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Quantitative transcriptomics offers a new way to obtain a detailed picture of freshly isolated cells. By direct isolation, the cells are unaffected by in vitro culture, and the isolation at cold temperatures maintains the cells relatively unaltered in phenotype by avoiding activation through receptor cross-linking or plastic adherence. Simultaneous analysis of several cell types provides the opportunity to obtain detailed pictures of transcriptomic differences between them. Here, we present such an analysis focusing on four human blood cell populations and compare those to isolated human skin mast cells. Pure CD19⁺ peripheral blood B cells, CD14⁺ monocytes, and CD4⁺ and CD8⁺ T cells were obtained by fluorescence-activated cell sorting, and KIT+ human connective tissue mast cells (MCs) were purified by MACS sorting from healthy skin. Detailed information concerning expression levels of the different granule proteases, protease inhibitors, Fc receptors, other receptors, transcription factors, cell signaling components, cytoskeletal proteins, and many other protein families relevant to the functions of these cells were obtained and comprehensively discussed. The MC granule proteases were found exclusively in the MC samples, and the T-cell granzymes in the T cells, of which several were present in both CD4⁺ and CD8⁺ T cells. High levels of CD4 were also observed in MCs and monocytes. We found a large variation between the different cell populations in the expression of Fc receptors, as well as for lipid mediators, proteoglycan synthesis enzymes, cytokines, cytokine receptors, and transcription factors. This detailed quantitative comparative analysis of more than 780 proteins of importance for the function of these populations can now serve as a good reference material for research into how these entities shape the role of these cells in immunity and tissue homeostasis.

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

The extended cleavage specificities of two hematopoietic serine proteases originating from the ray-finned fish, the spotted gar (Lepisosteus oculatus), have been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated using a panel of recombinant substrates. For one of the enzymes, the gar granzyme G, a strict preference for the aromatic amino acid Tyr was observed at the cleavable P1 position. Using a set of recombinant substrates showed that the gar granzyme G had a high selectivity for Tyr but a lower activity for cleaving after Phe but not after Trp. Instead, the second enzyme, gar DDN1, showed a high preference for Leu in the P1 position of substrates. This latter enzyme also showed a high preference for Pro in the P2 position and Arg in both P4 and P5 positions. The selectivity for the two Arg residues in positions P4 and P5 suggests a highly specific substrate selectivity of this enzyme. The screening of the gar proteome with the consensus sequences obtained by substrate phage display for these two proteases resulted in a very diverse set of potential targets. Due to this diversity, a clear candidate for a specific immune function of these two enzymes cannot yet be identified. Antisera developed against the recombinant gar enzymes were used to study their tissue distribution. Tissue sections from juvenile fish showed the expression of both proteases in cells in Peyer's patch-like structures in the intestinal region, indicating they may be expressed in T or NK cells. However, due to the lack of antibodies to specific surface markers in the gar, it has not been possible to specify the exact cellular origin. A marked difference in abundance was observed for the two proteases where gar DDN1 was expressed at higher levels than gar granzyme G. However, both appear to be expressed in the same or similar cells, having a lymphocyte-like appearance.

Mast cells (MCs) are known to have a pathological impact in a variety of settings, in particular in allergic conditions. There is also limited evidence implicating MCs in diabetes, raising the possibility that MC function may be influenced by alterations in glucose levels. However, it is not known whether MCs are directly affected by elevated glucose concentrations. Moreover, it is not known which glucose transporters that are expressed by MCs, and whether MCs are dependent on glucose transporters for activation. Here we addressed these issues. We show that MCs express high levels of both glucose transporter 1 (GLUT1/Slc2A1) and GLUT3 (Slc2A3). Further, we show that the inhibition of either GLUT1 or GLUT3 dampens both MC degranulation and cytokine induction in response to IgE receptor crosslinking, and that combined GLUT1 and GLUT3 inhibition causes an even more pronounced inhibition of these parameters. In contrast, the inhibition of GLUT1 or GLUT3, or combined GLUT1 and GLUT3 inhibition, had less impact on the ability of the MCs to respond to activation via compound 48/80. Elevated glucose concentrations did not affect MC viability, and had no stimulatory effect on MC responses to either IgE receptor crosslinking or compound 48/80. Altogether, these findings reveal that MCs are strongly dependent on glucose transport via GLUT1 and/or GLUT3 for optimal responses towards IgE-mediated activation, whereas MC functionality is minimally affected by elevated glucose levels. Based on these findings, antagonists of GLUT1 and GLUT3 may be considered for therapeutic intervention in allergic conditions.

The extended cleavage specificity of catfish granzyme-like II has been characterized using substrate phage display. The preference for particular amino acids at and surrounding the cleavage site was further validated by using a panel of recombinant substrates. This serine protease, which has previously been isolated as cDNA from a catfish natural killer-like cell line showed a preference for Ala in the P1 position of the substrate, and for multiple basic amino acids N-terminally of the cleavage site. A closely related zebrafish serine protease (zebrafish esterase-like) showed a very similar cleavage specificity, indicating an evolutionary conservation of this protease specificity among various fish species. Two catfish serine proteases, originating from NK-like cells, have now been isolated and characterized. One of them is highly specific met-ase with similar characteristics as the mammalian granzyme M. This enzyme may be involved in the induction of apoptosis in virus-infected cells, with a potential target in (catfish) caspase 6. In contrast to catfish granzyme-like I, the second enzyme analyzed here does not seem to have a direct counterpart in mammalian NK cells, and its role in the immune function of catfish NK cells is, therefore, still not known. However, this enzyme seems to be able to cleave a number of cytoskeletal proteins, indicating a separate strategy to induce apoptosis in target cells. Both of these enzymes are very interesting targets for further studies of their roles in catfish immunity, as enzymes with similar specificities have also been identified in zebrafish.

Granzymes A and K are two highly homologous serine proteases expressed by mammalian cytotoxic T cells (CTL) and natural killer cells (NK). Granzyme A is the most abundant of the different granzymes (gzms) expressed by these two cell types. Gzms A and K are found in all jawed vertebrates and are the most well conserved of all hematopoietic serine proteases. Their potential functions have been studied extensively for many years, however, without clear conclusions. Gzm A was for many years thought to serve as a key component in the defense against viral infection by the induction of apoptosis in virus-infected cells, similar to gzm B. However, later studies have questioned this role and instead indicated that gzm A may act as a potent inducer of inflammatory cytokines and chemokines. Gzms A and K form clearly separate branches in a phylogenetic tree indicating separate functions. Transcriptional analyses presented here demonstrate the presence of gzm A and K transcripts in both CD4⁺ and CD8⁺ T cells. To enable screening for their primary biological targets we have made a detailed analysis of their extended cleavage specificities. Phage display analysis of the cleavage specificity of the recombinant enzymes showed that both gzms A and K are strict tryptases with high selectivity for Arg over Lys in the P1 position. The major differences in the specificities of these two enzymes are located N-terminally of the cleavage site, where gzm A prefers small amino acids such as Gly in the P3 position and shows a relatively relaxed selectivity in the P2 position. In contrast, gzm K prefers large amino acids such as Phe, Tyr, and Trp in both the P2 and P3 positions and does not tolerate negatively charged residues in the P2 position. This major distinction in extended specificities is likely reflected also in preferred in vivo targets of these two enzymes. This information can now be utilized for high-precision screening of primary targets for gzms A and K in search of their highly conserved but still poorly defined functions in vertebrate immunity.

Background. Asthma is a chronic inflammatory disease with structural changes in the lungs defined as airway remodelling. Mast cell responses are important in asthma as they, upon activation, release mediators inducing bronchoconstriction, inflammatory cell recruitment, and often remodelling of the airways. As guinea pigs exhibit anatomical, physiological, and pharmacological features resembling human airways, including mast cell distribution and mediator release, we evaluated the effect of extracts from two common allergens, house dust mite (HDM) and cat dander (CDE), on histopathological changes and the composition of tryptase- and chymase-positive mast cells in the guinea pig lungs. Methods. Guinea pigs were exposed intranasally to HDM or CDE for 4, 8, and 12 weeks, and airway histology was examined at each time point. Hematoxylin and eosin, Picro-Sirius Red, and Periodic Acid-Schiff staining were performed to evaluate airway inflammation, collagen deposition, and mucus-producing cells. In addition, Astra blue and immunostaining against tryptase and chymase were used to visualize mast cells. Results. Repetitive administration of HDM or CDE led to the accumulation of inflammatory cells into the proximal and distal airways as well as increased airway smooth muscle mass. HDM exposure caused subepithelial collagen deposition and mucus cell hyperplasia at all three time points, whereas CDE exposure only caused these effects at 8 and 12 weeks. Both HDM and CDE induced a substantial increase in mast cells after 8 and 12 weeks of challenges. This increase was primarily due to mast cells expressing tryptase, but not chymase, thus indicating mucosal mast cells. Conclusions. We here show that exposure to HDM and CDE elicits asthma-like histopathology in guinea pigs with infiltration of inflammatory cells, airway remodelling, and accumulation of primarily mucosal mast cells. The results together encourage the use of HDM and CDE allergens for the stimulation of a clinically relevant asthma model in guinea pigs.

Cows produce saliva in very large quantities to lubricate and facilitate food processing. Estimates indicate an amount of 50-150 L per day. Human saliva has previously been found to contain numerous antibacterial components, such as lysozyme, histatins, members of the S-100 family and lactoferrin, to limit pathogen colonization. Cows depend on a complex microbial community in their digestive system for food digestion. Our aim here was to analyze how this would influence the content of their saliva. We therefore sampled saliva from five humans and both nose secretions and saliva from six cows and separated the saliva on SDS-PAGE gradient gels and analyzed the major protein bands with LC-MS/MS. The cow saliva was found to be dominated by a few major proteins only, carbonic anhydrase 6, a pH-stabilizing enzyme and the short palate, lung and nasal epithelium carcinoma-associated protein 2A (SPLUNC2A), also named bovine salivary protein 30 kDa (BSP30) or BPIFA2B. This latter protein has been proposed to play a role in local antibacterial response by binding bacterial lipopolysaccharides (LPSs) and inhibiting bacterial growth but may instead, according to more recent data, primarily have surfactant activity. Numerous peptide fragments of mucin-5B were also detected in different regions of the gel in the MS analysis. Interestingly, no major band on gel was detected representing any of the antibacterial proteins, indicating that cows may produce them at very low levels that do not harm the microbial flora of their digestive system. The nose secretions of the cows primarily contained the odorant protein, a protein thought to be involved in enhancing the sense of smell of the olfactory receptors and the possibility of quickly sensing potential poisonous food components. High levels of secretory IgA were also found in one sample of cow mouth drippings, indicating a strong upregulation during an infection. The human saliva was more complex, containing secretory IgA, amylase, carbonic anhydrase 6, lysozyme, histatins and a number of other less abundant proteins, indicating a major difference to the saliva of cows that show very low levels of antibacterial components, most likely to not harm the microbial flora of the rumen.

Equine asthma (EA) is a heterogenous, complex disease, with a significant negative impact on horse welfare and performance. EA and human asthma share fundamental similarities, making EA a useful model for studying the disease. One relevant sample type for investigating chronic lung inflammation is bronchoalveolar lavage fluid (BALF), which provides a snapshot of the immune cells present in the alveolar space. To investigate the immune cell landscape of the respiratory tract in horses with mild-to-moderate equine asthma (mEA) and healthy controls, single-cell RNA sequencing was conducted on equine BALF cells. We characterized the major immune cell populations present in equine BALF, as well as subtypes thereof. Interestingly, the most significantly upregulated gene discovered in cases of mEA was FKBP5, a chaperone protein involved in regulating the activity of the glucocorticoid receptor.