ATG5-regulated CCL2/MCP-1 production in myeloid cells selectively modulates anti-malarial CD4+ Th1 responses.

Parasite-specific CD4+ Th1 cell responses are the predominant immune effector for controlling malaria infection; however, the underlying regulatory mechanisms remain largely unknown. This study demonstrated that ATG5 deficiency in myeloid cells can significantly inhibit the growth of rodent blood-stage malarial parasites by selectively enhancing parasite-specific CD4+ Th1 cell responses. This effect was independent of ATG5-mediated canonical and non-canonical autophagy. Mechanistically, ATG5 deficiency suppressed FAS-mediated apoptosis of LY6G- ITGAM/CD11b+ ADGRE1/F4/80- cells and subsequently increased CCL2/MCP-1 production in parasite-infected mice. LY6G- ITGAM+ ADGRE1- cell-derived CCL2 selectively interacted with CCR2 on CD4+ Th1 cells for their optimized responses through the JAK2-STAT4 pathway. The administration of recombinant CCL2 significantly promoted parasite-specific CD4+ Th1 responses and suppressed malaria infection. Conclusively, our study highlights the previously unrecognized role of ATG5 in modulating myeloid cells apoptosis and sequentially affecting CCL2 production, which selectively promotes CD4+ Th1 cell responses. Our findings provide new insights into the development of immune interventions and effective anti-malarial vaccines.Abbreviations: ATG5: autophagy related 5; CBA: cytometric bead array; CCL2/MCP-1: C-C motif chemokine ligand 2; IgG: immunoglobulin G; IL6: interleukin 6; IL10: interleukin 10; IL12: interleukin 12; MFI: mean fluorescence intensity; JAK2: Janus kinase 2; LAP: LC3-associated phagocytosis; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; pRBCs: parasitized red blood cells; RUBCN: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; STAT4: signal transducer and activator of transcription 4; Th1: T helper 1 cell; Tfh: follicular helper cell; ULK1: unc-51 like kinase 1.

CD8+ Trms against malaria liver-stage: prospects and challenges.

Attenuated sporozoites provide a valuable model for exploring protective immunity against the malarial liver stage, guiding the design of highly efficient vaccines to prevent malaria infection. Liver tissue-resident CD8+ T cells (CD8+ Trm cells) are considered the host front-line defense against malaria and are crucial to developing prime-trap/target strategies for pre-erythrocytic stage vaccine immunization. However, the spatiotemporal regulatory mechanism of the generation of liver CD8+ Trm cells and their responses to sporozoite challenge, as well as the protective antigens they recognize remain largely unknown. Here, we discuss the knowledge gap regarding liver CD8+ Trm cell formation and the potential strategies to identify predominant protective antigens expressed in the exoerythrocytic stage, which is essential for high-efficacy malaria subunit pre-erythrocytic vaccine designation.

Malaria sporozoites evade host complement attack.

Complement is the first line of the host innate immune response against bacterial and viral infections; however, its role in the development of the malaria liver stage remains undefined. We found that sporozoite infection by either a mosquito bite or intravenous injection activated systemic complement, but neither depletion of C3 nor knockout of C3 had a significant effect on malaria liver stage development. Incubation of mouse serum with trypsin-treated sporozoites, but not naive sporozoites, led to the deposition of a membrane attack complex (MAC) on the surface of sporozoites and greatly reduced the number of exo-erythrocytic forms (EEF). Further studies have shown that the recruitment of complement H factor (CFH) may be associated with the prevention of MAC deposition on the surface of naïve sporozoites. Our data strongly suggest that sporozoites can escape complement attacks and provide us with a novel strategy to prevent malaria infection.

Calcipotriol Attenuates Form Deprivation Myopia Through a Signaling Pathway Parallel to TGF-ß2-Induced Increases in Collagen Expression.

Purpose: To determine the role of calcipotriol, a vitamin D3 analogue, in myopia development and altering the expression of scleral α1 chain of type I collagen (Col1α1) in mice. We also aimed to identify if the signaling pathway mediating the above changes is different from the one involved in transforming growth factor ß2 (TGF-ß2)-mediated increases of COL1A1 in cultured human scleral fibroblasts (HSFs). Methods: C57BL/6J mice were either intraperitoneally injected with calcipotriol and subjected to form deprivation (FD) or exposed to normal refractive development for 4 weeks. Scleral vitamin D receptor (Vdr) expression was knocked down using a Sub-Tenon's capsule injection of an adeno-associated virus-packaged short hairpin RNA (AAV8-shRNA). Refraction and biometric measurements evaluated myopia development. A combination of knockdown and induction strategies determined the relative contributions of the vitamin D3 and the TGF-ß2 signaling pathways in modulating COL1A1 expression in HSFs. Results: Calcipotriol injections suppressed FD-induced myopia (FDM), but it had no significant effect on normal refractive development. AAV8-shRNA injection reduced Vdr mRNA expression by 42% and shifted the refraction toward myopia (-3.15 ± 0.99D, means ± SEM) in normal eyes. In HSFs, VDR knockdown reduced calcipotriol-induced rises in COL1A1 expression, but it did not alter TGF-ß2-induced increases in COL1A1 expression. Additionally, TGF-ß2 augmented calcipotriol-induced rises in COL1A1 expression. TGF-ß receptor (TGFBRI/II) knockdown blunted TGF-ß2-induced increases in COL1A1 expression, whereas calcipotriol-induced increases in VDR and COL1A1 expression levels were unaltered. Conclusions: Scleral vitamin D3 inhibits myopia development in mice, potentially by activating a VDR-dependent signaling pathway and increasing scleral COL1A1 expression levels.

The distinctive role of membrane fibrinogen-like protein 2 in the liver stage of rodent malaria infections.

Viral infection often induce the expression of murine fibrinogen-like protein 2 (mFGL2) triggering immune coagulation, which causes severe liver pathogenesis via increased fibrin deposition and thrombosis in the microvasculature. We aimed to investigate the role of mFGL2 in the liver stage of malaria infections. We reveal that infection with malaria sporozoites also induces increased expression of mFGL2 and that this expression is primarily located within the liver Kupffer and endothelial cells. In addition, we report that inhibition of FGL2 has no significant effect on immune coagulation but increases the expression of inflammatory cytokines in the livers of infected mice. Interestingly, FGL2 deficiency had no significant impact on the development of liver stage malaria parasites or the pathogenesis of the infected liver. In contrast to viral infections, we conclude that mFGL2 does not contribute to either parasite development or liver pathology during these infections, revealing the unique features of this protein in liver-stage malaria infections.

Znhit1 Regulates p21Cip1 to Control Mouse Lens Differentiation.

Purpose: The transparency of the ocular lens is essential for refracting and focusing light onto the retina, and transparency is controlled by many factors and signaling pathways. Here we showed a critical role of chromatin remodeler zinc finger HIT-type containing 1 (Znhit1) in maintaining lens transparency. Methods: To explore the roles of Znhit1 in lens development, the cre-loxp system was used to generate lens-specific Znhit1 knockout mice (Znhit1Mlr10-Cre; Znhit1 cKO). Morphological changes in mice lenses were examined using hematoxylin and eosin staining. RNA sequencing (RNA-seq) and assay for transposase accessible chromatin using sequencing (ATAC-seq) were applied to screen transcriptome changes. Immunofluorescence staining were performed to assess proteins distribution and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were used for determining apoptosis. The mRNAs expression was examined by quantitative RT-PCR and proteins expression by Western blot. Results: Lens-specific conditional knockout mice had a severe cataract, microphthalmia phenotype, and seriously abnormal lens fiber cells differentiation. Deletion of Znhit1 in the lens resulted in decreased cell proliferation and increased cell apoptosis of the lens epithelia. ATAC-seq showed that Znhit1 deficiency increased chromatin accessibility of cyclin-dependent kinase inhibitors, including p57Kip2 and p21Cip1, and upregulated the expression of these genes in mRNA and protein levels. And we also showed that loss of Znhit1 lead to lens fibrosis by upregulating the expression of p21Cip1. Conclusions: Our findings suggested that Znhit1 is required for the survival of lens epithelial cells. The loss of Znhit1 leads to the overexpression of p21Cip1, further resulting in lens fibrosis, and impacted the establishment of lens transparency.

Scleral HIF-1α is a prominent regulatory candidate for genetic and environmental interactions in human myopia pathogenesis.

BACKGROUND: Myopia is a good model for understanding the interaction between genetics and environmental stimuli. Here we dissect the biological processes affecting myopia progression. METHODS: Human Genetic Analyses: (1) gene set analysis (GSA) of new genome wide association study (GWAS) data for 593 individuals with high myopia (refraction ≤ -6 diopters [D]); (2) over-representation analysis (ORA) of 196 genes with de novo mutations, identified by whole genome sequencing of 45 high-myopia trio families, and (3) ORA of 284 previously reported myopia risk genes. Contributions of the enriched signaling pathways in mediating the genetic and environmental interactions during myopia development were investigated in vivo and in vitro. RESULTS: All three genetic analyses showed significant enrichment of four KEGG signaling pathways, including amphetamine addiction, extracellular matrix (ECM) receptor interaction, neuroactive ligand-receptor interaction, and regulation of actin cytoskeleton pathways. In individuals with extremely high myopia (refraction ≤ -10 D), the GSA of GWAS data revealed significant enrichment of the HIF-1α signaling pathway. Using human scleral fibroblasts, silencing the key nodal genes within protein-protein interaction networks for the enriched pathways antagonized the hypoxia-induced increase in myofibroblast transdifferentiation. In mice, scleral HIF-1α downregulation led to hyperopia, whereas upregulation resulted in myopia. In human subjects, near work, a risk factor for myopia, significantly decreased choroidal blood perfusion, which might cause scleral hypoxia. INTERPRETATION: Our study implicated the HIF-1α signaling pathway in promoting human myopia through mediating interactions between genetic and environmental factors. FUNDING: National Natural Science Foundation of China grants; Natural Science Foundation of Zhejiang Province.

Opposing Effects of PPARα Agonism and Antagonism on Refractive Development and Form Deprivation Myopia in Guinea Pigs.

Purpose: To determine if drug-induced peroxisome proliferator-activated receptor α (PPARα) signal pathway modulation affects refractive development and myopia in guinea pigs. Methods: Pigmented guinea pigs were randomly divided into normal vision (unoccluded) and form deprivation myopia (FDM) groups. Each group received daily peribulbar injections of either a vehicle or (1) PPARα agonist, GW7647, clofibrate, or bezafibrate or (2) PPARα antagonist, GW6471, for 4 weeks. Baseline and posttreatment refraction and ocular biometric parameters were measured. Immunofluorescent staining of PPARα and two of its downstream readouts, cytosolic malic enzyme 1 (ME1) and apolipoproteinA II (apoA-II), was undertaken in selected scleral sections. Western blot analysis determined collagen type I expression levels. Results: GW6471 induced a myopic shift in unoccluded eyes, but had no effect on form-deprived eyes. Conversely, GW7647 inhibited FDM progression without altering unoccluded eyes. Bezafibrate and clofibrate had effects on refraction similar to those of GW7647 in unoccluded and form-deprived eyes. GW6471 downregulated collagen type I expression in unoccluded eyes whereas bezafibrate inhibited collagen type I decreases in form-deprived eyes. GW6471 also reduced the density of ME1- and apoA-II-stained cells in unoccluded eyes whereas bezafibrate increased apoA-II-positive cell numbers in form-deprived eyes. Conclusions: As GW7647 and GW6471 had opposing effects on myopia development, PPARα signaling modulation may be involved in this condition in guinea pigs. Fibrates are potential candidates for treating myopia since they reduced both FDM and the associated axial elongation. Bezafibrate also inhibited form deprivation-induced decreases in scleral collagen type I expression and the density of apoA-II expressing cells.

Experimental murine myopia induces collagen type Iα1 (COL1A1) DNA methylation and altered COL1A1 messenger RNA expression in sclera.

PURPOSE: To investigate whether myopia development is associated with changes of scleral DNA methylation in cytosine-phosphate-guanine (CpG) sites in the collagen 1A1 (COL1A1) promoter and messenger RNA (mRNA) levels following murine form deprivation myopia. METHODS: Fifty-seven C57BL/6 mice (postnatal day 23) were randomly assigned to four groups: (1) monocular form deprivation (MD) in which a diffuser lens was placed over one eye for 28 days; (2) normal controls without MD; (3) MD recovery in which the diffuser lens was removed for seven days; and (4) MD recovery normal controls. The DNA methylation pattern in COL1A1 promoter and exon 1 was determined by bisulfite DNA sequencing, and the COL1A1 mRNA level in sclera was determined by quantitative PCR. RESULTS: MD was found to induce myopia in the treated eyes. Six CpG sites in the promoter and exon 1 region of COL1A1 were methylated with significantly higher frequency in the treated eyes than normal control eyes (p<0.05), with CpG island methylation in MD-contralateral eyes being intermediate. Consistent with the CpG methylation, scleral COL1A1 mRNA was reduced by 57% in the MD-treated eyes compared to normal controls (p<0.05). After seven days of MD recovery, CpG methylation was significantly reduced (p=0.01). The methylation patterns returned to near normal level in five CpG sites, but the sixth was hypomethylated compared to normal controls. CONCLUSIONS: In parallel with the development of myopia and the reduced COL1A1 mRNA, the frequency of methylation in CpG sites of the COL1A1 promoter/exon 1 increased during MD and returned to near normal during recovery. Thus, hypermethylation of CpG sites in the promoter/exon 1 of COL1A1 may underlie reduced collagen synthesis at the transcriptional level in myopic scleras.