Human gut mycobiota tune immunity via CARD9-dependent induction of anti-fungal IgG antibodies.

Antibodies mediate natural and vaccine-induced immunity against viral and bacterial pathogens, whereas fungi represent a widespread kingdom of pathogenic species for which neither vaccine nor neutralizing antibody therapies are clinically available. Here, using a multi-kingdom antibody profiling (multiKAP) approach, we explore the human antibody repertoires against gut commensal fungi (mycobiota). We identify species preferentially targeted by systemic antibodies in humans, with Candida albicans being the major inducer of antifungal immunoglobulin G (IgG). Fungal colonization of the gut induces germinal center (GC)-dependent B cell expansion in extraintestinal lymphoid tissues and generates systemic antibodies that confer protection against disseminated C. albicans or C. auris infection. Antifungal IgG production depends on the innate immunity regulator CARD9 and CARD9+CX3CR1+ macrophages. In individuals with invasive candidiasis, loss-of-function mutations in CARD9 are associated with impaired antifungal IgG responses. These results reveal an important role of gut commensal fungi in shaping the human antibody repertoire through CARD9-dependent induction of host-protective antifungal IgG.

Fungal microbiota sustains lasting immune activation of neutrophils and their progenitors in severe COVID-19.

Gastrointestinal fungal dysbiosis is a hallmark of several diseases marked by systemic immune activation. Whether persistent pathobiont colonization during immune alterations and impaired gut barrier function has a durable impact on host immunity is unknown. We found that elevated levels of Candida albicans immunoglobulin G (IgG) antibodies marked patients with severe COVID-19 (sCOVID-19) who had intestinal Candida overgrowth, mycobiota dysbiosis and systemic neutrophilia. Analysis of hematopoietic stem cell progenitors in sCOVID-19 revealed transcriptional changes in antifungal immunity pathways and reprogramming of granulocyte myeloid progenitors (GMPs) for up to a year. Mice colonized with C. albicans patient isolates experienced increased lung neutrophilia and pulmonary NETosis during severe acute respiratory syndrome coronavirus-2 infection, which were partially resolved with antifungal treatment or by interleukin-6 receptor blockade. sCOVID-19 patients treated with tocilizumab experienced sustained reductions in C. albicans IgG antibodies titers and GMP transcriptional changes. These findings suggest that gut fungal pathobionts may contribute to immune activation during inflammatory diseases, offering potential mycobiota-immune therapeutic strategies for sCOVID-19 with prolonged symptoms.

Immune regulation by fungal strain diversity in inflammatory bowel disease.

The fungal microbiota (mycobiota) is an integral part of the complex multikingdom microbial community colonizing the mammalian gastrointestinal tract and has an important role in immune regulation1-6. Although aberrant changes in the mycobiota have been linked to several diseases, including inflammatory bowel disease3-9, it is currently unknown whether fungal species captured by deep sequencing represent living organisms and whether specific fungi have functional consequences for disease development in affected individuals. Here we developed a translational platform for the functional analysis of the mycobiome at the fungal-strain- and patient-specific level. Combining high-resolution mycobiota sequencing, fungal culturomics and genomics, a CRISPR-Cas9-based fungal strain editing system, in vitro functional immunoreactivity assays and in vivo models, this platform enables the examination of host-fungal crosstalk in the human gut. We discovered a rich genetic diversity of opportunistic Candida albicans strains that dominate the colonic mucosa of patients with inflammatory bowel disease. Among these human-gut-derived isolates, strains with high immune-cell-damaging capacity (HD strains) reflect the disease features of individual patients with ulcerative colitis and aggravated intestinal inflammation in vivo through IL-1ß-dependent mechanisms. Niche-specific inflammatory immunity and interleukin-17A-producing T helper cell (TH17 cell) antifungal responses by HD strains in the gut were dependent on the C. albicans-secreted peptide toxin candidalysin during the transition from a benign commensal to a pathobiont state. These findings reveal the strain-specific nature of host-fungal interactions in the human gut and highlight new diagnostic and therapeutic targets for diseases of inflammatory origin.

Immunoglobulins at the interface of the gut mycobiota and anti-fungal immunity.

The dynamic and complex community of microbes that colonizes the intestines is composed of bacteria, fungi, and viruses. At the mucosal surfaces, immunoglobulins play a key role in protection against bacterial and fungal pathogens, and their toxins. Secretory immunoglobulin A (sIgA) is the most abundantly produced antibody at the mucosal surfaces, while Immunoglobulin G (IgG) isotypes play a critical role in systemic protection. IgA and IgG antibodies with reactivity to commensal fungi play an important role in shaping the mycobiota and host antifungal immunity. In this article, we review the latest evidence that establishes a connection between commensal fungi and B cell-mediated antifungal immunity as an additional layer of protection against fungal infections and inflammation.

Inhaled Fine Particles Induce Alveolar Macrophage Death and Interleukin-1α Release to Promote Inducible Bronchus-Associated Lymphoid Tissue Formation.

Particulate pollution is thought to function as an adjuvant that can induce allergic responses. However, the exact cell types and immunological factors that initiate the lung-specific immune responses are unclear. We found that upon intratracheal instillation, particulates such as aluminum salts and silica killed alveolar macrophages (AMs), which then released interleukin-1α (IL-1α) and caused inducible bronchus-associated lymphoid tissue (iBALT) formation in the lung. IL-1α release continued for up to 2 weeks after particulate exposure, and type-2 allergic immune responses were induced by the inhalation of antigen during IL-1α release and iBALT formation, even long after particulate instillation. Recombinant IL-1α was sufficient to induce iBALTs, which coincided with subsequent immunoglobulin E responses, and IL-1-receptor-deficient mice failed to induce iBALT formation. Therefore, the AM-IL-1α-iBALT axis might be a therapeutic target for particulate-induced allergic inflammation.

Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation.

Agonists for TLR9 and stimulator of IFN genes (STING) offer therapeutic applications as both anti-tumor agents and vaccine adjuvants, though their clinical applications are limited; the clinically available TLR9 agonist is a weak IFN inducer and STING agonists induce undesired type 2 immunity. Yet, combining TLR9 and STING agonists overcame these limitations by synergistically inducing innate and adaptive IFNγ to become an advantageous type 1 adjuvant, suppressing type 2 immunity, in addition to exerting robust anti-tumor activities when used as a monotherapeutic agent for cancer immunotherapy. Here, we sought to decipher the immunological mechanisms behind the synergism mediated by TLR9 and STING agonists and found that their potent anti-tumor immunity in a Pan02 peritoneal dissemination model of pancreatic cancer was achieved only when agonists for TLR9 and STING were administered locally, and was via mechanisms involving CD4 and CD8 T cells as well as the co-operative action of IL-12 and type I IFNs. Rechallenge studies of long-term cancer survivors suggested that the elicitation of Pan02-specific memory responses provides protection against the secondary tumor challenge. Mechanistically, we found that TLR9 and STING agonists synergistically induce IL-12 and type I IFN production in murine APCs. The synergistic effect of the TLR9 and STING agonists on IL-12p40 was at protein, mRNA and promoter activation levels, and transcriptional regulation was mediated by a 200 bp region situated 983 bp upstream of the IL-12p40 transcription initiation site. Such intracellular transcriptional synergy may hold a key in successful cancer immunotherapy and provide further insights into dual agonism of innate immune sensors during host homeostasis and diseases.

ZBP1 governs the inflammasome-independent IL-1α and neutrophil inflammation that play a dual role in anti-influenza virus immunity.

Influenza A virus (IAV) triggers the infected lung to produce IL-1 and recruit neutrophils. Unlike IL-1ß, however, little is known about IL-1α in terms of its mechanism of induction, action and physiological relevance to the host immunity against IAV infection. In particular, whether Z-DNA-binding protein 1 (ZBP1), a key molecule for IAV-induced cell death, is involved in the IL-1α induction, neutrophil infiltration and the physiological outcome has not been elucidated. Here, we show in a murine model that the IAV-induced IL-1α is mediated solely by ZBP1, in an NLRP3-inflammasome-independent manner, and is required for the optimal IL-1ß production followed by the formation of neutrophil extracellular traps (NETs). During IAV infection, ZBP1 displays a dual role in anti-IAV immune responses mediated by neutrophils, resulting in either protective or pathological outcomes in vivo. Thus, ZBP1-mediated IL-1α production is the key initial step of IAV-infected NETs, regulating the duality of the consequent lung inflammation.

CD63-Mediated Antigen Delivery into Extracellular Vesicles via DNA Vaccination Results in Robust CD8+ T Cell Responses.

DNA vaccines are attractive immunogens for priming humoral and cellular immune responses to the encoded Ag. However, their ability to induce Ag-specific CD8+ T cell responses requires improvement. Among the strategies for improving DNA vaccine immunogenicity are booster vaccinations, alternate vaccine formulations, electroporation, and genetic adjuvants, but few, such as extracellular vesicles (EVs), target natural Ag delivery systems. By focusing on CD63, a tetraspanin protein expressed on various cellular membranes, including EVs, we examined whether a DNA vaccine encoding an Ag fused to CD63 delivered into EVs would improve vaccine immunogenicity. In vitro transfection with plasmid DNA encoding an OVA Ag fused to CD63 (pCD63-OVA) produced OVA-carrying EVs. Immunizations with the purified OVA-carrying EVs primed naive mice to induce OVA-specific CD4+ and CD8+ T cells, whereas immunization with EVs purified from cells transfected with control plasmids encoding OVA protein alone or a calnexin-OVA fusion protein delivered into the endoplasmic reticulum failed to do so. Vaccinating mice with pCD63-OVA induced potent Ag-specific T cell responses, particularly those from CD8+ T cells. CD63 delivery into EVs led to better CD8+ T cell responses than calnexin delivery into the endoplasmic reticulum. When we used a mouse tumor implantation model to evaluate pCD63-OVA as a therapeutic vaccine, the EV-delivered DNA vaccination significantly inhibited tumor growth compared with the control DNA vaccinations. These results indicate that EV Ag delivery via DNA vaccination offers a new strategy for eliciting strong CD8+ T cell responses to the encoded Ag, making it a potentially useful cancer vaccine.

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding.

Various bacteria are suggested to contribute to colorectal cancer (CRC) development, including pks+ E. coli which produce the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells. It remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells and its DNA to cause harm. Using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC, we found that pks+ E. coli drives CRC exacerbation and tissue invasion in a colibactin-dependent manner. Using isogenic mutant strains, we further demonstrate that CRC exacerbation critically depends on expression of the E. coli type-1 pilus adhesin FimH and the F9-pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. Together, we show that the oncogenic potential of pks+ E. coli critically depends on bacterial adhesion to host epithelial cells and is critically mediated by specific bacterial adhesins. Adhesin-mediated epithelial binding subsequently allows production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic avenues for the development of anti-adhesive therapies aiming at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC.

Safety and immunogenicity of a quadrivalent seasonal influenza vaccine adjuvanted with hydroxypropyl-ß-cyclodextrin: A phase 1 clinical trial.

OBJECTIVES: Hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), an oligosaccharide used as an excipient in pharmaceutical preparation, was recently reported to function as a vaccine adjuvant to co-administered antigens. In this study, we investigated the safety and immunogenicity of a seasonal influenza vaccine adjuvanted with HP-ß-CyD (FluCyD-vac) in healthy adults compared with those of a standard seasonal influenza vaccine (Flu-vac). METHODS: We conducted a single-blinded randomized phase 1 clinical trial study, and used two quadrivalent split seasonal influenza vaccines: FluCyD-vac containing 9 µg of HA/strain and 20% w/v of HP-ß-CyD, and Flu-vac containing 15 µg of hemagglutinin (HA)/strain only. All participants were randomly assigned to receive a single dose of Flu/CyD-vac or Flu-vac at a ratio of 2:1. We assessed solicited and unsolicited adverse events (AEs) and immune responses using hemagglutination inhibition (HI) titers. In addition, we assessed T-cell function in peripheral blood mononuclear cells (PBMCs), after stimulation with HA vaccine strains, using flow cytometry. RESULTS: Among 36 healthy volunteers enrolled in the study (FluCyD-vac, n = 24; Flu-vac, n = 12), FluCyD-vac was well tolerated. Most of the solicited AEs were mild local skin reactions at the injection site. No serious AEs were reported in either group. HI titers 21 days after vaccination with FluCyD-vac were comparable with those of Flu-vac and sufficient to meet international criteria, despite reduced HA antigen doses. When PBMCs were stimulated with the four HA antigens in the vaccine, tumor necrosis factor (TNF)-α-producing CD4+ T cells were enhanced in the FluCyD-vac group. CONCLUSION: FluCyD-vac was well-tolerated and immunogenic, despite containing 40% less HA antigens than Flu-vac. This study showed that HP-ß-CyD is a potentially safe, novel adjuvant for human influenza vaccine. CLINICAL TRIAL REGISTRY: UMIN000028530.

Mycobiota-induced IgA antibodies regulate fungal commensalism in the gut and are dysregulated in Crohn's disease.

Secretory immunoglobulin A (sIgA) plays an important role in gut barrier protection by shaping the resident microbiota community, restricting the growth of bacterial pathogens and enhancing host protective immunity via immunological exclusion. Here, we found that a portion of the microbiota-driven sIgA response is induced by and directed towards intestinal fungi. Analysis of the human gut mycobiota bound by sIgA revealed a preference for hyphae, a fungal morphotype associated with virulence. Candida albicans was a potent inducer of IgA class-switch recombination among plasma cells, via an interaction dependent on intestinal phagocytes and hyphal programming. Characterization of sIgA affinity and polyreactivity showed that hyphae-associated virulence factors were bound by these antibodies and that sIgA influenced C. albicans morphotypes in the murine gut. Furthermore, an increase in granular hyphal morphologies in patients with Crohn's disease compared with healthy controls correlated with a decrease in antifungal sIgA antibody titre with affinity to two hyphae-associated virulence factors. Thus, in addition to its importance in gut bacterial regulation, sIgA targets the uniquely fungal phenomenon of hyphal formation. Our findings indicate that antifungal sIgA produced in the gut can play a role in regulating intestinal fungal commensalism by coating fungal morphotypes linked to virulence, thereby providing a protective mechanism that might be dysregulated in patients with Crohn's disease.

IL-33 Is Essential for Adjuvant Effect of Hydroxypropyl-ß-Cyclodexrin on the Protective Intranasal Influenza Vaccination.

Vaccine adjuvants are traditionally used to augment and modulate the immunogenicity of vaccines, although in many cases it is unclear which specific molecules contribute to their stimulatory activity. We previously reported that both subcutaneous and intranasal administration of hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a pharmaceutical excipient widely used to improve solubility, can act as an effective adjuvant for an influenza vaccine. However, the mechanisms by which mucosal immune pathway is critical for the intranasal adjuvant activity of HP-ß-CD have not been fully delineated. Here, we show that intranasally administered HP-ß-CD elicits a temporary release of IL-33 from alveolar epithelial type 2 cells in the lung; notably, IL-33 expression in these cells is not stimulated following the use of other vaccine adjuvants. The experiments using gene deficient mice suggested that IL-33/ST2 signaling is solely responsible for the adjuvant effect of HP-ß-CD when it is administered intranasally. In contrast, the subcutaneous injection of HP-ß-CD and the intranasal administration of alum, as a damage-associated molecular patterns (DAMPs)-inducing adjuvant, or cholera toxin, as a mucosal adjuvant, enhanced humoral immunity in an IL-33-independent manner, suggesting that the IL-33/ST2 pathway is unique to the adjuvanticity of intranasally administered HP-ß-CD. Furthermore, the release of IL-33 was involved in the protective immunity against influenza virus infection which is induced by the intranasal administration of HP-ß-CD-adjuvanted influenza split vaccine. In conclusion, our results suggest that an understanding of administration route- and tissue-specific immune responses is crucial for the design of unique vaccine adjuvants.

Cyclic GMP-AMP Triggers Asthma in an IL-33-Dependent Manner That Is Blocked by Amlexanox, a TBK1 Inhibitor.

Extracellular host-derived DNA, as one of damage associated molecular patterns (DAMPs), is associated with allergic type 2 immune responses. Immune recognition of such DNA generates the second messenger cyclic GMP-AMP (cGAMP) and induces type-2 immune responses; however, its role in allergic diseases, such as asthma, has not been fully elucidated. This study aimed to determine whether cGAMP could induce asthma when used as an adjuvant. We intranasally sensitized mice with cGAMP together with house dust mite antigen (HDM), followed by airway challenge with HDM. We then assessed the levels of eosinophils in the broncho-alveolar lavage fluid (BALF) and serum HDM-specific antibodies. cGAMP promoted HDM specific allergic asthma, characterized by significantly increased HDM specific IgG1 and total IgE in the serum and infiltration of eosinophils in the BALF. cGAMP stimulated lung fibroblast cells to produce IL-33 in vitro, and mice deficient for IL-33 or IL-33 receptor (ST2) failed to develop asthma enhancement by cGAMP. Not only Il-33-/- mice, but also Sting-/-, Tbk1-/-, and Irf3-/-Irf7-/- mice which lack the cGAMP-mediated innate immune activation failed to increase eosinophils in the BALF than that from wild type mice. Consistently, intranasal and oral administration of amlexanox, a TBK1 inhibitor, decreased cGAMP-induced lung allergic inflammation. Thus, cGAMP functions as a type 2 adjuvant in the lung and can promote allergic asthma in manners that dependent on the intracellular STING/TBK1/IRF3/7 signaling pathway and the resultant intercellular signaling pathway via IL-33 and ST2 might be a novel therapeutic target for allergic asthma.

DAMP-Inducing Adjuvant and PAMP Adjuvants Parallelly Enhance Protective Type-2 and Type-1 Immune Responses to Influenza Split Vaccination.

Recently, it was reported that 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CyD), a common pharmaceutical additive, can act as a vaccine adjuvant to enhance protective type-2 immunogenicity to co-administered seasonal influenza split vaccine by inducing host-derived damage-associated molecular patterns (DAMPs). However, like most other DAMP-inducing adjuvants such as aluminum hydroxide (Alum), HP-ß-CyD may not be sufficient for the induction of protective type-1 (cellular) immune responses, thereby leaving room for improvement. Here, we demonstrate that a combination of HP-ß-CyD with a humanized TLR9 agonist, K3 CpG-ODN, a potent pathogen-associated molecular pattern (PAMP), enhanced the protective efficacy of the co-administered influenza split vaccine by inducing antigen-specific type-2 and type-1 immune responses, respectively. Moreover, substantial antigen-specific IgE induction by HP-ß-CyD, which can cause an allergic response to immunized antigen was completely suppressed by the addition of K3 CpG-ODN. Furthermore, HP-ß-CyD- and K3 CpG-ODN-adjuvanted influenza split vaccination protected the mice against lethal challenge with high doses of heterologous influenza virus, which could not be protected against by single adjuvant vaccines. Further experiments using gene deficient mice revealed the unique immunological mechanism of action in vivo, where type-2 and type-1 immune responses enhanced by the combined adjuvants were dependent on TBK1 and TLR9, respectively, indicating their parallel signaling pathways. Finally, the analysis of immune responses in the draining lymph node suggested that HP-ß-CyD promotes the uptake of K3 CpG-ODN by plasmacytoid dendritic cells and B cells, which may contributes to the activation of these cells and enhanced production of IgG2c. Taken together, the results above may offer potential clinical applications for the combination of DAMP-inducing adjuvant and PAMP adjuvant to improve vaccine immunogenicity and efficacy by enhancing both type-2 and type-1 immune responses in a parallel manner.

Intranasal hydroxypropyl-ß-cyclodextrin-adjuvanted influenza vaccine protects against sub-heterologous virus infection.

Intranasal vaccination with inactivated influenza viral antigens is an attractive and valid alternative to currently available influenza (flu) vaccines; many of which seem to need efficient and safe adjuvant, however. In this study, we examined whether hydroxypropyl-ß-cyclodextrin (HP-ß-CD), a widely used pharmaceutical excipient to improve solubility and drug delivery, can act as a mucosal adjuvant for intranasal flu vaccines. We found that intranasal immunization of mice with hemagglutinin split- as well as inactivated whole-virion influenza vaccine with HP-ß-CD resulted in secretion of antigen-specific IgA and IgGs in the airway mucosa and the serum as well. As a result, both HP-ß-CD adjuvanted-flu intranasal vaccine protected mice against lethal challenge with influenza virus, equivalent to those induced by experimental cholera toxin-adjuvanted ones. Of note, intranasal use of HP-ß-CD as an adjuvant induced significantly lower antigen-specific IgE responses than that induced by aluminum salt adjuvant. These results suggest that HP-ß-CD may be a potent mucosal adjuvant for seasonal and pandemic influenza vaccine.

RNA is an Adjuvanticity Mediator for the Lipid-Based Mucosal Adjuvant, Endocine.

Nasal vaccination has the potential to elicit systemic and mucosal immunity against pathogens. However, split and subunit vaccines lack potency at stimulating mucosal immunity, and an adjuvant is indispensable for eliciting potent mucosal immune response to nasal vaccines. Endocine, a lipid-based mucosal adjuvant, potentiates both systemic and mucosal immune responses. Although Endocine has shown efficacy and tolerability in animal and clinical studies, its mechanism of action remains unknown. It has been reported recently that endogenous danger signals are essential for the effects of some adjuvants such as alum or MF59. However, the contribution of danger signals to the adjuvanticity of Endocine has not been explored. Here, we show that RNA is likely to be an important mediator for the adjuvanticity of Endocine. Administration of Endocine generated nucleic acids release, and activated dendritic cells (DCs) in draining lymph nodes in vivo. These results suggest the possibility that Endocine indirectly activates DCs via damage-associated molecular patterns. Moreover, the adjuvanticity of Endocine disappeared in mice lacking TANK-binding kinase 1 (Tbk1), which is a downstream molecule of nucleic acid sensing signal pathway. Furthermore, co-administration of RNase A reduced the adjuvanticity of Endocine. These data suggest that RNA is important for the adjuvanticity of Endocine.

Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells.

Recent evidence suggest that a ß-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.