Ferritin heavy chain supports stability and function of the regulatory T cell lineage.

Regulatory T (TREG) cells develop via a program orchestrated by the transcription factor forkhead box protein P3 (FOXP3). Maintenance of the TREG cell lineage relies on sustained FOXP3 transcription via a mechanism involving demethylation of cytosine-phosphate-guanine (CpG)-rich elements at conserved non-coding sequences (CNS) in the FOXP3 locus. This cytosine demethylation is catalyzed by the ten-eleven translocation (TET) family of dioxygenases, and it involves a redox reaction that uses iron (Fe) as an essential cofactor. Here, we establish that human and mouse TREG cells express Fe-regulatory genes, including that encoding ferritin heavy chain (FTH), at relatively high levels compared to conventional T helper cells. We show that FTH expression in TREG cells is essential for immune homeostasis. Mechanistically, FTH supports TET-catalyzed demethylation of CpG-rich sequences CNS1 and 2 in the FOXP3 locus, thereby promoting FOXP3 transcription and TREG cell stability. This process, which is essential for TREG lineage stability and function, limits the severity of autoimmune neuroinflammation and infectious diseases, and favors tumor progression. These findings suggest that the regulation of intracellular iron by FTH is a stable property of TREG cells that supports immune homeostasis and limits the pathological outcomes of immune-mediated inflammation.

MIF is essential to the establishment of house dust mite-induced airway inflammation and tissue remodeling in mice.

Macrophage migration inhibitory factor (MIF) is present in high amounts in the BALF and serum of asthmatic patients, contributing to the pathogenesis of experimental asthma induced by OVA in mice. Whether MIF contributes to the physiopathology on a more complex and relevant asthma model has not been characterized. Mif-deficient (Mif-/- ) or WT mice treated with anti-MIF antibody were challenged multiple times using house dust mite (HDM) extract by the intranasal route. HDM-challenged Mif-/- mice presented decreased airway hyperresponsiveness, lung infiltration of eosinophils, mucus hypersecretion, and subepithelial fibrosis compared to HDM-challenged WT mice. Amounts of IL-4, IL-5, and IL-13 were decreased in the lungs of Mif-/- mice upon HDM challenges, but the increase of CCL11 was preserved, compared to HDM-challenged WT mice. We also observed increased numbers of group 2 innate lymphoid cells and Th2 cells in the BALF and mediastinal LNs (mLN)-induced challenged by HDM of WT mice, but not in HDM-challenged Mif-/- mice. Anti-MIF treatment abrogated the airway infiltration of eosinophils, mucus hypersecretion, and subepithelial fibrosis in the lungs of HDM-challenged mice. In conclusion, MIF ablation prevents the pathologic hallmarks of asthma in HDM-challenged mice, reinforcing the promising target of MIF for asthma therapy.

Self-complementary and tyrosine-mutant rAAV vectors enhance transduction in cystic fibrosis bronchial epithelial cells.

Recombinant adeno-associated virus (rAAV) vector platforms have shown considerable therapeutic success in gene therapy for inherited disorders. In cystic fibrosis (CF), administration of first-generation rAAV2 was safe, but clinical benefits were not clearly demonstrated. Therefore, next-generation vectors that overcome rate-limiting steps in rAAV transduction are needed to obtain successful gene therapy for this devastating disease. In this study, we evaluated the effects of single-strand or self-complementary (sc) rAAV vectors containing single or multiple tyrosine-to-phenylalanine (Y-F) mutations in capsid surface-exposed residues on serotypes 2, 8 or 9. For this purpose, CF bronchial epithelial (CFBE) cells were transduced with rAAV vectors, and the transgene expression of enhanced green fluorescence protein (eGFP) was analyzed at different time points. The effects of vectors on the cell viability, host cell cycle and in association with co-adjuvant drugs that modulate intracellular vector trafficking were also investigated. Six rAAV vectors demonstrated greater percentage of eGFP+ cells compared to their counterparts at days 4, 7 and 10 post-transduction: rAAV2 Y(272,444,500,730)F, with 1.95-, 3.5- and 3.06-fold increases; rAAV2 Y(252,272,444,500,704,730)F, with 1.65-, 2.12-, and 2-fold increases; scrAAV2 WT, with 1.69-, 2.68-, and 2.32-fold increases; scrAAV8 Y773F, with 57-, 6.06-, and 7-fold increases; scrAAV9 WT, with 7.47-, 4.64-, and 3.66-fold increases; and scrAAV9 Y446F, with 8.39-, 4.62-, and 4.4-fold increases. At days 15, 20, and 30 post-transduction, these vectors still demonstrated higher transgene expression than transfected cells. Although the percentage of eGFP+ cells reduced during the time-course analysis, the delta mean fluorescence intensity increased. These vectors also led to increased percentage of cells in G1-phase without eliciting any cytotoxicity. Prior administration of bortezomib or genistein did not increase eGFP expression in cells transduced with either rAAV2 Y(272,444,500,730)F or rAAV2 Y(252,272,444,500,704,730)F. In conclusion, self-complementary and tyrosine capsid mutations on rAAV serotypes 2, 8, and 9 led to more efficient transduction than their counterparts in CFBE cells by overcoming the intracellular trafficking and second-strand DNA synthesis limitations.

Variable ventilation improves pulmonary function and reduces lung damage without increasing bacterial translocation in a rat model of experimental pneumonia.

BACKGROUND: Variable ventilation has been shown to improve pulmonary function and reduce lung damage in different models of acute respiratory distress syndrome. Nevertheless, variable ventilation has not been tested during pneumonia. Theoretically, periodic increases in tidal volume (VT) and airway pressures might worsen the impairment of alveolar barrier function usually seen in pneumonia and could increase bacterial translocation into the bloodstream. We investigated the impact of variable ventilation on lung function and histologic damage, as well as markers of lung inflammation, epithelial and endothelial cell damage, and alveolar stress, and bacterial translocation in experimental pneumonia. METHODS: Thirty-two Wistar rats were randomly assigned to receive intratracheal of Pseudomonas aeruginosa (PA) or saline (SAL) (n = 16/group). After 24-h, animals were anesthetized and ventilated for 2 h with either conventional volume-controlled (VCV) or variable volume-controlled ventilation (VV), with mean VT = 6 mL/kg, PEEP = 5cmH2O, and FiO2 = 0.4. During VV, tidal volume varied randomly with a coefficient of variation of 30% and a Gaussian distribution. Additional animals assigned to receive either PA or SAL (n = 8/group) were not ventilated (NV) to serve as controls. RESULTS: In both SAL and PA, VV improved oxygenation and lung elastance compared to VCV. In SAL, VV decreased interleukin (IL)-6 expression compared to VCV (median [interquartile range]: 1.3 [0.3-2.3] vs. 5.3 [3.6-7.0]; p = 0.02) and increased surfactant protein-D expression compared to NV (2.5 [1.9-3.5] vs. 1.2 [0.8-1.2]; p = 0.0005). In PA, compared to VCV, VV reduced perivascular edema (2.5 [2.0-3.75] vs. 6.0 [4.5-6.0]; p < 0.0001), septum neutrophils (2.0 [1.0-4.0] vs. 5.0 [3.3-6.0]; p = 0.0008), necrotizing vasculitis (3.0 [2.0-5.5] vs. 6.0 [6.0-6.0]; p = 0.0003), and ultrastructural lung damage scores (16 [14-17] vs. 24 [14-27], p < 0.0001). Blood colony-forming-unit (CFU) counts were comparable (7 [0-28] vs. 6 [0-26], p = 0.77). Compared to NV, VCV, but not VV, increased expression amphiregulin, IL-6, and cytokine-induced neutrophil chemoattractant (CINC)-1 (2.1 [1.6-2.5] vs. 0.9 [0.7-1.2], p = 0.025; 12.3 [7.9-22.0] vs. 0.8 [0.6-1.9], p = 0.006; and 4.4 [2.9-5.6] vs. 0.9 [0.8-1.4], p = 0.003, respectively). Angiopoietin-2 expression was lower in VV compared to NV animals (0.5 [0.3-0.8] vs. 1.3 [1.0-1.5], p = 0.01). CONCLUSION: In this rat model of pneumonia, VV improved pulmonary function and reduced lung damage as compared to VCV, without increasing bacterial translocation.

Renal control of life-threatening malarial anemia.

Iron recycling prevents the development of anemia under homeostatic conditions. Whether iron recycling was co-opted as a defense strategy to prevent the development of anemia in response to infection is unclear. We find that in severe Plasmodium falciparum malaria, the onset of life-threatening anemia is associated with acute kidney injury (AKI), irrespective of parasite load. Using a well-established experimental rodent model of malaria anemia, we identify a transcriptional response that endows renal proximal tubule epithelial cells (RPTECs) with the capacity to store and recycle iron during P. chabaudi chabaudi (Pcc) infection. This response encompasses the induction of ferroportin 1/SLC40A1, which exports iron from RPTECs and counteracts AKI while supporting compensatory erythropoiesis and preventing the onset of life-threatening malarial anemia. Iron recycling by myeloid cells is dispensable to this protective response, suggesting that RPTECs provide an iron-recycling salvage pathway that prevents the pathogenesis of life-threatening malarial anemia.

c-MAF-dependent perivascular macrophages regulate diet-induced metabolic syndrome.

Macrophages are an essential part of tissue development and physiology. Perivascular macrophages have been described in tissues and appear to play a role in development and disease processes, although it remains unclear what the key features of these cells are. Here, we identify a subpopulation of perivascular macrophages in several organs, characterized by their dependence on the transcription factor c-MAF and displaying nonconventional macrophage markers including LYVE1, folate receptor 2, and CD38. Conditional deletion of c-MAF in macrophage lineages caused ablation of perivascular macrophages in the brain and altered muscularis macrophages program in the intestine. In the white adipose tissue (WAT), c-MAF­deficient perivascular macrophages displayed an altered gene expression profile, which was linked to an increased vascular branching. Upon feeding high-fat diet (HFD), mice with c-MAF­deficient macrophages showed improved metabolic parameters compared with wild-type mice, including less weight gain, greater glucose tolerance, and reduced inflammatory cell profile in WAT. These results define c-MAF as a central regulator of the perivascular macrophage transcriptional program in vivo and reveal an important role for this tissue-resident macrophage population in the regulation of metabolic syndrome.

Mesenchymal Stromal Cells From Emphysematous Donors and Their Extracellular Vesicles Are Unable to Reverse Cardiorespiratory Dysfunction in Experimental Severe Emphysema.

Although bone marrow-derived mesenchymal stromal cells (BM-MSCs) from patients with chronic obstructive pulmonary disease (COPD) appear to be phenotypically and functionally similar to BM-MSCs from healthy sources in vitro, the impact of COPD on MSC metabolism and mitochondrial function has not been evaluated. In this study, we aimed to comparatively characterize MSCs from healthy and emphysematous donors (H-MSCs and E-MSCs) in vitro and to assess the therapeutic potential of these MSCs and their extracellular vesicles (H-EVs and E-EVs) in an in vivo model of severe emphysema. For this purpose, C57BL/6 mice received intratracheal porcine pancreatic elastase once weekly for 4 weeks to induce emphysema; control animals received saline under the same protocol. Twenty-four hours after the last instillation, animals received saline, H-MSCs, E-MSCs, H-EVs, or E-EVs intravenously. In vitro characterization demonstrated that E-MSCs present downregulation of anti-inflammatory (TSG-6, VEGF, TGF-ß, and HGF) and anti-oxidant (CAT, SOD, Nrf2, and GSH) genes, and their EVs had larger median diameter and lower average concentration. Compared with H-MSC, E-MSC mitochondria also exhibited a higher respiration rate, were morphologically elongated, expressed less dynamin-related protein-1, and produced more superoxide. When co-cultured with alveolar macrophages, both H-MSCs and E-MSCs induced an increase in iNOS and arginase-1 levels, but only H-MSCs and their EVs were able to enhance IL-10 levels. In vivo, emphysematous mice treated with E-MSCs or E-EVs demonstrated no amelioration in cardiorespiratory dysfunction. On the other hand, H-EVs, but not H-MSCs, were able to reduce the neutrophil count, the mean linear intercept, and IL-1ß and TGF-ß levels in lung tissue, as well as reduce pulmonary arterial hypertension and increase the right ventricular area in a murine model of elastase-induced severe emphysema. In conclusion, E-MSCs and E-EVs were unable to reverse cardiorespiratory dysfunction, whereas H-EVs administration was associated with a reduction in cardiovascular and respiratory damage in experimental severe emphysema.

The Role of MIF on Eosinophil Biology and Eosinophilic Inflammation.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that participates in innate and adaptive immune responses. MIF contributes to the resistance against infection agents, but also to the cellular and tissue damage in infectious, autoimmune, and allergic diseases. In the past years, several studies demonstrated a critical role for MIF in the pathogenesis of type-2-mediated inflammation, including allergy and helminth infection. Atopic patients have increased MIF amounts in affected tissues, mainly produced by immune cells such as macrophages, Th2 cells, and eosinophils. Increased MIF mRNA and protein are found in activated Th2 cells, while eosinophils stock pre-formed MIF protein and secrete high amounts of MIF upon stimulation. In mouse models of allergic asthma, the lack of MIF causes an almost complete abrogation of the cardinal signs of the disease including mucus secretion, eosinophilic inflammation, and airway hyper-responsiveness. Additionally, blocking the expression of MIF in animal models leads to significant reduction of pathological signs of eosinophilic inflammation such as rhinitis, atopic dermatitis, eosinophilic esophagitis and helminth infection. A number of studies indicate that MIF is important in the effector phase of type-2 immune responses, while its contribution to Th2 differentiation and IgE production is not consensual. MIF has been found to intervene in different aspects of eosinophil physiology including differentiation, survival, activation, and migration. CD4+ T cells and eosinophils express CD74 and CXCR4, receptors able to signal upon MIF binding. Blockage of these receptors with neutralizing antibodies or small molecule antagonists also succeeds in reducing the signals of inflammation in experimental allergic models. Together, these studies demonstrate an important contribution of MIF on eosinophil biology and in the pathogenesis of allergic diseases and helminth infection.

Multiple doses of adipose tissue-derived mesenchymal stromal cells induce immunosuppression in experimental asthma.

In experimental house dust mite (HDM)-induced allergic asthma, therapeutic administration of a single dose of adipose tissue-derived mesenchymal stromal cells (MSCs) ameliorates lung inflammation but is unable to reverse remodeling. We hypothesized that multiple doses of MSCs might exert better therapeutic effects by reducing lung inflammation and remodeling but might also result in immunosuppressive effects in experimental asthma. HDM was administered intranasally in C57BL/6 mice. After the last HDM challenge, mice received two or three doses of MSCs (105 cells per day) or saline intravenously. An additional cohort of mice received dexamethasone as a positive control for immunosuppression. Two and three doses of MSCs reduced lung inflammation, levels of interleukin (IL)-4, IL-13, and eotaxin; total leukocyte, CD4+ T-cell, and eosinophil counts in bronchoalveolar lavage fluid; and total leukocyte counts in bone marrow, spleen, and mediastinal lymph nodes. Two and three doses of MSCs also reduced collagen fiber content and transforming growth factor-ß levels in lung tissue; however, the three-dose regimen was more effective, and reduced these parameters to control levels, while also decreasing α-actin content in lung tissue. Two and three doses of MSCs improved lung mechanics. Dexamethasone, two and three doses of MSCs similarly increased galectin levels, but only the three-dose regimen increased CD39 levels in the thymus. Dexamethasone and the three-dose, but not the two-dose regimen, also increased levels of programmed death receptor-1 and IL-10, while reducing CD4+ CD8low cell percentage in the thymus. In conclusion, multiple doses of MSCs reduced lung inflammation and remodeling while causing immunosuppression in HDM-induced allergic asthma.

Nanoparticle-based thymulin gene therapy therapeutically reverses key pathology of experimental allergic asthma.

Despite long-standing efforts to enhance care for chronic asthma, symptomatic treatments remain the only option to manage this highly prevalent and debilitating disease. We demonstrate that key pathology of allergic asthma can be almost completely resolved in a therapeutic manner by inhaled gene therapy. After the disease was fully and stably established, we treated mice intratracheally with a single dose of thymulin-expressing plasmids delivered via nanoparticles engineered to have a unique ability to penetrate the airway mucus barrier. Twenty days after the treatment, we found that all key pathologic features found in the asthmatic lung, including chronic inflammation, pulmonary fibrosis, and mechanical dysregulation, were normalized. We conducted tissue- and cell-based analyses to confirm that the therapeutic intervention was mediated comprehensively by anti-inflammatory and antifibrotic effects of the therapy. We believe that our findings open a new avenue for clinical development of therapeutically effective gene therapy for chronic asthma.

Serum from Asthmatic Mice Potentiates the Therapeutic Effects of Mesenchymal Stromal Cells in Experimental Allergic Asthma.

Asthma is a chronic inflammatory disease characterized by airway inflammation and remodeling, which can lead to progressive decline of lung function. Although mesenchymal stromal cells (MSCs) have shown beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been limited. Mounting evidence suggests that prior exposure of MSCs to specific inflammatory stimuli or environments can enhance their immunomodulatory properties. Therefore, we investigated whether stimulating MSCs with bronchoalveolar lavage fluid (BALF) or serum from asthmatic mice could potentiate their therapeutic properties in experimental asthma. In a house dust mite (HDM) extract asthma model in mice, unstimulated, asthmatic BALF-stimulated, or asthmatic serum-stimulated MSCs were administered intratracheally 24 hours after the final HDM challenge. Lung mechanics and histology; BALF protein, cellularity, and biomarker levels; and lymph-node and bone marrow cellularity were assessed. Compared with unstimulated or BALF-stimulated MSCs, serum-stimulated MSCs further reduced BALF levels of interleukin (IL)-4, IL-13, and eotaxin, total and differential cellularity in BALF, bone marrow and lymph nodes, and collagen fiber content, while increasing BALF IL-10 levels and improving lung function. Serum stimulation led to higher MSC apoptosis, expression of various mediators (transforming growth factor-ß, interferon-γ, IL-10, tumor necrosis factor-α-stimulated gene 6 protein, indoleamine 2,3-dioxygenase-1, and IL-1 receptor antagonist), and polarization of macrophages to M2 phenotype. In conclusion, asthmatic serum may be a novel strategy to potentiate therapeutic effects of MSCs in experimental asthma, leading to further reductions in both inflammation and remodeling than can be achieved with unstimulated MSCs. Stem Cells Translational Medicine 2019;8:301&312.

Eicosapentaenoic acid potentiates the therapeutic effects of adipose tissue-derived mesenchymal stromal cells on lung and distal organ injury in experimental sepsis.

BACKGROUND: Even though mesenchymal stromal cells (MSCs) mitigate lung and distal organ damage in experimental polymicrobial sepsis, mortality remains high. We investigated whether preconditioning with eicosapentaenoic acid (EPA) would potentiate MSC actions in experimental sepsis by further decreasing lung and distal organ injury, thereby improving survival. METHODS: In C57BL/6 mice, sepsis was induced by cecal hligation and puncture (CLP); sham-operated animals were used as control. Twenty-four hours after surgery, CLP mice were further randomized to receive saline, adipose tissue-derived (AD)-MSCs (105, nonpreconditioned), or AD-MSCs preconditioned with EPA for 6 h (105, EPA-preconditioned MSCs) intravenously. After 24 h, survival rate, sepsis severity score, lung mechanics and histology, protein level of selected biomarkers in lung tissue, cellularity in blood, distal organ damage, and MSC distribution (by technetium-99m tagging) were analyzed. Additionally, the effects of EPA on the secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-ß1 by MSCs were evaluated in vitro. RESULTS: Nonpreconditioned and EPA-preconditioned AD-MSCs exhibited similar viability and differentiation capacity, accumulated mainly in the lungs and kidneys following systemic administration. Compared to nonpreconditioned AD-MSCs, EPA-preconditioned AD-MSCs further reduced static lung elastance, alveolar collapse, interstitial edema, alveolar septal inflammation, collagen fiber content, neutrophil cell count as well as protein levels of interleukin-1ß and keratinocyte chemoattractant in lung tissue, and morphological abnormalities in the heart (cardiac myocyte architecture), liver (hepatocyte disarrangement and Kupffer cell hyperplasia), kidney (acute tubular necrosis), spleen (increased number of megakaryocytes and lymphocytes), and small bowel (villi architecture disorganization). EPA preconditioning of MSCs resulted in increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-ß). CONCLUSIONS: Compared to nonpreconditioned cells, EPA-preconditioned AD-MSCs yielded further reductions in the lung and distal organ injury, resulting in greater improvement in sepsis severity score and higher survival rate in CLP-induced experimental sepsis. This may be a promising therapeutic approach to improve outcome in septic patients.

Glutamine Therapy Reduces Inflammation and Extracellular Trap Release in Experimental Acute Respiratory Distress Syndrome of Pulmonary Origin.

The innate immune response plays an important role in the pathophysiology of acute respiratory distress syndrome (ARDS). Glutamine (Gln) decreases lung inflammation in experimental ARDS, but its impact on the formation of extracellular traps (ETs) in the lung is unknown. In a mouse model of endotoxin-induced pulmonary ARDS, the effects of Gln treatment on leukocyte counts and ET content in bronchoalveolar lavage fluid (BALF), inflammatory profile in lung tissue, and lung morphofunction were evaluated in vivo. Furthermore, ET formation, reactive oxygen species (ROS) production, glutathione peroxidase (GPx), and glutathione reductase (GR) activities were tested in vitro. Our in vivo results demonstrated that Gln treatment reduced ET release (as indicated by cell-free-DNA content and myeloperoxidase activity), decreased lung inflammation (reductions in interferon-γ and increases in interleukin-10 levels), and improved lung morpho-function (decreased static lung elastance and alveolar collapse) in comparison with ARDS animals treated with saline. Moreover, Gln reduced ET and ROS formation in BALF cells stimulated with lipopolysaccharide in vitro, but it did not alter GPx or GR activity. In this model of endotoxin-induced pulmonary ARDS, treatment with Gln reduced pulmonary functional and morphological impairment, inflammation, and ET release in the lung.

Impact of one versus two doses of mesenchymal stromal cells on lung and cardiovascular repair in experimental emphysema.

BACKGROUND: A single administration of mesenchymal stromal cells (MSCs) has been shown to reduce lung inflammation in experimental elastase-induced emphysema; however, effects were limited in terms of lung-tissue repair and cardiac function improvement. We hypothesized that two doses of MSCs could induce further lung and cardiovascular repair by mitigating inflammation and remodeling in a model of emphysema induced by multiple elastase instillations. We aimed to comparatively investigate the effects of one versus two doses of MSCs, administered 1 week apart, in a murine model of elastase-induced emphysema. METHODS: C57BL/6 mice were randomly divided into control (CTRL) and emphysema (E) groups. Mice in the E group received porcine pancreatic elastase (0.2 IU, 50 µL) intratracheally once weekly for four consecutive weeks; the CTRL animals received sterile saline (50 µL) using the same protocol. Three hours after the last instillation, the E group was further randomized to receive either saline (SAL) or murine MSCs (105 cells) intratracheally, in one or two doses (1 week apart). Fourteen days later, mice were euthanized, and all data analyzed. RESULTS: Both one and two doses of MSCs improved lung mechanics, reducing keratinocyte-derived chemokine and transforming growth factor-ß levels in lung homogenates, total cell and macrophage counts in bronchoalveolar lavage fluid (BALF), and collagen fiber content in airways and blood vessels, as well as increasing vascular endothelial growth factor in lung homogenates and elastic fiber content in lung parenchyma. However, only the two-dose group exhibited reductions in tumor necrosis factor-α in lung tissue, BALF neutrophil and lymphocyte count, thymus weight, and total cellularity, as well as CD8+ cell counts and cervical lymph node CD4+ and CD8+ T cell counts, as well as further increased elastic fiber content in the lung parenchyma and reduced severity of pulmonary arterial hypertension. CONCLUSIONS: Two doses of MSCs enhanced lung repair and improvement in cardiac function, while inducing T cell immunosuppression, mainly of CD8+ cells, in elastase-induced emphysema.

Critical role of CD4+ T cells and IFNγ signaling in antibody-mediated resistance to Zika virus infection.

Protective adaptive immunity to Zika virus (ZIKV) has been mainly attributed to cytotoxic CD8+ T cells and neutralizing antibodies, while the participation of CD4+ T cells in resistance has remained largely uncharacterized. Here, we show a neutralizing antibody response, dependent on CD4+ T cells and IFNγ signaling, which we detected during the first week of infection and is associated with reduced viral load in the brain, prevention of rapid disease onset and survival. We demonstrate participation of these components in the resistance to ZIKV during primary infection and in murine adoptive transfer models of heterologous ZIKV infection in a background of IFNR deficiency. The protective effect of adoptively transferred CD4+ T cells requires IFNγ signaling, CD8+ T cells and B lymphocytes in recipient mice. Together, this indicates the importance of CD4+ T cell responses in future vaccine design for ZIKV.

Eicosapentaenoic Acid Enhances the Effects of Mesenchymal Stromal Cell Therapy in Experimental Allergic Asthma.

Asthma is characterized by chronic lung inflammation and airway hyperresponsiveness. Despite recent advances in the understanding of its pathophysiology, asthma remains a major public health problem and, at present, there are no effective interventions capable of reversing airway remodeling. Mesenchymal stromal cell (MSC)-based therapy mitigates lung inflammation in experimental allergic asthma; however, its ability to reduce airway remodeling is limited. We aimed to investigate whether pre-treatment with eicosapentaenoic acid (EPA) potentiates the therapeutic properties of MSCs in experimental allergic asthma. Seventy-two C57BL/6 mice were used. House dust mite (HDM) extract was intranasally administered to induce severe allergic asthma in mice. Unstimulated or EPA-stimulated MSCs were administered intratracheally 24 h after final HDM challenge. Lung mechanics, histology, protein levels of biomarkers, and cellularity in bronchoalveolar lavage fluid (BALF), thymus, lymph nodes, and bone marrow were analyzed. Furthermore, the effects of EPA on lipid body formation and secretion of resolvin-D1 (RvD1), prostaglandin E2 (PGE2), interleukin (IL)-10, and transforming growth factor (TGF)-ß1 by MSCs were evaluated in vitro. EPA-stimulated MSCs, compared to unstimulated MSCs, yielded greater therapeutic effects by further reducing bronchoconstriction, alveolar collapse, total cell counts (in BALF, bone marrow, and lymph nodes), and collagen fiber content in airways, while increasing IL-10 levels in BALF and M2 macrophage counts in lungs. In conclusion, EPA potentiated MSC-based therapy in experimental allergic asthma, leading to increased secretion of pro-resolution and anti-inflammatory mediators (RvD1, PGE2, IL-10, and TGF-ß), modulation of macrophages toward an anti-inflammatory phenotype, and reduction in the remodeling process. Taken together, these modifications may explain the greater improvement in lung mechanics obtained. This may be a promising novel strategy to potentiate MSCs effects.

Bosutinib Therapy Ameliorates Lung Inflammation and Fibrosis in Experimental Silicosis.

Silicosis is an occupational lung disease for which no effective therapy exists. We hypothesized that bosutinib, a tyrosine kinase inhibitor, might ameliorate inflammatory responses, attenuate pulmonary fibrosis, and thus improve lung function in experimental silicosis. For this purpose, we investigated the potential efficacy of bosutinib in the treatment of experimental silicosis induced in C57BL/6 mice by intratracheal administration of silica particles. After 15 days, once disease was established, animals were randomly assigned to receive DMSO or bosutinib (1 mg/kg/dose in 0.1 mL 1% DMSO) by oral gavage, twice daily for 14 days. On day 30, lung mechanics and morphometry, total and differential cell count in alveolar septa and granuloma, levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IL-4, transforming growth factor (TGF)-ß, and vascular endothelial growth factor in lung homogenate, M1 and M2 macrophages, total leukocytes, and T cells in BALF, lymph nodes, and thymus, and collagen fiber content in alveolar septa and granuloma were analyzed. In a separate in vitro experiment, RAW264.7 macrophages were exposed to silica particles in the presence or absence of bosutinib. After 24 h, gene expressions of arginase-1, IL-10, IL-12, inducible nitric oxide synthase (iNOS), metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1, and caspase-3 were evaluated. In vivo, in silicotic animals, bosutinib, compared to DMSO, decreased: (1) fraction area of collapsed alveoli, (2) size and number of granulomas, and mononuclear cell granuloma infiltration; (3) IL-1ß, TNF-α, IFN-γ, and TGF-ß levels in lung homogenates, (4) collagen fiber content in lung parenchyma, and (5) viscoelastic pressure and static lung elastance. Bosutinib also reduced M1 cell counts while increasing M2 macrophage population in both lung parenchyma and granulomas. Total leukocyte, regulatory T, CD4+, and CD8+ cell counts in the lung-draining lymph nodes also decreased with bosutinib therapy without affecting thymus cellularity. In vitro, bosutinib led to a decrease in IL-12 and iNOS and increase in IL-10, arginase-1, MMP-9, and TIMP-1. In conclusion, in the current model of silicosis, bosutinib therapy yielded beneficial effects on lung inflammation and remodeling, therefore resulting in lung mechanics improvement. Bosutinib may hold promise for silicosis; however, further studies are required.

Human adipose tissue mesenchymal stromal cells and their extracellular vesicles act differentially on lung mechanics and inflammation in experimental allergic asthma.

BACKGROUND: Asthma is a chronic inflammatory disease that can be difficult to treat due to its complex pathophysiology. Most current drugs focus on controlling the inflammatory process, but are unable to revert the changes of tissue remodeling. Human mesenchymal stromal cells (MSCs) are effective at reducing inflammation and tissue remodeling; nevertheless, no study has evaluated the therapeutic effects of extracellular vesicles (EVs) obtained from human adipose tissue-derived MSCs (AD-MSC) on established airway remodeling in experimental allergic asthma. METHODS: C57BL/6 female mice were sensitized and challenged with ovalbumin (OVA). Control (CTRL) animals received saline solution using the same protocol. One day after the last challenge, each group received saline, 105 human AD-MSCs, or EVs (released by 105 AD-MSCs). Seven days after treatment, animals were anesthetized for lung function assessment and subsequently euthanized. Bronchoalveolar lavage fluid (BALF), lungs, thymus, and mediastinal lymph nodes were harvested for analysis of inflammation. Collagen fiber content of airways and lung parenchyma were also evaluated. RESULTS: In OVA animals, AD-MSCs and EVs acted differently on static lung elastance and on BALF regulatory T cells, CD3+CD4+ T cells, and pro-inflammatory mediators (interleukin [IL]-4, IL-5, IL-13, and eotaxin), but similarly reduced eosinophils in lung tissue, collagen fiber content in airways and lung parenchyma, levels of transforming growth factor-ß in lung tissue, and CD3+CD4+ T cell counts in the thymus. No significant changes were observed in total cell count or percentage of CD3+CD4+ T cells in the mediastinal lymph nodes. CONCLUSIONS: In this immunocompetent mouse model of allergic asthma, human AD-MSCs and EVs effectively reduced eosinophil counts in lung tissue and BALF and modulated airway remodeling, but their effects on T cells differed in lung and thymus. EVs may hold promise for asthma; however, further studies are required to elucidate the different mechanisms of action of AD-MSCs versus their EVs.

Effects of Bone Marrow Mesenchymal Stromal Cell Therapy in Experimental Cutaneous Leishmaniasis in BALB/c Mice Induced by Leishmania amazonensis.

Cutaneous leishmaniasis remains both a public health and a therapeutic challenge. To date, no ideal therapy for cutaneous leishmaniasis has been identified, and no universally accepted therapeutic regimen and approved vaccines are available. Due to the mesenchymal stromal cell (MSC) immunomodulatory capacity, they have been applied in a wide variety of disorders, including infectious, inflammatory, and allergic diseases. We evaluated the potential effects of bone marrow MSC therapy in a murine model of cutaneous leishmaniasis. In vitro, coculture of infected macrophages with MSC increased parasite load on macrophages in comparison with controls (macrophages without MSCs). In vivo, BALB/c mice were infected with 2 × 106Leishmania amazonensis (Josefa strain) promastigotes in the footpad. 7 and 37 days after infection, animals were treated with 1 × 105 MSCs, either intralesional (i.l.), i.e., in the same site of infection, or intravenously (i.v.), through the external jugular vein. Control animals received the same volume (50 µL) of phosphate-buffered saline by i.l. or i.v. routes. The lesion progression was assessed by its thickness measured by pachymetry. Forty-two days after infection, animals were euthanized and parasite burden in the footpad and in the draining lymph nodes was quantified by the limiting dilution assay (LDA), and spleen cells were phenotyped by flow cytometry. No significant difference was observed in lesion progression, regardless of the MSC route of administration. However, animals treated with i.v. MSCs presented a significant increase in parasite load in comparison with controls. On the other hand, no harmful effect due to MSCs i.l. administered was observed. The spleen cellular profile analysis showed an increase of IL-10 producing T CD4+ and TCD8+ cells in the spleen only in mice treated with i.v. MSC. The excessive production of IL-10 could be associated with the disease-aggravating effects of MSC therapy when intravenously administered. As a conclusion, in the current murine model of L. amazonensis-induced cutaneous disease, MSCs did not control the damage of cutaneous disease and, depending on the administration route, it could result in deleterious effects.

Therapeutic effects of bone marrow-derived mononuclear cells from healthy or silicotic donors on recipient silicosis mice.

BACKGROUND: Administration of bone marrow mononuclear cells (BMMCs) modulates lung inflammation and fibrosis in experimental silicosis. However, no studies have evaluated whether silicosis affects the efficacy of autologous BMMCs treatment. We hypothesized that BMMCs obtained from healthy or silicotic mice may improve lung function, but they might affect the inflammatory and fibrotic processes differently in experimental silicosis. METHODS: C57BL/6 mice were randomly divided into control (C) and silicosis (SIL) groups. Mice in the SIL group were instilled with silica particles intratracheally; the C animals received saline using the same protocol. On day 15, the animals were treated with saline (Sal) or BMMCs (2 × 106 cells) from healthy (BMMC-healthy) and silicotic (BMMC-sil) donors. Lung mechanics were measured, and lungs were collected for histology and molecular biology analysis. RESULTS: BMMCs obtained from healthy and silicotic donors presented similar percentages of cell populations. 99mTc-BMMCs tracking revealed preferential migration of cells to the liver, and only a few GFP+ BMMCs were observed in lung tissue 24 h after treatment, regardless of donor type. Both the SIL-BMMC-healthy and SIL-BMMC-sil groups showed improvement in lung function, a reduction in the fractional area of granuloma, and a decrease in the number of mononuclear and apoptotic cells in lung parenchyma. In addition, the number of F4/80+ macrophages, the levels of interleukin-1 beta and transforming growth factor beta, and collagen fiber content in granuloma were reduced in SIL-BMMC-healthy mice, whereas mRNA expression of MMP-9 and procollagen I and III was reduced in the SIL-BMMC-sil group. CONCLUSIONS: Administration of BMMCs from healthy and silicotic donors reduced lung inflammation and fibrosis, thus improving lung function. In addition, BMMC-healthy exhibited a greater improvement in lung morpho-functional changes in murine model of silicosis.