Ca2+-dependent repair of pneumolysin pores: A new paradigm for host cellular defense against bacterial pore-forming toxins

Pneumolysin (PLY), a key virulence factor of Streptococcus pneumoniae, permeabilizes eukaryotic cells by forming large trans-membrane pores. PLY imposes a puzzling multitude of diverse, often mutually excluding actions on eukaryotic cells. Whereas cytotoxicity of PLY can be directly attributed to the pore-mediated effects, mechanisms that are responsible for the PLY-induced activation of host cells are poorly understood. We show that PLY pores can be repaired and thereby PLY-induced cell death can be prevented. Pore-induced Ca2+ entry from the extracellular milieu is of paramount importance for the initiation of plasmalemmal repair. Nevertheless, active Ca2+ sequestration that prevents excessive Ca2+ elevation during the execution phase of plasmalemmal repair is of no less importance. The efficacy of plasmalemmal repair does not only define the fate of targeted cells but also intensity, duration and repetitiveness of PLY-induced Ca2+ signals in cells that were able to survive after PLY attack. Intracellular Ca2+ dynamics evoked by the combined action of pore formation and their elimination mimic the pattern of receptor-mediated Ca2+ signaling, which is responsible for the activation of host immune responses. Therefore, we postulate that plasmalemmal repair of PLY pores might provoke cellular responses that are similar to those currently ascribed to the receptor-mediated PLY effects. Our data provide new insights into the understanding of the complexity of cellular non-immune defense responses to a major pneumococcal toxin that plays a critical role in the establishment and the progression of life-threatening diseases. Therapies boosting plasmalemmal repair of host cells and their metabolic fitness might prove beneficial for the treatment of pneumococcal infections.

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis.

BACKGROUND Loss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor. AIM We hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood. METHODS To confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients. RESULTS Although neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37. CONCLUSIONS Collectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease.

Oxidative Stress and Ca2+ Release Events in Mouse Cardiomyocytes

Cellular oxidative stress, associated with a variety of common cardiac diseases, is well recognized to affect the function of several key proteins involved in Ca2+ signaling and excitation-contraction coupling, which are known to be exquisitely sensitive to reactive oxygen species. These include the Ca2+ release channels of the sarcoplasmic reticulum (ryanodine receptors or RyR2s) and the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Oxidation of RyR2s was found to increase the open probability of the channel, whereas CaMKII can be activated independent of Ca2+ through oxidation. Here, we investigated how oxidative stress affects RyR2 function and SR Ca2+ signaling in situ, by analyzing Ca2+ sparks in permeabilized mouse cardiomyocytes under a broad range of oxidative conditions. The results show that with increasing oxidative stress Ca2+ spark duration is prolonged. In addition, long and very long-lasting (up to hundreds of milliseconds) localized Ca2+ release events started to appear, eventually leading to sarcoplasmic reticulum (SR) Ca2+ depletion. These changes of release duration could be prevented by the CaMKII inhibitor KN93 and did not occur in mice lacking the CaMKII-specific S2814 phosphorylation site on RyR2. The appearance of long-lasting Ca2+ release events was paralleled by an increase of RyR2 oxidation, but also by RyR-S2814 phosphorylation, and by CaMKII oxidation. Our results suggest that in a strongly oxidative environment oxidation-dependent activation of CaMKII leads to RyR2 phosphorylation and thereby contributes to the massive prolongation of SR Ca2+ release events.

Keeping bugs in check: The mucus layer as a critical component in maintaining intestinal homeostasis

In the mammalian gastrointestinal tract the close vicinity of abundant immune effector cells and trillions of commensal microbes requires sophisticated barrier and regulatory mechanisms to maintain vital host-microbial interactions and tissue homeostasis. During co-evolution of the host and its intestinal microbiota a protective multilayered barrier system was established to segregate the luminal microbes from the intestinal mucosa with its potent immune effector cells, limit bacterial translocation into host tissues to prevent tissue damage, while ensuring the vital functions of the intestinal mucosa and the luminal gut microbiota. In the present review we will focus on the different layers of protection in the intestinal tract that allow the successful mutualism between the microbiota and the potent effector cells of the intestinal innate and adaptive immune system. In particular, we will review some of the recent findings on the vital functions of the mucus layer and its site-specific adaptations to the changing quantities and complexities of the microbiota along the (gastro-) intestinal tract. Understanding the regulatory pathways that control the establishment of the mucus layer, but also its degradation during intestinal inflammation may be critical for designing novel strategies aimed at maintaining local tissue homeostasis and supporting remission from relapsing intestinal inflammation in patients with inflammatory bowel diseases.

Molecular control of cardiac sodium homeostasis in health and disease.

INTRODUCTION Cardiac myocytes utilize three high-capacity Na transport processes whose precise function can determine myocyte fate and the triggering of arrhythmias in pathological settings. We present recent results on the regulation of all three transporters that may be important for an understanding of cardiac function during ischemia/reperfusion episodes. METHODS AND RESULTS Refined ion selective electrode (ISE) techniques and giant patch methods were used to analyze the function of cardiac Na/K pumps, Na/Ca exchange (NCX1), and Na/H exchange (NHE1) in excised cardiac patches and intact myocytes. To consider results cohesively, simulations were developed that account for electroneutrality of the cytoplasm, ion homeostasis, water homeostasis (i.e., cell volume), and cytoplasmic pH. The Na/K pump determines the average life-time of Na ions (3-10 minutes) as well as K ions (>30 minutes) in the cytoplasm. The long time course of K homeostasis can determine the time course of myocyte volume changes after ion homeostasis is perturbed. In excised patches, cardiac Na/K pumps turn on slowly (-30 seconds) with millimolar ATP dependence, when activated for the first time. In steady state, however, pumps are fully active with <0.2 mM ATP and are nearly unaffected by high ADP (2 mM) and Pi (10 mM) concentrations as may occur in ischemia. NCX1s appear to operate with slippage that contributes to background Na influx and inward current in heart. Thus, myocyte Na levels may be regulated by the inactivation reactions of the exchanger which are both Na- and proton-dependent. NHE1 also undergo strong Na-dependent inactivation, whereby a brief rise of cytoplasmic Na can cause inactivation that persists for many minutes after cytoplasmic Na is removed. This mechanism is blocked by pertussis toxin, suggesting involvement of a Na-dependent G-protein. Given that maximal NCX1- and NHE1-mediated ion fluxes are much greater than maximal Na/K pump-mediated Na extrusion in myocytes, the Na-dependent inactivation mechanisms of NCX1 and NHE1 may be important determinants of cardiac Na homeostasis. CONCLUSIONS Na/K pumps appear to be optimized to continue operation when energy reserves are compromised. Both NCX1 and NHE1 activities are regulated by accumulation of cytoplasmic Na. These principles may importantly control cardiac cytoplasmic Na and promote myocyte survival during ischemia/reperfusion episodes by preventing Ca overload.

Na(+), K(+), Cl(-), acid-base or H2O homeostasis in children with urinary tract infections: a narrative review.

Guidelines on the diagnosis and management of urinary tract infections in childhood do not address the issue of abnormalities in Na(+), K(+), Cl(-) and acid-base balance. We have conducted a narrative review of the literature with the aim to describe the underlying mechanisms of these abnormalities and to suggest therapeutic maneuvers. Abnormalities in Na(+), K(+), Cl(-) and acid-base balance are common in newborns and infants and uncommon in children of more than 3 years of age. Such abnormalities may result from factitious laboratory results, from signs and symptoms (such as excessive sweating, poor fluid intake, vomiting and passage of loose stools) of the infection itself, from a renal dysfunction, from improper parenteral fluid management or from the prescribed antimicrobials. In addition, two transient renal tubular dysfunctions may occur in infants with infectious renal parenchymal involvement: a reduced capacity to concentrate urine and pseudohypoaldosteronism secondary to renal tubular unresponsiveness to aldosterone that presents with hyponatremia, hyperkalemia and acidosis. In addition to antimicrobials, volume resuscitation with an isotonic solution is required in these children. In secondary pseudohypoaldosteronism, isotonic solutions (such as 0.9 % saline or lactated Ringer) correct not only the volume depletion but also the hyperkalemia and acidosis. In conclusion, our review suggests that in infants with infectious renal parenchymal involvement, non-renal and renal causes concur to cause fluid volume depletion and abnormalities in electrolyte and acid-base balance, most frequently hyponatremia.

TNF receptors regulate vascular homeostasis in zebrafish through a caspase-8, caspase-2 and P53 apoptotic program that bypasses caspase-3

Although it is known that tumor necrosis factor receptor (TNFR) signaling plays a crucial role in vascular integrity and homeostasis, the contribution of each receptor to these processes and the signaling pathway involved are still largely unknown. Here, we show that targeted gene knockdown of TNFRSF1B in zebrafish embryos results in the induction of a caspase-8, caspase-2 and P53-dependent apoptotic program in endothelial cells that bypasses caspase-3. Furthermore, the simultaneous depletion of TNFRSF1A or the activation of NF-κB rescue endothelial cell apoptosis, indicating that a signaling balance between both TNFRs is required for endothelial cell integrity. In endothelial cells, TNFRSF1A signals apoptosis through caspase-8, whereas TNFRSF1B signals survival via NF-κB. Similarly, TNFα promotes the apoptosis of human endothelial cells through TNFRSF1A and triggers caspase-2 and P53 activation. We have identified an evolutionarily conserved apoptotic pathway involved in vascular homeostasis that provides new therapeutic targets for the control of inflammation- and tumor-driven angiogenesis.

Gut Microbiota Orchestrates Energy Homeostasis during Cold.

Microbial functions in the host physiology are a result of the microbiota-host co-evolution. We show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase insulin sensitivity of the host and enable tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold, however, the body weight loss is attenuated, caused by adaptive mechanisms maximizing caloric uptake and increasing intestinal, villi, and microvilli lengths. This increased absorptive surface is transferable with the cold microbiota, leading to altered intestinal gene expression promoting tissue remodeling and suppression of apoptosis-the effect diminished by co-transplanting the most cold-downregulated strain Akkermansia muciniphila during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand.

The role of the sonic hedgehog signaling pathway in epidermal homeostasis

Non-melanoma skin cancer is the most frequently diagnosed malignancy in the United States of which basal cell carcinoma (BCC) accounts for 65%. It has recently been determined that deregulation of the sonic hedgehog (shh) pathway leads to the development of BCC. Shh, gli-1, gli-2 gli-3, ptc and smo are overexpressed in BCC and overexpression of these genes in the epidermis results in formation of BCC-like tumors. Despite these observations, the mechanisms by which the pathway controls epidermal homeostasis and the development of the malignant phentotype are unknown. This study assessed the role of the shh pathway in epidermal homeostasis through regulation of apoptosis and differentiation. ^ The anti-apoptotic protein, bcl-2 is overexpressed in BCC, however transcriptional regulators of bcl-2 in the epidermis are unknown. Transient transfection of primary keratinocytes with gli-1 resulted in an increase of bcl-2 expression. Database analysis revealed seven candidate gli binding sites on the bcl-2 promoter. Cotransfection of increasing amounts of gli-1 in keratinoycytes resulted in a corresponding dose-dependent increase in bcl-2 promoter luciferase activity. An N-terminal mutant of gli-3 inhibited gli-1 transactivation of the bcl-2 promoter. The region −428 to −420 was found to be important for gli-1 regulation through gel shift, luciferase assays and site-directed mutagenesis. ^ In order to assess the ability of the shh pathway to regulate keratinocyte differentiation, HaCaT keratinocytes overexpressing sonic hedgehog, were grown in organotypic raft culture. Overexpression of shh induced a basal cell phenotype compared to vector control, as evidenced by transmural staining of cytokeratin 14 and altered Ki67 staining. Shh also induced keratinocyte invasion into the underlying collagen. This was associated with increased phosphorylation of EGFR, jnk and raf and increased expression of c-jun, mmp-9 and Ki67. Interestingly, shh overexpression in HaCaTs did not induce the typical downstream effects of shh signaling, suggesting a gli-independent mechanism. Sonic hedgehog's ability to induce an invasive phenotype was found to be dependent on activation of the EGF pathway as inhibition of EGFR activity with AG1478 and c-225 was able to reduce the invasiveness of HaCaT shh keratinocytes, whereas treatment with EGF augmented the invasiveness of the HaCaT shh clones. ^ These studies reveal the importance of the sonic hedgehog pathway in epidermal homeostasis by regulation of apoptosis through bcl-2, and control of keratinocyte differentiation and invasion through activation of the EGF pathway. They further suggest potential mechanisms by which deregulation of the shh pathway may lead to the development of the malignant phenotype. ^

New insights into the role of the duodenal vitamin D receptor in calcium homeostasis

It is generally believed that 1,25(OH)2D3, bound to its receptor (VDR) contributes to calcium homeostasis by regulating active calcium absorption in the proximal small intestine. However, studying patients with hereditary vitamin D-resistant rickets (HVDRR) provided investigators with a better understanding of VDR's role in calcium homeostasis. HVDRR patients have inactivating mutations in the VDR, and as a consequence they develop hypocalcemia, hyperparathyroidism and severe rickets. However, these phenotypes can be corrected if the patients are given IV infusions of calcium or dietary calcium. This raises the question of what is the physiological significance of VDR-regulated active calcium absorption if calcium homeostasis can be restored independently of the VDR. ^ In order to distinguish the contribution of VDR in the proximal small intestine to overall calcium homeostasis, I generated transgenic mice expressing the human VDR (hVDR) exclusively in the proximal small intestine of mVDR-/- mice by using an hVDR-expressing transgene driven by the duodenal-specific adenosine deaminase enhancer (hVDR+/mVDR-/-). hVDR+/mVDR-/- mice expressed transcriptionally active hVDR only in the proximal small intestine and responded to 1,25(OH)2D3 by up-regulating expression of TRPV6 and calbindin D9K, genes involved in calcium absorption. Furthermore, ligated duodenal loop assays determined that calcium absorption in hVDR+/mVDR-/- mice was as responsive to 1,25(OH)2D3 as in WT mice. Despite having a functional hVDR in the proximal small intestine, hVDR+/mVDR-/- mice were hypocalcemic, had hyperparathyroidism, and were rachitic when fed a normal rodent diet at weaning, as were the mVDR-/- mice. However, when fed a high calcium, phosphorus, and lactose diet (rescue diet), the hVDR+/mVDR-/- mice responded more effectively than the mVDR-/- mice by down-regulation of parathyroid hormone production and by a greater increase in bone mineralization. Furthermore, when three-month-old rachitic mice were fed a rescue diet for 3 weeks, serum calcium and bone mineral content were normalized in hVDR+/mVDR-/- mice, but not in mVDR-/- mice. ^ In conclusion, hVDR expression enabled young mice to better use the rescue diet than mVDR-/- mice. Expression of transgenic hVDR also protected the ability of older mice to respond to the rescue diet despite the absence of the VDR elsewhere in the intestinal tract. I propose that because hVDR+/mVDR-/- mice responded better than mVDR-/- mice to the rescue diet, it is likely that VDR expression in the proximal small intestine is necessary in nutritional (insufficient dietary calcium) and physiological (age) conditions when passive calcium absorption is inadequate. ^

CARD9 functions as a key regulator in monocyte homeostasis and pathogen clearance

Mononuclear phagocytes are designed to neutralize systemic bacterial and fungal infections. However, the exact regulation of these functions are largely unknown. CARD9 was first identified as an immune-specific adaptor protein of unclear function. Here, we have found that Card9 is specifically expressed in monocyte-origin cell populations. To better understand the biological function of Card9, we have generated Card9-deficient (Card9-/-) mice. Hematologic profiling and histological analysis of Card9-/- mice revealed a decreased leukocyte/myeloid cell count, delayed monocyte maturation in bone marrow as well as monocyte counts in the peripheral blood. Upon M-CSF stimulation, Card9-/- macrophages further exhibit a partial loss in IKK phosphorylation. As a consequence, in vivo challenge with Listeria monocytogenes in Card9-/- mice results in a higher susceptibility to infection-associated inflammation and fatality. Collectively, these data suggest that CARD9 is required for monocyte development and function. ^ At the cellular level, Card9-/- macrophages are defective in killing Listeria and the production of pro-inflammatory cytokines. Molecular characterizations have further demonstrated that CARD9 inducibly interacts with NOD2, controls p38 MAPK activation, and regulates ROS production during Listeria infections. Cytotrap screening showed that CARD9 could physically associate with various g&barbelow;uanine e&barbelow;xchange f&barbelow;actor (GEF) proteins that are essential for regulating ROS production. In summary, we have first identified and provided genetic evidence that CARD9 functions as a novel regulator during monocyte development and serves as an essential protein adaptor for p38 MAPK activation during bacterial clearance processes in macrophages. ^

Depolarization-dependent synaptic potentiation and hyperexcitability induced by a Ca2+-independent trigger

Despite vast research efforts since Cajal's seminal thoughts on the adaptation of the nervous system, researchers have only recently begun to understand the diversity of forms of neuronal plasticity and its mechanisms. All known forms of activity-dependent neuronal plasticity utilize alterations in [Ca 2+]i as a signal of changes in the membrane voltage. Ca 2+ sensors trigger modifications in excitability or synaptic strength that last from seconds to weeks and presumably years. Intriguingly, Kunjilwar et al., (unpublished observations) discovered in peripheral sensory axons of Aplysia that the induction of depolarization-dependent long-term axonal hyperexcitability does not require Ca2+ transients. Here we show that induction of depolarization-dependent intermediate-term and long-term synaptic potentiation in Aplysia occurs in conditions that prevent Ca2+ entry through voltage-gated channels and elevation of [Ca2+]i. We found that the intermediate-term synaptic potentiation induced under conditions expected to prevent Ca 2+ transients is associated with increased excitability of sensory neuron axons near presynaptic terminals, suggesting that the synaptic potentiation involves a presynaptic locus. The Ca2+-independent intermediate- and long-term synaptic potentiation appeared similar to previously reported Ca2+-dependent modifications in Aplysia. ^

The role of MEKK3 in T cell homeostasis and IFNgamma production

MEKK3, a member of the MAP3K family, is involved in regulating multiple MAPK and NF-κB pathways. The MAPK and NF-κB signaling pathways are important in regulating T cell functions. MEKK3 is expressed through the development of T cell and also in subsets of T cell in the peripheral. However, the specific role of MEKK3 in T cell function is unknown. To reveal the in vivo function of MEKK3 in T cells, I have generated MEKK3 T cell conditional knock-out mice. Despite a normal thymus development in the conditional knock-out mice, I observed a decrease in the number of peripheral T-cells and impaired T-cell function in response to antigen stimulation. T cells undergo homeostatic proliferation under lymphopenia condition, a process called lymphopenia-induced proliferation (LIP). Using a LIP model, I demonstrated that the reduction of peripheral T cell number is largely due to a severe impairment of the self-antigen/MHC mediated T cell homeostasis. Upon anti-CD3 stimulation, the proliferation of MEKK3-deficient T cell is not significantly affected, but the production of IFNγ by naïve and effector CD4 T cells are markedly decreased. Interestingly, the IL-12/IL-18 driven IFNγ production and MAPK activation in MEKK3-deficient T cells is not affected, suggesting that MEKK3 selectively mediates the TCR induced MAPK signaling. Furthermore, I found that MEKK3 is activated by TCR stimulation in a RAC1/2 dependent manner, but not by IL-12/IL-18 stimulation. Finally, I showed that basal level of ERK and JNK activation is defective under LIP condition. I showed that the TCR induced ERK, JNK and p38 MAPK activation is also defective in MEKK3 deficient CD4 T cells. Taken together, my data demonstrate a crucial role of MEKK3 in T cell homeostasis and IFNγ production through regulating the TCR mediated MAPK pathway. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.