variable in dosing regimens, design and outcomes, meta-analyses suggest overall positive outcomes with respect to reduced asthma exacerbations and steroid requirements. The primary mechanism for increasing vitamin D levels in the body is through exposure of the skin to the ultraviolet B (UVB) component of ultraviolet radiation (UVR), most commonly from sun exposure. However, only a limited number of studies investigating the impact of UVR on the asthmatic response have been performed; these generally report on the impact of latitude as a surrogate of sun exposure, or address this in animal models. To the best of our knowledge no comprehensive trials to assess the impact of UVB radiation on asthma outcomes have been performed. Within this review we discuss observational and clinical studies in this field, and innate and adaptive immune mechanisms through which UVR and vitamin D may impact respiratory health, and asthma. We highlight the heterogeneity of asthmatic disease, which is likely to impact upon the efficacy of interventional studies, and briefly overview more recent findings relating to the impact of vitamin D/UVR on the development of asthma.

Urban Particulate Matter-Activated Human Dendritic Cells Induce the Expansion of Potent Inflammatory Th1, Th2, and Th17 Effector Cells.

Exposure to urban particulate matter (UPM) exacerbates asthmatic lung inflammation. Lung dendritic cells (DCs) are critical for stimulating T cell immunity and in maintaining airway tolerance, but they also react to airway UPM. The adjuvant role of UPM in enhancing primary immune responses by naive cells to allergen has been reported, but the direct effects of UPM-activated DCs on the functionality of human memory CD4 T cells (Tms), which constitute the majority of T cells in the lung, has not been investigated. Blood CD1c(+) DCs were purified and activated with UPM in the presence or absence of house dust mite or tetanus toxoid control antigen. 5-(and -6)-Carboxyfluorescein diacetate succinimidyl ester-labeled blood Tms were cocultured with autologous DCs, T cell proliferation and effector function were assessed using flow cytometry, and secreted cytokines were measured by combined bead array. UPM-DCs elicited IFN-γ and IL-13 secretion and induced proliferation in Tms isolated from both allergic patients with asthma and healthy control subjects, whereas only IL-13 was produced by Tms from patients with atopic asthma stimulated by house dust mite-loaded DCs. UPM-DCs drove the expansion and differentiation of a mixed population of Th1, Th2, and Th17 cell effectors through a mechanism that was dependent on major histocompatibility class II but not on cytokine-driven expansion. The data suggest that UPM not only has adjuvant properties but is also a source of antigen that stimulates the generation of Th2, Th1, and Th17 effector phenotypes, which have been implicated in both exacerbations of asthma and chronic inflammatory diseases.

1α,25-dihydroxyvitamin D3 acts via transforming growth factor-ß to up-regulate expression of immunosuppressive CD73 on human CD4+ Foxp3- T cells.

Vitamin D deficiency is associated with increased incidence and severity of various immune-mediated diseases. Active vitamin D (1α,25-dihydroxyvitamin D3; 1,25(OH)2 D3) up-regulates CD4(+) T-cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti-inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)2 D3 on expression of the downstream ecto-5'-nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor-ß (TGF-ß) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10(-8) to 10(-7) m, 1,25(OH)2 D3 significantly increased expression of CD73 on peripheral human CD4(+) T cells. Although 1,25(OH)2 D3 did not affect the mRNA expression of latent TGF-ß1 , 1,25(OH)2 D3 did up-regulate expression of TGF-ß-associated molecules [latency-associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin-1, thrombospondin-1 and αv integrin] which is likely to have contributed to the observed enhancement in TGF-ß bioactivity. CD73 was highly co-expressed with LAP and GARP following 1,25(OH)2 D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4(+) Foxp3(-) T cells, rather than CD4(+) Foxp3(+) T cells. Notably, neutralization of TGF-ß significantly impaired 1,25(OH)2 D3-mediated induction of CD73. Collectively, we show that 1,25(OH)2 D3 enhances expression of CD73 on CD4(+) Foxp3(-) T cells in a process that is at least partially TGF-ß-dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune-mediated disease.

Vitamin D influences asthmatic pathology through its action on diverse immunological pathways.

The prevalence of vitamin D insufficiency and deficiency has increased markedly in recent decades to current epidemic levels (Hyppönen E, et al. Am J Clin Nutr 2007;85:860-868). In parallel, there has been an increase in the incidence of a range of immune-mediated conditions ranging from cancer to autoimmune and respiratory diseases, including chronic obstructive pulmonary disease and asthma (Holick MF. N Engl J Med 2007;357:266-281; Finklea et al. Adv Nutr 2011;2:244-253). There is also an association with increased respiratory infections, which are the most common cause of asthma exacerbations (Finklea et al. Adv Nutr 2011;2:244-253). Together, this has resulted in considerable interest in the therapeutic potential of vitamin D to prevent and improve treatment of asthma and other respiratory diseases. To this end, data from clinical trials involving supplementation with active vitamin D, or more commonly a precursor, are starting to emerge. This review considers mechanisms by which vitamin D may act on the immune system to dampen inappropriate inflammatory responses in the airway while also promoting tolerance and antimicrobial defense mechanisms that collectively maintain respiratory health.

Immunoregulatory mechanisms of vitamin D relevant to respiratory health and asthma.

Vitamin D deficiency is prevalent among people with various immune-mediated conditions, including autoimmune diseases and asthma. Serum 25(OH)D levels inversely correlate with asthma severity, glucocorticoid responsiveness/dosage, and markers of pathogenesis, such as airway remodeling, IgE, and eosinophilia. Trials involving supplementation with active vitamin D or a precursor are beginning to emerge with variable results that, in part, reflect differences in study design. This review looks at the mechanisms by which vitamin D may protect against asthma, including increasing glucocorticoid responsiveness, skewing immune cells towards a regulatory phenotype, reducing the incidence of infections, airway remodeling, eosinophilia, and lowering the levels of IgE. Also discussed is the therapeutic potential for vitamin D, which is likely to be applicable to immune-mediated conditions beyond simply asthma.

1α,25-dihydroxyvitamin D3 in combination with transforming growth factor-ß increases the frequency of Foxp3⁺ regulatory T cells through preferential expansion and usage of interleukin-2.

A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune-mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3(+) CD4(+) T regulatory (Treg) cells when present at high concentrations or under strong T-cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3(+) Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3(+) Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10(-7) M). Stimulation of human CD4(+) T cells with a combination of 1,25(OH)2D3 and transforming growth factor-ß (TGF-ß) greatly increased the frequency of Foxp3(+) Treg cells, which is proposed to result from the preferential expansion of Foxp3(+) Treg cells, as compared with the Foxp3(-) effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin-2 by the Foxp3(+) Treg cells compared with Foxp3(-) effector T cells. In summary, modulation of the cytokine environment to one high in TGF-ß in the presence of 1,25(OH)2D3(10(-7) M) significantly increased Foxp3(+) Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases.