Sanmei Hu

Gonadal hormones and oxidative stress interaction differentially affects survival of male and female mice after lung Klebsiella Pneumoniae infection

Survival of mice after Klebsiella pneumoniae infection and phagocytosis by alveolar macrophages (AMs), in the presence or absence of ozone (O3) exposure prior to infection, is sex dependent. The objective of this work was to study the role of gonadal hormones, 5α-dihydrotestosterone (DHT) and 17β-estradiol (E2), on mouse survival after filtered air (FA) or O3 exposure. Gonadectomized female (G×F) and male (G×M) mice implanted with control or hormone pellets (DHT in G×F, or E2 in G×M), exposed to O3 (2 ppm, 3h) or FA, and infected with K. pneumoniae were monitored for survival. Survival in G×F was identical after FA or O3 exposure; in G×M O3 exposure resulted in lower survival compared to FA. In O3-exposed females, gonadectomy resulted in increased survival compared to intact females or to G×M+E2. A similar effect was observed in G×F+DHT. The combined negative effect of oxidative stress and hormone on survival was higher for E2. Gonadectomy eliminated (females) or minimized (males) the previously observed sex differences in survival in response to oxidative stress, and hormone treatment restored them. These findings indicate that gonadal hormones and/or oxidative stress have a significant effect on mouse survival.

Impact of sex and ozone exposure on the course of pneumonia in wild type and SP-A (−/−) mice

Female mice exhibited higher survival rate than males after pneumonia, with a reversal of this pattern following ozone exposure. Surfactant protein A (SP-A) plays an important role in innate immunity and SP-A (-/-) mice were more susceptible to pneumonia than wild type mice. Here, we investigated underlying mechanisms of the differential susceptibility of mice to pneumonia. Wild type and SP-A (-/-) C57BL/6J male and female mice were exposed to ozone or filtered air (FA) and then infected intratracheally with Klebsiella pneumoniae. Blood, spleen, and lung were analyzed for bacterial counts, lung and spleen weights, and sex hormone and cortisol levels were measured in plasma within two days post-infection. We found: 1) in the absence of ozone-induced oxidative stress, males had higher level of bacterial dissemination compared to females; ozone exposure decreased pulmonary clearance in both sexes and ozone-exposed females were more affected than males; 2) ozone exposure increased lung weight, but decreased spleen weight in both sexes, and in both cases ozone-exposed females were affected the most; 3) plasma cortisol levels in infected mice changed: ozone-exposed>FA-exposed, females>males, and infected>non-infected; 4) no major sex hormone differences were observed in the studied conditions; 5) differences between wild type and SP-A (-/-) mice were observed in some of the studied conditions. We concluded that reduced pulmonary clearance, compromised spleen response to infection, and increased cortisol levels in ozone-exposed females, and the higher level of lung bacterial dissemination in FA-exposed males, contribute to the previously observed survival outcomes.

Differences in the alveolar macrophage proteome in transgenic mice expressing human SP-A1 and SP-A2.

Surfactant protein A (SP-A) plays a number of roles in lung host defense and innate immunity. There are two human genes, SFTPA1 and SFTPA2, and evidence indicates that the function of SP-A1 and SP-A2 proteins differ in several respects. To investigate the impact of SP-A1 and SP-A2 on the alveolar macrophage (AM) phenotype, we generated humanized transgenic (hTG) mice on the SP-A knockout (KO) background, each expressing human SP-A1 or SP-A2. Using two-dimensional difference gel electrophoresis (2D-DIGE) we studied the AM cellular proteome. We compared mouse lines expressing high levels of SPA1, high levels of SP-A2, low levels of SP-A1, and low levels of SP-A2, with wild type (WT) and SP-A KO mice. AM from mice expressing high levels of SP-A2 were the most similar to WT mice, particularly for proteins related to actin and the cytoskeleton, as well as proteins regulated by Nrf2. The expression patterns from mouse lines expressing higher levels of the transgenes were almost the inverse of one another - the most highly expressed proteins in SP-A2 exhibited the lowest levels in the SP-A1 mice and vice versa. The mouse lines where each expressed low levels of SP-A1 or SP-A2 transgene had very similar protein expression patterns suggesting that responses to low levels of SP-A are independent of SP-A genotype, whereas the responses to higher amounts of SP-A are genotype-dependent. Together these observations indicate that in vivo exposure to SP-A1 or SP-A2 differentially affects the proteomic expression of AMs, with SP-A2 being more similar to WT.

SP-R210 (Myo18A) Isoforms as Intrinsic Modulators of Macrophage Priming and Activation

The surfactant protein (SP-A) receptor SP-R210 has been shown to increase phagocytosis of SP-A-bound pathogens and to modulate cytokine secretion by immune cells. SP-A plays an important role in pulmonary immunity by enhancing opsonization and clearance of pathogens and by modulating macrophage inflammatory responses. Alternative splicing of the Myo18A gene results in two isoforms: SP-R210S and SP-R210L, with the latter predominantly expressed in alveolar macrophages. In this study we show that SP-A is required for optimal expression of SP-R210L on alveolar macrophages. Interestingly, pre-treatment with SP-A prepared by different methods either enhances or suppresses responsiveness to LPS, possibly due to differential co-isolation of SP-B or other proteins. We also report that dominant negative disruption of SP-R210L augments expression of receptors including SR-A, CD14, and CD36, and enhances macrophages’ inflammatory response to TLR stimulation. Finally, because SP-A is known to modulate CD14, we used a variety of techniques to investigate how SP-R210 mediates the effect of SP-A on CD14. These studies revealed a novel physical association between SP-R210S, CD14, and SR-A leading to an enhanced response to LPS, and found that SP-R210L and SP-R210S regulate internalization of CD14 via distinct macropinocytosis-like mechanisms. Together, our findings support a model in which SP-R210 isoforms differentially regulate trafficking, expression, and activation of innate immune receptors on macrophages.

SH3GLB2/endophilin B2 regulates lung homeostasis and recovery from severe influenza A virus infection

New influenza A viruses that emerge frequently elicit composite inflammatory responses to both infection and structural damage of alveolar-capillary barrier cells that hinders regeneration of respiratory function. The host factors that relinquish restoration of lung health to enduring lung injury are insufficiently understood. Here, we investigated the role of endophilin B2 (B2) in susceptibility to severe influenza infection. WT and B2-deficient mice were infected with H1N1 PR8 by intranasal administration and course of influenza pneumonia, inflammatory, and tissue responses were monitored over time. Disruption of B2 enhanced recovery from severe influenza infection as indicated by swift body weight recovery and significantly better survival of endophilin B2-deficient mice compared to WT mice. Compared to WT mice, the B2-deficient lungs exhibited induction of genes that express surfactant proteins, ABCA3, GM-CSF, podoplanin, and caveolin mRNA after 7 days, temporal induction of CCAAT/enhancer binding protein CEBPα, β, and δ mRNAs 3–14 days after infection, and differences in alveolar extracellular matrix integrity and respiratory mechanics. Flow cytometry and gene expression studies demonstrated robust recovery of alveolar macrophages and recruitment of CD4+ lymphocytes in B2-deficient lungs. Targeting of endophilin B2 alleviates adverse effects of IAV infection on respiratory and immune cells enabling restoration of alveolar homeostasis.

An Electrochemical Biosensor for Rapid Detection of Pediatric Bloodstream Infections

Bloodstream infections are major contributing factors of morbidity and mortality among children. Precise and timely identification of causative agents can improve the clinical management and outcome of the infection, potentially saving lives. Electrochemical biosensors previously described by Gao et al. (2017) have the potential to deliver greater speed and discrimination. However, to date there are no data that determine whether the age of the host would cause bacteria to demonstrate different growth characteristics, or whether pediatric samples would behave differently using this electrochemical biosensor. The importance of this knowledge gap is clear: the preclinical testing phase of this line of research is limited by the relative lack of pediatric healthy blood volunteers to complete this work. Therefore, in this study we have applied this novel technology to diagnose bacteria spiked into pediatric blood and compared directly with adult blood samples. Only 180 µL of blood was utilized from both adult and pediatric volunteers and inoculated with Escherichia coli 67, and the signals generated at different time points were compared. We were able to demonstrate that the signals generated by adult and pediatric blood were not significantly different with this detection technology.