Brian R. Rosborough

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4+ Foxp3+ regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b+ cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4+ Foxp3+ Tregs, including an ST2L+ population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8+ IFN-γ+ cells. Also, despite reducing overall CD3+ cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3+ cells. Whereas control graft recipients displayed increases in systemic CD11b+ Gr-1hi cells, IL-33–treated recipients exhibited increased CD11b+ Gr-1int cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4+ Foxp3+ Tregs that underlie IL-33–mediated cardiac allograft survival.

IL-33 Is an Unconventional Alarmin That Stimulates IL-2 Secretion by Dendritic Cells To Selectively Expand IL-33R/ST2+ Regulatory T Cells

IL-33 is a recently characterized IL-1 family member that is proposed to function as an alarmin, or endogenous signal of cellular damage, as well as act as a pleiotropic cytokine. The ability of IL-33 to potentiate both Th1 and Th2 immunity supports its role in pathogen clearance and disease immunopathology. Yet, IL-33 restrains experimental colitis and transplant rejection by expanding regulatory T cells (Treg) via an undefined mechanism. We sought to determine the influence of IL-33 on hematopoietic cells that drives Treg expansion and underlies the therapeutic benefit of IL-33 administration. In this study, we identify a feedback loop in which conventional mouse CD11c+ dendritic cells (DC) stimulated by IL-33 secrete IL-2 to selectively expand IL-33R(ST2+)– suppressive CD4+Foxp3+ Treg. Interestingly, this occurs in the absence of classical DC maturation, and DC-derived (innate) IL-2 increases ST2 expression on both DC and interacting Treg. ST2+ Treg represent an activated subset of Foxp3+ cells, demonstrated to be ICOShighCD44high compared with their ST2− counterparts. Furthermore, although studies have shown that IL-33–exposed DC promote Th2 responses, we reveal that ST2+ DC are required for IL-33–mediated in vitro and in vivo Treg expansion. Thus, we have uncovered a relationship between IL-33 and innate IL-2 that promotes the selective expansion of ST2+ Treg over non-Treg. These findings identify a novel regulatory pathway driven by IL-33 in immune cells that may be harnessed for therapeutic benefit or for robust expansion of Treg in vitro and in vivo.

mTOR and GSK-3 shape the CD4+ T-cell stimulatory and differentiation capacity of myeloid DCs after exposure to LPS.

Prolonged inhibition of the kinase, mammalian target of rapamycin (mTOR), during myeloid dendritic cell (DC) generation confers resistance to maturation. Recently, however, mTOR inhibition immediately before Toll-like receptor ligation has been found to exert proinflammatory effects on myeloid cells, notably enhanced IL-12p40/p70 production. We show, for the first time, that mouse or human DCs generated under mTOR inhibition exhibit markedly enhanced IL-12p70 production after lipopolysaccharide (LPS) stimulation, despite impaired costimulatory molecule expression and poor T-cell stimulatory ability. Consistent with this finding, we reveal that increased IL-12p40 production occurs predominantly in CD86(lo) immature DCs. High IL-12p40/p70 production by CD86(lo) DC resulted from failed down-regulation of glycogen synthase kinase-3 (GSK-3) activity and could not be ascribed to enhanced Akt function. Despite high IL-12p70 secretion, rapamycin-conditioned, LPS-stimulated DCs remained poor T-cell stimulators, failing to enhance allogeneic Th1 cell responses. We also report that inhibition of GSK-3 impedes the ability of LPS-stimulated DCs to induce forkhead box p3 in CD4(+)CD25(-) T cells, as does the absence of IL-12p40/p70. Thus, GSK-3 activity in DC is regulated via signaling linked to mTOR and modulates their capacity both to produce IL-12p40/p70 and induce forkhead box p3 in CD4(+) T cells under inflammatory conditions.

Histones Activate the NLRP3 Inflammasome in Kupffer Cells during Sterile Inflammatory Liver Injury

Cellular processes that drive sterile inflammatory injury after hepatic ischemia/reperfusion (I/R) injury are not completely understood. Activation of the inflammasome plays a key role in response to invading intracellular pathogens, but mounting evidence suggests that it also plays a role in inflammation driven by endogenous danger-associate molecular pattern molecules released after ischemic injury. The nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) inflammasome is one such process, and the mechanism by which its activation results in damage and inflammatory responses following liver I/R is unknown. In this article, we report that both NLRP3 and its downstream target caspase-1 are activated during I/R and are essential for hepatic I/R injury, because both NLRP3 and caspase-1 knockout mice are protected from injury. Furthermore, inflammasome-mediated injury is dependent on caspase-1 expression in liver nonparenchymal cells. Although upstream signals that activate the inflammasome during ischemic injury are not well characterized, we show that endogenous extracellular histones activate the NLRP3 inflammasome during liver I/R through TLR9. This occurs through TLR9-dependent generation of reactive oxygen species. This mechanism is operant in resident liver Kupffer cells, which drive innate immune responses after I/R injury by recruiting additional cell types, including neutrophils and inflammatory monocytes. These novel findings illustrate a new mechanism by which extracellular histones and activation of NLRP3 inflammasome contribute to liver damage and the activation of innate immunity during sterile inflammation.

Hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection.

High‐mobility group box 1 (HMGB1) is an abundant chromatin‐associated nuclear protein and released into the extracellular milieu during liver ischemia‐reperfusion (I/R), signaling activation of proinflammatory cascades. Because the intracellular function of HMGB1 during sterile inflammation of I/R is currently unknown, we sought to determine the role of intracellular HMGB1 in hepatocytes after liver I/R. When hepatocyte‐specific HMGB1 knockout (HMGB1‐HC‐KO) and control mice were subjected to a nonlethal warm liver I/R, it was found that HMGB1‐HC‐KO mice had significantly greater hepatocellular injury after I/R, compared to control mice. Additionally, there was significantly greater DNA damage and decreased chromatin accessibility to repair with lack of HMGB1. Furthermore, lack of hepatocyte HMGB1 led to excessive poly(ADP‐ribose)polymerase 1 activation, exhausting nicotinamide adenine dinucleotide and adenosine triphosphate stores, exacerbating mitochondrial instability and damage, and, consequently, leading to increased cell death. We found that this was also associated with significantly more oxidative stress (OS) in HMGB1‐HC‐KO mice, compared to control. Increased nuclear instability led to a resultant increase in the release of histones with subsequently more inflammatory cytokine production and organ damage through activation of Toll‐like receptor 9. Conclusion: The lack of HMGB1 within hepatocytes leads to increased susceptibility to cellular death after OS conditions. (Hepatology 2014;59:1984–1997)

Cellular-specific role of toll-like receptor 4 in hepatic ischemia-reperfusion injury in mice.

Ischemia‐reperfusion (I/R) injury is a process whereby an initial hypoxic insult and subsequent return of blood flow leads to the propagation of innate immune responses and organ injury. The necessity of the pattern recognition receptor, Toll‐like receptor (TLR)4, for this innate immune response has been previously shown. However, TLR4 is present on various cell types of the liver, both immune and nonimmune cells. Therefore, we sought to determine the role of TLR4 in individual cell populations, specifically, parenchymal hepatocytes (HCs), myeloid cells, including Kupffer cells, and dendritic cells (DCs) subsequent to hepatic I/R. When HC‐specific (Alb‐TLR4−/−) and myeloid‐cell–specific (Lyz‐TLR4−/−) TLR4 knockout (KO) mice were subjected to warm hepatic ischemia, there was significant protection in these mice, compared to wild type (WT). However, the protection afforded in these two strains was significantly less than global TLR4 KO (TLR4−/−) mice. DC‐specific TLR4−/− (CD11c‐TLR4−/−) mice had significantly increased hepatocellular damage, compared to WT mice. Circulating levels of high‐mobility group box 1 (HMGB1) were significantly reduced in Alb‐TLR4−/− mice, compared to WT, Lyz‐TLR4−/−, CD11c‐TLR4−/− mice and equivalent to global TLR4−/− mice, suggesting that TLR4‐mediated HMGB1 release from HCs may be a source of HMGB1 after I/R. HCs exposed to hypoxia responded by rapidly phosphorylating the mitogen‐activated protein kinases, c‐Jun‐N‐terminal kinase (JNK) and p38, in a TLR4‐dependent manner; inhibition of JNK decreased release of HMGB1 after both hypoxia in vitro and I/R in vivo. Conclusion: These results provide insight into the individual cellular response of TLR4. The parenchymal HC is an active participant in sterile inflammatory response after I/R through TLR4‐mediated activation of proinflammatory signaling and release of danger signals, such as HMGB1. (HEPATOLOGY 2013)

IL-27 Production and STAT3-Dependent Upregulation of B7-H1 Mediate Immune Regulatory Functions of Liver Plasmacytoid Dendritic Cells

Plasmacytoid dendritic cells (pDCs) are highly specialized APCs that, in addition to their well-recognized role in anti-viral immunity, also regulate immune responses. Liver-resident pDCs are considerably less immunostimulatory than those from secondary lymphoid tissues and are equipped to promote immune tolerance/regulation through various mechanisms. IL-27 is an IL-12 family cytokine that regulates the function of both APCs and T cells, although little is known about its role in pDC immunobiology. In this study, we show that mouse liver pDCs express higher levels of IL-27p28 and EBV-induced protein 3 (Ebi3) compared with those of splenic pDCs. Both populations of pDCs express the IL-27Rα/WSX-1; however, only liver pDCs significantly upregulate expression of the coregulatory molecule B7 homolog-1 (B7-H1) in response to IL-27. Inhibition of STAT3 activation completely abrogates IL-27–induced upregulation of B7-H1 expression on liver pDCs. Liver pDCs treated with IL-27 increase the percentage of CD4+Foxp3+ T cells in MLR, which is dependent upon expression of B7-H1. pDCs from Ebi3-deficient mice lacking functional IL-27 show increased capacity to stimulate allogeneic T cell proliferation and IFN-γ production in MLR. Liver but not spleen pDCs suppress delayed-type hypersensitivity responses to OVA, an effect that is lost with Ebi3−/− and B7-H1−/− liver pDCs compared with wild-type liver pDCs. These data suggest that IL-27 signaling in pDCs promotes their immunoregulatory function and that IL-27 produced by pDCs contributes to their capacity to regulate immune responses in vitro and in vivo.

Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction

Mammalian target of rapamycin (mTOR) is an important, yet poorly understood integrative kinase that regulates immune cell function. mTOR functions in 2 independent complexes: mTOR complex (mTORC) 1 and 2. The immunosuppressant rapamycin (RAPA) inhibits mTORC1 but not mTORC2 and causes a paradoxical reduction in anti-inflammatory interleukin (IL) 10 and B7-homolog 1 (B7-H1) expression by dendritic cells (DCs). Using catalytic mTOR inhibitors and DCs lacking mTORC2, we show that restraint of signal transducer and activator of transcription 3-mediated IL-10 and B7-H1 expression during DC maturation involves a RAPA-insensitive and mTORC2-independent mTOR mechanism. Relatedly, catalytic mTOR inhibition promotes B7-H1-dependent and IL-1β-dependent DC induction of regulatory T cells (Tregs). Thus, we define an immunoregulatory pathway in which RAPA-sensitive mTORC1 in DCs promotes effector T-cell expansion and RAPA-insensitive mTORC1 restrains T(reg) induction. These findings identify the first known RAPA-insensitive mTOR pathway that is not mediated solely by mTORC2 and have implications for the use of catalytic mTOR inhibitors in inflammatory disease settings.

Histone deacetylase inhibition facilitates GM-CSF-mediated expansion of myeloid-derived suppressor cells in vitro and in vivo.

Chromatin‐modifying HDACi exhibit anti‐inflammatory properties that reflect their ability to suppress DC function and enhance regulatory T cells. The influence of HDACi on MDSCs, an emerging regulatory leukocyte population that potently inhibits T cell proliferation, has not been examined. Exposure of GM‐CSF‐stimulated murine BM cells to HDACi led to a robust expansion of monocytic MDSC (CD11b+Ly6C+F4/80intCD115+), which suppressed allogeneic T cell proliferation in a NOS‐ and HO‐1‐dependent manner with similar potency to control MDSCs. The increased yield of MDSCs correlated with blocked differentiation of BM cells and an overall increase in HSPCs (Lin–Sca‐1+c‐Kit+). In vivo, TSA enhanced the mobilization of splenic HSPCs following GM‐CSF administration and increased the number of CD11b+Gr1+ cells in BM and spleen. Increased numbers of Gr1+ cells, which suppressed T cell proliferation, were recovered from spleens of TSA‐treated mice. Overall, HDACi enhance MDSC expansion in vitro and in vivo, suggesting that acetylation regulates myeloid cell differentiation. These findings establish a clinically applicable approach to augment this rare and potent suppressive immune cell population and support a novel mechanism underlying the anti‐inflammatory action of HDACi.

Gut Bacteria Drive Kupffer Cell Expansion via MAMP-Mediated ICAM-1 Induction on Sinusoidal Endothelium and Influence Preservation-Reperfusion Injury after Orthotopic Liver Transplantation

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.