Browsing by Author "Rynda-Apple, Agnieszka"

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A Comprehensive Protocol for the Collection, Differentiation, Cryopreservation, and Resuscitation of Primary Murine Bone Marrow Derived Macrophages (BMDM)
(Informa UK Limited, 2024-08) Luu, Abby M.; Shepardson, Kelly M.; Rynda-Apple, Agnieszka
Background. The field of immunology has undoubtedly benefited from the in vitro use of cell lines for immunological studies; however, due to the “immortal” nature of many cell lines, they are not always the best model. Thus, direct collection and culture of primary cells from model organisms is a solution that many researchers utilize. To the best of our knowledge, there is not a singular protocol which encompasses the entire process of bone marrow cell collection through cryopreservation and resuscitation of cells from a murine model. Methods. Bone marrow cells were collected from mice with a C57BL6 genetic background. Cells were differentiated using L929 conditioned media. Cells were assessed using a combination of microscopy, differential staining, immunocytochemistry, and trypan blue. Results: Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Results. Primary murine BMDMs that underwent cryopreservation followed by resuscitation retained a high degree of viability. Furthermore, these BMDMs retained on overall ability to clear S. aureus. Conclusion. Crypopreserved and resuscitated primary murine BMDMs were viable and retained their pverall S. aureus clearance ability.
An ADAM17-Neutralizing Antibody Reduces Inflammation and Mortality While Increasing Viral Burden in a COVID-19 Mouse Model
(Frontiers Media SA, 2022-06) Hedges, Jodi F.; Snyder, Deann T.; Robison, Amanda; Grifka-Walk, Heather M.; Blackwell, Karlin; Shepardson, Kelly; Kominsky, Douglas; Rynda-Apple, Agnieszka; Walcheck, Bruce; Jutila, Mark A.
Angiotensin Converting Enzyme 2 (ACE2) is the primary cell entry receptor for SARS-CoV and SARS-CoV-2 viruses. A disintegrin and metalloproteinase 17 (ADAM17) is a protease that cleaves ectodomains of transmembrane proteins, including that of ACE2 and the proinflammatory cytokine TNF-α, from cell surfaces upon cellular activation. We hypothesized that blockade of ADAM17 activity would alter COVID-19 pathogenesis. To assess this pathway, we blocked the function of ADAM17 using the monoclonal antibody MEDI3622 in the K18-hACE2 transgenic mouse model of COVID-19. Antibody-treated mice were healthier, less moribund, and had significantly lower lung pathology than saline-treated mice. However, the viral burden in the lungs of MEDI3622-treated mice was significantly increased. Thus, ADAM17 appears to have a critical anti-viral role, but also may promote inflammatory damage. Since the inflammatory cascade is ultimately the reason for adverse outcomes in COVID-19 patients, there may be a therapeutic application for the MEDI3622 antibody.
IFNAR2 Is Required for Anti-influenza Immunity and Alters Susceptibility to Post-influenza Bacterial Superinfections
(2018-11) Shepardson, Kelly M.; Larson, Kyle; Johns, Laura L.; Stanek, Kayla; Cho, Hanbyul; Wellham, Julia; Henderson, Haley; Rynda-Apple, Agnieszka
Influenza virus infections particularly when followed by bacterial superinfections (BSI) result in significant morbidities and mortalities especially during influenza pandemics. Type I interferons (IFNs) regulate both anti-influenza immunity and host susceptibility to subsequent BSIs. These type I IFNs consisting of, among others, 14 IFN-α's and a single IFN-β, are recognized by and signal through the heterodimeric type I IFN receptor (IFNAR) comprised of IFNAR1 and IFNAR2. However, the individual receptor subunits can bind IFN-β or IFN-α's independently of each other and induce distinct signaling. The role of type I IFN signaling in regulating host susceptibility to both viral infections and BSI has been only examined with respect to IFNAR1 deficiency. Here, we demonstrate that despite some redundancies, IFNAR1 and IFNAR2 have distinct roles in regulating both anti-influenza A virus (IAV) immunity and in shaping host susceptibility to subsequent BSI caused by S. aureus. We found IFNAR2 to be critical for anti-viral immunity. In contrast to Ifnar1−/− mice, IAV-infected Ifnar2−/− mice displayed both increased and accelerated morbidity and mortality compared to WT mice. Furthermore, unlike IFNAR1, IFNAR2 was sufficient to generate protection from lethal IAV infection when stimulated with IFN-β. With regards to BSI, unlike what we found previously in Ifnar1−/− mice, Ifnar2−/− mice were not susceptible to BSI induced on day 3 post-IAV, even though absence of IFNAR2 resulted in increased viral burden and an increased inflammatory environment. The Ifnar2−/− mice similar to what we previously found in Ifnar1−/− mice were less susceptible than WT mice to BSI induced on day 7 post-IAV, indicating that signaling through a complete receptor increases BSI susceptibility late during clinical IAV infection. Thus, our results support a role for IFNAR2 in induction of anti-IAV immune responses that are involved in altering host susceptibility to BSI and are essential for decreasing the morbidity and mortality associated with IAV infection. These results begin to elucidate some of the mechanisms involved in how the individual IFNAR subunits shape the anti-viral immune response. Moreover, our results highlight the importance of examining the contributions of entire receptors, as individual subunits can induce distinct outcomes as shown here.
Immune suppressive activity of myeloid-derived suppressor cells in cancer requires inactivation of the type I interferon pathway
(Springer Science and Business Media LLC, 2021-03) Alicea-Torres, Kevin; Sanseviero, Emilio; Gui, Jun; Chen, Jinyun; Veglia, Filippo; Yu, Qiujin; Donthireddy, Laxminarasimha; Kossenkov, Andrew; Lin, Cindy; Fu, Shuyu; Mulligan, Charles; Nam, Brian; Masters, Gregory; Denstman, Fred; Bennett, Joseph; Hockstein, Neil; Rynda-Apple, Agnieszka; Nefedova, Yulia; Fuchs, Serge Y.; Gabrilovich, Dmitry I.
Myeloid-derived suppressor cells (MDSC) are pathologically activated neutrophils and monocytes with potent immune suppressive activity. These cells play an important role in accelerating tumor progression and undermining the efficacy of anti-cancer therapies. The natural mechanisms limiting MDSC activity are not well understood. Here, we present evidence that type I interferons (IFN1) receptor signaling serves as a universal mechanism that restricts acquisition of suppressive activity by these cells. Downregulation of the IFNAR1 chain of this receptor is found in MDSC from cancer patients and mouse tumor models. The decrease in IFNAR1 depends on the activation of the p38 protein kinase and is required for activation of the immune suppressive phenotype. Whereas deletion of IFNAR1 is not sufficient to convert neutrophils and monocytes to MDSC, genetic stabilization of IFNAR1 in tumor bearing mice undermines suppressive activity of MDSC and has potent antitumor effect. Stabilizing IFNAR1 using inhibitor of p38 combined with the interferon induction therapy elicits a robust anti-tumor effect. Thus, negative regulatory mechanisms of MDSC function can be exploited therapeutically.
Virus-like Particle-Induced Protection against MRSA Pneumonia Is Dependent on IL-13 and Enhancement of Phagocyte Function
(2012-07) Rynda-Apple, Agnieszka; Dobrinen, Erin; McAlpine, Mark; Read, Amanda; Harmsen, Ann L.; Richert, Laura E.; Calverley, Matthew; Pallister, Kyler; Voyich, Jovanka M.; Wiley, James A.; Johnson, Ben; Young, Mark J.; Douglas, Trevor; Harmsen, Allen G.
The importance of the priming of the lung environment by past infections is being increasingly recognized. Exposure to any given antigen can either improve or worsen the outcome of subsequent lung infections, depending on the immunological history of the host. Thus, an ability to impart transient alterations in the lung environment in anticipation of future insult could provide an important novel therapy for emerging infectious diseases. In this study, we show that nasal administration of virus-like particles (VLPs) before, or immediately after, lethal challenge with methicillin-resistant Staphylococcus aureus (MRSA) of mice i) ensures complete recovery from lung infection and near absolute clearance of bacteria within 12 hours of challenge, ii) reduces host response-induced lung tissue damage, iii) promotes recruitment and efficient bacterial clearance by neutrophils and CD11c+ cells, and iv) protects macrophages from MRSA-induced necrosis. VLP-mediated protection against MRSA relied on innate immunity. Complete recovery occurred in VLP-dosed mice with severe combined immunodeficiency, but not in wild-type mice depleted of either Ly6G+ or CD11c+ cells. Early IL-13 production associated with VLP-induced CD11c+ cells was essential for VLP-induced protection. These results indicate that VLP-induced alteration of the lung environment protects the host from lethal MRSA pneumonia by enhancing phagocyte recruitment and killing and by reducing inflammation-induced tissue damage via IL-13–dependent mechanisms.