Inflammasome activation and formation of ASC specks in patients with juvenile idiopathic arthritis.

Objective: The formation of large intracellular protein aggregates of the inflammasome adaptor ASC is a hallmark of inflammasome activation and characteristic of autoinflammation. Inflammasome activated cells release the highly proinflammatory cytokine IL-1ß in addition to ASC specks into the extracellular space. Autoinflammatory activity has been demonstrated in systemic JIA, however minimal data exist on the role of inflammasomes in other JIA subtypes. We therefore investigated, if pyroptotic cells are present in the circulation of oligo- and poly-articular JIA. Methods: Peripheral blood of JIA patients (n = 46) was investigated for ASC speck formation, a key step in inflammasome activation, by flow cytometry and immunofluorescence. Free ASC and proinflammatory cytokine levels were determined by ELISA and multiplex assay. Results: Oligo-articular JIA patients showed a significantly increased proportion of ASC speck+ monocytes compared to poly-articular JIA patients. In serum free ASC alone is not sufficient to assess inflammasome activity and does not correlate with ASC speck+ monocytes. Compared to control several cytokines were significantly elevated in samples of JIA patients. JIA serum containing antinuclear antibodies, incubated with ASC specks boosts a secondary inflammation by IL-1ß production in macrophages. Conclusion: For the first time, we detect ex vivo inflammasome activation by ASC speck formation in oligo- and poly-articular JIA patients. Most notably, inflammasome activation was significantly higher in oligo- compared to poly-articular JIA patients. This data suggests that inflammasome derived autoinflammation may have a greater influence in the previously thought autoimmune oligo-articular JIA patients.

Microarray-Based Analyses of Rhinovirus Species-Specific Antibody Responses in Exacerbated Pediatric Asthma in a German Pediatric Cohort.

Rhinoviruses (RV) account for a significant number of asthma exacerbations, and RV species C may be associated with a severe course in vulnerable patient groups. Despite important evidence on the role of RV reported by clinicians and life scientists, there are still unanswered questions regarding their influence on asthma exacerbation in young patients. Thus, we measured the RVspecies-specific IgG titers in our German pediatric exacerbation cohort using a microarray-based technology. For this approach, human sera of patients with exacerbated asthma and wheeze, as well as healthy control subjects (n = 136) were included, and correlation analyses were performed. Concordantly with previously published results, we observed significantly higher cumulative levels of RV species A-specific IgG (p = 0.011) and RV-C-specific IgG (p = 0.051) in exacerbated asthma group compared to age-matched controls. Moreover, atopic wheezers had increased RV-specific IgG levels for species A (p = 0.0011) and species C (p = 0.0009) compared to non-atopic wheezers. Hypothesizing that bacterial infection positively correlates with immune memory against RV, we included nasopharyngeal swab results in our analyses and detected limited correlations. Interestingly, the eosinophil blood titer positively correlated with RV-specific IgG levels. With these observations, we add important observations to the existing data regarding exacerbation in pediatric and adolescent medicine. We propose that scientists and clinicians should pay more attention to the relevance of RV species in susceptible pediatric patients.

Inhibition of TNF-α Restores Muscle Force, Inhibits Inflammation, and Reduces Apoptosis of Traumatized Skeletal Muscles.

BACKGROUND: Muscle injuries are common in humans and are often associated with irrecoverable damage and disability. Upon muscle injury, TNF-α signaling pathways modulate the healing process and are predominantly associated with tissue degradation. In this study we assumed that TNF-α inhibition could reduce the TNF-α-associated tissue degradation after muscle injury. MATERIALS AND METHODS: Therefore, the left soleus muscle of 42 male Wistar rats was injured using a standardized open muscle injury model. All rats were treated immediately after injury either with infliximab (single i.p. injection; 10 mg/kg b.w.) or saline solution i.p. Final measurements were conducted at day one, four, and 14 post injury. The muscle force, the muscle cell proliferation, the muscle cell coverage as well as the myofiber diameter served as read out parameters of our experiment. RESULTS: Systemic application of infliximab could significantly reduce the TNF-α levels in the injured muscle at day four upon trauma compared to saline treated animals. The ratio of muscle weight to body weight was increased and the twitch muscle force showed a significant rise 14 days after trauma and TNF-α inhibition. Quantification of myofiber diameter in the penumbra zone showed a significant difference between both groups at day one and four after injury, indicated by muscle hypertrophy in the infliximab group. Planimetric analysis of the injured muscle at day 14 revealed increased muscle tissue fraction in the infliximab group compared to the control animals. Muscle cell proliferation did not differ between both groups. CONCLUSIONS: These data provide evidence that the TNF-α blockade positively regulates the restauration of skeletal muscles upon injury.

Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood.

Inflammasome activation is linked to the aggregation of the adaptor protein ASC into a multiprotein complex, known as the ASC speck. Redistribution of cytosolic ASC to this complex has been widely used as a readout for inflammasome activation and precedes the downstream proteolytic release of the proinflammatory cytokines, IL-1ß and IL-18. Although inflammasomes are important for many diseases such as periodic fever syndromes, COVID-19, gout, sepsis, atherosclerosis and Alzheimer's disease, only a little knowledge exists on the precise and cell type specific occurrence of inflammasome activation in patient samples ex vivo. In this report, we provide detailed information about the optimal conditions to reliably identify inflammasome activated monocytes by ASC speck formation using a modified flow cytometric method introduced by Sester et al. in 2015. Since no protocol for optimal sample processing exists, we tested human blood samples for various conditions including anticoagulant, time and temperature, the effect of one freeze-thaw cycle for PBMC storage, and the fast generation of a positive control. We believe that this flow cytometric protocol will help researchers to perform high quality translational research in multicenter studies, and therefore provide a basis for investigating the role of the inflammasome in the pathogenesis of various diseases.

The mystery behind the nostrils - technical clues for successful nasal epithelial cell cultivation.

OBJECTIVES: Research involving the nose reveals important information regarding the morphology and physiology of the epithelium and its molecular response to agents. The role of nasal epithelial cells and other cell subsets within the nasal epithelium play an interesting translational split between experimental and clinical research studying respiratory disorders or pathogen reactions. With an additional technical manuscript including a detailed description of important technical aspects, tips, tricks, and nuances for a successful culturing of primary, human nasal epithelial cells (NAEPCs), we here aim to improve the process of communication between experimentalists and physicians, supporting the purpose of a fruitful work for future translational projects. METHODS: Based on previous work on various complex culture models of subject-derived NAEPCs, this additional manuscript harmonizes previously published facts combined with own experiences for a trouble-free implementation in laboratories. RESULTS: A well-designed experimental question is essential prior to the establishment of different NAEPCs culture models. The correct method of cell extraction from the nasal cavity is essential and represent an important basis for successful culture work. Prior enzymatic processing of biopsy specimens, cell culture materials, collagenization procedure, culture conditions, and choice of culture medium are some important practical notes that increase the quality of the culture. Moreover, protocols on imaging techniques including histologic and electron microscopy must be adapted for NAEPC culture. Adapted flow cytometric protocols and transepithelial electrical resistance measurements can add valuable information. OUTLOOK: A successful culturing of NAEPCs can provide an important basis for genetic studies and the implementation of omics-science, which is increasingly receiving broad attention in the scientific community. The common aim of in vitro 'mini-noses' will be a breakthrough in laboratories aiming to perform research under in vivo conditions. Here, organoid models are interesting models presenting a basis for translational studies.

Tim-3 is dispensable for allergic inflammation and respiratory tolerance in experimental asthma.

T cell immunoglobulin and mucin domain-containing molecule-3 (Tim-3) has been described as a transmembrane protein, expressed on the surface of various T cells as well as different cells of innate immunity. It has since been associated with Th1 mediated autoimmune diseases and transplantation tolerance studies, thereby indicating a possible role of this receptor in counter-regulation of Th2 immune responses. In the present study we therefore directly examined the role of Tim-3 in allergic inflammation and respiratory tolerance. First, Tim-3-/- mice and wild type controls were immunized and challenged with the model allergen ovalbumin (OVA) to induce an asthma-like phenotype. Analysis of cell numbers and distribution in the bronchoalveolar lavage (BAL) fluid as well as lung histology in H&E stained lung sections demonstrated a comparable degree of eosinophilic inflammation in both mouse strains. Th2 cytokine production in restimulated cell culture supernatants and serum IgE and IgG levels were equally increased in both genotypes. In addition, cell proliferation and the distribution of different T cell subsets were comparable. Moreover, analysis of both mouse strains in our respiratory tolerance model, where mucosal application of the model allergen before immunization, prevents the development of an asthma-like phenotype, revealed no differences in any of the parameters mentioned above. The current study demonstrates that Tim-3 is dispensable not only for the development of allergic inflammation but also for induction of respiratory tolerance in mice in an OVA-based model.

From Submerged Cultures to 3D Cell Culture Models: Evolution of Nasal Epithelial Cells in Asthma Research and Virus Infection.

Understanding the response to viral infection in the context of respiratory diseases is of significant importance. Recently, there has been more focus on the role of the nasal epithelium in disease modeling. Here, we provide an overview of different submerged, organotypic 3D and spheroid cell culture models of nasal epithelial cells, which were used to study asthma and allergy with a special focus on virus infection. In detail, this review summarizes the importance, benefits, and disadvantages of patient-derived cell culture models of nasal- and bronchial epithelial cells, including a comparison of these cell culture models and a discussion on why investigators should consider using nasal epithelial cells in their research. Exposure experiments, simple virus transduction analyses as well as genetic studies can be performed in these models, which may provide first insights into the complexity of molecular signatures and may open new doors for drug discovery and biomarker research.

The Rhinobiome of Exacerbated Wheezers and Asthmatics: Insights From a German Pediatric Exacerbation Network.

Although the nose, as a gateway for organism-environment interactions, may have a key role in asthmatic exacerbation, the rhinobiome of exacerbated children with asthma was widely neglected to date. The aim of this study is to understand the microbiome, the microbial immunology, and the proteome of exacerbated children and adolescents with wheeze and asthma. Considering that a certain proportion of wheezers may show a progression to asthma, the comparison of both groups provides important information regarding clinical and phenotype stratification. Thus, deep nasopharyngeal swab specimens, nasal epithelial spheroid (NAEsp) cultures, and blood samples of acute exacerbated wheezers (WH), asthmatics (AB), and healthy controls (HC) were used for culture (n = 146), 16 S-rRNA gene amplicon sequencing (n = 64), and proteomic and cytokine analyses. Interestingly, Proteobacteria were over-represented in WH, whereas Firmicutes and Bacteroidetes were associated with AB. In contrast, Actinobacteria commonly colonized HCs. Moreover, Staphylococcaceae, Enterobacteriaceae, Burkholderiaceae, Xanthobacteraceae, and Sphingomonadaceae were significantly more abundant in AB compared to WH and HC. The α-diversity analyses demonstrated an increase of bacterial abundance levels in atopic AB and a decrease in WH samples. Microbiome profiles of atopic WH differed significantly from atopic AB, whereby atopic samples of WH were more homogeneous than those of non-atopic subjects. The NAEsp bacterial exposure experiments provided a disrupted epithelial cell integrity, a cytokine release, and cohort-specific proteomic differences especially for Moraxella catarrhalis cultures. This comprehensive dataset contributes to a deeper insight into the poorly understood plasticity of the nasal microbiota, and, in particular, may enforce our understanding in the pathogenesis of asthma exacerbation in childhood.

B cell subsets are modulated during allergic airway inflammation but are not required for the development of respiratory tolerance in a murine model.

Allergic asthma is a widespread chronic inflammatory disease of the airways. The role of different B cell subsets in developing asthma and respiratory tolerance is not well known. Especially regulatory B (Breg) cells are proposed to be important in asthma regulation. Using wild-type (WT) and B cell-deficient (µMT) mice we investigated how B cells are affected by induction of allergic airway inflammation and respiratory tolerance and whether they are necessary to develop these conditions. WT mice with an asthma-like phenotype, characterized by increased airway hyper reactivity, eosinophilic airway inflammation, mucus hypersecretion and elevated Th2 cytokines, exhibited increased MHCII and CD23 expression on follicular mature B cells in lung, bronchial lymph nodes (bLN) and spleen, which contributed to allergen-specific T cell proliferation in vitro. Germinal center B cell numbers were elevated and associated with increased production of allergen-specific immunoglobulins especially in bLN. In contrast, respiratory tolerance clearly attenuated these B cell alterations and directly enhanced marginal zone precursor B cells, which induced regulatory T cells in vitro. However, µMT mice developed asthma-like and tolerized phenotypes like WT mice. Our data indicate that although B cell subsets are affected by asthma-like and respiratory tolerant phenotypes, B cells are not required for tolerance induction.

Dietary Restriction-Induced Alterations in Bone Phenotype: Effects of Lifelong Versus Short-Term Caloric Restriction on Femoral and Vertebral Bone in C57BL/6 Mice.

Caloric restriction (CR) is a well-described dietary intervention that delays the onset of aging-associated biochemical and physiological changes, thereby extending the life span of rodents. The influence of CR on metabolism, strength, and morphology of bone has been controversially discussed in literature. Thus, the present study evaluated whether lifelong CR versus short-term late-onset dietary intervention differentially affects the development of senile osteoporosis in C57BL/6 mice. Two different dietary regimens with 40% food restriction were performed: lifelong CR starting in 4-week-old mice was maintained for 4, 20, or 74 weeks. In contrast, short-term late-onset CR lasting a period of 12 weeks was commenced at 48 or 68 weeks of age. Control mice were fed ad libitum (AL). Bone specimens were assessed using microcomputed tomography (µCT, femur and lumbar vertebral body) and biomechanical testing (femur). Adverse effects of CR, including reduced cortical bone mineral density (Ct.BMD) and thickness (Ct.Th), were detected to some extent in senile mice (68+12w) but in particular in cortical bone of young growing mice (4+4w), associated with reduced femoral failure force (F). However, we observed a profound capacity of bone to compensate these deleterious changes of minor nutrition with increasing age presumably via reorganization of trabecular bone. Especially in lumbar vertebrae, lifelong CR lasting 20 or 74 weeks had beneficial effects on trabecular bone mineral density (Tb.BMD), bone volume fraction (BV/TV), and trabecular number (Tb.N). In parallel, lifelong CR groups showed reduced structure model index values compared to age-matched controls indicating a transformation of vertebral trabecular bone microarchitecture toward a platelike geometry. This effect was not visible in senile mice after short-term 12-week CR. In summary, CR has differential effects on cortical and trabecular bone dependent on bone localization and starting age. Our study underlines that bone compartments possess a lifelong capability to cope with changing nutritional influences.

Pneumococcal pneumonia suppresses allergy development but preserves respiratory tolerance in mice.

Colonization with Streptococcus pneumoniae (S. pneumoniae) is associated with an increased risk for recurrent wheeze and asthma. Killed S. pneumoniae showed some potential as an effective immunomodulatory therapy in a murine model of asthma. Murine studies demonstrated protection against allergic asthma by symbiotic bacteria via triggering regulatory T cell response: treatment with killed S. pneumoniae resulted in suppressed levels of allergen-specific Th2 cytokines, while early immunization generated a protective Th1 response. We investigated the impact of lung infection with live S. pneumoniae on both the development and maintenance of allergic airway inflammation and respiratory tolerance in mice. BALB/c mice were infected intratracheally with S. pneumoniae either prior to or after tolerance or allergy were induced, using ovalbumin (OVA) as model allergen. Infection of mice with S. pneumoniae prior to sensitization or after manifestation of allergic airway inflammation suppressed the development of an allergic phenotype as judged by reduced eosinophil counts in bronchoalveolar lavage fluid, decreased IgE serum levels and Th2 cytokines, relative to non-infected allergic control mice. In contrast, infection of mice with S. pneumoniae after manifestation of allergic airway inflammation combined with late mucosal re-challenge did not affect the allergic response. Moreover, induction and maintenance of respiratory tolerance to OVA challenge were not altered in S. pneumoniae-infected mice, demonstrating that mice remained tolerant to the model allergen and were protected from the development of allergic airway inflammation regardless of the time point of infection. Our results suggest that a bacterial infection may decrease the manifestation of an allergic phenotype not only prior to sensitization but also after manifestation of allergic airway inflammation in mice, whereas both, induction and maintenance of respiratory tolerance are not affected by pneumococcal pneumonia. These data may point to a role for undisturbed development and maintenance of mucosal tolerance for the prevention of allergic inflammation also in humans.

New TiAg composite coating for bone prosthesis engineering shows promising microvascular compatibility in the murine dorsal skinfold chamber model.

The incorporation of antimicrobial substances like silver into implant surface coatings is one promising concept against primary infections of endoprosthesis, especially for immunocompromised patients as well as against reinfection after revision operations. However, besides good antimicrobial and mechanical properties it is equally important that the implant material does not disturb the local microvascular perfusion of muscle tissue to enable microbial host defense and tissue repair processes. In this study the biocompatibility of a newly developed TiAg-composite coating applied on conventional titanium via physical vapor deposition was analysed. To evaluate the local microvascular and inflammatory response of striated muscle tissue upon implantation of TiAg-coated plates the murine dorsal skinfold chamber model was used. We repetitively examined local capillary and venular perfusion, endothelial integrity as well as leucocyte activation by intravital fluorescence microscopy at 1 h, 24 h as well as 3 and 7 days after implantation. TiAg-implants were well tolerated by the vascular system as indicated by intact functional capillary density and endothelial integrity compared to pure titanium plates and controls without a metal implant. Furthermore, quantification of rolling and adherent leucocytes did not reveal signs of inflammation upon TiAg-implantation.

Diabetes mellitus leads to accumulation of dendritic cells and nerve fiber damage of the subbasal nerve plexus in the cornea.

PURPOSE: To evaluate whether nerve fibers of the subbasal nerve plexus (SNP) and dendritic cells (DCs) are in association with each other leading to neuropathy in the diabetic cornea. METHODS: BALB/c mice were injected with streptozotocin (STZ) for 5 days for induction of diabetes mellitus (DM) or with vehicle solution (control). B6.VLep(ob/ob) (ob/ob) mice served as an obese and glucose-intolerant DM type 2 (DM II) model and lean B6.VLep(ob/+) (ob/+) mice as respective controls. Using in vivo corneal confocal microscopy (CCM), nerve fibers and DCs were quantified over a period of 9 weeks and additionally analyzed by in vitro immunofluorescence whole-mount staining. RESULTS: In STZ-diabetic mice, CCM revealed an increase of DC density (DCD) in contrast to controls, whereas nerve fiber density (NFD) was decreased with duration of DM. In ob/ob mice, DCD was 3-fold higher than in both ob/+ mice and STZ-diabetic mice. Whole-mount staining displayed CD11c(+) and major histocompatibility complex (MHC) class II(+) mature DCs in colocalization with class III ß-tubulin(+) nerve fibers in the cornea. CONCLUSIONS: Hyperglycemia leads to corneal DC infiltration, and obesity aggravates this immune response. The direct contact between DCs and the SNP can be assumed to be a trigger of nerve fiber damage and thus a contributing factor to polyneuropathy in diabetic corneas.

Vitamin D increases cellular turnover and functionally restores the skeletal muscle after crush injury in rats.

Insufficient skeletal muscle regeneration after injury often impedes the healing process and is accompanied by functional deficiencies or pain. The aim of our study was to provide evidence that vitamin D improves muscle healing after muscle injury. Therefore, we used male rats and induced an injury of the soleus muscle. After crush injury, animals received either 8.3 mg/kg (332,000 IU/kg) body weight vitamin D or vehicle solution, s.c. After assessment of muscle force at days 1, 4, 14, and 42 after injury, sampling of muscle tissue served for analysis of proliferation, apoptosis, satellite cells, and prolyl-4-hydroxylase-ß expression. Vitamin D application caused a significant increase in cell proliferation and a significant inhibition of apoptosis at day 4 after injury compared to control animals. The numbers of satellite cells were not influenced by the vitamin D application, but there was an increase in prolyl-4-hydroxylase-ß expression, indicative of increased extracellular matrix proteins. This cellular turnover resulted in a faster recovery of contraction forces at day 42 in the vitamin D group. Current data support the hypothesis that vitamin D promotes the regenerative process in injured muscle. Thus, vitamin D treatment may represent a promising therapy to optimize recovery after injury.

CD27 costimulation is not critical for the development of asthma and respiratory tolerance in a murine model.

CD27 is a costimulatory molecule of the TNFR family strongly expressed on activated CD4(+) and CD8(+) T lymphocytes. Binding with its ligand CD70, present on lymphocytes and DCs, leads to enhanced T cell activation and proliferation. Several other costimulatory molecules of the TNFR family like CD30, CD134 (OX40) or CD137 (4-1BB) have been shown to be critically involved in the development of asthma and/or respiratory tolerance. However, the role of CD27/CD70 signalling in these disease models has not been studied intensively. The aim of this study was to directly investigate the role of CD27 for the development of asthma and respiratory tolerance by comparative analysis of wild type (WT) and CD27(-/-) mice in the corresponding murine models. Ovalbumin (OVA)-sensitized and challenged CD27(-/-) mice developed comparably increased airway hyperreactivity (AHR), eosinophilic airway inflammation, mucus hypersecretion and elevated OVA-specific serum IgE levels in response to OVA sensitization as WT mice. In addition, Th2 cytokine production in spleen cell culture supernatants and proliferation of splenocytes after in vitro OVA restimulation was equally enhanced when derived from WT and CD27(-/-) mice. Furthermore, the absence of CD27 had no decisive impact on tolerance induction, so that WT and CD27(-/-) mice were comparably protected from asthma development by mucosal antigen application before sensitization. Our results suggest that CD27 costimulation is dispensable for a Th2 cell mediated allergic asthma response and respiratory tolerance induction in murine models.

Different role of CD30 in the development of acute and chronic airway inflammation in a murine asthma model.

CD30 is a costimulatory molecule of the TNF receptor superfamily, expressed on activated T and B cells. Previously, we have shown in a murine asthma model the crucial role of CD30 signaling for the development of this Th2-cell-mediated disease. In the present study, we investigated the role of CD30 in the maintenance of the immune response. In contrast to the acute model, in the chronic model CD30(-/-) mice developed a severe asthma-like phenotype with eosinophilic inflammation and high serum IgE levels. Collagen content, ECM protein deposition and proliferation of smooth muscle cells as signs for airway remodeling were equally increased in both CD30(-/-) and WT mice. Reduced expression of the costimulatory molecule OX40 on CD3(+) T cells in the acute and up-regulation in the chronic model indirectly supported a compensatory role of OX40 for CD30 signaling. In accordance, application of agonistic OX40 antibody restored the asthma phenotype in CD30(-/-) mice in the acute model, whereas chronic airway inflammation was reduced in the presence of an inhibitory anti-OX40 ligand antibody. These data demonstrate that the crucial role of CD30 signaling in the development of acute asthma may be taken over by other costimulatory molecules like OX40 after long-term exposure to the antigen.

Direct evidence for a critical role of CD30 in the development of allergic asthma.

BACKGROUND: CD30 is a costimulatory molecule belonging to the TNF receptor superfamily that is expressed on activated T and B cells. Several studies have demonstrated a positive correlation between expression of CD30 or increased levels of soluble CD30 and the development and severity of allergic diseases. However, thus far, the evidence for a role of CD30 in allergic diseases, such as asthma, is only indirect. OBJECTIVE: The aim of the study was to directly investigate the role of CD30 in a murine asthma model. METHODS: CD30-deficient (B6.129P2-Tnfrsf8(tm1Mak)/J) and wild-type (WT) mice were immunized to ovalbumin (OVA) to induce an asthma-like phenotype and compared in our murine asthma model. Moreover, CD30/CD30 ligand signaling was blocked in OVA-immunized WT animals by using mAbs against CD30 receptor and its ligand, CD153. RESULTS: The absence of CD30 in OVA-immunized CD30-deficient mice resulted in significantly reduced airway inflammation, serum IgE levels, and TH2 cytokine levels. The same effect was observed when CD30/CD153 signaling was blocked in OVA-immunized WT animals with mAbs against CD30 or CD30 ligand. CONCLUSION: Our results directly demonstrate that CD30/CD153 interaction plays an important role in the induction of TH2 cell-mediated allergic asthma. CLINICAL IMPLICATIONS: These findings provide evidence for a role of the costimulatory molecule CD30 in allergic asthma.