Complement Membrane Attack Complexes Assemble NLRP3 Inflammasomes Triggering IL-1 Activation of IFN-γ–Primed Human Endothelium

Abstract

Rationale: Complement activation contributes to multiple immune-mediated pathologies. In late allograft failure, donor-specific antibody deposits complement membrane attack complexes (MAC) on graft endothelial cells (ECs), substantially increasing their immunogenicity without causing lysis. Internalized MAC stabilize NIK (NF-κB [nuclear factor kappa-light-chain-enhancer of activated B cells]–inducing kinase) protein on Rab5+MAC+ endosomes, activating noncanonical NF-κB signaling. However, the link to increased immunogenicity is unclear. Objective: To identify mechanisms by which alloantibody and internalized MAC activate ECs to enhance their ability to increase T-cell responses. Methods and Results: In human EC cultures, internalized MAC also causes NLRP3 (NOD-like receptor family pyrin domain containing 3) translocation from endoplasmic reticulum to Rab5+MAC+NIK+ endosomes followed by endosomal NIK-dependent inflammasome assembly. Cytosolic NIK, stabilized by LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells), does not trigger inflammasome assembly, and ATP-triggered inflammasome assembly does not require NIK. IFN-γ (interferon-γ) primes EC responsiveness to MAC by increasing NLRP3, pro-caspase 1, and gasdermin D expression. NIK-activated noncanonical NF-κB signaling induces pro-IL (interleukin)-1β expression. Inflammasome processed pro-IL-1β, and gasdermin D results in IL-1β secretion that increases EC immunogenicity through IL-1 receptor signaling. Activation of human ECs lining human coronary artery grafts in immunodeficient mouse hosts by alloantibody and complement similarly depends on assembly of an NLRP3 inflammasome. Finally, in renal allograft biopsies showing chronic rejection, caspase-1 is activated in C4d + ECs of interstitial microvessels, supporting the relevance of the cell culture findings. Conclusions: In response to antibody-mediated complement activation, IFN-γ-primed human ECs internalize MAC, triggering both endosomal-associated NIK-dependent NLRP3 inflammasome assembly and IL-1 synthesis, resulting in autocrine/paracrine IL-1β-mediated increases in EC immunogenicity. Similar responses may underlie other complement-mediated pathologies.

Complement-activated interferon-γ–primed human endothelium transpresents interleukin-15 to CD8+ T cells

“…To determine whether IL-1 signaling was acting on ECs or T cells, we performed siRNA knockdown of p65 in ECs to inhibit their responses to IL-1β, and added exogenous IL-1β to EC/CD8 + Tem cell cocultures. We observed that siRNA knockdown of p65 significantly attenuated the IFN-γ production by CD8 + Tem cells in PBS, following which either PRA or control IgGsera injection (1 mouse per group of 4 for each combination) and grafts were harvested 24 hours later (12). We found that PRA induced both mouse complement deposition and inflammasome assembly in the EC lining, and that inflammasome assembly could be blocked by MCC950.…”

“…Viral transduction of ECs. Retroviral vector encoding mCherry-Rab5WT was generated, as described previously, by cutting mCherry-Rab5WT vector (Gia Voeltz, University of Colorado at Boulder, Boulder, Colorado, USA; Addgene plasmid 49201) at the BamH1 and Not1 sites and ligated into the retroviral expression vector pLZRSpBMN-Z (gift from Garry Nolan, Stanford University, Stanford, California, USA) (12). Retroviral vector encoding mCherry-Rab5DN was generated by using PCR and primer sequence GCGGCCGCTCAGT-TACTACAACACTGATT to introduce a 3′ Not1 restriction site using the mCherry-Rab5DN (S34N) vector (Sergio Grinstein, University of Toronto, Toronto, Ontario, Canada; Addgene plasmid 35139) as template and the insert was cut with BamH1 and Not1 and then ligated into pLZRSpBMN-Z.…”

“…These data collectively indicate that after IFN-γ upregulation of nuclear IL-15/ IL-15Rα complexes and upon PRA and complement activation, IL-15/IL-15Rα complexes translocate from the nucleus to the cell surface where they are available for transpresentation. complement activation and MAC signaling on human ECs, as would be expected with increased antibody binding (12). However, the actions of IFN-γ not only increased expression of MHC molecules, our rationale for using this pretreatment, but also induced expression of NLRP3, pro-caspase-1, and gasdermin D, actions that prime human ECs for NLRP3 inflammasome formation.…”

“…Endosomes containing internalized MACs recruit and stabilize NF-κB-inducing kinase (NIK), preventing its TRAF3-mediated polyubiquitinylation and rapid proteosomal degradation. Endosomal NIK activates 2 distinct responses: noncanonical NF-κB signaling to synthesize pro-IL-1β and assembly of an NLRP3 inflammasome that processes and secretes mature IL-1β (12). IFN-γ primes ECs to assemble NLRP3 inflammasomes and secrete IL-1 in response to MACs by increasing NLRP3, pro-caspase 1, and gasdermin D expression (12).…”

“…Endosomal NIK activates 2 distinct responses: noncanonical NF-κB signaling to synthesize pro-IL-1β and assembly of an NLRP3 inflammasome that processes and secretes mature IL-1β (12). IFN-γ primes ECs to assemble NLRP3 inflammasomes and secrete IL-1 in response to MACs by increasing NLRP3, pro-caspase 1, and gasdermin D expression (12). IL-1β feeds back on the ECs to induce proinflammatory genes through a canonical NF-κB pathway that increases the capacity of the ECs to activate alloreactive CD4 + Tem cells.…”