Jeppe Madura Larsen

IL-23 and TH17-mediated inflammation in human allergic contact dermatitis

BACKGROUND IL-17-producing T(H) (T(H)17) cells are key mediators of chronic inflammation in mice. Recent studies have implicated T(H)17-mediated inflammation in the pathogenesis of human autoimmune diseases; however, the involvement of T(H)17 cells in allergic disorders remains largely elusive. OBJECTIVE To investigate T(H)17-mediated inflammation in human beings with allergic contact dermatitis; in particular, the innate response of keratinocytes to contact allergen, the induction of allergen-specific T(H)17 cells, and the presence of T(H)17-related effector cells in inflamed skin. METHODS Human keratinocytes were stimulated with nickel in vitro followed by measurements of IL-23 and IL-12 production by quantitative PCR and ELISA. Allergen-specific memory T cells from the blood of individuals with nickel allergy and healthy controls were identified and characterized by using a short-term ex vivo assay. Nickel patch test lesions and normal skin were analyzed for the expression of T(H)17-related cells and molecules by using immunohistochemistry. RESULTS Keratinocytes were found to produce IL-23, but no detectable IL-12, in a response to nickel stimulation. Memory T cells isolated from peripheral blood of individuals with nickel allergy, but not healthy controls, contained T(H)17 and T(H)1 cells proliferating in response to nickel-pulsed DCs. Inflamed skin of nickel-challenged allergic individuals contained infiltrating neutrophils and cells expressing IL-17, IL-22, CCR6, and IL-22R. CONCLUSION Our results demonstrate the involvement of T(H)17-mediated immunopathology in human allergic contact dermatitis, including both innate and adaptive immune responses to contact allergens.

Divergent pro-inflammatory profile of human dendritic cells in response to commensal and pathogenic bacteria associated with the airway microbiota

Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3-5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota.

BCG stimulated dendritic cells induce an interleukin‐10 producing T‐cell population with no T helper 1 or T helper 2 bias in vitro

Mycobacterium bovis bacillus Calmette–Guérin (BCG) vaccine has been associated with beneficial effects on overall childhood mortality in low‐income countries; this cannot be explained merely by the prevention of tuberculosis (TB) deaths. The beneficial effects of BCG vaccine could be the result of either strengthening of pro‐inflammatory mechanisms, helping neonates to fight infections, or the induction of an immune‐regulatory network restricting overt inflammation and intense pathology. We aimed to study the effect of live BCG on the ability of dendritic cells (DCs) to polarize T‐cell responses. Monocyte‐derived DCs were matured in the presence or absence of BCG. The DC phenotype was assessed by CD83 expression, interleukin‐12 (IL‐12) and IL‐10 production, as well as for the ability to polarize T‐cell responses. Following stimulation with CD40 ligand, DCs matured in the presence of BCG showed enhanced IL‐10 and diminished IL‐12 production. These DCs primed naive T cells to develop into IL‐10‐producing T cells, with no T helper 1 or T helper 2 bias. These results suggest that BCG vaccination might result in the development of IL‐10‐producing DCs as well as IL‐10‐producing T cells that could contribute to restricting overt inflammation in infants exposed to pathogens and thus lead to lower infant mortality.

Protein Kinase C (PKC)α and PKCθ Are the Major PKC Isotypes Involved in TCR Down-Regulation

It is well known that protein kinase C (PKC) plays an important role in regulation of TCR cell surface expression levels. However, eight different PKC isotypes are present in T cells, and to date the particular isotype(s) involved in TCR down-regulation remains to be identified. The aim of this study was to identify the PKC isotype(s) involved in TCR down-regulation and to elucidate the mechanism by which they induce TCR down-regulation. To accomplish this, we studied TCR down-regulation in the human T cell line Jurkat, in primary human T cells, or in the mouse T cell line DO11.10 in which we either overexpressed constitutive active or dominant-negative forms of various PKC isotypes. In addition, we studied TCR down-regulation in PKC knockout mice and by using small interfering RNA-mediated knockdown of specific PKC isotypes. We found that PKCα and PKCθ were the only PKC isotypes able to induce significant TCR down-regulation. Both isotypes mediated TCR down-regulation via the TCR recycling pathway that strictly depends on Ser126 and the di-leucine-based receptor-sorting motif of the CD3γ chain. Finally, we found that PKCθ was mainly implicated in down-regulation of directly engaged TCR, whereas PKCα was involved in down-regulation of nonengaged TCR.

Chronic obstructive pulmonary disease and asthma-associated Proteobacteria, but not commensal Prevotella spp., promote Toll-like receptor 2-independent lung inflammation and pathology

Recent studies of healthy human airways have revealed colonization by a distinct commensal bacterial microbiota containing Gram‐negative Prevotella spp. However, the immunological properties of these bacteria in the respiratory system remain unknown. Here we compare the innate respiratory immune response to three Gram‐negative commensal Prevotella strains (Prevotella melaninogenica, Prevotella nanceiensis and Prevotella salivae) and three Gram‐negative pathogenic Proteobacteria known to colonize lungs of patients with chronic obstructive pulmonary disease (COPD) and asthma (Haemophilus influenzae B, non‐typeable Haemophilus influenzae and Moraxella catarrhalis). The commensal Prevotella spp. and pathogenic Proteobacteria were found to exhibit intrinsic differences in innate inflammatory capacities on murine lung cells in vitro. In vivo in mice, non‐typeable H. influenzae induced severe Toll‐like receptor 2 (TLR2)‐independent COPD‐like inflammation characterized by predominant airway neutrophilia, expression of a neutrophilic cytokine/chemokine profile in lung tissue, and lung immunopathology. In comparison, P. nanceiensis induced a diminished neutrophilic airway inflammation and no detectable lung pathology. Interestingly, the inflammatory airway response to the Gram‐negative bacteria P. nanceiensis was completely TLR2‐dependent. These findings demonstrate weak inflammatory properties of Gram‐negative airway commensal Prevotella spp. that may make colonization by these bacteria tolerable by the respiratory immune system.

Gene expression analysis of interferon-β treatment in multiple sclerosis

Treatment with interferon-β (IFN-β) induces the expression of hundreds of genes in blood mononuclear cells, and the expression of several genes has been proposed as a marker of the effect of treatment with IFN-β. However, to date no molecules have been identified that are stably induced by treatment with IFN-β. We use DNA microarrays to study gene expression in 10 multiple sclerosis (MS) patients who began de novo treatment with IFN-β. After the first injection of IFN-β, the expression of 74 out of 3428 genes changed at least two-fold and statistically significantly (after Bonferroni correction). In contrast, we observed no persisting effects of IFN-β on gene expression. Among the most strongly induced genes was MXA, which has been used in previous biomarker studies in MS. In addition, the study identified the induction of LGALS9 and TCIR1G, involved in negative regulation of T helper type I immunity and T-cell activation, as novel effects of IFN-β therapy in MS.

Children with asthma by school age display aberrant immune responses to pathogenic airway bacteria as infants

BACKGROUND Asthma is a highly prevalent chronic lung disease that commonly originates in early childhood. Colonization of neonatal airways with the pathogenic bacterial strains Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae is associated with increased risk of later childhood asthma. We hypothesized that children with asthma have an abnormal immune response to pathogenic bacteria in infancy. OBJECTIVE We aimed to assess the bacterial immune response in asymptomatic infants and the association with later development of asthma by age 7 years. METHODS The Copenhagen Prospective Studies on Asthma in Childhood birth cohort was followed prospectively, and asthma was diagnosed at age 7 years. The immune response to H influenzae, M catarrhalis, and S pneumoniae was analyzed in 292 infants using PBMCs isolated and stored since the age of 6 months. The immune response was assessed based on the pattern of cytokines produced and T-cell activation. RESULTS The immune response to pathogenic bacteria was different in infants with asthma by 7 years of age (P = .0007). In particular, prospective asthmatic subjects had aberrant production of IL-5 (P = .008), IL-13 (P = .057), IL-17 (P = .001), and IL-10 (P = .028), whereas there were no differences in T-cell activation or peripheral T-cell composition. CONCLUSIONS Children with asthma by school age exhibited an aberrant immune response to pathogenic bacteria in infancy. We propose that an abnormal immune response to pathogenic bacteria colonizing the airways in early life might lead to chronic airway inflammation and childhood asthma.

The immune response to Prevotella bacteria in chronic inflammatory disease

The microbiota plays a central role in human health and disease by shaping immune development, immune responses and metabolism, and by protecting from invading pathogens. Technical advances that allow comprehensive characterization of microbial communities by genetic sequencing have sparked the hunt for disease-modulating bacteria. Emerging studies in humans have linked the increased abundance of Prevotella species at mucosal sites to localized and systemic disease, including periodontitis, bacterial vaginosis, rheumatoid arthritis, metabolic disorders and low-grade systemic inflammation. Intriguingly, Prevotella abundance is reduced within the lung microbiota of patients with asthma and chronic obstructive pulmonary disease. Increased Prevotella abundance is associated with augmented T helper type 17 (Th17) -mediated mucosal inflammation, which is in line with the marked capacity of Prevotella in driving Th17 immune responses in vitro. Studies indicate that Prevotella predominantly activate Toll-like receptor 2, leading to production of Th17-polarizing cytokines by antigen-presenting cells, including interleukin-23 (IL-23) and IL-1. Furthermore, Prevotella stimulate epithelial cells to produce IL-8, IL-6 and CCL20, which can promote mucosal Th17 immune responses and neutrophil recruitment. Prevotella-mediated mucosal inflammation leads to systemic dissemination of inflammatory mediators, bacteria and bacterial products, which in turn may affect systemic disease outcomes. Studies in mice support a causal role of Prevotella as colonization experiments promote clinical and inflammatory features of human disease. When compared with strict commensal bacteria, Prevotella exhibit increased inflammatory properties, as demonstrated by augmented release of inflammatory mediators from immune cells and various stromal cells. These findings indicate that some Prevotella strains may be clinically important pathobionts that can participate in human disease by promoting chronic inflammation.

Cellular dynamics in the draining lymph nodes during sensitization and elicitation phases of contact hypersensitivity

Background:  The different role of various immunological effector cells in contact hypersensitivity (CHS) is receiving increased attention. During the past decade, the involvement of different cell types in CHS has been investigated by the use of antibody‐induced depletion of specific subtypes of immunological cells and by studying knockout mice lacking one or more of these immunological cell populations.

Consumer available permanent hair dye products cause major allergic immune activation in an animal model.

Background  p‐Phenylenediamine (PPD) and related substances are ingredients of more than two‐thirds of oxidative (permanent) hair dyes currently used. Although PPD is a potent skin sensitizer in predictive assays, the extent to which permanent hair dyes sensitize humans has been questioned due to the in‐use conditions, e.g. the presence of couplers in the hair dye gel and rapid oxidation using a developer.