Anaphylactic degranulation by mast cells requires the mobilization of inflammasome components.

The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1ß for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1ß was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1ß, resulting in severe inflammation.

A highly polarized TH2 bladder response to infection promotes epithelial repair at the expense of preventing new infections.

Urinary tract infections (UTIs) typically evoke prompt and vigorous innate bladder immune responses, including extensive exfoliation of the epithelium. To explain the basis for the extraordinarily high recurrence rates of UTIs, we examined adaptive immune responses in mouse bladders. We found that, following each bladder infection, a highly T helper type 2 (TH2)-skewed immune response directed at bladder re-epithelialization is observed, with limited capacity to clear infection. This response is initiated by a distinct subset of CD301b+OX40L+ dendritic cells, which migrate into the bladder epithelium after infection before trafficking to lymph nodes to preferentially activate TH2 cells. The bladder epithelial repair response is cumulative and aberrant as, after multiple infections, the epithelium was markedly thickened and bladder capacity was reduced relative to controls. Thus, recurrence of UTIs and associated bladder dysfunction are the outcome of the preferential focus of the adaptive immune response on epithelial repair at the expense of bacterial clearance.

α-Hemolysin promotes uropathogenic E. coli persistence in bladder epithelial cells via abrogating bacteria-harboring lysosome acidification.

There is a growing consensus that a significant proportion of recurrent urinary tract infections are linked to the persistence of uropathogens within the urinary tract and their re-emergence upon the conclusion of antibiotic treatment. Studies in mice and human have revealed that uropathogenic Escherichia coli (UPEC) can persist in bladder epithelial cells (BECs) even after the apparent resolution of the infection. Here, we found that, following the entry of UPEC into RAB27b+ fusiform vesicles in BECs, some bacteria escaped into the cytoplasmic compartment via a mechanism involving hemolysin A (HlyA). However, these UPEC were immediately recaptured within LC3A/B+ autophagosomes that matured into LAMP1+ autolysosomes. Thereafter, HlyA+ UPEC-containing lysosomes failed to acidify, which is an essential step for bacterial elimination. This lack of acidification was related to the inability of bacteria-harboring compartments to recruit V-ATPase proton pumps, which was attributed to the defragmentation of cytosolic microtubules by HlyA. The persistence of UPEC within LAMP1+ compartments in BECs appears to be directly linked to HlyA. Thus, through intravesicular instillation of microtubule stabilizer, this host defense response can be co-opted to reduce intracellular bacterial burden following UTIs in the bladder potentially preventing recurrence.

Loss of Bladder Epithelium Induced by Cytolytic Mast Cell Granules.

Programmed death and shedding of epithelial cells is a powerful defense mechanism to reduce bacterial burden during infection but this activity cannot be indiscriminate because of the critical barrier function of the epithelium. We report that during cystitis, shedding of infected bladder epithelial cells (BECs) was preceded by the recruitment of mast cells (MCs) directly underneath the superficial epithelium where they docked and extruded their granules. MCs were responding to interleukin-1ß (IL-1ß) secreted by BECs after inflammasome and caspase-1 signaling. Upon uptake of granule-associated chymase (mouse MC protease 4 [mMCPT4]), BECs underwent caspase-1-associated cytolysis and exfoliation. Thus, infected epithelial cells require a specific cue for cytolysis from recruited sentinel inflammatory cells before shedding.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

FK506-binding protein-like and FK506-binding protein 8 regulate dual leucine zipper kinase degradation and neuronal responses to axon injury.

The dual leucine zipper kinase (DLK) is a key regulator of axon regeneration and degeneration in response to neuronal injury; however, regulatory mechanisms of the DLK function via its interacting proteins are largely unknown. To better understand the molecular mechanism of DLK function, we performed yeast two-hybrid screening analysis and identified FK506-binding protein-like (FKBPL, also known as WAF-1/CIP1 stabilizing protein 39) as a DLK-binding protein. FKBPL binds to the kinase domain of DLK and inhibits its kinase activity. In addition, FKBPL induces DLK protein degradation through ubiquitin-dependent pathways. We further assessed other members in the FKBP protein family and found that FK506-binding protein 8 (FKBP8) also induced DLK degradation. We identified the lysine 271 residue in the kinase domain as a major site of DLK ubiquitination and SUMO3 conjugation and was thus responsible for regulating FKBP8-mediated proteasomal degradation that was inhibited by the substitution of the lysine 271 to arginine. FKBP8-mediated degradation of DLK is mediated by autophagy pathway because knockdown of Atg5 inhibited DLK destabilization. We show that in vivo overexpression of FKBP8 delayed the progression of axon degeneration and suppressed neuronal death after axotomy in sciatic and optic nerves. Taken together, this study identified FKBPL and FKBP8 as novel DLK-interacting proteins that regulate DLK stability via the ubiquitin-proteasome and lysosomal protein degradation pathways.

Stabilization of activated mast cells by ORAI1 inhibitor suppresses peanut-induced anaphylaxis and acute diarrhea.

Mast cell (MC) activation triggered by immunoglobulin E (IgE)-antigen crosslinking involves intracellular Ca2+ influx through the ORAI1 channel, which precedes granule exteriorization and de novo synthesis of mediators. Pharmacologically suppressing MCs via the inhibition of the ORAI1 Ca2+ channel may represent a potential strategy for preventing anaphylaxis. This study demonstrated that peanut-induced anaphylaxis in sensitized mice resulted in significant hypothermia and acute diarrhea. Utilizing the Mcpt5cre-DTA mouse model, we demonstrated that this anaphylactic response was mediated by IgE-antigen-induced MC activation. Prophylactic administration of MC suppressors was an effective means of preventing peanut-induced anaphylaxis. In addition, we observed the potent efficacy of an ORAI1 inhibitor in suppressing the FcεRI-mediated response of murine or human MCs, even when administered concurrently or post-allergen exposure. Mechanistically, the ORAI1 inhibitor was found to prevent the association of Synaptotagmin-2 with the SNARE complex. In an in vivo mouse model of peanut-induced anaphylaxis, the administration of the ORAI1 inhibitor after allergen challenge effectively suppressed allergic acute diarrhea and ameliorated anaphylaxis. Therefore, pharmacological intervention of ORAI1 channel inhibition in MCs represents a promising therapeutic avenue for the treatment of peanut-induced anaphylaxis and acute diarrhea in vivo.

Salmonella typhimurium impedes innate immunity with a mast-cell-suppressing protein tyrosine phosphatase, SptP.

The virulence of Salmonella is linked to its invasive capacity and suppression of adaptive immunity. This does not explain, however, the rapid dissemination of the pathogen after it breaches the gut. In our study, S. Typhimurium suppressed degranulation of local mast cells (MCs), resulting in limited neutrophil recruitment and restricting outflow of vascular contents into infection sites, thus facilitating bacterial spread. MC suppression was mediated by secreted effector protein (SptP), which shares structural homology with Yersinia YopH. SptP functioned by dephosphorylating the vesicle fusion protein N-ethylmalemide-sensitive factor and by blocking phosphorylation of Syk. Without SptP, orally challenged S. Typhimurium failed to suppress MC degranulation and exhibited limited colonization of the mesenteric lymph nodes. Administration of SptP to sites of E. coli infection markedly enhanced its virulence. Thus, SptP-mediated inactivation of local MCs is a powerful mechanism utilized by S. Typhimurium to impede early innate immunity.

A shared mechanism of multidrug resistance in laboratory-evolved uropathogenic Escherichia coli.

The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low AmpR and High AmpR, respectively. Whole-genome sequencing revealed that both Low and High AmpR strains contained mutations in the marR, acrR, and envZ genes. The High AmpR strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the AmpR strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the AmpR strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the AmpR strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.

Recurrent infections drive persistent bladder dysfunction and pain via sensory nerve sprouting and mast cell activity.

Urinary tract infections (UTIs) account for almost 25% of infections in women. Many are recurrent (rUTI), with patients frequently experiencing chronic pelvic pain and urinary frequency despite clearance of bacteriuria after antibiotics. To elucidate the basis for these bacteria-independent bladder symptoms, we examined the bladders of patients with rUTI. We noticed a notable increase in neuropeptide content in the lamina propria and indications of enhanced nociceptive activity. In mice subjected to rUTI, we observed sensory nerve sprouting that was associated with nerve growth factor (NGF) produced by recruited monocytes and tissue-resident mast cells. Treatment of rUTI mice with an NGF-neutralizing antibody prevented sprouting and alleviated pelvic sensitivity, whereas instillation of native NGF into naïve mice bladders mimicked nerve sprouting and pain behavior. Nerve activation, pain, and urinary frequency were each linked to the presence of proximal mast cells, because mast cell deficiency or treatment with antagonists against receptors of several direct or indirect mast cell products was each effective therapeutically. Thus, our findings suggest that NGF-driven sensory sprouting in the bladder coupled with chronic mast cell activation represents an underlying mechanism driving bacteria-independent pain and voiding defects experienced by patients with rUTI.

Microbiome in urological diseases: Axis crosstalk and bladder disorders.

Since the identification of the human urinary microbiome, numerous studies have characterized this microbial community and improved our knowledge of its association with urinary diseases. This association between urinary diseases and microbiota is not confined to the urinary microbiota; it is interconnected with the microbiota of other organs. The gastrointestinal, vaginal, kidney, and bladder microbiota all affect urinary diseases because they work with their respective organs to control the growth and operation of the immune, metabolic, and nervous systems through dynamic bidirectional communication along the bladder-centered axis. Therefore, disturbances in the microbial communities may result in the emergence of urinary diseases. In this review, we describe the increasing and intriguing evidence of complicated and critical relationships that may contribute to the development and progression of urinary diseases through disruption of the microbiota in various organs.

Enhancing adoptive T-cell therapy with fucoidan-based IL-2 delivery microcapsules.

Adoptive cell therapy (ACT) with antigen-specific T cells is a promising treatment approach for solid cancers. Interleukin-2 (IL-2) has been utilized in boosting the efficacy of ACT. However, the clinical applications of IL-2 in combination with ACT is greatly limited by short exposure and high toxicities. Herein, a complex coacervate was designed to intratumorally deliver IL-2 in a sustained manner and protect against proteolysis. The complex coacervate consisted of fucoidan, a specific IL-2 binding glycosaminoglycan, and poly-l-lysine, a cationic counterpart (FPC2). IL-2-laden FPC2 exhibited a preferential bioactivity in ex vivo expansion of CD8+T cells over Treg cells. Additionally, FPC2 was embedded in pH modulating injectable gel (FPC2-IG) to endure the acidic tumor microenvironment. A single intratumoral administration of FPC2-IG-IL-2 increased expansion of tumor-infiltrating cytotoxic lymphocytes and reduced frequencies of myeloid populations. Notably, the activation and persistency of tumor-reactive T cells were observed only in the tumor site, not in the spleen, confirming a localized effect of FPC2-IG-IL-2. The immune-favorable tumor microenvironment induced by FPC2-IG-IL-2 enabled adoptively transferred TCR-engineered T cells to effectively eradicate tumors. FPC2-IG delivery system is a promising strategy for T-cell-based immunotherapies.

The Urinary Microbiome: Role in Bladder Cancer and Treatment.

Commensal microbes have increasingly been found to be involved in the development and progression of cancer. The recent discovery of the urinary microbiome bolstered the notion that microbes might play a role in bladder cancer. Although microbial involvement in bladder neoplastic transformation and metastatic progression, except schisto somiasis, has not been established, accumulating research suggests that dysbiosis of the urinary microbiome can produce a chronically inflammatory urothelial microenvironment and lead to bladder cancer. In this review, we describe how the urinary microbiome might facilitate the development of bladder cancer by altering the host immune system and the kind of cytokines that are directly involved in these responses. We investigated the therapeutic possibilities of modulating the urinary microbiome, including immune checkpoint therapy. The responsiveness of patients to intravesical Bacillus Calmette-Guerin therapy was evaluated with respect to microbiome composition. We conclude by noting that the application of microbes to orchestrate the inflammatory response in the bladder may facilitate the development of treatments for bladder cancer.

Reactive oxygen species generated by NADPH oxidase 2 and 4 are required for chondrogenic differentiation.

Although generation of reactive oxygen species (ROS) by NADPH oxidases (Nox) is thought to be important for signal transduction in nonphagocytic cells, little is known of the role ROS plays in chondrogenesis. We therefore examined the possible contribution of ROS generation to chondrogenesis using both ATDC5 cells and primary chondrocytes derived from mouse embryos. The intracellular level of ROS was increased during the differentiation process, which was then blocked by treatment with the ROS scavenger N-acetylcysteine. Expression of Nox1 and Nox2 was increased upon differentiation of ATDC5 cells and primary mouse chondrocytes, whereas that of Nox4, which was relatively high initially, was decreased gradually during chondrogenesis. In developing limb, Nox1 and Nox2 were highly expressed in prehypertrophic and hypertrophic chondrocytes. However, Nox4 was highly expressed in proliferating chondrocytes and prehypertrophic chondrocytes. Depletion of Nox2 or Nox4 expression by RNA interference blocked both ROS generation and differentiation of ATDC5 cells, whereas depletion of Nox1 had no such effect. We also found that ATDC5 cells depleted of Nox2 or Nox4 underwent apoptosis. Further, inhibition of Akt phosphorylation along with subsequent activation of ERK was observed in the cells. Finally, depletion of Nox2 or Nox4 inhibited the accumulation of proteoglycan in primary chondrocytes. Taken together, our data suggest that ROS generated by Nox2 or Nox4 are essential for survival and differentiation in the early stage of chondrogenesis.

Tumor-Associated Mast Cells in Urothelial Bladder Cancer: Optimizing Immuno-Oncology.

Urothelial bladder cancer (UBC) is one of the most prevalent and aggressive malignancies. Recent evidence indicates that the tumor microenvironment (TME), including a variety of immune cells, is a critical modulator of tumor initiation, progression, evolution, and treatment resistance. Mast cells (MCs) in UBC are possibly involved in tumor angiogenesis, tissue remodeling, and immunomodulation. Moreover, tumor-infiltration by MCs has been reported in early-stage UBC patients. This infiltration is linked with a favorable or unfavorable prognosis depending on the tumor type and location. Despite the discrepancy of MC function in tumor progression, MCs can modify the TME to regulate the immunity and infiltration of tumors by producing an array of mediators. Nonetheless, the precise role of MCs in UBC tumor progression and evolution remains unknown. Thus, this review discusses some critical roles of MCs in UBC. Patients with UBC are treated at both early and late stages by immunotherapeutic methods, including intravenous bacillus Calmette-Guérin instillation and immune checkpoint blockade. An understanding of the patient response and resistance mechanisms in UBC is required to unlock the complete potential of immunotherapy. Since MCs are pivotal to understand the underlying processes and predictors of therapeutic responses in UBC, our review also focuses on possible immunotherapeutic treatments that involve MCs.

Nasal Immunization With Small Molecule Mast Cell Activators Enhance Immunity to Co-Administered Subunit Immunogens.

Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, small molecule mast cell activators may be a more cost-efficient adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. To identify novel mast cell activating compounds that could be evaluated for mucosal vaccine adjuvant activity, we employed high-throughput screening to assess over 55,000 small molecules for mast cell degranulation activity. Fifteen mast cell activating compounds were down-selected to five compounds based on in vitro immune activation activities including cytokine production and cellular cytotoxicity, synthesis feasibility, and selection for functional diversity. These small molecule mast cell activators were evaluated for in vivo adjuvant activity and induction of protective immunity against West Nile Virus infection in BALB/c mice when combined with West Nile Virus envelope domain III (EDIII) protein in a nasal vaccine. We found that three of the five mast cell activators, ST101036, ST048871, and R529877, evoked high levels of EDIII-specific antibody and conferred comparable levels of protection against WNV challenge. The level of protection provided by these small molecule mast cell activators was comparable to the protection evoked by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Thus, novel small molecule mast cell activators identified by high throughput screening are as efficacious as previously described mast cell activators when used as nasal vaccine adjuvants and represent next-generation mast cell activators for evaluation in mucosal vaccine studies.

ß-Defensin 2, an Antimicrobial Peptide, as a Novel Biomarker for Ulcerative Interstitial Cystitis; Can ß-Defensin 2 Suspect the Dysbiosis of Urine Microbiota?

As urine is not sterile, inflammatory reactions caused by dysbiosis of the urinary microbiota may induce interstitial cystitis. A study was conducted to determine whether ß-defensin 2 (BD-2), a specific antimicrobial peptide in the bladder, could be used as a novel diagnostic marker for ulcerative interstitial cystitis (IC). Urine samples from three female groups were examined: healthy controls (n = 34, Control group), non-Hunner type IC (n = 40, NHIC group), and Hunner type IC (n = 68, HIC group). Urine samples were collected via a transurethral catheter and assayed for BD-2 levels using enzyme linked immunosorbent assay. Under general or regional anesthesia, cystoscopy with diagnostic and therapeutic hydrodistension was performed in NHIC and HIC groups patients. These patients underwent a biopsy of the bladders. Based on the urinary specimens from 142 patients, BD-2 expression was found to be 18-fold higher in patients with Hunner type IC than in patients with non-Hunner type IC. The enhanced secretion of BD-2 exhibited a strong correlation with increased mast cell counts associated with bladder IC pathology. Enhanced urinary secretion of the antimicrobial peptide BD-2 from Hunner type IC patients associated with clinical phenotypes and demonstrated relatively robust levels to be used as a potential biomarker. Moreover, the increased urinary level of BD-2 may suggest a new possibility of biomarkers caused by dysbiosis of the urinary microbiota in ulcerative IC.

Novel mucosal adjuvant, mastoparan-7, improves cocaine vaccine efficacy.

Cocaine is one of the most potent and addictive psychostimulants known and there are no available pharmacotherapies to treat cocaine addiction. Here we describe a novel cocaine vaccine employing the mucosal adjuvant and mast cell-activating oligopeptide, mastoparan-7 (M7), to achieve optimal IgA antibody responses in mucosal secretions and effective induction of humoral immunity using a short immunization protocol. This formulation, using a hapten-carrier system to deliver cocaine as antigen, also reduced cocaine penetration of the blood brain barrier and protected mice from its psychoactive effects by reducing cocaine-induced locomotion. Surprisingly, the magnitude of cocaine-specific antibody titers induced by each adjuvant was not the major determinant of functional protection from cocaine challenge. A side-by-side comparison of the two haptens, cocaine and its analog GNC demonstrated that cocaine haptenation resulted in superior functional protection when used in combination with the novel mucosal adjuvant, M7. These results provide a new potential strategy for combatting cocaine addiction through mucosal vaccination.

Platelets trigger perivascular mast cell degranulation to cause inflammatory responses and tissue injury.

Platelet responses have been associated with end-organ injury and mortality following complex insults such as cardiac surgery, but how platelets contribute to these pathologies remains unclear. Our studies originated from the observation of microvascular platelet retention in a rat cardiac surgery model. Ensuing work supported the proximity of platelet aggregates with perivascular mast cells (MCs) and demonstrated that platelet activation triggered systemic MC activation. We then identified platelet activating factor (PAF) as the platelet-derived mediator stimulating MCs and, using chimeric animals with platelets defective in PAF generation or MCs lacking PAF receptor, defined the role of this platelet-MC interaction for vascular leakage, shock, and tissue inflammation. In application of these findings, we demonstrated that inhibition of platelet activation in modeled cardiac surgery blunted MC-dependent inflammation and tissue injury. Together, our work identifies a previously undefined mechanism of inflammatory augmentation, in which platelets trigger local and systemic responses through activation of perivascular MCs.

A humanized mouse model to study mast cells mediated cutaneous adverse drug reactions.

Recently a G-protein-coupled receptor, MAS Related GPR Family Member X2 (MRGPRX2), was identified as a specific receptor on human mast cells responsible for IgE independent adverse drug reactions (ADR). Although a murine homologue, Mrgprb2, has been identified for this receptor, its affinity for many ADR-causing drugs is poor making it difficult to undertake in vivo studies to examine mechanisms of ADR and to develop therapeutic strategies. Here, we have created humanized mice capable of generating MRGPRX2-expressing human MCs allowing for the study of MRGPRX2 MCs-mediated ADR in vitro as well as in vivo. Humanized mice were generated by hydrodynamic-injection of plasmids expressing human GM-CSF and IL-3 into NOD-scid IL2R-γ-/- strain of mice that had been transplanted with human hematopoietic stem cells. These GM/IL-3 humice expressed high numbers of tissue human MCs but the MRGPRX2 receptor expressed in MCs were limited to few body sites including the skin. Importantly, large numbers of MRGPRX2-expressing human MCs could be cultured from the bone marrow of GM/IL-3 humice revealing these mice to be an important source of human MCs for in vitro studies of MRGPRX2-related MCs activities. When GM/IL-3 humice were exposed to known ADR causing contrast agents (meglumine and gadobutrol), the humice were found to experience anaphylaxis analogous to the clinical situation. Thus, GM/IL-3 humice represent a valuable model for investigating in vivo interactions of ADR-causing drugs and human MCs and their sequelae, and these mice are also a source of human MRGPRX2-expressing MCs for in vitro studies.