Regulation of microglial responses after pediatric traumatic brain injury: exploring the role of SHIP-1.

Introduction: Severe traumatic brain injury (TBI) is the world's leading cause of permanent neurological disability in children. TBI-induced neurological deficits may be driven by neuroinflammation post-injury. Abnormal activity of SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) has been associated with dysregulated immunological responses, but the role of SHIP-1 in the brain remains unclear. The current study investigated the immunoregulatory role of SHIP-1 in a mouse model of moderate-severe pediatric TBI. Methods: SHIP-1+/- and SHIP-1-/- mice underwent experimental TBI or sham surgery at post-natal day 21. Brain gene expression was examined across a time course, and immunofluorescence staining was evaluated to determine cellular immune responses, alongside peripheral serum cytokine levels by immunoassays. Brain tissue volume loss was measured using volumetric analysis, and behavior changes both acutely and chronically post-injury. Results: Acutely, inflammatory gene expression was elevated in the injured cortex alongside increased IBA-1 expression and altered microglial morphology; but to a similar extent in SHIP-1-/- mice and littermate SHIP-1+/- control mice. Similarly, the infiltration and activation of CD68-positive macrophages, and reactivity of GFAP-positive astrocytes, was increased after TBI but comparable between genotypes. TBI increased anxiety-like behavior acutely, whereas SHIP-1 deficiency alone reduced general locomotor activity. Chronically, at 12-weeks post-TBI, SHIP-1-/- mice exhibited reduced body weight and increased circulating cytokines. Pro-inflammatory gene expression in the injured hippocampus was also elevated in SHIP-1-/- mice; however, GFAP immunoreactivity at the injury site in TBI mice was lower. TBI induced a comparable loss of cortical and hippocampal tissue in both genotypes, while SHIP-1-/- mice showed reduced general activity and impaired working memory, independent of TBI. Conclusion: Together, evidence does not support SHIP-1 as an essential regulator of brain microglial morphology, brain immune responses, or the extent of tissue damage after moderate-severe pediatric TBI in mice. However, our data suggest that reduced SHIP-1 activity induces a greater inflammatory response in the hippocampus chronically post-TBI, warranting further investigation.

Granulocyte Colony-Stimulating Factor is a Determinant of Severe Bronchopulmonary Dysplasia and Coincident Retinopathy.

Bronchopulmonary dysplasia (BPD), also called chronic lung disease of immaturity, afflicts approximately one third of all extremely premature infants, causing lifelong lung damage. There is no effective treatment other than supportive care. Retinopathy of prematurity (ROP), which impairs vision irreversibly, is common in BPD, suggesting a related pathogenesis. However, specific mechanisms of BPD and ROP are not known. Herein, a neonatal mouse hyperoxic model of coincident BPD and retinopathy was used to screen for candidate mediators, which revealed that granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3, was up-regulated significantly in mouse lung lavage fluid and plasma at postnatal day 14 in response to hyperoxia. Preterm infants with more severe BPD had increased plasma G-CSF. G-CSF-deficient neonatal pups showed significantly reduced alveolar simplification, normalized alveolar and airway resistance, and normalized weight gain compared with wild-type pups after hyperoxic lung injury. This was associated with a marked reduction in the intensity, and activation state, of neutrophilic and monocytic inflammation and its attendant oxidative stress response, and protection of lung endothelial cells. G-CSF deficiency also provided partial protection against ROP. The findings in this study implicate G-CSF as a pathogenic mediator of BPD and ROP, and suggest the therapeutic utility of targeting G-CSF biology to treat these conditions.

Enhanced Lyn Activity Causes Severe, Progressive Emphysema and Lung Cancer.

The epidemiological patterns of incident chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma are changing, with an increasing fraction of disease occurring in patients who are never-smokers or were not exposed to traditional risk factors. However, causative mechanism(s) are obscure. Overactivity of Src family kinases (SFKs) and myeloid cell-dependent inflammatory lung epithelial and endothelial damage are independent candidate mechanisms, but their pathogenic convergence has not been demonstrated. Here we present a novel preclinical model in which an activating mutation in Lyn, a nonreceptor SFK that is expressed in immune cells, epithelium, and endothelium-all strongly implicated in the pathogenesis of COPD-causes spontaneous inflammation, early-onset progressive emphysema, and lung adenocarcinoma. Surprisingly, even though activated macrophages, elastolytic enzymes, and proinflammatory cytokines were prominent, bone marrow chimeras formally demonstrated that myeloid cells were not disease initiators. Rather, lung disease arose from aberrant epithelial cell proliferation and differentiation, microvascular lesions within an activated endothelial microcirculation, and amplified EGFR (epidermal growth factor receptor) expression. In human bioinformatics analyses, LYN expression was increased in patients with COPD and was correlated with increased EGFR expression, a known lung oncogenic pathway, and LYN was linked to COPD. Our study shows that a singular molecular defect causes a spontaneous COPD-like immunopathology and lung adenocarcinoma. Furthermore, we identify Lyn and, by implication, its associated signaling pathways as new therapeutic targets for COPD and cancer. Moreover, our work may inform the development of molecular risk screening and intervention methods for disease susceptibility, progression, and prevention of these increasingly prevalent conditions.

The immunological link between neonatal lung and eye disease.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are two neonatal diseases of major clinical importance, arising in large part as a consequence of supplemental oxygen therapy used to promote the survival of preterm infants. The presence of coincident inflammation in the lungs and eyes of neonates receiving oxygen therapy indicates that a dysregulated immune response serves as a potential common pathogenic factor for both diseases. This review examines the current state of knowledge of immunological dysregulation in BPD and ROP, identifying similarities in the cellular subsets and inflammatory cytokines that are found in the alveoli and retina during the active phase of these diseases, indicating possible mechanistic overlap. In addition, we highlight gaps in the understanding of whether these responses emerge independently in the lung and retina as a consequence of oxygen exposure or arise because of inflammatory spill-over from the lung. As BPD and ROP are anatomically distinct, they are often considered discreet disease entities and are therefore treated separately. We propose that an improved understanding of the relationship between BPD and ROP is key to the identification of novel therapeutic targets to treat or prevent both conditions simultaneously.

An Experimental Model of Bronchopulmonary Dysplasia Features Long-Term Retinal and Pulmonary Defects but Not Sustained Lung Inflammation.

Bronchopulmonary dysplasia (BPD) is a severe lung disease that affects preterm infants receiving oxygen therapy. No standardized, clinically-relevant BPD model exists, hampering efforts to understand and treat this disease. This study aimed to evaluate and confirm a candidate model of acute and chronic BPD, based on exposure of neonatal mice to a high oxygen environment during key lung developmental stages affected in preterm infants with BPD. Neonatal C57BL/6 mouse pups were exposed to 75% oxygen from postnatal day (PN)-1 for 5, 8, or 14 days, and their lungs were examined at PN14 and PN40. While all mice showed some degree of lung damage, mice exposed to hyperoxia for 8 or 14 days exhibited the greatest septal wall thickening and airspace enlargement. Furthermore, when assessed at PN40, mice exposed for 8 or 14 days to supplemental oxygen exhibited augmented septal wall thickness and emphysema, with the severity increased with the longer exposure, which translated into a decline in respiratory function at PN80 in the 14-day model. In addition to this, mice exposed to hyperoxia for 8 days showed significant expansion of alveolar epithelial type II cells as well as the greatest fibrosis when assessed at PN40 suggesting a healing response, which was not seen in mice exposed to high oxygen for a longer period. While evidence of lung inflammation was apparent at PN14, chronic inflammation was absent from all three models. Finally, exposure to high oxygen for 14 days also induced concurrent outer retinal degeneration. This study shows that early postnatal exposure to high oxygen generates hallmark acute and chronic pathologies in mice that highlights its use as a translational model of BPD.

Links Between Inflammatory Bowel Disease and Chronic Obstructive Pulmonary Disease.

Inflammatory bowel disease (IBD) and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the gastrointestinal and respiratory tracts, respectively. These mucosal tissues bear commonalities in embryology, structure and physiology. Inherent similarities in immune responses at the two sites, as well as overlapping environmental risk factors, help to explain the increase in prevalence of IBD amongst COPD patients. Over the past decade, a tremendous amount of research has been conducted to define the microbiological makeup of the intestine, known as the intestinal microbiota, and determine its contribution to health and disease. Intestinal microbial dysbiosis is now known to be associated with IBD where it impacts upon intestinal epithelial barrier integrity and leads to augmented immune responses and the perpetuation of chronic inflammation. While much less is known about the lung microbiota, like the intestine, it has its own distinct, diverse microflora, with dysbiosis being reported in respiratory disease settings such as COPD. Recent research has begun to delineate the interaction or crosstalk between the lung and the intestine and how this may influence, or be influenced by, the microbiota. It is now known that microbial products and metabolites can be transferred from the intestine to the lung via the bloodstream, providing a mechanism for communication. While recent studies indicate that intestinal microbiota can influence respiratory health, intestinal dysbiosis in COPD has not yet been described although it is anticipated since factors that lead to dysbiosis are similarly associated with COPD. This review will focus on the gut-lung axis in the context of IBD and COPD, highlighting the role of environmental and genetic factors and the impact of microbial dysbiosis on chronic inflammation in the intestinal tract and lung.

Lung and Eye Disease Develop Concurrently in Supplemental Oxygen-Exposed Neonatal Mice.

Bronchopulmonary dysplasia (BPD) and retinopathy of prematurity (ROP) are two debilitating disorders that develop in preterm infants exposed to supplemental oxygen to prevent respiratory failure. Both can lead to lifelong disabilities, such as chronic obstructive pulmonary disease and vision loss. Due to the lack of a standard experimental model of coincident disease, the underlying associations between BPD and ROP are not well characterized. To address this gap, we used the robust mouse model of oxygen-induced retinopathy exposing C57BL/6 mice to 75% oxygen from postnatal day 7 to 12. The cardinal features of ROP were replicated by this strategy, and the lungs of the same mice were simultaneously examined for evidence of BPD-like lung injury, investigating both the short- and long-term effects of early-life supplemental oxygen exposure. At postnatal days 12 and 18, mild lung disease was evident by histopathologic analysis together with the expected vasculopathy in the inner retina. At later time points, the lung lesion had progressed to severe airspace enlargement and alveolar simplification, with concurrent thinning in the outer layer of the retina. In addition, critical angiogenic oxidative stress and inflammatory factors reported to be dysregulated in ROP were similarly impaired in the lungs. These data shed new light on the interconnectedness of these two neonatal disorders, holding potential for the discovery of novel targets to treat BPD and ROP.

IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization.

The contribution of mast cells in the microenvironment of solid malignancies remains controversial. Here we functionally assess the impact of tumor-adjacent, submucosal mast cell accumulation in murine and human intestinal-type gastric cancer. We find that genetic ablation or therapeutic inactivation of mast cells suppresses accumulation of tumor-associated macrophages, reduces tumor cell proliferation and angiogenesis, and diminishes tumor burden. Mast cells are activated by interleukin (IL)-33, an alarmin produced by the tumor epithelium in response to the inflammatory cytokine IL-11, which is required for the growth of gastric cancers in mice. Accordingly, ablation of the cognate IL-33 receptor St2 limits tumor growth, and reduces mast cell-dependent production and release of the macrophage-attracting factors Csf2, Ccl3, and Il6. Conversely, genetic or therapeutic macrophage depletion reduces tumor burden without affecting mast cell abundance. Therefore, tumor-derived IL-33 sustains a mast cell and macrophage-dependent signaling cascade that is amenable for the treatment of gastric cancer.

Regulation of hematopoietic cell signaling by SHIP-1 inositol phosphatase: growth factors and beyond.

SHIP-1 is a hematopoietic-specific inositol phosphatase activated downstream of a multitude of receptors including those for growth factors, cytokines, antigen, immunoglobulin and toll-like receptor agonists where it exerts inhibitory control. While it is constitutively expressed in all immune cells, SHIP-1 expression is negatively regulated by the inflammatory and oncogenic micro-RNA miR-155. Knockout mouse studies have shown the importance of SHIP-1 in various immune cell subsets and have revealed a range of immune-mediated pathologies that are engendered due to loss of SHIP-1's regulatory activity, impelling investigations into the role of SHIP-1 in human disease. In this review, we provide an overview of the literature relating to the role of SHIP-1 in hematopoietic cell signaling and function, we summarize recent reports that highlight the dysregulation of the SHIP-1 pathway in cancers, autoimmune disorders and inflammatory diseases, and lastly we discuss the importance of SHIP-1 in restraining myeloid growth factor signaling.

NFκB1 is essential to prevent the development of multiorgan autoimmunity by limiting IL-6 production in follicular B cells.

We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity of Nfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation, the formation of GC structures was severely disrupted in the Nfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels, which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 represses Il-6 transcription in Fo B cells, with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription of Il-6.Collectively, our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation of Il-6 gene expression in Fo B cells.

BAFF-driven autoimmunity requires CD19 expression.

B cell activating factor of the tumor necrosis factor family (BAFF or BLyS) is a critical factor for B cell survival and maturation. BAFF-transgenic (BAFF-Tg) mice develop autoimmunity that resembles systemic lupus erythematosus (SLE) in a T cell-independent but MyD88-dependent manner, implicating toll-like receptor (TLR) signaling. The specific B cell subtypes that make pro-inflammatory autoantibodies in BAFF-Tg mice are TLR-activated innate B cells known as marginal zone (MZ) and B1 B cells. These cells infiltrate the salivary glands and kidneys of diseased BAFF-Tg mice. However, loss of B1a or MZ B cells does not protect BAFF-Tg mice against disease, suggesting that B1b B cells might be the important pathogenic B cell subset. To test this hypothesis, we have generated BAFF-Tg mice that retained follicular B cells, but are deficient in B1a, B1b and MZ B cells, by crossing BAFF-Tg mice to CD19-deficient mice (BTg-CD19(-/-)). The BTg-CD19(-/-) mice did not produce autoantibodies and were protected from splenomegaly, kidney pathology and all signs of autoimmunity. This work suggests that B1b B cells, rather than MZ or B1a B cells, are sufficient and possibly required for the development of autoimmunity. Loss of the majority of innate-like B cells was able to protect BAFF-Tg mice from developing disease, so we can now conclude that autoimmunity induced by excessive BAFF production requires B1b B cells and CD19 signaling.

Interleukin-6 trans-signaling exacerbates inflammation and renal pathology in lupus-prone mice.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that is characterized by the production of antinuclear antibodies (ANAs) and leads to immune complex deposition in the kidneys and nephritis. Lyn tyrosine kinase is a regulator of antibody-mediated autoimmune disease, as evidenced by studies in gene-targeted mice and as suggested in genome-wide association studies in SLE. Like SLE patients, Lyn-deficient mice have increased levels of interleukin-6 (IL-6). Deletion of IL-6 from Lyn-deficient mice abrogates levels of inflammation, pathogenic autoantibodies, and nephritis. The purpose of this study was to assess the role of IL-6 trans-signaling in autoimmune disease by overexpressing soluble gp130Fc (sgp130Fc) in a mouse model. METHODS: The effect of overexpression of sgp130Fc on immune cell phenotypes was determined by flow cytometry in young and aged mice with lupus, and ANAs were measured by enzyme-linked immunosorbent assay. Glomerulonephritis was assessed by histopathologic analysis, by measuring the glomerular area and the blood urea nitrogen concentration, and by immunohistochemistry. Immunofluorescence defined renal immune complex and complement deposition. The acute-phase response was determined by quantitative real-time polymerase chain reaction. RESULTS: In contrast to removing IL-6, impaired IL-6 trans-signaling had little effect on many immune cell abnormalities in Lyn-/- mice. Pathogenic ANAs and kidney deposition of immune complexes were also unaltered by sgp130Fc. However, sgp130Fc overexpression led to diminished macrophage expansion, reduced glomerular leukocyte infiltration, reduced complement fixation, significantly attenuated glomerulonephritis, and improved renal function in Lyn-deficient mice. CONCLUSION: Our results reveal key roles of leukocytes, complement, and the innate immune system in mediating glomerulonephritis, and they implicate IL-6 trans-signaling in this process. We suggest that targeting this pathway may be an effective adjunct to B cell depletion in SLE treatment.

Attenuation of phosphoinositide 3-kinase δ signaling restrains autoimmune disease.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn(-/-)p110δ(+/KD) mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE.

New reagents for improved in vitro and in vivo examination of TGF-ß signalling.

Transforming growth factor-ß (TGF-ß) signalling controls many aspects of cell behaviour and is implicated as a key regulator in tumour formation and progression. However, evaluating levels of active TGF-ß in culture medium or patient plasma and gaining definitive information regarding the activity of downstream substrates such as Sma- and Mad-related protein 3 (Smad3) in vivo with accuracy and sensitivity has been problematic. Therefore, to overcome these technical issues we have created a NIH3T3 cell line with stable pCAGA(12)-luc expression that can now be utilised to detect TGF-ß activity with high sensitivity. In addition, we have created an adenoviral Smad3 luciferase reporter construct pAd.CAGA(12)-luc to successfully infect cells for in vitro assays, or prior to injection into mice and used to measure transcriptional activity in vivo. Thus, the NIH3T3-pCAGA(12)-luc cell line and the pAd.CAGA(12)-luc adenovirus will be extremely useful tools to measure TGF-ß signalling activity with far greater efficiency and reliability compared to original and currently used reagents.

Autoimmune disease in Lyn-deficient mice is dependent on an inflammatory environment established by IL-6.

Lyn-deficient mice develop Ab-mediated autoimmune disease resembling systemic lupus erythematosus where hyperactive B cells are major contributors to pathology. In this study, we show that an inflammatory environment is established in Lyn(-/-) mice that perturbs several immune cell compartments and drives autoimmune disease. Lyn(-/-) leukocytes, notably B cells, are able to produce IL-6, which facilitates hyperactivation of B and T cells, enhanced myelopoiesis, splenomegaly, and, ultimately, generation of pathogenic autoreactive Abs. Lyn(-/-) dendritic cells show increased maturation, but this phenotype is independent of autoimmunity as it is reiterated in B cell-deficient Lyn(-/-) mice. Genetic deletion of IL-6 on a Lyn-deficient background does not alter B cell development, plasma cell accumulation, or dendritic cell hypermaturation, suggesting that these characteristics are intrinsic to the loss of Lyn. However, hyperactivation of B and T cell compartments, extramedullary hematopoiesis, expansion of the myeloid lineage and autoimmune disease are all ameliorated in Lyn(-/-)IL-6(-/-) mice. Importantly, our studies show that although Lyn(-/-) B cells may be autoreactive, it is the IL-6-dependent inflammatory environment they engender that dictates their disease-causing potential. These findings improve our understanding of the mode of action of anti-IL-6 and B cell-directed therapies in autoimmune and inflammatory disease treatment.

A system for quantifying the patterning of the lymphatic vasculature.

The lymphatic vasculature is critical for immunity and interstitial fluid homeostasis, playing important roles in diseases such as lymphedema and metastatic cancer. Animal models have been generated to explore the role of lymphatics and lymphangiogenic growth factors in such diseases, and to study lymphatic development. However, analysis of lymphatic vessels has primary been restricted to counting lymphatics in two-dimensional tissue slices, due to a lack of more sophisticated methodologies. In order to accurately examine lymphatic dysfunction in these models, and analyse the effects of lymphangiogenic growth factors on the lymphatic vasculature, it is essential to quantify the morphology and patterning of the distinct lymphatic vessels types in three-dimensional tissues. Here, we describe a method for performing such analyses, integrating user-operated image-analysis software with an approach that considers important morphological, anatomical and patterning features of the distinct lymphatic vessel subtypes. This efficient, reproducible technique is validated by analysing healthy and pathological tissues.