Use of M-CSF for preventing or treating myeloid cytopenia and related complications

Abstract

The invention relates to methods and compositions comprising a macrophage colony stimulating factor (M-CSF) polypeptide or an agonist of the M-CSF receptor for preventing or treating myeloid cytopenia and related complications (such as infections) in a patient in need thereof (such as a patient undergoing hematopoietic stem cell transplantation).

Claims

1. A method of treating myeloid cytopenia and an associated viral, bacterial and/or fungal infection in a patient in need thereof, comprising: increasing production of myeloid cells expressing transcription factor PU.1 in said patient by administering to said patient a therapeutically effective amount of a macrophage colony stimulating factor (M-CSF) polypeptide or an interleukin-34 polypeptide, wherein said patient is undergoing or has undergone hematopoietic stem cell transplantation (HSCT), wherein expression of MafB in cells of a stem cell graft used for the HSCT was not previously modified, and wherein said myeloid cytopenia is neutropenia, monocytopenia and/or cytopenia of monocyte derived mononuclear phagocytes, wherein said myeloid cells comprise granulocyte/monocyte progenitor cells that give rise to granulocytes, monocytes and macrophages.

2. The method according to claim 1, wherein myeloid cytopenia is induced by a myeloablative therapy.

3. The method according to claim 1, wherein the M-CSF polypeptide has the sequence comprising or consisting of SEQ ID NO: 5.

4. The method according to claim 1, further comprising, prior to said increasing step, incubating a hematopoietic stem cell graft to be used for HSCT in the presence of the M-CSF polypeptide or the interleukin-34 polypeptide to produce primed cells and infusing the primed cells into said patient.

5. The method according to claim 1, wherein myeloid cytopenia and an associated viral infection is treated.

6. The method according to claim 5, wherein said viral infection is a cytomegalovirus infection.

7. A method for treating viral, bacterial and/or fungal infections associated with myeloid cytopenia in a patient that is undergoing or has undergone hematopoietic stem cell transplantation (HSCT), comprising: administering to said patient a therapeutically sufficient amount of an M-CSF polypeptide or an interleukin-34 polypeptide to cause increased production of myeloid cells expressing transcription factor PU.1 in said patient and thereby reduce viral, bacterial and/or viral infections in said patient, wherein expression of MafB in cells of a stem cell graft used for the HSCT was not previously modified, wherein said myeloid cells comprise granulocyte/monocyte progenitor cells.

8. The method of claim 7, wherein said viral, bacterial and/or fungal infection is a cytomegalovirus infection.

9. The method of claim 7, wherein said granulocyte/monocyte progenitor cells give rise to granulocytes, monocytes and macrophages.

10. The method of claim 7, wherein said M-CSF polypeptide comprises or consists of SEQ ID NO. 5.

Description

FIGURES

(1) FIG. 1: M-CSF activates the myeloid master regulator PU.1 in HSC.

(2) a-c) Representative FACS profile (a) and quantification of GFP expression in HSC (b) or CD150.sup.hiHSC (c) of PU.1-GFP reporter mice 16 h after control (PBS) or M-CSF injection. ***p=0.009; **p=0.03.

(3) d) Quantitative RT-PCR analysis of PU.1 expression normalized to GAPDH expression (R.U.) in sorted HSC after 16 h culture in the absence or presence of M-CSF, GM-CSF or G-CSF. Error bars show standard deviation of duplicates.

(4) FIG. 2: Continuous video-imaging of PU.1+ cell generation from individual PU.1 negative HSC.

(5) a) GFP-fluorescence intensity at 10 minute intervals (dots) and sliding median (lines) over 12 h observation time of 3 individual GFP negative sorted HSC from PU.1-GFP reporter mice after 18 h in M-CSF culture, representative of cells quantified in FIG. 2e (n=39). Green: cells activating GFP, black: cell remaining GFP negative.

(6) b) Still photos taken at times indicated by symbols in a) of fields with 2 representative HSC (cells A,B) showing activation of PU.1 at different time points. Cell C was outside of the shown field.

(7) c) Still photos taken at 40 min intervals over 8 h of 3 representative HSC in M-CSF culture without (cell C) or with activation of PU.1 (cells A,D), representative of cells quantified in FIG. 2e (n=39).

(8) d) Quantification of PU.1+ cells derived from PU.1 negative HSC (committed cells) with (n=39) or without M-CSF (n=42) as percentage of total cells after 24 h observation period. *p<0.1.

(9) e) Timing of PU.1 activation in PU.1 negative HSC of cells shown in d) over 24 h observation period.

(10) FIG. 3: M-CSF activates PU.1 and instructs myeloid identity in single HSC.

(11) a) Individual PU.1+ cells with a myeloid gene expression profile (blue) or expressing other lineage genes (white) as a percentage of total cells. *** p=0.009 (0 h), and 0.005 (-M-CSF).

(12) b) Experimental design for transplantation of sorted PU.1 and PU.1+ HSC from in vivo M-CSF primed CD45.2 PU.1-GFP mice into sub-lethally irradiated CD45.1 recipients and analysis of progeny cells after 2 weeks in the spleen.

(13) c,d) Representative FACS profiles (c) and quantification of the ratio (d) of donor GMP and MEP progenitors derived from transplanted PU.1 or PU.1+ HSC before or after M-CSF stimulation in vivo. **p=0.05, ***p=0.01, n=4, 8, 4.

(14) FIG. 4: M-CSF stimulates a reversible, PU.1-dependent myeloid differentiation preference in single HSC in vivo.

(15) a) Ratio of donor GMP to MEP progenitors in the spleens of sub-lethally irradiated recipients 2 weeks after transplantation of in vivo M-CSF primed or control HSC. Experimental design is shown in FIG. 6. ***p=0.003, n=8, 9.

(16) b) Donor contribution to blood of competitively reconstituted mice 4 weeks and 6 weeks after transplantation of M-CSF primed or control HSC, expressed as ratio of CD11b+ myeloid cells to platelets or CD19+ lymphoid cells. ***p=0.01, n=10.6, *p=0.07, n=6, 4.

(17) c) Donor contribution to Mac+ myeloid cells in the spleen of sub-lethally irradiated recipients 2 weeks after transplantation of control or M-CSF primed HSC with control (fl/fl) or deleted (/) PU.1 alleles. **p=0.05, n=6, 4, 5.

(18) FIG. 5: Differentiation potential of M-CSF induced PU.1+ cells.

(19) a) Experimental design for transplantation of sorted CD45.2 PU.1 HSC before and after induction of PU.1+ cells in M-CSF culture into sub-lethally irradiated CD45.1 recipients and analysis of progeny cells after 2 weeks in the spleen.

(20) b,c) Quantification of the ratio of donor GMP to MEP progenitors (b) and total GMP (c) derived from transplanted PU.1 HSC before or PU.1+ cells after M-CSF culture. **p=0.02, *p=0.07, n=6, 7.

(21) FIG. 6: Differentiation potential of M-CSF primed HSC.

(22) a) Experimental design for transplantation of in vivo M-CSF primed CD45.2 HSC into sub-lethally irradiated CD45.1 recipients and analysis of progeny cells after 2 weeks in the spleen or 4 weeks in the blood.

(23) b) Percentage of GMP and MEP progenitors in total donor cells derived from control (PBS) or M-CSF primed HSC in the spleen 2 weeks after transplantation.

(24) *p=0.1, **p=0.04, n=4, 4

(25) c) Quantification of the ratio of donor CD11b+ SSClo monocytes to CD19+ B-cells in the blood 4 weeks after transplantation. ***p=0.009, n=8, 4.

(26) FIG. 7: Competitive transplantation of M-CSF primed HSC

(27) a) Experimental design for competitive transplantation of FACS sorted in vivo M-CSF primed HSC (CD150+CD34-CD135-KSL) from actin-GFP CD45.2 mice together with CD45.2 competitor HSC into lethally irradiated CD45.1 recipients and analysis of blood cell contribution.

(28) b) Gating strategy for quantification of actin-GFP+ HSC derived platelets, lymphoid and myeloid blood cells.

(29) c) Donor contribution to blood of competitively reconstituted mice 4, 6 and 14 weeks after transplantation of M-CSF primed or control HSC, expressed as percentage of GFP+ donor cells in Mac+ myeloid, CD19+ B Cells, CD61+ Platelets (4, 6 and 14 weeks) and CD3e T Cells (14 weeks) and normalized to total GFP contribution in CD45.2 donor compartment. ** p=0.03, n=6, 4.

(30) FIG. 8: MCSF stimulation following Hematopoietic Stem and Progenitor Cells (HS/PC) transplantation protects against Pseudomonas aeruginosa infection (p<0.01).

(31) a) rhMCSF treatment

(32) b) Bac. virus mMCSF treatment

(33) FIG. 9: MCSF stimulation after HS/PC transplantation reduced bacterial tissue load (***P<0.01).

(34) FIG. 10: MCSF stimulation following HS/PC transplantation protects against Aspergillus fumigatus infection (p<0.01).

(35) a) rhMCSF treatment

(36) b) Bac. virus mMCSF treatment

EXAMPLE 1

M-CSF Instructs Myeloid Lineage Fate in Single Haemotopoietic Stem Cells

(37) Material & Methods

(38) Methods Summary:

(39) Flowcytometry, bone marrow transplantation and in vivo immunofluorescence of HSC were performed essentially as described.sup.3. Single cell nano-fluidics-based real-time PCR was performed using a BIOMARK HD system and 48.48 dynamic arrays (FLUIDIGM, CA, USA) and videomicroscopy analysis followed proposed standards.sup.24. Details of procedures and reagents are described in Supplementary Methods.

(40) Mice:

(41) CD45.1 and C57Bl/6 mice were obtained from Charles River. PU.1-GFP.sup.31 M-CSFR.sup./27 and PU.1.sup.fl/fl 32 mice have been described. Age- and sex-matched CD45.1 recipients that were reconstituted as described.sup.3 with CD45.2 foetal liver from wt or M-CSFR.sup./ embryos.sup.27 and PU.1.sup.fl/fl or PU.1.sup.fl/fl::MxCre bone marrow, were used to isolate CD150+CD34 KSLF HSC not earlier than 8 weeks after reconstitution. For in vivo injections the 10 g/mouse M-CSF, 5 mg/kg LPS (055:B5 E. coli) or sorted cells were injected in 100 l of PBS into the retro-orbital sinus. For HSC transplantation 400 CD150+CD34 KSLF HSC were sorted from CD45.2 mice and mixed with 100.000 Lin.sup.+ Sca.sup. CD45.1 carrier cells prior to injection into sub-lethally irradiated (4.5 Gy) CD45.1 recipient mice. For competitive transplantations, 1300 CD150+, CD34-KSLF HSC were isolated 16 h after control or 10 g M-CSF injection from actin-GFP CD45.2 mice.sup.33, mixed with equal numbers of CD45.2 competitor HSC and injected with 300.000 Lin.sup.+ Sca.sup. RC-lysed CD45.1 carrier cells into sub-lethally irradiated (4.5 Gy) CD45.1 recipients. Contribution to platelets, CD19+ B-cells and CD11b+ myeloid cells was analysed after 4 and 6 weeks in the blood from mice with at least 5% GFP+ donor cells. For PU.1 deletion PU.1.sup.fl/fl or PU.1.sup.fl/fl::MxCre reconstituted mice were intra-peritoneally injected with 5 g/g Polyinosinic:polycytidylic acid 7 and 9 days prior to control (PBS) or 10 g M-CSF injection. All mouse experiments were performed under specific pathogen-free conditions in accordance with institutional guidelines.

(42) FACS Analysis:

(43) For FACS sorting and analysis we used described staining protocols.sup.3 and published stem and progenitor cell definitions.sup.34, FACSCANTO BD LSRII and FACSARIA III equipment and DIVA software (BECTON-DICKINSON), analysing only populations with at least 200 events. For HSC analysis we used antibodies anti-CD34-FITC (clone RAM34, BD BIOSCIENCES), anti-CD135-PE (clone A2F10.1, BD BIOSCIENCES), anti-CD150-Pe-Cy7 (clone TC15-12F12.2, BIOLEGEND), anti-CD117-APC H7 (clone 2B8, BD BIOSCIENCES), anti-Sca-1-Pe-Cy5 (clone D7, BIOLEGEND), anti-CD48-APC (clone HM48-1, BIOLEGEND). Diverging from this or in addition we used antibodies anti-CD34 Alexa 700 (clone RAM34, BD BIOSCIENCES), anti-CD16/32 PE (clone 2.4G2, BD BIOSCIENCES), anti-CD11b PE-CF594 (clone M1/70, BD BIOSCIENCES), anti-CD19PE-Cy7 (clone 1D3, BD biosciences), anti CD45.2 APC (clone 104, BD BIOSCIENCES) and anti CD45.1 Pacific Blue (clone A20, BD BIOSCIENCES) for progenitor and blood cell analysis. LIVE/DEAD Fixable Violet Dead cell dye (Invitrogen) was used as viability marker.

(44) Intra-Splenic Injection of Sorted HSC and Fluorescence Microscopy:

(45) For analysis of HSC in vivo, 1500 to 7000 FACS sorted CD150.sup.+ CD34.sup. KSLF HSC were stained 10 min at 37 C. with 3 M CFSE (Invitrogen) in PBS/0.5% BSA, washed 3 in PBS/0.5% BSA and injected in 30 l PBS (containing or not 1 g of isotype control or AFS98 -M-CSFR antibody.sup.26 or 2 M GW2580, 10 M Ly29400, 10 M PD98059 or 2 M SU6656 inhibitors in 0.9% DMSO) into the spleen of anesthetized mice. After 24 h spleens were embedded in OCT (Tissue-Tek, Sakura) and frozen at 80 C. Cryostat sections (5 m) were prepared from the entire organ, dried and fixed 10 min in 4% PFA/PBS at room temperature (RT) and every 10.sup.th section was further processed. After washes in PBS, slides were blocked for 1 hour at RT in PBS/2% BSA/1% Donkey serum/1% FCS/0.1% saponin, incubated for 36 h at 4 C. with anti-PU.1 polyclonal antibody (Santa Cruz) in PBS/0.05% saponin (1:50), washed and incubated with secondary Alexa 546-donkey-anti-rabbit antibody (Molecular probes) in PBS/0.05% saponin (1:500). All immunofluorescence samples were mounted with ProLong Gold DAPI antifade (Molecular probes) and analyzed by multifluorescent microscopy on a Zeiss Axioplan 2. All CFSE+ cells were analysed for PU.1 expression up to 30 or 50 cells as indicated. Cell counts and staining were verified by a second trained microscopist blinded to sample identity. High-resolution photographs were obtained by confocal microscopy on a Leica SP5X.

(46) In Vitro Culture of HSC:

(47) CD150+CD34 KSLF HSC or CD150+CD34 CD48 KSLF HSC (single cells) were sorted into S-clone SF-03 medium (Sanko Jyunyaku) with 10% FBS supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (both Invitrogen) and cultivated in uncoated U-Shape 96 well plates (Greiner) in 100 l SCM, 20 ng/ml rSCF, 50 ng/ml rTPO+/100 ng/ml rM-CSF or 100 ng/ml rGM-CSF or 100 ng/ml rG-CSF. All cytokines were murine and from PeproTech. Cell viability was analyzed by AnnexinV and Propidium iodide FACS staining.sup.35.

(48) Quantitative Real Time PCR:

(49) Total RNA was isolated and reverse transcribed with MACS ONE-STEP T7 template kit (Miltenyi Biotec) and analysed by quantitative real-time PCR using TAQMAN Universal PCR Master Mix and a 7500 Fast Real Time PCR System sequence detection system (both Applied Biosystem), following the manufacturers' instructions.

(50) Single Cell Gene Expression Profiling:

(51) Single cells were sorted using the autoclone module on a FACSARIA III sorter (Becton-Dickinson) directly into 96 wells plate in the CELLS DIRECT Reaction Mix (Invitrogen). Individual cell lysis, cDNA synthesis and amplification was performed according to FLUIDIGM Advanced Development Protocol and single cell microfluidic real time PCR using Dynamic Array IFCs (BIOMARK FLUIDIGM) was performed by a trained technician of FLUIDIGM Inc. Preamplified products (22 cycles) were diluted 5-fold prior to analysis with Universal PCR Master Mix and inventoried TAQMAN gene expression assays (ABI) in 96.96 DYNAMIC ARRAY on a BIOMARK system (FLUIDIGM). Ct values were calculated from the system's software (BIOMARK Real-time PCR Analysis; FLUIDIGM) and filtered according to a set of quality control rules outlined below.

(52) Gene Filter:

(53) (a) For each gene, including controls, data with CtCall=FAILED and CtQuality<threshold were removed.

(54) (b) For each gene, including controls, CtValues>=32.0 were removed to filter out very low expression genes.

(55) (c) For each gene, including controls, genes with a difference of duplicate CtValues>=2.0 were considered inconsistent and removed.

(56) Sample Filter:

(57) (a) If the control gene (Gapdh) was not expressed or was removed according to gene filters (a-c), the whole sample was removed.

(58) (b) If the mean of the Ct values of all genes in a row was >=27.0 the whole sample row was removed.

(59) Time-Lapse Imaging and Analysis:

(60) Wherever possible our video microscopy protocols followed proposed guidelines.sup.24. In detail, FACS sorted CD150+ CD34 KSLF HCS from wt C57/Bl6 or GFP-negative CD150+ CD34 KSLF HSC from PU.1-GFP reporter mouse.sup.31 bone marrow were suspended in SCM supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin, 20 ng/ml rSCF, 50 ng/ml rTPO+/100 ng/ml rM-CSF and plated in Ibidi -slidesVI(0.4) (Biovalley SA, France). Time-lapse microscopy was performed using a Cell Observer system (Carl Zeiss Microscopy GmbH, Germany) at 37 C. and 5% CO2. Images were acquired every 10 minutes using 10 (A-plan 10/0.45 Ph1) or 40 (Plan-Apochromat 40/0.95 Korr M27) objectives in brightfield and fluorescence (GFP filters: EX BP 470/40; at 350 ms) with a COOLSNAP HQ2 monochrome camera (Photometrics) with a 22 binning and a metal halide 120W source for fluorescence illumination. For image analysis a matrix of 44 images was acquired for each time point. Images were stitched with AxioVision software (Carl Zeiss Microscopy GmbH, Germany) and processed with Fiji software.sup.36 using a slight rolling ball subtraction of background and 1 pixel Gaussian blur. For background subtraction of brightfield images, the median of z-projection was subtracted from the time-lapse stack. Single cell tracking was performed with basic commands of ImageJ.sup.37 and Fiji.sup.36 software and with specific tracking plugin MTrackJ.sup.38 in manual mode. Each cell was tracked manually frame-by-frame in the bright field channel and cross-controlled by two microscope specialists. Cells with non-standard morphology or size were rejected. The fluorescence signal was measured as the difference of maximum minus minimum intensity within a defined region of interest (ROI) around each cell. Cell properties and behaviour (cell division, cell death, position, fluorescence increase) were manually documented to build cumulated curves. R.sup.39 and Excel (Microsoft Corporation) software was used to manage data and build graphics.

(61) Statistical Analysis:

(62) P values were calculated by two-tailed non parametric Mann-Witney test for direct sample comparisons or Pearson's chi.sup.2 test for proportions (alpha=0.05). Whisker plots show median (lines), upper and lower quartiles (boxes) and extreme outliers (dotted whiskers).

(63) Results

(64) Lineage specific cytokines such as macrophage colony stimulating factor (M-CSF), can be strongly induced during physiological stress or infection.sup.10,11. and increase the production of mature cells from lineage-committed progenitors.sup.1,2. According to the prevailing model, however, they are generally not believed to directly influence differentiation decisions of haematopoietic stem cells (HSC).sup.9,12,13. Cell fate choice of HSC has traditionally been explained by stochastic models.sup.14. In this view transcriptional noise.sup.15 and random variations in competing lineage determining transcription factors lead to cross-antagonistic switches that initiate lineage choice.sup.4,5,6,7, whereas cytokines are thought to only act on the resulting progeny cells by stimulating their survival and proliferation.sup.8,9. A key example of such a master regulator is the transcription factor PU.1 that induces myelo-monocytic differentiation.sup.16,17. It is generally unknown whether external signals could drive the initial activation of such intrinsic master regulators. Since HSC deficient for the transcription factor MafB are sensitized to PU.1 activation in response to M-CSF.sup.3, we have investigated whether high systemic M-CSF levels could induce PU.1 and instruct myelo-monocytic fate in wt HSC without prior modification of transcription factor balance.

(65) We observed that lipopolysacharide (LPS), a strong mimetic of bacterial infection stimulating high systemic levels of M-CSF.sup.11, induced an up-regulation of GFP in long term HSC (CD117+sca+Lin-CD135-CD34-CD150+) of PU.1-GFP reporter mice.sup.18. Consistent with the expression of the M-CSF receptor (M-CSFR) in HSC.sup.3,19 direct intravenous injection of recombinant M-CSF also induced significantly increased PU.1 activation in HSC after 16 h (FIG. 1a,b). The treatment caused no significant change in M-CSFR or MafB expression, arguing against selection of myeloid primed HSC with high M-CSFR or low MafB levels. M-CSF also induced no change in the proportion of CD150.sup.hi HSC, reported to have myeloid lineage bias.sup.20, in GFP-positive or -negative HSC and activated PU.1 to a similar extent in CD150.sup.hi HSC (FIG. 1c) as in total HSC (FIG. 1a,b). Finally cultured CD150.sup.hi HSC revealed no proliferation or survival advantage in the presence of M-CSF. Together these data argued against selective amplification or survival of a pre-existing HSC sub-population and indicated that M-CSF could newly induce PU.1 expression in HSC.

(66) As shown in FIG. 1d, the M-CSF effect on stem cells was direct and specific, since FACS purified HSC showed increased PU.1 expression after 16 h in culture with M-CSF but not with GM-CSF or G-CSF, cytokines that may also be released during infection.sup.22. The observed changes in gene expression cannot be explained by M-CSF dependent selection of PU.1+ cells, as video-microscopy of cultured HSC showed no proliferation or survival advantage in M-CSF and PU.1 was induced before onset of cell division. Continuous observation of individual GFP-negative sorted HSC from PU.1-GFP mice by video imaging confirmed that M-CSF could induce PU.1 expression in previously PU.1 negative cells (FIG. 2a-c). We recorded the fate of HSC between 18 hours and 42 hours of culture, when both the induction of PU.1 in previously negative cells and the division of PU.1+ cells could theoretically occur. At the end of the 24 h observation period over two-fold more PU.1+ cells had developed in M-CSF than under control conditions (FIG. 2d) and backtracking the origin of these cells revealed that all PU.1+ cells were derived from previously PU.1 negative cells, but none from divisions of PU.1+ cells. Although the absence of PU.1+ cell division may be partially due to the phototoxic effects of GFP excitement.sup.23,24, we could conclude that the observed increase in PU.1+ cells was entirely due to M-CSF induced activation of the PU.1 reporter. These commitment events of PU.1 activation occurred 8 hours earlier and at a higher rate over the whole observation period in the presence of M-CSF (FIG. 2e). Our results indicated that M-CSF could directly increase PU.1 promoter activation in single, previously PU.1 negative HSC.

(67) To further investigate whether M-CSF induced PU.1 activation changed the cell identity of individual HSC we analyzed the mRNA expression profile of single cells by nanofluidic real time PCR on FLUIDIGM dynamic arrays. Consistent with their stem cell identity almost all freshly isolated HSC expressed stem and progenitor cell associated genes and about half expressed either no (lin-) or multiple lineage specific genes (mix). The remainder showed mainly megakaryocytic (Meg), megakaryocytic-erythroid (MegE) or myeloid lineage priming. Culture for 16 h without M-CSF led to an increased number of cells with a mixed lineage profile at the expense of Meg and lin-profiles. By contrast, culture in the presence of M-CSF induced a strong increase of cells with a myeloid gene expression signature. Consistent with the video microscopy results the increase in myeloid gene expression was associated with a doubling of the number of PU.1+ cells. Interestingly, this increase was entirely due to PU.1+ cells with a myeloid signature that did not express genes from any other lineage. By contrast, the number of PU.1+ cells that also expressed non-myeloid genes remained approximately constant (FIG. 3a). Together this indicated that M-CSF induced PU.1+ cells had assumed a myeloid cell identity. To evaluate whether this change in gene expression reflected a functional myeloid lineage choice in vivo we compared the differentiation potential of unstimulated PU.1 HSC to PU.1 and PU.1+ HSC after in vivo priming with M-CSF (FIG. 3b). Progenitor analysis in the spleen 2 weeks after transplantation of these populations revealed a higher ratio of granulocyte/macrophage progenitors (GMP) to megakaryocytic/erythroid progenitors (MEP) developing from PU.1+ HSC than from PU.1 HSC (FIG. 3c,d). We observed a similar increase in myeloid differentiation potential for PU.1+ cells derived from M-CSF stimulated PU.1 HSC in culture (FIG. 5a-c). Together these data showed that M-CSF induced PU.1 led to a myeloid cell fate change in single HSC.

(68) To further investigate, whether M-CSF could also induce a cell fate change of individual HSC in vivo, we transplanted CFSE-labelled HSC into the spleen, a site of extra-medullary haematopoiesis with adapted stem cell niches.sup.3,25, and analyzed expression of endogenous PU.1 protein by immuno-fluorescence in single HSC after 24 h. Whereas the vast majority of HSC were PU.1 negative immediately after transplantation, nearly all had activated PU.1 after transfer into spleens of LPS challenged hosts. This effect was principally dependent on M-CSF signalling as a blocking antibody against the M-CSF receptor.sup.26 strongly inhibited PU.1 activation. Furthermore, direct injection of recombinant M-CSF resulted in a similar strong induction of PU.1 in the transplanted HSC. This effect appeared to be entirely cell autonomous, as M-CSF receptor deficient (M-CSFR.sup./).sup.27 HSC showed no higher activation of PU.1 in M-CSF stimulated than control recipients. Similarly, small molecule inhibitors of the M-CSFR or PI3K, ERK and SRC kinases that signal downstream of the receptor.sup.28 also prevented induction of PU.1, consistent with the stimulation of transcriptional activators of the pu.1 gene by these pathways. Furthermore, transplantation of in vivo M-CSF primed CD45.2 HSC into sub-lethally irradiated CD45.1 recipients revealed an increased ratio of GMP to MEP progenitors in the spleen after 2 weeks (FIG. 4a, FIG. 6a,b) and an increased myeloid to lymphoid cell ratio in peripheral blood after 4 weeks (FIG. 6c). In competitive transplantation assays M-CSF primed HSC also showed a myeloid advantage compared to platelet and lymphoid contribution at 4 weeks in the blood that re-equilibrated after 6 weeks and did not compromise long-term multi-lineage contribution (FIG. 4b, FIG. 7). Finally, this myeloid differentiation preference of M-CSF primed HSC could be abolished by deletion of PU.1 (FIG. 4c). Together these results indicated that M-CSF could directly instruct a change in cell identity of single HSC in vivo that resulted in a reversible, PU.1-dependent myeloid differentiation preference.

(69) Our results show that under haematopoietic stress conditions of infection high systemic levels of M-CSF can directly instruct myeloid gene expression and differentiation preference of HSC. This challenges both the current view of cytokine action and how HSC make differentiation decision. Whereas cytokines are commonly thought to act on lineage-committed progenitors, we here show that stem cells are direct targets of lineage instruction by cytokines. HSC have been shown to proliferate in response to signals characteristic of bacterial.sup.29 or viral infections.sup.30 but without changing lineage specific gene expression or differentiation potential. In line with the prevailing paradigm of selective cytokine action it has been proposed that distinct stem cell subtypes could have a selective advantage in response to different stimuli.sup.21. Such a mechanism is difficult to distinguish from instructive mechanisms on a population basis. We have here employed multiple assays of single cell analysis in culture and in vivo in a time window before the onset of cell division to distinguish induced changes of lineage specification from selective mechanisms. These data indicate that M-CSF can directly change stem cell identity by activation of the myeloid master regulator PU.1 on the promoter, message and protein level, independently of selective survival or proliferation. The multi-lineage priming of gene expression in haematopoietic stem cells has generally been interpreted as indication that initial cell fate decisions are driven solely by stochastic fluctuations in the balance of lineage specific transcription factors.sup.4,5,6,12,13. Our data now indicate that cytokines can not only amplify random choices but also directly activate key regulators of lineage specification such as PU.1 to instruct lineage fate of haematopoietic stem cells to induce an insult tailored output of progeny. As M-CSF treatment can transiently increase the production of myeloid progeny without compromising stem cell activity, it may be useful to ameliorate myeloid cytopenias, particularly to protect patients from infection after stem cell transplantation.

EXAMPLES 2 & 3

Functional Impact of MCSF

(70) To study the functional impact of MCSF mediated HSC commitment during infections, two separate series of experiments were performed. In all experiments, after lethal irradiation of recipients, 2500 HS/PC with 200,000 cKit-carrier cells from donors were grafted. Uninfected and not transplanted mice served as controls to demonstrate that irradiation was lethal (dotted black line; FIGS. 8A, 8B and FIGS. 10A, 10B; n=12).

(71) Uninfected mice that received HS/PC transplants served as controls for efficient life saving transplantation (black line; FIGS. 8A, 8B and FIGS. 10A, 10B; n=8). Two other groups of mice received either 3 injections of MCSF (rhMCSF or Bacculo virus produced mMCSF) or PBS on the day of HS/PC transplantation.

(72) One-week post transplantation these mice were challenged with lethal doses of either the bacteria Pseudomonas aeruginosa or the opportunistic fungus Aspergillus fumigatus.

EXAMPLE 2

MCSF Stimulation Following HS/PC Transplantation Protects Against Bacterial Infection

(73) Material & Methods

(74) Mice:

(75) CD45.1 and C57Bl/6 mice were obtained from Charles River. 10-14 weeks old sex-matched CD45.2 recipients were reconstituted as described.sup.3 with bone marrow derived KSL (c-Kit(CD117)+, Sca+, Lin) HS/PC isolated from 6-8 weeks old CD45.1. For in vivo injections the indicated concentrations of M-CSF and/or sorted cells were injected in 100-200 l of PBS into the retro-orbital sinus. For HS/PC transplantation 2500 KLS HS/PC were sorted from CD45.1 mice and mixed with 200,000 cKit CD45.2 or cKit, Terr119+ carrier cells prior to injection into lethally irradiated (160 kV, 25 mA, 6.31 Gy) CD45.2 recipient mice. After irradiation all mice were given antibiotics in the drinking water to reduce the chance of opportunistic infection with other pathogens. (All mouse experiments were performed under specific pathogen-free conditions in accordance with institutional guidelines).

(76) Isolation of Hematopoietic Stem and Progenitor Cells (HS/PC) and cKit-Cells:

(77) Total bone marrow cells were depleted of mature cells by staining with biotinylated rat antimouse lineage antibody cocktail, followed by streptavidin immuno-magnetic micro beads (Miltenyi Biotec). Lineage negative cells were stained with HS/PC markers: anti-CD117-APC-H7 (clone 2B8, BD Biosciences), anti-Sca-1-PE-Cy5 (clone D7, Bio legend), Streptavidin-APC (eBioscience) and LIVE/DEAD Fixable Violet Dead cell dye (Invitrogen) as viability marker. HS/PC were sorted using FACSAriaIII equipment. For isolating cKit-carrier cells, whole bone marrow cells were depleted of cKit+ cells by staining with biotinylated antimouse CD117 (clone 2B8, BioLegend), followed by streptavidin immuno-magnetic micro beads (Miltenyi Biotec) and negative cells were sorted using automacs. For Terr119+ carrier cells, the cKit-cells were incubated with biotinylated anti-Ter119 followed by anti-biotin microbeads and positively sorted using automacs.

(78) M-CSF Treatment:

(79) Each mouse received three injections of 10 g M-CSF: 1 h before HS/PC transplantation; 6 h and 18 h post-transplantation. Human M-CSF (rhMCSF, Chiron Corporation Inc., USA, now part of Novartis AG) and Mouse M-CSF was expressed in baculovirus (Bac. virus mMCSF.sup.56) were used for the study.

(80) Infection with Pseudomonas aeruginosa:

(81) The P. aeruginosa PA14 strain was tagged with green fluorescent protein (GFP), as described elsewhere.sup.53,54. The GFP-tagged PA14 strain was cultured overnight at 37 C. in LB, diluted 1:100 in LB and grown for 3 hrs to reach bacterial exponential phase (3 to 4 OD600 nm). A volume of 100 l of a bacterial solution of 5103 CFU/ml diluted in PBS was further used for infection studies. One-week post-HS/PC transplantation mice were challenged by intra-peritoneal inoculation of 500 Colony Forming Units (CFUs) of bacteria in 100 L of sterile PBS.

(82) Bacterial Tissue Load Quantification:

(83) The infected mice were killed; the organs (spleen, lungs, liver and heart) were harvested and weighed. To determine CFUs per gram of tissue serial dilutions of tissue homogenates were prepared in PBS and plated on Pseudomonas Isolation Agar (PIA) (Difco laboratories) supplemented with appropriate antibiotics and incubated overnight at 37 C. The colonies were counted after 16-24 h.

(84) Results

(85) MCSF Stimulation Following HS/PC Transplantation Protects Against Pseudomonas aeruginosa Infection and Reduced Bacterial Tissue Load.

(86) Following irradiation and HS/PC transplantation, mice were infected with P. aeruginosa on Day8 (D8). Mice that were treated with human MCSF showed improved survival (triangle line; FIG. 8A; n=10) from 15.3% in untreated (square line) to 50% in MCSF treated mice (triangle line, FIGS. 8A and B; n=13). Mice that were treated with mouse MCSF showed further enhanced survival to 87.5% (triangle line; FIG. 8B; n=8).

(87) Furthermore in the mice succumbing to infection death was delayed in the M-CSF treated mice (FIGS. 8A and B).

(88) For analysis of bacterial load, mice were killed at 18 h after infection, tissue homogenates were prepared from spleen, lungs, liver and heart, and were plated to determine CFU. Eight to ten of the 13 mice in the untreated group were moribund compared with two to three of the 10 mice in the rhMCSF treated group. At this earlier time point, rhMCSF treated mice showed significant decrease in the tissue load of bacteria when compared with the untreated mice (FIG. 9) suggesting that increased survival was due to a reduction in bacterial load.

EXAMPLE 3

MCSF Stimulation Following HS/PC Transplantation Protects Against Fungal Infection

(89) Material & Methods

(90) Mice; Isolation of Hematopoietic Stem and Progenitor Cells (HS/PC) and cKit-Cells & M-CSF Treatment:

(91) As described above in EXAMPLE 2.

(92) Infection with Aspergillus Fumigates:

(93) Aspergillus fumigatus FGSC 1100 was provided by the Centre International de Ressources MicrobiennesChampignons Filamenteux (CIRM-CF, Marseille, France). For each experiment, cultures were grown on Malt agar medium (2% malt extract, 2% Bacto-agar DIFCO) for 5 days at 25 C. Conidial suspension was prepared in sterile saline according to Bitmansour et al. (2002).sup.55. One-week post-HS/PC transplantation mice were infected by intra-nasal inoculation of 2-410.sup.6 conidia in 20-40 L of sterile PBS.

(94) Fungal Culture of Infected Organs:

(95) The organs (lungs, liver, heart and spleen) were harvested, weighed and tissue homogenates were prepared in PBS. The homogenates were serially diluted and 200 L or each dilution was plated on Sabouraud dextrose agar (DIFCO). The plates were incubated at 25 C. and pictures were taken after 3-5 days.

(96) Results

(97) MCSF Stimulation Following HS/PC Transplantation Protects Against Aspergillus fumigatus Infection and Reduces Fungal Tissue Load.

(98) Post-irradiation and HS/PC transplantation, mice were infected with A. fumigatus on D8. Mice treated with human MCSF showed 40% survival (triangle line; FIG. 10A; n=10) compared to untreated mice, which showed only 10% survival (square line; FIGS. 10A and B; n=10). Interestingly, mice that were treated with mouse MCSF were further protected and showed 60% survival (triangle line; FIG. 10B; n=10). One among 10 mice in the untreated group that survived for 6 days after infection remained alive during the total 35-day observation period.

(99) Furthermore in the mice succumbing to infection death was slightly delayed from D9-D14 in untreated mice to D10-D15 in the rhMCSF treated group. For analysis of fungal load, mice were killed at 48 h after infection and tissue homogenates were prepared from lungs, liver and heart. Diluted homogenates ( 1/10 of lung, liver and heart; 1/100 of heart) were plated on Sabouraud dextrose agar plates to observe fungal colony growth. rhMCSF treated mice showed significant decrease in the tissue load of fungal colonies when compared with the untreated mice. Fungal tissue cultures were incubated beyond 96 h to verify the typical colony morphology of A. fumigatus.

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