GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression.

T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2 Cre and Tbx21 Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3-sufficient and Gata3-deficient CNS-infiltrating CD4+ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.

Crosstalk between ILC2s and Th2 cells varies among mouse models.

Type 2 T helper (Th2) cells and group 2 innate lymphoid cells (ILC2s) provide protection against helminth infection and are involved in allergic responses. However, their relative importance and crosstalk during type 2 immune responses are still controversial. By generating and utilizing mouse strains that are deficient in either ILC2s or Th2 cells, we report that interleukin (IL)-33-mediated ILC2 activation promotes the Th2 cell response to papain; however, the Th2 cell response to ovalbumin (OVA)/alum immunization is thymic stromal lymphopoietin (TSLP) dependent but independent of ILC2s. During helminth infection, ILC2s and Th2 cells collaborate at different phases of the immune responses. Th2 cells, mainly through IL-4 production, induce the expression of IL-25, IL-33, and TSLP, among which IL-25 and IL-33 redundantly promote ILC2 expansion. Thus, while Th2 cell differentiation can occur independently of ILC2s, activation of ILC2s may promote Th2 responses, and Th2 cells can expand ILC2s by inducing type 2 alarmins.

Gata3 ZsG and Gata3 ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

Recent advances in understanding the Th1/Th2 effector choice.

For over 35 years since Mosmann and Coffman proposed the seminal "type 1 T helper (Th1)/type 2 T helper (Th2)" hypothesis in 1986, the immunological community has appreciated that naïve CD4 T cells need to make important decisions upon their activation, namely to differentiate towards a Th1, Th2, Th17 (interleukin-17-producing T helper), follicular T helper (Tfh), or regulatory T cell (Treg) fate to orchestrate a variety of adaptive immune responses. The major molecular underpinnings of the Th1/Th2 effector fate choice had been initially characterized using excellent reductionist in vitro culture systems, through which the transcription factors T-bet and GATA3 were identified as the master regulators for the differentiation of Th1 and Th2 cells, respectively. However, Th1/Th2 cell differentiation and their cellular heterogeneity are usually determined by a combinatorial expression of multiple transcription factors, particularly in vivo, where dendritic cell (DC) and innate lymphoid cell (ILC) subsets can also influence T helper lineage choices. In addition, inflammatory cytokines that are capable of inducing Th17 cell differentiation are also found to be induced during typical Th1- or Th2-related immune responses, resulting in an alternative differentiation pathway, transiting from a Th17 cell phenotype towards Th1 or Th2 cells. In this review, we will discuss the recent advances in the field, focusing on some new players in the transcriptional network, contributions of DCs and ILCs, and alternative differentiation pathways towards understanding the Th1/Th2 effector choice in vivo.

Characterization of Islet Leukocyte Populations in Human and Murine Islets by Flow Cytometry.

An increasing body of evidence indicates that a local islet immune response is not only limited to type 1 diabetes, but also is associated with islet dysfunction in type 2 diabetes. Recently, the presence of pancreatic CD68+ macrophages within islet tissues was demonstrated by RT-PCR and immunohistochemical methods. However, the precise profile and activation status of intraislet leukocytes, which are present in both murine and human islets, are poorly defined. Here, we describe a detailed flow cytometry protocol designed to analyze both human and murine islets for intraislet leukocytes and leukocyte subsets. This approach permits the simultaneous identification of multiple intraislet leukocyte subsets, as well as their activation statuses. The use of flow cytometry-based approaches will advance the field of islet biology and help to identify unique changes in the immune cell composition that accompanies pathological islet inflammation and dysfunction in type 2 diabetes.

Atherosclerosis-Driven Treg Plasticity Results in Formation of a Dysfunctional Subset of Plastic IFNγ+ Th1/Tregs.

RATIONALE: Forkhead box P3+ T regulatory cells (Tregs) are key players in maintaining immune homeostasis. Evidence suggests that Tregs respond to environmental cues to permit or suppress inflammation. In atherosclerosis, Th1-driven inflammation affects Treg homeostasis, but the mechanisms governing this phenomenon are unclear. OBJECTIVE: Here, we address whether atherosclerosis impacts Treg plasticity and functionality in Apoe-/- mice, and what effect Treg plasticity might have on the pathology of atherosclerosis. METHODS AND RESULTS: We demonstrate that atherosclerosis promotes Treg plasticity, resulting in the reduction of CXCR3+ Tregs and the accumulation of an intermediate Th1-like interferon (IFN)-γ+CCR5+ Treg subset (Th1/Tregs) within the aorta. Importantly, Th1/Tregs arise in atherosclerosis from bona fide Tregs, rather than from T-effector cells. We show that Th1/Tregs recovered from atherosclerotic mice are dysfunctional in suppression assays. Using an adoptive transfer system and plasticity-prone Mir146a-/- Tregs, we demonstrate that elevated IFNγ+ Mir146a-/- Th1/Tregs are unable to adequately reduce atherosclerosis, arterial Th1, or macrophage content within Apoe-/- mice, in comparison to Mir146a+/+ Tregs. Finally, via single-cell RNA-sequencing and real-time -polymerase chain reaction, we show that Th1/Tregs possess a unique transcriptional phenotype characterized by coexpression of Treg and Th1 lineage genes and a downregulation of Treg-related genes, including Ikzf2, Ikzf4, Tigit, Lilrb4, and Il10. In addition, an ingenuity pathway analysis further implicates IFNγ, IFNα, interleukin-2, interleukin-7, CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), T-cell receptor, and Csnk2b-related pathways in regulating Treg plasticity. CONCLUSIONS: Atherosclerosis drives Treg plasticity, resulting in the accumulation of dysfunctional IFNγ+ Th1/Tregs that may permit further arterial inflammation and atherogenesis.

Smooth Muscle Cell-Derived Interleukin-17C Plays an Atherogenic Role via the Recruitment of Proinflammatory Interleukin-17A+ T Cells to the Aorta.

OBJECTIVE: Atherosclerosis is characterized by frequent communication between infiltrating leukocytes and vascular cells, through chemokine and cytokine networks. Interleukin-17C (IL-17C) is detectable within atherosclerotic lesions; however, the potential involvement of this cytokine has not been examined. Thus, we sought to investigate the role of IL-17C in atherosclerosis. APPROACH AND RESULTS: The expression of IL-17 cytokines was profiled within aortas of apolipoprotein E double knockout (Apoe(-/-)) mice, and Il17c expression was elevated. Flow cytometry experiments revealed a major population of aortic IL-17C-producing smooth muscle cells. Next, we generated Il17c(-/-)Apoe(-/-) mice and demonstrated that atherosclerotic lesion and collagen content was diminished within Western diet-fed Il17c(-/-)Apoe(-/-) aortas and aortic roots in comparison to Apoe(-/-) controls. Smooth muscle cells and fibroblasts were mainly responsible for the reduced Col1A1 expression in the aorta of Il17c(-/-)Apoe(-/-) mice. Importantly, IL-17C-treated Apoe(-/-) aortas showed upregulated Col1A1 expression ex vivo. Il17c(-/-)Apoe(-/-) mice displayed a proportional reduction in aortic macrophages, neutrophils, T cells, T helper 1 cells, and T regulatory cells, without corresponding changes in the peripheral immune composition. Examination of aortic IL-17A(+) T-cell receptor γδ T cells and Th17 cells demonstrated a stark reduction in the percentage and number of these subsets within Il17c(-/-)Apoe(-/-) versus Apoe(-/-) mice. Explanted 12-week Western diet-fed Apoe(-/-) aortas treated with IL-17C resulted in the induction of multiple vascular chemokines and cytokines. Th17 cells demonstrated attenuated migration toward supernatants from cultures of Il17c(-/-)Apoe(-/-) smooth muscle cells, and short-term homing experiments revealed diminished recruitment of Th17 cells to the aorta of Il17c(-/-)Apoe(-/-) recipients. CONCLUSIONS: Smooth muscle cell-derived IL-17C plays a proatherogenic role by supporting the recruitment of Th17 cells to atherosclerotic lesions.

CXCR6 regulates the recruitment of pro-inflammatory IL-17A-producing T cells into atherosclerotic aortas.

The adaptive immune response is involved in the development and progression of atherosclerosis and IL-17A(+) cells play a role in this disease. Although elevated number of CD4(+) IL-17A(+) (Th17) and IL-17A(+)TCRγδ(+) T cells are found within murine atherosclerotic aortas and human plaques, the mechanisms governing IL-17A(+) T-cell migration to atherosclerotic lesions are unclear. The chemokine receptor CXCR6 is expressed on several T-cell subsets and plays a pro-atherogenic role in atherosclerosis. Here, we used CXCR6-deficient (Cxcr6 (GFP/GFP) ) apolipoprotein E-deficient (Apoe (-/-) ) mice to investigate the involvement of CXCR6 in the recruitment IL-17A(+) T cells to atherosclerotic aortas. Flow cytometric analyses revealed reductions in Th17 and IL-17A(+)TCRγδ(+) T cells within aged Cxcr6 (GFP/GFP) Apoe (-/-) aortas, in comparison with age-matched Cxcr6 (GFP/+) Apoe (-/-) aortas. Although CXCR6-sufficient IL-17A(+) T cells efficiently migrated toward CXCL16, the migration of CXCR6-deficient IL-17A(+) T cells was abolished in transwell assays. Importantly, the recruitment of Cxcr6 (GFP/GFP) Apoe (-/-) IL-17A(+) T cells into the aortas of Apoe (-/-) recipients was markedly reduced in short-term adoptive transfer experiments. Altogether these results demonstrate an important role of CXCR6 in the regulation of pathological Th17 and IL-17A(+)TCRγδ(+) T-cell recruitment into atherosclerotic lesions.

Bariatric surgery decreases monocyte-platelet aggregates in blood: a pilot study.

Morbid obesity is accompanied by platelet hyperactivity, leading to thrombotic events including myocardial infarction and stroke. Bariatric surgery is an effective intervention to reduce cardiovascular risk in obesity. However, the effect of bariatric surgery on platelet function is largely unknown. This study investigated the effects of laparoscopic Roux-en-Y gastric bypass (RYGB) and laparoscopic adjustable gastric banding (LAGB) on prothrombotic monocyte-platelet aggregates (MPAs), markers of platelet activation in vivo. MPA were measured in whole blood by flow cytometry before surgery and 1 and 3 months after surgery. In non-obese healthy controls, MPA level is 13 ± 2 %. MPAs are elevated in morbidly obese subjects. RYGB (n = 12 patients) decreases MPAs 1 month after surgery by a weight-independent mechanism (56 ± 6 % presurgically vs 26 ± 8 % at 1 month, p <0.01). LAGB (n = 5 patients) has a smaller weight-dependent effect (49 ± 8 % presurgically vs 32 ± 6 % at 1 month, p > 0.05). Bariatric surgery may reduce thrombotic events by alleviation of platelet overactivity.

The dynamic lives of macrophage and dendritic cell subsets in atherosclerosis.

Atherosclerosis, the major pathological process through which arterial plaques are formed, is a dynamic chronic inflammatory disease of large- and medium-sized arteries in which the vasculature, lipid metabolism, and the immune system all play integral roles. Both the innate and adaptive immune systems are involved in the development and progression of atherosclerosis but myeloid cells represent the major component of the burgeoning atherosclerotic plaque. Various myeloid cells, including monocytes, macrophages (MΦs), and dendritic cells (DCs) can be found within the healthy and atherosclerotic arterial wall, where they can contribute to or regulate inflammation. However, the precise behaviors and functions of these cells in situ are still active areas of investigation that continue to yield exciting and surprising new data. Here, we review recent progress in understanding of the complex biology of MΦs and DCs, focusing particularly on the dynamic regulation of these subsets in the arterial wall and novel, emerging functions of these cells during atherogenesis.

Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes.

AIMS/HYPOTHESIS: Chronic inflammation in type 2 diabetes is proposed to affect islets as well as insulin target organs. However, the nature of islet inflammation and its effects on islet function in type 2 diabetes remain unclear. Moreover, the immune cell profiles of human islets in healthy and type 2 diabetic conditions are undefined. We aimed to investigate the correlation between proinflammatory cytokine expression, islet leucocyte composition and insulin secretion in type 2 diabetic human islets. METHODS: Human islets from organ donors with or without type 2 diabetes were studied. First and second phases of glucose-stimulated insulin secretion were determined by perifusion. The expression of inflammatory markers was obtained by quantitative PCR. Immune cells within human islets were analysed by FACS. RESULTS: Type 2 diabetic islets, especially those without first-phase insulin secretion, displayed higher CCL2 and TNFa expression than healthy islets. CD45(+) leucocytes were elevated in type 2 diabetic islets, to a greater extent in moderately functional type 2 diabetic islets compared with poorly functional ones, and corresponded with elevated ALOX12 but not with CCL2 or TNFa expression. T and B lymphocytes and CD11c(+) cells were detectable within both non-diabetic and type 2 diabetic islet leucocytes. Importantly, the proportion of B cells was significantly elevated within type 2 diabetic islets. CONCLUSIONS/INTERPRETATION: Elevated total islet leucocyte content and proinflammatory mediators correlated with islet dysfunction, suggesting that heterogeneous insulitis occurs during the development of islet dysfunction in type 2 diabetes. In addition, the altered B cell content highlights a potential role for the adaptive immune response in islet dysfunction.

Phenotypic and functional heterogeneity of macrophages and dendritic cell subsets in the healthy and atherosclerosis-prone aorta.

Atherosclerosis continues to be the leading cause of cardiovascular disease. Development of atherosclerosis depends on chronic inflammation in the aorta and multiple immune cells are involved in this process. Importantly, resident macrophages and dendritic cells (DCs) are present within the healthy aorta, but the functions of these cells remain poorly characterized. Local inflammation within the aortic wall promotes the recruitment of monocytes and DC precursors to the aorta and micro-environmental factors direct the differentiation of these emigrated cells into multiple subsets of macrophages and DCs. Recent data suggest that several populations of macrophages and DCs can co-exist within the aorta. Although the functions of M1, M2, Mox, and M4 macrophages are well characterized in vitro, there is a limited set of data on the role of these populations in atherogenesis in vivo. Recent studies on the origin and the potential role of aortic DCs provide novel insights into the biology of aortic DC subsets and prospective mechanisms of the immune response in atherosclerosis. This review integrates the results of experiments analyzing heterogeneity of DCs and macrophage subsets in healthy and diseased vessels and briefly discusses the known and potential functions of these cells in atherogenesis.

The IL-17A/IL-17RA axis plays a proatherogenic role via the regulation of aortic myeloid cell recruitment.

RATIONALE: Atherosclerosis is a disease of large- and medium-sized arteries that is characterized by chronic vascular inflammation. While the role of Th1, Th2, and T-regulatory subsets in atherogenesis is established, the involvement of IL-17A-producing cells remains unclear. OBJECTIVE: To investigate the role of the IL-17A/IL-17RA axis in atherosclerosis. METHODS AND RESULTS: We bred apolipoprotein-E-deficient (Apoe(-/-)) mice with IL-17A-deficient and IL-17 receptor A-deficient mice to generate Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) mice. Western diet fed Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) mice had smaller atherosclerotic plaques in the aortic arch and aortic roots, but showed little difference in plaque burden in the thoracoabdominal aorta in comparison with Apoe(-/-) controls. Flow cytometric analysis of Il17a(-/-)Apoe(-/-) and Il17ra(-/-)Apoe(-/-) aortas revealed that deficiency of IL-17A/IL-17RA preferentially reduced aortic arch, but not thoracoabdominal aortic T cell, neutrophil, and macrophage content in comparison with Apoe(-/-) aortic segments. In contrast to ubiquitous IL-17RA expression throughout the aorta, IL-17A was preferentially expressed within the aortic arch of WD-fed Apoe(-/-) mice. Deficiency of IL-17A or IL-17RA reduced aortic arch, but not thoracoabdominal aortic TNFα and CXCL2 expression. Aortic vascular IL-17RA supports monocyte adherence to explanted aortas in ex vivo adhesion assays. Short-term homing experiments revealed that the recruitment of adoptively transferred monocytes and neutrophils to the aortas of Il17ra(-/-)Apoe(-/-) mice is impaired in comparison with Apoe(-/-) recipients. CONCLUSIONS: The IL-17A/IL-17RA axis increases aortic arch inflammation during atherogenesis through the induction of aortic chemokines, and the acceleration of neutrophil and monocyte recruitment to this site.

Flow cytometry analysis of immune cells within murine aortas.

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.(1) The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.(2, 3) There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice(4). Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.(2) For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported(5,6). Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.(6) Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method(7) to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.