Lnk regulates integrin αIIbβ3 outside-in signaling in mouse platelets,leading to stabilization of thrombus development in vivo

The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk^–/– mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin αIIbβ3–mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk^–/– mice by using genetic depletion of Lnk and BM transplantation. Using FeCl₃- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk^–/– platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the β3 integrin subunit, and reduced binding of Fyn to integrin αIIbβ3. These results provide new insight into the mechanism of αIIbβ3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.     

PKCα regulates platelet granule secretion and thrombus formation in mice

Platelets are central players in atherothrombosis development in coronary artery disease. The PKC family provides important intracellular mechanisms for regulating platelet activity, and platelets express several members of this family, including the classical isoforms PKCα and PKCβ and novel isoforms PKCδ and PKCθ. Here, we used a genetic approach to definitively demonstrate the role played by PKCα in regulating thrombus formation and platelet function. Thrombus formation in vivo was attenuated in Prkca^–/– mice, and PKCα was required for thrombus formation in vitro, although this PKC isoform did not regulate platelet adhesion to collagen. The ablation of in vitro thrombus formation in Prkca^–/– platelets was rescued by the addition of ADP, consistent with the key mechanistic finding that dense-granule biogenesis and secretion depend upon PKCα expression. Furthermore, defective platelet aggregation in response to either collagen-related peptide or thrombin could be overcome by an increase in agonist concentration. Evidence of overt bleeding, including gastrointestinal and tail bleeding, was not seen in Prkca^–/– mice. In summary, the effects of PKCα ablation on thrombus formation and granule secretion may implicate PKCα as a drug target for antithrombotic therapy.     

A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule–delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.     

α-Ketoglutarate regulates acid-base balance through an intrarenal paracrine mechanism

Paracrine communication between different parts of the renal tubule is increasingly recognized as an important determinant of renal function. Previous studies have shown that changes in dietary acid-base load can reverse the direction of apical α-ketoglutarate (αKG) transport in the proximal tubule and Henle’s loop from reabsorption (acid load) to secretion (base load). Here we show that the resulting changes in the luminal concentrations of αKG are sensed by the αKG receptor OXGR1 expressed in the type B and non-A–non-B intercalated cells of the connecting tubule (CNT) and the cortical collecting duct (CCD). The addition of 1 mM αKG to the tubular lumen strongly stimulated Cl^–-dependent HCO₃^– secretion and electroneutral transepithelial NaCl reabsorption in microperfused CCDs of wild-type mice but not Oxgr1^–/– mice. Analysis of alkali-loaded mice revealed a significantly reduced ability of Oxgr1^–/– mice to maintain acid-base balance. Collectively, these results demonstrate that OXGR1 is involved in the adaptive regulation of HCO₃^– secretion and NaCl reabsorption in the CNT/CCD under acid-base stress and establish αKG as a paracrine mediator involved in the functional coordination of the proximal and the distal parts of the renal tubule.     

Strategic tuning of excited-state properties of electroluminescent materials with enhanced hot exciton mixing

Deep blue emitters with excellent stability, high quantum yield and multifunctionality are the major issues for full-color displays. In line with this, new multifunctional, thermally stable blue emitters viz., N-(4-(10-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-phenanthro[9,10-d]imidazol-2-yl)anthracen-9-yl)phenyl)-N-phenylbenzenamine (DPIAPPB) and 2-(10-(9H-carbazol-9-yl)anthracen-9-yl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-phenanthro[9,10-d]imidazole (CADPPI) with hybridized local charge transfer state (HLCT) and hot exciton properties have been synthesized. These molecules show high photoluminescence quantum yield (Φ_s/f): (DPIAPPB – 0.82/0.70 and CADPPI – 0.91/0.83). The CADPPI based device (EL – 467 nm) shows high efficiencies [η_c – 9.85 cd A⁻¹; η_p – 10.84 lm W⁻¹; η_ex – 4.78% at 2.8 V; CIE (0.15, 0.10)] compared to the DPIAPPB device (EL − 472 nm) [η_c – 6.56 cd A⁻¹; η_p – 6.16 lm W⁻¹; η_ex – 4.15% at 2.8 V with CIE (0.15, 0.12)]. The green device with CADPPI:Ir(ppy)₃ exhibits a maximum L – 59 012 cd m⁻²; η_ex – 16.8%; η_c – 37.3 cd A⁻¹; η_p – 39.8 lm W⁻¹ with CIE (0.30, 0.60) and the red device with CADPPI:Ir(MDQ)₂(acac) shows a maximum L – 43 456 cd m⁻²; η_ex – 21.9%; η_c – 36.0 cd A⁻¹; η_p – 39.6 lm W⁻¹ with CIE (0.64, 0.35).

The CADPPI:Ir(ppy)₃ device exhibits L – 90 12 cd m⁻²; η_ex – 18.8%; η_c − 27.3 cd A⁻¹; η_p – 29.8 lm W⁻¹; CIE (0.30, 0.60).     

Efficient electroluminescent hybridized local and charge-transfer host materials with small singlet–triplet splitting to enhance exciton utilization efficiency: excited state transition configuration

A series of efficient electroluminescent materials with dual carrier transport properties shows enhanced singlet exciton utilization (η_s) due to small singlet–triplet splitting (ΔE_ST). The strong orbital-coupling transitions of N-(4-(1-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-diphenyl-1H-imidazol-2-yl)naphthalen-4-yl)phenyl)-N-phenyl benzenamine (DDPB) exhibit deep blue emission at 435 nm (CIEy, 0.07) with an external quantum efficiency of 2.01%. The electroluminescent efficiencies of 2-(1-(9H-carbazol-9-yl)naphthalen-4-yl)-1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1H-phenanthro[9,10-d]imidazole (CDDPI) (L – 3992 cd m⁻²; η_ex – 3.01%; η_c – 2.56 cd A⁻¹; η_p – 2.12 lm W⁻¹) are higher than those of the N-(4-(1-(1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-H-phenanthro[9,10-d]imidazole-2-yl)naphthalen-4-yl)phenyl)-N-phenylbenzenamine (DBDPA) based device (L – 3015 cd m⁻²; η_ex – 2.85%; η_c – 2.01 cd A⁻¹; η_p – 1.92 lm W⁻¹). The blue emissive materials CDDPI and DBDPA are used as a host to construct green and red phosphorescent OLEDs: the green device based on CDDPI:Ir(ppy)₃ exhibits higher efficiencies (L – 8812 cd m⁻²; η_ex – 19.0%; η_c – 27.5 cd A⁻¹; η_p – 33.0 lm W⁻¹) at 2.7 V and the red device based on CDDPI:Ir(MQ)₂(acac) exhibits a maximum luminance of 39 661 cd m⁻² with excellent EL efficiencies [η_ex – 19.2%; η_c – 27.9 cd A⁻¹; η_p – 29.2 lm W⁻¹; CIE (0.64, 0.34)] compared with those of the DBDPA:Ir(MQ)₂(acac) based device [L – 37 621 cd m⁻²; η_ex – 18.5%; η_c – 25.2 cd A⁻¹; η_p – 25.8 lm W⁻¹; CIE (0.64, 0.34)].

CDDPI:Ir(ppy)₃ exhibits higher efficiencies: L = 8812 cd m⁻²; η_ex = 19.0%; η_c = 27.5 cd A⁻¹; η_p = 33.0 lm W⁻¹ at 2.7 V.     

Blood pressure homeostasis is maintained by a P311–TGF-β axis

P311 is an 8-kDa intracellular protein that is highly conserved across species and is expressed in the nervous system as well as in vascular and visceral smooth muscle cells. P311-null (P311^–/–) mice display learning and memory defects, but alterations in their vasculature have not been previously described. Here we report that P311^–/– mice are markedly hypotensive with accompanying defects in vascular tone and VSMC contractility. Functional abnormalities in P311^–/– mice resulted from decreased total and active levels of TGF-β1, TGF-β2, and TGF-β3 that arise as a specific consequence of decreased translation. Vascular hypofunctionality was fully rescued in vitro and in vivo by exogenous TGF-β1–TGF-β3. Conversely, P311-transgenic (P311^TG) mice had elevated levels of TGF-β1–TGF-β3 and subsequent hypertension. Consistent with findings attained in mouse models, arteries recovered from hypertensive human patients displayed increased P311 expression. Thus, we identified P311 as the first protein known to modulate TGF-β translation and the first pan-regulator of TGF-β expression under steady-state conditions. Together, our findings point to P311 as a critical blood pressure regulator and establish a potential link between P311 expression and the development of hypertensive disease.     

α1G-dependent T-type Ca2+ current antagonizes cardiac hypertrophy through a NOS3-dependent mechanism in mice

In noncontractile cells, increases in intracellular Ca²⁺ concentration serve as a second messenger to signal proliferation, differentiation, metabolism, motility, and cell death. Many of these Ca²⁺-dependent regulatory processes operate in cardiomyocytes, although it remains unclear how Ca²⁺ serves as a second messenger given the high Ca²⁺ concentrations that control contraction. T-type Ca²⁺ channels are reexpressed in adult ventricular myocytes during pathologic hypertrophy, although their physiologic function remains unknown. Here we generated cardiac-specific transgenic mice with inducible expression of α1G, which generates Ca_v3.1 current, to investigate whether this type of Ca²⁺ influx mechanism regulates the cardiac hypertrophic response. Unexpectedly, α1G transgenic mice showed no cardiac pathology despite large increases in Ca²⁺ influx, and they were even partially resistant to pressure overload–, isoproterenol-, and exercise-induced cardiac hypertrophy. Conversely, α1G^–/– mice displayed enhanced hypertrophic responses following pressure overload or isoproterenol infusion. Enhanced hypertrophy and disease in α1G^–/– mice was rescued with the α1G transgene, demonstrating a myocyte-autonomous requirement of α1G for protection. Mechanistically, α1G interacted with NOS3, which augmented cGMP-dependent protein kinase type I activity in α1G transgenic hearts after pressure overload. Further, the anti-hypertrophic effect of α1G overexpression was abrogated by a NOS3 inhibitor and by crossing the mice onto the Nos3^–/– background. Thus, cardiac α1G reexpression and its associated pool of T-type Ca²⁺ antagonize cardiac hypertrophy through a NOS3-dependent signaling mechanism.     

Suppression of KATP channel activity protects murine pancreatic β cells against oxidative stress

The enhanced oxidative stress associated with type 2 diabetes mellitus contributes to disease pathogenesis. We previously identified plasma membrane–associated ATP-sensitive K⁺ (K_ATP) channels of pancreatic β cells as targets for oxidants. Here, we examined the effects of genetic and pharmacologic ablation of K_ATP channels on loss of mouse β cell function and viability following oxidative stress. Using mice lacking the sulfonylurea receptor type 1 (Sur1) subunit of K_ATP channels, we found that, compared with insulin secretion by WT islets, insulin secretion by Sur1^–/– islets was less susceptible to oxidative stress induced by the oxidant H₂O₂. This was likely, at least in part, a result of the reduced ability of H₂O₂ to hyperpolarize plasma membrane potential and reduce cytosolic free Ca²⁺ concentration ([Ca²⁺]_c) in the Sur1^–/– β cells. Remarkably, Sur1^–/– β cells were less prone to apoptosis induced by H₂O₂ or an NO donor than WT β cells, despite an enhanced basal rate of apoptosis. This protective effect was attributed to upregulation of the antioxidant enzymes SOD, glutathione peroxidase, and catalase. Upregulation of antioxidant enzymes and reduced sensitivity of Sur1^–/– cells to H₂O₂-induced apoptosis were mimicked by treatment with the sulfonylureas tolbutamide and gliclazide. Enzyme upregulation and protection against oxidant-induced apoptosis were abrogated by agents lowering [Ca²⁺]_c. Sur1^–/– mice were less susceptible than WT mice to streptozotocin-induced β cell destruction and subsequent hyperglycemia and death, which suggests that loss of K_ATP channel activity may protect against streptozotocin-induced diabetes in vivo.     

PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice

Protein tyrosine phosphatase 1B (PTP1B) and SH2 domain–containing protein tyrosine phosphatase–2 (SHP2) have been shown in mice to regulate metabolism via the central nervous system, but the specific neurons mediating these effects are unknown. Here, we have shown that proopiomelanocortin (POMC) neuron–specific deficiency in PTP1B or SHP2 in mice results in reciprocal effects on weight gain, adiposity, and energy balance induced by high-fat diet. Mice with POMC neuron–specific deletion of the gene encoding PTP1B (referred to herein as POMC-Ptp1b^–/– mice) had reduced adiposity, improved leptin sensitivity, and increased energy expenditure compared with wild-type mice, whereas mice with POMC neuron–specific deletion of the gene encoding SHP2 (referred to herein as POMC-Shp2^–/– mice) had elevated adiposity, decreased leptin sensitivity, and reduced energy expenditure. POMC-Ptp1b^–/– mice showed substantially improved glucose homeostasis on a high-fat diet, and hyperinsulinemic-euglycemic clamp studies revealed that insulin sensitivity in these mice was improved on a standard chow diet in the absence of any weight difference. In contrast, POMC-Shp2^–/– mice displayed impaired glucose tolerance only secondary to their increased weight gain. Interestingly, hypothalamic Pomc mRNA and α–melanocyte-stimulating hormone (αMSH) peptide levels were markedly reduced in POMC-Shp2^–/– mice. These studies implicate PTP1B and SHP2 as important components of POMC neuron regulation of energy balance and point to what we believe to be a novel role for SHP2 in the normal function of the melanocortin system.     

Angiotensin-converting enzyme overexpression in myelomonocytes prevents Alzheimer’s-like cognitive decline

Cognitive decline in patients with Alzheimer’s disease (AD) is associated with elevated brain levels of amyloid β protein (Aβ), particularly neurotoxic Aβ_1–42. Angiotensin-converting enzyme (ACE) can degrade Aβ_1–42, and ACE overexpression in myelomonocytic cells enhances their immune function. To examine the effect of targeted ACE overexpression on AD, we crossed ACE^10/10 mice, which overexpress ACE in myelomonocytes using the c-fms promoter, with the transgenic APP_SWE/PS1_ΔE9 mouse model of AD (AD⁺). Evaluation of brain tissue from these AD⁺ACE^10/10 mice at 7 and 13 months revealed that levels of both soluble and insoluble brain Aβ_1–42 were reduced compared with those in AD⁺ mice. Furthermore, both plaque burden and astrogliosis were drastically reduced. Administration of the ACE inhibitor ramipril increased Aβ levels in AD⁺ACE^10/10 mice compared with the levels induced by the ACE-independent vasodilator hydralazine. Overall, AD⁺ACE^10/10 mice had less brain-infiltrating cells, consistent with reduced AD-associated pathology, though ACE-overexpressing macrophages were abundant around and engulfing Aβ plaques. At 11 and 12 months of age, the AD⁺ACE^10/WT and AD⁺ACE^10/10 mice were virtually equivalent to non-AD mice in cognitive ability, as assessed by maze-based behavioral tests. Our data demonstrate that an enhanced immune response, coupled with increased myelomonocytic expression of catalytically active ACE, prevents cognitive decline in a murine model of AD.     

IL-17 produced by neutrophils regulates IFN-γ–mediated neutrophil migration in mouse kidney ischemia-reperfusion injury

The IL-23/IL-17 and IL-12/IFN-γ cytokine pathways have a role in chronic autoimmunity, which is considered mainly a dysfunction of adaptive immunity. The extent to which they contribute to innate immunity is, however, unknown. We used a mouse model of acute kidney ischemia-reperfusion injury (IRI) to test the hypothesis that early production of IL-23 and IL-12 following IRI activates downstream IL-17 and IFN-γ signaling pathways and promotes kidney inflammation. Deficiency in IL-23, IL-17A, or IL-17 receptor (IL-17R) and mAb neutralization of CXCR2, the p19 subunit of IL-23, or IL-17A attenuated neutrophil infiltration in acute kidney IRI in mice. We further demonstrate that IL-17A produced by GR-1⁺ neutrophils was critical for kidney IRI in mice. Activation of the IL-12/IFN-γ pathway and NKT cells by administering α-galactosylceramide–primed bone marrow–derived DCs increased IFN-γ production following moderate IRI in WT mice but did not exacerbate injury or enhance IFN-γ production in either Il17a^–/– or Il17r^–/– mice, which suggested that IL-17 signaling was proximal to IFN-γ signaling. This was confirmed by the finding that IFN-γ administration reversed the protection seen in Il17a^–/– mice subjected to IRI, whereas IL-17A failed to reverse protection in Ifng^–/– mice. These results demonstrate that the innate immune component of kidney IRI requires dual activation of the IL-12/IFN-γ and IL-23/IL-17 signaling pathways and that neutrophil production of IL-17A is upstream of IL-12/IFN-γ. These mechanisms might contribute to reperfusion injury in other organs.     

The function of heme-regulated eIF2alpha kinase in murine iron homeostasis and macrophage maturation

Heme-regulated eIF2α kinase (HRI) plays an essential protective role in anemias of iron deficiency, erythroid protoporphyria, and β-thalassemia. In this study, we report that HRI protein is present in murine macrophages, albeit at a lower level than in erythroid precursors. Hri^–/– mice exhibited impaired macrophage maturation and a weaker antiinflammatory response with reduced cytokine production upon LPS challenge. The level of production of hepcidin, an important player in the pathogenesis of the anemia of inflammation, was significantly decreased in Hri^–/– mice, accompanied by decreased splenic macrophage iron content and increased serum iron content. Hepcidin expression was also significantly lower, with a concomitant increase in serum iron in Hri^–/– mice upon LPS treatment. We also demonstrated an impairment of erythrophagocytosis by Hri^–/– macrophages both in vitro and in vivo under chronic hemolytic anemia, providing evidence for the role of HRI in recycling iron from senescent red blood cells. This work demonstrates that HRI deficiency attenuates hepcidin expression and iron homeostasis in mice, indicating a potential role for HRI in the anemia of inflammation.     

Impairment of T and B Cell Development by Treatment with a Type I Interferon

Type I interferons α and β, naturally produced regulators of cell growth and differentiation, have been shown to inhibit IL-7–induced growth and survival of B cell precursors in vitro. After confirming an inhibitory effect on B lymphopoiesis in an ex vivo assay, we treated newborn mice with an active IFN-α2/α1 hybrid molecule to assess its potential for regulating B and T cell development in vivo. Bone marrow and splenic cellularity was greatly reduced in the IFN-α2/α1–treated mice, and B lineage cells were reduced by >80%. The bone marrow progenitor population of CD43⁺B220⁺HSA⁻ cells was unaffected, but development of the CD19⁺ pro–B cells and their B lineage progeny was severely impaired. Correspondingly, IL-7–responsive cells in the bone marrow were virtually eliminated by the interferon treatment. Thymus cellularity was also reduced by >80% in the treated mice. Phenotypic analysis of the residual thymocytes indicated that the inhibitory effect was exerted during the pro–T cell stage in differentiation. In IFN-α/β receptor^−/− mice, T and B cell development were unaffected by the IFN-α2/α1 treatment. The data suggest that type I interferons can reversibly inhibit early T and B cell development by opposing the essential IL-7 response.     

Rapid Interferon γ–dependent Clearance of Influenza A Virus and Protection from Consolidating Pneumonitis in Nitric Oxide Synthase 2–deficient Mice

Viral infection often activates the interferon (IFN)-γ–inducible gene, nitric oxide synthase 2 (NOS2). Expression of NOS2 can limit viral growth but may also suppress the immune system and damage tissue. This study assessed each of these effects in genetically deficient NOS2^−/− mice after infection with influenza A, a virus against which IFN-γ has no known activity. At inocula sufficient to cause consolidating pneumonitis and death in wild-type control mice, NOS2^−/− hosts survived with little histopathologic evidence of pneumonitis. Moreover, they cleared influenza A virus from their lungs by an IFN-γ–dependent mechanism that was not evident in wild-type mice. Even when the IFN-γ–mediated antiviral activity was blocked in NOS2^−/− mice with anti–IFN-γ mAb, such mice failed to succumb to disease. Further evidence that this protection was independent of viral load was provided by treating NOS2^+/+ mice with the NOS inhibitor, N ^ω-methyl-l-arginine (l-NMA). l-NMA prevented mortality without affecting viral growth. Thus, host NOS2 seems to contribute more significantly to the development of influenza pneumonitis in mice than the cytopathic effects of viral replication. Although NOS2 mediates some antiviral effects of IFN-γ, during influenza infection it can suppress another IFN-γ–dependent antiviral mechanism. This mechanism was observed only in the complete absence of NOS2 activity and appeared sufficient to control influenza A virus growth in the absence of changes in cytotoxic T lymphocyte activity.     

TAP-1 indirectly regulates CD4+ T cell priming in Toxoplasma gondii infection by controlling NK cell IFN-gamma production

To investigate if transporter associated with antigen processing (TAP)–1 is required for CD8⁺ T cell–mediated control of Toxoplasma gondii in vivo, we compared the resistance of TAP-1^−/−, CD8^−/−, and wild-type (WT) mice to infection with the parasite. Unexpectedly, TAP-1^−/− mice displayed greater susceptibility than CD8^−/−, β₂-microglobulin^−/− (β₂m^−/−), or WT mice to infection with an avirulent parasite strain. The decreased resistance of the TAP-1^−/− mice correlated with a reduction in the frequency of activated (CD62L^low CD44^hi) and interferon (IFN)-γ–producing CD4⁺ T cells. Interestingly, infected TAP-1^−/− mice also showed reduced numbers of IFN-γ–producing natural killer (NK) cells relative to WT, CD8^−/−, or β₂m^−/− mice, and after NK cell depletion both CD8^−/− and WT mice succumbed to infection with the same kinetics as TAP-1^−/− animals and displayed impaired CD4⁺ T cell IFN-γ responses. Moreover, adoptive transfer of NK cells obtained from IFN-γ^+/+, but not IFN-γ^−/−, animals restored the CD4⁺ T cell response of infected TAP-1^−/− mice to normal levels. These results reveal a role for TAP-1 in the induction of IFN-γ–producing NK cells and demonstrate that NK cell licensing can influence host resistance to infection through its effect on cytokine production in addition to its role in cytotoxicity.     

ESAT-6–dependent cytosolic pattern recognition drives noncognate tuberculosis control in vivo

IFN-γ is a critical mediator of host defense against Mycobacterium tuberculosis (Mtb) infection. Antigen-specific CD4⁺ T cells have long been regarded as the main producer of IFN-γ in tuberculosis (TB), and CD4⁺ T cell immunity is the main target of current TB vaccine candidates. However, given the recent failures of such a TB vaccine candidate in clinical trials, strategies to harness CD4-independent mechanisms of protection should be included in future vaccine design. Here, we have reported that noncognate IFN-γ production by Mtb antigen–independent memory CD8⁺ T cells and NK cells is protective during Mtb infection and evaluated the mechanistic regulation of IFN-γ production by these cells in vivo. Transfer of arenavirus- or protein-specific CD8⁺ T cells or NK cells reduced the mortality and morbidity rates of mice highly susceptible to TB in an IFN-γ–dependent manner. Secretion of IFN-γ by these cell populations required IL-18, sensing of mycobacterial viability, Mtb protein 6-kDa early secretory antigenic target–mediated (ESAT-6–mediated) cytosolic contact, and activation of NLR family pyrin domain–containing protein 3 (NLRP3) inflammasomes in CD11c⁺ cell subsets. Neutralization of IL-18 abrogated protection in susceptible recipient mice that had received noncognate cells. Moreover, improved Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine–induced protection was lost in the absence of ESAT-6–dependent cytosolic contact. Our findings provide a comprehensive mechanistic framework for antigen-independent IFN-γ secretion in response to Mtb with critical implications for future intervention strategies against TB.     

Endothelial LRP1 transports amyloid-β1–42 across the blood-brain barrier

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor–related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer’s disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER^T2 Lrp1^fl/fl mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [¹²⁵I] Aβ_1–42. Additionally, in the 5xFAD mouse model of AD, brain endothelial–specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD.     

In Vitro and In Vivo Evidence that Thrombospondin-1 (TSP-1) Contributes to Stirring- and Shear-Dependent Activation of Platelet-Derived TGF-β1

Thrombospondin 1 (TSP-1), which is contained in platelet α-granules and released with activation, has been shown to activate latent TGF-β1 in vitro, but its in vivo role is unclear as TSP-1-null (Thbs1^−/−) mice have a much less severe phenotype than TGF-β1-null (Tgfb1^−/−) mice. We recently demonstrated that stirring and/or shear could activate latent TGF-β1 released from platelets and have now studied these methods of TGF-β1 activation in samples from Thbs1^−/− mice, which have higher platelet counts and higher levels of total TGF-β1 in their serum than wild type mice. After either two hours of stirring or shear, Thbs1^−/− samples demonstrated less TGF-β1 activation (31% and 54% lower levels of active TGF-β1 in serum and platelet releasates, respectively). TGF-β1 activation in Thbs1^−/− mice samples was normalized by adding recombinant human TSP-1 (rhTSP-1). Exposure of platelet releasates to shear for one hour led to near depletion of TSP-1, but this could be prevented by preincubating samples with thiol-reactive agents. Moreover, replenishing rhTSP-1 to human platelet releasates after one hour of stirring enhanced TGF-β1 activation. In vivo TGF-β1 activation in carotid artery thrombi was also partially impaired in Thbs1^−/− mice. These data indicate that TSP-1 contributes to shear-dependent TGF-β1 activation, thus providing a potential explanation for the inconsistent in vitro data previously reported as well as for the differences in phenotypes of Thbs1^−/− and Tgfb1^−/− mice.