Drug Repurposing: The Anthelmintics Niclosamide and Nitazoxanide Are Potent TMEM16A Antagonists That Fully Bronchodilate Airways.

There is an unmet need in severe asthma where approximately 40% of patients exhibit poor ß-agonist responsiveness, suffer daily symptoms and show frequent exacerbations. Antagonists of the Ca2+-activated Cl- channel, TMEM16A, offers a new mechanism to bronchodilate airways and block the multiple contractiles operating in severe disease. To identify TMEM16A antagonists we screened a library of ∼580,000 compounds. The anthelmintics niclosamide, nitazoxanide, and related compounds were identified as potent TMEM16A antagonists that blocked airway smooth muscle depolarization and contraction. To evaluate whether TMEM16A antagonists resist use- and inflammatory-desensitization pathways limiting ß-agonist action, we tested their efficacy under harsh conditions using maximally contracted airways or airways pretreated with a cytokine cocktail. Stunningly, TMEM16A antagonists fully bronchodilated airways, while the ß-agonist isoproterenol showed only partial effects. Thus, antagonists of TMEM16A and repositioning of niclosamide and nitazoxanide represent an important additional treatment for patients with severe asthma and COPD that is poorly controlled with existing therapies. It is of note that drug repurposing has also attracted wide interest in niclosamide and nitazoxanide as a new treatment for cancer and infectious disease. For the first time we identify TMEM16A as a molecular target for these drugs and thus provide fresh insights into their mechanism for the treatment of these disorders in addition to respiratory disease.

Limited differential expression of miRNAs and other small RNAs in LPS-stimulated human monocytes.

Monocytes are a distinct subset of myeloid cells with diverse functions in early inflammatory immune modulation. While previous studies have surveyed the role of miRNA regulation on different myeloid cell lines and primary cultures, the time-dependent kinetics of inflammatory stimulation on miRNA expression and the relationship between miRNA-to-target RNA expression have not been comprehensively profiled in monocytes. In this study, we use next-generation sequencing and RT-PCR assays to analyze the non-coding small RNA transcriptome of unstimulated and lipopolysaccharide (LPS)-stimulated monocytes at 6 and 24 hours. We identified a miRNA signature consisting of five mature miRNAs (hsa-mir-146a, hsa-mir-155, hsa-mir-9, hsa-mir-147b, and hsa-mir-193a) upregulated by LPS-stimulated monocytes after 6 hours and found that most miRNAs were also upregulated after 24 hours of stimulation. Only one miRNA gene was down-regulated and no other small RNAs were found dysregulated in monocytes after LPS treatment. In addition, novel tRNA-derived fragments were also discovered in monocytes although none showed significant changes upon LPS stimulation. Interrogation of validated miRNA targets by transcriptomic analysis revealed that absolute expression of most miRNA targets implicating in innate immune response decreased over time in LPS-stimulated monocytes although their expression patterns along the treatment were heterogeneous. Our findings reveal a potential role by which selective miRNA upregulation and stable expression of other small RNAs enable monocytes to develop finely tuned cellular responses during acute inflammation.

Obstructive Lymphangitis Precedes Colitis in Murine Norovirus-Infected Stat1-Deficient Mice.

Murine norovirus (MNV) is an RNA virus that can prove lethal in mice with impaired innate immunity. We found that MNV-4 infection of Stat1-/- mice was not lethal, but produced a 100% penetrant, previously undescribed lymphatic phenotype characterized by chronic-active lymphangitis with hepatitis, splenitis, and chronic cecal and colonic inflammation. Lesion pathogenesis progressed from early ileal enteritis and regional dilated lymphatics to lymphangitis, granulomatous changes in the liver and spleen, and, ultimately, typhlocolitis. Lesion development was neither affected by antibiotics nor reproduced by infection with another enteric RNA virus, rotavirus. MNV-4 infection in Stat1-/- mice decreased expression of vascular endothelial growth factor (Vegf) receptor 3, Vegf-c, and Vegf-d and increased interferon (Ifn)-γ, tumor necrosis factor-α, and inducible nitric oxide synthase. However, anti-IFN-γ and anti-tumor necrosis factor-α antibody treatment did not attenuate the histologic lesions. Studies in Ifnαßγr-/- mice suggested that canonical signaling via interferon receptors did not cause MNV-4-induced disease. Infected Stat1-/- mice had increased STAT3 phosphorylation and expressed many STAT3-regulated genes, consistent with our findings of increased myeloid cell subsets and serum granulocyte colony-stimulating factor, which are also associated with increased STAT3 activity. In conclusion, in Stat1-/- mice, MNV-4 induces lymphatic lesions similar to those seen in Crohn disease as well as hepatitis, splenitis, and typhlocolitis. MNV-4-infected Stat1-/- mice may be a useful model to study mechanistic associations between viral infections, lymphatic dysfunction, and intestinal inflammation in a genetically susceptible host.

Murine norovirus inhibits B cell development in the bone marrow of STAT1-deficient mice.

Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1-/- hematopoietic cells and Stat1-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.

IL-17RA Signaling in Airway Inflammation and Bronchial Hyperreactivity in Allergic Asthma.

Asthma is a heterogeneous disease characterized by airway inflammation and hyperreactivity. IL-17 receptor A (IL-17RA) is a shared receptor subunit required for activity of IL-17 family cytokines, including IL-17A and IL-25. IL-17A and IL-25 induce different proinflammatory responses, and concentrations are elevated in subjects with asthma. However, the individual contributions of IL-17A and IL-25 to disease pathogenesis are unclear. We explored proinflammatory activities of the IL-17 pathway in models of pulmonary inflammation and assessed its effects on contractility of human bronchial airway smooth muscle. In two mouse models, IL-17RA, IL-17RB, or IL-25 blockade reduced airway inflammation and airway hyperreactivity. Individually, IL-17A and IL-25 enhanced contractility of human bronchial smooth muscle induced by methacholine or carbachol. IL-17A had more pronounced effects on methacholine-induced contractility in bronchial rings from donors with asthma compared with donors without asthma. Blocking the IL-17 pathway via IL-17RA may be a useful therapy for some patients with asthma by reducing pulmonary inflammation and airway hyperreactivity.

Butyrophilin-like 2 modulates B7 costimulation to induce Foxp3 expression and regulatory T cell development in mature T cells.

Naive T cell activation involves at least two signals from an APC, one through the TCR via interaction with peptide-MHC complexes and a second through ligation of CD28 with B7 ligands. Following activation, T cells upregulate a host of other membrane-bound costimulatory molecules that can either promote or inhibit further T cell maturation and proliferation. In some cases, it is necessary to attenuate T cell activation to prevent deleterious inflammation, and inhibitory members of the B7/butyrophilin family of ligands have evolved to balance the strong stimuli the activating B7 ligands confer. Human genetic association and in vitro studies have implicated one such ligand, BTNL2, in controlling inflammation at mucosal surfaces. In this study, we show that recombinant mouse BTNL2 modifies B7/CD28 signaling to promote expression of Foxp3, a transcription factor necessary for regulatory T cell (Treg) development and function. BTNL2 blocks Akt-mediated inactivation of Foxo1, a transcription factor necessary for Foxp3 expression. Immunophenotyping and gene profiling reveal that BTNL2-induced Treg share many properties with natural Treg, and in vivo they suppress enteritis induced by mouse effector T cells. These findings describe a mechanism by which environmental Ag-specific Tregs may be induced by APC expressing specific modulators of costimulatory signals.

The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination.

The basic helix-loop-helix transcription factor Olig1 promotes oligodendrocyte maturation and is required for myelin repair. We characterized an Olig1-regulated G protein-coupled receptor, GPR17, whose function is to oppose the action of Olig1. Gpr17 was restricted to oligodendrocyte lineage cells, but was downregulated during the peak period of myelination and in adulthood. Transgenic mice with sustained Gpr17 expression in oligodendrocytes exhibited stereotypic features of myelinating disorders in the CNS. Gpr17 overexpression inhibited oligodendrocyte differentiation and maturation both in vivo and in vitro. Conversely, Gpr17 knockout mice showed early onset of oligodendrocyte myelination. The opposing action of Gpr17 on oligodendrocyte maturation reflects, at least partially, upregulation and nuclear translocation of the potent oligodendrocyte differentiation inhibitors ID2/4. Collectively, these findings suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes via an ID protein-mediated negative regulation and may serve as a potential therapeutic target for CNS myelin repair.

Regulation of costimulation in the era of butyrophilins.

The butyrophilin and butyrophilin-like superfamily of molecules has garnered attention in the immunology world in the past few years as a result of the observation that the butyrophilin-like 2 molecule, BTNL2, can alter T cell responsiveness. Additional interest in this superfamily solidified following the discovery that genetic polymorphisms in BTNL2 are associated with predisposition to many human diseases. In this review, we will provide an overview of the members comprising the butyrophilin superfamily of molecules. We will then discuss BTNL2 immunomodulatory function, and BTNL2 structural associations with other costimulatory molecules. We will then draw your attention to some of the lesser-known butyrophilin superfamily members by describing the expression patterns of these molecules in human tissues and cells. And we will finish by hypothesizing on the potential influence on general immune homeostasis that might be mediated by this, thus-far little-studied, family of molecules.

BTNL2, a butyrophilin/B7-like molecule, is a negative costimulatory molecule modulated in intestinal inflammation.

Butyrophilin-like 2 (BTNL2) is a butyrophilin family member with homology to the B7 costimulatory molecules, polymorphisms of which have been recently associated through genetic analyses to sporadic inclusion body myositis and sarcoidosis. We have characterized the full structure, expression, and function of BTNL2. Structural analysis of BTNL2 shows a molecule with an extracellular region containing two sets of two Ig domains, a transmembrane region, and a previously unreported cytoplasmic tail. Unlike most other butyrophilin members, BTNL2 lacks the prototypical B30.2 ring domain. TaqMan and Northern blot analysis indicate BTNL2 is predominantly expressed in digestive tract tissues, in particular small intestine and Peyer's patches. Immunohistochemistry with BTNL2-specific Abs further localizes BTNL2 to epithelial and dendritic cells within these tissues. Despite its homology to the B7 family, BTNL2 does not bind any of the known B7 family receptors such as CD28, CTLA-4, PD-1, ICOS, or B and T lymphocyte attenuator. Because of its localization in the gut and potential role in the immune system, BTNL2 expression was analyzed in a mouse model of inflammatory bowel disease. BTNL2 is overexpressed during both the asymptomatic and symptomatic phase of the Mdr1a knockout model of spontaneous colitis. In functional assays, soluble BTNL2-Fc protein inhibits the proliferation of murine CD4(+) T cells from the spleen, mesenteric lymph node, and Peyer's patch. In addition, BTNL2-Fc reduces proliferation and cytokine production from T cells activated by anti-CD3 and B7-related protein 1. These data suggest a role for BTNL2 as a negative costimulatory molecule with implications for inflammatory disease.

Helicobacter bilis infection accelerates and H. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr1a-/-) mice.

mdr1a-deficient mice lack P-glycoprotein and spontaneously develop colitis with age. Helicobacter spp. are gram-negative organisms that have been associated with colitis in certain mouse strains, but Helicobacter spp. have been excluded as contributing to the spontaneous colitis that develops in mdr1a-/- mice. We wished to determine whether infection with either H. bilis or H. hepaticus would accelerate the development of inflammatory bowel disease (IBD) in mdr1a-/- mice. We found that H. bilis infection induced diarrhea, weight loss, and IBD in mdr1a-/- mice within 6 to 17 weeks post-inoculation and before the expected onset of spontaneous IBD. Histopathology of H. bilis-induced IBD included crypt hyperplasia, inflammatory cell infiltrates, crypt abscesses, and obliteration of normal gut architecture. Reverse transcription-polymerase chain reaction and Taqman analysis from colonic tissue showed increased transcripts for interferon-gamma and interleukin-10 from H. bilis-infected colitic mdr1a-/- mice. Additionally, mesenteric lymph nodes had increased cellularity with expansion of CD4+ and CD8+ T cells and B cells and increased proliferation to soluble H. bilis antigens with elaboration of interferon-gamma, tumor necrosis factor-alpha and interleukin-10. In contrast, H. hepaticus infection of mdr1a-/- mice did not accelerate disease but rather delayed the onset of spontaneous colitis which was milder in severity. mdr1a-/- mice infected with Helicobacter spp. may provide a useful tool to explore the pathogenesis of microbial-induced IBD in a model with a presumed epithelial cell "barrier" defect.