Minghe Ma

Disruption of the Cr2 Locus Results in a Reduction in B-1a Cells and in an Impaired B Cell Response to T-Dependent Antigen

Covalent attachment of activated products of the third component of complement to antigen enhances its immunogenicity, but the mechanism is not clear. This effect is mediated by specific receptors, mCR1 (CD35) and mCR2 (CD21), expressed primarily on B cells and follicular dendritic cells in mice. To dissect the role of mCR1 and mCR2 in the humoral response, we have disrupted the Cr2 locus to generate mice deficient in both receptors. The deficient mice (Cr2-/-) were found to have a reduction in the CD5+ population of peritoneal B-1 cells, although their serum IgM levels were within the range of normal mice. Moreover, Cr2-/- mice had a severe defect in their humoral response to T-dependent antigens that was characterized by a reduction in serum antibody titers and in the number and size of germinal centers within splenic follicles. Reconstitution of the deficient mice with bone marrow from MHC-matched Cr2+/+ donors corrected the defect, demonstrating that the defect was due to B cells themselves. These results indicate an obligatory role of B cell complement receptors in responses of the B cells to protein antigens.

Complement activation in factor D-deficient mice

To assess the contribution of the alternative pathway in complement activation and host defense and its possible role in the regulation of systemic energy balance in vivo, factor D-deficient mice were generated by gene targeting. The mutant mice have no apparent abnormality in development and their body weights are similar to those of factor D-sufficient littermates. Complement activation could not be initiated in the serum of deficient mice by the alternative pathway activators rabbit erythrocytes and zymosan. Surprisingly, injection of cobra venom factor (CVF) caused a profound and reproducible reduction in serum C3 levels, whereas, as expected, there was no C3 reduction in factor B-deficient mice treated similarly. Studies of C3 and factor B activation in vitro by CVF demonstrated that in factor D-deficient serum the α chain of C3 was cleaved gradually over a period of 60 min without detectable cleavage of factor B. CVF-dependent C3 cleavage in the deficient serum required the presence of Mg2+, whereas in normal mouse serum the presence of divalent cations was not required. These results suggest that in mouse proteolytic cleavage of factor B by factor D is not an absolute requirement for the zymogen to active enzyme conformational transition of CVF-bound factor B. Kinetics of opsonization of Streptococcus pneumoniae by C3 fragments was much slower in factor D-deficient serum, suggesting a significant contribution of the alternative pathway to antibacterial host defense early after infection.

Complement C4 Is Protective for Lupus Disease Independent of C3

The role of complement C3 in mediating systemic lupus erythematosus (SLE) was examined using a double-knockout C3nullC4null Fas (CD95)-deficient mouse model. Results from this study reveal significant lymphadenopathy, splenomegaly, elevated titers of anti-nuclear Abs and anti-dsDNA Abs, an increased number of anti-dsDNA-producing cells in ELISPOT assay, as well as severe glomerulonephritis in the double-deficient mice. Based on these clinical, serological, and histological parameters, we find that autoimmune disease in the double-knockout group is similar in severity to that in C4null lpr mice, but not to that in C3null lpr mice. The development of severe SLE in the absence of both classical and alternative complement pathways suggests that it is the absence of C4, and not the presence of C3, that is critical in SLE pathogenesis. Thus, complement C4 provides an important protective role against the development of SLE.

Redundant and Alternative Roles for Activating Fc Receptors and Complement in an Antibody-Dependent Model of Autoimmune Vitiligo

Complement and Fc receptor (FcR)-positive cells mediate effector functions of antibodies. Antibody-dependent immunity against the melanosome membrane glycoprotein gp75/tyrosinase-related protein-1 (TYRP-1) of melanocytes leads to autoimmune hypopigmentation (vitiligo) in mice. Hypopigmentation occurred in mice deficient in activating FcR containing the common gamma subunit (Fc gamma R gamma(-/-)) and in mice deficient in the C3 complement component. Mice doubly deficient in both Fc gamma R gamma and C3 did not develop hypopigmentation, suggesting that complement and Fc gamma R formed redundant mechanisms. Following passive immunization with antibody, no further adaptive immune responses were required. Chimeric Fc gamma R gamma(-/-),C3(-/-) mice reconstituted with bone marrow from either Fc gamma R gamma(-/-) or C3(-/-) mice or adoptively transferred with Fc gamma R gamma(+/-) macrophages did develop antibody-mediated hypopigmentation. Thus, either complement or macrophages expressing activating Fc gamma R can independently and alternatively mediate disease in a model of autoimmune vitiligo.

Complement C4 maintains peripheral B-cell tolerance in a myeloid cell dependent manner

The factors that allow self‐reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock‐in mouse strain, we identify a pathway by which B‐cell selection to nucleolar self‐antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B‐cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self‐reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self‐reactive B‐cell clones leading to increased spontaneous formation of GCs.

Uncoupling CD21 and CD19 of the B-cell coreceptor

Complement receptors (CRs) CD21 and CD35 form a coreceptor with CD19 and CD81 on murine B cells that when coligated with the B-cell receptor lowers the threshold of activation by several orders of magnitude. This intrinsic signaling role is thought to explain the impaired humoral immunity of mice bearing deficiency in CRs. However, CRs have additional roles on B cells independent of CD19, such as transport of C3-coated immune complexes and regulation of C4 and C3 convertase. To test whether association of CR with CD19 is necessary for their intrinsic activation-enhancing role, knockin mice expressing mutant receptors, Cr2Δ/Δgfp, that bind C3 ligands but do not signal through CD19 were constructed. We found that uncoupling of CR and CD19 significantly diminishes survival of germinal center B cells and secondary antibody titers. However, B memory is less impaired relative to mice bearing a complete deficiency in CRs on B cells. These findings confirm the importance of interaction of CR and CD19 for coreceptor activity in humoral immunity but identify a role for CR in B-cell memory independent of CD19.

Requirement for complement in antibody responses is not explained by the classic pathway activator IgM

Animals lacking complement factors C1q, C2, C3, or C4 have severely impaired Ab responses, suggesting a major role for the classic pathway. The classic pathway is primarily initiated by antigen–Ab complexes. Therefore, its role for primary Ab responses seems paradoxical because only low amounts of specific Abs are present in naive animals. A possible explanation could be that the classic pathway is initiated by IgM from naive mice, binding with sufficient avidity to the antigen. To test this hypothesis, a knock-in mouse strain, Cμ13, with a point mutation in the gene encoding the third constant domain of the μ-heavy chain was constructed. These mice produce IgM in which proline in position 436 is substituted with serine, a mutation previously shown to abrogate the ability of mouse IgM to activate complement. Unexpectedly, the Ab response to sheep erythrocytes and keyhole limpet hemocyanin in Cμ13 mice was similar to that in WT mice. Thus, although secreted IgM and the classic pathway activation are both required for the normal primary Ab response, this does not require that IgM activate C. This led us to test Ab responses in animals lacking one of three other endogenous activators of the classic pathway: specific intracellular adhesion molecule-grabbing nonintegrin R1, serum amyloid P component, and C-reactive protein. Ab responses were also normal in these animals.

Inhibition of JCPyV infection mediated by targeted viral genome editing using CRISPR/Cas9.

Progressive multifocal leukoencephalopathy (PML) is a debilitating disease resulting from infection of oligodendrocytes by the JC polyomavirus (JCPyV). Currently, there is no anti-viral therapeutic available against JCPyV infection. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) is a genome editing tool capable of introducing sequence specific breaks in double stranded DNA. Here we show that the CRISPR/Cas9 system can restrict the JCPyV life cycle in cultured cells. We utilized CRISPR/Cas9 to target the noncoding control region and the late gene open reading frame of the JCPyV genome. We found significant inhibition of virus replication and viral protein expression in cells recipient of Cas9 together with JCPyV-specific single-guide RNA delivered prior to or after JCPyV infection.

Identification and characterization of a novel broad spectrum virus entry inhibitor

ABSTRACT Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCE The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe ‘coincidence-detecting’ sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.