Dynamin2- and endothelial nitric oxide synthase-regulated invasion of bladder epithelial cells by uropathogenic Escherichia coli

Invasion of bladder epithelial cells by uropathogenic Escherichia coli (UPEC) contributes to antibiotic-resistant and recurrent urinary tract infections (UTIs), but this process is incompletely understood. In this paper, we provide evidence that the large guanosine triphosphatase dynamin2 and its partner, endothelial nitric oxide (NO) synthase (NOS [eNOS]), mediate bacterial entry. Overexpression of dynamin2 or treatment with the NO donor S-nitrosothiols increases, whereas targeted reduction of endogenous dynamin2 or eNOS expression with ribonucleic acid interference impairs, bacterial invasion. Exposure of mouse bladder to small molecule NOS inhibitors abrogates infection of the uroepithelium by E. coli, and, concordantly, bacteria more efficiently invade uroepithelia isolated from wild-type compared with eNOS(-/-) mice. E. coli internalization promotes rapid phosphorylation of host cell eNOS and NO generation, and dynamin2 S-nitrosylation, a posttranslational modification required for the bacterial entry, also increases during E. coli invasion. These findings suggest that UPEC escape urinary flushing and immune cell surveillance by means of eNOS-dependent dynamin2 S-nitrosylation and invasion of host cells to cause recurrent UTIs.     

Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.     

Toll-like receptor 4 is needed to restrict the invasion of Escherichia coli P4 into mammary gland epithelial cells in a murine model of acute mastitis

Mastitis, an inflammatory response of the mammary tissue to invading pathogenic bacteria, is a common disease in breast-feeding women and dairy animals. Escherichia coli is a leading cause of mastitis in dairy animals. During the course of the disease the host mounts a strong inflammatory response, but specific bacterial virulence factors have not yet been identified. Here we report the use of a murine mastitis model to investigate the innate inflammatory reaction of the mammary gland. We show that lipopolysaccharide (LPS) infusion induces mastitis in wild-type mice (C3H/HeN), but not in mice expressing mutated Toll-like receptor 4 (TLR4) (C3H/HeJ). The wild-type phenotype was restored by adoptive transfer of TLR4-expressing macrophages into the alveolar milk space of C3H/HeJ mice. In contrast to the LPS treatment, infection with E. coli P4 (ECP4) resulted in inflammation even in the absence of LPS/TLR4 signalling, indicating that additional factors play a role in the pathogenesis of the intact bacteria. Furthermore, in the absence of functional TLR4 the infecting ECP4 invade the epithelial cells with high efficiency, forming intracellular microcolonies. However, adoptive transfer with TLR4-expressing macrophages drastically reduced the epithelial invasion. Taken together, these results indicate that ECP4 has an invasive potential, which is restricted by alveolar macrophages in response to the LPS/TLR4 signalling.     

Type 1 pilus-mediated bacterial invasion of bladder epithelial cells

Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between alpha-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

Early Severe Inflammatory Responses to Uropathogenic E. coli Predispose to Chronic and Recurrent Urinary Tract Infection

Chronic infections are an increasing problem due to the aging population and the increase in antibiotic resistant organisms. Therefore, understanding the host-pathogen interactions that result in chronic infection is of great importance. Here, we investigate the molecular basis of chronic bacterial cystitis. We establish that introduction of uropathogenic E. coli (UPEC) into the bladders of C3H mice results in two distinct disease outcomes: resolution of acute infection or development of chronic cystitis lasting months. The incidence of chronic cystitis is both host strain and infectious dose-dependent. Further, development of chronic cystitis is preceded by biomarkers of local and systemic acute inflammation at 24 hours post-infection, including severe pyuria and bladder inflammation with mucosal injury, and a distinct serum cytokine signature consisting of elevated IL-5, IL-6, G-CSF, and the IL-8 analog KC. Mice deficient in TLR4 signaling or lymphocytes lack these innate responses and are resistant, to varying degrees, to developing chronic cystitis. Treatment of C3H mice with the glucocorticoid anti-inflammatory drug dexamethasone prior to UPEC infection also suppresses the development of chronic cystitis. Finally, individuals with a history of chronic cystitis, lasting at least 14 days, are significantly more susceptible to redeveloping severe, chronic cystitis upon bacterial challenge. Thus, we have discovered that the development of chronic cystitis in C3H mice by UPEC is facilitated by severe acute inflammatory responses early in infection, which subsequently are predisposing to recurrent cystitis, an insidious problem in women. Overall, these results have significant implications for our understanding of how early host-pathogen interactions at the mucosal surface determines the fate of disease.     

TLR4-initiated and cAMP-mediated abrogation of bacterial invasion of the bladder

The remarkable resistance of the urinary tract to infection has been attributed to its physical properties and the innate immune responses triggered by pattern recognition receptors lining the tract. We report a distinct TLR4 mediated mechanism in bladder epithelial cells (BECs) that abrogates bacterial invasion, a necessary step for successful infection. Compared to controls, uropathogenic type 1 fimbriated Escherichia coli and Klebsiella pneumoniae invaded BECs of TLR4 mutant mice in 10-fold or greater numbers. TLR4 mediated suppression of bacterial invasion was linked to increased intracellular cAMP levels which negatively impacted Rac-1 mediated mobilization of the cytoskeleton. Artificially increasing intracellular cAMP levels in BECs of TLR4 mutant mice restored resistance to type 1 fimbriated bacterial invasion. This finding reveals a novel function for TLR4 and another facet of bladder innate defense.     

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.     

Uropathogenic Escherichia coli invades host cells via an HDAC6-modulated microtubule-dependent pathway

Strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili that promote bacterial colonization and invasion of the bladder epithelium. Type 1 pilus-mediated interactions with host receptors, including alpha3beta1 integrin, trigger localized actin rearrangements that lead to internalization of adherent bacteria via a zipper-like mechanism. Here we report that type 1 pilus-mediated bacterial invasion of bladder cells also requires input from host microtubules and histone deacetylase 6 (HDAC6), a cytosolic enzyme that, by deacetylating alpha-tubulin, can alter the stability of microtubules along with the recruitment and directional trafficking of the kinesin-1 motor complex. We found that disruption of microtubules by nocodazole or vinblastine treatment, as well as microtubule stabilization by taxol, inhibited host cell invasion by UPEC, as did silencing of HDAC6 expression or pharmacological inhibition of HDAC6 activity. Invasion did not require two alternate HDAC6 substrates, Hsp90 and cortactin, but was dependent upon the kinesin-1 light chain KLC2 and an upstream activator of HDAC6, aurora A kinase. These results indicate that HDAC6 and microtubules act as vital regulatory elements during the invasion process, possibly via indirect effects on kinesin-1 and associated cargos.     

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.     

Toll-like receptor 4-mediated innate IL-10 activates antigen-specific regulatory T cells and confers resistance to Bordetella pertussis by inhibiting inflammatory pathology

Signaling through Toll-like receptors (TLR) activates dendritic cell (DC) maturation and IL-12 production, which directs the induction of Th1 cells. We found that the production of IL-10, in addition to inflammatory cytokines and chemokines, was significantly reduced in DCs from TLR4-defective C3H/HeJ mice in response to Bordetella pertussis. TLR4 was also required for B. pertussis LPS-induced maturation of DCs, but other B. pertussis components stimulated DC maturation independently of TLR4. The course of B. pertussis infection was more severe in C3H/HeJ than in C3H/HeN mice. Surprisingly, Ab- and Ag-specific IFN-gamma responses were enhanced at the peak of infection, whereas Ag-specific IL-10-producing T cells were significantly reduced in C3H/HeJ mice. This was associated with enhanced inflammatory cytokine production, cellular infiltration, and severe pathological changes in the lungs of TLR4-defective mice. Our findings suggest that TLR-4 signaling activates innate IL-10 production in response to B. pertussis, which both directly, and by promoting the induction of IL-10-secreting type 1 regulatory T cells, may inhibit Th1 responses and limit inflammatory pathology in the lungs during infection with B. pertussis.     

Immunomodulation via novel use of TLR4 by the filarial nematode phosphorylcholine-containing secreted product, ES-62

Filarial nematodes, parasites of vertebrates, including humans, secrete immunomodulatory molecules into the host environment. We have previously demonstrated that one such molecule, the phosphorylcholine-containing glycoprotein ES-62, acts to bias the immune response toward an anti-inflammatory/Th2 phenotype that is conducive to both worm survival and host health. For example, although ES-62 initially induces macrophages to produce low levels of IL-12 and TNF-alpha, exposure to the parasite product ultimately renders the cells unable to produce these cytokines in response to classic stimulators such as LPS/IFN-gamma. We have investigated the possibility that a TLR is involved in the recognition of ES-62 by target cells, because phosphorylcholine, a common pathogen-associated molecular pattern, appears to be responsible for many of the immunomodulatory properties of ES-62. We now demonstrate that ES-62-mediated, low level IL-12 and TNF-alpha production by macrophages and dendritic cells is abrogated in MyD88 and TLR4, but not TLR2, knockout, mice implicating TLR4 in the recognition of ES-62 by these cells and MyD88 in the transduction of the resulting intracellular signals. We also show that ES-62 inhibits IL-12 induction by TLR ligands other than LPS, bacterial lipopeptide (TLR2) and CpG (TLR9), via this TLR4-dependent pathway. Surprisingly, macrophages and dendritic cells from LPS-unresponsive, TLR4-mutant C3H/HeJ mice respond normally to ES-62. This is the first report to demonstrate that modulation of cytokine responses by a pathogen product can be abrogated in cells derived from TLR4 knockout, but not C3H/HeJ mice, suggesting the existence of a novel mechanism of TLR4-mediated immunomodulation.     

Impaired recognition by Toll-like receptor 4 is responsible for exacerbated murine Pneumocystis pneumonia

We investigated the effect of Toll-like receptor 4 (TLR4) on the progression of murine Pneumocystis pneumonia. TLR4-mutant C3H/HeJ and wild-type C3H/HeN mice were infected with Pneumocystis after depletion of CD4 T cells. Mutant mice lost body weight more quickly and showed exacerbated pulmonary injury even though there was no difference in Pneumocystis organism burden in the lung. Mutant mice showed reduced levels of IL-10, IL-12p40 and MIP-2 accompanied by elevated levels of TNF-alpha and IL-6 in the bronchoalveolar lavage fluid compared with those of wild-type mice 8 weeks after the infection. In response to stimulation with Pneumocystis antigen, the production of IL-10, IL-12p40 and MIP-2 by alveolar macrophages was partially impaired in mutant mice, while that in wild-type mice was suppressed by the anti-TLR4/MD-2 mAb, MTS510. Unlike the response to lipopolysaccharide stimulation, TLR4-reconstituted HEK293 cells showed no elevated NF-kappaB activation after stimulation with Pneumocystis antigen. Taken together, these findings suggest that recognition of Pneumocystis by TLR4 helps to regulate the host inflammatory responses through cytokine and chemokine production by alveolar macrophages.     

Clathrin, AP-2, and the NPXY-binding subset of alternate endocytic adaptors facilitate FimH-mediated bacterial invasion of host cells

The FimH adhesin, localized at the distal tips of type 1 pili, binds mannose-containing glycoprotein receptors like alpha3beta1 integrins and stimulates bacterial entry into target host cells. Strains of uropathogenic Escherichia coli (UPEC), the major cause of urinary tract infections, utilize FimH to invade bladder epithelial cells. Here we set out to define the mechanism by which UPEC enters host cells by investigating four of the major entry routes known to be exploited by invasive pathogens: caveolae, clathrin, macropinocytosis and secretory lysosomes. Using pharmacological inhibitors in combination with RNA interference against specific endocytic pathway components, mutant host cell lines and a mouse infection model system, we found that type 1 pili-dependent bacterial invasion of host cells occurs via a cholesterol- and dynamin-dependent phagocytosis-like mechanism. This process did not require caveolae or secretory lysosomes, but was modulated by calcium levels, clathrin, and cooperative input from the primary clathrin adaptor AP-2 and a subset of alternate adaptors comprised of Numb, ARH and Dab2. These alternate clathrin adaptors recognize NPXY motifs, as found within the cytosolic tail of beta1 integrin, suggesting a functional link between the engagement of integrin receptors by FimH and the clathrin-dependent uptake of type 1-piliated bacteria.     

QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli

Bacteria sense environmental cues and regulate gene expression accordingly so as to persist in diverse niches. QseC is a membrane sensor kinase shown in enterohemorrhagic Escherichia coli to respond to host and bacterial signals by phosphorylating the QseB response regulator at residue D51, resulting in QseB activation and presumably upregulation of virulence genes. We studied QseBC in uropathogenic E. coli (UPEC). UPEC establish infection by colonizing and invading bladder cells. After invasion, UPEC can escape into the cytoplasm where they can form intracellular bacterial communities. Deletion of qseC significantly attenuated intracellular bacterial community formation and virulence, whereas paradoxically qseB deletion did not impact pathogenesis. We found that QseB upregulates its own expression in the qseC mutant, arguing that it is activated even in the absence of QseC. However, expression of QseB, but not a QseB_D51A mutant, in the absence of QseC resulted in downregulation of type 1 pili, curli and flagella. We observed similar phenotypes with enterohemorrhagic E. coli , showing that this is not a UPEC-specific phenomenon. Target gene expression is restored when QseC is present. We discovered that QseC has phosphatase activity required for QseB dephosphorylation. Thus, the QseC phosphatase capacity is critical for modulating QseB activity and subsequent gene expression.     

Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that gammadelta T cell population was the major source of IL-17. Mice depleted of gammadelta T cells by mAb treatment or mice genetically lacking Vdelta1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vdelta1(+) gammadelta T cells as the source of IL-17. It was further revealed that gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although gammadelta T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of gammadelta T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.     

Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo.

The adaptive immune system has evolved distinct responses against different pathogens, but the mechanism(s) by which a particular response is initiated is poorly understood. In this study, we investigated the type of Ag-specific CD4(+) Th and CD8(+) T cell responses elicited in vivo, in response to soluble OVA, coinjected with LPS from two different pathogens. We used Escherichia coli LPS, which signals through Toll-like receptor 4 (TLR4) and LPS from the oral pathogen Porphyromonas gingivalis, which does not appear to require TLR4 for signaling. Coinjections of E. coli LPS + OVA or P. gingivalis LPS + OVA induced similar clonal expansions of OVA-specific CD4(+) and CD8(+) T cells, but strikingly different cytokine profiles. E. coli LPS induced a Th1-like response with abundant IFN-gamma, but little or no IL-4, IL-13, and IL-5. In contrast, P. gingivalis LPS induced Th and T cell responses characterized by significant levels of IL-13, IL-5, and IL-10, but lower levels of IFN-gamma. Consistent with these results, E. coli LPS induced IL-12(p70) in the CD8alpha(+) dendritic cell (DC) subset, while P. gingivalis LPS did not. Both LPS, however, activated the two DC subsets to up-regulate costimulatory molecules and produce IL-6 and TNF-alpha. Interestingly, these LPS appeared to have differences in their ability to signal through TLR4; proliferation of splenocytes and cytokine secretion by splenocytes or DCs from TLR4-deficient C3H/HeJ mice were greatly impaired in response to E. coli LPS, but not P. gingivalis LPS. Therefore, LPS from different bacteria activate DC subsets to produce different cytokines, and induce distinct types of adaptive immunity in vivo.     

Expression of toll-like receptor 2 on gamma delta T cells bearing invariant V gamma 6/V delta 1 induced by Escherichia coli infection in mice

We recently reported that the number of gamma delta T cells was increased after infection with Escherichia coli in C3H/HeN mice. We here showed that an i.p. injection with native lipid A derived from E. coli induced an increase of gamma delta T cells in the peritoneal cavity of LPS-responsive C3H/HeN mice and, albeit to a lesser degree, also in LPS-hyporesponsive C3H/HeJ mice. The purified gamma delta T cells from C3H/HeN and C3H/HeJ mice expressed a canonical TCR repertoire encoded by V gamma 6-J gamma 1/V delta 1-D delta 2-J delta 2 gene segments and proliferated in response to the native lipid A derived from E. coli in a TCR-independent manner. The lipid A-reactive gamma delta T cells bearing canonical V gamma 6/V delta 1 expressed Toll-like receptor (TLR) 2 mRNA, while TLR4 mRNA was undetectable. Treatment with a TLR2 anti-sense oligonucleotide resulted in hyporesponsiveness of the gamma delta T cells to the native lipid A. TLR2-deficient mice showed an impaired increase of the gamma delta T cells following injection of native lipid A. These results suggest that TLR2 is involved in the activation of canonical V gamma 6/V delta 1 T cells by native E. coli lipid A.