The Chlamydia trachomatis type III-secreted effector protein CteG induces centrosome amplification through interactions with centrin-2.

The centrosome is the main microtubule organizing center of the cell and is crucial for mitotic spindle assembly, chromosome segregation, and cell division. Centrosome duplication is tightly controlled, yet several pathogens, most notably oncogenic viruses, perturb this process leading to increased centrosome numbers. Infection by the obligate intracellular bacterium Chlamydia trachomatis (C.t.) correlates with blocked cytokinesis, supernumerary centrosomes, and multipolar spindles; however, the mechanisms behind how C.t. induces these cellular abnormalities remain largely unknown. Here we show that the secreted effector protein, CteG, binds to centrin-2 (CETN2), a key structural component of centrosomes and regulator of centriole duplication. Our data indicate that both CteG and CETN2 are necessary for infection-induced centrosome amplification, in a manner that requires the C-terminus of CteG. Strikingly, CteG is important for in vivo infection and growth in primary cervical cells but is dispensable for growth in immortalized cells, highlighting the importance of this effector protein to chlamydial infection. These findings begin to provide mechanistic insight into how C.t. induces cellular abnormalities during infection, but also indicate that obligate intracellular bacteria may contribute to cellular transformation events. Centrosome amplification mediated by CteG-CETN2 interactions may explain why chlamydial infection leads to an increased risk of cervical or ovarian cancer.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

IMPORTANCE: It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Toxicity Impacts on Human Adipose Mesenchymal Stem/Stromal Cells Acutely Exposed to Aroclor and Non-Aroclor Mixtures of Polychlorinated Biphenyl.

Polychlorinated biphenyl (PCB) accumulates in adipose where it may impact the growth and function of cells within the tissue. This is particularly concerning during adolescence when adipocytes expand rapidly. Herein, we sought to understand how exposure to PCB mixtures found in U.S. schools affects human adipose mesenchymal stem/stromal cell (MSC) health and function. We investigated how exposure to Aroclor 1016 and Aroclor 1254, as well as a newly characterized non-Aroclor mixture that resembles the PCB profile found in cabinets, Cabinet Mixture, affects adipose MSC growth, viability, and function in vitro. We found that exposure to all three mixtures resulted in two distinct types of toxicity. At PCB concentrations >20 µM, the majority of MSCs die, while at 1-10 µM, MSCs remained viable but display numerous alterations to their phenotype. At these sublethal concentrations, the MSC rate of expansion slowed and morphology changed. Further assessment revealed that PCB-exposed MSCs had impaired adipogenesis and a modest decrease in immunosuppressive capabilities. Thus, exposure to PCB mixtures found in schools negatively impacts the health and function of adipose MSCs. This work has implications for human health due to MSCs' role in supporting the growth and maintenance of adipose tissue.

RABL6A Regulates Schwann Cell Senescence in an RB1-Dependent Manner.

Schwann cells are normally quiescent, myelinating glia cells of the peripheral nervous system. Their aberrant proliferation and transformation underlie the development of benign tumors (neurofibromas) as well as deadly malignant peripheral nerve sheath tumors (MPNSTs). We discovered a new driver of MPNSTs, an oncogenic GTPase named RABL6A, that functions in part by inhibiting the RB1 tumor suppressor. RB1 is a key mediator of cellular senescence, a permanent withdrawal from the cell cycle that protects against cell immortalization and transformation. Based on the RABL6A-RB1 link in MPNSTs, we explored the hypothesis that RABL6A promotes Schwann cell proliferation and abrogates their senescence by inhibiting RB1. Using sequentially passaged normal human Schwann cells (NHSCs), we found that the induction of replicative senescence was associated with reduced expression of endogenous RABL6A. Silencing RABL6A in low passage NHSCs caused premature stress-induced senescence, which was largely rescued by co-depletion of RB1. Consistent with those findings, Rabl6-deficient MEFs displayed impaired proliferation and accelerated senescence compared to wildtype MEFs. These results demonstrate that RABL6A is required for maintenance of proper Schwann cell proliferation and imply that aberrantly high RABL6A expression may facilitate malignant transformation.

The Superantigen Toxic Shock Syndrome Toxin 1 Alters Human Aortic Endothelial Cell Function.

Staphylococcus aureus infective endocarditis (IE) is a fast-progressing and tissue-destructive infection of the cardiac endothelium. The superantigens (SAgs) toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin C (SEC), and the toxins encoded by the enterotoxin gene cluster (egc) play a novel and essential role in the etiology of S. aureus IE. Recent studies indicate that SAgs act at the infection site to cause tissue pathology and promote vegetation growth. The underlying mechanism of SAg involvement has not been clearly defined. In SAg-mediated responses, immune cell priming is considered a primary triggering event leading to endothelial cell activation and altered function. Utilizing immortalized human aortic endothelial cells (iHAECs), we demonstrated that TSST-1 directly activates iHAECs, as documented by upregulation of vascular and intercellular adhesion molecules (VCAM-1 and ICAM-1). TSST-1-mediated activation results in increased monolayer permeability and defects in vascular reendothelialization. Yet stimulation of iHAECs with TSST-1 fails to induce interleukin-8 (IL-8) and IL-6 production. Furthermore, simultaneous stimulation of iHAECs with TSST-1 and lipopolysaccharide (LPS) inhibits LPS-mediated IL-8 and IL-6 secretion, even after pretreatment with either of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß. IL-8 suppression is not mediated by TSST-1 binding to its canonical receptor major histocompatibility complex class II (MHC-II), supporting current evidence for a nonhematopoietic interacting site on SAgs. Together, the data suggest that TSST-1 differentially regulates cell-bound and secreted markers of endothelial cell activation that may result in dysregulated innate immune responses during S. aureus IE. Endothelial changes resulting from the action of SAgs can therefore directly contribute to the aggressive nature of S. aureus IE and development of life-threatening complications.

Connective Tissue Growth Factor Promotes Pulmonary Epithelial Cell Senescence and Is Associated with COPD Severity.

The purpose of this study was to determine whether expression of connective tissue growth factor (CTGF) protein in chronic obstructive pulmonary disease (COPD) is consistent in humans and animal models of COPD and to investigate the role of this protein in lung epithelial cells. CTGF in lung epithelial cells of ex-smokers with COPD was compared with ex-smokers without COPD by immunofluorescence. A total of twenty C57Bl/6 mice and sixteen non-human primates (NHPs) were exposed to cigarette smoke (CS) for 4 weeks. Ten mice of these CS-exposed mice and eight of the CS-exposed NHPs were infected with H3N2 influenza A virus (IAV), while the remaining ten mice and eight NHPs were mock-infected with vehicle as control. Both mRNA and protein expression of CTGF in lung epithelial cells of mice and NHPs were determined. The effects of CTGF overexpression on cell proliferation, p16 protein, and senescence-associated ß-galactosidase (SA-ß-gal) activity were examined in cultured human bronchial epithelial cells (HBECs). In humans, CTGF expression increased with increasing COPD severity. We found that protein expression of CTGF was upregulated in lung epithelial cells in both mice and NHPs exposed to CS and infected with IAV compared to those exposed to CS only. When overexpressed in HBECs, CTGF accelerated cellular senescence accompanied by p16 accumulation. Both CTGF and p16 protein expression in lung epithelia are positively associated with the severity of COPD in ex-smokers. These findings show that CTGF is consistently expressed in epithelial cells of COPD lungs. By accelerating lung epithelial senescence, CTGF may block regeneration relative to epithelial cell loss and lead to emphysema.

Diminished Phosphorylation of CREB Is a Key Event in the Dysregulation of Gluconeogenesis and Glycogenolysis in PCB126 Hepatotoxicity.

The dioxin-like PCB126 elicits toxicity in various target organs. In rat liver, an alteration in the transcript levels of several genes involved in glucose and fatty acid metabolism provides insights into the origin of its hepatotoxicity. To explore the mechanisms, male Sprague-Dawley rats, fed an AIN-93G diet, were injected with PCB126 (1 or 5 µmol/kg) or corn oil and euthanized after 2 weeks. PCB126 significantly decreased serum glucose levels and the transcript levels of genes of many gluconeogenic and glycogenolytic enzymes under the transcriptional control of a nuclear transcription factor, cAMP response element-binding protein (CREB). As a novel finding, we show that PCB126 significantly decreases CREB phosphorylation, which is important for regulating both gluconeogenesis and fatty acid oxidation in the liver and explains CREB's integrative effects on both carbohydrate and lipid metabolism in PCB126 toxicity.

Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.

Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer degeneration of multiple tissues. Patient-specific induced pluripotent stem (iPS) cells represent invaluable in vitro models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, which is accompanied by induction of the telomerase reverse transcriptase gene (TERT). We investigated whether defects in telomerase function would limit derivation and maintenance of iPS cells from patients with DC. Here we show that reprogrammed DC cells overcome a critical limitation in telomerase RNA component (TERC) levels to restore telomere maintenance and self-renewal. We discovered that TERC upregulation is a feature of the pluripotent state, that several telomerase components are targeted by pluripotency-associated transcription factors, and that in autosomal dominant DC, transcriptional silencing accompanies a 3' deletion at the TERC locus. Our results demonstrate that reprogramming restores telomere elongation in DC cells despite genetic lesions affecting telomerase, and show that strategies to increase TERC expression may be therapeutically beneficial in DC patients.

Novel Staphylococcus aureus Secreted Protein Alters Keratinocyte Proliferation and Elicits a Proinflammatory Response In Vitro and In Vivo.

Staphylococcus aureus is a leading cause of surgical site infections that results in increased hospital stays due to the development of chronic wounds. Little is known about factors involved in S. aureus' ability to prevent wounds from healing. We discovered a novel secreted protein produced by a surgical site isolate of S. aureus that prevents keratinocyte proliferation. The protein has a molecular weight of 15.7 kDa and an isoelectric point of 8.9. The cloned and purified protein has cytotoxic and proinflammatory properties, as shown in vitro and in vivo. Potent biological effects on keratinocytes and rabbit skin suggest that this protein may play an important role in preventing re-epithelialization. Its lack of homology to known exotoxins suggests that this protein is novel, and this observation is likely to open a new field of research in S. aureus exotoxins. Due to its cytotoxic activities, we call this new protein ε-cytotoxin.

Impact of Aging and a High-Fat Diet on Adipose-Tissue-Derived Extracellular Vesicle miRNA Profiles in Mice.

BACKGROUND: Middle-aged adults have the highest obesity rates, leading to significant health complications in later years. Obesity triggers the release of altered molecules, including extracellular vesicles (EVs) from excess adipose tissue (AT), contributing to various health complications. In this study, we assessed the effects of age and a high-fat diet on AT-derived EV miRNA profiles to understand their potential roles in aging and obesity. METHOD: C57BL/6 male mice were subjected to a normal chow diet (NCD) or a high-fat diet (HFD) for either 10-12 weeks (young mice, n = 10) or 50-61 weeks (middle-aged mice, n = 12). After evaluating metabolic characteristics, peri-gonadal white AT was isolated and cultured to obtain EVs. AT-derived EV miRNAs were profiled using a NanoString miRNA panel (n = 599). RESULTS: Middle-aged mice exhibited obesity regardless of diet. Young mice fed an HFD showed similar metabolic traits to middle-aged mice. In the NCD group, 131 differentially expressed miRNAs (DE-miRNAs) emerged in middle-aged mice compared to young mice, including miR-21, miR-148a, and miR-29a, associated with cancer, neuro/psychological disorders, and reproductive diseases. In the HFD group, 55 DE-miRNAs were revealed in middle-aged mice compared to young mice. These miRNAs were associated with significantly suppressed IGF1R activity. CONCLUSION: This study demonstrates the potential significant impact of miRNAs of AT EVs on aging- and obesity-related diseases.

The Impact of High Adiposity on Endometrial Progesterone Response and Metallothionein Regulation.

CONTEXT: Obesity is a disease with deleterious effects on the female reproductive tract, including the endometrium. OBJECTIVE: We sought to understand the effects of excess adipose on the benign endometrium. METHODS: A physiologic in vitro coculture system was developed, consisting of multicellular human endometrial organoids, adipose spheroids, and menstrual cycle hormones. Native human endometrial tissue samples from women with and without obesity were also analyzed. Benign endometrial tissues from premenopausal women ages 33 to 53 undergoing hysterectomy were obtained following written consent at Northwestern University Prentice Women's Hospital, Chicago, Illinois. Gene expression, protein expression, chromatin binding, and expression of DNA damage and oxidative damage markers were measured. RESULTS: Under high adiposity conditions, endometrial organoids downregulated endometrial secretory phase genes, suggestive of an altered progesterone response. Progesterone specifically upregulated the metallothionein (MT) gene family in the epithelial cells of endometrial organoids, while high adiposity significantly downregulated the MT genes. Silencing MT genes in endometrial epithelial cells resulted in increased DNA damage, illustrating the protective role of MTs. Native endometrium from women with obesity displayed increased MT expression and oxidative damage in the stroma and not in the epithelium, indicating the cell-specific impact of obesity on MT genes. CONCLUSION: Taken together, the in vitro and in vivo systems used here revealed that high adiposity or obesity can alter MT expression by decreasing progesterone response in the epithelial cells and increasing oxidative stress in the stroma.

Inhibition of human cytomegalovirus entry into mucosal epithelial cells.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

Upregulation of fatty acid synthesis genes in the livers of adolescent female rats caused by inhalation exposure to PCB52 (2,2',5,5'-Tetrachlorobiphenyl).

Elevated airborne PCB levels in older schools are concerning due to their health impacts, including cancer, metabolic dysfunction-associated steatotic liver disease (MASLD), cardiovascular issues, neurodevelopmental diseases, and diabetes. During a four-week inhalation exposure to PCB52, an air pollutant commonly found in school environments, adolescent rats exhibited notable presence of PCB52 and its hydroxylated forms in their livers, alongside changes in gene expression. Female rats exhibited more pronounced changes in gene expression compared to males, particularly in fatty acid synthesis genes regulated by the transcription factor SREBP1. In vitro studies with human liver cells showed that the hydroxylated metabolite of PCB52, 4-OH-PCB52, but not the parent compound, upregulated genes involved in fatty acid biosynthesis similar to in vivo exposure. These findings highlight the sex-specific effects of PCB52 exposure on livers, particularly in females, suggesting a potential pathway for increased MASLD susceptibility.

Effects of 28-day nose-only inhalation of PCB52 (2,2',5,5'-Tetrachlorobiphenyl) on the brain transcriptome.

A semi-volatile polychlorinated biphenyl (PCB) congener, PCB52, is present in the indoor air of schools; however, the effects of inhaled PCB52 on the brain have not been investigated. This study exposed male Sprague-Dawley rats at 39 days of age and female rats at 42 days of age to PCB52 for 4 hours per day over 28 consecutive days through nose-only inhalation. Neurobehavioral tests were conducted during the last 5 days of exposure. The total estimated PCB52 exposures after 28 days were 1080±20 µg/kg BW for male rats and 1140±10 µg/kg BW for female rats. PCB52 and its metabolites were detected by gas chromatography-tandem mass spectrometry in the brain, lung, and serum, with the lung showing the highest concentrations. PCB52 levels were higher in the brains of females than males. Males showed increased exploratory behavior compared to controls, whereas females exhibited decreased exploratory behavior compared to controls in the same tests. PCB52 exposure did not impact locomotor activity or working memory. Gene expression and pathway analysis in the striatum and cerebellum suggest that PCB52 inhalation causes mitochondrial dysfunction. No significant differences were observed by immunohistochemical evaluation in the density and percent area of total cells, astrocytes, or microglia in the striatum and cerebellar cortex. Our results indicate multilevel effects of inhaled PCB52 on the rat brain, from gene expression to behavioral effects.

Inhalation of 2,2',5,5'-tetrachlorobiphenyl (PCB52) causes changes to the gut microbiome throughout the gastrointestinal tract.

Polychlorinated biphenyls (PCBs), such as PCB52, are hazardous environmental contaminants present in indoor and outdoor environments. Oral PCB exposure affects the colon microbiome; however, it is unknown if inhalation of PCBs alters the intestinal microbiome. We hypothesize that sub-acute inhalation of PCB52 affects microbial communities depending on the location in the (GI) gastrointestinal tract and the local profiles of PCB52 and its metabolites present in the GI tract following mucociliary clearance and biliary or intestinal excretion. Sprague-Dawley rats were exposed via nose-only inhalation 4 h per day, 7 days per week, for 4 weeks to either filtered air or PCB52. After 28 days, differences in the microbiome and levels of PCB52 and its metabolites were characterized throughout the GI tract. PCB52 inhalation altered taxa abundances and predicted functions altered throughout the gut, with most alterations occurring in the large intestine. PCB52 and metabolite levels varied across the GI tract, resulting in differing PCB × microbiome networks. Thus, the presence of different levels of PCB52 and its metabolites in different parts of the GI tract has varying effects on the composition and predicted function of microbial communities. Future studies need to investigate whether these changes lead to adverse outcomes.

Updating "Dataset of transcriptomic changes that occur in human preadipocytes over a 3-day course of exposure to 3,3',4,4',5-Pentachlorobiphenyl (PCB126)" with additional data on exposure to 2,2',5,5'-tetrachlorobiphenyl (PCB52) or its 4-hydroxy metabolite (4-OH-PCB52).

Polychlorinated biphenyls (PCBs) were used extensively in building materials, including those used in schools. PCBs accumulate in fat, and exposure to PCBs is associated with the development of cancer, neurodevelopmental disorders, cardiovascular disease, obesity, and diabetes. The non-dioxin-like PCB congener, PCB52 (2,2',5,5'-tetrachlorobiphenyl), is found at one of the highest levels of any congener in school air. PCB52 is oxidized in the liver to hydroxylated forms, mainly 4-OH-PCB52 (2,2',5,5'-tetrachlorobiphenyl-4-ol). In a previous study, we reported on RNAseq data generated from exposure of human preadipocytes to the dioxin-like PCB congener, PCB126. In this new dataset, we used identical techniques to examine alterations in gene transcript levels in human preadipocytes exposed to PCB52 or 4-OH-PCB52 over a time course. This updated set of data provides a comprehensive transcriptional profile of changes that occur in preadipocytes exposed to PCB52 or 4-OH-PCB52 over time and allows for comparison of these changes between the parent compound and its hydroxy metabolite. The datasets will allow others to explore how PCB52 and 4-OH-PCB52 impact biological pathways in preadipocytes. Further studies can be performed to determine how these changes might lead to disease.

Hydroxylation markedly alters how the polychlorinated biphenyl (PCB) congener, PCB52, affects gene expression in human preadipocytes.

Polychlorinated biphenyls (PCBs) accumulate in adipose tissue and are linked to obesity and diabetes. The congener, PCB52 (2,2',5,5'-tetrachorobiphenyl), is found at high levels in school air. Hydroxylation of PCB52 to 4-OH-PCB52 (4-hydroxy-2,2',5,5'-tetrachorobiphenyl) may increase its toxicity. To understand PCB52's role in causing adipose dysfunction, we exposed human preadipocytes to PCB52 or 4-OH-PCB52 across a time course and assessed transcript changes using RNAseq. 4-OH-PCB52 caused considerably more changes in the number of differentially expressed genes as compared to PCB52. Both PCB52 and 4-OH-PCB52 upregulated transcript levels of the sulfotransferase SULT1E1 at early time points, but cytochrome P450 genes were generally not affected. A set of genes known to be transcriptionally regulated by PPARα were consistently downregulated by PCB52 at all time points. In contrast, 4-OH-PCB52 affected a variety of pathways, including those involving cytokine responses, hormone responses, focal adhesion, Hippo, and Wnt signaling. Sets of genes known to be transcriptionally regulated by IL17A or parathyroid hormone (PTH) were found to be consistently downregulated by 4-OH-PCB52. Most of the genes affected by PCB52 and 4-OH-PCB52 were different and, of those that were the same, many were changed in an opposite direction. These studies provide insight into how PCB52 or its metabolites may cause adipose dysfunction to cause disease.

Keratinocyte FABP5-VCP complex mediates recruitment of neutrophils in psoriasis.

One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.

Host Cationic Antimicrobial Molecules Inhibit S. aureus Exotoxin Production.

Innate immune molecules, including antimicrobial peptides (for example, defensins) and lysozyme, function to delay or prevent bacterial infections. These molecules are commonly found on mucosal and skin surfaces. Staphylococcus aureus is a common pathogen and causes millions of infections annually. It is well known that innate immune molecules, such as defensins and lysozyme, either poorly inhibit or do not inhibit the growth of S. aureus. Our current studies show that the α-defensin human neutrophil α-defensin-1 (HNP-1) and lysozyme inhibit exotoxin production, both hemolysins and superantigens, which are required for S. aureus infection. HNP-1 inhibited exotoxin production at concentrations as low as 0.001 µg/mL. Lysozyme inhibited exotoxin production at 0.05 to 0.5 µg/mL. Both HNP-1 and lysozyme functioned through at least one two-component system (SrrA/B). The ß-defensin human ß-defensin 1 (HBD-1) inhibited hemolysin but not superantigen production. The cation chelator S100A8/A9 (calprotectin), compared to EDTA, was tested for the ability to inhibit exotoxin production. EDTA at high concentrations inhibited exotoxin production; these were the same concentrations that interfered with staphylococcal growth. S100A8/A9 at the highest concentration tested (10 µg/mL) had no effect on S. aureus growth but enhanced exotoxin production. Lower concentrations had no effect on growth or exotoxin production. Lysostaphin is regularly used to lyse S. aureus. The lytic concentrations of lysostaphin were the only concentrations that also inhibited growth and exotoxin production. Our studies demonstrate that a major activity of innate defensin peptides and lysozyme is inhibition of staphylococcal exotoxin production but not inhibition of growth. IMPORTANCE Staphylococcus aureus causes large numbers of both relatively benign and serious human infections, which are mediated in large part by the organisms' secreted exotoxins. Since 1921, it has been known that lysozyme and, as shown later in the 1900s, other innate immune peptides, including human neutrophil α-defensin-1 (HNP-1) and human ß-defensin 1 (HBD-1), are either not antistaphylococcal or are only weakly inhibitory to growth. Our study confirms those findings but, importantly, shows that at subgrowth inhibitory concentrations, these positively charged innate immune peptides inhibit exotoxin production, including both hemolysins and the superantigen toxic shock syndrome toxin-1. The data show that the principal activity of innate immune peptides in the host is likely to be inhibition of exotoxin production required for staphylococcal mucosal or skin colonization rather than growth inhibition.

Native adiponectin plays a role in the adipocyte-mediated conversion of fibroblasts to myofibroblasts.

Adipocytes regulate tissues through production of adipokines that can act both locally and systemically. Adipocytes also have been found to play a critical role in regulating the healing process. To better understand this role, we developed a three-dimensional human adipocyte spheroid system that has an adipokine profile similar to in vivo adipose tissues. Previously, we found that conditioned medium from these spheroids induces human dermal fibroblast conversion into highly contractile, collagen-producing myofibroblasts through a transforming growth factor beta-1 (TGF-ß1) independent pathway. Here, we sought to identify how mature adipocytes signal to dermal fibroblasts through adipokines to induce myofibroblast conversion. By using molecular weight fractionation, heat inactivation and lipid depletion, we determined mature adipocytes secrete a factor that is 30-100 kDa, heat labile and lipid associated that induces myofibroblast conversion. We also show that the depletion of the adipokine adiponectin, which fits those physico-chemical parameters, eliminates the ability of adipocyte-conditioned media to induce fibroblast to myofibroblast conversion. Interestingly, native adiponectin secreted by cultured adipocytes consistently elicited a stronger level of α-smooth muscle actin expression than exogenously added adiponectin. Thus, adiponectin secreted by mature adipocytes induces fibroblast to myofibroblast conversion and may lead to a phenotype of myofibroblasts distinct from TGF-ß1-induced myofibroblasts.