Dectin-2 is a primary receptor for NLRP3 inflammasome activation in dendritic cell response to Histoplasma capsulatum.

Inflammasome is an intracellular protein complex that serves as cytosolic pattern recognition receptor (PRR) to engage with pathogens and to process cytokines of the interleukin-1 (IL-1) family into bioactive molecules. It has been established that interleukin-1ß (IL-1ß) is important to host defense against Histoplasma capsulatum infection. However, the detailed mechanism of how H. capsulatum induces inflammasome activation leading to IL-1ß production has not been studied. Here, we showed in dendritic cells (DCs) that H. capsulatum triggers caspase-1 activation and IL-1ß production through NLRP3 inflammasome. By reciprocal blocking of Dectin-1 or Dectin-2 in single receptor-deficient DCs and cells from Clec4n-/-, Clec7a-/-, and Clec7a-/-Clec4n-/- mice, we discovered that while Dectin-2 operates as a primary receptor, Dectin-1 serves as a secondary one for NLRP3 inflammasome. In addition, both receptors trigger Syk-JNK signal pathway to activate signal 1 (pro-IL-1ß synthesis) and signal 2 (activation of caspase-1). Results of pulmonary infection with H. capsulatum showed that CD103+ DCs are one of the major producers of IL-1ß and Dectin-2 and Dectin-1 double deficiency abolishes their IL-1ß response to the fungus. While K+ efflux and cathepsin B (but not ROS) function as signal 2, viable but not heat-killed H. capsulatum triggers profound lysosomal rupture leading to cathepsin B release. Interestingly, cathepsin B release is regulated by ERK/JNK downstream of Dectin-2 and Dectin-1. Our study demonstrates for the first time the unique roles of Dectin-2 and Dectin-1 in triggering Syk-JNK to activate signal 1 and 2 for H. capsulatum-induced NLRP3 inflammasome activation.

CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection.

Expression of a gene encoding a rice RING zinc-finger protein, OsRZFP34, enhances stomata opening.

By oligo microarray expression profiling, we identified a rice RING zinc-finger protein (RZFP), OsRZFP34, whose gene expression increased with high temperature or abscisic acid (ABA) treatment. As compared with the wild type, rice and Arabidopsis with OsRZFP34 overexpression showed increased relative stomata opening even with ABA treatment. Furthermore, loss-of-function mutation of OsRZFP34 and AtRZFP34 (At5g22920), an OsRZFP34 homolog in Arabidopsis, decreased relative stomata aperture under nonstress control conditions. Expressing OsRZFP34 in atrzfp34 reverted the mutant phenotype to normal, which indicates a conserved molecular function between OsRZFP34 and AtRZFP34. Analysis of water loss and leaf temperature under stress conditions revealed a higher evaporation rate and cooling effect in OsRZFP34-overexpressing Arabidopsis and rice than the wild type, atrzfp34 and osrzfp34. Thus, stomata opening, enhanced leaf cooling, and ABA insensitivity was conserved with OsRZFP34 expression. Transcription profiling of transgenic rice overexpressing OsRZFP34 revealed many genes involved in OsRZFP34-mediated stomatal movement. Several genes upregulated or downregulated in OsRZFP34-overexpressing plants were previously implicated in Ca(2+) sensing, K(+) regulator, and ABA response. We suggest that OsRZFP34 may modulate these genes to control stomata opening.

A novel RBD-protein/peptide vaccine elicits broadly neutralizing antibodies and protects mice and macaques against SARS-CoV-2.

The development of safe and effective vaccines to respond to COVID-19 pandemic/endemic remains a priority. We developed a novel subunit protein-peptide COVID-19 vaccine candidate (UB-612) composed of: (i) receptor binding domain of SARS-CoV-2 spike protein fused to a modified single-chain human IgG1 Fc; (ii) five synthetic peptides incorporating conserved helper and cytotoxic T lymphocyte (Th/CTL) epitopes derived from SARS-CoV-2 structural proteins (three from S2 subunit, one from membrane and one from nucleocapsid), and one universal Th peptide; (iii) aluminum phosphate as adjuvant. The immunogenicity and protective immunity induced by UB-612 vaccine were evaluated in four animal models: Sprague-Dawley rats, AAV-hACE2 transduced BALB/c mice, rhesus and cynomolgus macaques. UB-612 vaccine induced high levels of neutralizing antibody and T-cell responses, in all animals. The immune sera from vaccinated animals neutralized the SARS-CoV-2 original wild-type strains and multiple variants of concern, including Delta and Omicron. The vaccination significantly reduced viral loads, lung pathology scores, and disease progression after intranasal and intratracheal challenge with SARS-CoV-2 in mice, rhesus and cynomolgus macaques. UB-612 has been tested in primary regimens in Phase 1 and Phase 2 clinical studies and is currently being evaluated in a global pivotal Phase 3 clinical study as a single dose heterologous booster.

A multitope SARS-CoV-2 vaccine provides long-lasting B cell and T cell immunity against Delta and Omicron variants.

BackgroundThe Delta and Omicron variants of SARS-CoV-2 are currently responsible for breakthrough infections due to waning immunity. We report phase I/II trial results of UB-612, a multitope subunit vaccine containing S1-RBD-sFc protein and rationally designed promiscuous peptides representing sarbecovirus conserved helper T cell and cytotoxic T lymphocyte epitopes on the nucleocapsid (N), membrane (M), and spike (S2) proteins.MethodWe conducted a phase I primary 2-dose (28 days apart) trial of 10, 30, or 100 µg UB-612 in 60 healthy young adults 20 to 55 years old, and 50 of them were boosted with 100 µg of UB-612 approximately 7 to 9 months after the second dose. A separate placebo-controlled and randomized phase II study was conducted with 2 doses of 100 µg of UB-612 (n = 3,875, 18-85 years old). We evaluated interim safety and immunogenicity of phase I until 14 days after the third (booster) dose and of phase II until 28 days after the second dose.ResultsNo vaccine-related serious adverse events were recorded. The most common solicited adverse events were injection site pain and fatigue, mostly mild and transient. In both trials, UB-612 elicited respective neutralizing antibody titers similar to a panel of human convalescent sera. The most striking findings were long-lasting virus-neutralizing antibodies and broad T cell immunity against SARS-CoV-2 variants of concern (VoCs), including Delta and Omicron, and a strong booster-recalled memory immunity with high cross-reactive neutralizing titers against the Delta and Omicron VoCs.ConclusionUB-612 has presented a favorable safety profile, potent booster effect against VoCs, and long-lasting B and broad T cell immunity that warrants further development for both primary immunization and heterologous boosting of other COVID-19 vaccines.Trial RegistrationClinicalTrials.gov: NCT04545749, NCT04773067, and NCT04967742.FundingUBI Asia, Vaxxinity Inc., and Taiwan Centers for Disease Control, Ministry of Health and Welfare.

Immunization with apoptotic phagocytes containing Histoplasma capsulatum activates functional CD8(+) T cells to protect against histoplasmosis.

We have previously revealed the protective role of CD8(+) T cells in host defense against Histoplasma capsulatum in animals with CD4(+) T cell deficiency and demonstrated that sensitized CD8(+) T cells are restimulated in vitro by dendritic cells that have ingested apoptotic macrophage-associated Histoplasma antigen. Here we show that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently activated functional CD8(+) T cells whose contribution was equal to that of CD4(+) T cells in protection against Histoplasma challenge. Inhibition of macrophage apoptosis due to inducible nitric oxide synthase (iNOS) deficiency or by caspase inhibitor treatment dampened the CD8(+) T cell but not the CD4(+) T cell response to pulmonary Histoplasma infection. In mice subcutaneously immunized with viable Histoplasma yeasts whose CD8(+) T cells are protective against Histoplasma challenge, there was heavy granulocyte and macrophage infiltration and the infiltrating cells became apoptotic. In mice subcutaneously immunized with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic macrophages containing heat-killed Histoplasma, the CFSE-labeled macrophage material was found to localize within dendritic cells in the draining lymph node. Moreover, depleting dendritic cells in immunized CD11c-DTR mice significantly reduced CD8(+) T cell activation. Taken together, our results revealed that phagocyte apoptosis in the Histoplasma-infected host is associated with CD8(+) T cell activation and that immunization with apoptotic phagocytes containing heat-killed Histoplasma efficiently evokes a protective CD8(+) T cell response. These results suggest that employing apoptotic phagocytes as antigen donor cells is a viable approach for the development of efficacious vaccines to elicit strong CD8(+) T cell as well as CD4(+) T cell responses to Histoplasma infection.

NLRX1 Facilitates Histoplasma capsulatum-Induced LC3-Associated Phagocytosis for Cytokine Production in Macrophages.

LC3-associated phagocytosis (LAP) is an emerging non-canonical autophagy process that bridges signaling from pattern-recognition receptors (PRRs) to autophagic machinery. LAP formation results in incorporation of lipidated LC3 into phagosomal membrane (termed LAPosome). Increasing evidence reveals that LAP functions as an innate defense mechanism against fungal pathogens. However, the molecular mechanism involved and the consequence of LAP in regulating anti-fungal immune response remain largely unexplored. Here we show that Histoplasma capsulatum is taken into LAPosome upon phagocytosis by macrophages. Interaction of H. capsulatum with Dectin-1 activates Syk and triggers subsequent NADPH oxidase-mediated reactive oxygen species (ROS) response that is involved in LAP induction. Inhibiting LAP induction by silencing LC3α/ß or treatment with ROS inhibitor impairs the activation of MAPKs-AP-1 pathway, thereby reduces macrophage proinflammatory cytokine response to H. capsulatum. Additionally, we unravel the importance of NLRX1 in fungus-induced LAP. NLRX1 facilitates LAP by interacting with TUFM which associates with autophagic proteins ATG5-ATG12 for LAPosome formation. Macrophages from Nlrx1-/- mice or TUFM-silenced cells exhibit reduced LAP induction and LAP-mediated MAPKs-AP-1 activation for cytokine response to H. capsulatum. Furthermore, inhibiting ROS production in Nlrx1-/- macrophages almost completely abolishes H. capsulatum-induced LC3 conversion, indicating that both Dectin-1/Syk/ROS-dependent pathway and NLRX1-TUFM complex-dependent pathway collaboratively contribute to LAP induction. Our findings reveal new pathways underlying LAP induction by H. capsulatum for macrophage cytokine response.

Mucosa-Associated Lymphoid Tissue Lymphoma Translocation Protein 1 Positively Modulates Matrix Metalloproteinase-9 Production in Alveolar Macrophages upon Toll-Like Receptor 7 Signaling and Influenza Virus Infection.

Influenza A virus (IAV) infection causes significant morbidity and mortality worldwide. Matrix metalloproteinase-9 (MMP-9) degrades extracellular matrix and is involved in the pathology of influenza. It has been reported that MMP-9 mediates neutrophil migration in IAV infection. Whether alveolar macrophages, the first immune cells that encounter IAV, produce MMP-9, and the mechanism of its regulation have never been investigated. As Toll-like receptor 7 (TLR7) is one of the receptors in innate immune cells that recognize IAV, we used TLR7 agonists and IAV to stimulate alveolar macrophage MH-S cells, primary macrophages, and bone marrow neutrophils. Results showed that MMP-9 expression in macrophages is inducible by TLR7 agonists and IAV, yet, MMP-9 production by neutrophils is not inducible by either one of them. We hypothesized that MMP-9 production in macrophages is mediated through TLR7-NF-κB pathway and used microarray to analyze TLR7 agonist-induced NF-κB-related genes. Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), a positive regulator of NF-κB, is amongst the top highly induced genes. By use of MALT1 inhibitor (z-VRPR-fmk) and alveolar macrophages from MALT1-deficient mice, we found that MMP-9 production is MALT1-dependent. While MALT1 can act as a paracaspase in lymphocytes through degrading various signaling proteins, we discovered that MALT1 functions to reduce a negative regulator of NF-κB, cylindromatosis (CYLD), in alveolar macrophages. IAV-induced MMP-9, TNF, and IL-6 in lungs of MALT1-deficient mice are significantly lower than in wild-type mice after intratracheal infection. MALT1-deficient mice also have less body weight loss and longer survival after infection. Taken together, we demonstrated a novel role of MALT1 in regulating alveolar macrophage MMP-9 production whose presence exacerbates the severity of influenza.

Cell Intrinsic Galectin-3 Attenuates Neutrophil ROS-Dependent Killing of Candida by Modulating CR3 Downstream Syk Activation.

Invasive candidiasis is a leading cause of nosocomial bloodstream infection. Neutrophils are the important effector cells in host resistance to candidiasis. To investigate the modulation of neutrophil fungicidal function will advance our knowledge on the control of candidiasis. While recombinant galectin-3 enhances neutrophil phagocytosis of Candida, we found that intracellular galectin-3 downregulates neutrophil fungicidal functions. Co-immunoprecipitation and immunofluorescence staining reveal that cytosolic gal3 physically interacts with Syk in neutrophils after Candida stimulation. Gal3-/- neutrophils have higher level of Syk activation as well as greater abilities to generate reactive oxygen species (ROS) and kill Candida than gal3+/+ cells. While galectin-3 deficiency modulates neutrophil and macrophage activation and the recruitment of monocytes and dendritic cells, the deficiency does not affect the numbers of infiltrating neutrophils or macrophages. Galectin-3 deficiency ameliorates systemic candidiasis by reducing fungal burden, renal pathology, and mortality. Adoptive transfer experiments demonstrate that cell intrinsic galectin-3 negatively regulates neutrophil effector functions against candidiasis. Reducing galectin-3 expression or activity by siRNA or gal3 inhibitor TD139 enhances human neutrophil ROS production. Mice treated with TD139 have enhanced ability to clear the fungus. Our work unravels the mechanism by which galectin-3 regulates Syk-dependent neutrophil fungicidal functions and raises the possibility that blocking gal3 in neutrophils may be a promising therapeutic strategy for treating systemic candidiasis.

Distinct roles of complement receptor 3, Dectin-1, and sialic acids in murine macrophage interaction with Histoplasma yeast.

The yeast cells of dimorphic fungal pathogen Histoplasma reside primarily within the macrophages of an infected host; the interaction between the yeast and macrophage has a profound impact on host defense against the fungus. We used blocking antibodies and saccharides to identify the receptors that participate in the phagocytosis of and the cytokine response to Histoplasma. The phagocytosis and cytokine response results show that sialic acids on the macrophages were involved in the interaction between macrophages and Histoplasma. CR3, although not the only receptor involved, was responsible for phagocytosis and cytokine response. It is unclear which receptors other than CR3 are responsible for phagocytosis, but we did rule out the participation of TLR2, TLR4, MR, DC-SIGN/SIGNR1, FcgammaR, VLA-5, and Dectin-1. Even though Dectin-1 did not participate in phagocytosis, it collaborated with CR3 in the cytokine response to Histoplasma, suggesting that in the presence of phagocytic receptors, Histoplasma triggers cytokine signals through Dectin-1. Moreover, macrophage phagocytosis of and cytokine response to Histoplasma are Syk kinase-dependent. Our study delineated the distinct roles of CR3, Dectin-1, and sialic acids in the interaction with Histoplasma and suggested that multiple receptor use might be important to host defense against Histoplasma.