High Systemic Type I Interferon Activity is Associated with Active Class III/IV Lupus Nephritis

Abstract

Objective:

Previous studies suggest a link between high serum type I interferon (IFN) and lupus nephritis (LN). We determined whether serum IFN activity is associated with subtypes of LN and studied renal tissues and cells to understand the impact of IFN in LN.

Methods:

221 systemic lupus erythematosus (SLE) patients were studied. Serum IFN activity was measured by WISH bioassay. mRNA in-situ hybridization was used in renal tissue to measure expression of the representative IFN-induced gene, interferon-induced protein with tetratricopeptide repeats-1 (IFIT1), and the plasmacytoid dendritic cell (pDC) marker gene C-type lectin domain family-4 member C (CLEC4C or BDCA2). Podocyte cell line gene expression was measured by real-time PCR.

Results:

Class III/IV LN prevalence was significantly increased in patients with high serum IFN compared with those with low IFN (OR=5.48, p=4.0×10⁻⁷). In multivariate regression models, type I IFN was a stronger predictor of class III/IV LN than complement C3 or anti-dsDNA antibody, and could account for the association of these variables with LN. IFIT1 expression was increased in all classes of LN, but most in the glomerular areas of active class III/IV LN kidneys. IFIT1 expression was not closely co-localized with pDCs. IFN directly activated podocyte cell lines to induce chemokines and proapoptotic molecules.

Conclusion:

Systemic high IFN is involved in the pathogenesis of severe LN. We do not find co-localization of pDCs with IFN signature in renal tissue, and instead observe the greatest intensity of IFN signature in glomerular areas, which could suggest a blood source of IFN.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease caused by dysregulation of the immune system and characterized by autoantibody production and immune complex formation with multi-organ system involvement (1). It affects women 9 times more often than men, and generally diagnosed in early adulthood (2, 3). It is also well known that genetic factors play an important role in the development of SLE (1). Among the various organ manifestations of SLE, lupus nephritis (LN) represents one of the most serious which can progress to end-stage renal disease leading to increased morbidity and mortality. LN affects about 40% of SLE patients throughout their lifetime (3, 4). Despite recent advances in treatment, patients with LN still have higher morbidity and mortality compared with those without LN (4–6).

Type I interferon (IFN), in particular IFNα, plays a crucial role in the pathogenesis of SLE, having pleiotropic effects on various cell types and the potential to break immunologic self-tolerance (7, 8). IFNα induces dendritic cell differentiation to uptake self-antigens from dying cells and present them to CD4+ T cells, driving the autoimmune response (9). IFNα also induces B cell activation and differentiation including autoreactive B cells promoting autoantibody production (10). IFNα increases the production of B cell survival factors, such as B lymphocyte stimulator, from dendritic cells (10). Furthermore, in vitro studies show that antinuclear autoantibody immune complexes induce IFNα production in plasmacytoid dendritic cells (pDCs) via endosomal nucleic acid-sensing Toll-like receptors (TLRs) via their RNA or DNA component (11, 12), suggesting a feed-forward mechanism for antinuclear antibodies in enhancing IFNα production in SLE.

High serum IFNα activity is a heritable risk factor for SLE, as familial aggregation of high serum IFNα activity is observed in SLE families (13, 14), and some SLE-risk genetic variants which function in the IFNα pathway are gain-of-function in SLE patients (15–17). Serum IFNα activity is higher in patients with SLE compared with patients with other autoimmune diseases or healthy individuals (18–20). High levels of serum IFNα activity and increased IFN-inducible gene expression in peripheral blood mononuclear cells (PBMCs) are also associated with more severe disease, as well as with the presence of SLE-associated autoantibodies (19–23). Moreover, increased expression of IFNα-inducible genes in PBMCs is associated with LN, and the presence of antinuclear autoantibodies including anti-double-stranded DNA (anti-dsDNA) antibodies (21–24). However, the relationship between IFN and LN ISN/RPS histologic classes and activity/chronicity indices are less clear, and it is not known whether these relationships are independent of the strong association between type I IFN and autoantibodies such as anti-dsDNA. The source of IFN in renal disease is also unclear. While local production of IFN by pDCs in renal tissue would be possible, this has not been documented in recent single cell sequencing studies (25), despite a strong type I IFN signature in renal cells.

In this study, we determined whether serum type I IFN activity is associated with different classes of LN. We found that high serum type I IFN activity was significantly associated with class III/IV LN but not with other classes of LN, and this was independent of the presence of anti-dsDNA antibody and complement levels. We also found that the expression of the type I IFN-inducible gene, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) was significantly increased in class III/IV LN kidneys in a glomerular pattern which was not strongly co-localized with pDCs. Finally, in vitro experiments demonstrated that type I IFN induced proapoptotic molecules and chemokines in human podocytes. In aggregate these results suggest that systemic high type I IFN is involved in the pathogenesis of severe LN.

PATIENTS AND METHODS

Patients, samples, and clinical data

Serum samples and clinical data of 221 European-American SLE patients were obtained from the Mayo Clinic (Rochester). Patient’s ancestral background was determined by interview and self-report of the origins of the subject’s ancestors. Slides from kidney biopsy specimens for mRNA in situ hybridization experiments were obtained from NYU Medical Center. All patients fulfilled ≥4 of the American College of Rheumatology (ACR) revised criteria for the diagnosis of SLE (26). Patients provided informed consent and the study was approved by the Institutional Review Boards at the respective institutions (Mayo IRB# PR12-007618-01 and NYU IRB# S18-00489). Data regarding the presence or absence of ACR criteria for the diagnosis of SLE (26) and detailed clinical data were available. Samples were collected and studied at any point after the formal diagnosis of SLE. Non-lupus nephritis (LN) patients were defined as patients with SLE who never had kidney involvement, and LN patients are defined as SLE patients with kidney involvement based on renal biopsies who currently have LN or have a history of LN at the time of recruitment. Classes of LN were confirmed by renal biopsy review according to the classifications presented by the International Society of Nephrology/Renal Pathology Society (ISN/RPS) (27). Active LN is defined as renal domain ≥1 at least including urine protein ≥0.5g/24hr in systemic lupus erythematosus disease activity index 2000 (SLEDAI-2K) renal criteria (28).

Autoantibody measurement

Anti-nuclear antibodies (ANA) were measured by indirect immunofluorescence methods. Anti-dsDNA antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Anti-dsDNA antibody <30.0 IU/mL was considered negative and anti-dsDNA antibody>75.0 IU/mL was considered positive. Ranges between 30.0–75.0 IU/mL were considered as a borderline result and were re-analyzed using Crithidia luciliae immunofluorescence and the detectable fluorescence was considered positive. All the blood samples were assayed and analyzed in the same clinical laboratory at Mayo Clinic, and standard clinical cut-offs were used to define a positive result.

Serum type I IFN activity measurement

Type I IFN activity in sera was measured by performing an IFN bioassay as described previously (13, 29). Briefly, reporter cells (WISH cells, ATCC #CCL-25) were used to measure the ability of sera to cause type I IFN-induced gene transcription. WISH cells were incubated with patient sera for 6 hours and then lysed and 3 canonical type I IFN-induced transcripts (i.e. interferon-induced protein with tetratricopeptide repeats 1; IFIT1, myxovirus resistance 1; MX1 and protein kinase R; PKR) were measured by reverse transcriptase-polymerase chain reaction. Relative expression data from the 3 transcripts were then normalized by the healthy control data and presented as a type I IFN activity score (13). Pretreatment of sera with anti-IFN-alpha and anti-IFN-beta antibodies completely abrogates the IFN-induced gene expression observed in the assay, confirming that type I IFN is driving the readout observed. The WISH cells do not express type II IFN receptor or endosomal TLRs, and the assay output does not decrease in the presence of cycloheximide, ruling out autocrine loops and other parallel stimuli contributing to the gene expression observed.

mRNA in situ hybridization in kidney biopsy specimens

RNA in situ hybridization (ISH) for the IFN-induced gene IFIT1 and the pDC marker gene C-type lectin domain family 4 member C (CLEC4C, also known as BDCA2) was performed using RNAscope® 2.5 HD Red assay kit (Advanced Cell Diagnostics) and RNAscope probe specific to the gene encoding Homo sapiens (Hs) IFIT1 transcript variant 3 mRNA and Hs CLEC4C transcript variant 2 mRNA, according to the manufacturer’s instructions. Briefly, 5 μm formalin-fixed paraffin embedded biopsied kidney tissue sections were pretreated with heat and proteases prior to hybridization with the target oligo probes. Preamplifiers and amplifiers were sequentially hybridized to the target probe, and alkaline phosphatase-based amplifiers were then hybridized in final steps followed by detection using chromogenic substrate. Samples were quality-controlled for RNA integrity with an RNAscope probe specific to Hs-PPIB RNA and for background signal using bacterial negative control probe DapB. Specific RNA staining signal was identified as red, punctate dots. ISH for IFIT1 mRNA and CLEC4C mRNA was done in serial sections for every sample. Quantitative measurement of mRNA expression (i.e. IFIT1 and CLEC4C) and heatmap analysis was performed by Visiopharm Integrator System (Visiopharm).

Type I IFN-induced gene expression in human podocytes in vitro

A human immortalized podocyte cell line transformed with thermosensitive SV40 large T antigen (U19tsA58) and human telomerase (hTERT), was kindly provided by Dr. Jeffrey B. Kopp (NIH) (30). Briefly, podocyte cell line was first cultured in RPMI 1640 supplemented with 10% heat inactivated fetal bovine serum, insulin-transferrin-selenium G supplement and penicillin/streptomycin, in type 1 collagen-coated dishes under growth permissive condition (33°C) for cell expansion. Cells were then cultured under growth restricted condition (37°C) for 7 days for differentiation. After completing the differentiation step, cells were cultured in medium only or media with recombinant human (rh) IFNα (PBL Assay Science) 5000U/ml or rhIFNβ (PBL Assay Science) 5000U/ml for 24 hours. RNA extraction and cDNA synthesis were done according to the manufacturer’s instructions using the mRNA micro kit (QIAGEN) and subsequent cDNA synthesis was done by PrimeScript^™ II 1st strand cDNA synthesis kit (Takara/Clontech) according to the manufacturer’s instructions. qPCR was perfomed using 40 cycles on a StepOnePlus Real-Time PCR System (Applied Biosystems). TaqMan probes and primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs99999905_m1), BCL2-antagonist/killer 1 (BAK1, Hs00832876_g1) Bcl-2-associated X protein (BAK1, Hs00180269_m1), cathepsin L (CTSL, Hs00377632_m1), monocyte chemotactic protein-1 (MCP1, Hs00234140_m1), and C-X-C motif chemokine 10 (CXCL 10, Hs00171042_m1) were purchased from Applied Biosystems.

Statistical analysis

Data are summarized as mean ± SD or as median and interquartile (IQR) range as appropriate depending on the distribution of the data. Categorial analyses were performed using the chi-square test or the Fisher’s exact test, and quantitative analyses were performed using the 2-sample t-test, Mann-Whitney U test, or Kruskal-Wallis test again depending on the distribution of the data. Spearman’s rank order correlation analysis was performed to investigate the correlations among variables. A stepwise multivariate logistic regression was performed to investigate predictor variables and type I IFN activity as the outcome variable. P values < 0.05 for variables in the multivariate analysis were considered significant.

RESULTS

High serum type I IFN activity is associated with a higher prevalence of active class III/IV lupus nephritis

Clinical characteristics of 221 European-American SLE patients stratified by low and high levels of serum type I IFN activity are shown in Table 1. Mean ages at disease onset and at recruitment were significantly younger in the patients with high levels of serum type I IFN (IFN score ≥2) than those with low levels of serum type I IFN (IFN score<2) (p=0.0005 and p=0.0002, respectively), which was consistent with previous reports (14, 20). The presence of anti-dsDNA antibodies was associated with a high type I IFN measurement in the same sample (60.4% positive anti-dsDNA antibody in high type I IFN vs. 36.4% in low type I IFN, p=0.0025). Low complement C3 and C4 levels were also significantly associated with high levels of serum type I IFN in the patients with SLE (p=0.025 and p=0.030, respectively).

Table 1.

Clinical characteristics of European-American SLE patients stratified by low and high levels of serum type I IFN activity

	Total (n=221)	Low type I IFN (IFN score <2) (n=175)	High type I IFN (IFN score ≥2) (n=46)	P
Female, n (%)	188 (85.1)	149 (85.1)	39 (84.8)	NS
Age at recruitment, years	47.7 ± 16.3	49.8 ± 16.4	39.9 ±13.6	0.0002
Age of onset, years	36.1 ± 15.8	37.8 ± 16.0	29.9 ± 13.4	0.0005
Disease duration, years	11.6 ± 10.3	12.0 ± 10.7	10.0 ± 8.2	NS
ACR criteria*, n (IQR)	6 (5–7)	6 (5–7)	7 (6–8)	<0.0001
ANA, n (%)	218 (98.6)	174 (99.4)	44 (95.7)	NS
Anti-dsDNA antibody, n (%)	100 (41.7)	68 (36.4)	32 (60.4)	0.0025
Low complement C3, n (%)	29 (13.1)	18 (10.3)	11 (24.0)	0.0248
Low complement C4, n (%)	66 (29.9)	46 (26.3)	20 (43.5)	0.0298
Serum creatinine, mg/dL	0.93 ± 0.34	0.92 ± 0.31	0.98 ± 0.46	NS
Lupus nephritis†, n (%)	72 (32.6)	46 (26.3)	26 (56.5)	0.0002
Total SLEDAI score, (IQR)	2 (0–4)	2 (0–4)	4 (2–8)	<0.0001
Current medications:
Hydroxychloroquine, n (%)	171 (77.4)	140 (80.0)	31 (67.4)	NS
Cyclophosphamide, n (%)	2 (0.9)	2 (1.1)	0 (0.0)	NS
Azathioprine, n (%)	20 (9.0)	14 (8.0)	6 (13.0)	NS
Mycophenolate mofetil, n (%)	55 (24.9)	43 (24.6)	12 (26.1)	NS
Methotrexate, n (%)	24 (10.9)	20 (11.4)	4 (8.7)	NS
Prednisolone, mg/day (IQR)	0 (0–5)	0 (0–5)	5 (0–7.9)	0.0026

Data are means ± SD or median (IQR). P values <0.05 were considered significant. NS; not significant.

^*Number of ACR criteria fulfilled by the time of recruitment.

^†Lupus nephritis was diagnosed on the basis of renal biopsy findings.

ANA; anti-nuclear antibody, SLEDAI; systemic lupus erythematosus disease activity index.

SLE patients with high levels of serum type I IFN had a greater number of ACR clinical criteria and higher concurrent SLEDAI-2K scores than those with low levels of serum type I IFN (p<0.0001, respectively) (Table 1). In addition, serum type I IFN levels and SLEDAI-2K scores were positively correlated in our cross-sectional evaluation (r=0.28, p<0.0001) (Suppl. Figure 1). Furthermore, SLE patients with high levels of serum type I IFN had a higher incidence of LN (ever or current) (56.5%) than those with low levels of serum type I IFN (26.3%) (OR=3.65, p=0.0002 (Table 1). Medications were not significantly different in either group, except that the dose of prednisolone was higher in the patients with high levels of serum type I IFN than those with low levels of serum type I IFN (p=0.0026).

As shown in Table 2, the risk of class III/IV LN was strikingly increased in the patients with high levels of serum type I IFN than in those with low levels of serum type I IFN (OR=5.48, p=4.0×10⁻⁷) (Table 2). Specifically, 88.5% of patients with high IFN had proliferative LN compared to 58.7% with low IFN (Table 2).

Table 2.

SLE patients with high levels of type I IFN or with a positive result for anti-dsDNA antibody have a higher incidence of class III/IV lupus nephritis (LN)

Class of LN*	Low type I IFN (IFN score <2) (n=175)	High type I IFN (IFN score ≥2) (n=46)	OR (95% CI)	P value
LN (total)	46	26	3.65 (1.9–7.2)	1.8×10−4
Class III/IV LN	27	23	5.48 (2.7–11.1)	4.0×10−7
Non-class III/IV LN	19	3	0.54 (0.2–1.8)	0.42
Subtype of LN	Anti-dsDNA antibody (−) (n=127)	Anti-dsDNA antibody (+)† (n=94)	OR (95% CI)	P value
LN (total)	34	38	1.86 (1.1–3.3)	0.042
Class III/IV LN	18	32	3.13 (1.6–6.0)	6.1×10−4
Non-class III/IV LN	16	6	0.47 (0.2–1.3)	0.17
Subtype of LN	Low C3 (−) (n=192)	Low C3 (+)‡ (n=29)	OR (95% CI)	P value
LN (total)	56	16	2.99 (1.3–6.6)	9.6×10−3
Class III/IV LN	36	14	4.04 (1.8–9.1)	1.3×10−3
Non-class III/IV LN	20	2	0.64 (0.1–2.9)	0.75
Subtype of LN	Low C4 (−) (n=156)	Low C4(+)‡ (n=65)	OR (95% CI)	P value
LN (total)	47	25	1.45 (0.8–2.7)	0.27
Class III/IV LN	30	20	1.87 (0.9–3.6)	0.08
Non-class III/IV LN	17	5	0.68 (0.2–1.9)	0.62

^*Subtypes of LN were confirmed by renal biopsy review according to the classification of the International Society of Nephrology/Renal Pathology Society (ISN/RPS) guidelines [27].

^†Positivity of anti-dsDNA antibody and

^‡low complement C3 or C4 levels were defined as described in the Methods.

P values were calculated by chi-square test (Fisher’s exact test). P values <0.05 were considered significant. NS; not significant.

Positivity of anti-dsDNA antibody at the time of the blood draw was also significantly associated with a diagnosis of LN (OR=1.86) and more strongly associated with class III/IV LN (OR=3.13, p=6.1×10⁻⁴) in univariate analyses (Table 2). Similarly, low levels of complement C3 were significantly associated with a diagnosis of overall LN (OR=2.99), and more strongly associated with class III/IV nephritis (OR=4.04, p=1.3×10⁻³). Complement C4 levels were not associated with overall LN or Class.

Given that type I IFN, anti-dsDNA positivity, and low complement C3 are all correlated with each other (20), and are each associated with class III/IV LN in our univariate analysis above, we performed a multivariate regression to determine independent predictors of class III/IV nephritis. In a logistic regression model, we found that high levels of serum type I IFN was the independent predictor strongly associated with active class III/IV LN (p=0.044, OR=3.6, stepwise logistic regression) (Suppl Table 1). In contrast, anti-dsDNA antibody positivity and low complement C3 levels were not significantly associated with active class III/IV LN when type I IFN was included in the multivariate regression. This suggests that type I IFN could account for a large portion of the association of these variables with class III/IV LN in univariate analyses. While anti-dsDNA antibody positivity and low complement C3 were not completely redundant and overlapping with high type I IFN, it is striking that type I IFN was a stronger overall predictor than either of the other two more traditional biomarkers of LN.

Type I IFN-inducible gene expression is increased in active class III/IV lupus nephritis

To look for the effects of type I IFN in the affected tissue, we next performed mRNA in situ hybridization (ISH) to detect the expression levels of IFIT1, one of the major type I IFN-inducible genes, and C-type lectin domain family 4 member C (CLEC4C, also known as BDCA-2), a unique marker of pDCs, in kidney biopsy samples obtained from healthy controls and LN patients. Target mRNA expression was normalized by cell counts as assessed by nuclei. IFIT1 mRNA expression was significantly increased in class III/IV LN kidneys compared to healthy kidneys (p<0.05) (Fig. 1A; red dots and 1B). However, there were no statistical differences in IFIT1 expression between class III/IV LN and non-class III/IV (Fig. 1B). IFIT1 expression was observed in both glomerular lesions and interstitial lesions in class III/IV LN kidneys (Fig. 1A), but the glomerulus demonstrated greater staining when tissue was examined in overview heatmaps (Fig. 2). CLEC4C gene expression levels in tissue did not differ between LN classes (data not shown). While infiltrating pDCs were present in all LN classes, these cells were scarce and tended to be localized in the interstitial areas. Proximity analyses did not detect any significant proximity of pDCs to IFIT1 staining density (Fig. 3).

Figure 1. Expression of type I IFN-inducible gene IFIT1 is increased in class III/IV LN kidneys.

mRNA in situ hybridization (ISH) was performed. Briefly, 5 μm formalin-fixed paraffin embedded biopsied kidney tissue sections were pretreated with heat and proteases prior to hybridization with the target oligo probes. Preamplifiers and amplifiers were sequentially hybridized to the target probe, and alkaline phosphatase-based amplifiers were then hybridized in final steps followed by specific RNA detection using chromogenic substrate. (A) mRNA ISH for IFIT1 was performed using biopsied kidney samples. Pathological LN classes were classified according to the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of LN. Representative photomicrographs of mRNA ISH of glomerular lesions (upper panels) and interstitial lesions (lower panels) of the kidneys are shown for class IV LN and class V LN. Kidney specimens obtained from the non-cancerous lesion dissected from kidney clear cell carcinoma were used as controls. Each red dot shows the expression of IFIT1 mRNA. Bars indicate 50μm. (B) Scatter plot of IFIT1 mRNA expression ratio (normalized by the number of nucleus) in control kidneys, class III/IV LN kidneys and non-class III/IV LN kidneys are shown (n=4, 9 and 4, respectively). Data are expressed as median with interquartile range, *p<0.05. IFIT1, interferon-induced protein with tetratricopeptide repeats 1; LN, lupus nephritis

Figure 2. IFIT1 mRNA expression in LN kidney is higher in glomerulus lesion compared to that in interstitial lesion.

Quantitative measurement and heatmap analysis of mRNA IFIT1 expression in LN and control mRNA ISH samples were performed by Visiopharm Integrator System (Visiopharm). Representative figures of each LN subtype and control are shown. Heatmap analysis results are representative of 4 control kidneys, 9 class III/IV LN kidneys and 4 non-class III/IV LN kidneys. Red circle indicates glomerulus lesion.

Figure 3. No significant co-localization of pDCs with IFIT1 expression was observed in the renal tissue.

mRNA in situ hybridization (ISH) for CLEC4C and IFIT1 mRNA was performed in class III/IV LN kidneys and non-class III/IV LN kidneys (n=4 and 9, respectively). Representative figures of CLEC4C and IFIT1 mRNA expression density are shown in class IV LN kidney. In each sample, both CLEC4C and IFIT1 mRNA ISH are done in serial section. Each red dot indicates mRNA expression as indicated above. (A) Lesion with pDC (CLEC4C) infiltration. (B) Lesion without pDC (CLEC4C) infiltration. Black arrows indicate CLEC4C mRNA expression. Bars indicate 50μm. CLEC4C, C-type lectin domain family 4 member C; IFIT1, interferon-induced protein with tetratricopeptide repeats 1.

Type I IFN induces mRNA expression of proapoptotic molecules and chemokines in human podocytes

Based on the prominent expression of IFIT1 mRNA in the glomeruli of class III/IV LN (Fig. 1A) and previous reports indicating that podocyte injury is a pathologic feature of class III/IV LN (31, 32), we next determined the direct effect of type I IFN on human podocyte cell lines. IFNα and IFNβ both significantly induced mRNA expression of proapoptotic genes BAK1 and BAX compared to unstimulated podocytes at 24 hours (Fig. 4), with IFNβ inducing greater mRNA expression of BAK1 than IFNα. IFNα and IFNβ also significantly induced cathepsin L (CTSL) mRNA expression (Fig. 4), which degrades cytoskeleton related proteins such as CD2 associated protein during podocyte injury (33). Furthermore, IFNβ induced MCP1 and CXCL10 that are indispensable for recruiting inflammatory cells (monocytes/macrophages and pDCs) to inflamed tissues (34), whereas IFNα induced CXCL 10 alone (Fig. 4). These results indicate that type I IFN induces gene expression patterns that characterize podocyte injury in LN as well as chemokines that can result in inflammatory cell recruitment.

Figure 4. Type I IFN induces mRNA expressions of proapoptotic molecules, chemokines, and cathepsin L in human podocytes.

Human podocyte cell lines were first cultured under growth restricted condition (37°C) for 7 days for differentiation as described in the method. Cells were then stimulated with either recombinant human (rh) IFNα (5000 unit/mL), rhIFNβ (5000 unit/mL) or medium alone for 24 hours and RT-qPCR was performed to measure mRNA expressions for proapoptotic molecules BAK1 and BAX, chemokines CXCL10 and MCP1, and cathepsin L (CTSL). mRNA expression results are representative of 3 independent experiments. Data are expressed as means ± SEM (n=3 experiments). P values<0.05 were considered significant. *p<0.05 and **p<0.01.

DISCUSSION

In the present study, the large cohort allowed us to analyze detailed SLE patient clinical data within a single ancestral background, reducing the complexity as different ancestral backgrounds have a different prevalence of both clinical manifestations and high type I IFN (20). We show that high serum type I IFN activity is significantly associated with class III/IV LN but not with non-class III/IV LN in European-American SLE patients. We also demonstrate that the expression of type I IFN-inducible gene signature (i.e. IFIT1) is significantly increased in class III/IV LN kidneys. While pDCs were observed in the tissue, they did not correspond spatially to the type I IFN signature in tissue. Type I IFN induced mRNA expression of chemokines and molecules related to apoptosis and cytoskeleton function in human podocytes. Together, these results suggest a pathogenic role for systemic high type I IFN in severe class III/IV LN in SLE patients.

Our results are novel as we can separate the association of high serum type I IFN activity with active class III/IV LN in SLE as independent from anti-dsDNA antibody positivity or complement levels in regression models. This is important, as anti-dsDNA antibodies and low complement C3 are associated with LN (35–37), and are also associated with increased expression of type I IFN-inducible genes in PBMCs (21–23) and serum IFNα activity (20). In multivariate logistic regression, we found that the association between serum type I IFN activity and class III/IV LN is primary, and not secondary due to the presence of positive anti-dsDNA antibodies or low complement C3. Thus, our results suggest the importance of high type I IFN activity over anti-dsDNA antibodies in the pathogenesis of class III/IV LN.

Similar to recent single cell gene expression studies, we find increased type I IFN-inducible gene expression in the kidneys of LN patients. Using mRNA ISH, we could spatially map type I IFN signature in the tissue, providing the novel finding that the IFN signature was most prominent in glomerular areas of the kidney. IFIT1 mRNA was universally expressed in the glomeruli of class III/IV LN: in the mesangium, endothelium, epithelium, and infiltrating inflammatory cells. Together with the association of serum type I IFN activity with the presence of class III/IV LN, this suggests that high systemic type I IFN in SLE plays a roles in the pathogenesis of proliferative nephritis. Podocyte injury is a pathologic feature of class III/IV LN (31, 32). We found type I IFN induced expression of MCP1, CXCL10, and other molecules related to apoptosis and inflammation in human podocytes. Interestingly, IFN-β had a greater impact on some transcripts in the podocyte cell lines than did IFN-α, and this could be important pathologically. The relative contributions of IFN-α vs. IFN-β in human SLE are not currently well understood, but may contribute to the observed heterogeneity in patient manifestations. Because all of the glomerular cell types demonstrate an IFN signature in our study, future studies are needed to investigate the direct effect of type I IFN on other resident glomerular cells, i.e. mesangial cells, tubular, and endothelial cells in LN.

We investigated pDC infiltration in the biopsies because pDCs are a primary type I IFN-producing cell (34). Interestingly, we did not find any significant differences in pDC infiltration in the kidneys among any of the LN classes. However, IFIT1 expression in the glomeruli was significantly increased only in class III/IV LN but not in the other classes. There was no significant co-localization of pDCs with IFIT1 expression in the renal tissue. Thus, while pDCs can produce massive amounts of type I IFN after TLRs are ligated by nucleic acid-containing immune complexes (11), these data suggest that the initial source of the IFN signature in the kidney may not be the pDC. Other recent studies have also suggested alternate sources of the type I IFN signature in SLE (38). pDCs can play a number of other roles in immunity and inflammation, including inflammatory cytokine and chemokine production as well as antigen presentation and cross-presentation to CD4+ T cells (34), and it is possible that they could be functioning this way in LN.

Limitations of this study include the cross-sectional design which could not assess the fluctuation of variables over time. While patients were receiving various immunosuppressive agents, previous studies have shown that most of the immunosuppressive agents (i.e. hydroxychloroquine, azathioprine and mycophenolate mofetil) have not reduced type I IFN-induced gene expression in PBMCs when compared to SLE patients who were not receiving these agents (22). Further negating concerns regarding the influence of treatment, we did not find any association between commonly used medications and circulating type I IFN except for the well-described reduction associated with corticosteroids.

In conclusion, we have shown that high serum type I IFN activity is associated with active class III/IV LN in European-American SLE patients, which is independent of anti-dsDNA antibody status and complement levels. Second, we have shown that type I IFN-inducible gene expression is increased in class III/IV LN kidneys and not clearly related to pDC infiltration, and that type I IFN directly stimulates podocytes to induce chemokines and molecules related to podocyte injury. These results taken together suggest that systemic high type I IFN in SLE contributes to severe kidney involvement. While type I IFN receptor blockade has been shown to improve human SLE overall (39), we await trials of anti-IFN agents specifically focused on LN (40).