Nicole Stowell

Long-term activation of TLR3 by Poly(I:C) induces inflammation and impairs lung function in mice

BackgroundThe immune mechanisms associated with infection-induced disease exacerbations in asthma and COPD are not fully understood. Toll-like receptor (TLR) 3 has an important role in recognition of double-stranded viral RNA, which leads to the production of various inflammatory mediators. Thus, an understanding of TLR3 activation should provide insight into the mechanisms underlying virus-induced exacerbations of pulmonary diseases.MethodsTLR3 knock-out (KO) mice and C57B6 (WT) mice were intranasally administered repeated doses of the synthetic double stranded RNA analog poly(I:C).ResultsThere was a significant increase in total cells, especially neutrophils, in BALF samples from poly(I:C)-treated mice. In addition, IL-6, CXCL10, JE, KC, mGCSF, CCL3, CCL5, and TNFα were up regulated. Histological analyses of the lungs revealed a cellular infiltrate in the interstitium and epithelial cell hypertrophy in small bronchioles. Associated with the pro-inflammatory effects of poly(I:C), the mice exhibited significant impairment of lung function both at baseline and in response to methacholine challenge as measured by whole body plethysmography and an invasive measure of airway resistance. Importantly, TLR3 KO mice were protected from poly(I:C)-induced changes in lung function at baseline, which correlated with milder inflammation in the lung, and significantly reduced epithelial cell hypertrophy.ConclusionThese findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.

CCR3 antagonists: a potential new therapy for the treatment of asthma. Discovery and structure–activity relationships

CCR3 antagonist leads with IC(50) values in the microM range were converted into low nM binding compounds that displayed in vitro inhibition of human eosinophil chemotaxis induced by human eotaxin. In particular, 4-benzylpiperidin-1-yl-n-propylureas and erythro-3-(4-benzyl-2-(alpha-hydroxyalkyl)piperidin-1-yl)-n-propylureas (obtained via Beak reaction of N-BOC-4-benzylpiperidine) exhibited single digit nanomolar IC(50) values for CCR3.

Myelodysplasia and anemia of chronic disease in human tumor necrosis factor‐α transgenic mice

TNF‐α is a pleitropic cytokine that expresses both pro‐ and anti‐inflammatory activity and transgenic mice expressing human tumor necrosis factor‐α (TNF‐α) exhibit a progressive polyarthritis that models rheumatoid arthritis (RA). One of the common comorbidities of RA is anemia of chronic disease (ACD). The purpose of these experiments was to study the changes in the bone marrow and peripheral blood that accompany polyarthritis in TNF‐α transgenic mice in an effort to better understand the pathogenesis of myelodysplasia and ACD. Polychromatic cytometry, hematology and serum cytokine analysis were used to study the pathogenesis of ACD in human TNF‐α transgenic mice. Our hematological evaluation revealed a mild, compensated, microcytic hypochromic anemia, and monocytosis. In the bone marrow, we observed alterations in cell kinetics, decreased relative expression of transferrin receptor and increased apoptosis and cell death in several late precursor cell populations. Although significant levels of human TNF‐α were found in the serum, neither change in serum murine erythropoietin nor any significant difference observed in serum levels of murine IL‐β, IL‐5, IL‐6, IL‐10, IL‐12(p70), IL‐17, TNF‐α, IFNγ, GM‐CSF, MIP‐1αJE, MCP‐5 was observed. Tg197 mice develop a compensated, microcytic, hypochromic anemia, and a functional iron deficiency by 9 weeks of age. Changes in peripheral blood are reflected in alterations in cell kinetics, transferrin receptor expression and markedly increased apoptosis and cell death in the bone marrow indicating that TNF‐α may contribute to myelodysplasia in ACD. Moreover, since human TNF‐α can interact only with murine TNFR1, our data suggest that TNFR1 may play an important role in the development of ACD

Synthesis by native chemical ligation and crystal structure of human CCL2

The protein human CC chemokine ligand 2 (CCL2, also known as monocyte chemoattractant protein 1 or MCP‐1) has been synthesized using a combination of solid phase peptide synthesis (SPPS) and native chemical ligation (NCL). The thioester‐peptide segment was synthesized using the sulfonamide safety‐catch linker and 9‐fluorenylmethoxycarbonyl (Fmoc) SPPS, and pseudoproline dipeptides were used to facilitate the synthesis of both CCL2 fragments. After assembly of the full‐length peptide chain by NCL, a glutathione redox buffer was used to fold and oxidize the CCL2 protein. Synthetic human CCL2 binds to and activates the CCR2 receptor on THP‐1 cells, as expected. CCL2 was crystallized and the structure was determined by X‐ray diffraction at 1.9‐Å resolution. The structure of the synthetic protein is very similar to that of a previously reported structure of recombinant human CCL2, although the crystal form is different. The functional CCL2 dimer for the crystal structure reported here is formed around a crystallographic twofold axis. The dimer interface involves residues Val9‐Thr10‐Cys11, which form an intersubunit antiparallel β‐sheet. Comparison of the CCL2 dimers in different crystal forms indicates a significant flexibility of the quaternary structure. To our knowledge, this is one of the first crystal structures of a protein prepared using the sulfonamide safety‐catch linker and NCL.