Systemic Lupus Erythematosus (SLE) in (NZB x NZW)F1 Mice
(also a model of secondary Sjögren's syndrome)

Typical Results - Spontaneous SLE | Typical Results - Spontaneous Secondary Sjögren's Syndrome

(NZB x NZW)F1 mice (also referred to as NZBWF1 mice) spontaneously develop autoimmune diseases closely resembling SLE and secondary Sjögren's syndrome. We routinely run this model in female NZBWF1 mice.

Similar to human patients with SLE, these mice develop high concentrations of autoimmune anti-dsDNA and anti-nuclear antibodies, proteinuria, and immune complex glomerulonephritis. These mice have a strong type I interferon signature. Production of anti-nuclear and anti-dsDNA antibodies is detectable as early as 20 weeks of age. Mice develop extensive kidney pathology over a range of ages, typically following decline in body condition and several weeks of high proteinuria scores. As in human patients, disease in NZBWF1 mice is polygenic and spontaneous, making this an excellent model of human SLE, with very good predictive value for drug efficacy.

These mice also develop inflammation in the submandibular and lacrimal glands closely resembling secondary Sjögren's syndrome.

Treatment usually starts when mice are 21 to 24 weeks old, when initial signs of disease are normally present in some mice, but before severe proteinuria and irreversible kidney damage develop in most mice. By 42 weeks of age, 80 to 100% of untreated mice will develop glomerulonephritis.

Our standard in vivo readouts are proteinuria, body weight, and anti-dsDNA antibodies in serum or plasma. At the end of the study, we typically measure kidney and spleen weights, anti-dsDNA antibodies and blood urea nitrogen (BUN) in serum or plasma, and perform complete blood count (CBC) and histological analysis of kidneys. We can also perform immunofluorescence (IF) analysis of kidneys to evaluate IgG and C3 presence in the kidney cortex (glomeruli), flow cytometric analysis of splenocytes and kidney-infiltrating cells, and histological analysis of submandibular glands and lacrimal glands.

Please contact Hooke at or with questions or for a quotation.

Typical results - spontaneous SLE

Mice were treated from week 23 of life (Week 23) through the end of the study (Week 42). Proteinuria was measured once per week and body weight twice per week from Week 21. Serum was collected from all mice at Weeks 23 and 34 for anti-dsDNA IgG antibody measurement. At the end of the study serum was collected again for anti-dsDNA IgG antibody and blood urea nitrogen (BUN) measurement, kidneys and spleens were weighed, and left kidneys were collected for histological analysis.

Graphs below show mean + SEM or mean ± SEM, and tables show mean ± SD. * indicates p < 0.05, ** p < 0.01, *** p < 0.001.

Clinical readouts

As shown below, cyclophosphamide treatment inhibits disease development as evidenced by significantly lower proteinuria scores at the end of the study. Mice treated with cyclophosphamide typically experience an early spike in proteinuria.

Treatment	# mice	End proteinuria score	p value
Vehicle	14*	3.43 ± 0.81	-
Cyclophosphamide	15	1.97 ± 0.23	<0.001

*One (1) mouse was euthanized at Week 35 due to complications of lymphadenopathy and all readouts were therefore excluded after euthanasia.
End proteinuria scores compared using Wilcoxon's non-parametric test.

Tissue weights

Treatment	Kidney (mg)*	p value	Spleen (mg)	p value
Vehicle	225 ± 49	-	148 ± 49	-
Cyclophosphamide	192 ± 19	0.022	48 ± 6	<0.001

Tissue weights compared using 2-tailed Student's t-tests.
*For each mouse, the average weight of both kidneys was calculated and used for analysis.

BUN and anti-dsDNA measurements

Treatment	BUN in serum (mg/dL)	p value
Vehicle	64.5 ± 47.0	-
Cyclophosphamide	19.2 ± 3.4	<0.001

BUN in serum concentrations compared using 2-tailed Student's t-test.

anti-dsDNA IgG in serum (units/mL)
Treatment	Week 23 (baseline)	Week 34	p value	Week 42 (terminal)	p value
Vehicle	436 ± 458	1268 ± 1377	-	2189 ± 1517	-
Cyclophosphamide	436 ± 458	194 ± 110	0.006	191 ± 81	<0.001

Anti-dsDNA antibody concentrations in serum compared using 2-tailed Student's t-tests.

Histological analysis of kidneys

Kidney histological analysis - Total glomerular lesion

Kidney histological analysis - Total tubular and interstitial lesion

Kidney histological analysis - Total kidney lesion

Histological analysis of kidneys
Treatment	Total glomerular lesion	p value	Total tubular and interstitial lesion	p value	Total kidney lesion	p value
Vehicle	4.9 ± 2.7	-	1.1 ± 1.0	-	6.1 ± 3.6	-
Cyclophosphamide	0.4 ± 0.9	<0.001	0.0 ± 0.0	<0.001	0.4 ± 0.9	<0.001

Kidney lesion scores compared using Wilcoxon's non-parametric tests.

Periodic acid-Schiff (PAS) staining of kidneys

Illustrative images of Periodic acid-Schiff staining in kidneys

For a higher resolution image, click on the image above. Scale bars are 250 µm. Insets illustrate kidney transverse sections at the hilus (kidney cut in half, both halves embedded and sectioned), with red outlines indicating the locations of the magnified images.

IF analysis of IgG in kidney cortex

Mice were treated from Week 21 through the end of the study (Week 41). At the end of the study, kidneys were collected for IF analysis of IgG in the kidney cortex (glomeruli).

Graph shows mean ± SEM, and table shows mean ± SD. *** indicates p < 0.001.

Treatment	# mice	IgG⁺ area of kidney cortex (%)	p value
Vehicle	16	5.6 ± 4.6	-
Cyclophosphamide	12	0.2 ± 0.2	<0.001

Proportion of IgG⁺ area of kidney cortex compared using 2-tailed Student's t-test.

IgG in kidneys

Illustrative images of IgG in kidney cortex

For a higher resolution image, click on the image above. Scale bars are 50 µm. Images are magnified on the kidney cortex from transverse sections at the hilus.

Flow cytometric analysis of kidneys

Mice were treated from week 23 of life (Week 23) through the end of the study (Week 42). At the end of the study, right kidneys and spleens were collected from all remaining mice in the Vehicle group and from 7 representative mice in the Cyclophosphamide group for flow cytometric analysis.

All graphs below show mean ± SEM, and tables show mean ± SD. ** indicates p < 0.01, *** p < 0.001.

Proportions of CD45+ cells in live kidney cells

Proportions of B220+CD4- cells in CD45+ kidney cells

Flow cytometric analysis of kidneys - proportions of cells
Treatment	# mice	CD45⁺ in live cells (%)	p value	B220⁺CD4^- in CD45⁺ cells (%)	p value	B220^-CD4⁺ in CD45⁺ cells (%)	p value
Vehicle	10	25.2 ± 12.1	-	6.6 ± 3.1	-	25.1 ± 9.3	-
Cyclophosphamide	7	2.3 ± 0.3	<0.001	2.8 ± 0.4	0.009	34.8 ± 6.0	0.008

Cell population proportions compared using 2-tailed Student's t-tests.

Numbers of CD45+B220+CD4- cells in kidney cells

Flow cytometric analysis of kidneys - numbers of cells
Treatment	Total live (x10³)	p value	CD45⁺ (x10³)	p value	CD45⁺B220⁺ CD4^- (x10³)	p value	CD45⁺B220^-CD4⁺ (x10³)	p value
Vehicle	1664 ± 425	-	458 ± 313	-	24.4 ± 11.6	-	104 ± 71	-
Cycloph.	874 ± 219	<0.001	20 ± 5	0.002	0.6 ± 0.1	<0.001	7 ± 2	0.002

Cell numbers compared using 2-tailed Student's t-tests.

Flow plots of CD45⁺ cells

Vehicle

Cyclophosphamide

Flow cytometric analysis of spleens

All graphs below show mean ± SEM, and tables show mean ± SD. ** indicates p < 0.01, *** p < 0.001.

Proportions of CD45+ cells in live splenocytes

Proportions of B220+CD4- cells in CD45+ splenocytes

Flow cytometric analysis of spleens - proportions of cells
Treatment	# mice	CD45⁺ in live cells (%)	p value	B220⁺CD4^- in CD45⁺ cells (%)	p value	B220^-CD4⁺ in CD45⁺ cells (%)	p value
Vehicle	10	99.7 ± 0.7	-	55.9 ± 6.8	-	24.6 ± 2.8	-
Cyclophosphamide	7	98.9 ± 0.5	0.733	27.6 ± 4.3	<0.001	39.1 ± 12.8	<0.001

Cell population proportions compared using 2-tailed Student's t-tests.

Numbers of CD45+B220+CD4- cells in spleens

Flow cytometric analysis of spleens - numbers of cells
Treatment	Total live (x10⁶)	p value	CD45⁺ (x10⁶)	p value	CD45⁺B220⁺CD4^- (x10⁶)	p value	CD45⁺B220^-CD4⁺ (x10⁶)	p value
Vehicle	115.5 ± 27.9	-	114.4 ± 27.6	-	64.9 ± 19.5	-	28.0 ± 6.5	-
Cyclophosphamide	23.4 ± 7.1	<0.001	23.2 ± 7.0	<0.001	6.5 ± 2.6	<0.001	9.0 ± 2.5	<0.001

Cell numbers compared using 2-tailed Student's t-tests.

Flow plots of CD45⁺ cells

Vehicle

Cyclophosphamide

Typical results - spontaneous secondary Sjögren's syndrome

Mice were treated from week 21 of age (Week 21) through the end of the study (Week 41). At the end of the study, submandibular and lacrimal glands were collected for histological analysis and inflammatory foci per section counted.

Submandibular glands

Lacrimal glands

Graphs show mean ± SEM, and table shows mean ± SD. *** indicates p < 0.001.

		Submandibular glands		Lacrimal glands
Treatment	# mice	Inflammatory foci per section	p value	Inflammatory foci per section	p value
Vehicle	16	56.9 ± 19.2	-	22.1 ± 8.5	-
Cyclophosphamide	12	7.2 ± 8.5	<0.001	7.6 ± 8.9	<0.001

Numbers of inflammatory foci compared using 2-tailed Student's t-tests.

Hematoxylin and eosin (H&E) staining of submandibular glands

Submandibular gland hematoxylin and eosin staining illustrative images

For a higher resolution image, click on the image above. Scale bars are 250 µm. Insets illustrate longitudinal submandibular gland sections (2 glands) with red outlines indicating the locations of the magnified images.

Hematoxylin and eosin (H&E) staining of lacrimal glands

Lacrimal gland hematoxylin and eosin staining illustrative images

For a higher resolution image, click on the image above. Scale bars are 250 µm. Insets illustrate longitudinal lacrimal gland sections (2 glands) with red outlines indicating the locations of the magnified images.

Systemic Lupus Erythematosus (SLE) in (NZB x NZW)F1 Mice
(also a model of secondary Sjögren's syndrome)