A two-decade journey in identifying high mobility group box 1 (HMGB1) and procathepsin L (pCTS-L) as potential therapeutic targets for sepsis

Abstract

Introduction: Microbial infections and resultant sepsis are leading causes of death in hospitals, representing approximately 20% of total deaths worldwide. Despite the difficulties in translating experimental insights into effective therapies for often heterogenous patient populations, an improved understanding of the pathogenic mechanisms underlying experimental sepsis is still urgently needed. Sepsis is partly attributable to dysregulated innate immune responses manifested by hyperinflammation and immunosuppression at different stages of microbial infections.

Areas covered: Here we review our recent progress in searching for late-acting mediators of experimental sepsis and propose high mobility group box 1 (HMGB1) and procathepsin-L (pCTS-L) as potential therapeutic targets for improving outcomes of lethal sepsis and other infectious diseases.

Expert opinion: It will be important to evaluate the efficacy of HMGB1- or pCTS-L-targeting agents for the clinical management of human sepsis and other infectious diseases in future studies.

Keywords: HMGB1; Innate immune cells; Late-acting mediator; Pyroptosis; Sepsis; hyperinflammation; pCTS-L.

Figure 1.. Overall summary of pathogenic roles of late-acting mediators in dysregulated innate immune responses to lethal infections.

Microbe-derived pathogen-associated molecular patterns (PAMPs such as LPS) rely on cell surface pattern recognition receptors (PRRs such as TLR4) to activate innate immune cells to immediately release “early” proinflammatory mediators (such as TNF, IL-1β, and IFN-γ), which then stimulate hepatocytes and innate immune cells to synthesize and secrete an “intermediate” mediator, SAA. SAA then activate innate immune cells to release late-acting proinflammatory mediators such HMGB1 and pCTS-L, which bind cell surface PRRs such as TLR4 and RAGE to induce: i) the expression of cytokines/chemokines to trigger hyperinflammation; and ii) the expression of pro-Casp-11 to activate inflammasome and pyroptosis. The HMGB1- and pCTS-L-mediated hyperinflammation and pyroptosis-associated immunosuppression may adversely contribute to the pathogenesis of lethal sepsis. A panel of HMGB1- and pCTS-L-neutralizing mAbs may interrupt their interactions with TLR4 and RAGE, thereby impairing HMGB1- or pCTS-L-mediated dysregulated inflammation or immunosuppression to confer protection against lethal sepsis.

Figure 2.. Divergent roles of TLR4 and RAGE in the regulation of HMGB1-mediated hyperinflammation and immunosuppression.

Extracellular HMGB1 can differentially bind distinct PRRs (such as TLR4 and RAGE) with different affinities. Consequently, HMGB1 may induce divergent inflammatory responses that include immune cell migration (chemotaxis), immune activation (hyperinflammation), or pyroptosis-associated immunosuppression.

Figure 3.. Structural domains and amino acid sequence of murine and human precathepsin L (preCTS-L), procathepsin L (pCTS-L) and cathepsin L (CTS-L).

A nascent human and murine pre-cathepsin L (preCTS-L) are synthesized as a 333 or 334 amino acid precursor with an N-terminal 16- or 17-residue signal sequence (shown in blue). The signal sequence can direct the ribosome to the rough endoplasmic reticulum (ER) membrane to initiate the translocation of the growing peptide chain across the ER membrane, during which the signal peptide is cleaved off by a signal peptidase to release the pro-enzyme, procathepsin L (pCTS-L). Consequently, pCTS-L can be secreted extracellularly through the classical ER - Golgi exocytotic pathway, or alternatively cleaved in the endosome to release the cathepsin L (CTS-L), which is further proteolytically processed to produce the active enzyme consisting of a heavy and light chain. The epitope sequence (P13, residue 194–214) for a panel of protective anti-pCTS-L monoclonal antibodies (mAb2, mAb20 and mAb26) was underlined.

Figure 4.. Schematic models of pCTS-L interaction with a neutralizing mAb20 and two pattern recognition receptors (TLR4 and RAGE).

We used the Machine Learning based antibody modelling (ABodyBuilder-ML) to predict the three-dimensional structure (IMGT model) of a protective anti-pCTS-L mAb20, and then employed the ClusPro Web Server to predict possible structures of pCTS-L/mAb or pCTS-L/receptor complexes that exhibited the least Gibbs free energy. The epitope sequence for a panel of protective mAbs was marked on pCTS-L (as P13 in purple) to illustrate its relative position within pCTS-L/mAb or the pCTS-L/PRR complexes. Note the epitope sequence (P13) for protective mAbs could be docked onto the CDR loops of a protective mAb20 (Top Panel). However, this P13 epitope sequence could be sequestered into the hydrophobic crevices of TLR4 (Middle Panel) and positioned sideways in close proximity to the V-domain of RAGE (Bottom Panel). Thus, the engagement of the P13 epitope sequence by a protective mAb may competitively prevent the co-current engagement of pCTS-L with its PRRs such as TLR4 and RAGE.

Figure 5.. Potential effects of neutralizing monoclonal antibodies on the HMGB1-induced ACE2 expression, the physical interaction of SARS-CoV-2 S protein with host ACE2 receptor, or the CTS-L-mediated cleavage of SARS-CoV-2 S protein.

Upon ACE2 engagement, SARS-CoV-2 spike (S) protein undergoes conformational changes in a target cell through proteolytic cleavage at the S2’ site either by the TMPSS2 on host cytoplasmic membrane or the CTS-L in the acidic endosomes. This cleavage of the spike protein at the S2’ site by either enzyme similarly leads to exposure of the fusion peptide to cytoplasmic or endosomal membranes, initiating the fusion between viral and host cellular membranes to form fusion pore and consequent release of viral RNA to host cell cytosol for viral replication. It is important to test whether mAbs capable of inhibiting the proinflammatory activities of HMGB1 and pCTS-L or harmful interactions between viral S protein and host ACE2 receptor could limit viral infection in preclinical and clinical settings.