Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

Human intelectin-1 (hIntL-1) is a secreted glycoprotein capable of binding exocyclic 1,2-diols within surface glycans of human pathogens such as Streptococcus pneumoniae , Vibrio cholerae , and Helicobacter pylori . For the latter, lectin binding was shown to cause bacterial agglutination and increased phagocytosis, suggesting a role for hIntL-1 in pathogen surveillance.

Seasonal influenza epidemics represent a significant global health threat. The exacerbated immune response triggered by respiratory influenza virus infection causes severe pulmonary damage and contributes to substantial morbidity and mortality. Regulator of G-protein signaling 10 (RGS10) belongs to the RGS protein family that act as GTPase activating proteins for heterotrimeric G proteins to terminate signaling pathways downstream of G protein-coupled receptors. While RGS10 is highly expressed in immune cells, in particular monocytes and macrophages, where it has strong anti-inflammatory effects, its physiological role in the respiratory immune system has not been explored yet. Here, we show that Rgs10 negatively modulates lung immune and inflammatory responses associated with severe influenza H1N1 virus respiratory infection in a mouse model. In response to influenza A virus challenge, mice lacking RGS10 experience enhanced weight loss and lung viral titers, higher mortality and significantly faster disease onset. Deficiency of Rgs10 upregulates the levels of several proinflammatory cytokines and chemokines and increases myeloid leukocyte accumulation in the infected lung, markedly neutrophils, monocytes, and inflammatory monocytes, which is associated with more pronounced lung damage. Consistent with this, influenza-infected Rgs10-deficent lungs contain more neutrophil extracellular traps and exhibit higher neutrophil elastase activities than wild-type lungs. Overall, these findings propose a novel, in vivo role for RGS10 in the respiratory immune system controlling myeloid leukocyte infiltration, viral clearance and associated clinical symptoms following lethal influenza challenge. RGS10 also holds promise as a new, potential therapeutic target for respiratory infections.

Francisella tularensis (F. tularensis) is a facultative intracellular, Gram-negative bacterium that causes a fatal disease known as tularemia. Due to its extremely high virulence, ease of spread by aerosolization, and the potential to be used as a bioterror agent, F. tularensis is classified by the CDC as a Tier 1 Category A Select Agent. Previous studies have demonstrated the roles of inflammasome sensors; absent in melanoma 2 (AIM2) and NLRP3, in the generation of innate immune responses to F. tularensis infection. However, contributions of both the AIM2 and NLRP3 in the development of vaccine-induced adaptive immune responses against F. tularensis are not known. This study determined the contributions of Aim2 and Nlrp3-inflammasome sensors in vaccine-induced immune responses in a mouse model of respiratory tularemia. We developed a model to vaccinate the Aim2 and Nlrp3-deficient mice (Aim2-/- and Nlrp3-/-) using the emrA1 mutant of F. tularensis live vaccine strain (LVS). The results demonstrate that the innate immune responses in Aim2-/- and Nlrp3-/- mice vaccinated with the emrA1 mutant differ from their wild-type counterparts. However, despite these differences in the innate immune responses, both Aim2-/- and Nlrp3-/- mice are fully protected against an intranasal lethal challenge dose of F. tularensis LVS. Moreover, the lack of both Aim2 and Nlrp3 inflammasome sensors does not affect the production of the vaccination-induced antibody and cell-mediated responses. Overall, this study reports a novel finding that both Aim2 and Nlrp3 are dispensable for vaccination-induced immunity against respiratory tularemia caused by F. tularensis.

IntroductionMycobacterium tuberculosis (Mtb) is the primary cause of human tuberculosis (TB) and is currently the second most common cause of death due to a singleinfectious agent. The first line of defense against airborne pathogens, including Mtb, is the respiratory epithelium. One of the innate defenses used by respiratory epithelial cells to prevent microbial infection is an oxidative antimicrobial system consisting of the proteins, lactoperoxidase (LPO) and Dual oxidase 1 (Duox1), the thiocyanate anion (SCN-) and hydrogen peroxide (H2O2), which together lead to the generation of antimicrobial hypothiocyanite (OSCN-) in the airway lumen. OSCN- kills bacteria and viruses in vitro, but the role of this Duox1-based system in bacterial infections in vivo remains largely unknown. The goal of this study was to assess whether Duox1 contributes to the immune response against the unique respiratory pathogen, Mtb.MethodsDuox1-deficient (Duox1 KO) and wild-type (WT) mice were infected with Mtb aerosols and bacterial titers, lung pathology, cytokines and immune cell recruitment were assessed.Results and discussionMtb titers in the lung, spleen and liver were not different 30 days after infection between WT and Duox1 KO mice. Duox1 did not affect lung histology assessed at days 0, 30, and 90 post-Mtb infection. Mtb-infected Duox1 KO animals exhibited enhanced production of certain cytokines and chemokines in the airway; however, this response was not associated with significantly higher numbers of macrophages or neutrophils in the lung. B cell numbers were lower, while apoptosis was higher in the pulmonary lesions of Mtb-infected Duox1 KO mice compared to infected WT animals. Taken together, these data demonstrate that while Duox1 might influence leukocyte recruitment to inflammatory cell aggregates, Duox1 is dispensable for the overall clinical course of Mtb lung infection in a mouse model.

OBJECTIVES/GOALS: Ecotin is a potent periplasmic serin protease inhibitor that was first described in Escherichia coli (E. coli). Ecotin is known to inhibit a broad range of serine proteases including NE. Therefore, we hypothesized that ecotin can inhibit the NE activity present in CF clinical sputum samples. METHODS/STUDY POPULATION: To investigate the function of ecotin on CF sputum, orthologues from Campylobacter rectus and Campylobacter showae were recombinantly expressed and purified from E. coli to generate C. rectus ecotin, C. showae ecotin, and E. coli ecotin. Ecotin was added to CF sputum supernatant samples at various concentrations and NE enzymatic activity was measured by a fluorometric assay kit. In addition, CF sputum and healthy sputum was added to PMNs isolated from healthy donors for 3.5 hours and NE was measured with immunofluorescence staining. The sputum was washed two times prior to measuring NE released and on the PMNs, and PBMCs was used as a control. RESULTS/ANTICIPATED RESULTS: Our results indicate a clear inhibition of NE activity in CF sputum supernatant. C. showae ecotin showed the greatest inhibitory effect on NE activity in CF sputum supernatant. We also saw that CF sputum supernatant causing healthy PMNs to release more NE suggesting that NE in the CF airway may trigger more NE release from newly incoming PMNs to the lung. Our next steps will be to determine if ecotin can inhibit NE on the PMN surface and released from PMNs. DISCUSSION/SIGNIFICANCE: Neutrophil elastases are produced by PMNs to kill microbial pathogens in the lung, however in CF, the pathogens are unable to be killed by NE and instead causes severe lung damage. PMNs and NE has been shown to be elevated in the sputum of CF patients and there is currently no NE therapeutic inhibitor that has been effective in these patients.

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