Diabetes-specific microvascular disease is a leading cause of blindness, renal failure and nerve damage. Epidemiological data demonstrated that the high morbidity of T2DM occurs as a result of obesity and gradually develops into serious complications. To date, the mechanisms that underlie this observation are still ill-defined. In view of the effect of obesity on the gut microflora, Lepr^db/db mice underwent antibiotic treatment and microbiota transplants to modify the gut microbiome to investigate whether microbes are involved in the development of diabetic nephropathy (DN) and/or diabetic retinopathy (DR). The mouse feces were collected for bacterial 16S ribosomal RNA gene sequencing. Cytokines including TNF-α, TGF-β1, IFN-γ, IL-1β, IL-6, IL-17A, IL-10, and VEGFA were detected by enzyme-linked immunosorbent assay (ELISA), flow cytometry, real-time PCR and immunofluorescent assay. Eyes and kidney were collected for histopathological assay. Intestinal permeability was also detected using Evans Blue. The results showed that obesity influenced metabolic variables (including fast/fed glucose, insulin, and triglyceride), retinopathy and nephropathy, and the gut microbiota. Obesity mainly reduced the ratio of Bacteroidetes/Firmicutes and influenced relative abundance of Proteobacteria, Actinobacteria, and Spirochetes. Obesity also increased intestinal permeability, metabolic endotoxemia, cytokines, and VEGFA. Microbiota transplants confirm that obesity aggravates retinopathy and nephropathy through the gut microbiota. These findings suggest that obesity exacerbates retinopathy and nephropathy by inducing gut microbiota dysbiosis, which further enhanced intestinal permeability and chronic low-grade inflammation.

Background: Toxoplasma gondii infection during pregnancy can lead to fetal defect(s) or congenital complications. The inhibitory molecule B7-H4 expressed on decidual macrophages (dMφ) plays an important role in maternal–fetal tolerance. However, the effect of B7-H4 on the function of dMφ during T. gondii infection remains unclear. Methods: Changes in B7-H4 expression on dMφ after T. gondii infection were explored both in vivo and in vitro. B7-H4^-/- pregnant mice (pregnant mice with B7-H4 gene knockout) and purified primary human dMφ treated with B7-H4 neutralizing antibody were used to explore the role of B7-H4 signaling on regulating the membrane molecules, synthesis of arginine metabolic enzymes and cytokine production by dMφ with T. gondii infection. Also, adoptive transfer of dMφ from wild-type (WT) pregnant mice or B7-H4^-/- pregnant mice to infected B7-H4^-/- pregnant mice was used to examine the effect of B7-H4 on adverse pregnancy outcomes induced by T. gondii infection. Results: The results illustrated that B7-H4^-/- pregnant mice infected by T. gondii had poorer pregnancy outcomes than their wild-type counterparts. The expression of B7-H4 on dMφ significantly decreased after T. gondii infection, which resulted in the polarization of dMφ from the M2 toward the M1 phenotype by changing the expression of membrane molecules (CD80, CD86, CD163, CD206), synthesis of arginine metabolic enzymes (Arg-1, iNOS) and production of cytokines (IL-10, TNF-α) production. Also, we found that the B7-H4 downregulation after T. gondii infection increased iNOS and TNF-α expression mediated through the JAK2/STAT1 signaling pathway. In addition, adoptive transfer of dMφ from a WT pregnant mouse donor rather than from a B7-H4^-/- pregnant mouse donor was able to improve adverse pregnancy outcomes induced by T. gondii infection. Conclusions: The results demonstrated that the downregulation of B7-H4 induced by T. gondii infection led to the dysfunction of decidual macrophages and contributed to abnormal pregnancy outcomes. Moreover, adoptive transfer of B7-H4⁺ dMφ could improve adverse pregnancy outcomes induced by T. gondii infection. Graphical

Background: Women in early pregnancy infected by Toxoplasma gondii may have severe adverse pregnancy outcomes, such as spontaneous abortion and fetal malformation. The inhibitory molecule T cell immunoglobulin and mucin domain 3 (Tim-3) is highly expressed on decidual dendritic cells (dDCs) and plays an important role in maintaining immune tolerance. However, whether T. gondii infection can cause dDC dysfunction by influencing the expression of Tim-3 and further participate in adverse pregnancy outcomes is still unclear. Methods: An abnormal pregnancy model in Tim-3-deficient mice and primary human dDCs treated with Tim-3 neutralizing antibodies were used to examine the effect of Tim-3 expression on dDC dysfunction after T. gondii infection. Results: Following T. gondii infection, the expression of Tim-3 on dDCs was downregulated, those of the pro-inflammatory functional molecules CD80, CD86, MHC-II, tumor necrosis factor-α (TNF-α), and interleukin-12 (IL-12) were increased, while those of the tolerant molecules indoleamine 2,3-dioxygenase (IDO) and interleukin-10 (IL-10) were significantly reduced. Tim-3 downregulation by T. gondii infection was closely associated with an increase in proinflammatory molecules and a decrease in tolerant molecules, which further resulted in dDC dysfunction. Moreover, the changes in Tim-3 induced by T. gondii infection further reduced the secretion of the cytokine IL-10 via the SRC-signal transducer and activator of transcription 3 (STAT3) pathway, which ultimately contributed to abnormal pregnancy outcomes. Conclusions: Toxoplasma gondii infection can significantly downregulate the expression of Tim-3 and cause the aberrant expression of functional molecules in dDCs. This leads to dDC dysfunction, which can ultimately contribute to abnormal pregnancy outcomes. Further, the expression of the anti-inflammatory molecule IL-10 was significantly decreased by Tim-3 downregulation, which was mediated by the SRC-STAT3 signaling pathway in dDCs after T. gondii infection.

Background: Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression. Methods: LILRB4-deficient (LILRB4^−/−) pregnant mice infected with T. gondii with associated adverse pregnancy outcomes, and anti-LILRB4 neutralized antibodies-treated infected human dMDSCs were used in vivo and in vitro experiments, respectively. The aim was to investigate the effect of LILRB4 expression on dMDSC dysfunction induced by T. gondii infection. Results: Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection. Conclusions: The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection. Graphical