Turning off Asthma Attacks
Working in labs with human immune cells, Johns Hopkins researchers have identified a critical "off" switch used in a cell to turn on an immune response contributing to asthma attacks. The switch is made up of regulatory proteins controlling one of many immune signal pathways.
"Asthma patients are constantly firing through this pathway because their proteins are stuck in the 'on' position — without proper control by other proteins that shut down this reaction," according to Nicola Heller PhD, assistant professor of anesthesiology and critical care medicine at the Johns Hopkins University School of Medicine.
Asthma is related to an overabundance of one type of immune cell in the lungs called an M2 macrophage. In a non-asthmatic person, M2 macrophages activate and clean up inhaled allergens and foreign particles, then deactivate when the irritant is broken down.
However, asthma M2 cells and the chemical signals they emit linger and attrack other cells, causing inflammation and triggering an asthma attack. Over time, the lung is changed by M2 cell secretions and lung tissue is remodeled by irreversible obstructions driving poor lung function.
"If you prevent these cells from becoming M2 types, you can potentially stop continued inflammation and long-term structural changes."
Nicola Heller PhD, Assistant Professor, Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
The new research, reported Nov. 25 in the Journal of Biological Chemistry, investigated the role of two proteins, GRB10 and p70S6K, both controlled by the signaling pathway activating M2 cells.
In their previous work, also published in the Journal of Biological Chemistry Sept. 23, Heller's group has found: (A) the inflammatory pathway begins with interleukin 4 (IL-4), passed through a protein named IRS-2 before (B) activating M2 cells. People with allergies don't have proteins to stop IRS-2 making it more active and increasing formation of M2 cells in people with allergies.
In the new study, Heller's lab delved deeper into this IRS-2 pathway. By analyzing chemical changes of the IRS-2 protein, it determined IRS-2 appears in two different forms — "on" allowing signals to pass through — and "off" stopping signals from changing cells into M2 macrophages. In human white blood cells, two regulatory proteins — GRB10 and p70S6K — increased after IL-4 exposure.
In further experiments, they treated the immortalized white blood cells with both chemical and genetic blockers — small interfering RNA (siRNA) — designed to render either p70S6K or GRB10 nonfunctional. When they decreased GRB10 and p70S6K activity — they saw more of the "on" form of IRS-2. Therefore, these proteins are responsible for turning off IRS-2 and M2 downstream production.
"This confirmed for us that without properly functioning GRB10 and p70S6K, cells could not turn off IRS-2 signaling and M2 production."
Nicola Heller PhD
Heller's research team has already begun experiments exploring implications of their results, including looking at differences between cells from allergic and healthy individuals. They are testing the efficacy of an inhalable drug that mimics the function of GRB1 and p70S6K to shut off the development of M2 macrophages in the lungs of mice.
"One of the advantages of working with lung macrophages is they are some of the first cells to see anything put in an inhaler."
Nicola Heller PhD
The findings also have implications for treatment in cancer, as M2 macrophage cells play a regulatory role in tumor growth; as well as affecting fat deposition in disorders such as obesity.
'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.
Other researchers involved in this study include Kristi J. Warren, Xi Fang, Nagaraj M. Gowda and Joshua Thompson of the Johns Hopkins University School of Medicine.
Key words: allergy asthma macrophage mammalian target of rapamycin (mTOR) S6 kinase GRB10 IL-4 IRS-2 allergy p70S6K
This work was funded by the National Institutes of Health Pathway to Independence Award (K99/R00 HL096897).
Suppressor of Cytokine Signaling (SOCS) Regulates Interleukin-4 -activated Insulin Receptor "Substrate (IRS)-2 Tyrosine Phosphorylation in Monocytes and Macrophages via the Proteasome*
Sarah M. McCormick, Nagaraj Gowda, Jessie X. Fang and Nicola M. Heller
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.
Key words: allergy asthma macrophage monocyte phosphotyrosine signaling signal transduction insulin receptor substrate 2 interleukin-4 suppressor of cytokine signaling 1 ubiquination
Supported by a fellowship award from the Canadian Institute of Health Research.
This work was supported by National Institutes of Health Grant K99/R00 HL096897. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Received July 5, 2016.
Revision received August 8, 2016.
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Dec 12, 2016 Fetal Timeline Maternal Timeline News News Archive
Asthma inflammation begins when interleukin 4 (IL-4), passes through the IRS-2 protein
and activates M2 macrophage cells. However, in asthmatics the macrophages cannot turn off.
M2 cell secretions remodel lung tissue with irreversible obstructions, driving poor lung function.
Image Credit: Public Domain