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Early-Life Antibiotic Exposure Causes Intestinal Dysbiosis and Exacerbates Skin and Lung Pathology in Experimental Systemic Sclerosis
Accepted Manuscript Early life antibiotic exposure causes intestinal dysbiosis and exacerbates skin and lung pathology in experimental systemic sclerosis Heena Mehta, Philippe-Olivier Goulet, Shunya Mashiko, Jade Desjardins, Gemma Pérez, Martial Koenig, Jean-Luc Senécal, Marco Constante, Manuela M. Santos, Marika Sarfati PII:
S0022-202X(17)31854-7
DOI:
10.1016/j.jid.2017.06.019
Reference:
JID 938
To appear in:
The Journal of Investigative Dermatology
Received Date: 9 December 2016 Revised Date:
16 May 2017
Accepted Date: 15 June 2017
Please cite this article as: Mehta H, Goulet P-O, Mashiko S, Desjardins J, Pérez G, Koenig M, Senécal J-L, Constante M, Santos MM, Sarfati M, Early life antibiotic exposure causes intestinal dysbiosis and exacerbates skin and lung pathology in experimental systemic sclerosis, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.06.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Early life antibiotic exposure causes intestinal dysbiosis and exacerbates skin and lung pathology in experimental systemic
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sclerosis Heena Mehta1,*, Philippe-Olivier Goulet1,*, Shunya Mashiko1, Jade Desjardins1, Gemma Pérez2, Martial Koenig2, Jean-Luc Senécal2, Marco Constante3, Manuela M. Santos3, and Marika
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Sarfati1
Immunoregulation Laboratory, 2Laboratory for Research in Autoimmunity, and 3Nutrition and
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Microbiome Laboratory, Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, H2X 0A9, Canada.
Address correspondence to: Dr. Marika Sarfati, MD, PhD, Immunoregulation Laboratory, CRCHUM, Tour Viger (R12.424), 900 rue St Denis, Montréal, Québec H2X 0A9, Canada.
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Phone: 514-890-8000 ext. 26701; FAX: 514-412-7944; email: [email protected] These authors contributed equally to the study.
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Running title: Early life dysbiosis worsens experimental SSc
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ACCEPTED MANUSCRIPT ABSTRACT Patients with systemic sclerosis (SSc) display altered intestinal microbiota.
However, the
influence of intestinal dysbiosis on development of experimental SSc remains unknown.
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Topoisomerase I peptide-loaded dendritic cells (TOPOIA DCs) immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was
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withdrawn (earlyATB) or initiated (adultATB) during adulthood. TOPOIA DCs (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), pro-fibrotic Remarkably, earlyATB
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(Il13, Tweakr) and vascular function (Serpine1) gene expression.
exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13+ cells, mononuclear phagocytes and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life
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(earlyATB) being sufficient to cause long-term modification of the microbiota and a shift towards increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T cell responses were observed in earlyATB and lifelongATB but not
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adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin
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exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.
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ACCEPTED MANUSCRIPT INTRODUCTION Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology, but likely results from a combination of dysregulated immune response and environmental triggers in genetically
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predisposed individuals (Pattanaik et al., 2015). Patients with autoimmune disorders have an altered intestinal microbiome in relation to healthy controls (Hevia et al., 2014, Knip and Siljander, 2016, Yurkovetskiy et al., 2015). Modulation of gut microbiota in animal models is
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associated with amelioration of experimental autoimmune encephalitis (EAE) and adjuvantinduced arthritis (AIA), and reduced or accelerated development of type 1 diabetes (T1D)
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(Candon et al., 2015, Hansen et al., 2012, Hu et al., 2016, Hu et al., 2015, Johnson et al., 2015, Nieuwenhuis et al., 2000, Ochoa-Reparaz et al., 2009, Yokote et al., 2008). Microbes present at mucosal surfaces have been shown to have an enormous impact on human health. Several epidemiological observations report that altered gut microbiota during infancy (<6 months) is associated with altered development of immune system and increased risk of
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development of inflammatory and atopic disorders in humans (Abrahamsson et al., 2012, Fujimura et al., 2016, Penders et al., 2013). Animal studies have confirmed that modulation of gut microbiota during the early life period, in utero or perinatal, is crucial for influencing the
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development of inflammatory, autoimmune and atopic diseases (Hansen et al., 2012, Hu et al.,
2015).
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2016, Hu et al., 2015, Olszak et al., 2012, Russell et al., 2012, Russell et al., 2013, Zanvit et al.,
The gut-lung axis is well established (Samuelson et al., 2015), with intestinal dysbiosis influencing immune response generation not only at the local mucosa but also in pulmonary mucosal surfaces which in turn can shape immune responses in gut (Bazett et al., 2016, Ruane et al., 2016, Russell et al., 2012). While prior studies considered skin to harbour a unique resident microbiota uninfluenced by gut microbiota, more recent studies suggest a strong link between
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ACCEPTED MANUSCRIPT these two organ systems (Naik et al., 2012). For instance, a recent report on experimental psoriasis shows that intestinal dysbiosis induced during the perinatal period aggravates psoriasislike disease (Zanvit et al., 2015).
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Patients with SSc display a distinct intestinal microbiota when compared to healthy controls with increased levels of Bifodobacterium, Lactobacilllus, Fusobacterium and γ-Proteobacteria, and decreased Fecalibacterium and Clostridium (Volkmann et al., 2016). These observations suggest
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that an altered microbiota is either a cause or consequence of the disease process. The role of gut microbiota in SSc pathogenesis remains unknown and has not been investigated in experimental
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SSc. Therefore, we postulated that dysbiosis induced by antibiotic (ATB) exposure will affect skin and lung pathology development in topoisomerase I peptide-loaded dendritic cells (TOPOIA DCs)-induced SSc-like disease characterized by progressive and protracted skin and lung fibrosis (Mehta et al., 2016). In the present study, mice immunized with TOPOIA DCs were
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exposed to ATB for different periods in order to identify “a critical window of opportunity” for intervention to influence experimental SSc disease outcome at two distal sites, skin and lung.
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RESULTS
Repeated TOPOIA DCs immunization induces skin fibrosis associated with upregulated
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Col1a1, Il13, Tweakr and Serpine1 expression Skin fibrosis is a hallmark of patients with SSc. Repeated immunization with TOPOIA DCs induced an experimental disease that recapitulates the cardinal features of human dcSSc, which include inflammation, vasculopathy, and skin fibrosis (Mehta et al., 2016). As depicted in Fig 1, collagen 1 (Col1a1) gene expression by qRT-PCR was significantly augmented in skin of TOPOIA DCs (control group, no ATB treatment) relative to unpulsed DCs group, corroborating the increased hydroxyproline content reported at week 12 in this experimental SSc model (Mehta 4
ACCEPTED MANUSCRIPT et al., 2016). Expression of Il13 and Tweakr (Tnfsfr12a) as well as transcription factors Rorc and Foxp3 was significantly increased in control group when compared to unpulsed DCs, while Il6, Il33, Tgfb1, Tslp, Ifng and Il5 were not differentially regulated. In case of genes related to
for Mmp12, von Willebrand Factor (Vwf) and Timp1.
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vascular dysfunction, Serpine1 was significantly upregulated while no differences were observed
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Streptomycin exposure limited to early life period is sufficient to modify matrix and profibrotic skin gene expression profile
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Next, we investigated the influence of oral administration of streptomycin which targets both gram- positive and negative microbiota, on the relative skin gene expression profile in TOPOIA DCs immunized (control (noATB)) mice. Streptomycin is poorly absorbed through the gastrointestinal tract; therefore, streptomycin delivery through drinking water would limit the effect of ATB to the gut. As depicted in Fig 2a, for lifelongATB group, streptomycin exposure
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was initiated in utero and continued during the immunization period until experimental endpoint, while for earlyATB group antibiotic exposure was limited to the in utero and weaning period. In contrast, for the adultATB group, streptomycin exposure was initiated one week prior to start of
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immunization and continued until the end. In the lifelongATB group, Il33 and Il6 expression was
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upregulated, while in adultATB Col5a1, Il13 αSMA and Rorc was increased when compared to control (no ATB) group. Remarkably, ATB exposure limited to early life was associated with significant upregulation of Col5a1 and Il13 expression in skin (Fig 2b). Notably, Tslp and Mmp12 expression was significantly increased in all ATB treated groups. Comparison of relative fold change in gene expression among the different ATB groups further highlighted that Col5a1, Il13 and Tweakr were significantly elevated for both earlyATB and adultATB mice when compared to lifelongATB (Fig S1).
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ACCEPTED MANUSCRIPT Skin pathology was qualitatively examined by trichrome (Masson) stained sections (Fig 3a). Control (no ATB) group displayed skin fibrosis and inflammation when compared to unpulsed DCs immunized mice. Streptomycin exposure limited to early or adult life aggravated skin
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pathology with unorganized collagen bundles in the dermis, which corroborated the increase in Col5a1 gene expression. However, all streptomycin exposed mice displayed increased inflammation spread between epidermal, dermal and hypodermal layers. We next depicted the
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spatial distribution of the cellular content in skin at week 12 using immunohistochemistry (IHC) or immunofluorescence (IF) staining in the earlyATB group since there were no significant
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differences in the nature of cellular infiltration between different ATB-treated groups. Firstly, myofibroblasts, defined as αSMA-expressing cells, were confined to the dermal layer while F4/80+ macrophages were found mainly in the subcutaneous fat and panniculus carnosus (Fig 3b and c). Secondly, IL-13+ cells appeared to be present mainly in the deeper subcutaneous layer (Fig 3e). In fact, examination of sequential areas of the same skin section showed that IL-13+
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cells were not localized in the same areas as αSMA+ or F4/80+ stained areas. Rather, IL-13+ cells were detected in skin sites infiltrated by tryptase+ or CD34+ cells (Fig 3f and g). Two patterns of
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CD34 staining were observed, elongated cells which are likely fibrocytes, and large cells resembling mast cells since CD34 is a pan-mast cell marker (Fig 3g). Indeed, tryptase+ mast cells
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(which also stained with toluidine blue (not shown)) were found scattered in the dermal and deeper subcutaneous layers.
Taken collectively, these data provide evidence that ATB exposure limited to early life is sufficient to exacerbate skin pathology in experimental SSc.
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ACCEPTED MANUSCRIPT Streptomycin exposure limited to early life is sufficient to induce long-term microbial dysbiosis in TOPOIA DCs immunized mice In order to correlate exacerbated skin pathology observed in TOPOIA DCs immunized mice
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exposed to streptomycin for different time periods with microbial dysbiosis, we analyzed gut microbiota by 16S rRNA sequencing of fecal samples from untreated and ATB-treated immunized groups (Fig 2a). First, streptomycin administration resulted in a significant decrease
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in both species richness (Chao1 index) and evenness (Shannon index) parameters of gut microbial community in all three ATB-treated groups (Fig 4a). Among the ATB groups, the
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Chao1 index was the most reduced for the lifelongATB group while the Shannon diversity index was further decreased for adultATB. Secondly, Principal Coordinate Analysis (PCoA) on weighted UniFrac distances showed a significant separation only for earlyATB treated versus control (no ATB) groups (P=0.015 by pairwise weighted Adonis) (Fig 4b). Gut microbial dysbiosis in the ATB-treated groups was already evident at the phylum level, with a shift
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towards increased Bacteroidetes/Firmicutes ratio that was significant for the earlyATB group (Fig 4c). Furthermore, at the family level there was an over representation of Actinobacteria in the earlyATB group versus both lifelongATB and adultATB groups (P<0.0001, Kruskal-Wallis)
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(Fig 4d). At the species level (Fig 4e), a significant increase in Adlercreutzia and
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Dehalobacterium species, and decreased abundance of Mucispirillum schaedleri was observed in the earlyATB group when compared to other ATB groups. In case of the lifelongATB group, there was a significant enrichment of Bacteroides caccae, and for adultATB Ruminococcus gnavus was significantly decreased when compared to the control (no ATB) group. Interestingly, Mucispirillum schaedleri was the only species that was significantly reduced following immunization with TOPOIA DCs (control (no ATB) group). The significant decrease in this group in relation to unpulsed DCs was restored following lifelongATB or adultATB exposure but not in the earlyATB group (adjusted P<0.05 when compared to unpulsed DCs, Wilcoxon 7
ACCEPTED MANUSCRIPT followed by False Discovery Rate correction). Finally, ATB exposure irrespective of timing resulted in a general increase in the abundance of Parabacteroides species and an overall decrease in Bifidobacterium pseudolongum, Lactobacillus and Dorea species, and unclassified
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Coriobacteriaceae. Taken together, streptomycin exposure until experimental endpoint induced intestinal dysbiosis irrespective of treatment onset. Remarkably, exposure to streptomycin limited to early life
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caused a sustained long-term intestinal dysbiosis that did not revert to normal once the selection
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pressure of ATB was removed.
Streptomycin exposure in early life aggravates pulmonary fibrosis and alters pulmonary Th cell responses but does not elicit anti-topoisomerase I autoantibody response in TOPOIA DCs immunized mice
Patients with dcSSc develop anti-topoisomerase I (anti-TopoI) autoantibodies and lung fibrosis.
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Anti-TopoI autoantibody response was not induced at an early time point (week 12) in TOPOIAimmmunized mice control (no ATB) or ATB-treated groups, corroborating our previous findings which showed that anti-TopoI response is induced at a late time point (week 18) (Mehta et al.,
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2016) (Fig S2). Pulmonary fibrosis was significantly increased in the control (no ATB) when
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compared to unpulsed DCs group at week 12 (Fig 5a). As for skin pathology, we next looked for the critical window for streptomycin exposure that was associated with exacerbated lung fibrosis. Pulmonary fibrosis was significantly augmented in lifelongATB and earlyATB groups, but not adultATB, when compared to control group (Fig 5a). Qualitative analysis of collagen deposition by trichrome (Masson) staining of lung sections corroborated the hydroxyproline data at week 12 and indicated that lung fibrosis was observed around blood vessels and airways (Fig 5b). Interestingly, lung fibrosis was exacerbated for earlyATB and not lifelongATB group at an earlier time point (week 10) (Fig 5c). This suggested an accelerated onset of fibrosis for the 8
ACCEPTED MANUSCRIPT earlyATB group and an ongoing inflammatory response in this group since inflammation precedes fibrosis (Wick et al., 2013). Quantification of cellular content in inflamed lungs at this early time point revealed that the frequencies of CD31+ endothelial cells, CD11b+CD11c+
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antigen presenting cells and γδ+ T cells, which were associated with increased neutrophil infiltration, had augmented in lifelongATB but not earlyATB or adultATB-treated mice (Fig S3). We further examined lung IL-17A, IL-13 and IFNγ expression, all implicated in the regulation of
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fibrotic responses (Pattanaik et al., 2015), in control and ATB-treated groups at week 10. The frequency of IL-17A+ cells, CD4- and not CD4+ IL-17A+ T cells was significantly increased in
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lungs of TOPOIA DCs relative to unpulsed DCs immunized mice (Fig 5d). Although pulmonary IL-17 expression was not further augmented in ATB-treated mice groups, the source of IL-17A had shifted towards γδ+ T cells in all ATB-treated mice (Fig 5e). As seen for skin, IL-13 expression was upregulated in the control group relative to unpulsed DCs. In fact, the percentage of CD4+ IL-13+ as well as IFNγ+ T cells were significantly increased (Fig 5f). LifelongATB
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exposure maintained this CD4+ IL-13+ T cell response while the frequency of CD4+ IFNγ+ T cells was significantly reduced when compared to the control (no ATB) group. Interestingly,
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transient streptomycin exposure during early life (earlyATB) that already resulted in exacerbated fibrosis at week 10 (Fig 5c) was associated with sustained CD4+ IFNγ+ T cells but a decrease in
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CD4+ IL-13+ T cells (Fig 5f). Streptomycin treatment initiated during or limited to early life appeared to be a critical window to dysregulate pulmonary T cell responses and augment the susceptibility to lung fibrosis development in TOPOIA DCs-induced SSc-like disease.
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ACCEPTED MANUSCRIPT DISCUSSION In the present study, we showed that exposure of TOPOIA DCs immunized mice to streptomycin during different periods in life caused differential intestinal dysbiosis, altered tissue-specific
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immune responses, and exacerbated disease in skin and lungs. Most importantly, early life exposure, limited to in utero and perinatal/weaning period prior to immunization, appeared to be a critical window to aggravate skin pathology and accelerate lung fibrosis development in this
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experimental SSc model. EarlyATB treatment resulted in decreased intestinal microbial diversity and abundance that persisted at least 3 months after discontinuation of streptomycin. Our
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observation reinforces the concept that intestinal dysbiosis induced in early life increases the susceptibility to develop autoimmune disorder in adult life.
The shift towards increased
Bacteroidetes/Firmicutes ratio in the earlyATB group resembles dysbiosis observed in certain autoimmune and inflammatory disease models. Lupus prone mice and non-obese diabetic mice
Siljander, 2016).
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(T1D) display a microbiota that favour increased Bacteroidetes (Johnson et al., 2015, Knip and
In the present report, we showed an influence of intestinal dysbiosis, following exposure to a
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single antibiotic, on disease outcome simultaneously at two distal sites, skin and lungs. Intestinal
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dysbiosis caused by streptomycin differentially influences lung disease. For cystic fibrosis, a Th17-mediated disease, streptomycin exposure during early life results in protection from disease (Bazett et al., 2016). In case of allergic asthma, a typical Th2 disease, it results in no change in disease outcome (Russell et al., 2012), while for extrinsic allergic alveolitis, mediated by Th17/Th1 inflammation, it aggravates lung disease (Russell et al., 2015). Although the role of IL-17 seems contradictory in human and murine SSc studies, the data appear to be in line with a contribution of IL-17 to the inflammatory process, which is required for the development of fibrosis (Brembilla and Chizzolini, 2012, Okamoto et al., 2012, Truchetet et al., 2013, Yang et 10
ACCEPTED MANUSCRIPT al., 2014). Streptomycin exposure of TOPOIA DCs immunized group resulted in a shift to γδ+ T cells being a major source of IL-17A. In SSc patients, γδ+ T cells have an activated phenotype, are cytotoxic and pro-fibrotic (Giacomelli et al., 1998, Segawa et al., 2014, Ueda-Hayakawa et
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al., 2013). Furthermore, streptomycin exposure in experimental SSc caused exacerbated lung fibrosis, which was associated with dysregulated IL-13 and IFNγ responses. In fact, the temporal
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contribution of various Th subsets to SSc pathogenesis remains unclear. Interestingly, lung fibrosis was already aggravated at the time we measured pulmonary cytokine responses (week
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10) in earlyATB group, and fibrosis persisted at week 12, which is in line with the concept of waning inflammation prior to establishment of fibrosis (Gruschwitz and Vieth, 1997, Pattanaik et al., 2015, Wick et al., 2013). This suggests that modulation of gut microbiota during early life
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accelerated development of lung fibrosis.
Currently, there is no evidence for a direct influence of intestinal dysbiosis caused by oral antibiotics on skin microbiota. However, it must be stressed that each barrier site exposed to the
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external world harbours its own distinct microbiota, which can be modulated by local inflammation as has been demonstrated for lungs (Barfod et al., 2015), and is likely the case for
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skin as well. A prior study found an enrichment in Rhodotorula in skin of patients with early SSc compared to controls (Arron et al., 2014). All three antibiotic exposure groups had severe skin disease in terms of histological inflammation, and upregulation of Tslp expression. Our finding is corroborated in a study by Yockey et al that shows that skin TSLP is increased in germ-free and not conventionally housed mice only in the context of epithelial barrier breach (Yockey et al., 2013). In our study, ATB exposure for different time periods significantly reduced diversity and abundance of bacterial
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ACCEPTED MANUSCRIPT species. TSLP is increased in skin of SSc patients, and implicated in microvasculopathy and fibrosis (Christmann et al., 2013, Truchetet et al., 2016, Usategui et al., 2013). Therefore, skin pathology in ATB treated mice closely resembles SSc skin. Tslp and Rorc, characteristic of
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atopic dermatitis (AD) and psoriasis, were highly upregulated in our model. However, we were not able to detect type 2 (Il4, Il5) and type 3 (Il17a, Il22) genes which are hallmarks of AD and psoriasis respectively. Furthermore, Serpine1 and Tweakr skin expression was increased in
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TOPOIA DCs immunized mice relative to unpulsed DCs group but not regulated by ATB exposure. Serpine-1, which blocks fibrinolysis and promotes collagen deposition in SSc, is
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increased in the skin of SSc patients (Lemaire et al., 2016, Pattanaik et al., 2015). TWEAKR is associated with both inflammatory and fibroproliferative subsets of SSc patients (Sargent et al., 2016).
Skin Il13 expression was upregulated in both earlyATB and adultATB groups relative to control group, and IL-13+ cells appeared to co-localize with tryptase
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and CD34+ cells. It has been
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reported that CD34+ immature mast cells produce IL-13 in response to TSLP (Allakhverdi et al., 2011, Allakhverdi et al., 2009). In TSLPR-deficient mice, attenuated bleomycin-induced fibrosis is associated with decreased IL-13 expression (Usategui et al., 2013). IL-13 is increased in skin
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of bleomycin-induced fibrosis (Matsushita et al., 2004). Patients with SSc have increased IL-13
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expression in skin relative to healthy controls (Greenblatt et al., 2012). Gene polymorphisms in IL-13 and its receptor IL-13Ra2 are positively correlated with SSc and skin fibrosis (Granel et al., 2006a, Granel et al., 2006b). Global gene expression analysis demonstrated that Il13 is associated with the inflammatory SSc patient subset (Greenblatt et al., 2012, Sargent et al., 2016). However, IL-13 production by skin and lung mast cells warrants further investigation in our model.
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ACCEPTED MANUSCRIPT Collectively, our study provides evidence that early infancy is a critical period of life to alter the susceptibility to autoimmune disorder development. Hence, exposure to a single ATB, streptomycin, limited to in utero and perinatal/weaning period is sufficient to induce intestinal
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dysbiosis in adult life that is associated with exacerbated lung and skin pathology in experimental SSc. METHODS
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TOPOIA DCs-induced model of fibrosis and streptomycin exposure
Syngenic Balb/c mice were housed and bred in specific pathogen free facility of the CRCHUM
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and immunized at 5-7 weeks of age. All animal procedures were approved by the institutional committee for protection of animals of CHUM (CIPA-Comité Institutionnel de Protection des Animaux du CHUM). Preparation of TOPOIA10-26 peptide (CanPeptide) loaded bone marrow derived DCs and immunization protocol have been described in (Mehta et al., 2016). Streptomycin (CHUM Pharmacy) was administered via drinking water at a dose of 200 mg/L
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(Russell et al., 2012, Russell et al., 2015) as depicted in Fig 2a. Fibrosis in lungs and skin was quantified by the hydroxyproline assay (Mehta et al., 2016). Histopathologic assessment of skin and lungs
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Skin and lung tissue sections (5 µm) were examined by trichrome (Masson) stain (Mehta et al.,
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2017). IHC and IF staining was done on the Benchmark XT (Ventana) (Mehta et al., 2016) with the following primary antibodies: anti-αSMA (1/200, abcam ab5694), anti-IL-13 (1/100, abcam ab106732), anti-F4/80 (1/50, homemade BM8), and anti-CD34 (1/100, sc-7045) and mast cell tryptase (1/100, sc32889) from Santa Cruz Biotech. Secondary antibodies used include biotinylated polyclonal goat anti-rabbit (1/500, Dako E0432), rabbit anti-goat (1/300, Dako E0466) and goat anti-rat (1/300, Southern Biotech 3010-08), and goat anti-rabbit Alexa Fluor
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ACCEPTED MANUSCRIPT 488 (1/250, Molecular Probes A11008). All slides were examined in a blinded manner by HM and MS, and scanned as described in (Mehta et al., 2016). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
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Shaved 6 mm punch skin biopsy sample from near the site of injection was put in RNAlater (Qiagen) for 24 hours and processed as described in (Mehta et al., 2017). TaqMan probes for Col1a1, Col5a1, Acta2 (αSMA), Tnfrsf12a (Tweakr), Ifng, Il4, Il5, Il6, Il13, Il17a, Il22, Il33,
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Tgfb1, Tslp, Foxp3, Rorc, Mmp12, Serpine1, Vwf , Timp1 and Hprt (housekeeping gene) were purchased from Thermo Fisher. Relative expression data were multiplied by 1000 for graphical
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representation on a log scale. Fold change was calculated using the 2^ (-∆∆CT) method. Ex vivo culture and intracellular cytokine staining
In some experiments, at week 10 before CFA boost, lung cells were isolated, stimulated and stained as described in (Mehta et al., 2016). The following antibodies were purchased from
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Biolegend unless specified otherwise: CD45.2-APC-CY7 (104), CD4-PE-CY7 (RM4-5), IL17A-APC (TC11-18H10.1), IFNγ-PerCP (XMG1.2), γδ-PE (GL3, BD) and IL-13-Alexa-488 (eBio13A, eBioscience). Cells were acquired on the FACsAria II (BD) and data analyzed with
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Flowjo v7.6.4 (Tree Star).
Microbial DNA extraction and Illumina MiSeq sequencing
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Fecal samples were collected at the time of sacrifice (Fig. 2A), and total DNA was extracted with the Powersoil DNA extraction kit (MO BIO Laboratories Inc). V3-V4 region of the 16S ribosomal RNA (rRNA) was amplified. Sequencing was performed on the MiSeq platform (Miseq v2 reagent kit, 500 cycles PE – Illumina) at the Genome Québec Innovation Center (McGill University, Montréal). Microbiota analysis
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ACCEPTED MANUSCRIPT Forward and reverse gene sequences were aligned using the Paired-End Read merger (PEAR (Zhang et al., 2014)), and the QIIME (Caporaso et al., 2010) software was used to cluster the merged sequences to identify Operational Taxonomic Units (OTUs). The 97% similarity
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database was used to identify bacteria at the species level. OTUs were then filtered to remove taxa present in only one sample and taxa with less than 100 reads across all samples. Samples were confirmed to have a minimum of 1000 reads each.
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Chao1 and Shannon indices of alpha diversity were calculated using the R package vegan (Jari Oksanen and Solymos, 2015). To evaluate beta diversity, Principal Coordinate Analysis (PCoA)
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was performed with R package phyloseq (McMurdie and Holmes, 2012) and Adonis was calculated using the weighted UniFrac distance matrix, with the R package vegan (Jari Oksanen and Solymos, 2015). R (R Core Team, 2015) was used to generate graphical outputs. Statistical analysis
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R (R Core Team, 2015) and Prism v6 (GraphPad) were used to perform statistical analysis. Unpaired Student’s t-test with Welch’s correction or Mann-Whitney test was used to compare two groups and a P<0.05 was considered statistically significant. For microbiota analysis,
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ANOVA or Kruskal-Wallis test was performed followed by multiple comparisons with pairwise t test or Wilcoxon signed-rank test. False discovery rate (FDR) control based on the Benjamini-
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Hochberg method was used to correct for multiple testing and an adjusted P<0.05 was considered to be statistically significant. CONFLICT OF INTEREST The authors state no conflict of interest ACKNOWLEDGMENTS The authors thank all members of the histology platform at the Goodman Cancer Research Center, McGill University for staining of lung and skin sections, and CRCHUM molecular
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ACCEPTED MANUSCRIPT pathology core facility for scanning microscopy. This work was supported by the Canadian Institutes of Health Research (operating grant MOP-142211), and also by research grants from Sclérodermie Québec, Scleroderma Society of Ontario and Scleroderma Society of Canada.
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Donations were given by Mrs Gisele Sarrazin-Locas in support of the Laboratory for Research in Autoimmunity. JLS holds the University of Montréal Scleroderma Research Chair.
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HM, POG and MS designed the experiments. HM, POG, SM, MC, JD, GP performed the experiments. HM, POG, MC, MMS and MS analyzed the data. HM, POG, MC, MMS and MS
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wrote the manuscript, and HM, POG, MC, MMS, MK, JLS and MS reviewed it critically for intellectual content. All authors have given final approval of the presented version to be published. LEGEND TO FIGURES
Figure 1. Immunization with TOPOIA DCs induces skin fibrosis associated with
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upregulated Col1a1, Il13 and Tweakr gene expression At week 12, relative skin mRNA gene expression in control (no ATB) (TOPOIA DCs) group was examined by qRT-PCR. Data shown are mean±SEM and a pool of at least two experiments.
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*P<0.05 by unpaired Student’s t-test with Welch’s correction.
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Figure 2. Streptomycin exposure limited to early life period is sufficient to modify skin gene expression profile
a Immunization and streptomycin antibiotic (ATB) exposure schematic, and b comparison of relative skin mRNA gene expression by qRT-PCR in control (no ATB) and different ATB exposure groups at week 12. Data shown are mean±SEM and a pool of at least two experiments. Mean relative expression of each gene of ATB treated group was compared to corresponding gene of control (no ATB) by unpaired Student’s t-test with Welch’s correction. +P<0.05 and ++P<0.01. 16
ACCEPTED MANUSCRIPT Figure 3. Streptomycin exposure limited to early life period is sufficient to exacerbate skin pathology At week 12, a representative trichrome (Masson) stained skin sections from two experiments.
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EarlyATB group skin IHC for b αSMA, c F4/80, f mast cell tryptase and g CD34, and IF for d IL-13 isotype control and e IL-13. Arrows show positively stained areas. (g) fibrocyte (open arrow) and mast cell (closed arrow) Scale bar=100 µm, and for insets =10 µm.
intestinal dysbiosis in TOPOIA DCs immunized mice
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Figure 4. Streptomycin exposure limited to early life is sufficient to induce long-term
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a Chao1 and Shannon diversity indices, b Principal Coordinate Analysis (PCoA) on weighted UniFrac distance matrices show a significant separation only for earlyATB treated versus control (no ATB) groups. P<0.05 between earlyATB and control (no ATB) by pairwise weighted Adonis, c ratio of Bacteroidetes/Firmicutes phyla. +P<0.05 by Mann-Whitney test, relative
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abundances at d family, and e species levels. Data shown are a pool of two experiments. For (a) and (e), *,+,§P<0.05; ++,§§P<0.01 and +++, §§§P≤0.001 by ANOVA or Kruskal-Wallis test followed by multiple comparisons with pairwise t-test or Wilcoxon sign-ranked test. To control
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for multiple testing, false discovery rate control was done and an adjusted P<0.05 was considered significant.
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Figure 5. Streptomycin exposure in early life aggravates pulmonary fibrosis and alters pulmonary cytokine responses in TOPOIA DCs immunized mice Pulmonary fibrosis was quantified by hydroxyproline assay at weeks a 12 and c 10, and b representative lung cryosections from two experiments were examined at week 12 for peribronchial (open arrows) and perivascular (closed arrows) collagen and inflammation by trichrome (Masson) stain. Scale bar=500 µm. At week 10, d and f frequencies of ex vivo pulmonary IL-17A+, IL-13+ and IFNγ+ cells, and e representative FACS plots of percentages of
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S0022-202X(17)31854-7
DOI:
10.1016/j.jid.2017.06.019
Reference:
JID 938
To appear in:
The Journal of Investigative Dermatology
Received Date: 9 December 2016 Revised Date:
16 May 2017
Accepted Date: 15 June 2017
Please cite this article as: Mehta H, Goulet P-O, Mashiko S, Desjardins J, Pérez G, Koenig M, Senécal J-L, Constante M, Santos MM, Sarfati M, Early life antibiotic exposure causes intestinal dysbiosis and exacerbates skin and lung pathology in experimental systemic sclerosis, The Journal of Investigative Dermatology (2017), doi: 10.1016/j.jid.2017.06.019. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Early life antibiotic exposure causes intestinal dysbiosis and exacerbates skin and lung pathology in experimental systemic
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sclerosis Heena Mehta1,*, Philippe-Olivier Goulet1,*, Shunya Mashiko1, Jade Desjardins1, Gemma Pérez2, Martial Koenig2, Jean-Luc Senécal2, Marco Constante3, Manuela M. Santos3, and Marika
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Sarfati1
Immunoregulation Laboratory, 2Laboratory for Research in Autoimmunity, and 3Nutrition and
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Microbiome Laboratory, Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, H2X 0A9, Canada.
Address correspondence to: Dr. Marika Sarfati, MD, PhD, Immunoregulation Laboratory, CRCHUM, Tour Viger (R12.424), 900 rue St Denis, Montréal, Québec H2X 0A9, Canada.
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Phone: 514-890-8000 ext. 26701; FAX: 514-412-7944; email: [email protected] These authors contributed equally to the study.
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Running title: Early life dysbiosis worsens experimental SSc
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ACCEPTED MANUSCRIPT ABSTRACT Patients with systemic sclerosis (SSc) display altered intestinal microbiota.
However, the
influence of intestinal dysbiosis on development of experimental SSc remains unknown.
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Topoisomerase I peptide-loaded dendritic cells (TOPOIA DCs) immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was
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withdrawn (earlyATB) or initiated (adultATB) during adulthood. TOPOIA DCs (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), pro-fibrotic Remarkably, earlyATB
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(Il13, Tweakr) and vascular function (Serpine1) gene expression.
exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13+ cells, mononuclear phagocytes and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life
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(earlyATB) being sufficient to cause long-term modification of the microbiota and a shift towards increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T cell responses were observed in earlyATB and lifelongATB but not
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adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin
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exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.
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ACCEPTED MANUSCRIPT INTRODUCTION Systemic sclerosis (SSc) is an autoimmune disease of unknown etiology, but likely results from a combination of dysregulated immune response and environmental triggers in genetically
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predisposed individuals (Pattanaik et al., 2015). Patients with autoimmune disorders have an altered intestinal microbiome in relation to healthy controls (Hevia et al., 2014, Knip and Siljander, 2016, Yurkovetskiy et al., 2015). Modulation of gut microbiota in animal models is
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associated with amelioration of experimental autoimmune encephalitis (EAE) and adjuvantinduced arthritis (AIA), and reduced or accelerated development of type 1 diabetes (T1D)
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(Candon et al., 2015, Hansen et al., 2012, Hu et al., 2016, Hu et al., 2015, Johnson et al., 2015, Nieuwenhuis et al., 2000, Ochoa-Reparaz et al., 2009, Yokote et al., 2008). Microbes present at mucosal surfaces have been shown to have an enormous impact on human health. Several epidemiological observations report that altered gut microbiota during infancy (<6 months) is associated with altered development of immune system and increased risk of
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development of inflammatory and atopic disorders in humans (Abrahamsson et al., 2012, Fujimura et al., 2016, Penders et al., 2013). Animal studies have confirmed that modulation of gut microbiota during the early life period, in utero or perinatal, is crucial for influencing the
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development of inflammatory, autoimmune and atopic diseases (Hansen et al., 2012, Hu et al.,
2015).
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2016, Hu et al., 2015, Olszak et al., 2012, Russell et al., 2012, Russell et al., 2013, Zanvit et al.,
The gut-lung axis is well established (Samuelson et al., 2015), with intestinal dysbiosis influencing immune response generation not only at the local mucosa but also in pulmonary mucosal surfaces which in turn can shape immune responses in gut (Bazett et al., 2016, Ruane et al., 2016, Russell et al., 2012). While prior studies considered skin to harbour a unique resident microbiota uninfluenced by gut microbiota, more recent studies suggest a strong link between
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ACCEPTED MANUSCRIPT these two organ systems (Naik et al., 2012). For instance, a recent report on experimental psoriasis shows that intestinal dysbiosis induced during the perinatal period aggravates psoriasislike disease (Zanvit et al., 2015).
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Patients with SSc display a distinct intestinal microbiota when compared to healthy controls with increased levels of Bifodobacterium, Lactobacilllus, Fusobacterium and γ-Proteobacteria, and decreased Fecalibacterium and Clostridium (Volkmann et al., 2016). These observations suggest
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that an altered microbiota is either a cause or consequence of the disease process. The role of gut microbiota in SSc pathogenesis remains unknown and has not been investigated in experimental
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SSc. Therefore, we postulated that dysbiosis induced by antibiotic (ATB) exposure will affect skin and lung pathology development in topoisomerase I peptide-loaded dendritic cells (TOPOIA DCs)-induced SSc-like disease characterized by progressive and protracted skin and lung fibrosis (Mehta et al., 2016). In the present study, mice immunized with TOPOIA DCs were
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exposed to ATB for different periods in order to identify “a critical window of opportunity” for intervention to influence experimental SSc disease outcome at two distal sites, skin and lung.
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RESULTS
Repeated TOPOIA DCs immunization induces skin fibrosis associated with upregulated
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Col1a1, Il13, Tweakr and Serpine1 expression Skin fibrosis is a hallmark of patients with SSc. Repeated immunization with TOPOIA DCs induced an experimental disease that recapitulates the cardinal features of human dcSSc, which include inflammation, vasculopathy, and skin fibrosis (Mehta et al., 2016). As depicted in Fig 1, collagen 1 (Col1a1) gene expression by qRT-PCR was significantly augmented in skin of TOPOIA DCs (control group, no ATB treatment) relative to unpulsed DCs group, corroborating the increased hydroxyproline content reported at week 12 in this experimental SSc model (Mehta 4
ACCEPTED MANUSCRIPT et al., 2016). Expression of Il13 and Tweakr (Tnfsfr12a) as well as transcription factors Rorc and Foxp3 was significantly increased in control group when compared to unpulsed DCs, while Il6, Il33, Tgfb1, Tslp, Ifng and Il5 were not differentially regulated. In case of genes related to
for Mmp12, von Willebrand Factor (Vwf) and Timp1.
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vascular dysfunction, Serpine1 was significantly upregulated while no differences were observed
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Streptomycin exposure limited to early life period is sufficient to modify matrix and profibrotic skin gene expression profile
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Next, we investigated the influence of oral administration of streptomycin which targets both gram- positive and negative microbiota, on the relative skin gene expression profile in TOPOIA DCs immunized (control (noATB)) mice. Streptomycin is poorly absorbed through the gastrointestinal tract; therefore, streptomycin delivery through drinking water would limit the effect of ATB to the gut. As depicted in Fig 2a, for lifelongATB group, streptomycin exposure
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was initiated in utero and continued during the immunization period until experimental endpoint, while for earlyATB group antibiotic exposure was limited to the in utero and weaning period. In contrast, for the adultATB group, streptomycin exposure was initiated one week prior to start of
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immunization and continued until the end. In the lifelongATB group, Il33 and Il6 expression was
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upregulated, while in adultATB Col5a1, Il13 αSMA and Rorc was increased when compared to control (no ATB) group. Remarkably, ATB exposure limited to early life was associated with significant upregulation of Col5a1 and Il13 expression in skin (Fig 2b). Notably, Tslp and Mmp12 expression was significantly increased in all ATB treated groups. Comparison of relative fold change in gene expression among the different ATB groups further highlighted that Col5a1, Il13 and Tweakr were significantly elevated for both earlyATB and adultATB mice when compared to lifelongATB (Fig S1).
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ACCEPTED MANUSCRIPT Skin pathology was qualitatively examined by trichrome (Masson) stained sections (Fig 3a). Control (no ATB) group displayed skin fibrosis and inflammation when compared to unpulsed DCs immunized mice. Streptomycin exposure limited to early or adult life aggravated skin
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pathology with unorganized collagen bundles in the dermis, which corroborated the increase in Col5a1 gene expression. However, all streptomycin exposed mice displayed increased inflammation spread between epidermal, dermal and hypodermal layers. We next depicted the
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spatial distribution of the cellular content in skin at week 12 using immunohistochemistry (IHC) or immunofluorescence (IF) staining in the earlyATB group since there were no significant
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differences in the nature of cellular infiltration between different ATB-treated groups. Firstly, myofibroblasts, defined as αSMA-expressing cells, were confined to the dermal layer while F4/80+ macrophages were found mainly in the subcutaneous fat and panniculus carnosus (Fig 3b and c). Secondly, IL-13+ cells appeared to be present mainly in the deeper subcutaneous layer (Fig 3e). In fact, examination of sequential areas of the same skin section showed that IL-13+
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cells were not localized in the same areas as αSMA+ or F4/80+ stained areas. Rather, IL-13+ cells were detected in skin sites infiltrated by tryptase+ or CD34+ cells (Fig 3f and g). Two patterns of
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CD34 staining were observed, elongated cells which are likely fibrocytes, and large cells resembling mast cells since CD34 is a pan-mast cell marker (Fig 3g). Indeed, tryptase+ mast cells
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(which also stained with toluidine blue (not shown)) were found scattered in the dermal and deeper subcutaneous layers.
Taken collectively, these data provide evidence that ATB exposure limited to early life is sufficient to exacerbate skin pathology in experimental SSc.
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ACCEPTED MANUSCRIPT Streptomycin exposure limited to early life is sufficient to induce long-term microbial dysbiosis in TOPOIA DCs immunized mice In order to correlate exacerbated skin pathology observed in TOPOIA DCs immunized mice
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exposed to streptomycin for different time periods with microbial dysbiosis, we analyzed gut microbiota by 16S rRNA sequencing of fecal samples from untreated and ATB-treated immunized groups (Fig 2a). First, streptomycin administration resulted in a significant decrease
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in both species richness (Chao1 index) and evenness (Shannon index) parameters of gut microbial community in all three ATB-treated groups (Fig 4a). Among the ATB groups, the
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Chao1 index was the most reduced for the lifelongATB group while the Shannon diversity index was further decreased for adultATB. Secondly, Principal Coordinate Analysis (PCoA) on weighted UniFrac distances showed a significant separation only for earlyATB treated versus control (no ATB) groups (P=0.015 by pairwise weighted Adonis) (Fig 4b). Gut microbial dysbiosis in the ATB-treated groups was already evident at the phylum level, with a shift
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towards increased Bacteroidetes/Firmicutes ratio that was significant for the earlyATB group (Fig 4c). Furthermore, at the family level there was an over representation of Actinobacteria in the earlyATB group versus both lifelongATB and adultATB groups (P<0.0001, Kruskal-Wallis)
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(Fig 4d). At the species level (Fig 4e), a significant increase in Adlercreutzia and
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Dehalobacterium species, and decreased abundance of Mucispirillum schaedleri was observed in the earlyATB group when compared to other ATB groups. In case of the lifelongATB group, there was a significant enrichment of Bacteroides caccae, and for adultATB Ruminococcus gnavus was significantly decreased when compared to the control (no ATB) group. Interestingly, Mucispirillum schaedleri was the only species that was significantly reduced following immunization with TOPOIA DCs (control (no ATB) group). The significant decrease in this group in relation to unpulsed DCs was restored following lifelongATB or adultATB exposure but not in the earlyATB group (adjusted P<0.05 when compared to unpulsed DCs, Wilcoxon 7
ACCEPTED MANUSCRIPT followed by False Discovery Rate correction). Finally, ATB exposure irrespective of timing resulted in a general increase in the abundance of Parabacteroides species and an overall decrease in Bifidobacterium pseudolongum, Lactobacillus and Dorea species, and unclassified
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Coriobacteriaceae. Taken together, streptomycin exposure until experimental endpoint induced intestinal dysbiosis irrespective of treatment onset. Remarkably, exposure to streptomycin limited to early life
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caused a sustained long-term intestinal dysbiosis that did not revert to normal once the selection
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pressure of ATB was removed.
Streptomycin exposure in early life aggravates pulmonary fibrosis and alters pulmonary Th cell responses but does not elicit anti-topoisomerase I autoantibody response in TOPOIA DCs immunized mice
Patients with dcSSc develop anti-topoisomerase I (anti-TopoI) autoantibodies and lung fibrosis.
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Anti-TopoI autoantibody response was not induced at an early time point (week 12) in TOPOIAimmmunized mice control (no ATB) or ATB-treated groups, corroborating our previous findings which showed that anti-TopoI response is induced at a late time point (week 18) (Mehta et al.,
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2016) (Fig S2). Pulmonary fibrosis was significantly increased in the control (no ATB) when
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compared to unpulsed DCs group at week 12 (Fig 5a). As for skin pathology, we next looked for the critical window for streptomycin exposure that was associated with exacerbated lung fibrosis. Pulmonary fibrosis was significantly augmented in lifelongATB and earlyATB groups, but not adultATB, when compared to control group (Fig 5a). Qualitative analysis of collagen deposition by trichrome (Masson) staining of lung sections corroborated the hydroxyproline data at week 12 and indicated that lung fibrosis was observed around blood vessels and airways (Fig 5b). Interestingly, lung fibrosis was exacerbated for earlyATB and not lifelongATB group at an earlier time point (week 10) (Fig 5c). This suggested an accelerated onset of fibrosis for the 8
ACCEPTED MANUSCRIPT earlyATB group and an ongoing inflammatory response in this group since inflammation precedes fibrosis (Wick et al., 2013). Quantification of cellular content in inflamed lungs at this early time point revealed that the frequencies of CD31+ endothelial cells, CD11b+CD11c+
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antigen presenting cells and γδ+ T cells, which were associated with increased neutrophil infiltration, had augmented in lifelongATB but not earlyATB or adultATB-treated mice (Fig S3). We further examined lung IL-17A, IL-13 and IFNγ expression, all implicated in the regulation of
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fibrotic responses (Pattanaik et al., 2015), in control and ATB-treated groups at week 10. The frequency of IL-17A+ cells, CD4- and not CD4+ IL-17A+ T cells was significantly increased in
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lungs of TOPOIA DCs relative to unpulsed DCs immunized mice (Fig 5d). Although pulmonary IL-17 expression was not further augmented in ATB-treated mice groups, the source of IL-17A had shifted towards γδ+ T cells in all ATB-treated mice (Fig 5e). As seen for skin, IL-13 expression was upregulated in the control group relative to unpulsed DCs. In fact, the percentage of CD4+ IL-13+ as well as IFNγ+ T cells were significantly increased (Fig 5f). LifelongATB
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exposure maintained this CD4+ IL-13+ T cell response while the frequency of CD4+ IFNγ+ T cells was significantly reduced when compared to the control (no ATB) group. Interestingly,
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transient streptomycin exposure during early life (earlyATB) that already resulted in exacerbated fibrosis at week 10 (Fig 5c) was associated with sustained CD4+ IFNγ+ T cells but a decrease in
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CD4+ IL-13+ T cells (Fig 5f). Streptomycin treatment initiated during or limited to early life appeared to be a critical window to dysregulate pulmonary T cell responses and augment the susceptibility to lung fibrosis development in TOPOIA DCs-induced SSc-like disease.
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ACCEPTED MANUSCRIPT DISCUSSION In the present study, we showed that exposure of TOPOIA DCs immunized mice to streptomycin during different periods in life caused differential intestinal dysbiosis, altered tissue-specific
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immune responses, and exacerbated disease in skin and lungs. Most importantly, early life exposure, limited to in utero and perinatal/weaning period prior to immunization, appeared to be a critical window to aggravate skin pathology and accelerate lung fibrosis development in this
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experimental SSc model. EarlyATB treatment resulted in decreased intestinal microbial diversity and abundance that persisted at least 3 months after discontinuation of streptomycin. Our
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observation reinforces the concept that intestinal dysbiosis induced in early life increases the susceptibility to develop autoimmune disorder in adult life.
The shift towards increased
Bacteroidetes/Firmicutes ratio in the earlyATB group resembles dysbiosis observed in certain autoimmune and inflammatory disease models. Lupus prone mice and non-obese diabetic mice
Siljander, 2016).
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(T1D) display a microbiota that favour increased Bacteroidetes (Johnson et al., 2015, Knip and
In the present report, we showed an influence of intestinal dysbiosis, following exposure to a
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single antibiotic, on disease outcome simultaneously at two distal sites, skin and lungs. Intestinal
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dysbiosis caused by streptomycin differentially influences lung disease. For cystic fibrosis, a Th17-mediated disease, streptomycin exposure during early life results in protection from disease (Bazett et al., 2016). In case of allergic asthma, a typical Th2 disease, it results in no change in disease outcome (Russell et al., 2012), while for extrinsic allergic alveolitis, mediated by Th17/Th1 inflammation, it aggravates lung disease (Russell et al., 2015). Although the role of IL-17 seems contradictory in human and murine SSc studies, the data appear to be in line with a contribution of IL-17 to the inflammatory process, which is required for the development of fibrosis (Brembilla and Chizzolini, 2012, Okamoto et al., 2012, Truchetet et al., 2013, Yang et 10
ACCEPTED MANUSCRIPT al., 2014). Streptomycin exposure of TOPOIA DCs immunized group resulted in a shift to γδ+ T cells being a major source of IL-17A. In SSc patients, γδ+ T cells have an activated phenotype, are cytotoxic and pro-fibrotic (Giacomelli et al., 1998, Segawa et al., 2014, Ueda-Hayakawa et
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al., 2013). Furthermore, streptomycin exposure in experimental SSc caused exacerbated lung fibrosis, which was associated with dysregulated IL-13 and IFNγ responses. In fact, the temporal
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contribution of various Th subsets to SSc pathogenesis remains unclear. Interestingly, lung fibrosis was already aggravated at the time we measured pulmonary cytokine responses (week
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10) in earlyATB group, and fibrosis persisted at week 12, which is in line with the concept of waning inflammation prior to establishment of fibrosis (Gruschwitz and Vieth, 1997, Pattanaik et al., 2015, Wick et al., 2013). This suggests that modulation of gut microbiota during early life
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accelerated development of lung fibrosis.
Currently, there is no evidence for a direct influence of intestinal dysbiosis caused by oral antibiotics on skin microbiota. However, it must be stressed that each barrier site exposed to the
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external world harbours its own distinct microbiota, which can be modulated by local inflammation as has been demonstrated for lungs (Barfod et al., 2015), and is likely the case for
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skin as well. A prior study found an enrichment in Rhodotorula in skin of patients with early SSc compared to controls (Arron et al., 2014). All three antibiotic exposure groups had severe skin disease in terms of histological inflammation, and upregulation of Tslp expression. Our finding is corroborated in a study by Yockey et al that shows that skin TSLP is increased in germ-free and not conventionally housed mice only in the context of epithelial barrier breach (Yockey et al., 2013). In our study, ATB exposure for different time periods significantly reduced diversity and abundance of bacterial
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ACCEPTED MANUSCRIPT species. TSLP is increased in skin of SSc patients, and implicated in microvasculopathy and fibrosis (Christmann et al., 2013, Truchetet et al., 2016, Usategui et al., 2013). Therefore, skin pathology in ATB treated mice closely resembles SSc skin. Tslp and Rorc, characteristic of
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atopic dermatitis (AD) and psoriasis, were highly upregulated in our model. However, we were not able to detect type 2 (Il4, Il5) and type 3 (Il17a, Il22) genes which are hallmarks of AD and psoriasis respectively. Furthermore, Serpine1 and Tweakr skin expression was increased in
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TOPOIA DCs immunized mice relative to unpulsed DCs group but not regulated by ATB exposure. Serpine-1, which blocks fibrinolysis and promotes collagen deposition in SSc, is
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increased in the skin of SSc patients (Lemaire et al., 2016, Pattanaik et al., 2015). TWEAKR is associated with both inflammatory and fibroproliferative subsets of SSc patients (Sargent et al., 2016).
Skin Il13 expression was upregulated in both earlyATB and adultATB groups relative to control group, and IL-13+ cells appeared to co-localize with tryptase
+
and CD34+ cells. It has been
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reported that CD34+ immature mast cells produce IL-13 in response to TSLP (Allakhverdi et al., 2011, Allakhverdi et al., 2009). In TSLPR-deficient mice, attenuated bleomycin-induced fibrosis is associated with decreased IL-13 expression (Usategui et al., 2013). IL-13 is increased in skin
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of bleomycin-induced fibrosis (Matsushita et al., 2004). Patients with SSc have increased IL-13
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expression in skin relative to healthy controls (Greenblatt et al., 2012). Gene polymorphisms in IL-13 and its receptor IL-13Ra2 are positively correlated with SSc and skin fibrosis (Granel et al., 2006a, Granel et al., 2006b). Global gene expression analysis demonstrated that Il13 is associated with the inflammatory SSc patient subset (Greenblatt et al., 2012, Sargent et al., 2016). However, IL-13 production by skin and lung mast cells warrants further investigation in our model.
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ACCEPTED MANUSCRIPT Collectively, our study provides evidence that early infancy is a critical period of life to alter the susceptibility to autoimmune disorder development. Hence, exposure to a single ATB, streptomycin, limited to in utero and perinatal/weaning period is sufficient to induce intestinal
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dysbiosis in adult life that is associated with exacerbated lung and skin pathology in experimental SSc. METHODS
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TOPOIA DCs-induced model of fibrosis and streptomycin exposure
Syngenic Balb/c mice were housed and bred in specific pathogen free facility of the CRCHUM
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and immunized at 5-7 weeks of age. All animal procedures were approved by the institutional committee for protection of animals of CHUM (CIPA-Comité Institutionnel de Protection des Animaux du CHUM). Preparation of TOPOIA10-26 peptide (CanPeptide) loaded bone marrow derived DCs and immunization protocol have been described in (Mehta et al., 2016). Streptomycin (CHUM Pharmacy) was administered via drinking water at a dose of 200 mg/L
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(Russell et al., 2012, Russell et al., 2015) as depicted in Fig 2a. Fibrosis in lungs and skin was quantified by the hydroxyproline assay (Mehta et al., 2016). Histopathologic assessment of skin and lungs
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Skin and lung tissue sections (5 µm) were examined by trichrome (Masson) stain (Mehta et al.,
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2017). IHC and IF staining was done on the Benchmark XT (Ventana) (Mehta et al., 2016) with the following primary antibodies: anti-αSMA (1/200, abcam ab5694), anti-IL-13 (1/100, abcam ab106732), anti-F4/80 (1/50, homemade BM8), and anti-CD34 (1/100, sc-7045) and mast cell tryptase (1/100, sc32889) from Santa Cruz Biotech. Secondary antibodies used include biotinylated polyclonal goat anti-rabbit (1/500, Dako E0432), rabbit anti-goat (1/300, Dako E0466) and goat anti-rat (1/300, Southern Biotech 3010-08), and goat anti-rabbit Alexa Fluor
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ACCEPTED MANUSCRIPT 488 (1/250, Molecular Probes A11008). All slides were examined in a blinded manner by HM and MS, and scanned as described in (Mehta et al., 2016). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)
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Shaved 6 mm punch skin biopsy sample from near the site of injection was put in RNAlater (Qiagen) for 24 hours and processed as described in (Mehta et al., 2017). TaqMan probes for Col1a1, Col5a1, Acta2 (αSMA), Tnfrsf12a (Tweakr), Ifng, Il4, Il5, Il6, Il13, Il17a, Il22, Il33,
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Tgfb1, Tslp, Foxp3, Rorc, Mmp12, Serpine1, Vwf , Timp1 and Hprt (housekeeping gene) were purchased from Thermo Fisher. Relative expression data were multiplied by 1000 for graphical
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representation on a log scale. Fold change was calculated using the 2^ (-∆∆CT) method. Ex vivo culture and intracellular cytokine staining
In some experiments, at week 10 before CFA boost, lung cells were isolated, stimulated and stained as described in (Mehta et al., 2016). The following antibodies were purchased from
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Biolegend unless specified otherwise: CD45.2-APC-CY7 (104), CD4-PE-CY7 (RM4-5), IL17A-APC (TC11-18H10.1), IFNγ-PerCP (XMG1.2), γδ-PE (GL3, BD) and IL-13-Alexa-488 (eBio13A, eBioscience). Cells were acquired on the FACsAria II (BD) and data analyzed with
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Flowjo v7.6.4 (Tree Star).
Microbial DNA extraction and Illumina MiSeq sequencing
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Fecal samples were collected at the time of sacrifice (Fig. 2A), and total DNA was extracted with the Powersoil DNA extraction kit (MO BIO Laboratories Inc). V3-V4 region of the 16S ribosomal RNA (rRNA) was amplified. Sequencing was performed on the MiSeq platform (Miseq v2 reagent kit, 500 cycles PE – Illumina) at the Genome Québec Innovation Center (McGill University, Montréal). Microbiota analysis
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ACCEPTED MANUSCRIPT Forward and reverse gene sequences were aligned using the Paired-End Read merger (PEAR (Zhang et al., 2014)), and the QIIME (Caporaso et al., 2010) software was used to cluster the merged sequences to identify Operational Taxonomic Units (OTUs). The 97% similarity
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database was used to identify bacteria at the species level. OTUs were then filtered to remove taxa present in only one sample and taxa with less than 100 reads across all samples. Samples were confirmed to have a minimum of 1000 reads each.
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Chao1 and Shannon indices of alpha diversity were calculated using the R package vegan (Jari Oksanen and Solymos, 2015). To evaluate beta diversity, Principal Coordinate Analysis (PCoA)
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was performed with R package phyloseq (McMurdie and Holmes, 2012) and Adonis was calculated using the weighted UniFrac distance matrix, with the R package vegan (Jari Oksanen and Solymos, 2015). R (R Core Team, 2015) was used to generate graphical outputs. Statistical analysis
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R (R Core Team, 2015) and Prism v6 (GraphPad) were used to perform statistical analysis. Unpaired Student’s t-test with Welch’s correction or Mann-Whitney test was used to compare two groups and a P<0.05 was considered statistically significant. For microbiota analysis,
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ANOVA or Kruskal-Wallis test was performed followed by multiple comparisons with pairwise t test or Wilcoxon signed-rank test. False discovery rate (FDR) control based on the Benjamini-
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Hochberg method was used to correct for multiple testing and an adjusted P<0.05 was considered to be statistically significant. CONFLICT OF INTEREST The authors state no conflict of interest ACKNOWLEDGMENTS The authors thank all members of the histology platform at the Goodman Cancer Research Center, McGill University for staining of lung and skin sections, and CRCHUM molecular
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ACCEPTED MANUSCRIPT pathology core facility for scanning microscopy. This work was supported by the Canadian Institutes of Health Research (operating grant MOP-142211), and also by research grants from Sclérodermie Québec, Scleroderma Society of Ontario and Scleroderma Society of Canada.
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Donations were given by Mrs Gisele Sarrazin-Locas in support of the Laboratory for Research in Autoimmunity. JLS holds the University of Montréal Scleroderma Research Chair.
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HM, POG and MS designed the experiments. HM, POG, SM, MC, JD, GP performed the experiments. HM, POG, MC, MMS and MS analyzed the data. HM, POG, MC, MMS and MS
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wrote the manuscript, and HM, POG, MC, MMS, MK, JLS and MS reviewed it critically for intellectual content. All authors have given final approval of the presented version to be published. LEGEND TO FIGURES
Figure 1. Immunization with TOPOIA DCs induces skin fibrosis associated with
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upregulated Col1a1, Il13 and Tweakr gene expression At week 12, relative skin mRNA gene expression in control (no ATB) (TOPOIA DCs) group was examined by qRT-PCR. Data shown are mean±SEM and a pool of at least two experiments.
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*P<0.05 by unpaired Student’s t-test with Welch’s correction.
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Figure 2. Streptomycin exposure limited to early life period is sufficient to modify skin gene expression profile
a Immunization and streptomycin antibiotic (ATB) exposure schematic, and b comparison of relative skin mRNA gene expression by qRT-PCR in control (no ATB) and different ATB exposure groups at week 12. Data shown are mean±SEM and a pool of at least two experiments. Mean relative expression of each gene of ATB treated group was compared to corresponding gene of control (no ATB) by unpaired Student’s t-test with Welch’s correction. +P<0.05 and ++P<0.01. 16
ACCEPTED MANUSCRIPT Figure 3. Streptomycin exposure limited to early life period is sufficient to exacerbate skin pathology At week 12, a representative trichrome (Masson) stained skin sections from two experiments.
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EarlyATB group skin IHC for b αSMA, c F4/80, f mast cell tryptase and g CD34, and IF for d IL-13 isotype control and e IL-13. Arrows show positively stained areas. (g) fibrocyte (open arrow) and mast cell (closed arrow) Scale bar=100 µm, and for insets =10 µm.
intestinal dysbiosis in TOPOIA DCs immunized mice
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Figure 4. Streptomycin exposure limited to early life is sufficient to induce long-term
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a Chao1 and Shannon diversity indices, b Principal Coordinate Analysis (PCoA) on weighted UniFrac distance matrices show a significant separation only for earlyATB treated versus control (no ATB) groups. P<0.05 between earlyATB and control (no ATB) by pairwise weighted Adonis, c ratio of Bacteroidetes/Firmicutes phyla. +P<0.05 by Mann-Whitney test, relative
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abundances at d family, and e species levels. Data shown are a pool of two experiments. For (a) and (e), *,+,§P<0.05; ++,§§P<0.01 and +++, §§§P≤0.001 by ANOVA or Kruskal-Wallis test followed by multiple comparisons with pairwise t-test or Wilcoxon sign-ranked test. To control
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for multiple testing, false discovery rate control was done and an adjusted P<0.05 was considered significant.
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Figure 5. Streptomycin exposure in early life aggravates pulmonary fibrosis and alters pulmonary cytokine responses in TOPOIA DCs immunized mice Pulmonary fibrosis was quantified by hydroxyproline assay at weeks a 12 and c 10, and b representative lung cryosections from two experiments were examined at week 12 for peribronchial (open arrows) and perivascular (closed arrows) collagen and inflammation by trichrome (Masson) stain. Scale bar=500 µm. At week 10, d and f frequencies of ex vivo pulmonary IL-17A+, IL-13+ and IFNγ+ cells, and e representative FACS plots of percentages of
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ACCEPTED MANUSCRIPT CD4+ and γδ+ T cells after gating on IL-17A+ cells. Data shown are mean±SEM and a pool of at least two experiments. *,+,§P<0.05; **,++,§§P<0.01and §§§P≤0.001 by unpaired Student’s ttest with Welch’s correction.
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