RNA-sequencing reveals molecular and regional differences in the esophageal mucosa of achalasia patients.

Achalasia is an esophageal motility disorder characterized by the functional loss of myenteric plexus ganglion cells in the distal esophagus and lower esophageal sphincter. Histological changes have been reported in the esophageal mucosa of achalasia, suggesting its involvement in disease pathogenesis. Despite recent advances in diagnosis, our understanding of achalasia pathogenesis at the molecular level is very limited and gene expression profiling has not been performed. We performed bulk RNA-sequencing on esophageal mucosa from 14 achalasia and 8 healthy subjects. 65 differentially expressed genes (DEGs) were found in the distal esophageal mucosa of achalasia subjects and 120 DEGs were identified in proximal esophagus. Gene expression analysis identified genes common or exclusive to proximal and distal esophagus, highlighting regional differences in the disease. Enrichment of signaling pathways related to cytokine response and viral defense were observed. Increased infiltration of CD45+ intraepithelial leukocytes were seen in the mucosa of 38 achalasia patients compared to 12 controls. Novel insights into the molecular changes occurring in achalasia were generated in this transcriptomic study. Some gene changes observed in the mucosa of achalasia may be associated with esophagitis. Differences in DEGs between distal and proximal esophagus highlight the importance of better understanding regional differences in achalasia.

Endosomal Sequestration of TLR4 Antibody Induces Myeloid-Derived Suppressor Cells and Reverses Acute Type 1 Diabetes.

We previously showed that treating NOD mice with an agonistic monoclonal anti-TLR4/MD2 antibody (TLR4-Ab) reversed acute type 1 diabetes (T1D). Here, we show that TLR4-Ab reverses T1D by induction of myeloid-derived suppressor cells (MDSCs). Unbiased gene expression analysis after TLR4-Ab treatment demonstrated upregulation of genes associated with CD11b+Ly6G+ myeloid cells and downregulation of T-cell genes. Further RNA sequencing of purified, TLR4-Ab-treated CD11b+ cells showed significant upregulation of genes associated with bone marrow-derived CD11b+ cells and innate immune system genes. TLR4-Ab significantly increased percentages and numbers of CD11b+ cells. TLR4-Ab-induced CD11b+ cells, derived ex vivo from TLR4-Ab-treated mice, suppress T cells, and TLR4-Ab-conditioned bone marrow cells suppress acute T1D when transferred into acutely diabetic mice. Thus, the TLR4-Ab-induced CD11b+ cells, by the currently accepted definition, are MDSCs able to reverse T1D. To understand the TLR4-Ab mechanism, we compared TLR4-Ab with TLR4 agonist lipopolysaccharide (LPS), which cannot reverse T1D. TLR4-Ab remains sequestered at least 48 times longer than LPS within early endosomes, alters TLR4 signaling, and downregulates inflammatory genes and proteins, including nuclear factor-κB. TLR4-Ab in the endosome, therefore, induces a sustained, attenuated inflammatory response, providing an ideal "second signal" for the activation/maturation of MDSCs that can reverse acute T1D.