Unique Transcriptional Architecture in Airway Epithelial Cells and Macrophages Shapes Distinct Responses following Influenza Virus Infection Ex Vivo.

Airway epithelial cells and macrophages differ markedly in their responses to influenza A virus (IAV) infection. To investigate transcriptional responses underlying these differences, purified subsets of type II airway epithelial cells (ATII) and alveolar macrophages (AM) recovered from the lungs of mock- or IAV-infected mice at 9 h postinfection were subjected to RNA sequencing. This time point was chosen to allow for characterization of cell types first infected with the virus inoculum, prior to multicycle virus replication and the infiltration of inflammatory cells into the airways. In the absence of infection, AM predominantly expressed genes related to immunity, whereas ATII expressed genes consistent with their physiological roles in the lung. Following IAV infection, AM almost exclusively activated cell-intrinsic antiviral pathways that were dependent on interferon (IFN) regulatory factor 3/7 (IRF3/7) and/or type I IFN signaling. In contrast, IAV-infected ATII activated a broader range of physiological responses, including cell-intrinsic antiviral pathways, which were both independent of and dependent on IRF3/7 and/or type I IFN. These data suggest that transcriptional profiles hardwired during development are a major determinant underlying the different responses of ATII and AM to IAV infection.IMPORTANCE Airway epithelial cells (AEC) and airway macrophages (AM) represent major targets of influenza A virus (IAV) infection in the lung, yet the two cell types respond very differently to IAV infection. We have used RNA sequencing to define the host transcriptional responses in each cell type under steady-state conditions as well as following IAV infection. To do this, different cell subsets isolated from the lungs of mock- and IAV-infected mice were subjected to RNA sequencing. Under steady-state conditions, AM and AEC express distinct transcriptional activities, consistent with distinct physiological roles in the airways. Not surprisingly, these cells also exhibited major differences in transcriptional responses following IAV infection. These studies shed light on how the different transcriptional architectures of airway cells from two different lineages drive transcriptional responses to IAV infection.

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB.

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

The role of antigen presenting cells in the induction of HIV-1 latency in resting CD4(+) T-cells.

BACKGROUND: Combination antiretroviral therapy (cART) is able to control HIV-1 viral replication, however long-lived latent infection in resting memory CD4(+) T-cells persist. The mechanisms for establishment and maintenance of latent infection in resting memory CD4(+) T-cells remain unclear. Previously we have shown that HIV-1 infection of resting CD4(+) T-cells co-cultured with CD11c(+) myeloid dendritic cells (mDC) produced a population of non-proliferating T-cells with latent infection. Here we asked whether different antigen presenting cells (APC), including subpopulations of DC and monocytes, were able to induce post-integration latent infection in resting CD4(+) T-cells, and examined potential cell interactions that may be involved using RNA-seq. RESULTS: mDC (CD1c(+)), SLAN(+) DC and CD14(+) monocytes were most efficient in stimulating proliferation of CD4(+) T-cells during syngeneic culture and in generating post-integration latent infection in non-proliferating CD4(+) T-cells following HIV-1 infection of APC-T cell co-cultures. In comparison, plasmacytoid DC (pDC) and B-cells did not induce latent infection in APC-T-cell co-cultures. We compared the RNA expression profiles of APC subpopulations that could and could not induce latency in non-proliferating CD4(+) T-cells. Gene expression analysis, comparing the CD1c(+) mDC, SLAN(+) DC and CD14(+) monocyte subpopulations to pDC identified 53 upregulated genes that encode proteins expressed on the plasma membrane that could signal to CD4(+) T-cells via cell-cell interactions (32 genes), immune checkpoints (IC) (5 genes), T-cell activation (9 genes), regulation of apoptosis (5 genes), antigen presentation (1 gene) and through unknown ligands (1 gene). CONCLUSIONS: APC subpopulations from the myeloid lineage, specifically mDC subpopulations and CD14(+) monocytes, were able to efficiently induce post-integration HIV-1 latency in non-proliferating CD4(+) T-cells in vitro. Inhibition of key pathways involved in mDC-T-cell interactions and HIV-1 latency may provide novel targets to eliminate HIV-1 latency.

Pathogenic bacteria and TNF do not induce production of macrophage migration inhibitory factor (MIF) by human monocytes.

Elevated serum macrophage migration inhibitory factor (MIF) is associated with severe sepsis, but it is not clear whether bacteria stimulate synthesis of MIF by blood leukocytes directly or via induction of TNF. Here we assess production of MIF mRNA and protein by blood leukocytes from healthy human subjects (n=28) following exposure to bacteria commonly associated with sepsis (Escherichia coli and Streptococcus pneumoniae). Bacteria did not increase levels of MIF mRNA or secreted protein. CD14(+) monocytes were the main cell type producing MIF before and after stimulation. Exposure of leukocytes to TNF did not induce MIF. Hence elevated levels of serum MIF observed in sepsis may not reflect MIF produced by blood leukocytes stimulated directly by bacteria or TNF.

Understanding Factors That Modulate the Establishment of HIV Latency in Resting CD4+ T-Cells In Vitro.

Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.

Androgen receptor action in osteoblasts in male mice is dependent on their stage of maturation.

Androgen action via the androgen receptor (AR) is essential for normal skeletal growth and bone maintenance post-puberty in males; however, the molecular and cellular mechanisms by which androgens exert their actions in osteoblasts remains relatively unexplored in vivo. To identify autonomous AR actions in osteoblasts independent of AR signaling in other tissues, we compared the extent to which the bone phenotype of the Global-ARKO mouse was restored by replacing the AR in osteoblasts commencing at either the (1) proliferative or (2) mineralization stage of their maturation. In trabecular bone, androgens stimulated trabecular bone accrual during growth via the AR in proliferating osteoblasts and maintained trabecular bone post-puberty via the AR in mineralizing osteoblasts, with its predominant action being to inhibit bone resorption by decreasing the ratio of receptor activator of NF-κB ligand (RANKL) to osteoprotegerin (OPG) gene expression. During growth, replacement of the AR in proliferating but not mineralizing osteoblasts of Global-ARKOs was able to partially restore periosteal circumference, supporting the concept that androgen action in cortical bone to increase bone size during growth is mediated via the AR in proliferating osteoblasts. This study provides further significant insight into the mechanism of androgen action via the AR in osteoblasts, demonstrating that it is dependent on the stage of osteoblast maturation.

Polymorphisms in cytokine genes define subpopulations of HIV-1 patients who experienced immune restoration diseases.

OBJECTIVE: To further elucidate the immunopathogenesis of immune restoration diseases (IRD) in HIV patients responding to antiretroviral therapy and determine whether IRD associated with different opportunistic pathogens involve distinct immunopathological mechanisms. DESIGN: DNA samples from patients with a range of IRD were typed for polymorphic loci in genes encoding immune-mediators. METHODS: PCR-restriction fragment length polymorphism assays were used to type loci in the and genes. Alleles of a microsatellite in the CD30 promoter were determined by capillary electrophoresis. RESULTS: Only 8% of patients with IRD associated with a herpesvirus infection carried IL12B-3'UTR*2, compared with 42-54% of patients with other or no IRD. Patients with IRD arising from mycobacterial infection rarely carried IL6-174*C (36% versus 61-71%) and never carried TNFA-308*2 (0% versus 23-52%). TNFA-308*2 was carried by 52% of patients who experienced IRD associated with a herpesvirus infection, as several patients with exacerbations of cytomegalovirus retinitis carried this as part of a HLA-A2,B44 haplotype. Polymorphisms in and showed no distinct patterns. CONCLUSIONS: Distinct cytokine-mediated mechanisms contribute to IRD initiated by herpesvirus and mycobacterial infections.

Central MHC genes affect IgA levels in the human: reciprocal effects in IgA deficiency and IgA nephropathy.

This study investigates the hypothesis that alternative alleles of one or more genes in the central major histocompatibility complex (MHC) predispose carriers to IgA deficiency (IgAD) or IgA Nephropathy (IgAN). Australian caucasian IgAD, IgAN patients, and controls were typed at HLA loci, single nucleotide polymorphisms, and microsatellites in the MHC. Alleles of the D6S273 microsatellite exhibited strong associations with IgAD and IgAN. D6S273*129 and *139 were more frequent in IgAD and less frequent in IgAN patients than controls. The reverse was true for D6S273*133 and *131. Alleles of other microsatellites exhibited weak associations with IgAD or IgAN. D6S273*129 is found on the 65.1 ancestral haplotype [HLA-B14(65),DR1], which has been reported to be increased in IgAD, but the majority of IgAD patients with D6S273*129 did not have other alleles of the haplotype. D6S273*139 is characteristic of the 8.1 ancestral haplotype (HLA-A1,B8,DR3), which was common in IgAD and rare in IgAN patients. Further studies of the 8.1 haplotype in Australian, German and Spanish caucasian subjects revealed that HLA-DR3, in the absence of -B8, is not associated with IgAD. However -B8 is associated with IgAD in the absence of -DR3, consistent with a susceptibility locus in the central MHC. Provisional mapping within this region is discussed.

The septic shock associated HSPA1B1267 polymorphism influences production of HSPA1A and HSPA1B.

OBJECTIVE: To identify a functional polymorphic site(s) within HSPA1A and/or HSPA1B which is in linkage disequilibrium with the silent mutation HSPA1B1267A>G and explains its association with septic shock. SUBJECTS: The promoter region of HSPA1A and HSPA1B was sequenced in 100 healthy whites. Stimulation experiments were performed on 36 healthy subjects. MEASUREMENTS AND RESULTS: Sequencing the HSPA1A and HSPA1B promoter regions (approx. 500 bp upstream of the translation start site) identified ten novel and three known polymorphisms. Mononuclear cells were stimulated with lipopolysaccharide (10 microg/ml) for 4 and 8 h, and mRNA levels measured by reverse transcriptase polymerase chain reaction. Two polymorphisms, HSPA1A-27G>C and HSPA1A-327A>C, were found to be in strong linkage with HSPA1B1267A>G but, as with HSPA1B1267A>G, were not associated with stimulated mRNA levels. However, HSPA1B-179C>T, which is also in linkage with HSPA1B1267, was associated with stimulated HSPA1A and HSPA1B mRNA levels. Individuals homozygous for the C allele of HSPA1B-179C>T were associated with lower HSPA1A and HSPA1B mRNA levels than HSPA1B-179CT after 8 h lipopolysaccharide stimulation. CONCLUSIONS: HSPA1B-179C>T is in linkage disequilibrium with HSPA1B1267A>G and is associated with variable production of HSPA1B and HSPA1A production. This suggests that HSPA1B-179C>T affects HSP70 production and is a key determinant of individual susceptibility to a variety of infectious and inflammatory diseases.

Longitudinal assessment of thyroid function in pregnancy.

BACKGROUND: Trimester-specific reference intervals (RIs) for thyroid function tests are lacking for Beckman Dxl 800 analysers. We aimed to establish RIs for thyroid stimulating hormone (TSH), free thyroxine (fT4) and to track intraindividual changes in thyroid function throughout pregnancy. METHODS: One hundred and thirty healthy women without antithyroid peroxidase antibodies were followed longitudinally. Thyroid function was determined at trimester-1 (T1): 9-13 weeks; trimester-2 (T2): 22-26 weeks; trimester-3 (T3): 35-39 weeks and postpartum (PP): 8-12 weeks. A subgroup (n = 47) was used to track intraindividual changes using PP as non-pregnant state (baseline). RESULTS: For trimesters 1-3, TSH (median (2.5th, 5th, 95th and 97.5th percentile)) was 0.77 (0.03, 0.05, 2.33, 3.05), 1.17 (0.42, 0.47, 2.71, 3.36) and 1.35 (0.34, 0.42, 2.65, 2.83) mIU/L, respectively. Free T4 (mean (95%CI)) was 10.7 (5.9-15.5), 8.1 (4.9-11.3), 7.8 (4.5-11.0) pmol/L, respectively. In T2 and T3, 36% and 41% of the fT4 values, respectively, fell below the non-pregnancy lower normal limit. In the subgroup assessed for longitudinal changes, of the women with baseline TSH ≤ median, 71-75% remained at or below the corresponding median for trimesters 1-3. Of the women with baseline fT4 ≤ median, 69-81% also remained at or below the corresponding median for trimesters 1-3. High correlation was observed at different trimesters and baseline for TSH (Spearman's r: 0.593-0.846, P < 0.001) and for fT4 (r: 0.480-0.739, P < 0.001). CONCLUSIONS: Use of trimester-specific RIs would prevent misclassification of thyroid function during pregnancy. In the majority of women, TSH and fT4 tracked on the same side of the median distribution, from a non-pregnant baseline, throughout pregnancy.

Renal structure in normoalbuminuric and albuminuric patients with type 2 diabetes and impaired renal function.

OBJECTIVE: The structural basis of normoalbuminuric renal insufficiency in patients with type 2 diabetes remains to be elucidated. We compared renal biopsy findings in patients with type 2 diabetes and estimated glomerular filtration rate (eGFR) and measured GFR of <60 mL/min/1.73 m2, associated with either normo-, micro-, or macroalbuminuria. RESEARCH DESIGN AND METHODS: In patients with normo- (n = 8) or microalbuminuria (n = 6), renal biopsies were performed according to a research protocol. In patients with macroalbuminuria (n = 17), biopsies were performed according to clinical indication. Findings were categorized according to the Fioretto classification: category 1 (C1), normal/near normal; category 2 (C2), typical diabetic nephropathy (DN) with predominantly glomerular changes; and category 3 (C3), atypical with disproportionately severe interstitial/tubular/vascular damage and with no/mild diabetic glomerular changes. RESULTS: In our study population (mean eGFR 35 mL/min/1.73 m2), typical glomerular changes (C2) of DN were observed in 22 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 subjects with normoalbuminuria (P = 0.002). By contrast, predominantly interstitial or vascular changes (C3) were seen in only 1 of 23 subjects with micro- or macroalbuminuria compared with 3 of 8 normoalbuminuric subjects (P = 0.08). Mesangial area increased progressively from normal controls to patients with type 2 diabetes and normo-, micro-, and macroalbuminuria. Varying degrees of arteriosclerosis, although not necessarily the predominant pattern, were seen in seven of eight subjects with normoalbuminuria. CONCLUSIONS: Typical renal structural changes of DN were observed in patients with type 2 diabetes and elevated albuminuria. By contrast, in normoalbuminuric renal insufficiency, these changes were seen less frequently, likely reflecting greater contributions from aging, hypertension, and arteriosclerosis.

Dietary salt intake and mortality in patients with type 2 diabetes.

OBJECTIVE: Many guidelines recommend that patients with type 2 diabetes should aim to reduce their intake of salt. However, the precise relationship between dietary salt intake and mortality in patients with type 2 diabetes has not been previously explored. RESEARCH DESIGN AND METHODS: Six hundred and thirty-eight patients attending a single diabetes clinic were followed in a prospective cohort study. Baseline sodium excretion was estimated from 24-h urinary collections (24hU(Na)). The predictors of all-cause and cardiovascular mortality were determined by Cox regression and competing risk modeling, respectively. RESULTS: The mean baseline 24hU(Na) was 184 ± 73 mmol/24 h, which remained consistent throughout the follow-up (intraindividual coefficient of variation [CV] 23 ± 11%). Over a median of 9.9 years, there were 175 deaths, 75 (43%) of which were secondary to cardiovascular events. All-cause mortality was inversely associated with 24hU(Na), after adjusting for other baseline risk factors (P < 0.001). For every 100 mmol rise in 24hU(Na), all-cause mortality was 28% lower (95% CI 6-45%, P = 0.02). After adjusting for the competing risk of noncardiovascular death and other predictors, 24hU(Na) was also significantly associated with cardiovascular mortality (sub-hazard ratio 0.65 [95% CI 0.44-0.95]; P = 0.03). CONCLUSIONS: In patients with type 2 diabetes, lower 24-h urinary sodium excretion was paradoxically associated with increased all-cause and cardiovascular mortality. Interventional studies are necessary to determine if dietary salt has a causative role in determining adverse outcomes in patients with type 2 diabetes and the appropriateness of guidelines advocating salt restriction in this setting.

Alleles of the proximal promoter of BAT1, a putative anti-inflammatory gene adjacent to the TNF cluster, reduce transcription on a disease-associated MHC haplotype.

BACKGROUND: BAT1 belongs to the DEAD-box family of proteins, and is encoded in the central region of the MHC, a region containing genes affecting immunopathological disorders including Type 1 diabetes. We showed that BAT1 can reduce inflammatory cytokine production, supporting its candidacy as a disease gene. Here we examined the proximal promoter region of BAT1. RESULTS: Ten single nucleotide polymorphisms were identified in approximately 1.4 kb of sequence, defining at least seven alleles. Sections of the BAT1 promoter region were amplified from cells homozygous for the MHC haplotypes associated with susceptibility (HLA-A1, B8, DR3; 8.1 haplotype) and resistance (HLA-A3, B7, DR15; 7.1 haplotype) to diabetes and cloned into a promoter-less luciferase-encoding plasmid. Jurkat cells transiently transfected with fragments from the 8.1 haplotype exhibited a lower luciferase activity than those transfected with fragments from the 7.1 haplotype, indicating reduced transcription. The effect was clearest with the 520 bp immediately upstream of the transcriptional start site. Electrophoretic mobility shift assays using oligonucleotides spanning polymorphic sites within the 520 bp (proximal) promoter fragment showed haplotype-specific binding of nuclear proteins. CONCLUSIONS: In view of the anti-inflammatory role of BAT1, reduced production on a disease-associated haplotype constitutes a novel and self-consistent model for the effect of central MHC genes on disease.

Alleles carried at positions -819 and -592 of the IL10 promoter affect transcription following stimulation of peripheral blood cells with Streptococcus pneumoniae.

IL-10 inhibits the production of many pro-inflammatory cytokines. Polymorphisms in the IL10 gene promoter at positions -1082G-->A, -819C-->T and -592C-->A occur as three haplotypes, ATA, GCC and ACC. These influence several infectious and inflammatory diseases including community-acquired pneumonia, where a role for IL-10 is suggested by fluctuations in plasma levels of the cytokine. However, the effects of the haplotypes on IL-10 production are unclear. We stimulated peripheral blood mononuclear cells (PBMC) from at least five individuals homozygous for each of the three haplotypes with lipopolysaccharide (LPS, 10 microg/ml) or heat-killed Streptococcus pneumoniae (10(7)cfu/ml) and measured IL-10 mRNA by RT-PCR. Following S. pneumoniae stimulation, PBMC with the ATA haplotype had higher IL-10 mRNA levels than those with the GCC haplotype at 4 h (independent t-test; P=0.024), or the ACC haplotype at 4 h ( P<0.0001) and 8 h ( P=0.007). Following LPS stimulation, IL-10 mRNA levels were not significantly influenced by the IL10 haplotype, but similar trends were observed, consistent with the variable outcome of published studies. The results suggest that the -819 and/or -592 alleles affect transcription.