C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice methods
Aim. Evidence-backed execution summary for C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice methods from C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.
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mouse
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C9orf72 -/- mice exhibit lymphadenopathy and splenomegaly
reagent used in the protocol.
- Use
- During neurological testing, we unexpectedly noted palpable cervical masses in all C9orf72 -/- animals, but not in WT or C9orf72 +/- controls. Masses were palpable as early as 8 weeks of age and present in all null mice by 18 weeks, before onset of observed motor deficits. Upon dissection, the mass...
C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs
reagent used in the protocol.
- Use
- The enlargement of C9orf72 -/- spleens and LN suggests a disease process such as neoplasm or immune dysregulation, an unexpected finding given that ALS/FTD is not linked to such pathology in human patients. To address these possibilities, histopathology was conducted on spleen and LN from 8-60 week...
C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells
reagent used in the protocol.
- Use
- To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal...
IL-12, IL-17a, IL-10, and TNF are increased in C9orf72 -/- serum
reagent used in the protocol.
- Use
- To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was approximately 6-fold increased in C9orf72 -/- animals compared with controls. IL-10, IL-17a, and TNFα were also upregulated,...
C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells
reagent used in the protocol.
- Use
- An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleen...
C9orf72 -/- mice have high titers of autoantibodies
reagent used in the protocol.
- Use
- Expansions in plasma cells and transitioning B cells/plasmablasts can be associated with neoplasms such as multiple myeloma and plasmacytoma, in addition to autoimmunity. The spleen and LN of C9orf72 -/- animals were enlarged by infiltrates of F4/80 + foamy macrophages occupying appropriate regions for...
Aging C9orf72 -/- mice exhibit varying degrees of proliferative glomerulonephropathy
reagent used in the protocol.
- Use
- As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopathology. To further characterize renal changes in null mice, H&E stained kidney sections were analyzed and scored in five categories of disease...
C9orf72 -/- mice develop SLE-like disease
reagent used in the protocol.
- Use
- Systemic lupus erythematosis (SLE) is characterized by immune dysregulation affecting many organs of the body. C9orf72 -/- mice develop the lymphoid hyperplasia, anemia, and renal disease common in SLE patients and reminiscent of phenotypes observed in spontaneous mouse models of SLE such as the MRL/lpr...
C9orf72 -/- mice show mild motor deficits
Given the association of C9ORF72 to neuropathology, we performed clinical exams to determine whether loss of C9orf72 causes an ALS-like phenotype. At 40 weeks of age, C9orf72 -/- showed progressive weakness and collapse of hind limbs towards the lateral midline, with mild tremor and rigidity not observed...
- Use
- Given the association of C9ORF72 to neuropathology, we performed clinical exams to determine whether loss of C9orf72 causes an ALS-like phenotype. At 40 weeks of age, C9orf72 -/- showed progressive weakness and collapse of hind limbs towards the lateral midline, with mild tremor and rigidity not observed...
C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal...
- Use
- To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal...
RNAseq analyses reveal global inflammatory gene signatures in C9orf72 -/- mice
C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observed in monocytes, macrophages and DCs, with lower levels measured in lymphocytes. C9orf72 may indeed modulate the immune system, particularly...
- Use
- C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observed in monocytes, macrophages and DCs, with lower levels measured in lymphocytes. C9orf72 may indeed modulate the immune system, particularly...
C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells
An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleen...
- Use
- An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleen...
C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells
B cell (CD45 + B220 + CD19 + ) percentages were either unchanged or reduced in C9orf72 -/- spleen but increased in LN compared with controls. Similar to T cell populations, total B cell counts were increased overall in spleen and LN, reflective of lymphoid expansion (data not shown). Analysis of specific...
- Use
- B cell (CD45 + B220 + CD19 + ) percentages were either unchanged or reduced in C9orf72 -/- spleen but increased in LN compared with controls. Similar to T cell populations, total B cell counts were increased overall in spleen and LN, reflective of lymphoid expansion (data not shown). Analysis of specific...
C9orf72 -/- mice develop SLE-like disease
Increased autoantibody titers in lupus patients are positively correlated with an increased frequency of circulating T follicular helper (Tfh) cells. Interrogation of this specific cell population (CD4 + CXCR5 + CD44 + ICOS + PD-1 + Bcl-6 + ) in spleen, cervical LN, mesenteric LN, and blood by FACS analysis reveale...
- Use
- Increased autoantibody titers in lupus patients are positively correlated with an increased frequency of circulating T follicular helper (Tfh) cells. Interrogation of this specific cell population (CD4 + CXCR5 + CD44 + ICOS + PD-1 + Bcl-6 + ) in spleen, cervical LN, mesenteric LN, and blood by FACS analysis reveale...
Taqman expression analysis
Axillary and brachial lymph nodes, cervical lymph nodes, gonadal fat pad, frontal cortex, diaphragm, spinal cord, spleen, and thymus were dissected fresh into RNALater stabilization reagent (Qiagen) and stored at -20 °C. Tissues were homogenized in TRIzol and chloroform was used for phase separation...
- Use
- Axillary and brachial lymph nodes, cervical lymph nodes, gonadal fat pad, frontal cortex, diaphragm, spinal cord, spleen, and thymus were dissected fresh into RNALater stabilization reagent (Qiagen) and stored at -20 °C. Tissues were homogenized in TRIzol and chloroform was used for phase separation...
LacZ expression profiling
Mice were deeply anesthetized via Ketamine/Xylazine (120/5 mg/kg) intraperitoneal (IP) injection and fixed by cardiac perfusion using a 0.2% glutaraldehyde, 4% paraformaldehyde (PFA) solution. Brain, ribcage, lymph nodes, salivary glands, thymus, heart, lung, liver, spleen, stomach, kidney, intestine, urogenit...
- Use
- Mice were deeply anesthetized via Ketamine/Xylazine (120/5 mg/kg) intraperitoneal (IP) injection and fixed by cardiac perfusion using a 0.2% glutaraldehyde, 4% paraformaldehyde (PFA) solution. Brain, ribcage, lymph nodes, salivary glands, thymus, heart, lung, liver, spleen, stomach, kidney, intestine, urogenit...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical and graphical analyses were performed using GraphPad Prism software (version 3.0). Data were analyzed using unpaired Student's t -test, one-way analysis of variance (ANOVA) and non-parametric Mann-Whitney as indicated. Results were considered statistically significant at P values < 0.05.
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C9orf72 -/- mice show mild motor deficits
Given the association of C9ORF72 to neuropathology, we performed clinical exams to determine whether loss of C9orf72 causes an ALS-like phenotype. At 40 weeks of age, C9orf72 -/- showed progressive weakness and collapse of hind limbs towards the lateral midline, with mild tremor and rigidity not observed in WT or C9orf72 +/- (, data not shown). Open field observations demonstrated decreased locomotor behaviors and fewer rearing events in null mice compared with WT. CatWalk gait analyses also revealed signs of impaired lower interlimb coordination and reduced stride length with bradykinesia and dragging of hind limbs ( ). No difference between WT and C9orf72 -/- mice was observed in maximum time spent on the rotarod ( ).
C9orf72 -/- mice exhibit lymphadenopathy and splenomegaly
During neurological testing, we unexpectedly noted palpable cervical masses in all C9orf72 -/- animals, but not in WT or C9orf72 +/- controls. Masses were palpable as early as 8 weeks of age and present in all null mice by 18 weeks, before onset of observed motor deficits. Upon dissection, the masses proved to originate from cervical lymph node (LN) (, data not shown) and systemic lymphadenopathy was noted in certain null mice. Peyer's patches (PP) were also enlarged and splenomegaly was apparent by 8 weeks of age (, data not shown). By 36 weeks, C9orf72 -/- ceased gaining weight compared with WT and only 9 out of 17 survived to the end of the neurological assay period (60 weeks) (, data not shown).
C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs
The enlargement of C9orf72 -/- spleens and LN suggests a disease process such as neoplasm or immune dysregulation, an unexpected finding given that ALS/FTD is not linked to such pathology in human patients. To address these possibilities, histopathology was conducted on spleen and LN from 8-60 week old mice. The basic cellular organization of the enlarged C9orf72 -/- LN was preserved, with immunohistochemistry (IHC) identifying a B cell-rich rim (CD45R + ) arranged in follicles within the cortex and a T cell (CD3 + ) infiltrate between follicles and in the paracortex zone (data not shown). A mixed cell population consisting mostly of large round cells with variably distinct borders, a single round nucleus, and eosinophilic, foamy cytoplasm expanded the cortical and medullary nodal architecture. A similar cellular infiltrate was present in C9orf72 -/...
C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs
H&E and IHC analyses of additional organs from mice aged 8-60 weeks revealed a prominent F4/80 + population of elongated to angular cells in the liver and kidneys of null mice. This population was pronounced in C9orf72 -/- mouse liver at 8 weeks, though there was no evidence of associated liver disease (data not shown). Increased F4/80 + cell populations observed in C9orf72 -/- kidney were located primarily within the cortex, forming prominent cuffs around glomeruli and aggregates in the vicinity of the macula densa and adjacent tubules ( ). Increasing infiltrates of mixed leukocytes were also observed with age, accompanied by varying degrees of immune-mediated glomerular disease that was well established by 35-60 weeks. Inflammation was not present in brain or spinal cord tissue in any animals examined. These data implicate the spleen, LN and kidne...
C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal pathology but remain viable. Total CD45 + (leukocyte common antigen) cell counts were increased in all C9orf72 -/- tissues examined, consistent with the immune infiltration described above, however CD45 + percentages compared with total cell populations assayed were either unchanged or reduced compared with WT. To determine if homeostasis within leukocyte subsets was altered, we narrowed our focus using specific antibody panels. F4/80 + macrophages (CD45 + CD11b + F4/80 + Ly6G - ) were increased in the spleen, LN, kidney, and blood in C9orf72 -/ͨ...
IL-12, IL-17a, IL-10, and TNF are increased in C9orf72 -/- serum
To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was approximately 6-fold increased in C9orf72 -/- animals compared with controls. IL-10, IL-17a, and TNFα were also upregulated, although to a lesser extent. We did not observe changes in IL-1β, IL-2, or IL-4 suggesting this effect on cytokine secretion was not global. While there was a trend toward increased IL-6 in C9orf72 -/- serum compared with WT, this difference did not reach statistical significance. MCP-1 chemokine was significantly increased in female C9orf72 -/- animals and IFNγ was significantly increased in males with an increasing trend observed in females.
C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells
An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleens, LN, BM, blood and kidney from 30-35 week C9orf72 -/- and WT female mice, gating on CD45 leukocyte common antigen, CD4 or CD8, and various activation status markers. Percentages of CD45 + CD8 + and CD45 + CD4 + cells were reduced overall in C9orf72 -/- animals compared with controls, likely a consequence of myeloid expansion and consistent with RNA profiling results. Conversely, but reflective of expansion of lymphoid tissue and immune infiltration, total cell counts for these T cell populations were increased in null mice (data not shown). C...
Aging C9orf72 -/- mice exhibit varying degrees of proliferative glomerulonephropathy
As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopathology. To further characterize renal changes in null mice, H&E stained kidney sections were analyzed and scored in five categories of disease. Results showed significantly higher average scoring for membranoproliferative glomerulonephritis in C9orf72 -/- with evidence of occasional glomerulosclerosis, hyaline casts, basophilic tubules and interstitial mononuclear inflammation compared with WT controls ( ). Individual histopath scores are shown in. H&E staining of the renal cortex ( ) is representative of mild (middle row) to marked (bottom row) disease progression. Descriptively, mild changes consisted of glomerular enlargement with increased cellularity, and enlargement of Bowman's space. Mod...
Measurement outputs
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To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observe...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
B cell (CD45 + B220 + CD19 + ) percentages were either unchanged or reduced in C9orf72 -/- spleen but increased in LN compared with controls. Similar to T cell popul...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
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Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls.
from paperScoring or quantification
Quantify the primary readouts for this experiment: To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney...; C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observe...; An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf...; B cell (CD45 + B220 + CD19 + ) percentages were either unchanged or reduced in C9orf72 -/- spleen but increased in LN compared with controls. Similar to T cell popul....
from paperStatistical comparison
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney...; To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was a...; C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observe...; As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopatho...
from paperReporting output
Report representative outputs alongside summary comparisons for To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney..., C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observe..., An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf..., B cell (CD45 + B220 + CD19 + ) percentages were either unchanged or reduced in C9orf72 -/- spleen but increased in LN compared with controls. Similar to T cell popul....
inferred from protocolStructured statistical methods
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney...; To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was a...; C9orf72 ablation appears to cause a systemic immune response resulting in elevated inflammatory cytokines and myeloid cell expansion. High expression of C9orf72 has been observe...; As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopatho...
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Evidence quotes (8)
Given the association of C9ORF72 to neuropathology, we performed clinical exams to determine whether loss of C9orf72 causes an ALS-like phenotype. At 40 weeks of age, C9orf72 -/- showed progressive weakness and collapse of hind limbs towards the lateral midline, with mild tremor and rigidity not observed in WT or C9orf72 +/- (, data not shown). Open field observations demonstrated decreased locomotor behaviors and fewer rearing events in null mice compared with WT. CatWalk gait analyses also revealed signs of impaired lower interlimb coordination and reduced stride length with bradykinesia and dragging of hind limbs ( ). No difference between WT and C9orf72 -/- mice was observed in maximum time spent on the rotarod ( ).
During neurological testing, we unexpectedly noted palpable cervical masses in all C9orf72 -/- animals, but not in WT or C9orf72 +/- controls. Masses were palpable as early as 8 weeks of age and present in all null mice by 18 weeks, before onset of observed motor deficits. Upon dissection, the masses proved to originate from cervical lymph node (LN) (, data not shown) and systemic lymphadenopathy was noted in certain null mice. Peyer's patches (PP) were also enlarged and splenomegaly was apparent by 8 weeks of age (, data not shown). By 36 weeks, C9orf72 -/- ceased gaining weight compared with WT and only 9 out of 17 survived to the end of the neurological assay period (60 weeks) (, data not shown).
The enlargement of C9orf72 -/- spleens and LN suggests a disease process such as neoplasm or immune dysregulation, an unexpected finding given that ALS/FTD is not linked to such pathology in human patients. To address these possibilities, histopathology was conducted on spleen and LN from 8-60 week old mice. The basic cellular organization of the enlarged C9orf72 -/- LN was preserved, with immunohistochemistry (IHC) identifying a B cell-rich rim (CD45R + ) arranged in follicles within the cortex and a T cell (CD3 + ) infiltrate between follicles and in the paracortex zone (data not shown). A mixed cell population consisting mostly of large round cells with variably distinct borders, a single round nucleus, and eosinophilic, foamy cytoplasm expanded the cortical and medullary nodal architecture. A similar cellular infiltrate was present in C9orf72 -/- spleen, predominantly located within the red pulp, expanding the splenic architecture and corresponding to grossly increased spleen weights. These cells did not stain consistently with CD45R or CD3 but were strongly positive for the macrophage lineage marker F4/80. (, data not shown). Abund...
H&E and IHC analyses of additional organs from mice aged 8-60 weeks revealed a prominent F4/80 + population of elongated to angular cells in the liver and kidneys of null mice. This population was pronounced in C9orf72 -/- mouse liver at 8 weeks, though there was no evidence of associated liver disease (data not shown). Increased F4/80 + cell populations observed in C9orf72 -/- kidney were located primarily within the cortex, forming prominent cuffs around glomeruli and aggregates in the vicinity of the macula densa and adjacent tubules ( ). Increasing infiltrates of mixed leukocytes were also observed with age, accompanied by varying degrees of immune-mediated glomerular disease that was well established by 35-60 weeks. Inflammation was not present in brain or spinal cord tissue in any animals examined. These data implicate the spleen, LN and kidney as major sites of C9orf72 -/- immune pathology.
To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal pathology but remain viable. Total CD45 + (leukocyte common antigen) cell counts were increased in all C9orf72 -/- tissues examined, consistent with the immune infiltration described above, however CD45 + percentages compared with total cell populations assayed were either unchanged or reduced compared with WT. To determine if homeostasis within leukocyte subsets was altered, we narrowed our focus using specific antibody panels. F4/80 + macrophages (CD45 + CD11b + F4/80 + Ly6G - ) were increased in the spleen, LN, kidney, and blood in C9orf72 -/- mice, consistent with the F4/80 + infiltration observed by IHC ( ). Neutrophil (CD45 + CD11b + Ly6G + Ly6C int CD115 - ) and total monocyte (CD45 + CD11b + CD115 + ) percentages were also increased in C9orf72 -/- tissues compared with controls. Staining for Ly6G and Ly6C reveale...
To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was approximately 6-fold increased in C9orf72 -/- animals compared with controls. IL-10, IL-17a, and TNFα were also upregulated, although to a lesser extent. We did not observe changes in IL-1β, IL-2, or IL-4 suggesting this effect on cytokine secretion was not global. While there was a trend toward increased IL-6 in C9orf72 -/- serum compared with WT, this difference did not reach statistical significance. MCP-1 chemokine was significantly increased in female C9orf72 -/- animals and IFNγ was significantly increased in males with an increasing trend observed in females.
An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleens, LN, BM, blood and kidney from 30-35 week C9orf72 -/- and WT female mice, gating on CD45 leukocyte common antigen, CD4 or CD8, and various activation status markers. Percentages of CD45 + CD8 + and CD45 + CD4 + cells were reduced overall in C9orf72 -/- animals compared with controls, likely a consequence of myeloid expansion and consistent with RNA profiling results. Conversely, but reflective of expansion of lymphoid tissue and immune infiltration, total cell counts for these T cell populations were increased in null mice (data not shown). Co-staining CD8 + T cells with activation markers revealed increases in the early activation and effector memory T cell markers CD69 and CD44 in C9orf72 -/- spleen and kidney compared with controls. We observed significantly increased percentages of CD8 + T cells expressing PD-1, a co-inh...
As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopathology. To further characterize renal changes in null mice, H&E stained kidney sections were analyzed and scored in five categories of disease. Results showed significantly higher average scoring for membranoproliferative glomerulonephritis in C9orf72 -/- with evidence of occasional glomerulosclerosis, hyaline casts, basophilic tubules and interstitial mononuclear inflammation compared with WT controls ( ). Individual histopath scores are shown in. H&E staining of the renal cortex ( ) is representative of mild (middle row) to marked (bottom row) disease progression. Descriptively, mild changes consisted of glomerular enlargement with increased cellularity, and enlargement of Bowman's space. Moderate to severely affected animals also had tubular changes, increased interstitial leukocytic infiltration, thickened capillary walls, and proliferation of visceral (podocytes) and parietal epithelium. In 3/11 null mice, glomerulosclerosis was present, characterized by expansion of the mesangial ma...
Machine-readable layer
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"description": "Evidence-backed execution summary for C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice methods from C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice.",
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"name": "C9orf72 -/- mice show mild motor deficits",
"text": "Given the association of C9ORF72 to neuropathology, we performed clinical exams to determine whether loss of C9orf72 causes an ALS-like phenotype. At 40 weeks of age, C9orf72 -/- showed progressive weakness and collapse of hind limbs towards the lateral midline, with mild tremor and rigidity not observed in WT or C9orf72 +/- (, data not shown). Open field observations demonstrated decreased locomotor behaviors and fewer rearing events in null mice compared with WT. CatWalk gait analyses also revealed signs of impaired lower interlimb coordination and reduced stride length with bradykinesia and dragging of hind limbs ( ). No difference between WT and C9orf72 -/- mice was observed in maximum time spent on the rotarod ( )."
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"name": "C9orf72 -/- mice exhibit lymphadenopathy and splenomegaly",
"text": "During neurological testing, we unexpectedly noted palpable cervical masses in all C9orf72 -/- animals, but not in WT or C9orf72 +/- controls. Masses were palpable as early as 8 weeks of age and present in all null mice by 18 weeks, before onset of observed motor deficits. Upon dissection, the masses proved to originate from cervical lymph node (LN) (, data not shown) and systemic lymphadenopathy was noted in certain null mice. Peyer's patches (PP) were also enlarged and splenomegaly was apparent by 8 weeks of age (, data not shown). By 36 weeks, C9orf72 -/- ceased gaining weight compared with WT and only 9 out of 17 survived to the end of the neurological assay period (60 weeks) (, data not shown)."
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"name": "C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs",
"text": "The enlargement of C9orf72 -/- spleens and LN suggests a disease process such as neoplasm or immune dysregulation, an unexpected finding given that ALS/FTD is not linked to such pathology in human patients. To address these possibilities, histopathology was conducted on spleen and LN from 8-60 week old mice. The basic cellular organization of the enlarged C9orf72 -/- LN was preserved, with immunohistochemistry (IHC) identifying a B cell-rich rim (CD45R + ) arranged in follicles within the cortex and a T cell (CD3 + ) infiltrate between follicles and in the paracortex zone (data not shown). A mixed cell population consisting mostly of large round cells with variably distinct borders, a single round nucleus, and eosinophilic, foamy cytoplasm expanded the cortical and medullary nodal architecture. A similar cellular infiltrate was present in C9orf72 -/..."
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"name": "C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs",
"text": "H&E and IHC analyses of additional organs from mice aged 8-60 weeks revealed a prominent F4/80 + population of elongated to angular cells in the liver and kidneys of null mice. This population was pronounced in C9orf72 -/- mouse liver at 8 weeks, though there was no evidence of associated liver disease (data not shown). Increased F4/80 + cell populations observed in C9orf72 -/- kidney were located primarily within the cortex, forming prominent cuffs around glomeruli and aggregates in the vicinity of the macula densa and adjacent tubules ( ). Increasing infiltrates of mixed leukocytes were also observed with age, accompanied by varying degrees of immune-mediated glomerular disease that was well established by 35-60 weeks. Inflammation was not present in brain or spinal cord tissue in any animals examined. These data implicate the spleen, LN and kidne..."
},
{
"@type": "HowToStep",
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"name": "C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells",
"text": "To further interrogate the cellular infiltrate observed on histopathology, flow cytometric analysis was performed on spleen, cervical and mesenteric LN, PP, BM, blood and kidney from C9orf72 -/- and WT controls. Specific focus was on the 28-35 week time point in females, as the majority show renal pathology but remain viable. Total CD45 + (leukocyte common antigen) cell counts were increased in all C9orf72 -/- tissues examined, consistent with the immune infiltration described above, however CD45 + percentages compared with total cell populations assayed were either unchanged or reduced compared with WT. To determine if homeostasis within leukocyte subsets was altered, we narrowed our focus using specific antibody panels. F4/80 + macrophages (CD45 + CD11b + F4/80 + Ly6G - ) were increased in the spleen, LN, kidney, and blood in C9orf72 -/ͨ..."
},
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"position": 6,
"name": "IL-12, IL-17a, IL-10, and TNF are increased in C9orf72 -/- serum",
"text": "To further characterize the global effects of C9orf72 ablation, we measured various cytokines and chemokines in 8-60 week mouse serum (, data not shown). IL-12total was approximately 6-fold increased in C9orf72 -/- animals compared with controls. IL-10, IL-17a, and TNFα were also upregulated, although to a lesser extent. We did not observe changes in IL-1β, IL-2, or IL-4 suggesting this effect on cytokine secretion was not global. While there was a trend toward increased IL-6 in C9orf72 -/- serum compared with WT, this difference did not reach statistical significance. MCP-1 chemokine was significantly increased in female C9orf72 -/- animals and IFNγ was significantly increased in males with an increasing trend observed in females."
},
{
"@type": "HowToStep",
"position": 7,
"name": "C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells",
"text": "An increase in activation markers on monocytic cells is often accompanied by increases in lymphocyte activation parameters. In addition, the elevation in IL-12 observed in C9orf72 -/- serum could up regulate T cell activity. To quantify T cell populations, we performed flow cytometric analyses on spleens, LN, BM, blood and kidney from 30-35 week C9orf72 -/- and WT female mice, gating on CD45 leukocyte common antigen, CD4 or CD8, and various activation status markers. Percentages of CD45 + CD8 + and CD45 + CD4 + cells were reduced overall in C9orf72 -/- animals compared with controls, likely a consequence of myeloid expansion and consistent with RNA profiling results. Conversely, but reflective of expansion of lymphoid tissue and immune infiltration, total cell counts for these T cell populations were increased in null mice (data not shown). C..."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Aging C9orf72 -/- mice exhibit varying degrees of proliferative glomerulonephropathy",
"text": "As prefaced earlier, F4/80 + monocytes were present in high numbers in C9orf72 -/- kidneys ( ) with evidence of progressive glomerular disease observed by histopathology. To further characterize renal changes in null mice, H&E stained kidney sections were analyzed and scored in five categories of disease. Results showed significantly higher average scoring for membranoproliferative glomerulonephritis in C9orf72 -/- with evidence of occasional glomerulosclerosis, hyaline casts, basophilic tubules and interstitial mononuclear inflammation compared with WT controls ( ). Individual histopath scores are shown in. H&E staining of the renal cortex ( ) is representative of mild (middle row) to marked (bottom row) disease progression. Descriptively, mild changes consisted of glomerular enlargement with increased cellularity, and enlargement of Bowman's space. Mod..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "C9orf72 -/- mice show mild motor deficits"
},
{
"@type": "HowToTool",
"name": "C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells"
},
{
"@type": "HowToTool",
"name": "RNAseq analyses reveal global inflammatory gene signatures in C9orf72 -/- mice"
},
{
"@type": "HowToTool",
"name": "C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells"
},
{
"@type": "HowToTool",
"name": "C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells"
},
{
"@type": "HowToTool",
"name": "C9orf72 -/- mice develop SLE-like disease"
},
{
"@type": "HowToTool",
"name": "Taqman expression analysis"
},
{
"@type": "HowToTool",
"name": "LacZ expression profiling"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "C9orf72 -/- mice exhibit lymphadenopathy and splenomegaly"
},
{
"@type": "HowToSupply",
"name": "C9orf72 -/- mice display mixed inflammatory infiltrates in multiple organs"
},
{
"@type": "HowToSupply",
"name": "C9orf72 -/- lymphoid organs contain increased percentages of myeloid lineage cells"
},
{
"@type": "HowToSupply",
"name": "IL-12, IL-17a, IL-10, and TNF are increased in C9orf72 -/- serum"
},
{
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"name": "C9orf72 -/- mice have increased percentages of activated T lymphocytes and plasma cells"
},
{
"@type": "HowToSupply",
"name": "C9orf72 -/- mice have high titers of autoantibodies"
},
{
"@type": "HowToSupply",
"name": "Aging C9orf72 -/- mice exhibit varying degrees of proliferative glomerulonephropathy"
},
{
"@type": "HowToSupply",
"name": "C9orf72 -/- mice develop SLE-like disease"
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],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice",
"datePublished": "2016",
"author": [
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"@type": "Person",
"name": "Amanda Atanasio"
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"@type": "Person",
"name": "Vilma Decman"
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"name": "Derek White"
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"name": "Meg Ramos"
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{
"@type": "Person",
"name": "Burcin Ikiz"
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"@type": "Person",
"name": "Hoi-Ching Lee"
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"name": "Chia-Jen Siao"
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"name": "Susannah Brydges"
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{
"@type": "Person",
"name": "Elizabeth LaRosa"
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{
"@type": "Person",
"name": "Yu Bai"
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{
"@type": "Person",
"name": "Wen Fury"
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"name": "Patricia Burfeind"
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"name": "Jamie Orengo"
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"name": "Adelekan Oyejide"
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"name": "Michael Fralish"
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"name": "Wojtek Auerbach"
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"@type": "Person",
"name": "William Poueymirou"
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"name": "Jan Freudenberg"
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"name": "Guochun Gong"
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"name": "Brian Zambrowicz"
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"name": "David Valenzuela"
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"name": "George Yancopoulos"
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"identifier": "10.1038/srep23204"
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