Transcription factor Nr4a1 couples sympathetic and inflammatory cues in CNS-recruited macrophages to limit neuroinflammation.

The molecular mechanisms that link the sympathetic stress response and inflammation remain obscure. Here we found that the transcription factor Nr4a1 regulated the production of norepinephrine (NE) in macrophages and thereby limited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Lack of Nr4a1 in myeloid cells led to enhanced NE production, accelerated infiltration of leukocytes into the central nervous system (CNS) and disease exacerbation in vivo. In contrast, myeloid-specific deletion of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, protected mice against EAE. Furthermore, we found that Nr4a1 repressed autocrine NE production in macrophages by recruiting the corepressor CoREST to the Th promoter. Our data reveal a new role for macrophages in neuroinflammation and identify Nr4a1 as a key regulator of catecholamine production by macrophages.

Pharmacologically Enhanced Regulatory Hematopoietic Stem Cells Revert Experimental Autoimmune Diabetes and Mitigate Other Autoimmune Disorders.

Type 1 diabetes (T1D) is characterized by the loss of immune self-tolerance, resulting in an aberrant immune responses against self-tissue. A few therapeutics have been partially successful in reverting or slowing down T1D progression in patients, and the infusion of autologous hematopoietic stem cells (HSCs) is emerging as an option to be explored. In this study, we proposed to pharmacologically enhance by ex vivo modulation with small molecules the immunoregulatory and trafficking properties of HSCs to provide a safer and more efficacious treatment option for patients with T1D and other autoimmune disorders. A high-throughput targeted RNA sequencing screening strategy was used to identify a combination of small molecules (16,16-dimethyl PGE2 and dexamethasone), which significantly upregulate key genes involved in trafficking (e.g., CXCR4) and immunoregulation (e.g., programmed death ligand 1). The pharmacologically enhanced, ex vivo-modulated HSCs (regulatory HSCs [HSC.Regs]) have strong trafficking properties to sites of inflammation in a mouse model of T1D, reverted autoimmune diabetes in NOD mice, and delayed experimental multiple sclerosis and rheumatoid arthritis in preclinical models. Mechanistically, HSC.Regs reduced lymphocytic infiltration of pancreatic ß cells and inhibited the activity of autoreactive T cells. Moreover, when tested in clinically relevant in vitro autoimmune assays, HSC.Regs abrogated the autoimmune response. Ex vivo pharmacological modulation enhances the immunoregulatory and trafficking properties of HSCs, thus generating HSC.Regs, which mitigated autoimmune diabetes and other autoimmune disorders.

Cardif (MAVS) Regulates the Maturation of NK Cells.

Cardif, also known as IPS-1, VISA, and MAVS, is an intracellular adaptor protein that functions downstream of the retinoic acid-inducible gene I family of pattern recognition receptors. Cardif is required for the production of type I IFNs and other inflammatory cytokines after retinoic acid-inducible gene I-like receptors recognize intracellular antigenic RNA. Studies have recently shown that Cardif may have other roles in the immune system in addition to its role in viral immunity. In this study, we find that the absence of Cardif alters normal NK cell development and maturation. Cardif(-/-) mice have a 35% loss of mature CD27(-)CD11b(+) NK cells in the periphery. In addition, Cardif(-/-) NK cells have altered surface marker expression, lower cytotoxicity, decreased intracellular STAT1 levels, increased apoptosis, and decreased proliferation compared with wild-type NK cells. Mixed chimeric mice revealed that the defective maturation and increased apoptotic rate of peripheral Cardif(-/-) NK cells is cell intrinsic. However, Cardif(-/-) mice showed enhanced control of mouse CMV (a DNA ß-herpesvirus) by NK cells, commensurate with increased activation and IFN-γ production by these immature NK cell subsets. These results indicate that the skewed differentiation and altered STAT expression of Cardif(-/-) NK cells can result in their hyperresponsiveness in some settings and support recent findings that Cardif-dependent signaling can regulate aspects of immune cell development and/or function distinct from its well-characterized role in mediating cell-intrinsic defense to RNA viruses.

Nonclassical patrolling monocyte function in the vasculature.

Nonclassical patrolling monocytes are characterized by their unique ability to actively patrol the vascular endothelium under homeostatic and inflammatory conditions. Patrolling monocyte subsets (CX3CR1(high)Ly6C(-) in mouse and CX3CR1(high)CD14(dim)CD16(+) in humans) are distinct from the classical monocyte subsets (CCR2(high)Ly6C(+) in mouse and CCR2(high)CD14(+)CD16(-) in humans) and exhibit unique functions in the vasculature and inflammatory disease. Patrolling monocytes function in several disease settings to remove damaged cells and debris from the vasculature and have been associated with wound healing and the resolution of inflammation in damaged tissues. This review highlights the unique functions of these patrolling monocytes in the vasculature and during inflammation.

Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species.

UNLABELLED: Impaired T-cell responses in chronic hepatitis C virus (HCV) patients have been reported to be associated with the establishment of HCV persistent infection. However, the mechanism for HCV-mediated T-cell dysfunction is yet to be defined. Myeloid-derived suppressor cells (MDSCs) play a pivotal role in suppressing T-cell responses. In this study we examined the accumulation of MDSCs in human peripheral blood mononuclear cells (PBMCs) following HCV infection. We found that CD33(+) mononuclear cells cocultured with HCV-infected hepatocytes, or with HCV core protein, suppress autologous T-cell responses. HCV core-treated CD33(+) cells exhibit a CD14(+) CD11b(+/low) HLADR(-/low) phenotype with up-regulated expression of p47(phox) , a component of the NOX2 complex critical for reactive oxygen species (ROS) production. In contrast, immunosuppressive factors, arginase-1 and inducible nitric oxide synthase (iNOS), were not up-regulated. Importantly, treatment with an inactivator of ROS reversed the T-cell suppressive function of HCV-induced MDSCs. Lastly, PBMCs of chronic HCV patients mirror CD33(+) cells following treatment with HCV core where CD33(+) cells are CD14(+) CD11b(+) HLADR(-/low) , and up-regulate the expression of p47(phox). CONCLUSION: These results suggest that HCV promotes the accumulation of CD33(+) MDSC, resulting in ROS-mediated suppression of T-cell responsiveness. Thus, the accumulation of MDSCs during HCV infection may facilitate and maintain HCV persistent infection.

Extracellular hepatitis C virus core protein activates STAT3 in human monocytes/macrophages/dendritic cells via an IL-6 autocrine pathway.

Hepatitis C virus (HCV) infection is highly efficient in the establishment of persistent infection, which leads to the development of chronic liver disease and hepatocellular carcinoma. Impaired T cell responses with reduced IFN-γ production have been reported to be associated with persistent HCV infection. Extracellular HCV core is a viral factor known to cause HCV-induced T cell impairment via its suppressive effect on the activation and induction of pro-inflammatory responses by antigen-presenting cells (APCs). The activation of STAT proteins has been reported to regulate the inflammatory responses and differentiation of APCs. To further characterize the molecular basis for the regulation of APC function by extracellular HCV core, we examined the ability of extracellular HCV core to activate STAT family members (STAT1, -2, -3, -5, and -6). In this study, we report the activation of STAT3 on human monocytes, macrophages, and dendritic cells following treatment with extracellular HCV core as well as treatment with a gC1qR agonistic monoclonal antibody. Importantly, HCV core-induced STAT3 activation is dependent on the activation of the PI3K/Akt pathway. In addition, the production of multifunctional cytokine IL-6 is essential for HCV core-induced STAT3 activation. These results suggest that HCV core-induced STAT3 activation plays a critical role in the alteration of inflammatory responses by APCs, leading to impaired anti-viral T cell responses during HCV infection.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

Role of complement in immune regulation and its exploitation by virus.

Complement is activated during the early phase of viral infection and promotes destruction of virus particles as well as the initiation of inflammatory responses. Recently, complement and complement receptors have been reported to play an important role in the regulation of innate as well as adaptive immune responses during infection. The regulation of host immune responses by complement involves modulation of dendritic cell activity in addition to direct effects on T-cell function. Intriguingly, many viruses encode homologs of complement regulatory molecules or proteins that interact with complement receptors on antigen-presenting cells and lymphocytes. The evolution of viral mechanisms to alter complement function may augment pathogen persistence and limit immune-mediated tissue destruction. These observations suggest that complement may play an important role in both innate and adaptive immune responses to infection as well as virus-mediated modulation of host immunity.

The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus.

Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1, and Nr4a1-deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset.

Patrolling monocytes control tumor metastasis to the lung.

The immune system plays an important role in regulating tumor growth and metastasis. Classical monocytes promote tumorigenesis and cancer metastasis, but how nonclassical "patrolling" monocytes (PMo) interact with tumors is unknown. Here we show that PMo are enriched in the microvasculature of the lung and reduce tumor metastasis to lung in multiple mouse metastatic tumor models. Nr4a1-deficient mice, which specifically lack PMo, showed increased lung metastasis in vivo. Transfer of Nr4a1-proficient PMo into Nr4a1-deficient mice prevented tumor invasion in the lung. PMo established early interactions with metastasizing tumor cells, scavenged tumor material from the lung vasculature, and promoted natural killer cell recruitment and activation. Thus, PMo contribute to cancer immunosurveillance and may be targets for cancer immunotherapy.

HCV+ hepatocytes induce human regulatory CD4+ T cells through the production of TGF-beta.

BACKGROUND: Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4(+) regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown. METHODOLOGY/PRINCIPAL FINDINGS: HCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4(+) T cells. The production of IFN-gamma was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4(+) T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV(+) hepatocytes upregulated the production of TGF-beta and blockade of TGF-beta abrogated Treg phenotype and function. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.

Liver is able to activate naïve CD8+ T cells with dysfunctional anti-viral activity in the murine system.

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8(+) T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8(+) T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8(+) T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8(+) T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8(+) T cell response and accounts for the dysfunctional CD8(+) T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection.

Activation of the RNA-dependent protein kinase PKR promoter in the absence of interferon is dependent upon Sp proteins.

The protein kinase regulated by RNA (PKR) is interferon (IFN)-inducible and plays important roles in many cellular processes, including virus multiplication, cell growth, and apoptosis. The TATA-less PKR promoter possesses a novel 15-bp DNA element (kinase conserved sequence (KCS)) unique to the human and mouse PKR genes that is conserved in sequence and position. We found that Sp1 and Sp3 of the Sp family of transcription factors bind at the KCS element. Their involvement was analyzed in the activation of basal and IFN-inducible PKR promoter activity. Both the small and large isoforms of Sp3 co-purified with KCS protein binding activity (KBP) by using nuclear extracts from HeLa cells not treated with IFN. Two forms of the KCS-binding protein complex were demonstrated by electrophoretic mobility shift assay analysis; one contained Sp1 and the other Sp3. In mouse cells null for all Sp3 isoforms, PKR expression was reduced to approximately 50% that of wild-type cells in the absence of IFN. The IFN-inducible expression of PKR, however, was Sp3-independent but STAT1- and JAK1-dependent. Overexpression of Sp1 in human U cells resulted in increased PKR promoter activity. In Drosophila SL2 cells lacking Sp proteins, both Sp1 and Sp3 large but not small isoforms activated PKR promoter expression, with the Sp1-mediated activation dominant. Mutational analysis of the PKR promoter region indicated a cooperative interaction between two different Sp sites, one of which is within the KCS element. These results establish that, in the absence of IFN treatment, activation of PKR basal expression is mediated by Sp1 and Sp3 proteins in a cooperative manner.