The granulocyte colony stimulating factor pathway regulates autoantibody production in a murine induced model of systemic lupus erythematosus
© Lantow et al.; licensee BioMed Central Ltd. 2013
Received: 17 August 2012
Accepted: 3 April 2013
Published: 8 April 2013
An NZB-derived genetic locus (Sle2c2) that suppresses autoantibody production in a mouse model of induced systemic lupus erythematosus contains a polymorphism in the gene encoding the G-CSF receptor. This study was designed to test the hypothesis that the Sle2c2 suppression is associated with an impaired G-CSF receptor function that can be overcome by exogenous G-CSF.
Leukocytes from B6.Sle2c2 and B6 congenic mice, which carry a different allele of the G-CSF receptor, were compared for their responses to G-CSF. Autoantibody production was induced with the chronic graft-versus-host-disease (cGVHD) model by adoptive transfer of B6.bm12 splenocytes. Different treatment regimens varying the amount and frequency of G-CSF (Neulasta®) or carrier control were tested on cGVHD outcomes. Autoantibody production, immune cell activation, and reactive oxygen species (ROS) production were compared between the two strains with the various treatments. In addition, the effect of G-CSF treatment was examined on the production autoantibodies in the B6.Sle1.Sle2.Sle3 (B6.TC) spontaneous model of lupus.
B6.Sle2c2 and B6 leukocytes responded differently to G-CSF. G-CSF binding by B6.Sle2c2 leukocytes was reduced as compared to B6, which was associated with a reduced expansion in response to in vivo G-CSF treatment. G-CSF in vivo treatment also failed to mobilize bone-marrow B6.Sle2c2 neutrophils as it did for B6 neutrophils. In contrast, the expression of G-CSF responsive genes indicated a higher G-CSF receptor signaling in B6.Sle2c2 cells. G-CSF treatment restored the ability of B6.Sle2c2 mice to produce autoantibodies in a dose-dependent manner upon cGVHD induction, which correlated with restored CD4+ T cells activation, as well as dendritic cell and granulocyte expansion. Steady-state ROS production was higher in B6.Sle2c2 than in B6 mice. cGVHD induction resulted in a larger increase in ROS production in B6 than in B6.Sle2c2 mice, and this difference was eliminated with G-CSF treatment. Finally, a low dose G-CSF treatment accelerated the production of anti-dsDNA IgG in young B6.TC mice.
The different in vivo and in vitro responses of B6.Sle2c2 leukocytes are consistent with the mutation in the G-CSFR having functional consequences. The elimination of Sle2c2 suppression of autoantibody production by exogenous G-CSF indicates that Sle2c2 corresponds to a loss of function of G-CSF receptor. This result was corroborated by the increased anti-dsDNA IgG production in G-CSF-treated B6.TC mice, which also carry the Sle2c2 locus. Overall, these results suggest that the G-CSF pathway regulates the production of autoantibodies in murine models of lupus.
Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex etiology in which the production of pathogenic autoantibodies (autoAbs) results in cellular and tissue damage. Aside from B cells, which produce these autoAbs, and CD4+ T cells, which provide B cell help for the generation of class-switched, affinity maturated autoAbs, essentially every other immune cell subset has been implicated in SLE pathogenesis. The strong genetic basis of SLE is sustained by a large number of polymorphisms that have been identified in recent years through association studies in large cohorts of patients and controls . Mouse models of SLE have been used extensively to study both the cellular and genetic basis of SLE, and overall, the results obtained from these models have largely been validated in SLE patients. In particular, murine models have revealed a large number of SLE susceptibility genes, which are organized in the same three broad pathways: apoptosis and processing of apoptotic debris, toll-like receptor (TLR) signaling and type I IFN pathways, and lymphocyte activation in both SLE patients and SLE-prone mice [2, 3]. The genetic analysis of the NZM2410 mouse model has also shown the existence of both SLE-resistance and suppressor genes. Consequently, the SLE-resistant strain C57BL/6 (B6) carries susceptibility genes that were revealed when combined with either other susceptibility genes provided by the NZM2410 lupus-prone genome, or when subjected to a strong immune stimulation [4, 5].
The bm12- chronic graft vs host disease (cGVHD) model is a well-defined model of induced lupus in which B6.C-H2bm12 lymphocytes are transferred into H-2b B6 hosts. Within 3 weeks of transfer, mice develop lupus-like phenotypes including lymphocyte activation and anti-nuclear autoAbs, which are dependent on interactions between donor CD4+ T cells and host autoreactive B cells . We have shown that B6.Sle2c2 mice, which are B6 mice carrying an NZM2410 (NZB)-derived genomic region on the telomeric potion of the Sle2 locus, are profoundly resistant to bm12-cGVHD induction as compared to their B6 congenic controls . Using mixed bone-marrow (BM) chimeras and functional assays, we have shown that Sle2c2 suppression is mediated by BM-derived cells, but not by T cells, B cells, or dendritic cells (DCs). We mapped Sle2c2 resistance to a short genomic interval that contains Csf3r, the gene encoding for the granulocyte-colony stimulation factor (G-CSF) receptor (G-CSFR) . The NZM2410 allele of Csf3r (Csf3r N as opposed to the B6 allele Csf3r B ) carries a mutation in exon 10 (rs13477964) that results in a S378N substitution in its extracellular domain.
G-CSF is an essential factor in the recruitment and function of neutrophils . G-CSFR expression regulates GVHD in mice . Moreover, neutrophils have recently been implicated at multiple levels in the pathology of SLE or related systemic autoimmune diseases . Neutrophils inflict direct damage to the vasculature , the kidneys  and the skin  of lupus patients. They also contribute to SLE pathogenesis through their direct production of type I IFN , or through the highly immunogenic neutrophil extracellular traps (NETs) amplifying the production of either type I IFN  or IL-17 , although these later findings have been refuted in the MRL/lpr lupus-prone mouse model . G-CSF also controls the number of myeloid derived suppressor cells (MDSCs) , which are potent suppressors of alloreactive T cell responses in a GVHD model . In MRL/lpr mice, the percentage of MDSCs increases as disease progresses , corroborating our findings of an expanded GR1lo MDSC population in B6 mice with established cGVHD .
These studies lead us to hypothesize that the N378S mutation in the Csf3r gene impairs G-CSFR function. This mutation is located in the solvent-exposed part of the receptor in a putative fibronectin III domain outside the solved structure of the protein . It is therefore impossible at this point to predict its functional significance, although it has the potential to affect ligand binding. We hypothesized that the N378S mutation is responsible for the differential bm12-cGVHD response between B6 and B6.Sle2c2 mice, which we tested by comparing their response to exogenous G-CSF. We reasoned that if bm12-cGVHD resistance in B6.Sle2c2 mice was mediated by a gain of function in suppressive cells such as MDSCs, G-CSF treatment would induce bm12-cGVHD resistance in B6 mice. In contrast, if bm12-cGVHD resistance in B6.Sle2c2 mice was due to a loss of function or recruitment in inflammatory neutrophils, then G-CSF treatment would alleviate bm12-cGVHD resistance in B6.Sle2c2 mice, which would develop autoAbs and lymphocyte activation comparable to that of B6. Here we demonstrate a weaker binding of mouse G-CSF to B6.Sle2c2 leukocytes as compared to B6, as well as a defective expansion of B6.Sle2c2 myeloid cells and neutrophils in response to in-vivo G-CSF treatment. Furthermore, exogenous G-CSF restored bm12-cGVHD responses in B6.Sle2c2 mice. Finally, the B6.Sle1.Sle2.Sle3 congenic mice that display a full-blown spontaneous lupus as its NZM2410 parental strain carry the Sle2c2 locus . It was therefore predicted that G-CSF treatment would accelerate their autoAb production, which we showed in this study with a low-dose treatment. Overall these results suggest that Sle2c2 prevents the induction of systemic autoimmunity by impairing the production of inflammatory neutrophils due to a decreased ability of the G-CSFR to bind its ligand.
Materials and methods
The B6.Sle2c2 mice carrying an NZM2410 (NZB)-derived interval located between D4Mit11 and D4Mit72 have previously been described . B6 and B6.C-H2 bm12 /KhEg (bm12) were originally purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Cohorts of mice within an experiment were sex- and age-matched, but the reported phenotypes were neither affected by sex or age. The NZM2410-derived triple congenic mice B6.Sle1.Sle2.Sle3 (B6.TC) have been previously described . All animal protocols were approved by the Institutional Animal Care and Use Committee at the University of Florida.
cGVHD induction and G-CSF treatment
bm12-cGVHD was induced according to an established protocol . Briefly, 50 to 80 × 106 bm12 splenocytes were injected intraperitoneally into 2 to 4 month-old B6 or B6.Sle2c2 mice. Serum was collected for autoAb detection by ELISA as previously described  at day (d) 0, 7, 14 and 21 after induction. Antinuclear antibody (ANA) stains were conducted on slides containing fixed Hep-2 cells (Inova Diagnotics, San Diego, CA, USA) with mouse sera diluted 1:40, and revealed with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (Southern Biotech, Birmingham, AL, USA) at 1:50 dilution. Staining intensity was calculated with morphometry software (Metamorph, Molecular Devices, Sunnyvale, CA, USA) for a standardized level of green fluorescence averaged on 10 to 20 randomly selected Hep-2 cells per sample. Samples with an average staining intensity > 1 were considered positive. At d21, mice were sacrificed and splenocytes assessed by flow cytometry.
bm12-cGVHD-induced mice were treated with subcutaneous injections of 1.2, 12.0, or 120.0 ug pegylated human G-CSF (Neulasta®, huG-CSF, Amgen, Thousand Oaks, CA, USA), all starting at d1 of induction. Some huG-CSF injections were repeated every 7 d resulting in 4 different treatment regimens: one injection of 120 ug (120 × 1), two or three injections of 12 ug (12 × 2 and 12 × 3), and three injections of 1.2 ug (1.2 × 3). Control mice were injected with 5% dextrose as vehicle control. Each experimental group contained at least five recipient mice per treatment per strain, and control groups were performed side by side for each treatment group. Groups of four un-manipulated mice per strain were injected with huG-CSF and blood was collected every 2 to 3 d to compare the dose-response effect of G-CSF of peripheral blood leukocytes (PBLs) between strains. Serum mouse (m) G-CSF was measured by ELISA with reagents from Peprotech (Rocky Hills, NJ, USA) in sera diluted 1:50. B6.TC and B6 female mice were treated with six weekly injections of 1 ug of Neulasta or 5% dextrose starting at 2 months of age and were sacrificed at week 7. Serum was collected every two weeks and anti-dsDNA IgG, anti-chromatin IgG and total IgG were measured as previously described in sera diluted at 1:100 and 1:5000, respectively .
Splenocytes or PBLs were blocked with anti-CD16/CD32 (2.4G2), then stained with pre-titrated amounts of the following FITC-, phycoerythrin (PE)-, allophycocyanin-, or biotin-conjugated Abs: CD4 (RM4-5), CD69 (H1.2F3), CD44 (IM7), CD62L (MEL-14) B220 (RA3-6B2), CD86 (GL1), CD80 (16-10A1), CD22.2 (Cy34.1), I-ab (AF6-120.1), CD11b (M1/70), CD11c (HL3), Ly6C/G (GR-1/RB6-8C5) or isotype controls, all from BD Biosciences (San Diego, CA, USA). Biotinylated Abs were revealed by streptavidin-PercP-Cy5a. p-signal transducer and activator of transcription 3 (Stat3) (Y705) staining was performed after fixation and permeabilization (BD Cytofix/Cytoperm kit). G-CSF binding was performed by incubating splenocytes for 1 h with 25 ng of mG-CSF (ProSpec, East Brunswick, NJ, USA) biotinylated with a Biotin XX Microscale protein labeling kit (Molecular Probes, Eugene, OR, USA), detected by flow cytometry with PE-streptavidin. As negative controls, cells were incubated only with PE-streptavidin. ROS production was detected with 1 μM dihydrorhodamine 123 (DHR, Molecular Probes). Cell staining was analyzed using a FACScalibur or LSRFortessa (Becton Dickinson, Franklin Lakes, NJ, USA). At least 30,000 (PBLs) and 50,000 (splenocytes) events were acquired per sample, and dead cells were excluded based on scatter characteristics.
Gene expression analysis
Splenocytes and BM cells from B6 and B6.Sle2c2 mice (three each) were cultured in complete medium at a concentration of 106 cells/ml with 0.0, 0.1, or 1.0 ug/ml of Neulasta for 8 h. RNA was extracted and cDNA was synthesized as previously described . Expression of Itgam and Mpo relative to Gapdh was quantified by quantitative PCR measuring the incorporation of Sybergreen (Molecular Probes). The primer sequences were: Itgam 5'-CCC CAA TTA CGT AGC GAA TG-3'and 5'- TGC TGC GAA GAT CCT AGT TG -3'; Mpo 5'- TGC TCT CGA ACA AAG AGG GT-3' and 5'- CTT TGA CAG CCT GCA CGA-3'; Gapdh 5'- AGC TTG TCA TCA ACG GGA AG -3' and 5'- GTG GTT CAC ACC CAT CAC AA -3'. Results were calculated with the ΔΔ cycle threshold (Ct) method and normalized to the average values for untreated B6 samples.
Statistical analyses were performed with GraphPad Prism5, using the two-tailed unpaired t-test or Bonferroni multiple comparison test when multiple groups were compared. Non-parametric statistics were performed when data were not normally distributed. Graphs show mean and standard error of the mean (SEM), and statistical significance is represented as *P < 0.05, **P < 0.01, and ***P < 0.001.
B6.Sle2c2 leukocytes bind less G-CSF and expand less to exogenous G-CSF than B6
Exogenous G-CSF restores cGVHD responses in B6.Sle2c2 mice
The restoration of cGVHD response by G-CSF increases CD4+ T cell activation and expands the DC and neutrophil populations
The percentage of CD11b+ CD11c- GR1- macrophages was equivalent between B6 and Sle2c2 mice 3 weeks after cGVHD induction. huG-CSF treatments increased the percentage of macrophages in a dose-dependent manner similarly between these two strains without any correlation with autoAb production (data not shown). In contrast, the percentage of CD11b+ CD11c+ myeloid dendritic cells (DCs) was significantly lower in B6.Sle2c2 than in B6 mice, and the 12 × 2 treatment significantly expanded this cell population in the spleen of B6.Sle2c2 mice to a level similar to that of treated B6 mice (Figure 5B). The percentage of CD11b+ GR1hi neutrophils was significantly higher in un-manipulated B6.Sle2c2 than in B6 mice (3.97 ± 0.55% vs 2.50 ± 0.16%, P = 0.03). Three weeks after cGVHD induction, this difference was inverted, with B6.Sle2c2 values being significantly lower than B6 (Figure 5B). As in the blood of non-induced mice (Figure 1B), 12 × 2 treatment expanded the neutrophil population in the spleen and resulted in equivalent percentages in both strains. Finally, the percentage of CD11b+ GR1lo cells was similar in untreated and unmanipulated B6.Sle2c2 and B6 mice (0.49 ± 0.04 vs 0.50 ± 0.02%, P > 0.05), but three weeks after cGVHD induction, this population was significantly depleted in B6.Sle2c2 mice (Figure 5B). Also as in the blood of non-induced mice (Figure 1B), the 12 × 2 expanded the CD11b+ GR1lo population in B6.Sle2c2 mice three weeks after cGVHD induction and resulted in equivalent percentages in both strains (Figure 5B). Overall, these results showed that restoration of the cGVHD response in B6.Sle2c2 mice by huG-CSF treatment correlated with an increase in CD4+ T cell activation, and an expansion of the DC, neutrophils and CD11b+ GR1lo cell populations.
Reactive oxygen species (ROS) production is differentially up-regulated in the cGVHD response between B6.Sle2c2 and B6 mice
Exogenous G-CSF accelerates anti-dsDNA IgG in lupus-prone B6.TC mice
This study was performed to test the hypothesis that the S378N mutation in the Csf3r gene results in defective G-CSFR signaling and is responsible for the differential autoimmune response between B6 and B6.Sle2c2 mice. In our previous study, we speculated that bm12-cGVHD resistance in B6.Sle2c2 mice was mediated by enhanced MDSC function or number. However, G-CSF treatment did not induce bm12-cGVHD resistance in B6 mice, but instead eliminated bm12-cGVHD resistance in B6.Sle2c2 mice. We therefore conclude that B6.Sle2c2 mice are resistant to autoimmunity due to their incapacity to mobilize pro-inflammatory G-CSF-responsive BM-derived cells, most likely neutrophils. Indeed, we report here that B6.Sle2c2 leukocytes bind less G-CSF and expand less in response to in vivo G-CSF treatment. On the other hand, G-CSFR-responsive genes are expressed at higher levels in B6.Sle2c2 leukocytes, which present a greater STAT3 phosphorylation, indicating a higher level of G-CSFR signaling. Although the lower cell expansion to G-CSF and the higher G-CSFR signaling suggest opposing responses, it is possible that the latter is a steady-state compensatory response to the former. This hypothesis will require extensive comparison of G-CSFR signaling between the two alleles, including in cell lines transfected with G-CSFR constructs differing only at S378N.
The lower binding of G-CSF and impaired cellular expansion in response to exogenous G-CSF lead us to hypothesize that exogenous G-CSF would compensate for a loss of function phenotype. To this end, we indeed found that two huG-CSF treatment regimens restored the production of autoAbs to a level similar to that found in B6. These treatments also restored CD4+ T cell activation as well as DC and granulocyte expansion in B6.Sle2c2 mice to the same levels as in B6. This suggested that these cell populations are effectively modified by higher G-CSF availability and they are involved in the production of autoAbs in this model.
We also found that B6.Sle2c2 myeloid cells and granulocytes produced more ROS at steady-state than B6 cells. A recent study found a strong association between a coding mutation in the human NCF2 gene with SLE susceptibility, and determined that the disease associated allele was associated with a lower ROS production . This is consistent with decreased levels of Ncf1, a gene encoding for another unit of the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase complex, being associated with collagen-induced arthritis  and experimental allergic encephalomyelitis . Upon bm12-cGVHD induction, ROS production is upregulated to a greater level in the B6 than B6.Sle2c2 granulocytes. huG-CSF treatment increased ROS production in B6.Sle2c2 mice, which was concomitant with their production of autoAbs. These results suggest that the NZM2410 allele of Csf3r results in a higher ROS production at steady state levels of G-CSF that maintains CD4+ T cell tolerance, as it has been shown in other models . We showed here that bm12-cGVHD induction recruits inflammatory neutrophils, as is the case in human SLE , of which B6.Sle2c2 mice cannot achieve the same expansion as B6 mice unless exogenous G-CSF is provided. Signaling through the G-CSFR is critical for the development and function of inflammatory granulocytes, whereas G-CSFR deficiency is protective in collagen-induced arthritis . We therefore propose that impaired G-CSF binding and G-CSFR signaling is protective again systemic autoimmunity in two phases, first by producing higher levels of ROS, which maintains T cell tolerance, and second by failing to support the expansion of inflammatory neutrophils, which can amplify the autoimmune response either through the type I IFN or Th17 pathways .
We have previously reported that there is no difference in Csf3r expression between B6 and B6.Sle2c2 mice . Genomic sequencing of the Csf3r locus has identified a large number of intronic polymorphisms and four coding synonymous mutations, but S378N was the only coding non-synonymous mutation identified between the NZM2410 and B6 genomes (unpublished observations). None of the genes located within Sle2c2 locus have known functions in the G-CSF pathway. This implies that the differences that we report here between B6 and B6.Sle2c2 mice in their response to G-CSF are most likely due to the S378N polymorphism. This will, however, need to be formally addressed by stringent structure/function studies.
We have used the bm12-cGVHD model to test the hypothesis that Sle2c2 suppression involves the G-CSF pathway, as it is a very robust model that leads to a rapid synchronized production of autoAbs with very little inter-individual variation. However, we have evidence that Sle2c2 also suppresses spontaneous autoimmune responses in two NZM2410-derived models [30, 31] and we have now shown that a low-dose treatment with G-CSF accelerates the spontaneous production of anti-dsDNA IgG in B6.TC mice, which includes Sle2c2 .
An association between G-CSF treatment and disease exacerbation is supported by clinical findings, where G-CSF treatment of neutropenic SLE patients can be associated with severe flares . Moreover, depending on the dose, G-CSF treatment can either prevent or accelerate disease in the MRL/lpr mouse model of SLE . While these two reports did not explore the mechanism of disease exacerbation, we propose that it overlaps with the resistance or susceptibility to induced autoAb production mediated by the S378N G-CSFR polymorphism, and that the B6.Sle2c2 and B6.TC strains offer an excellent model to identify the cellular and molecular bases on how G-CSF modulates SLE pathogenesis.
Contrary to the results reported here, G-CSF treatment has been reported to promote reversal  or prevention  of type 1 diabetes in the NOD mouse. In this model, NADPH oxidase impairment through Ncf1-deficiency was associated with disease protection , contrary to the results obtained in SLE patients with NCF2-impaired function . These results suggest an inverse correlation between the protective effect of exogenous G-CSF and the protective effect of the NADPH oxidase products in autoimmune diseases. This has significant implications for devising strategies targeting the G-CSF pathways in opposite direction in diseases such as SLE and type I diabetes.
Exogenous G-CSF increases the production of autoAb in B6.Sle2c2 and B6.TC mice and both of these strains carry the same mutation in the G-CSFR. Furthermore, we have shown a defective mobilization of BM neutrophils in response to G-CSF treatment of B6.Sle2c2 mice as well as a different expression of G-CSF-responsive genes between B6 and B6.Sle2c2 mice. Overall, these data suggest that mice carrying the G-CSFR mutation are defective in mobilizing and/or activating inflammatory neutrophils, which inhibits the production of autoAbs. Further experiments will be required to demonstrate a direct role of neutrophils in this process.
chronic graft vs host disease
enzyme-linked immunosorbent assay
representative fluorescence-activated cell sorting
granulocyte-colony stimulation factor
granulocyte-colony stimulation factor receptor
myeloid-derived suppressor cell
mean fluorescence intensity
nicotinamide adenine dinucleotide phosphate-oxidase
neutrophil extracellular trap
peripheral blood leukocyte
polymerase chain reaction
reactive oxygen species
standard error of the mean
systemic lupus erythematosus
signal transducer and activator of transcription 3
The authors are grateful to Dr Clayton Mathews for stimulating discussions and to Nathalie Kanda for excellent animal care. Supported by the National Institutes of Health (RO1 AI068965 to LM and PO1 AI42288 to MAA).
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