MyD88-dependent signaling drives toll-like receptor-induced trained immunity

IntroductionDespite medical advances, infections frequently progress to the life-threatening clinical condition of sepsis, especially in immunocompromised patients. As evidenced by the ongoing COVID-19 pandemic, patients with advanced age or underlying co-morbidities such as chronic lung diseases, cancer, cardiovascular disease, or traumatic injury are at serious risk for life-threatening infections (1, 2). Hospital stays increase exposure to opportunistic pathogens which immunocompromised patients are unable to fight due to insufficient immune responses. Nosocomial infections affect two million patients and cause more than 90,000 deaths every year in the United States (3). Further complicating the matter, antibiotic resistance in microbes such as Pseudomonas aeruginosa and Staphylococcus aureus remains an ever-growing threat, with the Centers for Disease Control and Prevention reporting that nearly three million antibiotic resistant infections occur in the US each year (4). The development of new antibiotics is slow, with the most recent discovered in 1987, and the investment into new antibiotic research only temporarily addresses the situation given the unfortunate reality that pathogens will evolve to evade new antibiotics (4). Therefore, the development of novel protection strategies is an urgent priority, and immunomodulatory therapies hold strong potential to fill this need (5).Immune memory is classically thought to be a characteristic of the adaptive immune system, however recent reports show that the innate immune system also has the ability to develop memory of prior pathogen exposure. Evidence shows that innate leukocytes can be “trained” to mount a more robust immune response to subsequent infection, a phenomenon termed trained immunity (or innate immune training) (6). Much of the recent literature has focused on the ability of β-glucan, a component of the fungal cell wall, to induce trained immunity (7, 8). However, the capability of the Toll-like Receptor (TLR)-4 agonist lipopolysaccharide (LPS) to protect against subsequent infection was described as early as the 1950s (9). More recently, our research group has demonstrated that other TLR4 ligands, including the lipid A derivative compounds monophosphoryl lipid A (MPLA) and phosphorylated hexaacyl disaccharides (PHADs), increase leukocyte recruitment to sites of infection, protect against organ injury, and improve survival in a broad array of clinically relevant infections, and these effects extend up to two weeks (10–15). Our group has previously shown that macrophages are essential for the survival benefit conferred by MPLA; further, this protection is mediated, at least in part, by metabolic rewiring characterized by sustained augmentation of glycolysis and mitochondrial oxygen consumption capacities (13, 16).In a ligand-specific manner, activated TLRs recruit the adapter molecules myeloid differentiation primary response differentiation gene 88 (MyD88) or Toll/IL-1R (TIR) domain-containing adapter producing interferon-β (TRIF). Recruitment of MyD88 or TRIF elicits pathway-specific downstream signaling and gene transcription events, with MyD88 signaling culminating in production of pro-inflammatory mediators, and TRIF signaling resulting in production of Type I interferons. TLR4 uniquely signals through both the MyD88- and TRIF-dependent signaling cascades, but the respective contributions of these pathways in triggering trained immunity and host resistance to infection is unknown. Other investigators have shown that the efficacy of MPLA as a vaccine adjuvant is mediated through TRIF-dependent signaling (17). On the other hand, our lab has found that treatment with the MyD88-selective TLR9 agonist CpG, but not TRIF-selective TLR3 agonist Poly I:C, preserves core body temperature, increases leukocyte recruitment, and improves bacterial clearance to an acute intraperitoneal infectious challenge with Pseudomonas aeruginosa (12). In vitro analysis of antimicrobial functions of bone marrow-derived macrophages (BMDMs) showed that MPLA- and CpG-treated cells exhibited increased phagocytic and respiratory burst capacity. These data suggest that MyD88, rather than TRIF, is important for driving TLR-mediated training of innate immunity.In this study, we aimed to determine the relative contributions of MyD88- and TRIF-dependent signaling pathways and the role of macrophages in driving TLR-mediated infection resistance. Further, we sought to evaluate the role of these signaling pathways in stimulating metabolic reprogramming, a defining characteristic of trained immunity. We establish that MyD88-dependent, but not TRIF-dependent, signaling in macrophages is an essential driver of TLR-mediated trained immunity and resistance to infection. Using a combination of complimentary in vivo, in vitro, and ex vivo models, we show that macrophage metabolic rewiring is likewise MyD88-dependent and is associated with augmented antimicrobial immunity. We corroborated these findings using human monocyte-derived macrophages. These data advance current knowledge and provide insights that can be used to develop more specific TLR agonist-based immunotherapies aimed at inducing resistance to infection.ResultsMyD88, but not TRIF, activation is required for TLR-mediated protection against systemic S. aureus infectionWe previously showed that the TLR4 agonist monophosphoryl lipid A (MPLA) protects against a broad array of clinically relevant pathogens (10, 11, 13). As TLR4 triggers both MyD88- and TRIF-dependent signaling cascades, we sought to determine the relative contribution of these pathways in triggering trained immunity. Further, it was unknown whether activation of other TLRs can elicit trained immunity. To investigate these questions, wild type, MyD88-KO and TRIF-KO mice were treated with TLR agonists (20 µg i.v.) or vehicle for two consecutive days and were inoculated with S. aureus the following day (Figure 1A). Consistent with our previous findings, MPLA-treated wild type mice had significant survival benefit (80% survival) compared to vehicle-treated controls (Figure 1B). MPLA did not confer survival benefit in MyD88-KO (0% survival, (Figure 1C) but was effective in TRIF-KO mice (100% survival, (Figure 1D). The minimum lethal dose of S. aureus was adjusted for MyD88-KO mice due to increased sensitivity to infection, which resulted in similar blood bacteria loads in vehicle-treated mice from all three strains (Figure 1E). This was associated with a trend of decreased blood bacterial burden in MPLA-treated wild type and significantly decreased bacterial burden in TRIF-KO, but no difference in bacterial burden in MyD88-KO, mice.Figure 1 MYD88-activating CpG, but not TRIF-activating Poly I:C, confers resistance to S. aureus infection. (A) Mice were injected with TLR agonists (20 µg i.v.), or vehicle (Lactated Ringers) for two consecutive days prior to systemic challenge with S. aureus (i.v.). (B-D) Wild type (B), MyD88-KO (C), and TRIF-KO mice (D) were challenged with S. aureus and survival was monitored for 15 days (n=5-9/group). (E) Bacteremia was assessed 48h after infection. (F) Schematic of TLR agonist pathway specificity and downstream signaling. (G) Pathway specificity of MPLA, CpG, and Poly I:C were determined by Western blotting of protein isolated from wild type BMDMs treated for 24h with TLR agonists compared to unstimulated negative controls. (H) Survival was monitored for 15 days post-S. aureus inoculation (n = 20-21/group). (I) Bacteremia was assessed 48h after infection (n = 5-10). (J) Tissue bacterial load (CFU/gram) of lung and kidney was quantified at 72h post-infection (n = 8-10/group). (K) Mice were infected with 108 CFU S. aureus (i.v.) 1 day-post (1dp), 1 week-post (1wp) or 2 weeks-post (2wp) CpG treatment alongside vehicle-treated controls. (L) Survival was monitored for 15 days post-infection (n = 10-18/group). (M) Body weights were measured at baseline and after infection. (N, O) Bacteremia was assessed at 3h (N) and 48h (O) post-infection. Mean ± SEM are shown for body weight (M). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by a log-rank Mantel–Cox test for Kaplan–Meier survival plots or otherwise determined by ANOVA followed by Dunnett’s post-hoc multiple comparison test.Next, we treated mice with MyD88- and TRIF-pathway selective agonists prior to S. aureus infection. We chose the TLR9/MyD88-selective agonist
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