Article Figures & Data
Figures
- Figure 1. hSTING-N154S mice show impaired weight gain, paw swelling, and acral necrosis.
(A, B) Both female and male hSTING-N154S mice demonstrated a failure to gain weight starting at 8–10 wk of age. As discussed in the Results section, the “n” of mice used to calculate each time point was variable as all mice did not survive to endpoint (e.g., sacrifice due to the severity of disease); WT littermates were euthanized at these same time points as controls. (C) Generalized growth impairment was seen in hSTING-N154S mice (left) relative to WT littermates (right). (D–F) hSTING-N154S mice also developed progressive paw swelling that was first evident by ∼6 wk of age (red arrows). Paw thickness was determined by dorsoventral measurement (yellow arrow) using digital calipers. (G, H) hSTING-N154S mice developed tail inflammation and swelling with ensuing necrosis that lead to tail shortening (E). (H) These mice also exhibited losses of ear cartilage. For the paw thickness data, a one-way ANOVA with Tukey’s multiple comparisons post hoc test was used. ***P < 0.001 versus WT, n ≥ 13 per group.
- Figure S1. Paw thickness differences between the 1,501 and 1,504 hSTING-N154S (N154S) founder lines and representative histological sections of the lung, synovium, and quadriceps muscle from Vav1-hSTING-N154S mice.
(A) Caliper measurements of hind paw thickness of hSTING-N154S (closed circles) 1,501 (n = 5) and 1,504 (n = 9) sublines, and respective wild-type littermates (n = 3–5). Each symbol represents an individual mouse, and horizontal lines represent the mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by a Tukey’s multiple comparisons post hoc test between all groups. Significant differences between groups are denoted by *P < 0.05 or ***P < 0.001 as indicated. (B, C) Small very rare foci of leukocytic infiltrate in the lungs of hSTING-N154S mice (arrows), in a perivascular and peribronchiolar distribution. A focus of fibrosis (C) is occasionally associated with the infiltrate. (D) Representative tibiotalar joint section of a synovium from an hSTING-N154S mouse showing mild synovitis. (E) Representative section of an hSTING-N154S quadriceps muscle showing one of the occasional minute foci of infiltrating cells in an interstitial area (arrowhead) consistent with very mild myositis. Magnification: (B, C) 400×; (D, E) 100×.
- Figure 2. Paw inflammation in hSTING-N154S mice.
(A, B) Sections of representative hind paw digits from a WT littermate (A) and an hSTING-N154S mouse (B). The latter shows evidence of dermal edema, inflammatory infiltrates, and a region of necrosis (yellow asterisk). Infiltrates are also evident in skeletal muscle (arrows); there was also increased inflammatory cell accumulation within the bone marrow. (C) Higher magnification view of an hSTING-N154S digit showing marked myositis (arrows), dermal inflammatory infiltrate with edema (asterisk), and bone marrow necrosis (BM). (D, E) Paw inflammation in two different hSTING-N154S mice that had marked myositis associated with prominent inflammatory cell infiltrates, edema, and muscle fiber loss. Hematoxylin and eosin staining were used. Magnification: (A, B) 100×; (C) 200×; (D, E) 400×.
- Figure 3. Multiple arterial and venous thromboses in the paws of hSTING-N154S mice revealed by XRM and histopathology.
(A–C) Representative XRM imaging of MicrofilR-perfused forepaws from (A) a WT littermate and (B) an hSTING-N154S transgenic mouse showing venous dilation, thrombi, and multiple sites of vessel interruption (arrows). (C) Higher magnification view of the dilated veins in a forepaw from a transgenic mouse shows multiple venous thrombi (arrows). (D) Disrupted arteriole with transmural inflammatory infiltrates (arrows) and luminal fibrin deposition (asterisk). Note the absence of red blood cells in the lumen. (E) Paw arteriole showing complete occlusion of the lumen with collagenous (yellow) organization (asterisk) of the thrombus and residual fibrin (red). (F) Organizing paw arteriolar thrombosis (asterisk) showing a residual cleft of lumen containing red blood cells (arrow). This thrombus is older than the one in (D) with more mature collagen (green-yellow). (E, F) Internal elastic laminae were intact with no evidence of transmural vasculitis. (A–C) The orange areas in the paws are the result of incomplete decalcification. Stains: hematoxylin and eosin (D) and Movat pentachrome (E, F). Magnification: 400×.
- Figure S2. Intracellular staining for human STING in hematopoietic cells.
Single-cell suspensions were prepared from spleen and lung from hSTING-N154S, WT, and mSting-KO mice. (A, B) To examine Vav1-mutant hSTING expression, CD31+ endothelial cells that were isolated from the lung (A), and various splenic cell populations (CD3+, T cells; CD11b+, macrophages; and CD19+, B cells) (B), we used a human-specific STING fluor-conjugated antibody. (A) Representative dot plots show gating and relative proportion of CD31+ endothelial cells isolated from the lung tissues of mSting-KO, WT, and Vav1-hSTING-N154S mice. (B) Representative dot plots that show the gating and relative proportion of CD3+, CD11b+, and CD19+ cells. The spleen cells were stained with FITC-CD3, PerCPcy5.5-CD11b, and PE-CD19 and the lung cells were stained with PE-CD31, and PE-cy7-CD45. All antibodies were used at 1:200 dilutions. The cells were analyzed using a BD FACS cytometer; flow cytometry data were analyzed using FlowJo software (version 10.2).
- Figure 4. Western blot and intracellular staining show mutant human STING expression in splenic tissue and splenic cell populations.
(A) Representative dot plots showing the initial gating settings for the population of cells that were selected for FACS analyses (left panel) and the relative proportion of CD3+ and CD19+ cells (right panel) in a single-cell suspension of dissociated spleen. (B) Representative dot plots to show the percentage of CD3+ hSTING+ cells from the spleen of mSting-KO, WT, and Vav1-hSTING-N154S mice. Numbers below each gate are the percentage of cells within the corresponding gate. (C) Representative histogram showing human STING expression in the various splenic populations. CD3+ (T cells), CD11b+ (macrophages), and CD19+ (B cells) were obtained from the spleen; CD31+ endothelial cells were isolated from the lung. (D) Western blot detection of m/hSTING expression in splenic lysates using a polyclonal antibody that recognizes both mouse and human STING as described in the Results section. As positive controls, two human CRC lines known to express STING protein were used: HT29 and HCT116 (HCT). For spleen analyses, 40 μg of protein/lane and for CRC cell protein, 10 μg/lane were loaded. Arrow indicates the STING protein band in the human CRC lines.
- Figure 5. T-cell lymphopenia and type I interferon levels in hSTING-N154S mice.
(A–C) Whereas CD4+ T cell numbers were moderately reduced in the thymi of hSTING-N154S mice (A), there were marked reductions in the number of CD4+ and CD8+ cells in the spleens (B) and lymph nodes (C) when compared with WT mice. There were no differences in these T-cell populations when WT and mSting-KO mice were compared. One-way ANOVA with Tukey’s multiple comparisons post hoc tests were used to analyze group differences. WT and hSTING-N154S, n ≥ 10; mSting-KO, n = 4. Horizontal lines represent the mean ± SEM with significant differences denoted as *P < 0.05 or ***P < 0.001 versus WT. (D) Using quantitative RT-PCR analysis of splenic tissues, we observed that IFN-β transcripts were modestly increased in the hSTING-N154S mice (n = 7) relative to those in WT littermates (n = 7). (E) 13-plex Luminex assay of serum showed that mIFN-β levels were elevated in the sera of 8 of 13 hSTING-N154S mice (n = 13) compared with 4 of 10 WT littermates (n = 10). (F) Compared with WT littermates, there was also a significant increase in mIFN-α levels as detected via ELISA in the sera of hSTING-N154S mice (n = 12) (LumiKine Xpress mIFN-α ELISA kit). Horizontal lines represent the mean ± SEM serum concentrations (pg/ml) of murine IFN-β or IFN-α. Data are pooled from five independent experiments (n = 1–5 for each group). Unpaired t test was carried out between hSTING-N154S and WT groups where *P < 0.05.
- Figure 6. Increased cytokine levels in hSTING-N154S mice.
Serum cytokine levels in hSTING-N154S mice (n = 13) and WT littermate controls (n = 12) were measured by 31-plex murine cytokine array. Each symbol represents an individual mouse and horizontal lines represent the mean ± SEM of serum concentrations for each cytokine (pg/ml). Data are pooled from five independent experiments (n = 1–5 for each group). Unpaired t test was carried out between hSTING-N154S and WT groups where *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See Fig S3 for the remainder of the Luminex results from these mice.
- Figure S3. Systemic cytokine levels in hSTING-N154S mice.
Serum cytokine levels in hSTING-N154S mice (closed circles, n = 13) and WT littermate controls (open circles, n = 12) were measured by a Luminex 31-plex murine cytokine array. Each symbol represents an individual mouse, and horizontal lines represent the mean ± SEM serum concentration for each cytokine (pg/ml). Data were pooled from five independent experiments (n = 1–5 for each group). Unpaired t test was carried out between hSTING-N154S and WT groups where *P < 0.05.
- Figure 7. Lymphopenia in hSTING-N154S mice.
(A, B) Representative dot plots show gating and relative proportion of Ly6G+, CD19+, and CD4+ and CD8+ cells in single-cell suspensions of peripheral blood (A) and dissociated lung tissue (B) from hSTING-N154S mice and WT littermates that were analyzed by multicolour flow cytometry. Numbers above each gate are the percentage of cells within the corresponding gate. Regarding the intermediate CD19+/Ly6G staining (B), there are no known leukocytes that coexpress CD19 and Ly6G, and seeing that this population does not exist in the peripheral blood dot plots, this likely represents a lung parenchymal cell population. (C, D) Scatter plots showing the number of Ly6G+, CD19+, CD4+, and CD8a+ cells in peripheral blood (C) or dissociated lung tissue (D) from WT and hSTING-N154S mice. Each symbol represents an individual mouse and horizontal lines represent the mean ± SEM number of cells per mL of peripheral blood (C, n = 5) or per lung (D, n = 4) for each group. Data are pooled from two independent experiments (n = 1–3 for each group). Unpaired t test was used to compare between the two groups with P-values of <0.05 considered to be significant (*P < 0.05, **P < 0.01).
- Figure S4. Pulmonary macrophages in hSTING-N154S mice.
(A) Representative dot plots show gating and relative proportion of CD11bintF4/80+ alveolar macrophages in dissociated lung tissue (top row) and BAL fluid (bottom row) from hSTING-N154S mice and WT littermates analyzed by multicolour flow cytometry to quantify major leukocyte subsets. (B) Scatter plots showing mean ± SEM number of cells per lung (left) or per 500 μl of BAL fluid (right) (n = 4). Unpaired t test was used to compare data obtained from WT and hSTING-N154S mice. No significant differences were detected. Data are pooled from two independent experiments (n = 1–2 for each group).
- Figure S5. Detection of ANAs in the sera of hSTING-N154S transgenic or wild-type mice.
Note: 1 of the 15 ANA (+) hSTING-N154S mouse serums (titer: 1:1,280) was positive for myositis-associated autoantibodies against Jo-1, PL-7, and SRP.
- Figure S6. Autoantibodies detected in the sera of hSTING-N154S transgenic versus WT littermate control mice.
Autoantibodies to intracellular inflammatory myopathy (IM) and other connective tissue disease auto antigens as detected by addressable laser bead immunoassays showed increased reactivity to a variety of IM targets, but the highest titers were directed to Jo-1 (histidyl tRNA synthetase), PL-7 (threonyl tRNA synthetase), signal recognition particle (SRP), and U1-ribonucleoprotien (U1-RNP). Numerical values are expressed as median fluorescence index (MFI).
- Figure 8. hSTING-N154S vasculopathy is prevented by the lack of ifnar1.
(A–C) Representative photographs of male age-matched WT (left), hSTING-N154S (center), and hSTING-N154S/ifnar1-KO (right) mice showing differences in body size (A), ear pathology (B), and paw thickness (C). Arrows and arrowheads indicate differences in the hind paws and ears, respectively, of hSTING-N154S mice either in the presence (red) or absence (black) of ifnar1. (D–F) Scatter plots showing the differences in body weight (D), paw thickness (E), and spleen weight (F) amongst WT (open circles), hSTING-N154S (closed circles), and hSTING-N154S/ifnar1-KO (closed squares) male and female mice. (D–F) Each symbol represents an individual mouse (D–F), and horizontal lines represent the mean ± SEM. Spleen weight (F) was normalized to body weight (mg/g of body weight). (D–F) Statistical significance between data sets (D–F) was assessed by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test between all groups. Significant differences between WT (M/F, n = 10) and hSTING-N154S (M, n = 7; F, n = 8) or between hSTING-N154S and hSTING-N154S/Ifnar1-KO (M, n = 14; F, n = 16) mice are denoted by ***P < 0.001.
- Figure S7. No evidence of inflammation in the hSTING-N154S/Ifnar1-KO mice.
(A) There was a complete absence of paw inflammation in the hSTING-N154S/Ifnar1-KO mice as shown in this representative example. Note the lack of inflammatory infiltrates and edema in the dermis as well as the lack of myositis. H&E stain. Magnification: 100× generation of the hSTING-N154S transgenic mice. (B) A cDNA encoding human STING was synthesized so that it included the N154S mutation. The placement of the STING-N154S cDNA in relation to the 2.3/4.4(HS21/45) Vav1 gene promoter vector is shown. Restriction sites are H3, HindIII; Sf, SfiI; and N, Notl. Insertion was placed downstream of the Vav1 gene promoter to achieve expression of the mutant in hematopoietic cells. (C) The nucleotide sequence of the STING overexpressing cDNA. Purple sequences indicate the eliminated SfiI site and the blue AgC sequence the N154S point mutation; lowercase letters represent minor changes to the NM_198282 sequence so that it would match the consensus hSTING sequence.
- Figure 9. Absence of systemic hyper-cytokinemia in hSTING-N154S/Ifnar1-KO mice.
Serum cytokine levels (pg/ml) in age-matched WT, hSTING-N154S (N154S), and hSTING-N154S/Ifnar1-KO mice as measured by Luminex 31-plex murine cytokine array. Each symbol represents an individual mouse, and horizontal lines represent the mean ± SEM, n = 3 for WT and hSTING-N154S; n = 8 for hSTING-N154S/Ifnar1-KO. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey’s multiple comparisons post hoc test between all groups. Significant differences between hSTING-N154S mice and WT or hSTING-N154S/Ifnar1-KO denoted by *P < 0.05, **P < 0.01, ***P < 0.001; WT and hSTING-N154S/Ifnar1-KO differences denoted by φ P < 0.05. LIX, EOTAXIN, MIP-2, M-CSF, GM-CSF, IFNγ, VEGF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, and LIF were also measured; however, no differences were observed between the groups (data not shown).
