- Email: [email protected]
Make (No) Bones about Butyrate
Immunity
Previews in the peritoneum, suggesting that these chemokines may reach the vascular endothelium and override the requirement for neutrophil-derived CXCL2 or enhance ACKR1-dependent neutrophil extravasation. Interestingly, ACKR1-CXCL2 signaling did not affect neutrophil reverse transmigration, indicating that CXCL2 is a uni-directional chemokine affecting only extravasation but not re-entry into the circulation. Given that many neutrophils were found to exit inflamed tissues during the resolution of inflammation (Wang et al., 2017), blocking CXCL2 could generate an efflux of neutrophils from inflamed tissues in contrast to targeting other chemokines that might affect bidirectional trafficking. The non-redundant nature of CXCL2 in transmigration renders it a critical node in the regulation of inflammation and modulating its interactions with ACKR1 may serve as a selective approach to tune inflammation in a variety of medical contexts.
REFERENCES Bogoslowski, A., Butcher, E.C., and Kubes, P. (2018). Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 115, 2449–2454. Del Prete, A., Martı´nez-Mun˜oz, L., Mazzon, C., Toffali, L., Sozio, F., Za, L., Bosisio, D., Gazzurelli, L., Salvi, V., Tiberio, L., et al. (2017). The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage. Blood 130, 1223–1234. Finsterbusch, M., Voisin, M.B., Beyrau, M., Williams, T.J., and Nourshargh, S. (2014). Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF. J. Exp. Med. 211, 1307–1314.
by acting on macrophages, hepatocytes and myocytes. Nat. Med. 21, 239–247. Li, J.L., Lim, C.H., Tay, F.W., Goh, C.C., Devi, S., Malleret, B., Lee, B., Bakocevic, N., Chong, S.Z., Evrard, M., et al. (2016). Neutrophils self-regulate immune complex-mediated cutaneous inflammation through CXCL2. J. Invest. Dermatol. 136, 416–424. Ramos, C.D., Fernandes, K.S., Canetti, C., Teixeira, M.M., Silva, J.S., and Cunha, F.Q. (2006). Neutrophil recruitment in immunized mice depends on MIP-2 inducing the sequential release of MIP-1alpha, TNF-alpha and LTB(4). Eur. J. Immunol. 36, 2025–2034. Thiriot, A., Perdomo, C., Cheng, G., Novitzky-Basso, I., McArdle, S., Kishimoto, J.K., Barreiro, O., Mazo, I., Triboulet, R., Ley, K., et al. (2017). Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. BMC Biol. 15, 45.
Girbl, T., Lenn, T., Perez, L., Rolas, L., Barkaway, A., Thiriot, A., Del Fresno, C., Lynam, E., Hub, E., Thelen, M., et al. (2018). Distinct compartmentalization of the chemokines CXCL1 and CXCL2 and the atypical receptor ACKR1 determine discrete stages of neutrophil diapedesis. Immunity 49, this issue, 1062–1076.
Wan, W., Liu, Q., Lionakis, M.S., Marino, A.P., Anderson, S.A., Swamydas, M., and Murphy, P.M. (2015). Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoE-knockout mice. Cardiovasc. Res. 106, 478–487.
Li, P., Oh, D.Y., Bandyopadhyay, G., Lagakos, W.S., Talukdar, S., Osborn, O., Johnson, A., Chung, H., Maris, M., Ofrecio, J.M., et al. (2015). LTB4 promotes insulin resistance in obese mice
Wang, J., Hossain, M., Thanabalasuriar, A., Gunzer, M., Meininger, C., and Kubes, P. (2017). Visualizing the function and fate of neutrophils in sterile injury and repair. Science 358, 111–116.
Make (No) Bones about Butyrate Christina Begka1 and Benjamin J. Marsland1,* 1Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2018.12.005
In this issue of Immunity, Tyagi et al. (2018) report that the microbial metabolite butyrate orchestrates the interplay between regulatory T cells and CD8+ T cells, increasing Wnt signaling, and promoting bone formation in young mice. Microbial fermentation of dietary fibers in the colon produces short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, a group of microbial metabolites with immunomodulatory properties. SCFAs can act both locally and in the bone marrow, promoting the resolution of inflammatory disorders such as asthma (Trompette et al., 2014) while enhancing protective immunity against influenza (Trompette et al., 2018) in mice. Tyagi et al. (2018) now find that supplementation with the probiotic Lactobacillus rhamnosus GG (LGG) resulted in a butyrate-induced bone mass increase in
young mice. Butyrate mediated the expansion of regulatory T (Treg) cells, which instigated release of Wnt10b by CD8+ T cells and consequent activation of Wnt signaling in osteoblasts. Probiotics are widely available on the market and have been used in numerous clinical settings, albeit with limited success. Questions surrounding the immunomodulatory capacity of select probiotic strains and their dosing, viability, and localization within the gastrointestinal tract have led to a healthy skepticism. However, recent small-scale clinical studies with probiotics have shown
994 Immunity 49, December 18, 2018 ª 2018 Elsevier Inc.
encouraging results (Berni Canani et al., 2016) and certainly investigations with animal models are reporting efficacy and revealing detailed mechanisms of actions that might inform future clinical interventions (Schwarzer et al., 2016; Tyagi et al., 2018). Tyagi et al. (2018) found that oral delivery of LGG to mice increased butyrate concentrations in both the gut and serum, which in turn promoted extra-thymic Treg cell expansion, consistent with previous studies (Arpaia et al., 2013). Giving LGG to germ-free (GF) mice increased neither butyrate levels nor Treg cell numbers, indicating its effect
Immunity
Previews
Figure 1. Lactobacillus rhamnosus GG (LGG) and the Microbial Metabolite Butyrate Increase Bone Mass by Enhancing Wnt Signaling Administration of LGG to mice increases the abundance of Clostridia spp. in the gut with a consequent increase in both local and systemic butyrate. Butyrate either directly administered to mice or produced by the microbiome promotes the expansion of T regulatory (Treg) cells. TGF-b produced by Treg cells in the bone marrow increases the release of Wnt10b by bone marrow-resident CD8+ T cells leading to enhanced bone anabolism.
was dependent upon other constituents of the microbiota. To determine whether the impact of LGG supplementation on bone formation involved Treg cells, Tyagi et al. (2018) depleted Treg cells by delivery of anti-CD25 monoclonal antibodies or by using the DEREG mouse model, a strain where diphtheria toxin receptor is expressed under the control of the Foxp3 transcription factor promoter, thus allowing for selective depletion of Foxp3+ Treg cells upon diphtheria toxin administration to mice. Both of these treatments resulted in a loss of bone anabolic activity, revealing that the SCFA-mediated increase in bone mass was Treg cell dependent. The same group has previously reported that intermittent administration of parathyroid hormone (iPTH), which is used for osteoporosis treatment, promotes production of Wnt10b by bone marrow CD8+ T cells and activation of Wnt-mediated anabolic activity in pre-osteoblasts (Terauchi et al., 2009). In the current study, expression of the osteogenic Wnt ligand Wnt10b was increased in CD8+ T cells in the bone marrow of LGG- or butyrate-treated mice, but reduced in the absence of Treg cells. LGG or butyrate treatment of TCRb / mice, reconstituted with Wnt10b / CD8+ T cells and CD4+ Wnt10b+/+
T cells, failed to increase bone mass, pointing to a mechanistic link between the SCFA-mediated Treg pool and CD8+ T cell-induced Wnt signaling. Tyagi et al. (2018) next asked whether Treg cells would impact Wnt10b expression by CD8+ T cells directly; in vitro co-culture of Treg cells with CD8+ T cells increased Wnt10b mRNA expression in a manner reflective of the ratio of Treg cells to CD8+ T cells, which supports the conclusion that the butyrate-mediated expansion of Treg cells in vivo is related to a general expansion in cell number, as opposed to the differentiation of a particular subset of Treg cells. Wnt10b production is increased after CTLA-4-Igmediated inhibition of CD28 signaling (Roser-Page et al., 2014) in the context of T cell anergy. Tyagi et al. (2018) report that both LGG and butyrate treatments reduce the number of CD80+ and CD86+ mature dendritic cells, consequently limiting CD28 signaling in T cells as CD80/CD86 are its ligands. In addition, Tyagi et al. (2018) suggest that TGFb, not IL-10, from Treg cells blunts CD28 signaling in CD8+ T cells, as indicated by reduced levels of phosphorylated PI3K and pAKT, which are signaling molecules downstream of CD28. Although not addressed in detail, it is likely that the CD8+
T cells required TCR activation in order to produce Wnt10b, but the antigen specificity remains unclear. These latter finer details of the mechanism require further investigation and validation in vivo. How is Wnt10b expression regulated at a transcriptional level? Activated T cells form a complex between NFAT and AP-1, which facilitates the transcription of numerous genes, including those encoding inflammatory cytokines. Tyagi et al. (2018) suggest that Treg cells lead to reduced AP-1 production in bone marrow CD8+ T cells, which allows NFAT to instead complex with SMAD3 and consequently bind to the Wnt10b promoter. Overall, the authors proposed the following mechanistic links: LGG supplementation via butyrate expands the systemic Treg cell pool and through their TGFb production leads to increased nuclear SMAD3 concentrations in the adjacent CD8+ T cells and decreased expression of AP-1, with consequent binding of the NFAT:SMAD3 complex to the Wnt10b promoter (Figure 1). Work from our laboratory (Trompette et al., 2018) recently described the link between high-fiber diet and SCFA supplementation in activation of CD8+ T cells during influenza A virus infection. Butyrate, partly through free fatty acid Immunity 49, December 18, 2018 995
Immunity
Previews receptor 3 (FFAR3) signaling, altered the metabolic state of CD8+ T cells, resulting in an enhanced anti-viral response. Tyagi et al. (2018) found no direct effect of butyrate in CD8+ T cell-mediated Wnt10b expression, supportive of the necessity for a Treg-CD8+ T cell interplay in dampening T cell activation and promoting the Wnt pathway. In a recent study, Lucas et al. (2018) also suggested that oral SCFAs supplementation increased bone volume of mice in steady-state conditions, while hindering differentiation of osteoclasts and bone resorption in ovariectomized (OVX) mice, thus preventing bone volume loss. In contrast to Tyagi et al. (2018), Lucas et al. (2018) found mice that butyrate-treated RAG1 / maintained increased bone mass, suggesting that bone anabolic activity is T and B cell independent. Instead, propionate and butyrate treatment shifted the pre-osteoclasts metabolic reprogramming from oxidative phosphorylation to glycolysis, thus abrogating their differentiation. Participation of FFAR2 and FFAR3 SCFA receptors was dispensable during this metabolic process (Lucas et al., 2018). The discrepancies between the two studies were partially attributed by the authors to differences in experimental settings, including animal age and duration and doses of SCFA treatment. Moreover, different vendors account for distinct microbial communities, with the mice used for the Lucas et al. (2018) study harboring segmented filamentous bacteria (SFB), an acknowledged Th17 cell promoter, as opposed to the Tyagi et al. (2018) study, where mice were SFB negative. Future studies, with comparable experimental settings, are needed to assess the impact of SCFAs on the osteoblast-osteoclast balance and to clarify under which conditions the respective pathways are relevant. LGG treatment enhances the abundance of butyrate-producing Clostridia, and thus metabolites such as lactate, produced by LGG, are possible substrates for other bacteria to produce the butyrate (Berni Canani et al., 2016). These are important observations, particularly given the inter-individual heterogeneity of the gut microbiome in human populations—would the efficacy of
996 Immunity 49, December 18, 2018
LGG be dependent on the nature of a person’s microbiome? If so, cocktails of microbes might be required in order to elicit a protective effect, or in fact, bypassing microbes and utilizing metabolites might be most effective. A further important consideration revolves around not only the species of bacteria, but the strain. Mono-colonization of infant mice with different strains of Lactobacillus plantarum, for example, results in significantly different patterns of postnatal growth (Schwarzer et al., 2016), highlighting the point that any microbiota supplementation, or use of probiotics, requires profound knowledge of strainspecific characteristics. Dietary supplementation with fermentable fibers such as inulin or SCFAs such as butyrate have proven to be powerful and non-invasive interventions for attenuating inflammation-related pathologies, including asthma and menopausal-like bone resorption (Lucas et al., 2018; Trompette et al., 2018). The benefits of SCFA seen in inflammatory models are starting to be translated to humans; for example, a high-fiber diet was recently reported to improve the clinical outcome of patients with type 2 diabetes (T2D) (Zhao et al., 2018). Although most recognized for their anti-inflammatory action, SCFAs can also metabolically boost CD8+ T cell effector responses and protect against viral infection (Trompette et al., 2018). The impact of probiotic and SCFA supplementation in bone anabolism opens new horizons for exploiting mutualistic microbiota-host interactions in favor of maintaining juvenile growth under conditions of chronic malnutrition and prevention of osteoporosis (Schwarzer et al., 2016; Tyagi et al., 2018). The current literature, largely centered around mouse models, strongly supports SCFAs for the prevention of inflammatory diseases, promotion of antiviral immunity, and prevention of osteoporosis. Whether supplementation of people with SCFAs will prove to elicit the same health benefits seen in mice remains to be determined. Given the pleiotropic effects of SCFA on multiple cell types, care is certainly needed in order to avoid and monitor unexpected consequences; however, there is no question
that this is a promising avenue for continued research and development. REFERENCES Arpaia, N., Campbell, C., Fan, X., Dikiy, S., van der Veeken, J., deRoos, P., Liu, H., Cross, J.R., Pfeffer, K., Coffer, P.J., and Rudensky, A.Y. (2013). Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation. Nature 504, 451–455. Berni Canani, R., Sangwan, N., Stefka, A.T., Nocerino, R., Paparo, L., Aitoro, R., Calignano, A., Khan, A.A., Gilbert, J.A., and Nagler, C.R. (2016). Lactobacillus rhamnosus GG-supplemented formula expands butyrate-producing bacterial strains in food allergic infants. ISME J. 10, 742–750. Lucas, S., Omata, Y., Hofmann, J., Bo¨ttcher, M., Iljazovic, A., Sarter, K., Albrecht, O., Schulz, O., Krishnacoumar, B., Kro¨nke, G., et al. (2018). Short-chain fatty acids regulate systemic bone mass and protect from pathological bone loss. Nat. Commun. 9, 55. Roser-Page, S., Vikulina, T., Zayzafoon, M., and Weitzmann, M.N. (2014). CTLA-4Ig-induced T cell anergy promotes Wnt-10b production and bone formation in a mouse model. Arthritis Rheumatol. 66, 990–999. Schwarzer, M., Makki, K., Storelli, G., MachucaGayet, I., Srutkova, D., Hermanova, P., Martino, M.E., Balmand, S., Hudcovic, T., Heddi, A., et al. (2016). Lactobacillus plantarum strain maintains growth of infant mice during chronic undernutrition. Science 351, 854–857. Terauchi, M., Li, J.Y., Bedi, B., Baek, K.H., Tawfeek, H., Galley, S., Gilbert, L., Nanes, M.S., Zayzafoon, M., Guldberg, R., et al. (2009). T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling. Cell Metab. 10, 229–240. Trompette, A., Gollwitzer, E.S., Yadava, K., Sichelstiel, A.K., Sprenger, N., Ngom-Bru, C., Blanchard, C., Junt, T., Nicod, L.P., Harris, N.L., and Marsland, B.J. (2014). Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis. Nat. Med. 20, 159–166. Trompette, A., Gollwitzer, E.S., Pattaroni, C., Lopez-Mejia, I.C., Riva, E., Pernot, J., Ubags, N., Fajas, L., Nicod, L.P., and Marsland, B.J. (2018). Dietary fiber confers protection against flu by shaping Ly6c- patrolling monocyte hematopoiesis and CD8+ T cell metabolism. Immunity 48, 992– 1005.e8. Tyagi, A.M., Yu, M., Darby, T.M., Vaccaro, C., Li, J.Y., Owens, J.A., Hsu, E., Adams, J., Weitzmann, M.N., Jones, R.M., and Pacifici, R. (2018). The microbial metabolite butyrate stimulates bone formation via T regulatory cell-mediated regulation of WNT10B expression. Immunity 49, this issue, 1116–1131. Zhao, L., Zhang, F., Ding, X., Wu, G., Lam, Y.Y., Wang, X., Fu, H., Xue, X., Lu, C., Ma, J., et al. (2018). Gut bacteria selectively promoted by dietary fibers alleviate type 2 diabetes. Science 359, 1151–1156.
Previews in the peritoneum, suggesting that these chemokines may reach the vascular endothelium and override the requirement for neutrophil-derived CXCL2 or enhance ACKR1-dependent neutrophil extravasation. Interestingly, ACKR1-CXCL2 signaling did not affect neutrophil reverse transmigration, indicating that CXCL2 is a uni-directional chemokine affecting only extravasation but not re-entry into the circulation. Given that many neutrophils were found to exit inflamed tissues during the resolution of inflammation (Wang et al., 2017), blocking CXCL2 could generate an efflux of neutrophils from inflamed tissues in contrast to targeting other chemokines that might affect bidirectional trafficking. The non-redundant nature of CXCL2 in transmigration renders it a critical node in the regulation of inflammation and modulating its interactions with ACKR1 may serve as a selective approach to tune inflammation in a variety of medical contexts.
REFERENCES Bogoslowski, A., Butcher, E.C., and Kubes, P. (2018). Neutrophils recruited through high endothelial venules of the lymph nodes via PNAd intercept disseminating Staphylococcus aureus. Proc. Natl. Acad. Sci. USA 115, 2449–2454. Del Prete, A., Martı´nez-Mun˜oz, L., Mazzon, C., Toffali, L., Sozio, F., Za, L., Bosisio, D., Gazzurelli, L., Salvi, V., Tiberio, L., et al. (2017). The atypical receptor CCRL2 is required for CXCR2-dependent neutrophil recruitment and tissue damage. Blood 130, 1223–1234. Finsterbusch, M., Voisin, M.B., Beyrau, M., Williams, T.J., and Nourshargh, S. (2014). Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF. J. Exp. Med. 211, 1307–1314.
by acting on macrophages, hepatocytes and myocytes. Nat. Med. 21, 239–247. Li, J.L., Lim, C.H., Tay, F.W., Goh, C.C., Devi, S., Malleret, B., Lee, B., Bakocevic, N., Chong, S.Z., Evrard, M., et al. (2016). Neutrophils self-regulate immune complex-mediated cutaneous inflammation through CXCL2. J. Invest. Dermatol. 136, 416–424. Ramos, C.D., Fernandes, K.S., Canetti, C., Teixeira, M.M., Silva, J.S., and Cunha, F.Q. (2006). Neutrophil recruitment in immunized mice depends on MIP-2 inducing the sequential release of MIP-1alpha, TNF-alpha and LTB(4). Eur. J. Immunol. 36, 2025–2034. Thiriot, A., Perdomo, C., Cheng, G., Novitzky-Basso, I., McArdle, S., Kishimoto, J.K., Barreiro, O., Mazo, I., Triboulet, R., Ley, K., et al. (2017). Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. BMC Biol. 15, 45.
Girbl, T., Lenn, T., Perez, L., Rolas, L., Barkaway, A., Thiriot, A., Del Fresno, C., Lynam, E., Hub, E., Thelen, M., et al. (2018). Distinct compartmentalization of the chemokines CXCL1 and CXCL2 and the atypical receptor ACKR1 determine discrete stages of neutrophil diapedesis. Immunity 49, this issue, 1062–1076.
Wan, W., Liu, Q., Lionakis, M.S., Marino, A.P., Anderson, S.A., Swamydas, M., and Murphy, P.M. (2015). Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoE-knockout mice. Cardiovasc. Res. 106, 478–487.
Li, P., Oh, D.Y., Bandyopadhyay, G., Lagakos, W.S., Talukdar, S., Osborn, O., Johnson, A., Chung, H., Maris, M., Ofrecio, J.M., et al. (2015). LTB4 promotes insulin resistance in obese mice
Wang, J., Hossain, M., Thanabalasuriar, A., Gunzer, M., Meininger, C., and Kubes, P. (2017). Visualizing the function and fate of neutrophils in sterile injury and repair. Science 358, 111–116.
Make (No) Bones about Butyrate Christina Begka1 and Benjamin J. Marsland1,* 1Department of Immunology and Pathology, Central Clinical School, Monash University, Melbourne, VIC 3004, Australia *Correspondence: [email protected] https://doi.org/10.1016/j.immuni.2018.12.005
In this issue of Immunity, Tyagi et al. (2018) report that the microbial metabolite butyrate orchestrates the interplay between regulatory T cells and CD8+ T cells, increasing Wnt signaling, and promoting bone formation in young mice. Microbial fermentation of dietary fibers in the colon produces short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, a group of microbial metabolites with immunomodulatory properties. SCFAs can act both locally and in the bone marrow, promoting the resolution of inflammatory disorders such as asthma (Trompette et al., 2014) while enhancing protective immunity against influenza (Trompette et al., 2018) in mice. Tyagi et al. (2018) now find that supplementation with the probiotic Lactobacillus rhamnosus GG (LGG) resulted in a butyrate-induced bone mass increase in
young mice. Butyrate mediated the expansion of regulatory T (Treg) cells, which instigated release of Wnt10b by CD8+ T cells and consequent activation of Wnt signaling in osteoblasts. Probiotics are widely available on the market and have been used in numerous clinical settings, albeit with limited success. Questions surrounding the immunomodulatory capacity of select probiotic strains and their dosing, viability, and localization within the gastrointestinal tract have led to a healthy skepticism. However, recent small-scale clinical studies with probiotics have shown
994 Immunity 49, December 18, 2018 ª 2018 Elsevier Inc.
encouraging results (Berni Canani et al., 2016) and certainly investigations with animal models are reporting efficacy and revealing detailed mechanisms of actions that might inform future clinical interventions (Schwarzer et al., 2016; Tyagi et al., 2018). Tyagi et al. (2018) found that oral delivery of LGG to mice increased butyrate concentrations in both the gut and serum, which in turn promoted extra-thymic Treg cell expansion, consistent with previous studies (Arpaia et al., 2013). Giving LGG to germ-free (GF) mice increased neither butyrate levels nor Treg cell numbers, indicating its effect
Immunity
Previews
Figure 1. Lactobacillus rhamnosus GG (LGG) and the Microbial Metabolite Butyrate Increase Bone Mass by Enhancing Wnt Signaling Administration of LGG to mice increases the abundance of Clostridia spp. in the gut with a consequent increase in both local and systemic butyrate. Butyrate either directly administered to mice or produced by the microbiome promotes the expansion of T regulatory (Treg) cells. TGF-b produced by Treg cells in the bone marrow increases the release of Wnt10b by bone marrow-resident CD8+ T cells leading to enhanced bone anabolism.
was dependent upon other constituents of the microbiota. To determine whether the impact of LGG supplementation on bone formation involved Treg cells, Tyagi et al. (2018) depleted Treg cells by delivery of anti-CD25 monoclonal antibodies or by using the DEREG mouse model, a strain where diphtheria toxin receptor is expressed under the control of the Foxp3 transcription factor promoter, thus allowing for selective depletion of Foxp3+ Treg cells upon diphtheria toxin administration to mice. Both of these treatments resulted in a loss of bone anabolic activity, revealing that the SCFA-mediated increase in bone mass was Treg cell dependent. The same group has previously reported that intermittent administration of parathyroid hormone (iPTH), which is used for osteoporosis treatment, promotes production of Wnt10b by bone marrow CD8+ T cells and activation of Wnt-mediated anabolic activity in pre-osteoblasts (Terauchi et al., 2009). In the current study, expression of the osteogenic Wnt ligand Wnt10b was increased in CD8+ T cells in the bone marrow of LGG- or butyrate-treated mice, but reduced in the absence of Treg cells. LGG or butyrate treatment of TCRb / mice, reconstituted with Wnt10b / CD8+ T cells and CD4+ Wnt10b+/+
T cells, failed to increase bone mass, pointing to a mechanistic link between the SCFA-mediated Treg pool and CD8+ T cell-induced Wnt signaling. Tyagi et al. (2018) next asked whether Treg cells would impact Wnt10b expression by CD8+ T cells directly; in vitro co-culture of Treg cells with CD8+ T cells increased Wnt10b mRNA expression in a manner reflective of the ratio of Treg cells to CD8+ T cells, which supports the conclusion that the butyrate-mediated expansion of Treg cells in vivo is related to a general expansion in cell number, as opposed to the differentiation of a particular subset of Treg cells. Wnt10b production is increased after CTLA-4-Igmediated inhibition of CD28 signaling (Roser-Page et al., 2014) in the context of T cell anergy. Tyagi et al. (2018) report that both LGG and butyrate treatments reduce the number of CD80+ and CD86+ mature dendritic cells, consequently limiting CD28 signaling in T cells as CD80/CD86 are its ligands. In addition, Tyagi et al. (2018) suggest that TGFb, not IL-10, from Treg cells blunts CD28 signaling in CD8+ T cells, as indicated by reduced levels of phosphorylated PI3K and pAKT, which are signaling molecules downstream of CD28. Although not addressed in detail, it is likely that the CD8+
T cells required TCR activation in order to produce Wnt10b, but the antigen specificity remains unclear. These latter finer details of the mechanism require further investigation and validation in vivo. How is Wnt10b expression regulated at a transcriptional level? Activated T cells form a complex between NFAT and AP-1, which facilitates the transcription of numerous genes, including those encoding inflammatory cytokines. Tyagi et al. (2018) suggest that Treg cells lead to reduced AP-1 production in bone marrow CD8+ T cells, which allows NFAT to instead complex with SMAD3 and consequently bind to the Wnt10b promoter. Overall, the authors proposed the following mechanistic links: LGG supplementation via butyrate expands the systemic Treg cell pool and through their TGFb production leads to increased nuclear SMAD3 concentrations in the adjacent CD8+ T cells and decreased expression of AP-1, with consequent binding of the NFAT:SMAD3 complex to the Wnt10b promoter (Figure 1). Work from our laboratory (Trompette et al., 2018) recently described the link between high-fiber diet and SCFA supplementation in activation of CD8+ T cells during influenza A virus infection. Butyrate, partly through free fatty acid Immunity 49, December 18, 2018 995
Immunity
Previews receptor 3 (FFAR3) signaling, altered the metabolic state of CD8+ T cells, resulting in an enhanced anti-viral response. Tyagi et al. (2018) found no direct effect of butyrate in CD8+ T cell-mediated Wnt10b expression, supportive of the necessity for a Treg-CD8+ T cell interplay in dampening T cell activation and promoting the Wnt pathway. In a recent study, Lucas et al. (2018) also suggested that oral SCFAs supplementation increased bone volume of mice in steady-state conditions, while hindering differentiation of osteoclasts and bone resorption in ovariectomized (OVX) mice, thus preventing bone volume loss. In contrast to Tyagi et al. (2018), Lucas et al. (2018) found mice that butyrate-treated RAG1 / maintained increased bone mass, suggesting that bone anabolic activity is T and B cell independent. Instead, propionate and butyrate treatment shifted the pre-osteoclasts metabolic reprogramming from oxidative phosphorylation to glycolysis, thus abrogating their differentiation. Participation of FFAR2 and FFAR3 SCFA receptors was dispensable during this metabolic process (Lucas et al., 2018). The discrepancies between the two studies were partially attributed by the authors to differences in experimental settings, including animal age and duration and doses of SCFA treatment. Moreover, different vendors account for distinct microbial communities, with the mice used for the Lucas et al. (2018) study harboring segmented filamentous bacteria (SFB), an acknowledged Th17 cell promoter, as opposed to the Tyagi et al. (2018) study, where mice were SFB negative. Future studies, with comparable experimental settings, are needed to assess the impact of SCFAs on the osteoblast-osteoclast balance and to clarify under which conditions the respective pathways are relevant. LGG treatment enhances the abundance of butyrate-producing Clostridia, and thus metabolites such as lactate, produced by LGG, are possible substrates for other bacteria to produce the butyrate (Berni Canani et al., 2016). These are important observations, particularly given the inter-individual heterogeneity of the gut microbiome in human populations—would the efficacy of
996 Immunity 49, December 18, 2018
LGG be dependent on the nature of a person’s microbiome? If so, cocktails of microbes might be required in order to elicit a protective effect, or in fact, bypassing microbes and utilizing metabolites might be most effective. A further important consideration revolves around not only the species of bacteria, but the strain. Mono-colonization of infant mice with different strains of Lactobacillus plantarum, for example, results in significantly different patterns of postnatal growth (Schwarzer et al., 2016), highlighting the point that any microbiota supplementation, or use of probiotics, requires profound knowledge of strainspecific characteristics. Dietary supplementation with fermentable fibers such as inulin or SCFAs such as butyrate have proven to be powerful and non-invasive interventions for attenuating inflammation-related pathologies, including asthma and menopausal-like bone resorption (Lucas et al., 2018; Trompette et al., 2018). The benefits of SCFA seen in inflammatory models are starting to be translated to humans; for example, a high-fiber diet was recently reported to improve the clinical outcome of patients with type 2 diabetes (T2D) (Zhao et al., 2018). Although most recognized for their anti-inflammatory action, SCFAs can also metabolically boost CD8+ T cell effector responses and protect against viral infection (Trompette et al., 2018). The impact of probiotic and SCFA supplementation in bone anabolism opens new horizons for exploiting mutualistic microbiota-host interactions in favor of maintaining juvenile growth under conditions of chronic malnutrition and prevention of osteoporosis (Schwarzer et al., 2016; Tyagi et al., 2018). The current literature, largely centered around mouse models, strongly supports SCFAs for the prevention of inflammatory diseases, promotion of antiviral immunity, and prevention of osteoporosis. Whether supplementation of people with SCFAs will prove to elicit the same health benefits seen in mice remains to be determined. Given the pleiotropic effects of SCFA on multiple cell types, care is certainly needed in order to avoid and monitor unexpected consequences; however, there is no question
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