Inhibition of the Lectin Pathway of Complement Activation Reduces Acute Respiratory Distress Syndrome Severity in a Mouse Model of SARS-CoV-2 Infection.

Most patients with COVID-19 in the intensive care unit develop an acute respiratory distress syndrome characterized by severe hypoxemia, decreased lung compliance, and high vascular permeability. Activation of the complement system is a hallmark of moderate and severe COVID-19, with abundant deposition of complement proteins in inflamed tissue and on the endothelium during COVID-19. Using a transgenic mouse model of SARS-CoV-2 infection, we assessed the therapeutic utility of an inhibitory antibody (HG4) targeting MASP-2, a key enzyme in the lectin pathway. Treatment of infected mice with HG4 reduced the disease severity score and improved survival vs mice that received an isotype control antibody. Administration of HG4 significantly reduced the lung injury score, including alveolar inflammatory cell infiltration, alveolar edema, and alveolar hemorrhage. The ameliorating effect of MASP-2 inhibition on the severity of COVID-19 pathology is reflected by a significant reduction in the proinflammatory activation of brain microglia in HG4-treated mice.

Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

All 3 activation pathways of complement-the classic pathway (CP), the alternative pathway, and the lectin pathway (LP)- converge into a common central event: the cleavage and activation of the abundant third complement component, C3, via formation of C3-activating enzymes (C3 convertases). The fourth complement component, C4, and the second component, C2, are indispensable constituents of the C3 convertase complex, C4bC2a, which is formed by both the CP and the LP. Whereas in the absence of C4, CP can no longer activate C3, LP retains a residual but physiologically critical capacity to convert native C3 into its activation fragments, C3a and C3b. This residual C4 and/or C2 bypass route is dependent on LP-specific mannan-binding lectin-associated serine protease-2. By using various serum sources with defined complement deficiencies, we demonstrate that, under physiologic conditions LP-specific C4 and/or C2 bypass activation of C3 is mediated by direct cleavage of native C3 by mannan-binding lectin-associated serine protease-2 bound to LP-activation complexes captured on ligand-coated surfaces.-Yaseen, S., Demopulos, G., Dudler, T., Yabuki, M., Wood, C. L., Cummings, W. J., Tjoelker, L. W., Fujita, T., Sacks, S., Garred, P., Andrew, P., Sim, R. B., Lachmann, P. J., Wallis, R., Lynch, N., Schwaeble, W. J. Lectin pathway effector enzyme mannan-binding lectin-associated serine protease-2 can activate native complement C3 in absence of C4 and/or C2.

Inhibition of the lectin pathway of complement activation reduces LPS-induced acute respiratory distress syndrome in mice.

Acute respiratory distress syndrome (ARDS) is a life-threatening disorder with a high rate of mortality. Complement activation in ARDS initiates a robust inflammatory reaction that can cause progressive endothelial injury in the lung. Here, we tested whether inhibition of the lectin pathway of complement could reduce the pathology and improve the outcomes in a murine model of LPS-induced lung injury that closely mimics ARDS in human. In vitro, LPS binds to murine and human collectin 11, human MBL and murine MBL-A, but not to C1q, the recognition subcomponent of the classical pathway. This binding initiates deposition of the complement activation products C3b, C4b and C5b-9 on LPS via the lectin pathway. HG-4, a monoclonal antibody that targets MASP-2, a key enzyme in the lectin pathway, inhibited lectin pathway functional activity in vitro, with an IC50 of circa 10nM. Administration of HG4 (5mg/kg) in mice led to almost complete inhibition of the lectin pathway activation for 48hrs, and 50% inhibition at 60hrs post administration. Inhibition of the lectin pathway in mice prior to LPS-induced lung injury improved all pathological markers tested. HG4 reduces the protein concentration in bronchoalveolar lavage fluid (p<0.0001) and levels of myeloid peroxide (p<0.0001), LDH (p<0.0001), TNFα and IL6 (both p<0.0001). Lung injury was significantly reduced (p<0.001) and the survival time of the mice increased (p<0.01). From the previous findings we concluded that inhibition of the lectin pathway has the potential to prevent ARDS pathology.

Secondary Complement Deficiency Impairs Anti-Microbial Immunity to Klebsiella pneumoniae and Staphylococcus aureus During Severe Acute COVID-19.

A high incidence of secondary Klebsiella pneumoniae and Staphylococcus aureus infection were observed in patients with severe COVID-19. The cause of this predisposition to infection is unclear. Our data demonstrate consumption of complement in acute COVID-19 patients reflected by low levels of C3, C4, and loss of haemolytic activity. Given that the elimination of Gram-negative bacteria depends in part on complement-mediated lysis, we hypothesised that secondary hypocomplementaemia is rendering the antibody-dependent classical pathway activation inactive and compromises serum bactericidal activity (SBA). 217 patients with severe COVID-19 were studied. 142 patients suffered secondary bacterial infections. Klebsiella species were the most common Gram-negative organism, found in 58 patients, while S. aureus was the dominant Gram-positive organism found in 22 patients. Hypocomplementaemia was observed in patients with acute severe COVID-19 but not in convalescent survivors three months after discharge. Sera from patients with acute COVID-19 were unable to opsonise either K. pneumoniae or S. aureus and had impaired complement-mediated killing of Klebsiella. We conclude that hyperactivation of complement during acute COVID-19 leads to secondary hypocomplementaemia and predisposes to opportunistic infections.

Temporal regulation of Ig gene diversification revealed by single-cell imaging.

Rearranged Ig V regions undergo activation-induced cytidine deaminase (AID)-initiated diversification in sequence to produce either nontemplated or templated mutations, in the related pathways of somatic hypermutation and gene conversion. In chicken DT40 B cells, gene conversion normally predominates, producing mutations templated by adjacent pseudo-V regions, but impairment of gene conversion switches mutagenesis to a nontemplated pathway. We recently showed that the activator, E2A, functions in cis to promote diversification, and that G(1) phase of cell cycle is the critical window for E2A action. By single-cell imaging of stable AID-yellow fluorescent protein transfectants, we now demonstrate that AID-yellow fluorescent protein can stably localize to the nucleus in G(1) phase, but undergoes ubiquitin-dependent proteolysis later in cell cycle. By imaging of DT40 polymerized lactose operator-lambda(R) cells, in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that both the repair polymerase Poleta and the multifunctional factor MRE11/RAD50/NBS1 localize to lambda(R), and that lambda(R)/Poleta colocalizations occur predominately in G(1) phase, when they reflect repair of AID-initiated damage. We find no evidence of induction of gamma-H2AX, the phosphorylated variant histone that is a marker of double-strand breaks, and Ig gene conversion may therefore proceed by a pathway involving templated repair at DNA nicks rather than double-strand breaks. These results lead to a model in which Ig gene conversion initiates and is completed or nearly completed in G(1) phase. AID deaminates ssDNA, and restriction of mutagenesis to G(1) phase would contribute to protecting the genome from off-target attack by AID when DNA replication occurs in S phase.

E2A acts in cis in G1 phase of cell cycle to promote Ig gene diversification.

Rearranged Ig genes undergo diversification in sequence and structure initiated by the DNA deaminase, activation-induced deaminase. Ig genes must be transcribed for diversification to occur, but whether there are additional requirements for cis activation has not been established. Here we show, by chromatin immunoprecipitation, that the regulatory factor E2A associates with the rearranged Ig lambda(R) gene in the chicken DT40 B cell line, which performs constitutive Ig gene diversification. By analysis of a DT40 derivative in which polymerized lactose operator tags the rearranged lambda(R) gene, we show that E2A must function in cis to promote diversification and that stimulation of diversification in cis depends on the E2A activation domains. By direct imaging, we show that lambda(R)/E2A colocalizations are most prominent in G(1). We further show that expression of the E2A antagonist Id1 prevents lambda(R)/E2A colocalizations in G(1) and impairs diversification but not transcription of lambda(R). Thus, E2A acts in cis to promote Ig gene diversification, and G(1) phase is the critical window for E2A action.

Chromatin structure regulates gene conversion.

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vlambda pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vlambda donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vlambda array, and altered the outcome of Vlambda diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.

RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions.

BACKGROUND: Gene conversion depends upon the same factors that carry out more general process of homologous recombination, including homologous gene targeting and recombinational repair. Among these are the RAD51 paralogs, conserved factors related to the key recombination factor, RAD51. In chicken and other fowl, gene conversion (templated mutation) diversifies immunoglobulin variable region sequences. This allows gene conversion and recombinational repair to be studied using the chicken DT40 B cell line, which carries out constitutive gene conversion and provides a robust and physiological model for homology-directed repair in vertebrate cells. RESULTS: We show that DT40 contains constitutive nuclear foci of the repair factors RAD51D and XRCC2, consistent with activated homologous recombination. Single-cell imaging of a DT40 derivative in which the rearranged and diversifying immunoglobulin lambdaR light chain gene is tagged with polymerized lactose operator, DT40 PolyLacO-lambdaR, showed that RAD51D and XRCC2 localize to the diversifying lambdaR gene. Colocalizations correlate both functionally and physically with active immunoglobulin gene conversion. Ectopic expression of either RAD51D or XRCC2 accelerated the clonal rate of gene conversion, and conversion tracts were significantly longer in RAD51D than XRCC2 transfectants. CONCLUSION: These results demonstrate direct functions of RAD51D and XRCC2 in immunoglobulin gene conversion, and also suggest that modulation of levels of repair factors may be a useful strategy to promote gene correction in other cell types.

Antibody discovery ex vivo accelerated by the LacO/LacI regulatory network.

Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.

The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.

Targeted diversification of immunoglobulin variable regions is induced by activation-induced deaminase and may occur by either somatic hypermutation or gene conversion. MRE11-RAD50-NBS1 (MRN) is a ubiquitous and conserved nuclease complex critical for DNA break repair and is essential in class-switch recombination. Here we show that ectopic expression of NBS1, the regulatory subunit of MRN, accelerated hypermutation in the human B cell line Ramos and accelerated gene conversion in the chicken B cell line DT40. In both cases, accelerated diversification depended on MRN complex formation. These data suggest that MRN promotes DNA cleavage and/or mutagenic repair of lesions initiated by activation-induced deaminase, acting in the shared pathway of immunoglobulin gene diversification.

The effects of immunosuppressants on FAS-mediated activation-induced cell death in human T lymphocytes.

The effects of cyclosporin A (CsA) and methylprednisolone (MP) on Fas-mediated activation-induced cell death (FMAICD) of T lymphocytes were examined. T lymphocytes were activated with the immobilized anti-CD 3 and CD 28 monoclonal antibodies (MoAbs) (activation phase) and incubated further with the agonistic MoAb against Fas (death phase). Cell proliferation and DNA fragmentation were measured by XTT and diphenylamine assay. CsA in the activation phase inhibited DNA fragmentation mediated by anti-Fas MoAb but not MP. The combination of CsA and MP at the lower concentrations had little effect on FMAICD, although they had similar degrees of suppression on T lymphocyte proliferation as the maximum obtained by CsA or MP alone. In the death phase, MP induced apoptosis without 7C11 and CsA had no effects. These results indicate that the combination of CsA and MP at low concentrations could maintain FMAICD with the suppression on T lymphocyte proliferation.