Donor leukocyte trafficking during human ex vivo lung perfusion.

BACKGROUND: Ex vivo lung perfusion (EVLP) is used to assess and preserve lungs prior to transplantation. However, its inherent immunomodulatory effects are not completely understood. We examine perfusate and tissue compartments to determine the change in immune cell composition in human lungs maintained on EVLP. METHODS: Six human lungs unsuitable for transplantation underwent EVLP. Tissue and perfusate samples were obtained during cold storage and at 1-, 3- and 6-h during perfusion. Flow cytometry, immunohistochemistry, and bead-based immunoassays were used to measure leukocyte composition and cytokines. Mean values between baseline and time points were compared by Student's t test. RESULTS: During the 1st hour of perfusion, perfusate neutrophils increased (+22.2 ± 13.5%, p < 0.05), monocytes decreased (-77.5 ± 8.6%, p < 0.01) and NK cells decreased (-61.5 ± 22.6%, p < 0.01) compared to cold storage. In contrast, tissue neutrophils decreased (-22.1 ± 12.2%, p < 0.05) with no change in monocytes and NK cells. By 6 h, perfusate neutrophils, NK cells, and tissue neutrophils were similar to baseline. Perfusate monocytes remained decreased, while tissue monocytes remained unchanged. There was no significant change in B cells or T cell subsets. Pro-inflammatory cytokines (IL-1b, G-CSF, IFN-gamma, CXCL2, CXCL1 granzyme A, and granzyme B) and lymphocyte activating cytokines (IL-2, IL-4, IL-6, IL-8) increased during perfusion. CONCLUSIONS: Early mobilization of innate immune cells occurs in both perfusate and tissue compartments during EVLP, with neutrophils and NK cells returning to baseline and monocytes remaining depleted after 6 h. The immunomodulatory effect of EVLP may provide a therapeutic window to decrease the immunogenicity of lungs prior to transplantation.

IL-33 Precedes IL-5 in Regulating Eosinophil Commitment and Is Required for Eosinophil Homeostasis.

Eosinophils are important in the pathogenesis of many diseases, including asthma, eosinophilic esophagitis, and eczema. Whereas IL-5 is crucial for supporting mature eosinophils (EoMs), the signals that support earlier eosinophil lineage events are less defined. The IL-33R, ST2, is expressed on several inflammatory cells, including eosinophils, and is best characterized for its role during the initiation of allergic responses in peripheral tissues. Recently, ST2 expression was described on hematopoietic progenitor subsets, where its function remains controversial. Our findings demonstrate that IL-33 is required for basal eosinophil homeostasis, because both IL-33- and ST2-deficient mice exhibited diminished peripheral blood eosinophil numbers at baseline. Exogenous IL-33 administration increased EoMs in both the bone marrow and the periphery in wild-type and IL-33-deficient, but not ST2-deficient, mice. Systemic IL-5 was also increased under this treatment, and blocking IL-5 with a neutralizing Ab ablated the IL-33-induced EoM expansion. The homeostatic hypereosinophilia seen in IL-5-transgenic mice was significantly lower with ST2 deficiency despite similar elevations in systemic IL-5. Finally, in vitro treatment of bone marrow cells with IL-33, but not IL-5, led to specific early expansion of IL-5Rα-expressing precursor cells. In summary, our findings establish a basal defect in eosinophilopoiesis in IL-33- and ST2-deficient mice and a mechanism whereby IL-33 supports EoMs by driving both systemic IL-5 production and the expansion of IL-5Rα-expressing precursor cells.

Diet-induced obesity alters myeloid cell populations in naïve and injured lung.

BACKGROUND: There are pulmonary consequences to obesity, including increased prevalence of asthma, greater susceptibility to influenza, and possibly reduced susceptibility to lung injury. Although it is well established that obesity is associated with alterations to the immune system, little is known about obesity-associated changes to pulmonary immune cells. OBJECTIVES: We hypothesized that obesity would alter the inflammatory milieu in the unchallenged lung and circulation; thereby contributing to altered susceptibility to lung injury. METHODS: We used a murine model of diet-induced obesity and evaluated bone marrow and blood leukocytes at 3 months, and pulmonary leukocytes at 3 and 6 months for changes in their adhesion and chemokine receptors, markers of activation states, and cell numbers. We also evaluated the inflammatory response to LPS in obese mice. RESULTS: In the lung, diet-induced obesity was associated with increased leukocyte numbers over-time. Adhesion receptors were increased in a cell- and site-specific fashion, and there was an evolution of macrophage and neutrophil polarization toward M1 and N1, respectively. After LPS-challenge, obesity was associated with increased neutrophil recruitment to the lung with impaired migration into the alveolar space. Associated with these changes, obesity increased LFA-1 and ICAM-1 neutrophil expression and altered CXCL1 gradients. CONCLUSION: Our results highlight the effects of diet-induced obesity on the murine blood and lung leukocyte populations, including increases in adhesion receptor expression that may contribute to altered recruitment or retention within the lung. Translation of these findings to people with obesity will be critical for determining the basic inflammatory underpinnings of pulmonary disease susceptibility.

The immunology of food allergy.

Food allergies represent an increasingly prevalent human health problem, and therapeutic options remain limited, with avoidance being mainstay, despite its adverse effects on quality of life. A better understanding of the key immunological mechanisms involved in such responses likely will be vital for development of new therapies. This review outlines the current understanding of how the immune system is thought to contribute to prevention or development of food allergies. Drawing from animal studies, as well as clinical data when available, the importance of oral tolerance in sustaining immunological nonresponsiveness to food Ags, our current understanding of why oral tolerance may fail and sensitization may occur, and the knowledge of pathways that may lead to anaphylaxis and food allergy-associated responses are addressed.

TGF-ß1 limits the onset of innate lung inflammation by promoting mast cell-derived IL-6.

TGF-ß1 is an important suppressive mediator of inflammation, but it can also drive fibrosis and remodeling in the lung. In response to intratracheal LPS, neutrophils migrate into the lung, and TGF-ß1 was suggested to protect against the ensuing injury. However, the mechanisms for this protective role remain unknown. Using a model of acute lung injury, we demonstrate that TGF-ß1 decreases neutrophil numbers during the onset of injury. This was due to increased apoptosis rather than reduced migration. We demonstrate that TGF-ß1 does not directly regulate neutrophil apoptosis but instead functions through IL-6 to promote neutrophil clearance. rIL-6 is sufficient to promote neutrophil apoptosis and reduce neutrophilia in bronchoalveolar lavage fluid, while IL-6 increases rapidly following LPS-induced injury. Mast cells are a critical source of IL-6, because mast cell-deficient mice exhibit increased neutrophil numbers that are reduced by reconstitution with wild-type, but not IL-6(-/-), mast cells. Although IL-6 diminishes neutrophilia in mast cell-deficient mice, TGF-ß1 is ineffective, suggesting that these effects were mast cell dependent. Taken together, our findings establish a novel pathway through which TGF-ß1, likely derived from resident regulatory T cells, controls the severity and magnitude of early innate inflammation by promoting IL-6 from mast cells.

Pulmonary macrophage subpopulations in the induction and resolution of acute lung injury.

Macrophages are key orchestrators of the inflammatory and repair responses in the lung, and the diversity of their function is indicated by their polarized states and distinct subpopulations and localization in the lung. Here, we characterized the pulmonary macrophage populations in the interstitial and alveolar compartments during the induction and resolution of acute lung injury induced by Pseudomonas aeruginosa infection. We identified macrophage subpopulations and polarity according to FACS analysis of cell surface protein markers, combined with cell sorting for gene expression using real-time PCR. With these techniques, we validated a novel, alternatively activated (M2) marker (transferrin receptor), and we described three interstitial and alveolar macrophage subpopulations in the lung whose distribution and functional state evolved from the induction to resolution phases of lung injury. Together, these findings indicate the presence and evolution of distinct macrophage subsets in the lung that serve specific niches in regulating the inflammatory response and its resolution. Alterations in the balance and function of these subpopulations could lead to nonresolving acute lung injury.

Epilysin (matrix metalloproteinase-28) contributes to airway epithelial cell survival.

MMP28 is constitutively expressed by epithelial cells in many tissues, including the respiratory epithelium in the lung and keratinocytes in the skin. This constitutive expression suggests that MMP28 may serve a role in epithelial cell homeostasis. In an effort to determine its function in epithelial cell biology, we generated cell lines expressing wild-type or catalytically-inactive mutant MMP28 in two pulmonary epithelial cell lines, A549 and BEAS-2B. We observed that over-expression of MMP28 provided protection against apoptosis induced by either serum-deprivation or treatment with a protein kinase inhibitor, staurosporine. Furthermore, we observed increased caspase-3/7 activity in influenza-infected lungs from Mmp28-/- mice compared to wild-type mice, and this activity localized to the airway epithelium but was not associated with a change in viral load. Thus, we have identified a novel role of MMP28 in promoting epithelial cell survival in the lung.

Panel Optimization for High-Dimensional Immunophenotyping Assays Using Full-Spectrum Flow Cytometry.

Technological advancements in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system have led to the development of large flow cytometry panels reaching up to 43 colors at the single-cell level. However, as panel size and complexity increase, so too does the detail involved in designing and optimizing successful high-quality panels fit for downstream high-dimensional data analysis. In contrast to conventional flow cytometers, full-spectrum flow cytometers measure the entire emission spectrum of each fluorophore across all lasers. This allows for fluorophores with very similar emission maxima but unique overall spectral fingerprints to be used in conjunction, enabling relatively straightforward design of larger panels. Although a protocol for best practices in full-spectrum flow cytometry panel design has been published, there is still a knowledge gap in going from the theoretically designed panel to the necessary steps required for panel optimization. Here, we aim to guide users through the theory of optimizing a high-dimensional full-spectrum flow cytometry panel for immunophenotyping using comprehensive step-by-step protocols. These protocols can also be used to troubleshoot panels when issues arise. A practical application of this approach is exemplified with a 24-color panel designed for identification of conventional T-cell subsets in human peripheral blood. © 2021 Malaghan Institute of Medical Research, Cytek Biosciences. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation and evaluation of optimal spectral reference controls Support Protocol 1: Antibody titration Support Protocol 2: Changing instrument settings Basic Protocol 2: Unmixing evaluation of fully stained sample Basic Protocol 3: Evaluation of marker resolution Support Protocol 3: Managing heterogeneous autofluorescence Basic Protocol 4: Assessment of data quality using expert gating and dimensionality reduction algorithms.

Understanding Interleukin 33 and Its Roles in Eosinophil Development.

Over the last decade, significant interest in the contribution of three "epithelial-derived cytokines," such as thymic stromal lymphopoietin, interleukin 25, and interleukin 33 (IL-33), has developed. These cytokines have been strongly linked to the early events that occur during allergen exposures and how they contribute to the subsequent type 2 immune response. Of these three cytokines, IL-33 has proven particularly interesting because of the strong associations found between both it and its receptor, ST2, in several genome-wide association studies of allergic diseases. Further work has demonstrated clear mechanisms through which this cytokine might orchestrate allergic inflammation, including activation of several key effector cells that possess high ST2 levels, including mast cells, basophils, innate lymphoid cells, and eosinophils. Despite this, controversies surrounding IL-33 seem to suggest the biology of this cytokine might not be as simple as current dogmas suggest including: the relevant cellular sources of IL-33, with significant evidence for inducible expression in some hematopoietic cells; the mechanistic contributions of nuclear localization vs secretion; secretion and processing mechanisms; and the biological consequences of IL-33 exposure on different cell types. In this review, we will address the evidence for IL-33 and ST2 regulation over eosinophils and how this may contribute to allergic diseases. In particular, we focus on the accumulating evidence for a role of IL-33 in regulating hematopoiesis and how this relates to eosinophils as well as how this may provide new concepts for how the progression of allergy is regulated.

MMP28 promotes macrophage polarization toward M2 cells and augments pulmonary fibrosis.

Members of the MMP family function in various processes of innate immunity, particularly in controlling important steps in leukocyte trafficking and activation. MMP28 (epilysin) is a member of this family of proteinases, and we have found that MMP28 is expressed by macrophages and regulates their recruitment to the lung. We hypothesized that MMP28 regulates other key macrophage responses, such as macrophage polarization. Furthermore, we hypothesized that these MMP28-dependent changes in macrophage polarization would alter fibrotic responses in the lung. We examined the gene expression changes in WT and Mmp28-/- BMDMs, stimulated with LPS or IL-4/IL-13 to promote M1 and M2 cells, respectively. We also collected macrophages from the lungs of Pseudomonas aeruginosa-exposed WT and Mmp28-/- mice to evaluate changes in macrophage polarization. Lastly, we evaluated the macrophage polarization phenotypes during bleomycin-induced pulmonary fibrosis in WT and Mmp28-/- mice and assessed mice for differences in weight loss and total collagen levels. We found that MMP28 dampens proinflammatory macrophage function and promots M2 programming. In both in vivo models, we found deficits in M2 polarization in Mmp28-/- mice. In bleomycin-induced lung injury, these changes were associated with reduced fibrosis. MMP28 is an important regulator of macrophage polarization, promoting M2 function. Loss of MMP28 results in reduced M2 polarization and protection from bleomycin-induced fibrosis. These findings highlight a novel role for MMP28 in macrophage biology and pulmonary disease.