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Intranasal vaccines are anticipated to be powerful tools for combating many infectious diseases, including SARS- CoV-2, because they induce not only systemic immunity but also mucosal immunity at the site of initial infection. However, they are generally inefficient in inducing an antigen-specific immune response without adjuvants. Here, we developed an adjuvant-free intranasal vaccine platform that utilizes the preexisting immunity induced by previous infection or vaccination to enhance vaccine effectiveness. We made RBD-HA, a fusion of the receptor- binding domain (RBD) of spike derived from SARS-CoV-2 as a vaccine target with HA derived from influenza A virus (IAV) as a carrier protein. Intranasal immunization of previously IAV- infected mice with RBD-HA without an adjuvant elicited robust production of RBD-specific systemic IgG and mucosal IgA by utilizing both HA-specific preexisting IgG and [CD4.sup.+] T cells. Consequently, the mice were efficiently protected from SARS-CoV-2 infection. Additionally, we demonstrated the high versatility of this intranasal vaccine platform by assessing various vaccine antigens and preexisting immunity associated with a variety of infectious diseases. The results of this study suggest the promising potential of this intranasal vaccine platform to address problems associated with intranasal vaccines., Introduction The SARS-CoV-2 pandemic has accelerated vaccine development at an unprecedented rate. Several types of COVID-19 vaccines, including mRNA and adenovirus-vector vaccines expressing the SARS-CoV-2 spike glycoprotein, provide highly effective [...]

Salmonella species are among the world's most prevalent pathogens. Because the cell wall interfaces with the host, we designed a lipidomics approach to reveal pathogen-specific cell wall compounds. Among the molecules differentially expressed between Salmonella Paratyphi and S. Typhi, we focused on lipids that are enriched in S. Typhi, because it causes typhoid fever. We discovered a previously unknown family of trehalose phospholipids, 6,6'-diphosphatidyltrehalose (diPT) and 6-phosphatidyltrehalose (PT). Cardiolipin synthase B (ClsB) is essential for PT and diPT but not for cardiolipin biosynthesis. Chemotyping outperformed clsB homology analysis in evaluating synthesis of diPT. DiPT is restricted to a subset of Gram-negative bacteria: large amounts are produced by S. Typhi, lower amounts by other pathogens, and variable amounts by Escherichia coli strains. DiPT activates Mincle, a macrophage activating receptor that also recognizes mycobacterial cord factor (6,6'-trehalose dimycolate). Thus, Gram-negative bacteria show convergent function with mycobacteria. Overall, we discovered a previously unknown immunostimulant that is selectively expressed among medically important bacterial species.

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd-induced lipid substrates that define Gaucher's disease, Wolman's disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis-induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages., Introduction To benefit from immune cloaking and gain access to nutrients within host cells, intracellular bacteria must evade, withstand, or defeat host killing. Bacteria enter macrophages through phagocytosis, where infected [...]

Staphylococcus aureus is a prevalent pathogen in pneumonia and harbors glycolipids, which may serve as molecular patterns in Mincle (macrophage-inducible C-type lectin)–dependent pathogen recognition. We examined the role of Mincle in lung defense against S aureus in wild-type (WT), Mincle knockout (KO), and Mincle transgenic (tg) mice. Two glycolipids, glucosyl-diacylglycerol (Glc-DAG) and diglucosyl-diacylglycerol (Glc2-DAG), were purified, of which only Glc-DAG triggered Mincle reporter cell activation and professional phagocyte responses. Proteomic profiling revealed that Glc2-DAG blocked Glc-DAG–induced cytokine responses, thereby acting as inhibitor of Glc-DAG/Mincle signaling. WT mice responded to S aureus with a similar lung pathology as Mincle KO mice, most likely due to Glc2-DAG–dependent inhibition of Glc-DAG/Mincle signaling. In contrast, ectopic Mincle expression caused severe lung pathology in S aureus –infected mice, characterized by bacterial outgrowth and fatal pneumonia. Collectively, Glc2-DAG inhibits Glc-DAG/Mincle–dependent responses in WT mice, whereas sustained Mincle expression overrides Glc2-DAG–mediated inhibitory effects, conferring increased host susceptibility to S aureus. [ABSTRACT FROM AUTHOR]

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.

The electronic and vibrational structures of 1,2-benzanthracene-h12 (aBA-h12) and 1,2-benzanthracene-d12 (aBA-d12) were elucidated by analyzing fluorescence excitation spectra and dispersed fluorescence spectra in a supersonic jet on the basis of DFT calculation. We also observed the high-resolution and high-precision fluorescence excitation spectrum of the S 1 ← S 0 0 0 0 band, and determined the accurate rotational constants in the zero-vibrational levels of the S0 and S1 states. In this high-resolution measurement, we used a single-mode UV laser whose frequencies were controlled with reference to an optical frequency comb. The inertial defect is negligibly small, the molecule is considered to be planar, and the obtained rotational constants were well reproduced by the equation-of-motion coupled cluster singles and doubles (EOM-CCSD) calculation. Both a-type and b-type transitions are found to be included in the rotationally resolved spectrum, and the a-type contribution is dominant, that is, the transition moment is nearly parallel to the long axis of the aBA molecule. We concluded that the S1 state is mainly composed of the Φ(B) configuration. The observed fluorescence lifetime (106 ns) is considerably longer than that of the Φ(A) system, such as anthracene (18 ns). The transition moment for the lower state of mixed states becomes small, reflecting a near-cancelation of the contributions from the parts of the wavefunction corresponding to the two electronic configurations. The bandwidth of the S2 ← S0 transition is large, and the structure is complicated. It is attributed to vibronic coupling with the high vibrational levels of the S1 state. [ABSTRACT FROM AUTHOR]

C-type lectin receptors (CLRs) are a family of pattern recognition receptors, which detect a broad spectrum of ligands via small carbohydrate-recognition domains (CRDs). CLEC12A is an inhibitory CLR that recognizes crystalline structures such as monosodium urate crystals. CLEC12A also recognizes mycolic acid, a major component of mycobacterial cell walls, and suppresses host immune responses. Although CLEC12A could be a therapeutic target for mycobacterial infection, structural information on CLEC12A was not available. We report here the crystal structures of human CLEC12A (hCLEC12A) in ligand-free form and in complex with 50C1, its inhibitory antibody. 50C1 recognizes human-specific residues on the top face of hCLEC12A CRD. A comprehensive alanine scan demonstrated that the ligand-binding sites of mycolic acid and monosodium urate crystals may overlap with each other, suggesting that CLEC12A utilizes a common interface to recognize different types of ligands. Our results provide atomic insights into the blocking and ligand-recognition mechanisms of CLEC12A and leads to the design of CLR-specific inhibitors. [ABSTRACT FROM AUTHOR]

C-type lectin receptors (CLRs) expressed by myeloid cells constitute a versatile family of receptors that play a key role in innate immune recognition. Myeloid CLRs exhibit a remarkable ability to recognize an extensive array of ligands, from carbohydrates and beyond, and encompass pattern-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), and markers of altered self. These receptors, classified into distinct subgroups, play pivotal roles in immune recognition and modulation of immune responses. Their intricate signaling pathways orchestrate a spectrum of cellular responses, influencing processes such as phagocytosis, cytokine production, and antigen presentation. Beyond their contributions to host defense in viral, bacterial, fungal, and parasitic infections, myeloid CLRs have been implicated in non-infectious diseases such as cancer, allergies, and autoimmunity. A nuanced understanding of myeloid CLR interactions with endogenous and microbial triggers is starting to uncover the context-dependent nature of their roles in innate immunity, with implications for therapeutic intervention. C-type lectin receptors (CLRs) comprise a vast family of proteins, several of which play key roles in the immune system. In this article, Reis e Sousa et al. review the biology of CLRs expressed by myeloid cells and their role in innate immune responses to infection, commensal organisms, and tissue injury or dysfunction. [ABSTRACT FROM AUTHOR]

Dectin‐1 is a well‐characterized C‐type lectin receptor involved in anti‐fungal immunity through the recognition of polysaccharides; however, molecular mechanisms and outcomes initiated through self‐recognition have not been fully understood. Here, we purified a water‐soluble fraction from mouse liver that acts as a Dectin‐1 agonist. To address the physiological relevance of this recognition, we utilized sterile liver inflammation models. The CCl4‐induced hepatitis model showed that Dectin‐1 deficiency led to reduced inflammation through decreased inflammatory cell infiltration and lower pro‐inflammatory cytokine levels. Moreover, in a NASH model induced by streptozotocin and a high‐fat diet, hepatic inflammation and fibrosis were ameliorated in Dectin‐1‐deficient mice. The Dectin‐1 agonist activity was increased in the water‐soluble fraction from NASH mice, suggesting a potential pathogenic cycle between Dectin‐1 activation and hepatitis progression. In vivo administration of the fraction into mice induced hepatic inflammation. These results highlight a role of self‐recognition through Dectin‐1 that triggers hepatic innate immune responses and contributes to the exacerbation of inflammation in pathogenic settings. Thus, the blockade of this axis may provide a therapeutic option for liver inflammatory diseases. [ABSTRACT FROM AUTHOR]

Although chitin in fungal cell walls is associated with allergic airway inflammation, the precise mechanism underlying this association has yet to be elucidated. Here, we investigated the involvement of fungal chitin-binding protein and chitin in allergic airway inflammation. Recombinant Aspergillus fumigatus LdpA (rLdpA) expressed in Pichia pastoris was shown to be an O-linked glycoprotein containing terminal α-mannose residues recognized by the host C-type lectin receptor, Dectin-2. Chitin particles were shown to induce acute neutrophilic airway inflammation mediated release of interleukin-1α (IL-1α) associated with cell death. Furthermore, rLdpA–Dectin-2 interaction was shown to promote phagocytosis of rLdpA–chitin complex and activation of mouse bone marrow-derived dendritic cells (BMDCs). Moreover, we showed that rLdpA potently induced T helper 2 (Th2)-driven allergic airway inflammation synergistically with chitin, and Dectin-2 deficiency attenuated the rLdpA–chitin complex-induced immune response in vivo. In addition, we showed that serum LdpA-specific immunoglobulin levels were elevated in patients with pulmonary aspergillosis. Author summary: Asthma is the most common chronic disease among children in many countries. Although its fundamental causes are not completely understood, the inhalation of allergens, including fungal spores is a known risk factor for the development of asthma. Recent studies have shown that sensitization to the filamentous fungus, Aspergillus fumigatus, is associated with exacerbation of allergic asthma and other pulmonary diseases. Although chitin in fungal cell walls is associated with allergic airway inflammation, the precise mechanism underlying this association has yet to be elucidated. In this study, we showed that A. fumigatus chitin-binding glycoprotein, LysM domain protein A (LdpA), synergistically induces Dectin-2-mediated Th2-driven airway inflammation with chitin. Therefore, this study provided new insights into fungal-induced allergic airway diseases. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognize bacterial riboflavin–based metabolites as activating antigens. Although MAIT cells are found in tissues, it is unknown whether any host tissue–derived antigens exist. Here, we report that a sulfated bile acid, cholic acid 7-sulfate (CA7S), binds the nonclassical MHC class I protein MR1 and is recognized by MAIT cells. CA7S is a host-derived metabolite whose levels were reduced by more than 98% in germ-free mice. Deletion of the sulfotransferase 2a family of enzymes (Sult2a1-8) responsible for CA7S synthesis reduced the number of thymic MAIT cells in mice. Moreover, recognition of CA7S induced MAIT cell survival and the expression of a homeostatic gene signature. By contrast, recognition of a previously described foreign antigen, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil (5-OP-RU), drove MAIT cell proliferation and the expression of inflammatory genes. Thus, CA7S is an endogenous antigen for MAIT cells, which promotes their development and function. Editor's summary: Mucosal-associated invariant T (MAIT) cells are an innate-like T cell subset important for mucosal homeostasis, which recognize microbiota-derived vitamin B metabolites presented by the MHC-Ib molecule MR1. It is unclear, however, whether any host-derived ligands are also important for MAIT cell function and survival. Ito et al. report that the host-derived bile acid metabolite cholic acid 7-sulfate (CA7S) binds MR1 and is recognized by MAIT cells in mice. Moreover, genetic deletion of sulfotransferase 2a enzymes (Sult2a1-8) needed for its synthesis in mice reduced thymic MAIT cell numbers. A previously characterized vitamin B metabolite, 5-OP-RU, triggered human MAIT cell proliferation and the expression of inflammatory genes. By contrast, CA7S promoted human MAIT cell survival and a gene program associated with wound healing and immunoregulation. —Seth Thomas Scanlon [ABSTRACT FROM AUTHOR]