Monoclonal antibody-mediated neutralization of Clostridioides difficile toxin does not diminish induction of the protective innate immune response to infection.

Clostridioides difficile infection causes pathology that ranges in severity from diarrhea to pseudomembranous colitis. Toxin A and Toxin B are the two primary virulence factors secreted by C. difficile that drive disease severity. The toxins damage intestinal epithelial cells leading to a loss of barrier integrity and induction of a proinflammatory host response. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection. However, the impact of toxin neutralization on the induction and quality of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAbs therapy. At day 2 post-infection, C. difficile-infected, mAbs-treated mice had significantly less disease compared to isotype-treated mice despite remaining colonized with C. difficile. C. difficile-infected mAbs-treated mice still exhibited marked neutrophil infiltration and induction of a subset of proinflammatory cytokines within the intestinal lamina propria following infection that is comparable to isotype-treated mice. Furthermore, both mAbs and isotype-treated mice had an increase in IL-22-producing ILCs in the intestine following infection. MAbs-treated mice exhibited increased infiltration of eosinophils in the intestinal lamina propria, which has been previously reported to promote a protective host response following C. difficile infection. These findings show that activation of host protective mechanisms remain intact in the context of monoclonal antibody-mediated toxin neutralization.

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and development.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

IL-2 protects lupus-prone mice from multiple end-organ damage by limiting CD4-CD8- IL-17-producing T cells.

IL-2, a cytokine with pleiotropic effects, is critical for immune cell activation and peripheral tolerance. Although the therapeutic potential of IL-2 has been previously suggested in autoimmune diseases, the mechanisms whereby IL-2 mitigates autoimmunity and prevents organ damage remain unclear. Using an inducible recombinant adeno-associated virus vector, we investigated the effect of low systemic levels of IL-2 in lupus-prone MRL/Fas(lpr/lpr) (MRL/lpr) mice. Treatment of mice after the onset of disease with IL-2-recombinant adeno-associated virus resulted in reduced mononuclear cell infiltration and pathology of various tissues, including skin, lungs, and kidneys. In parallel, we noted a significant decrease of IL-17-producing CD3(+)CD4(-)CD8(-) double-negative T cells and an increase in CD4(+)CD25(+)Foxp3(+) immunoregulatory T cells (Treg) in the periphery. We also show that IL-2 can drive double-negative (DN) T cell death through an indirect mechanism. Notably, targeted delivery of IL-2 to CD122(+) cytotoxic lymphocytes effectively reduced the number of DN T cells and lymphadenopathy, whereas selective expansion of Treg by IL-2 had no effect on DN T cells. Collectively, our data suggest that administration of IL-2 to lupus-prone mice protects against end-organ damage and suppresses inflammation by dually limiting IL-17-producing DN T cells and expanding Treg.

Complement receptor of the immunoglobulin superfamily reduces murine lupus nephritis and cutaneous disease.

Complement activation takes place in autoimmune diseases and accounts for tissue inflammation. Previously, complement inhibition has been considered for the treatment of SLE. Complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the alternative pathway of complement and a soluble form reverses established inflammation and bone destruction in experimental autoimmune arthritis. We asked whether specific inhibition of the alternative pathway could inhibit autoimmunity and/or organ damage in lupus-prone mice. Accordingly, we treated lupus-prone MRL/lpr mice with a soluble form of CRIg (CRIg-Fc) and we found that it significantly diminished skin lesions, proteinuria and pyuria, and kidney pathology. Interestingly, serum levels of anti-DNA antibodies were not affected despite the fact that serum complement 3 (C3) levels increased significantly. Immunofluorescent staining of kidney tissues revealed a reduction in staining intensity for C3, IgG, and the macrophage marker Mac-2. Thus our data show that inhibition of the alternative pathway of complement controls skin and kidney inflammation even in the absence of an effect on the production of autoantibodies. We propose that CRIg should be considered for clinical trials in patients with systemic lupus erythematosus.

Orally available nucleoside analog UMM-766 provides protection in a murine model of orthopox disease.

Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.

Complement depletion protects lupus-prone mice from ischemia-reperfusion-initiated organ injury.

Ischemia-reperfusion (IR) injury causes a vigorous immune response that is amplified by complement activation, leading to local and remote tissue damage. Using MRL/lpr mice, which are known to experience accelerated tissue damage after mesenteric IR injury, we sought to evaluate whether complement inhibition mitigates organ damage. We found that complement depletion with cobra venom factor protected mice from local and remote lung tissue damage. Protection from injury was associated with less complement (C3) and membrane attack complex deposition, less neutrophil infiltration, and lower levels of local proinflammatory cytokine production. In addition, complement depletion was able to decrease the level of oxidative stress as measured by glutathione peroxidase 1 mRNA levels and superoxide dismutase activity. Furthermore, blockage of C5a receptor protected MRL/lpr mice from local tissue damage, but not from remote lung tissue damage. In conclusion, although treatments with cobra venom factor and C5a receptor antagonist were able to protect mice from local tissue damage, treatment with C5a receptor antagonist was not able to protect mice from remote lung tissue damage, implying that more factors contribute to the development of remote tissue damage after IR injury. These data also suggest that complement inhibition at earlier, rather than late, stages can have clinical benefit in conditions that are complicated with IR injury.

Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

Outer membrane vesicles: A bacterial-derived vaccination system.

Outer membrane vesicles (OMVs) are non-living spherical nanostructures that derive from the cell envelope of Gram-negative bacteria. OMVs are important in bacterial pathogenesis, cell-to-cell communication, horizontal gene transfer, quorum sensing, and in maintaining bacterial fitness. These structures can be modified to express antigens of interest using glycoengineering and genetic or chemical modification. The resulting OMVs can be used to immunize individuals against the expressed homo- or heterologous antigens. Additionally, cargo can be loaded into OMVs and they could be used as a drug delivery system. OMVs are inherently immunogenic due to proteins and glycans found on Gram negative bacterial outer membranes. This review focuses on OMV manipulation to increase vesiculation and decrease antigenicity, their utility as vaccines, and novel engineering approaches to extend their application.

Assessing donor-to-donor variability in human intestinal organoid cultures.

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high-confidence data using human primary cultures through thorough characterization of variability.

The IL-2 defect in systemic lupus erythematosus disease has an expansive effect on host immunity.

IL-2 production is decreased in systemic lupus erythematosus (SLE) patients and affects T cell function and other aspects of host immunity. Transcription factors regulating IL-2 production behave aberrantly in SLE T cells. In addition to IL-2 dysregulation, other IL-2 family members (IL-15 and IL-21) are abnormally expressed in SLE. Decreased IL-2 production in SLE patients leads to many immune defects such as decreased T(reg) production, decreased activation-induced cell death (AICD), and decreased cytotoxicity. IL-2 deficiency results in systemic dysregulation of host immune responses in patients suffering from SLE disease.

Colorectal cancer-associated anaerobic bacteria proliferate in tumor spheroids and alter the microenvironment.

Recent reports show that colorectal tumors contain microbiota that are distinct from those that reside in a 'normal' colon environment, and that these microbiota can contribute to cancer progression. Fusobacterium nucleatum is the most commonly observed species in the colorectal tumor microenvironment and reportedly influences disease progression through numerous mechanisms. However, a detailed understanding of the role of this organism in cancer progression is limited, in part due to challenges in maintaining F. nucleatum viability under standard aerobic cell culture conditions. Herein we describe the development of a 3-dimensional (3D) tumor spheroid model that can harbor and promote the growth of anaerobic bacteria. Bacteria-tumor cell interactions and metabolic crosstalk were extensively studied by measuring the kinetics of bacterial growth, cell morphology and lysis, cancer-related gene expression, and metabolomics. We observed that viable F. nucleatum assembles biofilm-like structures in the tumor spheroid microenvironment, whereas heat-killed F. nucleatum is internalized and sequestered in the cancer cells. Lastly, we use the model to co-culture 28 Fusobacterium clinical isolates and demonstrate that the model successfully supports co-culture with diverse fusobacterial species. This bacteria-spheroid co-culture model enables mechanistic investigation of the role of anaerobic bacteria in the tumor microenvironment.

A small-molecule screen identifies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection.

We developed a screening procedure to identify small-molecule compounds that altered infection by Listeria monocytogenes to gain insights into bacterial/host cellular processes required for intracellular pathogenesis. A small-molecule library of 480 compounds with known biological functions was screened, and 21 compounds that altered the L. monocytogenes infection of murine bone marrow-derived macrophages (BMM) were identified. The identified compounds affected various cellular functions, such as actin polymerization, kinase/phosphatase activity, calcium signaling, and apoptosis. Pimozide, an FDA-approved drug used to treat severe Tourette's syndrome and schizophrenia, was further examined and shown to decrease the bacterial uptake and vacuole escape of L. monocytogenes in BMM. The inhibitory effect of pimozide on internalization was not specific for L. monocytogenes, as the phagocytosis of other bacterial species (Bacillus subtilis, Salmonella enterica serovar Typhimurium, and Escherichia coli K12) was significantly inhibited in the presence of pimozide. The invasion and cell-to-cell spread of L. monocytogenes during the infection of nonprofessional phagocytic cells also was decreased by pimozide treatment. Although pimozide has been reported to be an antagonist of mammalian cell calcium channels, the infection of BMM in a calcium-free medium did not relieve the inhibitory effects of pimozide on L. monocytogenes infection. Our results provide a generalizable screening approach for identifying small-molecule compounds that affect cellular pathways that are required for intracellular bacterial pathogenesis. We also have identified pimozide, a clinically approved antipsychotic drug, as a compound that may be suitable for further development as a therapeutic for intracellular bacterial infections.

Microphysiological lung models to evaluate the safety of new pharmaceutical modalities: a biopharmaceutical perspective.

The lung is a complex organ; it is both the initial barrier for inhaled agents and the site of metabolism and therapeutic effect for a subset of systemically administered drugs. Comprised of more than 40 cell types that are responsible for various important functions, the lung's complexity contributes to the subsequent challenges in developing complex in vitro co-culture models (also called microphysiological systems (MPS), complex in vitro models or organs-on-a-chip). Although there are multiple considerations and limitations in the development and qualification of such in vitro systems, MPS exhibit great promise in the fields of pharmacology and toxicology. Successful development and implementation of MPS models may enable mechanistic bridging between non-clinical species and humans, and increase clinical relevance of safety endpoints, while decreasing overall animal use. This article summarizes, from a biopharmaceutical industry perspective, essential elements for the development and qualification of lung MPS models. Its purpose is to guide MPS developers and manufacturers to expedite MPS utilization for safety assessment in the biopharmaceutical industry.

CD62L is not a reliable biomarker for predicting PML risk in natalizumab-treated R-MS patients.

OBJECTIVE: To assess if the percentage of CD3(+)CD4(+)CD62L(+) cells in cryopreserved peripheral blood mononuclear cells (PBMCs) (here termed %CD62L) can predict risk of developing progressive multifocal leukoencephalopathy (PML) and better inform the physician for benefit-risk assessment of natalizumab treatment decisions in a global setting. METHODS: Cryopreserved PBMCs from 21 natalizumab-treated patients who developed PML and 104 matched natalizumab-treated patients with multiple sclerosis (MS) without PML collected as a part of Biogen clinical trials were retrospectively examined for CD3, CD4, CCR7, CD45RA, and CD62L by flow cytometry. RESULTS: In this cohort, %CD62L in natalizumab-treated patients did not predict PML risk. Natalizumab-treated patients with MS without PML showed highly variable %CD62L upon serial sampling. In the STRATA study, the distribution of %CD62L in samples collected more than 6 months before a PML diagnosis, at diagnosis, and in natalizumab-treated patients without PML overlapped. No statistical threshold for risk could be determined. In addition, we demonstrated that lymphocyte viability strongly affects %CD62L, supporting previous reports that %CD62L is inherently unstable following cryopreservation and is sensitive to sample collection. CONCLUSION: Data from this well-controlled cohort of natalizumab-treated patients indicate that %CD62L is not a biomarker of PML risk.

Regulatory pathways involved in the infection-induced production of IFN-gamma by NK cells.

The production of interferon gamma (IFN-gamma) by natural killer (NK) cells provides an innate mechanism of resistance to many intracellular pathogens. These events are regulated by multiple cytokines and transcription factors which have both positive and negative effects. This article reviews the role of cytokines, as well as costimulatory and signaling pathways, involved in NK cell responses associated with resistance to infection.

Costimulation in resistance to infection and development of immune pathology: lessons from toxoplasma.

Parasitic infections remain an important cause of disease worldwide, and it is important to understand how the immune system protects against these organisms. In addition, the study of how the immune system deals with different types of pathogens provides the opportunity to discern how innate and adaptive arms of the immune system interact to provide an integrated protective response. Costimulatory signals are an important element involved in the control of lymphocyte response, and this laboratory studies the role of the costimulatory molecules CD28 and ICOS in the events that lead to resistance to the opportunistic pathogen Toxoplasma gondii as well as the development of immune pathology associated with this infection.

The role of cytokines and their signaling pathways in the regulation of immunity to Toxoplasma gondii.

The development of a strong cellular immune response is critical for the control of the intracellular pathogen Toxoplasma gondii. This occurs by activation of a complex, integrated immune response, which utilizes cells of the innate and adaptive immune systems. In the last two decades there have been major advances in our understanding of the role of cytokines in the initiation and maintenance of protective immunity to T. gondii, and IFN-gamma has been identified as the major mediator of resistance to this pathogen. This article provides an overview of the biology of toxoplasmosis and focuses on the pivotal role of cytokines and their signaling pathways during infection. It also addresses the role of cytokines in modulating other immune functions that are critical in determining the balance between a protective and a pathological immune response.

Lupus-prone mice fail to raise antigen-specific T cell responses to intracellular infection.

Systemic lupus erythematosus (SLE) is characterized by multiple cellular abnormalities culminating in the production of autoantibodies and immune complexes, resulting in tissue inflammation and organ damage. Besides active disease, the main cause of morbidity and mortality in SLE patients is infections, including those from opportunistic pathogens. To understand the failure of the immune system to fend off infections in systemic autoimmunity, we infected the lupus-prone murine strains B6.lpr and BXSB with the intracellular parasite Toxoplasma gondii and survival was monitored. Furthermore, mice were sacrificed days post infection and parasite burden and cellular immune responses such as cytokine production and cell activation were assessed. Mice from both strains succumbed to infection acutely and we observed greater susceptibility to infection in older mice. Increased parasite burden and a defective antigen-specific IFN-gamma response were observed in the lupus-prone mice. Furthermore, T cell:dendritic cell co-cultures established the presence of an intrinsic T cell defect responsible for the decreased antigen-specific response. An antigen-specific defect in IFN- gamma production prevents lupus-prone mice from clearing infection effectively. This study reveals the first cellular insight into the origin of increased susceptibility to infections in SLE disease and may guide therapeutic approaches.

CaMK4-dependent activation of AKT/mTOR and CREM-α underlies autoimmunity-associated Th17 imbalance.

Tissue inflammation in several autoimmune diseases, including SLE and MS, has been linked to an imbalance of IL-17-producing Th (Th17) cells and Tregs; however, the factors that promote Th17-driven autoimmunity are unclear. Here, we present evidence that the calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased and required during Th17 cell differentiation. Isolation of naive T cells from a murine model of lupus revealed increased levels of CaMK4 following stimulation with Th17-inducing cytokines but not following Treg, Th1, or Th2 induction. Furthermore, naive T cells from mice lacking CaMK4 did not produce IL-17. Genetic or pharmacologic inhibition of CaMK4 decreased the frequency of IL-17-producing T cells and ameliorated EAE and lupus-like disease in murine models. Inhibition of CaMK4 reduced Il17 transcription through decreased activation of the cAMP response element modulator α (CREM-α) and reduced activation of the AKT/mTOR pathway, which is known to enhance Th17 differentiation. Importantly, silencing CaMK4 in T cells from patients with SLE and healthy individuals inhibited Th17 differentiation through reduction of IL17A and IL17F mRNA. Collectively, our results suggest that CaMK4 inhibition has potential as a therapeutic strategy for Th17-driven autoimmune diseases.

KN-93, an inhibitor of calcium/calmodulin-dependent protein kinase IV, promotes generation and function of Foxp3⁺ regulatory T cells in MRL/lpr mice.

OBJECTIVE: Foxp3(+) regulatory T cells (Treg) are pivotal for the maintenance of peripheral tolerance and prevent development of autoimmune diseases. We have reported that calcium/calmodulin-dependent protein kinase IV (CaMK4) deficient MRL/lpr mice display less disease activity by promoting IL-2 production and increasing the activity of Treg cells. To further define the mechanism of CaMK4 on Treg cells in systemic lupus erythematosus (SLE), we used the Foxp3-GFP reporter mice and treated them with KN-93, an inhibitor of CaMK4. METHODS: We generated MRL/lpr Foxp3-GFP mice to record Treg cells; stimulated naïve CD4(+) T cells from MRL/lpr Foxp3-GFP mice under Treg polarizing conditions in the absence or presence of KN-93; evaluated the number of GFP positive cells in lymphoid organs and examined skin and kidney pathology at 16 weeks of age. We also examined the infiltration of cells and recruitment of Treg cells in the kidney. RESULTS: We show that culture of MRL/lpr Foxp3-GFP T cells in the presence of KN-93 promotes Treg differentiation in a dose-dependent manner. Treatment of MRL/lpr Foxp3-GFP mice with KN-93 results in a significant induction of Treg cells in the spleen, peripheral lymph nodes and peripheral blood and this is accompanied by decreased skin and kidney damage. Notably, KN-93 clearly diminishes the accumulation of inflammatory cells along with reciprocally increased Treg cells in target organ. CONCLUSION: Our results indicate that KN-93 treatment enhances the generation of Treg cells in vitro and in vivo highlighting its potential therapeutic use for the treatment of human autoimmune diseases.