Tao Sai

IL-17C regulates the innate immune function of epithelial cells in an autocrine manner

Interleukin 17C (IL-17C) is a member of the IL-17 family that is selectively induced in epithelia by bacterial challenge and inflammatory stimuli. Here we show that IL-17C functioned in a unique autocrine manner, binding to a receptor complex consisting of the receptors IL-17RA and IL-17RE, which was preferentially expressed on tissue epithelial cells. IL-17C stimulated epithelial inflammatory responses, including the expression of proinflammatory cytokines, chemokines and antimicrobial peptides, which were similar to those induced by IL-17A and IL-17F. However, IL-17C was produced by distinct cellular sources, such as epithelial cells, in contrast to IL-17A, which was produced mainly by leukocytes, especially those of the TH17 subset of helper T cells. Whereas IL-17C promoted inflammation in an imiquimod-induced skin-inflammation model, it exerted protective functions in dextran sodium sulfate–induced colitis. Thus, IL-17C is an essential autocrine cytokine that regulates innate epithelial immune responses.

NRROS negatively regulates reactive oxygen species during host defence and autoimmunity.

Reactive oxygen species (ROS) produced by phagocytes are essential for host defence against bacterial and fungal infections. Individuals with defective ROS production machinery develop chronic granulomatous disease. Conversely, excessive ROS can cause collateral tissue damage during inflammatory processes and therefore needs to be tightly regulated. Here we describe a protein, we termed negative regulator of ROS (NRROS), which limits ROS generation by phagocytes during inflammatory responses. NRROS expression in phagocytes can be repressed by inflammatory signals. NRROS-deficient phagocytes produce increased ROS upon inflammatory challenges, and mice lacking NRROS in their phagocytes show enhanced bactericidal activity against Escherichia coli and Listeria monocytogenes. Conversely, these mice develop severe experimental autoimmune encephalomyelitis owing to oxidative tissue damage in the central nervous system. Mechanistically, NRROS is localized to the endoplasmic reticulum, where it directly interacts with nascent NOX2 (also known as gp91phox and encoded by Cybb) monomer, one of the membrane-bound subunits of the NADPH oxidase complex, and facilitates the degradation of NOX2 through the endoplasmic-reticulum-associated degradation pathway. Thus, NRROS provides a hitherto undefined mechanism for regulating ROS prodution—one that enables phagocytes to produce higher amounts of ROS, if required to control invading pathogens, while minimizing unwanted collateral tissue damage.

Furin-cleaved Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Is Active and Modulates Low Density Lipoprotein Receptor and Serum Cholesterol Levels

Background: Two forms of PCSK9, an intact and a furin cleaved form, circulate in blood. Results: Both forms, as highly purified recombinant proteins, are able to bind to and trigger degradation of LDL receptors and elevate serum cholesterol levels. Conclusion: Furin cleavage is not associated with a loss of PCSK9 polypeptides or a significant loss of function. Significance: LDL-c levels are controlled by both forms of PCSK9. Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg218-Gln219 in the surface-exposed “218 loop.” This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg218-Gln219 more efficiently than furin but also cleaves at Arg215-Phe216. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser153–Arg218), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Context-dependent signaling defines roles of BMP9 and BMP10 in embryonic and postnatal development

Many important signaling pathways rely on multiple ligands. It is unclear if this is a mechanism of safeguard via redundancy or if it serves other functional purposes. In this study, we report unique insight into this question by studying the activin receptor-like kinase 1 (ALK1) pathway. Despite its functional importance in vascular development, the physiological ligand or ligands for ALK1 remain to be determined. Using conventional knockout and specific antibodies against bone morphogenetic protein 9 (BMP9) or BMP10, we showed that BMP9 and BMP10 are the physiological, functionally equivalent ligands of ALK1 in vascular development. Timing of expression dictates the in vivo requisite role of each ligand, and concurrent expression results in redundancy. We generated mice (Bmp109/9) in which the coding sequence of Bmp9 replaces that of Bmp10. Surprisingly, analysis of Bmp109/9 mice demonstrated that BMP10 has an exclusive function in cardiac development, which cannot be substituted by BMP9. Our study reveals context-dependent significance in having multiple ligands in a signaling pathway.

Novel Staphylococcal Glycosyltransferases SdgA and SdgB Mediate Immunogenicity and Protection of Virulence-Associated Cell Wall Proteins

Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

Background: PILRα is an inhibitory receptor predominantly expressed in myeloid cells. Results: NPDC1 and COLEC12 are novel PILRα ligands. PILRα arginine residues 133 (mouse) and 126 (human) are critical contact residues. Conclusion: PILRα/ligand interactions involve a conserved domain in PILRα and a sialylated protein domain in the ligand. Significance: PILRα interacts with various ligands to alter myeloid cell function. Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

Increased Targeting of Donor Switch Region and IgE in Sγ1-Deficient B Cells

Ab class switch recombination involves a recombination between two repetitive DNA sequences known as switch (S) regions that vary in length, content, and density of the repeats. Abs expressed by B cells are diversified by somatic hypermutation and class switch recombination. Both class switch recombination and somatic hypermutation are initiated by activation-induced cytidine deaminase (AID), which preferentially recognizes certain hot spots that are far more enriched in the S regions. We found that removal of the largest S region, Sγ1 (10 kb), in mice can result in the accumulation of mutations and short-range intra-S recombination in the donor Sμ region. Furthermore, elevated levels of IgE were detected in trinitrophenol-OVA–immunized mice and in anti-CD40 plus IL-4–stimulated B cells in vitro. We propose that AID availability and targeting in part might be regulated by its DNA substrate. Thus, prominently transcribed S regions, such as Sγ1, might provide a sufficient sink for AID protein to titrate away AID from other accessible sites within or outside the Ig locus.

PILRα Negatively Regulates Mouse Inflammatory Arthritis

Paired Ig-like type 2 receptor (PILR)α inhibitory receptor and its counterpart PILRβ activating receptor are coexpressed on myeloid cells. In this article, we report that PILRα, but not PILRβ, is elevated in human rheumatoid arthritis synovial tissue and correlates with inflammatory cell infiltration. Pilrα−/− mice produce more pathogenic cytokines during inflammation and are prone to enhanced autoimmune arthritis. Correspondingly, engaging PILRα with anti-PILRα mAb ameliorates inflammation in mouse arthritis models and suppresses the production of proinflammatory cytokines. Our studies suggest that PILRα mediates an important inhibitory pathway that can dampen inflammatory responses.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination

Significance Switch (S) regions are repetitive DNA sequences. During an immune response, one of several S regions recombine with a donor switch (Sμ) that is constitutively “on,” resulting in the production of antibodies with new functions. Donor Sμ is large and very repeat-rich, while another switch, Sε, is less than half its size with a low density of repeats. We replaced Sε with Sμ in mice. These mice switch to Sε more effectively and produce high levels of IgE antibodies implicated in asthma, making this a useful model to study disease. In addition, placing Sμ outside of its native context revealed insights into how switches work. Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sμ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sμ. These results suggest that the sufficiency of Sμ to mediate IgH rearrangements may be influenced by context-dependent cues.

Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE.