In response to complement anaphylatoxin peptides C3a and C5a, human vascular endothelial cells migrate and mediate the activation of B-cells and polarization of T-cells.

The vascular endothelium has been discovered in the past several years to be important in shaping the cellular immune response. During the immune response the vascular endothelium is constantly perturbed by biologically potent molecules, including the complement activation peptides, C3a and C5a. Despite the importance of C3a and C5a in inflammation and immunity, their role in modulating lymphocyte function via activation of vascular endothelial cells is unknown. Accordingly, we investigated the regulated expression of the C3a and C5a receptors (complement anaphylatoxin C3a receptor [C3aR] and complement anaphylatoxin C5a receptor 1 [C5aR1]) on human umbilical vascular endothelial cells (HUVECs) and examined how C3a or C5a activation of HUVECs affects the activation and polarization of lymphatic cells. Our findings demonstrated that C3a and C5a increase C3aR and C5aR1 expression by HUVECs as well as directing their cellular transmigration and spreading through transwell filters. Moreover, C3a- or C5a-stimulated endothelial cells: (1) caused activation of B-lymphoblasts with significant increase in Fas Ligand (CD95L) (FasL), CD69, and IL-R1 expression, and (2) skewed T-lymphoblast cells toward a Th1 subtype, (CD4+ /CCR5+ ) that correlated with significant increase of IFN-γ. Collectively, these data indicate that C3a and C5a signaling is important in the activation and polarization of lymphocytes as they traffic through the vascular endothelium during the immune response.

The Second Receptor for C5a, C5aR2, Is Detrimental to Mice during Systemic Infection with Listeria monocytogenes.

Infection with Listeria monocytogenes is acquired through ingestion of contaminated foods and may lead to systemic infection and possible death, with an overall 20% mortality rate. Our previous work using C5aR1-/- mice and C3aR-/- mice demonstrated that C5aR1 and C3aR both play powerful anti-inflammatory and prosurvival roles during systemic infection with L. monocytogenes In our current study, we have examined the role of the third anaphylatoxin receptor, C5aR2, in the host immune response to systemic L. monocytogenes infection. C5aR2-/- mice had significantly lower bacterial burdens in the spleens and livers on both day 1 and 3 postinfection compared with C5aR2+/+ mice. The decreased bacterial burdens in the C5aR2-/- mice correlated with less liver damage and with improved survival of CD4+ and CD8+ T cells in the spleen on day 3 postinfection compared with C5aR2+/+ mice. C5aR2-/- mice also produced significantly less G-CSF, IL-6, and MCP-1 in the serum, spleen, and liver on day 1 postinfection compared with C5aR2+/+ mice. C5aR2-/- and C5aR2+/+ mice produced similar amounts of IFN-γ in their spleens on day 1 postinfection. Purified naive splenocytes from C5aR2-/- mice produced significantly more IFN-γ and IL-12p70 during in vitro infection with L. monocytogenes compared with splenocytes from C5aR2+/+ mice in an NF-κB-dependent manner. Induction of IL-12 and IFN-γ early during infection with L. monocytogenes is protective to the host, and we believe this innate increased ability to produce more IL-12 and IFN-γ provided early protection to the C5aR2-/- mice.

Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-ß signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.

SARS-CoV-2 restructures host chromatin architecture.

Some viruses restructure host chromatin, influencing gene expression, with implications for disease outcome. Whether this occurs for SARS-CoV-2, the virus causing COVID-19, is largely unknown. Here we characterized the 3D genome and epigenome of human cells after SARS-CoV-2 infection, finding widespread host chromatin restructuring that features widespread compartment A weakening, A-B mixing, reduced intra-TAD contacts and decreased H3K27ac euchromatin modification levels. Such changes were not found following common-cold-virus HCoV-OC43 infection. Intriguingly, the cohesin complex was notably depleted from intra-TAD regions, indicating that SARS-CoV-2 disrupts cohesin loop extrusion. These altered 3D genome/epigenome structures correlated with transcriptional suppression of interferon response genes by the virus, while increased H3K4me3 was found in the promoters of pro-inflammatory genes highly induced during severe COVID-19. These findings show that SARS-CoV-2 acutely rewires host chromatin, facilitating future studies of the long-term epigenomic impacts of its infection.

Caveolin-1 deficiency protects from pulmonary fibrosis by modulating epithelial cell senescence in mice.

Idiopathic pulmonary fibrosis is associated with a decreased expression of caveolin-1 (cav-1), yet its role remains unclear. To investigate the role of cav-1, we induced pulmonary fibrosis in wild-type (WT) and cav-1-deficient (cav-1(-/-)) mice using intratracheal instillation of bleomycin. Contrary to expectations, significantly less collagen deposition was measured in tissue from cav-1(-/-) mice than in their WT counterparts, consistent with reduced mRNA expression of procollagen1a2 and procollagen3a1. Moreover, cav-1(-/-) mice demonstrated 77% less α-smooth muscle actin staining, suggesting reduced mesenchymal cell activation. Levels of pulmonary injury, assessed by tenascin-C mRNA expression and CD44v10 detection, were significantly increased at Day 21 after injury in WT mice, an effect significantly attenuated in cav-1(-/-) mice. The apparent protective effect against bleomycin-induced fibrosis in cav-1(-/-) mice was attributed to reduce cellular senescence and apoptosis in cav-1(-/-) epithelial cells during the early phase of lung injury. Reduced matrix metalloproteinase (MMP)-2 and MMP-9 expressions indicated a low profile of senescence-associated secretory phenotype (SASP) in the bleomycin-injured cav-1(-/-) mice. However, IL-6 and macrophage inflammatory protein 2 were increased in WT and cav-1(-/-) mice after bleomycin challenge, suggesting that bleomycin-induced inflammatory response substantiated the SASP pool. Thus, loss of cav-1 attenuates early injury response to bleomycin by limiting stress-induced cellular senescence/apoptosis in epithelial cells. In contrast, decreased cav-1 expression promotes fibroblast activation and collagen deposition, effects that may be relevant in later stages of reparative response. Hence, therapeutic strategies to modulate the expression of cav-1 should take into account cell-specific effects in the regenerative responses of the lung epithelium to injury.

The pneumococcal serine-rich repeat protein is an intra-species bacterial adhesin that promotes bacterial aggregation in vivo and in biofilms.

The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273-341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122-166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.

SARS-CoV-2 Infection: Host Response, Immunity, and Therapeutic Targets.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has resulted in a global pandemic with severe socioeconomic effects. Immunopathogenesis of COVID-19 leads to acute respiratory distress syndrome (ARDS) and organ failure. Binding of SARS-CoV-2 spike protein to human angiotensin-converting enzyme 2 (hACE2) on bronchiolar and alveolar epithelial cells triggers host inflammatory pathways that lead to pathophysiological changes. Proinflammatory cytokines and type I interferon (IFN) signaling in alveolar epithelial cells counter barrier disruption, modulate host innate immune response to induce chemotaxis, and initiate the resolution of inflammation. Here, we discuss experimental models to study SARS-CoV-2 infection, molecular pathways involved in SARS-CoV-2-induced inflammation, and viral hijacking of anti-inflammatory pathways, such as delayed type-I IFN response. Mechanisms of alveolar adaptation to hypoxia, adenosinergic signaling, and regulatory microRNAs are discussed as potential therapeutic targets for COVID-19.

Sine oculis homeobox homolog 1 plays a critical role in pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with limited treatment options. The role of the developmental transcription factor Sine oculis homeobox homolog 1 (SIX1) in the pathophysiology of lung fibrosis is not known. IPF lung tissue samples and IPF-derived alveolar type II cells (AT2) showed a significant increase in SIX1 mRNA and protein levels, and the SIX1 transcriptional coactivators EYA1 and EYA2 were elevated. Six1 was also upregulated in bleomycin-treated (BLM-treated) mice and in a model of spontaneous lung fibrosis driven by deletion of Telomeric Repeat Binding Factor 1 (Trf1) in AT2 cells. Conditional deletion of Six1 in AT2 cells prevented or halted BLM-induced lung fibrosis, as measured by a significant reduction in histological burden of fibrosis, reduced fibrotic mediator expression, and improved lung function. These effects were associated with increased macrophage migration inhibitory factor (MIF) in lung epithelial cells in vivo following SIX1 overexpression in BLM-induced fibrosis. A MIF promoter-driven luciferase assay demonstrated direct binding of Six1 to the 5'-TCAGG-3' consensus sequence of the MIF promoter, identifying a likely mechanism of SIX1-driven MIF expression in the pathogenesis of lung fibrosis and providing a potentially novel pathway for targeting in IPF therapy.

Fulminant lung fibrosis in non-resolvable COVID-19 requiring transplantation.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) can lead to the development of acute respiratory distress syndrome (ARDS). In some patients with non-resolvable (NR) COVID-19, lung injury can progress rapidly to the point that lung transplantation is the only viable option for survival. This fatal progression of lung injury involves a rapid fibroproliferative response and takes on average 15 weeks from initial symptom presentation. Little is known about the mechanisms that lead to this fulminant lung fibrosis (FLF) in NR-COVID-19. METHODS: Using a pre-designed unbiased PCR array for fibrotic markers, we analyzed the fibrotic signature in a subset of NR-COVID-19 lungs. We compared the expression profile against control lungs (donor lungs discarded for transplantation), and explanted tissue from patients with idiopathic pulmonary fibrosis (IPF). Subsequently, RT-qPCR, Western blots and immunohistochemistry were conducted to validate and localize selected pro-fibrotic targets. A total of 23 NR-COVID-19 lungs were used for RT-qPCR validation. FINDINGS: We revealed a unique fibrotic gene signature in NR-COVID-19 that is dominated by a hyper-expression of pro-fibrotic genes, including collagens and periostin. Our results also show a significantly increased expression of Collagen Triple Helix Repeat Containing 1(CTHRC1) which co-localized in areas rich in alpha smooth muscle expression, denoting myofibroblasts. We also show a significant increase in cytokeratin (KRT) 5 and 8 expressing cells adjacent to fibroblastic areas and in areas of apparent epithelial bronchiolization. INTERPRETATION: Our studies may provide insights into potential cellular mechanisms that lead to a fulminant presentation of lung fibrosis in NR-COVID-19. FUNDING: National Institute of Health (NIH) Grants R01HL154720, R01DK122796, R01DK109574, R01HL133900, and Department of Defense (DoD) Grant W81XWH2110032 to H.K.E. NIH Grants: R01HL138510 and R01HL157100, DoD Grant W81XWH-19-1-0007, and American Heart Association Grant: 18IPA34170220 to H.K.-Q. American Heart Association: 19CDA34660279, American Lung Association: CA-622265, Parker B. Francis Fellowship, 1UL1TR003167-01 and The Center for Clinical and Translational Sciences, McGovern Medical School to X.Y.

Biofilm and planktonic pneumococci demonstrate disparate immunoreactivity to human convalescent sera.

BACKGROUND: Streptococcus pneumoniae (the pneumococcus) is the leading cause of otitis media, community-acquired pneumonia (CAP), sepsis, and meningitis. It is now evident that S. pneumoniae forms biofilms during nasopharyngeal colonization; the former which facilitates persistence, the latter, a prerequisite for subsequent development of invasive disease. Proteomic evaluation of S. pneumoniae suggests the antigen profile available for host-recognition is altered as a consequence of biofilm growth. This has potentially meaningful implications in regards to adaptive immunity and protection from disseminated disease. We therefore examined the antigen profile of biofilm and planktonic pneumococcal cell lysates, tested their reactivity with human convalescent sera and that generated against biofilm pneumococci, and examined whether immunization with biofilm pneumococci protected mice against infectious challenge. RESULTS: Biofilm pneumococci have dramatically altered protein profiles versus their planktonic counterparts. During invasive disease the humoral immune response is skewed towards the planktonic protein profile. Immunization with biofilm bacteria does not elicit a strong-cross-reactive humoral response against planktonic bacteria nor confer resistance against challenge with a virulent isolate from another serotype. We identified numerous proteins, including Pneumococcal serine-rich repeat protein (PsrP), which may serve as a protective antigens against both colonization and invasive disease. CONCLUSION: Differential protein production by planktonic and biofilm pneumococci provides a potential explanation for why individuals remain susceptible to invasive disease despite previous colonization events. These findings also strongly suggest that differential protein production during colonization and disease be considered during the selection of antigens for any future protein vaccine.

SARS-CoV-2 Restructures the Host Chromatin Architecture.

SARS-CoV-2 has made >190-million infections worldwide, thus it is pivotal to understand the viral impacts on host cells. Many viruses can significantly alter host chromatin 1 , but such roles of SARS-CoV-2 are largely unknown. Here, we characterized the three-dimensional (3D) genome architecture and epigenome landscapes in human cells after SARS-CoV-2 infection, revealing remarkable restructuring of host chromatin architecture. High-resolution Hi-C 3.0 uncovered widespread A compartmental weakening and A-B mixing, together with a global reduction of intra-TAD chromatin contacts. The cohesin complex, a central organizer of the 3D genome, was significantly depleted from intra-TAD regions, supporting that SARS-CoV-2 disrupts cohesin loop extrusion. Calibrated ChIP-Seq verified chromatin restructuring by SARS-CoV-2 that is particularly manifested by a pervasive reduction of euchromatin modifications. Built on the rewired 3D genome/epigenome maps, a modified activity-by-contact model 2 highlights the transcriptional weakening of antiviral interferon response genes or virus sensors (e.g., DDX58 ) incurred by SARS-CoV-2. In contrast, pro-inflammatory genes (e.g. IL-6 ) high in severe infections were uniquely regulated by augmented H3K4me3 at their promoters. These findings illustrate how SARS-CoV-2 rewires host chromatin architecture to confer immunological gene deregulation, laying a foundation to characterize the long-term epigenomic impacts of this virus.

The Streptococcus pneumoniae adhesin PsrP binds to Keratin 10 on lung cells.

Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island-encoded adhesin that mediates attachment to lung cells. It is a member of the serine-rich repeat protein family and the largest bacterial protein known. PsrP production by S. pneumoniae was confirmed by immunoblotting and a truncated version of the protein was determined to be glycosylated. Using isogenic psrP mutants complemented with various PsrP constructs and competitive inhibition assays with recombinant proteins, we determined that PsrP requires an extended SRR2 domain for function and that adhesion is mediated through amino acids 273-341 of its basic region (BR) domain. Affinity chromatography, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), fluorescent-activated cell sorting (FACS) and immunofluorescent colocalization studies determined that PsrP binds to Keratin 10 (K10) on the surface of lung but not nasopharyngeal epithelial cells. Unglycosylated K10 bound to wild type but not psrP deficient pneumococci; suggesting that unlike other serine-rich repeat proteins, PsrP-mediated adhesion is independent of lectin activity. Finally, mice immunized with recombinant (r)PsrP(BR) had significantly less bacteria in their blood and improved survival versus controls following intranasal challenge. We conclude that the BR domain of PsrP binds to K10 in a lectin-independent manner, that K10 is expressed on lung cells and that vaccination with rPsrP(BR) is protective against pneumococcal disease.

Circadian Clock and Complement Immune System-Complementary Control of Physiology and Pathology?

Mammalian species contain an internal circadian (i.e., 24-h) clock that is synchronized to the day and night cycles. Large epidemiological studies, which are supported by carefully controlled studies in numerous species, support the idea that chronic disruption of our circadian cycles results in a number of health issues, including obesity and diabetes, defective immune response, and cancer. Here we focus specifically on the role of the complement immune system and its relationship to the internal circadian clock system. While still an incompletely understood area, there is evidence that dysregulated proinflammatory cytokines, complement factors, and oxidative stress can be induced by circadian disruption and that these may feed back into the oscillator at the level of circadian gene regulation. Such a feedback cycle may contribute to impaired host immune response against pathogenic insults. The complement immune system including its activated anaphylatoxins, C3a and C5a, not only facilitate innate and adaptive immune response in chemotaxis and phagocytosis, but they can also amplify chronic inflammation in the host organism. Consequent development of autoimmune disorders, and metabolic diseases associated with additional environmental insults that activate complement can in severe cases, lead to accelerated tissue dysfunction, fibrosis, and ultimately organ failure. Because several promising complement-targeted therapeutics to block uncontrolled complement activation and treat autoimmune diseases are in various phases of clinical trials, understanding fully the circadian properties of the complement system, and the reciprocal regulation by these two systems could greatly improve patient treatment in the long term.

Distinct roles of CD28- and CD40 ligand-mediated costimulation in the development of protective immunity and pathology during Chlamydia muridarum urogenital infection in mice.

Infection with Chlamydia muridarum in the mouse urogenital tract can induce both protective immunity and inflammatory pathologies, which has been used as a model for understanding the immune and pathogenic mechanisms of C. trachomatis infection. We compared the roles of CD28- and CD40 ligand (CD40L)-mediated costimulation in C. muridarum infection. Mice with CD28 or CD80/CD86 gene knockout (KO) displayed an infection course similar to that of wild-type mice during both primary and secondary infection, suggesting that CD28-mediated costimulation is not required for protection against C. muridarum infection. However, mice deficient in CD40L or CD40 displayed a prolonged infection course after primary or secondary infection, suggesting that CD40-CD40L costimulation plays an essential role in the development of anti-C. muridarum immunity. Interestingly, the CD28- or CD80/CD86-deficient mice displayed significantly lower levels of inflammatory pathologies in the upper genital tracts after primary infection, although the attenuation in inflammation was no longer significant during secondary infection. However, the CD40L or CD40 KO mice developed inflammatory pathologies as severe as those in wild-type mice following either primary or secondary infection despite the obvious deficits in adaptive immunity in these KO mice. The resistance of CD28 or CD80/CD86 KO mice to chlamydial infection correlated with production of gamma interferon, while the development of inflammatory pathologies in CD40L or CD40 KO mice correlated with the production of other proinflammatory cytokines in mouse urogenital tracts during the early stages of the infection. These observations together suggest that C. muridarum-induced protective immunity and inflammatory pathologies can be mediated by distinct costimulatory signals.

Intracellular interleukin-1alpha mediates interleukin-8 production induced by Chlamydia trachomatis infection via a mechanism independent of type I interleukin-1 receptor.

Chlamydia trachomatis infection induces a wide array of inflammatory cytokines and chemokines, which may contribute to chlamydia-induced pathologies. However, the precise mechanisms by which Chlamydia induces cytokines remain unclear. Here we demonstrate that the proinflammatory cytokine interleukin-1alpha (IL-1alpha) plays an essential role in chlamydial induction of the chemokine IL-8. Cells deficient in IL-1alpha expression or IL-1alpha-competent cells treated with IL-1alpha-specific small interfering RNA failed to produce IL-8 in response to chlamydial infection. However, neutralization of extracellular IL-1alpha or blockade of or deficiency in type I IL-1 receptor (IL-1RI) signaling did not affect chlamydial induction of IL-8 in cells capable of producing IL-1alpha. These results suggest that IL-1alpha can mediate the chlamydial induction of IL-8 via an intracellular mechanism independent of IL-1RI, especially during the early stage of the infection cycle. This conclusion is further supported by the observations that expression of a transgene-encoded full-length IL-1alpha fusion protein in the nuclei enhanced IL-8 production and that nuclear localization of chlamydia-induced precursor IL-1alpha correlated with chlamydial induction of IL-8. Thus, we have identified a novel mechanism for chlamydial induction of the chemokine IL-8.

Caspase-1 contributes to Chlamydia trachomatis-induced upper urogenital tract inflammatory pathologies without affecting the course of infection.

Chlamydia trachomatis infection induces inflammatory pathologies in the upper genital tract, potentially leading to ectopic pregnancy and infertility in the affected women. Caspase-1 is required for processing and release of the inflammatory cytokines interleukin-1beta (IL-1beta), IL-18, and possibly IL-33. In the present study, we evaluated the role of caspase-1 in chlamydial infection and pathogenesis. Although chlamydial infection induced caspase-1 activation and processing of IL-1beta, mice competent and mice deficient in caspase-1 experienced similar courses of chlamydial infection in their urogenital tracts, suggesting that Chlamydia-activated caspase-1 did not play a significant role in resolution of chlamydial infection. However, when genital tract tissue pathologies were examined, the caspase-1-deficient mice displayed much reduced inflammatory damage. The reduction in inflammation was most obvious in the fallopian tube tissue. These observations demonstrated that although caspase-1 is not required for controlling chlamydial infection, caspase-1-mediated responses can exacerbate the Chlamydia-induced inflammatory pathologies in the upper genital tract, suggesting that the host caspase-1 may be targeted for selectively attenuating chlamydial pathogenicity without affecting the host defense against chlamydial infection.

Rottlerin inhibits chlamydial intracellular growth and blocks chlamydial acquisition of sphingolipids from host cells.

We report that rottlerin, a plant-derived compound known to inhibit various mammalian kinases, profoundly inhibited chlamydial growth in cell culture with a minimal inhibition concentration of 1 microM. The inhibition was effective even when rottlerin was added as late as the middle stage of chlamydial infection cycle, against multiple Chlamydia species, and in different host cell lines. Pretreatment of host cells with rottlerin prior to infection also blocked chlamydial growth, suggesting that rottlerin targets host factors. Moreover, rottlerin did not alter the chlamydial infection rate and did not directly target chlamydial protein synthesis and secretion. The rottlerin-mediated inhibition of chlamydial replication and inclusion expansion correlated well with the rottlerin-induced blockade of host cell sphingolipid trafficking from the Golgi apparatus into chlamydial inclusions. These studies not only allowed us to identify a novel antimicrobial activity for rottlerin but also allowed us to uncover a potential mechanism for rottlerin inhibition of chlamydial growth.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.

Senescent Cells Contribute to the Physiological Remodeling of Aged Lungs.

Age-associated decline in organ function governs life span. We determined the effect of aging on lung function and cellular/molecular changes of 8- to 32-month old mice. Proteomic analysis of lung matrix indicated significant compositional changes with advanced age consistent with a profibrotic environment that leads to a significant increase in dynamic compliance and airway resistance. The excess of matrix proteins deposition was associated modestly with the activation of myofibroblasts and transforming growth factor-beta signaling pathway. More importantly, detection of senescent cells in the lungs increased with age and these cells contributed toward the excess extracellular matrix deposition observed in our aged mouse model and in elderly human samples. Mechanistic target of rapamycin (mTOR)/AKT activity was enhanced in aged mouse lungs compared with those from younger mice associated with the increased expression of the histone variant protein, MH2A, a marker for aging and potentially for senescence. Introduction in the mouse diet of rapamycin, significantly blocked the mTOR activity and limited the activation of myofibroblasts but did not result in a reduction in lung collagen deposition unless it was associated with prevention of cellular senescence. Together these data indicate that cellular senescence significantly contributes to the extracellular matrix changes associated with aging in a mTOR 1-dependent mechanism.

Screening of stimulatory effects of dietary risk factors on mouse intestinal cell kinetics.

AIM: Although epidemiological and experimental studies validate influence of genetic, environmental and dietary factors in the causation of various types of cancers including colon, results from all these sources are inconclusive. Hypothesizing that high fat diet and obesity are among the major predisposing factors in the incidence of colon cancer, we evaluated the role of diet constituted with food material derived from a tropical plant, Tamarindus indica Linn (TI). METHODS: A two part randomized double-blind study was conducted employing inbred Swiss albino mice from a single generation for the whole investigation. One day-old neonates (n = 12) were subcutaneously administered with monosodium glutamate (MSG) to induce obesity (OB). At weaning these animals were maintained on modified AIN-76 diet supplemented with 10% TI and 10% fat bolus (w/w, TIFB) for 8 wk. Subsequently, in the second part of study, four groups of animals belonging to the same generation, age and gender (n = 12 per group), were maintained on: AIN-76 control diet (CD); AIN-76 mixed with 10% TI extract (TI); and, mixed with 10% TI and 10% FB (TIFB) for 8 wk, to determine intestinal crypt cell proliferation, functionally-specific enzyme activities, fermentation profile, and energy preferences. RESULTS: We observed a significant increase in the crypt cell production rate in distal colonic segment of experimental animals when compared with the controls. This segment also contained significantly low butyrate levels compared to control and TIFB groups. All the experimental groups showed a gross decrease in the enzyme activities viz., succinate dehydrogenase, acid-galactosidase and dipeptidyl amino peptidase IV demonstrating pathological stress caused by the test regimens, and an altered metabolic flux in the cellular environment. CONCLUSION: We have demonstrated a cumulative response to the three dietary factors, one of which (TI) is reported, herein, for the first time to modulate kinetics of large intestinal mucosa, contributing to total risk posed by these test agents.