Patients with systemic sclerosis show phenotypic and functional defects in neutrophils

BACKGROUND Systemic sclerosis (SSc) is a multiorgan autoimmune disease characterized by inflammation, vascular modification, and progressive fibrosis of the skin and several visceral organs. Innate and adaptive immune cells, including myeloid, B and T cells, are believed to be central to the pathogenesis of SSc. However, the role and functional state of neutrophil granulocytes (neutrophils) are ill-defined in SSc. METHODS We performed a prospective study of neutrophils freshly isolated from SSc patients and healthy donors (HD) by measuring in these neutrophils (i) functional cell surface markers, including CD16, CD62L, CD66b, CD66c, CXCR1, CXCR2, and CXCR4; (ii) cytokine-activated intracellular signal transducer and activator of transcription (STAT) pathways, such as phosphorylated STAT3 (pSTAT3), pSTAT5, and pSTAT6; (iii) production of neutrophil extracellular traps (NET) and intracellular myeloperoxidase (MPO); and (iv) phagocytosis of bacteria by the neutrophils. RESULTS Neutrophils of SSc patients expressed lower CD16 and CD62L and higher pSTAT3 and pSTAT6 compared to HD. Moreover, neutrophils of SSc patients lacked CXCR1 and CXCR2, the receptors responding to the potent neutrophil chemoattractant CXCL8. Neutrophils of SSc patients were also deficient in MPO levels, NET formation and phagocytosis of bacteria. CONCLUSIONS Neutrophils of patients with SSc display several functional defects affecting cell migration, NET formation, and phagocytosis of bacteria

IL-4 receptor engagement in human neutrophils impairs their migration and extracellular trap formation

Background Type 2 immunity serves to resist parasitic helminths, venoms, and toxins, but the role and regulation of neutrophils during type 2 immune responses are controversial. Helminth models suggested a contribution of neutrophils to type 2 immunity, whereas neutrophils are associated with increased disease severity during type 2 inflammatory disorders, such as asthma. Objective We sought to evaluate the effect of the prototypic type 2 cytokines IL-4 and IL-13 on human neutrophils. Methods Human neutrophils from peripheral blood were assessed without or with IL-4 or IL-13 for (1) expression of IL-4 receptor subunits, (2) neutrophil extracellular trap (NET) formation, (3) migration toward CXCL8 in vitro and in humanized mice, and (4) CXCR1, CXCR2, and CXCR4 expression, as well as (5) in nonallergic versus allergic subjects. Results Human neutrophils expressed both types of IL-4 receptors, and their stimulation through IL-4 or IL-13 diminished their ability to form NETs and migrate toward CXCL8 in vitro. Likewise, in vivo chemotaxis in NOD-scid-Il2rg−/− mice was reduced in IL-4–stimulated human neutrophils compared with control values. These effects were accompanied by downregulation of the CXCL8-binding chemokine receptors CXCR1 and CXCR2 on human neutrophils on IL-4 or IL-13 stimulation in vitro. Ex vivo analysis of neutrophils from allergic patients or exposure of neutrophils from nonallergic subjects to allergic donor serum in vitro impaired their NET formation and migration toward CXCL8, thereby mirroring IL-4/IL-13–stimulated neutrophils. Conclusion IL-4 receptor signaling in human neutrophils affects several neutrophil effector functions, which bears important implications for immunity in type 2 inflammatory disorders

Biased IL-2 signals induce Foxp3-rich pulmonary lymphoid structures and facilitate long-term lung allograft acceptance in mice

Transplantation of solid organs can be life-saving in patients with end-stage organ failure, however, graft rejection remains a major challenge. In this study, by pre-conditioning with interleukin-2 (IL-2)/anti-IL-2 antibody complex treatment biased toward IL-2 receptor α, we achieved acceptance of fully mismatched orthotopic lung allografts that remained morphologically and functionally intact for more than 90 days in immunocompetent mice. These allografts are tolerated by the actions of forkhead box p3 (Foxp3)$^{+}$ regulatory T (Treg) cells that home to the lung allografts. Although counts of circulating Treg cells rapidly return to baseline following cessation of IL-2 treatment, Foxp3$^{+}$ Treg cells persist in peribronchial and peribronchiolar areas of the grafted lungs, forming organized clusters reminiscent of inducible tertiary lymphoid structures (iTLS). These iTLS in lung allografts are made of Foxp3$^{+}$ Treg cells, conventional T cells, and B cells, as evidenced by using microscopy-based distribution and neighborhood analyses. Foxp3-transgenic mice with inducible and selective deletion of Foxp3$^{+}$ cells are unable to form iTLS in lung allografts, and these mice acutely reject lung allografts. Collectively, we report that short-term, high-intensity and biased IL-2 pre-conditioning facilitates acceptance of vascularized and ventilated lung allografts without the need of immunosuppression, by inducing Foxp3-controlled iTLS formation within allografts

Interleukin-4 receptor engagement impairs the migration and effector functions of neutrophils

Type 2 immunity serves to resist parasitic helminths, venoms, and toxins, but the role and regulation of neutrophils during type 2 immune responses are controversial. Helminth models suggested a contribution of neutrophils to type 2 immunity, whereas neutrophils are associated with increased disease severity during type 2 inflammatory disorders, such as asthma

The role of cytokines in T-cell memory in health and disease

Upon stimulation with their cognate antigen, naive T cells undergo proliferation and differentiation into effector cells, followed by apoptosis or survival as precursors of long-lived memory cells. These phases of a T-cell response and the ensuing maintenance of memory T cells are shaped by cytokines, most notably interleukin-2 (IL-2), IL-7, and IL-15 that share the common γ chain (γc ) cytokine receptor. Steady-state production of IL-7 and IL-15 is necessary for background proliferation and homeostatic survival of CD4+ and CD8+ memory T cells. During immune responses, augmented levels of IL-2, IL-15, IL-21, IL-12, IL-18, and type-I interferons determine the memory potential of antigen-specific effector CD8+ cells, while increased IL-2 and IL-15 cause bystander proliferation of heterologous CD4+ and CD8+ memory T cells. Limiting availability of γc cytokines, reduction in regulatory T cells or IL-10, and persistence of inflammation or cognate antigen can result in memory T cells, which fail to become cytokine-dependent long-lived cells. Conversely, increased IL-7 and IL-15 can expand memory T cells, including pathogenic tissue-resident memory T cells, as seen in lymphopenia and certain chronic-inflammatory disorders and malignancies. These abovementioned factors impact immunotherapy and vaccines directed at memory T cells in cancer and chronic infection

Establishment of a scalable microfluidic assay for characterization of population‐based neutrophil chemotaxis

Background: Regulation of neutrophil chemotaxis and activation plays crucial roles in immunity, and dysregulated neutrophil responses can lead to pathology as seen in neutrophilic asthma. Neutrophil recruitment is key for initiating immune defense and inflammation, and its modulation is a promising therapeutic target. Microfluidic technology is an attractive tool for characterization of neutrophil migration. Compared to transwell assays, microfluidic approaches could offer several advantages, including precis e control of defined chemokine gradients in space and time, automated quantitative analysis of chemotaxis, and high throughput. Methods: We established a microfluidic device for fully automated, quantitative assessment of neutrophil chemotaxis. Freshly isolated mouse neutrophils from bone marrow or human neutrophils from peripheral blood were assessed in real time using an epifluorescence microscope for their migration toward the potent chemoattractants C-X-C-motif ligand 2 (CXCL2) and CXCL8, without or with interleukin-4 (IL-4) pre-incubation. Results: Our microfluidic device allowed the precise and reproducible determination of the optimal CXCL2 and CXCL8 concentrations for mouse and human neutrophil chemotaxis, respectively. Furthermore, our microfluidic assay was able to measure the equilibrium and real-time dynamic effects of specific modulators of neutrophil chemotaxis. We demonstrated this concept by showing that IL-4 receptor signaling in mouse and human neutrophils inhibited their migration toward CXCL2 and CXCL8, respectively, and this inhibition was time-dependent. Conclusion: Collectively, our microfluidic device shows several advantages over traditional transwell migration assays and its design is amenable to future integration into multiplexed high-throughput platforms for screening of molecules that modulate the chemotaxis of different immune cells. Keywords: basic immunology; chemokines; inflammation; innate immunity

Regulation of neutrophils in type 2 immune responses

Type 2 immune responses contribute to the resistance to helminths and toxins as well as several physiological processes. Although they usually do not participate in type 2 immune responses, neutrophils have been shown in mice to enhance the anti-helminth response, but they also contribute to increased target tissue damage. Increased pathology and morbidity is also observed in type 2 immune-mediated disorders, such as allergic asthma, when neutrophils become a predominant subset of the infiltrate. How neutrophil recruitment is regulated during type 2 immune responses is now starting to become clear, with recent data showing that signaling via the prototypic type 2 cytokine interleukin-4 receptor mediates direct and indirect inhibitory actions on neutrophils in mice and humans

Nanoparticle-coupled topical methotrexate can normalize immune responses and induce tissue remodeling in psoriasis

Methotrexate (MTX) is an anti-proliferative drug used for treating inflammatory diseases including psoriasis. Nevertheless, its use in localized therapy is impeded due to poor transdermal penetration. We show that MTX coupled with gold-nanoparticles (GNPs) demonstrates superior anti-inflammatory efficacy compared to MTX-alone in an imiquimod (IMQ)-induced mouse model, significantly reducing γδ T cells, CD4+ T cells, and neutrophils. Furthermore, it was well tolerated upon systemic and topical administration. In an AGR129 human xenograft mouse model, two-week topical treatment with MTX-GNPs inhibited skin hyperplasia significantly better than topical calcipotriol-betamethasone (TCB) and led to profound tissue remodeling, involving upregulation of extracellular matrix reorganization and downregulation of cornification and keratinization processes. The number of resident T cells in the grafts as well as IL-17 production drastically decreased upon MTX-GNP treatment. While both MTX and MTX-GNPs directly prevented proliferation and induced apoptosis of T cells, suppression of cytokine production was a shared mechanism of GNP and MTX-GNPs. In conclusion, MTX-GNPs influence immune and stromal components of the skin, leading to potent inhibition of pathogenesis in preclinical psoriasis. MTX-GNPs surpass the efficacy of conventional MTX and standard of care, emerging as a non-steroidal, topical alternative for psoriasis treatment

Biased IL-2 signals induce Foxp3-rich pulmonary lymphoid structures and facilitate long-term lung allograft acceptance in mice

Transplantation of solid organs can be life-saving in patients with endstage organ failure, however, graft rejection remains a major challenge. In this study, by pre-conditioning with interleukin-2 (IL-2)/anti-IL-2 antibody complex treatment biased toward IL-2 receptor α, we achieved acceptance of fully mismatched orthotopic lung allografts that remained morphologically and functionally intact for more than 90 days in immunocompetent mice. These allografts are tolerated by the actions of forkhead box p3 (Foxp3)⁺ regulatory T (Treg) cells that home to the lung allografts. Although counts of circulating Treg cells rapidly return to baseline following cessation of IL-2 treatment, Foxp3⁺ Treg cells persist in peribronchial and peribronchiolar areas of the grafted lungs, forming organized clusters reminiscent of inducible tertiary lymphoid structures (iTLS). These iTLS in lung allografts are made of Foxp3⁺ Treg cells, conventional T cells, and B cells, as evidenced by using microscopy-based distribution and neighborhood analyses. Foxp3-transgenic mice with inducible and selective deletion of Foxp3⁺ cells are unable to form iTLS in lung allografts, and these mice acutely reject lung allografts. Collectively, we report that short-term, high-intensity and biased IL-2 pre-conditioning facilitates acceptance of vascularized and ventilated lung allografts without the need of immunosuppression, by inducing Foxp3-controlled iTLS formation within allografts.ISSN:2041-172

Dynamic prostate cancer transcriptome analysis delineates the trajectory to disease progression.

Comprehensive genomic studies have delineated key driver mutations linked to disease progression for most cancers. However, corresponding transcriptional changes remain largely elusive because of the bias associated with cross-study analysis. Here, we overcome these hurdles and generate a comprehensive prostate cancer transcriptome atlas that describes the roadmap to tumor progression in a qualitative and quantitative manner. Most cancers follow a uniform trajectory characterized by upregulation of polycomb-repressive-complex-2, G2-M checkpoints, and M2 macrophage polarization. Using patient-derived xenograft models, we functionally validate our observations and add single-cell resolution. Thereby, we show that tumor progression occurs through transcriptional adaption rather than a selection of pre-existing cancer cell clusters. Moreover, we determine at the single-cell level how inhibition of EZH2 - the top upregulated gene along the trajectory - reverts tumor progression and macrophage polarization. Finally, a user-friendly web-resource is provided enabling the investigation of dynamic transcriptional perturbations linked to disease progression