Sectm1a Facilitates Protection against Inflammation-Induced Organ Damage through Promoting TRM Self-Renewal

Abstract

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

Keywords: IL-4; LPS; Sectm1a; T helper cell; acute inflammation; proliferation; tissue-resident macrophage.

Graphical abstract

Figure 1

Loss of Sectm1a Aggravates Endotoxin-Induced Multi-organ Damage in Mice (A–C) After the administration of LPS (10 μg/g BW) to WT mice, the spleen (A), lung (B), and heart (C) were collected at indicated time points. Gene expression of Sectm1a in these tissues was determined by qRT-PCR (n = 4). (D–F) Sectm1a-KO mouse was generated as shown in Figure S2. At 24 h post-LPS (10 μg/g) or PBS injection, the spleen-to-body weight ratio (D) in WT and Sectm1a-KO mice was quantified, and the percentage increase (E) induced by LPS was calculated (n = 17–21). The ratio of spleen wet weight to dry weight (F) was also analyzed at the same time point post-LPS treatment (n = 6–9). (G) LPS-induced pulmonary vascular leakages were assessed by the extravasation of EB at 24 h post-LPS (10 μg/g) treatment. EB accumulation in different groups was normalized to control (n = 6–9). (H) Following 24 h of LPS (10 μg/g) treatment, ratios of lung wet weight to dry weight in WT and Sectm1a-KO mice were compared (n = 8–9). (I and J) Representative H&E staining images of lung sections at 24 h after PBS or LPS injection (I) are shown at ×200 original magnification. Scale bars, 50 μm. Lung injury scores (J) were assessed (n = 5–6). (K and L) Cardiovascular permeability was assessed by quantifying the extravasation of EB in the heart at 24 h post-treatment of PBS or LPS (10 μg/g). Blue represents DAPI, and red represents EB. Scale bars, 50 μm. n = 5–6. Data in all panels were pooled from at least two independent experiments. All results are presented as mean ± SEM and analyzed by Student’s t test, one-way ANOVA, or two-way ANOVA.

Figure 2

Sectm1a Deficiency Further Diminishes Tissue-Resident Macrophages in the Spleen and Lung after LPS Injection The whole spleen and lung tissues were collected from WT and Sectm1a-KO mice at 18 h post-PBS or LPS (10 or 20 μg/g) injection and subjected to flow cytometry analyses. (A) Representative flow cytometry plots of red pulp macrophages (CD45.2⁺ Lin⁻ CD11c⁻ F4/80²⁺ CD11b^low/− cells, blue gate) and splenic monocyte (CD45.2⁺ Lin⁻ CD11c⁻ F4/80⁺ CD11b²⁺ cells, red gate) following PBS or LPS (10 μg/g) treatment. Gating strategy is shown in Figure S3A. (B) Percentages of RPM population in WT and KO spleen were summarized following PBS or LPS treatment (n = 6). (C) The percentage change of the RPM population in the spleen of WT mice in response to different doses (10 μg/g, 20 μg/g) of LPS (n = 8–10). (D) Calculation of the percentage of splenic monocytes in WT and Sectm1a-KO mice after PBS or LPS (10 μg/g) treatment (n = 7–11). (E) Quantification percentages of released monocytes in WT and KO mice with the formula: Percentage of released monocytes = [(percentage of monocytes in PBS group) − (percentage of monocytes remaining in LPS group)]/(percentage of monocytes in PBS group) (n = 11). (F–H) Representative flow cytometry plots of AMs (F) (CD45.2⁺ Ly-6G⁻ CD3⁻ CD19⁻ MerTK⁺ CD64⁺ CD11c⁺ Siglec-F⁺ CD11b⁻) in the lung of WT and Sectm1a-KO mice after PBS or LPS injection. Gating strategy was shown in Figure S3B. Percentages of AMs in total live cells (G) and percentages of AMs in live cells excluding neutrophils (H) were quantified (n = 8). (I and J) Representative flow cytometry plots (I) of inflammatory monocytes (CD45.2⁺ Ly-6G⁻ CD3⁻ CD19⁻ MerTK⁻ Siglec-F⁻ CD64⁺ CD11b⁺ Ly-6C⁺) in the lung following PBS or LPS administration. Percentages of infiltrated inflammatory monocytes in total live cells (J) are shown (n = 5). (K) Flow cytometry showing the infiltration of neutrophil (CD45.2⁺ Ly-6G⁺ CD11b⁺ Ly-6C⁺) in the lung after LPS injection. (L) Percentages of infiltrated neutrophils in total live cells (n = 8).

Figure 3

Loss of Sectm1a Exacerbates LPS-Induced Reduction of Cardiac-Resident Macrophages (CRMs) in the Heart The whole hearts were collected from WT and Sectm1a-KO mice at 18 h after PBS or LPS (10 μg/g) injection and were subjected to flow cytometry analyses. (A and B) Representative flow cytometry plots of CRM (CD45.2⁺ Ly-6G⁻ F4/80⁺ CD64⁺ CD11b⁺ Ly-6C⁻) (green gate) in WT and Sectm1a-KO mice in response to endotoxin (A). Gating strategy is shown in Figure S3C. Percentages of CRM (B) in the total macrophage and monocyte population (n = 8). (C–E) Representative flow cytometry plots of inflammatory monocytes (CD45.2⁺ Ly-6G⁻ F4/80⁺ CD64⁺ CD11b⁺ Ly-6C⁺ MHCII⁻) in the heart following LPS administration (C). Percentages of infiltrated inflammatory monocytes (D) in the total macrophage and monocyte population (n = 8). Comparative analysis of the ratio of inflammatory monocytes to CRM (E) in WT and Sectm1a-KO mice after LPS injection (n = 8). (F and G) Representative flow cytometry plots (F) and ratios of CCR2⁺ to CCR2⁻ CRMs (G) in WT and Sectm1a-KO mice after PBS or LPS injection (n = 7). Gating strategy is shown in Figure S3C. (H and I) Flow cytometry showing the infiltration of neutrophils (CD45.2⁺ CD11b⁺ Ly-6G⁺ Ly-6C⁺) in the heart (H) after PBS or LPS injection. Percentages of infiltrated neutrophils in total leukocytes of heart (n = 8) (I). Data in all panels were pooled from at least two independent experiments. All results are presented as mean ± SEM and analyzed by Student’s t test or one-way ANOVA.

Figure 4

Sectm1a Deficiency Exacerbates Endotoxin-Induced Proliferation Suppression in Tissue-Resident Macrophages The spleen, lung, and heart in WT and Sectm1a-KO mice were collected and subjected to flow cytometry analysis at 16 h after PBS or LPS (10 μg/g) administration. (A, D, and G) Representative flow cytometry plots showing Ki-67⁺ RPMs (A), AMs (D), and CRMs (G), respectively. (B, E, and H) Percentages of Ki-67⁺ RPMs (B), AMs (E), and CRMs (H) after PBS or LPS injection (n = 5–10). (C) Immunofluorescent staining of spleen sections with F4/80 (blue) and Ki-67 (magenta) at 16 h after PBS or LPS treatment. Scale bar: 50 μm. (F and I) Quantification of mean fluorescence intensity (MFI) in Ki-67⁺ AMs (F) and CRMs (I) (n = 8–10). (J and K) Percentages of Ki-67⁺ cell population in CCR2⁻ (J) and CCR2⁺ (K) CRMs following PBS or LPS injection (n = 7–8). Data in all panels were pooled from at least two independent experiments. All results are presented as mean ± SEM and analyzed by Student’s t test or one-way ANOVA.

Figure 5

rSectm1a Promotes the Proliferation of Tissue-Resident Macrophages through Boosting the Expansion of T Helper Cells (A) RPM and naive CD4 T cell co-culture experiment design. (B) Representative bright-field image and immunofluorescence stain image of RPMs after co-culturing with IgG2a-treated or rSectm1a-treated naive CD4 T cells in the absence or presence of LPS (red represents F4/80; blue represents DAPI). Scale bars, 100 μm. (C) The proliferation of RPMs was assessed by counting cell numbers per microscopic field. All determinations were performed for at least three independent experiments (n = 8). (D and E) Representative immunofluorescence stain images (D) and quantification (E) of EdU-positive RPMs under co-culture condition following different treatments. Results are shown as means ± SEM of four independent experiments, and 200–300 cells were analyzed per experiment (n = 4). Scale bars, 100 μm. (F) Percentages of CD4⁺ IFN-γ⁺ T cell (Th1) and CD4⁺ IL-4⁺ T cell (Th2) after co-culture with RPMs in the absence or presence of LPS were shown in representative dot plots. (G–I) The intracellular cytokine assays for IFN-γ and IL-4 in CD4 T cell isolated from the spleen of WT or KO mice after LPS injection (G). (H and I) Percentages of CD4⁺ IL-4⁺ T cell (Th2) (H) and CD4⁺ IFN-γ⁺ T cell (Th1) (I) in vivo in response to endotoxin treatment (n = 4). (J–L) After co-culture, cytokine levels of IL-4 (J), IL-5 (K), and GM-CSF (L) in cell culture supernatants were measured by using Luminex technology (n = 3–4). (M–O) At 12 h post-LPS (10 μg/g) injection, cytokine levels of IL-4 (M), GM-CSF (N), and M-CSF (O) in sera were determined in WT and Sectm1a KO mice by using Luminex. (n = 5–8). All results are presented as mean ± SEM and analyzed by Student’s t test or one-way ANOVA.

Figure 6

GITR Is Critical for a Sectm1a-Mediated Regulatory Effect on the Activation of T Helper Cells (A–E) Following the same co-culture design in Figure 5A, RPMs isolated from WT mice were co-cultured with naive CD4 T cells isolated from GITR-KO mice. (A) Representative immunofluorescence stain image of RPMs after treatment (red represents F4/80, and blue represents DAPI). Scale bars, 50 μm. (B) The proliferation of RPMs was assessed by counting cell numbers per microscopic field. All determinations were performed for at least three independent experiments (n = 4). (C) Representative flow cytometry dot plots of CD4⁺ IFN-γ⁺ T cells (GITR^−/−) and CD4⁺ IL-4⁺ T cells (GITR^−/−) after co-culture with RPMs in the absence or presence of rSectm1a. (D and E) Percentages of CD4⁺ IL-4⁺ T cells (GITR^−/−) (D) and CD4⁺ IFN-γ⁺ T cells (GITR^−/−) (E) after co-culture (n = 4). (F–O) After intraperitoneal (i.p.) injection with LPS (10 μg/g), GITR-KO mice were immediately injected intravenously (i.v.) with IgG2a (125 μg/kg) (isotype) or rSectm1a (200 μg/kg). (F) Mouse survival was monitored for up to 96 h post-LPS treatment (n = 8–9). (G–O) At 18 h after last treatments, spleen and lung tissues were collected and subjected to flow cytometry experiments. (G–I) Representative flow cytometry plots (G) of RPMs (blue gate) and splenic monocytes (red gate) in GITR-KO mice with rSectm1a treatment or IgG2a treatment. The percentage of RPMs (H) and monocytes (I) in the live cells were calculated (n = 5–6). (J and K) Representative flow cytometry plots (J) and quantification (K) of AMs in the lung after different treatments. (L and M) Representative flow cytometry plots (L) and quantification (M) of inflammatory monocytes in the lung. (N and O) Representative flow cytometry plots (N) and quantification (O) of neutrophils in the lung. (P) After i.p. injection with LPS (15 μg/g), WT mice were immediately injected i.v. with IgG2a (125 μg/kg) (isotype) or rSectm1a (200 μg/kg) and monitored for survival up to 96 h post-LPS treatment (n = 10–16). (Q–W) WT mice were immediately injected i.v. with IgG2a (125 μg/kg) (isotype) or rSectm1a (200 μg/kg) following LPS (10 μg/g) i.p. injection. At 18 h after last treatments, the spleen and lung tissues were collected and subjected to flow cytometry experiments. (Q–S) Representative flow cytometry plots (Q) of RPMs (blue gate) and splenic monocyte (red gate) in WT mice with rSectm1a (or isotype) treatments and quantification of RPMs (R) and splenic monocytes (S) (n = 5). (T and U) Representative flow cytometry plots (T) and quantification of AMs (U) in the lung after different treatments (n = 5–6). (V and W) Representative flow cytometry plots (V) and quantification of neutrophils (W) in the lung (n = 5–6). Data in all panels were pooled from at least two independent experiments. All results are presented as mean ± SEM and analyzed by Mann-Whitney test, Student’s t test, or one-way ANOVA.

Figure 7

Tissue-Resident Macrophages Are Essential for Maintaining Local Vascular Integrity during Endotoxemia (A) Graphic illustration of mouse treatment scheme. To pre-deplete tissue-resident macrophages, we injected (i.v.) WT mice with clodronate-liposome (200 μL, clodronate 5 mg/mL). The control mice received vehicle-liposome (200 μL, no clodronate). After 3 days, mice were injected (i.p.) with LPS (5 μg/g). At 24 h after LPS injection, the spleen, lung, and heart were collected for the following experiments. (B) Representative flow cytometry plots showing the reduction of RPMs in the spleen (upper plot) and AMs in the lung (lower plot) on day 3 after clodronate liposome treatment. (C) The ratio of spleen wet weight to dry weight in control and macrophage pre-depleted mice was measured at 24 h after LPS treatment (n = 6). (D) Meanwhile, LPS-induced pulmonary vascular leakages were assessed by the extravasation of EB (n = 6). (E) Representative H&E staining images of lungs at 24 h after LPS injection were shown at ×200 original magnification; scale bars, 50 μm. (F) The lung injury scores were calculated (n = 5–6). (G and H) Cardiovascular permeability was assessed by quantifying the extravasation of EB in the heart at 24 h after LPS treatment. Frozen heart sections (G) were observed under a confocal LSM 710 microscope. Blue represents DAPI, and red represents EB; scale bars, 50 μm. The relative intensity of red fluorescence emitted by EB was quantified (H) with ImageJ software (n = 4–5). (I) Mouse cardiac endothelial cells (MCECs) (on the insert of transwell) were cultured in the presence or absence of RPMs on the bottom chamber for 4 days until the endothelial monolayer was established. Then fluorescein isothiocyanate (FITC)-dextran particle was added into the upper insert, and thrombin (3 U/mL) was used to stimulate MCECs for 1 h. The degree of EC monolayer leakage was determined by measuring the intensity of FITC-dextran fluorescence in the basolateral chamber (n = 4). (J–L) MCECs were cultured in the control medium (C-M) or RPM conditioned medium (RPM-M) for 1 day. qRT-PCR was used to determine mRNA levels of ZO-1 (J), VE-cadherin (K), and occludin (L), in the presence or absence of RPM conditioned medium (n = 4). (M) MCECs were cultured with control medium or RPM conditioned medium for 3 days to reach the monolayer on the coverslip. Representative images of immunofluorescent staining for ZO-1 (green) at 1 h after thrombin (3 U/mL) or PMA (40 ng/mL) treatment. White arrows indicate the integrity linear pattern of ZO-1. Yellow arrows present the fragmentations and depletions of ZO-1. Scale bars, 20 μm. All results are presented as mean ± SEM and analyzed by Student’s t test or one-way ANOVA.