A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

Background: A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1ß secretion and their contribution to the overall immune responses remains elusive. Objective: To investigate the mechanisms underlying the production of IL-1ß from rFlaA:Betv1 stimulated macrophages. Methods: Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1ß to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. Results: rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1ß secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1ß-expression in THP-1 macrophages. Finally, lack of IL-1ß positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1ß, IL-6, and TNF-α from peritoneal macrophages. Conclusion: The mechanisms contributing to rFlaA:Betv1-induced IL-1ß secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein.

Trained immune responses, based on metabolic and epigenetic changes in innate immune cells, are de facto innate immune memory and, therefore, are of great interest in vaccine development. In previous studies, the recombinant fusion protein rFlaA:Betv1, combining the adjuvant and toll-like receptor (TLR)5-ligand flagellin (FlaA) and the major birch pollen allergen Bet v 1 into a single molecule, significantly suppressed allergic sensitization in vivo while also changing the metabolism of myeloid dendritic cells (mDCs). Within this study, the immune-metabolic effects of rFlaA:Betv1 during mDC activation were elucidated. In line with results for other well-characterized TLR-ligands, rFlaA:Betv1 increased glycolysis while suppressing oxidative phosphorylation to different extents, making rFlaA:Betv1 a suitable model to study the immune-metabolic effects of TLR-adjuvanted vaccines. In vitro pretreatment of mDCs with cerulenin (inhibitor of fatty acid biosynthesis) led to a decrease in both rFlaA:Betv1-induced anti-inflammatory cytokine Interleukin (IL) 10 and T helper cell type (TH) 1-related cytokine IL-12p70, while the pro-inflammatory cytokine IL 1ß was unaffected. Interestingly, pretreatment with the glutaminase inhibitor BPTES resulted in an increase in IL-1ß, but decreased IL-12p70 secretion while leaving IL-10 unchanged. Inhibition of the glycolytic enzyme hexokinase-2 by 2-deoxyglucose led to a decrease in all investigated cytokines (IL-10, IL-12p70, and IL-1ß). Inhibitors of mitochondrial respiration had no effect on rFlaA:Betv1-induced IL-10 level, but either enhanced the secretion of IL-1ß (oligomycin) or decreased IL-12p70 (antimycin A). In extracellular flux measurements, mDCs showed a strongly enhanced glycolysis after rFlaA:Betv1 stimulation, which was slightly increased after respiratory shutdown using antimycin A. rFlaA:Betv1-stimulated mDCs secreted directly antimicrobial substances in a mTOR- and fatty acid metabolism-dependent manner. In co-cultures of rFlaA:Betv1-stimulated mDCs with CD4+ T cells, the suppression of Bet v 1-specific TH2 responses was shown to depend on fatty acid synthesis. The effector function of rFlaA:Betv1-activated mDCs mainly relies on glycolysis, with fatty acid synthesis also significantly contributing to rFlaA:Betv1-mediated cytokine secretion, the production of antimicrobial molecules, and the modulation of T cell responses.

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.

A Chinese herbal prescription Yiqi Jiedu decoction attenuates irradiation induced testis injury in mice.

OBJECTIVE: Yiqi Jiedu (YQJD) decoction is a Chinese herbal prescription, based on an experienced expert of traditional Chinese medicine. It is used for the injuries caused by radiotherapy. The current study was designed to investigate the protective effects of YQJD decoction on radiation damage of testis in mice, and to explore its potential mechanisms. METHODS: Mice were randomly divided into blank control group (Ctrl), model group (IR), positive drug group (IRA), and YQJD decoction group (IRY). After 10-day period intervention, they were whole-body irradiated with 2 Gy 60Co γ-rays and sacrificed on 7th day after irradiation. The indicators including the index and histopathology examination of testis, spermatogenic cell types and apoptosis, and the expression of TLR5, MyD88, NF-κB, TNF-α, IL-6 and Bcl-2 in testis. RESULTS: The testis atrophied significantly on 7th day of exposure to radiation, while YQJD decoction promoted the recovery of testis index and structure. Moreover, spermatogenic cell types and apoptosis had significant changes after irradiation. YQJD decoction protected the testicular function of spermatogenesis, as while as reduced the apoptosis rate of spermatogenic cells. In addition, RT-PCR and immunohistochemical analysis showed that YQJD decoction up-regulated the expression of TLR5 in testis. The levels of TLR5's downstream factors were also up-regulated in YQJD decoction group, which indicated that TLR5 signaling pathway might play an important role in the protective effects of YQJD decoction. CONCLUSIONS: The results showed that YQJD decoction attenuated irradiation induced testis injury in mice. Its potential mechanism was related to TLR5 signaling pathway.

R. nukuhivensis acts by reinforcing skin barrier function, boosting skin immunity and by inhibiting IL-22 induced keratinocyte hyperproliferation.

Rauvolfia nukuhivensis is a well-known plant used for its wide range of beneficial effects in Marquesas islands. It is made up of diverse indole alkaloids and is used as traditional medicine for skin application. The actual mechanism behind the virtue of this plant is still unknown. Hence, in this study we aimed at deciphering the impact of R. nukuhivensis on skin immune system in context of (1) homeostasis, (2) pathogen infection and (3) inflammation. Here we show that R. nukuhivensis enhances cellular metabolic activity and wound healing without inducing cellular stress or disturbing cellular homeostasis. It reinforces the epithelial barrier by up-regulating hBD-1. Nevertheless, in pathogenic stress, R. nukuhivensis acts by preparing the immune system to be reactive and effective directly. Indeed, it enhances the innate immune response by increasing pathogens sensors such as TLR5. Finally, R. nukuhivensis blocks IL-22 induced hyperproliferation via PTEN and Filaggrin up-regulation as well as BCL-2 downregulation. In conclusion, this study provides evidence on the several cutaneous application potentials of R. nukuhivensis such as boosting the immune response or in restoring the integrity of the epithelial barrier.

Amino acids 89-96 of Salmonella typhimurium flagellin represent the major domain responsible for TLR5-independent adjuvanticity in the humoral immune response.

Toll-like receptor 5 (TLR5) signaling in response to flagellin is dispensable for inducing humoral immunity, but alterations of aa 89-96, the TLR5 binding site, significantly reduced the adjuvanticity of flagellin. These observations indicate that the underlying mechanism remains incompletely understood. Here, we found that the native form of Salmonella typhimurium aa 89-96-mutant flagellin extracted from flagella retains some TLR5 recognition activity, indicating that aa 89-96 is the primary, but not the only site that imparts TLR5 activity. Additionally, this mutation impaired the production of IL-1ß and IL-18. Using TLR5KO mice, we found that aa 89-96 is critical for the humoral adjuvant effect, but this effect was independent of TLR5 activation triggered by this region of flagellin. In summary, our findings suggest that aa 89-96 of flagellin is not only the crucial site responsible for TLR5 recognition, but is also important for humoral immune adjuvanticity through a TLR5-independent pathway.

PMA induces vaccine adjuvant activity by the modulation of TLR signaling pathway.

Toll-like receptor (TLR) ligands are being developed for use as vaccine adjuvants and as immunomodulators because of their ability to stimulate innate and adaptive immune responses. Flagellin, a TLR5 ligand, was reported to show potent mucosal vaccine adjuvant activity. To identify ligands that potentiate the adjuvant activity of flagellin, we screened a plant library using HEK293T cells transiently cotransfected with phTLR5 and pNF- κ B-SEAP plasmids. The 90% EtOH extract from Croton tiglium showed significant NF- κ B transactivation in a TLR5-independent manner along with the increase of a flagellin activity. We have studied to characterize an active component from Croton tiglium and to elucidate the action mechanisms. Phorbol 12-myristate 13-acetate (PMA) was isolated as an active component of Croton tiglium by activity-guided fractionation, column chromatography, HPLC, NMR, and MS. PMA at a range of nM induced PKC-dependent NF- κ B activation and IL-8 production in both TLR5- and TLR5+ assay systems. In in vivo mouse vaccination model, PMA induced antigen-specific IgG and IgA antibody responses and increased IL-12 production corresponding to T cell responses in spleen lymphocytes. These results suggest that PMA would serve as an efficacious mucosal vaccine adjuvant.

Cryptotanshinone inhibits LPS-induced proinflammatory mediators via TLR4 and TAK1 signaling pathway.

Cryptotanshinone (CTN), one of the major constituents of tanshinones, was investigated for anti-inflammatory activity in the murine macrophage cell line RAW 264.7. CTN inhibited the production of nitric oxide (NO) production, as well as expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. Since CTN was considered as inhibiting LPS-triggered phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB activation, we consequently evaluated the expression of toll-like receptor 4 (TLR4) and CD14, as well as phosphorylation of TGF-ß-activated kinase 1 (TAK1). CTN reduced the expression of CD14 and TLR4, and suppressed LPS-induced phosphorylation of TAK1. Furthermore, CTN significantly increased the survival rate against LPS challenge in D-galactosamine-sensitized mice, which was in line with in vitro results. These results suggested that CD14/TLR4 and TAK1 might be the potential molecular targets for addressing the protective effects of CTN on LPS-induced inflammatory effects in macrophages.

TLR5 is not required for flagellin-mediated exacerbation of DSS colitis.

BACKGROUND: The two forms of human inflammatory bowel disease, Crohn's disease (CD) and ulcerative colitis (UC), are both associated with loss of tolerance to gut microbial antigens. The dominant antigen recognized by antibody and T-cell responses in patients with CD is bacterial flagellin. Flagellin is also the only known ligand for Toll-like receptor 5 (TLR5), a key protein in innate immunity. Although flagellin activates TLR5 to produce inflammatory responses in many cell types in the gut, there is conflicting evidence as to whether TLR5 is harmful or protective in CD and murine colitis models. A recent study found that administration of flagellin enemas to mice along with dextran sodium sulfate (DSS) made their colitis worse. METHODS: We sought to determine whether this exacerbation was due to TLR5 ligation, or to TLR5-independent adaptive immune responses to flagellin as an antigen, by using a transposon insertional mutant of the Escherichia coli H18 flagellin, 2H3, which lacks TLR5 stimulatory activity. RESULTS: We found that flagellin enemas produced only a mild exacerbation of DSS colitis, and that 2H3 was equivalent to or worse than wildtype flagellin. Moreover, we found that DSS colitis was more severe in TLR5(-/-) mice than wildtype C57BL/6 mice. CONCLUSIONS: Together, these results suggest that flagellin-mediated exacerbation of colitis is independent of TLR5.