Methods for generating non-transformed macrophage cell lines

Abstract

1. A method for producing continuously expanding, non-transformed porcine macrophages, the method comprising culturing a cell preparation from an organ obtained from a pig in a medium supplemented with GM-CSF, thereby allowing the cell population to differentiate into self-renewing, non-transformed macrophages.

Description

[Technical field]

[0001] The present invention relates generally to macrophages and, in particular, but not exclusively, to the establishment of GM-CSF-dependent, self-renewing, non-transformed porcine macrophages. [Background technology]

[0002] Macrophages are specialized cells involved in the detection, phagocytosis, and destruction of bacteria and other harmful organisms. They can also initiate inflammation by presenting antigens to T cells and releasing molecules (known as cytokines) that activate other cells.

[0003] Macrophages are important in the defense against infection, and studying these cells in vitro is key to understanding host-pathogen interactions and aiding in vaccine development.

[0004] Macrophages have very distinct properties depending on their development and organ distribution. Previously, all tissue-resident macrophages, including alveolar macrophages (AMs), were thought to be bone marrow hematopoietic stem cell (HSC)-derived cells with a limited life span. However, recent studies have demonstrated that most tissue-resident macrophages represent separate embryonic macrophage-derived lineages that are functionally distinct (1). Most tissue-resident macrophages are macrophage colony-stimulating factor (M-CSF)-driven self-renewing cells, whereas AMs are granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent, autonomously proliferating cells. AMs have unique properties among macrophages, such as susceptibility and response to pathogens (2).

[0005] Because transformed cell lines may not accurately represent primary cells, it is best to use primary cells to examine macrophages. Macrophage pathogenesis studies primarily use cells directly isolated from organs or M-CSF-derived macrophages produced ex vivo from bone marrow progenitors or peripheral blood mononuclear cells (PBMCs) (3). GM-CSF-derived macrophages from human PBMCs can be used to model human pulmonary AM (4). Nevertheless, these methods suffer from limited cell life span, limited availability, difficult genetic manipulation, and high donor-to-donor variability, which limits the use of these methods.

[0006] Mouse models containing macrophage systems are often used to study human and animal infections due to the relative ease of access to the tissue and experimentation in vivo, however, significant differences in macrophage responses and / or intracellular pathogen growth between different mammalian species limit the utility of mouse studies (5).

[0007] Recently, we established a novel, non-transformed, continuously growing mouse macrophage model (MPI cells) from fetal liver, which provides unlimited amounts of primary cells. This robust system faithfully recapitulates in vitro the murine AM-specific response to a variety of respiratory pathogens (6).

[0008] The study of macrophages in pigs is of great importance (5). Pigs are highly susceptible to a variety of viral (e.g., African swine fever virus [ASFV], porcine reproductive and respiratory syndrome virus [PRRSV], and influenza A virus [IAV]) and bacterial (e.g., Mycobacterium avium and Salmonella spp.) pathogens that grow in macrophages and / or significantly alter macrophage function (7-9). Some of these pathogens spread from pigs to humans and cause severe diseases in them (e.g., IAV) (9). Furthermore, pig models are increasingly used in biomedical research because the physiology of pigs and humans, including airway anatomy and function and disease susceptibility, is much closer than that of mice (10).

[0009] M-CSF can be used to differentiate porcine macrophages from monocytes, and detailed analysis of LPS-stimulated macrophages showed a close functional relationship between porcine and human MCSF-derived macrophages (5). However, these MCSF-derived macrophages need to be differentiated de novo from bone marrow and, like mouse and human cells, have a limited life span. Therefore, they are not suitable for the production of viral vaccines. Suitable, robust primary, non-transformed, continuous porcine macrophage cell lines are not available for research and vaccine production.

[0010] Such macrophage lines will be required to study important pathogens such as IAV, ASFV and PRRSV.

[0011] Humans and pigs are natural hosts of IAV, and the disease can spread in both directions between these species (9). IAV is an infectious disease that is the leading cause of death in humans. AM and innate immune responses play important roles in the pathogenesis of IAV infection (11). Pigs are susceptible to a wide range of IAV strains, and are considered to be a "mixing vessel" for these pathogens, since both avian and human IAV strains are propagated in pigs. Co-infection can also give rise to novel viruses, such as the triple (avian-human-swine) reassortant H1N1 strain responsible for the recent 2009 pandemic.

[0012] ASFV and PRRSV are pathogens of great economic importance. These viruses grow almost exclusively in macrophages, making it necessary to establish a permissive macrophage system in which they can grow continuously in order to study them efficiently and to develop vaccines (7, 8). ASFV causes a devastating Ebola-like hemorrhagic syndrome in pigs. Almost all infected animals die, with approximately 1.5 million pigs and 10,000 wild boars dying from the disease in 2018 and 2019. The lack of a vaccine limits disease control, and research is directed towards a deeper understanding of how the virus interacts with and modulates macrophages' function. This is important because potential vaccine strains with deleted anti-interferon viral genes induce a vigorous innate response that limits virus growth and prevents efficient vaccine production in normal macrophages (7).

[0013] PRRSV is one of the most economically important swine pathogens, endemic in most pork-producing countries, and pulmonary AM is the main target of the virus in natural infections (8). There are two distinct PRRSV species that are rapidly diversifying, and the highly pathogenic strains that have emerged can spread rapidly and cause devastating effects. Vaccination is crucial to control the disease, but available vaccines do not provide sufficient protection, and there is a high need to develop safer and more immunogenic vaccines, for which suitable in vitro systems are crucial. Summary of the Invention

[0014] The present invention relates to the establishment of a GM-CSF-derived, non-transformed, continuous porcine macrophage system.

[0015] A method for producing continuously expanding, non-transformed porcine macrophages involves culturing a cell preparation from an organ obtained from a pig in medium supplemented with GM-CSF, thereby allowing the cell population to differentiate into self-renewing, non-transformed macrophages.

[0016] One aspect of the invention provides a method for producing continuously growing, non-transformed porcine macrophages, the method comprising culturing a cell preparation from an organ obtained from a pig in a medium supplemented with GM-CSF, thereby allowing the cell population to differentiate into self-renewing, non-transformed macrophages.

[0017] The cell preparation may be cultured using feeder cells, such as mesenchymal feeder cells.

[0018] Macrophages prepared using this method may be characterized by expression of the porcine macrophage markers CD163 and CD172a.

[0019] The cell preparation may be based on fetal pig spleen cells.

[0020] The present invention also provides a macrophage obtained or obtainable by the method described and / or defined herein.

[0021] The present invention also provides macrophages obtained by the methods described herein for use in medicine and / or medical / pharmaceutical research.

[0022] The present invention also provides macrophages obtainable by the methods described herein for use in the manufacture of a vaccine.

[0023] The present invention also provides a porcine macrophage cell line that is GM-CSF derived, continuously growing, and non-transformed.

[0024] The macrophages may be characterized by expression of the porcine macrophage markers CD163 and CD172a.

[0025] The present invention also provides a continuously growing, non-transformed porcine macrophage system.

[0026] The system may be characterized by expression of the porcine macrophage markers CD163 and CD172a.

[0027] Some aspects and embodiments of the present invention are based on the principle or observation that continuously replicating, non-transformed macrophages can be obtained from porcine hematopoietic organs using GM-CSF and appropriate mesenchymal feeder cells.

[0028] The following data have been obtained in relation to said system:

[0029] 1. To differentiate macrophages, fetal porcine spleen cells were cultured with 20 μg / ml GM-CSF. After regular medium changes and supplementation with GM-CSF, cultures containing two distinct cell types were generated: flat mesenchymal or fibroblast-like cells, on top of which round loosely attached macrophage-like cells grew in clumps, and some of the latter cells were also suspended in the medium. The development of both cells required GM-CSF, since in its absence spleen cell cultures did not generate viable cells. These cultures could be maintained for at least one year with regular medium changes.

[0030] 2. We attempted to culture the floating macrophage-like cells separately with GM-CSF. These cells survived for several weeks but did not proliferate further. The isolated floating cells were also cultured with MCSF or GM-CSF and MCSF. These MCSF cultures initially showed strong cell proliferation, but after one week the cells stopped proliferating.

[0031] 3. Suspended macrophage-like cells were transferred to cultures of porcine fetal lung fibroblasts and, similar to the original cultures containing spleen-derived fibroblasts, attached and grew on lung fibroblast feeder cells. They grew in clumps and many were suspended in the medium. These cultures were maintained for over a year with regular medium changes.

[0032] 4. Suspension cells from lung fibroblast feeder cultures are cultured with GM-CSF, but not with feeder cells. These cells survive for a long time but do not proliferate further.

[0033] 5. Suspension cells isolated from feeder-containing cultures strongly express the porcine macrophage markers CD163 and CD172a.

[0034] 6. Suspended macrophages isolated from feeder-containing cultures generate a potent cytokine response to bacterial endotoxin (LPS) stimulation.

[0035] 7. Floating macrophages isolated from feeder-containing cultures can be passaged for at least four consecutive passages on mitomycin-treated STO fibroblast cell line feeder cells containing porcine GM-CSF. They proliferate continuously, doubling in approximately 3-4 days.

[0036] 8. Culture of fetal porcine bone marrow with GM-CSF produces cultures similar to those from spleen, containing mesenchymal / fibroblast-like adherent cells and floating macrophage-like cells, which, when transferred to mitomycin-treated STO fibroblasts, proliferate in the presence of GM-CSF but do so more slowly than similar cells from spleen.

[0037] 9. Isolated floating macrophages from feeder cultures have been transduced with a second generation lentivirus expressing the SV40 large T antigen tsA58 temperature sensitive mutant (Jat, PS & Sharp, PA (1989) Mol. Cell. Biol. 9, 1672-1681). The SV40 large T antigen tsA58 mutant is heat sensitive and can be used to generate conditionally immortalized cell lines (Jat, PS & Sharp, PA (1989) Mol. Cell. Biol. 9, 1672-1681). The resulting cells (known as PLTA58 cells) grow robustly without the need for feeder cells. These data represent a major difference from previous data in Takeuchi et al., Front Vet Sci, 4:132 (August 21, 2017). They found that unconditional cell immortalization of porcine kidney-derived macrophages could only be achieved by combined expression of telomerase protein and wild-type SV40 large T antigen.

[0038] 10. PLTA58 cells grow continuously without feeder cells. We have performed at least 20 passages since we established it in December 2021. We can regularly obtain at least 10 million cells from a T75 culture flask in one week of culture. We can freeze these cells and efficiently culture them again after thawing.

[0039] 11. PLTA58 cells are factor dependent and their proliferation is enhanced by GMCSF and / or MCSF.

[0040] 12. PLTA58 cells express porcine macrophage markers.

[0041] 13. PLTA58 cells can be efficiently stimulated with ligands that induce various innate responses, such as LPS (TLR4 ligand), Fsl-1 (TLR2 ligand), poly I:C (TLR3 ligand), and R848 (TLR7 / 8 ligand).

[0042] 14. PLTA58 cells support the growth of African swine fever virus.

[0043] The different aspects and embodiments of the invention may be used separately or together.

[0044] Further particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from the dependent claims may be combined with features of the independent claims as appropriate and in combinations other than as explicitly set out in the claims.

[0045] The invention is illustrated and described in more detail by way of example in the accompanying drawings, in which:

[0046] The exemplary embodiments are described in sufficient detail to enable those skilled in the art to embody and implement the systems and processes described herein, it is important to understand that the embodiments may be provided in many alternative forms and should not be construed as being limited to the examples set forth herein.

[0047] Accordingly, while the embodiments can be modified in various ways and can take various alternative forms, specific embodiments thereof are shown in the drawings and will be described by way of example below in detail, and there is no intention to limit the invention to the particular forms disclosed, but rather to cover all modifications, equivalents and alternatives falling within the scope of the appended claims.

[0048] Unless otherwise defined, all terms (including technical and scientific terms) used herein shall be interpreted in the conventional manner in the art. Furthermore, it will be understood that commonly used terms, unless expressly defined herein, should be interpreted in the conventional manner in the relevant art, and not in an idealized or overly formal manner.

[0049] All directional terminology used herein, such as upper, lower, radial, axial, etc., is used with respect to the drawings and should not be construed as limiting the invention. [Brief description of the drawings]

[0050] Figure 1: Continuously proliferating, cells derived from porcine GMCSF express markers characteristic of porcine macrophages.

[0051] Surface markers were detected on porcine macrophages by FACS using antibodies against the scavenger receptor CD163 and the signal regulatory protein αCD172a. Histograms show unstained (blue) and stained (red) cells.

[0052] Figure 2: In response to smooth and rough LPS, porcine macrophages produce levels of TNF-α similar to those of human macrophages.

[0053] Figure 3: TNF-α responses in human GMDM and porcine macrophages are highly dependent on LBP present in fetal bovine serum (FBS). Cells were stimulated with S-LPS (100 ng / ml) and R-LPS (100 ng / ml). Supernatants were collected 16 hours post-infection and TNF-α was measured by ELISA. Bars represent the mean ± SEM from triplicate samples.

[0054] Figure 4: Decreased production of IL-6 and TNF-α in MPI and porcine macrophages, respectively, upon repeated stimulation with LPS (LPS tolerance). (a) Cells were left unstimulated (N) or stimulated with 50 ng / ml S-LPS for 16 h (T), washed with PBS and replenished with medium (N) or 50 ng / ml LPS (N+L or T+L) for 24 h. Arrows indicate stimulation with LPS. Supernatants from MPI (b) and porcine macrophages (c) were analyzed by ELISA.

[0055] Figure 5: Comparable TNF-α cytokine production in response to S-LPS and IAV in porcine macrophages cultured under various conditions. Porcine cells were stimulated with influenza A virus strain Perth / 16 / 09 at an MOI of 3 and S-LPS (100 ng / ml). Supernatants were collected 16 hours post-infection and TNF-α was measured by ELISA. n=1.

[0056] Similarities between the responses of porcine macrophages cultured with M-CSF, GM-CSF, and GM-CSF / M-CSF after obtaining from lung fibroblast feeder cultures containing GM-CSF.

[0057] The first set of questions regarding the newly developed porcine macrophage model was aimed at comparing cells growing in different conditions. These data show that these porcine macrophages can be efficiently stimulated without feeder cells, but when cultured with the growth factors used.

[0058] Figure 6: Porcine macrophages elicit an early TNF-α response after challenge with lipopolysaccharide. Porcine cells were stimulated with IAV strain Perth / 16 / 09 at an MOI of 3 and S-LPS (100 ng / ml). Supernatants were collected 16 hours post-infection and TNF-α was measured by ELISA. n=1.

[0059] TNF-α cytokine production in porcine GM-CSF-derived macrophages stimulated with S-LPS and IAV is time-dependent.

[0060] The next experiment was on the significant proinflammatory cytokine production in porcine macrophages challenged with S-LPS and IAV at various time points. Figure 6 shows the early TNF-α production in porcine macrophages stimulated with S-LPS but not IAV. Also, the IAV response is lower compared to the response induced by endotoxin.

[0061] Figure 7: Porcine and MPI macrophages transduced with lentivirus expressing RFP. These data demonstrate that porcine macrophages, like mouse MPI macrophages, can be efficiently used as targets for recombinant protein expression by lentiviral vectors.

[0062] Figure 8: Long-term culture of porcine macrophages using GM-CSF isolated from feeder cells. Multinucleated giant cells characteristic of macrophages can be seen in the normal-sized cells.

[0063] Figure 9: Porcine macrophage cells growing on lung fibroblast feeder cells (A) and transferred, floating macrophages isolated without feeders (B).

[0064] FIG. 10: Porcine macrophages grown on an STO fibroblast cell line.

[0065] FIG. 11: Factor dependence of PLTA58 cells as demonstrated by cultures with or without growth factors (GMCSF and / or MCSF).

[0066] FIG. 12: FACS data for immortalized porcine cells.

[0067] PLTA58 cells express typical porcine macrophage markers.

[0068] Figure 13: PLTA58 cells stimulated with bacterial lipopolysaccharide (LPS).

[0069] FIG. 14: PLTA58 cells stimulated with poly I:C, Fsl-1 or R848.

[0070] Although illustrative embodiments of the present invention have been disclosed in detail herein with reference to the accompanying drawings, it will be understood that the invention is not limited to the exact embodiments shown, and various changes and modifications can be made by those skilled in the art without departing from the scope of the present invention, which is defined by the appended claims and equivalents thereof. References 1. Guilliams M, Mildner A and Yona S (2018) Heterogeneity in monocyte development and function. Immunity 49(4):595-613. 2. Hussell T and Bell TJ (2014) Alveolar macrophages: plasticity in a tissue-specific context. Nature reviews immunology 14(2):81-93. 3. Geissmann F, Gordon S, Hume DA, Mowat AM, and Randolph GJ (2010) Uncovering the heterogeneity of mononuclear phagocytes. Nature Reviews Immunology 10(6):453. 4. Akagawa KS et al. (2006) Functional heterogeneity of human monocyte-derived macrophages induced by colony-stimulating factors. Respirology 11:S32-S36. 5. Kapetanovic R et al. (2012) "Porcine bone marrow-derived macrophages resemble human macrophages in their response to bacterial lipopolysaccharide." The Journal of Immunology 188(7):3382-3394. 6. Fejer G et al. (2013) "A nontransformed GM-CSF-dependent macrophage lineage is a unique model for studying tissue macrophage function." Proceedings of the National Academy of Sciences 110(24):E2191-E2198. 7. Dixon L, Islam M, Nash R, and Reis A (2019) "African swine fever virus evasion of host defense", Virus research. 8. Singleton H, Graham SP, Bodman-Smith KB, Frossard JP, and Steinbach F (2016) Establishment of porcine monocyte-derived macrophage and dendritic cell lines to study interactions with PRRSV-1. Frontiers in microbiology 7:832. 9. Nelson MI and Vincent AL (2015) Reverse zoonosis of influenza to swine: new perspectives on the human-animal interface. Trends in microbiology 23(3):142-153. 10. Walters EM, Wells KD, Bryda EC, Schommer S, and Prather RS ​​(2017) Pig models, genomic tools and services for better understanding human health and disease. Lab animal 46(4):167. 11. Pulendran B and Maddur MS (2015) Innate immune sensing and response to influenza. Current topics in microbiology and immunology 386:23. 12. Lo Iacono M et al. (2018) Wharton's Jelly "Mesenchymal stromal cells support the expansion of cord blood-derived CD34+ cells and mimic the hematopoietic niche in a direct cell-cell contact culture system.", (SAGE Publications Sage CA:Los Angeles, CA).

Claims

1. 1. A method for producing continuously expanding, non-transformed porcine macrophages, comprising: i) culturing a cell preparation from an organ obtained from a pig in a medium supplemented with GM-CSF and differentiating the cell population into self-renewing, non-transformed macrophages, the method comprising: ii) transferring the suspension macrophage-like cells from the culture of step i) to a feeder cell culture, wherein the suspension cells isolated from the feeder cell-containing culture of step ii) strongly express porcine macrophage markers CD163 and CD172a.

2. 2. The method of claim 1, wherein the cell preparation of step i) is based on fetal pig spleen cells.

3. A method according to claim 1 or claim 2, wherein the feeder cell culture in step ii) is a fetal pig lung fibroblast culture.

4. The method of claim 1 or 2, wherein the feeder cells comprise mesenchymal feeder cells.

5. A method for producing a conditionally immortalized cell line of GM-CSF-dependent and inducibly transformed porcine macrophages, comprising a step of transducing floating macrophages from a feeder culture of step ii) described in any one of claims 1 to 4 with tsA58.

6. The method of claim 1, comprising the steps of: - Fetal pig spleen cells are cultured with GM-CSF to differentiate into macrophages. - By changing the medium and supplementing with GM-CSF, two different cell types are grown in culture: squamous mesenchymal or fibroblast-like cells and round, loosely attached macrophage-like cells growing in clumps on top of each other, with some of the latter cells floating in the medium. - Suspended macrophage-like cells were transferred into fetal porcine lung fibroblast cultures and allowed to attach and grow on lung fibroblast feeder cells, resulting in cell growth in clumps, with many of these cells floating in the medium, where the floating cells separated from the feeder-containing cultures strongly express the porcine macrophage markers CD163 and CD172a.

7. A method according to claim 6, comprising introducing a second-generation lentivirus expressing a temperature-sensitive mutant of SV40 large T antigen tsA58 into floating macrophages isolated from a feeder culture.

8. A macrophage obtained by the method according to any one of claims 1 to 7.

9. 9. The macrophage of claim 8 for use in medicine and / or medical / pharmaceutical research.

10. The macrophage of claim 8 for use in producing a vaccine.

11. 9. The macrophage of claim 8, which is capable of supporting the replication of one or more of IAV, ASFV, and PRRSV.

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