Pruritogens in pemphigoid diseases: Possible therapeutic targets for a burdensome symptom.

Pruritus is a hallmark feature in pemphigoid diseases, where it can be severe and greatly impact the quality of life of affected patients. Despite being a key symptom, the exact pathophysiological mechanisms involved in pruritus in pemphigoid are yet to be fully elucidated and effective therapies addressing them are limited. This review summarizes the present understanding of pruritus specific to pemphigoid diseases, especially the pruritogens that induce it, and the therapeutic options that have been explored so far. The majority of the available evidence is on bullous pemphigoid and epidermolysis bullosa acquisita. Histamine derived from basophils correlates with pruritus severity, with omalizumab demonstrating promising efficacy in pruritus for bullous pemphigoid. IL-4/-13 contribute to itch in bullous pemphigoid with dupilumab being evaluated in clinical trials. Other pruritogens of interest include substance P, tryptase, and thymic stromal lymphopoetin, with therapies targeting them requiring further investigation. Scratching behaviors contribute directly to blister formation through various mechanisms, such as pathological autoantibody recruitment, T helper cell type 1 polarization, and exposure of intracellular autoantigens. Treatments addressing these pathways may contribute to decreasing disease severity. Additional studies are needed to fully characterize how pruritus is regulated in pemphigoid diseases, to help pave the way to develop novel and effective therapeutics that will not only address pruritic symptoms but also decrease disease severity.

Pathological functions of granzyme B in inflammatory skin diseases.

Dysregulated skin immunity is a hallmark of many skin diseases such as atopic dermatitis, autoimmune blistering diseases, and interface dermatitis. Current treatment options for the inflammatory skin diseases are limited and sometimes ineffective, therefore further understanding of pathomechanisms in the inflammatory skin conditions is necessary to develop new therapeutic alternatives. Recent studies suggest that the serine protease, granzyme B, is a key mediator in multiple inflammatory skin diseases, implying that strategies targeting granzyme B may be an attractive treatment option for such diseases. Specifically, granzyme B exhibits not only an intracellular apoptotic function but also extracellular proteolytic roles in inflammatory skin diseases including infectious diseases, pemphigoid diseases, atopic dermatitis, alopecia areata, and interface dermatitis. In this review, we summarize the current understanding with respect to the functions of granzyme B in the pathomechanism of various inflammatory skin diseases and evaluate the possibility of therapeutics targeting granzyme B.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis.

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 × 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function.

The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. Here, we show that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-κB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, our results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila.

Host and Bacterial Factors Control Susceptibility of Drosophila melanogaster to Coxiella burnetii Infection.

Coxiella burnetii is the causative agent of Q fever, a zoonotic disease that threatens both human and animal health. Due to the paucity of experimental animal models, little is known about how host factors interface with bacterial components and affect pathogenesis. Here, we used Drosophila melanogaster, in conjunction with the biosafety level 2 (BSL2) Nine Mile phase II (NMII) clone 4 strain of C. burnetii, as a model to investigate host and bacterial components implicated in infection. We demonstrate that adult Drosophila flies are susceptible to C. burnetii NMII infection and that this bacterial strain, which activates the immune deficiency (IMD) pathway, is able to replicate and cause mortality in the animals. We show that in the absence of Eiger, the only known tumor necrosis factor (TNF) superfamily homolog in Drosophila, Coxiella-infected flies exhibit reduced mortality from infection. We also demonstrate that the Coxiella type 4 secretion system (T4SS) is critical for the formation of the Coxiella-containing vacuole and establishment of infection in Drosophila Altogether, our data reveal that the Drosophila TNF homolog Eiger and the Coxiella T4SS are implicated in the pathogenesis of C. burnetii in flies. The Drosophila/NMII model mimics relevant aspects of the infection in mammals, such as a critical role of host TNF and the bacterial T4SS in pathogenesis. Our work also demonstrates the usefulness of this BSL2 model to investigate both host and Coxiella components implicated in infection.

Invertebrate Iridescent Virus 6, a DNA Virus, Stimulates a Mammalian Innate Immune Response through RIG-I-Like Receptors.

Insects are not only major vectors of mammalian viruses, but are also host to insect-restricted viruses that can potentially be transmitted to mammals. While mammalian innate immune responses to arboviruses are well studied, less is known about how mammalian cells respond to viruses that are restricted to infect only invertebrates. Here we demonstrate that IIV-6, a DNA virus of the family Iridoviridae, is able to induce a type I interferon-dependent antiviral immune response in mammalian cells. Although IIV-6 is a DNA virus, we demonstrate that the immune response activated during IIV-6 infection is mediated by the RIG-I-like receptor (RLR) pathway, and not the canonical DNA sensing pathway via cGAS/STING. We further show that RNA polymerase III is required for maximal IFN-ß secretion, suggesting that viral DNA is transcribed by this enzyme into an RNA species capable of activating the RLR pathway. Finally, we demonstrate that the RLR-driven mammalian innate immune response to IIV-6 is functionally capable of protecting cells from subsequent infection with the arboviruses Vesicular Stomatitis virus and Kunjin virus. These results represent a novel example of an invertebrate DNA virus activating a canonically RNA sensing pathway in the mammalian innate immune response, which reduces viral load of ensuing arboviral infection.