Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation.

Neutrophils are the first immune cells recruited to sites of inflammation and infection. However, patients with allergic disorders such as atopic dermatitis show a paucity of skin neutrophils and are prone to bacterial skin infections, suggesting that allergic inflammation curtails neutrophil responses. Here we have shown that the type 2 cell signature cytokine interleukin-4 (IL-4) hampers neutrophil expansion and migration by antagonizing granulocyte colony-stimulating factor (G-CSF) and chemokine receptor-mediated signals. Cutaneous bacterial infection in mice was exacerbated by IL-4 signaling and improved with IL-4 inhibition, each outcome inversely correlating with neutrophil migration to skin. Likewise, systemic bacterial infection was worsened by heightened IL-4 activity, with IL-4 restricting G-CSF-induced neutrophil expansion and migration to tissues by affecting CXCR2-CXCR4 chemokine signaling in neutrophils. These effects were dependent on IL-4 acting through type 2 IL-4 receptors on neutrophils. Thus, targeting IL-4 might be beneficial in neutropenic conditions with increased susceptibility to bacterial infections.

Inflammation-Induced CCR7 Oligomers Form Scaffolds to Integrate Distinct Signaling Pathways for Efficient Cell Migration.

Host defense depends on orchestrated cell migration guided by chemokines that elicit selective but biased signaling pathways to control chemotaxis. Here, we showed that different inflammatory stimuli provoked oligomerization of the chemokine receptor CCR7, enabling human dendritic cells and T cell subpopulations to process guidance cues not only through classical G protein-dependent signaling but also by integrating an oligomer-dependent Src kinase signaling pathway. Efficient CCR7-driven migration depends on a hydrophobic oligomerization interface near the conserved NPXXY motif of G protein-coupled receptors as shown by mutagenesis screen and a CCR7-SNP demonstrating super-oligomer characteristics leading to enhanced Src activity and superior chemotaxis. Furthermore, Src phosphorylates oligomeric CCR7, thereby creating a docking site for SH2-domain-bearing signaling molecules. Finally, we identified CCL21-biased signaling that involved the phosphatase SHP2 to control efficient cell migration. Collectively, our data showed that CCR7 oligomers serve as molecular hubs regulating distinct signaling pathways.

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.

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.

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.

T-cell trafficking in the central nervous system.

To perform their distinct effector functions, pathogen-specific T cells have to migrate to target tissue where they recognize antigens and produce cytokines that elicit appropriate types of protective responses. Similarly, migration of pathogenic self-reactive T cells to target organs is an essential step required for tissue-specific autoimmunity. In this article, we review data from our laboratory as well as other laboratories that have established that effector function and migratory capacity are coordinately regulated in different T-cell subsets. We then describe how pathogenic T cells can enter into intact or inflamed central nervous system (CNS) to cause experimental autoimmune encephalomyelitis or multiple sclerosis. In particular, we elaborate on the role of CCR6/CCL20 axis in migration through the choroid plexus and the involvement of this pathway in immune surveillance of and autoimmunity in the CNS.

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.

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.

Much More Than IL-17A: Cytokines of the IL-17 Family Between Microbiota and Cancer.

The interleukin-(IL-)17 family of cytokines is composed of six members named IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17A is the prototype of this family, and it was the first to be discovered and targeted in the clinic. IL-17A is essential for modulating the interplay between commensal microbes and epithelial cells at our borders (i.e., skin and mucosae), and yet, for protecting us from microbial invaders, thus preserving mucosal and skin integrity. Interactions between the microbiota and cells producing IL-17A have also been implicated in the pathogenesis of immune mediated inflammatory diseases and cancer. While interactions between microbiota and IL-17B-to-F have only partially been investigated, they are by no means less relevant. The cellular source of IL-17B-to-F, their main targets, and their function in homeostasis and disease distinguish IL-17B-to-F from IL-17A. Here, we intentionally overlook IL-17A, and we focus instead on the role of the other cytokines of the IL-17 family in the interplay between microbiota and epithelial cells that may contribute to cancer pathogenesis and immune surveillance. We also underscore differences and similarities between IL-17A and IL-17B-to-F in the microbiota-immunity-cancer axis, and we highlight therapeutic strategies that directly or indirectly target IL-17 cytokines in diseases.

Nanoparticle-Coupled Topical Methotrexate Can Normalize Immune Responses and Induce Tissue Remodeling in Psoriasis.

Methotrexate (MTX) is an antiproliferative drug used for treating inflammatory diseases, including psoriasis. Nevertheless, its use in localized therapy is hindered because of poor transdermal penetration. We show that MTX coupled with gold nanoparticles (GNPs) demonstrates superior antiinflammatory efficacy than MTX alone in an imiquimod-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 and led to profound tissue remodeling, involving the upregulation of extracellular matrix reorganization and the downregulation of cornification and keratinization processes. The number of resident T cells in the grafts, as well as interleukin-17 production, drastically decreased upon MTX-GNP treatment. While both MTX and MTX-GNPs directly prevented the proliferation and induced apoptosis of T cells, the 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 the 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.

The Regulatory Effects of Interleukin-4 Receptor Signaling on Neutrophils in Type 2 Immune Responses.

Interleukin-4 (IL-4) receptor (IL-4R) signaling plays a pivotal role in type 2 immune responses. Type 2 immunity ensures several host-protective processes such as defense against helminth parasites and wound repair, however, type 2 immune responses also drive the pathogenesis of allergic diseases. Neutrophil granulocytes (neutrophils) have not traditionally been considered a part of type 2 immunity. While neutrophils might be beneficial in initiating a type 2 immune response, their involvement and activation is rather unwanted at later stages. This is evidenced by examples of type 2 immune responses where increased neutrophil responses are able to enhance immunity, however, at the cost of increased tissue damage. Recent studies have linked the type 2 cytokines IL-4 and IL-13 and their signaling via type I and type II IL-4Rs on neutrophils to inhibition of several neutrophil effector functions. This mechanism directly curtails neutrophil chemotaxis toward potent intermediary chemoattractants, inhibits the formation of neutrophil extracellular traps, and antagonizes the effects of granulocyte colony-stimulating factor on neutrophils. These effects are observed in both mouse and human neutrophils. Thus, we propose for type 2 immune responses that neutrophils are, as in other immune responses, the first non-resident cells to arrive at a site of inflammation or infection, thereby guiding and attracting other innate and adaptive immune cells; however, as soon as the type 2 cytokines IL-4 and IL-13 predominate, neutrophil recruitment, chemotaxis, and effector functions are rapidly shut off by IL-4/IL-13-mediated IL-4R signaling in neutrophils to prevent them from damaging healthy tissues. Insight into this neutrophil checkpoint pathway will help understand regulation of neutrophilic type 2 inflammation and guide the design of targeted therapeutic approaches for modulating neutrophils during inflammation and neutropenia.

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.