Fig 2: Myeloid EPOR deficiency aggravates inflammation in lung injury. (A) WT mice (n = 5) were treated with 3 mg/kg LPS (i.t.) at the indicated times (days 0, 1, 2, 3, and 5). The number of F4/80+EPOR+ macrophages in BALF was measured by FACS. (B) EPOR cKO and WT mice were challenged with 10 mg/kg LPS i.t., and survival was monitored (n = 6). (C) EPOR cKO and WT mice were treated with 3 mg/kg LPS (i.t.) at the indicated times. Change in body weight was monitored over 6 days (n = 4). (D) Lung tissue samples from mice with LPS administration for 48 h were dissected and subjected to H&E staining. Lung injury scores in each group were analyzed (n = 4). Scale bar: 150 µm. (E) EPOR cKO and WT mice were treated with 1 mg/kg eCIRP (i.t.). Lung tissue samples from mice with eCIRP administration for 48 h were dissected and subjected to H&E staining. Lung injury scores in each group were analyzed (n = 4). Scale bar: 150 µm. WT and EPOR cKO mice were treated with 3 mg/kg LPS (i.t.) at the indicated times (days 0, 1, 2, 3, and 5). (F) The MFI of CD80 and the percentage of CD206+ cells in BALF macrophages were measured by FACS at the indicated time (n = 3). (G) The concentrations of TNF-α, IL-6, and IL-10 in BALF at the indicated time was measured (n = 3). Data are representative of at least two independent experiments. Results were expressed as mean ± SD. *P < 0.05, **P < 0.01 compared to the WT group at indicated time. Statistics: Log-rank test (B) or unpaired two-tailed Student’s t-test (C–G). EPOR, erythropoietin receptor; LPS, lipopolysaccharide; eCIRP, extracellular cold-inducible RNA-binding protein; WT, wild type; BALF, bronchoalveolar lavage fluid; MFI, mean fluorescence intensity; FACS, fluorescence activated cell sorter.

Fig 3: Azapeptide MPE-001 regulates inflammatory profile of subretinal MPs and reduces photoreceptor degeneration. (A–H) CD36+/+ mice were illuminated for 5 days with blue light. Subcutaneous injections of 289 nmol/kg MPE-001 were administered after 1 day illumination and pursued daily for 7 consecutive days. (A) Upper panel: area of retinal cryosections were microdissected between ONL and RPE and visualized with green circles. Lower panel: bar graphs of IBA-1, iNOS, IL-6, IL-10 and IL-12 mRNA expression levels in microdissected retinal cryosections. Cytokine analysis were normalized to 18 s rRNA. (B) Subretinal MPs stained for F4/80 (red) and iNOS (green) on RPE flat mounts as assessed by confocal microscopy. (C) Subretinal MPs stained with IBA-1 (red) and IL-12 (green) on RPE flat mounts as assessed by confocal microscopy. (D) Subretinal MPs stained with anti-IBA-1 (green) and anti-CD206 (red) antibodies on RPE flat mounts as assessed by confocal microscopy. Scale bar: 15 µm. (E) Percentage of subretinal MPs (IBA-1+ or F4/80+) expressing INOS, IL-12 or CD206. (F) Confocal microscopy of neuroretinal flat mounts (photoreceptors side) and (G) retina cryosections from illuminated CD36+/+ mice treated or not with MPE-001 stained with fluorescein PNA (green) and anti-S-opsin (red) antibody; nuclei were counterstained with dapi (blue). Magnifications of white square show length of cone outer segment with S-opsin distribution. Scale bar: 10 µm. (H) Retinal cryosections stained with TUNEL (green). Nuclear layers were stained with DAPI (blue). Scale bar: 25 µm. (I) Percentage of TUNEL+ cells in ONL cryosections of the retina. In (A and E) unpaired t-test was performed. *P < 0.05, **P < 0.01 and ***P < 0.001 vs illuminated group (n = 3-4 mice/group). In I, one-way ANOVA test with Newman-Keuls for multiple comparison was performed. #P < 0.05 and ###P < 0.001 vs no illumination group. ? P < 0.05 vs illuminated group (n = 3-4 mice/group). Data are shown as mean ± S.E.M. RGC: Retinal Ganglion Cell. INL: Inner Nuclear Layer. ONL: Outer Nuclear Layer. RPE: Retinal Pigment Epithelium. CHR: Choroid.

Fig 4: Flow cytometry analysis of the peritoneal exudate cells from Ehrlichia-infected mice. (A) Flow cytometry gate strategy for the analysis of the peritoneal exudate cells were carried out as in Fig. 1. CD3-negative cells were plotted as CD11b × F4/80 for the analysis of peritoneal macrophages defined as F4/80+CD11b+ cells. (B) F4/80+CD11b+ cells were also addressed for the intracellular expression of iNOS as represented by flow histograms. Values represent percentages in the assigned regions. Quantification of the analyzed cell sub-populations in the different studied groups, naive, E. muris (EM) and Ixodes Ovatus Ehrlichia (IOE) are shown in each case. Values are expressed as mean and standard deviation of percentage. Asterisks represent relevant statistical difference between groups. Data is representative of three experimental sets performed individually with n = at least three mice per group in each experimental run.

Fig 5: Assessment of autophagic activity and mTORC1 signaling in macrophage polarization during infection with Ehrlichia. Assessment of autophagic activity in bone marrow-derived macrophages (BMM) infected with E. muris or IOE was carried out by western blot analysis. BMM were incubated or not with bacteria for 24 h prior to obtaining protein extracts from the different studied groups (E. muris-, IOE-infected and uninfected BMM). Flow cytometry was used to assess the impact of mTORC1 signaling in macrophage polarization via stimulation of BMM with mTORC1 inhibitor, rapamycin (Rapa) 10 μM, by the time of infection. (A) Measurement of LC3I and LC3II in the protein extracts accompanied by quantitative analysis of LC3II/LC3I ratio of conversion. Ratio of conversion was assessed after normalization of densitometry values based on the levels of β-actin. Levels of p62 and downstream targets of mTORC1 signaling, pS6 and p4E-BP1, in the considered protein extracts are also shown. Quantitative analyses expressed as relative densitometry are shown for each marker. Full-length blots are presented in Supplementary Fig. S6, from which data were cropped and assembled. (B) Flow cytometry dot plot showing region of considered BMM (CD11b+ F4/80+ cells gated on live cells) in which the percentage of MHC class IIhigh-expressing cells and the mean fluorescence intensity (MFI) of surface CD206 were measured. Values near the regions represent percentage and numbers above histograms indicate MFI values. (C) Quantitative analysis for flow cytometric determinations. All quantitative analyses are exhibited as mean and standard deviation. Asterisks represent relevant statistical difference between groups. Data is representative of at least two independent experiments.