Toll-like receptor 2 and 6 agonist fibroblast-stimulating lipopeptide increases expression and secretion of CXCL1 and CXCL2 by uveal melanocytes.

Fibroblast-stimulating lipopeptide (FSL-1) can activate Toll-like receptor 2 and 6 (TLR2/6), which recognize relevant molecules from gram-positive pathogens, fungus, and mycoplasma, and elevates the expression of CXCL1 and CXCL2, neutrophil chemoattractants, in certain types of cells. This effect has not previously been reported in the uveal melanocytes (UM). This study was designed to test the hypothesis that FSL-1 can induce the expression and secretion of CXCL1 and CXCL2 via activation of TLR2/6 in cultured human UM and producing an acute non-infectious uveitis reaction in the mouse. Flow cytometry and fluorescent immunostaining were used to measure the effect of FSL-1 on the expression of TLR2/6 in UM. Real time PCR and ELISA analysis were used to assess the ability of FSL-1 to elevate CXCL1/CXCL2 levels in cell lysates and conditioned media of UM, respectively. Flow cytometry measured phosphorylated MAPK and activated NF-κB signals in UM, with and without FSL-1 treatment. ELISA analysis tested the impact of various signal inhibitors (NF-κB, p38 MAPK, JNK1/2 and ERK1/2) and TLR2/6 antagonists on FSL-1-induced CXCL1/CXCL2 levels in cultured UM. The effects of neutralizing antibodies to TLR2 on FSL-1-induced mouse uveitis were tested in an experimental animal model. FSL-1 induced the expression of TLR2/6 proteins in cultured UM. FSL-1 significantly elevated the CXCL1 and CXCL2 proteins and mRNA levels in cultured UM time- and dose-dependently. FSL-1 mainly activated NF-κB, JNK, and expression of TLR2. FSL-1-induced expression of CXCL1 and CXCL2 was blocked by NF-κB, JNK, ERK inhibitors and TLR2 antagonists. Intravitreal injection of FSL-1 induced acute non-infectious mouse uveitis, which was significantly reduced in severity by a TLR2 antagonist. These results suggest that UM may play a role in the immune reaction, which targets invading pathogens, especially gram-positive bacteria. On the other hand, an excessive reaction to molecules from gram-positive bacteria may promote an inflammatory state of non-infectious uveitis.

Development of N-Acetylated Dipalmitoyl-S-Glyceryl Cysteine Analogs as Efficient TLR2/TLR6 Agonists.

Cancer vaccine is a promising immunotherapeutic approach to train the immune system with vaccines to recognize and eliminate tumors. Adjuvants are compounds that are necessary in cancer vaccines to mimic an infection process and amplify immune responses. The Toll-like receptor 2 and 6 (TLR2/TLR6) agonist dipalmitoyl-S-glyceryl cysteine (Pam2Cys) was demonstrated as an ideal candidate for synthetic vaccine adjuvants. However, the synthesis of Pam2Cys requires expensive N-protected cysteine as a key reactant, which greatly limits its application as a synthetic vaccine adjuvant in large-scaled studies. Here, we report the development of N-acetylated Pam2Cys analogs as TLR2/TLR6 agonists. Instead of N-protected cysteine, the synthesis utilizes N-acetylcysteine to bring down the synthetic costs. The N-acetylated Pam2Cys analogs were demonstrated to activate TLR2/TLR6 in vitro. Moreover, molecular docking studies were performed to provide insights into the molecular mechanism of how N-acetylated Pam2Cys analogs bind to TLR2/TLR6. Together, these results suggest N-acetylated Pam2Cys analogs as inexpensive and promising synthetic vaccine adjuvants to accelerate the development of cancer vaccines in the future.

A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia.

Regulation of iron metabolism and innate immunity are tightly interlinked. The acute phase response to infection and inflammation induces alterations in iron homeostasis that reduce iron supplies to pathogens. The iron hormone hepcidin is activated by such stimuli causing degradation of the iron exporter ferroportin and reduced iron release from macrophages, suggesting that hepcidin is the crucial effector of inflammatory hypoferremia. Here, we report the discovery of an acute inflammatory condition that is mediated by Toll-like receptors 2 and 6 (TLR2 and TLR6) and which induces hypoferremia in mice injected with TLR ligands. Stimulation of TLR2/TLR6 triggers profound decreases in ferroportin messenger RNA and protein expression in bone marrow-derived macrophages, liver, and spleen of mice without changing hepcidin expression. Furthermore, C326S ferroportin mutant mice with a disrupted hepcidin/ferroportin regulatory circuitry respond to injection of the TLR2/6 ligands FSL1 or PAM3CSK4 by ferroportin downregulation and a reduction of serum iron levels. Our findings challenge the prevailing role of hepcidin in hypoferremia and suggest that rapid hepcidin-independent ferroportin downregulation in the major sites of iron recycling may represent a first-line response to restrict iron access for numerous pathogens.

Pegylated bisacycloxypropylcysteine, a diacylated lipopeptide ligand of TLR6, plays a host-protective role against experimental Leishmania major infection.

TLRs recognize pathogen-expressed Ags and elicit host-protective immune response. Although TLR2 forms heterodimers with TLR1 or TLR6, recognizing different ligands, differences in the functions of these heterodimers remain unknown. In this study, we report that in Leishmania major-infected macrophages, the expression of TLR1 and TLR2, but not TLR6, increased; TLR2-TLR2 association increased, but TLR2-TLR6 association diminished. Lentivirus-expressed TLR1-short hairpin RNA (shRNA) or TLR2-shRNA administration reduced, but TLR6-shRNA increased L. major infection in BALB/c mice. Corroboratively, Pam3CSK4 (TLR1-TLR2 ligand) and peptidoglycan (TLR2 ligand) increased L. major infection but reduced TLR9 expression, whereas pegylated bisacycloxypropylcysteine (BPPcysMPEG; TLR2-TLR6 ligand) reduced L. major number in L. major-infected macrophages, accompanied by increased TLR9 expression, higher IL-12 production, and inducible NO synthase expression. Whereas MyD88, Toll/IL-1R adaptor protein, and TNFR-α-associated factor 6 recruitments to TLR2 were not different in Pam3CSK4-, peptidoglycan-, or BPPcysMPEG-treated macrophages, only BPPcysMPEG enhanced p38MAPK and activating transcription factor 2 activation. BPPcysMPEG conferred antileishmanial functions to L. major-infected BALB/c-derived T cells in a macrophage-T cell coculture and in BALB/c mice; the protection was TLR6 dependent and IL-12 dependent, and it was accompanied by reduced regulatory T cell number. BPPcysMPEG administration during the priming with fixed L. major protected BALB/c mice against challenge L. major infection; the protection was accompanied by low IL-4 and IL-10, but high IFN-γ productions and reduced regulatory T cells. Thus, BPPcysMPEG, a novel diacylated lipopeptide ligand for TLR2-TLR6 heterodimer, induces IL-12-dependent, inducible NO synthase-dependent, T-reg-sensitive antileishmanial protection. The data reveal a novel dimerization partner-dependent duality in TLR2 function.

TLR2/6 agonists and interferon-gamma induce human melanoma cells to produce CXCL10.

Clinical approaches to treat advanced melanoma include immune therapies, whose benefits depend on tumor-reactive T-cell infiltration of metastases. However, most tumors lack significant immune infiltration prior to therapy. Selected chemokines promote T-cell migration into tumors; thus, agents that induce these chemokines in the tumor microenvironment (TME) may improve responses to systemic immune therapy. CXCL10 has been implicated as a critical chemokine supporting T-cell infiltration into the TME. Here, we show that toll-like receptor (TLR) agonists can induce chemokine production directly from melanoma cells when combined with IFNγ treatment. We find that TLR2 and TLR6 are widely expressed on human melanoma cells, and that TLR2/6 agonists (MALP-2 or FSL-1) synergize with interferon-gamma (IFNγ) to induce production of CXCL10 from melanoma cells. Furthermore, melanoma cells and immune cells from surgical specimens also respond to TLR2/6 agonists and IFNγ by upregulating CXCL10 production, compared to treatment with either agent alone. Collectively, these data identify a novel mechanism for inducing CXCL10 production directly from melanoma cells, with TLR2/6 agonists +IFNγ and raise the possibility that intratumoral administration of these agents may improve immune signatures in melanoma and have value in combination with other immune therapies, by supporting T-cell migration into melanoma metastases.

The TLR-2/TLR-6 agonist macrophage-activating lipopeptide-2 augments human NK cell cytotoxicity when PGE2 production by monocytes is inhibited by a COX-2 blocker.

Macrophage-activating lipopeptide-2 (MALP-2) is a potent inducer of proinflammatory cytokine secretion by macrophages, monocytes, and dendritic cells. MALP-2 was reported to be involved in natural killer (NK) cell activation and ensuing tumor rejection. However, the mechanism of MALP-2-mediated NK cell activation remained unclear. Therefore, we studied the effects of MALP-2 on cultured human NK cells. We found that MALP-2 had no direct effect on NK cells. Instead, MALP-2 acted on monocytes and triggered the release of different molecules such as interleukin (IL)-1ß, IL-6, IL-10, IL-12, IL-15, interferon gamma-induced protein (IP-10), and prostaglandin (PG)-E2. Our data show that monocyte-derived IP-10 could significantly induce NK cell cytotoxicity as long as the immunosuppression by PGE2 is specifically inhibited by cyclooxygenase (COX)-2 blockade. In summary, our results show that MALP-2-mediated stimulation of monocytes results in the production of several mediators which, depending on the prevailing conditions, affect the activity of NK cells in various ways. Hence, MALP-2 administration with concurrent blocking of COX-2 can be considered as a promising approach in MALP-2-based adjuvant tumor therapies.

Cow-to-cow variation in fibroblast response to a toll-like receptor 2/6 agonist and its relation to mastitis caused by intramammary challenge with Staphylococcus aureus.

Staphylococcus aureus is a common cause of chronic mammary gland infections in dairy cattle. However, the inflammatory response and duration of infection following pathogen exposure is variable between individual animals. To investigate interanimal differences in immune response, dermal fibroblast cultures were established from skin biopsies collected from 50 early lactation Holstein cows. The fibroblasts ability to produce IL-8 in response to a 24-h treatment with a synthetic toll-like receptor 2/6 agonist (Pam2CSK4) was used to assign a response phenotype to the animals. Five high-responding and 5 low-responding animals were then selected for an intramammary challenge with S. aureus to evaluate differences in the inflammatory response, chronicity of infection, and development of antibodies to the pathogen. All animals exhibited clinical symptoms of mastitis at 24h postchallenge. Animals previously classified as high responders experienced a greater inflammatory response characterized by elevated levels of milk somatic cell count, IL-8, and BSA following the challenge compared with low responders. In addition, antibodies toward the challenge strain of S. aureus reached higher levels in whey from the challenged gland of high responders compared with low responders. Despite the antibody response, all 5 high responders were chronically infected for the 6-wk duration of the study, whereas 2 of the low responders cleared the infection, although 1 of these did become reinfected. The observed differences between animals classified as low and high responders based on their fibroblast responsiveness suggests that this cell type can be used to further examine the causes of interanimal variation in response to mammary infection.

Epigallocatechin gallate targeting of membrane type 1 matrix metalloproteinase-mediated Src and Janus kinase/signal transducers and activators of transcription 3 signaling inhibits transcription of colony-stimulating factors 2 and 3 in mesenchymal stromal cells.

BACKGROUND: CSF-2 and CSF-3 confer proangiogenic and immunomodulatory properties to mesenchymal stromal cells (MSCs). RESULTS: Transcriptional regulation of CSF-2 and CSF-3 in concanavalin A-activated MSCs requires MT1-MMP signaling and is inhibited by EGCG. CONCLUSION: The chemopreventive properties of diet-derived EGCG alter MT1-MMP-mediated intracellular signaling. SIGNIFICANCE: Pharmacological targeting of MSCs proangiogenic functions may prevent their contribution to tumor development. Epigallocatechin gallate (EGCG), a major form of tea catechins, possesses immunomodulatory and antiangiogenic effects, both of which contribute to its chemopreventive properties. In this study, we evaluated the impact of EGCG treatment on the expression of colony-stimulating factors (CSF) secreted from human bone marrow-derived mesenchymal stromal cells (MSCs), all of which also contribute to the immunomodulatory and angiogenic properties of these cells. MSCs were activated with concanavalin A (ConA), a Toll-like receptor (TLR)-2 and TLR-6 agonist as well as a membrane type 1 matrix metalloproteinase (MT1-MMP) inducer, which increased granulocyte macrophage-CSF (GM-CSF, CSF-2), granulocyte CSF (G-CSF, CSF-3), and MT1-MMP gene expression. EGCG antagonized the ConA-induced CSF-2 and CSF-3 gene expression, and this process required an MT1-MMP-mediated sequential activation of the Src and JAK/STAT pathways. Gene silencing of MT1-MMP expression further demonstrated its requirement in the phosphorylation of Src and STAT3, whereas overexpression of a nonphosphorylatable MT1-MMP mutant (Y573F) abrogated CSF-2 and CSF-3 transcriptional increases. Given that MSCs are recruited within vascularizing tumors and are believed to contribute to tumor angiogenesis, possibly through secretion of CSF-2 and CSF-3, our study suggests that diet-derived polyphenols such as EGCG may exert chemopreventive action through pharmacological targeting of the MT1-MMP intracellular signaling.

Toll-like receptor 2/6 agonist macrophage-activating lipopeptide-2 promotes reendothelialization and inhibits neointima formation after vascular injury.

OBJECTIVE: Reendothelialization after vascular injury (ie, balloon angioplasty or stent implantation) is clinically extremely relevant to promote vascular healing. We here investigated the therapeutic potential of the toll-like receptor 2/6 agonist macrophage-activating lipopeptide (MALP)-2 on reendothelialization and neointima formation in a murine model of vascular injury. APPROACH AND RESULTS: The left common carotid artery was electrically injured, and reendothelialization was quantified by Evans blue staining after 3 days. A single injection of MALP-2 (1 or 10 µg, IV) after vascular injury accelerated reendothelialization (P<0.001). Proliferation of endothelial cells at the wound margins determined by 5-ethynyl-2'-deoxyuridine incorporation was significantly higher in MALP-2-treated animals (P<0.05). Furthermore, wire injury-induced neointima formation of the left common carotid artery was completely prevented by a single injection of MALP-2 (10 µg, IV). In vitro, MALP-2 induced proliferation (BrdU incorporation) and closure of an artificial wound of endothelial cells (P<0.05) but not of smooth muscle cells. Protein array and ELISA analysis of isolated primary endothelial cells and ex vivo stimulated carotid segments revealed that MALP-2 stimulated the release of multiple growth factors and cytokines predominantly from endothelial cells. MALP-2 induced a strong activation of the mitogen-activated protein kinase cascade in endothelial cells, which was attenuated in smooth muscle cells. Furthermore, MALP-2 significantly enhanced circulating monocytes and hematopoietic progenitor cells. CONCLUSIONS: The toll-like receptor 2/6 agonist MALP-2 promotes reendothelialization and inhibits neointima formation after experimental vascular injury via enhanced proliferation and migration of endothelial cells. Thus, MALP-2 represents a novel therapeutic option to accelerate reendothelialization after vascular injury.

Toll-like receptor-2/6 and Toll-like receptor-9 agonists suppress viral replication but not airway hyperreactivity in guinea pigs.

Respiratory virus infections cause airway hyperreactivity (AHR). Preventative strategies for virus-induced AHR remain limited. Toll-like receptors (TLRs) have been suggested as a therapeutic target because of their central role in triggering antiviral immune responses. Previous studies showed that concurrent treatment with TLR2/6 and TLR9 agonists reduced lethality and the microbial burden in murine models of bacterial and viral pneumonia. This study investigated the effects of TLR2/6 and TLR9 agonist pretreatment on parainfluenza virus pneumonia and virus-induced AHR in guinea pigs in vivo. Synthetic TLR2/6 lipopeptide agonist Pam2CSK4 and Class C oligodeoxynucleotide TLR9 agonist ODN2395, administered in combination 24 hours before virus infection, significantly reduced viral replication in the lung. Despite a fivefold reduction in viral titers, concurrent TLR2/6 and TLR9 agonist pretreatment did not prevent virus-induced AHR or virus-induced inhibitory M2 muscarinic receptor dysfunction. Interestingly, the TLR agonists independently caused non-M2-dependent AHR. These data confirm the therapeutic antiviral potential of TLR agonists, while suggesting that virus inhibition may be insufficient to prevent virus-induced airway pathophysiology. Furthermore, TLR agonists independently cause AHR, albeit through a distinctly different mechanism from that of parainfluenza virus.

Synergistic interactions of TLR2/6 and TLR9 induce a high level of resistance to lung infection in mice.

Infectious pneumonias exact an unacceptable mortality burden worldwide. Efforts to protect populations from pneumonia have focused historically on antibiotic development and vaccine-enhanced adaptive immunity. However, we have reported recently that the lungs' innate defenses can be induced therapeutically by inhalation of a bacterial lysate that protects mice against otherwise lethal pneumonia. In this study, we tested in mice the hypothesis that TLRs are required for this antimicrobial phenomenon and found that resistance could not be induced in the absence of the TLR signaling adaptor protein MyD88. We then attempted to recapitulate the protection afforded by the bacterial lysate by stimulating the lung epithelium with aerosolized synthetic TLR ligands. Although most single or combination treatments yielded no protection, simultaneous treatment with ligands for TLR2/6 and TLR9 conferred robust, synergistic protection against virulent gram-positive and gram-negative pathogens. Protection was associated with rapid pathogen killing in the lungs, and pathogen killing could be induced from lung epithelial cells in isolation. Taken together, these data demonstrate the requirement for TLRs in inducible resistance against pneumonia, reveal a remarkable, unanticipated synergistic interaction of TLR2/6 and TLR9, reinforce the emerging evidence supporting the antimicrobial capacity of the lung epithelium, and may provide the basis for a novel clinical therapeutic that can protect patients against pneumonia during periods of peak vulnerability.

Toll-like receptor-2 agonist-allergen coupling efficiently redirects Th2 cell responses and inhibits allergic airway eosinophilia.

Toll-like receptor (TLR) agonists beneficially modulate allergic airway inflammation. However, the efficiency of TLR agonists varies considerably, and their exact cellular mechanisms (especially of TLR 2/6 agonists) are incompletely understood. We investigated at a cellular level whether the administration of the pharmacologically improved TLR2/6 agonist S-[2,3-bispalmitoyiloxy-(2R)-propyl]-R-cysteinyl-amido-monomethoxy polyethylene glycol (BPP) conjugated to antigenic peptide (BPP-OVA) could divert an existing Th2 response and influence airway eosinophilia. The effects of BPP-OVA on airway inflammation were assessed in a classic murine sensitization/challenge model and an adoptive transfer model, which involved the adoptive transfer of in vitro differentiated ovalbumin (OVA)-specific Th2 cells. Functional T-cell stimulation by lung dendritic cells (DCs) was determined both in vitro and in vivo, combined with a cytokine secretion analysis. A single mucosal application of BPP-OVA efficiently delivered antigen, led to TLR2-mediated DC activation, and resulted in OVA-specific T-cell proliferation via lung DCs in vivo. In alternative models of allergic airway disease, a single administration of BPP-OVA before OVA challenge (but not BPP alone) significantly reduced airway eosinophilia, most likely through altered antigen-specific T-cell stimulation via DCs. Analyses of adoptively transferred Th2-biased cells after BPP-OVA administration in vivo suggested that BPP-OVA guides antigen-specific Th2 cells to produce significantly higher amounts of IFN-γ upon allergen challenge. In conclusion, our data show for the first time that a single mucosal administration of a TLR 2/6 agonist-allergen conjugate can provoke IFN-γ responses in Th2-biased cells and alleviate allergic airway inflammation.

Atherosclerosis induced by endogenous and exogenous toll-like receptor (TLR)1 or TLR6 agonists.

Atherosclerosis is a chronic inflammatory vascular disease. Toll-like receptors (TLRs) are major initiators of inflammation. TLR2 promotes atherosclerosis in LDL receptor (LDLr)-deficient mice fed a high-fat diet (HFD). TLR2 forms heterodimers with TLR1 or TLR6 to enable inflammatory responses in the presence of distinct ligands. Here we asked whether TLR1 and/or TLR6 are required. We studied atherosclerotic disease using either TLR1- or TLR6-deficient mice. Deficiency of TLR1 or TLR6 did not diminish HFD-driven disease. When HFD-fed LDLr-deficient mice were challenged with Pam3 or MALP2, specific exogenous ligands of TLR2/1 or TLR2/6, respectively, atherosclerotic lesions developed with remarkable intensity in the abdominal segment of the descending aorta. In contrast to atherosclerosis induced by the endogenous agonists, these lesions were diminished by deficiency of either TLR1 or TLR6. The endogenous ligand(s) that arise from consumption of a HFD and promote disease via TLR2 are unknown. Either TLR1 or TLR6 are redundant for this endogenous ligand detection, or they are both irrelevant to endogenous ligand detection. However, the exogenous ligands Pam3 and MALP2 promote severe abdominal atherosclerosis in the descending aorta that is dependent on TLR1 and TLR6, respectively.

Stimulation of pulmonary immune responses by the TLR2/6 agonist MALP-2 and effect on melanoma metastasis to the lung.

Given that metastasized melanoma is a fatal disease in most cases, it is tempting to develop strategies to a priori prevent metastasis. We have stimulated the pulmonary innate immune system by macrophage-activating lipopeptide-2 (MALP-2), a specific agonist at Toll-like receptor (TLR) 2/6, and investigated its impact on experimental melanoma metastasis. In C57BL/6 mice, intratracheal application of MALP-2 induced a profound influx of neutrophils and macrophages into the lung, which peaked after 24 h (sixfold increase) and returned to baseline within 72 h. Further analysis revealed that MALP-2 also markedly induced VCAM-1 expression on pulmonary blood vessels. In vitro experiments demonstrated that this adhesion molecule mediates binding of B16F10 melanoma cells. Furthermore, in vivo or in vitro treatment with MALP-2 did not significantly affect the ability of immune cells to lyse melanoma cells. As a consequence, notwithstanding the profound pulmonary immune response induction and in contrast to conclusions drawn from some previous publications, the net extent of experimental metastasis did not change significantly, regardless of the application regimen of MALP-2 prior to, concomitant with or after tumor cell inoculation. Melanoma cells stably transfected with green fluorescent protein allowed tracking of early events after tumor cell dissemination and showed that MALP-2-mediated TLR2/6 activation did not interfere with pulmonary melanoma cell arrest. Likewise, boosting the immune induction after establishment of metastases did not change the clinical outcome. These unexpected results vividly counsel caution regarding predictions of immunomodulating therapies, as multiple intertwined effects may influence the net outcome.

Targeted Repolarization of Tumor-Associated Macrophages via Imidazoquinoline-Linked Nanobodies.

Tumor-associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as a promising target for the next generation of cancer immunotherapies. To repolarize their phenotype into a tumoricidal state, the Toll-like receptor 7/8 agonist imidazoquinoline IMDQ is site-specifically and quantitatively coupled to single chain antibody fragments, so-called nanobodies, targeting the macrophage mannose receptor (MMR) on TAMs. Intravenous injection of these conjugates result in a tumor- and cell-specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This is accompanied by a repolarization of TAMs towards a pro-inflammatory phenotype and an increase in anti-tumor T cell responses. Therefore, the therapeutic benefit of such nanobody-drug conjugates may pave the road towards effective macrophage re-educating cancer immunotherapies.

Host-directed therapy in foals can enhance functional innate immunity and reduce severity of Rhodococcus equi pneumonia.

Pneumonia caused by the intracellular bacterium Rhodococcus equi is an important cause of disease and death in immunocompromised hosts, especially foals. Antibiotics are the standard of care for treating R. equi pneumonia in foals, and adjunctive therapies are needed. We tested whether nebulization with TLR agonists (PUL-042) in foals would improve innate immunity and reduce the severity and duration of pneumonia following R. equi infection. Neonatal foals (n = 48) were nebulized with either PUL-042 or vehicle, and their lung cells infected ex vivo. PUL-042 increased inflammatory cytokines in BAL fluid and alveolar macrophages after ex vivo infection with R. equi. Then, the in vivo effects of PUL-042 on clinical signs of pneumonia were examined in 22 additional foals after intrabronchial challenge with R. equi. Foals infected and nebulized with PUL-042 or vehicle alone had a shorter duration of clinical signs of pneumonia and smaller pulmonary lesions when compared to non-nebulized foals. Our results demonstrate that host-directed therapy can enhance neonatal immune responses against respiratory pathogens and reduce the duration and severity of R. equi pneumonia.

Prophylactic intranasal administration of a TLR2/6 agonist reduces upper respiratory tract viral shedding in a SARS-CoV-2 challenge ferret model.

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.

A Toll-like receptor 2/6 agonist reduces allergic airway inflammation in chronic respiratory sensitisation to Timothy grass pollen antigens.

BACKGROUND: The hygiene hypothesis negatively correlates the microbial burden of the environment with the prevalence of T helper type 2 (Th2)-related disorders, e.g. allergy and asthma. This is explained by Th1 triggering through pathogen-associated molecular patterns via Toll-like receptors (TLRs). In this study, the biological effects of a TLR2/6 agonist as a potential treatment of allergic inflammation are explored. METHODS: In a model of chronic allergic airway inflammation induced by intranasal administration of Timothy grass pollen allergen extract, early TLR agonism and/or interferon (IFN)-gamma administration was compared to the therapeutic and immune-modulating effects of dexamethasone with regard to the cellular inflammation and cytokine profiles. RESULTS: Eosinophilic inflammation was clearly reduced by TLR2/6 agonism. This effect was also seen without simultaneous administration of IFN-gamma. However, lymphocyte counts were not affected among the different treatment groups. More precise determination of the lymphocyte-mediated immune reaction showed that TLR2/6 agonism induced neither CD4+foxp3+ regulatory T cells in draining lymph nodes nor a pronounced Th1 immune response. In contrast, dexamethasone reduced both sensitisation as well as allergic inflammation and, in addition, CD11c+ antigen-presenting cells in lymph nodes. Our data clearly point to the potential to rebalance Th2-skewed allergic immune responses by therapeutic TLR2/6 agonist administration. CONCLUSION: The use of the TLR2/6 agonist is a promising therapeutic approach in diseases with an imbalance in T cell responses, such as allergy and asthma.

Differential Response of Gestational Tissues to TLR3 Viral Priming Prior to Exposure to Bacterial TLR2 and TLR2/6 Agonists.

Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB.