A mechanism for the induction of type 2 immune responses by a protease allergen in the genital tract.

The genital mucosa is a barrier that is constantly exposed to a variety of pathogens, allergens, and external stimuli. Although both allergen exposure and parasite infections frequently occur in the genital area, the mechanism by which immune responses-particularly type 2 immunity-are induced has rarely been studied in the genital mucosa. Here, we demonstrate the induction of T helper type 2 (Th2) immunity in the genital mucosa in response to a model allergen, the protease papain. Intravaginal papain immunization induced type 2 immunity in a manner that was dependent on protease activity and the estrous phase of the mice. In addition, IL-33 was released from the vaginal epithelia after intravaginal papain immunization, leading to the activation of type 2 innate lymphoid cells (ILC2s). Moreover, the IL-33-MyD88 (myeloid differentiation primary response gene 88) signaling pathway was critical for the induction of type 2 immunity. We also found that Th2 differentiation in response to intravaginal papain treatment requires a specific dendritic cell (DC) subset that is controlled by interferon regulatory factor 4 (IRF4). These findings suggest that type 2 immunity is induced by a unique mechanism in the genital tract, which is an important, but often overlooked, barrier surface.

SARS-CoV-2 infection elucidates unique features of pregnancy-specific immunity.

Pregnancy is a risk factor for increased severity of SARS-CoV-2 and other respiratory infections. The mechanisms underlying this risk have not been well-established, partly due to a limited understanding of how pregnancy shapes immune responses. To gain insight into the role of pregnancy in modulating immune responses at steady state and upon perturbation, we collected peripheral blood mononuclear cells (PBMC), plasma, and stool from 226 women, including 152 pregnant individuals (n = 96 with SARS-CoV-2 infection and n = 56 healthy controls) and 74 non-pregnant women (n = 55 with SARS-CoV-2 and n = 19 healthy controls). We found that SARS-CoV-2 infection was associated with altered T cell responses in pregnant compared to non-pregnant women. Differences included a lower percentage of memory T cells, a distinct clonal expansion of CD4-expressing CD8 + T cells, and the enhanced expression of T cell exhaustion markers, such as programmed cell death-1 (PD-1) and T cell immunoglobulin and mucin domain-3 (Tim-3), in pregnant women. We identified additional evidence of immune dysfunction in severely and critically ill pregnant women, including a lack of expected elevation in regulatory T cell (Treg) levels, diminished interferon responses, and profound suppression of monocyte function. Consistent with earlier data, we found maternal obesity was also associated with altered immune responses to SARS-CoV-2 infection, including enhanced production of inflammatory cytokines by T cells. Certain gut bacterial species were altered in pregnancy and upon SARS-CoV-2 infection in pregnant individuals compared to non-pregnant women. Shifts in cytokine and chemokine levels were also identified in the sera of pregnant individuals, most notably a robust increase of interleukin-27 (IL-27), a cytokine known to drive T cell exhaustion, in the pregnant uninfected control group compared to all non-pregnant groups. IL-27 levels were also significantly higher in uninfected pregnant controls compared to pregnant SARS-CoV-2-infected individuals. Using two different preclinical mouse models of inflammation-induced fetal demise and respiratory influenza viral infection, we found that enhanced IL-27 protects developing fetuses from maternal inflammation but renders adult female mice vulnerable to viral infection. These combined findings from human and murine studies reveal nuanced pregnancy-associated immune responses, suggesting mechanisms underlying the increased susceptibility of pregnant individuals to viral respiratory infections.

Monocytes Contribute to IFN-ß Production via the MyD88-Dependent Pathway and Cytotoxic T-Cell Responses against Mucosal Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of respiratory viral infection in infants and children. However, little is known about the contribution of monocytes to antiviral responses against RSV infection. We identified the IFN-ß production of monocytes using IFN-ß/YFP reporter mice. The kinetic analysis of IFN-ß-producing cells in in vivo RSV-infected lung cells indicated that monocytes are recruited to the inflamed lung during the early phase of infection. These cells produced IFN-ß via the myeloid differentiation factor 88-mediated pathway, rather than the TLR7- or mitochondrial antiviral signaling protein-mediated pathway. In addition, monocyte-ablated mice exhibited decreased numbers of IFN-γ-producing and RSV Ag-specific CD8+ T cells. Collectively, these data indicate that monocytes play pivotal roles in cytotoxic T-cell responses and act as type I IFN producers during RSV infection.

Autophagic protein ATG5 controls antiviral immunity via glycolytic reprogramming of dendritic cells against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infections in infants. Macroautophagy/autophagy is a catalytic metabolic process required for cellular homeostasis. Although intracellular metabolism is important for immune responses in dendritic cells, the link between autophagy and immunometabolism remains unknown. Here, we show that the autophagy-related protein ATG5 regulates immunometabolism. Atg5-deficient mouse dendritic cells showed increased CD8A+ T-cell response and increased secretion of proinflammatory cytokines upon RSV infection. Transcriptome analysis showed that Atg5 deficiency alters the expression of metabolism-related genes. Atg5-deficient dendritic cells also showed increased activation of glycolysis and the AKT-MTOR-RPS6KB1 pathway and decreased mitochondrial activity, all of which are cellular signatures for metabolic activation. These cells also showed elevated CD8A+ T-cell priming and surface major histocompatibility complex (MHC) class I expression. Our results suggested that ATG5 regulated host immune responses by modulating dendritic cell metabolism. These findings may help develop potential antiviral therapies that alter host immunity by regulating autophagy and immunometabolism.Abbreviations : 2-DG: 2-deoxyglucose; AAK1: AP2 associated kinase 1; AKT: AKT serine/threonine kinase; AM: alveolar macrophage; ATG: autophagy; ATP: adenosine triphosphate; BAL: bronchoalveolar lavage; BMDC: bone marrow dendritic cell; CSF2/GM-CSF: colony-stimulating factor 2 (granulocyte-macrophage); CTL: cytotoxic T lymphocyte; ELISA: enzyme-linked immunosorbent assay; GFP: green fluorescent protein; GSEA: gene-set enrichment analysis; H-2Db: H-2 class I histocompatibility antigen, D-B alpha chain; H-2Kb: MHC class I H2-K-b; HIF1A: hypoxia-inducible factor 1 alpha; IFNG: interferon-gamma; IL: interleukin; ITGAX: integrin alpha X; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MHC: major histocompatibility complex; MTORC1: mammalian target of rapamycin kinase complex 1; PBS: phosphate-buffered saline; PFU: plaque-forming unit; RLR: retinoic acid-inducible-I-like receptor; ROS: reactive oxygen species; RPMI: Roswell Park Memorial Institute; RPS6KB1/S6K: ribosomal protein S6 kinase, polypeptide 1; RSV: respiratory syncytial virus; Th: T helper; TLR: toll-like receptor; Treg: regulatory T cells; UMAP: uniform manifold approximation and projection.

Multivalent DNA vaccine protects against genital herpes by T-cell immune induction in vaginal mucosa.

Genital herpes is one of the most common sexually transmitted infections (STIs), and it is mainly caused by the neurotropic herpes simplex virus (HSV-2). Not only does this infection cause ulcers, but HSV-2 can also stay in a latent state in the nervous system of the host throughout their lifespan. As a result, many people do not know that they harbor this infection. Moreover, HSV-2 serves as a major risk factor for human immunodeficiency virus (HIV) infection and can be transmitted to the fetus. Despite the high risk of infection and adverse effects, attempts at development of an effective vaccine for HSV-2 have not yet been successful. In this study, we developed a DNA vaccine for HSV-2 (SL-V20). This multivalent DNA vaccine effectively reduced the pathological symptoms of infection and induced efficient elimination of the virus in a mouse model. Intramuscular injection of SL-V20 led to induction of an HSV-2-specific T-cell response in the vagina, the major infection site, and in draining lymph organs. Dendritic cells (DCs), especially basic leucine zipper ATF-like transcription factor 3 (Baft3)+ DCs and partially interferon regulatory factor 4 (Irf4)+ DCs, were involved in this T-cell-mediated protective response, while B cells were dispensable for these prophylactic effects. This study demonstrates that SL-V20 offers a novel and effective vaccine against vaginal HSV-2 infection and may be applicable to patients, pending validation in clinical studies.

Autophagy protein ATG5 regulates CD36 expression and anti-tumor MHC class II antigen presentation in dendritic cells.

Macroautophagy/autophagy has been implicated in cytoplasmic and viral antigen presentation on major histocompatibility complex (MHC) class II molecules. However, the role of autophagy in the presentation of phagocytized tumor-associated antigens in vivo remains unclear. Following the administration of apoptotic tumor cells and in vivo chemotherapy, mice with a dendritic cell-specific deletion of Atg5, a key autophagy gene, exhibit reduced CD4+ T-cell priming but not CD8+ cytotoxic T-cell priming. Interestingly, Atg5-deficient dendritic cells have an elevated expression of scavenger receptor CD36 and show excessive lipid accumulation. Atg5-deficient dendritic cells increased CD36-dependent phagocytosis of apoptotic tumor cells. CD36 blockade ameliorates elevated phagocytosis and increases CD4+ T-cell priming in dendritic cells; intratumoral CD36 blockade inhibits tumor growth. Our results demonstrate that Atg5 is required for proper antigen phagocytosis and presentation to MHC class II via modulation of CD36 in dendritic cells and may be a future therapeutic target for anti-tumor therapy.Abbreviations: APC: antigen-presenting cell; ATG: autophagy-related; BMDC: bone marrow-derived dendritic cell; BODIPY: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene; CSFE: carboxyfluorescein diacetate succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole; IFNG/IFN-γ: interferon gamma; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MHC: major histocompatibility complex; NLDC: neonatal liver-derived dendritic cell; PDCD1/PD-1: programmed cell death 1; PI: propidium iodide; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; SERPINB/OVA: serine (or cysteine) peptidase inhibitor, clade B; TIMD4/TIM-4: T cell immunoglobulin and mucin domain containing 4.

Differential Role of Anti-Viral Sensing Pathway for the Production of Type I Interferon ß in Dendritic Cells and Macrophages Against Respiratory Syncytial Virus A2 Strain Infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory infectious disease in infants and young children. Dendritic cells (DCs) and macrophages (MACs) are known to play important roles in RSV recognition, and in the production of type I interferons (IFNs) and pro-inflammatory cytokine in RSV infection. Toll-like receptor 7 (TLR7), myeloid differentiation primary response 88 (MyD88), and mitochondrial antiviral-signaling protein (MAVS) are known to be important for the RSV sensing pathway in DCs and MACs. However, despite the critical roles of type I IFNs in the anti-RSV immune response, the pattern recognition receptors (PRRs) that are required for RSV sensing in DCs and MACs remain unclear. Here, we investigate the pathway activated by RSV A2 strain infection using an IFN-ß/YFP reporter mouse model to visualize IFN-ß-producing cells and in vitro RSV infection in bone marrow-derived DCs (BM-DCs) and macrophages (BM-DMs). We present our finding that MyD88, but not TLR7, are important for RSV recognition and type I IFN and pro-inflammatory production in DCs and MACs. MAVS-deficient BM-DCs and BM-DMs show impaired induction of IFN-ß production upon RSV stimulation, and this effect is RSV replication-dependent. Our study provides information on cell type-specific PRR requirements in innate immune responses against RSV infection.

Plasmacytoid Dendritic Cells Contribute to the Production of IFN-ß via TLR7-MyD88-Dependent Pathway and CTL Priming during Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of respiratory viral infection in infants and children, yet little is known about the antiviral response of plasmacytoid dendritic cells (pDCs) to RSV infection. We tracked the cellular source of interferon-ß using interferon-ß/yellow fluorescent protein (YFP) reporter mice and identified the signaling pathway activated by RSV that induces type I interferon production in pDCs and DCs. Results from in vitro analyses of RSV-stimulated bone marrow cells revealed that RSV induces interferon-ß production in both pDCs and DCs. Kinetic analyses of interferon-ß-producing cells in RSV-infected lung cells in vivo indicated that pDCs are rapidly recruited to sites of inflammation during infection. These cells produced interferon-ß via the TLR7-MyD88-mediated pathway and IFNα1R-mediated pathway rather than the MAVS-mediated pathway. Moreover, pDC-ablated mice exhibited decreased interferon-γ production and the antigen specificity of CD8+ T cells. Collectively, these data indicate that pDCs play pivotal roles in cytotoxic T lymphocyte (CTL) responses and are one of producers of type I interferon during RSV infection.

The autophagy Protein Atg5 Plays a Crucial Role in the Maintenance and Reconstitution Ability of Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) in bone marrow are pluripotent cells that can constitute the hematopoiesis system through self-renewal and differentiation into immune cells and red blood cells. To ensure a competent hematopoietic system for life, the maintenance of HSCs is tightly regulated. Although autophagy, a self-degradation pathway for cell homeostasis, is essential for hematopoiesis, the role of autophagy key protein Atg5 in HSCs has not been thoroughly investigated. In this study, we found that Atg5 deficiency in hematopoietic cells causes survival defects, resulting in severe lymphopenia and anemia in mice. In addition, the absolute numbers of HSCs and multiple-lineage progenitor cells were significantly decreased, and abnormal erythroid development resulted in reduced erythrocytes in blood of Vav_Atg5-/- mice. The proliferation of Lin-Sca-1+c-Kit+ HSCs was aberrant in bone marrow of Vav_Atg5-/- mice, and mature progenitors and terminally differentiated cells were also significantly altered. Furthermore, the reconstitution ability of HSCs in bone marrow chimeric mice was significantly decreased in the presence of Atg5 deficiency in HSCs. Mechanistically, impairment of autophagy-mediated clearance of damaged mitochondria was the underlying cause of the HSC functional defects. Taken together, these results define the crucial role of Atg5 in the maintenance and the reconstitution ability of HSCs.

Sox7 promotes high-grade glioma by increasing VEGFR2-mediated vascular abnormality.

High-grade glioma (HGG) is highly angiogenic, but antiangiogenic therapy has transient clinical benefit in only a fraction of patients. Vascular regulators of these heterogeneous responses remain undetermined. We found up-regulation of Sox7 and down-regulation of Sox17 in tumor endothelial cells (tECs) in mouse HGG. Sox7 deletion suppressed VEGFR2 expression, vascular abnormality, hypoxia-driven invasion, regulatory T cell infiltration, and tumor growth. Conversely, Sox17 deletion exacerbated these phenotypes by up-regulating Sox7 in tECs. Anti-VEGFR2 antibody treatment delayed tumor growth by normalizing Sox17-deficient abnormal vessels with high Sox7 levels but promoted it by regressing Sox7-deficient vessels, recapitulating variable therapeutic responses to antiangiogenic therapy in HGG patients. Our findings establish that Sox7 promotes tumor growth via vessel abnormalization, and its level determines the therapeutic outcome of VEGFR2 inhibition in HGG. In 189 HGG patients, Sox7 expression was heterogeneous in tumor vessels, and high Sox7 levels correlated with poor survival, early recurrence, and impaired vascular function, emphasizing the clinical relevance of Sox7 in HGG.

Transient Depletion of CD169+ Cells Contributes to Impaired Early Protection and Effector CD8+ T Cell Recruitment against Mucosal Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is a major cause of respiratory viral infections in infants and children. Alveolar macrophages (AMs) play a crucial role in combatting airborne pathogens, strongly express CD169, and are localized in the lung alveoli. Therefore, we used CD169-diphtheria toxin receptor (DTR) transgenic mice to explore the roles of CD169+ cells in immune responses to mucosal RSV infection. The administration of diphtheria toxin to CD169-DTR mice induced specific AM depletion and reduced the recruitment of Ly6Chi monocytes. Notably, CD169+ cell depletion reduced levels of innate cytokines, such as interferon-ß, IL-6, and TNF-α, in bronchoalveolar lavage fluid during RSV infection without affecting the production of proinflammatory chemokines. Moreover, the depletion of CD169+ cells increased the recruitment of inflammatory cells to the lung during the early stage of RSV infection, although not during the later stages of RSV infection. Furthermore, the depletion of CD169+ cells reduced the recruitment of effector CD8+ T cells to the lungs after RSV mucosal infection. Our findings suggest that modulating the number of CD169+ cells to enhance immune responses to RSV infection may be useful as a new therapeutic strategy.

Intratumoral depletion of regulatory T cells using CD25-targeted photodynamic therapy in a mouse melanoma model induces antitumoral immune responses.

Tumor immunotherapy aims to overcome the immunosuppressive microenvironment within tumors, and various approaches have been developed. Tumor-associated T regulatory cells (Tregs) suppress the activation and expansion of tumor antigen-specific effector T cells, thus, providing a permissive environment for tumor growth. Therefore, optimal strategies need to be established to deplete tumor-infiltrated Tregs because systemic depletion of Tregs can result in reduced anti-tumor effector cells and autoimmunity. Here, to selectively deplete Tregs in tumors, we intratumorally injected anti-CD25 antibodies conjugated to Chlorin e6 (Ce6), a photosensitizer that absorbs light to generate reactive oxygen species. Local depletion of tumor-associated Tregs with photodynamic therapy (PDT) inhibited tumor growth, which was likely due to the altered tumor immune microenvironment that was characterized by increased infiltration of CD8+ effector T cells and the expression of IFN-γ and CD107a, which is a cytolytic granule exocytosis marker in tumor tissues. Furthermore, PDT-induced intratumoral Treg depletion did not influence adaptive immune responses in a murine influenza infection model. Thus, our results show that intratumoral Treg-targeted PDT could specifically modulate tumor microenvironments by depleting Tregs and could be used as a novel cancer immunotherapy technique.