Enhanced IgG1 -mediated antibody response towards thymus-dependent immunization in CXCR1-deficient mice.

BACKGROUND: Chemokine receptors and their corresponding ligands are key players of immunity by regulation of immune cell differentiation and migration. CXCR1 is a high-affinity receptor for CXCL8. Differential expression of CXCR1 is associated with a variety of human pathologies including cancer and inflammatory diseases. While various studies have highlighted the importance of CXCR1-mediated CXCL8-sensing for neutrophil trafficking and function, its role in B-cell responses remains unsolved. Therefore, our aim was to investigate innate and adaptive antibody responses in CXCR1-deficient mice. METHODS: Cell populations of the spleen and the peritoneal cavity were identified and quantified via flow cytometry. To investigate thymus-independent (TI) and thymus-dependent (TD) antibody responses, mice were immunized intraperitoneally with TNP-Ficoll, Pneumovax23, and TNP-Chicken Gamma Globulin. Mice were bled before as well as 7 and 14 days after vaccination to collect serum. Serum antibody levels overtime were analyzed according to their specificity by enzyme-linked immunosorbent assay. B-1 cell functionality was examined by IL-5/IL-5Rα-dependent stimulation of peritoneal and splenic cells in vitro. To analyze CXCR1/2-expression, CD19+ splenocytes were enriched by magnetic-activated cell sorting before isolation of total RNA contents, followed by reverse transcription and real-time polymerase chain reaction. RESULTS: The distribution of natural B-1 cell populations was disturbed in the absence of CXCR1, while their responsiveness towards TI antigens and in vitro stimulation remained functional. Besides, CXCR1-deficiency was accompanied by increased frequencies of follicular B-2 cells in the spleen. Interestingly, these mice produced elevated levels of antigen-specific IgG1 upon TD immunization and harbored a significantly enlarged proportion of CXCR5-expressing T helper (H) cells. CXCR1-expression was detectable in CD19+ splenocytes derived from wild-type, but not CXCR1-deficient mice. CONCLUSION: Our data demonstrate a previously unknown relevance of CXCR1 for the production of specific IgG1 in response to vaccination. These findings identify CXCR1 as a promising candidate for future studies on the regulation of adaptive antibody responses.

Correction: mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo.

[This corrects the article DOI: 10.1371/journal.pone.0154001.].

Anthralin modulates the expression pattern of cytokeratins and antimicrobial peptides by psoriatic keratinocytes.

BACKGROUND: Psoriasis is an inflammatory skin disease with aberrant keratinocyte proliferation, presumably as a result of immune cell activation. Th17 cytokines like IL-17A and IL-22 are critically implicated in epidermal thickening, altered keratinocyte differentiation and production of innate factors such as antimicrobial peptides. Psoriasis treatment options include modern targeted therapies using anti-cytokine antibodies and traditional non-targeted treatments like anthralin (dithranol). While the mode of action of anti-cytokine antibodies is defined, the effects of topical anthralin on psoriatic skin are not fully understood. OBJECTIVE: This study aims to unravel the direct effects of anthralin on keratinocyte proliferation, differentiation and production of psoriasis-associated factors. METHODS: We tested the effects of anthralin on cell proliferation, cytokeratin expression and changes in the expression of antimicrobial peptides using primary keratinocytes and 3D psoriasis tissue models with and without stimulation of the psoriasis-promoting cytokines IL-17A and IL-22. Moreover, we compared the findings derived from monolayer and multilayer cultures to data derived from lesional skin of patients with psoriasis before and under treatment with anthralin. RESULTS: Our study shows that anthralin directly induces cell apoptosis in vitro in monolayer cultures but not in 3D psoriasis tissue models treated with IL-17A and IL-22. Yet, keratinocyte proliferation as determined by Ki-67 staining is impaired by anthralin in vivo. In lesional skin but not in 3D psoriasis tissue models anthralin rapidly normalizes cytokeratin (CK)16 expression. Furthermore, anthralin directly inhibits DEFB4 expression in vitro and in vivo, while other antimicrobial peptides and cytokines studied like IL-6 and IL-8 are regulated differently in vitro and in vivo. CONCLUSIONS: Our results show that anthralin directly regulates DEFB4A expression. However, its beneficial effects on psoriasis cannot be explained by direct effects on keratinocyte differentiation or cytokine expression.

Pseudomonas aeruginosa Airway Infection Recruits and Modulates Neutrophilic Myeloid-Derived Suppressor Cells.

Pseudomonas aeruginosa is an opportunistic pathogen that causes infections mainly in patients with cystic fibrosis (CF) lung disease. Despite innate and adaptive immune responses upon infection, P. aeruginosa is capable of efficiently escaping host defenses, but the underlying immune mechanisms remain poorly understood. Myeloid-derived suppressor cells (MDSCs) are innate immune cells that are functionally characterized by their potential to suppress T- and natural killer (NK)-cell responses. Here we demonstrate, using an airway in vivo infection model, that P. aeruginosa recruits and activates neutrophilic MDSCs, which functionally suppress T-cell responses. We further show that the CF gene defect (CF transmembrane conductance regulator, CFTR) modulates the functionality, but not the recruitment or generation of neutrophilic MDSCs. Collectively, we define a mechanism by which P. aeruginosa airway infection undermines host immunity by modulating neutrophilic MDSCs in vivo.

mRNA-Mediated Gene Supplementation of Toll-Like Receptors as Treatment Strategy for Asthma In Vivo.

Asthma is the most common chronic disease in childhood. Although several therapeutic options are currently available to control the symptoms, many drugs have significant side effects and asthma remains an incurable disease. Microbial exposure in early life reduces the risk of asthma and several studies have suggested protective effects of Toll-like receptor (TLR) activation. We showed previously that modified mRNA provides a safe and efficient therapeutic tool for in vivo gene supplementation. Since current asthma drugs do not take patient specific immune and TLR backgrounds into consideration, treatment with tailored mRNA could be an attractive approach to account for the patient's individual asthma phenotype. Therefore, we investigated the effect of a preventative treatment with combinations of Tlr1, Tlr2 and Tlr6 mRNA in a House Dust Mite-induced mouse model of asthma. We used chemically modified mRNA which is-in contrast to conventional viral vectors-non-integrating and highly efficient in gene transfer. In our study, we found that treatment with either Tlr1/2 mRNA or Tlr2/6 mRNA, but not Tlr2 mRNA alone, resulted in better lung function as well as reduced airway inflammation in vivo. The present results point to a potentially protective effect of TLR heterodimers in asthma pathogenesis.

CXCR4+ granulocytes reflect fungal cystic fibrosis lung disease.

Cystic fibrosis airways are frequently colonised with fungi. However, the interaction of these fungi with immune cells and the clinical relevance in cystic fibrosis lung disease are incompletely understood.We characterised granulocytes in airway fluids and peripheral blood from cystic fibrosis patients with and without fungal colonisation, non-cystic fibrosis disease controls and healthy control subjects cross-sectionally and longitudinally and correlated these findings with lung function parameters.Cystic fibrosis patients with chronic fungal colonisation by Aspergillus fumigatus were characterised by an accumulation of a distinct granulocyte subset, expressing the HIV coreceptor CXCR4. Percentages of airway CXCR4(+) granulocytes correlated with lung disease severity in patients with cystic fibrosis.These studies demonstrate that chronic fungal colonisation with A. fumigatus in cystic fibrosis patients is associated with CXCR4(+) airway granulocytes, which may serve as a potential biomarker and therapeutic target in fungal cystic fibrosis lung disease.

Pathogenic fungi regulate immunity by inducing neutrophilic myeloid-derived suppressor cells.

Despite continuous contact with fungi, immunocompetent individuals rarely develop pro-inflammatory antifungal immune responses. The underlying tolerogenic mechanisms are incompletely understood. Using both mouse models and human patients, we show that infection with the human pathogenic fungi Aspergillus fumigatus and Candida albicans induces a distinct subset of neutrophilic myeloid-derived suppressor cells (MDSCs), which functionally suppress T and NK cell responses. Mechanistically, pathogenic fungi induce neutrophilic MDSCs through the pattern recognition receptor Dectin-1 and its downstream adaptor protein CARD9. Fungal MDSC induction is further dependent on pathways downstream of Dectin-1 signaling, notably reactive oxygen species (ROS) generation as well as caspase-8 activity and interleukin-1 (IL-1) production. Additionally, exogenous IL-1ß induces MDSCs to comparable levels observed during C. albicans infection. Adoptive transfer and survival experiments show that MDSCs are protective during invasive C. albicans infection, but not A. fumigatus infection. These studies define an innate immune mechanism by which pathogenic fungi regulate host defense.

Regulatory T-cell impairment in cystic fibrosis patients with chronic pseudomonas infection.

RATIONALE: Patients with cystic fibrosis (CF) lung disease have chronic airway inflammation driven by disrupted balance of T-cell (Th17 and Th2) responses. Regulatory T cells (Tregs) dampen T-cell activation, but their role in CF is incompletely understood. OBJECTIVES: To characterize numbers, function, and clinical impact of Tregs in CF lung disease. METHODS: Tregs were quantified in peripheral blood and airway samples from patients with CF and from lung disease control patients without CF and healthy control subjects. The role of Pseudomonas aeruginosa and CF transmembrane conductance regulator (CFTR) in Treg regulation was analyzed by using in vitro and murine in vivo models. MEASUREMENTS AND MAIN RESULTS: Tregs were decreased in peripheral blood and airways of patients with CF compared with healthy controls or lung disease patients without CF and correlated positively with lung function parameters. Patients with CF with chronic P. aeruginosa infection had lower Tregs compared with patients with CF without P. aeruginosa infection. Genetic knockout, pharmacological inhibition, and P. aeruginosa infection studies showed that both P. aeruginosa and CFTR contributed to Treg dysregulation in CF. Functionally, Tregs from patients with CF or from Cftr(-/-) mice were impaired in suppressing conventional T cells, an effect that was enhanced by P. aeruginosa infection. The loss of Tregs in CF affected memory, but not naive Tregs, and manifested gradually with disease progression. CONCLUSIONS: Patients with CF who have chronic P. aeruginosa infection show an age-dependent, quantitative, and qualitative impairment of Tregs. Modulation of Tregs represents a novel strategy to rebalance T-cell responses, dampen inflammation, and ultimately improve outcomes for patients with infective CF lung disease.

The chemokine CCL18 characterises Pseudomonas infections in cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease is characterised by chronic Pseudomonas aeruginosa infection and leukocyte infiltration. Chemokines recruit leukocytes to sites of infection. Gene expression analysis identified the chemokine CCL18 as upregulated in CF leukocytes. We hypothesised that CCL18 characterises infection and inflammation in patients with CF lung disease. Therefore, we quantified CCL18 protein levels in the serum and airway fluids of CF patients and healthy controls, and studied CCL18 protein production by airway cells ex vivo. These studies demonstrated that CCL18 levels were increased in the serum and airway fluids from CF patients compared with healthy controls. Within CF patients, CCL18 levels were increased in P. aeruginosa-infected CF patients. CCL18 levels in the airways, but not in serum, correlated with severity of pulmonary obstruction in CF. Airway cells isolated from P. aeruginosa-infected CF patients produced significantly higher amounts of CCL18 protein compared with airway cells from CF patients without P. aeruginosa infection or healthy controls. Collectively, these studies show that CCL18 levels characterise chronic P. aeruginosa infection and pulmonary obstruction in patients with CF. CCL18 may, thus, serve as a potential biomarker and therapeutic target in CF lung disease.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

CXCR1 and CXCR2 haplotypes synergistically modulate cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease severity is largely independent on the CF transmembrane conductance regulator (CFTR) genotype, indicating the contribution of genetic modifiers. The chemokine receptors CXCR1 and CXCR2 have been found to play essential roles in the pathogenesis of CF lung disease. Here, we determine whether genetic variation of CXCR1 and CXCR2 influences CF lung disease severity. Genomic DNA of CF patients in Germany (n = 442) was analysed for common variations in CXCR1 and CXCR2 using a single-nucleotide polymorphism (SNP) tagging approach. Associations of CXCR1 and CXCR2 SNPs and haplotypes with CF lung disease severity, CXCR1 and CXCR2 expression, and neutrophil effector functions were assessed. Four SNPs in CXCR1 and three in CXCR2 strongly correlated with age-adjusted lung function in CF patients. SNPs comprising haplotypes CXCR1_Ha and CXCR2_Ha were in high linkage disequilibrium and patients heterozygous for the CXCR1-2 haplotype cluster (CXCR1-2_Ha) had lower lung function compared with patients with homozygous wild-type alleles (forced expiratory volume in 1 s ≤ 70% predicted, OR 7.24; p = 2.30 × 10(-5)). CF patients carrying CXCR1-2_Ha showed decreased CXCR1 combined with increased CXCR2 mRNA and protein expression, and displayed disturbed antibacterial effector functions. CXCR1 and CXCR2 genotypes modulate lung function and antibacterial host defence in CF lung disease.