In vitro gut microbiome models could provide timely and cost-efficient solutions to study microbiome responses to drugs. For this purpose, in vitro models that maintain the functional and compositional profiles of in vivo gut microbiomes would be extremely valuable. Here, we present a 96-deep well plate-based culturing model (MiPro) that maintains the functional and compositional profiles of individual gut microbiomes, as assessed by metaproteomics, while allowing a four-fold increase in viable bacteria counts. Comparison of taxon-specific functions between pre- and post-culture microbiomes shows a Pearson’s correlation coefficient r of 0.83 ± 0.03. In addition, we show a high degree of correlation between gut microbiome responses to metformin in the MiPro model and those in mice fed a high-fat diet. We propose MiPro as an in vitro gut microbiome model for scalable investigation of drug-microbiome interactions such as during high-throughput drug screening.

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

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The gut microbiome is a new target for therapeutics. In vitro high-throughput culture models could provide time-and-cost saving solutions to discover microbiome responses to drugs. Unfortunately, there has been no report of in vitro models capable of maintaining functional and compositional profiles resembling the in vivo gut microbiome. Here, we developed and validated a high-throughput culturing model named Mipro to maintain individuals' microbiomes. The Mipro model quintupled viable bacteria count while maintained the functional and compositional profiles of individuals' gut microbiomes.Comparison of taxon-specific functions between pre -and-post culture microbiomes showed Pearson's correlation coefficient r of 0.83 ± 0.03. Moreover, the Mipro model also exhibited a high degree of in vitro -in vivo correlation (Pearson's r of 0.68 ± 0.09) in microbial responses to metformin in mice fed a high-fat diet. Mipro provides a highly simulated gut microbiome for high-throughput investigation of drug-microbiome interactions.