MMP-9 cleaves SP-D and abrogates its innate immune functions in vitro

Possession of a properly functioning innate immune system in the lung is vital to prevent infections due to the ongoing exposure of the lung to pathogens. While mechanisms of pulmonary innate immunity have been well studied, our knowledge of how these systems are altered in disease states, leading to increased susceptibility to infections, is limited. One innate immune protein in the lung, the pulmonary collectin SP-D, has been shown to be important in innate immune defense, as well as clearance of allergens and apoptotic cells. MMP-9 is a protease with a wide variety of substrates, and has been found to be dysregulated in a myriad of lung diseases ranging from asthma to cystic fibrosis; in many of these conditions, there are decreased levels of SP-D. Our results indicate that MMP-9 is able to cleave SP-D in vitro and this cleavage leads to loss of its innate immune functions, including its abilities to aggregate bacteria and increase phagocytosis by mouse alveolar macrophages. However, MMP-9-cleaved SP-D was still detected in a solid-phase E. coli LPS-binding assay, while NE-cleaved SP-D was not. In addition, MMP-9 seems to cleave SP-D much more efficiently than NE at physiological levels of calcium. Previous studies have shown that in several diseases, including cystic fibrosis and asthma, patients have increased expression of MMP-9 in the lungs as well as decreased levels of intact SP-D. As patients suffering from many of the diseases in which MMP-9 is over-expressed can be more susceptible to pulmonary infections, it is possible that MMP-9 cleavage of SP-D may contribute to this phenotype

Small angle x-ray scattering with edge-illumination

Sensitivity to sub-pixel sample features has been demonstrated as a valuable capability of phase contrast x-ray imaging. Here, we report on a method to obtain angular-resolved small angle x-ray scattering distributions with edge-illumination- based imaging utilizing incoherent illumination from an x-ray tube. Our approach provides both the three established image modalities (absorption, differential phase and scatter strength), plus a number of additional contrasts related to unresolved sample features. The complementarity of these contrasts is experimentally validated by using different materials in powder form. As a significant application example we show that the extended complementary contrasts could allow the diagnosis of pulmonary emphysema in a murine model. In support of this, we demonstrate that the properties of the retrieved scattering distributions are consistent with the expectation of increased feature sizes related to pulmonary emphysema. Combined with the simplicity of implementation of edge-illumination, these findings suggest a high potential for exploiting extended sub-pixel contrasts in the diagnosis of lung diseases and beyond

DNase 2 Is the Main DNA-Degrading Enzyme of the Stratum Corneum

The cornified layer, the stratum corneum, of the epidermis is an efficient barrier to the passage of genetic material, i.e. nucleic acids. It contains enzymes that degrade RNA and DNA which originate from either the living part of the epidermis or from infectious agents of the environment. However, the molecular identities of these nucleases are only incompletely known at present. Here we performed biochemical and genetic experiments to determine the main DNase activity of the stratum corneum. DNA degradation assays and zymographic analyses identified the acid endonucleases L-DNase II, which is derived from serpinB1, and DNase 2 as candidate DNases of the cornified layer of the epidermis. siRNA-mediated knockdown of serpinB1 in human in vitro skin models and the investigation of mice deficient in serpinB1a demonstrated that serpinB1-derived L-DNase II is dispensable for epidermal DNase activity. By contrast, knockdown of DNase 2, also known as DNase 2a, reduced DNase activity in human in vitro skin models. Moreover, the genetic ablation of DNase 2a in the mouse was associated with the lack of acid DNase activity in the stratum corneum in vivo. The degradation of endogenous DNA in the course of cornification of keratinocytes was not impaired by the absence of DNase 2. Taken together, these data identify DNase 2 as the predominant DNase on the mammalian skin surface and indicate that its activity is primarily targeted to exogenous DNA

The baseline immunological and hygienic status of pigs impact disease severity of African swine fever.

African Swine Fever virus (ASFV) is a large double-enveloped DNA virus of the Asfarviridae family that causes a lethal hemorrhagic disease in domestic pigs and wild boars. Since 2007, a highly virulent genotype II strain has emerged and spread in Europe and South-East Asia, where millions of animals succumbed to the disease. Field- and laboratory-attenuated strains of ASFV cause highly variable clinical disease severity and survival, and mechanisms remain unclear. We hypothesized that the immunological and hygienic status of pigs is a determinant of ASF disease course. Here we compared the immunological profile at baseline and in response to ASFV infection in specific pathogen-free (SPF) and farm-raised Large White domestic pigs. At steady state, SPF pigs showed lower white blood cell counts and a lower basal inflammatory and antiviral transcriptomic profile compared to farm pigs, associated with profound differences in gut microbiome composition. After inoculation with a highly virulent ASFV genotype II strain (Armenia 2008), severe clinical signs, viremia and pro-inflammatory cytokines appeared sooner in SPF pigs, indicating a reduced capacity to control early virus replication. In contrast, during infection with an attenuated field isolate (Estonia 2014), SPF pigs presented a milder and shorter clinical disease with full recovery, whereas farm pigs presented severe protracted disease with 50% lethality. Interestingly, farm pigs showed higher production of inflammatory cytokines, whereas SPF pigs produced more anti-inflammatory IL-1ra early after infection and presented a stronger expansion of leukocytes in the recovery phase. Altogether, our data indicate that the hygiene-dependent innate immune status has a double-edge sword impact on immune responses in ASF pathogenesis. While the higher baseline innate immune activity helps the host in reducing initial virus replication, it promotes immunopathological cytokine responses, and delays lymphocyte proliferation after infection with an attenuated strain. Such effects should be considered for live vaccine development and vigilance

Phytochemical characteristics and the study of acute toxicity of extracts from fruit Рrunus domestica

The increased demand for modern phytopreparations is dictated by their wide application in the complex treatment of various diseases, the presence of low toxicity and the pharmacoeconomic component. Phytochemical analysis of medicinal plants reveals not only potential therapeutic effects, but also assumes their toxicological properties. In this context, the Prunus domestica, widely spread on the territory of our country and well known for its medicinal properties and applications in folk medicine, has attracted our attention. The aim of the work was to investigate the phytochemical composition of extracts obtained from fruit Prunus domestica and to study their toxic properties. Phytochemical analysis of the extracts obtained from fruits Prunus domestica was carried out according to the original method of extracting biologically active compounds, using instrumental research methods using modern laboratory analytical equipment. Studies of the safety of four extracts from Prunus domestica (liquid EC-1, EC-2 and dry CEPC, CEV) with the calculation of median lethal doses were carried out on mice and rats with the single intragastric and intraperitoneal routes of administration according to the method of T. V. Pastushenko. The degree of toxicity was determined according to the classification of K. K. Sidorov. Phytochemical study of extracts from fruit Prunus domestica provided information on the presence in their chemical composition of flavonoids, the sum of phenolic compounds, anthocyanins, neutral sugars, amino acids, organic and hydroxycinnamic acids. A higher content of the sum of phenolic compounds in the aqueous (ЕС-1) and alcoholic extracts (ЕС-2) as well as the presence of neutral sugars as products of hydrolysis of homopolysaccharides (СEV extract) and heteropolysaccharides (CEPC extract). Experimental studies proved that EC-1, EC-2, CEPC and SEV extracts had low toxicity beyond 15 000 mg/kg when administered orally and 3 000 mg/kg when administered intraperitoneally. The LD50 of the studied extracts of mice and rats after intraperitoneal injection was determined, which was more than 3 000 mg/kg. The chemical composition of biologically active compounds of extracts from the fruit Prunus domestica, which provides promising directions for further study of their pharmacological properties, has been investigated. Analysis of experimental data on acute toxicity of the studied extracts showed that they belong to the IV class of toxicity “relatively harmless substances", according to K. K. Sidorov classification. The LD50 of extracts from the fruit Prunus domestica for oral administration could not be determined, and the LD50 of the studied extracts for mice and rats after intraperitoneal administration was more than 3 000 mg/kg

Distinct gene expression patterns correlate with developmental and functional traits of iNKT subsets.

Invariant natural killer T (iNKT) cells comprise a subpopulation of innate lymphocytes developing in thymus. A new model proposes subdividing murine iNKT cells into iNKT1, 2 and 17 cells. Here, we use transcriptome analyses of iNKT1, 2 and 17 subsets isolated from BALB/c and C57BL/6 thymi to identify candidate genes that may affect iNKT cell development, migration or function. We show that Fcɛr1γ is involved in generation of iNKT1 cells and that SerpinB1 modulates frequency of iNKT17 cells. Moreover, a considerable proportion of iNKT17 cells express IL-4 and IL-17 simultaneously. The results presented not only validate the usefulness of the iNKT1/2/17-concept but also provide new insights into iNKT cell biology

Optimized intramuscular immunization with VSV-vectored spike protein triggers a superior immune response to SARS-CoV-2.

Immunization with vesicular stomatitis virus (VSV)-vectored COVID-19 vaccine candidates expressing the SARS-CoV-2 spike protein in place of the VSV glycoprotein relies implicitly on expression of the ACE2 receptor at the muscular injection site. Here, we report that such a viral vector vaccine did not induce protective immunity following intramuscular immunization of K18-hACE2 transgenic mice. However, when the viral vector was trans-complemented with the VSV glycoprotein, intramuscular immunization resulted in high titers of spike-specific neutralizing antibodies. The vaccinated animals were fully protected following infection with a lethal dose of SARS-CoV-2-SD614G via the nasal route, and partially protected if challenged with the SARS-CoV-2Delta variant. While dissemination of the challenge virus to the brain was completely inhibited, replication in the lung with consequent lung pathology was not entirely controlled. Thus, intramuscular immunization was clearly enhanced by trans-complementation of the VSV-vectored vaccines by the VSV glycoprotein and led to protection from COVID-19, although not achieving sterilizing immunity

Neutrophil extracellular trap formation requires OPA1-dependent glycolytic ATP production

Optic atrophy 1 (OPA1) is a mitochondrial inner membrane protein that has an important role in mitochondrial fusion and structural integrity. Dysfunctional OPA1 mutations cause atrophy of the optic nerve leading to blindness. Here, we show that OPA1 has an important role in the innate immune system. Using conditional knockout mice lacking Opa1 in neutrophils (Opa1(N Delta)), we report that lack of OPA1 reduces the activity of mitochondrial electron transport complex I in neutrophils. This then causes a decline in adenosine-triphosphate (ATP) production through glycolysis due to lowered NAD(+) availability. Additionally, we show that OPA1-dependent ATP production in these cells is required for microtubule network assembly and for the formation of neutrophil extracellular traps. Finally, we show that Opa1(N Delta) mice exhibit a reduced antibacterial defense capability against Pseudomonas aeruginosa.Peer reviewe

Myeloid conditional deletion and transgenic models reveal a threshold for the neutrophil survival factor Serpinb1

Serpinb1 is an inhibitor of neutrophil granule serine proteases cathepsin G, proteinase-3 and elastase. One of its core physiological functions is to protect neutrophils from granule protease-mediated cell death. Mice lacking Serpinb1a (Sb1a-/-), its mouse ortholog, have reduced bone marrow neutrophil numbers due to cell death mediated by cathepsin G and the mice show increased susceptibility to lung infections. Here, we show that conditional deletion of Serpinb1a using the Lyz2-cre and Cebpa-cre knock-in mice effectively leads to recombination-mediated deletion in neutrophils but protein-null neutrophils were only obtained using the latter recombinase-expressing strain. Absence of Serpinb1a protein in neutrophils caused neutropenia and increased granule permeabilization-induced cell death. We then generated transgenic mice expressing human Serpinb1 in neutrophils under the human MRP8 (S100A8) promoter. Serpinb1a expression levels in founder lines correlated positively with increased neutrophil survival when crossed with Sb1a-/- mice, which had their defective neutrophil phenotype rescued in the higher expressing transgenic line. Using new conditional and transgenic mouse models, our study demonstrates the presence of a relatively low Serpinb1a protein threshold in neutrophils that is required for sustained survival. These models will also be helpful in delineating recently described functions of Serpinb1 in metabolism and cancer

Functional Redundancy of Class I Phosphoinositide 3-Kinase (PI3K) Isoforms in Signaling Growth Factor-Mediated Human Neutrophil Survival

We have investigated the contribution of individual phosphoinositide 3-kinase (PI3K) Class I isoforms to the regulation of neutrophil survival using (i) a panel of commercially available small molecule isoform-selective PI3K Class I inhibitors, (ii) novel inhibitors, which target single or multiple Class I isoforms (PI3Kα, PI3Kβ, PI3Kδ, and PI3Kγ), and (iii) transgenic mice lacking functional PI3K isoforms (p110δKOγKO or p110γKO). Our data suggest that there is considerable functional redundancy amongst Class I PI3Ks (both Class IA and Class IB) with regard to GM-CSF-mediated suppression of neutrophil apoptosis. Hence pharmacological inhibition of any 3 or more PI3K isoforms was required to block the GM-CSF survival response in human neutrophils, with inhibition of individual or any two isoforms having little or no effect. Likewise, isolated blood neutrophils derived from double knockout PI3K p110δKOγKO mice underwent normal time-dependent constitutive apoptosis and displayed identical GM-CSF mediated survival to wild type cells, but were sensitized to pharmacological inhibition of the remaining PI3K isoforms. Surprisingly, the pro-survival neutrophil phenotype observed in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD) was resilient to inactivation of the PI3K pathway