NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

Exploratory studies with NX-13: oral toxicity and pharmacokinetics in rodents of an orally active, gut-restricted first-in-class therapeutic for IBD that targets NLRX1.

Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is an emerging therapeutic target for a spectrum of human diseases. NX-13 is a small molecule therapeutic designed to target and activate NLRX1 to induce immunometabolic changes resulting in lower inflammation and therapeutic responses in inflammatory bowel disease (IBD). This study investigates the safety of NX-13 in a seven-day, repeat-dose general toxicity study in male and female Sprague Dawley rats at oral doses of 500 and 1000 mg/kg. Weights, clinical signs, functional observational battery, clinical pathology and histopathology were used for evaluation. Daily oral dosing of NX-13 up to 1000 mg/kg did not result in any changes in weight, abnormal clinical signs or behavior. No significant differences were observed between treated and control rats in hematology or blood biochemistry. Histopathological evaluation of 12 tissues demonstrated no differences between controls and treated rats. There were no changes in weights of brain, heart, kidney, liver or spleen. Pharmacokinetic analysis of a single oral dose of NX-13 at 10 mg/kg in Sprague Dawley rats provided a maximum plasma concentration of 57 ng/mL at 0.5 h post-dose. Analysis of colon tissue after oral dosing with 1 and 10 mg/kg indicated high peak concentrations (10 and 100 µg/g, respectively) that scale in a dose-proportional manner. These experiments suggest that NX-13 is safe and well-tolerated in rats given oral doses as high as 1000 mg/kg with a favorable gastrointestinal localized pharmacokinetic profile, confirming NX-13 as a promising therapeutic for Crohn's disease and ulcerative colitis.

Oral Treatment with BT-11 Ameliorates Inflammatory Bowel Disease by Enhancing Regulatory T Cell Responses in the Gut.

Inflammatory bowel disease (IBD) is an expanding autoimmune disease afflicting millions that remains difficult to treat due to the accumulation of multiple immunological changes. BT-11 is an investigational new drug for IBD that is orally active, gut restricted, and targets the lanthionine synthetase C-like 2 immunometabolic pathway. CD25+ FOXP3+ CD4+ T cells are increased locally within the colon of BT-11-treated mice in Citrobacter rodentium and IL-10-/- mouse models of colitis. The maintained efficacy of BT-11 in the absence of IL-10 combined with the loss of efficacy when direct cell-cell interactions are prevented suggest that the regulatory T cell (Treg)-related elements of suppression are cell contact-mediated. When PD-1 is inhibited, both in vitro and in vivo, the efficacy of BT-11 is reduced, validating this assertion. The depletion of CD25+ cells in vivo abrogated the retention of therapeutic efficacy postdiscontinuation of treatment, indicating that Tregs are implicated in the maintenance of tolerance mediated by BT-11. Furthermore, the involvement of CD25 suggested a role of BT-11 in IL-2 signaling. Cotreatment with BT-11 and IL-2 greatly enhances the differentiation of CD25+ FOXP3+ cells from naive CD4+ T cells relative to either alone. BT-11 enhances phosphorylation of STAT5, providing a direct linkage to the regulation of FOXP3 transcription. Notably, when STAT5 is inhibited, the effects of BT-11 on the differentiation of Tregs are blocked. BT-11 effectively enhances the IL-2/STAT5 signaling axis to induce the differentiation and stability of CD25+ FOXP3+ cells in the gastrointestinal mucosa to support immunoregulation and immunological tolerance in IBD.

Activation of NLRX1 by NX-13 Alleviates Inflammatory Bowel Disease through Immunometabolic Mechanisms in CD4+ T Cells.

Inflammatory bowel disease (IBD) is a complex autoimmune disease with dysfunction in pattern-recognition responses, including within the NLR family. Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a unique NLR with regulatory and anti-inflammatory functions resulting in protection from IBD in mouse models. NX-13 is an orally active, gut-restricted novel drug candidate that selectively targets and activates the NLRX1 pathway locally in the gut. In vitro and in vivo efficacy of NLRX1 activation by NX-13 was examined. Oral treatment with NX-13 alleviates disease severity, colonic leukocytic infiltration, and cytokine markers of inflammation in three mouse models of IBD (dextran sulfate sodium, Mdr1a-/-, and CD45RBhi adoptive transfer). Treatment of naive CD4+ T cells with NX-13 in vitro decreases differentiation into Th1 and Th17 subsets with increased oxidative phosphorylation and decreased NF-κB activation and reactive oxygen species. With stimulation by PMA/ionomycin, TNF-α, or H2O2, PBMCs from ulcerative colitis patients treated with NX-13 had decreased NF-κB activity, TNF-α+ and IFN-γ+ CD4+ T cells and overall production of IL-6, MCP1, and IL-8. NX-13 activates NLRX1 to mediate a resistance to both inflammatory signaling and oxidative stress in mouse models and human primary cells from ulcerative colitis patients with effects on NF-κB activity and oxidative phosphorylation. NX-13 is a promising oral, gut-restricted NLRX1 agonist for treating IBD.

Solving Immunology?

Emergent responses of the immune system result from the integration of molecular and cellular networks over time and across multiple organs. High-content and high-throughput analysis technologies, concomitantly with data-driven and mechanistic modeling, hold promise for the systematic interrogation of these complex pathways. However, connecting genetic variation and molecular mechanisms to individual phenotypes and health outcomes has proven elusive. Gaps remain in data, and disagreements persist about the value of mechanistic modeling for immunology. Here, we present the perspectives that emerged from the National Institute of Allergy and Infectious Disease (NIAID) workshop 'Complex Systems Science, Modeling and Immunity' and subsequent discussions regarding the potential synergy of high-throughput data acquisition, data-driven modeling, and mechanistic modeling to define new mechanisms of immunological disease and to accelerate the translation of these insights into therapies.

Cooperation of Gastric Mononuclear Phagocytes with Helicobacter pylori during Colonization.

Helicobacter pylori, the dominant member of the human gastric microbiota, elicits immunoregulatory responses implicated in protective versus pathological outcomes. To evaluate the role of macrophages during infection, we employed a system with a shifted proinflammatory macrophage phenotype by deleting PPARγ in myeloid cells and found a 5- to 10-fold decrease in gastric bacterial loads. Higher levels of colonization in wild-type mice were associated with increased presence of mononuclear phagocytes and in particular with the accumulation of CD11b+F4/80hiCD64+CX3CR1+ macrophages in the gastric lamina propria. Depletion of phagocytic cells by clodronate liposomes in wild-type mice resulted in a reduction of gastric H. pylori colonization compared with nontreated mice. PPARγ-deficient and macrophage-depleted mice presented decreased IL-10-mediated myeloid and T cell regulatory responses soon after infection. IL-10 neutralization during H. pylori infection led to increased IL-17-mediated responses and increased neutrophil accumulation at the gastric mucosa. In conclusion, we report the induction of IL-10-driven regulatory responses mediated by CD11b+F4/80hiCD64+CX3CR1+ mononuclear phagocytes that contribute to maintaining high levels of H. pylori loads in the stomach by modulating effector T cell responses at the gastric mucosa.

NLRX1 Regulates Effector and Metabolic Functions of CD4+ T Cells.

Nucleotide oligomerization domain-like receptor X1 (NLRX1) has been implicated in viral response, cancer progression, and inflammatory disorders; however, its role as a dual modulator of CD4+ T cell function and metabolism has not been defined. The loss of NLRX1 results in increased disease severity, populations of Th1 and Th17 cells, and inflammatory markers (IFN-γ, TNF-α, and IL-17) in mice with dextran sodium sulfate-induced colitis. To further characterize this phenotype, we used in vitro CD4+ T cell-differentiation assays and show that NLRX1-deficient T cells have a greater ability to differentiate into an inflammatory phenotype and possess greater proliferation rates. Further, NLRX1-/- cells have a decreased responsiveness to immune checkpoint pathways and greater rates of lactate dehydrogenase activity. When metabolic effects of the knockout are impaired, NLRX1-deficient cells do not display significant differences in differentiation or proliferation. To confirm the role of NLRX1 specifically in T cells, we used an adoptive-transfer model of colitis. Rag2-/- mice receiving NLRX1-/- naive or effector T cells experienced increased disease activity and effector T cell populations, whereas no differences were observed between groups receiving wild-type or NLRX1-/- regulatory T cells. Metabolic effects of NLRX1 deficiency are observed in a CD4-specific knockout of NLRX1 within a Citrobacter rodentium model of colitis. The aerobic glycolytic preference in NLRX1-/- effector T cells is combined with a decreased sensitivity to immunosuppressive checkpoint pathways to provide greater proliferative capabilities and an inflammatory phenotype bias leading to increased disease severity.

Nonclinical Toxicology and Toxicokinetic Profile of an Oral Lanthionine Synthetase C-Like 2 (LANCL2) Agonist, BT-11.

BT-11 is an orally active, gut-restricted investigational therapeutic targeting the lanthionine synthetase C-like 2 pathway with lead indications in ulcerative colitis (UC) and Crohn disease (CD), 2 manifestations of inflammatory bowel disease (IBD). In 5 mouse models of IBD, BT-11 is effective at oral doses of 8 mg/kg. BT-11 was also efficacious at nanomolar concentrations in primary human samples from patients with UC and CD. BT-11 was tested under Good Laboratory Practice conditions in 90-day repeat-dose general toxicity studies in rats and dogs, toxicokinetics, respiratory, cardiovascular and central nervous system safety pharmacology, and genotoxicity studies. Oral BT-11 did not cause any clinical signs of toxicity, biochemical or hematological changes, or macroscopic or microscopic changes to organs in 90-day repeat-dose toxicity studies in rats and dogs at doses up to 1,000 mg/kg/d. Oral BT-11 resulted in low systemic exposure in both rats (area under the curve exposure from t = 0 to t = 8 hours [AUC0-8] of 216 h × ng/mL) and dogs (650 h × ng/mL) and rapid clearance with an average half-life of 3 hours. BT-11 did not induce changes in respiratory function, electrocardiogram parameters, or behavior with single oral doses of 1,000 mg/kg/d. There was no evidence of mutagenic or genotoxic potential for BT-11 up to tested limit doses using an Ames test, chromosomal aberration assay in human peripheral blood lymphocytes, or micronucleus assay in rats. Therefore, nonclinical studies show BT-11 to be a safe and well-tolerated oral therapeutic with potential as a potent immunometabolic therapy for UC and CD with no-observed adverse effect level >1,000 mg/kg in in vivo studies.

Novel Screening Tool for Stroke Using Artificial Neural Network.

BACKGROUND AND PURPOSE: The timely diagnosis of stroke at the initial examination is extremely important given the disease morbidity and narrow time window for intervention. The goal of this study was to develop a supervised learning method to recognize acute cerebral ischemia (ACI) and differentiate that from stroke mimics in an emergency setting. METHODS: Consecutive patients presenting to the emergency department with stroke-like symptoms, within 4.5 hours of symptoms onset, in 2 tertiary care stroke centers were randomized for inclusion in the model. We developed an artificial neural network (ANN) model. The learning algorithm was based on backpropagation. To validate the model, we used a 10-fold cross-validation method. RESULTS: A total of 260 patients (equal number of stroke mimics and ACIs) were enrolled for the development and validation of our ANN model. Our analysis indicated that the average sensitivity and specificity of ANN for the diagnosis of ACI based on the 10-fold cross-validation analysis was 80.0% (95% confidence interval, 71.8-86.3) and 86.2% (95% confidence interval, 78.7-91.4), respectively. The median precision of ANN for the diagnosis of ACI was 92% (95% confidence interval, 88.7-95.3). CONCLUSIONS: Our results show that ANN can be an effective tool for the recognition of ACI and differentiation of ACI from stroke mimics at the initial examination.

Translating nutritional immunology into drug development for inflammatory bowel disease.

PURPOSE OF REVIEW: To highlight recent advances in the understanding of nutritional immunology and in the development of novel therapeutics for inflammatory bowel disease (IBD). RECENT FINDINGS: We highlight the variety of factors that contribute to the interaction of the immune system and nutrition including the microbiome and the nervous system stimulation of the gut. We describe the potential for therapeutic development in IBD. Further, we review the cellular metabolic effects on immune activation and promising therapeutic targets. Finally, we show how the progression of understanding the role of lanthionine synthetase C-like 2 has encompassed both nutritional and therapeutic advances and led to the development of novel oral small molecule therapeutics for IBD. SUMMARY: Nutritional immunology and drug development research centered around immunoregulatory pathways can provide safer and more effective drugs while accelerating the path to cures.

Bistability analyses of CD4+ T follicular helper and regulatory cells during Helicobacter pylori infection.

T follicular helper (Tfh) cells are a highly plastic subset of CD4+ T cells specialized in providing B cell help and promoting inflammatory and effector responses during infectious and immune-mediate diseases. Helicobacter pylori is the dominant member of the gastric microbiota and exerts both beneficial and harmful effects on the host. Chronic inflammation in the context of H. pylori has been linked to an upregulation in T helper (Th)1 and Th17 CD4+ T cell phenotypes, controlled in part by the cytokine, interleukin-21. This study investigates the differentiation and regulation of Tfh cells, major producers of IL-21, in the immune response to H. pylori challenge. To better understand the conditions influencing the promotion and inhibition of a chronically elevated Tfh population, we used top-down and bottom-up approaches to develop computational models of Tfh and T follicular regulatory (Tfr) cell differentiation. Stability analysis was used to characterize the presence of two bi-stable steady states in the calibrated Tfh/Tfr models. Stochastic simulation was used to illustrate the ability of the parameter set to dictate two distinct behavioral patterns. Furthermore, sensitivity analysis helped identify the importance of various parameters on the establishment of Tfh and Tfr cell populations. The core network model was expanded into a more comprehensive and predictive model by including cytokine production and signaling pathways. From the expanded network, the interaction between TGFB-Induced Factor Homeobox 1 (Tgif1) and the retinoid X receptor (RXR) was displayed to exert control over the determination of the Tfh response. Model simulations predict that Tgif1 and RXR respectively induce and curtail Tfh responses. This computational hypothesis was validated experimentally by assaying Tgif1, RXR and Tfh in stomachs of mice infected with H. pylori.

Exploratory Studies With BT-11: A Proposed Orally Active Therapeutic for Crohn's Disease.

Lanthionine synthetase cyclase-like receptor 2 (LANCL2) is a novel therapeutic target for Crohn's disease (CD). BT-11 is a small molecule that binds LANCL2, is orally active, and has demonstrated therapeutic efficacy in 3 validated mouse models of colitis at doses as low as 8 mg/kg/d. Exploratory experiments evaluated BT-11 in male Harlan Sprague Dawley rats with a single oral dose of 500 mg/kg and 80 mg/kg/d for 14 days (n = 10 rats dosed/group). Treated and control rats were observed for behavioral detriments, and blood and tissues were collected for clinical pathology and histopathological examination. A functional observational battery demonstrated no differences between treated and control groups over multiple times of observation for quantal, categorical, and continuous end points, including posture, in cage activity, approach, response to touch, weight, grip strength, body temperature, and time on a rotarod. Histopathological examination of the brain, kidney, liver, adrenal gland, testes, stomach, small and large intestines, duodenum, pancreas, heart, lungs, spleen, thymus, and rib found no significant differences between the groups. Plasma enzymes associated with liver function were transiently elevated 2 to 4 days after the 500 mg/kg single dose but returned to normal values by 8 days and were not observed at any time in rats given 80 mg/kg/d for 14 days. One hour after oral administration of a single dose of 80 mg/kg, BT-11 had a maximal concentration of 21 ng/mL; the half-life was 3 hours. These experimental results demonstrated that BT-11 is well tolerated in rats, and, with further testing, may hold promise as an orally active therapeutic for CD.

Multiscale modeling of mucosal immune responses.

BACKGROUND: Computational techniques are becoming increasingly powerful and modeling tools for biological systems are of greater needs. Biological systems are inherently multiscale, from molecules to tissues and from nano-seconds to a lifespan of several years or decades. ENISI MSM integrates multiple modeling technologies to understand immunological processes from signaling pathways within cells to lesion formation at the tissue level. This paper examines and summarizes the technical details of ENISI, from its initial version to its latest cutting-edge implementation. IMPLEMENTATION: Object-oriented programming approach is adopted to develop a suite of tools based on ENISI. Multiple modeling technologies are integrated to visualize tissues, cells as well as proteins; furthermore, performance matching between the scales is addressed. CONCLUSION: We used ENISI MSM for developing predictive multiscale models of the mucosal immune system during gut inflammation. Our modeling predictions dissect the mechanisms by which effector CD4+ T cell responses contribute to tissue damage in the gut mucosa following immune dysregulation.Computational modeling techniques are playing increasingly important roles in advancing a systems-level mechanistic understanding of biological processes. Computer simulations guide and underpin experimental and clinical efforts. This study presents ENteric Immune Simulator (ENISI), a multiscale modeling tool for modeling the mucosal immune responses. ENISI's modeling environment can simulate in silico experiments from molecular signaling pathways to tissue level events such as tissue lesion formation. ENISI's architecture integrates multiple modeling technologies including ABM (agent-based modeling), ODE (ordinary differential equations), SDE (stochastic modeling equations), and PDE (partial differential equations). This paper focuses on the implementation and developmental challenges of ENISI. A multiscale model of mucosal immune responses during colonic inflammation, including CD4+ T cell differentiation and tissue level cell-cell interactions was developed to illustrate the capabilities, power and scope of ENISI MSM.

Pharmacophore modeling improves virtual screening for novel peroxisome proliferator-activated receptor-gamma ligands.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a nuclear hormone receptor involved in regulating various metabolic and immune processes. The PPAR family of receptors possesses a large binding cavity that imparts promiscuity of ligand binding not common to other nuclear receptors. This feature increases the challenge of using computational methods to identify PPAR ligands that will dock favorably into a structural model. Utilizing both ligand- and structure-based pharmacophore methods, we sought to improve agonist prediction by grouping ligands according to pharmacophore features, and pairing models derived from these features with receptor structures for docking. For 22 of the 33 receptor structures evaluated we observed an increase in true positive rate (TPR) when screening was restricted to compounds sharing molecular features found in rosiglitazone. A combination of structure models used for docking resulted in a higher TPR (40 %) when compared to docking with a single structure model (<20 %). Prediction was also improved when specific protein-ligand interactions between the docked ligands and structure models were given greater weight than the calculated free energy of binding. A large-scale screen of compounds using a marketed drug database verified the predictive ability of the selected structure models. This study highlights the steps necessary to improve screening for PPARγ ligands using multiple structure models, ligand-based pharmacophore data, evaluation of protein-ligand interactions, and comparison of docking datasets. The unique combination of methods presented here holds potential for more efficient screening of compounds with unknown affinity for PPARγ that could serve as candidates for therapeutic development.

Preclinical studies of amixicile, a systemic therapeutic developed for treatment of Clostridium difficile infections that also shows efficacy against Helicobacter pylori.

Amixicile shows efficacy in the treatment of Clostridium difficile infections (CDI) in a mouse model, with no recurrence of CDI. Since amixicile selectively inhibits the action of a B vitamin (thiamine pyrophosphate) cofactor of pyruvate:ferredoxin oxidoreductase (PFOR), it may both escape mutation-based drug resistance and spare beneficial probiotic gut bacteria that do not express this enzyme. Amixicile is a water-soluble derivative of nitazoxanide (NTZ), an antiparasitic therapeutic that also shows efficacy against CDI in humans. In comparative studies, amixicile showed no toxicity to hepatocytes at 200 µM (NTZ was toxic above 10 µM); was not metabolized by human, dog, or rat liver microsomes; showed equivalence or superiority to NTZ in cytochrome P450 assays; and did not activate efflux pumps (breast cancer resistance protein, P glycoprotein). A maximum dose (300 mg/kg) of amixicile given by the oral or intraperitoneal route was well tolerated by mice and rats. Plasma exposure (rats) based on the area under the plasma concentration-time curve was 79.3 h · µg/ml (30 mg/kg dose) to 328 h · µg/ml (100 mg/kg dose), the maximum concentration of the drug in serum was 20 µg/ml, the time to the maximum concentration of the drug in serum was 0.5 to 1 h, and the half-life was 5.6 h. Amixicile did not concentrate in mouse feces or adversely affect gut populations of Bacteroides species, Firmicutes, segmented filamentous bacteria, or Lactobacillus species. Systemic bioavailability was demonstrated through eradication of Helicobacter pylori in a mouse infection model. In summary, the efficacy of amixicile in treating CDI and other infections, together with low toxicity, an absence of mutation-based drug resistance, and excellent drug metabolism and pharmacokinetic metrics, suggests a potential for broad application in the treatment of infections caused by PFOR-expressing microbial pathogens in addition to CDI.

Systems modeling of molecular mechanisms controlling cytokine-driven CD4+ T cell differentiation and phenotype plasticity.

Differentiation of CD4+ T cells into effector or regulatory phenotypes is tightly controlled by the cytokine milieu, complex intracellular signaling networks and numerous transcriptional regulators. We combined experimental approaches and computational modeling to investigate the mechanisms controlling differentiation and plasticity of CD4+ T cells in the gut of mice. Our computational model encompasses the major intracellular pathways involved in CD4+ T cell differentiation into T helper 1 (Th1), Th2, Th17 and induced regulatory T cells (iTreg). Our modeling efforts predicted a critical role for peroxisome proliferator-activated receptor gamma (PPARγ) in modulating plasticity between Th17 and iTreg cells. PPARγ regulates differentiation, activation and cytokine production, thereby controlling the induction of effector and regulatory responses, and is a promising therapeutic target for dysregulated immune responses and inflammation. Our modeling efforts predict that following PPARγ activation, Th17 cells undergo phenotype switch and become iTreg cells. This prediction was validated by results of adoptive transfer studies showing an increase of colonic iTreg and a decrease of Th17 cells in the gut mucosa of mice with colitis following pharmacological activation of PPARγ. Deletion of PPARγ in CD4+ T cells impaired mucosal iTreg and enhanced colitogenic Th17 responses in mice with CD4+ T cell-induced colitis. Thus, for the first time we provide novel molecular evidence in vivo demonstrating that PPARγ in addition to regulating CD4+ T cell differentiation also plays a major role controlling Th17 and iTreg plasticity in the gut mucosa.

Oral Omilancor Treatment Ameliorates Clostridioides difficile Infection During IBD Through Novel Immunoregulatory Mechanisms Mediated by LANCL2 Activation.

BACKGROUND: Clostridioides difficile infection (CDI) is an opportunistic infection of the gastrointestinal tract, commonly associated with antibiotic administration, that afflicts almost 500 000 people yearly only in the United States. CDI incidence and recurrence is increased in inflammatory bowel disease (IBD) patients. Omilancor is an oral, once daily, first-in-class, gut-restricted, immunoregulatory therapeutic in clinical development for the treatment of IBD. METHODS: Acute and recurrent murine models of CDI and the dextran sulfate sodium-induced concomitant model of IBD and CDI were utilized to determine the therapeutic efficacy of oral omilancor. To evaluate the protective effects against C. difficile toxins, in vitro studies with T84 cells were also conducted. 16S sequencing was employed to characterize microbiome composition. RESULTS: Activation of the LANCL2 pathway by oral omilancor and its downstream host immunoregulatory changes decreased disease severity and inflammation in the acute and recurrence models of CDI and the concomitant model of IBD/CDI. Immunologically, omilancor treatment increased mucosal regulatory T cell and decreased pathogenic T helper 17 cell responses. These immunological changes resulted in increased abundance and diversity of tolerogenic gut commensal bacterial strains in omilancor-treated mice. Oral omilancor also resulted in accelerated C. difficile clearance in an antimicrobial-free manner. Furthermore, omilancor provided protection from toxin damage, while preventing the metabolic burst observed in intoxicated epithelial cells. CONCLUSIONS: These data support the development of omilancor as a novel host-targeted, antimicrobial-free immunoregulatory therapeutic for the treatment of IBD patients with C. difficile-associated disease and pathology with the potential to address the unmet clinical needs of ulcerative colitis and Crohn's disease patients with concomitant CDI.

Omilancor is an oral, gut-restricted first-in-class immunoregulatory therapeutic for the treatment of inflammatory bowel disease (IBD). This study demonstrates for the first time that omilancor provides therapeutic efficacy in models of acute and recurrent Clostridioides difficile infection (CDI), and concomitant CDI and IBD, by increasing regulatory T cell function while suppressing effector responses, plus modulating gut microbiome composition and preserving epithelial barrier function.

Treating Autoimmune Diseases With LANCL2 Therapeutics: A Novel Immunoregulatory Mechanism for Patients With Ulcerative Colitis and Crohn's Disease.

Lanthionine synthetase C-like 2 (LANCL2) therapeutics have gained increasing recognition as a novel treatment modality for a wide range of autoimmune diseases. Genetic ablation of LANCL2 in mice results in severe inflammatory phenotypes in inflammatory bowel disease (IBD) and lupus. Pharmacological activation of LANCL2 provides therapeutic efficacy in mouse models of intestinal inflammation, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, and psoriasis. Mechanistically, LANCL2 activation enhances regulatory CD4 + T cell (Treg) responses and downregulates effector responses in the gut. The stability and suppressive capacities of Treg cells are enhanced by LANCL2 activation through engagement of immunoregulatory mechanisms that favor mitochondrial metabolism and amplify IL-2/CD25 signaling. Omilancor, the most advanced LANCL2 immunoregulatory therapeutic in late-stage clinical development, is a phase 3 ready, first-in-class, gut-restricted, oral, once-daily, small-molecule therapeutic in clinical development for the treatment of UC and CD. In this review, we discuss this novel mechanism of mucosal immunoregulation and how LANCL2-targeting therapeutics could help address the unmet clinical needs of patients with autoimmune diseases, starting with IBD.

Oral LANCL2 therapeutics are a safe and effective treatment modality for the long-term management of autoimmune diseases, including UC and CD, without causing systemic immunosuppression. This review discusses in detail the immunoregulatory mechanisms of action of LANCL2 therapeutics. More specifically, the article describes how omilancor, a first-in-class, oral, once daily, gut-restricted LANCL2 therapeutic could help address the unmet clinical needs of patients with IBD and other immune-mediated diseases.

Helicobacter pylori infection in a pig model is dominated by Th1 and cytotoxic CD8+ T cell responses.

Helicobacter pylori infection is the leading cause for peptic ulcer disease and gastric adenocarcinoma. Mucosal T cell responses play an important role in mediating H. pylori-related gastric immunopathology. While induced regulatory T (iTreg) cells are required for chronic colonization without disease, T helper 1 (Th1) effector responses are associated with lower bacterial loads at the expense of gastric pathology. Pigs were inoculated with either H. pylori strain SS1 or J99. Phenotypic and functional changes in peripheral blood mononuclear cell (PBMC) populations were monitored weekly, and mucosal immune responses and bacterial loads were assessed up to 2 months postinfection. Both H. pylori strains elicited a Th1 response characterized by increased percentages of CD4(+)Tbet(+) cells and elevated gamma interferon (IFN-γ) mRNA in PBMCs. A subset of CD8(+) T cells expressing Tbet and CD16 increased following infection. Moreover, a significant increase in perforin and granzyme mRNA expression was observed in PBMCs of infected pigs, indicating a predominant cytotoxic immune response. Infiltration of B cells, myeloid cells, T cells expressing Treg- and Th17-associated transcription factors, and cytotoxic T cells was found in the gastric lamina propria of both infected groups. Interestingly, based on bacterial reisolation data, strain SS1 showed greater capacity to colonize and/or persist in the gastric mucosa than did strain J99. This novel pig model of infection closely mimics human gastric pathology and presents a suitable avenue for studying effector and regulatory responses toward H. pylori described in humans.

Mutations to essential orphan response regulator HP1043 of Helicobacter pylori result in growth-stage regulatory defects.

Helicobacter pylori establishes lifelong infections of the gastric mucosa, a niche considered hostile to most microbes. While responses to gastric acidity and local inflammation are understood, little is known as to how they are integrated into homeostatic control of cell division and growth-stage gene expression. Here we investigate the essential orphan response regulator HP1043, a member of the OmpR/PhoB subfamily of transcriptional regulators that is unique to the Epsilonproteobacteria and that lacks phosphorylation domains. To test the hypothesis that conformational changes in the homodimer might lead to defects in gene expression, we sought mutations that might alter DNA-binding efficiency. Two introduced mutations (C215S, C221S) C terminal to the DNA-binding domain of HP1043 (HP1043CC11) resulted in a 2-fold higher affinity for its own promoter by footprinting. Modeling studies with the crystal structure of HP1043 suggested that C215S might affect the helix-turn-helix domain. Genomic replacement of the hp1043 allele with the hp1043CC11 mutant allele resulted in a 2-fold decrease in protein levels, despite a dramatic increase in mRNA. The mutations did not affect in vitro growth rates or colonization efficiency in a mouse model. Proteomic profiling (CC11 mutant strain versus wild type) identified many expression differences, and quantitative PCR further revealed that 11 out of 12 examined genes had lost growth-stage regulation and that 6 of the genes contained HP1043 binding consensus sequences within the promoter regions (fur, cagA, cag23, flhA, flip, and napA). Our studies show that mutations that affect DNA-binding affinity can be used to identify new members of the HP1043 regulon.