extfieldsofstudy: Biology Medicine

text:
Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF

Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors.

Macrophages exposed to apoptotic cells counteract TGF-β-induced EMT in lung and kidney epithelial cells.
TGF-β 1 activation is a critical signaling element in EMT and plays a central role in pulmonary fibrosis pathogenesis. Thus, we assessed the impact of in vitro phagocyte exposure to apoptotic cells on TGF-β 1-induced EMT in murine AT II-like lung epithelial (LA-4) cells. TGF-β 1 exposure for 2-3 days caused LA-4 cells to undergo EMT, during which cells acquired a spindle-like shape ( Supplementary Fig.  S1a). Additionally, adherens junction protein E-cadherin expression was decreased, whereas the expression of N-cadherin and α -smooth muscle actin (SMA), a marker of myofibroblast differentiation, was upregulated ( Supplementary Fig. S1b-d). Treatment with conditioned medium derived from a murine macrophage cell line (RAW 264.7) exposed to apoptotic Jurkat cells for 20 h (ApoJ-exposed CM) inhibited TGF-β 1-induced EMT in LA-4 cells, based on morphologic cellular alteration ( Supplementary Fig. S1a) and EMT marker expression profiles at both the protein (Fig. 1a) and mRNA level ( Fig. 1b-d). These EMT marker changes weakened inversely as the conditioned medium was diluted 1:2 and 1:4 with X-VIVO medium ( Supplementary Fig. S1e). However, this inhibitory effect was not observed with conditioned media derived from co-culture with control, viable (ViaJ-exposed CM; Supplementary Fig. S1e) or necrotic Jurkat cells (NecJ-exposed CM). In addition, culture supernatant from apoptotic Jurkat cells alone did not induce an anti-EMT effect. Immunofluorescence using E-cadherin (red) and α -SMA (green) monoclonal antibodies was performed to validate EMT marker protein changes. Similar to the western data, the TGF-β 1-induced decrease in E-cadherin expression and increase in α -SMA expression in LA-4 cells were reversed by ApoJ-exposed CM, but not NecJ-exposed CM (Fig. 1e). We also confirmed the inhibitory effect of the ApoJ-exposed CM on TGF-β 1-induced EMT in primary mouse AT II cells (Fig. 1f) as well as HEK-293 human embryonic kidney epithelial cells ( Supplementary Fig. S2a).
To demonstrate that the anti-EMT effect of the conditioned medium was not restricted to apoptotic T cells, other apoptotic cell types, such as LA-4 epithelial cells, were exposed to RAW 264.7 cells, and the conditioned medium was added to LA-4 cells in the presence of TGF-β 1. That conditioned medium exerted similar inhibitory effects on EMT marker expression in LA-4 cells ( Supplementary Fig. S2b). Similar effects were also observed with apoptotic human Jurkat T cells and mouse thymocytes ( Supplementary Fig. S2c).
The loss of epithelial markers and acquisition of mesenchymal features is achieved through the well-orchestrated actions of the Snail, ZEB, and Basic helix-loop-helix transcription factor families 36,37 . Thus, we examined whether ApoJ-exposed CM inhibited enhanced expression of these transcription factors in LA-4 cells with TGF-β 1 treatment. The ApoJ-exposed CM inhibited TGF-β 1-induced mRNA expression of Snail1/2, Zeb1/2, and Twist1 in LA-4 cells (Fig. 2a-e), whereas the control, or NecJ-exposed CM did not affect transcription factor induction.

Macrophages exposed to apoptotic cells antagonize Smad-independent TGF-β1 signaling in LA-4 cells.
To further explore the intracellular signal transduction mechanism, we examined the effects of the conditioned medium on the canonical Smad-dependent TGF-β 1 signaling pathway. The ApoJ-exposed CM did not affect TGF-β 1-mediated phosphorylation of Smad2 and Smad3 in LA-4 cells( Supplementary Fig. S3a,b). Next, we examined Smad-independent TGF-β 1 signaling pathways, such as mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathways. Extracellular signal-regulated kinase (ERK) was enhanced at 5 min and then declined slightly, and p38 MAP kinase, and Akt phosphorylation was enhanced up to 6, and 8-12 h after TGF-β 1 stimulation, respectively, in LA-4 cells (Fig. 2f), whereas c-Jun N-terminal kinase (JNK) phosphorylation was not enhanced over the 24-h observation period (data not shown). ApoJ-exposed CM treatment partially inhibited TGF-β 1-induced phosphorylation of p38 MAP kinase and Akt (Fig. 2g,h); however, it had no effect on ERK phosphorylation (Supplementary Figure S3c). These data suggest that the bioactive mediators secreted from apoptosis-stimulated macrophages partially block Smad-independent TGF-β signaling, including the p38 MAP kinase and Akt pathways, in LA-4 cells.
Scientific RepoRts | 6:20992 | DOI: 10.1038/srep20992 Figure 1. Conditioned medium from RAW 264.7 cells exposed to apoptotic cells reduced TGF-β1-induced EMT in lung epithelial cells. RAW 264.7 cells were stimulated with apoptotic (ApoJ) or necrotic (NecJ) Jurkat cells for 20 h. Conditioned medium (CM) was added to LA-4 cells (a-e) or primary mouse alveolar type II epithelial (AT II) cells (f) in the absence or presence of 10 ng/ml TGF-β 1 for 72 h. (a,f) Immunoblots of total cell lysates were performed with anti-E-cadherin, N-cadherin, or α -SMA antibodies. Right: Densitometric analysis of the indicated EMT markers' relative abundances. (b-d) The amount of EMT markers' mRNA in LA-4 cell samples was analyzed by real-time PCR and normalized to that of Hprt mRNA. Values represent the mean ± s.e.m. of three independent experiments. *P < 0.05; compared with control or conditioned medium from RAW cells with ApoJ, or NecJ cells at 72 h after TGF-β 1 treatment; + P < 0.05 as indicated.  Conditioned medium from RAW 264.7 cells exposed to apoptotic cells reduced TGF-β1-induced EMT-regulating transcription factor expression and blocked Smad-independent TGF-β1 signaling in LA-4 cells. RAW 264.7 cells were stimulated with apoptotic (ApoJ) or necrotic (NecJ) Jurkat cells for 20 h. Conditioned medium (CM) was added to LA-4 cells in the absence or presence of 10 ng/ml TGF-β 1 for 72 h or the indicated time. (a-e) The amount of Snail1/2, Zeb1/2, and Twist1 mRNA in LA-4 cell samples was analyzed by real-time PCR and normalized to that of Hprt mRNA. (f-h) Western blot analysis of the relative amounts of total and phosphorylated ERK, p38 MAP kinase, and Akt protein in the indicated samples over time. Densitometric analysis of the relative phosphorylated protein abundances, normalized to that of total protein. Data in all bar graphs are the mean ± s.e.m. of three independent experiments. *P < 0.05 compared with control; + P < 0.05 as indicated.
COX-2-derived PGE 2 and PGD 2 secretion from macrophages induced by apoptotic cells mediates EMT inhibition. The COX-2/PGE 2 and PGD 2 pathways are potent EMT inhibitors in renal epithelial cells 32,39 . We showed that PGE 2 and PGD 2 production in RAW 264.7 cells is enhanced by exposure to apoptotic cells, predominantly via inducing COX-2 expression ( Supplementary Fig. S4a,b) 40 . Thus, we investigated whether COX-2-derived PGE 2 and PGD 2 , secreted from ApoJ-exposed macrophages, mediate anti-EMT effects in LA-4 cells. First, to determine the functional relevance of COX-2 signaling in macrophage-dependent anti-EMT effects, RAW 264.7 cells were pretreated with the highly selective COX-2 inhibitor NS-398 or COX-2-specific siRNA and incubated with apoptotic Jurkat cells for 20 h. NS-398 pre-treatment reversed the effect of conditioned medium on TGF-β 1-induced morphology (Fig. 4a). The RAW 264.7 cells were transfected with COX-2 or COX-1 specific siRNAs or with a negative-control siRNA and then were cultured for 6 or 48 h, respectively. The negative-control siRNA did not alter COX-2 and COX-1 protein amounts in cells ( Supplementary Fig. S4c,d). The abundances of COX-2 or COX-1 proteins were specifically decreased by ~100 or 90%, respectively, in cells transfected with COX-2 -or COX-1-specific siRNAs at 20 h after apoptotic cell exposure compared to those in naïve RAW264.7 cells. NS-398 or COX-2 gene knockdown reversed the inhibition of TGF-β 1-induced EMT in LA-4 cells, E-cadherin loss, and synthesis of α -SMA and N-cadherin at both the gene ( Supplementary Fig. S4e) and protein level (Fig. 4b,c). In contrast, COX-1 expression knockdown using COX-1-specific siRNA transfection did not reverse the inhibition of TGF-β 1-induced EMT in LA-4 cells ( Supplementary Fig. S4f). Moreover, NS-398 reversed reduction of TGF-β 1-induced mRNA expression of Zeb1/2, Snail1/2, and Twist1 in LA-4 cells by the ApoJ-exposed CM (Fig. 4d). The 15-lipoxygenase inhibitor PD-146176 did not reverse TGF-β -induced EMT inhibition by the conditioned medium in LA-4 cells (Supplementary Figure S4e; Fig. 4b,d). Quantitative morphometric analysis showed that NS-398, but not PD-146176, reversed the reduction of degree of elongated cell morphology or morphological index in LA4-cells exposed to ApoJ-exposed CM ( Supplementary Fig. S5a).

RhoA-dependent HGF secretion from macrophages programed by apoptotic cells mediates EMT inhibition.
Fibrotic remodeling-associated EMT in alveolar epithelial cells is negatively modulated by HGF 34 . We previously demonstrated that in vitro exposure of macrophages to apoptotic cells induced HGF mRNA and protein production via the RhoA-dependent pathway 40,41 . Thus, we examined the role of RhoA-dependent HGF secretion from RAW 264.7 cells exposed to apoptotic cells in anti-EMT effects. Inhibition of RhoA/Rho kinase signaling using the Rho kinase inhibitor Y-27632 in macrophages or inhibition of c-Met signaling using PHA-665752 in LA-4 cells reversed TGF-β 1-induced EMT inhibition by ApoJ-exposed CM, including reversing spindle-like cellular morphology (Fig. 6a) and reducing E-cadherin loss, synthesis of α -SMA and N-cadherin at the gene ( Supplementary Fig. S6a) and protein (Fig. 6b,c) level, and Zeb1/2, Snail1/2, and Twist1 mRNA expression downregulation in LA-4 cells (Fig. 6d). The conditioned medium from RAW 264.7 cells transfected with RhoA siRNA and exposed to apoptotic Jurkat cells also reversed TGF-β 1-induced EMT reduction in LA-4 cells (Fig. 6e). In addition, quantitative morphometric analysis showed that the reduced degree of elongated cell morphology in LA4-cells exposed to ApoJ-exposed CM was reversed by Y-27632 and PHA-665752 ( Supplementary  Fig. S6b). These data strongly suggest that RhoA-dependent HGF production from macrophages in response to apoptotic cells also partially mediates the anti-EMT effect of the conditioned medium via c-Met signaling in LA-4 cells.
Exogenous PGE 2 , PGD 2 , and HGF inhibit TGF-β1-induced EMT. To confirm that PGE 2 , PGD 2 , and HGF acted in a paracrine manner to induce the anti-EMT effects, we investigated the effect of these soluble mediators at basal (50, 7, and 150 pg/ml, respectively) and stimulation concentrations (150, 17, and 400 pg/ml, respectively) 40 on LA-4 cells. The conditioned medium derived from macrophages without apoptotic cells contains basal levels of PGE 2 , PGD 2 and HGF. As expected, each of these soluble mediators at basal concentration did not inhibit TGF-β 1-induced changes in EMT markers at both the gene (Supplementary Fig. S7a Fig. S7a-d). These findings suggest that they may have the same target molecules to inhibit TGF-β 1-induced signaling pathway.
To strengthen our conclusion that macrophage-mediated EMT inhibition is exerted partially through the blockage of Smad-independent TGF-β 1 signaling, we examined the effect of these soluble molecules on Smad-dependent of independent TGF-β 1 signaling in LA-4 cells. As shown in Supplementary Fig. S8), exogenous PGE 2 , PGD 2 , and HGF did not affect Smad-dependent TGF-β 1 signaling, but blocked TGF-β 1-induced phosphorylation of p38 MAP kinase and Akt in LA-4 cells. These data support our hypothesis that PGE 2 , PGD 2 , and HGF secreted by macrophages in response to apoptotic cells inhibit partially the EMT process through blockage of Smad-independent TGF-β signaling, including the p38 MAP kinase and Akt pathways. PGE 2 , PGD 2 , and HGF secretion from murine bone marrow-derived macrophages (BMDM) mediates the anti-EMT effects of the conditioned medium. In addition to RAW 264.7 macrophages, we also examined the interaction with primary isolated murine BMDM cultured in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and apoptotic or necrotic cells for 20 h. The conditioned medium derived from ApoJ-exposed BMDM substantially inhibited TGF-β 1-induced changes in EMT markers in LA-4 cells (Fig. 7a). This inhibitory effect was not observed with conditioned media derived from co-culture with control or necrotic Jurkat cells.
Similarly, treatment of BMDM with COX-2 or Rho kinase inhibitors or LA-4 cells with EP4, DP1, DP2, or c-Met antagonists reversed the conditioned medium-induced reduction of TGF-β 1-induced EMT markers at both the gene (Supplementary Fig. S9a-d) and protein level in LA-4 cells (Fig. 7b-e). The EP2 antagonist only reversed the E-cadherin mRNA and protein level reduction (Supplementary Fig. S9c; Fig. 7d). However, 15-lipoxygenase inhibitor PD146176 treatment of BMDM did not reverse the conditioned medium-induced reduction of TGF-β 1-induced EMT markers in LA-4 cells (Supplementary Fig. S9a; Fig. 7b). These data indicate that multiple bioactive mediators, such as COX-2/PGE 2 and /PGD 2 and RhoA signal-dependent HGF, block the EMT process in LA-4 cells via their specific receptors.
In vivo exposure to apoptotic cells prevents the EMT phenotype. Next, we examined the effect of apoptotic cells on EMT in the bleomycin-induced lung injury murine model. At 14 d following bleomycin treatment, primary AT II cells from mice exposed to apoptotic Jurkat cells changed morphology from an elongated  fibroblast-like shape to a typical rounded shape (Fig. 8a), and reduced E-cadherin mRNA and enhanced mesenchymal markers, such as vimentin and α-SMA, were reversed (Fig. 8b). However, instillation of viable Jurkat cells had no effect on EMT markers or morphology.
To confirm the EMT inhibitory effect of apoptotic cell instillation in alveolar epithelial cells after bleomycin treatment, we performed double immunofluorescence staining for α -SMA and fibroblast-specific protein-1 (FSP1) on lung tissue. E-cadherin/FSP1 double-positive cells, which reflect epithelial origin and a possible intermediate EMT-transition stage, were identified (Fig. 8c). Approximately 45% of the FSP1 positive fibroblasts were derived from lung epithelium 14 days after bleomycin treatment (Fig. 8d), indicating an apparent EMT phenomenon in mice following bleomycin treatment. Interestingly, both the number of epithelial-derived fibroblasts and FSP1 expression were reduced following apoptotic cell instillation (~26 % double-positive cells). Approximately 42% of the myofibroblasts expressing α -SMA in the interstitium co-localized with FSP1 (Fig. 8e,f). Apoptotic cell instillation decreased these markers and the number of double-positive cells in lung sections at 21 days after bleomycin treatment (~24% double-positive cells). Taken together, these data provide in vivo evidence that apoptotic cell instillation can prevent EMT and fibroblast activation in murine bleomycin-induced pulmonary fibrosis.
Previously, we demonstrated that COX-2 and HGF mRNA expression in alveolar macrophages and PGE 2 , and HGF production in BAL fluid from mice exposed to apoptotic Jurkat cells persistently enhanced up to 21 days after bleomycin treatment 30,31 . In the present study, we also found PGD 2 production in BAL fluid was enhanced following apoptotic cell instillation at 14 days after bleomycin treatment ( Supplementary Fig. S10a). We further investigated the role of macrophages for anti-EMT effects induced by apoptotic cell instillation. PGE 2 , PGD 2 , and HGF levels in alveolar macrophages taken from the bleomycin + apoptotic cell group at 14 days after bleomycin treatment were greater than those from the group of bleomycin + saline or viable Jurkat cells ( Supplementary Fig.  S10b-d). These data suggest that alveolar macrophages play a critical role for production of bioactive molecules in response to in vivo exposure to apoptotic cells, mediating anti-EMT process in alveolar epithelial cells. To confirm that PGE 2 -, PGD 2 , or HGF pathway is involved in anti-EMT effects induced by apoptotic cell instillation, the EP4, EP2, DP1, or c-Met antagonist was co-administered with apoptotic cells into the bleomycin-stimulated lungs. The enhanced E-cadherin, and the reduced vimentin, FSP1 and α -SMA mRNA and protein abundance in lung tissue by apoptotic cell instillation on days 14 or 21 after bleomycin treatment was reversed by AH-6809, AH-23848, BW-A868C ( Supplementary Fig. S10e,g), or PHA-665752 treatment ( Supplementary Fig. S10f,h). However, EP2 (AH-6809) showed inhibitory effect only on the enhanced E-cadherin mRNA and protein abundance by apoptotic cell instillation at 14 days after bleomycin treatment ( Supplementary Fig. S10e,g). These data strongly suggest that PGE 2 , PGD 2 , and HGF's pathways could mediate anti-EMT effects induced by exposure to apoptotic cells in vivo.

Discussion
Programming of macrophages by apoptotic cells may influence epithelial cell homeostasis during lung injury 28,42 . Recent studies provide evidence that TGF-β 1-induced EMT of alveolar epithelial cells may contribute to the formation of myofibroblasts in murine fibrotic lungs and IPF patient lungs [43][44][45][46][47][48] . In the present study, we used in vitro models of TGF-β 1-induced EMT in alveolar epithelial cells to determine whether interaction of phagocytes with apoptotic cells results in an anti-EMT effect. We first demonstrated that ApoJ-exposed CM inhibited TGF-β 1-induced EMT in LA-4 cells and primary mouse AT II cells as well as HEK-293 kidney epithelial cells. This inhibition in LA-4 cells was also observed when RAW 264.7 cells were exposed to different apoptotic cell types, such as LA-4 epithelial cells or primary mouse thymocytes, indicating a universal phenomenon that is cell-type independent. The TGF-β 1-induced EMT inhibition did not occur when LA-4 cells were incubated with viable-exposed or necrotic-exposed CM, indicating specificity for apoptotic cells. Importantly, this anti-EMT effect on LA-4 cells was confirmed using conditioned media derived from primary murine BMDM exposed to apoptotic Jurkat cells. Collectively, these data suggest that anti-EMT effects are primarily caused by trophic factors released by macrophages, acting in a paracrine manner on lung and kidney epithelial cells.
The expression of EMT core regulators, including Snail, Zeb, and Twist, is induced in response to TGF-β 1 through both Smad-dependent and -independent mechanisms 49 . Upon activation, these transcription factors repress epithelial marker gene expression and concomitantly activate mesenchymal gene expression. We demonstrated that ApoJ-exposed CM, but not NecJ-exposed CM, substantially suppressed TGF-β 1-induced mRNA expression of Snail1/2, Zeb1/2, and Twist1 in LA-4 cells. We further demonstrated that biological mediators in the ApoJ-exposed CM antagonize TGF-β 1 signaling by partially blocking intracellular Smad-independent signaling pathways, such as p38 MAP kinase and PI3k/Akt pathways, but not Smad-dependent signaling pathways. These data indicate that downregulation of Smad-independent signaling leads to the inactivation of transcription factors that bind to Snail1/2, Zeb1/2, and Twist1 promoters. However, because the inhibition of the p38 MAP kinase and PI3k/Akt pathways was not complete, another pathway may also be involved in controlling TGF-β 1-induced EMT progression in LA-4 cells Prostaglandins, including PGE 2 , PGD 2 , PGI 2 , PGF 2α , and thromboxane A 2 (TXA 2 ), are derived from arachidonic acid by constitutive COX-1 and inducible COX-2. Interestingly, the PGE 2 , PGD 2 , and PGI 2 synthesis PHA-665752). (f) PGE 2 (50 and 150 pg/ml), PGD 2 (7 and 17 pg/ml), or HGF (150 and 400 pg/ml) was added to LA-4 cell culture in the presence of TGF-β 1 for 48 h. (a) Morphological changes in the cells were examined by phase-contrast microscopy (Scale bars = 50 μ m). (b,c,e,f) Immunoblots of total cell lysates were performed with anti-E-cadherin, N-cadherin, or α -SMA antibodies. Right: Densitometric analysis of the indicated EMT markers' relative abundances. (d) The amounts of Snail1/2, Zeb1/2, and Twist1 mRNAs in LA-4 cell samples were analyzed by real-time PCR and normalized to that of Hprt mRNA. Values represent the mean ± s.e.m. of three independent experiments. *P < 0.05 compared with control; + P < 0.05 as indicated.
Scientific RepoRts | 6:20992 | DOI: 10.1038/srep20992   E-cadherin, vimentin, and α-SMA (b). The mRNA abundance of these EMT levels are enhanced in RAW 264.7 cells in response to apoptotic cells 14 . Data from our previous and present studies confirmed that COX-2-derived PGE 2 and PGD 2 are released from RAW 264.7 cells in response to apoptotic cells 40 . In addition, RAW 264.7 and primary peritoneal macrophages induce HGF production via RhoA/ Rho kinase-dependent signaling in response to apoptotic cells 41 . While somewhat controversial, COX-2/PGE 2 and HGF were demonstrated to be negative modulators of fibrotic remodeling-associated EMT 32,50,51 . Thus, we focused on demonstrating the role of these molecules in anti-EMT effects induced by ApoJ-exposed CM. Both pharmacologic inhibition of COX-2 activity and siRNA-mediated knockdown of COX-2 expression in RAW 264.7 cells 40 reversed the reduction of TGF-β 1-induced EMT by ApoJ-exposed CM in LA-4 cells. However, knockdown of COX-1 expression did not influence the anti-EMT effect induced by ApoJ-exposed CM. These findings suggest that COX-2, but not COX-1, is specifically involved in the anti-EMT effect induced by the interaction of macrophages with apoptotic cells.
Treatment with exogenous PGE 2 and PGD 2 , but not other prostaglandins, including PGI 2 , PGF 2α , and TXA 2 , has been demonstrated to inhibit TGF-β -induced EMT in renal epithelial cells 32,39 . Moreover, exogenous PGE 2 has been shown to suppress TGF-β -induced fibronectin and type 1 collagen α 2 (Col1A2) expression in human lung cancer A549 cells 50 . Considering the effect of EP1/2/3/4 agonists, the suppressive effect of PGE 2 on TGF-β 1-induced EMT may be mediated through the EP2 and EP4 receptors 50 . In our current study, the EP2 antagonist AH-6809 only partially reversed the reduction of TGF-β 1-induced EMT in LA-4 cells, whereas the antagonists of EP4, DP1, and DP2 reversed the effects on mesenchymal and epithelial markers as well as the transcriptional modulators Snail1/2, Zeb1/2, and Twist1. The importance of the PGE 2 -EP4 axis in limiting TGF-β 1-induced EMT has also been demonstrated using renal tubular epithelial cells isolated from wild-type and EP4 −/− mice 52 .
Previously, we reported that the interaction of apoptotic cells with macrophages upregulates COX-2/PGE 2 and HGF expression via a positive feedback loop in vitro and in vivo 40,53 . Interestingly, a COX-2-specific inhibitor, SC-59635, partially blocked the restoration of the epithelial phenotype, E-cadherin expression, and inhibition of α -SMA expression by exogenous HGF, indicating that COX-2 mediates the action of HGF 32 . Here, we demonstrated that blocking HGF production in macrophages using a Rho kinase inhibitor (Y-27632) or RhoA siRNA, or disrupting endogenous HGF signaling in LA-4 cells using a c-Met inhibitor (PHA-665752) strongly inhibited the reduction of TGF-β 1-induced EMT markers by the conditioned medium. Different molecular mechanisms for HGF's interference with EMT have been identified in different cell models. In renal tubular epithelial cells, HGF was shown to upregulate expression of the Smad co-repressor SnoN, which interacts with the translocated Smad complex and blocks the expression of Smad-dependent genes including integrin-linked kinase 33,35 . However, upregulation of SnoN protein levels by HGF was not observed in rat lung epithelial cells 34 . Instead, HGF mediated the upregulation of Smad7, an inhibitor of TGF-β signaling, in an ERK1/2-dependent manner. We confirmed that COX-2-derived PGE 2 and PGD 2 and HGF from primary murine BMDM exposed to apoptotic Jurkat cells acts in a paracrine manner to reduce TGF-β 1-induced EMT in LA-4 cells via their receptors, particularly EP4, DP1, DP2, and c-Met. Moreover, exogenous PGE 2 , PGD 2 , or HGF at the stimulated concentrations of 150, 17, and 400 pg/ ml, respectively, reduced TGF-β 1-induced EMT in LA-4 cells 40,41 . Interestingly, addition of all these mediators together at the stimulation concentrations to LA-4 cells did not result in synergistic effects. These findings support the concept that they may have the same target molecules to inhibit TGF-β 1-induced signaling pathway. Indeed, in consistent with ApoJ-exposed CM, exogenous PGE 2 , PGD 2 , or HGF blocked Smad-independent TGF-β 1 signaling, including the p38 MAP kinase and Akt pathways, but did not affect Smad-dependent TGF-β 1 signaling. The rodent bleomycin model of lung fibrosis allows the use of molecular tools to dissect the cellular and subcellular processes leading to fibrosis 54 . Our previous data emphasize that in vivo exposure to apoptotic cells has an anti-fibrotic effect, as assessed using lung hydroxyproline measurements and trichrome staining in the bleomycin-induced murine model 31,32 . In addition, we also demonstrated the reduction of fibroproliferation matrix markers, such as Col1A2 and fibronectin, and α -SMA protein in lung tissue following apoptotic cell instillation at days 14 and 21 after bleomycin treatment 53 . Our previous and present data demonstrated gradual enhancement of PGE 2 , PGD 2 , and HGF production in BAL fluid following apoptotic cells after bleomycin treatment 30,31 . In the present study, consistent with in vitro findings, PGE 2 , PGD 2 , and HGF levels were further enhanced in alveolar macrophages following in vivo exposure to apoptotic cells at 14 days after bleomycin treatment. These data suggest that alveolar macrophages are actively involved in anti-EMT process under in vivo condition. Furthermore, we found that apoptotic cell instillation in isolated AT II cells from mouse lungs 14 days after bleomycin treatment inhibited the EMT process, including morphological changes and mRNA expression of EMT markers, such as decreased E-cadherin and increased vimentin, and α-SMA. Apoptotic cell instillation may alter the induction of EMT and the differentiation capacity of lung fibroblasts in the alveolar interstitial space following bleomycin treatment, because immunofluorescence staining indicates reduced numbers of E-cadherin/FSP1 and α -SMA/FSP1 double-positive cells following apoptotic cell instillation. In consistent with markers was analyzed by real-time PCR in primary AT II cells from each group on day 14 after BLM treatment. Values represent the mean ± s.e.m. from five mice in each group. *P < 0.05 compared with control; + P < 0.05 as indicated. Immunofluorescence staining for E-cadherin (red), α -SMA (red), or fibroblast-specific protein-1 (S100A4, green) was performed in lung sections on days 14 (c) or 21 (e) following BLM treatment. Arrowheads indicate co-localization of E-cadherin or α -SMA in lung fibroblasts. The imaging medium was Vectashield fluorescence mounting medium containing DAPI. (c,e) Scale bars = 20 μ m. Representative images were obtained from three mice in each group. Graph representing the number of double positive cells of S100A4 and E-cadherin or α -SMA compared with the total S100A4 positive cell population in lung parenchyma on days 14 (d) or 21 (f) after bleomycin treatment. Mean of 5 high power fields per section ± s.e.m. from three mice in each group. *P < 0.05 compared with control; + P < 0.05 for BLM + ApoJ vs. BLM + Sal.
Scientific RepoRts | 6:20992 | DOI: 10.1038/srep20992 in vitro findings, inhibition of PGE 2 , PGD 2 , and HGF's pathways by co-administration of their specific receptor antagonists could also reverse the enhanced E-cadherin, and the reduced vimentin, FSP1 and α -SMA mRNA and protein abundance in lung tissue by exposure to apoptotic cells in vivo on days 14 or 21 after bleomycin treatment. Taken together, these data provide in vivo evidence that enhanced PGE 2 , PGD 2 , and HGF secretion in alveolar macrophages following apoptotic cell instillation protects against the EMT phenotype in AT II cells and fibroblast activation in murine bleomycin-induced lung fibrosis.
In summary, we have elucidated a novel role for macrophage interaction with apoptotic cells in antagonizing the TGF-β 1-induced EMT process via the signaling of multiple secreted bioactive mediators, including the COX-2-derived PGE 2 and PGD 2 and RhoA-dependent HGF. In addition, this study provides new insights into a possible mechanism for the anti-fibrotic effect of apoptotic cell instillation after bleomycin treatment. This effect is due to apoptotic cells' interference with EMT in alveolar epithelial cells, which would otherwise lead to fibroblast activation and ECM deposition. Considering that EMT is a major event in the pathogenesis of IPF and other fibrotic diseases in multiple organs, including the kidney, liver, and peritoneum, our data suggest that apoptotic cells could be used to develop effective new cell therapies to limit pathologic fibrosis in diverse organ diseases. Cell lines, culture, and stimulation. Murine RAW 264.7 macrophages (American Type Culture Collection (ATCC), Manassas, VA) were plated at a density of 10 5 cells/ml and incubated overnight in Dulbecco's modified Eagle's medium (DMEM, Media Tech Inc., Washington, DC, USA) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine,100 U/ml penicillin, and 100 μ g/ml streptomycin at 37 °C and 5% CO 2 . Prior to stimulation, the medium was replaced with serum-free X-VIVO medium (Lonza, Basel, Switzerland). The human leukemia T cell line, Jurkat, was obtained from ATCC and cultured in RPMI 1640 (Media Tech Inc.) containing 10% FBS. LA-4 cells were purchased from ATCC and grown in F12K medium (Lonza, Basel, Switzerland) containing 15% heat-inactivated FBS at 37 °C in 5% CO 2 .

Reagents
Isolation and Culture of Primary Cells. BMDM were differentiated from bone marrow myeloid stem cells of C57BL/6 mice as described previously 55 . After 7-10 days in culture with L929 complement DMEM, BMDM differentiation was confirmed by FACS analysis using anti-CD11b. Individual thymocytes were isolated from 3-to 4-week-old BALB/c mice by mincing the thymus through a 70-μ m pore size cell strainer (BD Biosciences, Bedford, MA, USA). Primary murine AT II cells were isolated from BALB/c mice and purified using a modification of published methods 56,57 . The purity of AT II cells was typically > 90%, as assessed using pro-SP-C immunofluorescence staining.
Induction of cell death. Human Jurkat T lymphocytes, LA-4 lung epithelial cells, and murine thymocytes were exposed to ultraviolet irradiation at 254 nm for 10 min followed by incubation in RPMI-1640 with 10% FBS for 2 h at 37 °C and 5% CO 2. Evaluation of nuclear morphology using light microscopy on Wright-Giemsa-stained samples indicated that the irradiated cells were approximately 70-80% apoptotic 40 . Lysed (necrotic) Jurkat T cells were obtained by multiple freeze-thaw cycles 38 . Apoptosis and necrosis were confirmed by Annexin V-FITC/ propidium iodide (BD Biosciences, San Jose, CA) staining followed by flow cytometric analysis on a FACSCalibur system (BD Biosciences) 28 . Incubation of epithelial cells with conditioned medium. LA-4 and HEK-293 cells were plated in 6-well culture plates (2 × 10 5 cells/well) and cultured overnight in 200 μ l RPMI 1640 or DMEM, respectively, containing 10% FBS. Primary AT II cells were plated and cultured on type 1 collagen-coated culture plates (1 × 10 6 cells/well) for 48 h. Cells were treated for 24-72 h with conditioned medium from macrophages in the presence of 10 ng/ml TGF-β 1 28 . In some experiments, 10 μ M AH-6809, AH-23848, BW-A868C, or BAY-u3405 or 250 nM PHA-665752 was used to antagonize EP2, EP4, DP1, DP2, or c-Met, respectively. The antagonist was added 1 h before the addition of the conditioned medium with 10 ng/ml TGF-β 1.

Immunoblot analysis.
To detect the expression of epithelial and mesenchymal markers, cells were lysed in 0.5% Triton X-100-containing lysis buffer and resolved on a 10% SDS-PAGE gel prior to transfer onto nitrocellulose. Membranes were blocked at room temperature with Tris-buffered saline containing 3% BSA and then incubated at room temperature with various anti-mouse primary antibodies and probed with mouse anti-mouse HRP-conjugated secondary antibody. Bands were visualized using enhanced chemiluminescence.
Enzyme-linked immunosorbent assay (ELISA) measurement. Culture supernatants were collected and PGD 2 concentration was measured using an EIA kit according to the manufacturer's instructions.
Quantitative real-time PCR (qPCR). Gene expression was analyzed by real-time qPCR on a StepOnePlus system (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). For each qPCR assay, a total of 50 ng cDNA was used. Primer sets for PCR-based amplifications were designed using Primer Express software. The primers used were as follows (name: forward primer, reverse primer). Immunocytochemistry. Cells were grown on coverslips and cultured for the indicated time and conditions, followed by fixation with 2% formaldehyde (formalin) for 10 min. Cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) and stained with goat antibody against mouse E-cadherin (Abcam), α -SMA (Abcam), or goat IgG isotype control (all from Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Cells were stained with rabbit polyclonal anti E-cadherin antibody (1:250 dilution) and mouse monoclonal anti-α -SMA antibody (1:250 dilution). Subsequently, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (1:400; Jackson ImmunoReseach) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (Molecular Probes, Life Technologies). The slides were mounted with Vectashield Mounting Medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA). All slides were imaged with a confocal microscope (LSM5 PASCAL, Carl Zeiss).
Quantification of elongated cell morphology. Morphological changes in the cells examined by phase-contrast microscopy were quantitated by measuring the lengths of the major and minor cell axes using Zeiss software (Zeiss LSM Image Browser 59 . The ratios of the major axis to the minor axis of cells were used to determine the degree of elongated cell morphology. For each experiment, at least 30 cells of each cell type were measured. Data were statistically analyzed using analysis of variance followed by Tukey's post hoc test. Animal protocols. Specific pathogen-free male C57BL/6 mice (Orient Bio, Sungnam, Korea) weighing 20~25 g were used for all experiments. The Animal Care Committee of the Ewha Medical Research Institute approved the experimental protocol (N0. 11-0176). Mice were cared for and handled in accordance with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals. Mouse pharyngeal aspiration was used to administer a test solution containing bleomycin (5 U/kg body weight in 30 μ l) 42,60 . Two days after bleomycin treatment, saline alone or 1 × 10 7 apoptotic or viable Jurkat cells in 50 μ l saline were administered intratracheally through pharyngeal aspiration 61,62 . Mice were euthanized on days 14 and 21 following bleomycin treatment.
Bronchoalveolar lavage (BAL) cells, lung tissue, and cell counts. BAL was performed through a tracheal cannula using 0.7 ml aliquots of ice-cold Ca 2+ /Mg 2+ -free phosphate-buffered medium (145 mM NaCl, 5 mM KCl, 1.9 mM NaH 2 PO 4 , 9.35 mM Na 2 HPO 4 , and 5.5 mM dextrose; pH 7.4) to a total of 3.5 ml for each mouse. BAL samples were centrifuged at 500 × g for 5 min at 4 °C, and cell pellets were washed and resuspended in phosphate-buffered medium. Cell counts were determined using an electronic Coulter Counter fitted with a cell sizing analyzer (Coulter Model ZBI with a channelizer 256; Coulter Electronics, Bedfordshire, UK). Alveolar macrophages were identified by their characteristic cell diameters. After BAL, lungs were removed, immediately frozen in liquid nitrogen, and stored at − 70 °C.
Preparation of alveolar macrophages. Alveolar macrophages were isolated as described previously, with slight modifications 16,32 . In brief, suspended alveolar macrophages were over 95% viable as determined by trypan blue dye exclusion. Alveolar macrophages (5 × 10 5 per well in 12-well plates) were cultured in serum-free X-vivo medium for 60 min. Nonadherent cells were removed by washing three times before isolation of total RNA. Approximately 90-95% of the plastic-adherent cells were morphologically macrophages.
Enzyme-linked immunosorbent assay (ELISA). PGE 2 , PGD 2 , and HGF were measured from the 18 h supernatants of cultured alveolar macrophages or BAL fluid using a specific ELISA kit as per the manufacturer's instructions (R&D Systems, Minneapolis, MN).
Immunohistochemistry. Four-μ m-thick sections were obtained from formalin-fixed, paraffin-embedded tissues. Slides were deparaffinized twice in xylene and rehydrated through graded ethanol solutions to distilled water. Sections were incubated with primary antibodies against E-cadherin, α -SMA, FSP1, or control rabbit IgG at room temperature for 30 min. Cells were incubated with Texas Red-conjugated anti-mouse secondary antibody and FITC-conjugated goat anti-rat secondary antibody (Vector Laboratories, Inc.). The sections were washed with Tris-buffered saline between all steps. The sections were mounted in Vectashield Mounting Medium with DAPI. All slides were imaged using a confocal microscope.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
The role of doxycycline in the therapy of multidrug-resistant E. coli – an in vitro study

This study assessed the in vitro antibacterial activity of combinations of amikacin and doxycycline or tigecycline against multidrug-resistant E. coli isolates. Twenty-four different pulsotypes, including 10 extended-spectrum β-lactamase (ESBL)-, 10 carbapenem-resistant, 2 New Delhi Metallo-beta-lactamase (NDM)- and 2 Klebsiella pneumoniae carbapenemase (KPC)-E. coli isolates were collected. All 24 isolates were susceptible to amikacin and tigecycline. Only 30% of ESBL and 50% of carbapenem-resistant E. coli were susceptible to doxycycline. Both of the NDM-E. coli had a MIC of 64 μg/ml. The checkerboard method showed that for the ESBL- and carbapenem-resistant E. coli, the synergistic effects of amikacin/doxycycline were 80% and 90%, respectively. For the two KPC- and two NDM-E. coli, the FIC index of amikacin/doxycycline were 0.5/0.375 and 0.5/0.281, respectively. For the ESBL- and carbapenem-resistant E. coli isolates, the combinations of amikacin and doxycycline exhibited synergistic activities against 80%, and 80% and 10% vs 60%, and 80% and 10% of the isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. The synergistic effect seems to be similar for doxycycline and tigecycline based combinations with amikacin. In conclusion, the antibacterial activity of doxycycline can be enhanced by the addition of amikacin and is observed against most multidrug-resistant E. coli isolates.

The in vitro antibacterial activity of antibiotic combinations assessed by the broth method.
The in vitro determination of the inhibitory effect of combination regimens followed the time-killing method was defined by the CLSI 17 . In brief, bacterial suspensions were diluted to concentrations 5.0× 10 5 colony-forming units (CFU)/mL in fresh Mueller-Hinton broth. Drug concentrations of amikacin, tigecycline and doxycycline were adjusted to those of 1xMIC, 1/2xMIC, and 1/4xMIC. Each drug alone and the combination of amikacin and tigecycline or doxycycline were tested. Bacterial counts were measured at 24 h by enumerating the colonies in 10-fold serially diluted specimens of 100 μ L aliquots plated on the nutrient agar (Difco Laboratories, Sparks, MD) at 37 °C.
Definitions Synergy was defined as a ≥ 2-log 10 decrease in the CFU/ml between the combination and its most active constituent after 24 h and the number of surviving organisms in the presence of the combination must be ≥2 log 10 CFU/ml below the starting inoculum. Bacteriostatic activities were defined as the presence of ≥ 2 log 10 , but < 3 log 10 reductions, and bactericidal activities were defined as the presence of ≥ 3 log 10 reductions in the CFU/mL at 24 h, relative to the initial inoculum 17 . All experiments were performed in duplicate.
The in vitro antibacterial activity of antibiotic combinations assessed by the checkerboard method. To evaluate the effect of the combinations, the fractional inhibitory concentration (FIC) was calculated for each combination by the broth microdilution technique as recommended by the CLSI and as previously described 14,18,19 . Briefly, the 96-well microdilution plates were inoculated with each test organism to yield the appropriate density (10 5 CFU/ml) in 100 μ l of Mueller-Hinton broth (MHB) and incubated at 35 °C in ambient air for 24 h. One well with no antibiotic was used as a positive growth control on each plate. The plates were read for visual turbidity, and the results were recorded at 35 °C in ambient air using a magnifying mirror reader after 24 h of incubation, as turbidity in the wells indicated the growth of the microorganism. The MIC was determined as the well in the microtiter plate with the lowest drug concentration at which there was no visible growth. The following formulas were used to calculate the FIC index: FIC of drug A = MIC of drug A in combination/MIC of drug A alone, FIC of drug B = MIC of drug B in combination/MIC of drug B alone, and FIC index = FIC of drug A + FIC of drug B. Synergy was defined as a FIC index ≤ 0.5, indifference was defined as a FIC index > 0.5 but ≤ 4, and antagonism was defined as a FIC index > 4 20 . All drug combinations were performed repeatedly to validate the data.
The detection of β-Lactamase genes. Plasmid DNA was extracted as templates and polymerase chain reaction (PCR) was used to amplify CTX-M, TEM, IMI, IMP, VIM, KPC, OXA and NDM using specific primers as previously published [21][22][23] . For AmpC genes, the following primers were used: (a) CMY-2-forward (TTT TCA AGA ATG CGC CAG GC), CMY-2-reverse (CTG CTG CTG ACA GCC TCT TT); and (b) DHA-1-forward (CTG ATG AAA AAA TCG TTA TC) and DHA-1-reverse (ATT CCA GTG CAC TCA AAA TA). For SHV genes, the following primers were used: (a) SHV-forward (GAT CCA CTA TCG CCA GCA GG) and SHV-reverse (ACC ACA ATG CGC TCT GC TTT G); and (b) SHV-12-forward (ATG CGT TAT ATT CGC CTG TG) and SHV-12-reverse (TTAGCGTTGCCAGTGCTCG). Amplicons were purified with PCR clean-up kits (Roche Diagnostics, GmbH, Penzberg, Germany) and were sequenced on an ABI PRISM3730 sequencer analyzer (Applied Biosystems, Foster City, CA, USA). Pulsed-field gel electrophoresis. PFGE was performed as described previously 24 with a CHEF DR II apparatus (Bio-Rad Laboratories, Hercules, Calif.). In brief, the DNA in the plugs was digested with XbaI, and electrophoresis was performed in a 1% agarose gel (in 0.5x TBE [Tris-borate-EDTA] buffer). The electrophoretic conditions used were as follows: initial switch time, 2.0 s; final switch time, 35.0 s; run time, 21 h; gradient, 6 V/cm; angle, 120°; and temperature, 14 °C. The bacteriophage lambda ladder pulsed-field grade (PFG) and low-range PFG molecular weight markers were loaded onto all gels. The PFGE patterns were visually examined and interpreted according to the criteria of Tenover et al. 25 . The similarities of the PFGE profiles of each strain were compared using a Dice coefficient at 1.0% of tolerance and 0.8% of optimization. Figure 1 shows the PFGE profile of the enrolled 10 ESBL-, 10 carbapenem-resistant-, two KPC-2 producing and 2 NDM-E. coli isolates (one was NDM-1, and the other was NDM-5), and all of them had different PFGE profiles. Table 1 shows their MIC values and the susceptible rates of amikacin, doxycycline, and tigecycline. All of the 24 E. coli isolates were susceptible to amikacin and tigecycline. However, only 30% of ESBL E. coli and 50% of carbapenem-resistant E. coli were susceptible to doxycycline. For doxycycline, both of the NDM positive E. coli had MICs of 64 μ g/ml, and in contrast, both KPC-producing E. coli had MIC values ≤ 2 μ g/ml.

Results
The ESBL and carbapenemase genes detected among the clinical isolates are presented in Table 2. For ten ESBL E. coli isolates, genes encoding CTX-M were detected for all isolates. Additionally, genes encoding TEM and CMY were detected for three and two isolates, respectively. For ten carbapenem-resistant E. coli isolates, genes encoding CMY were detected for all isolates. However, genes encoding CTX-M and TEM were detected for four and two isolates, respectively. For two KPC-producing E. coli isolates, genes encoding CMT, TEM, and CTX-M were detected for one isolate. For two NDM positive E. coli isolates, both had the gene encoding CMT and TEM, and one had the KPC-2 gene.
The results of the checkerboard methods are shown in Table 3. For the ESBL E. coli, the FIC 50/90 of doxycycline and the tigecycline-based combination were 0.375/0.563 and 0.5/0.563, respectively. The synergistic effects of amikacin/doxycycline and amikacin/tigecycline were 80% and 60%, respectively. For carbapenem-resistant E. coli, the FIC 50/90 of doxycycline and the tigecycline-based combination were 0.375/0.5, and 0.5/0.563, respectively. The synergistic effects of amikacin/doxycycline and amikacin/tigecycline were 90% and 80%, respectively. For both, there was no antagonism among the two combinations. For the two KPC E. coli and the two NDM E. coli, the FIC index values of amikacin/doxycycline were 0.5/0.375 and 0.5/0.281, respectively, and the FIC index values of amikacin/tigecycline were 0.375/0.5 and 0.265/0.312, respectively.
The in vitro activities of the combination of amikacin and doxycycline at the drug concentrations of 1xMIC, 1/2xMIC and 1/4x MIC against each isolate are shown in Table 4. For ESBL E. coli, the reduction of CFU at 24 hours ranged from 2.99 to 4.2, 0.05-4.2, and 0.29-4.08 log 10 , at concentrations of 1x, 1/2x and 1/4xMIC, respectively. The combinations of amikacin and doxycycline exhibited bactericidal effects against 90%, 70%, and 10% of the tested isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. These combinations were synergistic against 80%, 80%, and 10% of the isolates at the concentrations of 1x, 1/2x and 1/4xMIC, respectively. For carbapenem-resistant E. coli isolates, the reduction of CFU at 24 hours ranged from 0. 28    and 0.73-2.00 log 10, at concentrations of 1x, 1/2x and 1/4xMIC, respectively. The combinations of amikacin and doxycycline exhibited bactericidal effects against 90%, 50%, and 10% of the tested isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. These combinations were synergistic against 60%, 80%, and 10% of the isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. For KPC E. coli, at the concentration of the 1x MIC combination, one of two strains had a synergistic effect, and the reduction of the CFU at 24 hours compared to the initial inoculum was 3.79 log 10 and was − 3.94 compared to most active antibiotic. At the concentration of 1/2x MIC, both strains had synergistic effects, and the reduction of the CFU at 24 hours compared to the initial inoculum was 3.79/2.10 log 10 and was 6.45/4.49 compared to the most active antibiotic. Both strains had synergistic effects.
The NDM strain combinations of amikacin and doxycycline were not performed because the MIC of doxycycline was too high.
The in vitro activities of combinations of amikacin and tigecycline at the drug concentrations of 1xMIC, 1/2xMIC and 1/4x MIC against each isolate are also shown in Table 4. For ESBL-E. coli, the reduction of CFU at 24 hours ranged from 2.18 to 3.72, 1.77-3.72 and 0.87-3.68 log 10, at concentrations of 1x, 1/2x and 1/4xMIC, respectively. The combinations of amikacin and tigecycline exhibited bactericidal effects against 90%, 90%, and 20% of the tested isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. These combinations were synergistic against 50%, 100%, and 20% of the isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. For carbapenem-resistant E. coli, the reduction of CFU at 24 hours ranged from 1.12 to 3.82, 2.00-3.82 and 0.30-1.56 log 10, at concentrations of 1x, 1/2x and 1/4xMIC, respectively. The combinations of amikacin and tigecycline exhibited bactericidal effects against 90%, 70%, and 0% of the tested isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. These combinations were synergistic against 30%, 100%, and 0% of the isolates at concentrations of 1x, 1/2x and 1/4xMIC, respectively. For KPC E. coli, at the combined concentrations of 1x MIC and 1/4 x MIC, both two strains have no synergistic effect. At the combined concentration of 1/2x MIC, both strains had synergistic effects and with a value of 2.56/6.15 log 10 compared to most active antibiotic. The reduction of CFU at 24 hours compared to the initial inoculum was 3.73/3.53 log 10 , exhibiting a bactericidal effect. One of the two NDM strains at the combined concentration of 1x MIC had a synergistic effect, and the reduction of CFU at 24 hours compared to the initial inoculum was 4.00 log 10 was − 2.38/0 log 10 compares to the most active antibiotic. At the combined concentration of 1/2x MIC, both strains had a synergistic effect, and the reduction of CFU at 24 hours compared to the initial inoculum was 4.00/3.68 log 10 and was 6.45/6.58 log 10 compared to the most active antibiotic. However, no synergistic effect was noted at the combined 1/4 x MIC.

Discussion
Antibiotic combination therapy has become the possible resolution for the treatment of severe multidrug resistant organism infections, and various antibiotic combination regimens for treating multidrug resistant E. coli have been recommended based on in vitro and in vivo studies. However, research investigating the in vitro antibacterial activity of the combinations of an aminoglycoside (amikacin) and tigecycline or doxycycline against multidrug-resistant E. coli isolates is scarce. This is the first study to assess this type of combined antibiotic regimen against multidrug-resistant E. coli, including ESBL-, carbapenem-resistant, NDM-and KPC-producing E. coli isolates. Based on this in vitro study, we have several significant findings. Most important, although tigecycline and amikacin displayed greater in vitro activities against multidrug-resistant E. coli than doxycycline, the synergistic effect seems to be similar between the combination of doxycycline and amikacin and the combination of tigecycline plus amikacin. As doxycycline is safe, inexpensive, and almost universally availability, further large in vitro and in vivo studies are warranted to clarify its role as a new adjunctive therapy to improve the outcomes of multidrug-resistant E. coli infections.
Although doxycycline is a cheap antimicrobial agent, it exhibits a broad spectrum of activity against different pathogens, including Gram-negative bacteria, and remains as a useful or even drug of choice in the treatment of many infectious diseases 26,27 . Even in this era of the increasing prevalence of multidrug-resistant organism infections, doxycycline is efficacious against multidrug-resistant A. baumannii 28 , Pseudomonas aeruginosa 29 , and Stenotrophomonas maltophilia 30 . In this first study investigating the in vitro activity of doxycycline against multidrug-resistant E. coli, we found that most clinical isolates, including seven (70%) ESBL-, five (50%) carbapenem-resistant and two (100%) NDM -E. coli, were not susceptible to doxycycline. However, even sub-inhibitory concentrations of an aminoglycoside combined with doxycycline can exhibit synergistic activities against more than 80% of tested isolates. For this combination, using 1/2xMIC of doxycycline (2 μ g/mL, which is achievable in serum) produces the best synergism 7,31 . Therefore, our findings indicate the potential role of doxycycline-containing combinations in the management of multidrug-resistant E. coli infections.
Tigecycline, the first glycylcycline, exhibits potent activity against a wide range of clinically significant gram-positive and gram-negative bacteria, including multidrug-resistant strains (e.g., oxacillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and ESBL-producing Enterobacteriaceae), and  Table 3. Continued anaerobes (e.g., Bacteroides spp) 32 . Like several previous studies [33][34][35] , we found that the MIC values of tigecycline against all tested isolates remained low (≤ 1 μ g/mL), and all of the tested isolates were susceptible to tigecycline. However, in vitro activity does not equate to an in vivo response, and the current suggested dosage of tigecycline for adults only achieves low serum concentrations; therefore, tigecycline cannot be recommend for the treatment of bloodstream infections, even those caused by so called "tigecycline-susceptible" isolates. To overcome this barrier to the treatment of critical conditions and the emergent tigecycline-resistant strains, tigecycline-containing combinations have been proposed as possible solutions. In this study, we found that using 1/2xMIC of tigecycline (0.5 μ g/mL) in combination with sub-inhibitory concentrations of an aminoglycoside, synergism can be achieved for all of the 24 tested isolates. However, if we use 1/4xMICs of tigecycline (0.25 μ g/mL) in combination with 1/4xMICs of an aminoglycoside, synergism was found for only two of the tested isolates. A previous study showed that the serum attainable concentration of tigecycline was only 0.38 and 0.93 μ g/mL after a single dose injection of 50 mg and 100 mg tigecycline, respectively 36 . Therefore, if we formulate tigecycline-containing combination regimens based on the recommended dosages (100 mg loading, followed by 50 mg every 12 h), we can obtain the synergistic effect with tigecycline and amikacin despite low serum levels of tigecycline (< 1 μ g/mL).

The results of the checkerboard method of amikacin-based combinations with doxycycline and tigecycline against 10 extended-spectrum β-lactamase (ESBL)-, 10 carbapenem-resistant-(CRE), 2 New Delhi Metallo-beta-lactamase (NDM)-and 2 Klebsiella pneumoniae carbapenemase (KPC)-producing
In this study, we found an unusual association between NDM-1 and KPC-2 in one E. coli isolates, and it is the first detection of this combination in Taiwan. As previously reported 37 , this isolate should be multi-drug resistant against most antibiotics, excluding tigecycline. Previous studies only found this double carbapenemase-producer in K. pneumoniae, E. cloacae, Citrobacter freundii and Enterobacter hormaechei isolates from Brazil, Pakistan, China, and India [38][39][40][41][42] . However, we did not find the mutation of outer membrane porin (Omp) in KPC or NDM-producing isolates. As previous reports [43][44][45] , we found that the mutation of OmpA, OmpC, or OmpF was only presented in carbapenem-resistant strains. Overall, all of these findings indicate the worldwide emergence of double, or even multiple, carbapenemase-producing bacteria among Enterobacteriacae, including in Taiwan.
Finally, recent studies 46,47 showed that the different resistance mechanisms of multidrug-resistant organisms may influence the synergistic effects of combination therapy. For carbapenem-resistant K. pneumoniae, Laishram et al. 46 found that isolates producing NDM carbapenemase alone showed significantly more synergy than isolates producing OXA-48-like carbapenemase. Furthermore, Hong et al. 47 found that clinical isolates of KPC-producing K. pneumoniae with high porin expression were more responsive to a combination of colistin-doripenm-ertapenem than isolates with low expression (100% [8/8] vs 0% [0/4]; p = 0.002). In this study of limited clinical isolates, we did not assess whether the MDR E. coli with different resistant mechanisms had different responses to antibiotic combination therapy. However, further investigations are warranted to clarify this issue.
In conclusion, despite the lower susceptible rate of doxycycline, the antibacterial activity of such an ancient antimicrobial agent can be enhanced by the addition of amikacin. The synergistic effect of such combinations seems to be as effective as the tigecycline/amikacin combination against most multidrug-resistant E. coli isolates, and warrants further in vivo investigation to confirm their therapeutic efficacy.  Table 4. The log change (log 10 CFU/ml) from the starting inoculum and the most active single agent after 24 h of incubation with different concentrations of antibiotics combinations including 1x, 1/2x and 1/4x MICs of amikacin, doxycycline and tigecycline for 10 extended-spectrum β-lactamase (ESBL)-, 10 carbapenem-resistant-, 2 New Delhi Metallo-beta-lactamase (NDM)-and 2 Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli isolates. -cidal refers to the bactericidal effect and -static refers to the bacteriostatic effect. ND refers to not done. a -cidal refers to bactericidal effect and -static refers to bacteriostatic effect. ND refers to not done.


text:
High tau levels in cerebrospinal fluid predict nursing home placement and rapid progression in Alzheimer’s disease

Background Increased concentrations of cerebrospinal fluid (CSF) total tau (t-tau) and phosphorylated tau, as well as decreased amyloid-β 42 peptide, are biomarkers of Alzheimer’s disease (AD) pathology, but few studies have shown an association with AD progression rate. We hypothesized that high CSF tau, as a marker of ongoing neurodegeneration, would predict a more aggressive course of AD, using time to nursing home placement (NHP) as the main outcome. Methods Our sample inlcuded 234 patients with mild cognitive impairment (MCI) due to AD (n = 134) or mild to moderate AD (n = 100) who underwent lumbar puncture at a memory clinic and were followed for 2–11 years (median 4.9 years). Results Individuals with CSF t-tau in the highest quartile (≥900 ng/L) had a higher ratio of NHP, both in the total cohort and in patients with MCI only (adjusted HR 2.17 [95 % CI 1.24–3.80]; HR 2.37 [95 % CI 1.10–5.09], respectively), than the lowest quartile. The association between high t-tau levels and future steep deterioration was confirmed in analyses with conversion to moderate dementia (HR 1.66; 95 % CI 1.08–2.56), rapid decline in Mini Mental State Examination score (≥4-point drop/12 months), and dying in severe dementia as outcomes. Conclusions To our knowledge, this is the first study to show that high CSF t-tau levels predict early NHP and conversion to moderate dementia in an AD cohort. Selecting patients with high CSF t-tau, indicating more aggressive neurodegeneration and steeper decline, for AD immunotherapy trials might increase the possibility of showing contrast between active treatment and placebo. Electronic supplementary material The online version of this article (doi:10.1186/s13195-016-0191-0) contains supplementary material, which is available to authorized users.

Background
Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. It is characterized by a gradual cognitive deterioration, loss of function in activities of daily living (ADL), poor quality of life, and shorter survival [1]. At the stage of mild cognitive impairment (MCI), patients are still autonomous. Subsequently, mild dementia is followed by moderate dementia, defined as loss of ability to perform personal activities of daily living (PADL). Further disease progression increases the burden on both patients and caregivers and eventually leads to nursing home placement (NHP), with a tremendous impact on societal costs [2]. The rate of progression is highly variable between patients, supposedly reflecting the intensity of the neurodegenerative process [3,4]. It is of utmost importance to learn more about predictors of rapid decline in order to develop treatment aimed at slowing down the disease process.
Aggregation of misfolded amyloid-β (Aβ) is an early pathogenic event in AD. It is believed to mediate the formation of abnormally phosphorylated tau in neurofibrillary tangles [5]. These two processes are reflected by decreased amyloid-β 42 peptide (Aβ42) and increased phosphorylated tau (p-tau) in the cerebrospinal fluid (CSF) [6], while high CSF total tau (t-tau) is a marker of ongoing axonal degeneration [7,8]. These three biomarkers are predictors of conversion from the MCI stage to AD mild dementia; that is, they separate MCI due to AD from MCI non-AD cases on a group level [9,10]. However, in patients with established AD, levels remain stable from the prodromal/MCI stage onward [11]. Whether the magnitude of CSF t-tau may predict future speed of progression later in AD is less well understood.
Recent findings suggest that tau pathology can propagate between neurons [12,13], and immunotherapy trials targeting tau have recently started [14]. We previously reported that high CSF tau can predict rapid cognitive decline and risk of dying in patients with severe dementia [15]. The aim of the present study was to investigate whether high CSF tau levels also would predict time to NHP. Factors known to promote early institutionalization, such as single living, education level, and baseline severity of cognitive impairment were taken into account. Further, we analyzed whether high CSF tau was related to three other proxies of progression rate: time to conversion to moderate dementia, rapid cognitive decline as measured by repeated evaluation with the Mini Mental State Examination (MMSE) [16], and risk of dying in patients with severe dementia.

Study population and diagnostic procedures
We conducted a retrospective study, and the cohort consisted of all patients referred to the Memory Clinic at the Uppsala University Hospital, where all five authors work as physicians. Participants underwent a lumbar puncture (LP) as part of the diagnostic procedure between 2003 and 2011 and were diagnosed with AD (n = 234). Computed tomography or magnetic resonance imaging scans, caregiver interviews, thorough cognitive assessments, and in some cases regional glucose uptake by positron emission tomography were other parts of the diagnostic workup. The diagnosis of probable AD dementia was set according to the National Institute of Neurological and Communicative Disorders and Stroke/ Alzheimer's Disease and Related Disorders Association criteria [17] and the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, criteria [18]. Patients with neuroimaging findings suggesting a mixture of AD and cerebrovascular pathology were not included. CSF samples were collected by LP in polypropylene tubes following standardized procedures. Samples with clear visual blood contamination were excluded. All CSF samples were centrifuged and aliquoted in polypropylene tubes, and all samples were frozen once before analyses, which were done continuously as part of routine clinical practice. CSF concentrations of t-tau, p-tau, and Aβ42 were determined using sandwich enzyme-linked immunosorbent assays (ELISAs) at the Clinical Neurochemistry Laboratory, University of Gothenburg, Mölndal, Sweden [8,19,20]. Assays were performed by experienced, boardcertified laboratory technicians strictly following procedures approved by the Swedish Board for Accreditation and Conformity Assessment. To reduce variability over time and between batches, strict laboratory procedures were followed, including regular calibration of pipettes and preventive service of instruments (ELISA readers). For each assay, specified acceptance criteria for the calibration curve of an ELISA plate were applied for approval of each individual run, including optical density (OD) of the blank <0.1, OD of the highest calibrator between 1.2 and 3.0, OD of lowest calibrator larger than OD of the blank, and coefficient of variance in percent below 15 % for each calibrator (run in duplicate). In addition, internal quality control (QC) samples consisting of aliquots of pooled CSF (with low and high biomarker levels) were run on each ELISA plate together with patient CSF samples. The QC samples were placed at the beginning and the end of each ELISA plate, and had to fall within a prespecified target value set by repeated analyses for each ELISA format. Last, to ensure conformance between ELISA batches, a batch-bridging procedure to compare old with new batches was performed using a panel of individual (n > 20) CSF samples. If a systematic difference was identified, the new batch was rejected and another batch was evaluated.
The CSF biomarker results were taken into account in the diagnostic procedure, together with all other information. Ninety-eight percent of the patients had at least one CSF biomarker outside the normal range. The original diagnoses were continuously reevaluated at follow-up visits for a minimum of 2 years, and all diagnostic workup was scrutinized by one of the authors (MDG) to ensure accuracy. Mild dementia was defined as impaired performance in instrumental ADL only, and moderate dementia was defined as loss of function in PADL (i.e., needing assistance with either of grooming, bathing, dressing, toileting, feeding, or ambulation). One hundred patients fulfilled the AD dementia criteria at baseline, and one hundred thirty-four individuals were at the MCI stage [21] at baseline and converted to AD dementia during follow-up. These 234 patients formed the study population and were followed for a maximum of 11 years to May 2014 or to the date of death.

Outcomes
The digital medical record system kept at the Uppsala County Council is common to all health care provided in this area, including the vast majority of the general practitioners (GPs) at the health centers and dementia group living facilities, which enabled follow-up from diagnosis to death. Most patients were followed at the Memory Clinic every 6-12 months during the first years after diagnosis, and they were subsequently followed at health centers and eventually at the nursing homes.
Dates of moving to a nursing home were captured from the records, as were information on education level and living conditions. Descriptions of the patients' ADL function, cognitive deterioration, and medical status, as documented by the physicians at the Memory Clinic, by the GPs, and in the nursing homes also by registered nurses or occupational therapists, were collected from the medical records. Date of conversion to moderate dementia was defined as the date of the first documentation of impaired ability to perform PADL. Baseline MMSE scores were retrieved from the testing that most closely corresponded to the time of LP (within 1-3 months). Since this was a clinical study, the intervals of the clinical follow-up and MMSE retesting after baseline varied according to the individual patient's needs. Decline in MMSE was calculated as the difference between the baseline score and the last available test score divided by time in months, and we defined rapid cognitive decline as a ≥4-point decline in MMSE per 12 months. Nine patients had a shorter time span than 6 months between baseline and last MMSE. Dying in severe dementia was identified as death after a prolonged decline over months at the end stage of AD and being immobilized and dependent in all PADL, in contrast to premature death due to other causes.

Statistics
Cox proportional hazards models were used to assess the HRs for NHP, conversion to moderate dementia, and dying in severe dementia. Eight individuals lacked sufficient information to determine cause of death and were censored as nonresponders at the date of death. Another 11 patients moved from Uppsala County Council and were censored as nonresponders for conversion to moderate dementia/NHP when they moved and as nonresponders at the date of death. Patients lacking MMSE scores at follow-up (n = 21) were withdrawn from the analyses of rapid decline. ORs of rapid cognitive decline were determined by logistic regression. All analyses were performed in crude models and the same multivariate models, adjusted for age, sex, living condition, education level, baseline mild to moderate dementia stage, and MMSE score. MCI was referenced when the models were adjusted for mild to moderate dementia. In addition, separate analyses were conducted with subjects with baseline mild to moderate dementia and MCI due to AD, respectively. The CSF biomarker levels in univariate models and in stratified analyses (MCI and mild to moderate dementia) for conversion from mild to moderate dementia were entered with a split at median. In all other analyses, the CSF biomarker levels were entered as quartiles. Student's t test was used for comparing normally distributed variables between groups. The level of statistical significance was set to p = 0.05. All statistics and figures were produced using Statistica version 10 software (StatSoft, Tulsa, OK, USA).

Results
Baseline demographics and clinical characteristics are shown in Table 1, and a flowchart is shown in Fig. 1. Concentrations of CSF biomarkers did not differ between the disease stages. All but nine patients were treated with cholinesterase inhibitors and/or memantine during part of the follow-up period. Apolipoprotein E genotypes were available for only 82 individuals (ε4/4, n = 25; ε4/3, n = 39; ε4/2, n = 1; ε4 noncarriers, n = 17), who were therefore not included in the analyses. During follow-up, 149 patients converted to moderate dementia and 112 patients were admitted to NHP; all had converted to moderate dementia before this event. The times from baseline to NHP (mean ± SD) were 4.3 ± 1.9 years in the MCI group (n = 54), 3.1 ± 1.3 years in the mild dementia group (n = 46), and 1.3 ± 1.0 years in the moderate dementia group (n = 12). Baseline CSF t-tau greater than the median value was associated with a higher risk of NHP (Table 2), with a dose-dependent relationship in crude and adjusted models ( Table 3, Fig. 2). Individuals in the highest quartile of CSF t-tau (≥900 ng/L) experienced the highest risk, which could also be seen when including subjects with MCI only (adjusted HR 2.37, 95 % CI 1.10-5.09). The corresponding risk for patients with baseline mild AD was HR 3.11 (95 % CI 1. 35-7.13) in crude analyses, which did not reach statistical significance in the multivariate model. High p-tau  Table 2, Additional file 1: Table S1, and Additional file 2: Table S2).
Similarly, subjects in the highest quartile of t-tau had higher rates of conversion to moderate dementia (Table 3, Fig. 3). CSF t-tau above the median was associated with increased rate of conversion to moderate dementia, both in MCI (adjusted HR 1.66, 95 % CI 1.08-2.56) and in mild dementia (adjusted HR 1.80, 95 % CI 1.02-3.20). These associations remained in crude and multivariate models including only cohabiting patients. There were no consistent associations between CSF Aβ42 or p-tau concentrations and this outcome in crude or adjusted models ( Table 2,  Additional file 1: Table S1, and Additional file 2: Table S2).
Our previous findings in 196 subjects of this cohort, followed for a shorter period [15], were confirmed in this extended population. Setting the lowest quartile of CSF ttau as a reference, adjusted OR for rapid cognitive decline in the highest quartile was 3.04 (95 % CI 1.16-8.99) (  (Table 3). Further, subjects in the highest quartile of p-tau had an increased HR of death in severe dementia compared with participants in the three lowest quartiles (adjusted HR 1.88, 95 % CI 1.03-3.45); otherwise, p-tau was not significantly related to these outcomes (Additional file 1: Table S1). CSF Aβ42 was associated with neither rapid cognitive decline nor death in severe dementia (Additional file 2: Table S2). None of the CSF biomarkers was related to all-cause mortality.

Discussion
To our knowledge, this is the first report that a high CSF concentration of t-tau predicts an earlier need of institutionalization (NHP) and conversion to moderate AD dementia, two solid markers of disease burden and societal costs. These associations were dose-dependent and present already at the stage of MCI due to AD. Moreover, they were independent of age, sex, education level, and living conditions as well as of baseline cognitive and functional status. We were able to capture these two outcomes in a memory clinic cohort through a long follow-up with virtually no loss. Further, subjects with AD who had high CSF t-tau had a steeper cognitive decline and a higher risk of dying in severe dementia, which confirms and expands previous reports [15,22,23]. In our previous study [14], CSF levels of p-tau were associated with an increased risk of rapid cognitive decline. In the present study, CSF Aβ42 and p-tau showed no consistent associations with the rate of decline.
Our results fit well with the current pathophysiological model, where decreased Aβ42 in CSF reflecting amyloid accumulation in the brain is a very early event, which is stable from the presymptomatic stage onward [24]. The density of amyloid plaques at autopsy is poorly correlated with clinical status, whereas there is a closer correlation between the extent of tangles and cognitive dysfunction [25,26]. CSF t-tau and p-tau have been shown to increase already in the asymptomatic stage or  in the MCI stage in both familiar and sporadic AD [27,28], and they remain constant thereafter [11]. CSF t-tau is classified as a marker of neuronal injury in the National Institute on Aging-Alzheimer's Association criteria [29]. The magnitude of increase in CSF tau levels is very high in disorders with malignant degeneration, such as Creutzfeldt-Jakob disease [30,31]. However, in pure tauopathies such as progressive supranuclear paralysis and corticobasal degeneration, which generally have a faster progression than AD, t-tau in CSF is only moderately increased [32]. This suggests the existence of specific mechanisms behind AD tauopathy. In addition to a passive release from dying neurons, there is accumulating evidence that tau is actively secreted and transmitted between synaptically connected neurons in a prion-like manner [12,13]. Hence, high CSF t-tau in AD may reflect not only a high passive release of aggregated tau from dying neurons but also a high fraction of "active" tau and the intensity of AD spreading, which also adds support to new therapeutic approaches acting in the extracellular space [33]. Our finding that high CSF t-tau, rather than p-tau, is associated with AD progression rate is well in concordance with this model. One of the authors (LK) reviewed all published studies (1756 in November 2015) resulting from a PubMed search using the keywords "Alzheimer's disease," "cerebrospinal fluid," and "tau." Similar to our findings, the vast majority of the cross-sectional studies described no association between tau levels and disease stage. Moreover, most longitudinal studies focused on distinguishing AD from nonprogressive AD (i.e., non-AD, cases in MCI populations), where abnormal biomarkers, as expected, predict conversion to AD. Regarding CSF biomarkers and progression in the dementia stage of AD, we found seven longitudinal studies, and the results were contradictory. Two studies with a follow-up period of approximately 3 years showed that high t-tau predicted steeper decline in MMSE score [22,23], but the results from the other five studies were not clear-cut [34,35] or negative [36][37][38], in this respect, for patients with AD dementia. The association between CSF biomarkers and loss of ADL has been assessed in two reports from the Alzheimer's Disease Neuroimaging Initiative cohort, with no clear evidence of any relation with high CSF tau in patients who had reached the dementia stage [39,40]. No study had NHP or conversion to moderate dementia as outcomes, which likely reflects the lack of health care systems enabling long-term follow-up. Caregiver burden, including loss of PADL and behavioral and psychological symptoms of dementia (BPSD), underlie the decision for NHP, as does the health of the spouse. In patients living alone, potential hazards, together with BPSD and the quality of the home care, are important factors. Interestingly, one report described a relationship between high CSF tau and psychosis in AD [41].
Only a few studies have assessed the relationship with AD mortality. In a small study, high CSF tau was associated with shorter 6-year survival [42]. Cluster analyses of a larger cohort showed a relationship between very high CSF t-tau and low Aβ42 and mortality [34]. In contrast, high tau was associated with shorter survival in dementia with Lewy bodies, but not in AD, in a cohort followed for up to 7 years [43]. Mortality is not an ideal marker of progression, since many patients have a "preterm" death due to non-AD-related causes, which attenuates possible relationships. In a recent study done in southern Sweden with up to 12 years of follow-up [44], researchers found no relationship between high t-tau and early death caused by dementia. However, misdiagnosis cannot be excluded, since data were collected from a registry. In the present study, it was possible to capture "death in severe dementia" by scrutinizing patients' status through the medical records.
CSF analyses have been a part of the diagnostic procedures at Swedish memory clinics since the beginning of the 2000s [2]. Forty percent of 17,000 newly diagnosed patients in the National Swedish Dementia Registry (SveDem; 2007-2011), where 92 % were assessed at a specialist unit, underwent LP [45]. Our cohort is likely to be representative of patients with AD in the younger age group who consult memory clinics for diagnostic workup. Further, the cohort is probably representative of participants in AD clinical trials, who are recruited mainly from a memory clinic, and a coliving spouse is generally needed. However, it differs from a general AD population with respect to younger age, higher education (21 %, reflecting the setting in a university city), and cohabiting status. Twenty-four percent were living alone, which is less than in a general Swedish dementia population, where 47 % live alone at the time of diagnosis (data from SveDem, 50,000 patients, [2007][2008][2009][2010][2011][2012][2013][2014][2015]. Patients with AD living together with a spouse are likely to be diagnosed at an earlier time point than those who live by themselves. Since low education and solitary living are factors associated with early NHP, our results would be an underestimation of the true associations. NHP might be a valid marker of rapid decline in our population, since costs of nursing home care in Sweden are provided by the authorities and are available regardless of income. However, the timing of NHP differs between countries, depending on ethnicity, social structure, and socioeconomic status.
The major strength of this study is the long follow-up period of a large AD cohort, where the diagnostic workup was scrutinized and reevaluated. Virtually all subjects had at least one positive CSF biomarker, and loss to follow-up was minimal. We were able to capture dates of NHP and the approximate time point for conversion to moderate dementia, as well as to evaluate the medical conditions at the time of death. This was possible because of the personal identity numbers assinged to all Swedish citizens and the sharing of digital medical records by all health care units, including the dementia group living facilities. Another strength of the cohort is the clinical setting at a memory clinic rather than a research program with highly selected patients. Time to NHP was chosen as a robust major outcome, and the association between increased CSF t-tau and rapid deterioration was confirmed using three other outcomes reflecting factors of vital importance for patients, caregivers, and societal costs.
The present study also has limitations. One is the lack of systematic follow-up at predefined time points for the cognitive and functional assessments, as well as the lack of formal ADL and BPSD scales. Instead, intervals varied according to the individual patient's needs; for example, assessments of MMSE and ADL were more frequent in rapid decliners. This would only create more contrast between the groups, however.

Conclusions
This study provides evidence of a biomarker that already at the stage of MCI due to AD is a marker of future deterioration rate. Our findings of an association between rapid decline and high CSF t-tau, rather than Aβ42 or p-tau, are in accordance with the understanding of tau as a marker of aggressive neurodegeneration, including active interneuronal tau propagation. Further prospective studies using standardized measurements are needed to confirm the predictive value of t-tau in CSF. During the past few years, research on AD biomarkers has been focused on identifying preclinical cases that may benefit from future disease-halting treatment. However, clinical research ought to focus more on patients with rapid decline, in view of the very low quality of life of patients and caregivers and the high societal costs. Previous immunotherapy studies may have failed in part due to misdiagnosis and/or slower than expected disease progression in the highly selected participants. Clinical trials evaluating the efficacy of potential AD diseasemodifying drugs should take into consideration the large interindividual variation of progression rate and may benefit from selecting participants with high CSF tau levels.
To our knowledge, this is the first study to show that high CSF t-tau levels predict early NHP and conversion to moderate dementia in an AD cohort. Selecting patients with high CSF t-tau, indicating a more aggressive neurodegeneration and steeper decline, for AD immunotherapy trials might increase the possibility of showing contrast between active treatment and placebo.

Additional files
Additional file 1: Table S1. Crude and adjusted Cox proportional hazards ratios (95 % CI) of nursing home placement, conversion to moderate dementia, and death in severe dementia; crude and adjusted OR (95 % CI) of rapid cognitive decline according to quartiles of CSF p-tau. (DOCX 15 kb) Additional file 2: Table S2. Crude and adjusted Cox proportional hazards ratios (95 % CI) of nursing home placement, conversion to moderate dementia, and death in severe dementia; crude and adjusted OR (95 % CI) of rapid cognitive decline according to quartiles of CSF amyloid-β 42