As early secreted antigenic target of 6 kDa (ESAT-6) of Mycobacterium tuberculosis (Mtb) is an essential virulence factor and macrophages are critical for tuberculosis infection and immunity, we studied ESAT-6 stimulated IL-6 production by macrophages. ESAT-6 stimulated significantly higher IL-6 secretion by murine bone marrow derived macrophages (BMDM) compared to culture filtrate protein 10 kDa (CFP10) and antigen 85A. Polymyxin B, an LPS blocker, did not affect ESAT-6 stimulated macrophage IL-6 production. ESAT-6 but not Pam3CSK4 induced IL-6 by TLR2 knockout BMDM. ESAT-6 induced phosphorylation and DNA binding of STAT3 and this was blocked by STAT3 inhibitors but not by rapamycin. STAT3 inhibitors suppressed ESAT-6-induced IL-6 transcription and secretion without affecting cell viability. This was confirmed by silencing STAT3 in macrophages. Blocking neither IL-6Rα/IL-6 nor IL-10 affected ESAT-6-induced STAT3 activation and IL-6 production. Infection of BMDM and human macrophages with Mtb with esat-6 deletion induced diminished STAT3 activation and reduced IL-6 production compared to wild type and esat-6 complemented Mtb strains. Administration of ESAT-6 but not CFP10 induced STAT3 phosphorylation and IL-6 expression in the mouse lungs, consistent with expression of ESAT-6, IL-6 and phosphorylated-STAT3 in Mtb-infected mouse lungs. We conclude that ESAT-6 stimulates macrophage IL-6 production through STAT3 activation.

PurposeDefects in neural crest development are a major contributing factor in corneal dysgenesis, but little is known about the genetic landscape during corneal development. The purpose of this study was to provide a detailed transcriptome profile and evaluate changes in gene expression during mouse corneal development.MethodsRNA sequencing was used to uncover the transcriptomic profile of periocular mesenchyme (pNC) isolated at embryonic day (E) 10.5 and corneas isolated at E14.5 and E16.5. The spatiotemporal expression of several differentially expressed genes was validated by in situ hybridization.ResultsAnalysis of the whole-transcriptome profile between pNC and embryonic corneas identified 3815 unique differentially expressed genes. Pathway analysis revealed an enrichment of differentially expressed genes involved in signal transduction (retinoic acid, transforming growth factor-β, and Wnt pathways) and transcriptional regulation.ConclusionsOur analyses, for the first time, identify a large number of differentially expressed genes during progressive stages of mouse corneal development. Our data provide a comprehensive transcriptomic profile of the developing cornea. Combined, these data serve as a valuable resource for the identification of novel regulatory networks crucial for the advancement of studies in congenital defects, stem cell therapy, bioengineering, and adult corneal diseases.

Early secreted antigenic target of 6 kDa (ESAT-6), the major virulence factor of Mycobacterium tuberculosis, affects host immunity and the formation of granulomas likely through inflammatory cytokines. To understand its role in this regard further, we investigated the effect of ESAT-6 on macrophages by determining the production of macrophage chemoattractant protein (MCP)-1, a major chemokine associated with tuberculosis pathogenesis, by murine bone marrowderived macrophages (BMDMs) and its regulation by protein kinases and cytokines. The results revealed that ESAT-6, but not Ag85A and culture filtrate protein 10 kDa (CFP10), induced MCP-1 production by BMDMs dose and time dependently. Inhibition of p38 but not other mitogen-activated protein kinases (MAPK) and PI3K further enhanced ESAT-6-induced MCP-1 production by BMDMs. Inhibition of p38 MAPK enhanced ESAT-6-induced MCP-1 mRNA accumulation without affecting mRNA stability. ESAT-6 also induced TNF-a from BMDMs and MCP-1 from mouse lung epithelial cells, and these were suppressed by p38 MAPK inhibition, implying cytokine-and cell-specific effect of p38 MAPK inhibition on ESAT-6-induced MCP-1 by macrophages. Pretreatment of BMDMs with IL-4, but not other cytokines (IL-2, IL-10, TNF-a, IFN-c and IL-1a) further elevated ESAT-6-stimulated MCP-1 production although IL-4 did not induce MCP-1 without ESAT-6. Both p38 MAPK inhibitor and IL-4 did not show additive effect on ESAT-6-induced MCP-1 protein level despite such effect on MCP-1 mRNA level was evident. In conclusion, these results indicate a specific role for both p38 MAPK and IL-4 in ESAT-6-induced MCP-1 production by macrophages and suggest a pathway with significance in tuberculosis pathogenesis.

During development, cells aggregate at tissue boundaries to form normal tissue architecture of organs. However, how cells are segregated into tissue precursors remains largely unknown. Cornea development is a perfect example of this process whereby neural crest cells aggregate in the periocular region prior to their migration and differentiation into corneal cells. Our recent RNA-Seq analysis identified upregulation of Nephronectin (Npnt) transcripts during early stages of corneal development where its function has not been investigated. We found that Npnt mRNA and protein are expressed by various ocular tissues including the migratory periocular neural crest (pNC), which also express the integrin alpha 8 (Itga8) receptor. Knockdown of either Npnt or Itga8 attenuated cornea development, whereas overexpression of Npnt resulted in cornea thickening. Moreover, overexpression of Npnt variants lacking RGD binding sites did not affect corneal thickness. Neither the knockdown or augmentation of Npnt caused significant changes in cell proliferation, suggesting that Npnt directs pNC migration into the cornea. In vitro analyses showed that Npnt promotes pNC migration from explanted periocular mesenchyme, which requires Itga8, focal adhesion kinase (FAK) and Rho kinase (ROCK). Combined, these data suggest that Npnt augments cell migration into the presumptive cornea ECM by functioning as a substrate for Itgα8-positive pNC cells.

Corneal defects are a major cause of blindness, but treatment options remain limited. Advancements require improved understanding of molecular processes involved in corneal development. Corneal development is an intricate process that involves migration, proliferation, and differentiation of embryonic neural stem cells known as neural crest cells. However, very little is known about the molecular mechanisms involved in these processes. Our recent studies identified Nephronectin, an extracellular matrix protein, in the developing cornea. The current study is to determine whether Nephronectin signals through ErbB proteins (ErbB1, ErbB2, ErbB3) during corneal development. To test the hypothesis that neural crest cells co‐localize and interact with nephronectin through ErbB proteins during corneal development, we first isolated cDNA from corneal tissues, then identified the expression of the ErbB genes by reverse transcription polymerase chain reaction (RT‐PCR), and finally determined their localization by in situ hybridization. Our initial analysis by RT‐PCR indicates that ErbB1 is highly expressed during corneal development. Next, we synthesized RNA probes using a cloning vector to analyze their spatial expression on paraffin‐embedded embryonic corneal sections. Our preliminary analyses showed that ErbB1 is expressed in the presumptive corneal stroma and epithelium, suggesting a potential role of Nephronectin/ErbB1 signaling during cell migration, proliferation, or differentiation into corneal cells. Additionally, ErbB2 and ERbB3 are expressed in the epithelium, indicating a role in proliferation. Identifying signaling pathways that govern corneal development can lead to better strategies to repair corneal defects.Support or Funding InformationResearch reported in this abstract was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.