G-protein-coupled receptor P2Y10 facilitates chemokine-induced CD4 T cell migration through autocrine/paracrine mediators.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, play a central role in the regulation of T cell migration. Various GPCRs are upregulated in activated CD4 T cells, including P2Y10, a putative lysophospholipid receptor that is officially still considered an orphan GPCR, i.e., a receptor with unknown endogenous ligand. Here we show that in mice lacking P2Y10 in the CD4 T cell compartment, the severity of experimental autoimmune encephalomyelitis and cutaneous contact hypersensitivity is reduced. P2Y10-deficient CD4 T cells show normal activation, proliferation and differentiation, but reduced chemokine-induced migration, polarization, and RhoA activation upon in vitro stimulation. Mechanistically, CD4 T cells release the putative P2Y10 ligands lysophosphatidylserine and ATP upon chemokine exposure, and these mediators induce P2Y10-dependent RhoA activation in an autocrine/paracrine fashion. ATP degradation impairs RhoA activation and migration in control CD4 T cells, but not in P2Y10-deficient CD4 T cells. Importantly, the P2Y10 pathway appears to be conserved in human T cells. Taken together, P2Y10 mediates RhoA activation in CD4 T cells in response to auto-/paracrine-acting mediators such as LysoPS and ATP, thereby facilitating chemokine-induced migration and, consecutively, T cell-mediated diseases.

Control of myeloid cell density in barrier tissues.

The interface between the mammalian host and its environment is formed by barrier tissues, for example, of the skin, and the respiratory and the intestinal tracts. On the one hand, barrier tissues are colonized by site-adapted microbial communities, and on the other hand, they contain specific myeloid cell networks comprising macrophages, dendritic cells, and granulocytes. These immune cells are tightly regulated in function and cell number, indicating important roles in maintaining tissue homeostasis and immune balance in the presence of commensal microorganisms. The regulation of myeloid cell density and activation involves cell-autonomous 'single-loop circuits' including autocrine mechanisms. However, an array of microenvironmental factors originating from nonimmune cells and the microbiota, as well as the microanatomical structure, impose additional layers of regulation onto resident myeloid cells. This review discusses models integrating these factors into cell-specific programs to instruct differentiation and proliferation best suited for the maintenance and renewal of immune homeostasis in the tissue-specific environment.

TNFSF15 Promotes Antimicrobial Pathways in Human Macrophages and These Are Modulated by TNFSF15 Disease-Risk Variants.

BACKGROUND & AIMS: TNFSF15 genetic variants leading to increased TNF superfamily member 15 (TNFSF15) expression confer risk for inflammatory bowel disease (IBD), and TNFSF15 is being explored as a therapeutic target in IBD patients. Although the focus for TNFSF15-mediated inflammatory outcomes has been predominantly on its action on T cells, TNFSF15 also promotes inflammatory outcomes in human macrophages. Given the critical role for macrophages in bacterial clearance, we hypothesized that TNFSF15 promotes antimicrobial pathways in human macrophages and that macrophages from TNFSF15 IBD risk carriers with higher TNFSF15 expression have an advantage in these antimicrobial outcomes. METHODS: We analyzed protein expression, signaling, bacterial uptake, and intracellular bacterial clearance in human monocyte-derived macrophages through flow cytometry, enzyme-linked immunosorbent assay, and gentamicin protection. RESULTS: Autocrine/paracrine TNFSF15 interactions with death receptor 3 (DR3) were required for optimal levels of pattern-recognition-receptor (PRR)-induced bacterial clearance in human macrophages. TNFSF15 induced pyruvate dehydrogenase kinase 1-dependent bacterial uptake and promoted intracellular bacterial clearance through reactive oxygen species, nitric oxide synthase 2, and autophagy up-regulation. The TNFSF15-initiated TNF receptor-associated factor 2/receptor-interacting protein kinase 1/RIP3 pathway was required for mitogen-activated protein kinase and nuclear factor-κB activation, and, in turn, induction of each of the antimicrobial pathways; the TNFSF15-initiated Fas-associated protein with death domain/mucosa-associated lymphoid tissue lymphoma translocation protein 1/caspase-8 pathway played a less prominent role in antimicrobial functions, despite its key role in TNFSF15-induced cytokine secretion. Complementation of signaling pathways or antimicrobial pathways restored bacterial uptake and clearance in PRR-stimulated macrophages where TNFSF15:DR3 interactions were inhibited. Monocyte-derived macrophages from high TNFSF15-expressing rs6478108 TT IBD risk carriers in the TNFSF15 region showed increased levels of the identified antimicrobial pathways. CONCLUSIONS: We identify that autocrine/paracrine TNFSF15 is required for optimal PRR-enhanced antimicrobial pathways in macrophages, define mechanisms regulating TNFSF15-dependent bacterial clearance, and determine how the TNFSF15 IBD risk genotype modulates these outcomes.

An Autocrine Circuit of IL-33 in Keratinocytes Is Involved in the Progression of Psoriasis.

IL-33 is constitutively expressed in the skin. Psoriasis is a common skin inflammatory disease. The roles of IL-33 in psoriasis have not been well-elucidated. We identified that keratinocytes (KCs) are the predominant cells expressing IL-33 and its receptor, suppression of tumorigenicity 2, in the skin. KCs actively released IL-33 on psoriasis inflammatory stimuli and induced psoriasis-related cytokine, chemokine, and inflammatory molecules genes transcription in KCs in an autocrine manner. IL-33‒specific deficiency in KCs ameliorated imiquimod-induced psoriatic dermatitis. In addition, intradermal injection of recombinant IL-33 alone induced psoriasis-like dermatitis, which is attributed to the transcriptional upregulation of genes enriched in IL-17, TNF, and chemokine signaling pathway in KCs on recombinant IL-33 stimulation. Our data demonstrate that the autocrine circuit of IL-33 in KCs promotes the progression of psoriatic skin inflammation, and IL-33 is a potential therapeutic target for psoriasis.

miR-21a in exosomes from Lewis lung carcinoma cells accelerates tumor growth through targeting PDCD4 to enhance expansion of myeloid-derived suppressor cells.

In patients with lung cancer, myeloid-derived suppressor cells (MDSCs) have been reported to be significantly increased. Tumor-derived exosomes (TDEs) from various cancers played a critical role in MDSC induction. However, studies on the molecular mechanism underlying MDSC expansion induced by exosomes from lung cancer cells are still limited. In this study, we demonstrated that LLC-Exo accelerated tumor growth along with a significant accumulation of MDSCs in mouse tumor model. miRNA profiling showed that miR-21a was enriched in LLC-Exo. The depletion of miR-21a in LLC-Exo leads to the loss of their ability to induce MDSC expansion. Further results showed that miR-21a of LLC-Exo induced MDSC expansion via downregulation of the programmed cell death protein 4 (PDCD4) protein. The results of gain-and loss-of-function experiments validated that PDCD4 function as a critical inhibitor to negatively regulate expansion of MDSCs via inhibition Il-6 production in bone marrow cells. In addition, our data showed that exosomes derived from human lung cancer cell lines expressing miR-21a, also induced expansion of MDSCs in human CD14+ monocytes in vitro. Overall, our results demonstrated that miR-21a enriched in lung carcinoma cell-derived exosomes could promote functional expansion of MDSCs through targeting PDCD4.

Connecting secretome to hematopoietic stem cell phenotype shifts in an engineered bone marrow niche.

Hematopoietic stem cells (HSCs) primarily reside in the bone marrow, where they receive external cues from their local microenvironment. The complex milieu of biophysical cues, cellular components and cell-secreted factors regulates the process by which HSC produce the blood and immune system. We previously showed direct coculture of primary murine hematopoietic stem and progenitor cells with a population of marrow-derived mesenchymal stromal and progenitor cells (MSPCs) in a methacrylamide-functionalized gelatin (GelMA) hydrogel improves hematopoietic progenitor maintenance. However, the mechanism by which MSPCs influenced HSC fate decisions remained unknown. Herein, we report the use of proteomic analysis to correlate HSC phenotype to a broad candidate pool of 200 soluble factors produced by combined mesenchymal and hematopoietic progeny. Partial least squares regression (PLSR), along with an iterative filter method, identified TGFß-1, MMP-3, c-RP and TROY as positively correlated with HSC maintenance. Experimentally, we then observe exogenous stimulation of HSC monocultures in GelMA hydrogels with these combined cytokines increases the ratio of hematopoietic progenitors to committed progeny after a 7-day culture 7.52 ± 3.65-fold compared to non-stimulated monocultures. Findings suggest a cocktail of the downselected cytokines amplifies hematopoietic maintenance potential of HSCs beyond that of MSPC-secreted factors alone. This work integrates empirical and computation methods to identify cytokine combinations to improve HSC maintenance within an engineered HSC niche, suggesting a route toward identifying feeder-free culture platforms for HSC expansion. Insight Hematopoietic stem cells within an artificial niche receive maintenance cues in the form of soluble factors from hematopoietic and mesenchymal progeny. Applying a proteomic regression analysis, we identify a reduced set of soluble factors correlated to maintenance of a hematopoietic phenotype during culture in a biomaterial model of the bone marrow niche. We identify a minimum factor cocktail that promotes hematopoietic maintenance potential in a gelatin-based culture, regardless of the presence of mesenchymal feeder cells. By combining empirical and computational methods, we report an experimentally feasible number of factors from a large dataset, enabling exogenous integration of soluble factors into an engineered hematopoietic stem cell for enhanced maintenance potential of a quiescent stem cell population.

IL23 induces IL23R recycling and amplifies innate receptor-induced signalling and cytokines in human macrophages, and the IBD-protective IL23R R381Q variant modulates these outcomes.

OBJECTIVE: The interleukin (IL)23 pathway contributes to IBD pathogenesis and is being actively studied as a therapeutic target in patients with IBD. Unexpected outcomes in these therapeutic trials have highlighted the importance of understanding the cell types and mechanisms through which IL23 regulates immune outcomes. How IL23 regulates macrophage outcomes and the consequences of the IL23R R381Q IBD-protective variant on macrophages are not well defined; macrophages are key players in IBD pathogenesis and inflammation. DESIGN: We analysed protein and RNA expression, signalling and localisation in human monocyte-derived macrophages (MDMs) through western blot, ELISA, real-time PCR, flow cytometry, immunoprecipitation and microscopy. RESULTS: IL23R was critical for optimal levels of pattern-recognition receptor (PRR)-induced signalling and cytokines in human MDMs. In contrast to the coreceptor IL12Rß1, IL23 induced dynamic IL23R cell surface regulation and this required clathrin and dynamin-mediated endocytosis and endocytic recycling-dependent pathways; these pathways were essential for IL23R-mediated outcomes. The IBD-protective IL23R R381Q variant showed distinct outcomes. Relative to IL23R R381, HeLa cells expressing IL23R Q381 showed decreased IL23R recycling and reduced assembly of IL23R Q381 with Janus kinase/signal transducer and activator of transcription pathway members. In MDMs from IL23R Q381 carriers, IL23R accumulated in late endosomes and lysosomes on IL23 treatment and cells demonstrated decreased IL23R- and PRR-induced signalling and cytokines relative to IL23R R381 MDMs. CONCLUSION: Macrophage-mediated inflammatory pathways are key contributors to IBD pathogenesis, and we identify an autocrine/paracrine IL23 requirement in PRR-initiated human macrophage outcomes and in human intestinal myeloid cells, establish that IL23R undergoes ligand-induced recycling, define mechanisms regulating IL23R-induced signalling and determine how the IBD-protective IL23R R381Q variant modulates these processes.

Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature.

With multifactorial etiologies, combined with disease heterogeneity and a lack of suitable diagnostic markers and therapy, endometriosis remains a major reproductive health challenge. Extracellular vesicles (EVs) have emerged as major contributors of disease progression in several conditions, including a variety of cancers; however, their role in endometriosis pathophysiology has remained elusive. Using next-generation sequencing of EVs obtained from endometriosis patient tissues and plasma samples compared with controls, we have documented that patient EVs carry unique signatures of miRNAs and long noncoding RNAs (lncRNAs) reflecting their contribution to disease pathophysiology. Mass spectrophotometry-based proteomic analysis of EVs from patient plasma and peritoneal fluid further revealed enrichment of specific pathways, as well as altered immune and metabolic processes. Functional studies in endometriotic epithelial and endothelial cell lines using EVs from patient plasma and controls clearly indicate autocrine uptake and paracrine cell proliferative roles, suggestive of their involvement in endometriosis. Multiplex cytokine analysis of cell supernatants in response to patient and control plasma-derived EVs indicate robust signatures of important inflammatory and angiogenic cytokines known to be involved in disease progression. Collectively, these findings suggest that endometriosis-associated EVs carry unique cargo and contribute to disease pathophysiology by influencing inflammation, angiogenesis, and proliferation within the endometriotic lesion microenvironment.

Follicular B2 Cell Activation and Class Switch Recombination Depend on Autocrine C3ar1/C5ar1 Signaling in B2 Cells.

The involvement of complement in B2 cell responses has been regarded as occurring strictly via complement components in plasma. In this study, we show that Ab production and class switch recombination (CSR) depend on autocrine C3a and C5a receptor (C3ar1/C5ar1) signaling in B2 cells. CD40 upregulation, IL-6 production, growth in response to BAFF or APRIL, and AID/Bcl-6 expression, as well as follicular CD4+ cell CD21 production, all depended on this signal transduction. OVA immunization of C3ar1-/-C5ar1-/- mice elicited IgM Ab but no other isotypes, whereas decay accelerating factor (Daf1)-/- mice elicited more robust Ab production and CSR than wild-type (WT) mice. Comparable differences occurred in OVA-immunized µMT recipients of WT, C3ar1-/-C5ar1-/- , and Daf1-/- B2 cells and in hen egg lysozyme-immunized µMT recipients of MD4 B2 cells on each genetic background. B2 cells produced factor I and C3 and autophosphorylated CD19. Immunized C3-/-C5-/- recipients of WT MD4 bone marrow efficiently produced Ab. Thus, B2 cell-produced complement participates in B2 cell activation.

IL-4/IFN-γ inflammatory cytokine profile induces a deficient regulation of the IL-1ß/IL-1RI/EP2/COX-2 pathway in nasal mucosa.

BACKGROUND: We hypothesized that the peculiar mixed interleukin-4 (IL-4/Th2) and interferon gamma INF-γ (INF-γ/Th1) inflammatory milieu found in the airways of patients with aspirin-exacerbated respiratory disease (AERD) is responsible for the altered regulation of the IL-1ß/IL-1RI-/EP2/COX-2 autocrine loop also found in these patients. The objective of the study is to demonstrate that IL-4 and INF-γ cytokines, are capable of inducing in healthy nasal mucosa (NM) the dysregulation of the autocrine loop of COX reported in AERD. SUBJECTS AND METHODS: Fibroblasts were obtained from NM (n = 8). To evaluate the role of IL-4 and IFN-γ on the autocrine loop, fibroblasts were incubated with or without IL-1ß, in the presence or absence of IL-4 and/or IFN-γ for 48 h. After this period, the expression of EP2, EP3, EP4, IL-1RI, COX-2 and mPGES-1 was measured by Western blot. RESULTS: Stimulation of fibroblasts with IL-1ß significantly increased the expression of EP2, but had no effects on EP3 and EP4 expression Incubation with IL-4 or IFN-γ alone was not able to modify the expression of any of the components of the autocrine loop. In contrast, co-treatment with IL-4 and IFN-γ was able to significantly inhibit IL-1ß-induced EP2, IL-1RI, COX-2 and mPGES-1. CONCLUSION: These results suggest that the mixed Th1/Th2 inflammatory pattern found in the airways of AERD patients might be responsible for the altered regulation of the COX pathway also reported in these asthma patients.

Cholecystokinin Downregulates Psoriatic Inflammation by Its Possible Self-Regulatory Effect on Epidermal Keratinocytes.

Cholecystokinin (CCK) is a peptide hormone that functions in digestive organs and the CNS. We previously showed that CCK downregulates peripheral pruritus by suppressing degranulation of mast cells. In this study, we demonstrated that CCK octapeptide (CCK8) was constitutively expressed in the epidermis of normal skin, whereas its expression was lost in acanthotic lesions of psoriasis. In contrast, CCKA receptor (CCKAR), a high-affinity receptor for CCK, was constitutively expressed in the epidermis of psoriatic skin lesions. Expression of CCK was also reduced in skin lesions of an imiquimod (IMQ)-induced psoriatic mouse model. Notably, the expression level of CCK inversely correlated with the severity of epidermal inflammation, raising the possibility that CCK from epidermal keratinocytes suppresses the psoriatic inflammation. To verify this hypothesis, we investigated the effects of sulfated CCK octapeptide (CCK8S) on the development of IMQ-induced psoriatic inflammation. i.p. injection of CCK8S suppressed the IMQ-induced psoriatic inflammation accompanied by reduced mRNA expression of IL-17, IL-22, and IL-6 but not of IL-23. The suppressive effect of CCK8S was completely restored by administration of CCKAR antagonist. In vitro studies showed that exogenous CCK8S suppressed IL-6 production in CCKAR-expressing cultured human keratinocytes, and blocking the endogenous CCK signaling with CCKAR antagonist markedly enhanced IL-6 production. When keratinocytes were stimulated with IL-17, the expression of endogenous CCK was significantly decreased. These findings suggest that CCK physiologically functions as a negative regulator of keratinocyte-based inflammation in an autocrine or paracrine manner, although decreased CCK may pathologically contribute to continuous and aggravated skin lesions such as psoriasis.

Autocrine VEGF signalling on M2 macrophages regulates PD-L1 expression for immunomodulation of T cells.

M2-polarized macrophages, on one hand, can promote tumour vascularization by producing proangiogenic factors, such as vascular endothelial growth factor (VEGF). On the other hand, the expression of VEGF receptors (VEGFR) in this cell lineage was also reported. Although the function of VEGF/VEGFR axis plays a pivotal role in macrophages infiltration and angiogenesis, however, there is still lack of the direct evidence to show the role of VEGF as an autocrine operating in M2 macrophages, particularly for immunomodulation. In our study, we surprisingly discovered that M2 macrophages polarized by baicalin can simultaneously express VEGF and its receptors. Taking advantage of this unique culture system, we were able to investigate the biological activity of M2 macrophages in response to the autocrine VEGF milieu. Our results showed that the expression of programmed death-ligand 1 (PD-L1) on M2 macrophages was significantly up-regulated in autocrine VEGF milieu. Through the blockade of autocrine VEGF signalling, PD-L1 expression on M2 macrophages was dramatically down-regulated. Furthermore, transplantation of PD-L1+ M2 macrophage stimulated by autocrine VEGF into allogeneic mice significantly suppressed host CD4+ /CD8+ T cells in the peripheral blood and increased CD4+ CD25+ regulatory T cells in the bone marrow. In conclusion, our findings provide a novel biological basis to support the current successful strategy using combined VEGF/PD-1 signalling blockade in cancer therapy.

Candida albicans ß-Glucan Differentiates Human Monocytes Into a Specific Subset of Macrophages.

ß-Glucan derived from cell walls of Candida albicans is a potent immune modulator. It has been shown to induce trained immunity in monocytes via epigenetic and metabolic reprogramming and to protect from lethal sepsis if applied prior to infection. Since ß-glucan-trained monocytes have not been classified within the system of mononuclear phagocytes we analyzed these cells metabolically, phenotypically and functionally with a focus on monocyte-to-macrophage differentiation and compared them with naïve monocytes and other types of monocyte-derived cells such as classically (M1) or alternatively (M2) activated macrophages and monocyte-derived dendritic cells (moDCs). We show that ß-glucan inhibits spontaneous apoptosis of monocytes independent from autocrine or paracrine M-CSF release and stimulates monocyte differentiation into macrophages. ß-Glucan-differentiated macrophages exhibit increased cell size and granularity and enhanced metabolic activity when compared to naïve monocytes. Although ß-glucan-primed cells expressed markers of alternative activation and secreted higher levels of IL-10 after lipopolysaccharide (LPS), their capability to release pro-inflammatory cytokines and to kill bacteria was unaffected. Our data demonstrate that ß-glucan priming induces a population of immune competent long-lived monocyte-derived macrophages that may be involved in immunoregulatory processes.

From fever to immunity: A new role for IGFBP-6?

Fever is a fundamental response to infection and a hallmark of inflammatory disease, which has been conserved and shaped through millions of years of natural selection. Although fever is able to stimulate both innate and adaptive immune responses, the very nature of all the molecular thermosensors, the timing and the detailed mechanisms translating a physical trigger into a fundamental biological response are incompletely understood. Here we discuss the consequence of hyperthermic stress in dendritic cells (DCs), and how the sole physical input is sensed as an alert stimulus triggering a complex transition in a very narrow temporal window. Importantly, we review recent findings demonstrating the significant and specific changes discovered in gene expression and in the metabolic phenotype associated with hyperthermia in DCs. Furthermore, we discuss the results that support a model based on a thermally induced autocrine signalling, which rewires and sets a metabolism checkpoint linked to immune activation of dendritic cells. Importantly, in this context, we highlight the novel regulatory functions discovered for IGFBP-6 protein: induction of chemotaxis; capacity to increase oxidative burst and degranulation of neutrophils, ability to induce metabolic changes in DCs. Finally, we discuss the role of IGFBP-6 in autoimmune disease and how novel mechanistic insights could lead to exploit thermal stress-related mechanisms in the context of cancer therapy.

Purinergic P2X4 receptors and mitochondrial ATP production regulate T cell migration.

T cells must migrate in order to encounter antigen-presenting cells (APCs) and to execute their varied functions in immune defense and inflammation. ATP release and autocrine signaling through purinergic receptors contribute to T cell activation at the immune synapse that T cells form with APCs. Here, we show that T cells also require ATP release and purinergic signaling for their migration to APCs. We found that the chemokine stromal-derived factor-1α (SDF-1α) triggered mitochondrial ATP production, rapid bursts of ATP release, and increased migration of primary human CD4+ T cells. This process depended on pannexin-1 ATP release channels and autocrine stimulation of P2X4 receptors. SDF-1α stimulation caused localized accumulation of mitochondria with P2X4 receptors near the front of cells, resulting in a feed-forward signaling mechanism that promotes cellular Ca2+ influx and sustains mitochondrial ATP synthesis at levels needed for pseudopod protrusion, T cell polarization, and cell migration. Inhibition of P2X4 receptors blocked the activation and migration of T cells in vitro. In a mouse lung transplant model, P2X4 receptor antagonist treatment prevented the recruitment of T cells into allograft tissue and the rejection of lung transplants. Our findings suggest that P2X4 receptors are therapeutic targets for immunomodulation in transplantation and inflammatory diseases.

Juxtacrine Signaling Inhibits Antitumor Immunity by Upregulating PD-L1 Expression.

Programmed death-ligand 1 (PD-L1) is a well-known immune checkpoint protein that helps cancer cells evade immune response. Anti-PD-L1 immune therapy has been approved for the treatment of several advanced human cancers. Therefore, further understanding of the regulatory mechanisms of PD-L1 is critical to improve PD-L1-targeting immunotherapy. Recent studies indicated that contact-dependent pathways may regulate anticancer immunity, highlighting the importance of cell contact-induced signaling in cancer immunity. Here, we show that tumor cell contact upregulates PD-L1 expression and reduces T-cell-mediated cell killing through the membrane receptor tyrosine kinase ephrin receptor A10 (EphA10), which is not expressed in normal tissues except testis and is known to mediate cell contact-dependent juxtacrine signaling. Knockout of EphA10 in tumor cells increased T-cell-mediated antitumor immunity in syngeneic mouse models. EphA10 expression also correlated positively with PD-L1 in human breast tumor tissues. Together, our data reveal that in addition to paracrine/autocrine signaling, cell contact-mediated juxtacrine signaling also promotes PD-L1 expression, implying that tumor cells may escape immune surveillance via this mechanism and that targeting EphA10 to boost antitumor immunity may be a new immune checkpoint blockade strategy for female patients with breast cancer.Significance: Regulation of PD-L1 expression by cell contact-mediated signaling promotes immune escape in breast cancer and may lead to the development of an immunotherapy with less adverse effects in female patients. Cancer Res; 78(14); 3761-8. ©2018 AACR.

An Autocrine TNFα-Tumor Necrosis Factor Receptor 2 Loop Promotes Epigenetic Effects Inducing Human Treg Stability In Vitro.

A crucial issue for Treg-based immunotherapy is to maintain a bona fide Treg phenotype as well as suppressive function during and after ex vivo expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation in vitro, in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg in vitro. Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the FOXP3 gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα-TNFR2 underlying the maintenance of Treg stability in vitro. Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα-TNFR2 loop plays an important role in endorsing Treg stability.

Evidence for autocrine and paracrine regulation of allergen-induced mast cell mediator release in the guinea pig airways.

Mast cells play an essential role in immediate type hypersensitivity reactions and in chronic allergic diseases of the airways, including asthma. Mast cell mediator release can be modulated by locally released autacoids and circulating hormones, but surprisingly little is known about the autocrine effects of mediators released upon mast cell activation. We thus set out to characterize the autocrine and paracrine effects of mast cell mediators on mast cell activation in the guinea pig airways. By direct measures of histamine, cysteinyl-leukotriene and thromboxane release and with studies of allergen-evoked contractions of airway smooth muscle, we describe a complex interplay amongst these autacoids. Notably, we observed an autocrine effect of the cysteinyl-leukotrienes acting through cysLT1 receptors on mast cell leukotriene release. We confirmed the results of previous studies demonstrating a marked enhancement of mast cell mediator release following cyclooxygenase inhibition, but we have extended these results by showing that COX-2 derived eicosanoids inhibit cysteinyl-leukotriene release and yet are without effect on histamine release. Given the prominent role of COX-1 inhibition in aspirin-sensitive asthma, these data implicate preformed mediators stored in granules as the initial drivers of these adverse reactions. Finally, we describe the paracrine signaling cascade leading to thromboxane synthesis in the guinea pig airways following allergen challenge, which occurs indirectly, secondary to cysLT1 receptor activation on structural cells and/ or leukocytes within the airway wall, and a COX-2 dependent synthesis of the eicosanoid. The results highlight the importance of cell-cell and autocrine interactions in regulating allergic responses in the airways.

Plant-to-plant communication triggered by systemin primes anti-herbivore resistance in tomato.

Plants actively respond to herbivory by inducing various defense mechanisms in both damaged (locally) and non-damaged tissues (systemically). In addition, it is currently widely accepted that plant-to-plant communication allows specific neighbors to be warned of likely incoming stress (defense priming). Systemin is a plant peptide hormone promoting the systemic response to herbivory in tomato. This 18-aa peptide is also able to induce the release of bioactive Volatile Organic Compounds, thus also promoting the interaction between the tomato and the third trophic level (e.g. predators and parasitoids of insect pests). In this work, using a combination of gene expression (RNA-Seq and qRT-PCR), behavioral and chemical approaches, we demonstrate that systemin triggers metabolic changes of the plant that are capable of inducing a primed state in neighboring unchallenged plants. At the molecular level, the primed state is mainly associated with an elevated transcription of pattern -recognition receptors, signaling enzymes and transcription factors. Compared to naïve plants, systemin-primed plants were significantly more resistant to herbivorous pests, more attractive to parasitoids and showed an increased response to wounding. Small peptides are nowadays considered fundamental signaling molecules in many plant processes and this work extends the range of downstream effects of this class of molecules to intraspecific plant-to-plant communication.

miR-146a modulates autoreactive Th17 cell differentiation and regulates organ-specific autoimmunity.

Autoreactive CD4 T cells that differentiate into pathogenic Th17 cells can trigger autoimmune diseases. Therefore, investigating the regulatory network that modulates Th17 differentiation may yield important therapeutic insights. miR-146a has emerged as a critical modulator of immune reactions, but its role in regulating autoreactive Th17 cells and organ-specific autoimmunity remains largely unknown. Here, we have reported that miR-146a-deficient mice developed more severe experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis (MS). We bred miR-146a-deficient mice with 2D2 T cell receptor-Tg mice to generate 2D2 CD4 T cells that are deficient in miR-146a and specific for myelin oligodendrocyte glycoprotein (MOG), an autoantigen in the EAE model. miR-146a-deficient 2D2 T cells induced more severe EAE and were more prone to differentiate into Th17 cells. Microarray analysis revealed enhancements in IL-6- and IL-21-induced Th17 differentiation pathways in these T cells. Further study showed that miR-146a inhibited the production of autocrine IL-6 and IL-21 in 2D2 T cells, which in turn reduced their Th17 differentiation. Thus, our study identifies miR-146a as an important molecular brake that blocks the autocrine IL-6- and IL-21-induced Th17 differentiation pathways in autoreactive CD4 T cells, highlighting its potential as a therapeutic target for treating autoimmune diseases.