Functional In Vivo Imaging of Macrophages.

Resident tissue macrophages (RTMs) are specialized phagocytes that are widely distributed throughout the body and are responsible for maintaining homeostasis. Recent advances in experimental techniques have enabled us to gain a greater insight into the actual in vivo biology of RTMs by observing their spatiotemporal dynamics directly in their native environment. Here, we detail a method for live tracking macrophages in a prototypical stromal tissue with high spatial and temporal resolution and great experimental versatility. Our approach builds on a custom intravital imaging platform and straightforward surgical preparation to gain access to an intact stromal compartment in order to analyze the morphological and behavioral dynamics of RTMs at single-cell resolution before and after experimental intervention. Furthermore, our versatile approach can also be utilized for live visualization of intracellular signaling and even for tracking cell organelles at subcellular resolution, and can be combined with downstream analyses such as multiplex confocal imaging, providing a unique insight into macrophage biology in vivo.

Stunning of neutrophils accounts for the anti-inflammatory effects of clodronate liposomes.

Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.

The Mechanism of Action of Antigen Processing Independent T Cell Epitopes Designed for Immunotherapy of Autoimmune Diseases.

Immunotherapy with antigen-processing independent T cell epitopes (apitopes) targeting autoreactive CD4+ T cells has translated to the clinic and been shown to modulate progression of both Graves' disease and multiple sclerosis. The model apitope (Ac1-9[4Y]) renders antigen-specific T cells anergic while repeated administration induces both Tr1 and Foxp3+ regulatory cells. Here we address why CD4+ T cell epitopes should be designed as apitopes to induce tolerance and define the antigen presenting cells that they target in vivo. Furthermore, we reveal the impact of treatment with apitopes on CD4+ T cell signaling, the generation of IL-10-secreting regulatory cells and the systemic migration of these cells. Taken together these findings reveal how apitopes induce tolerance and thereby mediate antigen-specific immunotherapy of autoimmune diseases.

Death in the sauna-vitality markers for heat exposure.

In sauna-associated deaths, the vitality of heat exposure is of great importance. Two case reports address this. First, we present the case of a 77-year-old man who was found dead in the sauna of his family home. When found, the sauna door was closed, and the sauna indicated a temperature of 78 °C. The body had already begun to decay and was partially mummified when it was found. In the other case, a 73-year-old woman was found dead in the sauna by her husband. In this case, the sauna door was also closed. The sauna was still in operation at a temperature of approximately 70 °C. Epidermal detachments were found. In both autopsies and their follow-up examinations, there were no indications of a cause of death competing with heat shock. The expression of heat shock proteins in kidneys and lungs and the expression of aquaporin 3 in skin were investigated to detect pre-mortal temperature influences.

Fire exposure after lethal hypothermia.

A 49-year-old woman was found dead in her apartment after a fire began in her building. During the forensic autopsy, 3rd to 4th degree burns were found on the woman's body, but there were no indications that she was alive when the fire started. Interestingly, hemorrhagic gastric mucosa erosions, as well as bloody contents in the esophagus, stomach, and intestines, were observed. However, the source of the bleeding could not be found. The cause of death was therefore determined to be hypothermia with postmortem fire exposure. The cause of the hypothermia could possibly have been high internal blood loss. The organs showed early signs of putrefaction. It was theorized that the woman had not died immediately before the fire began, but rather a few days before. Examination of heat shock proteins (HSPs) to evaluate premortem thermal influences did not reveal HSP 27, 60, or 70 expression in renal tissue, possibly because of the putrefaction. However, Sudan staining of this tissue revealed a fatty degeneration of renal tubular cells. Opposing temperature influences, as in this case, are rather rare and require thorough investigations.

Natural cardiac death after stent implantation with iatrogenic injury of a coronary artery.

In forensic practice, autopsies are regularly carried out in cases of suspected medical malpractice to determine whether a treatment resulted in death. Intraoperative deaths, as well as deaths shortly after an operation, can be particularly suspicious as iatrogenic. We report a case of a 75-year-old woman with a complaint of intermittent angina pectoris who underwent cardiac catheterization. Intra-interventionally, coronary artery dissection occurred and was stabilized by the placement of two stents. After this procedure, the patient suffered from chest pain. At 5.5 h after the procedure ended, the woman suddenly and unexpectedly died. At forensic autopsy, a hemopericardium with cardiac tamponade was found to have been caused by the rupture of a myocardial infarction that was several days old and had remained clinically unrecognized. This case report illustrates the importance of forensic autopsies in terms of external quality assurance in medicine.

Chimeric antigen receptor T cells for the treatment of cancer and the future of preclinical models for predicting their toxicities.

Chimeric antigen receptor T-cell therapy has achieved highly promising results in clinical trials, particularly in B-cell malignancies. However, reports of serious adverse events including a number of patient deaths have raised concerns about safety of this treatment. Presently available preclinical models are not designed for predicting toxicities seen in human patients. Besides choosing the right animal model, careful considerations must be taken in chimeric antigen receptor T-cell design and the amount of T cells infused. The development of more sophisticated in vitro models and humanized mouse models for preclinical modeling and toxicity tests will help us to improve the design of clinical trials in cancer immunotherapy.

Myeloid-derived suppressor cells mediate tolerance induction in autoimmune disease.

In multiple sclerosis (MS) T cells aberrantly recognize self-peptides of the myelin sheath and attack the central nervous system (CNS). Antigen-specific peptide immunotherapy, which aims to restore tolerance while avoiding the use of non-specific immunosuppressive drugs, is a promising approach to combat autoimmune disease, but the cellular mechanisms behind successful therapy remain poorly understood. Myeloid-derived suppressor cells (MDSCs) have been studied intensively in the field of cancer and to a lesser extent in autoimmunity. Because of their suppressive effect on the immune system in cancer, we hypothesized that the development of MDSCs and their interaction with CD4+ T cells could be beneficial for antigen-specific immunotherapy. Hence, changes in the quantity, phenotype and function of MDSCs during tolerance induction in our model of MS were evaluated. We reveal, for the first time, an involvement of a subset of MDSCs, known as polymorphonuclear (PMN)-MDSCs, in the process of tolerance induction. PMN-MDSCs were shown to adopt a more suppressive phenotype during peptide immunotherapy and inhibit CD4+ T-cell proliferation in a cell-contact-dependent manner, mediated by arginase-1. Moreover, increased numbers of tolerogenic PMN-MDSCs, such as observed over the course of peptide immunotherapy, were demonstrated to provide protection from disease in a model of experimental autoimmune encephalomyelitis.

Impact of peripheral myeloid cells on amyloid-ß pathology in Alzheimer's disease-like mice.

Although central nervous system-resident microglia are believed to be ineffective at phagocytosing and clearing amyloid-ß (Aß), a major pathological hallmark of Alzheimer's disease (AD), it has been suggested that peripheral myeloid cells constitute a heterogeneous cell population with greater Aß-clearing capabilities. Here, we demonstrate that the conditional ablation of resident microglia in CD11b-HSVTK (TK) mice is followed by a rapid repopulation of the brain by peripherally derived myeloid cells. We used this system to directly assess the ability of peripheral macrophages to reduce Aß plaque pathology and therefore depleted and replaced the pool of resident microglia with peripherally derived myeloid cells in Aß-carrying APPPS1 mice crossed to TK mice (APPPS1;TK). Despite a nearly complete exchange of resident microglia with peripheral myeloid cells, there was no significant change in Aß burden or APP processing in APPPS1;TK mice. Importantly, however, newly recruited peripheral myeloid cells failed to cluster around Aß deposits. Even additional anti-Aß antibody treatment aimed at engaging myeloid cells with amyloid plaques neither directed peripherally derived myeloid cells to amyloid plaques nor altered Aß burden. These data demonstrate that mere recruitment of peripheral myeloid cells to the brain is insufficient in substantially clearing Aß burden and suggest that specific additional triggers appear to be required to exploit the full potential of myeloid cell-based therapies for AD.

Extra-thymically induced T regulatory cell subsets: the optimal target for antigen-specific immunotherapy.

Antigen-specific immunotherapy aims to selectively restore tolerance to innocuous antigens in cases of autoimmune or allergic disease, without the need for general immune suppression. Although the principle of antigen-specific immunotherapy was discovered more than a century ago, its clinical application to date is limited, particularly in the control of autoimmunity. This has resulted mainly from a lack of in-depth understanding of the underlying mechanism. More recently, the differentiation of extra-thymically induced T regulatory (Treg) cell subsets has been shown to be instrumental in peripheral tolerance induction. Two main types of inducible Treg cells, interleukin-10-secreting or Foxp3(+) , have now been described, each with distinct characteristics and methods of therapeutic induction. It is crucial, therefore, to identify the suitability of either subset in the control of specific immune disorders. This review explores their natural function, the known mechanisms of therapeutic differentiation of either subset as well as their in vivo functionality and discusses new developments that may aid their use in antigen-specific immunotherapy, with a focus on autoimmune disease.

Inhibition of IL-12/IL-23 signaling reduces Alzheimer's disease-like pathology and cognitive decline.

The pathology of Alzheimer's disease has an inflammatory component that is characterized by upregulation of proinflammatory cytokines, particularly in response to amyloid-ß (Aß). Using the APPPS1 Alzheimer's disease mouse model, we found increased production of the common interleukin-12 (IL-12) and IL-23 subunit p40 by microglia. Genetic ablation of the IL-12/IL-23 signaling molecules p40, p35 or p19, in which deficiency of p40 or its receptor complex had the strongest effect, resulted in decreased cerebral amyloid load. Although deletion of IL-12/IL-23 signaling from the radiation-resistant glial compartment of the brain was most efficient in mitigating cerebral amyloidosis, peripheral administration of a neutralizing p40-specific antibody likewise resulted in a reduction of cerebral amyloid load in APPPS1 mice. Furthermore, intracerebroventricular delivery of antibodies to p40 significantly reduced the concentration of soluble Aß species and reversed cognitive deficits in aged APPPS1 mice. The concentration of p40 was also increased in the cerebrospinal fluid of subjects with Alzheimer's disease, which suggests that inhibition of the IL-12/IL-23 pathway may attenuate Alzheimer's disease pathology and cognitive deficits.

Neuroprotective function for ramified microglia in hippocampal excitotoxicity.

BACKGROUND: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration. METHODS: Mouse organotypic hippocampal slice cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia. RESULTS: Treatment of mouse organotypic hippocampal slice cultures with 10-50 µM N-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA. CONCLUSIONS: Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.