The linear ubiquitin assembly complex (LUBAC) is a multi-protein E3 ubiquitin ligase complex composed of two stabilizing proteins, the Heme-Oxidized Iron responsive element binding protein 2 ubiquitin Ligase-1L (HOIL-1L) and Shank-Associated RH domain-Interacting Protein (SHARPIN), and a catalytic component, HOIL-1-Interacting Protein (HOIP)32–34 (Fig. 3). The proteins within the heteromeric complex contain multiple domains for interactions within the complex, ubiquitin binding, as well as catalytic activity.32–36 LUBAC is an essential regulator of NF-κB activation and has been shown to act as a molecular rheostat, regulating the amplitude of the epithelial-driven inflammatory response dung IAV infection.35,36

Fig. 3.

LUBAC is necessary for NF-κB dependent inflammation

LUBAC covalently attaches linear ubiquitin chains to NEMO, which facilitates the recruitment of additional IKK complexes. Stably docked IKK complexes result in the efficient transautophosphorylation and activation of proximal IKKα/β, followed by the phosphorylation and degradation of IκBα. NF-κB translocates to the nucleus to stimulate transcription of inflammatory genes.

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The respiratory epithelium actively participates in the first line of defense against pathogens by orchestrating host innate immunity.23,37–39 As IAV replicates within the respiratory epithelium cells, the cytosolic pattern recognition receptor (PRR), RIG-I, is activated and initiates formation of a signaling platform to which LUBAC is recruited. LUBAC covalently attaches Met-1 linked linear ubiquitin chains to the NF-κB essential modulator (NEMO), a component of the inhibitor of NF-κB (IκB) kinase (IKK) complex along with IKKα and IKKβ.40,41 Due to the high affinity of NEMO's ubiquitin binding domain for linear chains, linear ubiquitination of NEMO facilitates the recruitment of additional IKK complexes, which results in the efficient trans-autophosphorylation and activation of proximal IKKβ followed by the phosphorylation and degradation of IκBα and robust NF-kB activation (Fig. 3).34,35,40,42,43

Recent reports show that destabilization of respiratory epithelial LUBAC, via loss of the non-catalytic component HOIL-1L, dampens the host response during severe influenza and promotes survival with reduced lung injury as well as reduced viral titers. However, when LUBAC activity is abolished through deletion of HOIP, the alveolar epithelia driven inflammatory response is inhibited and mortality is increased. These findings highlight the fine line between an excessive and an inadequate immune response and suggest that therapeutic modulation of LUBAC activity may be crucial, as it functions as a rheostat regulating the amplitude of the host response to IAV infection.

LUBAC covalently attaches linear ubiquitin chains to NEMO, which facilitates the recruitment of additional IKK complexes. Stably docked IKK complexes result in the efficient transautophosphorylation and activation of proximal IKKα/β, followed by the phosphorylation and degradation of IκBα. NF-κB translocates to the nucleus to stimulate transcription of inflammatory genes.

Current anti-influenza strategies are limited to yearly vaccination or administration of antiviral drugs, however, short therapeutic windows, viral mutation, and resistance to current therapies limit their effectiveness. Despite available vaccination and anti-viral drugs, the most recent pandemic in 2009 resulted in an estimated 151,700–575,400 deaths in its first year of circulation worldwide.44 The pandemic strain contained a novel assortment of viral genes not previously identified in animal or human populations. From its first detection in April 2009, it was only 3 months until resistance to anti-viral drugs was reported, and it took an additional 3 months before the first vaccine offering protection from the pandemic strain was administered.45 In addition to emerging pandemic strains, between 291,000 and 646,000 people worldwide die from seasonal influenza-related respiratory illnesses each year.46 Novel mutations and reassortments of the virus will inevitably lead to the next IAV pandemic; therefore, the use and development of therapeutics that target conserved host pathways, rather than the virus itself, hold promise to curtail the impact of viral infection. Moreover, a heterogeneous response to IAV with the same virulence exists within the population, suggesting that host factors play a crucial role regulating the host response and determining the severity of lung injury.2,3,47 Additionally, experimental evidence from human studies and animal models of severe IAV show that viral titers do not always correlate with severity of disease, but rather ARDS induced “cytokine storm” is the major driver of morbidity and mortality.4,28,38,48

Severe IAV infection is associated with inflammatory cytokines in humans and mice. Due to their pleiotropic and redundant effects, targeting of individual cytokines may not be a suitable approach to reduce pathology during IAV infection. Instead, dampening of the immune response may be more effective, as was the case with LUBAC destabilization noted above.36 FDA-approved anti-inflammatory drugs, including corticosteroids and statins, have been proposed for the treatment of “cytokine storm” associated with severe IAV infection.49 Moreover, current data regarding their efficacy is limited to mouse models and retrospective patient observations.49,50 Corticosteroids have been shown to be effective in limiting the inflammation in some lung pathologies.49–51 However, observational studies of the impact of corticosteroid treatment of IAV-infected patients suggest against their use; with administration associated with higher incidence of hospital-acquired pneumonia, longer duration of mechanical ventilation, and increased mortality.50 Similarly, clinical evidence does not support corticosteroid treatment for COVID-19 lung injury.52 Statins are another class of drugs recognized for their ability to dampen inflammation.49 While experimental evidence from mouse models using statins during IAV infection have been inconclusive regarding their benefit,49,53,54 retrospective analysis of patient data suggests an association between statin treatment and lower IAV mortality rates.55 These examples highlight the need for new avenues of drug discovery and validations, as no currently available immune modulators have convincingly demonstrated their ability to improve outcomes during influenza infection.

LUBAC represents a potential new target for limiting the pathological inflammation that occurs during IAV infection. Several chemical inhibitors as well as peptides that bind HOIP have been used to inhibit LUBAC activity in cell culture56–59 and in vitro assays60,61 and support the specific targetablility of LUBAC (Fig. 4). Currently, LUBAC inhibitors fall into two categories: those that target the catalytic activity of HOIP (i.e. BAY117082, Gliotoxin, HOIPIN) or those that disrupt the interaction between LUBAC components to destabilize the complex (i.e. stapled peptides). BAY117082, a small molecule commonly used as an inhibitor of NF-κB activation. It has been observed that treatment of RAW 264.7 macrophages with BAY117082 prevented IL-1 stimulated formation of linear ubiquitin chains. Further investigation revealed that BAY117082 irreversibly inhibits LUBAC through a chemical reaction with cysteine residues in the active site of HOIP, the catalytic unit of LUBAC.58 While BAY117082 represents a potent inhibitor of LUBAC activity, it targets multiple components of the ubiquitin system, including inhibition of E2 ubiquitin conjugating enzymes, and possible proteasome inhibition.58 As such, use of BAY117082 is not suitable for the study of LUBAC-dependent physiological functions or therapeutic targeting of LUBAC activity in disease. Gliotoxin, a fungal metabolite, was identified in a high-throughput screening for LUBAC inhibitors using a time-resolved FRET-based screening system. While gliotoxin is known to have multiple cellular targets, it is able to inhibit LUBAC activity and downstream activation of NF-κB at 10x lower concentrations.61 Gliotoxin's strong, irreversible binding to the catalytic site of HOIP makes it a selective inhibitor of LUBAC activity.61 Interestingly, the potency of gliotoxin has been shown to vary between cell types, with myeloid and lymphoid cells being more sensitive to gliotoxin-mediated NF-κB inhibition than epithelial cells.61,62 However, this irreversible inhibition may quench the inflammatory response and increase susceptibility to secondary infections. HOIPINs are synthetic small molecules that reversibly inhibit LUBAC though targeting of HOIP activity, displaying both LUBAC specificity as well as low cytotoxicity. Several derivatives have been made with varying degrees of efficacy (HOIPIN-1-8), with HOIPIN-8 showing significantly enhanced ability to prevent LUBAC-mediated NF-κB activation in response to TNF-α without cytotoxicity compared to the other derivatives in vitro.57 Conversely, stapled α-helical peptides developed based on specific LUBAC structures disrupt interactions necessary for stable complex formation.56,63 Stapled peptides are a class of synthetic macrocycles where the secondary α-helix structure is stabilized by the introduction of a hydrophobic bridge or “staple” that rigidifies specific areas to inhibit protein:protein interactions.56 Stapled peptides based on the HOIP ubiquitin binding domain have been shown to successfully inhibit LUBAC activity in vitro by disrupting its interaction with HOIL-1L and destabilizing the overall complex.56,63 While several inhibitors of LUBAC have been developed and shown promise in vitro, no data is available detailing their efficacy in vivo. Further investigation in to these compounds which target LUBAC stability to modulate the degree of LUBAC activity is warranted as they may be therapeutically beneficial for the treatment of hyper-inflammatory response during viral infection, where a graded host response is necessary.

Fig. 4.

Schematic representation of the small molecule inhibitors (BAY117082, Gliotoxin, HOIPIN1-8) that target the catalytic domain of HOIP and the stapled α-helical peptides which disrupt the interaction between LUBAC components to destabilize the complex.

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