Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica

Citation: McGill, J. L., Rusk, R. A., Guerra-Maupome, M., Briggs, R. E., & Sacco, R. E. (2016). Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica. Plos One, 11(3), 20. doi:10.1371/journal.pone.0151083Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and Upsilon delta T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing Upsilon delta T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and Upsilon delta T cells in the pathogenesis of BRDC

Familial pneumothorax: towards precision medicine.

One in 10 patients suffering from primary spontaneous pneumothoraces has a family history of the disorder. Such familial pneumothoraces can occur in isolation, but can also be the presentation of serious genetic disorders with life-threatening vascular or cancerous complications. As the pneumothorax frequently precedes the more dangerous complications by many years, it provides an opportunity to intervene in a focused manner, permitting the practice of precision medicine. In this review, we will discuss the clinical manifestations and underlying biology of the genetic causes of familial pneumothorax

The Malaria Testing and Treatment Market in Kinshasa, Democratic Republic of the Congo, 2013

Background The Democratic Republic of Congo (DRC) is one of the two most leading contributors to the global burden of disease due to malaria. This paper describes the malaria testing and treatment market in the nation’s capital province of Kinshasa, including availability of malaria testing and treatment and relative anti-malarial market share for the public and private sector. Methods A malaria medicine outlet survey was conducted in Kinshasa province in 2013. Stratified multi-staged sampling was used to select areas for the survey. Within sampled areas, all outlets with the potential to sell or distribute anti-malarials in the public and private sector were screened for eligibility. Among outlets with anti-malarials or malaria rapid diagnostic tests (RDT) in stock, a full audit of all available products was conducted. Information collected included product information (e.g. active ingredients, brand name), amount reportedly distributed to patients in the past week, and retail price. Results In total, 3364 outlets were screened for inclusion across Kinshasa and 1118 outlets were eligible for the study. Among all screened outlets in the private sector only about one in ten (12.1%) were stocking quality-assured Artemisinin-based Combination Therapy (ACT) medicines. Among all screened public sector facilities, 24.5% had both confirmatory testing and quality-assured ACT available, and 20.2% had sulfadoxine-pyrimethamine (SP) available for intermittent preventive therapy during pregnancy (IPTp). The private sector distributed the majority of anti-malarials in Kinshasa (96.7%), typically through drug stores (89.1% of the total anti-malarial market). Non-artemisinin therapies were the most commonly distributed anti-malarial (50.1% of the total market), followed by non quality-assured ACT medicines (38.5%). The median price of an adult quality-assured ACT was $6.59, and more expensive than non quality-assured ACT ($3.71) and SP ($0.44). Confirmatory testing was largely not available in the private sector (1.1%). Conclusions While the vast majority of anti-malarial medicines distributed to patients in Kinshasa province are sold within the private sector, availability of malaria testing and appropriate treatment for malaria is alarmingly low. There is a critical need to improve access to confirmatory testing and quality-assured ACT in the private sector. Widespread availability and distribution of non quality-assured ACT and non-artemisinin therapies must be addressed to ensure effective malaria case management

Bovine Gamma Delta T Cells Contribute to Exacerbated IL-17 Production in Response to Co-Infection with Bovine RSV and Mannheimia haemolytica.

Human respiratory syncytial virus (HRSV) is a leading cause of severe lower respiratory tract infection in children under five years of age. IL-17 and Th17 responses are increased in children infected with HRSV and have been implicated in both protective and pathogenic roles during infection. Bovine RSV (BRSV) is genetically closely related to HRSV and is a leading cause of severe respiratory infections in young cattle. While BRSV infection in the calf parallels many aspects of human infection with HRSV, IL-17 and Th17 responses have not been studied in the bovine. Here we demonstrate that calves infected with BRSV express significant levels of IL-17, IL-21 and IL-22; and both CD4 T cells and γδ T cells contribute to this response. In addition to causing significant morbidity from uncomplicated infections, BRSV infection also contributes to the development of bovine respiratory disease complex (BRDC), a leading cause of morbidity in both beef and dairy cattle. BRDC is caused by a primary viral infection, followed by secondary bacterial pneumonia by pathogens such as Mannheimia haemolytica. Here, we demonstrate that in vivo infection with M. haemolytica results in increased expression of IL-17, IL-21 and IL-22. We have also developed an in vitro model of BRDC and show that co-infection of PBMC with BRSV followed by M. haemolytica leads to significantly exacerbated IL-17 production, which is primarily mediated by IL-17-producing γδ T cells. Together, our results demonstrate that calves, like humans, mount a robust IL-17 response during RSV infection; and suggest a previously unrecognized role for IL-17 and γδ T cells in the pathogenesis of BRDC

Primers used for qPCR.

<p>Primers used for qPCR.</p

WC1.1⁺ γδ T cells produce IL-17 in response to BRSV.

<p>γδ T cells were purified from the peripheral blood of BRSV vaccinated or control animals by FACS based upon their expression of the γδ T cell receptor and either expression of WC1.1, WC1.2 or lack of WC1. Cells were cultured in the presence of autologous APC ± BRSV for 6 days. Cell culture supernatants were then analyzed by ELISA for IL-17. (B) Cell culture supernatants from (A) were also diluted 1:1 and added to BT cells for 24 hours. After 24 hours, BT were analyzed by qPCR for expression of IL-8. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results are pooled from two independent experiments. Data represent means ± SEM.</p

Cell culture supernatants from BRSV stimulated PBMC induce expression of IL-8, MUC5B and MUC5AC by BT cells.

<p>PBMC from control (nonvaccinated) and BRSV vaccinated cows were isolated and stimulated with heat-killed BRSV for 6 days as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151083#pone.0151083.g002" target="_blank">Fig 2</a>. Cell culture supernatants were then diluted 1:1 in cMEM and added to confluent BT cells in 96 well plates for 24 hours. RNA was the isolated from the BT and analyzed by qPCR for expression of IL-8 (A), MUC5B (B) and MUC5AC (C). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.</p

IL-17 and Th17 responses from BRSV vaccinated cattle.

<p>Peripheral blood was collected from cows receiving annual vaccinations with a multivalent vaccine containing live-attenuated BRSV (n = 8), or from control cows that were not included in the vaccination program due to inclusion in another study (n = 6). PMBC were isolated and stimulated with BRSV for 24 hours or 6 days, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151083#pone.0151083.g001" target="_blank">Fig 1</a>. RNA was isolated from the cells and analyzed by qPCR for expression of IL-17 (A, left panel), IL-21 (B) and IL-22 (C). Cell culture supernatants were also analyzed by ELISA for IL-17 (A, right panel). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. Results were pooled from two independent experiments. Data represent means ± SEM.</p

Both CD4 T cells and γδ T cells produce IL-17 in response to BRSV.

<p>PBMC were isolated from control or BRSV vaccinated cows and labeled with Cell Trace Violet. Cells were then cultured for 6 days with BRSV. On day 6, CD4 T cells (A) and γδ T cells (B) were analyzed for virus-specific proliferation as measured by Cell Trace Violet dilution. Representative flow plots are shown in A and B. Aggregate results are shown in C. (D) CD4 T cells and γδ T cells from BRSV vaccinated or nonvaccinated animals were isolated by MACS and cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. (E) CD4 T cells and γδ T cells were MACS purified from peripheral blood of calves infected or not with BRSV strain 375 for 7 days. Purified cells were cultured in the presence of autologous APC ± BRSV. After 6 days, cell culture supernatants were analyzed by ELISA for IL-17. For A-C, background levels of proliferation were subtracted and results are presented as change over mock. Results are pooled from two independent experiments. Data represent means ± SEM.</p

IL-17 and Th17 responses in the lungs and peripheral blood of calves infected with BRSV.

<p>Calves (n = 8) were infected via aerosol inoculation with BRSV Strain 375 (RSV) as described in Materials and Methods. Control calves remained uninfected (n = 8). On day 7 post-infection, the animals were sacrificed and the lungs analyzed by qPCR for expression of IL-17, IL-21 and IL-22 (A) and IL-8 (B). In separate experiments, PBMC were isolated from control (n = 8) and BRSV infected calves (n = 8) on day 7-post infection. PBMC were stimulated with BRSV for 24 hours and then analyzed for expression of IL-17 by qPCR (C); or for 6 days and then cell culture supernatants were analyzed by ELISA for expression of IL-17 (D). For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to samples from uninfected control calves. Results were pooled from a total of three independent experiments. Data represent means ± SEM.</p