Age and Gender Adjusted Comparison of Clinical Features between Severe Cases Infected with H7N9 and H1N1pdm Influenza A in Jiangsu Province, China

<div><p>Background</p><p>Influenza H7N9 and H1N1pdm can cause severe human infections. It is important to investigate the distinguishing clinical features between these two diseases. Several studies have compared the differences in general, however, age and gender adjusted comparisons may be more useful and informative to the health professionals.</p><p>Methods</p><p>A total of 184 severe H1N1pdm patients and 37 severe H7N9 patients from Jiangsu Province were included in this analysis to perform age and gender adjusted comparison of clinical features.</p><p>Results</p><p>After adjusting age and gender, no significant differences in chronic medical conditions or treatment were found between severely ill patients with H7N9 and H1N1pdm. Severely ill patients with H7N9 had significantly longer interval from onset of illness to neuraminidase inhibitor treatment and to death. They were more likely to have complications such as acute respiratory distress syndrome (ARDS), liver and renal dysfunctions, and had a significantly higher risk of death.</p><p>Conclusion</p><p>Our results suggests that age and gender should be adjusted as important confounding factors when comparing the clinical features between severe H7N9 and H1N1pdm patients to avoid any misunderstanding regarding the differences between these two diseases particularly in terms of clinical severity and prognosis.</p></div

Age distribution of severe patients infected with influenza H7N9 and H1N1pdm in Jiangsu Province, China.

<p>Age distribution of severe patients infected with influenza H7N9 and H1N1pdm in Jiangsu Province, China.</p

Distribution of selected variables between severe H7N9 and H1N1pdm patients.

<p>^a Patients with missing data were excluded for each variable.</p><p>^b Pearson chi-square test was used for comparing proportions and continuity correction or Fisher’s Exact Test was used if appropriate. Mann-Whitney U test was used for comparing medians.</p><p>^c Age and gender were adjusted using General Linear Model (for BMI) or Cox proportional hazards model (for selected time durations) for continuous variables and Logistic regression model for categorical variables.</p><p>^d Interquartile range (IQR), Frequency (FREQ), Body mass index (BMI)</p><p>Distribution of selected variables between severe H7N9 and H1N1pdm patients.</p

Image_1_Tissue-Resident Macrophages Limit Pulmonary CD8 Resident Memory T Cell Establishment.TIFF

Tissue resident memory CD8 T cells (TRM) serve as potent local sentinels and contribute significantly to protective immunity against intracellular mucosal pathogens. While the molecular and transcriptional underpinnings of TRM differentiation are emerging, how TRM establishment is regulated by other leukocytes in vivo is largely unclear. Here, we observed that expression of PPAR-γ in the myeloid compartment was a negative regulator of CD8 TRM establishment following influenza virus infection. Interestingly, myeloid deficiency of PPAR-γ resulted in selective impairment of the tissue-resident alveolar macrophage (AM) compartment during primary influenza infection, suggesting that AM are likely negative regulators of CD8 TRM differentiation. Indeed, influenza-specific CD8 TRM cell numbers were increased following early, but not late ablation of AM using the CD169-DTR model. Importantly, these findings were specific to the parenchyma of infected tissue as circulating memory T cell frequencies in lung and TCM and TEM in spleen were largely unaltered following macrophage ablation. Further, the magnitude of the effector response could not explain these observations. These data indicate local regulation of pulmonary TRM differentiation is alveolar macrophage dependent. These, findings could aid in vaccine design aimed at increasing TRM density to enhance protective immunity, or deflating their numbers in conditions where they cause overt or veiled chronic pathologies.</p

Table_1_Rhein Suppresses Lung Inflammatory Injury Induced by Human Respiratory Syncytial Virus Through Inhibiting NLRP3 Inflammasome Activation via NF-κB Pathway in Mice.docx

Rhein is one of active anthraquinone components in traditional Chinese herbal medicine Rheum palmatum L., possessing anti-inflammatory, antioxidant, antitumor, antiviral, and hepatoprotective activities. Human respiratory syncytial virus (RSV), a common virus, is able to result in pneumonia and bronchitis, which usually can be seen in infants. However, so far the effects of Rhein on RSV-induced pneumonia are still unknown. As the NLRP3 inflammasome is activated excessively, it is able to lead to inflammatory response and tissue injury in most viral infection process (including RSV infection) of respiratory tract. Therefore, we designed experiments to reveal whether Rhein can treat RSV-induced pneumonia by inhibiting NLRP3 inflammasome activation. In present research, we established the pneumonia model of BALB/C mice caused by RSV. First of all, the pathology of lung tissue and the weight of mice were evaluated, and the corresponding lung index was calculated. Additionally, the expression of pro-inflammatory mediators in serum and lung tissues, and related proteins (NLRP3, ASC and Caspase-1) of NLRP3 inflammasome and NF-κB pathway were detected by Enzyme-linked immunosorbent assay (ELISA), Real-time PCR (RT-PCR), Immunohistochemistry (IHC), and Western blot (WB), respectively. The determination of lung index and lung tissue pathological evaluation revealed that Rhein was able to alleviate lung infection and injury caused by RSV. The results of ELISA showed that Rhein was able to reduce the release of pro-inflammatory cytokines in the serum and lung tissues of RSV-induced BALB/c mice, including IL-1β, IL-6, TNF-α, IL-18, and IL-33. Additionally, it was revealed that Rhein inhibited the immune inflammatory response of RSV-infected mice, which was likely to be associated with the inhibition the NLRP3 inflammasome activation via NF-κB pathway. To sum up, our results indicated that Rhein may inhibit RSV-induced pulmonary inflammatory response effectively; meanwhile, it is emphasized that Rhein therapy is likely to be a promising treatment on the RSV-infected lung inflammation and avoidance of lung tissue damage.</p

Myeloid PPAR-γ deficiency leads to unresolved persistent fibrosis.

(A) WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were infected with influenza virus. H&E staining of lung sections from WT or PPAR-γ cKO mice at day 60 p.i. (B) WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were treated with saline (mock) or infected with influenza as indicated. Collagen content in the whole lungs from indicated groups mice was measured by Hydroxyproline assay at day 60 p.i. (C) WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were infected with influenza virus. Lung fibrotic genes expression was measured by QIAGEN Fibrosis RT2 RT-PCR array at day 60 p.i. Red dots, genes upregulated in PPAR-γ cKO lungs, green dots, genes downregulated in PPAR-γ cKO lungs. (D) WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were treated with saline (mock) or infected with influenza virus as indicated. Collagen content in the lungs from indicated groups mice was measured by Hydroxyproline assay at day 90 p.i. (E) WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were infected with influenza virus. Lung fibrotic genes expression was measured by QIAGEN Fibrosis RT2 RT-PCR array at day 90 p.i. Red dots, genes upregulated in PPAR-γ cKO lungs, green dots, genes downregulated in PPAR-γ cKO lungs. Data are representative of two independent experiments (3–5 mice/group/experiment). Statistical differences are indicated. *, P < 0.05 (one-way ANOVA).</p

Myeloid PPAR-γ deficiency causes impaired lung inflammation resolution and enhanced collagen deposition.

WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were infected with influenza virus. Mice were sacrificed for various analysis at 30 d.p.i. (A) Host morbidity (% of initial weight) was monitored every other day till 30 d.p.i. (B) H.E stainning of lung sections collected from WT or PPAR-γ cKO mice. Low magnitude of whole left lung section image and high magnitude of indicated section images are depicted. (C) Lung cells from WT or PPAR-γ cKO mice were stained with CD64, Ly6G, Siglec F and CD11b and analyzed by flow cytometry. CD64+ macrophages were then subdivided into Siglec Fhi CD11blow and Siglec Flow CD11bhi populations. (D) Total numbers of CD64+ macrophages in the lungs from WT or PPAR-γ cKO mice were enumerated by flow cytometry. (E) Percentages of Siglec Fhi CD11blow or Siglec Flow CD11bhi macrophage populations were determined by flow cytometry. (F) Il1b, Tgfb1, Il6 and Ccl2 gene expression levels in the lungs from WT or PPAR-γ cKO mice were determined by realtime RT-PCR. (G) ATII gene, Sftpb and Abca3, expression levels in the lungs from WT or PPAR-γ cKO mice were determined by realtime RT-PCR. (H) Collagen content in the lungs from WT or PPAR-γ cKO mice was measured by Hydroxyproline assay. Data are representative of at least two independent experiments (n = 2–4 mice/group/experiment). Statistical differences are indicated. *, P < 0.05 (two tailed t-test).</p

Mice with myeloid PPAR-γ deficiency were more resistant to bleomycin damage.

WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were inoculated with bleomycin (0.072 Unit/mouse). (A) Host morbidity (% of initial weight loss) was monitored following bleomycin treatment. (B) Collagen content in whole lungs from WT or PPAR-γ cKO mice was measured by Hydroxyproline assay at 42 days after bleomycin treatment. Data are representative of two independent experiments (3–5 mice/group/experiment). Statistical differences are indicated. *, P < 0.05. ns, not significant. (A. t test or B. one-way ANOVA).</p

PPAR-γ deficient macrophages exhibited pro-inflammatory and pro-fibrotic gene expression.

WT (Ppargfl/fl) or PPAR-γ cKO (PpargΔLyz2) mice were infected with influenza. Mice were sacrificed for macrophage isolation at 30 d.p.i. (A) Lung cells were stained with anti-CD45, anti-Ly6G, anti-MerTK and anti-CD64. MerTK+/CD64+ lung macrophages in CD45+ Ly6G- cells were sorted by flow cytometry for realtime RT-PCR analysis of gene expression. (B) Expression of Tnf, Ccl2, Il1b and Arg1 in lung macrophages (isolated from pooled 3–4 mice/group/experiment) by realtime RT-PCR. (C) Expression of growth factors Igf1, Pdgfa, Tgfb and Ctgf in sorted lung macrophages (isolated from pooled 3–4 mice/group/experiment) by realtime RT-PCR. (D) Expression of several tissue remodeling enzymes and fibrotic genes Mmp2, Mmp8, Mmp12, Mmp7, Mmp9 and Timp1 in sorted lung macrophages (isolated from pooled 3–4 mice/group/experiment) by realtime RT-PCR. (E) Heat-map expression of the pro-inflammatory and profibrotic genes shown in B-D in lung alveolar macrophages from adult WT or conditional PPAR-γ-deficient mice (PpargΔCD11c) in published microarray dataset (GSE60249). Data are representative of two independent experiments of pooled lung macrophages from 3–4 mice except (E).</p

Macrophages from human pulmonary fibrosis patients exhibited diminished PPARG and dysregulated pro-inflammatory and pro-fibrotic gene expression.

PPARG, pro-inflammatory and pro-fibrotic gene expression in lung macrophages isolated from 61 healthy volunteers or 23 pulmonary fibrosis patients in the GSE49072 microarray datasets. (A) PPARG expression in lung alveolar macrophages from healthy or pulmonary fibrosis patients. (B) MMP7, MMP12, TIMP1, CCL2 or PDGFA expression in lung alveolar macrophages from healthy or pulmonary fibrosis patients. Statistical values are indicated (two-tailed Mann-Whitney test).</p