Cyclosporine A Induces MicroRNAs Controlling Innate Immunity during Renal Bacterial Infection.

Urinary tract infections (UTIs) mainly due to uropathogenic Escherichia coli (UPEC) are one of the most frequent complications in kidney-transplanted patients, causing significant morbidity. However, the mechanisms underlying UTI in renal grafts remain poorly understood. Here, we analysed the effects of the potent immunosuppressive agent cyclosporine A (CsA) on the activation of collecting duct cells that represent a preferential site of adhesion and translocation for UPEC. CsA induced the inhibition of lipopolysaccharide- induced activation of collecting duct cells due to the downregulation of the expression of TLR4 via the microRNA Let-7i. Using an experimental model of ascending UTI, we showed that the pretreatment of mice with CsA prior to infection induced a marked fall in cytokine production by collecting duct cells, neutrophil recruitment, and a dramatic rise of bacterial load, but not in infected TLR4-defective mice kidneys. This effect was also observed in CsA-treated infected kidneys, where the expression of Let-7i was increased. Treatment with a synthetic Let-7i mimic reproduced the effects of CsA. Conversely, pretreatment with an anti-Let-7i antagonised the effects of CsA and rescued the innate immune response of collecting duct cells against UPEC. Thus, the utilisation of an anti-Let-7i during kidney transplantation may protect CsA-treated patients from ascending bacterial infection.

Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4(-/-) MCDs, but not in Tlr5(-/-) MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5(-/-) than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5(-/-) kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post-infected Tlr5(-/-) kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.

Calcineurin/NFAT signaling and innate host defence: a role for NOD1-mediated phagocytic functions.

The calcineurin/nuclear factor of activated T cells (NFATs) signaling pathway plays a central role in T cell mediated adaptive immune responses, but a number of recent studies demonstrated that calcineurin/NFAT signaling also plays a key role in the control of the innate immune response by myeloid cells. Calcineurin inhibitors, such as cyclosporine A (CsA) and tacrolimus (FK506), are commonly used in organ transplantation to prevent graft rejection and in a variety of immune diseases. These immunosuppressive drugs have adverse effects and significantly increase host's susceptibility towards bacterial or fungal infections. Recent studies highlighted the role of NFAT signaling in fungal infection and in the control of the pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 1 (NOD1), which predominantly senses invasive Gram-negative bacteria and mediates neutrophil phagocytic functions. This review summarises some of the current knowledge concerning the role of NFAT signaling in the innate immune response and the recent advances on NFAT-dependent inhibition of NOD1-mediated innate immune response caused by CsA, which may contribute to sensitizing transplant recipients to bacterial infection.

Neonatal immune adaptation of the gut and its role during infections.

The intestinal tract is engaged in a relationship with a dense and complex microbial ecosystem, the microbiota. The establishment of this symbiosis is essential for host physiology, metabolism, and immune homeostasis. Because newborns are essentially sterile, the first exposure to microorganisms and environmental endotoxins during the neonatal period is followed by a crucial sequence of active events leading to immune tolerance and homeostasis. Contact with potent immunostimulatory molecules starts immediately at birth, and the discrimination between commensal bacteria and invading pathogens is essential to avoid an inappropriate immune stimulation and/or host infection. The dysregulation of these tight interactions between host and microbiota can be responsible for important health disorders, including inflammation and sepsis. This review summarizes the molecular events leading to the establishment of postnatal immune tolerance and how pathogens can avoid host immunity and induce neonatal infections and sepsis.

Cyclosporine A impairs nucleotide binding oligomerization domain (Nod1)-mediated innate antibacterial renal defenses in mice and human transplant recipients.

Acute pyelonephritis (APN), which is mainly caused by uropathogenic Escherichia coli (UPEC), is the most common bacterial complication in renal transplant recipients receiving immunosuppressive treatment. However, it remains unclear how immunosuppressive drugs, such as the calcineurin inhibitor cyclosporine A (CsA), decrease renal resistance to UPEC. Here, we investigated the effects of CsA in host defense against UPEC in an experimental model of APN. We show that CsA-treated mice exhibit impaired production of the chemoattractant chemokines CXCL2 and CXCL1, decreased intrarenal recruitment of neutrophils, and greater susceptibility to UPEC than vehicle-treated mice. Strikingly, renal expression of Toll-like receptor 4 (Tlr4) and nucleotide-binding oligomerization domain 1 (Nod1), neutrophil migration capacity, and phagocytic killing of E. coli were significantly reduced in CsA-treated mice. CsA inhibited lipopolysaccharide (LPS)-induced, Tlr4-mediated production of CXCL2 by epithelial collecting duct cells. In addition, CsA markedly inhibited Nod1 expression in neutrophils, macrophages, and renal dendritic cells. CsA, acting through inhibition of the nuclear factor of activated T-cells (NFATs), also markedly downregulated Nod1 in neutrophils and macrophages. Silencing the NFATc1 isoform mRNA, similar to CsA, downregulated Nod1 expression in macrophages, and administration of the 11R-VIVIT peptide inhibitor of NFATs to mice also reduced neutrophil bacterial phagocytosis and renal resistance to UPEC. Conversely, synthetic Nod1 stimulating agonists given to CsA-treated mice significantly increased renal resistance to UPEC. Renal transplant recipients receiving CsA exhibited similar decrease in NOD1 expression and neutrophil phagocytosis of E. coli. The findings suggest that such mechanism of NFATc1-dependent inhibition of Nod1-mediated innate immune response together with the decrease in Tlr4-mediated production of chemoattractant chemokines caused by CsA may contribute to sensitizing kidney grafts to APN.

MicroRNA-146a-mediated downregulation of IRAK1 protects mouse and human small intestine against ischemia/reperfusion injury.

Intestinal ischemia/reperfusion (I/R) injury causes inflammation and tissue damage and is associated with high morbidity and mortality. Uncontrolled activation of the innate immune system through toll-like receptors (Tlr) plays a key role in I/R-mediated tissue damage but the underlying mechanisms have not been fully resolved. Here, we identify post-transcriptional upregulation of the essential Tlr signalling molecule interleukin 1 receptor-associated kinase (Irak) 1 as the causative mechanism for post-ischemic immune hyper-responsiveness of intestinal epithelial cells. Increased Irak1 protein levels enhanced epithelial ligand responsiveness, chemokine secretion, apoptosis and mucosal barrier disruption in an experimental intestinal I/R model using wild-type, Irak1(-/-) and Tlr4(-/-) mice and ischemic human intestinal tissue. Irak1 accumulation under hypoxic conditions was associated with reduced K48 ubiquitination and enhanced Senp1-mediated deSUMOylation of Irak1. Importantly, administration of microRNA (miR)-146a or induction of miR-146a by the phytochemical diindolylmethane controlled Irak1 upregulation and prevented immune hyper-responsiveness in mouse and human tissue. These findings indicate that Irak1 accumulation triggers I/R-induced epithelial immune hyper-responsiveness and suggest that the induction of miR-146a offers a promising strategy to prevent I/R tissue injury.

Establishment of intestinal homeostasis during the neonatal period.

The intestinal mucosa faces the challenge of regulating the balance between immune tolerance towards commensal bacteria, environmental stimuli and food antigens on the one hand, and induction of efficient immune responses against invading pathogens on the other hand. This regulatory task is of critical importance to prevent inappropriate immune activation that may otherwise lead to chronic inflammation, tissue disruption and organ dysfunction. The most striking example for the efficacy of the adaptive nature of the intestinal mucosa is birth. Whereas the body surfaces are protected from environmental and microbial exposure during fetal life, bacterial colonization and contact with potent immunostimulatory substances start immediately after birth. In the present review, we summarize the current knowledge on the mechanisms underlying the transition of the intestinal mucosa during the neonatal period leading to the establishment of a stable, life-long host-microbial homeostasis. The environmental exposure and microbial colonization during the neonatal period, and also the influence of maternal milk on the immune protection of the mucosa and the role of antimicrobial peptides, are described. We further highlight the molecular mechanisms of innate immune tolerance in neonatal intestinal epithelium. Finally, we link the described immunoregulatory mechanisms to the increased susceptibility to inflammatory and infectious diseases during the neonatal period.

A role for collecting duct epithelial cells in renal antibacterial defences.

Urinary tract infections (UTIs), which are mainly due to uropathogenic Escherichia coli (UPEC), occur via the retrograde ascent of the bacteria along the urinary tract system. The adhesion and invasion mechanisms of UPEC have been extensively studied in bladder epithelial cells, but less is known about the role of renal tubule epithelial cells (RTEC) in renal antibacterial defences. This review considers recent advances in the understanding of the role of RTECs in inducing an innate immune response mediated by Toll-like receptors (TLRs) in experimental UTI. Collecting duct cells are a preferential site of adhesion of UPEC colonizing the kidneys. Epithelial TLR4 activation induces an inflammatory response and the recruitment of lipid rafts to the plasma membrane, both of which facilitate the transcytosis of non-cytolytic UPEC strains across intact collecting duct cell layers to invade the renal interstitium. Arginine vasopressin, which regulates water absorption in the collecting duct, also acts as a potent modulator of the TLR4-mediated intrarenal innate response caused by UPEC. The role of epithelial TLR5 in renal host defences is also discussed. These findings highlight the role of RTECs in triggering the innate immune response in the context of ascending UTIs.

Potentiation of epithelial innate host responses by intercellular communication.

The epithelium efficiently attracts immune cells upon infection despite the low number of pathogenic microbes and moderate levels of secreted chemokines per cell. Here we examined whether horizontal intercellular communication between cells may contribute to a coordinated response of the epithelium. Listeria monocytogenes infection, transfection, and microinjection of individual cells within a polarized intestinal epithelial cell layer were performed and activation was determined at the single cell level by fluorescence microscopy and flow cytometry. Surprisingly, chemokine production after L. monocytogenes infection was primarily observed in non-infected epithelial cells despite invasion-dependent cell activation. Whereas horizontal communication was independent of gap junction formation, cytokine secretion, ion fluxes, or nitric oxide synthesis, NADPH oxidase (Nox) 4-dependent oxygen radical formation was required and sufficient to induce indirect epithelial cell activation. This is the first report to describe epithelial cell-cell communication in response to innate immune activation. Epithelial communication facilitates a coordinated infectious host defence at the very early stage of microbial infection.

miR-146a mediates protective innate immune tolerance in the neonate intestine.

After birth, the intestinal mucosa undergoes a dramatic transition from a sterile protected site to an environmentally exposed and permanently colonized surface. The mechanisms that facilitate this transition are ill defined. Here, we demonstrate that microRNA-146a-mediated translational repression and proteolytic degradation of the essential Toll-like receptor (TLR) signaling molecule interleukin 1 receptor associated kinase 1 (IRAK1) is sufficient to induce intestinal epithelial innate immune tolerance and provide protection from bacteria-induced epithelial damage in neonates. Despite low IRAK1 protein levels, continuous TLR4- and IRAK1-dependent signal transduction induced by intraepithelial endotoxin persistence during the neonatal period maintains tolerance through sustained miR-146a expression. Strikingly, it additionally facilitates transcription of a distinct set of genes involved in cell survival, differentiation, and homeostasis. Thus, our results identify the underlying molecular mechanisms of intestinal epithelial innate immune tolerance during the neonatal period and characterize tolerance as an active condition involved in the establishment of intestinal mucosal homeostasis.

Contribution of renal tubule epithelial cells in the innate immune response during renal bacterial infections and ischemia-reperfusion injury.

The epithelial cells that line the renal tubule are sometimes severely injured in the course of inflammatory kidney diseases. These renal tubule epithelial cells (RTECs) express some of the Toll-like receptors (TLRs) of the innate immune system. A number of studies have implicated RTECs, together with bone marrow-derived cells, in triggering an innate immune response to bacterial infection and/or ischemic stress. RTECs expressing TLR4, which recognizes lipopolysaccharide (LPS), contribute to defending the host against ascending urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPECs). Activation of TLR2 and TLR4 signaling by endogenous damage-associated molecular patterns controls the inflammatory responses of RTECs and cell apoptosis in kidneys subjected to ischemia/reperfusion (I/R) injury. This review will consider some recent advances in understanding of the role of RTECs in inducing the innate immune response in experimental models of ascending UTIs and renal I/R injury. Arginine vasopressin, which regulates renal water absorption, has been shown to act as a potent modulator of the innate response in collecting duct cells, a preferred intrarenal site for UPEC adhesion. The activation of the mitogen-associated protein kinase ERK1/2 in post-hypoxic RTECs has also been shown to be selectively regulated by TLR2 via the serine-threonine protein phosphatase 5, which is associated with the endoplasmic reticulum resident heat shock protein, gp96, which acts as a master chaperone of TLRs. These findings provide further support for the concept that RTECs are actively involved in triggering the innate immune response, at least in the context of ascending UTIs and I/R injury.

O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense in murine intestinal epithelial cells.

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.

TLR4- and TLR2-mediated B cell responses control the clearance of the bacterial pathogen, Leptospira interrogans.

Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira interrogans that are transmitted by asymptomatic infected rodents. Leptospiral lipoproteins and LPS have been shown to stimulate murine cells via TLRs 2 and 4. Host defense mechanisms remain obscure, although TLR4 has been shown to be involved in clearing Leptospira. In this study, we show that double (TLR2 and TLR4) knockout (DKO) mice rapidly died from severe hepatic and renal failure following Leptospira inoculation. Strikingly, the severe proinflammatory response detected in the liver and kidney from Leptospira-infected DKO mice appears to be independent of MyD88, the main adaptor of TLRs. Infection of chimeric mice constructed with wild-type and DKO mice, and infection of several lines of transgenic mice devoid of T and/or B lymphocytes, identified B cells as the crucial lymphocyte subset responsible for the clearance of Leptospira, through the early production of specific TLR4-dependent anti-Leptospira IgMs elicited against the leptospiral LPS. We also found a protective tissue compartmentalized TLR2/TLR4-mediated production of IFN-gamma by B and T lymphocytes, in the liver and kidney, respectively. In contrast, the tissue inflammation observed in Leptospira-infected DKO mice was further characterized to be mostly due to B lymphocytes in the liver and T cells in the kidney. Altogether these findings demonstrate that TLR2 and TLR4 play a key role in the early control of leptospirosis, but do not directly trigger the inflammation induced by pathogenic Leptospira.

TLR4 facilitates translocation of bacteria across renal collecting duct cells.

Uropathogenic Escherichia coli (UPEC) are the most frequent causes of urinary tract infections and pyelonephritis. Renal medullary collecting duct (MCD) cells are the intrarenal site to which UPEC strains prefer to adhere and initiate an inflammatory response, but the ability of UPEC strains to translocate across impermeant MCD cells has not been demonstrated definitively. Here, several UPEC strains adhered to the apical surface and translocated across confluent murine inner MCD cells grown on filters. UPEC strains expressing cytolytic and vacuolating cytotoxins disrupted the integrity of cell layers, whereas noncytolytic UPEC strains passed through the cell layers without altering tight junctions. Apical-to-basal transcellular translocation was dramatically reduced after extinction of Toll-like receptor 4 (TLR4) and the lipid raft marker caveolin-1 by small interfering RNA. Furthermore, disruption of lipid raft integrity by filipin III and methyl-beta-cyclodextrin significantly reduced both the transcellular translocation of UPEC across murine inner MCD cell layers and the stimulation of proinflammatory mediators. Bacterial translocation was also significantly reduced in primary cultures of TLR4-deficient mouse MCD cells compared with MCD cells from wild-type mice. Benzyl alcohol, an anesthetic that enhances membrane fluidity, favored the recruitment of caveolin-1 in lipid rafts and increased the translocation of UPEC across cultured TLR4-deficient MCD cells. These findings demonstrate that the transcellular translocation of UPEC strains across impermeant layers of MCD cells may occur through lipid rafts via a TLR4-facilitated process.

Hormonal control of the renal immune response and antibacterial host defense by arginine vasopressin.

Ascending urinary tract infection (UTI) and pyelonephritis caused by uropathogenic Escherichia coli (UPEC) are very common infections that can cause severe kidney damage. Collecting duct cells, the site of hormonally regulated ion transport and water absorption controlled by vasopressin, are the preferential intrarenal site of bacterial adhesion and initiation of inflammatory response. We investigated the effect of the potent V2 receptor (V2R) agonist deamino-8-D-arginine vasopressin (dDAVP) on the activation of the innate immune response using established and primary cultured collecting duct cells and an experimental model of ascending UTI. dDAVP inhibited Toll-like receptor 4-mediated nuclear factor kappaB activation and chemokine secretion in a V2R-specific manner. The dDAVP-mediated suppression involved activation of protein phosphatase 2A and required an intact cystic fibrosis transmembrane conductance regulator Cl- channel. In vivo infusion of dDAVP induced a marked fall in proinflammatory mediators and neutrophil recruitment, and a dramatic rise in the renal bacterial burden in mice inoculated with UPECs. Conversely, administration of the V2R antagonist SR121463B to UPEC-infected mice stimulated both the local innate response and the antibacterial host defense. These findings evidenced a novel hormonal regulation of innate immune cellular activation and demonstrate that dDAVP is a potent modulator of microbial-induced inflammation in the kidney.

Renal collecting duct epithelial cells react to pyelonephritis-associated Escherichia coli by activating distinct TLR4-dependent and -independent inflammatory pathways.

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lps(d)) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lps(n)) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-alpha still remained greater in UPEC-infected than in naive C3H/HeJ (Lps(d)) mice. Using primary cultures of microdissected Lps(n) MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-beta-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lps(n) MCDs leads to the activation of NF-kappaB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.