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Type 2 (T2) asthma is characterized by airflow limitations and elevated levels of blood and sputum eosinophils, fractional exhaled nitric oxide, IgE, and periostin. While eosinophils are associated with exacerbations, the contribution of eosinophils to lung inflammation, remodeling and function remains largely hypothetical.To determine the effect of T2 cytokines IL-4, IL-13 and IL-5 on eosinophil biology and compare the impact of depleting just eosinophils versus inhibiting all aspects of T2 inflammation on airway inflammation.Human eosinophils or endothelial cells stimulated with IL-4, IL-13 or IL-5 were assessed for gene changes or chemokine release.Mice exposed to house dust mite extract received anti-IL-4Rα (dupilumab), anti-IL-5 or control antibodies and were assessed for changes in lung histological and inflammatory endpoints.IL-4 or IL-13 stimulation of human eosinophils and endothelial cells induced gene expression changes related to granulocyte migration; whereas, IL-5 induced changes reflecting granulocyte differentiation.In a mouse model, blocking IL-4Rα improved lung function by impacting multiple effectors of inflammation and remodeling, except peripheral eosinophil counts, thereby disconnecting blood eosinophils from airway inflammation, remodeling and function. Blocking IL-5 globally reduced eosinophil counts but did not impact inflammatory or functional measures of lung pathology. Whole lung transcriptome analysis revealed that IL-5 or IL-4Rα blockade impacted eosinophil associated genes, whereas IL-4Rα blockade also impacted genes associated with multiple cells, cytokines and chemokines, mucus production, cell:cell adhesion and vascular permeability.Eosinophils are not the sole contributor to asthma pathophysiology or lung function decline and emphasizes the need to block additional mediators to modify lung inflammation and impact lung function.

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a two-hour time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes (ISGs) as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling datasets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5’ untranslated regions (5’UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5’UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

Background: Dupilumab, a fully human monoclonal antibody that binds IL-4Raand inhibits signaling of both IL-4 and IL-13, has shown robust efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma and atopic dermatitis. Aim: To distinguish the roles of IL-4 and IL-13 in driving type 2 inflammation by comparing IL-4 vs IL-13 vs dual IL-4/13 blockade. Methods: Human mast cells, eosinophils or endothelial cells were treated with IL-4 or IL-13 and assessed for cytokine/chemokine release and/or FceR1aexpression. Humanized mice were exposed to house dust mite thrice weekly for 4 weeks and received twice‐weekly injections of dupilumab, anti‐IL-4, IL-13Rα2 fusion protein, control antibodies, or no antibody. Results: IL-4 and IL-13 differentially induced different patterns of cytokine release from human endothelial cells, mast cells and eosinophils. Similarly, IL-4 and IL-13 to a lesser extent, induced mast cell FceR1aexpression. In a murine asthma model, antigen specific responses were IL-4-dependent, whereas mucus production was IL-13-dependent. Lung eosinophil infiltration and cytokine/chemokine induction were dependent on both IL-4 and IL-13. Notably, dual receptor blockade with dupilumab, but not individual ligand blockade, prevented all of these type 2 associated pathologies. Conclusion: IL-4 and IL-13 act independently and synergistically to drive type 2 inflammation highlighting the need for dual blockade. This could explain in part the benefit of dupilumab across multiple type 2 diseases and key mechanistic differences between IL-4 and IL-13.

Rationale: Several biologics directed against effectors of type 2 inflammation have been approved, highlighting the need for a mechanistic comparison. Aim: To understand the contribution of the main type 2 cytokines to lung pathology, we performed a comparison of IL‐4/IL‐13 dual blockade using dupilumab, a fully human monoclonal antibody that binds IL-4Ra, versus IL‐5 blockade. Methods: Mice were exposed to house dust mite for 4 weeks and received twice‐weekly subcutaneous injections of dupilumab or anti‐IL‐5. Human eosinophils or endothelial cells were treated with IL-4, IL-13 or IL-5 and assessed for cytokine/chemokine release and/or integrin expression. Results: In a humanized murine asthma model, IL-4Ra blockade prevented antigen specific responses, lung cellular infiltration (including eosinophils), mucus production and lung function decline, while IL‐5 blockade only reduced eosinophil levels in the circulation and lung tissue but did not significantly impact any of the other endpoints. IL-4Ra blockade prevented entry of eosinophils into the lung by blunting chemotaxis and activation of the endothelium, while IL-5 did not impact the endothelium. Conclusion: In this mouse model of asthma, dupilumab protected from lung function decline by having a broader impact on lung inflammation and remodeling than IL-5 blockade. Notably, dupilumab blocked eosinophil infiltration into lung tissue without affecting circulating eosinophil levels, an observation suggesting that tissue (but not circulating) eosinophils contribute to disease pathology. These data provide evidence of IL‐4 and IL‐13 as key drivers of type 2 inflammation and key mechanistic differences between IL‐4Rα blockade versus IL‐5 inhibition.

Background Dupilumab, a fully human monoclonal antibody that binds IL‐4Rα and inhibits signaling of both IL‐4 and IL‐13, has shown efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma, atopic dermatitis, and chronic sinusitis with nasal polyposis. We sought to provide a comprehensive analysis of the redundant and distinct roles of IL‐4 and IL‐13 in type 2 inflammation and report dupilumab mechanisms of action. Methods Using primary cell assays and a mouse model of house dust mite–induced asthma, we compared IL‐4 vs IL‐13 vs IL‐4Rα blockers. Results Intranasal administration of either IL‐4 or IL‐13 confers an asthma‐like phenotype in mice by inducing immune cell lung infiltration, including eosinophils, increasing cytokine/chemokine expression and mucus production, thus demonstrating redundant functions of these cytokines. We further teased out their respective contributions using human in vitro culture systems. Then, in a mouse asthma model by comparing in head‐to‐head studies, either IL‐4 or IL‐13 inhibition to dual IL‐4/IL‐13 inhibition, we demonstrate that blockade of both IL‐4 and IL‐13 is required to broadly block type 2 inflammation, which translates to protection from allergen‐induced lung function impairment. Notably, only dual IL‐4/IL‐13 blockade prevented eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils, contributes to disease pathology. Conclusions Overall, these data support IL‐4 and IL‐13 as key drivers of type 2 inflammation and help provide insight into the therapeutic mechanism of dupilumab, a dual IL‐4/IL‐13 blocker, in multiple type 2 diseases., The IL‐4Rα antibody, dupilumab, binds IL‐4Rα with high affinity, directly blocks IL‐4 and IL‐13/IL‐13Rα1 complex binding to IL‐4Rα, and thereby prevents IL‐4Rα‐mediated signaling induced by both IL‐4 and IL‐13. IL‐4 and IL‐13 play distinct and overlapping roles and blockade of both cytokines is required to broadly block type 2 inflammation, which translates to protection from allergen‐induced lung function impairment. Only dual IL‐4/IL‐13 blockade with dupilumab prevents eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils contribute to disease pathology.

Immunoglobulin E (IgE) plays an important role in allergic diseases. Nevertheless, the source of IgE serological memory remains controversial. We reexamined the mechanism of serological memory in allergy using a dual reporter system to track IgE+ plasma cells in mice. Short-term allergen exposure resulted in the generation of IgE+ plasma cells that resided mainly in secondary lymphoid organs and produced IgE that was unable to degranulate mast cells. In contrast, chronic allergen exposure led to the generation of long-lived IgE+ plasma cells that were primarily derived from sequential class switching of IgG1, accumulated in the bone marrow, and produced IgE capable of inducing anaphylaxis. IgE+ plasma cells were found in the bone marrow of human allergic, but not nonallergic donors, and allergen-specific IgE produced by these cells was able to induce mast cell degranulation when transferred to mice. These data demonstrate that long-lived IgE+ bone marrow plasma cells arise during chronic allergen exposure and establish serological memory in both mice and humans.

Bacterial secretion systems play a central role in interfering with host inflammatory responses to promote replication in tissue sites. Many intracellular bacteria utilize secretion systems to promote their uptake and survival within host cells. An intracellular niche can help bacteria avoid killing by phagocytic cells, and may limit host sensing of bacterial components. Secretion systems can also play an important role in limiting host sensing of bacteria, by translocating proteins that disrupt host immune signaling pathways. Extracellular bacteria, on the other hand, utilize secretion systems to prevent uptake by host cells and maintain an extracellular niche. Secretion systems, in this case, limit sensing and inflammatory signaling which can occur as bacteria replicate and release bacterial products in the extracellular space. In this review, we will cover the common mechanisms used by intracellular and extracellular bacteria to modulate innate immune and inflammatory signaling pathways, with a focus on translocated proteins of the type III and type IV secretion systems.

Internalization of microorganisms by phagocytic cells usually results in restriction and death of the microbe. Most pathogens are able to cause disease because they have the ability to inactivate, bypass, or exploit the phagocytic machinery of the host. Here we describe common strategies that allow pathogens to bypass phagocytic killing. These strategies include inactivation of phagocytes, construction of intracellular replication vacuoles, destruction of phagocytic compartments, and manipulation of host cell autophagic machinery.

OBJECTIVE Nominally resistant mouse strains such as C57BL/6 (B6) harbor latent type 1 diabetes susceptibility genes uncovered in outcross to disease-susceptible NOD mice. However, identification of possible recessively acting B6-derived susceptibility genes is limited because very few F2 progeny derived from outcrossing this strain with NOD develop spontaneous autoimmune diabetes. Thus, we assessed whether a transgenic T-cell receptor (TCR) disease transfer model allowed the mapping of recessively acting B6 genetic loci that in the proper context contribute to diabetes. RESEARCH DESIGN AND METHODS CD8 T-cells transgenically expressing the diabetogenic AI4 TCR were transferred into 91 (NODxB6.H2g7)F1xB6.H2g7 first-backcross (BC1) females. A genome-wide scan was performed for loci affecting clinical diabetes and insulitis severity. RESULTS A major locus on chromosome 11 in tight linkage with the marker D11Mit48 (logarithm of odds score = 13.2) strongly determined whether BC1 progeny were susceptible to AI4 T-cell–mediated diabetes. Mice homozygous versus heterozygous for B6 markers of this chromosome 11 genetic locus were, respectively, highly susceptible or resistant to AI4-induced insulitis and diabetes. The genetic effect is manifest by host CD4 T-cells. Microarray analyses of mRNA transcript expression identified a limited number of candidate genes. CONCLUSIONS The distal region of chromosome 11 in B6 mice harbors a previously unrecognized recessively acting gene(s) that can promote autoreactive diabetogenic CD8 T-cell responses. Future identification of this gene(s) may further aid the screening of heterogeneous humans at future risk for diabetes, and might also provide a target for possible disease interventions.

Many pathogens, particularly those that require their host for survival, have devised mechanisms to subvert the host immune response in order to survive and replicate intracellularly. Legionella pneumophila, the causative agent of Legionnaires' disease, promotes intracellular growth by translocating proteins into its host cytosol through its type IV protein secretion machinery. At least 5 of the bacterial translocated effectors interfere with the function of host cell elongation factors, blocking translation and causing the induction of a unique host cell transcriptional profile. In addition, L. pneumophila also interferes with translation initiation, by preventing cap-dependent translation in host cells. We demonstrate here that protein translation inhibition by L. pneumophila leads to a frustrated host MAP kinase response, where genes involved in the pathway are transcribed but fail to be translated due to the bacterium-induced protein synthesis inhibition. Surprisingly, few pro-inflammatory cytokines, such as IL-1α and IL-1β, bypass this inhibition and get synthesized in the presence of Legionella effectors. We show that the selective synthesis of these genes requires MyD88 signaling and takes place in both infected cells that harbor bacteria and neighboring bystander cells. Our findings offer a perspective of how host cells are able to cope with pathogen-encoded activities that disrupt normal cellular process and initiate a successful inflammatory response., Author Summary Translation inhibition is a common virulence mechanism used by a number of pathogens (e.g. Diphtheria Toxin, Shiga Toxin and Pseudomonas Exotoxin A). It has been a mystery how host cells mount a pathogen-specific response and clear infection under conditions where protein synthesis is blocked by pathogens. Using Legionella pneumophila as a model, a bacterium that efficiently blocks the host protein translation machinery, we show here that the innate immune system has devised a mechanism to cope with translation inhibition by selectively synthesizing proteins that are required for inflammation.

Cathepsin B has been shown to not only reside within endo-lysosomes of intestinal epithelial cells, but it was also secreted into the extracellular space of intestinal mucosa in physiological and pathological conditions. In an effort to further investigate the function of this protease in the intestine, we generated a transgenic mouse model that would enable us to visualize the localization of cathepsin B in vivo. Previously we showed that the A33-antigen promoter could be successfully used in vitro in order to express cathepsin B-green fluorescent protein chimeras in cells that co-expressed the intestine-specific transcription factor Cdx1. In this study an analog approach was used to express chi-meric cathepsin B specifically in the intestine of transgenic animals. No overt phenotype was observed for the transgenic mice that reproduced normally. Biochemical and morphological studies confirmed that the overall intestinal phenotype including the structure and polarity of this tissue as well as cell numbers and differentiation states were not altered in the A33-CathB-EGFP mice when compared to wild type animals. However, transgenic expression of chimeric cathepsin B could not be visualized because it was not translated in situ although the transgene was maintained over several generations.