Introduction

Interleukin (IL)-17 is a proinflammatory cytokine that is produced largely by a unique CD4+ T-helper (Th) subset called Th17 cells. The development of Th17 cells is suppressed by interferon (IFN)-γ produced by Th1 cells, suggesting cross-regulation between Th17 and Th1 cells. Thus, this study analyzed the balance of CD4+ Th17 and Th1 cell responses in peripheral blood from patients with systemic lupus erythematosus (SLE) and healthy subjects.

Methods

Twenty-five adult patients with SLE and 26 healthy subjects matched for gender and age (± 2 years) were recruited. Peripheral blood mononuclear cells (PBMCs) from patients and healthy subjects were stimulated for 4 h ex vivo with phorbol myristate acetate (PMA) and ionomycin. The frequency of CD4+ T cells producing IL-17 and/or IFN-γ was measured by using flow cytometry. Expression of Th17-associated chemokine receptors CCR4 and CCR6 on CD4+ T cells as well as plasma levels of Th17-polarizing cytokines were assessed. Disease activity was evaluated by the SLE disease activity index score (SLEDAI). Unpaired t test and Pearson correlation were used for statistical analyses.

Results

Patients with SLE had an increased frequency of CD4+IL-17+ T cells compared with healthy subjects. However, the frequency of CD4+IFN-γ+ T cells was similar between the two groups, indicating an altered balance of Th17 and Th1 cell responses in SLE. Patients with SLE also had an increased frequency of CD4+CCR4+CCR6+ T cells that are known to produce IL-17. The frequency of CD4+IL-17+ T cells and CD4+CCR4+CCR6+ T cells correlated with disease activity. In measuring plasma levels of the Th17-polarizing cytokines, levels of IL-6 were higher in patients with SLE than in healthy subjects, although levels of IL-1β, IL-21, IL-23, and transforming growth factor (TGF)-β were not different between the two groups.

Conclusions

We demonstrate an enhanced Th17 cell response that correlates with disease activity in patients with SLE, suggesting a role for IL-17 in the pathogenesis of lupus. Our data indicate that the mechanisms involved in balancing Th1 and Th17 regulation, as well as in producing IL-6, are aberrant in SLE, leading to an increased Th17 response. We suggest that CCR4 and CCR6 expression on CD4+ T cells should be considered as markers of disease activity, and that IL-17 blocking may offer a therapeutic target in SLE.

IL-1b signaling augments continued splenic monocyte supply during acute inflammation.

Monocytes (Mo) and macrophages (MΦ) are emerging therapeutic targets in malignant, cardiovascular, and autoimmune disorders. Targeting of Mo/MΦ and their effector functions without compromising innate immunity’s critical defense mechanisms first requires addressing gaps in knowledge about the life cycle of these cells. Here we studied the source, tissue kinetics, and clearance of Mo/MΦ in murine myocardial infarction, a model of acute inflammation after ischemic injury. We found that a) Mo tissue residence time was surprisingly short (20 h); b) Mo recruitment rates were consistently high even days after initiation of inflammation; c) the sustained need of newly made Mo was fostered by extramedullary monocytopoiesis in the spleen; d) splenic monocytopoiesis was regulated by IL-1β; and e) the balance of cell recruitment and local death shifted during resolution of inflammation. Depending on the experimental approach, we measured a 24 h Mo/MΦ exit rate from infarct tissue between 5 and 13% of the tissue cell population. Exited cells were most numerous in the blood, liver, and spleen. Abrogation of extramedullary monocytopoiesis proved deleterious for infarct healing and accelerated the evolution of heart failure. We also detected rapid Mo kinetics in mice with stroke. These findings expand our knowledge of Mo/MΦ flux in acute inflammation and provide the groundwork for novel anti-inflammatory strategies for treating heart failure.

Th17 cells produce IL-17 that plays an important role in host defense. However, little is known about whether aging affects human Th17 cells. Here we demonstrated that healthy elderly people (age≥65) had a decreased frequency of IL-17-producing cells in memory CD4+ T cells compared to healthy young people (age≤40) while both groups had similar frequencies of IFN-γ-producing cells in the same memory cell subset as measured by flow cytometry. In contrast, the healthy elderly had increased differentiation of IL-17-producing effector cells but not IFN-γ-producing cells from naïve CD4+ T cells compared to the healthy young. The results of ELISA also showed similar findings with increased IL-17 production from naïve CD4+ T cells and decreased IL-17 production from memory CD4+ T cells in the elderly compared to the young. These findings indicate that aging differentially affects naïve and memory Th17 cell responses in humans.

In this study, the antibacterial effect was evaluated to determine the benefits of high speed drying (HSD) and far-infrared radiation drying (FIR) compared to the freeze drying (FD) method. Citrus press-cakes (CPCs) are released as a by-product in the citrus processing industry. Previous studies have shown that the HSD and FIR drying methods are much more economical for drying time and mass drying than those of FD, even though FD is the most qualified drying method. The disk diffusion assay was conducted, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined with methanol extracts of the dried CPCs against 11 fish and five food-related pathogenic bacteria. The disk diffusion results indicated that the CPCs dried by HSD, FIR, and FD prevented growth of all tested bacteria almost identically. The MIC and MBC results showed a range from 0.5-8.0 mg/mL and 1.0-16.0 mg/mL respectively. Scanning electron microscopy indicated that the extracts changed the morphology of the bacteria cell wall, leading to destruction. These results suggest that CPCs dried by HSD and FIR showed strong antibacterial activity against pathogenic bacteria and are more useful drying methods than that of the classic FD method in CPCs utilization.

Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.

Objectives

The aim of this study was to explore post-MI myocardial inflammation.

Background

Innate immune cells are centrally involved in infarct healing and are emerging therapeutic targets in cardiovascular disease, however; clinical tools to assess their presence in tissue are scarce. Furthermore, it is currently not known if the non-ischemic remote zone recruits monocytes.

Methods

Acute inflammation was followed in mice with coronary ligation by 18FDG PET/MRI, FACS, PCR and histology.

Results

Gd-DTPA enhanced infarcts showed high 18FDG uptake on day 5 after MI. Cell depletion and isolation data confirmed that this largely reflected inflammation; CD11b+ cells had 4-fold higher 18FDG uptake than the infarct tissue from which they were isolated (P<0.01). Surprisingly, there was considerable monocyte recruitment in the remote myocardium (~104/mg myocardium, 5.6-fold increase, P<0.01), a finding mirrored by macrophage infiltration in remote myocardium of patients with acute MI. Temporal kinetics of cell recruitment were slower than in the infarct, with peak numbers on day 10 after ischemia. Quantitative PCR showed robust increase of recruiting adhesion molecules and chemokines in remote myocardium (e.g. 12-fold increase of MCP-1), although levels were always lower than in the infarct. Finally, matrix metalloproteinase activity was significantly increased in non-infarcted myocardium, suggesting that monocyte recruitment to the remote zone may contribute to post MI dilation.

Conclusion

These studies shed light on the innate inflammatory response in remote myocardium after myocardial infarction.

Zinc enhances TCR signaling in part by inhibiting Shp-1 recruitment to the TCR synapse.

Zinc is a trace element that is essential for innate and adaptive immune responses. In addition to being a structural element of many proteins, zinc also functions as a neurotransmitter and an intracellular messenger. Temporal or spatial changes in bioavailable zinc may influence the activity of several enzymes, including kinases and phosphatases. We provide evidence that zinc functions as an ionic signaling molecule after T cell activation. Cytoplasmic zinc concentrations increased within 1 min after T cell receptor (TCR) triggering, in particular in the subsynaptic compartment. The increase depended on the extracellular zinc concentrations and was inhibited by silencing zinc transporter Zip6. Increased zinc influx reduced the recruitment of SHP-1 to the TCR activation complex, augmented ZAP70 phosphorylation and sustained calcium influx. By calibrating TCR activation thresholds, increased extracellular zinc bioavailability facilitated the induction of T cell proliferative responses to suboptimal stimuli.

Rationale

Monocytes recruited to ischemic myocardium originate from a reservoir in the spleen, and the release from their splenic niche relies on angiotensin-II (Ang-II) signaling.

Objective

Since monocytes are centrally involved in tissue repair after ischemia, we here hypothesized that early ACE inhibitor therapy impacts healing after myocardial infarction partly via effects on monocyte traffic.

Methods and Results

In a mouse model of permanent coronary ligation, Enalapril arrested the release of monocytes from the splenic reservoir and consequently reduced their recruitment into the healing infarct by 45%, as quantified by flow cytometry of digested infarcts. Time-lapse intravital microscopy revealed that Enalapril reduces monocyte motility in the spleen. In vitro migration assays and Western blotting showed that this was caused by reduced signaling through the Ang-II receptor subtype 1. We then studied the long-term consequences of blocked splenic monocyte release in atherosclerotic apoE-/- mice, in which infarct healing is impaired due to excessive inflammation in the cardiac wound. Enalapril improved histological healing biomarkers and reduced inflammation in infarcts measured by fluorescence molecular tomography (FMT-CT) of proteolytic activity. ACE inhibition improved MRI-derived ejection fraction by 14% on day 21, despite initially comparable infarct size. In apoE-/- mice, ischemia reperfusion injury resulted in larger infarct size, enhanced monocyte recruitment and was reversible by Enalapril treatment. Splenectomy reproduced anti-inflammatory effects of Enalapril.

Conclusion

This study suggests that benefits of early ACE inhibition after MI can partially be attributed to its potent anti-inflammatory impact on the splenic monocyte reservoir.

The ability to mount adaptive immune responses to vaccinations and viral infections declines with increasing age. To identify mechanisms leading to immunosenescence, primary CD4 T cell responses were examined in 60- to 75-year-old individuals lacking overt functional defects. Transcriptome analysis indicated a selective defect in zinc homeostasis. CD4 T cell activation was associated with zinc influx via the zinc transporter Zip6, leading to increased free cytoplasmic zinc and activation of negative feedback loops, including the induction of zinc-binding metallothioneins. In young adults, activation-induced cytoplasmic zinc concentrations declined after 2 days to below prestimulation levels. In contrast, activated naïve CD4 T cells from older individuals failed to downregulate cytoplasmic zinc, resulting in excessive induction of metallothioneins. Activation-induced metallothioneins regulated the redox state in activated T cells and accounted for an increased proliferation of old CD4 T cells, suggesting that regulation of T cell zinc homeostasis functions as a compensatory mechanism to preserve the replicative potential of naïve CD4 T cells with age.

Rheumatoid arthritis (RA) is characterized by premature immune aging with accumulation of degenerate T cells deficient for CD28. Gene expression profiling of CD4+CD28− and CD4+CD28+ T cells to discover disease-promoting activities of CD28− T cells identified expression of CD70 as a most striking difference. Hence, CD70 was significantly more expressed in CD4 T cells from RA patients compared to age-matched controls (P<0.006). The underlying mechanism was a failure to repress CD70 expression after activation-dependent induction. This defect in RA was not related to differential promoter demethylation. CD70 on bystander CD4+CD28− T cells functioned by lowering the threshold for T-cell activation; admixture of CD4+CD28− T cells augmented TCR-induced responses of autologous naïve CD4+CD28+ T cells, particularly of low avidity T cells. The data support a model where CD70 expressed on T cells causes degeneracy in T-cell responses and undermines tolerance mechanisms that normally control T-cell autoreactivity.

Abstract

The ability to mount adaptive immune responses to vaccinations and viral infections declines with increasing age. To identify mechanisms leading to immunosenescence, primary CD4 T cell responses were examined in 60- to 75-year-old individuals lacking overt functional defects. Transcriptome analysis indicated a selective defect in zinc homeostasis. CD4 T cell activation was associated with zinc influx via the zinc transporter Zip6, leading to increased free cytoplasmic zinc and activation of negative feedback loops, including the induction of zinc-binding metallothioneins. In young adults, activation-induced cytoplasmic zinc concentrations declined after 2 days to below prestimulation levels. In contrast, activated naïve CD4 T cells from older individuals failed to downregulate cytoplasmic zinc, resulting in excessive induction of metallothioneins. Activation-induced metallothioneins regulated the redox state in activated T cells and accounted for an increased proliferation of old CD4 T cells, suggesting that regulation of T cell zinc homeostasis functions as a compensatory mechanism to preserve the replicative potential of naïve CD4 T cells with age.

Naïve and memory CD4 and CD8 T cells represent a highly dynamic system with constant homeostatic and antigen-driven proliferation, influx, and loss of T cells. Thymic activity dwindles with age and essentially ceases in the later decades of life, severely constraining the generation of new T cells. Homeostatic control mechanisms are very effective to maintain a large and diverse subset of naïve CD4 T cells for many years up to the 8th decade of life, but eventually and abruptly fail at about the age of 75 years. In contrast, the CD8 T cell compartment is more unstable, with progressive diminution of naïve T cells and increasing loss of diversity already during mid adulthood. Vaccination strategies need to aim at developing a broad repertoire of memory T cells before the critical time period when the naïve CD4 T-cell repertoire collapses. Research efforts need to aim at understanding the homeostatic control mechanisms to ultimately expand the time period of repertoire stability.

With increasing age, the competence of the immune system to fight infections and tumors declines. Age-dependent changes have been mostly described for human CD8 T cells, raising the question of whether the response patterns for CD4 T cells are different. Gene expression arrays of memory CD4 T cells yielded a similar age-induced fingerprint as has been described for CD8 T cells. In cross-sectional studies, the phenotypic changes were not qualitatively different for CD4 and CD8 T cells, but occurred much more frequently in CD8 T cells. Homeostatic stability partially explained this lesser age sensitivity of CD4 T cells. With aging, naïve and central memory CD8 T cells were lost at the expense of phenotypically distinct CD8 effector T cells, while effector CD4 T cells did not accumulate. However, phenotypic shifts on central memory T cells were also more pronounced in CD8 T cells. This distinct stability in cell surface marker expression can be reproduced in vitro. The data show that CD8 T cells are age sensitive by at least two partially independent mechanisms: fragile homeostatic control and gene expression instability in a large set of regulatory cell surface molecules.