Microbial pathogens induce neurodegeneration in Alzheimer’s disease mice: protection by microglial regulation

Background Neurodegeneration is considered the consequence of misfolded proteins’ deposition. Little is known about external environmental effects on the neurodegenerative process. Infectious agent-derived pathogen-associated molecular patterns (PAMPs) activate microglia, key players in neurodegenerative diseases. We hypothesized that systemic microbial pathogens may accelerate neurodegeneration in Alzheimer’s disease (AD) and that microglia play a central role in this process. Methods We examined the effect of an infectious environment and of microbial Toll-like receptor (TLR) agonists on cortical neuronal loss and on microglial phenotype in wild type versus 5xFAD transgenic mice, carrying mutated genes associated with familial AD. Results We examined the effect of a naturally bred environment on the neurodegenerative process. Earlier and accelerated cortical neuron loss occurred in 5xFAD mice housed in a natural (“dirty”) environment than in a specific-pathogen-free (SPF) environment, without increasing the burden of Amyloid deposits and microgliosis. Neuronal loss occurred in a microglia-rich cortical region but not in microglia-poor CA regions of the hippocampus. Environmental exposure had no effect on cortical neuron density in wild-type mice. To model the neurodegenerative process caused by the natural infectious environment, we injected systemically the bacterial endotoxin lipopolysaccharide (LPS), a TLR4 agonist PAMP. LPS caused cortical neuronal death in 5xFAD, but not wt mice. We used the selective retinoic acid receptor α agonist Am580 to regulate microglial activation. In primary microglia isolated from 5xFAD mice, Am580 markedly attenuated TLR agonists-induced iNOS expression, without canceling their basic immune response. Intracerebroventricular delivery of Am580 in 5xFAD mice reduced significantly the fraction of (neurotoxic) iNOS + microglia and increased the fraction of (neuroprotective) TREM2 + microglia. Furthermore, intracerebroventricular delivery of Am580 prevented neurodegeneration induced by microbial TLR agonists. Conclusions Exposure to systemic infections causes neurodegeneration in brain regions displaying amyloid pathology and high local microglia density. AD brains exhibit increased susceptibility to microbial PAMPs’ neurotoxicity, which accelerates neuronal death. Microglial modulation protects the brain from microbial TLR agonist PAMP-induced neurodegeneration.

B cells imprint adoptively transferred CD8+ T cells with enhanced tumor immunity

BackgroundAdoptive T cell transfer (ACT) therapy improves outcomes in patients with advanced malignancies, yet many individuals relapse due to the infusion of T cells with poor function or persistence. Toll-like receptor (TLR) agonists can invigorate antitumor T cell responses when administered directly to patients, but these responses often coincide with toxicities. We posited that TLR agonists could be repurposed ex vivo to condition T cells with remarkable potency in vivo, circumventing TLR-related toxicity.MethodsIn this study we investigated how tumor-specific murine CD8+ T cells and human tumor infiltrating lymphocytes (TILs) are impacted when expanded ex vivo with the TLR9 agonist CpG.ResultsHerein we reveal a new way to reverse the tolerant state of adoptively transferred CD8+ T cells against tumors using TLR-activated B cells. We repurposed the TLR9 agonist, CpG, commonly used in the clinic, to bolster T cell—B cell interactions during expansion for ACT. T cells expanded ex vivo from a CpG-treated culture demonstrated potent antitumor efficacy and prolonged persistence in vivo. This antitumor efficacy was accomplished without in vivo administration of TLR agonists or other adjuvants of high-dose interleukin (IL)-2 or vaccination, which are classically required for effective ACT therapy. CpG-conditioned CD8+ T cells acquired a unique proteomic signature hallmarked by an IL-2RαhighICOShighCD39low phenotype and an altered metabolic profile, all reliant on B cells transiently present in the culture. Likewise, human TILs benefitted from expansion with CpG ex vivo, as they also possessed the IL-2RαhighICOShighCD39low phenotype. CpG fostered the expansion of potent CD8+ T cells with the signature phenotype and antitumor ability via empowering a direct B–T cell interaction. Isolated B cells also imparted T cells with the CpG-associated phenotype and improved tumor immunity without the aid of additional antigen-presenting cells or other immune cells in the culture.ConclusionsOur results demonstrate a novel way to use TLR agonists to improve immunotherapy and reveal a vital role for B cells in the generation of potent CD8+ T cell-based therapies. Our findings have immediate implications in the clinical treatment of advanced solid tumors.

Immunotherapy with Cell-Based Biological Drugs to Cure HIV-1 Infection

Since its discovery 35 years ago, there have been no therapeutic interventions shown to enable full HIV-1 remission. Combined antiretroviral therapy (cART) has achieved the sustained control of HIV-1 replication, however, the life-long treatment does not eradicate long-lived latently infected reservoirs and can result in multiple side effects including the development of multidrug-resistant escape mutants. Antibody-based treatments have emerged as alternative approaches for a HIV-1 cure. Here, we will review clinical advances in coreceptor-targeting antibodies, with respect to anti-CCR5 antibodies in particular, which are currently being generated to target the early stages of infection. Among the Env-specific antibodies widely accepted as relevant in cure strategies, the potential role of those targeting CD4-induced (CD4i) epitopes of the CD4-binding site (CD4bs) in eliminating HIV-1 infected cells has gained increasing interest and will be presented. Together, with approaches targeting the HIV-1 replication cycle, we will discuss the strategies aimed at boosting and modulating specific HIV-1 immune responses, highlighting the harnessing of TLR agonists for their dual role as latency reverting agents (LRAs) and immune-modulatory compounds. The synergistic combinations of different approaches have shown promising results to ultimately enable a HIV-1 cure.

CpG Oligodeoxynucleotides for Anticancer Monotherapy from Preclinical Stages to Clinical Trials

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered by CpG ODN has been shown to activate plasmacytoid dendritic cells (pDCs) and cytotoxic T lymphocytes (CTLs), enhancing T cell-mediated antitumor immunity. However, the extent of antitumor immunity carried by TLR agonists has not been optimized individually or in combinations with cancer vaccines, resulting in a decreased preference for TLR agonists as adjuvants in clinical trials. Although various combination therapies involving CpG ODNs have been applied in clinical trials, none of the CpG ODN-based drugs have been approved by the FDA, owing to the short half-life of CpG ODNs in serum that leads to low activation of natural killer cells (NK cells) and CTLs, along with increases of pro-inflammatory cytokine productions. This review summarized the current innovation on CpG ODNs that are under clinical investigation and explored the future direction for CpG ODN-based nanomedicine as an anticancer monotherapy.

Magnitude and Breadth of Antibody Cross-reactivity Induced by Recombinant Influenza Hemagglutinin Trimer Vaccine Is Enhanced by Combination Adjuvants

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Of all the formulations tested, a combination of CpG, MPLA and AddaVAX (termed “IVAX-1”) yielded the greatest breadth and magnitude of Ab responses, particularly against the HA1 region (which includes the variable head domain) of HA. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNg gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Quercetin Preserves Oral Cavity Health by Mitigating Inflammation and Microbial Dysbiosis

Failure to attenuate inflammation coupled with consequent microbiota changes drives the development of bone-destructive periodontitis. Quercetin, a plant-derived polyphenolic flavonoid, has been linked with health benefits in both humans and animals. Using a systematic approach, we investigated the effect of orally delivered Quercetin on host inflammatory response, oral microbial composition and periodontal disease phenotype. In vivo, quercetin supplementation diminished gingival cytokine expression, inflammatory cell infiltrate and alveolar bone loss. Microbiome analyses revealed a healthier oral microbial composition in Quercetin-treated versus vehicle-treated group characterized by reduction in the number of pathogenic species including Enterococcus, Neisseria and Pseudomonas and increase in the number of non-pathogenic Streptococcus sp. and bacterial diversity. In vitro, Quercetin diminished inflammatory cytokine production through modulating NF-κB:A20 axis in human macrophages following challenge with oral bacteria and TLR agonists. Collectively, our findings reveal that Quercetin supplement instigates a balanced periodontal tissue homeostasis through limiting inflammation and fostering an oral cavity microenvironment conducive of symbiotic microbiota associated with health. This proof of concept study provides key evidence for translational studies to improve overall health.

Platelet Toll-like Receptors in Healthy and Acute Myocardial Infarction Subjects

<p>Platelet activation is pathological in acute myocardial infarction (AMI). Despite treatment with dual anti-platelet therapy (DAPT), platelet activation can continue to occur post-AMI and has been linked to an increased risk of recurrent cardiovascular events. Toll-like receptors (TLRs) are important innate immune receptors, and platelets are known to express a subset of TLRs. The functional significance of these platelet-TLR pathways in AMI has not been fully examined but may contribute to persistent post-AMI platelet activation. Platelet-TLR expression, TLR-mediated platelet activation and the platelet effect on leukocyte responses to TLR stimulation were examined in this thesis. Platelet-TLR expression and TLR-mediated platelet activation was examined for a subset of these receptors (TLR1, 2, 4, 6 and 9) in healthy subjects and in AMI subjects on DAPT. We observed an increase in platelet expression of TLR1, 4 and 9 in AMI platelets compared to healthy subjects. Further investigation into platelet-TLR9 expression showed an increase in expression upon platelet activation in healthy, but not AMI, subjects. We observed direct, dose-dependent platelet activation in response to Pam3CSK4 (TLR2/1 agonist) and ODN2009 (TLR9 agonist) in healthy subjects and in AMI on DAPT. For both cohorts, platelets were also directly activated by a high dose of LPS (TLR4 agonist) but were not directly activated by FSL-1 (TLR6 agonist). These results demonstrate that some (TLR1, 2, 4 and 9), but not other (TLR6), platelet-TLR pathways can cause platelet activation in AMI despite treatment with potent anti-platelet therapy. For the results described above, we were unable to assess TLR-mediated platelet activation in the absence of anti-platelet therapy in AMI subjects as these drugs are administered before or immediately upon presentation to hospital. It was therefore not possible to exclude the possibility that DAPT was providing a degree of inhibition of platelet activation in AMI patients. To address this, we determined the extent to which aspirin monotherapy or DAPT could inhibit platelet activation in response to TLR2/1, TLR4 and TLR9 stimulation in a cross-over study in healthy subjects. We demonstrated that DAPT only modestly inhibited, and aspirin monotherapy did not inhibit, platelet activation in response to all TLR agonists tested and platelets still became potently activated despite treatment with anti-platelet agents. These platelet-TLRs represent intact on-treatment platelet activation pathways. Lastly, we determined the extent to which platelets modulate leukocyte responses to TLR2/1, TLR2/6 and TLR4 stimulation. Platelets were able to reduce neutrophil responses to TLR stimulation, and modulated PBMC cytokine and chemokine production in a complex manner following stimulation with LPS and FSL-1. The presence of platelets did not change cytokine/chemokine production in response to Pam3CSK4, demonstrating a TLR agonist-specific manner of platelet modulation. We further investigated the effect of platelets on neutrophil responses to TLR stimulation. With platelets, neutrophil activation was attenuated, and phagocytic activity was increased in unstimulated cultures and in response to various doses of Pam3CSK4 and FSL-1. Neutrophil elastase secretion was attenuated in unstimulated cultures and in response to low-dose stimulation with all three TLR agonists. We show that platelets can both augment and attenuate various markers of neutrophil function. Together, this work indicates that platelets express functional TLR pathways that can differentially regulate a number of thrombotic and inflammatory responses in healthy subjects and in subjects with AMI.</p>

Platelet Toll-like Receptors in Healthy and Acute Myocardial Infarction Subjects

<p>Platelet activation is pathological in acute myocardial infarction (AMI). Despite treatment with dual anti-platelet therapy (DAPT), platelet activation can continue to occur post-AMI and has been linked to an increased risk of recurrent cardiovascular events. Toll-like receptors (TLRs) are important innate immune receptors, and platelets are known to express a subset of TLRs. The functional significance of these platelet-TLR pathways in AMI has not been fully examined but may contribute to persistent post-AMI platelet activation. Platelet-TLR expression, TLR-mediated platelet activation and the platelet effect on leukocyte responses to TLR stimulation were examined in this thesis. Platelet-TLR expression and TLR-mediated platelet activation was examined for a subset of these receptors (TLR1, 2, 4, 6 and 9) in healthy subjects and in AMI subjects on DAPT. We observed an increase in platelet expression of TLR1, 4 and 9 in AMI platelets compared to healthy subjects. Further investigation into platelet-TLR9 expression showed an increase in expression upon platelet activation in healthy, but not AMI, subjects. We observed direct, dose-dependent platelet activation in response to Pam3CSK4 (TLR2/1 agonist) and ODN2009 (TLR9 agonist) in healthy subjects and in AMI on DAPT. For both cohorts, platelets were also directly activated by a high dose of LPS (TLR4 agonist) but were not directly activated by FSL-1 (TLR6 agonist). These results demonstrate that some (TLR1, 2, 4 and 9), but not other (TLR6), platelet-TLR pathways can cause platelet activation in AMI despite treatment with potent anti-platelet therapy. For the results described above, we were unable to assess TLR-mediated platelet activation in the absence of anti-platelet therapy in AMI subjects as these drugs are administered before or immediately upon presentation to hospital. It was therefore not possible to exclude the possibility that DAPT was providing a degree of inhibition of platelet activation in AMI patients. To address this, we determined the extent to which aspirin monotherapy or DAPT could inhibit platelet activation in response to TLR2/1, TLR4 and TLR9 stimulation in a cross-over study in healthy subjects. We demonstrated that DAPT only modestly inhibited, and aspirin monotherapy did not inhibit, platelet activation in response to all TLR agonists tested and platelets still became potently activated despite treatment with anti-platelet agents. These platelet-TLRs represent intact on-treatment platelet activation pathways. Lastly, we determined the extent to which platelets modulate leukocyte responses to TLR2/1, TLR2/6 and TLR4 stimulation. Platelets were able to reduce neutrophil responses to TLR stimulation, and modulated PBMC cytokine and chemokine production in a complex manner following stimulation with LPS and FSL-1. The presence of platelets did not change cytokine/chemokine production in response to Pam3CSK4, demonstrating a TLR agonist-specific manner of platelet modulation. We further investigated the effect of platelets on neutrophil responses to TLR stimulation. With platelets, neutrophil activation was attenuated, and phagocytic activity was increased in unstimulated cultures and in response to various doses of Pam3CSK4 and FSL-1. Neutrophil elastase secretion was attenuated in unstimulated cultures and in response to low-dose stimulation with all three TLR agonists. We show that platelets can both augment and attenuate various markers of neutrophil function. Together, this work indicates that platelets express functional TLR pathways that can differentially regulate a number of thrombotic and inflammatory responses in healthy subjects and in subjects with AMI.</p>

An Overview of Recent Insights into the Response of TLR to SARS-CoV-2 Infection and the Potential of TLR Agonists as SARS-CoV-2 Vaccine Adjuvants

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to coronavirus disease (COVID-19), a global health pandemic causing millions of deaths worldwide. However, the immunopathogenesis of COVID-19, particularly the interaction between SARS-CoV-2 and host innate immunity, remains unclear. The innate immune system acts as the first line of host defense, which is critical for the initial detection of invading pathogens and the activation and shaping of adaptive immunity. Toll-like receptors (TLRs) are key sensors of innate immunity that recognize pathogen-associated molecular patterns and activate downstream signaling for pro-inflammatory cytokine and chemokine production. However, TLRs may also act as a double-edged sword, and dysregulated TLR responses may enhance immune-mediated pathology, instead of providing protection. Therefore, a proper understanding of the interaction between TLRs and SARS-CoV-2 is of great importance for devising therapeutic and preventive strategies. The use of TLR agonists as vaccine adjuvants for human disease is a promising approach that could be applied in the investigation of COVID-19 vaccines. In this review, we discuss the recent progress in our understanding of host innate immune responses in SARS-CoV-2 infection, with particular focus on TLR response. In addition, we discuss the use of TLR agonists as vaccine adjuvants in enhancing the efficacy of COVID-19 vaccine.