Publications

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective 1,2 . This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury 2,3 . Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context 4–12 . However, how neuro–immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the Na V 1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro–immune interactions has potential to treat non-healing tissues in which dysregulated neuro–immune interactions impair tissue healing.

Signal transducer and activator of transcription-3 (Stat3) is one of the most important molecules involved in the initiation of liver development and regeneration. In order to investigate the hepatoprotective effects of Stat3, we examined whether Stat3 protects against Fas-mediated liver injury in the mouse. A constitutively activated form of Stat3 (Stat3-C) was adenovirally overexpressed in mouse liver by intravenous injection, and then a nonlethal dose of Fas agonist (Jo2) was injected intraperitoneally into the mouse (0.3 μg/g body wt). Stat3-C dramatically suppressed both apoptosis and necrosis induced by Jo2. In contrast, liver-specific Stat3-knockout mice failed to survive following Jo2 injection. Stat3-C upregulated expression of FLICE inhibitor protein (FLIP), Bcl-XL, and Bcl-2, and accordingly downregulated activities of FLICE and caspase-3 that were redox-independent. Interestingly, Stat3-C also upregulated the redox-associated protein redox factor-1 (Ref-1) and reduced apoptosis in liver following Jo2 injection by suppressing oxidative stress and redox-sensitive caspase-3 activity. These findings indicate that Stat3 activation protects against Fas-mediated liver injury by inhibiting caspase activities in redox-dependent and -independent mechanisms.

Inflammation is counterbalanced by anti-inflammatory cytokines such as IL-10, in which Stat3 mediates the signaling pathway. In this study, we demonstrate that resident macrophages, but not other cell types, are important targets of IL-10 in a murine model of acute peritonitis. Injection of thioglycollate i.p. induced a considerable number of neutrophils and macrophages in the peritoneum, which was significantly augmented in mice with a cell-type specific disruption of the Stat3 gene in macrophages and neutrophils (LysMcre/Stat3flox/− mice). The augmented leukocyte infiltration was accompanied by increased peritoneal levels of TNF-α, MIP-2, KC chemokine (KC), and MCP-1/CCL2. Stat3 was tyrosine phosphorylated in peritoneal resident macrophages as well as infiltrating leukocytes in the littermate controls, suggesting that Stat3 in either or both of these cells might play a regulatory role in inflammation. The peritoneal levels of TNF-α, MIP-2, KC, and MCP-1 were similarly elevated in LysMcre/Stat3flox/− mice rendered leukopenic by cyclophosphamide treatment as compared with the controls. Adoptive transfer of resident macrophages from LysMcre/Stat3flox/− mice into the control littermates resulted in increases in the peritoneal level of TNF-α, MIP-2, KC, and MCP-1 after i.p. injection of thioglycollate. Under these conditions, control littermates harboring LysMcre/Stat3flox/− macrophages exhibited an augmented leukocyte infiltration relative to those received control macrophages. Taken together, these data provide evidence that resident macrophages, but not other cell types, play a regulatory role in inflammation through a Stat3 signaling pathway. Stat3 in resident macrophages appears to function as a repressor protein in this model of acute inflammation.

The ability of TLR agonists to promote adaptive immune responses is attributed to their ability to robustly activate innate immunity. However, it has been observed that, for adjuvants in actual use in research and vaccination, TLR signaling is dispensable for generating humoral immunity. Here, we examined the role of TLR5 and MyD88 in promoting innate and humoral immunity to flagellin using a prime/boost immunization regimen. We observed that eliminating TLR5 greatly reduced flagellin-induced cytokine production, except for IL-18, and ablated DC maturation but did not significantly impact flagellin's ability to promote humoral immunity. Elimination of MyD88, which will ablate signaling through TLR and IL-1beta/IL-18 generated by Nod-like receptors, reduced, but did not eliminate flagellin's promotion of humoral immunity. In contrast, loss of the innate immune receptor for profilin-like protein (PLP), TLR11, greatly reduced the ability of PLP to elicit humoral immunity. Together, these results indicate that, firstly, the degree of innate immune activation induced by TLR agonists may be in great excess of that needed to promote humoral immunity and, secondly, there is considerable redundancy in mechanisms that promote the humoral immune response upon innate immune recognition of flagellin. Thus, it should be possible to design innate immune activators that are highly effective vaccine adjuvants yet avoid the adverse events associated with systemic TLR activation.

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1 −/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4 −/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.

Leishmania species present unusual challenges to the immune system with their capacity to downregulate inflammatory responses as well as their ability to live within macrophages. Although toll-like receptor (TLR) pathways have been implicated in the recognition of several classes of pro-inflammatory microbes, it is not known if pathogens with anti-inflammatory properties activate the host response through this family of proteins. In this study, Leishmania major stimulation of cytokine promoter-luciferase reporter constructs was examined in transfected macrophages to detect early signs of cellular activation. L. major selectively activated the promoter region of IL-1α, but not IL-6, IL-8, IL-10, or an NF-κB reporter. IL-1α mRNA expression was also stimulated by L. major, although at lower levels than lipopolysacharide-stimulated macrophages. No IL-1α protein was detectable in stimulated cell lysates or culture supernatants. Transfection of macrophages with a dominant-negative version of myeloid differentiation factor 88 (MyD88), an adaptor protein which interacts with TLRs, inhibited activation of the IL-1α promoter. Furthermore, stimulation of IL-1α RNA expression by L. major was inhibited in peritoneal macrophages from MyD88–/– as compared to MyD88+/+ mice. These observations indicate that L. major stimulates IL-1α promoter activity and mRNA expression in macrophages through MyD88-dependent pathways. However, additional anti-inflammatory pathways must also be activated which downregulate transcription and ultimately inhibit translation of the IL-1α protein. Examination of promoter activation is a powerful tool for understanding the early events in macrophage activation for anti-inflammatory pathogens such as Leishmania that have mechanisms to downregulate transcription and translation.

Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E2 (PGE2) induces angiogenesis through the prostaglandin E2 receptor (EP1­–4). PGE2 is not only known to induce angiogenesis but also known to modulate function and differentiation of regulatory T cells (Tregs). We evaluated effect of Tregs on angiogenesis by using acute hind limb ischemia model.

Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF) is an adaptor molecule that is recruited to TLR3 and -4 upon agonist stimulation and triggers activation of IFN regulatory factor 3 (IRF3) and expression of type 1 IFNs, which are critical for cellular antiviral responses. We show that Akt is a downstream molecule of TRIF/TANK-binding kinase 1 (TBK1) and plays an important role in the activation of IRF3 by TLR3 and -4 agonists. Blockade of Akt by a dominant-negative mutant or by short interfering RNA decreased IRF3 activation and IFN-β expression induced by polyinosinic:polycytidylic acid [poly(I:C)], LPS, TRIF, and TBK1. Association of endogenous TBK1 and Akt was observed in macrophages when stimulated with poly(I:C) and LPS. In vitro kinase assays combined with reversed-phase liquid chromatography mass spectrometry analysis showed that TBK1 enhanced phosphorylation of Akt on Ser(473), whereas knockdown of TBK1 expression by short interfering RNA in macrophages decreased poly(I:C)- and LPS-induced Akt phosphorylation. Embryonic fibroblasts derived from TBK1 knockout mice also showed impaired Akt phosphorylation in response to poly(I:C) and LPS. To our knowledge, our results demonstrate a new regulatory mechanism for Akt activation mediated by TBK1 and a novel role of Akt in TLR-mediated immune responses.

Toll-like receptors recognize pathogen-associated molecular patterns, activate innate immunity, and consequently modulate adaptive immunity in response to infections. TLRs are also expressed on T cells, and it has been shown that T cell activation is modulated by TLR ligands. However, the functions of TLRs on Th1 and Th2 effector cells and the molecular mechanisms underlying TLR-mediated activation are not fully understood. We analyzed TLR functions and downstream signaling events in both effector T cells. In mouse Th1 cells the stimulation by TLR2 but not by other TLRs directly induced IFN-γ production, cell proliferation, and cell survival without TCR stimulation, and these effects were greatly enhanced by IL-2 or IL-12 through the enhanced activation of MAPKs. In contrast, no TLR affected the function of effector Th2 cells. These results identify TLR2 as a new specific activator of Th1 cell function and imply the involvement in Th1-mediated responses.

No abstract is provided for this article.

Recent results indicate a significant contribution of innate immune signaling to maintain mucosal homeostasis, but the precise underlying signal transduction pathways are ill-defined. By comparative analysis of intestinal epithelial cells isolated from conventionally raised and germ-free mice, as well as animals deficient in the adaptor molecules MyD88 and TRIF, the TLR3 and TLR4, as well as the type I and III IFN receptors, we demonstrate significant TLR-mediated signaling under homeostatic conditions. Surprisingly, homeostatic expression of Reg3γ and Paneth cell enteric antimicrobial peptides critically relied on TRIF and, in part, TLR3 but was independent of IFN receptor signaling. Reduced antimicrobial peptide expression was associated with significantly lower numbers of Paneth cells and a reduced Paneth cell maturation and differentiation factor expression in TRIF mutant compared with wild-type epithelium. This phenotype was not transferred to TRIF-sufficient germ-free animals during cohousing. Low antimicrobial peptide expression in TRIF-deficient mice caused reduced immediate killing of orally administered bacteria but was not associated with significant alterations in the overall composition of the enteric microbiota. The phenotype was rapidly restored in a TRIF-independent fashion after transient epithelial damage. Our results identify TRIF signaling as a truly homeostatic pathway to maintain intestinal epithelial barrier function revealing fundamental differences in the innate immune signaling between mucosal homeostasis and tissue repair.