System-wide immunohistochemical analysis of protein co-localization

Background

The analysis of co-localized protein expression in a tissue section is often conducted with immunofluorescence histochemical staining which is typically visualized in localized regions. On the other hand, chromogenic immunohistochemical staining, in general, is not suitable for the detection of protein co-localization. Here, we developed a new protocol, based on chromogenic immunohistochemical stain, for system-wide detection of protein co-localization and differential expression.

Methodology/Principal Findings

In combination with a removable chromogenic stain, an efficient antibody stripping method was developed to enable sequential immunostaining with different primary antibodies regardless of antibody''s host species. Sections were scanned after each staining, and the images were superimposed together for the detection of protein co-localization and differential expression. As a proof of principle, differential expression and co-localization of glutamic acid decarboxylase67 (GAD67) and parvalbumin proteins was examined in mouse cortex.

Conclusions/Significance

All parvalbumin-containing neurons express GAD67 protein, and GAD67-positive neurons that do not express parvalbumin were readily visualized from thousands of other neurons across mouse cortex. The method provided a global view of protein co-localization as well as differential expression across an entire tissue section. Repeated use of the same section could combine assessments of co-localization and differential expression of multiple proteins.     

CCR4 is a key modulator of innate immune responses

CCR4 is recognized as a key receptor in Th2-associated immune processes, although very little is known about its role in innate immunity. Previous studies reported increased resistance to LPS-induced lethality in CCR4(-/-) mice compared with wild-type mice. This study demonstrates that CCR4(-/-) mice are similarly resistant to challenge with other TLR agonists, as well as bacterial peritonitis. Resistance was associated with enhanced early leukocyte recruitment, increased TLR expression, a skewed type 2 cytokine/chemokine profile, and improved bacterial clearance. Macrophages from CCR4(-/-) mice exhibited many features consistent with alternative activation, including elevated secretion of type 2 cytokines/chemokines and the found in inflammatory zone 1 (FIZZ1) protein. MyD88-dependent NF-kappaB signaling was significantly down-regulated in CCR4(-/-) macrophages, whereas p38 MAPK and JNK activation were conversely increased. These data stress the importance of CCR4 in macrophage differentiation and innate immune responses to pathogens, as well as the involvement of chemokine receptor expression in TLR signaling regulation.     

SHARPIN is a key regulator of immune and inflammatory responses

Mice with spontaneous mutations in the Sharpin gene develop chronic proliferative dermatitis that is characterized by eosinophilic inflammation of the skin and other organs with increased expression of type 2 cytokines and dysregulated development of lymphoid tissues. The mutant mice share phenotypic features with human hypereosinophilic syndromes. The biological function of SHARPIN and how its absence leads to such a complex inflammatory phenotype in mice are poorly understood. However, recent studies identified SHARPIN as a novel modulator of immune and inflammatory responses. The emerging mechanistic model suggests that SHARPIN functions as an important adaptor component of the linear ubiquitin chain assembly complex that modulates activation of NF-κB signalling pathway, thereby regulating cell survival and apoptosis, cytokine production and development of lymphoid tissues. In this review, we will summarize the current understanding of the ubiquitin-dependent regulatory mechanisms involved in NF-κB signalling, and incorporate the recently obtained molecular insights of SHARPIN into this pathway. Recent studies identified SHARPIN as an inhibitor of β1-integrin activation and signalling, and this may be another mechanism by which SHARPIN regulates inflammation. Furthermore, the disrupted lymphoid organogenesis in SHARPIN-deficient mice suggests that SHARPIN-mediated NF-κB regulation is important for de novo development of lymphoid tissues.     

System-wide mapping of peptide-GPCR interactions in C. elegans

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Bacterial glycans: key mediators of diverse host immune responses

Recent studies have shown that the synthesis of various polysaccharides by bacteria can induce immune responses that are beneficial to the bacterium, the host, or both. Here, we discuss the diverse interactions between bacterial glycans and the host immune system.     

Regulation of murine IgE responses: induction of long-lived inhibition of allergen-specific responses is genetically restricted.

We previously reported that administration of high Mr glutaraldehyde-polymerized ovalbumin (OA-POL) 10 days prior to OA Al(OH)3 immunization results in 85 to 99% inhibition of primary and secondary anti-OA IgE responses and 10(2)- to 10(4)-fold increases in OA-specific IgG2a production in each of 14 inbred and 1 outbred murine strains tested. Administration of unmodified OA under the same conditions fails to inhibit IgE synthesis and yields only minor increases in IgG2a production. In the present report, the genetic restrictions placed on the capacity of this modified allergen to elicit long-lived reciprocal regulation of specific IgE and IgG2a responses were examined. Virtually permanent, antigen-specific inhibition of IgE production (greater than 90% for greater than 22 months) was elicited in C57B1/6 mice following administration of a single course of OA-POL. This inhibition was paralleled by substantial (250-fold) increases in specific IgG2a production and was dependent on the activity of extremely long-lived regulatory CD4 T cells. In contrast, BALB/c mice failed to maintain an IgE unresponsive state beyond 10-12 weeks and exhibited transient and less intense increases in IgG2a production. Examination of MHC and Igh congenic strains revealed that the induction of long-term split tolerance by these modified allergens is under multigenic control and is not solely attributable to MHC, Igh, or background genes.     

Meiotic maturation: receptor trafficking is the key

Meiotic maturation and ovulation rates in Caenorhabditis elegans are regulated by a sperm-released gradient of major sperm protein (MSP). Recent work has provided insights into the modulation of the MSP signal by the trafficking of its receptor in oocytes.     

Context is key: A comment on Herczeg et al. 2019

In the last several years, there has been a surge in the number of studies addressing the causes and consequences of among‐individual variation in cognitive ability and behavioural plasticity. Here, we use a recent publication by Herczeg et al. (2019: 32(3), 218–226) to highlight three shortcomings common to this newly emerging field. In their study, Herczeg et al. attempted to link variation in cognitive ability and behavioural plasticity by testing whether selection lines of guppies (Poecilia reticulata) that differ in relative brain size also differ in behavioural plasticity, as might be expected if the costs to plasticity are predominantly derived from the cost of developing large brains. First, residual brain size may not be a suitable proxy for ‘cognitive ability’. Recent work has shown that intraspecific variation in cognitive ability can be better understood by considering variation in the specific brain areas responsible for the relevant behaviours as opposed to whole‐brain mass. Second, the measure of behavioural plasticity, habituation, is unlikely to fulfil the assumptions that plasticity is both adaptive and costly. Finally, we point out several misconceptions regarding animal personality that continue to contribute to the choice of traits that are not well aligned with study objectives. Elucidating the mechanisms underlying among‐individual variation in cognition and behavioural plasticity within populations requires integration between behavioural ecology and comparative cognition, and the study system developed by Herczeg et al. has the potential to provide important mechanistic insights. We hope that by articulating and critically appraising the underlying assumptions that are common in these traditionally separate disciplines, a strong foundation can emerge to allow for more fruitful integration of these fields.     

Human memory: The hippocampus is the key

A recent study of brain-damaged patients with various degrees of amnesia provides compelling new evidence that the hippocampus plays a vital role in the laying down of new memories. Why existing memories are also affected by hippocampal damage is particularly puzzling.     

The cell pushes back: The Arp2/3 complex is a key orchestrator of cellular responses to environmental forces

The Arp2/3 complex orchestrates the formation of branched actin networks at the interface between the cytoplasm and membranes. Although it is widely appreciated that these networks are useful for scaffolding, creating pushing forces and delineating zones at the membrane interface, it has only recently come to light that branched actin networks are mechanosensitive, giving them special properties. Here, we discuss recent advances in our understanding of how Arp2/3-generated actin networks respond to load forces and thus allow cells to create pushing forces in responsive and tuneable ways to effect cellular processes such as migration, invasion, phagocytosis, adhesion and even nuclear and DNA damage repair.     

Terrestrial insects and climate change: adaptive responses in key traits

Understanding and predicting how adaptation will contribute to species' resilience to climate change will be paramount to successfully managing biodiversity for conservation, agriculture, and human health‐related purposes. Making predictions that capture how species will respond to climate change requires an understanding of how key traits and environmental drivers interact to shape fitness in a changing world. Current trait‐based models suggest that low‐ to mid‐latitude populations will be most at risk, although these models focus on upper thermal limits, which may not be the most important trait driving species' distributions and fitness under climate change. In this review, we discuss how different traits (stress, fitness and phenology) might contribute and interact to shape insect responses to climate change. We examine the potential for adaptive genetic and plastic responses in these key traits and show that, although there is evidence of range shifts and trait changes, explicit consideration of what underpins these changes, be that genetic or plastic responses, is largely missing. Despite little empirical evidence for adaptive shifts, incorporating adaptation into models of climate change resilience is essential for predicting how species will respond under climate change. We are making some headway, although more data are needed, especially from taxonomic groups outside of Drosophila, and across diverse geographical regions. Climate change responses are likely to be complex, and such complexity will be difficult to capture in laboratory experiments. Moving towards well designed field experiments would allow us to not only capture this complexity, but also study more diverse species.     

Bacterial-epithelial contact is a key determinant of host innate immune responses to enteropathogenic and enteroaggregative Escherichia coli

Background

Enteropathogenic (EPEC) and Enteroaggregative (EAEC) E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s) remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains.

Methodology

Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS) negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence.

Results

Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti-inflammatory response.

Conclusion

Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbe''s ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis.     

Intrinsic radiation sensitivity: cellular signaling is the key

The concept that the balance between DNA damage and repair determines intrinsic radiation sensitivity has dominated radiobiology for several decades. There is undeniably a cause- effect relationship between radiation-induced molecular alterations in the genomic DNA and cellular consequences. In the last decade, however, it has become obvious that the chromatin context affects the fate of damaged DNA and that cellular signaling is an important factor in defining intrinsic radiation sensitivity. Damaged DNA is the site of signal generation; however, alternative signaling at the plasma membrane is triggered: Reactive oxygen species (ROS) inactivate phosphatases and consequently cause activation of kinases localized at the plasma membrane; this includes ligand-independent activation of receptor kinases. Cells with an apparently functional DNA repair system may show increased radiation sensitivity due to deficiencies in specific kinases essential for repair activation and checkpoint control. Other signals that determine intrinsic radiosensitivity may affect proneness to apoptosis, the balance between DNA damage fixation and repair, and the translocation of proteins participating in the response to ionizing radiation. Interplay between the various signals decides the extent to which the repair of radiation-inflicted damage is supported or limited; in some cell types, this includes DNA-damage-independent processes guided by plasma membrane-generated signaling. Cellular signaling in the context of specific subcellular structures is the key to understanding how the molecular effects of radiation are expressed as biological consequences in various cell types. A systems approach should bring us closer to this end.