Amber Kaplan

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary ‘‘shotgun’’ and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. ‘‘Shotgun’’ lipidomics data revealed that… Show more

Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary ‘‘shotgun’’ and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. ‘‘Shotgun’’ lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity. Show less

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-β is antimicrobial. Parts of the IFN-β molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-β can directly kill Staphylococcus aureus. Further, a mutant S. aureus that is more… Show more

Type I IFNs are a cytokine family essential for antiviral defense. More recently, type I IFNs were shown to be important during bacterial infections. In this article, we show that, in addition to known cytokine functions, IFN-β is antimicrobial. Parts of the IFN-β molecular surface (especially helix 4) are cationic and amphipathic, both classic characteristics of antimicrobial peptides, and we observed that IFN-β can directly kill Staphylococcus aureus. Further, a mutant S. aureus that is more sensitive to antimicrobial peptides was killed more efficiently by IFN-β than was the wild-type S. aureus, and immunoblotting showed that IFN-β interacts with the bacterial cell surface. To determine whether specific parts of IFN-β are antimicrobial, we synthesized IFN-β helix 4 and found that it is sufficient to permeate model prokaryotic membranes using synchrotron x-ray diffraction and that it is sufficient to kill S. aureus. These results suggest that, in addition to its well-known signaling activity, IFN-β may be directly antimicrobial and be part of a growing family of cytokines and chemokines, called kinocidins, that also have antimicrobial properties. Show less

In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade… Show more

In response to tissue injury, hyaluronan (HA) polymers are cleaved by host hyaluronidases, generating small fragments that ligate Toll-like receptors (TLRs) to elicit inflammatory responses. Pathogenic bacteria such as group B Streptococcus (GBS) express and secrete hyaluronidases as a mechanism for tissue invasion, but it is not known how this activity relates to immune detection of HA. We found that bacterial hyaluronidases secreted by GBS and other Gram-positive pathogens degrade pro-inflammatory HA fragments to their component disaccharides. In addition, HA disaccharides block TLR2/4 signaling elicited by both host-derived HA fragments and other TLR2/4 ligands, including lipopolysaccharide. Application of GBS hyaluronidase or HA disaccharides reduced pulmonary pathology and pro-inflammatory cytokine levels in an acute lung injury model. We conclude that breakdown of host-generated pro-inflammatory HA fragments to disaccharides allows bacterial pathogens to evade immune detection and could be exploited as a strategy to treat inflammatory diseases. Show less

The importance of type I IFNs in the host response to viral infection is well established; however, their role in bacterial infection is not fully understood. Several bacteria (both Gram-positive and -negative) have been shown to induce IFN-b production in myeloid cells, but this IFN-b is not always beneficial to the host.We examined whether Staphylococcus aureus induces IFN-b from myeloid phagocytes, and if so, whether it is helpful or harmful to the host to do so. We found that S. aureus… Show more

The importance of type I IFNs in the host response to viral infection is well established; however, their role in bacterial infection is not fully understood. Several bacteria (both Gram-positive and -negative) have been shown to induce IFN-b production in myeloid cells, but this IFN-b is not always beneficial to the host.We examined whether Staphylococcus aureus induces IFN-b from myeloid phagocytes, and if so, whether it is helpful or harmful to the host to do so. We found that S. aureus poorly induces IFN-b production compared with other bacteria. S. aureus is highly resistant to degradation in the phagosome because it is resistant to lysozyme. Using a mutant that is more sensitive to lysozyme, we show that phagosomal degradation and release of intracellular ligands is essential for induction of IFN-b and inflammatory chemokines downstream of IFN-b. Further, we found that adding exogenous IFN-b during S. aureus infection (in vitro and in vivo) was protective. Together, the data demonstrate that failure to induce IFN-b production during S. aureus infection contributes to pathogenicity. Show less

Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare, and many… Show more

Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare, and many model ligands are studied in soluble form, outside of their microbial context. By comparing a wild-type strain of Staphylococcus aureus and a lysozyme-sensitive mutant, we have been able directly to address the role of degradation of live bacteria by mouse macrophages in determining the overall innate cellular inflammatory response. Our investigations revealed a biphasic response to S. aureus that consisted of an initial signal resulting from the engagement of surface TLR2, followed by a later, second wave on inflammatory gene induction. This second wave of inflammatory signaling was dependent on and correlated with the timing of bacterial degradation in phagosomes. We found that TLR2 signaling followed by TLR2/TLR9 signaling enhanced sensitivity to small numbers of bacteria. We further found that treating wild-type bacteria with the peptidoglycan synthesis-inhibiting antibiotic vancomycin made S. aureus more susceptible to degradation and resulted in increased inflammatory responses, similar to those observed for mutant degradation-sensitive bacteria. Show less

The vitamin D-activating enzyme 1a-hydroxylase (CYP27B1) and vitamin D receptor (VDR) support anti-inflammatory responses to vitamin D in many tissues. Given the high basal expression of CYP27B1 and VDR in trophoblastic cells from the placenta, we hypothesized that anti-inflammatory effects of vitamin D may be particularly important in this organ. Pregnant wild type (WT) mice i.p. injected with LPS showed elevated expression of mouse Cyp27b1 (4-fold) and VDR (6-fold). Similar results were also… Show more

The vitamin D-activating enzyme 1a-hydroxylase (CYP27B1) and vitamin D receptor (VDR) support anti-inflammatory responses to vitamin D in many tissues. Given the high basal expression of CYP27B1 and VDR in trophoblastic cells from the placenta, we hypothesized that anti-inflammatory effects of vitamin D may be particularly important in this organ. Pregnant wild type (WT) mice i.p. injected with LPS showed elevated expression of mouse Cyp27b1 (4-fold) and VDR (6-fold). Similar results were also obtained after ex vivo treatment of WT placentas with LPS. To assess the functional impact of this, we carried out ex vivo studies using placentas 2/2 for fetal (trophoblastic) Cyp27b1 or VDR. Vehicle-treated 2/2 placentas showed increased expression of IFN-g and decreased expression of IL-10 relative to +/+ placentas. LPS-treated 2/2 placentas showed increased expression of TLR2, IFN-g, and IL-6. Array analyses identified other inflammatory factors that are dysregulated in Cyp27b12/2 versus Cyp27b1+/+ placentas after LPS challenge. Data highlighted enhanced expression of IL-4, IL-15, and IL-18, as well as several chemokines and their receptors, in Cyp27b12/2 placentas. Similar results for IL-6 expression were observed with placentas 2/2 for trophoblastic VDR. Finally, ex vivo treatment of WT placentas with the substrate for Cyp27b1, 25-hydroxyvitamin D3, suppressed LPS-induced expression of IL-6 and the chemokine Ccl11. These data indicate that fetal (trophoblastic) vitamin D plays a pivotal role in controlling placental inflammation. In humans, this may be a key factor in placental responses to infection and associated adverse outcomes of pregnancy. Show less

In aquatic ecosystems, dense populations of snails can shed millions of digenean trematode cercariae every day. These short-lived, free-living larvae are rich in energy and present a potential resource for consumers. We investigated whether estuarine fishes eat cercariae shed by trematodes of the estuarine snail Cerithidea californica. In aquaria we presented cercariae from 10 native trematode species to 6 species of native estuarine fishes. Many of these fishes readily engorged on cercariae… Show more

In aquatic ecosystems, dense populations of snails can shed millions of digenean trematode cercariae every day. These short-lived, free-living larvae are rich in energy and present a potential resource for consumers. We investigated whether estuarine fishes eat cercariae shed by trematodes of the estuarine snail Cerithidea californica. In aquaria we presented cercariae from 10 native trematode species to 6 species of native estuarine fishes. Many of these fishes readily engorged on cercariae. To determine if fishes ate cercariae in the field, we collected the most common fish species, Fundulus parvipinnis (California killifish), from shallow water on rising tides when snails shed cercariae. Of 61 killifish, 3 had recognizable cercariae in their gut. Because cercariae are common in this estuary, they could be frequent sources of energy for small fishes. In turn, predation on cercariae by fishes (and other predators) could also reduce the transmission success of trematodes. Show less

West Nile virus (WNV), a mosquito-transmitted single stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7-/-) and myeloid differentiation factor 88-deficient (Myd88-/-) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45+ leukocytes and CD11b+macrophages failed to home to… Show more

West Nile virus (WNV), a mosquito-transmitted single stranded RNA (ssRNA) flavivirus, causes human disease of variable severity. We investigated Toll-like receptor 7-deficient (Tlr7-/-) and myeloid differentiation factor 88-deficient (Myd88-/-) mice, which both have defective recognition of ssRNA, and found increased viremia and susceptibility to lethal WNV infection. Despite increased tissue concentrations of most innate cytokines, CD45+ leukocytes and CD11b+macrophages failed to home to WNV-infected cells and infiltrate into target organs of Tlr7-/- mice. Tlr7-/- mice and macrophages had reduced interleukin- 12 (IL-12) and IL-23 responses after WNV infection, and mice deficient in IL-12 p40 and IL-23 p40 (Il12b-/-) or IL-23 p19 (Il23a-/-), but not IL-12 p35 (Il12a-/-), responded similarly to Tlr7-/- mice, with increased susceptibility to lethal WNV encephalitis. Collectively, these results demonstrate that TLR7 and IL-23-dependent WNV responses represent vital host defense mechanism that operates by affecting immune cell homing to infected target cells. Show less

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta.… Show more

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is a potent inducer of the antimicrobial protein cathelicidin, CAMP (LL37). In macrophages this response is dependent on intracrine synthesis of 1,25(OH)2D from precursor 25-hydroxyvitamin D (25OHD), catalyzed by the enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1). In view of the fact that trophoblastic cells also express abundant CYP27B1, we postulated a similar intracrine pathway for induction of CAMP in the placenta. Analysis of placenta explants, primary cultures of human trophoblast, and the 3A trophoblastic cell line treated with 1,25(OH)2D (1–100 nM) revealed dose-dependent induction of CAMP similar to that observed with primary cultures of human macrophages. Also consistent with macrophages, induction of trophoblastic CAMP was enhanced via intracrine conversion of 25OHD to 1,25(OH)2D. However, in contrast to macrophages, induction of CAMP by vitamin D in trophoblasts was not enhanced by costimulation with Toll-like receptor ligands, such as lipopolysaccharide. Despite this, exposure to vitamin D metabolites significantly enhanced antibacterial responses in trophoblastic cells: 3A cells infected with Escherichia coli showed decreased numbers of bacterial colony-forming units compared with vehicle-treated controls when treated with 25OHD (49.6% 6 10.9%) or 1,25(OH)2D (45.4% 6 9.2%), both P , 0.001. Treatment with 25OHD (1–100 nM) or 1,25(OH)2D (0.1–10 nM) also protected 3A cells against cell death following infection with E. coli (13.6%–26.9% and 22.3%–40.2% protection, respectively). These observations indicate that 1,25(OH)2D can function as an intracrine regulator of CAMP in trophoblasts, and may thus provide a novel mechanism
for activation of innate immune responses in the placenta. Show less

Group B streptococcus (GBS) is one of the leading causes of neonatal infection; however the molecular mechanisms involved are not clearly known. Here we used high and low hemolytic GBS isolates and mutant GBS that lacks b-hemolysin expression and showed that GBS infection or exposure to GBS hemolysin extract induces primary human trophoblast, placental fibroblast and JEG3 trophoblast cell line death, and that GBS-induced trophoblast death was b-hemolysin dependent. The fibroblasts and… Show more

Group B streptococcus (GBS) is one of the leading causes of neonatal infection; however the molecular mechanisms involved are not clearly known. Here we used high and low hemolytic GBS isolates and mutant GBS that lacks b-hemolysin expression and showed that GBS infection or exposure to GBS hemolysin extract induces primary human trophoblast, placental fibroblast and JEG3 trophoblast cell line death, and that GBS-induced trophoblast death was b-hemolysin dependent. The fibroblasts and trophoblasts provide an innate immune barrier between fetal and maternal circulation in the placenta. These data suggest that GBS may disrupt this barrier to invade fetal circulation. Show less