Clostridioides difficile Binary Toxin Binding Component Increases Virulence in a Hamster Model.

Background: Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods: To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results: Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions: Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection.

Dupilumab Use Is Associated With Protection From Coronavirus Disease 2019 Mortality: A Retrospective Analysis.

We previously found that type 2 immunity promotes coronavirus disease 2019 (COVID-19) pathogenesis in a mouse model. To test relevance to human disease, we used electronic health record databases and determined that patients on dupilumab (anti-interleukin [IL]-4R monoclonal antibody that blocks IL-13 and IL-4 signaling) at the time of COVID-19 infection had lower mortality.

Safety and Efficacy of Dupilumab for the Treatment of Hospitalized Patients With Moderate to Severe Coronavirus Disease 2019: A Phase 2a Trial.

Background: Based on studies implicating the type 2 cytokine interleukin 13 (IL-13) as a potential contributor to critical coronavirus disease 2019 (COVID-19), this trial was designed as an early phase 2 study to assess dupilumab, a monoclonal antibody that blocks IL-13 and interleukin 4 signaling, for treatment of inpatients with COVID-19. Methods: We conducted a phase 2a randomized, double-blind, placebo-controlled trial (NCT04920916) to assess the safety and efficacy of dupilumab plus standard of care vs placebo plus standard of care in mitigating respiratory failure and death in those hospitalized with COVID-19. Results: Forty eligible subjects were enrolled from June to November of 2021. There was no statistically significant difference in adverse events nor in the primary endpoint of ventilator-free survival at day 28 between study arms. However, for the secondary endpoint of mortality at day 60, there were 2 deaths in the dupilumab group compared with 5 deaths in the placebo group (60-day survival: 89.5% vs 76.2%; adjusted hazard ratio [HR], 0.05 [95% confidence interval {CI}, .004-.72]; P = .03). Among subjects who were not in the intensive care unit (ICU) at randomization, 3 subjects in the dupilumab arm were admitted to the ICU compared to 6 in the placebo arm (17.7% vs 37.5%; adjusted HR, 0.44 [95% CI, .09-2.09]; P = .30). Last, we found evidence of type 2 signaling blockade in the dupilumab group through analysis of immune biomarkers over time. Conclusions: Although the primary outcome of day 28 ventilator-free survival was not reached, adverse events were not observed and survival was higher in the dupilumab group by day 60. Clinical Trials Registration: NCT04920916.

Safety and Efficacy of Dupilumab for the Treatment of Hospitalized Patients with Moderate to Severe COVID 19: A Phase IIa Trial.

Background: A profound need remains to develop further therapeutics for treatment of those hospitalized with COVID-19. Based on data implicating the type 2 cytokine interleukin (IL)-13 as a significant factor leading to critical COVID-19, this trial was designed to assess dupilumab, a monoclonal antibody that blocks IL-13 and IL-4 signaling, for treatment of inpatients with COVID-19. Methods: We conducted a phase IIa randomized double-blind placebo-controlled trial to assess the safety and efficacy of dupilumab plus standard of care versus placebo plus standard of care in mitigating respiratory failure and death in those hospitalized with COVID-19. Subjects were followed prospectively for 60 days. The primary endpoint was the proportion of patients alive and free of invasive mechanical ventilation at 28 days. Findings: Forty eligible subjects were enrolled from June to November of 2021. There was no difference in adverse events nor in ventilator free survival at day 28 between study arms. However, for the secondary endpoint of mortality at day 60, subjects randomized to dupilumab had a higher survival rate compared to the placebo group (89.5% vs 76.2%, adjusted HR 0.05, 95% CI: 0.0-0.72, p=0.03). There were fewer subjects admitted to the ICU in the dupilumab group compared to placebo (33.3% vs 66.7%; adjusted HR 0.44, 95% CI: 0.09-2.09, p=0.30). Lastly, we saw downstream evidence of IL-4 and IL-13 signaling blockade in the dupilumab group through analysis of immune biomarkers over time. Interpretation: Dupilumab was well tolerated and improved 60-day survival in patients hospitalized with moderate to severe COVID-19. Trial Registration: This trial is registered with ClinicalTrials.gov, NCT04920916 . Funding: Virginia Biosciences Health Research Corporation, PBM C19, Henske Family Foundation, National Institutes of Health, National Cancer Institute.

The IL-33-ILC2 pathway protects from amebic colitis.

Entamoeba histolytica is a pathogenic protozoan parasite that causes intestinal colitis, diarrhea, and in some cases, liver abscess. Through transcriptomics analysis, we observed that E. histolytica infection was associated with increased expression of IL-33 mRNA in both the human and murine colon. IL-33, the IL-1 family cytokine, is released after cell injury to alert the immune system of tissue damage. Treatment with recombinant IL-33 protected mice from amebic infection and intestinal tissue damage; moreover, blocking IL-33 signaling made mice more susceptible to amebiasis. IL-33 limited the recruitment of inflammatory immune cells and decreased the pro-inflammatory cytokine IL-6 in the cecum. Type 2 immune responses were upregulated by IL-33 treatment during amebic infection. Interestingly, administration of IL-33 protected RAG2-/- mice but not RAG2-/-γc-/- mice, demonstrating that IL-33-mediated protection required the presence of innate lymphoid cells (ILCs). IL-33 induced recruitment of ILC2 but not ILC1 and ILC3 in RAG2-/- mice. At baseline and after amebic infection, there was a significantly higher IL13+ILC2s in C57BL/J mice, which are naturally resistant to amebiasis, than CBA/J mice. Adoptive transfer of ILC2s to RAG2-/-γc-/- mice restored IL-33-mediated protection. These data reveal that the IL-33-ILC2 pathway is an important host defense mechanism against amebic colitis.

IL-13 is a driver of COVID-19 severity.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here, we report that elevated IL-13 was associated with the need for mechanical ventilation in 2 independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab, a mAb that blocks IL-13 and IL-4 signaling, had less severe disease. In SARS-CoV-2-infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, hyaluronan synthase 1 (Has1) was the most downregulated gene, and accumulation of the hyaluronan (HA) polysaccharide was decreased in the lung. In patients with COVID-19, HA was increased in the lungs and plasma. Blockade of the HA receptor, CD44, reduced mortality in infected mice, supporting the importance of HA as a pathogenic mediator. Finally, HA was directly induced in the lungs of mice by administration of IL-13, indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and HA has important implications for therapy of COVID-19 and, potentially, other pulmonary diseases. IL-13 levels were elevated in patients with severe COVID-19. In a mouse model of the disease, IL-13 neutralization reduced the disease and decreased lung HA deposition. Administration of IL-13-induced HA in the lung. Blockade of the HA receptor CD44 prevented mortality, highlighting a potentially novel mechanism for IL-13-mediated HA synthesis in pulmonary pathology.

IL-13 is a driver of COVID-19 severity.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here we report that elevated interleukin-13 (IL-13) was associated with the need for mechanical ventilation in two independent patient cohorts. In addition, patients who acquired COVID-19 while prescribed Dupilumab had less severe disease. In SARS-CoV-2 infected mice, IL-13 neutralization reduced death and disease severity without affecting viral load, demonstrating an immunopathogenic role for this cytokine. Following anti-IL-13 treatment in infected mice, in the lung, hyaluronan synthase 1 (Has1) was the most downregulated gene and hyaluronan accumulation was decreased. Blockade of the hyaluronan receptor, CD44, reduced mortality in infected mice, supporting the importance of hyaluronan as a pathogenic mediator, and indicating a new role for IL-13 in lung disease. Understanding the role of IL-13 and hyaluronan has important implications for therapy of COVID-19 and potentially other pulmonary diseases.

Type 2 cytokines IL-4 and IL-5 reduce severe outcomes from Clostridiodes difficile infection.

Clostridiodes difficile infection (CDI) is the leading cause of hospital-acquired gastrointestinal infections in the U.S. While the immune response to C. difficile is not well understood, it has been shown that severe disease is accompanied by high levels of infiltrating immune cells and pro-inflammatory cytokine production. This study tests the roles of two type 2 cytokines, IL-4 and IL-5, in mediating protection in a murine model of disease. Administration of IL-5 protected from mortality due to CDI, and both IL-4 and IL-5 were protective against severe disease symptoms. Together, the results from this study increase our understanding of how type 2 immune signaling processes are protective from severe C. difficile infection.

Evaluation of K18-hACE2 Mice as a Model of SARS-CoV-2 Infection.

Murine models of SARS-CoV-2 infection are critical for elucidating the biological pathways underlying COVID-19. Because human angiotensin-converting enzyme 2 (ACE2) is the receptor for SARS-CoV-2, mice expressing the human ACE2 gene have shown promise as a potential model for COVID-19. Five mice from the transgenic mouse strain K18-hACE2 were intranasally inoculated with SARS-CoV-2 Hong Kong/VM20001061/2020. Mice were followed twice daily for 5 days and scored for weight loss and clinical symptoms. Infected mice did not exhibit any signs of infection until day 4, when no other obvious clinical symptoms other than weight loss were observed. By day 5, all infected mice had lost around 10% of their original body weight but exhibited variable clinical symptoms. All infected mice showed high viral titers in the lungs as well as altered lung histology associated with proteinaceous debris in the alveolar space, interstitial inflammatory cell infiltration, and alveolar septal thickening. Overall, these results show that the K18-hACE2 transgenic background can be used to establish symptomatic SARS-CoV-2 infection and can be a useful mouse model for COVID-19.

The Inflammasome and Type-2 Immunity in Clostridium difficile Infection.

Clostridium difficile (reclassified as " Clostridioides ") is the leading cause of hospital-acquired infections in the United States, and is associated with high-patient mortality and high rates of recurrence. Inflammasome priming and activation by the bacterial toxins, TcdA , TcdB , and C. difficile transferase (CDT), initiates a potent immune response that is characterized by interleukin- (IL) 8, IL-1ß, and neutrophil recruitment, and is required for pathogen killing. However, it is becoming clearer that a strong inflammatory response during C. difficile infection can result in host tissue damage, and is associated with worse patient outcome. Recent work has begun to show that a type-2 immune response, most often associated with helminth infections, allergy, and asthma, may be protective during C. difficile infection. While the mechanisms through how this response protect are still unclear, there is evidence that it is mediated through eosinophil activity. This chapter will review the immune response to C. difficile, how the inflammasome signaling during infection can deleterious to the host, as well as the current understanding of a protective type-2 immunity. Understanding the host immune response may help to provide insight into novel approaches to prognosis markers, as well as how treat patient C. difficile infection without, or in addition to, antibiotics.