Classical Pathway Stimulus in Complement-Mediated Thrombotic Microangiopathy

Medicine Life Sciences Hematology Immunology
Complement-mediated thrombotic microangiopathy Classical pathway IgM Complement biosensors Diagnosis Therapeutic monitoring Complement inhibition

Utilizing Complement Biosensors to Explore Novel Mechanisms in Complement-Mediated Thrombotic Microangiopathy

Background and Necessity

Complement-mediated thrombotic microangiopathy (CM-TMA) is a thrombotic microangiopathy caused by complement system dysregulation, with complex clinical manifestations including subtypes such as atypical hemolytic uremic syndrome (aHUS). Research indicates that approximately 40-50% of CM-TMA patients do not carry driving mutations or specific autoantibodies in complement pathways, necessitating further exploration of its pathological mechanisms. The main treatment approach for CM-TMA involves using C5 inhibitors like eculizumab to inhibit the terminal complement pathway. While this strategy has shown efficacy, some patients continue to exhibit abnormal complement activity, with clinical relapses or incomplete remission, raising interest in its diverse etiology.

The complement system is a key component of the innate immune system. Traditionally, CM-TMA is thought to be primarily driven by dysregulation of the alternative pathway (AP), leaving the role of the classical pathway (CP) in CM-TMA underexplored. Current in vitro diagnostic methods, including the modified Ham test (mHam) and human microvascular endothelial cell (HMEC-1) deposition assays, can detect complement abnormalities but have limited diagnostic sensitivity and clinical utility. Developing more precise tools and exploring unknown pathological mechanisms are critical goals for advancing CM-TMA research.

Publication Source

This study, conducted collaboratively by researchers from Johns Hopkins University and Cleveland Clinic, includes key authors such as Michael A. Cole, Nikhil Ranjan, and Gloria F. Gerber. Published in the December 12, 2024 issue of the journal Blood, the research builds on a long-standing focus on complement-related diseases to investigate the role of the classical complement pathway in CM-TMA.

Methods and Experimental Framework

The study employs an autonomously bioluminescent HEK293 cell line as a complement biosensor, combined with CRISPR gene-editing technology, to create a model for studying the disease mechanisms, analyzing patient sera, and evaluating the effectiveness of multiple complement inhibitors. The study is structured as follows:

1. Creation and Validation of Complement Biosensors

  • Autonomous bioluminescent HEK293 cells (LiveLight HEK293) were used as the foundation.
  • CRISPR technology was employed to individually delete three membrane-bound complement regulatory proteins: CD55 (DAF), CD59, and CD46 (MCP). This resulted in PIGA knockout cells (PIGAKO), CD46 knockout cells (CD46KO), and a triple-knockout cell line (DKO).
  • Real-time monitoring of cellular metabolic health assessed complement activation levels based on the bioluminescence signal.

2. Patient Serum Collection and Grouping

  • The study analyzed 41 serum samples from the Johns Hopkins Complement Registry, including acute CM-TMA patients, remission samples, six acute TTP (thrombotic thrombocytopenic purpura) patients, and 19 healthy controls.
  • Samples were categorized into acute and remission phases. Acute samples were collected within 14 days of initial clinical manifestation, with no prior complement inhibitor treatment.

3. Experimental Design and Detection of Complement Activation

  • Real-time bioluminescent mHam test: Cells were incubated with patient sera, with classical pathway (Sutimlimab) and alternative pathway (Ach-5548, etc.) inhibitors added, to dynamically measure complement activity.
  • Flow cytometry: After serum incubation, C3c and C4d complements deposited on cell surfaces were measured to evaluate the relative contributions of the CP and AP.
  • Immunoglobulin modification experiments: Selective reduction of IgM with dithiothreitol (DTT) or cleavage of IgG with IgG-degrading enzyme (IDES) was used to explore the immune drivers of the disease.

Results

1. Classical Pathway-Driven Complement Activation

This study is the first to identify significant IgM-mediated CP activation in CM-TMA, particularly during acute disease states. Real-time bioluminescent tests demonstrated: - Complement activity on CD46KO and PIGAKO cells was markedly higher in acute patients compared to healthy controls (p < 0.01). - Sutimlimab (C1s inhibitor) provided robust protection against complement-mediated cytotoxicity, while AP inhibitors like Ach-5548 showed limited efficacy.

2. IgM’s Central Role in Classical Pathway Activation

Using DTT reduction and IDES cleavage experiments, IgM was confirmed as the primary surface activator of the CP, with IgG playing a secondary role: - DTT pretreatment reduced complement activity by about 85%, while IDES-treated serum did not exhibit similar effects. - Adding IgM isolated from patient serum to healthy control serum reproduced complement activation characteristics consistent with CM-TMA, underscoring IgM’s pathogenicity.

3. Amplification via the Alternative Pathway

While the CP serves as a primary trigger, the AP contributes to complement activity via amplification. However, “triple alternative pathway blockade” (inhibiting factors B, D, and C3) failed to fully suppress complement enzymatic reactions in some patients, confirming the dominant role of CP activation.

4. Therapeutic Monitoring via Biosensors

The study demonstrated the potential use of the bioluminescent mHam test to monitor the efficacy of complement inhibitors. Persistent complement activity in some patients indicated the need for higher doses or combined inhibition of the CP.

Significance and Innovations

1. Challenging Traditional Understanding

Previously, research focused on AP dysregulation. This study demonstrates the significant role of the CP in CM-TMA, providing a new explanation for cases without pathogenic mutations.

2. Diagnostic and Therapeutic Applications

The novel complement biosensor displayed high sensitivity in differentiating CM-TMA from TTP. Its use in tracking treatment response can aid in optimizing doses of C5 or C1s inhibitors.

3. Disruption of IgM Immunologic Tolerance

The study suggests polyreactive or autoreactive IgM as a key immunopathological feature of CM-TMA, offering new insights for studying other complement-related autoimmune disorders.

Summary and Future Directions

Through innovative complement biosensors and comprehensive experimental validation, this study advances the understanding of CM-TMA’s pathogenesis, refining diagnostics and therapeutic strategies. Larger cohort studies are needed for further validation and to correlate ongoing complement activity with clinical outcomes. Exploring the role of CP activation in other complement-related diseases remains a promising avenue for future research. Ultimately, this study lays a solid foundation for achieving precision medicine in CM-TMA diagnosis and treatment.