A host-directed macrocyclic peptide therapeutic for MDR gram negative bacterial infections.

The emergence of infections by carbapenem resistant Enterobacteriaceae (CRE) pathogens has created an urgent public health threat, as carbapenems are among the drugs of last resort for infections caused by a growing fraction of multi-drug resistant (MDR) bacteria. There is global consensus that new preventive and therapeutic strategies are urgently needed to combat the growing problem of MDR bacterial infections. Here, we report on the efficacy of a novel macrocyclic peptide, minimized theta-defensin (MTD)-12813 in CRE sepsis. MTD12813 is a theta-defensin inspired cyclic peptide that is highly effective against CRE pathogens K. pneumoniae and E. coli in vivo. In mouse septicemia models, single dose administration of MTD12813 significantly enhanced survival by promoting rapid host-mediated bacterial clearance and by modulating pathologic cytokine responses, restoring immune homeostasis, and preventing lethal septic shock. The peptide lacks direct antibacterial activity in the presence of mouse serum or in peritoneal fluid, further evidence for its indirect antibacterial mode of action. MTD12813 is highly stable in biological matrices, resistant to bacterial proteases, and nontoxic to mice at dose levels 100 times the therapeutic dose level, properties which support further development of the peptide as a first in class anti-infective therapeutic.

Deep learning for detection and segmentation of artefact and disease instances in gastrointestinal endoscopy.

The Endoscopy Computer Vision Challenge (EndoCV) is a crowd-sourcing initiative to address eminent problems in developing reliable computer aided detection and diagnosis endoscopy systems and suggest a pathway for clinical translation of technologies. Whilst endoscopy is a widely used diagnostic and treatment tool for hollow-organs, there are several core challenges often faced by endoscopists, mainly: 1) presence of multi-class artefacts that hinder their visual interpretation, and 2) difficulty in identifying subtle precancerous precursors and cancer abnormalities. Artefacts often affect the robustness of deep learning methods applied to the gastrointestinal tract organs as they can be confused with tissue of interest. EndoCV2020 challenges are designed to address research questions in these remits. In this paper, we present a summary of methods developed by the top 17 teams and provide an objective comparison of state-of-the-art methods and methods designed by the participants for two sub-challenges: i) artefact detection and segmentation (EAD2020), and ii) disease detection and segmentation (EDD2020). Multi-center, multi-organ, multi-class, and multi-modal clinical endoscopy datasets were compiled for both EAD2020 and EDD2020 sub-challenges. The out-of-sample generalization ability of detection algorithms was also evaluated. Whilst most teams focused on accuracy improvements, only a few methods hold credibility for clinical usability. The best performing teams provided solutions to tackle class imbalance, and variabilities in size, origin, modality and occurrences by exploring data augmentation, data fusion, and optimal class thresholding techniques.

Lactobacillus reuteri DSM 17938 feeding of healthy newborn mice regulates immune responses while modulating gut microbiota and boosting beneficial metabolites.

Early administration of Lactobacillus reuteri DSM 17938 (LR) prevents necrotizing enterocolitis and inhibits regulatory T-cell (Treg)-deficiency-associated autoimmunity in mice. In humans, LR reduces crying time in breastfed infants with colic, modifies severity in infants with acute diarrheal illnesses, and improves pain in children with functional bowel disorders. In healthy breastfed newborns with evolving microbial colonization, it is unclear if early administration of LR can modulate gut microbiota and their metabolites in such a way as to promote homeostasis. We gavaged LR (107 colony-forming units/day, daily) to C57BL/6J mice at age of day 8 for 2 wk. Both male and female mice were investigated in these experiments. We found that feeding LR did not affect clinical phenotype or inflammatory biomarkers in plasma and stool, but LR increased the proportion of Foxp3+ regulatory T cells (Tregs) in the intestine. LR also increased bacterial diversity and the relative abundance of p_Firmicutes, f_Lachnospiraceae, f_Ruminococcaceae, and genera Clostridium and Candidatus arthromitus, while decreasing the relative abundance of p_Bacteriodetes, f_Bacteroidaceae, f_Verrucomicrobiaceae, and genera Bacteroides, Ruminococcus, Akkermansia, and Sutterella. Finally, LR exerted a major impact on the plasma metabolome, upregulating amino acid metabolites formed via the urea, tricarboxylic acid, and methionine cycles and increasing tryptophan metabolism. In conclusion, early oral administration of LR to healthy breastfed mice led to microbial and metabolic changes which could be beneficial to general health.NEW & NOTEWORTHY Oral administration of Lactobacillus reuteri DSM 17938 (LR) to healthy breastfed mice promotes intestinal immune tolerance and is linked to proliferation of beneficial gut microbiota. LR upregulates plasma metabolites that are involved in the urea cycle, the TCA cycle, methionine methylation, and the polyamine pathway. Herein, we show that LR given to newborn mice specifically increases levels of tryptophan metabolites and the purine nucleoside adenosine that are known to enhance tolerance to inflammatory stimuli.

Human LAP+GARP+FOXP3+ regulatory T cells attenuate xenogeneic graft versus host disease.

Adoptive transfer of regulatory T cells (FOXP3+ Tregs) has been developed as a potential curative immune therapy to prevent and treat autoimmune and graft-versus-host diseases (GVHD). A major limitation that has hindered the use of Treg immunotherapy in humans is the difficulty of consistently isolating and obtaining highly purified Tregs after ex vivo expansion. Methods: We isolated bona fide Tregs from expansion cultures based on their selective surface expression of latency-associated peptide (LAP). The TCR Vß diversity and intracellular cytokine production of Tregs were determined by flow cytometer. The TSDR methylation was determined by epigenetic human FOXP3 qPCR Assay. Their in vitro and in vivo potency was confirmed with suppression assay and humanized xenogeneic GVHD (xGVHD) murine model, respectively. Results: LAP+ repurification results in >90% LAP+FOXP3+ Tregs, leaving behind FOXP3- and FOXP3+ nonTregs within the LAP- population. After 4-week expansion, the LAP+ Tregs were >1 billion cells, highly suppressive and anergic in vitro, >90% demethylated in the TSDR and able to maintain TCR Vß diversity. In the xGVHD model, exogenous CD25-PBMC administered alone results in a median survival of 32 days. The co-transfer of LAP+ Tregs increased median survival to 47 days, while the LAP parent (CD25+) and LAP- nonTregs had median survival of 39 and 31 days, respectively. Conclusions: These preclinical data together provide evidence that LAP+ Tregs are highly purified with fully suppressive function for cell therapy. This population results in a more effective and safer product for immunotherapy to treat GVHD and provides the necessary preclinical data for transition into a clinical trial with LAP+ Tregs to prevent or treat GVHD and other autoimmune diseases.

Macrocyclic θ-defensins suppress tumor necrosis factor-α (TNF-α) shedding by inhibition of TNF-α-converting enzyme.

Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as adisintegrin and metalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases.

Adenosine A2A Receptor Deletion Blocks the Beneficial Effects of Lactobacillus reuteri in Regulatory T-Deficient Scurfy Mice.

The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF[Formula: see text]) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases.

Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency-induced autoimmunity via adenosine A2A receptors.

Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell-driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency-mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota-inosine-A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction.

Rhesus θ-defensin-1 (RTD-1) exhibits in vitro and in vivo activity against cystic fibrosis strains of Pseudomonas aeruginosa.

OBJECTIVES: Chronic endobronchial infections with Pseudomonas aeruginosa contribute to bronchiectasis and progressive loss of lung function in patients with cystic fibrosis. This study aimed to evaluate the therapeutic potential of a novel macrocyclic peptide, rhesus θ-defensin-1 (RTD-1), by characterizing its in vitro antipseudomonal activity and in vivo efficacy in a murine model of chronic Pseudomonas lung infection. METHODS: Antibacterial testing of RTD-1 was performed on 41 clinical isolates of P. aeruginosa obtained from cystic fibrosis patients. MIC, MBC, time-kill and post-antibiotic effects were evaluated following CLSI-recommended methodology, but using anion-depleted Mueller-Hinton broth. RTD-1 was nebulized daily for 7 days to cystic fibrosis transmembrane conductance regulator (CFTR) F508del-homozygous mice infected using the agar bead model of chronic P. aeruginosa lung infection. In vivo activity was evaluated by change in lung bacterial burden, airway leucocytes and body weight. RESULTS: RTD-1 exhibited potent in vitro bactericidal activity against mucoid and non-mucoid strains of P. aeruginosa (MIC90 = 8 mg/L). Cross-resistance was not observed when tested against MDR and colistin-resistant isolates. Time-kill studies indicated very rapid, concentration-dependent bactericidal activity of RTD-1 with ≥3 log10 cfu/mL reductions at concentrations ≥4× MIC. No post-antibiotic effect was observed. In vivo, nebulized treatment with RTD-1 significantly decreased lung P. aeruginosa burden (mean difference of -1.30 log10 cfu; P = 0.0061), airway leucocytes (mean difference of -0.37 log10; P = 0.0012) and weight loss (mean difference of -12.62% at day 7; P < 0.05) when compared with controls. CONCLUSIONS: This study suggests that RTD-1 is a promising potential therapeutic agent for cystic fibrosis airway disease.

FoxP3⁺ regulatory T cells attenuate experimental necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) results from severe intestinal inflammation in premature infants. FoxP3(+) regulatory T cells (Tregs) are central to gut homeostasis. While Treg proportions are significantly reduced in the ileums of premature infants with NEC, it is unknown whether they play a critical function in preventing NEC. This study investigated Treg development in newborn rat pups and their role in experimental NEC induction. Utilizing an established rat model of experimental NEC, the ontogeny of T cells and Tregs in newborn pups was characterized by flow cytometry. To investigate the functions of Tregs, newborn pups were given Tregs harvested from adult rats prior to NEC induction to assess clinical improvement and mechanisms of immune regulation. The results revealed that there were few Treg numbers in the terminal ileums of newborn rats and 8-fold reduction after NEC. Adoptive transfer of Tregs significantly improved weight loss, survival from 53% to 88%, and NEC incidence from 87% to 35%. The Tregs modulated the immune response as manifested in reduced CD80 expression on antigen presenting cells and decreased T cell activation within the mesenteric lymph nodes. These findings suggest that while Tregs are present in the intestines, their numbers might be insufficient to dampen the excessive inflammatory state in NEC. Adoptive transfer of Tregs attenuates the severity of NEC by limiting the immune response. Strategies to enhance Tregs have a therapeutic potential in controlling the development of NEC.

Sam68 modulates the promoter specificity of NF-κB and mediates expression of CD25 in activated T cells.

CD25, the alpha chain of the interleukin-2 receptor, is expressed in activated T cells and has a significant role in autoimmune disease and tumorigenesis; however, the mechanisms regulating transcription of CD25 remain elusive. Here we identify the Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel non-Rel component in the nuclear factor-kappaB (NF-κB) complex that confers CD25 transcription. Our results demonstrate that Sam68 has an essential role in the induction and maintenance of CD25 in T cells. T-cell receptor engagement triggers translocation of the inhibitor of NF-κB kinase alpha (IKKα) from the cytoplasm to the nucleus, where it phosphorylates Sam68, causing complex formation with NF-κB in the nucleus. These findings reveal the important roles of KH domain-containing components and their spatial interactions with IKKs in determining the binding targets of NF-κB complexes, thus shedding novel insights into the regulatory specificity of NF-κB.

Dominant gain-of-function STAT1 mutations in FOXP3 wild-type immune dysregulation-polyendocrinopathy-enteropathy-X-linked-like syndrome.

BACKGROUND: Mutations in signal transducer and activator of transcription (STAT) 1 cause a broad spectrum of disease, ranging from severe viral and bacterial infections (amorphic alleles) to mild disseminated mycobacterial disease (hypomorphic alleles) to chronic mucocutaneous candidiasis (CMC; hypermorphic alleles). The hypermorphic mutations are also associated with arterial aneurysms, autoimmunity, and squamous cell cancers. OBJECTIVE: We sought to investigate the role of STAT1 gain-of-function mutations in phenotypes other than CMC. METHODS: We initially screened patients with CMC and autoimmunity for STAT1 mutations. We functionally characterized mutations in vitro and studied immune profiles and regulatory T (Treg) cells. After our initial case identifications, we explored 2 large cohorts of patients with wild-type forkhead box protein 3 and an immune dysregulation-polyendocrinopathy-enteropathy-X-linked (IPEX)-like phenotype for STAT1 mutations. RESULTS: We identified 5 children with polyendocrinopathy, enteropathy, and dermatitis reminiscent of IPEX syndrome; all but 1 had a variety of mucosal and disseminated fungal infections. All patients lacked forkhead box protein 3 mutations but had uniallelic STAT1 mutations (c.629 G>T, p.R210I; c.1073 T>G, p.L358W, c.796G>A; p.V266I; c.1154C>T, T385M [2 patients]). STAT1 phosphorylation in response to IFN-γ, IL-6, and IL-21 was increased and prolonged. CD4(+) IL-17-producing T-cell numbers were diminished. All patients had normal Treg cell percentages in the CD4(+) T-cell compartment, and their function was intact in the 2 patients tested. Patients with cells available for study had normal levels of IL-2-induced STAT5 phosphorylation. CONCLUSIONS: Gain-of-function mutations in STAT1 can cause an IPEX-like phenotype with normal frequency and function of Treg cells.

T regulatory cells in cancer: recent advances and therapeutic potential.

IMPORTANCE OF THE FIELD: The active suppression of immune responses against tumor is a major barrier to the likely success of cancer immunotherapy. There is now compelling evidence implicating T regulatory cells (Tregs) as being key players driving immune suppression. Elevated frequencies of Tregs within the peripheral circulation and tumor microenvironment of cancer patients correlate with poor prognosis and reduced survival. Understanding the mechanism of Treg elevation is critical for the development of new approaches aiming to modulate the frequency and function of Tregs to enhance the efficacy of cancer immune-based therapies. AREAS COVERED IN THIS REVIEW: This review focuses on current knowledge concerning Tregs in cancer and discusses putative mechanisms which underlie the expansion of Tregs in cancer patients. Additionally, we review current strategies to deplete/suppress Treg activity, the limitations of these strategies and future perspective for improving their efficacy. WHAT THE READER WILL GAIN: An insight of the current aspects concerning Treg subsets in cancer and an overview of recent advances in the identification of Treg-specific markers, in addition to the potential strategies to target Tregs for enhancing antitumor immunity. TAKE HOME MESSAGE: Mechanisms by which Treg functions modulate the immune response to tumors are becoming further understood. However, specific markers to tumor-specific/induced Tregs are yet to be clearly identified, which is a major limitation in optimizing strategies to specifically target Tregs in cancer. Despite this, strategies aimed at modulating Tregs in patients are providing some early encouraging results supporting the overall concept and indicating that further studies are clearly warranted.

Selective expression of latency-associated peptide (LAP) and IL-1 receptor type I/II (CD121a/CD121b) on activated human FOXP3+ regulatory T cells allows for their purification from expansion cultures.

Although adoptive transfer of regulatory T cells (Foxp3(+) Tregs) has proven to be efficacious in the prevention and treatment of autoimmune diseases and graft-versus-host disease in rodents, a major obstacle for the use of Treg immunotherapy in humans is the difficulty of obtaining a highly purified preparation after ex vivo expansion. We have identified latency-associated peptide (LAP) and IL-1 receptor type I and II (CD121a/CD121b) as unique cell-surface markers that distinguish activated Tregs from activated FOXP3(-) and FOXP3(+) non-Tregs. We show that it is feasible to sort expanded FOXP3(+) Tregs from non-Tregs with the use of techniques for magnetic bead cell separation based on expression of these 3 markers. After separation, the final product contains greater than 90% fully functional FOXP3(+) Tregs. This novel protocol should facilitate the purification of Tregs for both cell-based therapies as well as detailed studies of human Treg function in health and disease.

Therapeutic potential of FOXP3(+) regulatory T cells and their interactions with dendritic cells.

FOXP3(+) regulatory T cells, a unique subset of T cells, are critical for orchestrating an immune response and preventing self-reactivity. With the increasing prevalence and unsatisfactory treatment of autoimmunity, allergic diseases, cancer and chronic infections, much attention has been focused on understanding their mechanisms of action in order to manipulate their function. One goal is to develop drugs or biologics that can enhance or abrogate their functions. Another approach is to utilize Tregs in adoptive cell-based therapy to treat autoimmune diseases or transplant-related complications. This review will focus on their therapeutic potential and mechanisms of action, particularly their interaction with dendritic cells.