Michal Itan

Paired immunoglobulin-like receptor A is an intrinsic, self-limiting suppressor of IL-5–induced eosinophil development

Eosinophilia is a hallmark characteristic of T helper type 2 (TH2) cell–associated diseases and is critically regulated by the central eosinophil growth factor interleukin 5 (IL-5). Here we demonstrate that IL-5 activity in eosinophils was regulated by paired immunoglobulin-like receptors PIR-A and PIR-B. Upon self-recognition of β2-microglobulin (β2M) molecules, PIR-B served as a permissive checkpoint for IL-5–induced development of eosinophils by suppressing the proapoptotic activities of PIR-A, which were mediated by the Grb2-Erk-Bim pathway. PIR-B–deficient bone marrow eosinophils underwent compartmentalized apoptosis, resulting in decreased blood eosinophilia in naive mice and in mice challenged with IL-5. Subsequently, Pirb−/− mice displayed impaired aeroallergen-induced lung eosinophilia and induction of lung TH2 cell responses. Collectively, these data uncover an intrinsic, self-limiting pathway regulating IL-5–induced expansion of eosinophils, which has broad implications for eosinophil-associated diseases.

CD300f associates with IL-4 receptor α and amplifies IL-4–induced immune cell responses

Significance IL-4 receptor (R) α is a critical component in IL-4– and IL-13–mediated signaling and subsequent effector functions such as those observed in allergy. Thus, it is a primary therapeutic target in diseases such as atopic dermatitis and asthma. Despite extensive studies, it is unknown whether an additional receptor system exists that may act to amplify IL-4Rα signaling and subsequent IL-4/IL-13–induced responses. We now report that CD300f is physically associated with IL-4Rα and potently amplifies IL-4Rα–induced responses in vitro and in vivo. Our results establish CD300f as a previously unidentified IL-4Rα coreceptor. To the best of our knowledge, this is the first report of an additional receptor that serves to amplify the IL-4 signaling pathway. IL-4 receptor (R) α, the common receptor chain for IL-4 and IL-13, is a critical component in IL-4– and IL-13–mediated signaling and subsequent effector functions such as those observed in type 2 inflammatory responses. Nonetheless, the existence of intrinsic pathways capable of amplifying IL-4Rα–induced responses remains unknown. In this study, we identified the myeloid-associated Ig receptor CD300f as an IL-4–induced molecule in macrophages. Subsequent analyses demonstrated that CD300f was colocalized and physically associated with IL-4Rα. Using Cd300f−/− cells and receptor cross-linking experiments, we established that CD300f amplified IL-4Rα–induced responses by augmenting IL-4/IL-13–induced signaling, mediator release, and priming. Consistently, IL-4– and aeroallergen-treated Cd300f−/− mice displayed decreased IgE production, chemokine expression, and inflammatory cell recruitment. Impaired responses in Cd300f−/− mice were not due to the inability to generate a proper Th2 response, because IL-4/IL-13 levels were markedly increased in allergen-challenged Cd300f−/− mice, a finding that is consistent with decreased cytokine consumption. Finally, CD300f expression was increased in monocytes and eosinophils obtained from allergic rhinitis patients. Collectively, our data highlight a previously unidentified role for CD300f in IL-4Rα–induced immune cell responses. These data provide new insights into the molecular mechanisms governing IL-4Rα–induced responses, and may provide new therapeutic tools to target IL-4 in allergy and asthma.

CMRF35-like molecule 1 (CLM-1) regulates eosinophil homeostasis by suppressing cellular chemotaxis

Eosinophil accumulation in health and disease is a hallmark characteristic of mucosal immunity and type 2 helper T cell (Th2) inflammation. Eotaxin-induced CCR3 (chemokine (C-C motif) receptor 3) signaling has a critical role in eosinophil chemotactic responses. Nevertheless, the expressions of immunoreceptor tyrosine-based inhibitory motif-bearing receptors such as CMRF35-like molecule-1 (CLM-1) and their ability to govern eosinophil migration are largely unknown. We now report that CLM-1 (but not CLM-8) is highly and distinctly expressed by colonic and adipose tissue eosinophils. Furthermore, Clm1−/− mice display elevated baseline tissue eosinophilia. CLM-1 negatively regulated eotaxin-induced eosinophil responses including eosinophil chemotaxis, actin polymerization, calcium influx, and extracellular signal-regulated kinase (ERK)-1/2, but not p38 phosphorylation. Addition of CLM-1 ligand (e.g., phosphatidylserine) rendered wild-type eosinophils hypochemotactic in vitro and blockade of CLM-1/ligand interactions rendered wild-type eosinophils hyperchemotactic in vitro and in vivo in a model of allergic airway disease. Interestingly, suppression of cellular recruitment via CLM-1 was specific to eosinophils and eotaxin, as leukotriene B4 (LTB4)- and macrophage inflammatory protein-1α (MIP-1α)-induced eosinophil and neutrophil migration were not negatively regulated by CLM-1. Finally, peripheral blood eosinophils obtained from allergic rhinitis patients displayed elevated CLM-1/CD300f levels. These data highlight CLM-1 as a novel regulator of eosinophil homeostasis and demonstrate that eosinophil accumulation is constantly governed by CLM-1, which negatively regulates eotaxin-induced eosinophil responses.

MicroRNA profiling reveals opposing expression patterns for miR-511 in alternatively and classically activated macrophages

Abstract Background: Macrophages are heterogeneous cells, which possess pleotropic effector and immunoregulatory functions. The phenotypic diversity of macrophages is best exemplified by the ability of IL-4 or IL-13, two key cytokines in asthma to promote macrophages into a suppressive/anti-inflammatory phenotype (e.g. alternatively activated or M2) whereas exposure to IFN-γ followed by microbial trigger renders macrophages pro-inflammatory (e.g. classically activated or M1). Intriguingly, only limited data exists regarding the expression of miRNA in M2 macrophages. Objective: To define the miRNA profile of M2 and M1 macrophages. Methods: Bone marrow-derived macrophages were activated to classically and alternatively activated states using IL-4, IL-13 or IFN-γ followed by Escherichia coli stimulation. Thereafter, an unbiased miRNA “mining” approach was utilized and the expression of several miRNAs was validated following in-vitro and in-vivo macrophage activation (qPCR). miR-511 over-expression was performed followed by global transcriptional and bioinformatic analyses. Results: We report unique miRNA expression profiles in M2 and M1 macrophages involving multiple miRNAs. Among these miRNAs, we established that miR-511 is increased in macrophages following IL-4- and IL-13-stimulation and decreased in M1 macrophages both in-vitro and in-vivo. Increased miR-511 expression was sufficient to induce marked transcriptional changes in macrophages. Interestingly, bioinformatics analyses revealed that miR-511 altered the expression of gene products that are associated with hallmark alternatively activated macrophage functions, such as cellular proliferation, wound healing responses and inflammation. Conclusions: Our data establish miR-511 as a bona fide M2-associated miRNA. These data may have significant implications in asthma where the expression of IL-4 and IL-13 are highly increased.

A key requirement for CD300f in innate immune responses of eosinophils in colitis

Eosinophils are traditionally studied in the context of type 2 immune responses. However, recent studies highlight key innate immune functions for eosinophils especially in colonic inflammation. Surprisingly, molecular pathways regulating innate immune activities of eosinophil are largely unknown. We have recently shown that the CD300f is highly expressed by colonic eosinophils. Nonetheless, the role of CD300f in governing innate immune eosinophil activities is ill-defined. RNA sequencing of 162 pediatric Crohn’s disease patients revealed upregulation of multiple Cd300 family members, which correlated with the presence of severe ulcerations and inflammation. Increased expression of CD300 family receptors was also observed in active ulcerative colitis (UC) and in mice following induction of experimental colitis. Specifically, the expression of CD300f was dynamically regulated in monocytes and eosinophils. Dextran sodium sulfate (DSS)-treated Cd300f−/− mice exhibit attenuated disease activity and histopathology in comparison with DSS-treated wild type (WT). Decreased disease activity in Cd300f−/− mice was accompanied with reduced inflammatory cell infiltration and nearly abolished production of pro-inflammatory cytokines. Monocyte depletion and chimeric bone marrow transfer experiments revealed a cell-specific requirement for CD300f in innate immune activation of eosinophils. Collectively, we uncover a new pathway regulating innate immune activities of eosinophils, a finding with significant implications in eosinophil-associated gastrointestinal diseases.

CD300f:IL-5 cross-talk inhibits adipose tissue eosinophil homing and subsequent IL-4 production

Eosinophils and their associated cytokines IL-4 and IL-5 are emerging as central orchestrators of the immune-metabolic axis. Herein, we demonstrate that cross-talk between the Ig-superfamily receptor CD300f and IL-5 is a key checkpoint that modifies the ability of eosinophils to regulate metabolic outcomes. Generation of Il5Tg/Cd300f−/− mice revealed marked and distinct increases in eosinophil levels and their production of IL-4 in the white and brown adipose tissues. Consequently, Il5Tg/Cd300f−/− mice had increased alternatively activated macrophage accumulation in the adipose tissue. Cd300f−/− mice displayed age-related accumulation of eosinophils and macrophages in the adipose tissue and decreased adipose tissue weight, which was associated with decreased diet-induced weight gain and insulin resistance. Notably, Il5Tg/CD300f−/− were protected from diet-induced weight gain and glucose intolerance. These findings highlight the cross-talk between IL-5 receptor and CD300f as a novel pathway regulating adipose tissue eosinophils and offer new entry points for therapeutic intervention for obesity and its complications.

Transcriptome profiling of mouse colonic eosinophils reveals a key role for eosinophils in the induction of s100a8 and s100a9 in mucosal healing

Eosinophils are bone marrow-derived cells that have been largely implicated in Th2-associated diseases. Recent data highlights a key role for eosinophils in mucosal innate immune responses especially in the gastrointestinal (GI) tract, which is one of the largest eosinophil reservoirs in the body. Although eosinophils express and synthesize a plethora of proteins that can mediate their effector activities, the transcriptome signature of eosinophils in mucosal inflammation and subsequent repair has been considerably overlooked. We demonstrate that eosinophils are recruited to the colon in acute inflammatory stages where they promote intestinal inflammation and remain in substantial numbers throughout the mucosal healing process. Microarray analysis of primary colonic eosinophils that were sorted at distinct stages of mucosal inflammation and repair revealed dynamic regulation of colonic eosinophil mRNA expression. The clinically relevant genes s100a8 and s100a9 were strikingly increased in colonic eosinophils (up to 550-fold and 80-fold, respectively). Furthermore, local and systemic expression of s100a8 and s100a9 were nearly diminished in eosinophil-deficient ΔdblGATA mice, and were re-constituted upon adoptive transfer of eosinophils. Taken together, these data may provide new insight into the involvement of eosinophils in colonic inflammation and repair, which may have diagnostic and therapeutic implications.

Eosinophils support adipocyte maturation and promote glucose tolerance in obesity

Accumulating data have indicated a fundamental role of eosinophils in regulating adipose tissue homeostasis. Here, we performed whole-genome RNA sequencing of the small intestinal tract, which suggested the presence of impaired lipid metabolism in eosinophil-deficient ΔdblGATA mice. ΔdblGATA mice fed a high-fat diet (HFD) showed reduced body fat mass, impaired enlargement of adipocytes, decreased expression of adipogenic genes, and developed glucose intolerance. HFD induced accumulation of eosinophils in the perigonadal white adipose tissue. Concordantly, adipocyte-differentiated 3T3-L1 cells promoted the migration of eosinophils through the expression of CCL11 (eotaxin-1) and likely promoted their survival through the expression of interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor. HFD-fed ΔdblGATA mice showed increased infiltration of macrophages, CD4+ T-cells, and B-cells, increased expression of interferon-γ, and decreased expression of IL-4 and IL-13 in white adipose tissue. Interferon-γ treatment significantly decreased lipid deposition in adipocyte-differentiated 3T3-L1 cells, while IL-4 treatment promoted lipid accumulation. Notably, HFD-fed ΔdblGATA mice showed increased lipid storage in the liver as compared with wild-type mice. We propose that obesity promotes the infiltration of eosinophils into adipose tissue that subsequently contribute to the metabolic homeostasis by promoting adipocyte maturation.

Author Correction: Transcriptome profiling of mouse colonic eosinophils reveals a key role for eosinophils in the induction of s100a8 and s100a9 in mucosal healing

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.