Nicola M. Heller

Regulation of inflammation by interleukin-4: a review of "alternatives".

Studies of IL‐4 have revealed a wealth of information on the diverse roles of this cytokine in homeostatic regulation and disease pathogenesis. Recent data suggest that instead of simple linear regulatory pathways, IL‐4 drives regulation that is full of alternatives. In addition to the well‐known dichotomous regulation of Th cell differentiation by IL‐4, this cytokine is engaged in several other alternative pathways. Its own production involves alternative mRNA splicing, yielding at least two functional isoforms: full‐length IL‐4, encoded by the IL‐4 gene exons 1–4, and IL‐4δ2, encoded by exons 1, 3, and 4. The functional effects of these two isoforms are in some ways similar but in other ways quite distinct. When binding to the surface of target cells, IL‐4 may differentially engage two different types of receptors. By acting on macrophages, a cell type critically involved in inflammation, IL‐4 induces the so‐called alternative macrophage activation. In this review, recent advances in understanding these three IL‐4‐related branch points—alternative splicing of IL‐4, differential receptor engagement by IL‐4, and differential regulation of macrophage activation by IL‐4—are summarized in light of their contributions to inflammation.

Commentary: IL-4 and IL-13 receptors and signaling

Interleukin (IL)-4 and IL-13 were discovered approximately 30years ago and were immediately linked to allergy and atopic diseases. Since then, new roles for IL-4 and IL-13 and their receptors in normal gestation, fetal development and neurological function and in the pathogenesis of cancer and fibrosis have been appreciated. Studying IL-4/-13 and their receptors has revealed important clues about cytokine biology and led to the development of numerous experimental therapeutics. Here we aim to highlight new discoveries and consolidate concepts in the field of IL-4 and IL-13 structure, receptor regulation, signaling and experimental therapeutics.

estrogen Signaling Modulates Allergic inflammation and Contributes to Sex Differences in Asthma

Asthma is a chronic airway inflammatory disease that affects ~300 million people worldwide. It is characterized by airway constriction that leads to wheezing, coughing, and shortness of breath. The most common treatments are corticosteroids and β2-adrenergic receptor antagonists, which target inflammation and airway smooth muscle constriction, respectively. The incidence and severity of asthma is greater in women than in men, and women are more prone to develop corticosteroid-resistant or “hard-to-treat” asthma. Puberty, menstruation, pregnancy, menopause, and oral contraceptives are known to contribute to disease outcome in women, suggesting a role for estrogen and other hormones impacting allergic inflammation. Currently, the mechanisms underlying these sex differences are poorly understood, although the effect of sex hormones, such as estrogen, on allergic inflammation is gaining interest. Asthma presents as a heterogeneous disease. In typical Th2-type allergic asthma, interleukin (IL)-4 and IL-13 predominate, driving IgE production and recruitment of eosinophils into the lungs. Chronic Th2-inflammation in the lung results in structural changes and activation of multiple immune cell types, leading to a deterioration of lung function over time. Most immune cells express estrogen receptors (ERα, ERβ, or the membrane-bound G-protein-coupled ER) to varying degrees and can respond to the hormone. Together these receptors have demonstrated the capacity to regulate a spectrum of immune functions, including adhesion, migration, survival, wound healing, and antibody and cytokine production. This review will cover the current understanding of estrogen signaling in allergic inflammation and discuss how this signaling may contribute to sex differences in asthma and allergy.

Alternative activation-skewed microglia/macrophages promote hematoma resolution in experimental intracerebral hemorrhage

Microglia/macrophages (MMΦ) are highly plastic phagocytes that can promote both injury and repair in diseased brain through the distinct function of classically activated and alternatively activated subsets. The role of MMΦ polarization in intracerebral hemorrhage (ICH) is unknown. Herein, we comprehensively characterized MMΦ dynamics after ICH in mice and evaluated the relevance of MMΦ polarity to hematoma resolution. MMΦ accumulated within the hematoma territory until at least 14days after ICH induction. Microglia rapidly reacted to the hemorrhagic insult as early as 1-1.5h after ICH and specifically presented a protective alternatively activated phenotype. Substantial numbers of activated microglia and newly recruited monocytes also assumed an early alternatively activated phenotype, but the phenotype gradually shifted to a mixed spectrum over time. Ultimately, markers of MMΦ classic activation dominated at the chronic stage of ICH. We enhanced MMΦ alternative activation by administering intraperitoneal injections of rosiglitazone, and subsequently observed elevations in CD206 expression on brain-isolated CD11b+ cells and increases in IL-10 levels in serum and perihematomal tissue. Enhancement of MMΦ alternative activation correlated with hematoma volume reduction and improvement in neurologic deficits. Intraventricular injection of alternative activation signature cytokine IL-10 accelerated hematoma resolution, whereas microglial phagocytic ability was abolished by IL-10 receptor neutralization. Our results suggest that MMΦ respond dynamically to brain hemorrhage by exhibiting diverse phenotypic changes at different stages of ICH. Alternative activation-skewed MMΦ aid in hematoma resolution, and IL-10 signaling might contribute to regulation of MMΦ phagocytosis and hematoma clearance in ICH.

Enhanced resolution of experimental ARDS through IL-4-mediated lung macrophage reprogramming.

Despite intense investigation, acute respiratory distress syndrome (ARDS) remains an enormous clinical problem for which no specific therapies currently exist. In this study, we used intratracheal lipopolysaccharide or Pseudomonas bacteria administration to model experimental acute lung injury (ALI) and to further understand mediators of the resolution phase of ARDS. Recent work demonstrates macrophages transition from a predominant proinflammatory M1 phenotype during acute inflammation to an anti-inflammatory M2 phenotype with ALI resolution. We tested the hypothesis that IL-4, a potent inducer of M2-specific protein expression, would accelerate ALI resolution and lung repair through reprogramming of endogenous inflammatory macrophages. In fact, IL-4 treatment was found to offer dramatic benefits following delayed administration to mice subjected to experimental ALI, including increased survival, accelerated resolution of lung injury, and improved lung function. Expression of the M2 proteins Arg1, FIZZ1, and Ym1 was increased in lung tissues following IL-4 treatment, and among macrophages, FIZZ1 was most prominently upregulated in the interstitial subpopulation. A similar trend was observed for the expression of macrophage mannose receptor (MMR) and Dectin-1 on the surface of alveolar macrophages following IL-4 administration. Macrophage depletion or STAT6 deficiency abrogated the therapeutic effect of IL-4. Collectively, these data demonstrate that IL-4-mediated therapeutic macrophage reprogramming can accelerate resolution and lung repair despite delayed use following experimental ALI. IL-4 or other therapies that target late-phase, proresolution pathways may hold promise for the treatment of human ARDS.

Suppressor of Cytokine Signaling (SOCS)1 Regulates Interleukin-4 (IL-4)-activated Insulin Receptor Substrate (IRS)-2 Tyrosine Phosphorylation in Monocytes and Macrophages via the Proteasome.

Allergic asthma is a chronic lung disease initiated and driven by Th2 cytokines IL-4/-13. In macrophages, IL-4/-13 bind IL-4 receptors, which signal through insulin receptor substrate (IRS)-2, inducing M2 macrophage differentiation. M2 macrophages correlate with disease severity and poor lung function, although the mechanisms that regulate M2 polarization are not understood. Following IL-4 exposure, suppressor of cytokine signaling (SOCS)1 is highly induced in human monocytes. We found that siRNA knockdown of SOCS1 prolonged IRS-2 tyrosine phosphorylation and enhanced M2 differentiation, although siRNA knockdown of SOCS3 did not affect either. By co-immunoprecipitation, we found that SOCS1 complexes with IRS-2 at baseline, and this association increased after IL-4 stimulation. Because SOCS1 is an E3 ubiquitin ligase, we examined the effect of proteasome inhibitors on IL-4-induced IRS-2 phosphorylation. Proteasomal inhibition prolonged IRS-2 tyrosine phosphorylation, increased ubiquitination of IRS-2, and enhanced M2 gene expression. siRNA knockdown of SOCS1 inhibited ubiquitin accumulation on IRS-2, although siRNA knockdown of SOCS3 had no effect on ubiquitination of IRS-2. Monocytes from healthy and allergic individuals revealed that SOCS1 is induced by IL-4 in healthy monocytes but not allergic cells, whereas SOCS3 is highly induced in allergic monocytes. Healthy monocytes displayed greater ubiquitination of IRS-2 and lower M2 polarization than allergic monocytes in response to IL-4 stimulation. Here, we identify SOCS1 as a key negative regulator of IL-4-induced IRS-2 signaling and M2 differentiation. Our findings provide novel insight into how dysregulated expression of SOCS increases IL-4 responses in allergic monocytes, and this may represent a new therapeutic avenue for managing allergic disease.

Estrogen Signaling Contributes to Sex Differences in Macrophage Polarization during Asthma

Allergic asthma is a chronic Th2 inflammation in the lungs that constricts the airways and presents as coughing and wheezing. Asthma mostly affects boys in childhood and women in adulthood, suggesting that shifts in sex hormones alter the course of the disease. Alveolar macrophages have emerged as major mediators of allergic lung inflammation in animal models as well as humans. Whether sex differences exist in macrophage polarization and the molecular mechanism(s) that drive differential responses are not well understood. We found that IL-4–stimulated bone marrow–derived and alveolar macrophages from female mice exhibited greater expression of M2 genes in vitro and after allergen challenge in vivo. Alveolar macrophages from female mice exhibited greater expression of the IL-4Rα and estrogen receptor (ER) α compared with macrophages from male mice following allergen challenge. An ERα-specific agonist enhanced IL-4–induced M2 gene expression in macrophages from both sexes, but more so in macrophages from female mice. Furthermore, IL-4–stimulated macrophages from female mice exhibited more transcriptionally active histone modifications at M2 gene promoters than did macrophages from male mice. We found that supplementation of estrogen into ovariectomized female mice enhanced M2 polarization in vivo upon challenge with allergen and that macrophage-specific deletion of ERα impaired this M2 polarization. The effects of estrogen are long-lasting; bone marrow–derived macrophages from ovariectomized mice implanted with estrogen exhibited enhanced IL-4–induced M2 gene expression compared with macrophages from placebo-implanted littermates. Taken together, our findings suggest that estrogen enhances IL-4–induced M2 gene expression and thereby contributes to sex differences observed in asthma.

The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2

Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.

β-Catenin is required for the differentiation of iNKT2 and iNKT17 cells that augment IL-25-dependent lung inflammation.

BackgroundInvariant Natural Killer T (iNKT) cells have been implicated in lung inflammation in humans and also shown to be a key cell type in inducing allergic lung inflammation in mouse models. iNKT cells differentiate and acquire functional characteristics during development in the thymus. However, the correlation between development of iNKT cells in the thymus and role in lung inflammation remains unknown. In addition, transcriptional control of differentiation of iNKT cells into iNKT cell effector subsets in the thymus during development is also unclear. In this report we show that β-catenin dependent mechanisms direct differentiation of iNKT2 and iNKT17 subsets but not iNKT1 cells.MethodsTo study the role for β-catenin in lung inflammation we utilize mice with conditional deletion and enforced expression of β-catenin in a well-established mouse model for IL-25-dependen lung inflammation.ResultsSpecifically, we demonstrate that conditional deletion of β-catenin permitted development of mature iNKT1 cells while impeding maturation of iNKT2 and 17 cells. A role for β-catenin expression in promoting iNKT2 and iNKT17 subsets was confirmed when we noted that enforced transgenic expression of β-catenin in iNKT cell precursors enhanced the frequency and number of iNKT2 and iNKT17 cells at the cost of iNKT1 cells. This effect of expression of β-catenin in iNKT cell precursors was cell autonomous. Furthermore, iNKT2 cells acquired greater capability to produce type-2 cytokines when β-catenin expression was enhanced.DiscussionThis report shows that β-catenin deficiency resulted in a profound decrease in iNKT2 and iNKT17 subsets of iNKT cells whereas iNKT1 cells developed normally. By contrast, enforced expression of β-catenin promoted the development of iNKT2 and iNKT17 cells. It was important to note that the majority of iNKT cells in the thymus of C57BL/6 mice were iNKT1 cells and enforced expression of β-catenin altered the pattern to iNKT2 and iNKT17 cells suggesting that β-catenin may be a major factor in the distinct pathways that critically direct differentiation of iNKT effector subsets.ConclusionsThus, we demonstrate that β-catenin expression in iNKT cell precursors promotes differentiation toward iNKT2 and iNKT17 effector subsets and supports enhanced capacity to produce type 2 and 17 cytokines which in turn augment lung inflammation in mice.

New perspectives on the regulation of type II inflammation in asthma

Asthma is a chronic inflammatory disease of the lungs which has been thought to arise as a result of inappropriately directed T helper type-2 (Th2) immune responses of the lungs to otherwise innocuous inhaled antigens. Current asthma therapeutics are directed towards the amelioration of downstream consequences of type-2 immune responses (i.e. β-agonists) or broad-spectrum immunosuppression (i.e. corticosteroids). However, few approaches to date have been focused on the primary prevention of immune deviation. Advances in molecular phenotyping reveal heterogeneity within the asthmatic population with multiple endotypes whose varying expression depends on the interplay between numerous environmental factors and the inheritance of a broad range of susceptibility genes. The most common endotype is one described as “type-2-high” (i.e. high levels of interleukin [IL]-13, eosinophilia, and periostin). The identification of multiple endotypes has provided a potential explanation for the observations that therapies directed at typical Th2 cytokines (IL-4, IL-5, and IL-13) and their receptors have often fallen short when they were tested in a diverse group of asthmatic patients without first stratifying based on disease endotype or severity. However, despite the incorporation of endotype-dependent stratification schemes into clinical trial designs, variation in drug responses are still apparent, suggesting that additional genetic/environmental factors may be contributing to the diversity in drug efficacy. Herein, we will review recent advances in our understanding of the complex pathways involved in the initiation and regulation of type-2-mediated immune responses and their modulation by host factors (genetics, metabolic status, and the microbiome). Particular consideration will be given to how this knowledge could pave the way for further refinement of disease endotypes and/or the development of novel therapeutic strategies for the treatment of asthma .