BATF2 prevents T-cell-mediated intestinal inflammation through regulation of the IL-23/IL-17 pathway.

Inappropriate activation of the IL-23 signaling pathway causes chronic inflammation through the induction of immunopathological Th17 cells in several tissues including the intestine, whereas adequate Th17 responses are essential for host defense against harmful organisms. In the intestinal lamina propria, IL-23 is primarily produced by innate myeloid cells including dendritic cells (DCs) and macrophages (Mϕs). However, the molecular mechanisms underlying the regulation of IL-23 production by these cells remains poorly understood. In this study, we demonstrated that BATF2 regulates intestinal homeostasis by inhibiting IL-23-driven T-cell responses. Batf2 was highly expressed in intestinal innate myeloid subsets, such as monocytes, CD11b+ CD64+ Mϕs and CD103+ DCs. Batf2-/- mice spontaneously developed colitis and ileitis with altered microbiota composition. In this context, IL-23, but not TNF-α and IL-10, was produced in high quantities by intestinal CD11b+ CD64+ Mϕs from Batf2-/- mice compared with wild-type mice. Moreover, increased numbers of IFN-γ+, IL-17+ and IFN-γ+ IL-17+ CD4+ T cells, but not IL-10+ CD4+ T cells, accumulated in the colons and small intestines of Batf2-/- mice. In addition, RORγt-expressing innate lymphoid cells were increased in Batf2-/- mice. Batf2-/-Rag2-/- mice showed a reduction in intestinal inflammation present in Batf2-/- mice. Furthermore, the high numbers of intestinal IL-17+ and IFN-γ+ IL-17+ CD4+ T cells were markedly reduced in Batf2-/- mice when introducing Il23a deficiency, which was associated with the abrogation of intestinal inflammation. These results indicated that BATF2 in innate myeloid cells is a key molecule for the suppression of IL-23/IL-17 pathway-mediated adaptive intestinal pathology.

Single-cell RNA sequencing reveals the expansion of circulating tissue-homing B cell subsets in secondary acute dengue viral infection.

The roles of antibodies secreted by subsets of B cells in dengue virus (DENV) infection have been extensively studied, yet, the contribution of tissue-homing B cells to antiviral immunity remains unclear. In this study, we performed a comprehensive analysis of B cell subpopulations in peripheral blood samples from DENV-infected patients using single-cell RNA-sequencing (scRNA-seq) datasets and flow cytometry. We showed that plasma cells (PCs) and plasmablasts (PBs) were the predominant B cell populations during the acute phase of secondary natural DENV infection, but not in convalescent phase nor in healthy controls. Interestingly, these cells expressed proliferation, adhesion, and tissue-homing genes, including SELPLG, a homing marker of the skin, the initial infected site of DENV. Flow cytometry analysis confirmed a significant upregulation of cell surface expression of a cutaneous lymphocyte-associated antigen (CLA) encoded by SELPLG in PCs and PBs, compared to naive and memory B cells from the same patients. The analysis of an independent single-cell B-cell receptor sequencing (scBCR-seq) dataset of DENV-infected patients revealed that the peripheral blood PCs and PBs exhibited the highest clonal expansion in secondary DENV infection compared to other B cell subsets. These clonally expanded cells also expressed the highest levels of tissue-homing genes, including SELPLG. In addition, by utilizing a public scRNA-seq dataset of SARS-CoV2 infection, we demonstrated the upregulation of several tissue-homing genes in PCs and PBs. Our study provides evidence for the potential roles of tissue-homing B cell subsets in the context of immune responses against viral infections in humans.

Computational workflow for investigating highly variable genes in single-cell RNA-seq across multiple time points and cell types.

Here, we present a computational approach for investigating highly variable genes (HVGs) associated with biological pathways of interest, across multiple time points and cell types in single-cell RNA-sequencing (scRNA-seq) data. Using public dengue virus and COVID-19 datasets, we describe steps for using the framework to characterize the dynamic expression levels of HVGs related to common and cell-type-specific biological pathways over multiple immune cell types. For complete details on the use and execution of this protocol, please refer to Arora et al.1.

Single-cell temporal analysis of natural dengue infection reveals skin-homing lymphocyte expansion one day before defervescence.

Effective clinical management of acute dengue virus (DENV) infection relies on the timing of suitable treatments during the disease progression. We analyzed single-cell transcriptomic profiles of the peripheral blood mononuclear cell samples from two DENV patients, collected daily during acute phase and also at convalescence. Key immune cell types demonstrated different dynamic responses over the course of the infection. On the day before defervescence (Day -1), we observed the peak expression of several prominent genes in the adaptive immunological pathways. We also characterized unique effector T cell clusters that expressed skin-homing signature genes at Day -1, whereas upregulation of skin and gut homing genes was also observed in plasma cells and plasmablasts during the febrile period. This work provides an overview of unique molecular dynamics that signify the entry of the critical phase, and the findings could improve the patient management of DENV infection.

Innate Lymphoid Cells Activation and Transcriptomic Changes in Response to Human Dengue Infection.

Background: Dengue virus (DENV) infection has a global impact on public health. The clinical outcomes (of DENV) can vary from a flu-like illness called dengue fever (DF), to a more severe form, known as dengue hemorrhagic fever (DHF). The underlying innate immune mechanisms leading to protective or detrimental outcomes have not been fully elucidated. Helper innate lymphoid cells (hILCs), an innate lymphocyte recently discovered, functionally resemble T-helper cells and are important in inflammation and homeostasis. However, the role of hILCs in DENV infection had been unexplored. Methods: We performed flow cytometry to investigate the frequency and phenotype of hILCs in peripheral blood mononuclear cells from DENV-infected patients of different disease severities (DF and DHF), and at different phases (febrile and convalescence) of infection. Intracellular cytokine staining of hILCs from DF and DHF were also evaluated by flow cytometry after ex vivo stimulation. Further, the hILCs were sorted and subjected to transcriptome analysis using RNA sequencing. Differential gene expression analysis was performed to compare the febrile and convalescent phase samples in DF and DHF. Selected differentially expressed genes were then validated by quantitative PCR. Results: Phenotypic analysis showed marked activation of all three hILC subsets during the febrile phase as shown by higher CD69 expression when compared to paired convalescent samples, although the frequency of hILCs remained unchanged. Upon ex vivo stimulation, hILCs from febrile phase DHF produced significantly higher IFN-γ and IL-4 when compared to those of DF. Transcriptomic analysis showed unique hILCs gene expression in DF and DHF, suggesting that divergent functions of hILCs may be associated with different disease severities. Differential gene expression analysis indicated that hILCs function both in cytokine secretion and cytotoxicity during the febrile phase of DENV infection. Conclusions: Helper ILCs are activated in the febrile phase of DENV infection and display unique transcriptomic changes as well as cytokine production that correlate with severity. Targeting hILCs during early innate response to DENV might help shape subsequent immune responses and potentially lessen the disease severity in the future.

Novel salivary gland allergens from tropical mosquito species and IgE reactivity in allergic patients.

BACKGROUND: Mosquito allergy is common in tropical countries but remains under-diagnosed. This may be due to the lack of knowledge and diagnostic tools for tropical mosquito allergens. OBJECTIVE: We aimed to characterize allergens from tropical mosquito species and investigate IgE reactivity in mosquito-allergic patients to the salivary gland proteins from these mosquitoes. METHODS: Salivary gland extract (SGE) from 4 mosquito species, highly distributed in the tropics, including Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, and Anopheles dirus b, were studied. SGE-specific IgE and IgG ELISA were developed, and serum from 64 mosquito-allergic and 22 non-allergic healthy control subjects was assayed. Further investigations using IgE-immunoblots followed by mass spectrometry analysis were performed to identify and characterize allergens from each species. RESULTS: Mosquito-allergic subjects have detectable serum IgE to SGE derived from local mosquito species, while the IgE levels to Aedes communis using commercially available ELISA were mostly minimal. IgE-immunoblot analysis and mass spectrometry identified 5 novel mosquito allergens from A. albopictus (Aed al 2, Aed al 3), C. quinquefasciatus (Cul q 2.01, Cul q 3), and A. dirus b (Ano d 2). Interestingly, 4 of the 5 new allergens belong to the D7 protein family. CONCLUSIONS & CLINICAL RELEVANCE: Five novel allergens from 3 tropical mosquito species were characterized. The majority of mosquito-allergic subjects who live in the tropics have IgE reactivity to these allergens. Our study paves the way for the development of diagnostic tests, component-resolved diagnostics, and future immunotherapy for mosquito allergy in tropical countries.

Neutrophil Activation and Early Features of NET Formation Are Associated With Dengue Virus Infection in Human.

The involvement of the immune system in the protection and pathology of natural dengue virus (DENV) has been extensively studied. However, despite studies that have referred to activation of neutrophils in DENV infections, the exact roles of neutrophils remain elusive. Here, we explored the phenotypic and functional responses of neutrophils in a cohort of adult dengue patients. Results indicated that during an acute DENV infection, neutrophils up-regulate CD66b expression, and produce a more robust respiratory response as compared with that in convalescent or healthy individuals; this confirmed in vivo neutrophil activation during DENV infection. Spontaneous decondensation of nuclei, an early event of neutrophil extracellular trap (NET) formation, was also markedly increased in cells isolated from DENV-infected patients during the acute phase of the infection. In vitro incubation of NETs with DENV-2 virus significantly decreased DENV infectivity. Interestingly, increased levels of NET components were found in the serum of patients with more severe disease form-dengue hemorrhagic fever (DHF), but not uncomplicated dengue fever, during the acute phase of the infection. Levels of pro-inflammatory cytokines IL-8 and TNFα were also increased in DHF patients as compared with those in healthy and DF subjects. This suggested that NETs may play dual roles during DENV infection. The increased ability for NET formation during acute DENV infection appeared to be independent of PAD4-mediated histone H3 hyper-citrullination. Our study suggests that neutrophils are involved in immunological responses to DENV infection.

iNKT Cells and Their Potential Lipid Ligands during Viral Infection.

Invariant natural killer T (iNKT) cells are a unique population of lipid-reactive CD1d-restricted innate-like T lymphocytes. Despite being a minor population, they serve as an early source of cytokines and promote immunological crosstalk thus bridging innate and adaptive immunity. Diseases ranging from allergy, autoimmunity, and cancer, as well as infectious diseases, including viral infection, have been reported to be influenced by iNKT cells. However, it remains unclear how iNKT cells are activated during viral infection, as virus-derived lipid antigens have not been reported. Cytokines may activate iNKT cells during infections from influenza and murine cytomegalovirus, although CD1d-dependent activation is evident in other viral infections. Several viruses, such as dengue virus, induce CD1d upregulation, which correlates with iNKT cell activation. In contrast, herpes simplex virus type 1 (HSV-1), human immunodeficiency virus (HIV), Epstein-Barr virus, and human papilloma virus promote CD1d downregulation as a strategy to evade iNKT cell recognition. These observations suggest the participation of a CD1d-dependent process in the activation of iNKT cells in response to viral infection. Endogenous lipid ligands, including phospholipids as well as glycosphingolipids, such as glucosylceramide, have been proposed to mediate iNKT cell activation. Pro-inflammatory signals produced during viral infection may stimulate iNKT cells through enhanced CD1d-dependent endogenous lipid presentation. Furthermore, viral infection may alter lipid composition and inhibit endogenous lipid degradation. Recent advances in this field are reviewed.