Dual inhibition of complement and Toll-like receptors as a novel approach to treat inflammatory diseases-C3 or C5 emerge together with CD14 as promising targets

Abstract

The host is protected by pattern recognition systems, including complement and TLRs, which are closely cross-talking. If improperly activated, these systems might induce tissue damage and disease. Inhibition of single downstream proinflammatory cytokines, such as TNF, IL-1β, and IL-6, have failed in clinical sepsis trials, which might not be unexpected, given the substantial amounts of mediators involved in the pathogenesis of this condition. Instead, we have put forward a hypothesis of inhibition at the recognition phase by "dual blockade" of bottleneck molecules of complement and TLRs. By acting upstream and broadly, the dual blockade could be beneficial in conditions with improper or uncontrolled innate immune activation threatening the host. Key bottleneck molecules in these systems that could be targets for inhibition are the central complement molecules C3 and C5 and the important CD14 molecule, which is a coreceptor for several TLRs, including TLR4 and TLR2. This review summarizes current knowledge of inhibition of complement and TLRs alone and in combination, in both sterile and nonsterile inflammatory processes, where activation of these systems is of crucial importance for tissue damage and disease. Thus, dual blockade might provide a general, broad-acting therapeutic regimen against a number of diseases where innate immunity is improperly activated.

Keywords: inflammation; innate immunity; therapy.

Figure 1.. The complement system.

The complement system can be activated through 3 pathways (top), all converging to the cleavage of C3 to generate C3a and C3b (middle). The classic pathway (CP) is typically activated by antibodies, but also, pentraxins (PTX), including C-reactive protein (CRP), serum amyloid P component (SAP), and PTX3 can activate C1q. The lectin pathway (LP) is activated through recognition of carbohydrates by mannose-binding lectin (MBL), ficolins, and collectins. Furthermore, LP activation may be mediated through IgM antibodies, e.g., directed against damaged self-antigens. The alternative pathway (AP) is activated by foreign or damaged own cells, facilitated by the continuous spontaneous hydrolysis of C3. AP also has an important function in the complement system, providing an amplification loop that enhances C3 activation independent of which pathway is initially activated. This effect is mainly a result of properdin (FP), the only positive regulator in the complement system, which stabilizes the C3 convertase. Activation of C3 leads to formation of a C5 convertase, cleaving C5 into C5a and C5b. The anaphylatoxins C3a and C5a bind to the receptors C3aR, C5aR1, and C5aR2, leading to downstream production of inflammatory mediators (bottom). C5b initiates the formation of the TCC, which forms the membrane attack complex (MAC) if inserted into a membrane (bottom). This may lead to lysis of bacteria and cells or in sublytic doses to activation of cells. The cleavage and inactivation of C3b generate iC3b, bind to complement receptors 3 (CR3; CD11b/CD18) and 4 (CR4; CD11c/CD18), facilitating phagocytosis, oxidative burst, and downstream inflammation (right). The complement system is tightly regulated by soluble inhibitors (yellow), including C1 inhibitor (C1-INH), factor H (FH), factor I (FI), C4-binding protein (C4BP), anaphylatoxin inhibitor (AI) inactivating the anafylatoxins (e.g., C5a to C5adesArg), vitronectin (Vn), and clusterin (Cl), keeping the continuous low-grade activation in the fluid phase in check. Host cell membranes are equipped with a number of inhibitors to protect them against attack by complement (right), including membrane cofactor protein (MCP; CD46), CR1 (CD35), decay accelerating factor (DAF; CD55), controlling C4 and C3 activation, and CD59 protecting against final assembly of the C5b-9 complex. Some attractive targets for therapeutic inhibition are indicated with black arrowheads, e.g., specific CP activation by one of the C1qrs components, specific LP activation by MBL and MASP intervention, and specific AP activation by neutralizing factor D (FD), which will attenuate the amplification of the system induced by all initial activation mechanisms. The inhibition of C3 is the broadest possible strategy, whereas inhibition of C5 cleavage will clock both the inflammatory potent C5a fragment and formation of the inflammatory and lytic C5b-9 complex. Alternatively, C5a can be inhibited, preserving the C5b-9 pathway, or the anaphylatoxin receptors can be blocked to prevent signaling. In particular, the blocking of C5aR1 will attenuate inflammation, whereas the effect of blocking C3aR and C5aR2 receptors is to be studied in more detail, as they might have more anti-inflammatory effects.

Figure 2.. The TLRs.

TLRs are transmembrane proteins recognizing conserved patterns of microbial structures, as well as damage self-molecules. Ten TLRs have been described in humans, the first 9 with defined ligands. TLR1, -2, -4, -5, and -6 are plasma membrane receptors, whereas TLR3, -7, -8, and -9 are intracellular, located to the endosomal membrane. TLR2 heterodimerizes with TLR1 or -6, whereas all others homodimerize. TLR4 is translocated from the plasma membrane, where it serves as the LPS receptor, with MD2 and CD14 as coreceptors, to the endosomal membrane. All TLRs, except TLR3, use MyD88 as one of their adaptor proteins. TLR2, -4, and -5 signal through IL-1R-associated kinases (IRAKs) and TNFR-associated factor 6 (TRAF6) to activate NF-κB to produce proinflammatory cytokines, whereas TLR7, -8, and -9 activate IRF7, and intracellular TLR4 activates IRF3 to produce IFN-α and -β. CD14 is a coreceptor for several of the TLRs. It has been known for years that TLR4 and TLR2 use CD14, but recently, CD14 has been described as a coreceptor, at least for mice, also for TLR3, -7, and -9. Some attractive targets for therapeutic inhibition are indicated with black arrowheads. Neutralization, usually using mAb, of both sCD14 and membrane-bound CD14 will inhibit LPS binding to TLR4. CD14 is a cofactor for a number of TLRs—TLR2 being the best documented—thus, CD14 acts as a potent molecule to target several TLR members. Specific TLR inhibitors, including the lipid A antagonist eritoran, blocks the TLR4/MD2 complex, and the humanized anti-TLR2 antibody prevents the dimerization of TLR2 with TLR1 and TLR6. A number of other specific inhibitors of both membrane-bound and intracellular TLRs and their signaling molecules are under development. HSP, heat shock protein; HMGB1, high mobility group box 1; MAL, MyD88 adapter-like; Pam3CSK4, palmitoyl-3-cysteine-serine-lysine-4; TBK1, TANK-binding kinase 1; IKKɛ, IκB kinase ɛ.

Figure 3.. The “double blockade” of bottleneck recognition molecules at innate immune recognition.

An upstream approach for inhibition of inflammation achieved by targeting the key complement molecules C3 or C5 and the CD14 molecule of the TLR family are proposed. Activation through all initial complement pathways converges at C3 and C5, and blocking of the bottleneck molecule C5 inhibits formation of the potent anaphylatoxin C5a, which is a main contributor in the pathogenesis of a number of disease conditions. As CD14 serves as a coreceptor for several of the TLRs, including the important TLR4 and TLR2, it might be regarded as a bottleneck molecule in the TLR family. Combined inhibition of these molecules will reduce the downstream inflammatory response substantially.