Immune Sentinels Research Articles

TOLL-LIKE RECEPTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Toll-like receptors belong to the pattern recognition receptors family present on a variety of immune cells including normal and malignant B-cells. They act as immediate molecular sentinels of innate immunity but also act as a molecular bridge between the innate and the adaptive immune response; distinct Toll-like receptors are able to bind specific pattern molecules of bacteria, viruses and autoantigens. In this review we will briefly introduce the Toll-like receptor family and their expression pattern, signaling and function in the B cell context; following we will summarize the published data on TLR in chronic lymphocytic leukemia, and we will discuss their emerging role in the modulation of leukemia pathobiology.

Immune surveillance in the central nervous system

The CNS, which consists of the brain and spinal cord, is continuously monitored by resident microglia and blood-borne immune cells such as macrophages, dendritic cells and T cells to detect for damaging agents that would disrupt homeostasis and optimal functioning of these vital organs. Further, the CNS must balance between vigilantly detecting for potentially harmful factors and resolving any immunological responses that in themselves can create damage if left unabated. We discuss the physiological roles of the immune sentinels that patrol the CNS, the molecular markers that underlie their surveillance duties, and the consequences of interrupting their functions following injury and infection by viruses such as JC virus, human immunodeficiency virus, herpes simplex virus and West Nile virus.

α-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a well-known mediator of skin pigmentation. More recently, it has been shown that α-MSH also exerts a strong anti-inflammatory and immunosuppressive activity. To elucidate the mechanisms underlying α-MSH-induced immunosuppression, we investigated whether α-MSH affects dendritic cell/T cell communication, as especially this interaction has an important role in the regulation of immune responses. Here, we show that α-MSH, by binding to MC-1R, induced tolerogenic dendritic cells, which were capable of expanding CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in vitro, as well as in vivo. Notably, those α-MSH-induced Tregs were functional as they efficiently inhibited cutaneous contact allergy and ongoing psoriasis-like skin inflammation in mice. Furthermore, α-MSH induced tolerogenic dendritic cells capable of generating functional Tregs in human blood. Interestingly, human Tregs expanded via α-MSH-stimulated dendritic cells suppressed the proliferation and cytokine secretion of pathogenic T-helper-17 (Th17) cells from individuals with psoriasis. Taken together, these data indicate that α-MSH induced immunosuppressive Tregs in vitro and in vivo, which inhibited disease progression in a mouse model of psoriasis-like skin inflammation and suppressed the activation and proliferation of effector T cells from subjects with psoriasis.

Immune response in melanoma: an in-depth analysis of the primary tumor and corresponding sentinel lymph node

AbstractThe sentinel lymph node is the initial site of metastasis. Downregulation of antitumor immunity has a role in nodal progression. Our objective was to investigate the relationship between immune modulation and sentinel lymph node positivity, correlating it with outcome in melanoma patients. Lymph node/primary tissues from melanoma patients prospectively accrued and followed at New York University Medical Center were evaluated for the presence of regulatory T cells (Foxp3+) and dendritic cells (conventional: CD11c+, mature: CD86+) using immunohistochemistry. Primary melanoma immune cell profiles from sentinel lymph node-positive/-negative patients were compared. Logistic regression models inclusive of standard-of-care/immunological primary tumor characteristics were constructed to predict the risk of sentinel lymph node positivity. Immunological responses in the positive sentinel lymph node were also compared with those in the negative non-sentinel node from the same nodal basin and matched negative sentinel lymph node. Decreased immune response was defined as increased regulatory T cells or decreased dendritic cells. Associations between the expression of these immune modulators, clinicopathological variables, and clinical outcome were evaluated using univariate/multivariate analyses. Primary tumor conventional dendritic cells and regression were protective against sentinel lymph node metastasis (odds ratio=0.714, 0.067; P=0.0099, 0.0816, respectively). Antitumor immunity was downregulated in the positive sentinel lymph node with an increase in regulatory T cells compared with the negative non-sentinel node from the same nodal basin (P=0.0005) and matched negative sentinel lymph node (P=0.0002). The positive sentinel lymph node also had decreased numbers of conventional dendritic cells compared with the negative sentinel lymph node (P<0.0001). Adding sentinel lymph node regulatory T cell expression improved the discriminative power of a recurrence risk assessment model using clinical stage. Primary tumor regression was associated with prolonged disease-free (P=0.025) and melanoma-specific (P=0.014) survival. Our results support an assessment of local immune profiles in both the primary tumor and sentinel lymph node to help guide therapeutic decisions.

Type I Interferon Regulates the Dynamics of Innate Myeloid Sentinel Cells During Persistent Viral Infection (169.7)

Abstract Viral infections of brain usually trigger inflammatory responses that give rise to a wide range of pathological outcomes. To gain novel insights into how the brain responds to a virus as it establishes persistence, we utilized genomic and two-photon imaging approaches to study a pure innate immune response to a lymphocytic choriomeningitis virus (LCMV) infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cells dynamics in the brain of CX3CR1-GFP mice lacking LCMV-specific T cells. Within the first two weeks of infection, brain resident myeloid cells were highly activated and responded directly by sequestering DsRed-labeled viral antigen. At the genomic level, this triggered massive changes in gene expression that were attenuated over time (likely due to inhibition by viral nucleoprotein) and were all directly linked to type I interferon (IFN-I) signaling. Surprisingly, in the absence of IFN-I signaling, no genes were differentially expressed in the nervous system despite the presence of persistent virus. In addition, two-photon imaging studies revealed that IFN-I receptor deficient innate immune sentinels were completely unresponsive to the establishment of a persistent viral infection. These data demonstrate that IFN-I drives all innate immune activity in the brain following LCMV infection, and deficiency in IFN-I signaling renders LCMV invisible in its murine host.

Resident CD141 (BDCA3)+ dendritic cells in human skin produce IL-10 and induce regulatory T cells that suppress skin inflammation

Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141(+) DDCs). CD141(+) DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D(3) (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141(+) DDCs from human blood DCs. These CD141(+) DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141(+) DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141(+) DDC-like cells have potential clinical use for their capacity to induce immune tolerance.

Non-canonical alternatives: What a macrophage is 4

Macrophages play pleiotropic, niche-specific roles in all tissues and organs. As immune sentinels, tissue macrophages regulate immune activation and inflammation; in turn, their function is modulated by inflammatory mediators deriving from such activation. Recent papers have established unanticipated roles for interleukin 4 and the alternative activation of tissue macrophages in the organismal response to diverse environmental stressors.

Are plasmacytoid dendritic cells the misguided sentinels of malarial immunity?

Dendritic cells (DCs), the sentinels of immunity, reside in almost every organ of the body. These cells are responsible for initiating immune responses against infectious agents. DCs are divided into different subsets based on their biological functions, with plasmacytoid DCs (pDCs) and conventional DCs (cDCs) being two major populations. The ability of DCs to protect against malaria infection was recently questioned when pDCs were reported to be a reservoir for rodent Plasmodium spp. in the spleen. This opinion article explores how the occupation of pDCs by the parasite may corrupt immunity against malaria.

Quantitative Measurements in 3-Dimensional Datasets of Mouse Lymph Nodes Resolve Organ-Wide Functional Dependencies

Deep tissue imaging has become state of the art in biology, but now the problem is to quantify spatial information in a global, organ-wide context. Although access to the raw data is no longer a limitation, the computational tools to extract biologically useful information out of these large data sets is still catching up. In many cases, to understand the mechanism behind a biological process, where molecules or cells interact with each other, it is mandatory to know their mutual positions. We illustrate this principle here with the immune system. Although the general functions of lymph nodes as immune sentinels are well described, many cellular and molecular details governing the interactions of lymphocytes and dendritic cells remain unclear to date and prevent an in-depth mechanistic understanding of the immune system. We imaged ex vivo lymph nodes isolated from both wild-type and transgenic mice lacking key factors for dendritic cell positioning and used software written in MATLAB to determine the spatial distances between the dendritic cells and the internal high endothelial vascular network. This allowed us to quantify the spatial localization of the dendritic cells in the lymph node, which is a critical parameter determining the effectiveness of an adaptive immune response.

사리장 처리에 의한 수지상세포의 성숙 유도

Dendritic cells (DCs) are professional antigen-presenting cells playing key roles in immune sentinels as initiators of T-cell responses against microbial pathogens and tumors. Sarijang, a folk sauce containing extracts of Rhynchosia nulubilis, Ulmus davidiana roots, Allium sativum, and Rhus Verniaiflura bark, has been used as a nonspecific immunostimulant for cancer patients. However, little is known about its immunomodulating effects or their mechanisms. In this study, we investigated whether sarijang induces phenotypic and functional maturation of DCs. For this study, murine bone marrow-derived myeloid DCs were cultured in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF), and the generated immature DCs were stimulated with sarijang or lipopolysaccharide (LPS). Our data indicated that sarijang significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) as well as major histocompatibility complex (MHC) II, as did LPS. The results provide new insight into the immunopharmacology of sarijang and suggest a novel approach to the manipulation of DC for therapeutic application.

ESAT-6 induced COX-2 expression involves coordinated interplay between PI3K and MAPK signaling

Macrophages, as sentinels of robust host immunity, are key regulators of innate immune responses against invading mycobacteria; however, pathogenic mycobacteria survive in the infected host by subverting host innate immunity. Infection dependent expression of early secreted antigenic target protein 6 (ESAT-6) by Mycobacterium tuberculosis is strongly correlated with subversion of innate immune responses against invading mycobacteria. As a part of multifaceted immunity to mycobacterial infection, induced expression of cyclooxygenase-2 (COX-2) may act as an important influencing factor towards effective host immunity. In the current investigation, we demonstrate that ESAT-6 triggers COX-2 expression both in vitro and in vivo in a TLR2 dependent manner. Signaling perturbation data suggest that signaling dynamics of PI3K and p38 and JNK1/2 MAPK assume critical importance in ESAT-6 triggered expression of COX-2 in macrophages. Interestingly, ESAT-6 triggered PI3K-MAPK signaling axis holds the capacity to regulate coordinated activation of NF-κB and AP-1. Overall, current investigation provides mechanistic insights into ESAT-6 induced COX-2 expression and unravels TLR2 mediated interplay of PI3K and MAPK signaling axis as a rate-determining step during intricate host immune responses. These findings would serve as a paradigm to understand pathogenesis of mycobacterial infection and clearly pave a way towards development of novel therapeutics.

The Role of Sugars in Dendritic Cell Trafficking

Dendritic cells (DCs) are crucial components of the immune response, strategically positioned as immune sentinels. Complex trafficking and accurate positioning of DCs are indispensable for both immunity and tolerance. This is particularly evident for their therapeutic application where an unmet clinical need exists for DCs with improved migratory capacity upon adoptive transfer into patients. One critical step that directs the trafficking of DCs throughout the body is their egress from the vasculature, starting with their adhesive interactions with vascular endothelium under shear flow. Both tethering and rolling rely on interactions mediated by specific glycans attached to glycoproteins and glycolipids present on the DC surface. In DCs, surface glycosylation, including the expression of selectin ligands, changes significantly depending on the local microenvironment and the functional state of the cells. These changes have been documented and have potential implications in important cell functions such as migration. In this article, we review the glycobiological aspects in the context of DC interaction with endothelium, and offer insights on how it can be applied to modulate DC applicability in therapy.

The anti-microbial peptide LL-37 modulates immune responses in the palatine tonsils where it is exclusively expressed by neutrophils and a subset of dendritic cells

Antimicrobial peptides are essential elements of epithelial defense against invading micro-organisms. The palatine tonsils are positioned at the entry of the airway and the gut and as such are ideally situated to act as immune sentinels in the pharynx protecting against microbial invasion. Tonsils express a number of antimicrobial peptides including hCAP18/LL-37. Here we clearly define the expression of hCAP18/LL-37 in the tonsils showing unequivocally that hCAP18/LL-37 is mainly expressed by infiltrating neutrophils and follicular CD11c+CD13+HLA-DR+ dendritic cells, rarely by macrophages, and never by the epithelium itself. To explore possible functions for follicle-derived LL-37, we stimulated tonsil mononuclear cells with LL-37 in vitro and observed the secretion of the proinflammatory cytokines CCL5 and CXCL9, expression of IFN-γ and MX-1 and down-regulation of chemokine receptors CCR4 and CCR6 which are involved in tissue-selective T cell trafficking. Taken together, these data illustrate new potential immunoregulatory functions for hCAP18/LL-37 in the tonsils.

Kidney Dendritic Cells Induce Innate Immunity against Bacterial Pyelonephritis

Dendritic cells (DCs) are the most abundant immune cells in the kidney and form an intricate network in the tubulointerstitium, suggesting that they may play an important role in interstitial infections such as pyelonephritis. Here, we optimized a murine pyelonephritis model by instilling uropathogenic Escherichia coli two times at a 3-hour interval, which produced an infection rate of 84%. By 3 hours after the second instillation, resident kidney DCs began secreting the chemokine CXCL2, which recruits neutrophilic granulocytes. During the time studied, DCs remained responsible for most of the CXCL2 production. Neutrophils began infiltrating the kidney 3 hours after the second instillation and phagocytozed bacteria. Macrophages followed 3 hours later and contributed much less to both CXCL2 production and bacterial phagocytosis. To investigate whether DCs recruit neutrophils into the kidney for antibacterial defense, we used CD11c.DTR mice allowing conditional depletion of CD11c(+) dendritic cells. The absence of CD11c(+) DCs markedly delayed neutrophil recruitment and bacterial clearance. In conclusion, these findings suggest that the tubulointerstitial dendritic cell network serves an innate immune sentinel function against bacterial pyelonephritis.

Impact of immune modulation on sentinel lymph node positivity and outcome in melanoma patients.

8561 Background: The interplay between melanoma and the immune system is multifactorial. Immune modulation is a key factor in melanoma metastasis, but its impact on patient management and potential prognostic relevance at the time of primary melanoma diagnosis are not well studied. We investigated the relationship between immune modulation and sentinel lymph node (SLN) positivity and correlated it with outcome in melanoma patients. Methods: We examined T regulatory cells (Tregs: Foxp3+), myeloid dendritic cells (myDCs: CD11c+), and mature dendritic cells (maDCs: CD86+) in lymph node (LN) and primary tissues from 84 melanoma patients prospectively accrued and followed up at New York University Medical Center using immunohistochemistry to detect Foxp3, CD11c, and CD86. The immune response in positive SLN biopsies (SLN+) was compared to that in negative SLN biopsies (SLN-) from patients matched for age, sex, primary tumor thickness/ulceration and negative LNs (LN-) from the same nodal basins of SLN+ patients. Immune cells were quantified as the absolute number in a representative HPF. Decreased immune response was defined as increased Tregs, decreased myDCs, or decreased maDCs. Associations between the expression of these immune modulators, clinicopathologic variables, and outcome were evaluated using univariate and multivariate analyses. Results: Anti-tumor immune response was downregulated both in the SLN+ (n=31) compared to the SLN- (n=53) with significant increase in Tregs (p=0.0002) and decrease in myDCs (p=0.00002) and in the 31 SLN+ compared to the LN- from the same basin with significant increase in Tregs (p=0.0005). Logistic regression models revealed that myDCs and maDCs in the primary tissue were associated with SLN status (p=0.001, 0.01, respectively). Adding Treg expression in the SLN as a predictor variable for recurrence improved the odds ratio in the logistic regression model for stage III from 5.99 to 10.42. Conclusions: Immune response is progressively downregulated with increased tumor involvement of the LNs. Our data support immunosuppression in the SLN as a predictor of worse outcome and suggest that evaluation of local immune profiles can guide the selection of therapy in the adjuvant setting.

Denditic cells and regulatory T cells: development, crosstalk and consequences (152.31)

Abstract Dendritic cells (DCs) play a major role of immune sentinels of our body. These cells capture, process and presents both foreign and self antigens to lymphocytes that activate immune responses or maintain immune tolerance. Despite a growing interest, their behavior remains mysterious. We have characterized in vivo at the cellular and molecular level, their development, their homeostasis and their intercellular communication with natural regulatory T cells (Tregs). We had previously found that Tregs were essential for maintaining normal numbers of dendritic cells (DCs): when Tregs are depleted, there is a compensatory Flt3-dependent increase in DCs. We now show that Treg homeostasis is maintained in vivo by the existence of a feedback regulatory loop between DCs and Tregs. We find that loss of DCs leads to a loss of Tregs, and that the remaining Tregs exhibit decreased Foxp3 expression. The DC-dependent loss in Tregs leads to an increase in the number of T cells producing inflammatory cytokines, such as interferon gamma and interleukin 17. Conversely, increasing the number of DCs leads to increased Treg division and accumulation by a mechanism that requires major histocompatibility complex II expression on DCs. The increase in Tregs induced by DC expansion is sufficient to prevent type 1 autoimmune diabetes and IBD, which suggests that interference with this feedback loop will create new opportunities for immune-based therapies.

Dynamics of an innate myeloid cell response to acute CNS viral infection (162.16)

Abstract Viral infections of central nervous system (CNS) often trigger an inflammatory response that gives rise to a wide range of pathological outcomes. The CNS is equipped with an elaborate network of innate immune sentinels that routinely serve as first responders to these infections. The meninges and perivascular spaces are inhabited by specialized macrophages, and the brain parenchyma contains microglia - the most abundant CNS myeloid cell. To gain novel insights into how myeloid cells respond to an acute CNS viral infection, we utilized genomics and two-photon imaging approaches to study a pure innate response to LCMV infection. This was accomplished by monitoring temporal gene expression patterns and myeloid cell dynamics in the brains of CX3CR1-GFP mice lacking LCMV-specific T cells. Two-photon imaging studies revealed that CNS myeloid sentinels responded significantly to acute infection by sequestering DsRed-labeled virus. Microglia but not peripheral myeloid cells proliferated following acute infection and responded directly to viral antigen by reducing process complexity, suggesting a localized response to the presence of virus. However, at the genomic level, acute infection triggered massive changes in CNS gene expression with the majority of genes being linked to innate viral control and the type I interferon system. These data suggest that localized detection of virus by CNS myeloid sentinels can translate into engagement of an extensive anti-viral program.

Disparate Antiviral Responses in Molluscum contagiosum Virus–Induced Skin Lesions

Molluscum contagiosum virus (MCV) is a poxvirus that causes tumor-like skin lesions. New evidence indicates that plasmacytoid dendritic cells, type I interferon production, and interferon-induced dendritic cells have prominent roles in anti-MCV responses, and these features characterize the inflammatory response in lesions that will likely undergo spontaneous regression.

Construction and Evaluation of a Combined Cyclophosphamide/Nanoparticle Anticancer Vaccine

Tumor immunotherapy is a rapidly emerging form of cancer treatment. In the current study, a nanoparticle-based vaccine was constructed and the efficacy was assessed through analysis of immune cell populations, tumor growth rates, and metastasis. The vaccine was fabricated through encapsulation of plasmid DNA encoding the tumor-associated antigen Mage-b, and the TLR9 agonist CpG oligodeoxynucleotides by a biodegradable polymer, poly(L,D-lactic-coglycolic acid) (PLGA). The size and shape of the nanoparticles suggested that they were an appropriate size for uptake by professional antigen presenting cells; dendritic cells. Furthermore, effects of the immunopotentiating drug cyclo-phosphamide was included to decrease systemic populations of regulatory T cells (Treg); immune system sentinels that down-regulate immune responses. The vaccine was assessed using the 4T1 murine mammary carcinoma model which is a model for stage IV breast cancer. The combined cyclophosphamide/nanoparticle vaccine was shown to significantly reduce 4T1 tumor growth rates and lung metastasis in female BALB/c mice.

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation

Mast cells are tissue-resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis. In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR(-/-) and control mast cells were engrafted into mast cell-deficient WBB6 F1-Kit(w) /Kit(W-v) (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR(-/-) animals and in murine and human mast cell cultures. Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR(-/-) mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR(-/-) mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin-8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR(-/-) mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis. Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a-modulated expression of FcγR.