Airway inflammation and dysbiosis in antibody deficiency despite the presence of IgG.

BACKGROUND: Patients with antibody deficiency suffer chronic respiratory symptoms, recurrent exacerbations, and progressive airways disease despite systemic replacement of IgG. Little is known about the respiratory tract biology of these patients. OBJECTIVE: We sought to measure immunoglobulin levels, inflammatory cytokines, and mediators of tissue damage in serum and sputum from patients with antibody deficiency and healthy controls; to analyze the respiratory microbiome in the same cohorts. METHODS: We obtained paired sputum and serum samples from 31 immunocompetent subjects and 67 antibody-deficient patients, the latter divided on computed tomography scan appearance into "abnormal airways" (bronchiectasis or airway thickening) or "normal airways." We measured inflammatory cytokines, immunoglobulin levels, neutrophil elastase, matrix-metalloproteinase-9, urea, albumin, and total protein levels using standard assays. We used V3-V4 region 16S sequencing for microbiome analysis. RESULTS: Immunodeficient patients had markedly reduced IgA in sputum but higher concentrations of IgG compared with healthy controls. Inflammatory cytokines and tissue damage markers were higher in immunodeficient patients, who also exhibited dysbiosis with overrepresentation of pathogenic taxa and significantly reduced alpha diversity compared with immunocompetent individuals. These differences were seen regardless of airway morphology. Sputum matrix-metalloproteinase-9 and elastase correlated inversely with alpha diversity in the antibody-deficient group, as did sputum IgG, which correlated positively with several inflammatory markers, even after correction for albumin levels. CONCLUSIONS: Patients with antibody deficiency, even with normal lung imaging, exhibit inflammation and dysbiosis in their airways despite higher levels of IgG compared with healthy controls.

Enhanced Pro-apoptotic Effects of Fe(II)-Modified IVIG on Human Neutrophils.

Mild modification of intravenous immunoglobulin (IVIG) has been reported to result in enhanced polyspecificity and leveraged therapeutic effects in animal models of inflammation. Here, we observed that IVIG modification by ferrous ions, heme or low pH exposure, shifted the repertoires of specificities in different directions. Ferrous ions exposed Fe(II)-IVIG, but not heme or low pH exposed IVIG, showed increased pro-apoptotic effects on neutrophil granulocytes that relied on a FAS-dependent mechanism. These effects were also observed in human neutrophils primed by inflammatory mediators or rheumatoid arthritis joint fluid in vitro, or patient neutrophils ex vivo from acute Crohn's disease. These observations indicate that IVIG-mediated effects on cells can be enhanced by IVIG modification, yet specific modification conditions may be required to target specific molecular pathways and eventually to enhance the therapeutic potential.

Granulocyte death mediated by specific antibodies in intravenous immunoglobulin (IVIG).

Granulocytes are key effector cells of the innate immune system that cause substantial by-stander tissue damage in a broad range of inflammatory disorders. Specific antibodies with the potential to regulate granulocyte survival are present in polyclonal intravenous immunoglobulin (IVIG) preparations and may contribute to the anti-inflammatory effects of high-dose IVIG therapy. Here, we discuss idiosyncratic characteristics of antibodies to FAS and Siglecs contained in IVIG pertaining to granulocyte death regulation and highlight implications for research and clinical practice.

IVIG regulates the survival of human but not mouse neutrophils.

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab')2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

Native California soils are selective reservoirs for multidrug-resistant bacteria.

Soil bacteria can exhibit extensive antibiotic resistomes and act as reservoirs of important antibiotic resistance traits. However, the geographic sources and evolutionary drivers of resistance traits are poorly understood in these natural settings. We investigated the prevalence, spatial structure and evolutionary drivers of multidrug resistance in natural populations of Bradyrhizobium, a cosmopolitan bacterial lineage that thrives in soil and aquatic systems as well as in plant and human hosts. We genotyped > 400 isolates from plant roots and soils across California and assayed 98 of them for resistance traits against 17 clinically relevant antibiotics. We investigated the geographic and phylogenetic structure of resistance traits, and analysed correlations of resistance with strain abundance, host infection capacity and in vitro fitness. We found: (i) multidrug resistance at all sites, (ii) subsets of resistance traits that are spatially structured and (iii) significant associations between resistance traits and increased strain abundance or host infection capacity. Our results highlight multiple selective factors that can result in the spread of resistance traits in native Bradyrhizobium populations.

Development of the First Fluorescence Screening Assay for the SLC39A2 Zinc Transporter.

Zinc is an essential micronutrient that is crucial for many vital cellular functions such as DNA and protein synthesis, metabolism, and intracellular signaling. Therefore, the intracellular zinc concentration is tightly regulated by zinc transporters and zinc-binding proteins. The members of the SCL39 transporter family transport zinc into the cytosol. The SLC39A2 (hZIP2) protein is highly expressed in prostate epithelial cells and was found to be involved in prostate cancer development. Thus far, there is no specific modulator available for the SLC39 transporters. The aim of this study was to develop a screening assay for compound screening targeting hZIP2. Employing the pIRES2-DsRed Express 2 bicistronic vector, we detected human ZIP2 expression at the plasma membrane in transiently transfected HEK293 cells. Using the FLIPR Tetra fluorescence plate reader, we demonstrated that ZIP2 transports Cd(2+) with an apparent K(m) value of 53.96 nM at an extracellular pH of 6.5. The cadmium influx via hZIP2 was inhibited by zinc in a competitive manner. We found that hZIP2 activity can be measured using cadmium in the range of 0.1 to 10 µM with our assay. In summary, for the first time we developed an assay for human ZIP2 that can be adapted to other zinc transporters.

Human TRPV5 and TRPV6: key players in cadmium and zinc toxicity.

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.