Fig 2: Ifp35-/- mice have attenuated clinical symptoms after lethal influenza virus infection(A–C) Body weight loss (percent) (A), clinical score (B), and survival rate (percent) (C) of C57BL/6J wild-type mice and Ifp35-/- mice infected with PR8 (2LD50); n = 12.(D) H&E staining of the lung tissue. Lymphocyte and macrophage infiltration in the multifocal lesions is indicated by black arrow. Fibrin exudation in the alveolar space and alveolar septum is indicated by a red arrow. Degenerative and desquamated bronchial epithelial cells are indicated by a green arrow. Scale bars, 100 µm. The picture is shown as original magnifications ×100.(E) The pathological score of the lung tissue in Ifp35-/- mice.(F and G) Serum TNF and IL-6 (pg/mL) in mice 3 days after PR8 infection, detected by ELISA; n = 5.(H) qRT-PCR results of PR8 viral genome RNA (nucleoprotein, NP) in the lung tissue of C57BL/6J wild-type mice and Ifp35-/- mice 3 days after infection; n = 6.Data in (E)–(H) are mean values ± SEM. Significance in (A) and (B) was calculated with two-way ANOVA with Sidak’s post-test. Significance in (C) was determined using log rank test. Significance in (E)–(H) was assessed by Mann-Whitney U test. *p < 0.05,**p < 0.01, ***p < 0.001; ns, no significance.

Fig 3: Cholesterol metabolism drives regulatory B cell function.a, b Kinetics of IL10 mRNA transcript (a) and IL-10-secreted protein (b) expression at various time points in human B cells after TLR9 stimulation (n = 4 for mRNA or n = 4–5 for protein). IL10 mRNA was measured by qRT-PCR, and calculated relative to 18S. c Schematic protocol for the co-culture for T and B cells. Briefly, human B cells were stimulated through TLR9 ± atorvastatin (AT) ± mevalonate (MA) overnight, before washing and addition to anti-CD3/28 stimulated human CD4+ T cells for 4 days ± aIL-10 antibody. d IFN? production in human CD4+ T cells after co-culture with autologous TLR9-activated B cells (n = 6, pval = 0.03 and 0.02). IFN? suppression was calculated by ((x - y)/x)*100 where x = IFN? production by T cells alone, y = IFN? production in co-cultured T cells. e IL-10 expression in human B cells after stimulation through TLR9 ± AT ± MA (n = 7, pval = 0.003). f IL-10 mRNA expression over time in human B cells after stimulation through TLR9 ± AT ± MA (n = 4). g TNF expression in human B cells after stimulation through TLR9 ± AT ± MA (n = 7). h IL6 or LTA mRNA expression, relative to 18S, in human B cells after stimulation through TLR9 ± AT (n = 4). Each data point represents individual donors. All data presented are mean ± SD. Statistical testing in all figures was done by a Friedman’s test with Dunns’s multiple comparisons test, or for (f) a mixed-effects model with Dunnett’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, and all significant values are shown.

Fig 4: solTNFR1 and solTNFR2 are increased in COVID-19 patients. Soluble forms of TNF, TNFR1, and TNFR2 were quantified in the sera of COVID-19 patients and healthy donors (HD) by ELISA sandwich assay. A general comparison of solTNF, solTNFR1, and solTNFR2 levels between HD and COVID-19 was performed (A–C, respectively). In addition, a more detailed analysis between HD and groups of COVID-19 (mild, moderate, and severe) was performed to confirm if solTNF, solTNFR1, and solTNFR2 levels were different (D–F, respectively). Data are presented as median with interquartile range (solTNF: HD n = 16, mild n = 14, moderate n = 29, and severe n = 50; solTNFR1: HD n = 16, mild n = 13, moderate n = 24, and severe n = 49; solTNFR2: HD n = 18, mild n = 15, moderate n = 29, and severe n = 53), **** p < 0.0001, ** p < 0.01.

Fig 5: NPWT materials do not require direct contact with cells to suppress inflammatory activation. (a) Schematic of the transwell experiments, explaining the indirect culture setup and the workflow. (b) ELISA results of the transwell culture experiments for TNF-a and MCP-1 production by the macrophages. (c) qRT-PCR results of the transwell culture experiments for TNF, CCL2, and IL1B by the macrophages. RPL37A was used a housekeeping gene for normalization. Both experiments used empty transwell (no material) controls. All bar plots are represented as the mean of 3 independent experiments, with error indicating the S.E.M. Abbreviations are as follows: VC—Veraflo Cleanse, GF—Granufoam, CT—Cotton gauze, Ctrl—No material control. * indicates p < 0.05 as tested using one-way ANOVA with multiple comparisons.