CD5L as a promising biological therapeutic for treating sepsis

Sepsis, a life-threatening condition arising from the body's overwhelming response to infection, results in multiple organ failure and high mortality rates. Current treatments focus on infection control and supportive care, lacking effective immunomodulatory strategies. This research explores CD5L, a circulating protein with reported roles in immune regulation and inflammation resolution, as a potential therapeutic agent for sepsis. CD5L's involvement in various pathologies suggests its potential as a diagnostic or prognostic marker, but its precise biological role in infection and sepsis remains unclear. The variability in reported serum CD5L levels across different studies, coupled with the inability of standard assays to differentiate between active and inactive forms, hinders a comprehensive understanding. In healthy individuals, CD5L is primarily bound to IgM, rendering it biologically inactive. However, disease states may induce dissociation, leading to the release of active CD5L. The impact of this active form is also controversial, potentially both beneficial (promoting infection control and inflammation resolution) or detrimental (aggravating disease progression). Its dual nature as a pattern recognition receptor (PRR), recognizing both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), further complicates the picture. CD5L’s reported anti-inflammatory and tissue-repairing properties also add to the complexity. Given this multifaceted role, this study aimed to clarify CD5L's involvement in sepsis using mouse models, focusing on the efficacy of recombinant CD5L (rCD5L) as a therapeutic intervention.

Existing literature highlights the inconsistent and often contradictory roles of CD5L in various disease contexts. Studies have shown CD5L to serve as a prognostic or diagnostic biomarker in diverse clinical scenarios, including infection, cancer, and autoimmune diseases. However, the lack of standardized assays to measure biologically active CD5L has led to conflicting interpretations. Most studies focus on total serum CD5L, disregarding the biologically relevant free form. The research also explores CD5L’s reported functions as a pattern recognition receptor (PRR), able to bind to both PAMPs and DAMPs, influencing phagocytosis and inflammation. While some research suggests a protective role in combating infections, others highlight its potential for exacerbating inflammation. These inconsistencies necessitate further research to clarify CD5L's functional role in infection and inflammatory diseases, particularly in sepsis, where inflammation plays a central role.

This study employed genetically modified mice and experimental models of sepsis to investigate the effects of CD5L. CD5L-deficient mice were generated using CRISPR/Cas9 gene editing technology, targeting the SRCR domain 1-encoding exon 3 of the *Cd5l* gene. These mice, along with wild-type (WT) controls, were subjected to cecal ligation and puncture (CLP), a widely used polymicrobial sepsis model, at both attenuated (medium-grade) and full-scale (high-grade) severities. To assess therapeutic potential, rCD5L was administered intraperitoneally (IP) or intravenously (IV) to WT mice after CLP induction. A parallel LPS-induced endotoxic shock model was used to examine CD5L's role in sterile inflammation. A comprehensive range of analyses was conducted, including survival studies, bacterial load quantification (CFU counts), immune cell phenotyping using flow cytometry (leukocyte recruitment, neutrophil and macrophage analysis), cytokine and chemokine profiling using multiplex assays (inflammatory response analysis), transcriptomic analysis of peritoneal cells via RNA sequencing (gene expression and pathway analysis), and histopathological examination of major organs (organ damage assessment). Finally, size-exclusion chromatography and Western blotting were used to distinguish between free and IgM-bound CD5L to assess the biological activity of the protein. Specific methodologies included ELISA for CD5L, CXCL1, LPS, AST, and creatinine quantification; RT-qPCR for gene expression analysis; immunofluorescence for HMGB1 staining; and various flow cytometry protocols for immune cell profiling and phagocytosis assays, including in vivo and in vitro phagocytosis assays using pHrodo™ Red *E. coli* BioParticles™ and cecal bacteria. Data analysis involved various statistical tests including log-rank (Mantel-Cox), Mann-Whitney, two-tailed unpaired t-tests with Welch's correction, one-way ANOVA with Dunnett's correction, two-way ANOVA with Šídák's correction, and Spearman's rank correlation. High-dimensional data analysis including t-SNE and LDA were employed to understand the complex interplay of different factors.

CD5L-deficient mice showed significantly increased susceptibility to sepsis in a medium-grade CLP model, with over 60% mortality compared to 100% survival in WT mice. These mice exhibited impaired neutrophil recruitment, elevated blood bacteremia, and higher bacterial counts in multiple organs. Transcriptomic analysis revealed a heightened pro-inflammatory transcriptional profile in CD5L-deficient mice, reflecting a loss of immune response control. Therapeutic administration of rCD5L in WT mice with high-grade CLP dramatically improved survival rates, reaching over 70% with IV administration. rCD5L treatment effectively reduced bacterial loads in several organs, especially in the blood and liver with IV administration. Moreover, rCD5L significantly lowered endotoxin and HMGB1 (DAMP) levels, demonstrating its anti-inflammatory effect. In an LPS-induced endotoxic shock model, CD5L-deficient mice displayed significantly higher mortality, highlighting CD5L's crucial role in controlling inflammation. In this model, rCD5L treatment dramatically improved survival, achieving close to 80% survival with a higher dose of rCD5L. Further mechanistic investigations revealed that rCD5L significantly increased CXCL1 levels, a chemokine known for its role in neutrophil chemotaxis and activation. Interestingly, while rCD5L did not significantly affect macrophage phagocytosis, it enhanced neutrophil phagocytosis. The IV administration of rCD5L led to both increased neutrophil recruitment and increased neutrophil activation, demonstrating enhanced efficacy compared to IP administration.

The findings strongly support the hypothesis that CD5L plays a crucial role in combating sepsis. The significantly reduced survival and impaired immune responses in CD5L-deficient mice underscore the protein's protective role. The remarkable efficacy of rCD5L in rescuing WT mice from lethal sepsis highlights its therapeutic potential. The superior performance of IV rCD5L administration suggests that targeting circulating neutrophils for activation and recruitment is crucial for effective sepsis management. The observed increase in CXCL1, a key neutrophil chemoattractant, further supports this mechanism. This study's results contradict a previous report showing that rCD5L aggravated sepsis, likely due to differing treatment timing. Administering rCD5L after the initial inflammatory burst, as opposed to concurrently, allowed the natural immune response to proceed unimpeded, while mitigating the later uncontrolled systemic inflammation. This strategy may be vital in avoiding the suppression of beneficial early immune responses. Overall, the study presents compelling evidence for the therapeutic efficacy of rCD5L in sepsis, warranting further exploration as a potential treatment for human patients. The findings suggest that the free, bioactive form of CD5L is essential for its therapeutic effect, providing insights for future drug development.

This study demonstrates the remarkable therapeutic potential of CD5L in experimental sepsis models. The significant improvement in survival rates, reduction of bacterial burden, and decrease in inflammatory mediators following rCD5L treatment highlight its efficacy. The detailed mechanistic insights into CD5L's role in neutrophil recruitment, activation, and phagocytosis provide a strong foundation for future translational research. Future studies should focus on optimizing rCD5L administration protocols and investigating the precise mechanisms of neutrophil activation and CXCL1 regulation. Clinical trials are warranted to assess the safety and efficacy of rCD5L as a novel therapeutic strategy for sepsis in humans.

This study used mouse models, which may not fully recapitulate the complexities of human sepsis. While the CLP and LPS models are widely used, they may not entirely reflect the diverse etiology and pathogenesis of human sepsis. Further research is needed to confirm these findings in larger animal models that more closely mimic human physiology and disease progression. The study primarily focused on acute sepsis; further investigation is necessary to determine rCD5L's efficacy in chronic or recurrent sepsis. Finally, while the study identified the importance of the free form of CD5L, further research is required to completely delineate the mechanisms involved in CD5L’s interaction with IgM and its influence on CD5L’s bioavailability and clinical efficacy.