Single-stranded oligonucleotides that inhibit endocytosis
阅读说明：本技术 抑制胞吞作用的单链寡核苷酸 (Single-stranded oligonucleotides that inhibit endocytosis ) 是由 彼得·约尔弗 安娜-莉娜·玛丽·斯佩兹 亚历山德拉·玛利亚·顿达尔斯卡 于 2018-09-06 设计创作，主要内容包括：本发明提供了用于预防或治疗受试者的流感病毒感染的单链寡核苷酸(ssON),其中单链寡核苷酸是抑制胞吞作用的单链寡核苷酸。本发明还提供了一种单链寡核苷酸(ssON),所述单链寡核苷酸(ssON)包含具有与表1中列出的SEQ ID NO:13-21中的任一序列或其互补序列共有至少60％序列同一性的序列的多核苷酸或由该多核苷酸组成。(The present invention provides a single stranded oligonucleotide (ssON) for use in the prevention or treatment of an influenza virus infection in a subject, wherein the single stranded oligonucleotide is a single stranded oligonucleotide that inhibits endocytosis. The invention also provides a single stranded oligonucleotide (ssON) comprising or consisting of a polynucleotide having a sequence that shares at least 60% sequence identity with any one of the sequences set forth in SEQ ID NOs 13 to 21, or the complement thereof, set forth in table 1.)
1. A single stranded oligonucleotide (ssON) for use in the prevention or treatment of an influenza virus infection in a subject,
wherein the single stranded oligonucleotide is a single stranded oligonucleotide that inhibits endocytosis.
2. The ssON for use according to claim 1, wherein the ssON is an ssON that inhibits clathrin-mediated endocytosis and/or caveolin-dependent endocytosis.
3.The ssON for use according to claim 1 or 2, wherein the ssON is a ssON that modulates Toll-like receptor signaling, preferably wherein the ssON is an ssON that inhibits TLR3 signaling.
4. The ssON for use according to any of claims 1-3, wherein said ssON inhibits endocytosis and thereby prevents entry of influenza virus into a cell.
5. The ssON for use according to any of claims 1-4, wherein said ssON is at least 25 nucleotides in length.
6. The ssON for use according to any of claims 1-5, wherein (i) at least 90% of the internucleotide linkages of said ssON are phosphorothioate internucleotide linkages; or (ii) said ssON comprises at least 4 phosphorothioate internucleotide linkages and at least 4 2' -O-methyl modifications.
7. The ssON for use according to any one of claims 1-6, wherein said ssON does not comprise a CpG motif.
8. The ssON for use according to any of claims 1-7, wherein:
(a) the ssON is at least 25 nucleotides in length;
(b) (i) at least 90% of the internucleotide linkages of said ssON are phosphorothioate internucleotide linkages; or (ii) said ssON comprises at least 4 phosphorothioate internucleotide linkages and at least 4 2' -O-methyl modifications; and
(c) the ssON does not contain any CpG motifs.
9. The ssON for use according to any one of claims 1-8, wherein said ssON comprises at least 6 phosphorothioate internucleotide linkages and at least 6 2' -O-methyl modifications.
10. The ssON for use according to any of claims 1-9, wherein all internucleotide linkages in the ssON are phosphorothioate internucleotide linkages.
11. The ssON for use according to any of claims 1-10, wherein said ssON is between 20 and 70 nucleotides or between 25 and 70 nucleotides in length.
12. The ssON for use according to any of claims 1-11, wherein said ssON is between 28 and 70 nucleotides in length.
13.The ssON for use according to any of claims 1-10, wherein said ssON is between 25 and 35 nucleotides in length.
14. The ssON for use according to any one of claims 1-13, wherein no more than 16 consecutive nucleotides of said ssON are complementary to any human mRNA sequence.
15. The ssON for use according to any of claims 1-14, wherein said ssON is not self-complementary.
16. The ssON for use according to any of claims 1-15, wherein the monosaccharide in said ssON is selected from the group consisting of 2 '-deoxyribose and 2' -O-methylribose.
17. The ssON for use according to any of claims 1-16, wherein said ssON does not bind to an influenza-specific gene or polypeptide.
18. The ssON for use according to any of claims 1-17, wherein the ssON does not comprise TTAGGG.
19. The ssON for use according to any one of claims 1-18, wherein said ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence.
20. The ssON for use of any of claims 1-19, wherein said ssON comprises at least 3 different nucleotides selected from the group consisting of A, C, G and T, or at least 4 different nucleotides selected from the group consisting of A, C, G and T.
21. The ssON for use according to any of claims 1-20, wherein the ssON comprises or consists of a polynucleotide having any one of SEQ ID NOs 1-24 listed in table 1 or a complement thereof, optionally wherein the ssON comprises or consists of a polynucleotide having any one of SEQ ID NOs 1, 3, 4, 7, 8, 10, 11, 13-22 and 24 or a complement thereof.
22. The ssON for use according to any of claims 1-21, wherein said ssON comprises or consists of a polynucleotide having a sequence that shares at least 60% sequence identity with any one of the sequences set forth in SEQ ID NOs 1-24, or the complement thereof, listed in table 1.
23. The ssON for use according to any of claims 1-22, wherein said ssON comprises or consists of a polynucleotide having any one of the sequences set forth in SEQ ID NOs 1-24 listed in table 1, or a complement thereof, wherein up to 10 nucleotides are substituted by another nucleotide.
Use of ssON in the manufacture of a medicament for preventing or treating an influenza virus infection in a subject,
wherein the ssON is an endocytosis-inhibiting ssON.
25. A method for preventing or treating an influenza virus infection in a subject, the method comprising administering ssON to the subject,
wherein the ssON is an endocytosis-inhibiting ssON.
26. The use according to claim 24 or the method according to claim 25, wherein said ssON is as defined in any of claims 1-23.
27. The ssON for use according to any one of claims 1-24 and 26, or the method according to claim 25 or 26, wherein said influenza virus is an influenza a virus.
28. The ssON for use according to any one of claims 1-24, 26 and 27, or the method according to any one of claims 25-27, wherein the influenza virus is an influenza virus that uses clathrin-mediated endocytosis and/or caveolin-dependent endocytosis to enter a host cell.
29. The ssON for use according to any of claims 1-24 and 26-28, or the method according to any of claims 25-28, wherein said ssON is administered as a monotherapy.
30. The ssON for use according to any of claims 1-24 and 26-28, or the method according to any of claims 25-28, wherein the subject is administered an additional therapeutic agent.
31. The ssON for use according to any one of claims 1-24 and 26-29, or the method according to any one of claims 25-29, wherein the subject has not been administered poly (I: C).
32. A single stranded oligonucleotide (ssON) comprising or consisting of a polynucleotide having a sequence that shares at least 60% sequence identity with any one of the sequences set forth in table 1, seq id NOs 13 to 21, or the complement thereof.
33. The ssON of claim 32, wherein the ssON comprises or consists of a polynucleotide having a sequence that shares at least 85% sequence identity with any one of SEQ ID NOs 13-21, or the complement thereof, listed in table 1.
34. The ssON of claim 32 or 33, wherein the ssON comprises or consists of a polynucleotide having any one of the sequences set forth in SEQ ID NOs 13-21 listed in table 1, or a complement thereof, wherein up to 10 nucleotides are substituted by another nucleotide.
35. The ssON of any of claims 32-34, wherein the ssON comprises or consists of a polynucleotide having any one of the sequences set forth in table 1, seq id NOs 13-21, or a complement thereof.
36. The ssON of any of claims 32-35, wherein the ssON comprises at least 6 phosphorothioate internucleotide linkages and at least 6 2' -O-methyl modifications.
37. The ssON of any of claims 32-36, wherein all internucleotide linkages in the ssON are phosphorothioate internucleotide linkages.
38. The ssON of any of claims 32-37, wherein the ssON is between 15 and 70 nucleotides or between 25 and 70 nucleotides in length.
39. The ssON of any of claims 32-38, wherein the ssON is between 28 and 70 nucleotides in length.
40. The ssON of any of claims 32-38, wherein the ssON is between 25 and 35 nucleotides in length.
41. The ssON of any of claims 32-40, wherein no more than 16 contiguous nucleotides of the ssON are complementary to any human mRNA sequence.
42. The ssON of any of claims 32-41, wherein the ssON is not self-complementary.
43. The ssON of any of claims 32-42, wherein the ssON does not comprise TTAGGG.
44. The ssON of any one of claims 32-43, wherein the ssON does not comprise at least one GGGG sequence and/or does not comprise at least one GGG sequence.
45. The ssON of any of claims 32-44, wherein the monosaccharide in the ssON is selected from the group consisting of 2 '-deoxyribose and 2' -O-methylribose.
46. The ssON of any of claims 32-45, wherein the ssON does not bind to an influenza-specific gene or polypeptide.
47. The ssON of any of claims 32-46, wherein the ssON inhibits endocytosis.
48. The ssON of any of claims 32-47, for use in medicine.
49. A pharmaceutical composition comprising the ssON of any one of claims 32-47 and a pharmaceutically acceptable carrier.
50. A composition comprising the ssON of any one of claims 32-47 and an additional therapeutic agent.
51. The ssON according to any of claims 32-47, for use in treating or preventing a disease or condition of the skin and/or subcutaneous tissue.
52. Use of the ssON of any of claims 32-47 in the manufacture of a medicament for treating or preventing a disease or condition of the skin and/or subcutaneous tissue.
53. A method of preventing or treating a disease or condition of the skin and/or subcutaneous tissue, the method comprising administering to a subject the ssON of any one of claims 32-47.
54. The use of claim 51 or 52, or the method of claim 53, wherein the disease or condition is dermatitis and/or eczema.
55. The use according to any one of claims 51, 52 and 54, or the method according to claim 53 or 54, wherein the disease or condition is atopic dermatitis and/or pruritus.
56. The use of any one of claims 51, 52, 54 and 55, or the method of any one of claims 53-55, wherein infection is associated with the disease or condition.
57. The use of any one of claims 51, 52, and 54-56, or the method of any one of claims 53-56, wherein the subject is administered an additional therapeutic agent, optionally wherein the additional therapeutic agent is an anti-inflammatory agent or an anti-pruritic agent.
58. A method for identifying a ssON for use in treating or preventing an influenza virus infection, the method comprising:
(a) providing a test ssON; and is
(b) Assessing whether the test ssON inhibits endocytosis;
wherein the test ssON that inhibits endocytosis is an ssON useful in the treatment or prevention of influenza virus infection.
59. The method of claim 57, wherein said method further comprises testing said test ssoN in an animal model of influenza virus infection.
Example 1: extracellular single-stranded oligonucleotides modulate endocytic uptake and downstream Toll-like receptor response
SUMMARY
In order to maintain essential cellular functions and control the uptake of endosomal molecules, the endocytic pathway needs to be tightly regulated. However, there is a lack of understanding of extracellular molecules that regulate endocytic uptake. In this study, we demonstrated that single stranded oligonucleotides (ssons) inhibit some endocytic pathways in human monocyte-derived cells (modcs) without affecting others. Both single-stranded DNA and RNA confer endocytosis inhibition. It is affected by concentration and ssON length. ssON-mediated inhibition modulates downstream cellular signaling of Pattern Recognition Receptors (PRRs) localized in the affected endosomal pathway. The introduction of non-natural linkages and/or modified nucleosides is not necessary for inhibition, but is tolerated and may be necessary in order to maintain efficacy over a longer period of time. We provide evidence that ssON exerts an immunomodulatory effect following intradermal injection in macaques. These studies reveal the regulatory role of extracellular ssons in the endocytic uptake of Toll-like receptor (TLR) ligands and provide a mechanistic explanation for their immune regulation. The ssON-mediated endocytosis interference (SOMIE) was identified as a regulatory process that temporarily suppressed TLR3/4/7 signaling, thereby avoiding excessive immune responses.
Introduction to the design reside in
Endocytic mechanisms control the lipid and protein composition of the plasma membrane, thereby regulating how the cell interacts and communicates with its surrounding environment. In order to respond properly and affect their environment, cells must tightly regulate plasma membrane composition and uptake of ligands into endosomes (Doherty and McMahon, 2009). For example, the removal of cell surface receptors has made possible long-term sensitivity to specific ligands (Irannejad and Zastrow, 2014). However, endocytosis not only negatively regulates the interaction with the external environment. These processes also control basic mechanisms such as mitosis, cell migration, and immunoregulatory processes such as the initiation of innate immune responses and antigen presentation (Doherty and McMahon, 2009). Immunomodulation stems from the fact that: many pathogens use the endocytic pathway to mediate their internalization (Cossart and Helenius, 2014). Likewise, uptake of nucleic acids released from microorganisms or dead cells by endocytosis can activate TLRs localized in endosomes (Kawai and Akira, 2010).
Although there is much understanding of the different endocytic structures of the cargo, it is not clear how the specific mechanisms by which these cargo are engaged, internalized, and how their uptake is regulated. More and more endocytic pathways are reported in the literature, to varying degrees described and illustrated. In this study, it has been evaluated whether extracellular ssON modulates three distinct pathways: clathrin-mediated endocytosis (CME), caveolin-dependent endocytosis (CDE), and megakaryocytic action (MPC) (Doherty and McMahon, 2009).
CME is mediated by small (100nm diameter) vesicles with a coating consisting of clathrin. Clathrin-coated vesicles (CCVs) are found in almost all cells and form a region of the plasma membrane known as the clathrin-coated fossa. These CCVs are responsible for removing GCPR from the outer plasma membrane, reducing their signaling, and desensitizing cells to specific ligands. In addition, these crypts can concentrate receptors responsible for receptor-mediated endocytosis of ligands such as low density lipoprotein, transferrin, and several other ligands (Kumari et al, 2010), including dsRNA and its analogs pI: C (Itoh et al, 2008).
CDE is one of the most extensively studied endocytic pathways that are not clathrin-coated. It is present on the surface of many cell types and consists of caveolin proteins and a bilayer rich in cholesterol and glycolipids. The pits are small (about 50nm in diameter) bottle-like depressions in the film. The cryptic pathway is responsible for endocytosis of ligands such as albumin, tetanus toxin, and cholera toxin subunit b (ctb) (Kumari et al, 2010).
MPC occurs in a highly wrinkled region of the plasma membrane, which is then pinched off into the cell to form larger vesicles (0.5-5 μm in diameter) filled with large amounts of extracellular fluid and molecules. Uptake indicators include liquid phase markers such as dextran (Doherty and McMahon, 2009).
Nucleic acid sensing TLRs are uniquely targeted to endosomes and stimulate moDC maturation in vitro (Isomura et al, 2005). TLR3, TLR7, TLR8 and TLR9 initiate an immune response to infection by recognizing microbial nucleic acids in endosomes. This localization of TLRs in different intracellular compartments plays an important role in activating responses to foreign nucleic acids while maintaining tolerance to self-nucleic acids (Roers et al, 2016). TLR signaling constitutes a defense mechanism during viral infection in response to nucleic acids present in, for example, the viral genome or intermediates thereof during the replication cycle (Amarante and Watanabe, 2010; Vercammen et al, 2008). The dsRNA analogue pI: C is often used as a TLR3 agonist, and it has been shown to be dependent on CME to enter the endocytic pathway (Itoh et al, 2008).
To assess whether extracellular ssons have the ability to modulate endocytic pathways, we used herein a cell model system that allows fluorescence-based uptake studies using endocytic markers (e.g., transferrin CTB and dextran), as well as downstream cellular pathways from "signaling endosomes" (Scita and Di fiere, 2010; Sorkin and Zastrow, 2009). Cell signaling from endosomes occurs against receptors such as NOTCH family members, tumor
Dendritic Cells (DCs) are antigen presenting cells that integrate stimuli from the environment, resulting in immune activation or induction and maintenance of immune tolerance. DCs consist of developmentally and functionally distinct subsets that differentially modulate T cell responses, acting as messengers between the innate immune system and the adaptive immune system (Merad et al, 2013). DC subsets are present in tissues in contact with the external environment, such as the skin and respiratory system, and the lining of the intestine. DCs exhibit a wide variety of endocytic pathways due to their role as external environmental sensors. In addition, as messengers between the innate and adaptive immune systems, they respond to several trigger molecules, such as TLRs that sense ONs, that are taken up and processed in the endocytic pathway. In contrast, DNA containing a repetitive TTAGGG motif derived from mammalian telomeres can suppress the immune response (Gursel et al, 2003). Synthetic ss-DNA molecules with immunosuppressive functions are being studied in preclinical models and differ in size, sequence and nucleotide backbone, but their mechanism of action is often poorly understood (Bayik et al, 2016).
It is well established that ON does not pass passively through the plasma membrane, and intracellular delivery requires transfection reagents or other membrane-permeable methods (Sharma and Watts, 2015; Silva et al, 2015). However, free ONs are thought to be taken up by integrins and/or scavenger receptors (Juliano and Carver, 2015). To our knowledge, it is not clear whether there is a difference in cellular uptake between ssON and dsON. Here, we compared the endocytic capacity of ssons with different lengths and sequences. We show that ssons 35 bases in length are not endocytosed by the madc, but rather inhibit endocytosis of transferrin, CTB and the dsRNA analogue polyinosine-polycytidylic acid (pI: C), but do not inhibit dextran uptake. The introduction of non-natural phosphorothioate linkages (PS) increased the inhibitory effect, but the naturally occurring natural Phosphodiester (PO) ssON and RNA pI: C purified from the cells induced IL-6 production in short-term cultures.
We previously reported that CpG-containing ssons inhibit TLR3 signaling in primary human monocyte-derived cells (modcs) expressing TLR3/4/8 but lacking
Results
Extracellular ssON inhibits the endocytosis of transferrin and CTB
Here, we investigated whether extracellular ssON could inhibit the uptake of common endocytic pathway markers in human modcs (without any transfection method or delivery system). Human mocc cells were treated with endocytic uptake markers (transferrin, CTB and dextran and LDL) for 45min at +37 ℃ or +4 ℃ with or without 0.5 μ M ssON (sequence in table a). At +37 ℃, transferrin, CTB and dextran as well as LDL were taken up as measured by flow cytometry (fig. 1A-D, J). The fluorescent signals of transferrin and CTB were significantly suppressed by the addition of 0.5 μ M ssON35 PO (fig. 1A, B, G and H). Furthermore, we found that the fluorescence uptake signal of transferrin and CTB as well as LDL was completely blocked by the addition of 0.5 μ M modified ssON35 PS (fig. 1C, D, G, H and J), while MPCs remained unaffected by ssON35 PS treatment, as demonstrated by the same uptake of dextran at +37 ℃ with or without the addition of 0.5 μ M ssON35 PS (fig. 1E). No endocytosis occurred at +4 ℃ showing that transferrin and CTB uptake was completely inhibited by the addition of 0.5. mu.M ssON35 PS (at 37 ℃) (FIGS. 1A, B, G and H). ssON35 PS itself showed identical uptake at +4 ℃ and +37 ℃, showing that fluorescently labeled ssON35 PS stayed extracellular and were not internalized into the endocytic pathway (fig. 1F and I). Taken together, these results show that both native ssON PO and ssON PS can significantly inhibit CME and CDE, but MPC remains unaffected, suggesting that ssON plays a regulatory role in cargo-to-endosome uptake.
Length requirement for the ability of extracellular ssON to inhibit TLR3 ligand uptake and subsequent TLR3 activation in modcs
C, a dsRNA analogue, is a TLR3 agonist that is taken up by CME (Itoh et al, 2008). We next ask the question: whether ssON35 PS is able to block pI: C mediated maturation of modcs and, if it is, by inhibiting endocytic uptake (fig. 2). C-dependent moDC maturation was monitored 48 hours post-treatment by measuring the expression of co-stimulatory molecules CD86, CD83 and CD80 in CD1a positive cells (CD83 and CD80 data not shown), and the secretion of the proinflammatory cytokine IL-6 at 24 hours post-treatment. We found that ssON significantly blocked pI C-mediated induction of CD86, EC, in a concentration-dependent manner50Between 100 and 200nM (FIG. 2A, B). We also detected that ssON35 PS significantly inhibited pI: C-mediated IL-6 secretion by moDC (fig. 2C). Similar to the uptake of endosomal markers transferrin and CTB, inhibition of IL-6 was enhanced by stability modification independent of ssON (fig. 2D). When makingWith the naturally occurring PO backbone, inhibition was absent 48 hours after initial treatment (data not shown). However, when studying inhibition at an earlier time point, ssON with a PO backbone has the same potency as PS ssON. This effect lasted 3 hours and remained present after 4 hours (fig. 2D). After 5 hours, the effect completely disappeared (data not shown). Microscopy studies also demonstrated that uptake of fluorescently labeled pI: C in modcs was effectively blocked by the addition of 0.5 μ M ssON35 PS (fig. 2E, F).
Then, we ask the question: whether the observed inhibition of endocytosis and downstream TLR3 activation can be correlated with a particular ssON sequence. We compared three different ssON sequences (ssON 35PS, ssON GtA PS and ssON ComplPS; Table A), which all show the same inhibitory effect in madC treated with pI: C for 48 hours (FIG. 2G). ssON GtA is based on the parental sequence ssON35, but all guanosine (G) bases are replaced by adenosine (a), while ssON Compl is the complement of
After concluding that inhibition is not strictly dependent on the ssON sequence, we next evaluated whether ssON length affects potency. We found that the shorter ssON did not have the same inhibitory effect as the longer ssON, with a length cut-off between 20 and 25 bases (fig. 2H, I). Microscopy studies showed that fluorescently labeled
Taken together, these data demonstrate that ssON is able to inhibit the endocytic uptake and downstream effects of the TLR3 agonist pI C in a concentration-dependent manner, and that these effects are not associated with a particular ssON sequence or motif. ssON length does affect inhibition and has a cut-off between 20-25 bases.
Kinetics of ssON-mediated inhibition of TLR signaling
If extracellular ssON is capable of inhibiting certain endocytic pathways, the biokinetics should follow the endosomal uptake studies previously performed in modcs (Choi et al, 2013; Smole et al, 2015). Therefore, we assessed the kinetics of activation and inhibition of mocc maturation by exposing cells to TLR3 agonists pI: C or inhibitory ssON35 PS, and subsequently pulsing the cells with ssON35 PS/pI: C over a period ranging from 5 minutes to 24 hours. Co-stimulation of CD86 expression and IL-6 secretion is considered to be a marker of pI C-induced maturation of the mocCs. Exposure of cells to pI: C followed by ssON35 PS pulse showed that inhibition was present for up to 50 minutes and then rapidly decreased and disappeared about 2h after the initial pI: C treatment (fig. 3A, B). MoDC treated in the reverse manner, i.e., treated with ssON35 PS first, and then pulsed with pI: C, showed no signs of maturation or cytokine production up to 24 hours after the initial priming (FIG. 3C, D). Addition of ssON35 PS inhibited TLR3 activation for up to 50-60min after agonist exposure (fig. 3A). According to previous studies, human modcs take about 35-60min to endocytose extracellular compounds (Smole et al, 2015). In summary, our findings indicate that addition of ssON35 PS cannot inhibit pic-induced moDC maturation in cases where the TLR3 agonist is endocytosed and initiation of TLR3 signaling has been initiated. However, at an earlier point in time, ssON35 PS suppresses this process.
Prevention of pI: C mediated cell death in human mocCs by ssON
pI C can induce human apoptosis (Sun et al, 2011). To assess whether ssON could prevent this, the modcs were treated with pI: C with or without ssON35 PS addition and apoptosis was measured by Live/Dead cell staining kit using flow cytometry. Cells treated with pI: C alone had about a 20% reduction in viability. By adding 0.5 μ M ssON35 PS, the frequency of cell death decreased back to the level of untreated cells 48 hours after the initial treatment (fig. 3E).
Inhibition of pI C uptake occurs against ssON, not dsON
The data we provide here demonstrate that the ability of extracellular ssON to inhibit endocytic uptake is dependent ON the ON length and not ON its sequence (figure 2). To assess whether the introduction of dsON affected endocytic uptake, ssON35 PS was kept at a constant concentration (0.5 μ M) while increasing concentrations of the complementary strand (
Inhibition of ssON occurs against TLR3 and TLR7 localized in endosomes, but does not inhibit membrane-permeable TLRs Effect of agonists
Since TLRs that sense ssON are dependent on endocytosis and signaling occurs from signaling endosomes (Sorkin and Zastrow,2009), we next evaluated whether ssON has the ability to block TLR-induced moDC maturation by membrane-permeable TLR agonists. R848 is a membrane-permeable TLR7/8 agonist and therefore does not rely on endocytosis to activate TLR signaling (Govindaraj et al, 2011). CL307 is a TLR7 agonist coupled to spermine and therefore its cellular uptake is dependent on endocytosis (Soulet et al, 2002). modcs lack the expression of TLR7 that induces ssON, so these experiments were performed in Peripheral Blood Mononuclear Cells (PBMCs). PBMCs were treated with pI C (TLR3 agonist), CL307(TLR7 agonist) or R848 (membrane permeable TLR7/8 agonist) in the absence or presence of ssON35 PS (fig. 3G). ssON35 PS significantly reduced IL-6 secretion from PBMC treated with pI: C and CL307, but not after R848 stimulation. These data demonstrate that extracellular ssON35 PS has the ability to inhibit TLR-induced pro-inflammatory IL-6 secretion only when the agonist relies on endocytosis to reach its receptor.
ssON-mediated inhibition of TLR4 signaling is dependent on endosomal uptake
TLR4 responds to lipopolysaccharide stimulation and has two distinct signaling pathways (Kagan et al, 2008; Rajaiah et al, 2015; Tatematsu et al, 2016). One is mediated by MyD88 and occurs at the plasma membrane, leading to NFkB activation. The second pathway is mediated by TRIF, stimulating a strong interferon response. TRIF-mediated signaling occurs in clathrin-coated endosomes and is therefore CME dependent (FIG. 4A). Therefore, we concluded that the addition of ssON35 PS together with TLR4 agonists should primarily inhibit the interferon response of modcs, as extracellular ssON inhibits the CME pathway (fig. 1). To validate this hypothesis, we treated modcs with the TLR4 agonist LPS and ssON35 PS simultaneously. Signalling through the MyD 88-dependent pathway is assessed by secretion of IL-6 and IL-10, and the TRIF-dependent pathway is assessed by secretion of IL-29 and CXCL10 (Rosadini et al, 2015). Secretion of IL-6 and IL-10 downstream of the MyD 88-dependent pathway remained unaffected when the modcs were exposed to LPS and ssON35 PS ssON (fig. 4B and C), but we found that CXCL10 and IL-29 were significantly inhibited (fig. 4D and 4E). This demonstrates that the addition of ssON35 PS selectively inhibits one of two pathways derived from the same TLR. One is dependent on endocytosis and the other is not. To confirm that LPS was taken up into cells, and ssON inhibited this uptake, we exposed modcs to Alexa-488 labeled LPS and quantified uptake by flow cytometry. We found that the fluorescence signal was significantly increased after incubation at 37 ℃ compared to 4 ℃ and, in addition, LPS uptake was significantly inhibited in the presence of ssON35 PS at 37 ℃ (fig. 4F and G). LPS uptake was confirmed by microscopy and also shown to be inhibited in the presence of ssON at 37 ℃ (fig. 4H and I).
Inhibition of internal body uptake can be achieved with both DNA and RNA
To elucidate whether the ability to inhibit endocytic uptake is unique to DNA, or whether RNA also has this activity, we synthesized 35mer ssON RNA and DNA analogs (table a). A native, unmodified 35-mer RNA had no effect on endocytic uptake by the mocC (data not shown). However, ON DNA analogue 2' OMe and total RNA purified from modcs had the ability to inhibit endosomal uptake (fig. 5A and B). pI C-induced IL-6 secretion was reduced by ssON 2' OMe PS to a level comparable to ssON35 PS (FIG. 5C). Using the same assay, total RNA (totRNA) purified with a cut-off filter below 200b reduced IL-6 production in a concentration-dependent manner (FIG. 5D). These data show that ssons with the ability to inhibit endocytic uptake of human modcs can be derived from DNA or RNA.
Administration of ssON35 PS modulates dsRNA-mediated inflammation in cynomolgus monkey skin
To assess whether stabilized ssON35 PS mediates effects in vivo, we measured the overall intrinsic response to pI: C in the presence or absence of ssON35 PS by transcriptomic profiling of skin biopsies taken from the injection site. We found that intradermal injection of pI: C resulted in the induction of multiple innate immune response genes compared to PBS (fig. 6A). Gene Ontology (Gene Ontology, GO) -annotation of genes with significantly regulated pI: C after injection (p <0.05) showed Gene enrichment associated with GO process "defense response" (false discovery rate (FDR) q-value: 2.85E-48) and "immune system process" (FDR q-value: 1.14E-47) compared to PBS control. Furthermore, GO function "cytokine activity" and "chemokine activity" were enriched (FDR q values: 7.95E-14 and 2.25E-10, respectively). To gain more insight into which genes in the skin showed modulated expression, we compared the group receiving only intradermal injections of pI: C with the group receiving pI: C/ssON and calculated the fold change between the two groups and the significance of the difference (fig. 6B). The results show that chemokines and genes associated with inflammatory conditions were among the most downregulated genes when pI: C/ssON 35PS co-administration was compared to pI: C treatment alone (FIG. 6C upper panel). It can be noted that expression of IL-6 was in the most downregulated gene (negative FC >2p <0.05) (fig. 6D), supporting in vitro data (fig. 2C). We also verified IL-6 secretion by analyzing aliquots of filtered dermal supernatant collected without additional in vitro stimulation (fig. 6E). Significant induction of IL-6 was detected after pI: C treatment (p <0.05), and induction of IL-6 was reduced after pI: C/ssON 35PS treatment. Taken together, our data show that pI: C induces a significant inflammatory feature locally in cynomolgus monkey skin, a phenomenon that is suppressed by co-administration of ssON35 PS.
Experimental procedures
Single-stranded oligonucleotides
Fully PS substituted ssON35 PS and 2' OMe PS were prepared from Integrated DNA Technologies. Other modified oligonucleotides were purchased from Eurofins. For the sequence, see table a. Total RNA was purified from modcs using RNeasy total RNA purification kit according to the manufacturer's instructions (Qiagen).
TLR ligands and labeled endosomal markers
The TLR3 ligand high molecular weight pI C (25. mu.g/mL unless otherwise noted), TLR7 ligand CL307, and TLR7/8 ligand R848 were all purchased from Invivogen. An endosomal marker; transferrin-
Uptake study of MoDC
Human monocytes (1mL/10mL buffy coat; StemShell Technologies) were negatively selected from buffy coat using the RosetteSep monoclonal enzyme kit and 5 × 10 in RPMI 1640 (all from Invitrogen Life Technologies) supplemented with 10% FCS, 1mM sodium pyruvate, 10mM HEPES, 2mM L-glutamine, and 1% streptomycin and penicillin5Density of individual cells/mL with GM-CSF (250 ng/mL; PeproTech) and IL-4(6.5 ng/mL; R&D Systems) for 6 days to differentiate human monocytes into modcs. The modcs were exposed to endosomal markers and ssON in intact 10% RPMI medium (or serum-free medium for PO ON uptake studies) ON ice and then transferred to 37 ℃ for 45 min. After incubation, cells were placed back on ice, washed and fixed, and uptake was monitored by flow cytometry. CME was monitored by uptake of 50. mu.g/ml Alexa 647-labeled transferrin. CDE was monitored by 1. mu.g/ml Alexa 488-labeled cholera toxin B, and MPC was monitored by 0.5mg/ml Texas-Red-labeled dextran. Endocytic uptake of TLR4 was monitored by 1 μ g of Alexa 488-labeled LPS. Uptake of LDL was measured by quantifying uptake of DiL-labeled LDL (molecular probes).
MoDC maturation study
Human monocytes were differentiated as described above. The modcs were exposed to pI: C and ssON and maturation was assessed after 48 hours using monoclonal antibodies (abs) targeting CD1a and the moDC maturation markers CD86, CD83 and CD80 (all abs from BDBiosciences). Flow cytometry sample data were obtained on fortessa (bd biosciences) and analyzed with FlowJo software (TreeStar).
Cytokine/chemokine secretion study
Supernatants were collected from cells at specific time points after treatment (pI: C, CL307, R848(Invivogen) or LPS (Sigma)) and cytokine/chemokine secretion was measured by standard ELISA according to the manufacturer's instructions (Mabtech; IL-6, IL-10 and IL-29. Life technologies: CXCL 10). The protein amount was monitored by 3,3',5,5' -Tetramethylbenzidine (TMB) absorbance at 450nm and is given in pg/ml or relative absorbance units.
Wide field microscopy
The modcs were allowed to adhere to poly-L-lysine coated glass slides in intact RPMI medium without phenol red for 2-4 h. Cells were treated with the indicated ligands at 37 ℃ for 45min in the presence or absence of ssON35 PS. Unbound ligand was washed away and cells were covered with whole medium and images were obtained in a wide field Cell Observer microscope using a 40X lens at 37 ℃. Images were obtained and analyzed using the
Kinetic analysis of TLR3 inhibition
To elucidate the kinetics behind the observed inhibition of TLR3 signaling, the modcs were exposed to 25 μ g/ml pI: C/0.5 μ M ssON35 PS and then pulsed with 0.5 μ M ssON35 PS or pI: C, respectively, for a period of from 5 minutes to 24 hours. Cells were analyzed by flow cytometry for expression of the moDC maturation markers CD80 and CD86 48 hours after initial exposure. In addition, at 48 hours after the treatment, supernatants were collected from the cells, and secretion of proinflammatory IL-6 was measured by elisa (mabtech).
Viability test
Surviving cells were selected from the total cell population 48 hours after initial treatment with 25. mu.g/ml pI: C/0.5. mu.M ssON35 PS. Surviving cells were assessed using the Live/Dead fixable near IR staining kit according to the manufacturer's protocol (Life technologies). Sample data was obtained using fortessa (bd biosciences) and analyzed using FlowJo software (TreeStar corporation).
Mo by dsONDC maturation Studies
5 μ M ssON35 PS was preincubated with 1-10 μ M complementary ssON35 PO in PBS at 55 ℃ and then left at ambient temperature for 30 minutes. The ON mixture was added to MoDC at a final concentration of 0.5. mu.M ssoN35 PS along with 25. mu.g/ml pI: C. MoDC maturation was assessed as described above.
Animals and injections
Adult cynomolgus monkeys (Macaca fascicularis) were treated according to the European NHP nursing guidelines (European guidelines for NHP care) (EU Directive N63/2010) (N18). The study was approved and approved by the CEA Ethical Animal Committee (Ethical Animal Committee of the CEA) "acceptance d' Ethiqueen Exp acceptance Animal", according to the designation 12-013, which was registered by the French institute (French research Ministry) according to number 44. Animals were treated under anesthesia and injected intradermally in the left upper and right lateral flank with 170 μ g pI: C alone or with 170 μ g ssON in 100 μ L PBS or PBS alone. Skin biopsies (8 mm diameter) were collected from anesthetized animals 24 hours after injection.
Cytokine secretion assay in macaques
To assess Cytokine and chemokine production directly ex vivo from macaque skin biopsies, aliquots of filtered dermal supernatant were collected and measured on a Bio-plex device (Bio-Rad) using MILLIPLEX MAP NHP Cytokine Magnetic Bead pad (Millipore) according to the manufacturer's instructions.
Microarray analysis
Using Tissue
Statistical analysis
The non-parametric Mann-Whitney test was used to compare the presented data. The number of donors and replicates is illustrated in each figure legend. P value: not significant (n.s) P > 0.05; p is less than or equal to 0.05; p is less than or equal to 0.01; p is less than or equal to 0.001.