In vitro enteroid-derived three-dimensional tissue model of human small intestinal epithelium with innate immune responses

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

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Long Non-coding RNAs RN7SK and GAS5 Regulate Macrophage Polarization and Innate Immune Responses

Frontiers in Immunology ◽

10.3389/fimmu.2020.604981 ◽

2020 ◽

Vol 11 ◽

Author(s):

Imran Ahmad ◽

Araceli Valverde ◽

Raza Ali Naqvi ◽

Afsar R. Naqvi

Keyword(s):

Immune Responses ◽

Epigenetic Regulation ◽

Innate Immune ◽

Differentially Expressed ◽

Immune Functions ◽

Innate Immune Responses ◽

Antigen Uptake ◽

Non Coding Rnas

Macrophages (Mφ) are immune cells that exhibit remarkable functional plasticity. Identification of novel endogenous factors that can regulate plasticity and innate immune functions of Mφ will unravel new strategies to curb immune-related diseases. Long non-coding RNAs (lncRNAs) are a class of endogenous, non-protein coding, regulatory RNAs that are increasingly being associated with various cellular functions and diseases. Despite their ubiquity and abundance, lncRNA-mediated epigenetic regulation of Mφ polarization and innate immune functions is poorly studied. This study elucidates the regulatory role of lncRNAs in monocyte to Mφ differentiation, M1/M2 dichotomy and innate immune responses. Expression profiling of eighty-eight lncRNAs in monocytes and in vitro differentiated M2 Mφ identified seventeen differentially expressed lncRNAs. Based on fold-change and significance, we selected four differentially expressed lncRNAs viz., RN7SK, GAS5, IPW, and ZFAS1 to evaluate their functional impact. LncRNA knockdown was performed on day 3 M2 Mφ and the impact on polarization was assessed on day 7 by surface marker analysis. Knockdown of RN7SK and GAS5 showed downregulation of M2 surface markers (CD163, CD206, or Dectin) and concomitant increase in M1 markers (MHC II or CD23). RN7SK or GAS5 knockdown showed no significant impact on CD163, CD206, or CD23 transcripts. M1/M2 markers were not impacted by IPW or ZFAS1 knockdown. Functional regulation of antigen uptake/processing and phagocytosis, two central innate immune pathways, by candidate lncRNA was assessed in M1/M2 Mφ. Compared to scramble, enhanced antigen uptake and processing were observed in both M1/M2 Mφ transfected with siRNA targeting GAS5 and RN7SK but not IPW and ZFAS1. In addition, knockdown of RN7SK significantly augmented uptake of labelled E. coli in vitro by M1/M2 Mφ, while no significant difference was in GAS5 silencing cells. Together, our results highlight the instrumental role of lncRNA (RN7SK and GAS5)-mediated epigenetic regulation of macrophage differentiation, polarization, and innate immune functions.

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Physicochemical and Morphological Transformations of Chitosan Nanoparticles across the Gastrointestinal Tract and Cellular Toxicity in an In Vitro Model of the Small Intestinal Epithelium

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.9b05506 ◽

2019 ◽

Vol 68 (1) ◽

pp. 358-368 ◽

Cited By ~ 6

Author(s):

Zhongyuan Guo ◽

Xiaoqiong Cao ◽

Glen M DeLoid ◽

Kaarunya Sampathkumar ◽

Kee Woei Ng ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Intestinal Epithelium ◽

In Vitro Model ◽

Chitosan Nanoparticles ◽

Cellular Toxicity ◽

Small Intestinal Epithelium ◽

Small Intestinal ◽

Vitro Model ◽

Morphological Transformations

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The Absence of Hck, Fgr, and Lyn Tyrosine Kinases Augments Lung Innate Immune Responses to Pneumocystis murina

Infection and Immunity ◽

10.1128/iai.01441-08 ◽

2009 ◽

Vol 77 (5) ◽

pp. 1790-1797 ◽

Cited By ~ 13

Author(s):

Michael P. Nelson ◽

Allison E. Metz ◽

Shaoguang Li ◽

Clifford A. Lowell ◽

Chad Steele

Keyword(s):

Immune Responses ◽

Alveolar Macrophages ◽

Tyrosine Kinases ◽

Innate Immune ◽

Innate Immune Responses ◽

Independent Manner ◽

Intratracheal Administration ◽

Monocyte Chemoattractant Protein 1 ◽

Immunoglobulin Receptor

ABSTRACT Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn−/− mice 3 days postchallenge, although the level was less than that observed in Hck−/− Fgr−/− Lyn−/− mice. A correlate to augmented clearance of P. murina in Hck−/− Fgr−/− Lyn−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.

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Evolutionary and structural analysis of SARS-CoV-2 specific evasion of host immunity

Genes and Immunity ◽

10.1038/s41435-020-00120-6 ◽

2020 ◽

Vol 21 (6-8) ◽

pp. 409-419

Author(s):

Irfan Hussain ◽

Nashaiman Pervaiz ◽

Abbas Khan ◽

Shoaib Saleem ◽

Huma Shireen ◽

...

Keyword(s):

Immune Responses ◽

Nonstructural Protein ◽

Critical Role ◽

Clinical Manifestations ◽

Host Immunity ◽

Innate Immune ◽

Host Immune System ◽

Innate Immune Responses ◽

In Vivo Models

AbstractThe outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading fast worldwide. There is a pressing need to understand how the virus counteracts host innate immune responses. Deleterious clinical manifestations of coronaviruses have been associated with virus-induced direct dysregulation of innate immune responses occurring via viral macrodomains located within nonstructural protein-3 (Nsp3). However, no substantial information is available concerning the relationship of macrodomains to the unusually high pathogenicity of SARS-CoV-2. Here, we show that structural evolution of macrodomains may impart a critical role to the unique pathogenicity of SARS-CoV-2. Using sequence, structural, and phylogenetic analysis, we identify a specific set of historical substitutions that recapitulate the evolution of the macrodomains that counteract host immune response. These evolutionary substitutions may alter and reposition the secondary structural elements to create new intra-protein contacts and, thereby, may enhance the ability of SARS-CoV-2 to inhibit host immunity. Further, we find that the unusual virulence of this virus is potentially the consequence of Darwinian selection‐driven epistasis in protein evolution. Our findings warrant further characterization of macrodomain-specific evolutionary substitutions in in vitro and in vivo models to determine their inhibitory effects on the host immune system.

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High-Resolution Profiling of Innate Immune Responses by Porcine Dendritic Cell Subsets in vitro and in vivo

Frontiers in Immunology ◽

10.3389/fimmu.2020.01429 ◽

2020 ◽

Vol 11 ◽

Cited By ~ 1

Author(s):

Gaël Auray ◽

Stephanie C. Talker ◽

Irene Keller ◽

Sylvie Python ◽

Markus Gerber ◽

...

Keyword(s):

Dendritic Cell ◽

High Resolution ◽

Immune Responses ◽

Innate Immune ◽

Innate Immune Responses ◽

Dendritic Cell Subsets

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Cellular Composition and Differentiation Signaling in Chicken Small Intestinal Epithelium

Animals ◽

10.3390/ani9110870 ◽

2019 ◽

Vol 9 (11) ◽

pp. 870

Author(s):

Haihan Zhang ◽

Dongfeng Li ◽

Lingbin Liu ◽

Ling Xu ◽

Mo Zhu ◽

...

Keyword(s):

Intestinal Epithelium ◽

Intestinal Development ◽

Cell Culture Model ◽

Proliferation Zone ◽

Small Intestinal Epithelium ◽

Culture Model ◽

Different Types ◽

Development And Differentiation ◽

Small Intestinal

The small intestine plays an important role for animals to digest and absorb nutrients. The epithelial lining of the intestine develops from the embryonic endoderm of the embryo. The mature intestinal epithelium is composed of different types of functional epithelial cells that are derived from stem cells, which are located in the crypts. Chickens have been widely used as an animal model for researching vertebrate embryonic development. However, little is known about the molecular basis of development and differentiation within the chicken small intestinal epithelium. This review introduces processes of development and growth in the chicken gut, and compares the cellular characteristics and signaling pathways between chicken and mammals, including Notch and Wnt signaling that control the differentiation in the small intestinal epithelium. There is evidence that the chicken intestinal epithelium has a distinct cellular architecture and proliferation zone compared to mammals. The establishment of an in vitro cell culture model for chickens will provide a novel tool to explore molecular regulation of the chicken intestinal development and differentiation.

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