Targeting DCs for Tolerance Induction: Don’t Lose Sight of the Neutrophils

Chronic inflammatory disorders (CID), such as autoimmune diseases, are characterized by overactivation of the immune system and loss of immune tolerance. T helper 17 (Th17) cells are strongly associated with the pathogenesis of multiple CID, including psoriasis, rheumatoid arthritis, and inflammatory bowel disease. In line with the increasingly recognized contribution of innate immune cells to the modulation of dendritic cell (DC) function and DC-driven adaptive immune responses, we recently showed that neutrophils are required for DC-driven Th17 cell differentiation from human naive T cells. Consequently, recruitment of neutrophils to inflamed tissues and lymph nodes likely creates a highly inflammatory loop through the induction of Th17 cells that should be intercepted to attenuate disease progression. Tolerogenic therapy via DCs, the central orchestrators of the adaptive immune response, is a promising strategy for the treatment of CID. Tolerogenic DCs could restore immune tolerance by driving the development of regulatory T cells (Tregs) in the periphery. In this review, we discuss the effects of the tolerogenic adjuvants vitamin D3 (VD3), corticosteroids (CS), and retinoic acid (RA) on both DCs and neutrophils and their potential interplay. We briefly summarize how neutrophils shape DC-driven T-cell development in general. We propose that, for optimization of tolerogenic DC therapy for the treatment of CID, both DCs for tolerance induction and the neutrophil inflammatory loop should be targeted while preserving the potential Treg-enhancing effects of neutrophils

Dexamethasone, but Not Vitamin D or A, Dampens the Inflammatory Neutrophil Response to Protect At-risk COVID-19 Patients

Dexamethasone (DEX) was the first drug shown to save lives of critically ill coronavirus disease 2019 (COVID-19) patients suffering from respiratory distress. A hyperactivated state of neutrophils was found in COVID-19 patients compared to non-COVID pneumonia cases. Given the beneficial effects of DEX in COVID-19 patients, we investigated the effects of DEX and of other immunomodulatory drugs vitamin D3 (VD3) and retinoic acid (RA) on neutrophil function. DEX, but not VD3 or RA, significantly inhibited all tested aspects of neutrophil function, e.g., degranulation, intracellular ROS production, CXCL8 release and NETosis. Interestingly, RA displayed the opposite effect by significantly increasing both CXCL8 and NET release by neutrophils. Taken together, these data suggest that the lower COVID-19 mortality in DEX-treated patients may in part be due to the dampening effect of DEX on the inflammatory neutrophil response, which could prevent neutrophil plugs with NETS in the lungs and other inflamed organs of patients

VSTM1-v2 does not drive human Th17 cell differentiation: A replication study

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human myeloid cells. We previously showed that dendritic cell (DC)-driven Th17 cell differentiation of human naive CD4+ T cells requires presence of neutrophils, which is inhibited by SIRL-1 ligation. VSTM1-v2 is a soluble isoform of SIRL-1, which was previously proposed to function as a Th17 polarizing cytokine. Here, we investigated the effect of VSTM1-v2 on DC-driven Th17 cell development. Neutrophils induced DC-driven Th17 cell differentiation, which was not enhanced by VSTM1-v2. Similarly, we found no effect of VSTM1-v2 on cytokine-driven Th17 cell development. Thus, our results do not support a role for VSTM1-v2 in Th17 cell differentiation

Vitamin D3 Priming of Dendritic Cells Shifts Human Neutrophil-Dependent Th17 Cell Development to Regulatory T Cells

Vitamin D3 (VD3) is a potential adjuvant for use in tolerogenic vaccine formulations that target dendritic cells (DCs) for the treatment of chronic inflammatory disorders, e.g., autoimmune diseases. These disorders are often associated with enhanced activity of IL-17-producing T helper 17 (Th17) cells which develop in a DC-driven and neutrophil-dependent fashion. Here, we investigated the effect of VD3 on Candida albicans-specific human T-cell differentiation, since C. albicans is a model pathogen for Th17 cell development. VD3 priming of DCs restricted neutrophil-dependent Th17 cell development and neutrophil-independent Th1 cell formation from naive CD4+ T cells. In line with this, the production of Th1/Th17-polarizing cytokines IL-12 and IL-23 by DCs was reduced by VD3 priming. Development of both FoxP3+CD127lowCD25+ Tregs and IL-10-producing T cells was significantly enhanced in VD3-primed conditions, even in the presence of neutrophils. ICOS+ Tregs, major IL-10 producers, CD69+FoxP3+, and TIGIT+FoxP3+ Tregs were significantly induced by VD3 priming as well. Our data support the potential use of VD3 as an adjuvant to induce tolerance in the treatment of autoimmune disorders, including those in which neutrophils are involved in pathogenesis, since we show that Treg development is enhanced by VD3 even in the presence of neutrophils, while Th17 cell development is restricted

VSTM1-v2 does not bind to leukocytes.

Erythrocyte-lysed whole blood was incubated with His-tagged VSTM1-v2 or sLAIR-1ecto (10–50 μg/mL), followed by detection with anti-His-AF647. (A) Lymphocytes (lower population), monocytes (middle population) and granulocytes (upper population) were gated based on forward scatter (FSC) and sideward scatter (SSC), followed by gating on single cells and AF-647+ cells. (B) Shown are representative dot plots of lymphocytes stained with 50 μg/mL VSTM1-v2 and/or anti-His-AF647. (C-E) The graphs show the quantification of the percentage of AF647+ cells of lymphocytes (C), monocytes (D), or granulocytes (E). Data are shown as mean ± SD of two donors. (TIF)</p

VSTM1-v2 does not enhance Th17 cell differentiation and activation.

CD4+ T cells were stimulated by C. albicans-activated moDCs, with or without autologous neutrophils (A-C), or with antibodies and a polarizing cytokine mix (D), with or without addition of 10 or 100 ng/mL VSTM1-v2. Intracellular IL-17 expression was determined by flow cytometry. (A) Schematic representation of the co-culture system of moDC-driven Th17 cell differentiation and activation. (B) Representative dot plots of the percentage of IL-17+ cells after co-culture of naive CD4+ T cells. The y-axis indicates the fluorescence intensity of the IL-17A staining, while the x-axis indicates the forward scatter (FSC). (C) The percentage of IL-17+ cells after co-culture of naive CD4+ T cells (n = 10), each donor represented by a different color, or memory CD4+ T cells (n = 3; mean ± SD). (D) The percentage of IL-17+ cells after stimulation of total CD4+ T cells with anti-CD3, anti-CD28, and a Th17 polarizing mix, mean ± SD, n = 3. Statistical significance was determined using a Friedman test with Dunn’s correction (C, D). ** p biorender.com.</p

Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli

Efficient neutrophil activation requires two simultaneous activating stimuli

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli

Efficient neutrophil activation requires two simultaneous activating stimuli

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli

Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli

Neutrophils are abundantly present in the synovium and synovial fluid of patients suffering from arthritis. Neutrophils can be activated by a multitude of stimuli and the current dogma states that this is a two-step process, consisting of a priming step followed by an activation step. Considering that neutrophil activation occurs in an inflammatory environment, where multiple stimuli are present, we argue that a two-step process is highly unlikely. Here, we indeed demonstrate that neutrophils require simultaneous ligation of two different receptors for efficient activation. We isolated human peripheral blood neutrophils and cultured them with various combinations of stimuli (GM-CSF, fMLF, TNF, and LPS). Next, we evaluated essential neutrophil functions, including degranulation and ROS production using flow cytometry, mediator release using ELISA, NETosis by a live cell imaging method, phagocytosis by imaging flow cytometry, and extracellular vesicle (EV) release quantified by high-resolution flow cytometry. Exposure of neutrophils to any combination of stimuli, but not to single stimuli, resulted in significant degranulation, and mediator and EV release. Furthermore, ROS production increased substantially by dual stimulation, yet appeared to be more dependent on the type of stimulation than on dual stimulation. Phagocytosis was induced to its maximum capacity by a single stimulus, while NETosis was not induced by any of the used physiological stimuli. Our data indicate that neutrophil activation is tightly regulated and requires activation by two simultaneous stimuli, which is largely independent of the combination of stimuli