Widespread alterations in systemic immune profile are linked to lung function heterogeneity and airway microbes in cystic fibrosis.

BACKGROUND: Excessive inflammation and recurrent airway infections characterize people with cystic fibrosis (pwCF), a disease with highly heterogeneous clinical outcomes. How the overall immune response is affected in pwCF, its relationships with the lung microbiome, and the source of clinical heterogeneity have not been fully elucidated. METHODS: Peripheral blood and sputum samples were collected from 28 pwCF and an age-matched control group. Systemic immune cell subsets and surface markers were quantified using multiparameter flow cytometry. Lung microbiome composition was reconstructed using metatranscriptomics on sputum samples, and microbial taxa were correlated to circulating immune cells and surface markers expression. RESULTS: In pwCF, we found a specific systemic immune profile characterized by widespread hyperactivation and altered frequencies of several subsets. These included substantial changes in B-cell subsets, enrichment of CD35+/CD49d+ neutrophils, and reduction in dendritic cells. Activation markers and checkpoint molecule expression levels differed from healthy subjects. CTLA-4 expression was increased in Tregs and, together with impaired B-cell subsets, correlated with patients' lung function. Concentrations and frequencies of key immune cells and marker expression correlated with the relative abundance of commensal and pathogenic bacteria in the lungs. CONCLUSION: The CF-specific immune signature, involving hyperactivation, immune dysregulation with alteration in Treg homeostasis, and impaired B-cell function, is a potential source of lung function heterogeneity. The activity of specific microbes contributes to disrupting the balance of the immune response. Our data provide a unique foundation for identifying novel markers and immunomodulatory targets to develop the future of cystic fibrosis treatment and management.

Refined analytical pipeline for the pharmacodynamic assessment of T-cell responses to vaccine antigens.

Pharmacodynamic assessment of T-cell-based cancer immunotherapies often focus on detecting rare circulating T-cell populations. The therapy-induced immune cells in blood-derived clinical samples are often present in very low frequencies and with the currently available T-cell analytical assays, amplification of the cells of interest prior to analysis is often required. Current approaches aiming to enrich antigen-specific T cells from human Peripheral Blood Mononuclear Cells (PBMCs) depend on in vitro culturing in presence of their cognate peptides and cytokines. In the present work, we improved a standard, publicly available protocol for T-cell immune analyses based on the in vitro expansion of T cells. We used PBMCs from healthy subjects and well-described viral antigens as a model system for optimizing the experimental procedures and conditions. Using the standard protocol, we first demonstrated significant enrichment of antigen-specific T cells, even when their starting frequency ex vivo was low. Importantly, this amplification occurred with high specificity, with no or neglectable enrichment of irrelevant T-cell clones being observed in the cultures. Testing of modified culturing timelines suggested that the protocol can be adjusted accordingly to allow for greater cell yield with strong preservation of the functionality of antigen-specific T cells. Overall, our work has led to the refinement of a standard protocol for in vitro stimulation of antigen-specific T cells and highlighted its reliability and reproducibility. We envision that the optimized protocol could be applied for longitudinal monitoring of rare blood-circulating T cells in scenarios with limited sample material.

Influence of Prior Imaging Information on Diagnostic Accuracy for Focal Skeletal Processes-A Retrospective Analysis of the Consistency between Biopsy-Verified Imaging Diagnoses.

INTRODUCTION: Comparing imaging examinations with those previously obtained is considered mandatory in imaging guidelines. To our knowledge, no studies are available on neither the influence, nor the sequence, of prior imaging and reports on diagnostic accuracy using biopsy as the reference standard. Such data are important to minimize diagnostic errors and to improve the preparation of diagnostic imaging guidelines. The aim of our study was to provide such data. MATERIALS AND METHODS: A retrospective cohort of 216 consecutive skeletal biopsies from patients with at least 2 different imaging modalities (X-ray, CT and MRI) performed within 6 months of biopsy was identified. The diagnostic accuracy of the individual imaging modality was assessed. Finally, the possible influence of the sequence of imaging modalities was investigated. RESULTS: No significant difference in the accuracy of the imaging modalities was shown, being preceded by another imaging modality or not. However, the sequence analyses indicate sequential biases, particularly if MRI was the first imaging modality. CONCLUSION: The sequence of the imaging modalities seems to influence the diagnostic accuracy against a pathology reference standard. Further studies are needed to establish evidence-based guidelines for the strategy of using previous imaging and reports to improve diagnostic accuracy.

Analysis of complement deposition and processing on Chlamydia trachomatis.

Chlamydia trachomatis (C. trachomatis) is the leading cause of sexually transmitted bacterial infections worldwide, with over 120 million annual cases. C. trachomatis infections are associated with severe reproductive complications in women such as extrauterine pregnancy and tubal infertility. The infections are often long lasting, associated with immunopathology, and fail to elicit protective immunity which makes recurrent infections common. The immunological mechanisms involved in C. trachomatis infections are only partially understood. Murine infection models suggest that the complement system plays a significant role in both protective immunity and immunopathology during primary Chlamydia infections. However, only limited structural and mechanistic evidence exists on complement-mediated immunity against C. trachomatis. To expand our current knowledge on this topic, we analyzed global complement deposition on C. trachomatis using comprehensive in-depth mass spectrometry-based proteomics. We show that factor B, properdin, and C4b bind to C. trachomatis demonstrating that C. trachomatis-induced complement activation proceeds through at least two activation pathways. Complement activation leads to cleavage and deposition of C3 and C5 activation products, causing initiation of the terminal complement pathway and deposition of C5b, C6, C7, C8, C9 on C. trachomatis. Interestingly, using immunoelectron microscopy, we show that C5b-9 deposition occurred sporadically and only in rare cases formed complete lytic terminal complexes, possibly caused by the presence of the negative regulators vitronectin and clusterin. Finally, cleavage analysis of C3 demonstrated that deposited C3b is degraded to the opsonins iC3b and C3dg and that this complement opsonization facilitates C. trachomatis binding to human B-cells.

Proteomic analysis of synovial fluid from rheumatic arthritis and spondyloarthritis patients.

BACKGROUND: The aetiologies and pathogeneses of the joint diseases rheumatoid arthritis (RA) and spondyloarthritis (SpA) are still not fully elucidated. To increase our understanding of the molecular pathogenesis, we analysed the protein composition of synovial fluid (SF) from rheumatoid arthritis (RA) and spondyloarthritis (SpA) patients. METHODS: Fifty-six synovial fluid samples (RA, n = 32; SpA, n = 24) were digested with trypsin, and the resulting peptides were separated by liquid chromatography and analysed by tandem mass spectrometry. Additionally, the concentration of cell-free DNA (cfDNA) in the synovial fluid was measured, and plasma C-reactive protein (CRP) was determined. RESULTS: Three hundred thirty five proteins were identified within the SF. The more abundant proteins seen in RA SF were inflammatory proteins, including proteins originating from neutrophil granulocytes, while SpA SF had less inflammatory proteins and a higher concentration of haptoglobin. The concentration of cell-free DNA in the SF increased together with proteins that may have originated from neutrophils. Plasma CRP levels in both RA and SpA, correlated to other acute phase reactants. CONCLUSIONS: The proteomic results underline that neutrophils are central in the RA pathology but not in SpA, and even though inhibitors of neutrophils (migration, proteinase inhibitors) were present in the SF it was not sufficient to interrupt the disease process.

Opsonophagocytosis of Chlamydia pneumoniae by Human Monocytes and Neutrophils.

The human respiratory tract pathogen Chlamydia pneumoniae, which causes mild to severe infections, has been associated with the development of chronic inflammatory diseases. To understand the biology of C. pneumoniae infections, several studies have investigated the interaction between C. pneumoniae and professional phagocytes. However, these studies have been conducted under nonopsonizing conditions, making the role of opsonization in C. pneumoniae infections elusive. Thus, we analyzed complement and antibody opsonization of C. pneumoniae and evaluated how opsonization affects chlamydial infectivity and phagocytosis in human monocytes and neutrophils. We demonstrated that IgG antibodies and activation products of complement C3 and C4 are deposited on the surface of C. pneumoniae elementary bodies when incubated in human serum. Complement activation limits C. pneumoniae infectivity in vitro and has the potential to induce bacterial lysis by the formation of the membrane attack complex. Coculture of C. pneumoniae and freshly isolated human leukocytes showed that complement opsonization is superior to IgG opsonization for efficient opsonophagocytosis of C. pneumoniae in monocytes and neutrophils. Neutrophil-mediated phagocytosis of C. pneumoniae was crucially dependent on opsonization, while monocytes retained minor phagocytic potential under nonopsonizing conditions. Complement opsonization significantly enhanced the intracellular neutralization of C. pneumoniae in peripheral blood mononuclear cells and neutrophils and almost abrogated the infectious potential of C. pneumoniae In conclusion, we demonstrated that complements limit C. pneumoniae infection in vitro by interfering with C. pneumoniae entry into permissive cells by direct complement-induced lysis and by tagging bacteria for efficient phagocytosis in both monocytes and neutrophils.

Complement mediated Klebsiella pneumoniae capsule changes.

The Gram-negative bacterium Klebsiella pneumoniae is an opportunistic pathogen, which can cause life-threatening infections such as sepsis. Worldwide, emerging multidrug resistant K. pneumoniae infections are challenging to treat, hence leading to increased mortality. Therefore, understanding the interactions between K. pneumoniae and the immune system is important to develop new treatment options. We characterized ten clinical K. pneumoniae isolates obtained from blood of bacteremia patients. The interaction of the isolates with human serum was investigated to elucidate how K. pneumoniae escapes the host immune system, and how complement activation by K. pneumoniae changed the capsule structure. All K. pneumoniae isolates activated the alternative complement pathway despite serum resistance of seven isolates. One serum sensitive isolate activated two or all three pathways, and this isolate was lysed and had numerous membrane attack complexes in the outer membrane. However, we also found deposition of complement components in the capsule of serum resistant isolates resulting in morphological capsule changes and capsule shedding. These bacteria did not lyse, and no membrane attack complex was observed despite deposition of C5b-9 within the capsule, indicating that the capsule of serum resistant K. pneumoniae isolates is a defense mechanism against complement-mediated lysis.

Immunobiology of monocytes and macrophages during Chlamydia trachomatis infection.

Infections caused by the intracellular bacterium Chlamydia trachomatis are a global health burden affecting more than 100 million people annually causing damaging long-lasting infections. In this review, we will present and discuss important aspects of the interaction between C. trachomatis and monocytes/macrophages.

Proteomic analysis of lipopolysaccharide activated human monocytes.

Monocytes are key mediators of innate immunity and comprise an important cellular defence against invading pathogens. However, exaggerated or dysregulated monocyte activation can lead to severe immune-mediated pathology such as sepsis or chronic inflammatory diseases. Thus, detailed insight into the molecular mechanisms of monocyte activation is essential to understand monocyte-driven inflammatory pathologies. We therefore investigated the global protein changes in human monocytes during lipopolysaccharide (LPS) activation to mimic bacterial activation. Purified human monocytes were stimulated with LPS for 17 h and analyzed by state-of-the-art liquid chromatography tandem mass spectrometry (LC-MS/MS). The label-free quantitative proteome analysis identified 2746 quantifiable proteins of which 101 had a statistically significantly different abundance between LPS-stimulated cells and unstimulated controls. Additionally, 143 proteins were exclusively identified in either LPS stimulated cells or unstimulated controls. Functional annotation clustering demonstrated that LPS, most significantly, regulates proteasomal- and lysosomal proteins but in opposite directions. Thus, seven proteasome subunits were upregulated by LPS while 11 lysosomal proteins were downregulated. Both systems are critically involved in processing of proteins for antigen-presentation and together with LPS-induced regulation of CD74 and tapasin, our data suggest that LPS can skew monocytic antigen-presentation towards MHC class I rather than MHC class II. In summary, this study provides a sensitive high throughput protein analysis of LPS-induced monocyte activation and identifies several LPS-regulated proteins not previously described in the literature which can be used as a source for future studies.

Complement C3 opsonization of Chlamydia trachomatis facilitates uptake in human monocytes.

Chlamydia trachomatis is an obligate intracellular bacterium that causes severe infections, which can lead to infertility and ectopic pregnancy. Although both innate and adaptive immune responses are elicited during chlamydial infection the bacterium succeeds to evade host defense mechanisms establishing chronic infections. Thus, studying the host-pathogen interaction during chlamydial infection is of importance to understand how C. trachomatis can cause chronic infections. Both the complement system and monocytes play essential roles in anti-bacterial defense, and, therefore, we investigated the interaction between the complement system and the human pathogens C. trachomatis D and L2. Complement competent serum facilitated rapid uptake of both chlamydial serovars into monocytes. Using immunoelectron microscopy, we showed that products of complement C3 were loosely deposited on the bacterial surface in complement competent serum and further characterization demonstrated that the deposited C3 product was the opsonin iC3b. Using C3-depleted serum we confirmed that complement C3 facilitates rapid uptake of chlamydiae into monocytes in complement competent serum. Complement facilitated uptake did not influence intracellular survival of C. trachomatis or C. trachomatis-induced cytokine secretion. Hence, C. trachomatis D and L2 activate the complement system leading to chlamydial opsonization by iC3b and subsequent phagocytosis, activation and bacterial elimination by human monocytes.