Protective Role of the Toll-Like Receptor 5 Agonist KMRC011 against Murine Colitis Induced by Citrobacter rodentium and Dextran Sulfate Sodium.

This study aimed to identify the therapeutic ability of a novel toll-like receptor (TLR) 5 agonist, KMRC011, on ulcerative colitis induced by Citrobacter rodentium and dextran sulfate sodium in a C57BL/6N mouse model. Ulcerative colitis was induced in the mice by the oral administration of 1% dextran sulfate sodium in sterile drinking water for seven days ad libitum, followed by C. rodentium infection on the seventh day by intra-gastric administration (DSS-CT group). KMRC011 was administered intramuscularly at both 24 h and 15 min before (Treatment 1 group), and at both 15 min and 24 h after (Treatment 2 group) the C. rodentium infection. The length of the large intestine and histopathological counts were significantly greater and mucosal thickness was significantly thinner in the Treatment 1 group compared to the DSS-CT and Treatment 2 groups. Il-6 and Il-10 mRNA expression levels were upregulated, while Ifn-γ and Tnf-α mRNA expression levels were significantly downregulated in the Treatment 1 group, compared to the DSS-CT group. NF-κB p65 expression level was elevated due to ulcerative colitis in the DSS-CT group, but was significantly downregulated in the Treatment 1 group. Overall, KMRC011 showed protective effects against murine colitis by inhibiting NF-κB signaling.

Clinical evaluation of toll-like receptor-5 agonist for radiation-induced oral mucositis in beagle dogs.

This study aimed to evaluate the clinical safety and validate the radiomitigative effect of KMRC011, against radiation-induced oral mucositis in beagle dogs. Clinical safety was evaluated by assessing tolerability, complete blood tests, and plasma biochemistry after drug administration. The radiomitigative effect of KMRC011 was evaluated macropathologically and histopathologically after inducing oral mucositis iatrogenically using 20 Gy irradiation. The plasma concentration of interleukin-6 was measured via enzyme-linked immunosorbent assay, as a biomarker of KMRC011 bioreactivity. Decreased tolerability, increased neutrophil count, hepatic enzyme concentration, C-reactive protein concentration, and interleukin-6 concentration after the administration was observed and ceased within 24 h without additional treatment. Although all animals included in the present study developed severe mucositis in the late course of the study, animals administered KMRC011 showed less erythema, ulcer, inflammatory infiltration. These results suggest that KMRC011 may be used as an adjuvant for radiotherapy without severe adverse effects, especially during short-term radiotherapy, such as hypofractionated radiotherapy or stereotactic radiotherapy.

BACH2 in TRegs Limits the Number of Adipose Tissue Regulatory T Cells and Restrains Type 2 Immunity to Fungal Allergens.

FoxP3+ regulatory T cells (Tregs) are essential for self-tolerance and moderating tissue-damaging inflammation. Tregs that develop and mature in the thymus are classified as central Tregs or effector Tregs based on whether Tregs predominately inhabit secondary lymphoid organs (central Tregs) or tissues (effector Tregs). By generating mice that are conditionally deficient for Bach2 in peripheral Tregs, we have examined the role of Bach2 in regulating Treg homeostasis and effector functions. Unlike global and T cell-specific Bach2-deficient mice, Treg-specific Bach2 ablation did not result in unprovoked TH2 inflammation in the lungs. However, Bach2 deficiency in Tregs led to augmented expressions of IRF4, BATF, and GATA3 and a significant increase in the accumulation of ST2 (IL-33R)+ve effector Tregs in the spleen and visceral adipose tissue (VAT) but not in the lungs. Enhanced Bach2-deficient Treg numbers in VAT was not linked to hyperresponsiveness to exogenous IL-33 in vivo. Most strikingly, Treg-specific Bach2 deficiency resulted in enhanced fungal protease-induced Type 2 allergic inflammation in the lungs, with no detectable effects on Type 1 responses to systemic or respiratory viral infections. In summary, we ascribe vital roles for Bach2 in peripheral Tregs: as a transcriptional checkpoint to limit precocious differentiation into effector Tregs in lymphoid tissues and as a regulator of the functional program that restrains Type 2 but not Type 1 inflammation in lungs. Results presented in this manuscript implicate dysregulated Tregs in the pathogenesis of airway hypersensitivities, asthma, and other allergic disorders.

Vaccine adjuvants to engage the cross-presentation pathway.

Adjuvants are indispensable components of vaccines for stimulating optimal immune responses to non-replicating, inactivated and subunit antigens. Eliciting balanced humoral and T cell-mediated immunity is paramount to defend against diseases caused by complex intracellular pathogens, such as tuberculosis, malaria, and AIDS. However, currently used vaccines elicit strong antibody responses, but poorly stimulate CD8 cytotoxic T lymphocyte (CTL) responses. To elicit potent CTL memory, vaccines need to engage the cross-presentation pathway, and this requirement has been a crucial bottleneck in the development of subunit vaccines that engender effective T cell immunity. In this review, we focus on recent insights into DC cross-presentation and the extent to which clinically relevant vaccine adjuvants, such as aluminum-based nanoparticles, water-in oil emulsion (MF59) adjuvants, saponin-based adjuvants, and Toll-like receptor (TLR) ligands modulate DC cross-presentation efficiency. Further, we discuss the feasibility of using carbomer-based adjuvants as next generation of adjuvant platforms to elicit balanced antibody- and T-cell based immunity. Understanding of the molecular mechanism of DC cross-presentation and the mode of action of adjuvants will pave the way for rational design of vaccines for infectious diseases and cancer that require balanced antibody- and T cell-based immunity.

Vaccine-induced systemic and mucosal T cell immunity to SARS-CoV-2 viral variants.

The first-generation COVID-19 vaccines have been effective in mitigating severe illness and hospitalization, but recurring waves of infections are associated with the emergence of SARS-CoV-2 variants that display progressive abilities to evade antibodies, leading to diminished vaccine effectiveness. The lack of clarity on the extent to which vaccine-elicited mucosal or systemic memory T cells protect against such antibody-evasive SARS-CoV-2 variants remains a critical knowledge gap in our quest for broadly protective vaccines. Using adjuvanted spike protein­based vaccines that elicit potent T cell responses, we assessed whether systemic or lung-resident CD4 and CD8 T cells protected against SARS-CoV-2 variants in the presence or absence of virus-neutralizing antibodies. We found that 1) mucosal or parenteral immunization led to effective viral control and protected against lung pathology with or without neutralizing antibodies, 2) protection afforded by mucosal memory CD8 T cells was largely redundant in the presence of antibodies that effectively neutralized the challenge virus, and 3) "unhelped" mucosal memory CD8 T cells provided no protection against the homologous SARS-CoV-2 without CD4 T cells and neutralizing antibodies. Significantly, however, in the absence of detectable virus-neutralizing antibodies, systemic or lung-resident memory CD4 and "helped" CD8 T cells provided effective protection against the relatively antibody-resistant B1.351 (ß) variant, without lung immunopathology. Thus, induction of systemic and mucosal memory T cells directed against conserved epitopes might be an effective strategy to protect against SARS-CoV-2 variants that evade neutralizing antibodies. Mechanistic insights from this work have significant implications in the development of T cell­targeted immunomodulation or broadly protective SARS-CoV-2 vaccines.

A first-in-human study of KMRC011, a potential treatment for acute radiation syndrome, to explore tolerability, pharmacokinetics, and pharmacodynamics.

KMRC011 is a novel Toll-like receptor 5 agonist under development as a treatment for acute radiation syndrome (ARS). The aim of this first-in-human study was to investigate the tolerability, pharmacokinetics, and pharmacodynamics of a single intramuscular dose of KMRC011 in healthy subjects. A randomized, single-blind, placebo-controlled, single dose-escalation study was conducted with the starting dose of 5 µg. Eight (4 only for 5 µg cohort) subjects per cohort were randomly assigned to KMRC011 or placebo in a 3:1 ratio. Dose-limiting toxicity (DLT) was assessed throughout the study. Serum concentrations of KMRC011, granulocyte colony-stimulating factor (G-CSF), and interleukin-6 (IL-6) were measured up to 48 h postdose. Based on safety review, the dose of KMRC011 escalated up to 20 µg, and consequently, a total of 4 dose levels (5, 10, 15, and 20 µg) were explored. The most common adverse event was injection site reaction, showing no dose-related trend. Three DLTs (2 cases of hepatic enzyme increased and 1 of pyrexia) were observed; 1 in the 15 µg cohort and 2 in the 20 µg cohort. A developed method could not detect any KMRC011 in serum. KMRC011 15 µg and 20 µg showed significant increases of G-CSF, IL-6, and absolute neutrophil counts, compared with the placebo. A single intramuscular administration of KMRC011 ranging from 5 to 15 µg was tolerated in healthy subjects. Doses of KMRC011 equal to or greater than 15 µg exerted TLR5 agonist-like activities by increasing serum G-CSF and IL-6. It suggests that KMRC011 has the potential for a treatment for ARS.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (TRMs) is a goal of T cell-based vaccines against respiratory viral pathogens, such as influenza A virus (IAV). C-C chemokine receptor type 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 TRMs in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (TC1/TC17/TH1/TH17) IAV nucleoprotein-specific lung TRMs to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced TRM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendritic cells (DCs), and monocyte-derived dendritic cells internalized and processed vaccine antigen in lungs. We found that basic leucine zipper ATF-like transcription factor 3 (BATF3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127hi, KLRG-1lo, OX40+ve CD62L+ve, and mucosally imprinted CD69+ve CD103+ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung TRMs induced by CCR2 deficiency was linked to dampened expression of T-bet but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses, and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced TRMs. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens, including IAV and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IMPORTANCE While antibody-based immunity to influenza A virus (IAV) is type and subtype specific, lung- and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of antiviral lung-resident memory T cells following intranasal vaccination. These findings suggest that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses, such as IAV and SARS-CoV-2.

Reaction of a Molybdenum Bis(dinitrogen) Complex with Carbon Dioxide: A Combined Experimental and Computational Investigation.

Refluxing Mo(CO)6 in the presence of the phosphine-functionalized α-diimine ligand Ph2PPrDI allowed for substitution and formation of the dicarbonyl complex, (Ph2PPrDI)Mo(CO)2. Oxidation with I2 followed by heating resulted in further CO dissociation and isolation of the corresponding diiodide complex, (Ph2PPrDI)MoI2. Reduction of this complex under a N2 atmosphere afforded the corresponding bis(dinitrogen) complex, (Ph2PPrDI)Mo(N2)2. The solid-state structures of all three compounds were found to feature a tetradentate chelate and cis-monodentate ligands. Notably, the addition of CO2 to (Ph2PPrDI)Mo(N2)2 is proposed to result in head-to-tail CO2 coupling to generate the corresponding metallacycle and ultimately a mixture of (Ph2PPrDI)Mo(CO)2 and the bis(oxo) dimer, [(κ3-Ph2PPrDI)Mo(O)(µ-O)]2. Computational studies have been performed to gain insight into the reaction and evaluate the importance of cis-coordination sites for selective head-to-tail CO2 reductive coupling, CO deinsertion, disproportionation, and stepwise CO2 deinsertion.

CCR2 Regulates Vaccine-Induced Mucosal T-Cell Memory to Influenza A Virus.

Elicitation of lung tissue-resident memory CD8 T cells (T RM s) is a goal of T-cell based vaccines against respiratory viral pathogens such as influenza A virus (IAV). Chemokine receptor 2 (CCR2)-dependent monocyte trafficking plays an essential role in the establishment of CD8 T RM s in lungs of IAV-infected mice. Here, we used a combination adjuvant-based subunit vaccine strategy that evokes multifaceted (T C 1/T C 17/T H 1/T H 17) IAV nucleoprotein-specific lung T RM s, to determine whether CCR2 and monocyte infiltration are essential for vaccine-induced T RM development and protective immunity to IAV in lungs. Following intranasal vaccination, neutrophils, monocytes, conventional dendrtitic cells (DCs) and monocyte-derived DCs internalized and processed vaccine antigen in lungs. We also found that Basic Leucine Zipper ATF-Like Transcription Factor 3 (BATF-3)-dependent DCs were essential for eliciting T cell responses, but CCR2 deficiency enhanced the differentiation of CD127 HI /KLRG-1 LO , OX40 +ve CD62L +ve and mucosally imprinted CD69 +ve CD103 +ve effector and memory CD8 T cells in lungs and airways of vaccinated mice. Mechanistically, increased development of lung T RM s, induced by CCR2 deficiency was linked to dampened expression of T-bet, but not altered TCF-1 levels or T cell receptor signaling in CD8 T cells. T1/T17 functional programming, parenchymal localization of CD8/CD4 effector and memory T cells, recall T cell responses and protective immunity to a lethal IAV infection were unaffected in CCR2-deficient mice. Taken together, we identified a negative regulatory role for CCR2 and monocyte trafficking in mucosal imprinting and differentiation of vaccine-induced T RM s. Mechanistic insights from this study may aid the development of T-cell-based vaccines against respiratory viral pathogens including IAV and SARS-CoV-2. IMPORTANCE: While antibody-based immunity to influenza A virus (IAV) is type and sub-type specific, lung and airway-resident memory T cells that recognize conserved epitopes in the internal viral proteins are known to provide heterosubtypic immunity. Hence, broadly protective IAV vaccines need to elicit robust T-cell memory in the respiratory tract. We have developed a combination adjuvant-based IAV nucleoprotein vaccine that elicits strong CD4 and CD8 T cell memory in lungs and protects against H1N1 and H5N1 strains of IAV. In this study, we examined the mechanisms that control vaccine-induced protective memory T cells in the respiratory tract. We found that trafficking of monocytes into lungs might limit the development of anti-viral lung-resident memory T cells, following intranasal vaccination. These findings suggested that strategies that limit monocyte infiltration can potentiate vaccine-induced frontline T-cell immunity to respiratory viruses such as IAV and SARS-CoV-2.

IgLON5 Regulates the Adhesion and Differentiation of Myoblasts.

IgLON5 is a cell adhesion protein belonging to the immunoglobulin superfamily and has important cellular functions. The objective of this study was to determine the role played by IgLON5 during myogenesis. We found IgLON5 expression progressively increased in C2C12 myoblasts during transition from the adhesion to differentiation stage. IgLON5 knockdown (IgLON5kd) cells exhibited reduced cell adhesion, myotube formation, and maturation and reduced expressions of different types of genes, including those coding for extracellular matrix (ECM) components (COL1a1, FMOD, DPT, THBS1), cell membrane proteins (ITM2a, CDH15), and cytoskeletal protein (WASP). Furthermore, decreased IgLON5 expression in FMODkd, DPTkd, COL1a1kd, and ITM2akd cells suggested that IgLON5 and these genes mutually control gene expression during myogenesis. IgLON5 immunoneutralization resulted in significant reduction in the protein level of myogenic markers (MYOD, MYOG, MYL2). IgLON5 expression was higher in the CTX-treated gastrocnemius mice muscles (day 7), which confirmed increase expression of IgLON5 during muscle. Collectively, these results suggest IgLON5 plays an important role in myogenesis, muscle regeneration, and that proteins in ECM and myoblast membranes form an interactive network that establishes an essential microenvironment that ensures muscle stem cell survival.

Combination Adjuvants Affect the Magnitude of Effector-Like Memory CD8 T Cells and Protection against Listeriosis.

The development of T cell-based subunit protein vaccines against diseases such as tuberculosis and malaria remains a challenge for immunologists. Here, we have identified a nanoemulsion adjuvant, Adjuplex (ADJ), which enhanced dendritic cell (DC) cross-presentation and elicited effective memory T cell-based immunity to Listeria monocytogenes. We further evaluated whether cross-presentation induced by ADJ can be combined with the immunomodulatory effects of Toll-like receptor (TLR) agonists (CpG or glucopyranosyl lipid adjuvant [GLA]) to evoke systemic CD8 T cell-based immunity to L. monocytogenes. Mechanistically, vaccination with ADJ, alone or in combination with CpG or GLA, augmented activation and antigen uptake by CD103+ migratory and CD8α+ resident DCs and upregulated CD69 expression on B and T lymphocytes in vaccine-draining lymph nodes. By engaging basic leucine zipper ATF-like transcription factor 3-dependent cross-presenting DCs, ADJ potently elicited effector CD8 T cells that differentiated into granzyme B-expressing CD27LO effector-like memory CD8 T cells, which provided effective immunity to L. monocytogenes in the spleen and liver. CpG or GLA alone did not elicit effector-like memory CD8 T cells and induced moderate protection in the spleen but not in the liver. Surprisingly, combining CpG or GLA with ADJ reduced the number of ADJ-induced memory CD8 T cells and compromised protective immunity to L. monocytogenes, especially in the liver. Taken together, the data presented in this study provide a glimpse of protective CD8 T cell memory differentiation induced by a nanoemulsion adjuvant and demonstrate the unexpected negative effects of TLR signaling on the magnitude of CD8 T cell memory and protective immunity to L. monocytogenes, a model intracellular pathogen.

Carbomer-based adjuvant elicits CD8 T-cell immunity by inducing a distinct metabolic state in cross-presenting dendritic cells.

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a virus and a systemic intracellular bacterial infection. Studies to understand the metabolic and molecular basis for ADJ's effect on antigen cross-presentation by dendritic cells (DCs) revealed several unique and distinctive mechanisms. ADJ-stimulated DCs produced IL-1ß and IL-18, suggestive of inflammasome activation, but in vivo activation of CD8 T cells was unaffected in caspase 1-deficient mice. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, ADJ modulated multiple steps in the cytosolic pathway of cross-presentation by enabling accumulation of degraded antigen, reducing endosomal acidity and promoting antigen localization to early endosomes. Further, by increasing ROS production and lipid peroxidation, ADJ promoted antigen escape from endosomes to the cytosol for degradation by proteasomes into peptides for MHC I loading by TAP-dependent pathways. Furthermore, we found that induction of lipid bodies (LBs) and alterations in LB composition mediated by ADJ were also critical for DC cross-presentation. Collectively, our model challenges the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have strong implications in the rational development of safe and effective immune adjuvants to potentiate robust T-cell based immunity.

Programming Multifaceted Pulmonary T Cell Immunity by Combination Adjuvants.

Induction of protective mucosal T cell memory remains a formidable challenge to vaccinologists. Using a combination adjuvant strategy that elicits potent CD8 and CD4 T cell responses, we define the tenets of vaccine-induced pulmonary T cell immunity. An acrylic-acid-based adjuvant (ADJ), in combination with Toll-like receptor (TLR) agonists glucopyranosyl lipid adjuvant (GLA) or CpG, promotes mucosal imprinting but engages distinct transcription programs to drive different degrees of terminal differentiation and disparate polarization of TH1/TC1/TH17/TC17 effector/memory T cells. Combination of ADJ with GLA, but not CpG, dampens T cell receptor (TCR) signaling, mitigates terminal differentiation of effectors, and enhances the development of CD4 and CD8 TRM cells that protect against H1N1 and H5N1 influenza viruses. Mechanistically, vaccine-elicited CD4 T cells play a vital role in optimal programming of CD8 TRM and viral control. Taken together, these findings provide further insights into vaccine-induced multifaceted mucosal T cell immunity with implications in the development of vaccines against respiratorypathogens, including influenza virus and SARS-CoV-2.

KMRC011, an agonist of toll-like receptor 5, mitigates irradiation-induced tissue damage and mortality in cynomolgus monkeys.

In the study here, the potential applicability of KMRC011 - an agonist of toll-like receptor-5 - as a countermeasure for radiation toxicities was evaluated. Following a single 5.5 Gy total body irradiation (TBI, surface absorbed dose = 7 Gy) of Co60 γ-rays, mortality rates and degrees of pathological lesions that developed over 80 days were compared in monkeys that received TBI only and a group that was injected once with KMRC011 (10 µg/kg) after TBI. Compared to the TBI-only hosts (80%), the death rate was significantly improved by the use of KMRC011 (40%), all deaths in both groups occurred in the period from Days 19-24 post-TBI. Further analysis of monkeys that survived until the end of the experiment showed that AST and ALT levels were elevated only in the TBI group, and that radiation-induced tissue damage was alleviated by the KMRC011 injection. Additionally, expression of cell death-related proteins was lower in tissues from the KMRC011-treated hosts than in those in the TBI-only group. Other measured parameters, including body weight, food uptake, and hematological values did not significantly differ between the two groups over the entire period. The results of this study, thus demonstrate that KMRC011 could potentially be used as a medical countermeasure for the treatment of acute radiation exposure.

Polymeric Pathogen-Like Particles-Based Combination Adjuvants Elicit Potent Mucosal T Cell Immunity to Influenza A Virus.

Eliciting durable and protective T cell-mediated immunity in the respiratory mucosa remains a significant challenge. Polylactic-co-glycolic acid (PLGA)-based cationic pathogen-like particles (PLPs) loaded with TLR agonists mimic biophysical properties of microbes and hence, simulate pathogen-pattern recognition receptor interactions to safely and effectively stimulate innate immune responses. We generated micro particle PLPs loaded with TLR4 (glucopyranosyl lipid adjuvant, GLA) or TLR9 (CpG) agonists, and formulated them with and without a mucosal delivery enhancing carbomer-based nanoemulsion adjuvant (ADJ). These adjuvants delivered intranasally to mice elicited high numbers of influenza nucleoprotein (NP)-specific CD8+ and CD4+ effector and tissue-resident memory T cells (TRMs) in lungs and airways. PLPs delivering TLR4 versus TLR9 agonists drove phenotypically and functionally distinct populations of effector and memory T cells. While PLPs loaded with CpG or GLA provided immunity, combining the adjuvanticity of PLP-GLA and ADJ markedly enhanced the development of airway and lung TRMs and CD4 and CD8 T cell-dependent immunity to influenza virus. Further, balanced CD8 (Tc1/Tc17) and CD4 (Th1/Th17) recall responses were linked to effective influenza virus control. These studies provide mechanistic insights into vaccine-induced pulmonary T cell immunity and pave the way for the development of a universal influenza and SARS-CoV-2 vaccines.

Radioprotective effect of newly synthesized toll-like receptor 5 agonist, KMRC011, in mice exposed to total-body irradiation.

Exposure to ionizing radiation leads to severe damages in radiosensitive organs and induces acute radiation syndrome, including effects on the hematopoietic system and gastrointestinal system. In this study, the radioprotective ability of KMRC011, a novel toll-like receptor 5 (TLR5) agonist, was investigated in C57BL6/N mice exposed to lethal total-body gamma-irradiation. In a 30-day survival study, KMRC011-treated mice had a significantly improved survival rate compared with control after 11 Gy total-body irradiation (TBI), and it was found that the radioprotective activity of KMRC011 depended on its dosage and repeated treatment. In a 5-day short-term study, we demonstrated that KMRC011 treatment stimulated cell proliferation and had an anti-apoptotic effect. Furthermore, KMRC011 increased the expressions of genes related to DNA repair, such as Rad21, Gadd45b, Sod2 and Irg1, in the small intestine of lethally irradiated mice. Interestingly, downregulation of NF-κB p65 in the mouse intestine by KMRC011 treatment was observed. This data indicated that KMRC011 exerted a radioprotective activity partially by regulating NF-κB signaling. Finally, peak expression levels of G-CSF, IL-6, IFN-γ, TNF-α and IP-10 induced by KMRC011 treatment were different depending on the route of administration and type of cytokine. These cytokines could be used as candidate biomarkers for the evaluation of KMRC011 clinical efficacy. Our data indicated that KMRC011 has radioprotective activity in lethally irradiated mice and may be developed as a therapeutic agent for radioprotection.

Repeated injection of KMRC011, a medical countermeasure for radiation, can cause adverse health effects in cynomolgus monkeys.

High-dose radiation-induced tissue damage is a major limiting factor in the medical application of nuclear technology. Herein, we tested 28-day repeated-dose toxicity of KMRC011, an agonist of toll-like receptor (TLR) 5, which is being developed as a medical countermeasure for radiation, using cynomolgus monkeys. KMRC011 (0.01, 0.02 or 0.04 mg/kg/day) was intramuscularly injected once daily for 4 weeks, and each two monkeys in both control and 0.04 mg/kg/day group were observed for an additional 2-week recovery period. There were no dose-related toxicological changes in mortality, clinical observations, body weight, food consumption, ophthalmological findings, electrocardiographs, coagulation, serum chemistry, organ weights, or urinalysis and urine chemistry. Although treatment-related changes, such as increased white blood cells, increased absolute and relative neutrophils, decreased relative lymphocytes and inflammatory lesions, were noted in the maximum dose group, these findings were not observed after the 2-week recovery period. Further, we considered that the kidneys and heart may be target organs of TLR5 agonists, as well as the spleen, and that autophagic signals can be triggered in tissue damage and the repair process. Importantly, accumulation of p62 protein, an indicator of autophagy, and a decrease of caveolin-1 protein, a regulator of TLR5 protein half-life, were found in both tissues from the highest dose group. Therefore, we conclude that the no-observed-adverse-effect level for KMRC011 may be greater than 0.04 mg/kg/day in male and female monkeys. Additionally, we propose that further studies are needed to identify the molecular signals, which are related to KMRC011-induced adverse effects.

TLR5 binding and activation by KMRC011, a flagellin-derived radiation countermeasure.

Entolimod (CBLB502) is a flagellin-derived radiation countermeasure currently under clinical trial. Entolimod exerts radioprotective activity by directly interacting with TLR5, an innate immune receptor, using the conserved domains of flagellin. Entolimod was designed to contain an artificially introduced N-terminal region that is not related to drug effects and might trigger unexpected toxic immunogenic reactions in humans. To refine the entolimod drug design, we engineered entolimod into KMRC011 by removing its ancillary region. The TLR5 binding and activating capacities of KMRC011 were assessed through biophysical and cellular analyses. KMRC011 forms an exceptionally stable complex with TLR5 at a 1:1 molar ratio with an equilibrium dissociation constant of ∼100 pM and potently activates TLR5. Moreover, alanine scanning mutagenesis identified the R90 and E114 residues of KMRC011 as a TLR5 activation hotspot. Further comparative analysis demonstrated that KMRC011 binds and activates TLR5 in a mode similar to that of entolimod. Thus, we propose that KMRC011 can be used in place of entolimod as a second-generation radiation countermeasure that shows none of the immunogenic side effects derived from the entolimod ancillary region.

PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis.

Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase; it forms diverse heterotrimeric holoenzymes that counteract kinase actions. Using a peptidome that tiles the disordered regions of the human proteome, we identified proteins containing [LMFI]xx[ILV]xEx motifs that serve as interaction sites for B'-family PP2A regulatory subunits and holoenzymes. The B'-binding motifs have important roles in substrate recognition and in competitive inhibition of substrate binding. With more than 100 novel ligands identified, we confirmed that the recently identified LxxIxEx B'α-binding motifs serve as common binding sites for B' subunits with minor variations, and that S/T phosphorylation or D/E residues at positions 2, 7, 8 and 9 of the motifs reinforce interactions. Hundreds of proteins in the human proteome harbor intrinsic or phosphorylation-responsive B'-interaction motifs, and localize at distinct cellular organelles, such as midbody, predicting kinase-facilitated recruitment of PP2A-B' holoenzymes for tight spatiotemporal control of phosphorylation at mitosis and cytokinesis. Moroever, Polo-like kinase 1-mediated phosphorylation of Cyk4/RACGAP1, a centralspindlin component at the midbody, facilitates binding of both RhoA guanine nucleotide exchange factor (epithelial cell transforming sequence 2 (Ect2)) and PP2A-B' that in turn dephosphorylates Cyk4 and disrupts Ect2 binding. This feedback signaling loop precisely controls RhoA activation and specifies a restricted region for cleavage furrow ingression. Our results provide a framework for further investigation of diverse signaling circuits formed by PP2A-B' holoenzymes in various cellular processes.

Structural hierarchy controlling dimerization and target DNA recognition in the AHR transcriptional complex.

The aryl hydrocarbon receptor (AHR) belongs to the PAS (PER-ARNT-SIM) family transcription factors and mediates broad responses to numerous environmental pollutants and cellular metabolites, modulating diverse biological processes from adaptive metabolism, acute toxicity, to normal physiology of vascular and immune systems. The AHR forms a transcriptionally active heterodimer with ARNT (AHR nuclear translocator), which recognizes the dioxin response element (DRE) in the promoter of downstream genes. We determined the crystal structure of the mammalian AHR-ARNT heterodimer in complex with the DRE, in which ARNT curls around AHR into a highly intertwined asymmetric architecture, with extensive heterodimerization interfaces and AHR interdomain interactions. Specific recognition of the DRE is determined locally by the DNA-binding residues, which discriminates it from the closely related hypoxia response element (HRE), and is globally affected by the dimerization interfaces and interdomain interactions. Changes at the interdomain interactions caused either AHR constitutive nuclear localization or failure to translocate to nucleus, underlying an allosteric structural pathway for mediating ligand-induced exposure of nuclear localization signal. These observations, together with the global higher flexibility of the AHR PAS-A and its loosely packed structural elements, suggest a dynamic structural hierarchy for complex scenarios of AHR activation induced by its diverse ligands.