Salmonella enterica Serovar Typhimurium Induces NAIP/NLRC4- and NLRP3/ASC-Independent, Caspase-4-Dependent Inflammasome Activation in Human Intestinal Epithelial Cells.

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes diseases ranging from gastroenteritis to systemic infection and sepsis. Salmonella uses type III secretion systems (T3SS) to inject effectors into host cells. While these effectors are necessary for bacterial invasion and intracellular survival, intracellular delivery of T3SS products also enables detection of translocated Salmonella ligands by cytosolic immune sensors. Some of these sensors form multimeric complexes called inflammasomes, which activate caspases that lead to interleukin-1 (IL-1) family cytokine release and pyroptosis. In particular, the Salmonella T3SS needle, inner rod, and flagellin proteins activate the NAIP/NLRC4 inflammasome in murine intestinal epithelial cells (IECs), which leads to restriction of bacterial replication and extrusion of infected IECs into the intestinal lumen, thereby preventing systemic dissemination of Salmonella. While these processes are quite well studied in mice, the role of the NAIP/NLRC4 inflammasome in human IECs remains unknown. Unexpectedly, we found the NAIP/NLRC4 inflammasome is dispensable for early inflammasome responses to Salmonella in both human IEC lines and enteroids. Additionally, NLRP3 and the adaptor protein ASC are not required for inflammasome activation in Caco-2 cells. Instead, we observed a necessity for caspase-4 and gasdermin D pore-forming activity in mediating inflammasome responses to Salmonella in Caco-2 cells. These findings suggest that unlike murine IECs, human IECs do not rely on NAIP/NLRC4 or NLRP3/ASC inflammasomes and instead primarily use caspase-4 to mediate inflammasome responses to Salmonella pathogenicity island 1 (SPI-1)-expressing Salmonella.

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches.

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3' to P6' was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Laser-facilitated epicutaneous immunotherapy with hypoallergenic beta-glucan neoglycoconjugates suppresses lung inflammation and avoids local side effects in a mouse model of allergic asthma.

BACKGROUND: Allergen-specific immunotherapy via the skin targets a tissue rich in antigen-presenting cells, but can be associated with local and systemic side effects. Allergen-polysaccharide neoglycogonjugates increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects. OBJECTIVE: We investigated the immunogenicity, allergenicity, and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA). METHODS: The biological activity of LamOVA was characterized in vitro using bone marrow-derived dendritic cells. Immunogenicity and therapeutic efficacy were analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin, and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T-cell polarization were assessed. RESULTS: Conjugation of laminarin to ovalbumin reduced its IgE binding capacity fivefold and increased its immunogenicity threefold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with higher levels of therapy-induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA. CONCLUSION: Conjugation of ovalbumin to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser-generated micropores is safe and equally effective compared to SCIT with alum, without the need for adjuvant.

IL-31 transgenic mice show reduced allergen-induced lung inflammation.

Interleukin-31 (IL-31) is a Th2 cell-derived cytokine that has been closely linked to pruritic skin inflammation. More recently, enhanced IL-31 serum levels have also been observed in patients with allergic rhinitis and allergic asthma. Therefore, the main aim of this study was to unravel the contribution of IL-31 to allergen-induced lung inflammation. We analyzed lung inflammation in response to the timothy grass (Phleum pratense) pollen allergen Phl p 5 in C57BL/6 wild-type (wt) mice, IL-31 transgenic (IL-31tg) mice, and IL-31 receptor alpha-deficient animals (IL-31RA-/- ). IL-31 and IL-31RA levels were monitored by qRT-PCR. Cellular infiltrate in bronchoalveolar lavage fluid (BALF) and lung tissue inflammation, mucus production as well as epithelial thickness were measured by flow cytometry and histomorphology. While allergen challenge induced IL-31RA expression in lung tissue of wt and IL-31tg mice, high IL-31 expression was exclusively observed in lung tissue of IL-31tg mice. Upon Phl p 5 challenge, IL-31tg mice showed reduced numbers of leukocytes and eosinophils in BALF and lung tissue as well as diminished mucin expression and less pronounced epithelial thickening compared to IL-31RA-/- or wt animals. These findings suggest that the IL-31/IL-31RA axis may regulate local, allergen-induced inflammation in the lungs.

The NAIP/NLRC4 inflammasome in infection and pathology.

In this review we give an overview of the NAIP/NLRC4 activation mechanism as well as the described roles of this inflammasome, with a focus on in vivo infection and pathology. After ligand recognition by NAIP sensor proteins the NAIP/NLRC4 inflammasome forms through oligomerization with the NLRC4 adaptor to activate Caspase-1. The activating ligands are intracellular bacterial flagellin or type-3 secretion system components, delivered by pathogens. In vivo experiments indicate a role in macrophages during lung, spleen and liver infection and systemic sepsis like conditions, as well as in intestinal epithelial cells. Upon NAIP/NLRC4 activation in the intestine, epithelial cell extrusion is triggered in addition to the canonical inflammasome outcomes of cytokine cleavage and pyroptosis. Human patients with auto-activating mutations in NLRC4 present with an autoinflammatory syndrome including enterocolitis. Although one of the better understood inflammasomes in terms of mechanism, tissue specific functions of NAIP/NLRC4 are only beginning to be understood.

Nitrated food proteins induce a regulatory immune response associated with allergy prevention after oral exposure in a Balb/c mouse food allergy model.

BACKGROUND: Food allergy is associated with a high personal health and economic burden. For immunomodulation toward tolerance, food compounds could be chemically modified, for example, by posttranslational protein nitration, which also occurs via diet-derived nitrating agents in the gastrointestinal tract. OBJECTIVE: We sought to analyze the effect of pretreatment with nitrated food proteins on the immune response in a mouse food allergy model and on human monocyte-derived dendritic cells (moDCs) and PBMCs. METHODS: The model allergen ovalbumin (OVA) was nitrated in different nitration degrees, and the secondary structures of proteins were determined by circular dichroism (CD). Allergy-preventive treatment with OVA, nitrated OVA (nOVA), and maximally nitrated OVA (nOVAmax) were performed before mice were immunized with or without gastric acid-suppression medication. Antibody levels, regulatory T-cell (Treg) numbers, and cytokine levels were evaluated. Human moDCs or PBMCs were incubated with proteins and evaluated for expression of surface markers, cytokine production, and proliferation of Th2 as well as Tregs. RESULTS: In contrast to OVA and nOVA, the conformation of nOVAmax was substantially changed. nOVAmax pretreated mice had decreased IgE as well as IgG1 and IgG2a levels and Treg numbers were significantly elevated, while cytokine levels remained at baseline level. nOVAmax induced a regulatory DC phenotype evidenced by a decrease of the activation marker CD86 and an increase in IL-10 production and was associated with a higher proliferation of memory Tregs. CONCLUSION: Oral pretreatment with highly nitrated proteins induces a tolerogenic immune response in the food allergy model and in human immune cells.

Why we haven't died out yet: changes in women's mimic reactions to visual erotic stimuli during their menstrual cycles.

From an evolutionary point of view, female sexual desire contributes greatly to the success of reproduction by coordinating sexual behavior. It is known that female sexual desire fluctuates with the menstrual cycle. However, little is known about the role of basic emotions during menstrual cycle. We designed a facial EMG study to investigate facial expressions of joy during the menstrual cycle. 35 healthy women underwent 2 EMG sessions (T1 and T2). T1 took place in the follicular phase, T2 in the luteal phase. IAPS pictures of nude men (erotic stimuli) or of animals (control stimuli) were presented at both sessions. The activity of musculus zygomaticus major (responsible for expressing joy) was measured. We tested the hypothesis that zygomaticus activity is more pronounced in the follicular phase than in the luteal phase. The main result was that during the follicular phase, significantly more zygomaticus reactions were observed than during the luteal phase. This effect was restricted only to erotic stimuli. We concluded that an increased positive emotional responsiveness to erotic stimuli during the follicular phase is an important precondition for the probability of sexual activity during the conceptive days and thus for the success of reproduction.