Immune defenses in the urinary tract.

Recent advances in preclinical modeling of urinary tract infections (UTIs) have enabled the identification of key facets of the host response that influence pathogen clearance and tissue damage. Here, we review new insights into the functions of neutrophils, macrophages, and antimicrobial peptides in innate control of uropathogens and in mammalian infection-related tissue injury and repair. We also discuss novel functions for renal epithelial cells in innate antimicrobial defense. In addition, epigenetic modifications during bacterial cystitis have been implicated in bladder remodeling, conveying susceptibility to recurrent UTI. In total, contemporary work in this arena has better defined host processes that shape UTI susceptibility and severity and might inform the development of novel preventive and therapeutic approaches for acute and recurrent UTI.

A host receptor enables type 1 pilus-mediated pathogenesis of Escherichia coli pyelonephritis.

Type 1 pili have long been considered the major virulence factor enabling colonization of the urinary bladder by uropathogenic Escherichia coli (UPEC). The molecular pathogenesis of pyelonephritis is less well characterized, due to previous limitations in preclinical modeling of kidney infection. Here, we demonstrate in a recently developed mouse model that beyond bladder infection, type 1 pili also are critical for establishment of ascending pyelonephritis. Bacterial mutants lacking the type 1 pilus adhesin (FimH) were unable to establish kidney infection in male C3H/HeN mice. We developed an in vitro model of FimH-dependent UPEC binding to renal collecting duct cells, and performed a CRISPR screen in these cells, identifying desmoglein-2 as a primary renal epithelial receptor for FimH. The mannosylated extracellular domain of human DSG2 bound directly to the lectin domain of FimH in vitro, and introduction of a mutation in the FimH mannose-binding pocket abolished binding to DSG2. In infected C3H/HeN mice, type 1-piliated UPEC and Dsg2 were co-localized within collecting ducts, and administration of mannoside FIM1033, a potent small-molecule inhibitor of FimH, significantly attenuated bacterial loads in pyelonephritis. Our results broaden the biological importance of FimH, specify the first renal FimH receptor, and indicate that FimH-targeted therapeutics will also have application in pyelonephritis.

Morphologic design of nanostructures for enhanced antimicrobial activity.

Despite significant progress in synthetic polymer chemistry and in control over tuning the structures and morphologies of nanoparticles, studies on morphologic design of nanomaterials for the purpose of optimizing antimicrobial activity have yielded mixed results. When designing antimicrobial materials, it is important to consider two distinctly different modes and mechanisms of activity-those that involve direct interactions with bacterial cells, and those that promote the entry of nanomaterials into infected host cells to gain access to intracellular pathogens. Antibacterial activity of nanoparticles may involve direct interactions with organisms and/or release of antibacterial cargo, and these activities depend on attractive interactions and contact areas between particles and bacterial or host cell surfaces, local curvature and dynamics of the particles, all of which are functions of nanoparticle shape. Bacteria may exist as spheres, rods, helices, or even in uncommon shapes (e.g., box- and star-shaped) and, furthermore, may transform into other morphologies along their lifespan. For bacteria that invade host cells, multivalent interactions are involved and are dependent upon bacterial size and shape. Therefore, mimicking bacterial shapes has been hypothesized to impact intracellular delivery of antimicrobial nanostructures. Indeed, designing complementarities between the shapes of microorganisms with nanoparticle platforms that are designed for antimicrobial delivery offers interesting new perspectives toward future nanomedicines. Some studies have reported improved antimicrobial activities with spherical shapes compared to non-spherical constructs, whereas other studies have reported higher activity for non-spherical structures (e.g., rod, discoid, cylinder, etc.). The shapes of nano- and microparticles have also been shown to impact their rates and extents of uptake by mammalian cells (macrophages, epithelial cells, and others). However, in most of these studies, nanoparticle morphology was not intentionally designed to mimic specific bacterial shape. Herein, the morphologic designs of nanoparticles that possess antimicrobial activities per se and those designed to deliver antimicrobial agent cargoes are reviewed. Furthermore, hypotheses beyond shape dependence and additional factors that help to explain apparent discrepancies among studies are highlighted.

Morphologic Design of Silver-Bearing Sugar-Based Polymer Nanoparticles for Uroepithelial Cell Binding and Antimicrobial Delivery.

Platelet-like and cylindrical nanostructures from sugar-based polymers are designed to mimic the aspect ratio of bacteria and achieve uroepithelial cell binding and internalization, thereby improving their potential for local treatment of recurrent urinary tract infections. Polymer nanostructures, derived from amphiphilic block polymers composed of zwitterionic poly(d-glucose carbonate) and semicrystalline poly(l-lactide) segments, were constructed with morphologies that could be tuned to enhance uroepithelial cell binding. These nanoparticles exhibited negligible cytotoxicity, immunotoxicity, and cytokine adsorption, while also offering substantial silver cation loading capacity, extended release, and in vitro antimicrobial activity (as effective as free silver cations) against uropathogenic Escherichia coli. In comparison to spherical analogues, cylindrical and platelet-like nanostructures engaged in significantly higher association with uroepithelial cells, as measured by flow cytometry; despite their larger size, platelet-like nanostructures maintained the capacity for cell internalization. This work establishes initial evidence of degradable platelet-shaped nanostructures as versatile therapeutic carriers for treatment of epithelial infections.

Sex Differences in Population Dynamics during Formation of Kidney Bacterial Communities by Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC), the primary etiologic agent of urinary tract infections (UTIs), encounters a restrictive population bottleneck within the female mammalian bladder. Its genetic diversity is restricted during establishment of cystitis because successful UPEC must invade superficial bladder epithelial cells prior to forming clonal intracellular bacterial communities (IBCs). In this study, we aimed to understand UPEC population dynamics during ascending pyelonephritis, namely, formation of kidney bacterial communities (KBCs) in the renal tubular lumen and nucleation of renal abscesses. We inoculated the bladders of both male and female C3H/HeN mice, a background which features vesicoureteral reflux; we have previously shown that in this model, males develop severe, high-titer pyelonephritis and renal abscesses much more frequently than females. Mice were infected with 40 isogenic, PCR-tagged ("barcoded") UPEC strains, and tags remaining in bladder and kidneys were ascertained at intervals following infection. In contrast to females, males maintained a majority of strains within both the bladder and kidneys throughout the course of infection, indicating only a modest host-imposed bottleneck on overall population diversity during successful renal infection. Moreover, the diverse population in the infected male kidneys obscured any restrictive bottleneck in the male bladder. Finally, using RNA in situ hybridization following mixed infections with isogenic UPEC bearing distinct markers, we found that despite their extracellular location (in the urinary space), KBCs are clonal in origin. This finding indicates that even with bulk reflux of infected bladder urine into the renal pelvis, successful ascension of UPEC to establish the tubular niche is an uncommon event.

HOME2 Study: Household Versus Personalized Decolonization in Households of Children With Methicillin-Resistant Staphylococcus aureus Skin and Soft Tissue Infection-A Randomized Clinical Trial.

BACKGROUND: A household approach to decolonization decreases skin and soft tissue infection (SSTI) incidence, though this is burdensome and costly. As prior SSTI increases risk for SSTI, we hypothesized that the effectiveness of decolonization measures to prevent SSTI when targeted to household members with prior year SSTI would be noninferior to decolonizing all household members. METHODS: Upon completion of our 12-month observational Household Observation of Methicillin-resistant Staphylococcus aureus in the Environment (HOME) study, 102 households were enrolled in HOME2, a 12-month, randomized noninferiority trial. Pediatric index patients with community-associated methicillin-resistant Staphylococcus aureus (MRSA) SSTI, their household contacts, and pets were enrolled. Households were randomized 1:1 to the personalized (decolonization performed only by household members who experienced SSTI during the HOME study) or household (decolonization performed by all household members) approaches. The 5-day regimen included hygiene education, twice-daily intranasal mupirocin, and daily bleach-water baths. At 5 follow-up visits in participants' homes, swabs to detect S. aureus were collected from participants, environmental surfaces, and pets; incident SSTIs were ascertained. RESULTS: Noninferiority of the personalized approach was established for the primary outcome 3-month cumulative SSTI: 23 of 212 (10.8%) participants reported SSTI in household approach households, while 23 of 236 (9.7%) participants reported SSTI in personalized approach households (difference in proportions, -1.1% [95% confidence interval, -6.7% to 4.5%]). In multivariable analyses, prior year SSTI and baseline MRSA colonization were associated with cumulative SSTI. CONCLUSIONS: The personalized approach was noninferior to the household approach in preventing SSTI. Future studies should interrogate longer durations of decolonization and/or decontamination of the household environment to reduce household MRSA burden. CLINICAL TRIALS REGISTRATION: NCT01814371.

Sex effects in pyelonephritis.

Urinary tract infections (UTIs) are generally considered a disease of women. However, UTIs affect females throughout the lifespan, and certain male populations (including infants and elderly men) are also susceptible. Epidemiologically, pyelonephritis is more common in women but carries increased morbidity when it does occur in men. Among children, high-grade vesicoureteral reflux is a primary risk factor for upper-tract UTI in both sexes. However, among young infants with UTI, girls are outnumbered by boys; risk factors include posterior urethral valves and lack of circumcision. Recent advances in mouse models of UTI reveal sex differences in innate responses to UTI, which vary somewhat depending on the system used. Moreover, male mice and androgenized female mice suffer worse outcomes in experimental pyelonephritis; evidence suggests that androgen exposure may suppress innate control of infection in the urinary tract, but additional androgen effects, as well as non-hormonal sex effects, may yet be specified. Among other intriguing directions, recent experiments raise the hypothesis that the postnatal testosterone surge that occurs in male infants may represent an additional factor driving the higher incidence of UTI in males under 6 months of age. Ongoing work in contemporary models will further illuminate sex- and sex-hormone-specific effects on UTI pathogenesis and immune responses.

Incidence and treatment of hemophagocytic lymphohistiocytosis in hospitalized children with Ehrlichia infection.

We report a large cohort of pediatric patients with human monocytic ehrlichiosis (HME), enabling an estimated incidence of secondary hemophagocytic lymphohistiocytosis (HLH) in hospitalized children with HME. Among 49 children with PCR-confirmed Ehrlichia infection, 8 (16%) met current criteria for HLH. Those with HLH had more significant hematologic abnormalities and longer durations from symptom onset to admission and definitive anti-infective therapy. Among these eight, three received chemotherapy plus doxycycline, one of whom died; the other five were treated with doxycycline without chemotherapy, and all survived without HLH recurrence. Our findings demonstrate that antimicrobial therapy alone can successfully resolve Ehrlichia-associated HLH.

High Levels of Cyclic Di-GMP in Klebsiella pneumoniae Attenuate Virulence in the Lung.

The bacterial second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) has been shown to influence the expression of virulence factors in certain pathogenic bacteria, but little is known about its activity in the increasingly antibiotic-resistant pathogen Klebsiella pneumoniae Here, the expression in K. pneumoniae of a heterologous diguanylate cyclase increased the bacterial c-di-GMP concentration and attenuated pathogenesis in murine pneumonia. This attenuation remained evident in mice lacking the c-di-GMP sensor STING, indicating that the high c-di-GMP concentration exerted its influence not on host responses but on bacterial physiology. While serum resistance and capsule expression were unaffected by the increased c-di-GMP concentration, both type 3 and type 1 pili were strongly upregulated. Importantly, attenuation of K. pneumoniae virulence by high c-di-GMP levels was abrogated when type 1 pilus expression was silenced. We conclude that increased type 1 piliation may hamper K. pneumoniae virulence in the respiratory tract and that c-di-GMP signaling represents a potential therapeutic target for antibiotic-resistant K. pneumoniae in this niche.

Androgen exposure potentiates formation of intratubular communities and renal abscesses by Escherichia coli.

Females across their lifespan and certain male populations are susceptible to urinary tract infections (UTI). The influence of female vs. male sex on UTI is incompletely understood, in part because preclinical modeling has been performed almost exclusively in female mice. Here, we employed established and new mouse models of UTI with uropathogenic Escherichia coli (UPEC) to investigate androgen influence on UTI pathogenesis. Susceptibility to UPEC UTI in both male and female hosts was potentiated with 5α-dihydrotestosterone, while males with androgen receptor deficiency and androgenized females treated with the androgen receptor antagonist enzalutamide were protected from severe pyelonephritis. In androgenized females and in males, UPEC formed dense intratubular, biofilm-like communities, some of which were sheltered from infiltrating leukocytes by the tubular epithelium and by peritubular fibrosis. Abscesses were nucleated by small intratubular collections of UPEC first visualized at five days postinfection and briskly expanded over the subsequent 24 hours. Male mice deficient in Toll-like receptor 4, which fail to contain UPEC within abscesses, were susceptible to lethal dissemination. Thus, androgen receptor activation imparts susceptibility to severe upper-tract UTI in both female and male murine hosts. Visualization of intratubular UPEC communities illuminates early renal abscess pathogenesis and the role of abscess formation in preventing dissemination of infection. Additionally, our study suggests that androgen modulation may represent a novel therapeutic route to combat recalcitrant or recurrent UTI in a range of patient populations.

Synthesis, characterization, in vitro SAR and in vivo evaluation of N,N'bisnaphthylmethyl 2-alkyl substituted imidazolium salts against NSCLC.

Alkyl- and N,N'-bisnaphthyl-substituted imidazolium salts were tested in vitro for their anti-cancer activity against four non-small cell lung cancer cell lines (NCI-H460, NCI-H1975, HCC827, A549). All compounds had potent anticancer activity with 2 having IC50 values in the nanomolar range for three of the four cell lines, a 17-fold increase in activity against NCI-H1975 cells when compared to cisplatin. Compounds 1-4 also showed high anti-cancer activity against nine NSCLC cell lines in the NCI-60 human tumor cell line screen. In vitro studies performed using the Annexin V and JC-1 assays suggested that NCI-H460 cells treated with 2 undergo an apoptotic cell death pathway and that mitochondria could be the cellular target of 2 with the mechanism of action possibly related to a disruption of the mitochondrial membrane potential. The water solubilities of 1-4 was over 4.4mg/mL using 2-hydroxypropyl-ß-cyclodextrin as a chemical excipient, thereby providing sufficient solubility for systemic administration.

Androgens Enhance Male Urinary Tract Infection Severity in a New Model.

Urinary tract infections (UTIs) occur predominantly in females but also affect substantial male patient populations; indeed, morbidity in complicated UTI is higher in males. Because of technical obstacles, preclinical modeling of UTI in male mice has been limited. We devised a minimally invasive surgical bladder inoculation technique that yields reproducible upper and lower UTI in both male and female mice, enabling studies of sex differences in these infections. Acute uropathogenic Escherichia coli (UPEC) cystitis in C57BL/6 and C3H/HeN males recapitulated the intracellular bacterial community pathway previously shown in females. However, surgically infected females of these strains exhibited more robust bladder cytokine responses and more efficient UPEC control than males. Compared with females, C3H/HeN males displayed a striking predilection for chronic cystitis, manifesting as persistent bacteriuria, high-titer bladder bacterial burdens, and chronic inflammation. Furthermore, males developed more severe pyelonephritis and 100% penetrant renal abscess (a complication that is rare in female mice). These phenotypes were sharply abrogated after castration but restored with exogenous testosterone, suggesting that male susceptibility to UTI is strongly influenced by androgen exposure. These data substantiate the long-standing presumption that anatomic differences in urogenital anatomy confer protection from UTI in males; however, as clinically observed, male sex associated with more severe UTI once these traditional anatomic barriers were bypassed. This study introduces a highly tractable preclinical model for interrogating sex differences in UTI susceptibility and pathogenesis, and illuminates an interplay between host sex and UTI that is more complex than previously appreciated.

Klebsiella pneumoniae FimK Promotes Virulence in Murine Pneumonia.

Klebsiella pneumoniae, a chief cause of nosocomial pneumonia, is a versatile and commonly multidrug-resistant human pathogen for which further insight into pathogenesis is needed. We show that the pilus regulatory gene fimK promotes the virulence of K. pneumoniae strain TOP52 in murine pneumonia. This contrasts with the attenuating effect of fimK on urinary tract virulence, illustrating that a single factor may exert opposing effects on pathogenesis in distinct host niches. Loss of fimK in TOP52 pneumonia was associated with diminished lung bacterial burden, limited innate responses within the lung, and improved host survival. FimK expression was shown to promote serum resistance, capsule production, and protection from phagocytosis by host immune cells. Finally, while the widely used K. pneumoniae model strain 43816 produces rapid dissemination and death in mice, TOP52 caused largely localized pneumonia with limited lethality, thereby providing an alternative tool for studying K. pneumoniae pathogenesis and control within the lung.

Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli.

During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly L-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr(-/-)mice recapitulated the derepressed PMN recruitment observed previously in Ido1(-/-)mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis.

Cathelicidin augments epithelial receptivity and pathogenesis in experimental Escherichia coli cystitis.

BACKGROUND: Cathelicidin is a proposed defender against infection of the urinary tract via its antimicrobial properties, but its activity has not been delineated in a dedicated cystitis model. METHODS: Female C57Bl/6 mice, wild type or deficient in cathelin-related antimicrobial peptide (CRAMP; an ortholog of the sole human cathelicidin, LL-37), were infected transurethrally with the cystitis-derived uropathogenic Escherichia coli (UPEC) strain UTI89. Infection course was evaluated by bladder titers, intracellular bacterial community quantification, and histological analysis. Immune responses and resolution were characterized through cytokine profiling, microscopy, and quantitation of epithelial recovery from exfoliation. RESULTS: CRAMP-deficient mice exhibited significantly lower bladder bacterial loads and fewer intracellular bacterial communities during acute cystitis. Although differences in bacterial titers were evident as early as 1 hour after infection, CRAMP-deficient mice showed no baseline alterations in immune activation, uroepithelial structure, apical expression of uroplakins (which serve as bacterial receptors), or intracellular bacterial growth rate. CRAMP-deficient hosts demonstrated less intense cytokine responses, diminished neutrophil infiltration, and accelerated uroepithelial recovery. CONCLUSIONS: Mice lacking the antimicrobial peptide cathelicidin experienced less severe infection than wild-type mice in a well-established model of cystitis. Although CRAMP exhibits in vitro antibacterial activity against UPEC, it may enhance UPEC infection in the bladder by promoting epithelial receptivity and local inflammation.

Imidazolium salts as small-molecule urinary bladder exfoliants in a murine model.

We present a novel family of small-molecule urinary bladder exfoliants that are expected to be of great value in preclinical studies of urologic conditions and have improved potential for translation compared with prior agents. There is broad urologic interest in the therapeutic potential of such exfoliating agents. The primary agent used in preclinical models, the cationic peptide protamine sulfate (PS), has limited translational potential due to concerns including systemic adverse reactions and bladder tissue injury. Intravesical application of a safe, systemically nontoxic exfoliant would have potential utility in the eradication of Escherichia coli and other uropathogens that reside in the bladder epithelium following cystitis, as well as in chronic bladder pain and bladder cancer. Here, we introduce a family of imidazolium salts with potent and focused exfoliating activity on the bladder epithelium. Synthesis and purification were straightforward and scalable, and the compounds exhibited prolonged stability in lyophilized form. Most members of the compound family were cytotoxic to cultured uroepithelial cells, with >10-fold differences in potency across the series. Upon topical (intravesical) administration of selected compounds to the murine bladder, complete epithelial exfoliation was achieved with physiologically relevant imidazolium concentrations and brief contact times. The exfoliative activity of these compounds was markedly improved in comparison to PS, as assessed by microscopy, immunofluorescence, and immunoblotting for uroplakins. Bladder uroepithelium regenerated within days to yield a histologically normal appearance, and no toxicity was observed. Finally, the chemical scaffold offers an opportunity for inclusion of antimicrobials or conjugation with chemotherapeutic or other moieties.

Intracellular lifestyles and immune evasion strategies of uropathogenic Escherichia coli.

Paradigms in the pathogenesis of urinary tract infections have shifted dramatically as a result of recent scientific revelations. Beyond extracellular colonization of the bladder luminal surface, as traditional clinical thinking would hold, uropathogenic bacteria direct a complex, intracellular cascade that shelters bacteria from host defenses and leads to persistent bacterial residence within the epithelium. After epithelial invasion, many organisms are promptly expelled by bladder epithelial cells; a minority establish a niche in the cytoplasm that results in the development of biofilm-like intracellular bacterial communities and serves as the primary location for bacterial expansion. Exfoliation of the superficial epithelial layer acts to reduce the bacterial load but facilitates chronic residence of small nests of bacteria that later reemerge to cause some episodes of recurrent cystitis, a familiar clinical scenario in otherwise healthy women. Advances in both in vitro and animal models of cystitis promise to provide insights into the bacterial and host transcriptional and biochemical pathways that define these pathogenic stages.