Molly A. Ingersoll

Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy

The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.

G‐CSF induction early in uropathogenic Escherichia coli infection of the urinary tract modulates host immunity

Uropathogenic Escherichia coli (UPEC), the causative agent of approximately 85% of urinary tract infections (UTI), is a major health concern primarily affecting women. During infection, neutrophils infiltrate the bladder, but the mechanism of recruitment is not well understood. Here, we investigated the role of UPEC‐induced cytokine production in neutrophil recruitment and UTI progression. We first examined the kinetics of cytokine expression during UPEC infection of the bladder, and their contribution to neutrophil recruitment. We found that UPEC infection induces expression of several pro‐inflammatory cytokines including granulocyte colony‐stimulating factor (G‐CSF, CSF‐3), not previously known to be involved in the host response to UTI. G‐CSF induces neutrophil emigration from the bone marrow; these cells are thought to be critical for bacterial clearance during infection. Upon neutralization of G‐CSF during UPEC infection, we found fewer circulating neutrophils, decreased neutrophil infiltration into the bladder and, paradoxically, a decreased bacterial burden in the bladder. However, depletion of G‐CSF resulted in a corresponding increase in macrophage‐activating cytokines, such as monocyte chemotactic protein‐1 (MCP‐1, CCL‐2) and Il‐1β, which may be key in host response to UPEC infection, potentially resolving the paradoxical decreased bacterial burden. Thus, G‐CSF acts in a previously unrecognized role to modulate the host inflammatory response during UPEC infection.

Bacillus Calmette-Guérin Strain Differences Have an Impact on Clinical Outcome in Bladder Cancer Immunotherapy

BACKGROUND Whether the commonly used bacillus Calmette-Guérin (BCG) strains Connaught and Tice confer different treatment responses in non-muscle-invasive bladder cancer (NMIBC) is unknown. OBJECTIVES To compare clinical efficacy, immunogenicity, and genetics of BCG Connaught and Tice. DESIGN, SETTING, AND PARTICIPANTS A prospective randomized single-institution trial with treatment of 142 high-risk NMIBC patients with BCG Connaught or Tice. INTERVENTION Patients were randomized to receive six instillations of BCG Connaught or Tice. For experimental studies, BCG strains were compared in C57Bl/6 mice. Bladders and lymphoid tissues were analyzed by cytometry and the latter cultivated to detect live BCG. BCG genomic DNA was sequenced and compared with reference genomes. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Recurrence-free survival was the primary end point of the clinical study. The Kaplan-Meier estimator was used for estimating survival and time-to-event end points. Nonparametric tests served for the analysis of the in vivo results. RESULTS AND LIMITATIONS Treatment with BCG Connaught conferred significantly greater 5-yr recurrence-free survival compared with treatment with BCG Tice (p=0.0108). Comparable numbers of patients experienced BCG therapy-related side effects in each treatment group (p=0.09). In mice, BCG Connaught induced stronger T-helper cell 1-biased responses, greater priming of BCG-specific CD8(+) T cells, and more robust T-cell recruitment to the bladder than BCG Tice. Genome sequencing of the BCG strains revealed candidate genes potentially involved in the differential clinical responses. CONCLUSIONS BCG strain may have an impact on treatment outcome in NMIBC immunotherapy. PATIENT SUMMARY We compared the efficacy of two commonly used bacillus Calmette-Guérin (BCG) strains for the treatment of NMIBC and found that treatment with BCG Connaught prevented recurrences more efficiently than BCG Tice. Comparison of the immunogenicity of the two strains in mice indicated superior immunogenicity of BCG Connaught. We also identified genetic differences that may explain the differential efficacy of the Connaught and Tice BCG strains. TRIAL REGISTRATION NCT00003779.

Pathogenicity islands of Shigella

Shigella species are the causative agents of bacillary dysentery. Signs of disease range from mild diarrhea to a severe form of disease including fever, abdominal cramps, and stools containing blood, pus and mucus. Shigella are primarily human pathogens but can produce disease symptoms in other primates (Sansonetti 1992). After ingestion, shigellae traverse the intestinal epithelial barrier through specialized cells, called M-cells, at the level of the colon (Wassef et al. 1989). These cells transport antigens, including enteric pathogens, across the epithelium. Following transcytosis, micro-organisms gain access to lymphoid follicles containing resident tissue macrophages (Jarry et al. 1989; Soestayo et al. 1990). After phagocytosis, shigellae rapidly destroy the membrane of the phagosome and are liberated into the host cell cytoplasm (Finlay and Falkow 1988; Maurelli and Sansonetti 1988).

Characterization of a Novel Murine Model of Staphylococcus saprophyticus Urinary Tract Infection Reveals Roles for Ssp and SdrI in Virulence

ABSTRACT Staphylococcus saprophyticus, an obligate human pathogen, is the most common Gram-positive causative agent of urinary tract infection (UTI) in young, healthy women. Despite the clinical importance of S. saprophyticus, little is known about how it causes disease in the urinary tract or how the host responds to the infection. Here we established an in vivo model to study both host and bacterial factors contributing to S. saprophyticus UTI. Using this model, we show that S. saprophyticus preferentially infects C3H/HeN murine kidneys instead of the bladder, a trait observed for multiple clinical isolates. Bacterial persistence in the kidneys was observed in C3H/HeN mice but not in C57BL/6 mice, indicating that host factors strongly contribute to the ability of S. saprophyticus to cause UTI. Using C3H/HeN mice as a model, histologic and immunofluorescence analyses of infected tissues revealed that S. saprophyticus induced epithelial cell shedding in the bladder and an inflammatory response characterized by macrophage and neutrophil infiltration in the bladder and kidneys. The inflammatory response correlated with increased production of proinflammatory cytokines and chemokines in both the bladder and the kidneys. Finally, we observed that the putative S. saprophyticus virulence factors Ssp and SdrI were important for persistence, but not for initial colonization, in the murine urinary tract. Thus, we characterized both host and bacterial factors involved in progression of S. saprophyticus UTI, and we describe a useful model system for studying factors involved in the pathogenesis of this Gram-positive uropathogen.

The ShiA protein encoded by the Shigella flexneri SHI-2 pathogenicity island attenuates inflammation

Shigella spp. are the aetiologic agents of dysentery, a severe diarrhoeal syndrome characterized by acute inflammation in the colon. The inflammatory response, which includes recruitment of polymorphonuclear leukocytes (PMN), damages the colonic mucosa and exacerbates the infection. Shigella encodes a pathogenicity island (PAI), SHI‐2, which is localized in a region of the chromosome linked to the induction of inflammation. Surprisingly, SHI‐2 deletion mutants induce a stronger inflammatory response than wild‐type Shigella as measured by increased villus blunting, increased PMN infiltration and induction of apoptosis in a rabbit ileal loop model of shigellosis. Mutational analysis mapped the hyper‐inflammatory phenotype to a single gene, shiA. Similar to SHI‐2 deletion mutants, infection with a shiA mutant strain induces dramatically elevated levels of inflammation when compared to the wild‐type strain. Furthermore, infection with a wild‐type strain containing multiple copies of shiA results in fewer infiltrating PMN and apoptotic cells, as well as preservation of a normal villus architecture at the site of infection, thus acting in a dominant fashion over the pro‐inflammatory mechanisms of Shigella. The molecular mechanism of action of ShiA is independent of any in vitro phenotype associated with Shigella virulence. Our data suggest that ShiA allows Shigella to attenuate the host inflammatory response in a novel manner.

ShiA abrogates the innate T-cell response to Shigella flexneri infection.

ABSTRACT Shigella spp. are the causative agent of bacillary dysentery. Infection results in acute colonic injury due to the host inflammatory response. The mediators of the damage, infiltrating polymorphonuclear leukocytes (PMN), also resolve the infection. Shigella flexneris virulence effectors are encoded on its large virulence plasmid and on pathogenicity islands in the chromosome. The SHI-2 pathogenicity island encodes the virulence factor ShiA, which down-regulates Shigella-induced inflammation. In the rabbit ileal loop model, infection with a shiA null strain (ΔshiA) induces a more severe inflammation than wild-type infection. Conversely, a Shigella strain that overexpresses ShiA (ShiA+) is less inflammatory than the wild-type strain. To determine the host responses modulated by ShiA, we performed infection studies using the mouse lung model, which recapitulates the phenotypes observed in the rabbit ileal loop model. Significantly, ShiA+ strain-infected mice cleared the bacteria and survived infection, while wild-type- and ΔshiA strain-infected mice could not clear the bacteria and ultimately died. Surprisingly, microarray analysis of infected lungs revealed the regulation of genes involved in innate T-cell responses to infection. Immunohistochemistry showed that wild-type- and ΔshiA strain-infected animals have greater numbers of PMN and T cells in their lungs over the course of infection than ShiA+ strain-infected animals. These results suggest that the T-cell innate response is suppressed by ShiA in Shigella infections.

Considerations on the use of urine markers in the management of patients with low-/intermediate-risk non–muscle invasive bladder cancer

OBJECTIVES Many molecular assays for bladder cancer diagnosis and surveillance have been developed over the past several decades. However, none of these markers have been routinely implemented into clinical decision making. Beyond their potential for screening high-risk populations, urine markers likely have the greatest potential in the follow-up of patients with non-muscle invasive bladder cancer (NMIBC). METHODS Here, we discuss the current options and limitations of the use of urine markers for patient surveillance, focusing on patients with low-/intermediate-risk NMIBC. RESULTS As these patients have a very low risk of tumor progression, the primary goal of surveillance is detection of recurrent disease. Although urine cytology seems to be limited to detection of few patients who would develop high-grade tumors, we conclude that the use of markers with high sensitivity for low-grade disease for patient follow-up has the potential to decrease the frequency of urethrocystoscopy without compromising patient prognosis. Because a single marker may not have sufficient sensitivity for detection of low-grade tumors, different scenarios, e.g., multitesting and reflex or sequential approaches, are discussed. CONCLUSIONS There is consensus that currently available markers have the potential to support clinical decision making in follow-up of patients with low-/intermediate-risk NMIBC. In light of our analysis, further additional randomized controlled studies to effectively assess the clinical usefulness of modern urine markers are required.

Considerations on the use of urine markers in the management of patients with high-grade non–muscle-invasive bladder cancer

OBJECTIVE Diagnosis and surveillance of high risk non muscle-invasive bladder cancer (NMIBC) represent specific challenges to urologists. In contrast to low/intermediate risk tumors, these tumors recur more frequently. A significant number will eventually progress to muscle-invasive bladder cancer, a life threatening disease requiring extensive therapeutic efforts. Although clinical risk factors have been identified that may predict tumor recurrence and progression, additional biomarkers are desperately needed to improve tumor diagnosis and guide clinical management of these patients. In this article, the role of molecular urine markers in the management of high risk NMIBC is analyzed. METHODS In this context, several potential indications (diagnostic, prognostic, predictive) were identified and the requirements for molecular markers were defined. In addition, current knowledge within the different indications was summarized. RESULTS Significant progress has been made in the last decade studying the impact of molecular urine markers in patients with high risk NMIBC. CONCLUSIONS Although we may not be ready for the inclusion of molecular markers in clinical decision-making, and many questions remain unanswered, recent studies have identified situations in which the use of molecular markers in particular in high grade tumors may prove beneficial for patient diagnosis and surveillance.

Macrophages Subvert Adaptive Immunity to Urinary Tract Infection

Urinary tract infection (UTI) is one of the most common bacterial infections with frequent recurrence being a major medical challenge. Development of effective therapies has been impeded by the lack of knowledge of events leading to adaptive immunity. Here, we establish conclusive evidence that an adaptive immune response is generated during UTI, yet this response does not establish sterilizing immunity. To investigate the underlying deficiency, we delineated the naïve bladder immune cell compartment, identifying resident macrophages as the most populous immune cell. To evaluate their impact on the establishment of adaptive immune responses following infection, we measured bacterial clearance in mice depleted of either circulating monocytes, which give rise to macrophages, or bladder resident macrophages. Surprisingly, mice depleted of resident macrophages, prior to primary infection, exhibited a nearly 2-log reduction in bacterial burden following secondary challenge compared to untreated animals. This increased bacterial clearance, in the context of a challenge infection, was dependent on lymphocytes. Macrophages were the predominant antigen presenting cell to acquire bacteria post-infection and in their absence, bacterial uptake by dendritic cells was increased almost 2-fold. These data suggest that bacterial uptake by tissue macrophages impedes development of adaptive immune responses during UTI, revealing a novel target for enhancing host responses to bacterial infection of the bladder.