Co-Culture of Human Bronchial Fibroblasts and CD4+ T Cells Increases Th17 Cytokine Signature

Background

Airway inflammation is an important characteristic of asthma and has been associated with airway remodelling and bronchial hyperreactivity. The mucosal microenvironment composed of structural cells and highly specialised extracellular matrix is able to amplify and promote inflammation. This microenvironment leads to the development and maintenance of a specific adaptive response characterized by Th2 and Th17. Bronchial fibroblasts produce multiple mediators that may play a role in maintaining and amplifying this response in asthma.

Objective

To investigate the role of bronchial fibroblasts obtained from asthmatic subjects and healthy controls in regulating Th17 response by creating a local micro-environment that promotes this response in the airways.

Methods

Human bronchial fibroblasts and CD4⁺T cells were isolated from atopic asthmatics and non-atopic healthy controls. CD4⁺T were co-cultured with bronchial fibroblasts of asthmatic subjects and healthy controls. RORc gene expression was detected by qPCR. Phosphorylated STAT-3 and RORγt were evaluated by western blots. Th17 phenotype was measured by flow cytometry. IL-22, IL17, IL-6 TGF-β and IL1-β were assessed by qPCR and ELISA.

Results

Co-culture of CD4⁺T cells with bronchial fibroblasts significantly stimulated RORc expression and induced a significant increase in Th17 cells as characterized by the percentage of IL-17⁺/CCR6⁺ staining in asthmatic conditions. IL-17 and IL-22 were increased in both normal and asthmatic conditions with a significantly higher amount in asthmatics compared to controls. IL-6, IL-1β, TGF-β and IL-23 were significantly elevated in fibroblasts from asthmatic subjects upon co-culture with CD4⁺T cells. IL-23 stimulates IL-6 and IL-1β expression by bronchial fibroblasts.

Conclusion

Interaction between bronchial fibroblasts and T cells seems to promote specifically Th17 cells profile in asthma. These results suggest that cellular interaction particularly between T cells and fibroblasts may play a pivotal role in the regulation of the inflammatory response in asthma.     

SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity.     

Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl–methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.     

Facultative response to a kleptoparasite by the cooperatively breeding pied babbler

In many cases of interspecific kleptoparasitism, hosts developdefensive behaviors to minimize the impact of kleptoparasites.Because vigilance and defensive behaviors are often costly,selection should favor hosts that adjust the amount of investmentneeded to minimize losses to kleptoparasitism. However, examplesof such facultative responses are rare. Here, we investigatethe response of cooperatively breeding pied babblers (Turdoidesbicolor) to the drongo (Dicrurus adsimilis), an avian kleptoparasitethat regularly follows pied babbler groups, often giving alarmcalls to alert the group to predators but also occasionallygiving false alarm calls in order to steal food items. We showthat pied babbler response to drongos varies markedly accordingto babbler group size. In small groups, where there are fewindividuals available to act as sentinels, babblers sentinelless when a drongo is present and respond strongly to drongoalarm calls. However, in large groups, where there are manyindividuals available to participate in predator vigilance,babblers sentinel more often when a drongo is present, rarelyrespond to drongo alarm calls, and aggressively displace drongos,with a consequent decline in the number of successful kleptoparasitismevents. This behavior represent a facultative response to akleptoparasite according to the costs versus benefits of toleratingtheir presence.     

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5–6 months old Assaf lambs using Al(OH)₃ or Escherichia coli lipopolysaccharide + inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%–85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.     

The expression of essential components for human influenza virus internalisation in Vero and MDCK cells

MDCK and Vero cell lines have been used as substrates for influenza virus replication. However, Vero cells produced lower influenza virus titer yield compared to MDCK. Influenza virus needs molecules for internalisation of the virus into the host cell, such as influenza virus receptor and clathrin. Human influenza receptor is usually a membrane protein containing Sia(α2,6) Gal, which is added into the protein in the golgi apparatus by α2,6 sialyltransferase (SIAT1). Light clathrin A (LCA), light clathrin B (LCB) and heavy clathrin (HC) are the main components needed for virus endocytosis. Therefore, it is necessary to compare the expression of SIAT1 and clathrin in Vero and MDCK cells. This study is reporting the expression of SIAT1 and clathrin observed in both cells with respect to the levels of (1) RNA by using RT-PCR, (2) protein by using dot blot analysis and confocal microscope. The results showed that Vero and MDCK cells expressed both SIAT1 and clathrin proteins, and the expression of SIAT1 in MDCK was higher compared to Vero cells. On the other hand, the expressions of LCA, LCB and HC protein in MDCK cells were not significantly different to Vero cells. This result showed that the inability of Vero cells to internalize H1N1 influenza virus was possibly due to the lack of transmembrane protein receptor which contained Sia(α2,6) Gal.     

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

Vitamin D3 supplementation during pregnancy and lactation for women living with HIV in Tanzania: A randomized controlled trial

BackgroundObservational studies suggest that vitamin D deficiency among people living with HIV is associated with a greater risk of disease progression and death. Low levels of vitamin D in pregnancy are also associated with poor fetal and infant growth. Therefore, vitamin D supplementation may improve clinical outcomes for pregnant women living with HIV and improve fetal and postnatal growth for their infants.Methods and findingsWe conducted a randomized, triple-blind, placebo-controlled trial of vitamin D₃ supplementation among pregnant and lactating women living with HIV in Dar es Salaam, Tanzania (ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02305927","term_id":"NCT02305927"}}NCT02305927). Participants were randomized with 1:1 allocation stratified by study clinic to receive either daily 3,000 IU vitamin D₃ supplements or matching placebo supplements from the second trimester of pregnancy (12–27 weeks) until 1 year postpartum. The primary outcomes were (i) maternal HIV progression or death, (ii) small-for-gestational-age (SGA) live births (<10th percentile), and (iii) infant stunting at 1 year of age (length-for-age z-score < −2). We also examined the effect of vitamin D₃ supplementation on secondary maternal and infant health outcomes, maternal and infant serum 25-hydroxyvitamin D (25[OH]D) concentrations, and maternal hypercalcemia. An intent-to-treat analysis was used as the primary analytic approach. We enrolled 2,300 pregnant women between June 15, 2015, and April 17, 2018, and follow-up of mothers and infants was completed on October 20, 2019. There were 1,148 pregnant women randomly assigned to the vitamin D₃ group, and 1,152 to the placebo group. The proportion of mothers lost to follow-up at 1 year postpartum was 6.6% in the vitamin D₃ group (83 of 1,148) and 6.6% in the placebo group (76 of 1,152). The proportion of children lost to follow-up at 1 year of age was 5.5% in the vitamin D₃ group (59 of 1,074 live births) and 5.2% in the placebo group (57 of 1,093 live births). There was no difference in the risk of maternal HIV progression or death, with 166 events during 1,461 person-years of follow-up in the vitamin D₃ group and 141 events during 1,469 person-years of follow-up in the placebo group (hazard ratio 1.21, 95% CI 0.97 to 1.52, p = 0.09). There was no difference in the risk of SGA birth between the vitamin D₃ (229 SGA births among 1,070 live births) and placebo groups (236 SGA births among 1,091 live births) (relative risk 1.03, 95% CI 0.87 to 1.22, p = 0.70). There was also no difference in the risk of infant stunting at 1 year of age between the vitamin D₃ (407 events among 867 infants) and placebo groups (413 events among 873 infants) (relative risk 1.00, 95% CI 0.92 to 1.10, p = 0.95). In terms of adverse events, no cases of maternal hypercalcemia were identified. One hypersensitivity reaction to the trial supplements occurred for a pregnant woman in the placebo group. A limitation of our study is that our findings may not be generalizable to HIV-negative pregnant women or contexts where severe vitamin D deficiency is prevalent.ConclusionsThe trial findings do not support routine vitamin D supplementation for pregnant and lactating women living with HIV in Tanzania.Trial registrationClinicalTrials.gov Identifier: {"type":"clinical-trial","attrs":{"text":"NCT02305927","term_id":"NCT02305927"}}NCT02305927.

Christopher R. Sudfeld and colleagues, investigate the impact of vitamin D supplementation on clinical outcomes for pregnant women living with HIV, and growth outcomes for their infants.