C4b-binding protein inhibits particulate- and crystalline-induced NLRP3 inflammasome activation.

Dysregulated NLRP3 inflammasome activation drives a wide variety of diseases, while endogenous inhibition of this pathway is poorly characterised. The serum protein C4b-binding protein (C4BP) is a well-established inhibitor of complement with emerging functions as an endogenously expressed inhibitor of the NLRP3 inflammasome signalling pathway. Here, we identified that C4BP purified from human plasma is an inhibitor of crystalline- (monosodium urate, MSU) and particulate-induced (silica) NLRP3 inflammasome activation. Using a C4BP mutant panel, we identified that C4BP bound these particles via specific protein domains located on the C4BP α-chain. Plasma-purified C4BP was internalised into MSU- or silica-stimulated human primary macrophages, and inhibited MSU- or silica-induced inflammasome complex assembly and IL-1ß cytokine secretion. While internalised C4BP in MSU or silica-stimulated human macrophages was in close proximity to the inflammasome adaptor protein ASC, C4BP had no direct effect on ASC polymerisation in in vitro assays. C4BP was also protective against MSU- and silica-induced lysosomal membrane damage. We further provide evidence for an anti-inflammatory function for C4BP in vivo, as C4bp-/- mice showed an elevated pro-inflammatory state following intraperitoneal delivery of MSU. Therefore, internalised C4BP is an inhibitor of crystal- or particle-induced inflammasome responses in human primary macrophages, while murine C4BP protects against an enhanced inflammatory state in vivo. Our data suggests C4BP has important functions in retaining tissue homeostasis in both human and mice as an endogenous serum inhibitor of particulate-stimulated inflammasome activation.

Circadian Clock and Complement Immune System-Complementary Control of Physiology and Pathology?

Mammalian species contain an internal circadian (i.e., 24-h) clock that is synchronized to the day and night cycles. Large epidemiological studies, which are supported by carefully controlled studies in numerous species, support the idea that chronic disruption of our circadian cycles results in a number of health issues, including obesity and diabetes, defective immune response, and cancer. Here we focus specifically on the role of the complement immune system and its relationship to the internal circadian clock system. While still an incompletely understood area, there is evidence that dysregulated proinflammatory cytokines, complement factors, and oxidative stress can be induced by circadian disruption and that these may feed back into the oscillator at the level of circadian gene regulation. Such a feedback cycle may contribute to impaired host immune response against pathogenic insults. The complement immune system including its activated anaphylatoxins, C3a and C5a, not only facilitate innate and adaptive immune response in chemotaxis and phagocytosis, but they can also amplify chronic inflammation in the host organism. Consequent development of autoimmune disorders, and metabolic diseases associated with additional environmental insults that activate complement can in severe cases, lead to accelerated tissue dysfunction, fibrosis, and ultimately organ failure. Because several promising complement-targeted therapeutics to block uncontrolled complement activation and treat autoimmune diseases are in various phases of clinical trials, understanding fully the circadian properties of the complement system, and the reciprocal regulation by these two systems could greatly improve patient treatment in the long term.

In response to complement anaphylatoxin peptides C3a and C5a, human vascular endothelial cells migrate and mediate the activation of B-cells and polarization of T-cells.

The vascular endothelium has been discovered in the past several years to be important in shaping the cellular immune response. During the immune response the vascular endothelium is constantly perturbed by biologically potent molecules, including the complement activation peptides, C3a and C5a. Despite the importance of C3a and C5a in inflammation and immunity, their role in modulating lymphocyte function via activation of vascular endothelial cells is unknown. Accordingly, we investigated the regulated expression of the C3a and C5a receptors (complement anaphylatoxin C3a receptor [C3aR] and complement anaphylatoxin C5a receptor 1 [C5aR1]) on human umbilical vascular endothelial cells (HUVECs) and examined how C3a or C5a activation of HUVECs affects the activation and polarization of lymphatic cells. Our findings demonstrated that C3a and C5a increase C3aR and C5aR1 expression by HUVECs as well as directing their cellular transmigration and spreading through transwell filters. Moreover, C3a- or C5a-stimulated endothelial cells: (1) caused activation of B-lymphoblasts with significant increase in Fas Ligand (CD95L) (FasL), CD69, and IL-R1 expression, and (2) skewed T-lymphoblast cells toward a Th1 subtype, (CD4+ /CCR5+ ) that correlated with significant increase of IFN-γ. Collectively, these data indicate that C3a and C5a signaling is important in the activation and polarization of lymphocytes as they traffic through the vascular endothelium during the immune response.

A Preclinical Safety Study of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells for Macular Degeneration.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

Complement Receptor 1 (CR1/CD35)-expressing retinal pigment epithelial cells as a potential therapy for age-related macular degeneration.

The purpose of this study was to identify a membrane-bound complement inhibitor that could be overexpressed on retinal pigment epithelial cells (RPE) providing a potential therapy for age-related macular degeneration (AMD). This type of therapy may allow replacement of damaged RPE with cells that are able to limit complement activation in the retina. Complement Receptor 1 (CR1) is a membrane-bound complement inhibitor commonly found on erythrocytes and immune cells. In this study, QPCR and flow cytometry data demonstrated that CR1 is not well-expressed by RPE, indicating that its overexpression may provide extra protection from complement activation. To screen CR1 for this ability, a stable CR1-expressing ARPE19 line was created using a combination of antibiotic selection and FACS. Cell-based assays were used to demonstrate that addition of CR1 inhibited deposition of complement proteins C3b and C6 on the transfected line. In the end, this study identifies CR1 as a complement inhibitor that may be overexpressed on stem cell-derived RPE to create a potential "enhanced" cell therapy for AMD. A combination cell/complement therapy may create transplantable RPE better suited to avoid complement-mediated lysis and limit chronic inflammation in the retina.

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections.

The complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar-/- mice, whereas C5ar1-/- and C5ar2-/- mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1-/- mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8Δ71-73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a "super-agonist" peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.

Derivation and characterization of the human embryonic stem cell line CR-4: Differentiation to human retinal pigment epithelial cells.

The CR-4 human embryonic stem cell line was derived from the inner cell mass of a developing blastocyst. This cell line has been adapted to grow in feeder-free conditions and is especially well-suited for differentiation to retinal pigment epithelium. The line demonstrates a normal human 46,XX female karyotype. Pluripotency was assessed through qRT-PCR for expression of NANOG, OCT-4, and SOX-2. A teratoma assay was performed and results were positive for all three germ layers. Testing for Mycoplasma was negative.

The Complement Anaphylatoxins C5a and C3a Suppress IFN-ß Production in Response to Listeria monocytogenes by Inhibition of the Cyclic Dinucleotide-Activated Cytosolic Surveillance Pathway.

Listeria monocytogenes is an intracellular Gram-positive bacterium that induces expression of type I IFNs (IFN-α/IFN-ß) during infection. These cytokines are detrimental to the host during infection by priming leukocytes to undergo L. monocytogenes-mediated apoptosis. Our previous studies showed that C5aR1-/- and C3aR-/- mice are highly susceptible to L. monocytogenes infection as a result of increased IFN-ß-mediated apoptosis of major leukocyte cell populations, including CD4+ and CD8+ T cells. However, the mechanisms by which C3a and C5a modulate IFN-ß expression during L. monocytogenes infection were not examined in these initial investigations. Accordingly, we report in this article that C5a and C3a suppress IFN-ß production in response to L. monocytogenes via cyclic di-AMP (c-di-AMP), a secondary messenger molecule of L. monocytogenes, in J774A.1 macrophage-like cells and in bone marrow-derived dendritic cells (BMDCs). Moreover, C5a and C3a suppress IFN-ß production by acting through their respective receptors, because no inhibition was seen in C5aR1-/- or C3aR-/- BMDCs, respectively. C5a and C3a suppress IFN-ß production in a manner that is dependent on Bruton's tyrosine kinase, p38 MAPK, and TANK-binding kinase 1 (TBK1), as demonstrated by the individual use of Bruton's tyrosine kinase, p38 MAPK, and TBK1 inhibitors. Pretreatment of cells with C5a and C3a reduced the expression of the IFN-ß signaling molecules DDX41, STING, phosphorylated TBK1, and phosphorylated p38 MAPK in wild-type BMDCs following treatment with c-di-AMP. Collectively, these data demonstrate that C3a and C5a, via direct signaling through their specific receptors, suppress IFN-ß expression by modulation of a distinct innate cytosolic surveillance pathway involving DDX41, STING, and other downstream molecular targets of L. monocytogenes-generated c-di-AMP.

Targeted Disruption of the ß2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

UNLABELLED: Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of ß2-microglobulin on both gene alleles in hESCs. Because ß2-microglobulin serves as the HLA-I light chain, disruption of the ß2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced ß2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with ß2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, ß2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that ß2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. SIGNIFICANCE: This study reports the generation of a novel ß2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells.

Regulation of autoimmune germinal center reactions in lupus-prone BXD2 mice by follicular helper T cells.

BXD2 mice spontaneously develop autoantibodies and subsequent glomerulonephritis, offering a useful animal model to study autoimmune lupus. Although initial studies showed a critical contribution of IL-17 and Th17 cells in mediating autoimmune B cell responses in BXD2 mice, the role of follicular helper T (Tfh) cells remains incompletely understood. We found that both the frequency of Th17 cells and the levels of IL-17 in circulation in BXD2 mice were comparable to those of wild-type. By contrast, the frequency of PD-1+ CXCR5+ Tfh cells was significantly increased in BXD2 mice compared with wild-type mice, while the frequency of PD-1+ CXCR5+ Foxp3+ follicular regulatory T (Tfr) cells was reduced in the former group. The frequency of Tfh cells rather than that of Th17 cells was positively correlated with the frequency of germinal center B cells as well as the levels of autoantibodies to dsDNA. More importantly, CXCR5+ CD4+ T cells isolated from BXD2 mice induced the production of IgG from naïve B cells in an IL-21-dependent manner, while CCR6+ CD4+ T cells failed to do so. These results together demonstrate that Tfh cells rather than Th17 cells contribute to the autoimmune germinal center reactions in BXD2 mice.

The C5a anaphylatoxin receptor (C5aR1) protects against Listeria monocytogenes infection by inhibiting type 1 IFN expression.

Listeria monocytogenes is a major cause of mortality resulting from food poisoning in the United States. In mice, C5 has been genetically linked to host resistance to listeriosis. Despite this genetic association, it remains poorly understood how C5 and its activation products, C5a and C5b, confer host protection to this Gram-positive intracellular bacterium. In this article, we show in a systemic infection model that the major receptor for C5a, C5aR1, is required for a normal robust host immune response against L. monocytogenes. In comparison with wild-type mice, C5aR1(-/-) mice had reduced survival and increased bacterial burden in their livers and spleens. Infected C5aR1(-/-) mice exhibited a dramatic reduction in all major subsets of splenocytes, which was associated with elevated caspase-3 activity and increased TUNEL staining. Because type 1 IFN has been reported to impede the host response to L. monocytogenes through the promotion of splenocyte death, we examined the effect of C5aR1 on type 1 IFN expression in vivo. Indeed, serum levels of IFN-α and IFN-ß were significantly elevated in L. monocytogenes-infected C5aR1(-/-) mice. Similarly, the expression of TRAIL, a type 1 IFN target gene and a proapoptotic factor, was elevated in NK cells isolated from infected C5aR1(-/-) mice. Treatment of C5aR1(-/-) mice with a type 1 IFNR blocking Ab resulted in near-complete rescue of L. monocytogenes-induced mortality. Thus, these findings reveal a critical role for C5aR1 in host defense against L. monocytogenes through the suppression of type 1 IFN expression.

The receptor for the complement C3a anaphylatoxin (C3aR) provides host protection against Listeria monocytogenes-induced apoptosis.

Listeria monocytogenes is a Gram-positive intracellular bacterium that is acquired through tainted food and may lead to systemic infection and possible death. Despite the importance of the innate immune system in fighting L. monocytogenes infection, little is known about the role of complement and its activation products, including the potent C3a anaphylatoxin. In a model of systemic L. monocytogenes infection, we show that mice lacking the receptor for C3a (C3aR(-/-)) are significantly more sensitive to infection compared with wild-type mice, as demonstrated by decreased survival, increased bacterial burden, and increased damage to their livers and spleens. The inability of the C3aR(-/-) mice to clear the bacterial infection was not caused by defective macrophages or by a reduction in cytokines/chemokines known to be critical in the host response to L. monocytogenes, including IFN-γ and TNF-α. Instead, TUNEL staining, together with Fas, active caspase-3, and Bcl-2 expression data, indicates that the increased susceptibility of C3aR(-/-) mice to L. monocytogenes infection was largely caused by increased L. monocytogenes-induced apoptosis of myeloid and lymphoid cells in the spleen that are required for ultimate clearance of L. monocytogenes, including neutrophils, macrophages, dendritic cells, and T cells. These findings reveal an unexpected function of C3a/C3aR signaling during the host immune response that suppresses Fas expression and caspase-3 activity while increasing Bcl-2 expression, thereby providing protection to both myeloid and lymphoid cells against L. monocytogenes-induced apoptosis.

A site-specific genetic modification for induction of pluripotency and subsequent isolation of derived lung alveolar epithelial type II cells.

Human induced pluripotent stem cells (hiPSCs) have great therapeutic potential in repairing defective lung alveoli. However, genetic abnormalities caused by vector integrations and low efficiency in generating hiPSCs, as well as difficulty in obtaining transplantable hiPSC-derived cell types are still major obstacles. Here we report a novel strategy using a single nonviral site-specific targeting vector with a combination of Tet-On inducible gene expression system, Cre/lox P switching gene expression system, and alveolar epithelial type II cell (ATIIC)-specific Neomycin(R) transgene expression system. With this strategy, a single copy of all of the required transgenes can be specifically knocked into a site immediately downstream of ß-2-microglobulin (B2M) gene locus at a high frequency, without causing B2M dysfunction. Thus, the expression of reprogramming factors, Oct4, Sox2, cMyc, and Klf4, can be precisely regulated for efficient reprogramming of somatic cells into random integration-free or genetic mutation-free hiPSCs. The exogenous reprogramming factor transgenes can be subsequently removed after reprogramming by transient expression of Cre recombinase, and the resulting random integration-free and exogenous reprogramming factor-free hiPSCs can be selectively differentiated into a homogenous population of ATIICs. In addition, we show that these hiPSC-derived ATIICs exhibit ultrastructural characteristics and biological functions of normal ATIICs. When transplanted into bleomycin-challenged mice lungs, hiPSC-derived ATIICs efficiently remain and re-epithelialize injured alveoli to restore pulmonary function, preventing lung fibrosis and increasing survival without tumorigenic side effect. This strategy allows for the first time efficient generation of patient-specific ATIICs for possible future clinical applications.

Receptor for complement peptide C3a: a therapeutic target for neonatal hypoxic-ischemic brain injury.

Complement is an essential component of inflammation that plays a role in ischemic brain injury. Recent reports demonstrate novel functions of complement in normal and diseased CNS, such as regulation of neurogenesis and synapse elimination. Here, we examined the role of complement-derived peptide C3a in unilateral hypoxia-ischemia (HI), a model of neonatal HI encephalopathy. HI injury was induced at postnatal day 9 (P9), and loss of hippocampal tissue was determined on P31. We compared WT mice with transgenic mice expressing C3a under the control of glial fibrillary acidic protein promoter, which express biologically active C3a only in CNS and without the requirement of a priori complement activation. Further, we injected C3a peptide into the lateral cerebral ventricle of mice lacking the C3a receptor (C3aR) and WT mice and assessed HI-induced memory impairment 41 d later. We found that HI-induced tissue loss in C3a overexpressing mice was reduced by 50% compared with WT mice. C3a peptide injected 1 h after HI protected WT but not C3aR-deficient mice against HI-induced memory impairment. Thus, C3a acting through its canonical receptor ameliorates behavioral deficits after HI injury, and C3aR is a novel therapeutic target for the treatment of neonatal HI encephalopathy.

Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.

C5a receptors are found in the central nervous system (CNS), on both neurons and glia. However, the origin of the C5a, which activates these receptors, is unclear. In the present study, we show that primary cultured mouse cortical neurons constitutively express C5, the precursor of C5a, and express the classical receptor for C5a, CD88. With cell ischemia caused by 12 h glucose deprivation, or oxygen-glucose deprivation (OGD), neurons demonstrated increased apoptosis, up-regulation of CD88, and increased levels of C5a in the media. Exogenous murine C5a (100 nM) added to the neuronal cultures resulted in apoptosis, without affecting cell necrosis. Pretreatment of the cells with the specific CD88 receptor antagonist PMX53 (100 nM) significantly blocked ischemia-induced apoptosis (∼50%), and neurons from CD88(-/-) mice were similarly protected. In a murine model of stroke, using middle cerebral artery occlusion (MCAO), we found that C5a levels in the brain increased; this also occurred in cerebral slice cultures exposed to OGD. CD88(-/-) mice subjected to MCAO had significantly reduced infarct volumes and improved neurological scores. Taken together, our results demonstrate that neurons in the CNS have the capability to generate C5a following ischemic stress, and this has the potential to activate their C5a receptors, with deleterious consequences.

Negative regulation of pulmonary Th17 responses by C3a anaphylatoxin during allergic inflammation in mice.

Activation of complement is one of the earliest immune responses to exogenous threats, resulting in various cleavage products including anaphylatoxin C3a. In addition to its contribution to host defense, C3a has been shown to mediate Th2 responses in animal models of asthma. However, the role of C3a on pulmonary Th17 responses during allergic inflammation remains unclear. Here, we show that mice deficient in C3a receptor (C3aR) exhibited (i) higher percentages of endogenous IL-17-producing CD4(+) T cells in the lungs, (ii) higher amounts of IL-17 in the bronchoalveolar lavage fluid, and (iii) more neutrophils in the lungs than wild-type mice when challenged with intranasal allergens. Moreover, adoptive transfer experiments showed that the frequencies of antigen-specific IL-17-producing CD4(+) T cells were significantly higher in the lungs and bronchial lymph nodes of C3aR-deficient recipients than those of wild-types recipients. Bone-marrow reconstitution study indicated that C3aR-deficiency on hematopoietic cells was required for the increased Th17 responses. Furthermore, C3aR-deficient mice exhibited increased percentages of Foxp3(+) regulatory T cells; however, depletion of these cells minimally affected the induction of antigen-specific Th17 cell population in the lungs. Neutralization of IL-17 significantly reduced the number of neutrophils in bronchoalveolar lavage fluid of C3aR-deficient mice. Our findings demonstrate that C3a signals negatively regulate antigen-specific Th17 responses during allergic lung inflammation and the size of Foxp3(+) regulatory T cell population in the periphery.

A defect in the synthesis of Interferon-γ by the T cells of Complement-C5 deficient mice leads to enhanced susceptibility for tuberculosis.

Interferon-γ (IFNγ) plays a major role during host defense against Mycobacterium tuberculosis (Mtb). T cells produce IFNγ in response to IL-12 and IL-18 secreted from Mtb infected macrophages. IFNγ in turn, induces nitric oxide secretion in macrophages that kills Mtb. IFNγ knockout mice are thus hyper-susceptible to tuberculosis. We reported earlier that Complement-C5 deficient (C5(-/-)) congenic mice are more susceptible to tuberculosis and showed reduced IL-12 synthesis in their macrophages. Using C5(-/-) congenic mice that carry a deletion in the C5 gene and the wild type C5(+/+) mice, we demonstrate here that, the C5(-/-) derived CD3(+) T cells, have an additional defect in the synthesis of IFNγ. C5(-/-) T cells produced lower levels of IFNγ upon stimulation by antigen presenting cells (APCs) infected with Mtb or when stimulated directly with a combination of IL-12 and IL-18. The latter was in part due to a reduced phosphorylation of STAT4 following IL-12/IL-18 stimulation. Addition of C5a peptide to IL-12/IL-18 partially restored STAT4 phosphorylation and IFNγ synthesis in C5(-/-) T cells indicating that IL-12/IL-18 mediated signaling within CD3(+) T cells involves C5a peptide. Finally, C5(-/-) T cells derived from M. bovis BCG or Mtb infected mice showed a reduced expression of T-bet (T-box expressed in T cells) transcription factor, which correlated well with a reduced T cell secretion of IFNγ. Since T-bet mediated IFNγ synthesis facilitates Th1 expansion, C5(-/-) mouse derived T cells appear to have an intrinsic defect in the production of IFNγ, which is related to C5 deficiency and this may explain their increased susceptibility to infection with Mtb and BCG.

Therapeutic potential of lung epithelial progenitor cells derived from embryonic and induced pluripotent stem cells.

Embryonic stem (ES) cells derived from preimplantation blastocysts and induced pluripotent stem (iPS) cells generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide a possible unlimited source of cells that could be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. Because of inherent difficulties in deriving endodermal cells from undifferentiated cell cultures, applications using lung epithelial cells derived from ES and iPS cells have lagged behind similar efforts devoted to other tissues, such as the heart and spinal cord. However, during the past several years, significant advances in culture, differentiation, and purification protocols, as well as in bioengineering methodologies, have fueled enthusiasm for the development of stem cell-based lung therapeutics. This article provides an overview of recent research achievements and discusses future technical challenges that must be met before the promise of stem cell applications for lung disease can be realized.

Transplantation of human embryonic stem cell-derived alveolar epithelial type II cells abrogates acute lung injury in mice.

Respiratory diseases are a major cause of mortality and morbidity worldwide. Current treatments offer no prospect of cure or disease reversal. Transplantation of pulmonary progenitor cells derived from human embryonic stem cells (hESCs) may provide a novel approach to regenerate endogenous lung cells destroyed by injury and disease. Here, we examine the therapeutic potential of alveolar type II epithelial cells derived from hESCs (hES-ATIICs) in a mouse model of acute lung injury. When transplanted into lungs of mice subjected to bleomycin (BLM)-induced acute lung injury, hES-ATIICs behaved as normal primary ATIICs, differentiating into cells expressing phenotypic markers of alveolar type I epithelial cells. Without experiencing tumorigenic side effects, lung injury was abrogated in mice transplanted with hES-ATIICs, demonstrated by recovery of body weight and arterial blood oxygen saturation, decreased collagen deposition, and increased survival. Therefore, transplantation of hES-ATIICs shows promise as an effective therapeutic to treat acute lung injury.

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds.

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl](1/2) at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys(36)-Cys(49) and two disulfide bonds formed by two pair of consecutive cysteines, Cys(22)-Cys(23) and Cys(56)-Cys(57), a unique disulfide structure of polypeptide that has not been documented previously.