Amanda McFarlane

Cathelicidins have direct antiviral activity against respiratory syncytial virus in vitro and protective function in vivo in mice and humans.

Respiratory syncytial virus (RSV) is a leading cause of respiratory tract infection in infants, causing significant morbidity and mortality. No vaccine or specific, effective treatment is currently available. A more complete understanding of the key components of effective host response to RSV and novel preventative and therapeutic interventions are urgently required. Cathelicidins are host defense peptides, expressed in the inflamed lung, with key microbicidal and modulatory roles in innate host defense against infection. In this article, we demonstrate that the human cathelicidin LL-37 mediates an antiviral effect on RSV by inducing direct damage to the viral envelope, disrupting viral particles and decreasing virus binding to, and infection of, human epithelial cells in vitro. In addition, exogenously applied LL-37 is protective against RSV-mediated disease in vivo, in a murine model of pulmonary RSV infection, demonstrating maximal efficacy when applied concomitantly with virus. Furthermore, endogenous murine cathelicidin, induced by infection, has a fundamental role in protection against disease in vivo postinfection with RSV. Finally, higher nasal levels of LL-37 are associated with protection in a healthy human adult RSV infection model. These data lead us to propose that cathelicidins are a key, nonredundant component of host defense against pulmonary infection with RSV, functioning as a first point of contact antiviral shield and having additional later-phase roles in minimizing the severity of disease outcome. Consequently, cathelicidins represent an inducible target for preventative strategies against RSV infection and may inform the design of novel therapeutic analogs for use in established infection.

Combination peptide immunotherapy based on T‐cell epitope mapping reduces allergen‐specific IgE and eosinophilia in allergic airway inflammation

Peptide immunotherapy using soluble peptides containing allergen‐derived immunodominant T‐cell epitopes holds therapeutic promise for allergic asthma. Previous studies in BALB/c mice using the immunodominant peptide epitope of chicken ovalbumin (p323–339) have been unable to demonstrate therapeutic effects in ovalbumin‐induced allergic airway inflammation. We have previously shown that intravenous application of p323–339 can effectively tolerise p323–339‐reactive T cells in a non‐allergic model in C57BL/6 mice. This study aimed to assess the effects of using p323–339 immunotherapy in a C57BL/6 model of ovalbumin‐induced allergic airway inflammation, identify any additional epitopes recognized by the ovalbumin‐responsive T‐cell repertoire in C57BL/6 mice and assess the effects of combination peptide immunotherapy in this model. Ovalbumin‐reactive T‐cell lines were generated from ovalbumin‐immunized C57BL/6 mice and proliferative responses to a panel of overlapping peptides covering the ovalbumin sequence were assessed. Soluble peptides (singly or combined) were administered intravenously to C57BL/6 mice before the induction of ovalbumin‐induced allergic airway inflammation. Peptide immunotherapy using the 323–339 peptide alone did not reduce the severity of allergic airway inflammation. An additional immunodominant T‐cell epitope in ovalbumin was identified within the 263–278 sequence. Combination peptide immunotherapy, using the 323–339 and 263–278 peptides together, reduced eosinophilia in the bronchoalveolar lavage and ovalbumin‐specific IgE, with apparent reductions in interleukin‐5 and interleukin‐13. Characterization of the T‐cell response to a model allergen has allowed the development of combination peptide immunotherapy with improved efficacy in allergic airway inflammation. This model holds important potential for future mechanistic studies using peptide immunotherapy in allergy.

Enteric helminth-induced type I interferon signaling protects against pulmonary virus infection through interaction with the microbiota

Background: Helminth parasites have been reported to have beneficial immunomodulatory effects in patients with allergic and autoimmune conditions and detrimental consequences in patients with tuberculosis and some viral infections. Their role in coinfection with respiratory viruses is not clear. Objective: Here we investigated the effects of strictly enteric helminth infection with Heligmosomoides polygyrus on respiratory syncytial virus (RSV) infection in a mouse model. Methods: A murine helminth/RSV coinfection model was developed. Mice were infected by means of oral gavage with 200 stage 3 H polygyrus larvae. Ten days later, mice were infected intranasally with either RSV or UV‐inactivated RSV. Results: H polygyrus–infected mice showed significantly less disease and pulmonary inflammation after RSV infection associated with reduced viral load. Adaptive immune responses, including TH2 responses, were not essential because protection against RSV was maintained in Rag1−/− and Il4r&agr;−/− mice. Importantly, H polygyrus infection upregulated expression of type I interferons and interferon‐stimulated genes in both the duodenum and lung, and its protective effects were lost in both Ifnar1−/− and germ‐free mice, revealing essential roles for type I interferon signaling and microbiota in H polygyrus–induced protection against RSV. Conclusion: These data demonstrate that a strictly enteric helminth infection can have remote protective antiviral effects in the lung through induction of a microbiota‐dependent type I interferon response. Graphical abstract Figure. No Caption available.

Effector and central memory T helper 2 cells respond differently to peptide immunotherapy

Significance Peptide immunotherapy (PIT) of ongoing allergy must control “memory” T helper 2 (Th2) cells. Memory T cells can be subdivided into effector memory T cells (Tem), which seem to be involved in immediate immune responses, and central memory T cells (Tcm), which are thought to provide long-term memory. We show that PIT can control allergic lung disease more effectively when the disease is driven by Tem Th2 cells, rather than by Tcm Th2 cells. PIT-treated Tcm remained more responsive to allergen, with a greater capacity to produce inflammatory Th2 cytokines in the lung. These were suppressed in PIT-treated Tem. These differences are important for clinical translation of PIT, because Tcm may be particularly dominant in some seasonal allergic conditions, such as hay fever. Peptide immunotherapy (PIT) offers realistic prospects for the treatment of allergic diseases, including allergic asthma. Much is understood of the behavior of naive T cells in response to PIT. However, treatment of patients with ongoing allergic disease requires detailed understanding of the responses of allergen-experienced T cells. CD62L expression by allergen-experienced T cells corresponds to effector/effector memory (CD62Llo) and central memory (CD62Lhi) subsets, which vary with allergen exposure (e.g., during, or out with, pollen season). The efficacy of PIT on different T helper 2 (Th2) cell memory populations is unknown. We developed a murine model of PIT in allergic airway inflammation (AAI) driven by adoptively transferred, traceable ovalbumin-experienced Th2 cells. PIT effectively suppressed AAI driven by unfractionated Th2 cells. Selective transfer of CD62Lhi and CD62Llo Th2 cells revealed that these two populations behaved differently from one another and from previously characterized (early deletional) responses of naive CD4+ T cells to PIT. Most notably, allergen-reactive CD62Llo Th2 cells were long-lived within the lung after PIT, before allergen challenge, in contrast to CD62Lhi Th2 cells. Despite this, PIT was most potent against CD62Llo Th2 cells in protecting from AAI, impairing their ability to produce Th2 cytokines, whereas this capacity was heightened in PIT-treated CD62Lhi Th2 cells. We conclude that Th2 cells do not undergo an early deletional form of tolerance after PIT. Moreover, memory Th2 subsets respond differently to PIT. These findings have implications for the clinical translation of PIT in different allergic scenarios.

Respiratory syncytial virus infection influences tight junction integrity

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air–liquid interface (ALI) and infected with RSV and ultraviolet (UV)‐irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV‐irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post‐infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down‐regulated claudin‐1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin‐1 and claudin‐7 at protein level.

Expression of the B cell differentiation factor BAFF and chemokine CXCL13 in a murine model of Respiratory Syncytial Virus infection

HighlightsRSV infection increases B cell growth factor BAFF and CXCL13 chemokine expression.Lung BAFF was increased at 2 and 7 days post infection, CXCL13 at 1, 2 and 7 days.These cytokines may have an important role in local airway responses to RSV. &NA; Innate immune responses are known to influence the subsequent development of adaptive immunity. We have previously shown that RSV infection of human airway epithelial cells results in production of the B cell growth factor, BAFF. To better understand how the airway responds to RSV infection by production of this and other factors to support or enhance local B cell responses to infection, we analysed the lung expression of BAFF and B cell homeostatic chemokines CXCL12, CXCL13, CCL19 and CCL21 in a murine model of RSV infection. Following infection with A2 strain RSV, the highest RSV N gene expression was observed at day 4 after challenge with virus. In contrast, two peaks of elevated BAFF expression at days 2 and 7 were observed. CXCL13 was significantly elevated at days 1, 2 and 7. CXCL12, CCL19 and CCL21 were expressed within lung tissue from control and RSV challenged animals but no significant difference in expression was found. Immunofluorescence showed BAFF to be present throughout the tissue however CXCL13 expression was localized to cell rich areas probably constituting lymphoid aggregates. Our results define the kinetics of B cell chemoattractant and growth factor expression during RSV infection and indicate an important role for these cytokines in the airway response to RSV infection.

Modulation of influenza virus mediated immunopathology; the role for SOCS proteins

Background: Polyvalent vaccination represents a recent attempt to improve the effectiveness of lung cancer immunotherapy. This study aimed to investigate whether a gene expression pattern of tumor-associated antigens (TAA) would exist indicating that their use will be most appropriate for the polyvalent vaccination of Caucasian non-small cell lung carcinoma (NSCLC) patients. We examined the concomitant expression of genes belonging to different TAA families for which expression frequencies either have never been detected in NSCLC or vary widely in the literature. Methods: Tumor material from 23 patients with NSCLC (12 adenocarcinomas, 8 squamous cell carcinomas, 3 bronchoalveolar carcinomas) was examined. mRNA transcripts were detected for 5 genes of the survivin family, 5 MAGE-A genes as well as the genes of human telomerase reverse transcriptase (hTERT) and p53, by the use of quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) or semiquantitative RT-PCR. Results: 15/23 (65%) and 8/23 (35%) tumor samples were found expressing 6-11 and 2-5 out of the 12 examined TAAs, respectively, at levels >1% of the testis reference sample. The most prevalent TAA patterns observed were those of survivin standard (survivinstd)/survivin-2B expressed by 22/23 (95.5%) tumor samples and of survivin-std/survivin-2B/hTERT expressed by 19/23 (82.5%) tumor samples. The expression levels of the survivin-std gene strongly positively correlated to those of the survivin-2B (p=0.001) and the hTERT genes (p=0.031). The number of concomitantly expressed genes was found to be positively correlated to the age of the patients (p=0.001) and the tumor size (p=0.048). Conclusion: This study provides evidence that, in Caucasian patients with NSCLC, highly prevalent expression patterns of TAA genes, predominantly of overexpressed TAAs, do exist. This result implies that the combined use of these TAA could help in designing more effective NSCLC immunotherapeutic protocols.Purpose/Objective: Rheumatoid arthritis (RA) is a chronic autoimmune disease that leads to joint destruction. The recruitment of effectors cells, including monocytes to the joint space is an important step in RA progression and is mediated by chemokines (Ch) and their receptors (ChR). MicroRNAs are a recently discovered class of posttranscriptional regulators. Many members of the miR family are implicated in the regulation of cell movement and migration. Our previous study showed miR-155 is upregulated in RA synovial fluid (SF) monocytes suggesting that this miR may be involved in activation of these cells, including their migration into joint space. We hypothesized that miR-155 could regulates migration of monocytes in RA by modulating the expression of the chemokine and chemokine receptor system. Materials and methods: Peripheral blood (PB) CD14+ cells from healthy controls (HC) and RA patients were transfected with miR-155 mimic or scramble mimic using N-TER nanoparticles and cultured for 48 h. TaQman Low Density Array and multiplex assay was used to evaluate ChR expression and Ch production, respectively. Similar analysis was carried out on bone marrow monocytes (BMM) from miR-155-/- and WT mice. In addition, absolute copy numbers of miR- 155 transcripts in PB and SF CD14+ of RA and HC were assessed by QPCR. Results: PB and SF monocytes in RA patients showed higher copy number of miR-155 compared to HC. Overexpression of miR-155 in HC and RA monocytes did not affect the production of CCL2, CCL7, CCL21, CXCL5, CXCL8, CXCL7, CXCL10 and CX3CL1. In contrast, overexpression of miR-155 induced the production of chemokines such as CCL4, CCL5 and CCL22 in RA monocytes and CCL3 in both RA and HC. Analysis of chemokine receptors in BMM of miR-155-/- and WT mice revealed significantly higher levels of CCR1, CCR2, CCR5 and CXCR4 in miR-155 deficient cells suggesting that miR-155 can act as a negative regulator of these receptors in homeostatic state. As expected, TLR-4 ligand significantly suppressed expression of these receptors in both WT and miR-155-/- cells. Analysis of 3’UTRs of Ch/ ChR (TargetScan) suggests that miR-155 is likely interfering with signaling pathways implicated in Ch/ChR system expression. Conclusions: Deregulation of miR-155 in RA monocytes can contribute to the production of pro-inflammatory chemokines by these cells and to their accumulation at sites of inflammation.Purpose/Objective: Sphingosine kinase (SPHKs), SphK1 and SphK2, have been identified to phosphorylate sphingosine into sphingosine-1- phosphate (S1P). They are involved in a wide variety of cellular responses. S1P acts via S1P Receptors, S1PR1, S1PR2, S1PR3, S1PR4 and S1PR5, all of which can be bound and activated specifically by S1P. A defect either in S1P signalling or S1PRs has been associated with many pathologies. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by high levels of proinflammatory cytokine production. Elevated SPHK1, S1P, and S1P1 have been reported in RA synovium. S1P signalling via S1P1 promotes synoviocyte proliferation, increases COX-2 expression and prostaglandin E2 production. This study comprehensively evaluated expression of SPHK1/2 and S1PRs in RA patients compare to healthy controls (HC) and osteoarthritis (OA) in peripheral blood (PB) and synovial tissues, respectively. Materials and methods: mRNA and protein expression of SPHK1/2 and SIPRs were examined in neutrophils, monocytes and T lymphocytes of peripheral blood of 10 HC and RA patients, who met the diagnostic criteria of 2010 ARC / EULAR by QPCR and FACS, respectively. Competitive ELISA assessed SIP in serum of RA patients with remission and relapse and HC. We also performed SPHK 1/2 and SIPRs immunohistochemistry in synovial tissue from 4 RA/ OA patients. Results: S1P was three times high in RA than those observed in HC, also was statistically higher in RA patient with relapse than remission. Intracellular expression of hSPHK1 in RA patients, with opposed to HC, was up regulated 1.4-folds in monocytes and T- lymphocytes with significance expression in CD4T cells. hS1P1 and hS1P3 exhibited a similar expression were up-regulated in neutrophils, while, hS1P5 was statistical high in T cells. In contrast, hS1P4 was down regulated in all sorted cells particularly in CD4T cells. As opposed to OA synovial tissue, RA synovial tissues were strongly positive for hSPHK1 and hS1P1, 3 expressions. Quantitative analysis showed, SPHK1 and hS1P3 are expressed in lining, sub lining and vascular endothelial layer, while hS1P1 expressed mainly in lining and sub lining layers of the RA synovial tissue compared with OA. Conclusions: These results suggest that SPHKs/S1P and its S1PRs might play a role in RA pathogenesis. The clinical significance of S1P as a biomarker for disease activity deserves further attention.