Stimulator of interferon genes (STING) activation exacerbates experimental colitis in mice

Detection of cytoplasmic DNA by the host’s innate immune system is essential for microbial and endogenous pathogen recognition. In mammalian cells, an important sensor is the stimulator of interferon genes (STING) protein, which upon activation by bacterially-derived cyclic dinucleotides (cDNs) or cytosolic dsDNA (dsDNA), triggers type I interferons and pro-inflammatory cytokine production. Given the abundance of bacterially-derived cDNs in the gut, we determined whether STING deletion, or stimulation, acts to modulate the severity of intestinal inflammation in the dextran sodium sulphate (DSS) model of colitis. DSS was administered to Tmem173gt (STING-mutant) mice and to wild-type mice co-treated with DSS and a STING agonist. Colitis severity was markedly reduced in the DSS-treated Tmem173gt mice and greatly exacerbated in wild-type mice co-treated with the STING agonist. STING expression levels were also assessed in colonic tissues, murine bone marrow derived macrophages (BMDMs), and human THP-1 cells. M1 and M2 polarized THP-1 and murine BMDMs were also stimulated with STING agonists and ligands to assess their responses. STING expression was increased in both murine and human M1 polarized macrophages and a STING agonist repolarized M2 macrophages towards an M1-like subtype. Our results suggest that STING is involved in the host’s response to acutely-induced colitis.

chronic carbon tetrachloride administration to wild-type mice resulted in early endoplasmic reticulum stress, type I interferon production, hepatocyte apoptosis and liver fibrosis, whereas hepatocyte death and fibrosis in similarly treated mice lacking STING was prevented 22 . There is also evidence that STING deficiency reduces pro-inflammatory cytokine production and arthritis scores in a murine model of cytosolic self-DNA-mediated autoimmunity 23 . In addition, STING gain-of-function mutations lead to an auto-inflammatory disease, typically manifested in infancy, that exhibits a severe acral vasculopathy and lung inflammation, amongst other features 24 .
Thus, STING appears able to serve a protective function, involved in host defense against viruses and bacteria and injury repair, or alternatively, as a potentially pathogenic pro-inflammatory mediator depending on the nature and severity of the insult. While there is much evidence that excessive STING activation plays a role in triggering various disease states, including autoimmunity and autoinflammatory disease 19,23,24 , the role of STING as a regulator of the response of the host to colitis remains largely undefined. Hence, we examined the role of STING activation and/or its deficiency in the DSS-induced model of experimental colitis. While this model does not replicate all aspects of human colitis, it does share some similarities including epithelial barrier dysfunction, cytokine dysregulation, and mucosal pathology and thus has proven very useful when examining the functions of genes and mediators involved in the intestinal immune response to injury and bacterial invasion of colonic tissue 25 .
While it was possible that treatments with a STING agonist could have improved the inflammation associated with DSS-colitis, we hypothesized that it would have pro-inflammatory effects. Herein we identified that DSS-treated normal mice that were co-treated with a STING agonist developed a dramatic worsening of colitis whereas similarly treated mice that lacked the STING gene had a reduction in disease severity. We also observed a decreased expression of the macrophage activation marker Iba-1 in response to DSS in these STING-KO mice and that STING activation of murine BMDMs by a STING agonist or STING ligands re-polarized M2 macrophages towards a pro-inflammatory M1-like subtype. Lastly, DSS-induced colitis, as well as pharmacological activation of STING in vitro, were found to increase STING protein expression.

Results
Agonist-induced STING activation worsened, whereas STING deficiency reduced weight loss and colonic shortening in response to DSS. Although all mice that received DSS experienced drops in body weight relative to tap water-treated controls, weight loss was aggravated in the WT mice treated with the murine STING agonist DMXAA 26 , but was significantly reduced in the Tmem173 gt mice (Fig. 1a-d). Weight changes in the non-DSS treated WT mice that received 5 mg/kg/day of DMXAA alone via i.p. injection were not significantly different when compared to tap water-treated controls (data not shown). As with weight loss, colonic shortening occurred in all DSS-treated mice, however, the degree of colonic shortening in the DSS-treated WT mice that were co-treated with DMXAA was significantly greater (Fig. 2a). In contrast, DSS-induced colonic shortening in Tmem173 gt mice, either with or without DMXAA treatment, was considerably less. This indicated that the exacerbation of DSS-induced colitis was being driven by STING activation.
Agonist-induced STING activation exacerbated DSS-induced colonic damage and inflammation. We next examined histological sections using a standardized, commonly employed method for assessing intestinal pathology. This incorporates both epithelial damage and the degree of inflammatory infiltrate to assign a score for colonic damage, that is, the histological activity index (HAI). Histologically, unlike the report by Canesso et al. 17 , we did not observe any significant colonic mucosal abnormalities (eg. crypt depth, Fig. 2b or goblet cell numbers, Fig. 2c) when sections from untreated WT and Tmem173 gt mice were compared. The HAI was elevated in the DSS-treated mice groups when compared to respective tap water-treated groups of mice ( Fig. 3ad), however, the degree of colonic damage, hypertrophy, infiltration, and muscle thickening with edema in the DSS-treated wild-type mice that had also been treated with the STING agonist was significantly worse (Fig. 4a-c). In addition, no exacerbation of DSS-induced colitis was detected in Tmem173 gt mice that had received daily injections of DMXAA (Fig. 4c). Furthermore, epithelial damage and crypt cell losses in DSS-treated Tmem173 gt mice were markedly reduced (Fig. 4d). Brush border membrane and goblet cell losses were accentuated in DSS-treated WT mice that were also treated with DMXAA (Fig. 4d). Consistent with STING being the principal target of DMXAA in mice, colonic damage was attenuated in the DMXAA-plus DSS-treated Tmem173 gt mice. In addition, there were no overt signs of hemorrhagic necrosis observed in the colons of these mice.

Colitis-induced expression of the macrophage activation marker Iba-1 was reduced in
Tmem173 gt mice. Since the presence or absence of STING protein regulated the severity of DSS-induced colitis, we investigated whether inflammation might regulate the numbers of activated macrophages. We thus stained paraffin sections of DSS-exposed colons with Iba-1, a protein expressed in macrophages, microglia (and possibly in neutrophils), that is upregulated during activation, to assess whether STING activation or absence would influence Iba-1 expression in these colons. There were no overt observable differences in the expression of Iba-1 when tap water-treated WT and Tmem173 gt tissues were compared (Fig. 5a,b). Though no formal measurements were utilized, colonic inflammation appears to have increased the number of activated macrophages (as per visual assessment of Iba-1 staining) in all DSS-treated groups ( Fig. 5c-f). However, this increase in Iba-1 staining was markedly attenuated in the Tmem173 gt mice as compared to WT mice that received similar treatments ( Fig. 5c-f). STING protein expression is increased by colitis and M1 polarization of murine or human THP-1-derived macrophages. STING protein expression was detected in colonic tissue lysates obtained from control WT mice, but not from Tmem173 gt mice (Fig. 6a). We also observed that STING protein expression in the colonic lysates from the WT mice that had been administered DSS was significantly increased relative to that found in the WT littermates that received tap water only (Fig. 6b,d). In addition, STING protein expression levels were only detected in the colonic lysates obtained from DSS-treated WT mice, but not from Tmem173 gt mice (Fig. 6c). Since whole colonic tissue sections were analyzed, it was not possible to discern which cell types were contributing to the STING protein levels. However, since M1 polarized macrophages have a key role in the pathogenesis of DSS-induced colitis 27,28 we investigated whether these cells exhibited increased STING protein levels in response to M1-polarizing stimuli. Indeed, STING protein levels were dramatically increased under M1 polarizing conditions (Fig. 6e,f,h,i). In contrast, both M0 (no polarizing factors) or M2 polarized macrophages demonstrated much lower STING levels (Fig. 6e,f,h). Similarly, STING levels were dramatically increased in PMA-differentiated human THP-1-derived macrophages in response to M1 polarizing stimuli, while M2 polarization led to no significant change in STING expression levels (Fig. 6g,j). Thus, M1 polarization of human or murine cells with LPS and IFNγ was associated with a substantial increase in STING protein expression, a finding that might predict an increased sensitivity of M1 polarized cells to either bacterially-or endogenously-derived cDNs.
Treatment of BMDMs with a STING agonist induces a pro-inflammatory subtype. We next sought to explain why DSS colitis was exacerbated by co-treatment with a STING agonist. While it has been shown that M1 polarization can exacerbate DSS-induced colitis, the associated inflammation was attenuated by genetic manipulations favouring differentiation of the M2 phenotype 27,29,30 . Previously, we showed that the activation of the STING pathway with a STING agonist was able to induce the repolarization of murine M2 macrophages, both in vitro and in vivo, towards an M1-like phenotype 11 which possibly explains the adverse effect of DMXAA on colitis severity. Weight losses associated with DSS-induced colitis in Tmem17 Gt and wild-type mice. (a) Weight losses were exacerbated in the DSS mice that were co-treated with the murine STING agonist, DMXAA, and attenuated in the Tmem173 Gt group of mice. To examine the effects of a given treatment regime on weight change, we isolated groups and compared the effects of (b) DSS-induced colitis either alone or following cotreatments with (c) 5 mg/kg or (d)10 mg/kg of DMXAA (i.p.). Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n ≥ 8 mice per group with differences denoted by *P < 0.05,**P < 0.01, ***P < 0.001 relative to DSS-treated WT.
Based on expression of the murine M1 marker iNOS, and M2 marker, Arg-1, macrophages from Tmem173 gt mice polarized in vitro into both the M1 and M2 phenotypes similar to WT macrophages ( Fig. 7a,b). Thus, cells lacking STING were able to respond to M1 (IFNγ plus LPS) or M2 (IL-4) polarizing stimuli. In contrast, Tmem173 gt macrophages treated with a STING agonist were hypo-responsive (eg. failed to generate IFNβ following DMXAA treatment) when compared to that secreted by WT macrophages (Fig. 7c). Similar results were observed using the natural STING ligands, bacterial c-di-AMP or mammalian 2′3′-cGAMP, as the production of the cytokine was only observed in the WT macrophages (Fig. 7d).
Importantly, and in keeping with our previous study on 129/SvJ macrophages 11 , WT M2-polarized C57BL/6 macrophages treated with 2′3′-cGAMP or c-di-AMP also underwent M1-like repolarization. This was demonstrated by a decrease in the M2 macrophage markers, Arg-1 and Fizz1 (Fig. 8a,b), and an increase in the expression levels of potential markers of M1-like activity (including IFNβ, CXCL-10, iNOS and IL-12p40 transcripts) in the M2 population (Fig. 8c,d). While effective delivery of these two cDNs into macrophages required lipofectamine, we previously showed that this treatment does not activate or polarize these cells 11 . Thus, one possible explanation as to why the co-administration of DSS and a STING agonist led to a dramatic worsening of colitis, is that the STING agonists increased pro-inflammatory activities in the M1 cells and repolarized M2 cells towards an M1-like phenotype.

Discussion
To determine if STING signalling was involved in the host response to colonic inflammation, we examined, in the context of an experimental mouse model of colitis, whether STING activation, or conversely, STING-deficiency, would affect disease severity. While it was plausible that treatments with a STING agonist could have improved the inflammation associated with DSS-induced colitis-based upon results in which its deficiency was observed to exacerbate inflammation 14,17 -we hypothesized that it would have pro-inflammatory effects. Indeed, we found that the DSS-treated WT mice that were co-treated with the murine STING agonist DMXAA, developed a dramatic worsening of colitis. In contrast, similarly treatedTmem173 gt mice had reduced disease severity as well as decreased expression of the macrophage/microglia activation marker, Iba-1. We also found that STING activation of murine BMDMs by DMXAA or STING ligands (including the bacterial second shortening in all treatment groups, but relative to DSS-treated WT mice, was significantly greater in the WT DSS mice that had also received DMXAA. In contrast, this effect was significantly reduced in both the Tmem173 gt and the Tmem173 gt mice co-treated with the STING agonist. Black-filled dots are for WT, or WT + 5 or + 10 mg/kg DMXAA; white-clear dots are for the Tmem173 gt receiving similar treatments. Statistical significance between data sets was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values are means ± SEM, n ≥ 8 mice per group, ***P < 0.001 longer relative to DSS-treated WT. Analysis of (b) crypt depth or (c) goblet cell numbers in the colons of the untreated WT and TMEM173 Gt mice. To assess crypt depth, 4 random locations from 6 untreated WT or TMEM173 Gt mice (n = 24 total) measurements were plotted. Upon statistical comparison, there were no significant differences in crypt height or goblet cell numbers when sections from untreated WT and Tmem173 gt mice were compared. Goblet cell counts represent the number of goblet cells per 200 um 2 area, n = 12, Student's T-test was used to determine possible differences between data sets. www.nature.com/scientificreports www.nature.com/scientificreports/ messenger c-di-AMP and the non-canonical ligand 2′3′-cGAMP), re-polarized M2 macrophages towards a pro-inflammatory M1-like subtype. Of note, this effect was greatly dampened in the Tmem173 gt macrophages. In addition, DSS-induced colitis, as well as pharmacological activation of STING in vitro, increased STING protein expression levels. Collectively, these findings suggest that STING is capable of modulating inflammatory processes within the distal intestinal tract.
We found that the expression of Iba-1 in the intestinal macrophages of mice was significantly influenced by the presence (or absence) of STING and on STING activation following treatments with a murine STING agonist. Previous studies have shown that expression of Iba-1 is increased in the colonic tissues of mice in a model of trinitrobenzene sulfonic acid-induced acute colitis as well as in the acute rejection of cardiac allografts 31,32 . Furthermore, in a study of 47 IBD patients in various stages of disease (e.g. active vs. remission), it was reported that colonic Iba-1 expression was higher in the patients with active disease 33 . There are reports that Iba-1 expression was increased following addition of the pro-inflammatory cytokines IL-1β and TNF-α in a rat macrophage cell line as well as in mice with bleomycin-induced acute lung injury 34,35 . In our study, colitis appears to have increased Iba-1 in all DSS-treated groups, however, this was markedly reduced in the Tmem173 gt mice when compared to WT mice that received similar treatments. This finding is of significance as it suggests that STING activation could be involved in the activation of colonic macrophages during the acute phase of colitis.
Previously, we reported that M2 polarized murine macrophages treated with 2′3′-cGAMP or c-di-AMP, repolarized into a M1-pro-inflammatory subtype which suggests that the STING pathway has a role in re-educating M2 macrophages towards a more pro-inflammatory phenotype 11 . In the current study, we found that macrophages derived from Tmem173 gt mice polarized in vitro into both M1 and M2 phenotypes similar to that found in WT mice. This indicated that loss of STING did not affect polarization in response to IFN-γ and LPS or IL-4. However, and consistent with the loss of activation events dependent on the STING pathway, the Tmem173 gt macrophages treated with either DMXAA or various STING ligands did not respond to the same extent as the WT macrophages (eg. loss of the M2 markers Arg-1 and Fizz1 and increased expression of the M1 markers CXCL-10, iNOS and IL-12p40) and lacked IFN-β induction. This function of STING activation may have accounted for the www.nature.com/scientificreports www.nature.com/scientificreports/ dramatic worsening of DSS colitis we observed following DMXAA administration as the inhibition of M2 polarization has shown to worsen colitis, whereas the promotion of M2 (or inhibition of M1) polarization attenuates inflammation 28,36,37 .
We also investigated whether in vitro polarization of macrophages might have an effect on STING protein expression within M0, M1 or M2 polarized murine and human macrophages. We observed that STING protein levels were greatly augmented in both human and murine M1-polarized macrophages relative to both non-polarized and M2-polarized macrophages. It is plausible that macrophage activation by bacterially-derived products could function during colitis, at least in part, to promote the up-regulation of STING protein expression. High levels of STING protein might conceivably sensitize macrophages to limited amounts of cDNs.
That we did not see the complete prevention of DSS colitis in the Tmem173 gt mice was not unexpected given the known participation of various PAMP receptors, as well as the inflammasome, in cells of the innate immune system 2,16 . Also, with regard to the latter, there was an intriguing report that showed that cDNs can stimulate the NLRP3 inflammasome in a STING-independent manner 38 . Thus, in the DSS model, STING-mediated effects are obviously acting in concert with various other pro-inflammatory pathway effectors. However, in view of the multifactorial processes involved in the pathogenesis of colitis, the degree of disease inhibition in the setting of STING deficiency was striking.
While there is clinical data stating that IFN-β is a beneficial therapeutic for the treatment of ulcerative colitis, there are also reports that this therapy in multiple sclerosis or chronic hepatitis patients can worsen disease, or even spontaneously induce IBD [39][40][41] . In addition, it was reported that the treatment of DSS-treated mice with a Lactobacillus acidophilus strain that constitutively expressed IFN-β, increased disease severity 42 . There is also evidence that the loss of the IFNAR1, a critical component of the IFN signaling pathway 43,44 , does not exacerbate or induce colitis 45 . This receptor activates intracellular signal transduction in response to all type I interferons including IFN-β and the various IFN-α subtypes 46 . For example, Tschurtschenthaler et al. showed that mice deficient in ifnar1 (in intestinal epithelial cells) did not exhibit spontaneous inflammation or increased severity in Figure 4. Administration of a STING agonist exacerbates DSS-induced colitis. Sections were harvested from DSS-treated WT mice that were co-treated with (a) 5 mg/kg/day of DMXAA or from (b) WT or (c) Tmem173 gt mice co-treated with 10 mg/kg/day of DMXAA every other day. (d) Inflammation-as assessed using the histological activity index (HAI), an index that provides a damage score-in the WT mice that were administered DMXAA was more severe than that of DMXAA-treated Tmem173 gt mice. Note the loss of the epithelial brush border (black arrowheads) and goblet cells (yellow arrowheads). Colonic damage in the Tmem173 gt mice cotreated with DMXAA was markedly reduced when compared to both WT groups. Sections were harvested on day 7. PAS stain. All sections imaged using a 20x objective; black bars at the bottom left of the panels represent 200 μM. Statistical significance was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values for (D) are the means ± SEM, n ≥ 7 mice per group, ***P < 0.001 relative to both the WT(5) and WT(10) groups of mice. (2019) 9:14281 | https://doi.org/10.1038/s41598-019-50656-5 www.nature.com/scientificreports www.nature.com/scientificreports/ DSS colitis compared with ifnar1 +/+ mice 45 . Finally, a Cochrane database review by Seow et al. 47 found no support for Type I IFN therapy for the induction of remission in patients with ulcerative colitis and recommended that efficacy and safety should be evaluated prior to continuation or initiation of clinical trials.
STING may be involved in homeostatic processes that regulate both inflammation and carcinogenesis 14,15 . For example, chronic STING activation may alter the predisposition to carcinogenesis as noted by Xia et al. in which they observed that STING signaling is suppressed in a wide variety of cancers 48 . Moreover, Zhu et al. 14 reported that the presence of STING attenuated inflammation-induced colorectal cancer in mice. Superficially, this appeared to contrast with our findings in which STING deficiency was protective during DSS-induced colitis and that treatments with a STING-agonist dramatically exacerbated disease severity. A few possibilities could account for the apparent discrepancy in these findings, such as differences in the microbiota between facilities, the DSS used (eg. source, concentration, lot, etc.), and more likely, that the disease induction models employed were Figure 5. DSS-induced colitis increased the numbers of activated macrophages in WT mice. To assess macrophage activation, paraffin sections from the various treatment groups were stained with Iba-1, a protein upregulated during activation of macrophages and possibly neutrophils (black arrows). Representative sections harvested from vehicle-treated (a) WT or (b) Tmem173 gt mice, or from DSS-treated (c) WT or (d) Tmem173 gt mice or from DSS treated (e) WT or (f) Tmem173 gt mice that were co-treated with DMXAA (5 mg/kg). Although there were no discernable differences in macrophage activation between the untreated WT and Tmem173 gt groups of mice, Iba-1 staining of colonic tissues from the DSS-treated WT and DSS-treated WT mice co-treated with DMXAA looked to be increased when compared with similarly-treated Tmem173 gt mice. All sections were harvested on day 7 from mice treated with 3% DSS. Anti-Iba-1 immuno-histochemical stain. Sections imaged using a 20x objective. www.nature.com/scientificreports www.nature.com/scientificreports/ different. While our model employed DSS treatment over 7 days, in the Zhu et al. study, mice were injected with azoxymethane (AOM), a DNA damaging agent that also has effects on the gut microbiome 49,50 prior to the initiation of DSS exposure. Thus, the basal homeostatic state of the gut lining prior to DSS exposure was likely to be different in the two models. To further add to the ambiguity, a recent study by Canesso et al. observed that STING was required for intestinal homeostasis and the control of inflammation and that the loss of STING worsened DSS-induced colitis 17 , whereas in contrast, Ahn et al. reported that STING-deficient mice are rescued from spontaneous colitis in IL-10 deficient mice 18 . In summary, our studies suggest that STING acts as a pro-inflammatory mediator of DSS-induced colitis, and this is supported by the observation that colitis was dramatically worsened when the mice were co-treated with a STING agonist.
The possibility that human STING responds poorly to bacterially derived cDNs has been raised 26 , however, this study was limited to peripheral blood mononuclear cells (PBMCs) derived from only 2 individuals. The STING expression levels could potentially have been a factor as some variants have low STING protein and reduced TMEM173 transcript levels which have shown to be unresponsive respond to cDNs 51 . For example, there is evidence that STING alleles have variable responses to bacterially-derived cDNs within the human population 52,53 and that sizable fractions of the human population are homozygous for the following STING allele: R71H-G230A-R293Q. In view of the fact that there are multiple polymorphisms within the STING cDN binding and dimerization domains [52][53][54] , it would appear premature to conclude that all human STING variants would show impaired responses to bacterially-derived cDNs.
These results support our hypothesis that STING activation plays a role in the development and the degree of inflammation in the DSS model of colitis. Whether colonic epithelial damage allows free cDNs in the luminal fluid to access cells of the immune system, or whether bacterial phagocytosis is required to deliver cDNs or bacterial dsDNA remains an open question. The development of human STING-specific antagonists is an area of active investigation, particularly for the treatment of inflammatory and autoimmune diseases. To this end, screening efforts in the lab of Ablasser et al. 55 , recently identified molecules that appear to suppress STING activation. If our findings can be extrapolated to gut inflammation in humans, then the next stage would be to further develop and utilize STING antagonists as therapeutics in an attempt to limit the degree and to promote the resolution of intestinal inflammation. Conversely, given the adverse impact of the murine STING agonist on DSS-induced colitis in mice, and if this phenomenon develops in humans, then caution is warranted as the therapeutic administration of STING agonists could exacerbate existing or underlying inflammatory disease(s).

Methods
Mice. These studies were conducted in accordance with the guidelines established by the Canadian Council of Animal Care, with all protocols approved by the Health Sciences Animal Care Committee at the University of Calgary. Male mice (8-10 wks of age) were fed standard laboratory chow, allowed water ad libitum, and were housed in a double barrier unit within individual micro-isolator cages (Techniplast) having HEPA filter lids with individual filtered air and water supplies in a room maintained at 22 ± 1 °C, 65-75% humidity, and a 12-hr light/ dark cycle. Male C57BL/6J mice were selected for these studies as they are generally considered more susceptible than females to DSS (increased severity, increased hyperplasia) 56 and are largely the sex selected for these studies. In addition, while there are studies that suggest that Crohn's disease is more prevalent in females, in ulcerative colitis population-based studies, no significant differences have been shown 57,58 . However, we acknowledge that there could be differences regarding the role of the STING protein and the sex of the mice.
Tmem173 gt C57BL/6J mice (Jackson Laboratories, USA) do not produce IFN-β in response to cDNs or Listeria monocytogenes infection 1 . These mutant mice carry a missense mutation in exon 6 of the transmembrane protein 173 (Tmem173 or Sting) gene, which results in an isoleucine-to-asparagine change at amino acid 199, in the C-terminus of the protein. C57BL/6J mice from the same commercial source were used as the wild-type controls.
Homozygous Tmem173 gt and wild-type mice were administered 3% dextran sulfate sodium (DSS) in their drinking water for 7 days. A second group were treated with 5 or 10 mg/kg/day i.p of DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist that specifically binds to murine but not human STING 26 , concomitant with DSS treatment. Mice were monitored daily to assess water consumption, changes in body weight, stool consistency, and for the presence of gross fecal blood.
Histology and immunohistochemistry. Colonic samples (3-4 cm distal to the cecum) were fixed in 10% neutral buffered formalin, embedded in paraffin, and then stained with either hematoxylin and eosin (H&E) or periodic acid-Schiff (PAS)(Calgary Laboratory Services, AB, Canada). Damage was assessed using a previously described histological activity index (HAI) 59 . In brief: epithelial damage, 0 = none, 1 = minimal loss of goblet cells (GCs), 2 = extensive loss of GCs, 3 = loss of crypt and extensive loss of GCs, 4 = extensive crypt and GC loss. Infiltration, 0 = none, 1 = crypt base, 2 = muscularis mucosa, 3 = extensive muscularis mucosa and edema, 4 = submucosal. Colonic crypt depth was measured in 4 locations from 6 untreated WT and TMEM173 Gt mice; n = 24 total measurements plotted. To assess possible differences in goblet cells, the number of goblet cells per 200 um 2 area were also counted from the untreated WT and TMEM173 Gt mice; n = 12.
Paraffin sections were also stained with an antibody against Iba-1 (ionized calcium-binding adapter molecule 1) that is expressed by activated macrophages. Sections were blocked (2% normal goat serum) for 2 hrs at room temperature (RT) and then incubated with the anti-Iba-1 (1:200, rabbit polyclonal, Wako #019-19741) in blocking buffer overnight at 4 °C. The sections were then washed and incubated with biotinylated anti-rabbit IgG antibody (1:200) in blocking buffer for 2 hrs at RT, then incubated with Elite ABC reagent Reagent A (1:100) and Reagent B (1:100) for 1 hr each, and then incubated in peroxidase solution.
immunoblotting. For STING protein expression, colonic homogenates (10% w/v), or macrophage lysates, were prepared in extraction buffer (0.15 M NaCl, 5 mM EDTA, 1% Triton-X 100, 10 mM Tris-HCl, pH 7.4) with the addition of a protease inhibitor cocktail (Complete, Roche Diagnostic GmbH). Protein concentrations were determined by Bradford assay. Proteins (50 μg) were separated on 12% SDS-polyacrylamide gels and transferred onto PVDF membranes. Membranes were blocked in 5% BSA/TBST and then incubated with an anti-STING rabbit polyclonal antibody (1:2500 dilution, Cell Signaling Technology Inc.) for 24 hr at 4 °C. An HRP-conjugated  . (a,b) Using iNOS/NOS2 mRNA induction as a marker for M1 cells, and Arg-1 mRNA induction as a marker for the M2 subtype, we observed no differences in the expression of either marker when these cells from the WT and Tmem173 gt were compared. (c) Increased IFN-β expression or secretion in response to STING agonists (or cytoplasmic DNA exposure) is a commonly used marker to show that the STING pathway has been activated. In this regard, when M1 or M2 polarized cells from the WT or Tmem17 Gt were incubated for 6-hr with DMXAA, it was observed that IFN-β mRNA expression was only induced in the WT macrophages. (d) Similarly, when the 3 subtypes of macrophages were exposed to the non-canonical ligand 2′3′-cGAMP or the bacterial second messenger c-di-AMP, IFN-β expression was increased solely in the WT macrophage polarized subsets. Significance was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values represent the means ± SEM of the fold changes in the respective mRNAs; *P < 0.05, **P < 0.01, or ***P < 0.001 vs. the corresponding Tmem17 Gt group (eg. WT M1, DMXAA treated vs. Tmem17 Gt M1, DMXAA treated). For macrophage polarization: M0, untreated; M1, 50 ng/ml of LPS and IFNγ; M2, 40 ng/ml IL-4 for 48 hrs. STING agonists: DMXAA 20 μg/ml; c-di-AMP 20 μg/ml plus lipofectamine; 2′3′-cGAMP 20 μg/ml plus lipofectamine for 6 hrs. Data was normalized to β-actin mRNA and experimental transcripts expressed as the relative fold change in each mRNA selected. (2019) 9:14281 | https://doi.org/10.1038/s41598-019-50656-5 www.nature.com/scientificreports www.nature.com/scientificreports/ donkey anti-rabbit secondary antibody was used. Membranes were then stripped and re-probed with an anti-β-actin antibody as a loading control (Sigma-Aldrich, USA). STING protein bands were visualized with SuperSignal ® West Pico chemiluminescence substrate and then the signal density was quantified using Image J (1.52K National Institute of Health, USA). Semi-quantitative analyses were performed by measuring the relative abundance of the murine or human STING protein which was then normalized to β-actin. The background was subtracted from all densitometric measurements.
Macrophage isolation and polarization. Using aseptic technique, femurs were harvested, and the bone marrow flushed from C57BL/6 and Tmem173 gt mice for macrophage polarization studies. The BMDMs were expanded in culture for 7 d in the presence of L-929 mouse fibroblast conditioned medium as a source of M-CSF. (c-f) Moreover, IFN-β, CXCL-10, iNOS and IL-12p40 mRNAs were also increased at either the 6 hr or the 24 hr timepoint post-cDN exposure. All cDN treatments were carried out using lipofectamine permeabilization of the cultured cells. Significance was assessed by one-way ANOVA followed by Tukey's multiple comparisons post-hoc test between all groups. Values represent the means ± SEM of the fold changes in the respective mRNAs; *P < 0.05, **P < 0.01, ***P < 0.001 vs. the control groups treated with IL-4 alone. Data was normalized to β-actin mRNA and experimental transcripts expressed as the relative fold change in each mRNA tested.