2.1 Animals

C57BL/6 background Lyn^{− /−} (L^−/−) mice^{12, 15} and G-CSF^−/− (G^−/−) mice^{7, 24} have been described. Lyn^−/−G-CSF^{− /−} (LG^−/−) mice were generated by intercrossing and genotyping, and were ostensibly healthy and fertile. C57BL/6 (C57) controls were from Monash Animal Services SPF facility (Clayton, Australia), where all mice were housed from weaning. Mice were analyzed at 36 weeks of age, when autoimmune kidney pathology is severe in Lyn^−/− mice.¹⁵ Research was conducted under the Australian Code for the Care and Use of Animals for Scientific Purposes, and approval was obtained from the Alfred Research Alliance Animal Ethics Committee (E/1688/2016/M).

2.2 Quantification of G-CSF in mouse sera

Serum from aged mice was analyzed by custom Bio-Plex Pro Assay Kit (Bio-Rad) as per the manufacturer's instructions. Results were revealed using Bio-Plex 200 System with the Bio-Plex Manager 4 software.

2.3 RT-PCR

RNA from lung tissue was obtained by Trizol/Chloroform extraction and converted to complementary DNA (cDNA) using the FireScript RT kit (Solis Biodyne) as per manufacturer's instructions. RT-PCR used validated primers for Csf3 ⁴⁵ and 18S ⁴⁶ with PowerUp SYBR Green Master Mix (Applied Biosystems) as per manufacturer's instructions for 40 cycles under single-plex conditions (QuantStudio 7; Life Technologies). RT-negative and template-negative controls were included to validate cDNA-specific amplification. Samples were run in triplicate and cycle threshold values were determined by automatic threshold analysis (QuantStudio Software), from which the triplicate average was used to determine relative gene expression using the 2^−ΔΔCt method, with 18S as the housekeeping control.

2.4 Flow cytometry

BM cells were flushed from the femurs and single-cell suspensions of spleen were prepared by extrusion. Red blood cells were lysed with ACK lysis buffer, Fcγ receptors were blocked using Fc Block (clone 2.4G2), and fluorophore-conjugated monoclonal antibodies were used to stain cells. Labeled cells were analyzed by flow cytometry using an LSRFortessa X-20 Instrument (BD Biosciences). Viable cells were gated by fluorogold exclusion and single cells were further assessed as follows: erythrocytes: Ter119⁺CD71^±; BM neutrophils: Lineage(B220+NK1.1+Thy1.2+CD115+)⁻c-Kit⁻SiglecF⁻CD11b⁺Ly6G⁺; splenic neutrophils: CD11b⁺CD115⁻SiglecF⁻Ly6G⁺; monocytes: CD11b⁺CD115⁺, further subdivided as classical (Ly6C⁺CD43⁻) or nonclassical (Ly6C⁻CD43⁺); B cells: B220⁺CD19⁺, further subdivided as follicular (CD21/35^loCD23⁺), marginal zone (CD21/35^hiCD23⁻) or transitional (CD21/35⁻CD23⁻); plasma cells: B220^loCD138^hi; CD4⁺ T cells: CD3ε⁺CD4⁺; CD8⁺ T cells: CD3ε⁺CD8α⁺. CD4+ and CD8⁺ T cells were further subdivided as effector memory (CD44⁺CD62L⁻). Total cell numbers were derived from cell counts after red blood cell lysis and cell proportions were classified by flow cytometry. The expression of cell surface activation markers was determined by normalizing their geometric mean fluorescence intensity (gMFI) on gated cell populations by dividing the gMFI of individual samples by the mean gMFI of the C57BL/6 group within each experiment, allowing data from multiple experiments to be pooled.

2.5 Detection of autoantibodies by enzyme-linked immunosorbent assay

MaxiSorp 96-well immunoplates (Nunc) were treated overnight with 50 µl/well of 50 µg/ml calf thymus DNA in phosphate-buffered saline (PBS) (Sigma-Aldrich). Coated plates were washed with PBS/0.5% Tween-20, blocked with assay diluent (BD) for 60 min, and then serum samples were added. An in-house high-titer antinuclear antibody reference serum was used to generate a standard curve for derivation of arbitrary titer values. After incubation (2 h, room temp), plates were washed and goat anti-mouse immunoglobulin G (H + L)-horseradish peroxidase (Southern Biotech) detection antibody was added to the wells for a further 60 min. Colorimetric detection was achieved with TMB chromogenic substrates A and B (BD), and the reaction was stopped with 1.5M sulfuric acid. Plates were read on a MultiSkan GO microplate spectrophotometer (Thermo Fisher Scientific) at 450 nm with wavelength correction at 595 nm. Relative titers were established by plotting sample optical density values against the standard curve.

2.6 Kidney histology

Mouse kidneys were fixed in 10% neutral-buffered formalin for 24 h, transferred to ethanol, and processed. Paraffin-embedded sections of 3 µm were set on SuperFrost slides and stained with Periodic Acid–Schiff. Sections were imaged using an Olympus BX-51 light microscope (Olympus Australia), taking more than eight images of the kidney cortex/sample, capturing at least 30 glomeruli per kidney. Polygon tool tracing with ImageJ software (NIH, 1.52d) was used to quantify the area of each glomerulus. A custom disease score describing the changes to glomerular cellularity (mesangial, endothelial and infiltrating) and morphology was derived based on the typical disease presentation of Lyn^{− /−} mice and classified as follows: 0: Normal glomerular cellularity and morphology; 1: Mild cellular expansion and/or early-stage disrupted glomerular morphology that includes lobularity and/or Bowman's space enlargement; 2: Advanced cellular expansion with distinct disruption to glomerular morphology, pyknosis and/or karyorrhexis; 3: Severe cellular expansion/consolidation with advanced lobularity or fragmentation and/or enlargement of the Bowman's space with cellular infiltration (with or without crescent formation) and/or periglomerular cellular expansion; 4: End-stage glomerular destruction, progressive or complete loss of cellularity, and/or distinct glomerular morphology. Segmental necrosis was scored as the proportion of glomeruli exhibiting necrotic lesions per kidney, as determined by diffuse or patches of anuclear pink staining. At least 30 glomeruli were analyzed per kidney for each parameter, with the mean value taken to represent each sample.

2.7 Human participants and clinical assessments

Patients aged 18 and over, fulfilling the 1997 American College of Rheumatology revised criteria or the Systemic Lupus International Collaborating Clinic criteria for SLE classification,^{47, 48} already enrolled in the Australian Lupus Registry & Biobank,⁴⁹ were recruited for this study from the Monash Lupus Clinic (Clayton, Victoria, Australia) between June 2015 and September 2017. As part of the prospective collection of demographic and clinical data, disease activity was assessed using the hybrid SLE Disease Activity Index 2000 (SLEDAI-2K).⁵⁰ Renal-specific disease activity was assessed using the four renal domain components of the SLEDAI-2K (proteinuria, hematuria, pyuria, and urinary cast), as described⁵¹; an SLE patient was considered to have active renal disease if at least one of these four renal items of the SLEDAI-2K was positive. HCs consisted of healthy subjects aged 18 and above who were recruited between February and August 2017 from Monash Medical Centre (Clayton, Victoria, Australia). All study participants gave written informed consent. This study was approved by the Monash Health Human Research Ethics Committee (13019A).

2.8 Quantification of G-CSF from human sera

Quantification of serum G-CSF concentration was conducted by Crux Biolabs (Bayswater, Victoria) using Quantibody® platform (Raybiotech), as part of a commercial 40-plex panel (Human Inflammation Array 3: QAH-INF-3), as per the manufacturer's protocol. An arbitrary value of the upper limit of quantification (ULOQ) and half the lower limit of quantification (LLOQ) was assigned to serum G-CSF values falling above and below the ULOQ and LLOQ, respectively.

2.9 Statistical analyses

For mouse studies, statistical significance was determined using Mann–Whitney nonparametric U test (for two groups) or Kruskal–Wallis H test, followed by Dunn's multiple comparisons test (for four groups), with horizontal bars on data representing the median ± interquartile range (IQR). Specific comparisons between C57BL/6 and G-CSF^−/− groups and Lyn^−/− and Lyn^−/−G-CSF^−/− groups were also conducted using Mann–Whitney nonparametric U test. Horizontal bars represent median ± IQR where the data are displayed on a linear scale or geometric mean ± geometric SD where logarithmic data are displayed. Significance by Mann–Whitney test (represented by #) and Dunn's multiple comparisons test (represented by *) is denoted by p > .05 (not significant) not stated, not stated, p < .05 # or *, p < .01 ## or **, p < .001 ### or ***, p < .0001 #### or ****. Mouse data analyses utilized GraphPad Prism software (version 8.0.2). For human studies, serum G-CSF concentrations were transformed into a binary variable as being either detectable or not detectable, using the LLOQ as a threshold. Comparison of proportions were assessed using Pearson's χ ² test. Comparison of concentrations were conducted using Mann–Whitney nonparametric U test, with box and whisker plots representing the median, IQR, and range. All p values are numerically displayed, with a p < .05 considered statistically significant. Human data analyses utilized R software (version 4.0.2).

3.1 Serum G-CSF levels are elevated in Lyn-deficient mice

Previous studies have shown that G-CSF levels are elevated in Lyn^−/−IL-10^−/− mice and in mice lacking Lyn in DCs.^{17, 18} To clarify if levels were elevated in aged Lyn^−/− mice, we measured serum G-CSF, finding that levels were significantly higher in 36-week-old mice, compared with healthy C57BL/6 mice (Figure S1A).

3.2 Lyn-deficient mice lacking G-CSF maintain hallmarks of systemic inflammation

To determine whether G-CSF contributes to the pathogenesis of lupus in Lyn^−/− mice, we generated Lyn^−/−G-CSF^−/− mice that were aged to 36 weeks and assessed alongside age-matched C57BL/6 controls and single-mutant Lyn^{− /−} and G-CSF^−/− mice for hallmarks of inflammation and pathology. RT-PCR of lung tissue confirmed that expression of the G-CSF gene, Csf3, was not detected in Lyn^−/−G-CSF^−/− mice (Figure S1B). When compared with Lyn^−/− mice, Lyn^−/−G-CSF^−/− mice exhibited similar splenomegaly (Figure S1C), loss of splenic cellularity (Figure S1D), and splenic erythropoiesis, as indicated by the expansion of splenic erythroblasts (Ter119⁺CD71^±) (Figure S1E,F), indicating that G-CSF does not grossly influence inflammation in Lyn^−/− mice.

3.3 Neutrophil expansion, but not activation, is moderated in Lyn^−/−G-CSF^−/− mice

We next assessed the impact of G-CSF deficiency on neutrophil development in Lyn^−/− mice. In line with previous studies,²⁴ mice deficient in G-CSF exhibited impaired BM neutrophil numbers, which were similarly reduced in Lyn^−/−G-CSF^−/− mice (Figure 1A,B). In addition, the expression of the neutrophil maturation marker CXCR2 was similarly reduced on BM neutrophils from both G-CSF^−/− and Lyn^−/−G-CSF^−/− mice (Figure 1C), indicating that G-CSF-deficiency is dominant in the BM.

In spleen, neutrophil numbers were largely normalized in aged Lyn^−/−G-CSF^{− /−} mice, being similar to those of C57BL/6 mice and augmented above the neutropenia of G-CSF^−/− mice; however, they remained significantly below the neutrophil expansion observed in aged Lyn^−/− mice (Figure 1D,E). Interestingly, splenic neutrophils from aged Lyn^−/−G-CSF^−/− mice exhibited a striking loss of CD62L expression (Figure 1F) and modest upregulation of CD11b (Figure 1G), indicative of activation and in line with that in Lyn^−/− mice. However, this trait appears to be intrinsic to Lyn-deficiency, as it was observed in young mice before disease onset (Figure S2). Thus, despite moderating neutrophil expansion, G-CSF-deficiency did not impact neutrophil activation in the periphery of Lyn^−/− mice.

3.4 G-CSF deficiency confers minor moderation of monocyte expansion in Lyn^−/− mice

As G-CSF can also act on monocytes, this compartment was examined. As reported, aged Lyn^−/− mice exhibited an expansion of monocytes^{15, 19}; however, this was significantly, albeit partially, moderated in Lyn^−/−G-CSF^−/− mice (Figure 2A,B). A notable shift in monocyte polarization was observed in both Lyn^−/− and Lyn^−/−G-CSF^−/− mice away from homeostatic classical monocytes (Ly6C⁺CD43⁻), which were more abundant in aged C57BL/6 mice, toward inflammatory nonclassical monocytes (Ly6C⁻CD43⁺) (Figure 2C,D), with expansion of both subsets observed in Lyn^−/− and Lyn^−/−G-CSF^−/− mice (Figure 2E). Interestingly, this inflammatory shift was also present, but to a lesser degree, in G-CSF^−/− mice. Collectively, these data indicate that deficiency of G-CSF has a minor impact on monocyte expansion, but it does not attenuate monocyte polarization in Lyn^−/− mice.

3.5 G-CSF does not influence the B cell and plasma cell compartments of Lyn^−/− mice

Lyn^−/− mice exhibit B cell developmental defects and show B cell hyperactivation, which facilitates their autoimmune pathology, and thus the impact of G-CSF-deficiency on this compartment was examined. Aged Lyn^−/− mice exhibited typical B cell lymphopenia (CD19⁺B220⁺), which persisted in Lyn^−/−G-CSF^−/− mice; however, numbers of B cells were slightly recovered (Figure 3A,B). In addition, both Lyn^−/− and Lyn^−/−G-CSF^−/− mice showed reduced follicular B cells (CD21/35^loCD23⁺), complete loss of marginal zone B cells (CD21/35^hiCD23⁻), and expansion of the transitional B cell compartment (CD21/35⁻CD23⁻) (Figure 3C,D). Similarly, follicular B cells of Lyn^−/−G-CSF^−/− mice exhibited enhanced expression of MHCII (Figure 3E) as well as CD80 and CD86 (Figure 3F), as observed in Lyn^−/− mice. Whereas G-CSF-deficient mice displayed a minor expansion of plasma cells, marked plasmacytosis was equivalently observed in both Lyn^−/− and Lyn^−/−G-CSF^−/− mice (Figures 3A and 3G), with plasma cells from both genotypes exhibiting similarly augmented expression of the immunoregulatory FcγRII/III (Figure 3H). Overall, this indicates that G-CSF is not implicated in the developmental defects or hyperactivation of the B cell and plasma cell compartments in Lyn^−/− mice.

3.6 G-CSF does not alter the DC phenotype of Lyn^−/− mice

DCs are strongly associated with the lupus disease process in Lyn^−/− mice¹⁸ and in human SLE,²⁵ and G-CSF is implicated in modulation of DC function and the promotion of anergic T cells.²⁶ Phenotyping of conventional DCs (cDCs) of aged mice revealed that both Lyn^−/− and Lyn^−/−G-CSF^−/− mice exhibited a significant deficit in the splenic cDC1 subset (CD11c^hiMHCII⁺CD8α⁺CD172α⁻), whereas the cDC2 subset (CD11c^hiMHCII⁺CD8α^-CD172α⁺) was unaffected (Figure S3A–C). On further examination, expression levels of CD86 were normal on cDC1, but elevated on cDC2 of both Lyn^−/− and Lyn^−/−G-CSF^−/− mice (Figure S3D and Figure 3E), indicative of a hypermature phenotype. These data suggest that G-CSF deficiency does not alter the cDC phenotype of Lyn^−/− mice.

3.7 CD4⁺ effector memory T cells are expanded in Lyn^−/−G-CSF^{− /−} mice

T cells are implicated in the pathogenesis of autoimmune disease in Lyn^−/− mice, where they display inflammation-driven hyperactivation,^{15, 19, 27} and, therefore, we examined whether G-CSF influenced this phenotype. Unexpectedly, aged G-CSF^−/− mice exhibited an expansion of CD4⁺ T cells, and contrary to the well-established CD4⁺ T cell lymphopenia observed in aged Lyn^−/− mice, Lyn^−/−G-CSF^−/− mice had normal numbers of CD4⁺ T cells, comparable to C57BL/6 controls (Figure 4A,B). CD4⁺ T cells from both Lyn^−/− and Lyn^−/−G-CSF^−/− displayed a similarly activated phenotype with greater proportions of cells expressing the activation markers CD25 (Figure 4C) and CD69 (Figure 4D). Although phenotypic subsetting revealed that the CD4⁺ T cells of both Lyn^−/− and Lyn^−/−G-CSF^−/− mice were skewed toward an effector memory (CD44⁺CD62L⁻) phenotype (Figure 4E,F), an expansion of numbers was only seen in Lyn^−/−G-CSF^−/− mice due to their increase in total CD4⁺ T cells, compared with Lyn^−/− mice (Figure 4G).

Examination of CD8⁺ T cells revealed no differences in CD8⁺ T cell lymphopenia, skewing to an effector memory phenotype (CD44⁺CD62L⁻) and enhanced CD69 expression between Lyn^−/− and Lyn^−/−G-CSF^−/− mice (Figure S4A–D).

3.8 G-CSF deficiency exacerbates autoimmunity and kidney pathology in Lyn^−/− mice

We next assessed the contribution of G-CSF to autoimmunity and kidney pathology in Lyn-deficient mice. Autoantibody titers were minimal in aged G-CSF^−/− mice, comparable to those of C57BL/6 mice (Figure 5A). Although variable, Lyn^−/− mice developed, on average, autoantibody titers that were 398 times greater than those detected in C57BL/6 mice, which were further augmented in Lyn^−/−G-CSF^−/− mice, being five times greater than titers in Lyn^−/− mice (Figure 5A). Kidney pathology was assessed via three independent histopathological measures: scoring of glomerular hypercellularity and dysregulation of tissue morphology; assessing frequency of glomeruli exhibiting necrotic lesions (segmental necrosis); and measuring glomerular expansion (cross-sectional area). These histopathological studies showed that kidneys from aged G-CSF^−/− mice were healthy with glomeruli exhibiting a normal rounded morphology with only mild subcapsular space enlargement, no indication of cellular infiltration (median tissue score 0.39, IQR: 0.26–0.46) with negligible segmental necrosis, and standard cross-sectional area (median area 4396 μm², IQR: 3801–4335), comparable to aged-matched C57BL/6 mice (median tissue score 0.33, IQR: 0.25–0.46; median area 3888 μm², IQR: 3451–4666) (Figure 5B–E). Conversely, both aged Lyn^−/− mice and Lyn^−/−G-CSF^−/− mice exhibited varying degrees of glomerular injury, denoted as moderate, advanced, and extreme, which correlate with the described scoring criteria of 0–4 (Figure 5B). Lyn^−/−G-CSF^−/− mice exhibited further glomerular hypercellularity and dysregulation of tissue morphology (median tissue score 2.65, IQR: 2.47–2.77) with markedly increased occurrence of segmental necrosis (median necrosis freq. 11.62%, IQR: 2.15–18.28) and enhanced glomerular expansion (median area 7106 μm², IQR: 6035–7704), compared with Lyn^−/− mice (median tissue score 2.14, IQR: 1.94–2.30; median necrosis freq. 0.0%, IQR: 0.00–2.14; median area 5471 μm², IQR: 5079–6247) (Figure 5B–E). Collectively, these data suggest that deficiency of G-CSF augments the development of autoimmunity and resultant kidney pathology in Lyn^−/− mice.

3.9 Serum G-CSF levels are not increased in patients with lupus or those with active renal disease

To determine whether G-CSF is implicated in human disease, G-CSF was measured in the serum of 198 SLE patients and 38 healthy controls (HCs) (Table S1). G-CSF was detectable in 84.3% (167/198) of SLE patients and 92.1% (35/38) of HC (p = .21, Pearson's χ ² test), and there was no significant difference in serum G-CSF concentrations between patients with SLE (median: 377.1 pg/ml, IQR: 133.6–1067.2) and HC (median: 440.5 pg/ml, IQR: 192.2–1799.2) (Figure 6A). Furthermore, there was no difference in the proportion of SLE patients with detectable serum G-CSF concentrations between SLE patients with active (82.6%, 38/46) or inactive (84.9%, 129/152) renal disease (p = .71, Pearson's χ ² test), nor in the serum G-CSF concentrations between SLE patients with active (median: 404.0 pg/ml, IQR: 115.4–990.8) or inactive (median: 371.4 pg/ml, IQR: 161.5–1350.2) renal disease (Figure 6B). This indicates that serum G-CSF levels do not correlate with lupus incidence or kidney disease activity in humans.