Inflammatory Cytokines in Middle Ear Effusion of Patients With Asthma, Chronic Rhinosinusitis With Nasal Polyps With or Without NSAID Intolerance.

OBJECTIVE: To measure the inflammatory cytokines of middle ear effusion (MEE) in otitis media (OM) associated with asthma and chronic rhinosinusitis with nasal polyps (CRSwNP) with or without nonsteroidal anti-inflammatory drug (NSAID) sensitivity to strengthen our assumption that OM is part of the same inflammatory entity. The potential individual differences between MEE inflammatory cytokines could be used in clinical practice for more individual characterization of the inflammation. STUDY DESIGN: Case-control study. SETTING: Tertiary referral center. PATIENTS: Convenience sample of 24 case patients with otitis media with effusion (OME) or chronic otitis media (COM), asthma, and CRSwNP, 14 of whom had NSAID intolerance, and 8 controls with OME but no history of asthma, CRSwNP, or NSAID intolerance. INTERVENTION: Diagnostic. MAIN OUTCOME AND MEASURE: Inflammatory cytokines including interleukins (IL)-4, IL-5, IL-6, IL-13, and interferon gamma (IFN-γ) in middle ear effusion. RESULTS: The MEE mass fractions of IL-5 (p = 0.003) and IFN-γ (p = 0.048) were higher among our case patients with OME/COM than among the controls. For IL-4 and IL-13, the mass fractions were also higher among the case patients than the controls, but this difference was not statistically significant (p = 0.199 and p = 0.617, respectively). We found no difference between the IL-6 mass fractions of the groups. We found notable heterogeneity in individual patients' cytokine levels. CONCLUSIONS: According to our findings, OM, when present, should be considered part of the respiratory inflammatory process associated with asthma and CRSwNP. The individual differences in MEE cytokine levels could be useful as biomarkers.

Interaction of mediators and effector cells in cashew nut-induced anaphylaxis.

BACKGROUND: The underlying mechanisms of an immediate food-induced allergic reaction involve mast cell degranulation and recruitment of other effector cells, such as lymphocytes, eosinophils, and basophils. How the interaction of various mediators and cells results in anaphylaxis is not fully understood. OBJECTIVE: To evaluate changes in platelet-activating factor (PAF), platelet-activating factor acetylhydrolase (PAF-AH), tryptase, eosinophils, basophils, and eosinophil cationic protein (ECP) in cashew nut-induced anaphylaxis. METHODS: Open cashew nut challenges were performed on 106 children (aged 1-16 years), sensitized to cashew nut, with earlier allergic reaction to cashew nut or no known exposure. PAF, PAF-AH, tryptase, ECP, eosinophils, and basophils were measured at 4 time points. RESULTS: Of 72 challenges with positive results, 34 were defined as anaphylactic. Eosinophil count decreased progressively during an anaphylactic reaction at all 4 time points (P < .005*) compared with baseline. Although significant PAF elevation was observed 1 hour from moderate-to-severe reaction (P = .04*), PAF seemed to peak especially in anaphylaxis but did not achieve statistical significance. PAF peak ratio (peak PAF/baseline PAF) was significantly greater in anaphylactic reactions compared with the no-anaphylaxis group (P = .008*). Maximal percentage change in eosinophils revealed negative correlation to severity score and PAF peak ratio (Spearman's rho -0.424 and -0.516, respectively). Basophils decreased significantly in moderate-to-severe reactions and in anaphylaxis (P < .05*) compared with baseline. Delta-tryptase (peak tryptase minus baseline) did not differ significantly between anaphylaxis and the no-anaphylaxis subgroups (P = .05). CONCLUSION: PAF is a specific anaphylaxis biomarker. Marked decline of eosinophils during anaphylaxis may be related to robust secretion of PAF reflecting migration of eosinophils to target tissues.

Long-term changes in milk component immunoglobulins reflect milk oral immunotherapy outcomes in Finnish children.

BACKGROUND: Milk oral immunotherapy (OIT) may increase the amount of milk protein that can be ingested without triggering an allergic reaction. It is important to understand why some patients benefit from the treatment while others do not. OBJECTIVE: The aim was to define the differences in the milk allergen component-specific (casein, α-lactalbumin, ß-lactoglobulin) immunoglobulin (sIg [sIgE, sIgG4, and sIgA]) levels relative to the long-term outcomes of milk OIT. METHODS: In this long-term, open-label follow-up study, 286 children started milk OIT between 2005 and 2015. Follow-up data were collected at two points: the post-buildup phase and long term (range 1-11 years, median 6 years). Comparisons of sIg levels were made among three outcome groups of self-reported long-term milk consumption (high-milk dose, low-milk dose, and avoidance). RESULTS: A total of 168 (59%) of the 286 patients on OIT participated. Most patients (57%) were in the high-dose group; here, 80% of these patients had a baseline casein sIgE value less than 28 kUA/L, they had the lowest casein sIgE levels at all time (p < .001), their casein sIgG4/IgE levels increased, and long-term casein sIgA was highest compared with the low-dose and avoidance groups (p = .02). Low-milk dose group had the highest casein sIgG4/IgE levels in long term (p = .002). CONCLUSION: The baseline Ig profiles and responses to milk OIT differed depending on long-term milk consumption. Lower casein sIgE levels were associated with better outcome. Milk casein sIgA differed in the long term among high-milk consumers.

Optimizing tools for evaluating challenge outcomes in children with cashew nut allergy.

BACKGROUND: The incidence of cashew nut anaphylaxis is increasing and there is a need for accurate diagnostic tests. Age-specific cutoffs in children are lacking. Changes in serum tryptase levels are not well documented in pediatric food allergy, except in anaphylaxis. OBJECTIVE: To evaluate the ability of various tests to diagnose cashew nut allergy and to predict reaction severity. We also investigated changes in tryptase and their correlation to reaction severity. METHODS: We performed an open cashew nut challenge on 106 children (aged 1-16 years), who were sensitized to cashew nut with either previous allergic reaction to cashew nut or no known exposure. We analyzed the accuracy of Ana o 3 immunoglobulin E (IgE), cashew nut IgE, skin prick test, basophil activation test (BAT), and combinations thereof to diagnose cashew nut allergy and to predict reaction severity. Tryptase level was measured at the baseline and during an allergic reaction. RESULTS: A total of 72 children had positive challenge outcomes. Ana o 3 IgE seemed to be the best single test to diagnose cashew allergy, with a 0.97 kU/L cutoff exhibiting 94.1% specificity and 61.1% sensitivity. Though BAT values of at least 22.8% best predicted reaction severity, with 91.7% specificity and 60.7% sensitivity, the cutoffs were age-specific. Tryptase levels increased substantially 1 to 2 hours after the first allergic symptoms compared with baseline. CONCLUSION: Ana o 3 IgE seems to be the best diagnostic test in pediatric cashew nut allergy, and test combinations do not seem to improve the diagnostics. Cutoffs are age-specific. BAT is promising in predicting reaction severity. Tryptase levels should be measured 1 to 2 hours after initiation of an allergic reaction.

High Prevalence of Sensitization to Mites and Insects in Greenhouses Using Biologic Pest Control.

BACKGROUND: Mites and insects are widely used as biologic pest control in greenhouses. A few studies have reported sensitization to mites among greenhouse workers, but the prevalence of sensitization to pest control insects is not known. OBJECTIVE: We aimed to determine the prevalence of IgE-mediated sensitization to pests and their control organisms in the population of exposed greenhouse workers and the relationship between sensitization and allergic symptoms. METHODS: In a cross-sectional study, we interviewed 117 tomato and cucumber greenhouse workers from eight companies that use biologic pest control. Sensitization to nine organisms was assessed by serum-specific IgE measurement. We also measured fractional exhaled nitric oxide. RESULTS: The prevalence of specific sensitization to pests and pest control organisms was 50%; to mites, 29%; and to insects, 46%. Of the individual species, Macrolophus pygmaeus insect sensitization had the highest prevalence (46%). Asthma symptoms were significantly associated with sensitization to pest and pest control organisms (odds ratio [OR] = 3.9; 95% confidence interval [CI], 1.2-12.5) and with a fractional exhaled nitric oxide level of 25 ppb or greater (OR = 4.8; 95% CI, 1.7-13.8), indicating eosinophilic airway inflammation. Southeast Asian origin was significantly associated with sensitization (OR = 5.1; 95% CI, 2.1-12.1) and rhinitis (OR = 2.8; 95% CI, 1.2-6.3). CONCLUSIONS: Tomato and cucumber greenhouse workers were commonly sensitized to predatory insect M pygmaeus and pest control mites. Our findings stress the importance of surveilling and preventing work-related allergic diseases among greenhouse workers.

A Randomized, Open-Label Trial of Hen's Egg Oral Immunotherapy: Efficacy and Humoral Immune Responses in 50 Children.

BACKGROUND: Egg allergy is the second most common food allergy in children. Persistent food allergy increases the risk of anaphylaxis and reduces the quality of life. OBJECTIVE: To determine the efficacy of oral immunotherapy (OIT) with raw egg white powder and study its effects on humoral responses in children with persistent egg allergy. METHODS: Fifty children aged 6 to 17 years with egg allergy, diagnosed by double-blind, placebo-controlled food challenge, were randomized 3:2 to 8 months of OIT with a maintenance dose of 1 g of egg white protein or 6 months of avoidance after which the avoidance group crossed over to OIT. We examined changes in IgE, IgG4, and IgA concentrations to Gal d 1-4 during OIT compared with avoidance and assessed clinical reactivity at 8 and 18 months. RESULTS: After 8 months, 22 of 50 children (44%) on OIT and 1 of 21 (4.8%) on egg avoidance were desensitized to the target dose, 23 of 50 (46%) were partially desensitized (dose <1 g), and 5 of 50 (10%) discontinued. IgG4 concentrations to Gal d 1-4 and IgA to Gal d 1-2 increased significantly, whereas IgE to Gal d 2 decreased. A heatmap analysis of the IgE patterns revealed 3 distinct clusters linked with the clinical outcome. High baseline egg white-specific IgE and polysensitization to Gal d 1-4 related with failure to achieve the maintenance dose at 8 months. After 18 months of treatment, 36 of 50 patients (72%) were desensitized and 8 of 50 (16%) partially desensitized. CONCLUSIONS: OIT with raw egg enables liberation of egg products into the daily diet in most patients. Subjects with high egg white-specific IgE concentrations and sensitization to multiple egg allergen components at baseline benefit from prolonged treatment.

Integrative Transcriptomics Reveals Activation of Innate Immune Responses and Inhibition of Inflammation During Oral Immunotherapy for Egg Allergy in Children.

We previously reported the results of a randomized, open-label trial of egg oral immunotherapy (OIT) in 50 children where 44% were desensitized and 46% were partially desensitized after 8 months of treatment. Here we focus on cell-mediated molecular mechanisms driving desensitization during egg OIT. We sought to determine whether changes in genome-wide gene expression in blood cells during egg OIT correlate with humoral responses and the clinical outcome. The blood cell transcriptome of 50 children receiving egg OIT was profiled using peripheral blood mononuclear cell (PBMC) samples obtained at baseline and after 3 and 8 months of OIT. We identified 467 differentially expressed genes (DEGs) after 3 or 8 months of egg OIT. At 8 months, 86% of the DEGs were downregulated and played a role in the signaling of TREM1, IL-6, and IL-17. In correlation analyses, Gal d 1-4-specific IgG4 antibodies associated positively with DEGs playing a role in pathogen recognition and antigen presentation and negatively with DEGs playing a role in the signaling of IL-10, IL-6, and IL-17. Desensitized and partially desensitized patients had differences in their antibody responses, and although most of the transcriptomic changes were shared, both groups had also specific patterns, which suggest slower changes in partially desensitized and activation of NK cells in the desensitized group. OIT for egg allergy in children inhibits inflammation and activates innate immune responses regardless of the clinical outcome at 8 months. Changes in gene expression patterns first appear as posttranslational protein modifications, followed by more sustained epigenetic gene regulatory functions related to successful desensitization.

Serum chitinase-like protein YKL-40 is linked to small airway function in children with asthmatic symptoms.

BACKGROUND: Lung function impairment among asthmatic children begins in early life, and biomarkers for identifying this impairment are needed. The chitinase-like protein YKL-40 has been associated with asthma and lung function in adults, but studies in children have yielded conflicting results. We evaluated the potential of YKL-40 and other systemic biomarkers for identifying lung function deficits in children with asthmatic symptoms. METHODS: We determined the levels of serum YKL-40, periostin, and high-sensitivity C-reactive protein (hs-CRP) from the blood samples of 49 children with asthmatic symptoms. Lung function was assessed with impulse oscillometry (IOS) and spirometry, combined with an exercise challenge and a bronchodilator test. Fractional exhaled nitric oxide was measured at multiple flow rates. RESULTS: Serum levels of YKL-40 showed significant correlations with most IOS indices at baseline (P = .008-.039), but there was no association between YKL-40 and spirometry parameters. Neither periostin nor hs-CRP were associated with baseline lung function. Children with a significant response in either the exercise challenge or the bronchodilator test had increased serum levels of YKL-40 (P = .003) and periostin (P = .035). YKL-40 correlated significantly with the blood neutrophil count (rs  = .397, P = .005) but was not associated with biomarkers of eosinophilic inflammation. CONCLUSION: Serum YKL-40 is a potential biomarker for lung function deficits in children with asthmatic symptoms. These deficits appear to be focused on small airways and may remain undetected with spirometry.

A ß2-Integrin/MRTF-A/SRF Pathway Regulates Dendritic Cell Gene Expression, Adhesion, and Traction Force Generation.

ß2-integrins are essential for immune system function because they mediate immune cell adhesion and signaling. Consequently, a loss of ß2-integrin expression or function causes the immunodeficiency disorders, Leukocyte Adhesion Deficiency (LAD) type I and III. LAD-III is caused by mutations in an important integrin regulator, kindlin-3, but exactly how kindlin-3 regulates leukocyte adhesion has remained incompletely understood. Here we demonstrate that mutation of the kindlin-3 binding site in the ß2-integrin (TTT/AAA-ß2-integrin knock-in mouse/KI) abolishes activation of the actin-regulated myocardin related transcription factor A/serum response factor (MRTF-A/SRF) signaling pathway in dendritic cells and MRTF-A/SRF-dependent gene expression. We show that Ras homolog gene family, member A (RhoA) activation and filamentous-actin (F-actin) polymerization is abolished in murine TTT/AAA-ß2-integrin KI dendritic cells, which leads to a failure of MRTF-A to localize to the cell nucleus to coactivate genes together with SRF. In addition, we show that dendritic cell gene expression, adhesion and integrin-mediated traction forces on ligand coated surfaces is dependent on the MRTF-A/SRF signaling pathway. The participation of ß2-integrin and kindlin-3-mediated cell adhesion in the regulation of the ubiquitous MRTF-A/SRF signaling pathway in immune cells may help explain the role of ß2-integrin and kindlin-3 in integrin-mediated gene regulation and immune system function.

Beta2-Integrins and Interacting Proteins in Leukocyte Trafficking, Immune Suppression, and Immunodeficiency Disease.

Beta2-integrins are complex leukocyte-specific adhesion molecules that are essential for leukocyte (e.g., neutrophil, lymphocyte) trafficking, as well as for other immunological processes such as neutrophil phagocytosis and ROS production, and T cell activation. Intriguingly, however, they have also been found to negatively regulate cytokine responses, maturation, and migratory responses in myeloid cells such as macrophages and dendritic cells, revealing new, and unexpected roles of these molecules in immunity. Because of their essential role in leukocyte function, a lack of expression or function of beta2-integrins causes rare immunodeficiency syndromes, Leukocyte adhesion deficiency type I, and type III (LAD-I and LAD-III). LAD-I is caused by reduced or lost expression of beta2-integrins, whilst in LAD-III, beta2-integrins are expressed but dysfunctional because a major integrin cytoplasmic regulator, kindlin-3, is mutated. Interestingly, some LAD-related phenotypes such as periodontitis have recently been shown to be due to an uncontrolled inflammatory response rather than to an uncontrolled infection, as was previously thought. This review will focus on the recent advances concerning the regulation and functions of beta2-integrins in leukocyte trafficking, immune suppression, and immune deficiency disease.

Filamin A Is Required for Optimal T Cell Integrin-Mediated Force Transmission, Flow Adhesion, and T Cell Trafficking.

T cells traffic from the bloodstream into tissues to perform their functions in the immune system and are therefore subjected to a range of different mechanical forces. Integrins are essential for T cell trafficking into the tissues, as they mediate firm adhesion between the T cell and the endothelium under shear flow conditions. In addition, integrins are important for the formation of the contact between the T cell and the APC required for T cell activation. The actin-binding protein filamin A (FlnA) provides an important link between the integrin and the actin cytoskeleton. FlnA has been reported to function as an integrin inhibitor by competing with talin. However, its role in regulating integrin-dependent immune functions in vivo is currently poorly understood. In this study, we have investigated the role of FlnA in T cells, using T cell-specific FlnA knockout mice. We report that FlnA is required for the formation of strong integrin-ligand bonds under shear flow and for the generation of integrin-mediated T cell traction forces on ligand-coated hydrogels. Consequently, absence of FlnA leads to a reduction in T cell adhesion to integrin ligands under conditions of shear flow, as well as reduced T cell trafficking into lymph nodes and sites of skin inflammation. In addition, FlnA is not needed for T cell activation in vivo, which occurs in shear-free conditions in lymphoid organs. Our results therefore reveal a role of FlnA in integrin force transmission and T cell trafficking in vivo.

Filamin A Regulates Neutrophil Adhesion, Production of Reactive Oxygen Species, and Neutrophil Extracellular Trap Release.

Neutrophils are of fundamental importance in the early immune response and use various mechanisms to neutralize invading pathogens. They kill endocytosed pathogens by releasing reactive oxygen species in the phagosome and release neutrophil extracellular traps (NETs) into their surroundings to immobilize and kill invading micro-organisms. Filamin A (FlnA) is an important actin cross-linking protein that is required for cellular processes involving actin rearrangements, such cell migration. It has also been shown to negatively regulate integrin activation and adhesion. However, its role in the regulation of ß2 integrin-dependent adhesion, as well as in other cellular functions in neutrophils, is poorly understood. Using a transgenic mouse model in which FlnA is selectively depleted in myeloid cells, such as neutrophils, we show that FlnA negatively regulates ß2 integrin adhesion to complement component iC3b and ICAM-1 in shear-free, but not shear-flow, conditions. FlnA deletion does not affect phagocytosis of Escherichia coli or Staphylococcus aureus or their intracellular killing. However, FlnA negatively regulates production of reactive oxygen species upon cell activation. Conversely, neutrophil activation through TLR4, as well as through activation by the Gram-negative bacteria E. coli, results in reduced NET production in FlnA-depleted neutrophils. Thus, FlnA is a negative regulator of ß2 integrin-dependent cell adhesion and reactive oxygen species production but is required for NET production in primary murine neutrophils.

Anti-Inflammatory Effects of Metformin Irrespective of Diabetes Status.

RATIONALE: The diabetes mellitus drug metformin is under investigation in cardiovascular disease, but the molecular mechanisms underlying possible benefits are poorly understood. OBJECTIVE: Here, we have studied anti-inflammatory effects of the drug and their relationship to antihyperglycemic properties. METHODS AND RESULTS: In primary hepatocytes from healthy animals, metformin and the IKKß (inhibitor of kappa B kinase) inhibitor BI605906 both inhibited tumor necrosis factor-α-dependent IκB degradation and expression of proinflammatory mediators interleukin-6, interleukin-1ß, and CXCL1/2 (C-X-C motif ligand 1/2). Metformin suppressed IKKα/ß activation, an effect that could be separated from some metabolic actions, in that BI605906 did not mimic effects of metformin on lipogenic gene expression, glucose production, and AMP-activated protein kinase activation. Equally AMP-activated protein kinase was not required either for mitochondrial suppression of IκB degradation. Consistent with discrete anti-inflammatory actions, in macrophages, metformin specifically blunted secretion of proinflammatory cytokines, without inhibiting M1/M2 differentiation or activation. In a large treatment naive diabetes mellitus population cohort, we observed differences in the systemic inflammation marker, neutrophil to lymphocyte ratio, after incident treatment with either metformin or sulfonylurea monotherapy. Compared with sulfonylurea exposure, metformin reduced the mean log-transformed neutrophil to lymphocyte ratio after 8 to 16 months by 0.09 U (95% confidence interval, 0.02-0.17; P=0.013) and increased the likelihood that neutrophil to lymphocyte ratio would be lower than baseline after 8 to 16 months (odds ratio, 1.83; 95% confidence interval, 1.22-2.75; P=0.00364). Following up these findings in a double-blind placebo controlled trial in nondiabetic heart failure (trial registration: NCT00473876), metformin suppressed plasma cytokines including the aging-associated cytokine CCL11 (C-C motif chemokine ligand 11). CONCLUSION: We conclude that anti-inflammatory properties of metformin are exerted irrespective of diabetes mellitus status. This may accelerate investigation of drug utility in nondiabetic cardiovascular disease groups. CLINICAL TRIAL REGISTRATION: Name of the trial registry: TAYSIDE trial (Metformin in Insulin Resistant Left Ventricular [LV] Dysfunction). URL: https://www.clinicaltrials.gov. Unique identifier: NCT00473876.

Blood cell integrins and diseases.

Integrins are adhesion molecules on the surface of cells. In blood cells they are responsible for rapid changes during adhesion of the cell to the endothelium. Deficiency or defective function of integrins will result in severe illnesses. Surprisingly, certain variants of integrins are associated with an increased risk of developing SLE. In autoimmune diseases and as a result of organ transplantations integrins participate in reactions in which leukocytes attack the body's own tissues. This has resulted in the development of drugs in antibody form for inhibition of the action of integrins. These drugs may, however, exhibit severe adverse effects.

Mice Lacking beta2-Integrin Function Remain Glucose Tolerant in Spite of Insulin Resistance, Neutrophil Infiltration and Inflammation.

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.

Optimal T Cell Activation and B Cell Antibody Responses In Vivo Require the Interaction between Leukocyte Function-Associated Antigen-1 and Kindlin-3.

Kindlin-3 is an important integrin regulator that is mutated in the rare genetic disorder, leukocyte adhesion deficiency type III, a disorder characterized by defective neutrophil trafficking and platelet function, leading to recurrent bacterial infections and bleeding. Kindlin-3 is also known to regulate T cell adhesion in vitro and trafficking in vivo, but whether the integrin/kindlin interaction regulates T or B cell activation in vivo is unclear. In this study, we used TTT/AAA ß2-integrin knock-in (KI) mice and TCR-transgenic (OT-II) KI mice, in which the integrin/kindlin connection is disrupted, to investigate the role of the integrin/kindlin interaction in T cell activation. We show that basal T cell activation status in these animals in vivo is normal, but they display reduced T cell activation by wild-type Ag-loaded dendritic cells in vitro. In addition, T cell activation in vivo is reduced. We also show that basal Ab levels are normal in TTT/AAA ß2-integrin KI mice, but B cell numbers in lymph nodes and IgG and IgM production after immunization are reduced. In conclusion, we show that the integrin/kindlin interaction is required for trafficking of immune cells, as well as for T cell activation and B cell Ab responses in vivo. These results imply that the immunodeficiency found in leukocyte adhesion deficiency type III patients, in addition to being caused by defects in neutrophil function, may be due, in part, to defects in lymphocyte trafficking and activation.

Functional Beta2-Integrins Restrict Skin Inflammation In Vivo.

Beta2-integrins and the important integrin regulator kindlin-3 are essential for leukocyte trafficking, but the role of beta2-integrins in regulating inflammation is still incompletely understood. Here, we have investigated skin inflammation in a mouse model where the kindlin-3 binding site in the beta2-integrin has been mutated (TTT/AAA-beta2-integrin knock-in), leading to expressed but dysfunctional integrins. We show that, surprisingly, neutrophil trafficking into the inflamed skin in a contact hypersensitivity model is normal in these mice, although trafficking of T cells and eosinophils into the skin is reduced. Instead, expression of dysfunctional integrins leads to increased mast cell and dendritic cell numbers in the skin, increased inflammatory cytokine production in the inflamed skin in vivo, and in mast cells in vitro. Furthermore, expression of dysfunctional integrins leads to increased dendritic cell activation and migration to lymph nodes and increased Th1 responses in vivo. Therefore, the kindlin-3/integrin interaction is important for trafficking of T cells and eosinophils but not absolutely required for neutrophil trafficking into the inflamed skin. Functional beta2-integrins also have a major role in restricting the immune response in the inflamed skin and lymph nodes in vivo, likely through effects on mast cell and dendritic cell numbers and activation.

Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

BACKGROUND: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma. METHODS: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation. RESULTS: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages. CONCLUSIONS: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Loss of beta2-integrin-mediated cytoskeletal linkage reprogrammes dendritic cells to a mature migratory phenotype.

The actin cytoskeleton has been reported to restrict signalling in resting immune cells. Beta2-integrins, which mediate adhesion and cytoskeletal organization, are emerging as negative regulators of myeloid cell-mediated immune responses, but the molecular mechanisms involved are poorly understood. Here, we show that loss of the interaction between beta2-integrins and kindlin-3 abolishes the actin-linkage of integrins and the GM-CSF receptor in dendritic cells. This leads to increased GM-CSF receptor/Syk signalling, and to the induction of a transcriptional programme characteristic of mature, migratory dendritic cells, accumulation of migratory dendritic cells in lymphoid organs, and increased Th1 immune responses in vivo. We observe increased GM-CSF responses and increased survival in neutrophils where the interaction between integrin and the cytoskeleton is disrupted. Thus, ligand-reinforced beta2-integrin tail interactions restrict cytokine receptor signalling, survival, maturation and migration in myeloid cells and thereby contribute to immune homeostasis in vivo.

Acinetobacter species in the skin microbiota protect against allergic sensitization and inflammation.

BACKGROUND: The human commensal microbiota interacts in a complex manner with the immune system, and the outcome of these interactions might depend on the immune status of the subject. OBJECTIVE: Previous studies have suggested a strong allergy-protective effect for Gammaproteobacteria. Here we analyze the skin microbiota, allergic sensitization (atopy), and immune function in a cohort of adolescents, as well as the influence of Acinetobacter species on immune responses in vitro and in vivo. METHODS: The skin microbiota of the study subjects was identified by using 16S rRNA sequencing. PBMCs were analyzed for baseline and allergen-stimulated mRNA expression. In in vitro assays human monocyte-derived dendritic cells and primary keratinocytes were incubated with Acinetobacter lwoffii. Finally, in in vivo experiments mice were injected intradermally with A lwoffii during the sensitization phase of the asthma protocol, followed by readout of inflammatory parameters. RESULTS: In healthy subjects, but not in atopic ones, the relative abundance of Acinetobacter species was associated with the expression of anti-inflammatory molecules by PBMCs. Moreover, healthy subjects exhibited a robust balance between anti-inflammatory and TH1/TH2 gene expression, which was related to the composition of the skin microbiota. In cell assays and in a mouse model, Acinetobacter species induced strong TH1 and anti-inflammatory responses by immune cells and skin cells and protected against allergic sensitization and lung inflammation through the skin. CONCLUSION: These results support the hypothesis that skin commensals play an important role in tuning the balance of TH1, TH2, and anti-inflammatory responses to environmental allergens.