Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.A.B.G.-R. is supported by the Sara Borrell Program (CD11/00165) and CIBER de Enfermedades Cardiovasculares (CB16/11/00286). H.R. was supported by a short-term EMBO fellowship (EMBOSTF7204). I.J.M. was supported by a Marie-Sklodowska-Curie postdoctoral fellowship (PIEF-GA-2012-330728). N.M. is supported by Swiss National Science Foundation grant 31003A_15972 and the European Research Council (starting grant 337703–zebra–Heart). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovacio´ n, y Universidades (MCNU), and the Pro CNIC Foundation AGRADECIENTOS: ProCNIC; Severo Ochoa (SEV-2015-0505)S

Differential modulation of pulmonary caspases: Is this the key to Ureaplasma-driven chronic inflammation?

Although accepted agents in chorioamnionitis and preterm birth, the role of Ureaplasma species (spp.) in inflammation-driven morbidities of prematurity, including the development of bronchopulmonary dysplasia, remains controversial. To add to scarce in vitro data addressing the pro-inflammatory capacity of Ureaplasma spp., pulmonary epithelial-like A549 cells and human pulmonary microvascular endothelial cells (HPMEC) were incubated with Ureaplasma (U.) urealyticum, U. parvum, and Escherichia coli lipopolysaccharide (LPS). Ureaplasma isolates down-regulated caspase mRNA levels in A549 cells (caspase 8: p<0.001, 9: p<0.001, vs. broth), while increasing caspase protein expression, enzyme activity, and cell death in HPMEC (active caspase 3: p<0.05, caspase 8: p<0.05, active caspase 9: p<0.05, viability: p<0.05). LPS, contrarily, induced caspase mRNA expression in HPMEC (caspase 3: p<0.01, 4: p<0.001, 5: p<0.001, 8: p<0.001, vs. control), but not in A549 cells, and did not affect enzyme activity or protein levels in either cell line. LPS, but neither Ureaplasma isolate, enhanced mRNA expression of pro-inflammatory interleukin (IL)-6 in both A549 (p<0.05, vs. control) and HPMEC (p<0.001) as well as tumor necrosis factor-α (p<0.01), IL-1β (p<0.001), and IL-8 (p<0.05) in HPMEC. We are therefore the first to demonstrate a differential modulation of pulmonary caspases by Ureaplasma spp. in vitro. Ureaplasma-driven enhanced protein expression and activity of caspases in pulmonary endothelial cells result in cell death and may cause structural damage. Down-regulated caspase mRNA in pulmonary epithelial cells, contrarily, may indicate Ureaplasma-induced inhibition of apoptosis and prevent effective immune responses. Both may ultimately contribute to chronic Ureaplasma colonization and long-term pulmonary inflammation

<i>Ureaplasma</i>-driven mRNA expression of pro-inflammatory cytokines in A549 cells and HPMEC.

In A549 cells, mRNA levels of TNF-α (a), IL-1β (b), IL-6 (c), and IL-8 (d) were assessed via qRT-PCR after 4 and 30 h stimulation. Similarly, mRNA expression in HPMEC was determined for TNF-α (e), IL-1β (f), IL-6 (g), and IL-8 (h). Data are presented as means ± SD from n ≥ 3 independent experiments. LPS stimulated cells were compared vs. control, cells exposed to Ureaplasma isolates vs. control and vs. broth. * p p p p Ureaplasma urealyticum serovar 8, SV3: Ureaplasma parvum serovar 3.</p

<i>Ureaplasma</i>-driven caspase mRNA and protein responses in HPMEC.

Following an incubation period of 4 and 30 h, mRNA levels of caspase (CASP) 3 (a), caspase 4 (b), caspase 5 (c), caspase 8 (d), and caspase 9 (e) were obtained via qRT-PCR, and relative expression was calculated using the ΔΔCT method. The percentage of viable, active caspase 3 (f), caspase 8 (g), and active caspase 9 (h) positive HPMEC was determined by flow cytometry after 24 h stimulation (the respective gating strategy is illustrated in S1 Fig). Data are presented as means ± SD from n ≥ 3 independent experiments. Cells stimulated with LPS were compared vs. control, Ureaplasma exposed cells vs. control and vs. broth. * p p p p p Ureaplasma urealyticum serovar 8, SV3: Ureaplasma parvum serovar 3.</p

Cascade of caspase activation and potential pathways influenced by <i>Ureaplasma</i> spp.

Simplified scheme (a) depicting caspase activation processes. Multiple trigger factors can initiate apoptosis and pyroptosis. Under engagement of several additional proteins not mentioned here, initiator caspases 4 and 5 for pyroptosis or 8 and 9 for apoptosis are produced and activated. These subsequently activate effector caspases 1 or 3. Caspases are activated by cleavage or dimerization, often followed by a maturational process. Control mechanisms confine programmed cell death [26, 27, 43–45]. The potential influence of Ureaplasma spp. on apoptosis according to our results is illustrated in (b). Pathways affected by Ureaplasma isolates are marked in red. In A549 cells, Ureaplasma-triggered reduction of caspase 8 and 9 mRNA may result in an absent increase in protein production and active caspases. Ultimately, effector caspase 3 remains unactivated and apoptosis is impaired. In HPMEC, Ureaplasma spp. seem to enhance caspase 8 protein and caspase 9 activity. Both caspases may subsequently activate effector caspase 3 and induce apoptosis. It remains to be determined if the caspase 3 activation observed is directly due to Ureaplasma or a consequence of initiator caspase activation. Hypothesized underlying mechanisms Ureaplasma isolates may engage are indicated with a dashed line. Ⱶ inhibit / down-regulate; ← activate / up-regulate. CASP: caspase; IFN: interferon; LPS: lipopolysaccharide; NK: natural killer; TLR: toll-like receptor; TNF: tumor necrosis factor. Illustrations: https://smart.servier.com/.</p

Primers for qRT-PCR.

Primers for qRT-PCR.</p

<i>Ureaplasma</i>-driven cell death in pulmonary epithelial and endothelial cells.

Following a 24 h stimulation period, numbers of dead cells were determined for different conditions using flow cytometry and a viability dye staining dead cells. Results for A549 cells are shown in (a), whereas (b) depicts the percentage of dead HPMEC. Data are presented as means ± SD and were obtained from n = 3 individual experiments. Cells stimulated with LPS were compared vs. control, cells exposed to Ureaplasma spp. vs. control and vs. broth. * p p Ureaplasma urealyticum serovar 8, SV3: Ureaplasma parvum serovar 3.</p

<i>Ureaplasma</i>-driven caspase mRNA and protein responses in A549 cells.

After 4 and 30 h stimulation of A549 cells, mRNA levels of caspase (CASP) 3 (a), caspase 4 (b), caspase 5 (c), caspase 8 (d), and caspase 9 (e) were assessed via qRT-PCR, and relative expression was calculated using the ΔΔCT method. After 24 h stimulation, the percentage of viable, active caspase 3 (f), caspase 8 (g), and active caspase 9 (h) positive A549 cells was determined by flow cytometry (the respective gating strategy is illustrated in S1 Fig). Data are shown as means ± SD and were obtained from n ≥ 3 independent experiments. Cells stimulated with LPS were compared vs. control, cells exposed to Ureaplasma isolates vs. control and vs. broth. * p p p p p p Ureaplasma urealyticum serovar 8, SV3: Ureaplasma parvum serovar 3.</p