GENETIC ABLATION OF THE C-TYPE LECTIN RECEPTOR CLEC2D INCREASES PERITONITIS MORTALITY, INFLAMMATION AND PHYSIOLOGY WITHOUT DIMINISHING ORGAN INJURY.

BACKGROUND: Sepsis accounts for substantial morbidity and mortality motivating investigators to continue the search for pathways and molecules driving the pathogenesis of the disease. The current study examined if the novel C-type Lectin Receptor (CLR), Clec2d, plays a significant role in the pathogenesis of sepsis. METHODS: Clec2d knockout (KO) mice were fully backcrossed onto the C57\BL6 background. Acute endotoxemia was induced with an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Sepsis was induced in two different models, Cecal Ligation and Puncture (CLP) and Pseudomonas aeruginosa pneumonia. Both models were treated with antibiotics and fluid resuscitation. In the sepsis models, physiologic and hematologic measurements were measured at 24 hours by collecting a small sample of peripheral blood. Mortality was followed for 14 days. RESULTS: A total of 197 mice were studied, 58 wild type (WT) and 54 knock-out (KO) in the LPS model; 27 wild type and 21 KO mice in the CLP model; and 22 WT and 15 KO mice in the pneumonia model. Clec2d KO mice had greater mortality in the LPS and CLP studies but not the pneumonia model. There were significant differences in multiple parameters determined 24 hours post sepsis between mice who would subsequently died and those lived. Consistent with previous reports in the CLP model, higher concentrations of IL-6, increased numbers of peripheral blood lymphocytes and greater renal injury were found in the dying mice. In contrast, in the pneumonia model IL-6 was higher in the surviving mice, however, the IL-6 levels in the pneumonia model (0.6 ± 0.3 ng/ml mean ± SEM) were less than 2% of the IL-6 levels of mice that died in the CLP model (41 ± 9 ng/ml, mean ± SEM). There were no differences in the lymphocyte count or renal injury between living and dying mice in the pneumonia model. In both sepsis models dying mice had lower heart rates, respiratory rates, and body temperatures. These values were also lower in the KO mice compared to the WT in CLP, but the breath rate and body temperature were increased in the KO pneumonia mice. CONCLUSION: The C-type lectin receptor Clec2d plays a complicated role in the pathogenesis of sepsis which varies with source of infection as demonstrated in the models used to study the disease. These data highlight the heterogeneity of the responses to sepsis and provide further evidence that a single common pathway driving sepsis organ injury and death likely does not exist.

Immune Sensing of Cell Death through Recognition of Histone Sequences by C-Type Lectin-Receptor-2d Causes Inflammation and Tissue Injury.

The immune system monitors the health of cells and is stimulated by necrosis. Here we examined the receptors and ligands driving this response. In a targeted screen of C-type lectin receptors, a Clec2d reporter responded to lysates from necrotic cells. Biochemical purification identified histones, both free and bound to nucleosomes or neutrophil extracellular traps, as Clec2d ligands. Clec2d recognized poly-basic sequences in histone tails and this recognition was sensitive to post-translational modifications of these sequences. As compared with WT mice, Clec2d-/- mice exhibited reduced proinflammatory responses to injected histones, and less tissue damage and improved survival in a hepatotoxic injury model. In macrophages, Clec2d localized to the plasma membrane and endosomes. Histone binding to Clec2d did not stimulate kinase activation or cytokine production. Rather, histone-bound DNA stimulated endosomal Tlr9-dependent responses in a Clec2d-dependent manner. Thus, Clec2d binds to histones released upon necrotic cell death, with functional consequences to inflammation and tissue damage.

Uric acid as a danger signal in gout and its comorbidities.

Uric acid is a waste product of purine catabolism. This molecule comes to clinical attention when it nucleates to form crystals of monosodium urate (MSU) in joints or other tissues, and thereby causes the inflammatory disease of gout. Patients with gout frequently suffer from a number of comorbid conditions including hypertension, diabetes mellitus and cardiovascular disease. Why MSU crystals trigger inflammation and are associated with comorbidities of gout has been unclear, but recent studies provide new insights into these issues. Rather than simply being a waste product, uric acid could serve a pathophysiological role as a local alarm signal that alerts the immune system to cell injury and helps to trigger both innate and adaptive immune responses. The inflammatory component of these immune responses is caused when urate crystals trigger both inflammasome-dependent and independent pathways to generate the proinflammatory cytokine IL-1. The resulting bioactive IL-1 stimulates the inflammation of gout and might contribute to the development of other comorbidities. Surprisingly, the same mechanisms underlie the inflammatory response to a number of irritant particles, many of which also cause disease. These new insights help to explain the pathogenesis of gout and point to potential new therapeutic targets for this and other sterile inflammatory diseases.

Targeting thymic epithelia AR enhances T-cell reconstitution and bone marrow transplant grafting efficacy.

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

Androgen receptor influences on body defense system via modulation of innate and adaptive immune systems: lessons from conditional AR knockout mice.

Upon insult, such as infection or tissue injury, the innate and adaptive immune systems initiate a series of responses to defend the body. Recent studies from immune cell-specific androgen receptor (AR) knockout mice demonstrated that androgen and its receptor (androgen/AR) play significant roles in both immune regulations. In the innate immunity, androgen/AR is required for generation and proper function of neutrophils; androgen/AR also regulates wound healing processes through macrophage recruitment and proinflammatory cytokine production. In adaptive immunity, androgen/AR exerts suppressive effects on development and activation of T and B cells. Removal of such suppression causes thymic enlargement and excessive export of immature B cells. Altogether, androgen/AR plays distinct roles in individual immune cells, and targeting androgen/AR may help in treatment and management of immune-related diseases.

The role of androgen and androgen receptor in skin-related disorders.

Androgen and androgen receptor (AR) may play important roles in several skin-related diseases, such as androgenetic alopecia and acne vulgaris. Current treatments for these androgen/AR-involved diseases, which target the synthesis of androgens or prevent its binding to AR, can cause significant adverse side effects. Based on the recent studies using AR knockout mice, it has been suggested that AR and androgens play distinct roles in the skin pathogenesis, and AR seems to be a better target than androgens for the treatment of these skin diseases. Here, we review recent studies of androgen/AR roles in several skin-related disorders, including acne vulgaris, androgenetic alopecia and hirsutism, as well as cutaneous wound healing.

Innate and adaptive immune responses to cell death.

The immune system plays an essential role in protecting the host against infections and to accomplish this task has evolved mechanisms to recognize microbes and destroy them. In addition, it monitors the health of cells and responds to ones that have been injured and killed, even if this occurs under sterile conditions. This process is initiated when dying cells expose intracellular molecules that can be recognized by cells of the innate immune system. As a consequence of this recognition, dendritic cells are activated in ways that help to promote T-cell responses to antigens associated with the dying cells. In addition, macrophages are stimulated to produce the cytokine interleukin-1 that then acts on radioresistant parenchymal cells in the host in ways that drive a robust inflammatory response. In addition to dead cells, a number of other sterile particles and altered physiological states can similarly stimulate an inflammatory response and do so through common pathways involving the inflammasome and interleukin-1. These pathways underlie the pathogenesis of a number of diseases.

Monocyte/macrophage androgen receptor suppresses cutaneous wound healing in mice by enhancing local TNF-alpha expression.

Cutaneous wounds heal more slowly in elderly males than in elderly females, suggesting a role for sex hormones in the healing process. Indeed, androgen/androgen receptor (AR) signaling has been shown to inhibit cutaneous wound healing. AR is expressed in several cell types in healing skin, including keratinocytes, dermal fibroblasts, and infiltrating macrophages, but the exact role of androgen/AR signaling in these different cell types remains unclear. To address this question, we generated and studied cutaneous wound healing in cell-specific AR knockout (ARKO) mice. General and myeloid-specific ARKO mice exhibited accelerated wound healing compared with WT mice, whereas keratinocyte- and fibroblast-specific ARKO mice did not. Importantly, the rate of wound healing in the general ARKO mice was dependent on AR and not serum androgen levels. Interestingly, although dispensable for wound closure, keratinocyte AR promoted re-epithelialization, while fibroblast AR suppressed it. Further analysis indicated that AR suppressed wound healing by enhancing the inflammatory response through a localized increase in TNF-alpha expression. Furthermore, AR enhanced local TNF-alpha expression via multiple mechanisms, including increasing the inflammatory monocyte population, enhancing monocyte chemotaxis by upregulating CCR2 expression, and enhancing TNF-alpha expression in macrophages. Finally, targeting AR by topical application of a compound (ASC-J9) that degrades AR protein resulted in accelerated healing, suggesting a potential new therapeutic approach that may lead to better treatment of wound healing.

Immune suppressive activity and lack of T helper differentiation are differentially regulated in natural regulatory T cells.

The mechanism for controlling Th cytokine expression in natural regulatory T (nTreg) cells is unclear. Here, it was found that under polarizing conditions Foxp3 did not affect Th1 cell, partially inhibited Th17 cell, but greatly inhibited Th2 cell differentiation of conventional CD4 T cells. Under the polarizing conditions, nTreg cells failed to differentiate into Th2 and Th17 cells, but differentiated into IFN-gamma-producing cells. Such Foxp3-transduced CD4 T cells and nTreg cells expressed T-bet, GATA-3, or retinoic acid-related orphan receptor (ROR)gammat, and retroviral GATA-3 and RORgammat could not induce Th2 and Th17 differentiation from nTreg cells. However, regardless of their cytokine profiles, the Foxp3-transduced CD4 T cells and nTreg cells remained immune suppressive. These results suggested that it is possible to convert pathogenic Th cells to Treg-like cells for therapeutic application. In conclusion, our studies show that Foxp3 is sufficient for immune suppression, whereas the inhibition of cytokine expression requires additional mechanisms.

Neutropenia with impaired host defense against microbial infection in mice lacking androgen receptor.

Neutrophils, the major phagocytes that form the first line of cell-mediated defense against microbial infection, are produced in the bone marrow and released into the circulation in response to granulocyte-colony stimulating factor (G-CSF). Here, we report that androgen receptor knockout (ARKO) mice are neutropenic and susceptible to acute bacterial infection, whereas castration only results in moderate neutrophil reduction in mice and humans. Androgen supplement can restore neutrophil counts via stabilizing AR in castrated mice, but not in ARKO and testicular feminization mutant (Tfm) mice. Our results show that deletion of the AR gene does not influence myeloid lineage commitment, but significantly reduces the proliferative activity of neutrophil precursors and retards neutrophil maturation. CXCR2-dependent migration is also decreased in ARKO neutrophils as compared with wild-type controls. G-CSF is unable to delay apoptosis in ARKO neutrophils, and ARKO mice show a poor granulopoietic response to exogenous G-CSF injection. In addition, AR can restore G-CSF-dependent granulocytic differentiation upon transduction into ARKO progenitors. We further found that AR augments G-CSF signaling by activating extracellular signal-regulated kinase 1/2 and also by sustaining Stat3 activity via diminishing the inhibitory binding of PIAS3 to Stat3. Collectively, our findings demonstrate an essential role for AR in granulopoiesis and host defense against microbial infection.

Susceptibility to autoimmunity and B cell resistance to apoptosis in mice lacking androgen receptor in B cells.

Estrogens have been linked to a higher female incidence of autoimmune diseases. The role of androgen and the androgen receptor (AR) in autoimmune diseases, however, remains unclear. Here we report that the lack of AR in B cells in different strains of mice, namely general AR knockout, B cell-specific AR knockout, and naturally occurring testicular feminization mutation AR-mutant mice, as well as castrated wild-type mice, results in increased B cells in blood and bone marrow. Analysis of the targeted mice, together with bone marrow transplantation using Rag1(-/-) recipients, overexpression of retrovirally encoded AR-cDNA, and small interfering RNA-mediated AR mRNA knockdown approaches also show that the B cell expansion results from resistance to apoptosis and increased proliferation of bone marrow precursor B cells, accompanied by changes in several key modulators related to apoptosis, such as Fas/FasL signals, caspases-3/-8, nuclear factor-kappaB, and Bcl-2. We also show that the effects of AR loss are, in part, B cell intrinsic. Mice bearing AR-deficient B cells show increased levels of serum IgG2a and IgG3 as well as basal double-stranded DNA-IgG antibodies and are more vulnerable to development of collagen-induced arthritis. Together, these data indicate that androgen/AR play a crucial role in B cell homeostasis and tolerance. Therapies targeting AR might provide an alternative strategy with which to battle autoimmune diseases.

Androgen receptor is a new potential therapeutic target for the treatment of hepatocellular carcinoma.

BACKGROUND & AIMS: Androgen effects on hepatocellular carcinoma (HCC) remain controversial and androgen ablation therapy to treat HCC also leads to inconsistent results. Here we examine androgen receptor (AR) roles in hepatocarcinogenesis using mice lacking AR in hepatocytes. METHODS: By using the Cre-Lox conditional knockout mice model injected with carcinogen, we examined the AR roles in hepatocarcinogenesis. We also tested the possible roles of AR in cellular oxidative stress and DNA damage sensing/repairing systems. By using AR degrading compound, ASC-J9, or AR-small interference RNA, we also examined the therapeutic potentials of targeting AR in HCC. RESULTS: We found AR expression was increased in human HCC compared with normal livers. We also found mice lacking hepatic AR developed later and less HCC than their wild-type littermates with comparable serum testosterone in both male and female mice. Addition of functional AR in human HCC cells also resulted in the promotion of cell growth in the absence or presence of 5alpha-dihydrotestosterone. Mechanistic dissection suggests that AR may promote hepatocarcinogenesis via increased cellular oxidative stress and DNA damage, as well as suppression of p53-mediated DNA damage sensing/repairing system and cell apoptosis. Targeting AR directly via either AR-small interference RNA or ASC-J9 resulted in suppression of HCC in both ex vivo cell lines and in vivo mice models. CONCLUSIONS: Our data point to AR, but not androgens, as a potential new and better therapeutic target for the battle of HCC.

Induction of costimulation of human CD4 T cells by tumor necrosis factor-related apoptosis-inducing ligand: possible role in T cell activation in systemic lupus erythematosus.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has recently been shown to induce costimulation of mouse T cells in conjunction with signals from the T cell receptor. This study was undertaken to investigate TRAIL-induced costimulation of human T cells in order to determine the role of TRAIL-induced T cell activation in human systemic lupus erythematosus (SLE). METHODS: An in vitro T cell stimulation system with immobilized anti-CD3 and recombinant TRAIL receptor DR4-Fc proteins was used to activate human T cells purified from healthy individuals and from patients with SLE. The T cells were stimulated in vitro to assay their proliferation response by (3)H-thymidine incorporation, and their cytokine production by enzyme-linked immunosorbent assay. Activation of p38 MAPK after TRAIL stimulation was detected with specific anti-phospho-p38 MAPK monoclonal antibodies in Western blots. RESULTS: Enhanced T cell proliferation and increased interleukin-2 and interferon-gamma (IFNgamma) production were demonstrated in human T cells after stimulation with immobilized DR4-Fc and anti-CD3 in vitro. TRAIL engagement selectively activated human CD4, rather than CD8, T cells and augmented IFNgamma production. Activation of p38 MAPK was detected after TRAIL-induced T cell activation. T cells isolated from patients with SLE demonstrated a stronger response to TRAIL-induced costimulation, in terms of proliferation and increased up-regulation of CD25 after activation, when compared with T cells from healthy subjects. CONCLUSION: TRAIL engagement induces costimulation of human CD4 T cells via a p38 MAPK-dependent pathway. The results suggest that enhanced reactivity of T cells to autoantigens as a result of TRAIL-induced costimulation may play a role in the development of human autoimmune diseases.