Assessment of the Effectiveness of Team-based Learning Activities on Learning Outcomes in the Undergraduate Immunology Classroom.

Immunology is inherently interdisciplinary. Understanding how the immune system functions requires knowledge from several scientific disciplines, including molecular biology, cellular biology, genetics, and biochemistry. Furthermore, immunology is conceptually complex, requiring the identification of a plethora of immune components and mastery of a large volume of new vocabulary. These attributes can pose challenges to student learning in the undergraduate immunology classroom. Team-based learning (TBL) is a pedagogical method used to increase student engagement in learning, improve student collaboration, and develop communication skills. In a variety of educational settings, TBL activities have been shown to foster a deeper understanding of complex topics, increase student confidence in course content, and improve learning outcomes. In this study, we examined differences in the impact of traditional lecture versus TBL activities on student learning outcomes for four different topics presented in an undergraduate adaptive immunity course composed largely of academically high-performing students. We matched content across two student cohorts, delivered via team-based learning methodology (T cell development and Ab-mediated functions) and traditional lecture (B cell development and T cell effector functions). Student learning was assessed using content questions across a range of Bloom's taxonomy levels, which demonstrated that the TBL activities did not improve examination performance over lecture-based learning in this course. However, students found this learning tool to be valuable, indicating that the TBL activities assisted with preparation for examinations and provided a necessary opportunity to address misconceptions.

An oral commensal attenuates Pseudomonas aeruginosa-induced airway inflammation and modulates nitrite flux in respiratory epithelium.

IMPORTANCE: Respiratory infections are a leading cause of morbidity and mortality in people with cystic fibrosis (CF). These infections are polymicrobial in nature with overt pathogens and other colonizing microbes present. Microbiome data have indicated that the presence of oral commensal bacteria in the lungs is correlated with improved outcomes. We hypothesize that one oral commensal, Streptococcus parasanguinis, inhibits CF pathogens and modulates the host immune response. One major CF pathogen is Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterium with intrinsic drug resistance and an arsenal of virulence factors. We have previously shown that S. parasanguinis inhibits P. aeruginosa in vitro in a nitrite-dependent manner through the production of reactive nitrogen intermediates. In this study, we demonstrate that while this mechanism is evident in a cell culture model of the CF airway, an alternative mechanism by which S. parasanguinis may improve outcomes for people with CF is through immunomodulation.

Commensal colonization reduces Pseudomonas aeruginosa burden and subsequent airway damage.

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Interestingly, oral streptococcal colonization has been associated with stable CF lung function. The most abundant streptococcal species found in stable patients, Streptococcus salivarius, has been shown to downregulate pro-inflammatory cytokines in multiple colonization models. However, no studies have demonstrated how S. salivarius potentially improves lung function. Our lab previously demonstrated that the P. aeruginosa exopolysaccharide Psl promotes S. salivarius biofilm formation in vitro, suggesting a possible mechanism by which S. salivarius is incorporated into the CF airway microbial community. In this study, we demonstrate that co-infection of rats leads to enhanced S. salivarius colonization and reduced P. aeruginosa colonization. Histological scores for tissue inflammation and damage are lower in dual-infected rats compared to P. aeruginosa infected rats. Additionally, pro-inflammatory cytokines IL-1ß, IL-6, CXCL2, and TNF-α are downregulated during co-infection compared to P. aeruginosa single-infection. Lastly, RNA sequencing of cultures grown in synthetic CF sputum revealed that P. aeruginosa glucose metabolism genes are downregulated in the presence of S. salivarius, suggesting a potential alteration in P. aeruginosa fitness during co-culture. Overall, our data support a model in which S. salivarius colonization is promoted during co-infection with P. aeruginosa, whereas P. aeruginosa airway bacterial burden is reduced, leading to an attenuated host inflammatory response.

Oral Commensal Streptococci: Gatekeepers of the Oral Cavity.

Oral commensal streptococci are primary colonizers of the oral cavity. These streptococci produce many adhesins, metabolites, and antimicrobials that modulate microbial succession and diversity within the oral cavity. Often, oral commensal streptococci antagonize cariogenic and periodontal pathogens such as Streptococcus mutans and Porphyromonas gingivalis, respectively. Mechanisms of antagonism are varied and range from the generation of hydrogen peroxide, competitive metabolite scavenging, the generation of reactive nitrogen intermediates, and bacteriocin production. Furthermore, several oral commensal streptococci have been shown to alter the host immune response at steady state and in response to oral pathogens. Collectively, these features highlight the remarkable ability of oral commensal streptococci to regulate the structure and function of the oral microbiome. In this review, we discuss mechanisms used by oral commensal streptococci to interact with diverse oral pathogens, both physically and through the production of antimicrobials. Finally, we conclude by exploring the critical roles of oral commensal streptococci in modulating the host immune response and maintaining health and homeostasis.

Piecing Complement Together with LEGO Bricks: Impacts on Interest, Confidence, and Learning in the Immunology Classroom.

Teaching and learning complex molecular cascades can often be challenging. In immunology, students struggle to visualize immunological processes, such as activation of the complement system, which involves three separate cascades leading to multiple effector functions. Offering learning activities that use tangible modeling can help students learn conceptually difficult content by fostering a visual understanding of concepts, as well as instill confidence and interest in the material. In this article, we describe a learning activity using LEGO bricks that demonstrates the activation of the classical, lectin, and alternative complement pathways and formation of the membrane attack complex. In both an introductory and advanced immunology course, we investigated the effect of the activity on student learning and subject confidence. Performance on examination questions about complement demonstrated that the LEGO activity improved learning in a naive student population (students in introductory immunology), but not in a previously informed student population (students in advanced immunology). In addition, self-reported confidence in the content was significantly higher in students who completed the LEGO activity in the advanced course, but not the introductory course, compared with those who did not do the activity. Students in both courses who did the activity had a positive perception of the activity, with a majority of students reporting that they enjoyed the activity and had more interest in the complement system.

A Commensal Streptococcus Dysregulates the Pseudomonas aeruginosa Nitrosative Stress Response.

Chronic infections in the cystic fibrosis (CF) airway are composed of both pathogenic and commensal bacteria. However, chronic Pseudomonas aeruginosa infections are the leading cause of lung deterioration in individuals with CF. Interestingly, oral commensals can translocate to the CF lung and their presence is associated with improved lung function, presumably due to their ability to antagonize P. aeruginosa. We have previously shown that one commensal, Streptococcus parasanguinis, produces hydrogen peroxide that reacts with nitrite to generate reactive nitrogen intermediates (RNI) which inhibit P. aeruginosa growth. In this study, we sought to understand the global impact of commensal-mediated RNI on the P. aeruginosa transcriptome. RNA sequencing analysis revealed that S. parasanguinis and nitrite-mediated RNI dysregulated expression of denitrification genes in a CF isolate of P. aeruginosa compared to when this isolate was only exposed to S. parasanguinis. Further, loss of a nitric oxide reductase subunit (norB) rendered an acute P. aeruginosa isolate more susceptible to S. parasanguinis-mediated RNI. Additionally, S. parasanguinis-mediated RNI inactivated P. aeruginosa aconitase activity. Lastly, we report that P. aeruginosa isolates recovered from CF individuals are uniquely hypersensitive to S. parasanguinis-mediated RNI compared to acute infection or environmental P. aeruginosa isolates. These findings illustrate that S. parasanguinis hinders the ability of P. aeruginosa to respond to RNI, which potentially prevents P. aeruginosa CF isolates from resisting commensal and host-induced RNI in the CF airway.

Pseudomonas aeruginosa polysaccharide Psl supports airway microbial community development.

Pseudomonas aeruginosa dominates the complex polymicrobial cystic fibrosis (CF) airway and is a leading cause of death in persons with CF. Oral streptococcal colonization has been associated with stable CF lung function. However, no studies have demonstrated how Streptococcus salivarius, the most abundant streptococcal species found in individuals with stable CF lung disease, potentially improves lung function or becomes incorporated into the CF airway biofilm. By utilizing a two-species biofilm model to probe interactions between S. salivarius and P. aeruginosa, we discovered that the P. aeruginosa exopolysaccharide Psl promoted S. salivarius biofilm formation. Further, we identified a S. salivarius maltose-binding protein (MalE) that is required for promotion of biofilm formation both in vitro and in a Drosophila melanogaster co-infection model. Finally, we demonstrate that promotion of dual biofilm formation with S. salivarius is common among environmental and clinical P. aeruginosa isolates. Overall, our data supports a model in which S. salivarius uses a sugar-binding protein to interact with P. aeruginosa exopolysaccharide, which may be a strategy by which S. salivarius establishes itself within the CF airway microbial community.

Nitrite Triggers Reprogramming of the Oral Polymicrobial Metabolome by a Commensal Streptococcus.

Commensal streptococci regulate health and homeostasis within oral polymicrobial communities. Remarkably, high salivary nitrite concentrations have also been associated with improved health in the oral cavity. We previously demonstrated that nitrite assists hydrogen peroxide-producing oral commensal streptococci in regulating homeostasis via the generation of reactive nitrogen species (RNS), which have antimicrobial activity on oral pathogens. However, it is unknown how nitrite and commensal streptococci work in concert to influence the metabolome of oral polymicrobial communities. In this study, we report that nitrite aids commensal streptococci in the inhibition of multi-kingdom pathogens that reside in distinct oral niches, which supports commensal dominance. More importantly, we show that commensal streptococci utilize nitrite to drive the metabolic signature of multispecies biofilms in a manner that supports commensal metabolism and resistance to RNS, and restricts metabolic processes that are required for pathogen virulence. Taken together, our study provides insight into how commensal streptococci use nitrite to trigger shifts in the oral polymicrobial metabolome to support health and homeostasis.

Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells.

Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30-90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16-65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 (P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction (P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1-10 µM Y-27632 (P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.