CD1d expression and invariant natural killer T-cell numbers are reduced in patients with upper gastrointestinal cancers and are further impaired by commonly used chemotherapies.

Esophageal and gastric cancers collectively cause over 1.1 million deaths annually and only 20-30% of patients respond favorably to current therapies. Cellular therapies using invariant natural killer T (iNKT) cells are showing promise for patients with other cancers; therefore, we investigated if these cells are altered in esophageal and gastric cancer patients. Flow cytometric analysis of peripheral blood from 139 patients revealed that iNKT cells are depleted from patients with esophageal and gastric adenocarcinoma and esophageal squamous cell carcinoma, both before and after treatment. Interrogation of the KMPlot database of transcriptomic data from 876 gastric cancer patients revealed that low CD1d expression is associated with poor prognosis. These observations suggest that therapies that boost CD1d expression and iNKT cell responses may benefit these patients. However, we found that chemotherapies used for esophageal and gastric cancers have adverse effects on iNKT cells in vitro. Cisplatin caused a significant reduction of CD1d expression by esophageal tumor cell lines. Cisplatin, 5-fluorouracil and carboplatin induced dose-dependent apoptosis in primary lines of iNKT cells and inhibited CD1d-dependent interferon-γ production and cytolytic degranulation by viable iNKT cells. Interestingly, cisplatin increased granzyme B and perforin production and decreased the production of the granzyme B inhibitor PI9, which protects cytotoxic cells from self-damage by granzyme B. Thus, cisplatin-induced apoptosis of iNKT cells may be mediated in part by altering granzyme B and PI9 expression. Our data suggest that iNKT cell-based immunotherapies may benefit patients with gastrointestinal cancers, but may be negatively affected by chemotherapies used for these cancers.

Type 1 IFN Induction by Cytosolic Nucleic Acid Is Intact in Neonatal Mononuclear Cells, Contrasting Starkly with Neonatal Hyporesponsiveness to TLR Ligation Due to Independence from Endosome-Mediated IRF3 Activation.

Two million infants die each year from infectious diseases before they reach 12 mo; many of these diseases are vaccine preventable in older populations. Pattern recognition receptors represent the critical front-line defense against pathogens. Evidence suggests that the innate immune system does not fully develop until puberty, contributing to impaired response to infection and impaired vaccine responses in neonates, infants, and children. The activity of the pattern recognition receptor family of cytosolic nucleic acid (CNA) sensors in this pediatric population has not been reported. We show that in direct contrast to weak TLR-induced type I IFN in human cord blood mononuclear cells, cord blood mononuclear cells are capable of initiating a potent response to CNA, inducing both antiviral type I IFN and, unexpectedly, proinflammatory TNF-α. A deficiency in Rab11-GTPase endosome formation and consequent lack of IRF3 activation in neonatal monocytes is at least in part responsible for the marked disparity in TLR-induced IFN production between neonatal and adult monocytes. CNA receptors do not rely on endosome formation, and therefore, these responses remain intact in neonates. Heightened neonatal responses to CNA challenge are maintained in children up to 2 y of age and, in marked contrast to TLR4/9 agonists, result in IL-12p70 and IFN-γ generation. CNA sensors induce robust antiviral and proinflammatory pathways in neonates and children and possess great potential for use as immunostimulants or vaccine adjuvants for targeted neonatal and pediatric populations to promote cell-mediated immunity against invasive infectious disease.

Novel thioglycoside analogs of α-galactosylceramide stimulate cytotoxicity and preferential Th1 cytokine production by human invariant natural killer T cells.

Invariant natural killer T (iNKT) cells recognize glycolipid antigens bound to CD1d molecules on antigen-presenting cells. Therapeutic activation of iNKT cells with the xenogeneic glycolipid α-galactosylceramide (α-GalCer) can prevent and reverse tumor growth in murine models, but clinical trials using α-GalCer-stimulated human iNKT cells have shown limited efficacy. We synthesized a series of thioglycoside analogs of α-GalCer with different substituents to the galactose residue and found that two of these compounds, XZ7 and XZ11, bound to CD1d-transfected HeLa cells and activated lines of expanded human iNKT cells. Both compounds stimulated cytolytic degranulation by iNKT cells and while XZ7 preferentially stimulated the production of the antitumor cytokine interferon-γ (IFN-γ), XZ11 preferentially stimulated interleukin-4 (IL-4) production. This biased T helper type 1 effector profile of XZ7 was also evident when iNKT were stimulated with dendritic cells presenting this glycolipid. Separate analysis of the responses of CD4+, CD8α+ and CD4-CD8- iNKT cells indicated that XZ7 preferentially activated CD8α+ iNKT cells, and to a lesser degree, CD4-CD8- iNKT cells. The partial agonist effect of glycolipid XZ7, inducing cytotoxicity and IFN-γ production but not IL-4 production, indicates that specific protumour activities of iNKT cells can be abolished, while preserving their antitumor activities, by introducing structural modifications to α-GalCer. Since XZ7 was much less potent than α-GalCer as an iNKT cell agonist, it is unlikely to be superior to α-GalCer as a therapeutic agent for cancer, but may serve as a parent compound for developing more potent structural analogs.

Platelet microaggregation in sepsis examined by quartz crystal microbalance with dissipation technology.

Antiplatelet therapies remain an area of potential interest for the treatment of sepsis; however, studies of platelet aggregation in sepsis have yielded conflicting results. We examined platelet aggregation patterns in patients with septic shock using quartz crystal microbalance with dissipation technology, a microfluidic device capable of measuring platelet microaggregate formation under flow conditions. Platelet aggregation was increased in the washed platelet samples of septic patients. Conversely, these same platelets aggregated less than healthy controls when examined in their plasma.

Trained immunity is induced in humans after immunization with an adenoviral vector COVID-19 vaccine.

BackgroundHeterologous effects of vaccines are mediated by "trained immunity," whereby myeloid cells are metabolically and epigenetically reprogrammed, resulting in heightened responses to subsequent insults. Adenovirus vaccine vector has been reported to induce trained immunity in mice. Therefore, we sought to determine whether the ChAdOx1 nCoV-19 vaccine (AZD1222), which uses an adenoviral vector, could induce trained immunity in vivo in humans.MethodsTen healthy volunteers donated blood on the day before receiving the ChAdOx1 nCoV-19 vaccine and on days 14, 56, and 83 after vaccination. Monocytes were purified from PBMCs, cell phenotype was determined by flow cytometry, expression of metabolic enzymes was quantified by RT-qPCR, and production of cytokines and chemokines in response to stimulation ex vivo was analyzed by multiplex ELISA.ResultsMonocyte frequency and count were increased in peripheral blood up to 3 months after vaccination compared with their own prevaccine controls. Expression of HLA-DR, CD40, and CD80 was enhanced on monocytes for up to 3 months following vaccination. Moreover, monocytes had increased expression of glycolysis-associated enzymes 2 months after vaccination. Upon stimulation ex vivo with unrelated antigens, monocytes produced increased IL-1ß, IL-6, IL-10, CXCL1, and MIP-1α and decreased TNF, compared with prevaccine controls. Resting monocytes produced more IFN-γ, IL-18, and MCP-1 up to 3 months after vaccination compared with prevaccine controls.ConclusionThese data provide evidence for the induction of trained immunity following a single dose of the ChAdOx1 nCoV-19 vaccine.FundingThis work was funded by the Health Research Board (EIA-2019-010) and Science Foundation Ireland Strategic Partnership Programme (proposal ID 20/SPP/3685).

The E3 ubiquitin ligase Ro52 negatively regulates IFN-beta production post-pathogen recognition by polyubiquitin-mediated degradation of IRF3.

Induction of type I IFNs is a fundamental cellular response to both viral and bacterial infection. The role of the transcription factor IRF3 is well established in driving this process. However, equally as important are cellular mechanisms for turning off type I IFN production to limit this response. In this respect, IRF3 has previously been shown to be targeted for ubiquitin-mediated degradation postviral detection to turn off the IFN-beta response. In this study, we provide evidence that the E3 ligase Ro52 (TRIM21) targets IRF3 for degradation post-pathogen recognition receptor activation. We demonstrate that Ro52 interacts with IRF3 via its C-terminal SPRY domain, resulting in the polyubiquitination and proteasomal degradation of the transcription factor. Ro52-mediated IRF3 degradation significantly inhibits IFN-beta promoter activity, an effect that is reversed in the presence of the proteasomal inhibitor MG132. Specific targeting of Ro52 using short hairpin RNA rescues IRF3 degradation following polyI:C-stimulation of HEK293T cells, with a subsequent increase in IFN-beta production. Additionally, shRNA targeting of murine Ro52 enhances the production of the IRF3-dependent chemokine RANTES following Sendai virus infection of murine fibroblasts. Collectively, this demonstrates a novel role for Ro52 in turning off and thus limiting IRF3-dependent type I IFN production by targeting the transcription factor for polyubiquitination and subsequent proteasomal degradation.

Innate Lymphocyte Th1 and Th17 Responses in Elderly Hospitalised Patients with Infection and Sepsis.

Background: the role of innate immunity in human sepsis must be fully clarified to identify potential avenues for novel immune adjuvant sepsis therapies. Methods: A prospective observational study was performed including patients with sepsis (septic group), infection without sepsis (infection group), and healthy controls (control group) in the setting of acute medical wards and intensive care units in a 1000-bed university hospital. A total of 42 patients with sepsis, 30 patients with infection, and 30 healthy controls were studied. The differentiation states of circulating mucosal associated invariant T (MAIT) cells and Natural Killer T (NKT) cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentiated (CD45RA+, CD197-). The differentiation states of circulating gamma-delta T lymphocytes were characterised as naive (CD45RA+, CD27+), central memory (CD45RA-, CD27+), effector memory (CD45RA-, CD27-), or terminally differentiated (CD45RA+, CD27-). The expression of IL-12 and IL-23 receptors, the transcription factors T-Bet and RORγt, and interferon-γ and IL-17a were analysed. Results: MAIT cell counts were lower in the septic group (p = 0.002) and the infection group (p < 0.001) than in the control group. The MAIT cell T-Bet expression in the infection group was greater than in the septic group (p = 0.012). The MAIT RORγt expression in the septic group was lower than in the control group (p = 0.003). The NK cell counts differed in the three groups (p < 0.001), with lower Natural Killer (NK) cell counts in the septic group (p < 0.001) and in the infection group (p = 0.001) than in the control group. The NK cell counts increased in the septic group in the 3 weeks following the onset of sepsis (p = 0.028). In lymphocyte stimulation experiments, fewer NK cells expressed T-Bet in the septic group than in the infection group (p = 0.002), and fewer NK cells expressed IFN-γ in the septic group than in the control group (p = 0.002). The NKT cell counts were lower in the septic group than both the control group (p = 0.05) and the infection group (p = 0.04). Fewer NKT cells expressed T-Bet in the septic group than in the infection group (p = 0.004). Fewer NKT cells expressed RORγt in the septic group than in the control group (p = 0.003). Fewer NKT cells expressed IFN-γ in the septic group than in both the control group (p = 0.002) and the infection group (p = 0.036). Conclusion: The clinical presentation of infection and or sepsis in patients is linked with a mosaic of changes in the innate lymphocyte Th1 and Th17 phenotypes. The manipulation of the innate lymphocyte phenotype offers a potential avenue for immune modulation in patients with sepsis.

Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells.

Uncoupling protein 2 (UCP2) regulates glucose-stimulated insulin secretion in pancreatic beta-cells. UCP2 content, measured by calibrated immunoblot in INS-1E insulinoma cells (a pancreatic beta-cell model) grown in RPMI medium, and INS-1E mitochondria, was 2.0 ng/million cells (7.9 ng/mg mitochondrial protein). UCP2 content was lower in cells incubated without glutamine and higher in cells incubated with 20 mM glucose, and varied from 1.0-4.4 ng/million cells (2.7-14.5 ng/mg mitochondrial protein). This dynamic response to nutrients was achieved by varied expression rates against a background of a very short UCP2 protein half-life of about 1 h.

Dysregulated T helper type 1 (Th1) and Th17 responses in elderly hospitalised patients with infection and sepsis.

OBJECTIVE: The role of Th1 and Th17 lymphocyte responses in human infection and sepsis of elderly patients has yet to be clarified. DESIGN: A prospective observational study of patients with sepsis, infection only and healthy controls. SETTING: The acute medical wards and intensive care units in a 1000 bed university hospital. PATIENTS: 32 patients with sepsis, 20 patients with infection, and 20 healthy controls. Patients and controls were older than 65 years of age. Patients with recognised underlying immune compromise were excluded. METHODS: Phenotype, differentiation status and cytokine production by T lymphocytes were determined by flow cytometry. MEASUREMENTS: The differentiation states of circulating CD3+, CD4+, and CD8+ T cells were characterised as naive (CD45RA+, CD197+), central memory (CD45RA-, CD197+), effector memory (CD45RA-, CD197-), or terminally differentated (CD45RA+, CD197-). Expression of IL-12 and IL-23 receptors, and the transcription factors T-bet and RORγt, was analysed in circulating T lymphocytes. Expression of interferon- γ and IL-17A were analysed following stimulation in vitro. RESULTS: CD4+ T cells from patients with infection predominantly expressed effector-memory or terminally differentiated phenotypes but CD4+ T cells from patients with severe sepsis predominantly expressed naive phenotypes (p<0.0001). CD4+ T cells expressing IL-23 receptor were lower in patients with sepsis compared to patients with infection alone (p = 0.007). RORγt expression by CD4+ T cells was less frequent in patients with sepsis (p<0.001), whereas T-bet expressing CD8+ T cells that do not express RORγt was lower in the sepsis patients. HLA-DR expression by monocytes was lower in patients with sepsis. In septic patients fewer monocytes expressed IL-23. CONCLUSION: Persistent failure of T cell activation was observed in patients with sepsis. Sepsis was associated with attenuated CD8+Th1 and CD4+Th17 based lymphocyte response.

Human Vδ3+ γδ T cells induce maturation and IgM secretion by B cells.

This study tested the hypothesis that the Vδ3 subset of human γδ T cells, like their Vδ2 counterparts, can influence differentiation, antibody secretion and cytokine production by B cells. Vδ3 T cells constitute a minor subset of peripheral blood lymphocytes but are enriched in the liver and gut and are expanded in patients with cytomegalovirus activation and B cell chronic lymphocytic leukemia. They have been reported to include MHC class I and CD1d restricted cells. Like Vδ2 T cells, they are capable of maturing dendritic cells into cytokine-producing antigen presenting cells, making them potential targets for dendritic cell-based immunotherapies. Since it is unknown if Vδ3 T cells can also provide B cell help, we investigated if Vδ3 T cells can promote B cell differentiation, antibody secretion and cytokine production in vitro. Vδ3 T cells were sorted from healthy human blood and expanded using phytohemagglutinin and cultured with freshly isolated human B cells. We found that Vδ3 T cells and B cells reciprocally induced expression of maturation markers CD40, CD86 and HLA-DR but not TH1, TH2 or TH17 cytokines. Furthermore, Vδ3 T cells promoted the release of IgM, but not IgG, IgA or IgE by B cells. These data demonstrate, for the first time, a reciprocal activating relationship between Vδ3 T cells and B cells, which could prove a useful target for cellular immunotherapy.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

Cold acclimation and oxygen consumption in the thymus.

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (approximately 1.7-fold) and GDP binding (approximately 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (approximately 1.5-fold), GDP-inhibitable (approximately 1.3-fold) and palmitate activatable (approximately 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue approximately 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.

A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1.

Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.

Lack of activation of UCP1 in isolated brown adipose tissue mitochondria by glucose-O-ω-modified saturated fatty acids of various chain lengths.

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.

On the mechanism of mitochondrial uncoupling protein 1 function.

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.

Identification of a functioning mitochondrial uncoupling protein 1 in thymus.

We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent B(max) and K(D) values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus.