Margarita Martinez-Moczygemba

Pulmonary alveolar proteinosis caused by deletion of the GM-CSFRα gene in the X chromosome pseudoautosomal region 1

Pulmonary alveolar proteinosis (PAP) is a rare lung disorder in which surfactant-derived lipoproteins accumulate excessively within pulmonary alveoli, causing severe respiratory distress. The importance of granulocyte/macrophage colony-stimulating factor (GM-CSF) in the pathogenesis of PAP has been confirmed in humans and mice, wherein GM-CSF signaling is required for pulmonary alveolar macrophage catabolism of surfactant. PAP is caused by disruption of GM-CSF signaling in these cells, and is usually caused by neutralizing autoantibodies to GM-CSF or is secondary to other underlying diseases. Rarely, genetic defects in surfactant proteins or the common β chain for the GM-CSF receptor (GM-CSFR) are causal. Using a combination of cellular, molecular, and genomic approaches, we provide the first evidence that PAP can result from a genetic deficiency of the GM-CSFR α chain, encoded in the X-chromosome pseudoautosomal region 1.

Interleukins-4, -5, and -13: emerging therapeutic targets in allergic disease.

For the first time, allergic diseases have emerged as major public health concerns. Highly effective therapies for allergic disease now exist, but are plagued by serious side effects and the fact that a significant minority of patients remains unresponsive. Studies from many laboratories have established that T helper type 2 (T(H)2) cytokines contribute importantly to diseases such as asthma, and therapeutic strategies that target the key T(H)2 cytokines are of potential benefit in allergic disease. In this article, we will review the biology of the T(H)2 cytokines interleukin (IL)-4, IL-5, and IL-13 and their receptors, and will consider several novel strategies to neutralize these molecules in human and experimental asthma. While promising, newer therapies face a gauntlet of developmental challenges, but offer the hope of reducing allergic diseases once again to minor public health concerns.

JAK kinases control IL-5 receptor ubiquitination, degradation, and internalization

IL‐5, IL‐3, and GM‐CSF are related hematopoietic cytokines, which regulate the function of myeloid cells and are mediators of the allergic inflammatory response. These cytokines signal through heteromeric receptors containing a specific α chain and a shared signaling chain, βc. Previous studies demonstrated that the ubiquitin (Ub) proteasome degradation pathway was involved in signal termination of the βc‐sharing receptors. In this study, the upstream molecular events leading to proteasome degradation of the IL‐5 receptor (IL‐5R) were examined. By using biochemical and flow cytometric methods, we show that JAK kinase activity is required for βc ubiquitination and proteasome degradation but only partially required for IL‐5R internalization. Furthermore, we demonstrate the direct ubiquitination of the βc cytoplasmic domain and identify lysine residues 566 and 603 as sites of βc ubiquitination. Lastly, we show that ubiquitination of the βc cytoplasmic domain begins at the plasma membrane, increases after receptor internalization, and is degraded by the proteasome after IL‐5R internalization. We propose an updated working model of IL‐5R down‐regulation, whereby IL‐5 ligation of its receptor activates JAK2/1 kinases, resulting in βc tyrosine phosphorylation, ubiquitination, and IL‐5R internalization. Once inside the cell, proteasomes degrade the βc cytoplasmic domain, and the truncated receptor complex is terminally degraded in the lysosomes. These data establish a critical role for JAK kinases and the Ub/proteasome degradation pathway in IL‐5R down‐regulation.

Proteasomal regulation of βc signaling reveals a novel mechanism for cytokine receptor heterotypic desensitization

IL-5, IL-3, and GM-CSF are hematopoietic cytokines that are key mediators of the allergic inflammatory response. The receptors for these three cytokines consist of a cytokine-specific alpha (Ralpha) chain and a shared common beta (betac) chain. Herein, we demonstrate that agonistic ligation of these receptor subunits rapidly induces proteasomal degradation of the betac, but not the Ralpha, cytoplasmic domain, resulting in termination of signal transduction and yielding a truncated betac isoform ligated to the Ralpha subunit. Proteasomal degradation of the betac cytoplasmic domain was also a prerequisite for endocytosis and lysosomal degradation of the ligated receptor subunits. Moreover, proteasome-dependent termination of signaling induced by one betac-engaging cytokine resulted in cellular desensitization to signal transduction by subsequent stimulation with another betac-engaging cytokine. These data provide the first evidence for ligand-dependent proteasomal degradation of the betac cytoplasmic domain, and they establish a novel mechanism for heterotypic desensitization of shared cytokine receptor signaling.

Separate endocytic pathways regulate IL-5 receptor internalization and signaling

Eosinophils are critically dependent on IL‐5 for their activation, differentiation, survival, and augmentation of cytotoxic activity. We previously showed that the cytoplasmic domain of the hematopoietic receptor, βc, which is shared by IL‐5, IL‐3, and GM‐CSF, is directly ubiquitinated and degraded by the proteasomes in a JAK2‐dependent manner. However, studies describing the spatial distribution, endocytic regulation, and trafficking of βc‐sharing receptors in human eosinophils are currently lacking. Using deconvolution microscopy and biochemical methods, we clearly demonstrate that IL‐5Rs reside in and are internalized by clathrin‐ and lipid raft‐dependent endocytic pathways. Microscopy analyses in TF1 cells and human eosinophils revealed significant colocalization of βc, IL‐5Rα, and Cy3‐labeled IL‐5 with transferrin‐ (clathrin) and cholera toxin‐B‐ (lipid raft) positive vesicles. Moreover, whereas internalized IL‐5Rs were detected in both clathrin‐ and lipid raft‐positive vesicles, biochemical data revealed that tyrosine phosphorylated, ubiquitinated, and proteasome‐degraded IL‐5Rs partitioned to the soluble, nonraft fractions (clathrin‐containing). Lastly, we show that optimal IL‐5‐induced signaling requires entry of activated IL‐5Rs into the intracellular compartment, as coimmunoprecipitation of key signaling molecules with the IL‐5R was completely blocked when either endocytic pathway was inhibited. These data provide the first evidence that IL‐5Rs segregate and traffic into two distinct plasma membrane compartments, and they further establish that IL‐5R endocytosis regulates signaling both positively and negatively.

Shiga toxins induce autophagy leading to differential signalling pathways in toxin‐sensitive and toxin‐resistant human cells

The bacterial virulence factors Shiga toxins (Stxs) are expressed by Shigella dysenteriae serotype 1 and certain Escherichia coli strains. Stxs are protein synthesis inhibitors and induce apoptosis in many cell types. Stxs induce apoptosis via prolonged endoplasmic reticulum stress signalling to activate both extrinsic and intrinsic pathways in human myeloid cells. Studies have shown that autophagy, a lysosome‐dependent catabolic process, may be associated with activation of pro‐survival or death processes. It is currently unknown if autophagy contributes to apoptosis or protects cells from Stxs. To study cellular responses to Stxs, we intoxicated toxin‐sensitive cells (THP‐1 and HK‐2 cells), and toxin‐resistant cells (primary human monocyte‐derived macrophages) and examined toxin intracellular trafficking and autophagosome formation. Stxs translocated to different cell compartments in toxin‐resistant versus toxin‐sensitive cells. Confocal microscopy revealed autophagosome formation in both toxin‐resistant and toxin‐sensitive cells. Proteolytic cleavage of Atg5 and Beclin‐1 plays pivotal roles in switching non‐cytotoxic autophagy to cell death signalling. We detected cleaved forms of Atg5 and Beclin‐1 in Stx‐treated toxin‐sensitive cells, while cleaved caspases, calpains, Atg5 and Beclin‐1 were not detected in toxin‐resistant primary human monocytes and macrophages. These findings suggest that toxin sensitivity correlates with caspase and calpain activation, leading to Atg5 and Beclin‐1 cleavage.

Three Lysine Residues in the Common β Chain of the Interleukin-5 Receptor Are Required for Janus Kinase (JAK)-dependent Receptor Ubiquitination, Endocytosis, and Signaling

Background: A complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. Results: We identified three βc lysine residues that are required for JAK kinase binding to the receptor. Conclusion: JAK kinase binding to βc via 3 lysines is essential for receptor ubiquitination. Significance: We provide new mechanistic details regarding IL-5R biology, which is important for understanding eosinophil physiology. Eosinophils are multifunctional leukocytes implicated in the pathogenesis of numerous inflammatory diseases including allergic asthma and hypereosinophilic syndrome. Eosinophil physiology is critically dependent on IL-5 and the IL-5 receptor (IL-5R), composed of a ligand binding α chain (IL-5Rα), and a common β chain, βc. Previously, we demonstrated that the βc cytoplasmic tail is ubiquitinated and degraded by proteasomes following IL-5 stimulation. However, a complete understanding of the role of βc ubiquitination in IL-5R biology is currently lacking. By using a well established, stably transduced HEK293 cell model system, we show here that in the absence of ubiquitination, βc subcellular localization, IL-5-induced endocytosis, turnover, and IL-5R signaling were significantly impaired. Whereas ubiquitinated IL-5Rs internalized into trafficking endosomes for their degradation, ubiquitination-deficient IL-5Rs accumulated on the cell surface and displayed blunted signaling even after IL-5 stimulation. Importantly, we identified a cluster of three membrane-proximal βc lysine residues (Lys457, Lys461, and Lys467) whose presence was required for both JAK1/2 binding to βc and receptor ubiquitination. These findings establish that JAK kinase binding to βc requires the presence of three critical βc lysine residues, and this binding event is essential for receptor ubiquitination, endocytosis, and signaling.

Therapeutic Strategies for Harnessing Human Eosinophils in Allergic Inflammation, Hypereosinophilic Disorders, and Cancer

The eosinophil is a multifunctional granulocyte best known for providing host defense against parasites. Paradoxically, eosinophils are also implicated in the pathogenesis of allergic inflammation, asthma, and hypereosinophilic syndromes. Emerging evidence also supports the potential for harnessing the cytotoxic power of eosinophils and redirecting it to kill solid tumors. Central to eosinophil physiology is interleukin-5 (IL-5) and its receptor (IL-5R) which is composed of a ligand-specific alpha chain (IL-5Rα) and the common beta chain (βc). Eosinophil activation can lead to their degranulation, resulting in rapid release of an arsenal of tissue-destructive proinflammatory mediators and cytotoxic proteins that can be both beneficial and detrimental to the host. This review discusses eosinophil immunobiology and therapeutic strategies for targeting of IL-5 and IL-5R, as well as the potential for harnessing eosinophil cytotoxicity as a tumoricide.

Immune Dysregulation in the Pathogenesis of Pulmonary Alveolar Proteinosis

Pulmonary alveolar proteinosis (PAP) is a rare disease of the lung characterized by the accumulation of surfactant-derived lipoproteins within pulmonary alveolar macrophages and alveoli, resulting in respiratory insufficiency and increased infections. The disease is caused by a disruption in surfactant catabolism by alveolar macrophages due to loss of functional granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. The underlying molecular mechanisms causing deficiencies in GM-CSF signaling are as follows: 1) high levels of neutralizing GM-CSF autoantibodies observed in autoimmune PAP; 2) mutations in CSF2RA, the gene encoding the α chain of the GM-CSF receptor, observed in hereditary PAP; and 3) reduced numbers and function of alveolar macrophages as a result of other clinical diseases seen in secondary PAP. Recent studies investigating the biology of GM-CSF have revealed that not only does this cytokine have an indispensable role in lung physiology, but it is also a critical regulator of innate immunity and lung host defense.

Bacillus anthracis Spore Surface Protein BclA Mediates Complement Factor H Binding to Spores and Promotes Spore Persistence.

Spores of Bacillus anthracis, the causative agent of anthrax, are known to persist in the host lungs for prolonged periods of time, however the underlying mechanism is poorly understood. In this study, we demonstrated that BclA, a major surface protein of B. anthracis spores, mediated direct binding of complement factor H (CFH) to spores. The surface bound CFH retained its regulatory cofactor activity resulting in C3 degradation and inhibition of downstream complement activation. By comparing results from wild type C57BL/6 mice and complement deficient mice, we further showed that BclA significantly contributed to spore persistence in the mouse lungs and dampened antibody responses to spores in a complement C3-dependent manner. In addition, prior exposure to BclA deletion spores (ΔbclA) provided significant protection against lethal challenges by B. anthracis, whereas the isogenic parent spores did not, indicating that BclA may also impair protective immunity. These results describe for the first time an immune inhibition mechanism of B. anthracis mediated by BclA and CFH that promotes spore persistence in vivo. The findings also suggested an important role of complement in persistent infections and thus have broad implications.