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.

Tetraspanin CD53 modulates lymphocyte trafficking but not systemic autoimmunity in Lyn-deficient mice.

The leukocyte-restricted tetraspanin CD53 has been shown to promote lymphocyte homing to lymph nodes (LNs) and myeloid cell recruitment to acutely inflamed peripheral organs, and accelerate the onset of immune-mediated disease. However, its contribution in the setting of chronic systemic autoimmunity has not been investigated. We made use of the Lyn-/- autoimmune model, generating Cd53-/- Lyn-/- mice, and compared trafficking of immune cells into secondary lymphoid organs and systemic autoimmune disease development with mice lacking either gene alone. Consistent with previous observations, absence of CD53 led to reduced LN cellularity via reductions in both B and T cells, a phenotype also observed in Cd53-/- Lyn-/- mice. In some settings, Cd53-/- Lyn-/- lymphocytes showed greater loss of surface L-selectin and CD69 upregulation above that imparted by Lyn deficiency alone, indicating that absence of these two proteins can mediate additive effects in the immune system. Conversely, prototypical effects of Lyn deficiency including splenomegaly, plasma cell expansion, elevated serum immunoglobulin M and anti-nuclear antibodies were unaffected by CD53 deficiency. Furthermore, while Lyn-/- mice developed glomerular injury and showed elevated glomerular neutrophil retention above than that in wild-type mice, absence of CD53 in Lyn-/- mice did not alter these responses. Together, these findings demonstrate that while tetraspanin CD53 promotes lymphocyte trafficking into LNs independent of Lyn, it does not make an important contribution to development of autoimmunity, plasma cell dysfunction or glomerular injury in the Lyn-/- model of systemic autoimmunity.

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.

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.

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.

Genetic interdependence of Lyn and negative regulators of B cell receptor signaling in autoimmune disease development.

Ab-mediated autoimmune disease is multifaceted and may involve many susceptibility loci. The majority of autoimmune patients are thought to have polymorphisms in a number of genes that interact in different combinations to contribute to disease pathogenesis. Studies in mice and humans have implicated the Lyn protein tyrosine kinase as a regulator of Ab-mediated autoimmune disease. To examine whether haploinsufficiency of Lyn gives rise to cellular and clinical manifestations of autoimmune disease, we evaluated the phenotype of Lyn(+/-) mice. We find that their B cell compartment is significantly perturbed, with reduced numbers of marginal zone and transitional stage 2 B cells, expansion of plasma cells, downregulation of surface IgM, and upregulation of costimulatory molecules. Biochemical studies show that Lyn(+/-) B cells have defects in negative regulation of signaling, whereas Lyn(+/-) mice develop IgG autoantibodies and glomerulonephritis with age. Because Lyn has a pivotal role in the activation of inhibitory phosphatases, we generated mice harboring double heterozygous loss-of-function mutations in Lyn and SHP-1 or Lyn and SHIP-1. Partial inactivation of SHP-1 or SHIP-1 amplifies the consequence of Lyn haploinsufficiency, leading to an accelerated development of autoantibodies and disease. Our data also reveal that the BALB/c background is protective against autoimmune-mediated glomerulonephritis, even in the face of high titer autoantibodies, whereas the C57BL/6 background is susceptible. This study demonstrates that Lyn is a haploinsufficient gene in autoimmune disease and importantly shows that quantitative genetic variation in Lyn-regulated pathways can mirror the complete loss of a single critical inhibitory molecule.

Activated eosinophils in early-life impair lung development and promote long-term lung damage.

Exaggeration of type 2 immune responses promotes lung inflammation and altered lung development; however, eosinophils, despite expansion in the postnatal lung, have not been specifically assessed in the context of neonatal lung disease. Furthermore, early-life factors including prematurity and respiratory infection predispose infants to chronic obstructive pulmonary disease later in life. To assess eosinophils in the developing lung and how they may contribute to chronic lung disease, we generated mice harboring eosinophil-specific deletion of the negative regulatory enzyme SHIP-1. This increased the activity and number of pulmonary eosinophils in the developing lung, which was associated with impaired lung development, expansion of activated alveolar macrophages (AMφ), multinucleated giant cell formation, enlargement of airspaces, and fibrosis. Despite regression of eosinophils following completion of lung development, AMφ-dominated inflammation persisted, alongside lung damage. Bone marrow chimera studies showed that SHIP-1-deficient eosinophils were not sufficient to drive inflammatory lung disease in adult steady-state mice but once inflammation and damage was present, it could not be resolved. Depletion of eosinophils during alveolarization alleviated pulmonary inflammation and lung pathology, demonstrating an eosinophil-intrinsic effect. These results show that the presence of activated eosinophils during alveolarization aggravates AMφs and promotes sustained inflammation and long-lasting lung pathology.

Development of severe colitis is associated with lung inflammation and pathology.

Inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis are chronic relapsing diseases that affect the gastrointestinal tract, most commonly the colon. A link between the gut and the lung is suggested since patients with IBD have an increased susceptibility for chronic inflammatory lung disease. Furthermore, in the absence of overt lung disease, IBD patients have worsened lung function and more leukocytes in sputum than healthy individuals, highlighting a conduit between the gut and lung in disease. To study the gut-lung axis in the context of IBD, we used TCRδ-/- mice, which are highly susceptible to dextran sulfate sodium (DSS) due to the importance of γδ T cells in maintenance of barrier integrity. After induction of experimental colitis using DSS, the lungs of TCRδ-/- mice exhibited signs of inflammation and mild emphysema, which was not observed in DSS-treated C57BL/6 mice. Damage to the lung tissue was accompanied by a large expansion of neutrophils in the lung parenchyma and an increase in alveolar macrophages in the lung wash. Gene expression analyses showed a significant increase in Csf3, Cxcl2, Tnfa, and Il17a in lung tissue in keeping with neutrophil infiltration. Expression of genes encoding reactive oxygen species enzymes and elastolytic enzymes were enhanced in the lungs of both C57BL/6 and TCRδ-/- mice with colitis. Similarly, surfactant gene expression was also enhanced, which may represent a protective mechanism. These data demonstrate that severe colitis in a susceptible genetic background is sufficient to induce lung inflammation and tissue damage, providing the research community with an important tool for the development of novel therapeutics aimed at reducing co-morbidities in IBD patients.

Regulation of Microglial Signaling by Lyn and SHIP-1 in the Steady-State Adult Mouse Brain.

Chronic neuroinflammation and glial activation are associated with the development of many neurodegenerative diseases and neuropsychological disorders. Recent evidence suggests that the protein tyrosine kinase Lyn and the lipid phosphatase SH2 domain-containing inositol 5' phosphatase-1 (SHIP-1) regulate neuroimmunological responses, but their homeostatic roles remain unclear. The current study investigated the roles of Lyn and SHIP-1 in microglial responses in the steady-state adult mouse brain. Young adult Lyn-/- and SHIP-1-/- mice underwent a series of neurobehavior tests and postmortem brain analyses. The microglial phenotype and activation state were examined by immunofluorescence and flow cytometry, and neuroimmune responses were assessed using gene expression analysis. Lyn-/- mice had an unaltered behavioral phenotype, neuroimmune response, and microglial phenotype, while SHIP-1-/- mice demonstrated reduced explorative activity and exhibited microglia with elevated activation markers but reduced granularity. In addition, expression of several neuroinflammatory genes was increased in SHIP-1-/- mice. In response to LPS stimulation ex vivo, the microglia from both Lyn-/- and SHIP-1-/- showed evidence of hyper-activity with augmented TNF-α production. Together, these findings demonstrate that both Lyn and SHIP-1 have the propensity to control microglial responses, but only SHIP-1 regulates neuroinflammation and microglial activation in the steady-state adult brain, while Lyn activity appears dispensable for maintaining brain homeostasis.

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.

Loss of STAT6 promotes autoimmune disease and atopy on a susceptible genetic background.

Atopy and autoimmunity are usually considered opposed immunological manifestations. Lyn(-/-) mice develop lupus-like autoimmune disease yet have coexistent intrinsic allergic traits and are prone to severe, persistent asthma induced exogenously. Recently it has been proposed that the Th2 environment and IgE auto-Abs promotes autoimmune disease in Lyn(-/-) mice. To examine this apparent contradiction, we derived Lyn(-/-) mice with a null mutation in STAT6, a regulator of Th2 immunity that integrates signaling from the IL-4/IL-13 receptor complex. Atopy and spontaneous peritoneal eosinophilia, characteristic of Lyn(-/-) mice, were lost in young Lyn(-/-)STAT6(-/-) mice; however, autoimmune disease was markedly exacerbated. At a time-point where Lyn(-/-) mice showed only mild autoimmune disease, Lyn(-/-)STAT6(-/-) mice had maximal titres of IgG and IgA auto-Abs, impaired renal function, myeloid expansion and a highly activated T cell compartment. Remarkably, low level IgE auto-Abs but not IgG1 auto-Abs were a feature of some aged Lyn(-/-)STAT6(-/-) mice. Furthermore, aged Lyn(-/-)STAT6(-/-) mice showed dramatically increased levels of serum IgE but minimal IgG1, suggesting that class-switching to IgE can occur in the absence of an IgG1 intermediate. The results show that Lyn-deficient mice can overcome the effects of disabling Th2 immunity, highlighting the importance of Lyn in controlling Th2 responses. Our data also indicates that, under certain conditions, STAT6-independent factors can promote IgE class-switching. This work has important clinical implications as many experimental therapies designed for the treatment of asthma or atopy are based on targeting the STAT6 axis, which could potentially reveal life endangering autoimmunity or promote atopy in susceptible individuals.

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.

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.

CTLA4Ig alters the course of autoimmune disease development in Lyn-/- mice.

Lyn-deficient (Lyn(-/-)) mice develop an age-dependent autoimmune disease similar to systemic lupus erythematosus, characterized by the production of IgG anti-nuclear Ab. To determine the extent to which this autoimmune phenotype is driven by T cell costimulation, we generated Lyn(-/-) mice expressing a soluble form of the T cell inhibitory molecule, CTLA4 (CTLA4Ig). Surprisingly, although CTLA4Ig prevented myeloid hyperplasia, splenomegaly and IgG anti-nuclear Ab production in Lyn(-/-) mice, it did not inhibit immune complex deposition and tissue destruction in the kidney. In fact, regardless of CTLA4Ig expression, Lyn(-/-) serum contained elevated titers of IgA anti-nuclear Ab, although generally IgA deposition in the kidney was only revealed in the absence of self-reactive IgG. This demonstrated that activation of autoreactive B cell clones in Lyn(-/-) mice can still occur despite impaired costimulation. Indeed, CTLA4Ig did not alter perturbed Lyn(-/-) B cell development and behavior, and plasma cell frequencies were predominantly unaffected. These results suggest that when self-reactive B cell clones are unimpeded in acquiring T cell help, they secrete pathogenic IgG autoantibodies that trigger the fulminant autoimmunity normally observed in Lyn(-/-) mice. The absence of these IgG immune complexes reveals an IgA-mediated axis of autoimmunity that is not sufficient to cause splenomegaly or extramedullary myelopoiesis, but which mediates destructive glomerulonephritis. These findings have implications for the understanding of the basis of Ab-mediated autoimmune diseases and for their treatment with CTLA4Ig.

Loss of CD11b Accelerates Lupus Nephritis in Lyn-Deficient Mice Without Disrupting Glomerular Leukocyte Trafficking.

Systemic lupus erythematosus (SLE) is a complex, heterogeneous autoimmune disease. A common manifestation, lupus nephritis, arises from immune complex deposition in the kidney microvasculature promoting leukocyte activation and infiltration, which triggers glomerular damage and renal dysfunction. CD11b is a leukocyte integrin mainly expressed on myeloid cells, and aside from its well-ascribed roles in leukocyte trafficking and phagocytosis, it can also suppress cytokine production and autoreactivity. Genome-wide association studies have identified loss-of-function polymorphisms in the CD11b-encoding gene ITGAM that are strongly associated with SLE and lupus nephritis; however, it is not known whether these polymorphisms act alone to induce disease or in concert with other risk alleles. Herein we show using Itgam-/- mice that loss of CD11b led to mild inflammatory traits, which were insufficient to trigger autoimmunity or glomerulonephritis. However, deficiency of CD11b in autoimmune-prone Lyn-deficient mice (Lyn-/-Itgam-/- ) accelerated lupus-like disease, driving early-onset immune cell dysregulation, autoantibody production and glomerulonephritis, impacting survival. Migration of leukocytes to the kidney in Lyn-/- mice was unhindered by lack of CD11b. Indeed, kidney inflammatory macrophages were further enriched, neutrophil retention in glomerular capillaries was increased and kidney inflammatory cytokine responses were enhanced in Lyn-/-Itgam-/- mice. These findings indicate that ITGAM is a non-monogenic autoimmune susceptibility gene, with loss of functional CD11b exacerbating disease without impeding glomerular leukocyte trafficking when in conjunction with other pre-disposing genetic mutations. This highlights a primarily protective role for CD11b in restraining inflammation and autoimmune disease and provides a potential therapeutic avenue for lupus treatment.

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.

Perturbation of the CD4 T cell compartment and expansion of regulatory T cells in autoimmune-prone Lyn-deficient mice.

Regulatory T cells (Tregs) are a subset of T lymphocytes that are responsible for suppressing the function of other immune cells, and preventing potentially harmful autoimmune responses. Studies in autoimmune-prone mice and human autoimmune diseases have shown reduced Treg number or function as a causative factor for the apparent loss of tolerance that contributes to disease. We have found that Lyn-deficient mice, which develop high titers of autoantibodies with age, have a perturbed Treg compartment. Contrary to what has been observed in some strains of autoimmune-prone mice, aged Lyn-deficient mice have increased numbers of Tregs. This expansion occurs in the presence of elevated serum IL-2 and diminished TGF-beta. Despite expansion of the Treg compartment, Lyn-deficient mice succumb at approximately 1 year of age due to immune complex-mediated glomerulonephritis. We have shown that Lyn is not expressed in Tregs or indeed in any T cell subset, suggesting that the expansion and apparent functional deficiency in Tregs in Lyn-deficient mice is due to extrinsic factors rather than an intrinsic Treg defect. Indeed, using an in vivo colitis model, we have shown that Lyn-deficient Tregs can suppress inflammation. These results suggest that Tregs are expanding in Lyn-deficient mice in an effort to control the autoimmune disease but are simply overwhelmed by the disease process. This study highlights the role of the inflammatory setting in autoimmune disease and its consideration when contemplating the use of Tregs as an autoimmune therapy.

The Role of Innate Lymphoid Cells in Chronic Respiratory Diseases.

The lung is a vital mucosal organ that is constantly exposed to the external environment, and as such, its defenses are continuously under threat. The pulmonary immune system has evolved to sense and respond to these danger signals while remaining silent to innocuous aeroantigens. The origin of the defense system is the respiratory epithelium, which responds rapidly to insults by the production of an array of mediators that initiate protection by directly killing microbes, activating tissue-resident immune cells and recruiting leukocytes from the blood. At the steady-state, the lung comprises a large collection of leukocytes, amongst which are specialized cells of lymphoid origin known as innate lymphoid cells (ILCs). ILCs are divided into three major helper-like subsets, ILC1, ILC2 and ILC3, which are considered the innate counterparts of type 1, 2 and 17 T helper cells, respectively, in addition to natural killer cells and lymphoid tissue inducer cells. Although ILCs represent a small fraction of the pulmonary immune system, they play an important role in early responses to pathogens and facilitate the acquisition of adaptive immunity. However, it is now also emerging that these cells are active participants in the development of chronic lung diseases. In this mini-review, we provide an update on our current understanding of the role of ILCs and their regulation in the lung. We summarise how these cells and their mediators initiate, sustain and potentially control pulmonary inflammation, and their contribution to the respiratory diseases chronic obstructive pulmonary disease (COPD) and asthma.