ImmunoHorizons最新文献

Atopic dermatitis (AD) is characterized by dysregulated T cell immunity and skin microbiome dysbiosis with predominance of Staphylococcus aureus, which is associated with exacerbating AD skin inflammation. Specific glycosylation patterns of S. aureus cell wall structures amplify skin inflammation through interaction with Langerhans cells (LCs). Nevertheless, the role of LCs in AD remains poorly characterized. Here, we performed single cell RNA sequencing of primary epidermal LCs and dermal T cells, isolated from skin biopsies of AD patients and healthy control subjects, alongside specific glycoanalysis of S. aureus strains isolated from the AD lesions. Our findings revealed 4 LC subpopulations ie, 2 steady-state clusters [LC1 and LC1H] and 2 proinflammatory/matured subsets [LC2 and migratory LCs]. The latter 2 subsets were enriched in AD skin. AD LCs showed enhanced expression of C-type lectin receptors, the high-affinity IgE receptor, and activation of prostaglandin and leukotriene biosynthesis pathways, upregulated transcriptional signatures related to T cell activation pathways, and increased expression of CCL17 compared with healthy LCs. Correspondingly, T helper 2 and T regulatory cell populations were increased in AD lesions. Complementary, we performed bulk RNA sequencing of primary LCs stimulated with the S. aureus strains isolated from the AD lesions, which showed upregulation of T helper 2-related pathways. Our study provides proof-of-concept for a role of LCs in connecting the S. aureus-T cell axis in the AD inflammatory cycle.

Respiratory syncytial virus (RSV) is a major contributor to morbidity and mortality in infants. We developed an in vitro model of human respiratory infection to study cellular immune responses to RSV in infants, children, and adults. The model includes human lung epithelial A549 cells or human fetal lung fibroblasts infected with a clinical strain of RSV at a multiplicity of infection of 0.3, cocultured with human cord blood mononuclear cells (CBMCs) or peripheral blood mononuclear cells (PBMCs). Mononuclear cells were collected at multiple ages ranging from birth to adulthood. After 20 h of incubation, flow cytometry was used to measure CBMC/PBMC responses to RSV. A549s were more permissive to RSV and when infected produced more CCL5, CCL11, and CXCL9; less CSF-3, CXCL10, interleukin (IL)-1α, IL-1RA, and IL-6; and similar CCL2, CCL3, CCL4, CCL7, CXCL1, CXCL11, IL-1β, IL-7, IL-8, and tumor necrosis factor α compared with fibroblasts; A594s were used for subsequent experiments. CBMCs/PBMCs upregulated multiple markers of activation, maturation, and degranulation upon exposure to RSV-infected A549s. Interferon γ expression in natural killer, CD4, and CD8 cells and CD107a expression in natural killer cells showed a gradual increase from infancy to adulthood. IL-12 expression in dendritic cells and monocytes was highest in adult PBMCs. Our in vitro model of human RSV infection recapitulated the expected bias away from T helper 1 and effector responses to RSV infection in infancy and revealed changes in innate and adaptive RSV-specific cellular immune responses over time.

Dysregulated differentiation of naïve CD4+ T cells into T helper 17 (Th17) cells is likely a key factor predisposing to many autoimmune diseases. Therefore, better understanding how Th17 differentiation is regulated is essential to identify novel therapeutic targets and strategies to identify individuals at high risk of developing autoimmunity. Here, we extend our prior work using chemical inhibitors to provide mechanistic insight into a novel regulator of Th17 differentiation, the kinase dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). We generated a conditional knockout mouse model to validate DYRK1A as a regulator of Th17 differentiation that acts in a dose-dependent fashion at least in part by modulating interleukin (IL)-6 signaling through multiple mechanisms. We identified a new role for DYRK1A in regulating surface expression of IL-6 receptor subunits in naïve CD4+ T cells, consistent with DYRK1A's impact on Th17 differentiation. Physiologic relevance is supported by findings in people with Down syndrome, in which increased expression of DYRK1A, encoded on chromosome 21, is linked to increased IL-6 responsiveness. Our findings highlight DYRK1A as a druggable target of broad therapeutic and prognostic interest in autoimmunity and immune function.

The differentiation and functionality of virus-specific T cells during acute viral infections are crucial for establishing long-term protective immunity. While numerous molecular regulators impacting T cell responses have been uncovered, the role of cellular prion proteins (PrPc) remains underexplored. Here, we investigated the impact of PrPc deficiency on the differentiation and function of virus-specific T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong acute infection model. Our findings reveal that Prnp-/- mice exhibit a robust expansion of virus-specific CD8+ T cells, with similar activation profiles as wild-type mice during the early stages of infection. However, Prnp-/- mice had higher frequencies and numbers of virus-specific memory CD8+ T cells, along with altered differentiation profiles characterized by increased central and effector memory subsets. Despite similar proliferation rates early during infection, Prnp-/- memory CD8+ T cells had decreased proliferation compared with their wild-type counterparts. Additionally, Prnp-/- mice had higher numbers of cytokine-producing memory CD8+ T cells, indicating a more robust functional response. Furthermore, Prnp-/- mice had increased virus-specific CD4+ T cell responses, suggesting a broader impact of PrPc deficiency on T cell immunity. These results unveil a previously unrecognized role for PrPc in regulating the differentiation, proliferation, and functionality of virus-specific T cells, providing valuable insights into immune system regulation by prion proteins during viral infections.

CD73 is ubiquitously expressed and regulates critical functions across multiple organ systems. The sequential actions of CD39 and CD73 accomplish the conversion of adenosine triphosphate to adenosine and shift the adenosine triphosphate-driven proinflammatory immune cell milieu toward an anti-inflammatory state. This immunological switch is a major mechanism by which regulatory T (Treg) cells control inflammation. Foxp3 engages in Treg development and function. Foxp3 mutations result in the scurfy (SF) mouse phenotype and a rapidly lethal lymphoproliferative syndrome. We generated double knockout (KO) mouse (CD73KOSF) by breeding heterozygous Foxp3sf/J females to CD73KO male mice to remove host CD73. We initially aimed to use these mice to identify a specific probiotic-CD73 effect, previously shown for Limosilactobacillus reuteri DSM 17938. We expected CD73 deletion to enhance the severity of autoimmunity in SF mice. However, we unexpectedly observed that KO of host CD73 in SF mice clinically reduced the severity of autoimmunity including reduced ear thickness, increased ear size, and less deformed ears, along with less dry and brittle skin. KO of CD73 in SF mice significantly reduced the numbers of CD4+ and CD8+T cells in spleen and blood. We identified that KO of CD73 in SF mice reduced the numbers of T cells in the thymus compared with those in SF mice, indicating that the milder clinical phenotype may be due to reduced central and peripheral lymphoproliferation. These new findings suggest targeting CD73 could improve T cell-mediated dermatitis, one of the most common symptoms in Treg deficiency-associated primary immune deficiencies.

Influenza virus infects millions each year, contributing greatly to human morbidity and mortality. Upon viral infection, pathogen-associated molecular patterns activate pattern recognition receptors on host cells, triggering an immune response. The CD209 protein family, homologs of DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), is thought to modulate immune responses to viruses. The effects of the mouse functional DC-SIGN homolog CD209d/e on the lung immune responses during influenza viral infection are not known. Therefore, we generated mice that lack both CD209d and e isoforms to determine the role in influenza viral infection. We infected wild-type and CD209d/e gene-deficient (CD209d/e-/-) mice with influenza virus and measured the cellular response in bronchoalveolar lavage, the expression of proinflammatory cytokines, antiviral genes, toll-like receptors (TLRs) in the lung, and lung pathology. We found CD209d/e-/- mice had decreased viral burden, TLR3 and TLR9 expression, interferon response, macrophages in bronchoalveolar lavage, and parenchymal lung inflammation compared with control mice. We also found less influenza viral uptake in alveolar macrophages and bone marrow-derived macrophages isolated from CD209d/e-/- mice when compared with control mice. We further investigated the role CD209d/e by treating bone marrow-derived macrophages from control and CD209d/e-/- mice with TLR agonists. We found that lacking CD209d/e decreased the expression of TLR3, TLR9, RIG1, STAT1, and STAT2 compared with controls. Collectively these results show that CD209d/e plays an important role in viral sensing/uptake and inflammatory immune responses during influenza viral infection.

Autoimmune pediatric neurologic diseases have variable phenotypes and presentations, making diagnosis challenging. The pathologic mechanisms are also distinct, including cell-mediated and Ab-mediated autoimmunity, paraneoplastic syndromes, and postinfectious processes. In recent years a number of studies have described the characteristics of the autoantibodies involved in a number of these diseases. Some of the described Abs use a restricted set of variable gene segments. We sought to compare the Ab characteristics of autoantibodies related to some of the more common disorders to discover whether specific Ab signatures are universally associated with neuroautoimmune diseases. We initially performed a literature review to summarize the Ab characteristics of autoantibodies related to some of the more common disorders, including N-methyl-d-aspartate receptor (NMDAR) and leucine-rich, glioma-inactivated 1 (LGI-1). Next, we performed data analysis from selected studies that sequenced Ig genes to further characterize NMDAR and LGI-1 autoantibodies including CDR3 length distribution, variable gene sequence usage, and isotype use. We found that CDR3 length of NMDAR autoantibodies was normally distributed whereas the CDR3 length distribution of LGI-1 autoantibodies was skewed, suggesting that there is no global structural restriction on types of autoantibodies that can cause encephalitis. We also found that IgG1-IgG3 were the main NMDAR autoantibody isotypes detected, while IgG4 was the major isotype used in autoantibodies from LGI-1 encephalitis. These findings are useful for our understanding of autoimmune encephalitis and will help facilitate better diagnosis and treatment of these conditions in the future.

Chronic suppurative otitis media (CSOM) is a neglected disease that afflicts 330 million people worldwide and is the most common cause of permanent hearing loss among children in the developing world. Previously, we discovered that outer hair cell (OHC) loss occurred in the basal turn of the cochlea and that macrophages are the major immune cells associated with OHC loss in CSOM. Macrophage-associated cytokines are upregulated. Specifically, CCL-2, an important member of the MCP family, is elevated over time following middle ear infection. CCR2 is a common receptor of the MCP family and the unique receptor of CCL2. CCR2 knockout mice (CCR2-/-) have been used extensively in studies of monocyte activation in neurodegenerative diseases. In the present study, we investigated the effect of CCR2 deletion on the cochlear immune response and OHC survival in CSOM. The OHC survival rate was 84 ± 12.5% in the basal turn of CCR2+/+ CSOM cochleae, compared with was 63 ± 19.9% in the basal turn of CCR2-/- CSOM cochleae (p ≤ 0.05). Macrophage numbers were significantly reduced in CCR2-/- CSOM cochleae compared with CCR2+/+ CSOM cochleae (p ≤ 0.001). In addition, CCL7 was upregulated, whereas IL-33 was downregulated, in CCR2-/- CSOM cochleae. Finally, the permeability of the blood-labyrinth barrier in the stria vascularis remained unchanged in CCR2-/- CSOM compared with CCR2+/+ CSOM. Taken together, the data suggest that CCR2 plays a protective role through cochlear macrophages in the CSOM cochlea.

Human PBMC-based assays are often used as biomarkers for the diagnosis and prognosis of disease, as well as for the prediction and tracking of response to biological therapeutics. However, the development and use of PBMC-based biomarker assays is often limited by poor reproducibility. Complex immunological assays can be further complicated by variation in cell handling before analysis, especially when using cryopreserved cells. Variation in postthaw viability is further increased if PBMC isolation and cryopreservation are done more than a few hours after collection. There is currently a lack of evidence-based standards for the minimal PBMC viability or "fitness" required to ensure the integrity and reproducibility of immune cell-based assays. In this study, we use an "induced fail" approach to examine the effect of thawed human PBMC fitness on four flow cytometry-based assays. We found that cell permeability-based viability stains at the time of thawing did not accurately quantify cell fitness, whereas a combined measurement of metabolic activity and early apoptosis markers did. Investigation of the impact of different types and levels of damage on PBMC-based assays revealed that only when cells were >60-70% live and apoptosis negative did biomarker values cease to be determined by cell fitness rather than the inherent biology of the cells. These data show that, to reproducibly measure immunological biomarkers using cryopreserved PBMCs, minimal acceptable standards for cell fitness should be incorporated into the assay protocol.