Probiotic treatment with viable α-galactosylceramide-producing Bacteroides fragilis reduces diabetes incidence in female nonobese diabetic mice.

BACKGROUND: We aimed to investigate whether alpha-galactosylceramide (α-GalCer)-producing Bacteroides fragilis could induce natural killer T (NKT) cells in nonobese diabetic (NOD) mice and reduce their diabetes incidence. METHODS: Five-week-old female NOD mice were treated orally with B. fragilis, and islet pathology and diabetes onset were monitored. Immune responses were analyzed by flow cytometry and multiplex technology. Effects of ultraviolet (UV)-killed α-GalCer-producing B. fragilis and their culture medium on invariant NKT (iNKT) cells were tested ex vivo on murine splenocytes, and the immunosuppressive capacity of splenocytes from B. fragilis-treated NOD mice were tested by adoptive transfer to nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice. RESULTS: B. fragilis reduced the diabetes incidence from 69% to 33% and the percent of islets with insulitis from 40% to 7%, which doubled the serum insulin level compared with the vehicle-treated control mice. Furthermore, the early treatment reduced proinflammatory mediators in the serum, whereas the proportion of CD4+ NKT cell population was increased by 33%. B. fragilis growth media stimulated iNKT cells and anti-inflammatory M2 macrophages ex vivo in contrast to UV-killed bacteria, which had no effect, strongly indicating an α-GalCer-mediated effect. Adoptive transfer of splenocytes from B. fragilis-treated NOD mice induced a similar diabetes incidence as splenocytes from untreated NOD mice. CONCLUSIONS: B. fragilis induced iNKT cells and M2 macrophages and reduced type 1 diabetes in NOD mice. The protective effect seemed to be more centered on gut-pancreas interactions rather than a systemic immunosuppression. B. fragilis should be considered for probiotic use in individuals at risk of developing type 1 diabetes.

The Alteration of Circulating Invariant Natural Killer T, γδT, and Natural Killer Cells after Ischemic Stroke in Relation to Clinical Outcomes: A Prospective Case-Control Study.

The adaptive response occurs only after 7-10 days of antigen presentation. Nevertheless, the autoreactive T cells infiltrate the stroke lesion within the first 48 h. Thus, we hypothesized that the unconventional lymphocytes as invariant natural killer T cells (iNKT) and γδT cells that share immediate innate and delayed adaptive response features are involved in acute stroke pathophysiology. We assessed prospectively the quantity of circulating iNKT cells, γδT cells, and NK cells with flow cytometry in 52 subjects within three months after stroke, and we compared the results with those obtained in age-, sex-, and vascular risk factor-matched controls. We studied lymphocyte parameters regarding clinical outcomes, infarct volume, stroke-associated infection (SAI), and burden risk factors. The reduced number of circulating γδT cells and decreased percentage of the Vδ2 subset in the acute phase of stroke correlated with worse neurological status in the recovery phase. In subjects treated with thrombolysis and those who developed SAI, a lower percentage of γδT cells in the 90-day follow-up was observed. An increased percentage of iNKT cells in the acute and subacute phases of stroke was observed, and it was related to the worse clinical status. The circulating NK cells do not change temporarily or affect the outcomes after stroke. It seems that γδT cells play a long-lasting role in ischemic stroke, mainly related to the Vδ2 subset. The role of iNKT cells appears to be detrimental, especially in the acute and subacute phases of stroke. The effect of circulating NK cells on the outcome after stroke seems negligible.

Lipidomic scanning of self-lipids identifies headless antigens for natural killer T cells.

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

NKT cells promote Th1 immune bias to dengue virus that governs long-term protective antibody dynamics.

NKT cells are innate-like T cells, recruited to the skin during viral infection, yet their contributions to long-term immune memory to viruses are unclear. We identified granzyme K, a product made by cytotoxic cells including NKT cells, as linked to induction of Th1-associated antibodies during primary dengue virus (DENV) infection in humans. We examined the role of NKT cells in vivo using DENV-infected mice lacking CD1d-dependent (CD1ddep) NKT cells. In CD1d-KO mice, Th1-polarized immunity and infection resolution were impaired, which was dependent on intrinsic NKT cell production of IFN-γ, since it was restored by adoptive transfer of WT but not IFN-γ-KO NKT cells. Furthermore, NKT cell deficiency triggered immune bias, resulting in higher levels of Th2-associated IgG1 than Th1-associated IgG2a, which failed to protect against a homologous DENV rechallenge and promoted antibody-dependent enhanced disease during secondary heterologous infections. Similarly, Th2 immunity, typified by a higher IgG4/IgG3 ratio, was associated with worsened human disease severity during secondary infections. Thus, CD1ddep NKT cells establish Th1 polarity during the early innate response to DENV, which promotes infection resolution, memory formation, and long-term protection from secondary homologous and heterologous infections in mice, with consistent associations observed in humans. These observations illustrate how early innate immune responses during primary infections can influence secondary infection outcomes.

Cytokine responses of CD4+ T cells and NKT cells to periodontitis-associated bacteria in individuals with or without periodontitis.

AIM: Periodontitis is an inflammatory disease driven by opportunistic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum, where T-cell and NKT-cell responses to these bacteria in patients with periodontitis grade B or C are not fully elucidated. The objective is to determine if exaggerated proinflammatory Th-cell responses to periodontitis-associated bacteria, but not commensal bacteria, is a characteristic of increased periodontitis grade. METHODS: Mononuclear cells from patients with periodontitis grade C (n = 26) or grade B (n = 33) and healthy controls (HCs; n = 26) were stimulated with P. gingivalis, F. nucleatum or the commensal bacteria, Staphylococcus epidermidis and Cutibacterium acnes. Cytokine production by different T-cell populations and FOXP3-expression by regulatory T cells were assessed by flow cytometry. RESULTS: Compared to HCs, grade C patients had decreased frequencies of interleukin (IL)-10-producing CD4+ T cells before stimulation (p = .02) and increased frequencies of IFN-y-producing CD4+ T cells after stimulation with P. gingivalis (p = .0019). Grade B patients had decreased frequencies of FOXP3+ CD4+ T cells before (p = .030) before and after stimulation with anti-CD2/anti-CD3/anti-CD28-loaded beads (p = .047), P. gingivalis (p = .013) and S. epidermidis (p = .018). Clinical attachment loss correlated with the frequencies of IFN-y-producing Th1 cells in P. gingivalis- and F. nucleatum-stimulated cultures in grade B patients (p = .023 and p = .048, respectively) and with the frequencies of Th17 cells in P. gingivalis-stimulated cultures (p = .0062) in grade C patients. Patients with periodontitis grade C or grade B showed lower frequencies of IL-10-producing NKT cells than HCs in unstimulated cultures (p = .0043 and p = .027 respectively). CONCLUSIONS: Both periodontitis groups showed decreased frequencies of immunoregulatory T-cell and NKT cell subsets at baseline. Clinical attachment loss correlated with P. gingivalis-induced Th17-responses in grade C patients and with Th1-responses in grade B patients when cells were stimulated with P. gingivalis, supporting that dysregulated pro-inflammatory T-cell responses to periodontitis-associated bacteria contribute to the pathogenesis of periodontitis.

Tim-4 alleviates acute hepatic injury by modulating homeostasis and function of NKT cells.

T-cell immunoglobulin and mucin domain-containing molecule 4 (Tim-4) is an immune checkpoint molecule, which involves in numerous inflammatory diseases. Tim-4 is mainly expressed on antigen-presenting cells. However, increasing evidence has shown that Tim-4 is also expressed on natural killer T (NKT) cells. The role of Tim-4 in maintaining NKT cell homeostasis and function remains unknown. In this study, we explored the effect of Tim-4 on NKT cells in acute liver injury. This study found that Tim-4 expression on hepatic NKT cells was elevated during acute liver injury. Tim-4 deficiency enhanced IFN-γ, TNF-α expression while impaired IL-4 production in NKT cells. Loss of Tim-4 drove NKT-cell effector lineages to be skewed to NKT1 subset. Furthermore, Tim-4 KO mice were more susceptible to α-Galactosylceramide (α-GalCer) challenge. In conclusion, our findings indicate that Tim-4 plays an important role in regulating homeostasis and function of NKT cells in acute liver injury. Therefore, Tim-4 might become a new regulator of NKT cells and a potential target for the therapy of acute hepatitis.

Unraveling Crucial Mitochondria-Related Genes in the Transition from Ulcerative Colitis to Colorectal Cancer.

Purpose: To clarify the significance of mitochondria-related differentially expressed genes (MTDEGs) in UC carcinogenesis through a bioinformatics analysis and provide potential therapeutic targets for patients with UC associated colorectal cancer. Methods: Microarray GSE37283 was utilized to investigate differentially expressed genes (DEGs) in UC and UC with neoplasia (UCN). MTDEGs were identified by intersecting DEGs with human mitochondrial genes. Utilizing LASSO and random forest analyses, we identified three crucial genes. Subsequently, using ROC curve to investigate the predictive ability of three key genes. Following, three key genes were confirmed in AOM/DSS mice model by Real-time PCR. Finally, single-sample gene set enrichment analysis (ssGSEA) was employed to explore the correlation between the hub genes and immune cells infiltration in UC carcinogenesis. Results: The three identified hub MTDEGs (HMGCS2, MAVS, RDH13) may exhibit significant diagnostic specificity in the transition from UC to UCN. Real-time PCR assay further confirmed that the expressions of HMGCS2 and RDH13 were significantly downregulated in UCN mice than that in UC mice. ssGSEA analysis revealed the hub genes were highly associated with CD56dim natural killer cells. Conclusion: RDH13, HMGCS2, and MAVS may become diagnostic indicators and potential biomarkers for UCN. Our research has the potential to enhance our understanding of the mechanisms underlying carcinogenesis in UC.

Intratumoral NKT cell accumulation promotes antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is a potentially lethal disease lacking effective treatments. Its immunosuppressive tumor microenvironment (TME) allows it to evade host immunosurveillance and limits response to immunotherapy. Here, using the mouse KRT19-deficient (sgKRT19-edited) PDA model, we find that intratumoral accumulation of natural killer T (NKT) cells is required to establish an immunologically active TME. Mechanistically, intratumoral NKT cells facilitate type I interferon (IFN) production to initiate an antitumor adaptive immune response, and orchestrate the intratumoral infiltration of T cells, dendritic cells, natural killer cells, and myeloid-derived suppressor cells. At the molecular level, NKT cells promote the production of type I IFN through the interaction of their CD40L with CD40 on myeloid cells. To evaluate the therapeutic potential of these observations, we find that administration of folinic acid to mice bearing PDA increases NKT cells in the TME and improves their response to anti-PD-1 antibody treatment. In conclusion, NKT cells have an essential role in the immune response to mouse PDA and are potential targets for immunotherapy.

Features of the CD1 gene family in rodents and the uniqueness of the immune system of naked mole-rat.

Cluster of Differentiation 1 (CD1) proteins are widely expressed throughout jawed vertebrates and present lipid antigens to specific CD1-restricted T lymphocytes. CD1 molecules play an important role in immune defense with the presence or absence of particular CD1 proteins frequently associated with the functional characteristics of the immune system. Here, we show the evolution of CD1 proteins in the Rodentia family and the diversity among its members. Based on the analysis of CD1 protein-coding regions in rodent genomes and the reconstruction of protein structures, we found that Heterocephalus glaber represents a unique member of the suborder Hystricomorpha with significant changes in protein sequences and structures of the CD1 family. Multiple lines of evidence point to the absence of CD1d and CD1e and probably a dysfunctional CD1b protein in Heterocephalus glaber. In addition, the impact of CD1d loss on the CD1d/Natural killer T (NKT) cell axis in the naked mole-rat and its potential implications for immune system function are discussed in detail.

An Adjuvanted Vaccine-Induced Pathogenesis Following Influenza Virus Infection.

An incomplete Freund's adjuvant elicited an overt pathogenesis in vaccinated mice following the intranasal challenge of A/California/07/2009 (H1N1) virus despite the induction of a higher specific antibody titer than other adjuvanted formulations. Aluminum hydroxide adjuvants have not induced any pathogenic signs in a variety of formulations with glycolipids. A glycolipid, α-galactosyl ceramide, improved a stimulatory effect of distinct adjuvanted formulations on an anti-influenza A antibody response. In contrast to α-galactosyl ceramide, its synthetic analogue C34 was antagonistic toward a stimulatory effect of an aluminum hydroxide adjuvant on a specific antibody response. The aluminum hydroxide adjuvant alone could confer complete vaccine-induced protection against mortality as well as morbidity caused by a lethal challenge of the same strain of an influenza A virus. The research results indicated that adjuvants could reshape immune responses either to improve vaccine-induced immunity or to provoke an unexpected pathogenic consequence. On the basis of these observations, this research connotes the prominence to develop a precision adjuvant for innocuous vaccination aimed at generating a protective immunity without aberrant responses.