Toxoplasma ROP16Ⅰ/Ⅲ ameliorated inflammatory bowel diseases via inducing M2 phenotype of macrophages

BACKGROUND Inflammatory bowel disease（IBD） is characterized by chronic and non-specific inflammation of the intestinal mucosa and mainly includes ulcerative colitis and Crohn’s disease.AIM To explore the beneficial effect of Toxo ROP16Ⅰ/Ⅲ-induced M2 phynotype macrophages in homeostasis of IBDs through downregulation of M1 inflammatory cells.METHODS RAW264.7 macrophages stimulated by lipopolysaccharide（LPS）（M1 cells） were co-cultured with Caco-2 cells as an inflammatory model of IBD in vitro.The expression of Toxo ROP16Ⅰ/Ⅲ was observed in RAW264.7 macrophages that were transfected with p EGFP-rop16Ⅰ/Ⅲ.The phenotypes of M2 and M1 macrophage cells were assessed by quantitative real-time reverse transcriptase polymerase chain reaction and the expression of tumor necrosis factor（TNF）-α,interleukin（IL）-1β,IL-6,transforming growth factor（TGF）-β1,IL-10,inducible nitric oxide synthase（i NOS）,and arginase-1（Arg-1） was detected.The expression of i NOS,Arg-1,signal transducer and activator of transcription 3（Stat3）,p-Stat3,Stat6,pStat6,programmed death ligand-2（PD-L2）,caspase-3,-8,and-9 was analyzed by Western blotting,and Griess assays were performed to detect nitric oxide（NO）.TNF-α,IL-1β,IL-6,TGF-β1,and IL-10 expression in the supernatants was detected by enzyme-linked immunosorbent assay,and Caco-2 cell apoptosis was determined by flow cytometry after mixing M1 cells with M2 cells in a Caco-2 cell co-culture system.RESULTS M1 cells exhibited significantly increased production of i NOS,NO,TNF-α,IL-1β,and IL-6,while Toxo ROP16Ⅰ/Ⅲ induced macrophage bias to M2 cells in vitro,showing increased expression of Arg-1,IL-10 and TGF-β1 and elevated production of p-Stat3 and p-Stat6.The mixed M1 and M2 cell culture induced by Toxo ROP16 Ⅰ/Ⅲ exhibited decreased production of NO and i NOS and upregulated expression of Arg-1 and PD-L2.Accordingly,Caco-2 cells became apoptotic,and apoptosis-associated proteins such as caspase-3,-8 and-9 were dampened during co-culture of M1 and M2 cells.Flow cytometry analysis showed that co-culture of M1 cells with Caco-2 cells facilitated the apoptosis of Caco-2 cells,but co-culture of M1 and M2 cells alleviated Caco-2 cell apoptosis.CONCLUSION Toxo ROP16 Ⅰ/Ⅲ-induced M2 macrophages inhibited apoptosis of Caco-2 cells caused by M1 macrophages.This finding may help gain a better understanding of the underlying mechanism and represent a promising therapeutic strategy for IBDs.

Endothelial progenitor cell-conditioned medium promotes angiogenesis and is neuroprotective after spinal cord injury

Endothelial progenitor cells secrete a variety of growth factors that inhibit inflammation, promote angiogenesis and exert neuroprotective effects. Therefore, in this study, we investigated whether endothelial progenitor cell-conditioned medium might have therapeutic effectiveness for the treatment of spinal cord injury using both in vitro and in vivo experiments. After primary culture of bone marrow-derived macrophages, lipopolysaccharide stimulation was used to classically activate macrophages to their proinflammatory phenotype. These cells were then treated with endothelial progenitor cell-conditioned medium or control medium. Polymerase chain reaction was used to determine mR NA expression levels of related inflammatory factors. Afterwards, primary cultures of rat spinal cord neuronal cells were prepared and treated with H2O2and either endothelial progenitor cell-conditioned medium or control medium. Hoechst 33258 and propidium iodide staining were used to calculate the proportion of neurons undergoing apoptosis. Aortic ring assay was performed to assess the effect of endothelial progenitor cell-conditioned medium on angiogenesis. Compared with control medium, endothelial progenitor cell-conditioned medium mitigated the macrophage inflammatory response at the spinal cord injury site, suppressed apoptosis, and promoted angiogenesis. Next, we used a rat model of spinal cord injury to examine the effects of the endothelial progenitor cell-conditioned medium in vivo. The rats were randomly administered intraperitoneal injection of PBS, control medium or endothelial progenitor cell-conditioned medium, once a day, for 6 consecutive weeks. Immunohistochemistry was used to observe neuronal morphology. Terminal deoxynucleotidyl transferase-mediated d UTP nick-end labeling assay was performed to detect the proportion of apoptotic neurons in the gray matter. The Basso, Beattie and Bresnahan Locomotor Rating Scale was used to evaluate the recovery of motor function of the bilateral hind limbs after spinal cord injury. Compared with the other two groups, the number of axons was increased, cavities in the spinal cord were decreased, the proportion of apoptotic neurons in the gray matter was reduced, and the Basso, Beattie and Bresnahan score was higher in the endothelial progenitor cell-conditioned medium group. Taken together, the in vivo and in vitro results suggest that endothelial progenitor cell-conditioned medium suppresses inflammation, promotes angiogenesis, provides neuroprotection, and promotes functional recovery after spinal cord injury.

nduction of M2-1ike macrophages in recipient NOD-scid mice by allogeneic donor CD4＋CD25＋ regulatory T cells

CD4＋CD25＋ regulatory T cells （Tregs） play an important role in maintaining host immune tolerance via regulation of the phenotype and function of the innate and adaptive immune cells. Whether allogeneic CD4＋CD25＋ Tregs can regulate recipient mouse macrophages is unknown. The effect of allogeneic donor CD4＋CD25＋ Tregs on recipient mouse resident F4/80＋macrophages was investigated using a mouse model in which allogeneic donor CD4＋CD25＋ Tregs were adoptively transferred into the peritoneal cavity of host NOD-scid mice. The phenotype and function of the recipient macrophages were then assayed. The peritoneal F4/80＋ macrophages in the recipient mice that received the allogeneic CD4＋CD25＋ Tregs expressed significantly higher levels of CD23 and programmed cell death-ligand I（PD-L1） and lower levels of CD80, CD86, CD40 and MHC II molecules compared to the mice that received either allogeneic CD4＋CD25- T cells （Teffs） or no cells. The resident F4/80＋ macrophages of the recipient mice injected with the allogeneic donor CD4＋CD25＋ Tregs displayed significantly increased phagocytosis of chicken red blood cells （cRBCs） and arginase activity together with increased IL-IO production, whereas these macrophages also showed decreased immunogenicity and nitric oxide （NO） production. Blocking arginase partially but significantly reversed the effects of CD4＋CD25＋ Tregs with regard to the induction of the M2 macrophages in vivo. Therefore, the allogeneic donor CD4＋CD25＋ Tregs can induce the M2 macrophages in recipient mice at least in part via an arginase pathway. We have provided in vivo evidence to support the unknown pathways by which allogeneic donor CD4＋CD25＋ Tregs regulate innate immunity in recipient mice by promoting the differentiation of M2 macrophages.

The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation

The transcription factor PU.1 is involved in regulation of macrophage differentiation and maturation.However,the role of PU.1 in alternatively activated macrophage(AAM)and asthmatic inflammation has yet been investigated.Here we report that PU.1 serves as a critical regulator of AAM polarization and promotes the pathological progress of asthmatic airway inflammation.In response to the challenge of DRA(dust mite,ragweed,and Aspergillus)allergens,conditional PU.1-deficient(PU/ER(T)^(+/-))mice displayed attenuated allergic airway inflammation,including decreased alveolar eosinophil infiltration and reduced production of IgE,which were associated with decreased mucous glands and goblet cell hyperplasia.The reduced asthmatic inflammation in PU/ER(T)^(+/-) mice was restored by adoptive transfer of IL-4-induced wild-type(WT)macrophages.Moreover,after treating PU/ER(T)^(+/-) mice with tamoxifen to rescue PU.1 function,the allergic asthmatic inflammation was significantly restored.In vitro studies demonstrate that treatment of PU.1-deficient macrophages with IL-4 attenuated the expression of chitinase 3-like 3(Ym-1)and resistin-like molecule alpha 1(Fizz-1),two specific markers of AAM polarization.In addition,PU.1 expression in macrophages was inducible in response to IL-4 challenge,whichwas associated with phosphorylation of signal transducer and activator of transcription 6(STAT6).Furthermore,DRAchallenge in sensitized mice almost abrogated gene expression of Ym-1 and Fizz-1 in lung tissues of PU/ER(T)^(+/-) mice compared with WT mice.These data,all together,indicate that PU.1 plays a critical role in AAM polarization and asthmatic inflammation.

Local administration of liposomal-based Plekhf1 gene therapy attenuates pulmonary fibrosis by modulating macrophage polarization

Idiopathic pulmonary fibrosis(IPF)is a fatal interstitial lung disease with limited therapeutic options.Macrophages,particularly alternatively activated macrophages(M2),have been recognized to contribute to the pathogenesis of pulmonary fibrosis.Therefore,targeting macrophages might be a viable therapeutic strategy for IPF.Herein,we report a potential nanomedicinebased gene therapy for IPF by modulating macrophage M2 activation.In this study,we illustrated that the levels of pleckstrin homology and FYVE domain containing 1(Plekhf1)were increased in the lungs originating from IPF patients and PF mice.Further functionality studies identified the pivotal role of Plekhf1 in macrophage M2 activation.Mechanistically,Plekhf1 was upregulated by IL-4/IL-13 stimulation,after which Plekhf1 enhanced PI3K/Akt signaling to promote the macrophage M2 program and exacerbate pulmonary fibrosis.Therefore,intratracheal administration of Plekhf1 siRNA-loaded liposomes could effectively suppress the expression of Plekhf1 in the lungs and notably protect mice against BLM-induced lung injury and fibrosis,concomitant with a significant reduction in M2 macrophage accumulation in the lungs.In conclusion,Plekhf1 may play a crucial role in the pathogenesis of pulmonary fibrosis,and Plekhf1 siRNA-loaded liposomes might be a promising therapeutic approach against pulmonary fibrosis.