MyD88 and beyond: a perspective on MyD88-targeted therapeutic approach for modulation of host immunity

MyD88 was originally known to be involved in signal transduction from TLR and IL-1R family members with exception of TLR3 [6, 17, 20, 29, 30]. In 2006, Sun and Ding reported that MyD88 is also involved in transmitting signals induced by interferon-γ (IFN-γ) in macrophages and demonstrated that MyD88 deficiency results in diminished production of TNF-α- and IFN-γ-inducible protein 10 (IP-10) treated with IFN-γ [31]. These results demonstrated that the involvement of MyD88 goes beyond the signal transduction process other than TLR-IL-1R family members. Subsequently, Liu et al. reported that the deficiency in MHC class II resulted in impaired TLR triggered production of pro-inflammatory cytokines and protected mice from an otherwise lethal challenge with TLR ligands and live Gram-negative bacteria [32]. This study also concluded that both the TLR- and MHC-mediated responses engage MyD88 [33]. MHC class II molecules are known to activate various cellular functions in immune and non-immune cells when cross-linked by antibody or superantigens [34‐36]. Staphylococcal enterotoxins (SEs) including SEB also known as superantigens, upon exposure through non-enteric route such as inhalation, cause a life-threatening toxic shock syndrome (TSS). The profound clinical consequences of SEB-induced TSS often leads to organ failure and death which are due to an excessive pro-inflammatory cytokine response such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) [37, 38]. SEB binding to MHC class II molecules on antigen-presenting cells and cross-linking to T cell receptors triggered the event. The results from our laboratory demonstrated that MyD88 gene–knockout mice (MyD88^−/−) were resistant to staphylococcal enterotoxin A (SEA) [39] or SEB-induced toxic shock [40] and showed a reduced level of pro-inflammatory cytokines in serum. In contrast, the potent cytokine response of wild-type mice was significant and lethal. It has also been demonstrated that SEA or SEB exposure activated MHC class II-linked MyD88-mediated pro-inflammatory cytokine signaling in human monocytes [41]. In human primary cells, MyD88 gene silencing using siRNA followed by SEB or SEB plus LPS stimulation results in decreased transcriptional activation of MyD88 and lower expression of IL-1β [42]. These data validate that in addition to TLR-IL-1R family of receptors, MyD88 is also an essential signaling component engaged in SEB-induced pro-inflammatory cytokine responses.

While recruitment of MyD88 is a prerequisite for inflammatory signaling and convergence point of multiple pro-inflammatory cytokines, dysregulation or overstimulation leads to inflammation-associated syndromes with severe pathological consequences to host; thus, MyD88 appears to be a unique target for therapeutic intervention of severe pro-inflammatory cytokine signaling. It has been shown that the BB-loop region acts as the mediator of the homo (adaptor-adaptor)- and hetero (receptor-adaptor)dimerization that is necessary for the function of TIR domains to induce MyD88-mediated signaling [6, 17, 43]. Bartfai et al. showed that a synthetic molecule, hydrocinnamoyl-L-valyl-pyrrolidine, (AS1), mimicking the BB-loop of the TIR domain, representing consensus the amino acid sequence of RDVLPGT (aa196-202) disrupted TLR/IL-1R signaling as shown in Fig. 2 [44]. The mimetic blocked IL-1 signaling by disrupting MyD88 and IL-1R association and reduced fever associated with inflammation in mice. By application of structure-based approach a small molecule compound1 mimicking the BB-loop of TIR domain of MyD88 was initially designed which did interfere MyD88-mediated signaling (Fig. 2) [42]. Later, a modified dimeric compound EM-163 synthesized from compound1 joined by an aromatic benzene ring and found to be more effective than compound I which attenuated TNF-α, IFN-γ, IL-6, IL-2, and IL-1β in human primary cells with exposure to SEB. Pretreatment of EM-163 completely abrogated TNF-α, IFN-γ, IL-6, IL-12p70, and IL-1β and protected BALB/c mice from toxic shock–induced deaths from lethal SEB challenge and remained healthy [45]. EM-163 treatment also protected mice from post-exposure to SEB. Also, pretreatment of EM-163 to C57BL/6 mice also completely abrogated TNF-α, IFN-γ, IL-6, IL-12p70, and IL-1β responses and protected from SEA lethal challenge. Furthermore, modification of EM-163 was made utilizing compound 1 [42, 46] by covalent linkage using nonpolar cyclohexane [47, 48]. Dimeric compound 4210 was synthesized in which two modules of compound 1 were covalently linked by a non-polar cyclohexane ring so that a six-member heterocycle ring will increase flexibility for binding to the exposed BB-loop of the TIR domain to interact with the target domain of MyD88. Compound 4210 strongly inhibited the production of pro-inflammatory cytokines in human primary cells to SEB or LPS extracted from Francisella tularensis, Escherichia coli, or Burkholderia mallei. Consistent with cytokine inhibition, in a ligand-induced cell-based reporter assay, compound 4210 inhibited B. mallei or LPS-induced MyD88-mediated NF-kB-dependent expression of reporter activity. Furthermore, results from a newly expressed MyD88 revealed that dimeric compound 4210 inhibits MyD88 dimer formation which is critical for pro-inflammatory signaling and a single administration of compound 4210 in mice showed complete protection from lethal toxin challenge. BB-loop mimetic inhibited homodimerization of MyD88 through TIR domain interaction and blocked MyD88-mediated signaling [47]. In addition to compound 4210, tri-peptide derivative compound 7, synthesized to mimic a key BB‐loop region involved in (TIR) domain interactions, was a potent, stable, and drug‐like small molecule and was shown to attenuate pro-inflammatory cytokines in human peripheral blood mononuclear cells and bronchial epithelial cells challenged with a live vaccine strain of F. tularensis [49]. It appears that small molecules which target TIR domain interactions in MyD88‐dependent TLR signaling represent a promising strategy toward host‐directed therapeutics against infection‐induced inflammation-associated sepsis.

Besides, BB-loop mimetic using an additional alternative approach by building a protein–protein dimeric docking model of the TIR-domain of MyD88, Olson et al. identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to identifying a molecule T5910047 that inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures [50]. Subsequently, based on the structure similarity, compound T6167923 was identified from the PubChem database which was found to be more potent with improved drug-like properties and capable of inhibiting MyD88 homodimer formation critical for the MyD88-mediated pro-inflammatory signaling and completely protected mice from toxic shock–induced death [50, 51].

Mario M et al. reported a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a hepta-peptide in the BB-loop of the MyD88-TIR domain, which inhibited IL-1R/TLR signaling by interfering with MyD88 homodimerization of the TIR domains and did not affect homodimerization of the death domains. Oral administration of ST2825 dose-dependently inhibited IL-1beta-induced production of IL-6 in treated mice [52]. ST2825 also suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling, thus, suggesting that it may have therapeutic potential in the treatment of chronic inflammatory diseases [53, 54]. Van Tassell et al. reported that pharmacologic inhibition of MyD88 in vivo attenuates pathologic left ventricular (LV) dilation and hypertrophy in a mouse model of non-reperfused acute myocardial infarction (AMI) independent of infarction in mouse. Data suggest that pharmacologic MyD88 inhibition protects against pathologic LV remodeling without altering infarct scar formation. The report suggests that MyD88 may be a viable target for pharmacologic inhibition in AMI [54].

A spontaneous and sustained activation of MyD88-mediated via NF-κB signaling is associated with inflammation-induced cancer. Xie et al. reported that TLR/MyD88 signaling may be a therapeutic target for colitis-associated cancer (CAC) intervention and designed a MyD88 inhibitor TJ-M2010-5, which was shown to bind to the TIR domain of MyD88 to interfere with its homo-dimerization, and the TLR/MyD88 signal pathway. In a mouse model of azoxymethane/dextran sodium sulfate (AOM/DSS)—induced (CAC) in combination with TJ-M2010-5 administration, the anti-inflammation-related cancer effect of MyD88 inhibitor was investigated in vivo, and MyD88 inhibitors may be a promising therapeutic modality for treating patients with CAC [55].

A mutation associated with nearly 1/3 of human diffuse large B cell lymphomas (DLBCLs) has been identified within MyD88 DLBCLs bearing MyD88L265P. This mutation correlates with tumor cell proliferation and survival involving spontaneous and sustained activation of NF-κB signaling. Protein–protein docking model and in silico identified MyD88 inhibitor derived small molecule compound T6167923 showed inhibition MyD88 dimer formation and were able to inhibit cell proliferation of DLBCLs bearing the MyD88L265P as measured by iso-thermal shift assay. Future studies defining the molecular mechanism of this mutation with additional human patient tumor isolates will inform and propel development of novel therapeutics to counteract both inflammation as well as tumor formation [56].

Chronic obstructive pulmonary disease (COPD) is characterized by emphysema, small airway remodeling, pulmonary hypertension, and chronic bronchitis. Treatment with corticoids has no effects on COPD. Acute exacerbations triggered by infections play an important pathogenic role in COPD. Recent pandemic with COVID-19 disease associated with SARS-CoV-2 infections causes respiratory failure with severe lung inflammation. Thus, there is a clear unmet medical need that requires effective and safe anti-inflammatory or disease-modifying therapies for these critical conditions which are lacking. The critical role of MyD88 in pro-inflammatory signaling associated with severe inflammation especially in chronic lung diseases such as chronic obstructive pulmonary disease (COPD) is summarized in a recent review [51]. The discovery and development of pharmacological inhibitors of MyD88 signaling suggest MyD88 as a drug target to treat respiratory diseases, which may represent a significant therapeutic progress [51]. The proof of concept that therapeutic targeting of MyD88 may be feasible and first preclinical data are highly promising and open a great opportunity to treat exacerbations of COPD and other chronic respiratory diseases. However, extensive preclinical investigations and risk analyses are required with careful evaluation of reduced host resistance and opportunistic infections. In regard to the COVID-19 disease, the role of MyD88 has yet to be undetermined, although it is known that crushing pro-inflammatory cytokine storm is the main reason for lung pathology. Thus, it has been demonstrated that inhibition of MyD88 led to limit pro-inflammatory cytokines like TNF-α and IL-1β [47]. Since MyD88 is a critical signaling adaptor protein, at the convergence of multiple pro-inflammatory pathways involve in inducing host innate immunity, though speculative, pharmacologic inhibition of MyD88 in TIR blockage may likely provide limiting pro-inflammatory cytokines and possibly alleviate lung-associated damage.

Since the discovery of MyD88, a considerable progress has been made on the understanding of MyD88-linked antiviral type I IFN response and other pro-inflammatory cytokine responses with many viral and bacterial infections including its spatiotemporal regulation and function. The consequence of early strong and effective innate immune responses largely leads to overall host immunity including activation of various immune cells. The balance between host immune control and viral immune evasion is pivotal to viral pathogenesis. An unbalanced immune response, characterized by a weak production of type I IFNs and an intensified release of unbalanced pro-inflammatory cytokines, contributes to the severe forms of the many viral diseases including the current COVID-19 pandemic with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection [85, 86]. The modest IFN response could explain why viremia peaks at early stages of the disease, at the time of symptoms appearance, and not around 7 to 10 days following symptoms, like during SARS-CoV and MERS-CoV infections. Also markedly elevated plasma levels of pro-inflammatory cytokines including IL-6 and chemokines have been detected in patients with COVID-19 [87, 88], associated with severe pathology and impaired lung functions. While it is known that dysregulation/over-activation of MyD88 contributes to exacerbated inflammatory response, the so-called cytokine storm, and in patients with SARS-CoV-2 infections, provides evidence that SARS-CoV-2 pathogenesis is at least partially controlled by innate immune signaling, although it is yet undetermined whether upregulation of MyD88 is linked with impaired type IFN response with SARS-CoV-2 infection. Recent research highlights that key components of innate immune signaling pathways including host factor such as MyD88 regulate exacerbated inflammatory response [83]. It has also been reported that with many virus infections, upregulation of MyD88 is associated with the impairment type IFN response, and MyD88 inhibition improved type IFN response and suppressed virus replication and improved survival in a mouse model of multiple viral diseases [80, 83, 89‐91]. Generally, the balance between the host type I IFN induction and the ability of virus to spread is determined in the first few hours of virus replication. While viral proteins are known to exert an effect on the interference of type I IFN induction and evade host antiviral innate immune effector mechanism, further detailed mechanistic studies are required to determine whether the IFN antagonists are identified in SARS-CoV-2 [85]. Also, earlier studies highlight the importance of TLR adaptor signaling in generating a balanced protective immune response to highly pathogenic SARS coronavirus infections and in particular TRIF-mediated immune signaling contributes to a protective innate immune response [86]. Therefore, it is yet unknown whether upregulation of intracellular host cell factor(s) included MyD88 which is the convergence point of pro-inflammatory signaling pathways, responsible for severe pro-inflammatory cytokines while dampening the antiviral type I IFN response through sequestration of IRFs from TRIF pathways. A strong type I IFN induction at the early stage of infection is indispensable for vertebrates to control viral infections because modulation of innate immune responses in a balanced manner promotes antigen presentation and natural killer cell functions [92]. Impaired T cell response leads to lymphopenia, and functional exhaustion of CD4⁺ and CD8⁺ T cells is associated with COVID-19 [93] which can result from IFN production deficiency. IFNs are also known to be important regulators of the development of regulatory T cells (T_reg), and T_reg cell counts in patients with COVID-19 have been shown to inversely correlate with disease severity [87, 94]. While dysregulated MyD88-mediated pro-inflammatory signaling leads to cytokine storm, the production of antiviral type I IFNs is reportedly blunted; thus, targeting MyD88 in limiting the inflammatory response and allowing more TRIF-mediated immune signaling for augmenting antiviral type I IFN response would be highly desirable. Although small molecule identified with type 1 IFN–inducing properties by high-throughput screening has been reported, however, the molecular target and the mechanism-associated IFN induction is yet unknown (Fig. 3) [95]. Our laboratory demonstrated that a lead inhibitor of MyD88, compound 4210 functions as an immune modulating molecule through deactivation of MyD88, adjusts biological pathways and accelerates type I and potentially type III IFN signaling centered on the activation of IRFs for strong host antiviral IFN response [83]. Similar to compound 4210, other small-molecule inhibitors of MyD88 (blocker of TIR-domain-homodimerization of MyD88) such as T6167923 and S5 [96, 97] have also shown type I–inducing properties (unpublished data). Since imbalanced host response to SARS-CoV-2 drives the development of severity of COVID-19 disease [88], it is anticipated that pharmacologic inhibition of MyD88 would likely induce an antiviral type I IFN as well as providing TIR blockade in limiting pro-inflammatory cytokines. Thus, MyD88-targeted disease-modifying therapy tangling these two-pronged antiviral mechanisms may be a potential post-exposure therapy in tackling COVID-19 disease.

The proof-of-concept on MyD88-targeted therapeutic approach in controlling inflammatory diseases has been validated, and even first phase clinical trial against COPD was proved to be successful [51]; reviewed by Padova et al.]. Also, recent research revealed underlying mechanisms of MyD88 in the impairment of antiviral type IFN signaling through MyD88-IRF interaction which influences MyD88-independent alternate pathways of immune signaling with many virus infections. Recent report on therapeutic inhibition of MyD88 in restoring host antiviral type IFN response in MyD88-inpedendent pathways has been described in vitro with several viral infections and also in mice models of viral diseases. These encouraging data suggest that host-directed therapy in enhancing the host immune response is feasible by stimulating mechanisms that are involved in host defense, particularly target pathways that are perturbed as a consequence of pathogen exposure.

In addition, conventional antimicrobials that directly target pathogens must continue, but additional alternate approaches are also required particularly in the absence of broad-spectrum therapeutics or vaccination strategies effective for bacterial and viral infections. Development of novel host-targeted therapeutic treatment models, a complementing strategy with small molecules targeting host factors, offers a benefit for broad-spectrum host-directed therapeutic approach. Over the years, the development of such therapeutic agents targeting the host factor(s) that are critically involved in regulating the function of the immune system and/or other cellular processes are very limited. The critical barrier toward the development of such host-directed therapeutic is the identification and/or validation of appropriate therapeutic target(s) that play a critical role during a broad range of emerging/re-emerging as well as opportunistic viral/bacterial infections. In the case of targeting hyper-inflammation and imbalanced immune responses, treatment should be focused on symptomatic rather than causal, which likely reduces exacerbated tissue damage in infectious diseases. Therefore, targeting host factor critical regulating host overall immunity would likely enhance the immune response by stimulating mechanisms that are involved in host defense against the pathogen, particularly target pathways that are perturbed as a consequence of pathogen exposure and contribute to hyper-inflammation that lead to dysbalanced responses at the site of pathology [98]. In addition, anti-infectives that directly target the pathogen, adjunct therapy targeting host factors such as MyD88, a central component in immunological pathways, in the induction or adjusting host immunity will add an extra advantage. A short-term treatment with MyD88 inhibitors combined with canonical anti-infectives will provide untapped opportunities for restoring overall host immunity.

Attenuation of the MyD88 signaling pathways as an anti-inflammatory strategy is clearly beneficial. In this review, our discussion focused mostly on MyD88, and its role in the regulation of host-immune signaling pathways in the context of reducing exacerbated inflammation to enhance immunomodulation and/or balance host reactions at the site of pathology likely holds promise for the selective and symptomatic treatment of infectious diseases. Based on the available published evidence, the role of immune response to SARS-CoV-2 infection indicates that the host immune response plays an important role in controlling SARS-CoV-2 infection and the immune dysregulation can significantly modify the clinical outcomes of affected patients [99]. Recently, Hadadji et al. reported that impaired type I IFN activity (characterized by no IFN-β and low IFN-α production) and exacerbated inflammatory responses in severe COVID-19 patients [100] provide insights into the treatment of severe COVID-19 through induction/adjusting host type I IFN response. The concept of targeting a major host factor such as MyD88 affecting dual pathway directly such as exacerbated inflammation-causing lung pathology and respiratory distress and indirectly restricting antiviral type I IFN response is certainly acceptable. Modulation of host immunity to clinical outcome particularly those with pandemic potential may have a great therapeutic value in particular to COVID-19. Recent reports showed that patients treated with IFN-alpha -2b did not show evidence of a cytokine storm, one of the dangerous responses observed in some COVID patients [101]. In experimental animal model, treatment with MyD88 inhibitor increased IFN-β and reduced inflammatory cytokine response which showed promising results likely achieving these dual goals. Targeting the host factors such as MyD88 and pathways in innate immune modulation such type I IFN and also type III IFN to restrict productive replication virus and spread offers the opportunity for broad-spectrum antiviral drugs. But more in-depth academic and pharmaceutical research is required for the successful developments of inhibitors of MyD88 dimerization.

Taken together, the data presented in this review suggest that the pharmacological blockade with a short-term treatment with MyD88 inhibitors showed type I IFN induction while reducing acute exacerbation of various inflammatory cytokines. Given that overall imbalance in host immune system with inflammatory diseases including toxic shock, COPD as well as COVID-19 which is more to do with controlling inflammation, MyD88-targeted therapy would likely provide an advantage in treating severe inflammatory responses. At present, available data suggest the benefit of MyD88-targeted therapy with broad-spectrum antiviral type I IFN inducing properties; future in-depth efforts should be focused for the suitability of this approach in deploying to complex diseases like COPD and COVID-19 in limiting inflammation-associated syndrome to infection.