Intestinal Epithelial Co-Culture Sensitivity to Pro-Inflammatory Stimuli and Polyphenols Is Medium-Independent.

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1ß (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

Gold nanoparticle distribution in advanced in vitro and ex vivo human placental barrier models.

BACKGROUND: Gold nanoparticles (AuNPs) are promising candidates to design the next generation NP-based drug formulations specifically treating maternal, fetal or placental complications with reduced side effects. Profound knowledge on AuNP distribution and effects at the human placental barrier in dependence on the particle properties and surface modifications, however, is currently lacking. Moreover, the predictive value of human placental transfer models for NP translocation studies is not yet clearly understood, in particular with regards to differences between static and dynamic exposures. To understand if small (3-4 nm) AuNPs with different surface modifications (PEGylated versus carboxylated) are taken up and cross the human placental barrier, we performed translocation studies in a static human in vitro co-culture placenta model and the dynamic human ex vivo placental perfusion model. The samples were analysed using ICP-MS, laser ablation-ICP-MS and TEM analysis for sensitive, label-free detection of AuNPs. RESULTS: After 24 h of exposure, both AuNP types crossed the human placental barrier in vitro, although in low amounts. Even though cellular uptake was higher for carboxylated AuNPs, translocation was slightly increased for PEGylated AuNPs. After 6 h of perfusion, only PEGylated AuNPs were observed in the fetal circulation and tissue accumulation was similar for both AuNP types. While PEGylated AuNPs were highly stable in the biological media and provided consistent results among the two placenta models, carboxylated AuNPs agglomerated and adhered to the perfusion device, resulting in different cellular doses under static and dynamic exposure conditions. CONCLUSIONS: Gold nanoparticles cross the human placental barrier in limited amounts and accumulate in placental tissue, depending on their size- and/or surface modification. However, it is challenging to identify the contribution of individual characteristics since they often affect colloidal particle stability, resulting in different biological interaction in particular under static versus dynamic conditions. This study highlights that human ex vivo and in vitro placenta models can provide valuable mechanistic insights on NP uptake and translocation if accounting for NP stability and non-specific interactions with the test system.

Influence of epidermal growth factor (EGF) and hydrocortisone on the co-culture of mature adipocytes and endothelial cells for vascularized adipose tissue engineering.

The composition of vascularized adipose tissue is still an ongoing challenge as no culture medium is available to supply adipocytes and endothelial cells appropriately. Endothelial cell medium is typically supplemented with epidermal growth factor (EGF) as well as hydrocortisone (HC). The effect of EGF on adipocytes is discussed controversially. Some studies say it inhibits adipocyte differentiation while others reported of improved adipocyte lipogenesis. HC is known to have lipolytic activities, which might result in mature adipocyte dedifferentiation. In this study, we evaluated the influence of EGF and HC on the co-culture of endothelial cells and mature adipocytes regarding their cell morphology and functionality. We showed in mono-culture that high levels of HC promoted dedifferentiation and proliferation of mature adipocytes, whereas EGF seemed to have no negative influence. Endothelial cells kept their typical cobblestone morphology and showed a proliferation rate comparable to the control independent of EGF and HC concentration. In co-culture, HC promoted dedifferentiation of mature adipocytes, which was shown by a higher glycerol release. EGF had no negative impact on adipocyte morphology. No negative impact on endothelial cell morphology and functionality could be seen with reduced EGF and HC supplementation in co-culture with mature adipocytes. Taken together, our results demonstrate that reduced levels of HC are needed for co-culturing mature adipocytes and endothelial cells. In co-culture, EGF had no influence on mature adipocytes. Therefore, for the composition of vascularized adipose tissue constructs, the media with low levels of HC and high or low levels of EGF can be used.

In vitro co-culture models for the assessment of orthopedic antibacterial biomaterials.

The antibacterial biofunctionality of bone implants is essential for the prevention and treatment of implant-associated infections (IAI). In vitro co-culture models are utilized to assess this and study bacteria-host cell interactions at the implant interface, aiding our understanding of biomaterial and the immune response against IAI without impeding the peri-implant bone tissue regeneration. This paper reviews existing co-culture models together with their characteristics, results, and clinical relevance. A total of 36 studies were found involving in vitro co-culture models between bacteria and osteogenic or immune cells at the interface with orthopedic antibacterial biomaterials. Most studies (∼67%) involved co-culture models of osteogenic cells and bacteria (osteo-bac), while 33% were co-culture models of immune cells and bacterial cells (im-bac). All models involve direct co-culture of two different cell types. The cell seeding sequence (simultaneous, bacteria-first, and cell-first) was used to mimic clinically relevant conditions and showed the greatest effect on the outcome for both types of co-culture models. The im-bac models are considered more relevant for early peri-implant infections, whereas the osteo-bac models suit late infections. The limitations of the current models and future directions to develop more relevant co-culture models to address specific research questions are also discussed.

Understanding fibroblast-immune cell interactions via co-culture models and their role in asthma pathogenesis.

Asthma is a chronic lung disease involving airway inflammation and fibrosis. Fibroblasts are the main effector cells important for lung tissue production which becomes abnormal in asthmatics and is one of the main contributors to airway fibrosis. Although fibroblasts were traditionally viewed solely as structural cells, they have been discovered to be highly active, and involved in lung inflammatory and fibrotic processes in asthma. In line with this, using 2D and 3D in vitro co-culture models, a complex interaction between lung fibroblasts and various immune cells important for the pathogenesis of asthma have been recently uncovered. Hence, in this review, we provide the first-ever summary of various studies that used 2D and 3D in vitro co-culture models to assess the nature of aberrant immune cell-fibroblast interactions and their contributions to chronic inflammation and fibrotic mechanisms in asthma pathogenesis.

In Vitro Bacterial Competition of Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli against Coagulase-Negative Staphylococci from Bovine Mastitis Milk.

Intramammary infection (IMI) from the environment and infected quarters can cause co-infection. The objective of this study was to determine the ability of coagulase-negative staphylococci (CNS) to survive in the same environment as Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli as major pathogens. In total, 15 and 242 CNS strains were used in Experiment I and Experiment II, respectively. Both experiments were separated into three conditions: culture with CNS 24 h before (PRIOR), after (AFTER), and at the same time (EQUAL). The lack of a clear zone, regardless of size, was determined to be the key to the survival of both. The CNS species' percentages of survival against major pathogens were tested using Fisher's exact test. Differences in the percentages of survival were evident among the CNS species in all conditions. For the PRIOR condition, all CNS mostly survived when living with major strains; however, S. chromogenes could degrade S. agalactiae. Although most CNS strains were degraded in the AFTER and EQUAL conditions, some strains of S. hominis and S. simulans could resist S. aureus and S. agalactiae. In conclusion, some specific strains of CNS are able to survive in an environment with major pathogens. Research into the survival strains may indicate that the concept of novel bacteria with bacteriolytic capabilities might be possible as a novel mastitis treatment.

Tumor Cells Modulate Macrophage Phenotype in a Novel In Vitro Co-Culture Model of the NSCLC Tumor Microenvironment.

INTRODUCTION: Macrophage phenotype in the tumor microenvironment correlates with prognosis in NSCLC. Immunosuppressive macrophages promote tumor progression, whereas proinflammatory macrophages may drive an antitumor immune response. How individual NSCLCs affect macrophage phenotype is a major knowledge gap. METHODS: To systematically study the impact of lung cancer cells on macrophage phenotypes, we developed an in vitro co-culture model that consisted of molecularly and clinically annotated patient-derived NSCLC lines, human cancer-associated fibroblasts, and murine macrophages. Induced macrophage phenotype was studied through quantitative real-time polymerase chain reaction and validated in vivo using NSCLC xenografts through quantitative immunohistochemistry and clinically with The Cancer Genome Atlas (TCGA)-"matched" patient tumors. RESULTS: A total of 72 NSCLC cell lines were studied. The most frequent highly induced macrophage-related gene was Arginase-1, reflecting an immunosuppressive M2-like phenotype. This was independent of multiple clinicopathologic factors, which also did not affect M2:M1 ratios in matched TCGA samples. In vivo, xenograft tumors established from high Arginase-1-inducing lines (Arghi) had a significantly elevated density of Arg1+ macrophages. Matched TCGA clinical samples to Arghi NSCLC lines had a significantly higher ratio of M2:M1 macrophages (p = 0.0361). CONCLUSIONS: In our in vitro co-culture model, a large panel of patient-derived NSCLC lines most frequently induced high-expression Arginase-1 in co-cultured mouse macrophages, independent of major clinicopathologic and oncogenotype-related factors. Arghi cluster-matched TCGA tumors contained a higher ratio of M2:M1 macrophages. Thus, this in vitro model reproducibly characterizes how individual NSCLC modulates macrophage phenotype, correlates with macrophage polarization in clinical samples, and can serve as an accessible platform for further investigation of macrophage-specific therapeutic strategies.

Development of an Advanced Multicellular Intestinal Model for Assessing Immunomodulatory Properties of Anti-Inflammatory Compounds.

Intestinal inflammation is the collective term for immune system-mediated diseases of unknown, multifactorial etiology, with often complex interactions between genetic and environmental factors. To mechanistically investigate the effect of treatment with compounds possessing immunomodulating properties in the context of intestinal inflammation, we developed an immunocompetent in vitro triculture intestinal model consisting of a differentiated intestinal epithelial layer (Caco-2/HT29-MTX) and immunocompetent cells (differentiated THP-1). The triculture mimicked a healthy intestine with stable barrier integrity. Lipopolysaccharide treatment triggered a controlled and reversible inflammatory state, resulting in significant impairment of barrier integrity and release of pro-inflammatory cytokines and chemokines, which are known hallmarks of intestinal inflammation. Treatment with known anti-inflammatory reference compounds (TPCA-1 and budenoside) prevented the induction of an inflammatory state; the decreasing triculture responses to this treatment measured by cytokine release, transepithelial electric resistance (TEER), and epithelial layer permeability proved the suitability of the intestinal model for anti-inflammatory drug screening. Finally, selected tobacco alkaloids (nicotine and anatabine (R/S and S forms)) were tested in the in vitro triculture for their potential anti-inflammatory properties. Indeed, naturally occurring alkaloids, such as tobacco-derived alkaloids, have shown substantial anti-inflammatory effects in several in vitro and in vivo models of inflammation, gaining increasing interest. Similar to the anti-inflammatory reference compounds, one of the tobacco alkaloids under investigation partially prevented the decrease in the TEER and increase in permeability and reduced the release of pro-inflammatory cytokines and chemokines. Taken together, these data confirm that our in vitro model is suitable for screening potential anti-inflammatory compounds in the context of intestinal inflammation.

The Effect of a Polyester Nanofibrous Membrane with a Fibrin-Platelet Lysate Coating on Keratinocytes and Endothelial Cells in a Co-Culture System.

Chronic wounds affect millions of patients worldwide, and it is estimated that this number will increase steadily in the future due to population ageing. The research of new therapeutic approaches to wound healing includes the development of nanofibrous meshes and the use of platelet lysate (PL) to stimulate skin regeneration. This study considers a combination of a degradable electrospun nanofibrous blend of poly(L-lactide-co-ε-caprolactone) and poly(ε-caprolactone) (PLCL/PCL) membranes (NF) and fibrin loaded with various concentrations of PL aimed at the development of bioactive skin wound healing dressings. The cytocompatibility of the NF membranes, as well as the effect of PL, was evaluated in both monocultures and co-cultures of human keratinocytes and human endothelial cells. We determined that the keratinocytes were able to adhere on all the membranes, and their increased proliferation and differentiation was observed on the membranes that contained fibrin with at least 50% of PL (Fbg + PL) after 14 days. With respect to the co-culture experiments, the membranes with fibrin with 20% of PL were observed to enhance the metabolic activity of endothelial cells and their migration, and the proliferation and differentiation of keratinocytes. The results suggest that the newly developed NF combined with fibrin and PL, described in the study, provides a promising dressing for chronic wound healing purposes.

Ongoing inflammation enhances the toxicity of engineered nanomaterials: Application of an in vitro co-culture model of the healthy and inflamed intestine.

Chronic inflammatory conditions can negatively impact intestinal barrier function and affect the epithelium's interaction with nano-sized materials. We demonstrate the application of a Caco-2/THP-1 co-culture mimicking the intestine in healthy (i.e. stable) or inflamed state in nanotoxicological research. The co-cultures were exposed to non-toxic concentrations of silver nanoparticles (AgNPs) or silver nitrate (AgNO3) for 24 h. The barrier integrity and cytokine release as well as necrotic and apoptotic cell death were investigated. AgNPs and AgNO3 most strongly affected the inflamed co-culture. Higher concentrations of AgNPs induced a significant increase in barrier integrity in the inflamed but not the stable co-culture. Necrotic and apoptotic cell death was detected in both conditions but were significantly more pronounced in the inflamed condition. The exposure to AgNO3 affected barrier integrity in all experimental set-ups, but caused nuclear condensation only in the Caco-2 monoculture and the inflamed co-culture. AgNPs reduced the release of monocyte chemoattractant protein-1 in the stable model. Clear differences were observed in the effects of AgNPs and AgNO3 in relation to the model's health status. The results suggest an increased vulnerability of the inflamed epithelial barrier towards AgNPs underlining the importance to consider the intestinal health status in the safety assessment of nanomaterials.

Synthesis and in vitro evaluation of cyclodextrin hyaluronic acid conjugates as a new candidate for intestinal drug carrier for steroid hormones.

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric ß-cyclodextrin derivates is described, which result from the attachment of a hydrophilic ß-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized ß-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (ß-estradiol, dexamethasone) and compared to monomeric ß-cyclodextrin derivates regarding solubilization and complexation efficiency. The ß-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of ß-estradiol, higher solubilities could be achieved by complexation with both synthesized ß-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized ß-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like ß-estradiol.

Vaccine-Mediated Mechanisms Controlling Francisella tularensis SCHU S4 Growth in a Rat Co-Culture System.

Francisella tularensis causes the severe disease tularemia. In the present study, the aim was to identify correlates of protection in the rat co-culture model by investigating the immune responses using two vaccine candidates conferring distinct degrees of protection in rat and mouse models. The immune responses were characterized by use of splenocytes from naïve or Live vaccine strain- (LVS) or ∆clpB/∆wbtC-immunized Fischer 344 rats as effectors and bone marrow-derived macrophages infected with the highly virulent strain SCHU S4. A complex immune response was elicited, resulting in cytokine secretion, nitric oxide production, and efficient control of the intracellular bacterial growth. Addition of LVS-immune splenocytes elicited a significantly better control of bacterial growth than ∆clpB/∆wbtC splenocytes. This mirrored the efficacy of the vaccine candidates in the rat model. Lower levels of IFN-γ, TNF, fractalkine, IL-2, and nitrite were present in the co-cultures with ∆clpB/∆wbtC splenocytes than in those with splenocytes from LVS-immunized rats. Nitric oxide was found to be a correlate of protection, since the levels inversely correlated to the degree of protection and inhibition of nitric oxide production completely reversed the growth inhibition of SCHU S4. Overall, the results demonstrate that the co-culture assay with rat-derived cells is a suitable model to identify correlates of protection against highly virulent strains of F. tularensis.

What Have In Vitro Co-Culture Models Taught Us about the Contribution of Epithelial-Mesenchymal Interactions to Airway Inflammation and Remodeling in Asthma?

As the lung develops, epithelial-mesenchymal crosstalk is essential for the developmental processes that drive cell proliferation, differentiation, and extracellular matrix (ECM) production within the lung epithelial-mesenchymal trophic unit (EMTU). In asthma, a number of the lung EMTU developmental signals have been associated with airway inflammation and remodeling, which has led to the hypothesis that aberrant activation of the asthmatic EMTU may lead to disease pathogenesis. Monoculture studies have aided in the understanding of the altered phenotype of airway epithelial and mesenchymal cells and their contribution to the pathogenesis of asthma. However, 3-dimensional (3D) co-culture models are needed to enable the study of epithelial-mesenchymal crosstalk in the setting of the in vivo environment. In this review, we summarize studies using 3D co-culture models to assess how defective epithelial-mesenchymal communication contributes to chronic airway inflammation and remodeling within the asthmatic EMTU.

High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model.

Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

2D and 3D In Vitro Co-Culture for Cancer and Bone Cell Interaction Studies.

Co-culture assays are used to study the mutual interaction between cells in vitro. This chapter describes 2D and 3D co-culture systems used to study cell-cell signaling crosstalk between cancer cells and bone marrow adipocytes, osteoblasts, osteoclasts, and osteocytes. The chapter provides a step-by-step guide to the most commonly used cell culture techniques, functional assays, and gene expression.

Osteoinductive and anti-inflammatory properties of chitosan-based scaffolds for bone regeneration.

Current bone implants based on new biomaterials may cause a foreign body reaction (FBR) around the implant itself thus prolonging the healing time following bone fractures. In this paper, biomimetic chitosan-based scaffolds promoting bone tissue regeneration and controlling inflammatory response are proposed. First, the anti-inflammatory potential of scaffolds on hMSCs stimulated by lipopolysaccharide (LPS) was investigated by dosing the levels of some interleukins and oxidative stress metabolites (IL-1ß, IL-10 and nitrites) involved in immune response. Then, to mimic the inflammation process at osteoporotic site, the effect of scaffolds was evaluated on in vitro co-culture model based on osteoblasts and macrophages stimulated by LPS. Results demonstrated that bioactivated scaffolds are able to i) inhibit synthesis of inflammatory mediators such as IL-1ß; ii) reduce oxidative stress metabolites; and iii) promote anti-inflammatory markers generation (IL-10) in hMSCs. Finally, bioactivated scaffolds show an anti-inflammatory activity also on in vitro co-cultures, which better mimic in vivo damaged bone microenvironment.

Human Co- and Triple-Culture Model of the Alveolar-Capillary Barrier on a Basement Membrane Mimic.

The development of an in vitro model resembling the alveolar-capillary barrier might be a highly beneficial tool to study lung physiology as well as the immune response of the lung to infection or after exposure to nanoparticles. This study is based on an in vitro alveolar barrier developed on a basement membrane mimic, composed of ultrathin nanofiber meshes generated via electrospinning using bioresorbable poly(ɛ-caprolactone). As cellular components, NCI H441, resembling the alveolar epithelial cells, and ISO-HAS-1, an endothelial cell line, were used to perform bipolar coculture experiments for a total cultivation period of 14 days. In addition to immunohistochemical and immunofluorescent studies, transepithelial electrical resistance (TER) and transport capabilities of the in vitro model system were investigated. Alveolar barrier function could be clearly determined for the postulated bipolar coculture system on the basement membrane mimic, since TER increased during the course of bipolar cultivation. Furthermore, to gain first insights into possible lung inflammatory reactions in vitro, this coculture model was further expanded by a human leukemia monocyte cell line (THP-1). This triple-culture system was able to maintain adequately the barrier properties of the bipolar coculture, thus making this in vitro model consisting of epithelial, endothelial, and immune cells on a basement membrane mimic a promising basis for further studies in tissue engineering.

Inhibition of Low-Grade Inflammation by Anthocyanins after Microbial Fermentation in Vitro.

The anti-inflammatory effects of anthocyanins (ACNs) on vascular functions are discussed controversially because of their low bioavailability. This study was performed to determine whether microorganism (MO)-fermented ACNs influence vascular inflammation in vitro. Therefore, MO growth media were supplemented with an ACN-rich grape/berry extract and growth responses of Escherichia coli, E. faecalis and H. alvei, as well as ACN fermentation were observed. MO supernatants were used for measuring the anti-inflammatory effect of MO-fermented ACNs in an epithelial-endothelial co-culture transwell system. After basolateral enrichment (240 min), endothelial cells were stimulated immediately or after 20 h with TNF-α. Afterwards, leukocyte adhesion, expression of adhesion molecules and cytokine release were measured. Results indicate that E. coli, E. faecalis and H. alvei utilized ACNs differentially concomitant with different anti-inflammatory effects. Whereas E. coli utilized ACNs completely, no anti-inflammatory effects of fermented ACNs were observed on activated endothelial cells. In contrast, ACN metabolites generated by E. faecalis and H. alvei significantly attenuated low-grade stimulated leukocyte adhesion, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine secretion (IL-8 and IL-6), as well as NF-κB mRNA expression with a more pronounced effect of E. faecalis than H. alvei. Thus, MO-fermented ACNs have the potential to reduce inflammation.

An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy.

Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naïve, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, ΔclpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naïve splenocytes, ΔclpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the ΔclpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the ΔclpB-immune splenocytes also contained higher levels of IFN-γ, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-γ, TNF-α, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS-/- mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

Application of rapid in vitro co-culture system of macrophages and T-cell subsets to assess the immunogenicity of dogs vaccinated with live attenuated Leishmania donovani centrin deleted parasites (LdCen-/-).

BACKGROUND: Live attenuated Leishmania donovani parasites as LdCen(-/-) were shown to confer protective immunity against Leishmania infection in mice, hamsters, and dogs. Strong immunogenicity in dogs vaccinated with LdCen(-/-) has been previously reported, including increased antibody response favoring Th1 response lymphoproliferative responses, CD4(+) and CD8(+) T-cells activation, increased levels of Th1 and reduction of Th2 cytokines, in addition to a significant reduction in parasite burden after 18 and 24 months post virulent parasite challenge. METHODS: Aimed at validating a new method using in vitro co-culture systems with macrophages and purified CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells of immunized dogs with both LdCen(-/-) and Leishmune® to assess microbicide capacity of macrophages and the immune response profile as the production of IFN-γ, TNF-α, IL-12, IL-4 and IL-10 cytokines. RESULTS AND DISCUSSION: Our data showed co-cultures of macrophages and purified T-cells from dogs immunized with LdCen(-/-) and challenged with L. infantum were able to identify high microbicidal activity, especially in the co-culture using CD4(+) T-cells, as compared to the Leishmune® group. Similarly, co-cultures with CD8(+) T-cells or CD4(+):CD8(+) T-cells in both experimental groups were able to detect a reduction in the parasite burden in L. infantum infected macrophages. Moreover, co-cultures using CD4(+) or CD8(+) or CD4(+):CD8(+) T-cells from immunized dogs with both LdCen(-/-) and Leishmune® were able to identify higher levels of IFN-γ and IL-12 cytokines, reduced levels of IL-4 and IL-10, and a higher IFN-γ/IL-10 ratio. While the highest IFN-γ levels and IFN-γ/IL-10 ratio were the hallmarks of LdCen(-/-) group in the co-culture using CD4(+) T-cells, resulting in strong reduction of parasitism, the Leishmune® immunization presented a differential production of TNF-α in the co-culture using CD4(+):CD8(+) T-cells. CONCLUSION: The distinct conditions of co-culture systems were validated and able to detect the induction of immune protection. The method described in this study applied a new, more accurate approach and was able to yield laboratory parameters useful to test and monitor the immunogenicity and efficacy of Leishmania vaccines in dogs.