Ines Ambite

Background: Finding new ways of treating bacterial infections is essential. The NlpD protein, which inhibits RNA polymerase II (Pol II), has shown therapeutic efficacy against urinary tract infection. This study investigated the mechanism of Pol II inhibition and protection by NlpD and its LytM peptide. Methods: Recombinant NlpD and LytM were screened for interactions with constituents of the Pol II complex, using AlphaFold predictions and protein interaction technology. Treatment effects were quantified in infected tissues and regulated host response pathways identified by genome-wide transcriptomics analysis in models of acute pyelonephritis and acute cystitis in Irf3−/− and Asc−/− mice, respectively. Results: LytM was shown to interact with constituents of the Pol II multiprotein complex, inhibiting the CDK12 kinase from phosphorylating the Pol II subunit RPB1 and disrupting Pol II complex formation by interfering with the interaction between PAF1C and RPB1. The protection by LytM against acute pyelonephritis was accompanied by a reduction in gene expression in infected kidneys from >1900 significantly regulated genes (fold change >6) in the placebo group to about 150 in LytM-treated mice. The inhibition of gene expression in infected kidneys particularly targeted the excessive innate immune response. A similar effect was observed in acute cystitis. Bacterial clearance was accelerated in both model by LytM treatment, with effects against antibiotic-sensitive and resistant Escherichia coli strains. Conclusions: The results suggest that inhibiting the disease response of the host, using NlpD or LytM, may offer an efficient alternative to antibiotics in these models.

Cystitis is a bacterial infection of the bladder that occurs in about half of women at least once in their lifetime. Antibiotics such as nitrofurantoin are used to treat cystitis, but antibiotic resistance is a concern, especially for recurrent infections. Here we report an open-label, randomized, single-centre, phase 2 study to analyse the acute and long-term safety and efficacy of the IL-1 receptor antagonist anakinra, compared with nitrofurantoin, in recurrent cystitis. A total of 30 adult female patients with a documented history of recurrent cystitis and a current acute cystitis episode were randomized in a 2:1 ratio to treatment with anakinra ( n = 20) or nitrofurantoin ( n = 10) for 5 days. Primary and secondary efficacy end-points were reached, defined as the reduction in typical symptoms, measured by the acute cystitis symptom score (day 5), longitudinal symptom scores, recurrence rates, quality of life, gene expression analysis and microbiology at follow-up on days 15 and 30 and at 6 months. Symptom scores were decreased in the anakinra ( P < 0.001) and nitrofurantoin ( P < 0.001) arms after 5 days and remained low after 15 days, 30 days and 6 months. Recurrences were less frequent after 6 months in both treatment groups compared with the 6-month pre-enrolment history ( P < 0.001 for anakinra and P = 0.004 for nitrofurantoin), and the quality of life was increased, without adverse effects. Immune gene expression was rapidly inhibited in the anakinra-treated patients but not in the nitrofurantoin group. Targeted innate immune inhibition therapy shows non-inferiority to nitrofurantoin in patients with recurrent acute cystitis. German Clinical Trials Register ID: DRKS00025964 .

Background: Infections trigger complex immune responses, with conserved as well as disease-specific characteristics. Methods: Proteomic screening technology and gene expression analysis were used here to define the immune response in infants, with their first episode of febrile urinary tract infection. Urine and peripheral blood samples were obtained at enrollment and at follow-up, after 6 months. Results: Pair-wise proteomic analysis of urine samples detected a broad local cytokine response in urine; 20 of the 24 proteins were strongly activated during acute infection compared with follow-up. Functional profiling identified the response as a cytokine storm, and major innate immune response regulators and effector molecules were activated, such as IL-1α, IL-1β, IL-1RA, IL-33, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, GM-CSF, IL-6, IL-17, TNF-α and IFN-γ. In addition, the adaptive immune response was activated, including CD40L, IL-2, Granzyme B, IL-10, IL-15 and PD-L1. Gene expression analysis of peripheral blood RNA detected a systemic cytokine storm profile, which was more pronounced in infants with renal involvement, defined by positive acute dimercaptosuccinic acid scans, than in infants with febrile urinary tract infection without renal involvement. Conclusions: Local and systemic hyperactivation of innate immunity characterizes acute pyelonephritis, a common and severe bacterial infection in childhood and a significant cause of urosepsis and mortality in adults. The results define a transient cytokine storm response, resembling that induced during severe acute respiratory syndrome coronavirus 2 infection, as characteristic of acute pyelonephritis, rather than individual protein biomarkers.

The membranous ER spans the entire cell, creating a network for the biosynthesis of proteins and lipids, cell-wide communication, and nuclear delivery of molecules, including therapeutic agents. Here, we identify a novel ER response triggered by the tumoricidal complex alpha1-oleate, defined by a loss of peripheral ER structure, extensive ER vesiculation. Alpha1-oleate was present in the ER-derived vesicle membranes, also decorated by ER-resident and ER-interacting proteins, calnexin and ORP3, and in their lumen, also enriched for KDEL, confirming their ER origin distinct from lipid droplets. Rapid nuclear uptake of the complex constituents resulted in diffuse nuclear staining, and the asymmetrical perinuclear enrichment of the collapsing ER with its content of alpha1-oleate created large invaginations lined by the ER, inner nuclear membrane markers, and lamin nucleoskeleton. In parallel, a change in nuclear shape resulted in a volcano-like structure. This newly discovered, potent ER response to alpha1-oleate may have evolved to package ER-associated cellular contents in the nuclei of dying tumor cells, thus sequestering toxic cell debris associated with apoptotic cell death.

This study investigated the molecular basis of disease severity in acute pyelonephritis (APN), a common and potentially life-threatening bacterial infection. Two cohorts of infants with febrile urinary tract infection were included. Renal involvement was defined by DMSA scans and molecular disease determinants by gene expression analysis and proteomic screens, at diagnosis and after 6 mo. Innate immune hyper-activation, systemically and locally in the urinary tract, was defined as a cytokine storm. Neutrophil degranulation and renal toxicity genes were strongly regulated, with overexpression in the APN group (first DMSA+). Adaptive immune attenuation in the APN group further supported the notion of an immune imbalance. DNA exome genotyping identified APN and febrile urinary tract infection as genetically distinct and scarring associated genes, but the activation of renal toxicity genes during acute infection was unrelated to the development of renal scarring. The results define APN as a hyper-inflammatory disorder with the characteristics of a cytokine storm combined with adaptive immune attenuation. The findings are consistent with innate immune dysfunctions and neutrophil disorders identified as determinants of APN susceptibility in genetic models.

Background: The tumoricidal complex alpha1‐oleate targets bladder cancer cells, triggering rapid, apoptosis‐like tumor cell death. Clinical effects of alpha1‐oleate were recently observed in patients with non‐muscle invasive bladder cancer (NMIBC), using a randomized, placebo‐controlled study protocol.Aims: To investigate if there are dose‐dependent effects of alpha1‐oleate.Materials and Methods: Here, patients with NMIBC were treated by intravesical instillation of increasing concentrations of alpha1‐oleate (1.7, 8.5, or 17 mM) and the treatment response was defined relative to a placebo group.Results: Strong, dose‐dependent anti‐tumor effects were detected in alpha1‐oleate treated patients for a combination of molecular and clinical indicators; a complete or partial response was detected in 88% of tumors treated with 8.5 mM compared to 47% of tumors treated with 1.7 mM of alpha1‐oleate. Uptake of alpha1‐oleate by the tumor triggered rapid shedding of tumor cells into the urine and cell death by an apoptosis‐like mechanism. RNA sequencing of tissue biopsies confirmed the activation of apoptotic cell death and strong inhibition of cancer gene networks, including bladder cancer related genes. Drug‐related side effects were not recorded, except for local irritation at the site of instillation.Discussion and Conclusions: These dose‐dependent anti‐tumor effects of alpha1‐oleate are promising and support the potential of alpha1‐oleate treatment in patients with NMIBC.

Background: The molecular content of urine is defined by filtration in the kidneys and by local release from tissues lining the urinary tract. Pathological processes and different therapies change the molecular composition of urine and a variety of markers have been analyzed in patients with bladder cancer. The response to BCG immunotherapy and chemotherapy has been extensively studied and elevated urine concentrations of IL‐1RA, IFN‐α, IFN‐γ TNF‐α, and IL‐17 have been associated with improved outcome.Methods: In this study, the host response to intravesical alpha 1‐oleate treatment was characterized in patients with non‐muscle invasive bladder cancer by proteomic and transcriptomic analysis.Results: Proteomic profiling detected a significant increase in multiple cytokines in the treatment group compared to placebo. The innate immune response was strongly activated, including IL‐1RA and pro‐inflammatory cytokines in the IL‐1 family (IL‐1α, IL‐1β, IL‐33), chemokines (MIP‐1α, IL‐8), and interferons (IFN‐α2, IFN‐γ). Adaptive immune mediators included IL‐12, Granzyme B, CD40, PD‐L1, and IL‐17D, suggesting broad effects of alpha 1‐oleate treatment on the tumor tissues.Conclusions: The cytokine response profile in alpha 1‐oleate treated patients was similar to that reported in BCG treated patients, suggesting a significant overlap. A reduction in protein levels at the end of treatment coincided with inhibition of cancer‐related gene expression in tissue biopsies, consistent with a positive treatment effect. Thus, in addition to killing tumor cells and inducing cell detachment, alpha 1‐oleate is shown to activate a broad immune response with a protective potential.

Innate immunity is essential for the anti-microbial defense, but excessive immune activation may cause severe disease. In this study, immunotherapy was shown to prevent excessive innate immune activation and restore the anti-bacterial defense. E. coli-infected Asc^−/− mice develop severe acute cystitis, defined by IL-1 hyper-activation, high bacterial counts, and extensive tissue pathology. Here, the interleukin-1 receptor antagonist (IL-1RA), which inhibits IL-1 hyper-activation in acute cystitis, was identified as a more potent inhibitor of inflammation and NK1R- and substance P-dependent pain than cefotaxime. Furthermore, IL-1RA treatment inhibited the excessive innate immune activation in the kidneys of infected Irf3^−/− mice and restored tissue integrity. Unexpectedly, IL-1RA also accelerated bacterial clearance from infected bladders and kidneys, including antibiotic-resistant E. coli, where cefotaxime treatment was inefficient. The results suggest that by targeting the IL-1 response, control of the innate immune response to infection may be regained, with highly favorable treatment outcomes, including infections caused by antibiotic-resistant strains.

Bladder cancer is common and one of the most costly cancer forms, due to a lack of curative therapies. Recently, clinical safety and efficacy of the alpha1‐oleate complex was demonstrated in a placebo‐controlled study of nonmuscle invasive bladder cancer. Our study investigated if long‐term therapeutic efficacy is improved by repeated treatment cycles and by combining alpha1‐oleate with low‐dose chemotherapy. Rapidly growing bladder tumors were treated by intravesical instillation of alpha1‐oleate, Epirubicin or Mitomycin C alone or in combination. One treatment cycle arrested tumor growth, with a protective effect lasting at least 4 weeks in mice receiving 8.5 mM of alpha1‐oleate alone or 1.7 mM of alpha‐oleate combined with Epirubicin or Mitomycin C. Repeated treatment cycles extended protection, defined by a lack of bladder pathology and a virtual absence of bladder cancer‐specific gene expression. Synergy with Epirubicin was detected at the lower alpha1‐oleate concentration and in vitro, alpha1‐oleate was shown to enhance the uptake and nuclear translocation of Epirubicin, by tumor cells. Effects at the chromatin level affecting cell proliferation were further suggested by reduced BrdU incorporation. In addition, alpha1‐oleate triggered DNA fragmentation, defined by the TUNEL assay. The results suggest that bladder cancer development may be prevented long‐term in the murine model, by alpha1‐oleate alone or in combination with low‐dose Epirubicin. In addition, the combination of alpha1‐oleate and Epirubicin reduced the size of established tumors. Exploring these potent preventive and therapeutic effects will be of immediate interest in patients with bladder cancer.

Innovative solutions are needed for the treatment of bacterial infections, and a range of antibacterial molecules have been explored as alternatives to antibiotics. A different approach is to investigate the immune system of the host for new ways of making the antibacterial defence more efficient. However, the immune system has a dual role as protector and cause of disease: in addition to being protective, increasing evidence shows that innate immune responses can become excessive and cause acute symptoms and tissue pathology during infection. This role of innate immunity in disease suggests that the immune system should be targeted therapeutically, to inhibit over-reactivity. The ultimate goal is to develop therapies that selectively attenuate destructive immune response cascades, while augmenting the protective antimicrobial defence but such treatment options have remained underexplored, owing to the molecular proximity of the protective and destructive effects of the immune response. The concept of innate immunomodulation therapy has been developed successfully in urinary tract infections, based on detailed studies of innate immune activation and disease pathogenesis. Effective, disease-specific, immunomodulatory strategies have been developed by targeting specific immune response regulators including key transcription factors. In acute pyelonephritis, targeting interferon regulatory factor 7 using small interfering RNA or treatment with antimicrobial peptide cathelicidin was protective and, in acute cystitis, targeting overactive effector molecules such as IL-1β, MMP7, COX2, cAMP and the pain-sensing receptor NK1R has been successful in vivo. Furthermore, other UTI treatment strategies, such as inhibiting bacterial adhesion and vaccination, have also shown promise.