The basic immunology of asthma.

In many asthmatics, chronic airway inflammation is driven by IL-4-, IL-5-, and IL-13-producing Th2 cells or ILC2s. Type 2 cytokines promote hallmark features of the disease such as eosinophilia, mucus hypersecretion, bronchial hyperresponsiveness (BHR), IgE production, and susceptibility to exacerbations. However, only half the asthmatics have this "type 2-high" signature, and "type 2-low" asthma is more associated with obesity, presence of neutrophils, and unresponsiveness to corticosteroids, the mainstay asthma therapy. Here, we review the underlying immunological basis of various asthma endotypes by discussing results obtained from animal studies as well as results generated in clinical studies targeting specific immune pathways.

A bispecific antibody strategy to target multiple type 2 cytokines in asthma.

BACKGROUND: Asthma is a chronic inflammatory airway disease in which innate and adaptive immune cells act together to cause eosinophilic inflammation, goblet cell metaplasia (GCM), and bronchial hyperreactivity (BHR). In clinical trials using biologicals against IL-4 receptor (IL-4R) α or IL-5, only a subset of patients with moderate-to-severe asthma responded favorably, suggesting that distinct pathophysiologic mechanisms are at play in subgroups of patients called endotypes. However, the effect of multiple cytokine blockade using bispecific antibodies has not been tested. OBJECTIVE: We sought to target simultaneously the IL-4, IL-13, and IL-5 signaling pathways with a novel IL-4Rα/IL-5-bispecific antibody in a murine house dust mite (HDM) model of asthma. METHODS: Two mAbs neutralizing IL-4Rα and IL-5 were generated by using a llama-based antibody platform. Their heavy and light chains were then cotransfected in mammalian cells, resulting in a heterogeneous antibody mixture from which the bispecific antibody was isolated by using a dual anti-idiotypic purification process. C57BL/6J mice were finally sensitized and challenged to HDM extracts and treated during challenge with the antibodies. RESULTS: We successfully generated and characterized the monospecific and bispecific antibodies targeting IL-4Rα and IL-5. The monospecific antibodies could suppress eosinophilia, IgE synthesis, or both, whereas only the IL-4Rα/IL-5-bispecific antibody and the combination of monospecific antibodies additionally inhibited GCM and BHR. CONCLUSION: Type 2 cytokines act synergistically to cause GCM and BHR in HDM-exposed mice. These preclinical results show the feasibility of generating bispecific antibodies that target multiple cytokine signaling pathways as superior inhibitors of asthma features, including the difficult-to-treat GCM.

The mucosal adjuvant cholera toxin B instructs non-mucosal dendritic cells to promote IgA production via retinoic acid and TGF-ß.

It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-ß or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-ß signaling inhibitor or neutralizing anti-TGF-ß was added, demonstrating the involvement of RA and TGF-ß in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant.

An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma.

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation.

Mechanism of action of clinically approved adjuvants.

Aluminum-containing adjuvants continue to be the most widely used adjuvants for human use. In the last year a major breakthrough has been the realization that alum adjuvant triggers an ancient pathway of innate recognition of crystals in monocytes and triggers them to become immunogenic dendritic cells, nature's adjuvant. This recognition can occur directly, via the triggering of the NALP3 inflammasome by alum crystals, or indirectly through release of the endogenous danger signal uric acid. It is also clear now that adjuvants trigger the stromal cells at the site of injection, leading to the necessary chemokines that attract the innate immune cells to the site of injection. How exactly these pathways interact remains to be determined.

Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome.

Adjuvants are vaccine additives that stimulate the immune system without having any specific antigenic effect of itself. In this study we show that alum adjuvant induces the release of IL-1beta from macrophages and dendritic cells and that this is abrogated in cells lacking various NALP3 inflammasome components. The NALP3 inflammasome is also required in vivo for the innate immune response to OVA in alum. The early production of IL-1beta and the influx of inflammatory cells into the peritoneal cavity is strongly reduced in NALP3-deficient mice. The activation of adaptive cellular immunity to OVA-alum is initiated by monocytic dendritic cell precursors that induce the expansion of Ag-specific T cells in a NALP3-dependent way. We propose that, in addition to TLR stimulators, agonists of the NALP3 inflammasome should also be considered as vaccine adjuvants.

Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells.

Alum (aluminum hydroxide) is the most widely used adjuvant in human vaccines, but the mechanism of its adjuvanticity remains unknown. In vitro studies showed no stimulatory effects on dendritic cells (DCs). In the absence of adjuvant, Ag was taken up by lymph node (LN)-resident DCs that acquired soluble Ag via afferent lymphatics, whereas after injection of alum, Ag was taken up, processed, and presented by inflammatory monocytes that migrated from the peritoneum, thus becoming inflammatory DCs that induced a persistent Th2 response. The enhancing effects of alum on both cellular and humoral immunity were completely abolished when CD11c(+) monocytes and DCs were conditionally depleted during immunization. Mechanistically, DC-driven responses were abolished in MyD88-deficient mice and after uricase treatment, implying the induction of uric acid. These findings suggest that alum adjuvant is immunogenic by exploiting "nature's adjuvant," the inflammatory DC through induction of the endogenous danger signal uric acid.

Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells.

Extracellular ATP serves as a danger signal to alert the immune system of tissue damage by acting on P2X or P2Y receptors. Here we show that allergen challenge causes acute accumulation of ATP in the airways of asthmatic subjects and mice with experimentally induced asthma. All the cardinal features of asthma, including eosinophilic airway inflammation, Th2 cytokine production and bronchial hyper-reactivity, were abrogated when lung ATP levels were locally neutralized using apyrase or when mice were treated with broad-spectrum P2-receptor antagonists. In addition to these effects of ATP in established inflammation, Th2 sensitization to inhaled antigen was enhanced by endogenous or exogenous ATP. The adjuvant effects of ATP were due to the recruitment and activation of lung myeloid dendritic cells that induced Th2 responses in the mediastinal nodes. Together these data show that purinergic signaling has a key role in allergen-driven lung inflammation that is likely to be amenable to therapeutic intervention.

Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function.

Inhalation of iloprost, a stable prostacyclin (PGI(2)) analog, is a well-accepted and safe treatment for pulmonary arterial hypertension. Although iloprost mainly acts as a vasodilator by binding to the I prostanoid (IP) receptor, recent evidence suggests that signaling via this receptor also has antiinflammatory effects through unclear mechanisms. Here we show in a murine model of asthma that iloprost inhalation suppressed the cardinal features of asthma when given during the priming or challenge phase. As a mechanism of action, iloprost interfered with the function of lung myeloid DCs, critical antigen-presenting cells of the airways. Iloprost treatment inhibited the maturation and migration of lung DCs to the mediastinal LNs, thereby abolishing the induction of an allergen-specific Th2 response in these nodes. The effect of iloprost was DC autonomous, as iloprost-treated DCs no longer induced Th2 differentiation from naive T cells or boosted effector cytokine production in primed Th2 cells. These data should pave the way for a clinical effectiveness study using inhaled iloprost for the treatment of asthma.