Anthony A. Horner

Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

IFN-αβ Promote Priming of Antigen-Specific CD8+ and CD4+ T Lymphocytes by Immunostimulatory DNA-Based Vaccines

Immunostimulatory sequence (ISS) DNA containing unmethylated CpG dinucleotides stimulate NK and APC to secrete proinflammatory cytokines, including IFN-αβ and -γ, TNF-α, and IL-6 and -12, and to express costimulatory surface molecules such as CD40, B7-1, and B7-2. Although ISS DNA has little direct effect on T cells by these criteria, immunization of wild-type mice with ISS DNA and OVA results in Ag-specific CTL and Th1-type T helper activity. This investigation examines the mechanisms by which ISS DNA primes CD8+ and CD4+ lymphocyte activities. In this report we demonstrate that ISS DNA regulates the expression of costimulatory molecules and TAP via a novel autocrine or paracrine IFN-αβ pathway. Coordinated regulation of B7 costimulation and TAP-dependent cross-presentation results in priming of Ag-specific CD8+ CTL, whereas CD40, B7, and IL-12 costimulation is required for priming of CD4+ Th cells by ISS-based vaccines.

Immunostimulatory DNA-Based Vaccines Elicit Multifaceted Immune Responses Against HIV at Systemic and Mucosal Sites

Immunostimulatory DNA sequences (ISS, also known as CpG motifs) are pathogen-associated molecular patterns that are potent stimulators of innate immunity. We tested the ability of ISS to act as an immunostimulatory pathogen-associated molecular pattern in a model HIV vaccine using gp120 envelope protein as the Ag. Mice immunized with gp120 and ISS, or a gp120:ISS conjugate, developed gp120-specific immune responses which included: 1) Ab production; 2) a Th1-biased cytokine response; 3) the secretion of β-chemokines, which are known to inhibit the use of the CCR5 coreceptor by HIV; 4) CTL activity; 5) mucosal immune responses; and 6) CD8 T cell responses that were independent of CD4 T cell help. Based on these results, ISS-based immunization holds promise for the development of an effective preventive and therapeutic HIV vaccine.

DNA-based immunotherapeutics for the treatment of allergic disease.

Summary: Allergic diseases are a growing health concern in industrialized countries. Despite a number of effective therapeutic options for the prevention and treatment of the pathophysiologic responses which characterize allergic diseases, the induction of true allergen desensitization remains an elusive therapeutic goal. Only immunotherapy (IT) has been shown to have any effect on the underlying hypersensitivities which mediate allergic reactions, and traditional protein‐based allergen IT has a limited scope of efficacy. However, a number of reagents collectively termed DNA‐based immunotherapeutics have proven highly effective in both the prevention and reversal of Th2‐mediated hypersensitivity states in mouse models of allergic disease. Four basic DNA‐based immunotherapeutic modalities have been used for these studies. These include immunization with gene vaccines, allergen mixed with immunostimulatory oligodeoxynucleotide (ISS‐ODN), and physical allergen–ISS‐ODN conjugates (AIC), as well as immunomodulation with ISS‐ODN alone. Results from many laboratories have generated guarded optimism that DNA‐based immunotherapeutics may be effective for the reversal of allergic hypersensitivity states in humans, and several clinical trials have already been initiated. This review will focus on our present understanding of the biological activities of DNA‐based immunotherapeutics and their application to the treatment of allergic diseases.

Invariant NKT cells are required for airway inflammation induced by environmental antigens

House dust contains antigens capable of activating mouse and human iNKT cells, contributing to allergen-induced airway inflammation.

Intranasal Immunotherapy Is More Effective Than Intradermal Immunotherapy for the Induction of Airway Allergen Tolerance in Th2-Sensitized Mice

Immunotherapy (IT) by injection more readily induces clinical tolerance to stinging insects than to respiratory allergens. However, while systemic immunization induces adaptive responses systemically, the induction of mucosal immunity generally requires local Ag exposure. Taken together, these observations suggest that the poor success rate of systemic IT for asthma could be a consequence of inadequate immune modulation in the airways. In support of this position, investigations presented in this report demonstrate that allergen IT more effectively induces airway allergen tolerance in Th2-sensitized mice, when delivered by the intranasal (i.n.) vs the intradermal (i.d.) route. Moreover, compared with native allergen, allergen immunostimulatory sequence oligodeoxynucleotide conjugate proved to be a more effective i.n. IT reagent for protecting allergic mice from airway hypersensitivity responses. Furthermore, for both native allergen and allergen immunostimulatory sequence oligodeoxynucleotide conjugate, i.n. and i.d. IT delivery were similarly effective in modulating systemic immune profiles in Th2-sensitized mice, while only i.n. IT had significant immunomodulatory activity on B and T cell responses in the airways. The present investigations may be the first to suggest that i.n. IT is more effective than i.d. IT for the treatment of asthma. Furthermore, our results suggest that modulating airway rather than systemic immunity may be the more important therapeutic target for the induction of clinical tolerance to respiratory allergens.

Antigen–immunostimulatory oligonucleotide conjugates: mechanisms and applications

Summary:  Conjugation of protein antigen with immunostimulatory oligonucleotides creates a potent immunogen. Physical linking of oligonucleotides to antigen enhances antigen uptake and targets the adjuvant properties of the oligonucleotides to the antigen‐presenting cell. In addition, the conjugated oligonucleotides appear to have improved immunostimulatory abilities compared to free oligonucleotides, presumably due to enhanced activation of Toll‐like receptor 9. Immunization with these conjugate preparations elicits antigen‐specific antibody responses, a T‐helper cell 1‐biased cytokine profile from CD4 T cells, and CD8 cytotoxic T‐lymphocyte activity that is CD4 independent. The humoral and cellular immune responses induced by these conjugates suggest they can be used to create effective vaccines against infectious pathogens and tumors and to beneficially modulate allergic responses. Indeed, recent clinical trial data show symptom relief and immunomodulation of the allergic response in patients with allergic rhinitis. This review considers the mechanisms of action of antigen–oligonucleotide conjugates and discusses available data regarding their use for the prevention and treatment of infectious, oncologic, and allergic diseases.

Allergen-independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis

While effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS‐ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty‐four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS‐ODN or control oligodeoxynucleotide (C‐ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS‐ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS‐ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C‐ODN. In addition, splenocytes from ISS‐ODN but not C‐ODN treated mice displayed attenuated OVA‐specific interleukin (IL)‐4, IL‐5, and IL‐13 but increased interferon‐γ responses. Finally, ISS‐ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2‐biased immune parameters towards Th1 dominance. As ISS‐ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS‐ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.

Update on toll-like receptor-directed therapies for human disease.

Innate responses to microbes are mediated in large part by toll-like receptors (TLRs), which recognise a diverse range of molecules produced by viruses, bacteria and fungi. Great effort has been directed towards translating this knowledge into the development of new therapies for a wide spectrum of diseases, including infectious, malignant, autoimmune and allergic diseases. This review will provide a brief update on completed, ongoing and planned clinical trials of TLR ligand-based therapies for the treatment of diseases in humans.

Airway House Dust Extract Exposures Modify Allergen-Induced Airway Hypersensitivity Responses by TLR4-Dependent and Independent Pathways

TLR ligands and other allergen-nonspecific immunostimulatory molecules are ubiquitous in ambient air and have profound modulatory activities in animal models of allergic asthma. However, several of these molecules have been shown to promote exaggerated Th2-biased airway hypersensitivity responses (AHRs), whereas others attenuate the asthmatic phenotype. Therefore, it has proven difficult to extrapolate experimental results with purified molecules toward a more general understanding of the allergen-nonspecific immunomodulatory influence of living environments on the natural history of allergic asthma. These investigations determined how regular and intermittent airway exposures to an unpurified, but sterile house dust extract standard (HDEst) affected the OVA-specific AHR and immune status of previously Th2-sensitized mice. Low-dose daily and high-dose intermittent HDEst exposures modulated ongoing AHRs considerably, reducing eosinophil recruitment and methacholine responsiveness, while increasing neutrophilic inflammation. However, only daily airway delivery of low-dose HDEst attenuated OVA-specific Th2 cytokine production and Th2-biased AHRs to allergen challenge 1 mo later. Finally, whereas LPS mimicked many of the immunomodulatory characteristics of HDEst in this murine asthma model, daily airway HDEst delivery was highly effective in attenuating the AHR of OVA/alum-sensitized TLR4-deficient mice. Taken together, these investigations provide direct evidence that living environments contain allergen-nonspecific immunostimulatory molecules that influence the airway hypersensitivity status of allergen-sensitized mice by TLR4-dependent and independent mechanisms.