CD4+ T cell-associated cytokines induce a chronic pro-inflammatory phenotype in induced pluripotent stem cell-derived midbrain astrocytes.

Astrocytes are mediators of homeostasis but contribute to neuroinflammation in Parkinson's disease (PD). Mounting evidence suggests involvement of peripheral immune cells in PD pathogenesis. Therefore, this study aimed to determine the potential role of peripheral immune secreted cytokines in modulating midbrain astrocyte reactivity. Human iPSC-derived midbrain astrocytes were exposed to 5% and 10% CD4+ T cell conditioned media (CD4CM) for 24 h, 72 h, and 7 days to assess chronic exposure. Additionally, astrocytes were exposed to the Th17 cell cytokine, IL-17A (10 ng/mL), alone and in combination with TNF-α (0.3 ng/mL) to assess potential synergistic effects of both cytokines at 24 h, 72 h, and 7 days. CD4CM induced acute and chronic alterations in midbrain astrocytes. Increased NFκB translocation to the nucleus, increased expression of the pro-inflammatory genes, IL-1ß, CXCL10 at 24 h, C3, LCN2, IL-6 at 24 and 48 h, as well as an increase in their release of pro-inflammatory cytokines IL-6 and CXCL10 at both these time points were observed. A synergistic response to the combination of IL-17A and TNF-α on increasing inflammatory gene expression and cytokine release occurred. IL-17A and TNF-α increased intensity of S100ß at 24 h, decreased nuclear area and increased circularity of astrocytes at 72 h. A synergistic effect on γH2AX intensity at 72 h and an increase in LDH release at 7 days was observed. Our results demonstrate that IL-17A and TNF-α act synergistically, enhancing midbrain astrocyte reactivity to a similar degree as CD4CM. This highlights the importance of the peripheral immune secreted cytokines in increasing the reactivity status of midbrain astrocytes, implicating their role in PD.

Human Macrophages Activate Bystander Neutrophils' Metabolism and Effector Functions When Challenged with Mycobacterium tuberculosis.

Neutrophils are dynamic cells, playing a critical role in pathogen clearance; however, neutrophil infiltration into the tissue can act as a double-edged sword. They are one of the primary sources of excessive inflammation during infection, which has been observed in many infectious diseases including pneumonia and active tuberculosis (TB). Neutrophil function is influenced by interactions with other immune cells within the inflammatory lung milieu; however, how these interactions affect neutrophil function is unclear. Our study examined the macrophage-neutrophil axis by assessing the effects of conditioned medium (MΦ-CM) from primary human monocyte-derived macrophages (hMDMs) stimulated with LPS or a whole bacterium (Mycobacterium tuberculosis) on neutrophil function. Stimulated hMDM-derived MΦ-CM boosts neutrophil activation, heightening oxidative and glycolytic metabolism, but diminishes migratory potential. These neutrophils exhibit increased ROS production, elevated NET formation, and heightened CXCL8, IL-13, and IL-6 compared to untreated or unstimulated hMDM-treated neutrophils. Collectively, these data show that MΦ-CM from stimulated hMDMs activates neutrophils, bolsters their energetic profile, increase effector and inflammatory functions, and sequester them at sites of infection by decreasing their migratory capacity. These data may aid in the design of novel immunotherapies for severe pneumonia, active tuberculosis and other diseases driven by pathological inflammation mediated by the macrophage-neutrophil axis.

Metformin has anti-inflammatory effects and induces immunometabolic reprogramming via multiple mechanisms in hidradenitis suppurativa.

BACKGROUND: Targeting immunometabolism has shown promise in treating autoimmune and inflammatory conditions. Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease involving painful lesions in apocrine gland-bearing skin. Therapeutic options for HS are limited and often ineffective; thus, there is a pressing need for improved treatments. To date, metabolic dysregulation has not been investigated in HS. As HS is highly inflammatory, we hypothesized that energy metabolism is dysregulated in these patients. Metformin, an antidiabetic drug, which is known to impact on cellular metabolic and signalling pathways, has been shown to have anti-inflammatory effects in cancer and arthritis. While metformin is not licensed for use in HS, patients with HS taking metformin show improved clinical symptoms. OBJECTIVE: To assess the effect and mechanism of action of metformin in HS. METHODS: To assess the effect of metformin in vivo, we compared the immune and metabolic profiles of peripheral blood mononuclear cells (PBMCs) of patients with HS taking metformin vs. those not taking metformin. To examine the effect of metformin treatment ex vivo, we employed a skin explant model on skin biopsies from patients with HS not taking metformin, which we cultured with metformin overnight. We used enzyme-linked immunosorbent assays, multiplex cytokine assays and quantitative real-time polymerase chain reaction (RT-PCR) to measure inflammatory markers, and Seahorse flux technology and quantitative RT-PCR to assess glucose metabolism. RESULTS: We showed that metabolic pathways are dysregulated in the PBMCs of patients with HS vs. healthy individuals. In metformin-treated patients, these metabolic pathways were restored and their PBMCs had reduced inflammatory markers following long-term metformin treatment. In the skin explant model, we found that overnight culture with metformin reduced inflammatory cytokines and chemokines and glycolytic genes in lesions and tracts of patients with HS. Using in vitro assays, we found that metformin may induce these changes via the NLR family pyrin domain containing 3 (NLRP3) inflammasome and the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) pathway, which is linked to glycolysis and protein synthesis. CONCLUSIONS: Our study provides insight into the mechanisms of action of metformin in HS. The anti-inflammatory effects of metformin support its use as a therapeutic agent in HS, while its effects on immunometabolism suggest that targeting metabolism is a promising therapeutic option in inflammatory diseases, including HS.

Targeting the NLRP3 inflammasome reduces inflammation in hidradenitis suppurativa skin.

BACKGROUND: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life. OBJECTIVES: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies. METHODS: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays. RESULTS: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1ß and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1ß and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation. CONCLUSIONS: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications.

Targeting bioenergetics prevents CD4 T cell-mediated activation of synovial fibroblasts in rheumatoid arthritis.

OBJECTIVES: We investigated the reciprocal relationship linking fibroblast-like synoviocytes (FLS) and T lymphocytes in the inflamed RA synovium and subsequently targeted cellular metabolic pathways in FLS to identify key molecular players in joint inflammation. METHODS: RA FLS were cultured with CD4 T cells or T cell conditioned medium (CD4CM); proliferation, expression of adhesion molecules and intracellular cytokines were examined by flow cytometry. FLS invasiveness and secreted cytokines were measured by transwell matrigel invasion chambers and ELISA, while metabolic profiles were determined by extracellular Seahorse flux analysis. Gene expression was quantified by real-time quantitative RT-PCR. RESULTS: Our results showed mutual activation between CD4 T cells and FLS, which resulted in increased proliferation and expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 by both CD4 T cells and FLS. Furthermore, interaction between CD4 T cells and FLS resulted in an increased frequency of TNF-α+, IFN-γ+ and IL-17A+ CD4 T cells and augmented TNF-α, IFN-γ, IL-17A, IL-6, IL-8 and VEGF secretion. Moreover, CD4CM promoted invasiveness and boosted glycolysis in FLS while downregulating oxidative phosphorylation, effects paralleled by increased glucose transporters GLUT1 and GLUT3; key glycolytic enzymes GSK3A, HK2, LDHA and PFKFB3; angiogenic factor VEGF and MMP-3 and MMP-9. Importantly, these effects were reversed by the glycolytic inhibitor 2-DG and AMP analogue 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). CONCLUSION: This study demonstrates that CD4 T cells elicit an aggressive phenotype in FLS, which subsequently upregulate glycolysis to meet the increased metabolic demand. Accordingly, 2-DG and AICAR prevent this activation, suggesting that glycolytic manipulation could have clinical implications for RA treatment.

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.

Innate lymphoid cell (ILC) subsets are enriched in the skin of patients with hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic relapsing inflammatory skin disease manifested as painful inflamed lesions including deep-seated nodules, abscesses and sinus tracts. The exact aetiology of HS is unclear. Recent evidence suggests that immune dysregulation plays a crucial role in pathogenesis and disease progression. Innate lymphoid cells (ILC) are a recently identified immune cell subset involved in mediating immunity, however their role in HS has not yet been investigated. Three distinct subsets of ILC- ILC1, ILC2 and ILC3 have been described, and these are involved in skin tissue homeostasis and pathologic inflammation associated with autoimmunity and allergic diseases. In this study, we analysed by multiparameter flow cytometry the frequencies of ILC subsets in skin and peripheral blood mononuclear cells (PBMC) of HS patients and compared these to healthy control subjects and psoriasis patients. The absolute numbers of total ILC and subsets thereof were significantly reduced in the blood of HS patients relative to healthy controls. However, when patients were stratified according to treatment, this reduction was no longer observed in patients undergoing anti-TNF treatment. In HS lesional skin the absolute numbers of ILC were significantly increased relative to control skin. Furthermore, the frequencies of total ILC as well as ILC2 and ILC3 were significantly higher in non-lesional than lesional HS skin. This study analysed for the first time the presence of ILC subsets in the blood and skin of HS patients. Our findings suggest that ILC may participate in HS pathogenesis.

Differential Regulation of Human Treg and Th17 Cells by Fatty Acid Synthesis and Glycolysis.

In this study we examined the metabolic requirements of human T helper cells and the effect of manipulating metabolic pathways in Th17 and Treg cells. The Th17:Treg cell axis is dysregulated in a number of autoimmune or inflammatory diseases and therefore it is of key importance to identify novel strategies to modulate this axis in favor of Treg cells. We investigated the role of carbohydrate and fatty acid metabolism in the regulation of human memory T helper cell subsets, in order to understand how T cells are regulated at the site of inflammation where essential nutrients including oxygen may be limiting. We found that Th17 lineage cells primarily utilize glycolysis, as glucose-deprivation and treatment with rapamycin resulted in a reduction in these cells. On the other hand, Treg cells exhibited increased glycolysis, mitochondrial respiration, and fatty acid oxidation, whereas Th17 cells demonstrated a reliance upon fatty acid synthesis. Treg cells were somewhat reliant on glycolysis, but to a lesser extent than Th17 cells. Here we expose a fundamental difference in the metabolic requirements of human Treg and Th17 cells and a possible mechanism for manipulating the Th17:Treg cell axis.

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.

CD3ε Expression Defines Functionally Distinct Subsets of Vδ1 T Cells in Patients With Human Immunodeficiency Virus Infection.

Human γδ T cells expressing the Vδ1 T cell receptor (TCR) recognize self and microbial antigens and stress-inducible molecules in a major histocompatibility complex-unrestricted manner and are an important source of innate interleukin (IL)-17. Vδ1 T cells are expanded in the circulation and intestines of patients with human immunodeficiency virus (HIV) infection. In this study, we show that patients with HIV have elevated frequencies, but not absolute numbers, of circulating Vδ1 T cells compared to control subjects. This increase was most striking in the patients with Candida albicans co-infection. Using flow cytometry and confocal microscopy, we identify two populations of Vδ1 T cells, based on low and high expression of the ε chain of the CD3 protein complex responsible for transducing TCR-mediated signals (denoted CD3εlo and CD3εhi Vδ1 T cells). Both Vδ1 T cell populations expressed the CD3 ζ-chain, also used for TCR signaling. Using lines of Vδ1 T cells generated from healthy donors, we show that CD3ε can be transiently downregulated by activation but that its expression is restored over time in culture in the presence of exogenous IL-2. Compared to CD3εhi Vδ1 T cells, CD3εlo Vδ1 T cells more frequently expressed terminally differentiated phenotypes and the negative regulator of T cell activation, programmed death-1 (PD-1), but not lymphocyte-activation gene 3, and upon stimulation in vitro, only the CD3εhi subset of Vδ1 T cells, produced IL-17. Thus, while HIV can infect and kill IL-17-producing CD4+ T cells, Vδ1 T cells are another source of IL-17, but many of them exist in a state of exhaustion, mediated either by the induction of PD-1 or by downregulation of CD3ε expression.

Human Natural Killer cell expression of ULBP2 is associated with a mature functional phenotype.

NKG2D is an important activating receptor expressed on NK cells. Ligands (termed NKG2DL) for this receptor include ULBP1-6, MICA and MICB in humans; they are upregulated in stressed, cancerous or infected cells where they engage NKG2D to induce NK cell cytotoxicity and cytokine production. Expression of NKG2DL on effector cells has been described in mice and more recently in human cells. We confirm that NK cell lines and IL-2 stimulated primary human NK cells also express the NKG2DL, ULBP2. However, expression of ULBP2 was not a result of transfer from a non-NK cell to an NK cell and in contrast to recent reports we saw no evidence that ULBP2 expression targeted these NK cells for fratricide or for cytotoxicity by NKG2D-expressing, non-NK effector cells. ULBP2 expression was however linked to expression of mature CD57(+) NK cells. In particular, expression of ULBP2 was strongest on those NK cells that had evidence of recent activation and proliferation. We suggest that ULBP2 could be used to identify recently activated "mature" NK cells. Defining this phenotype would be useful for understanding the ontogeny on human NK cells.

Human Vδ2(+) γδ T Cells Differentially Induce Maturation, Cytokine Production, and Alloreactive T Cell Stimulation by Dendritic Cells and B Cells.

Human γδ T cells expressing the Vγ9Vδ2 T cell receptor can induce maturation of dendritic cells (DC) into antigen-presenting cells (APC) and B cells into antibody-secreting plasma cells. Since B cells are capable of presenting antigens to T cells, we investigated if Vγ9Vδ2 T cells can influence antigen-presentation by these cells. We report that Vγ9Vδ2 T cells induced expression of CD86, HLA-DR, and CD40 by B cells and stimulated the release of IL-4, IL-6, TNF-α, and IgG, IgA, and IgM. Vγ9Vδ2 T cells also augmented the ability of B cells to stimulate proliferation but not IFN-γ or IL-4 release by alloreactive T cells. In contrast, Vγ9Vδ2 T cells induced expression of CD86 and HLA-DR and the release of IFN-γ, IL-6, and TNF-α by DC and these DC stimulated proliferation and IFN-γ production by conventional T cells. Furthermore, CD86, TNF-α, IFN-γ, and cell contact were found to be important in DC activation by Vγ9Vδ2 T cells but not in the activation of B cells. These data suggest that Vγ9Vδ2 T cells can induce maturation of B cells and DC into APC, but while they prime DC to stimulate T helper 1 (TH1) responses, they drive maturation of B cells into APC that can stimulate different T cell responses. Thus, Vγ9Vδ2 T cells can control different arms of the immune system through selective activation of B cells and DC in vitro, which may have important applications in immunotherapy and for vaccine adjuvants.

HIV-1 Tat clade-specific cytokine induction in monocytes/macrophages is not evidenced in total or Vγ9Vδ2 T lymphocytes.

HIV-1 Tat exhibits clade-specific cytokine induction in monocytes. We investigated if Tat clades A-D can alter tumour necrosis factor (TNF)-α and interferon (IFN)-γ production by total and Vγ9Vδ2 T cells in vitro. Tat clade B, but not C, augmented TNF-α production by THP-1 cells. However, Tat clades A-D did not affect TNF-α or IFN-γ production or secretion by resting or activated conventional and Vγ9Vδ2 T cells. Therefore, transactivation of cytokines by Tat is immune cell-specific.