Characterization of an Osmr Conditional Knockout Mouse Model

Oncostatin M (OSM) is a member of the interleukin-6 (IL-6) family of cytokines and has been found to have distinct anti-inflammatory and pro-inflammatory properties in various cellular and disease contexts. OSM signals through two receptor complexes, one of which includes OSMRβ. To investigate OSM-OSMRβ signaling in adult hematopoiesis, we utilized the readily available conditional Osmrfl/fl mouse model B6;129-Osmrtm1.1Nat/J, which is poorly characterized in the literature. This model contains loxP sites flanking exon 2 of the Osmr gene. We crossed Osmrfl/fl mice to interferon-inducible Mx1-Cre, which is robustly induced in adult hematopoietic cells. We observed complete recombination of the Osmrfl allele and loss of exon 2 in hematopoietic (bone marrow) as well as non-hematopoietic (liver, lung, kidney) tissues. Using a TaqMan assay with probes downstream of exon 2, Osmr transcript was lower in the kidney but equivalent in bone marrow, lung, and liver from Osmrfl/fl Mx1-Cre versus Mx1-Cre control mice, suggesting that transcript is being produced despite loss of this exon. Western blots show that liver cells from Osmrfl/fl Mx1-Cre mice had complete loss of OSMR protein, while bone marrow, kidney, and lung cells had reduced OSMR protein at varying levels. RNA-seq analysis of a subpopulation of bone marrow cells (hematopoietic stem cells) finds that some OSM-stimulated genes, but not all, are suppressed in Osmrfl/fl Mx1-Cre cells. Together, our data suggest that the B6;129-Osmrtm1.1Nat/J model should be utilized with caution as loss of Osmr exon 2 has variable and tissue-dependent impact on mRNA and protein expression.


INTRODUCTION
OSM, belonging to the IL-6 family, plays an important role in development, malignancy, and homeostasis of various tissue types [1][2][3][4][5] .OSM is primarily produced by mature activated hematopoietic cells such as monocytes, T lymphocytes, neutrophils, macrophages, and dendritic cells 4,6,7 .OSM was first isolated based on its ability to inhibit the proliferation of melanoma tumor cells, while having no significant effect on normal human fibroblasts 6 .Due to this unique characteristic, it was given the name oncostatin M, where "onco" referred to cancer, "statin" to its ability to inhibit cell proliferation, and "M" to melanoma.Despite the initial identification of OSM as a tumor suppressor, a pro-inflammatory role of OSM has been reported in colon cancer, breast cancer, pancreatic cancer, myeloma, brain tumors, chronic lymphocytic leukemia, hepatoblastoma and COVID-19 1,3,8 .OSM, like IL-6, has been linked to tumor growth and is often found in high concentrations in the serum and tumor sites of patients and animal models of cancer 1,3,8 .Significantly increased Osm transcript production has also been associated with aging in both the human thymus and mouse hematopoietic stem cells (HSCs) compared to young tissues 9,10 .Human OSM interacts with two distinct receptor complexes termed type I and type II.The type I receptor complex contains leukemia inhibitory factor receptor alpha (LIFR) and interleukin 6 signal transducer (IL6ST; GP130), while the type II receptor complex contains oncostatin M receptor (OSMR) and IL6ST 4,[11][12][13] .While recent studies have shown that OSM has a lower affinity for the type I receptor complex than the type II receptor complex 14,15 , much remains unknown about how OSM interacts with these complexes in different contexts.
An additional complexity in studying OSM signaling is that while human OSM is known to signal through both the type I and type II receptor complexes, mouse OSM is reported to signal only through the type II receptor complex [16][17][18] .
To understand the role of OSM signaling in aging and disease in a physiologically relevant in vivo setting, mouse knockout models are essential.Two existing mouse models are commonly used to investigate loss of receptor complex II signaling through knockout of Osmr.The first, Osmr tm1Mtan , was reported by Tanaka et al. in 2003  19 .This model disrupts the first coding exon of Osmr by knock-in of a lacZ and neomycin expression cassette into the proximal region and is non-conditional in nature 19 .These Osmr -/-mice were viable and fertile, born at expected Mendelian ratios, and had normal adult body weights 19 .Knockout of Osmr was confirmed by loss of mRNA in lung tissue 19 .This model exhibited hematopoiesis phenotypes showing reduced numbers of erythroid and megakaryocytic progenitors and their mature counterparts 19 .However, these were attributed to both cell-autonomous and cell non-autonomous effects of Osmr -/-on hematopoietic progenitor cells and non-hematopoietic cells in the bone marrow microenvironment 19 , which are best resolved by the use of a conditional knockout model.The second existing model, B6;129-Osmr tm1.1Nat /J, is a conditional knockout model in which loxP sites flank the first coding exon of the Osmr gene.
Additionally, adjacent to the second loxP site is an Frt site remaining after excision of a PGK-neomycin selectable marker.This model was originally designed to study Osmr in the retina and is available in The Jackson Laboratory's mouse repository; however, characterization of the model has not been published by the donating investigators.To investigate cell-autonomous effects of Osmr conditional knockout on hematopoiesis, we obtained the B6;129-Osmr tm1.1Nat /J (referred to as Osmr fl/fl ) model and crossed these mice to an interferon-inducible Mx1-Cre allele 20 , which is commonly used in the study of adult hematopoiesis.We characterized Osmr fl/fl Mx1-Cre mice using classical methods such as the development of a PCR reaction to evaluate recombination of the Osmr fl allele, real-time PCR and RNA sequencing for Osmr mRNA transcript, and Western blotting to evaluate OSMR at the protein level.We investigated both hematopoietic tissue (bone marrow) as well as non-hematopoietic tissues (liver, lung, kidney) in which OSMR is also expressed.Our results demonstrate variable alterations in mRNA and protein expression in various tissues isolated from Osmr fl/fl Mx1-Cre compared to control Mx1-Cre mice.

Osmr fl/fl Mx1-Cre Mice
To generate an inducible Osmr knockout model, the B6;129-Osmr tm1.1Nat /J strain was crossed to Mx1-Cre 20 .Mx1-Cre is an interferon-inducible cre recombinase that is highly efficient in expression in progenitor and mature cells of the adult hematopoietic lineages.Thus, in the Osmr fl/fl Mx1-Cre model, the Osmr fl allele should remain unrecombined with wild-type expression at the transcript and protein levels prior to administration of the double-stranded RNA poly(I:C), which then induces an interferon response and expression of cre recombinase.After crossing B6;129-Osmr tm1.1Nat /J to Mx1-Cre to generate Osmr fl/fl Mx1-Cre and control Mx1-Cre animals, we genotyped three control and three experimental animals alongside positive (parental Osmr fl/fl and Mx1-Cre mice) and negative (wild-type C57BL/6J) controls.At the Osmr locus, genomic DNA from Osmr fl/fl Mx1-Cre mice contained a loxP site (230bp) and genomic DNA from control Mx1-Cre mice did not contain a loxP site (170bp), recognized by primers flanking exon 2 (Figure 1A).Genomic DNA from all Osmr fl/fl Mx1-Cre and control Mx1-Cre mice were also positive for the transgenic cre allele (100bp) (Figure 1B).
Following injection with poly(I:C) to induce Mx1-Cre-mediated recombination of the Osmr fl locus, we isolated DNA from bone marrow, lung, liver, and kidney tissues.These tissues were selected based on expression of both Osmr and Mx1 transcripts using publicly available databases including the human protein atlas [21][22][23] , Mouse Genome Informatics 24 and The Bgee suite 24 .A recombination PCR assay was designed to assess Osmr fl recombination efficiency.The primers generate a product spanning the loxP-flanked exon 2 and bind to both the wild-type and modified Osmr loci generating product sizes of ~860bp for the floxed (fl) allele, 738bp for the wild-type (wt) allele and ~300bp for the recombined () allele.This PCR assay detected a ~300bp recombined allele in all tissue samples from Osmr fl/fl Mx1-Cre mice (Figure 1C), demonstrating that recombination of the Osmr fl locus occurred efficiently following poly(I:C) injection in the bone marrow, lung, liver, and kidney.

Mx1-Cre Mice
We next evaluated levels of Osmr transcript in cells from the lung, liver, kidney, and bone marrow of Osmr fl/fl Mx1-Cre and control Mx1-Cre animals.Following harvest, cells were flash-frozen and used for RNA extraction, cDNA synthesis and semi-quantitative real-time PCR.TaqMan probes annealing to a target amplicon spanning Osmr exons 17 and 18, downstream of exon 2 deletion, were used.In control Mx1-Cre mice, Osmr transcript was detected at a low level in lung cells and robustly detected in liver, kidney, and bone marrow (Figure 2A).In Osmr fl/fl Mx1-Cre mice, Osmr transcript in lung and liver was detected at similar levels as in control Mx1-Cre mice.In the kidney, Osmr transcript was significantly reduced in Osmr fl/fl Mx1-Cre mice compared to control Mx1-Cre.In the bone marrow, Osmr transcript trended toward a decrease in Osmr fl/fl Mx1-Cre mice compared to control Mx1-Cre.
Given this tissue-dependent variation in Osmr transcript levels, we performed western blotting with a published OSMR antibody 14,16,25,26  Given the reduction in OSMR protein detected in bone marrow lysates of Osmr fl/fl Mx1-Cre mice, we utilized this model to investigate Osmr signaling in hematopoietic stem cells (HSCs) which are a small subset of bone marrow hematopoietic cells.HSCs were prospectively isolated from the bone marrow of mice using fluorescence-activated cell sorting.HSCs from Osmr fl/fl Mx1-Cre and control Mx1-Cre mice were placed into serumfree media with or without 500ng/mL of recombinant murine OSM or vehicle control for 60min, then flash frozen for RNA sequencing (RNA-seq).Significantly differentially expressed genes were defined based on stringent cutoffs (p < 0.01 and log2(FC) > 3 or < -3) (Figure 3A, Supplemental Table 1).Consistent with our real-time PCR analysis of bone marrow cells, no difference in Osmr transcript abundance was observed in Osmr fl/fl Mx1-Cre HSCs compared to control Mx1-Cre HSCs (Figure 3B).We removed predicted genes and pseudogenes from this list, resulting in a total of 91 significantly differentially expressed genes between Osmr fl/fl Mx1-Cre and control Mx1-Cre HSCs.Of these, 59 genes were increased in expression and 32 were decreased in expression in HSCs but not induced in Osmr fl/fl Mx1-Cre HSCs were enriched for signatures of extracellular matrix disassembly, G1/S transition, endonuclease, and hydrolase activity (Figure 3E).In contrast, the 10 genes that were induced by OSM in control Mx1-Cre HSCs but repressed by OSM in Osmr fl/fl Mx1-Cre HSCs were enriched for signatures of epithelial to mesenchymal transition, endothelial cell proliferation and ion channel binding (Figure 3F).These data suggest that a subset of genes transcriptionally upregulated in response to OSM signaling in control Mx1-Cre HSCs are no longer upregulated in response to OSM in Osmr fl/fl Mx1-Cre HSCs, consistent with genetic knockout and functional loss of OSMR.However, as a subset of OSM signaling target genes are increased in expression in Osmr fl/fl Mx1-Cre HSCs, and other genes are no longer induced by OSM signaling, these results suggest that there is an incomplete loss of OSMRβ and/or that these genes are not bona fide targets of OSM-OSMRβ signaling.

DISCUSSION
Our data show that Osmr fl/fl Mx1-Cre mice have near-complete recombination of the genomic Osmr fl locus in bone marrow, lung, liver, and kidney tissue.However, the extent of reduction in Osmr transcript and OSMRβ protein expression in Osmr fl/fl Mx1-Cre tissues compared to Mx1-Cre tissues was dependent upon the tissue type examined.These findings are consistent with a previous study of the B6;129-Osmr tm1.1Nat /J model showing reduced OSMRβ, as opposed to a complete knockout, in K5+ epidermis 14,16,25,26 .Additionally, despite the lack of observable differences in Osmr transcript expression in HSCs isolated from Osmr fl/fl Mx1-Cre compared to Mx1-Cre mice, we observed alterations in the levels of other transcripts.Focusing on genes induced by OSM signaling in control Mx1-Cre HSCs, we found that only some of these genes are no longer induced by OSM signaling in Osmr fl/fl Mx1-Cre HSCs.We interpret this data to mean that residual OSMRβ expression in Osmr fl/fl Mx1-Cre HSCs permits some OSM signaling to occur.In addition, HSCs with reduction or loss of OSMRβ appear to incur compensatory activation of OSM-stimulated genes indicating a more complex, tissue-dependent regulation of OSM-OSMR signaling than was previously understood.
Future studies will be necessary to understand the mechanisms underlying tissuedependent differences in Osmr transcript abundance and OSMRβ protein abundance in the B6;129-Osmr tm1.1Nat /J model.It is possible that tissue-dependent differences in transcript isoforms and/or splicing underly differences in abundance of transcript and protein.For example, transcripts lacking exon 2 may be sufficient to produce OSMRβ protein in the lung but not the liver or bone marrow.The appearance of two high molecular weight bands in the OSMRβ western blot in the lung but not in other tissues is consistent with multiple protein products being present in this tissue.In the bone marrow, a lower molecular weight band was visible in the OSMRβ western blot, which may indicate a shorter OSMRβ isoform that is still produced in Osmr fl/fl Mx1-Cre cells.
Utilizing mouse models to understand the role of OSM signaling in human pathophysiology is essential but is an inherent challenge due to the previously characterized differences in receptor binding between mice and humans.Other studies have reported novel and innovative tools to aid in overcoming this challenge.For example, with the knowledge that specific charged residues in the AB loop of OSM are crucial for selection of binding to receptor complex I versus complex II, "humanized" murine OSM variants have been generated to gain more accurate insights into the role of human OSM in mouse models 16 .In addition, a potent and specific inhibitor of OSM signaling has been engineered using a receptor fusion protein that contains the ligand binding domains of murine OSMRβ and murine GP130 27 .This construct can be delivered into cells using viral vectors and has been used in multiple studies investigating the hematopoietic stem cell niche in the bone marrow and the role of OSM in irritable bowel disease 26,28 .Using this construct to generate transgenic animals would be very useful for future in vivo studies.Moving forward, we advise that other tools be utilized in parallel to the B6;129-Osmr tm1.1Nat /J model and/or that studies include a detailed characterization of Osmr transcript and protein levels in the tissue and cell type of focus.

Western Blotting for OSMR
Total protein was extracted from snap frozen tissues with a Tissue Lyser II (Qiagen) using 5mm Bead Beater balls (Qiagen) after the addition of 50mM HEPES pH 7.5 (Gibco) and 6M urea (Sigma-Aldrich) at 1.5mL per 200mg tissue.Insoluble material was spun out at 21,000 x g for 20m.Total protein concentration of each sample was determined by microBCA (ThermoFisher).SDS-PAGE gel samples were prepared.Two 4-15% Mini-PROTEAN TGX Precast, 1.0mm, 15-well, SDS-PAGE Gel (Bio-Rad) were loaded with 25ug total protein per well.The gels were run at 150V for 1h at room temperature in Tris•Glycine•SDS buffer (Bio-Rad) then transferred overnight at 4°C at 40mAmp to 0.22mm PVDF (Bio-Rad) in Tris•Glycine buffer (Bio-Rad) with 10% methanol.The blots were placed in Block (2% BSA + 4% Dry Milk) at room temperature for 1h.The blots were then placed in primary antibody Polyclonal Goat IgG anti-mouse OSMR (R&D systems AF662) diluted in fresh block at 1:2500 and incubated on an orbital shaker at 4°C overnight.Then the blots were washed with TBS with 0.03% Tween 20 (TBS-T) for 15min and three more washes for 5 minutes each.Then the blots were placed in Peroxidase AffiniPure Bovine Anti-Goat IgG (H+L)(Jackson Immuno Research) diluted 1:5,000 in block.The blots rotated for 1h at room temperature and then were washed with TBS-T for 15min and three more washes for 5 minutes each.
The blot was then developed with (ThermoFisher) SuperSignal West Pico Plus reagents (ThermoFisher) and visualized with a Syngene G-Box.The blots were then stripped with Restore™ PLUS Western Blot Stripping Buffer (ThermoFisher) at room temperature for 5min.The blots were re-blocked and then incubated with -actin (Cell Signaling Technology clone D6A8) at 1:5000 for 1h at room temperature.Then the blots were washed with TBS-T for 15min and three more washes for 5 minutes each.Then the blots were placed in Goat Anti-Rabbit IgG (H+L)-HRP (Bio-Rad) diluted 1:5,000 in block.
The blots rotated for 1h at room temperature and then were washed with TBS-T for 15min and three more washes for 5 minutes each.The blot was then developed with

Supplemental Table 1. Differential Gene Expression Analysis of
to quantify protein expression in the lung, liver, kidney, and bone marrow isolated from Osmr fl/fl Mx1-Cre and control Mx1-Cre mice.OSMR protein expression showed modest reduction in the lung of Osmr fl/fl Mx1-Cre mice compared to control Mx1-Cre mice (Figure 2B-C).OSMR expression in the liver was undetectable in Osmr fl/fl Mx1-Cre mice, and OSMR expression in the kidney and bone marrow were reduced to very low levels in Osmr fl/fl Mx1-Cre mice.Together, these data suggest that Osmr fl/fl Mx1-Cre mice have reduced OSMR in cell lysates prepared from lung, liver, kidney, and bone marrow and that the level of reduction in OSMR protein is tissue dependent.Exploring Transcriptional Alterations in Osmr fl/fl Mx1-Cre Hematopoietic Stem Cells (HSCs) Osmr fl/fl Mx1-Cre HSCs.The top differentially expressed genes based on fold change included increased expression of Gbp2b, Myh3, Cplx3, Gpr31c and decreased expression of Wdpcp, Lgalsl, Tesk2, Tigar.Functional annotation of differentially expressed genes in Osmr fl/fl Mx1-Cre compared to control Mx1-Cre HSCs identified enrichment of signatures related to proliferation, actin binding, RNA Pol II activity and protein binding, and depletion of signatures related to smoothened pathway signaling, intraciliary transport, and protein phosphorylation (Figure 3C).These data suggest that modest transcriptional differences are observed in HSCs isolated from Osmr fl/fl Mx1-Cre compared to Mx1-Cre mice, despite lack of detectable differences in Osmr transcript itself.Next, we utilized our RNA-seq dataset to explore how control Mx1-Cre versus Osmr fl/fl Mx1-Cre HSCs respond to stimulation with recombinant OSM.Using the same stringent cutoffs defined above to identify significantly upregulated genes, control Mx1-Cre HSCs had 37 genes increased in expression following OSM stimulation compared to vehicle treatment (Figure 3D, Supplemental Table 2).Examining these 37 genes in Osmr fl/fl Mx1-Cre HSCs stimulated with OSM (Supplemental Table 3) revealed several patterns; (I) 17 genes were no longer induced by OSM in Osmr fl/fl Mx1-Cre HSCs, (II) 6 genes were induced by OSM in both control Mx1-Cre and Osmr fl/fl Mx1-Cre HSCs, and (III) 10 genes were induced by OSM in control Mx1-Cre HSCs but repressed by OSM in Osmr fl/fl Mx1-Cre HSCs.The 17 genes that were induced by OSM in control Mx1-Cre

Figure 2 .
Figure 2. Osmr fl/fl Mx1-Cre Mice Have Variable and Tissue-Dependent Osmr Expression at the Transcript and Protein Levels.(A) Relative Osmr expression assessed by real-time PCR in lung, liver, kidney, and bone marrow from Mx1-Cre control and Osmr fl/fl Mx1-Cre mice (n = 3-4).Bars show low to high values with a line at the mean.*p < 0.05 by paired t test.(B) Western blots of cell lysates from lung, liver, kidney, and bone marrow probed with antibodies against OSMR and β-actin.(C)

C
Figure 2