Adenosine metabolized from extracellular ATP promotes type 2 immunity through triggering A2BAR signaling in intestinal epithelial cells

Intestinal nematode parasites can cross the epithelial barrier, causing tissue damage and release of danger-associated molecular patterns (DAMPs) that may promote host protective type 2 immunity. We investigate whether adenosine binding to the A2B adenosine receptor (A2BAR) on intestinal epithelial cells (IECs) plays an important role. Specific blockade of IEC A2BAR inhibits the host protective memory response to the enteric helminth, Heligmosomoides polygyrus bakeri (Hpb), including disruption of gran-uloma development at the host-parasite interface. Memory T cell development is blocked during the primary response, and transcriptional analyses reveal profound impairment of IEC activation. Extracel-lular ATP is visualized 24 h after inoculation and is shown in CD39-deficient mice to be critical for the adenosine production mediating the initiation of type 2 immunity. Our studies indicate a potent adeno-sine-mediated IEC pathway that, along with the tuft cell circuit, is critical for the activation of type 2 immunity

Protocol for immunofluorescence staining of murine helminth-infected intestinal and lung tissues

Summary: Preserving structurally intact tissue through proper tissue fixation and cryopreservation minimizes tissue autolysis, desiccation, and enzymatic degradation. In this protocol, we describe the use of an optimized fresh freezing technique that cryopreserves the tissue and favors the retention of the natural protein structure of antigens. We also detail the use of cold, low-concentration paraformaldehyde (PFA) fixation to enhance tissue morphology and detection of fluorescent proteins, such as GFP and TdTomato, in tissues of genetically engineered mice.For complete details on the use and execution of this protocol, please refer to Chen et al. (2022)1 and El-Naccache et al. (2022).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Expression of SNAIL and Par3 in BJAB and BC-3 tumors in mouse xenograft model.

<p>(A, B) NOD/SCID mice were injected with ten millions of BJAB and BC-3 cells intraperitoneal. (C, D). After 5 weeks, the mice were scarified and tumor dissected for analysis of transcripts and proteins of SNAIL and Par3 in these tumors. (E) Immunohistochemistry were performed on BJAB and BC-3 generated tumors. Here we used primary antibodies against LANA, E-cadherin and Par3. DAPI was used for nuclear staining. (F) H and E staining was shown for a similar group of tumor tissues capitalize to Fig 8F.</p

Status of EMTs in KSHV infected PBMCs and Par3 knockdown cells.

<p>(A) PBMCs were subjected to infection with KSHV and analyzed using days post-infection for the screening of epithelial (E-cadherin, Zo-1, Dsp) and mesenchymal (Snail, Lef1, B-catenin, Cdh2). qRT-PCR was performed for the transcript analysis of EMTs (epithelial to mesenchymal markers). (B) HEK293-KSHV-shControl and HEK-293KSHV-shPar3 cells were used to study these EMTs. qRT-PCR was performed to determine the fold changes for EMTs and to confirm the Par3 knockdown in HEK-293KSHV stable cell lines. (C) BJAB-shContol and BJAB-shPar3 cells were generated to study these EMTs. qRT-PCR was performed to analyze the fold changes for EMTs and to confirm Par3 expression in transiently transfected BJAB cells.</p

LANA stabilizes Par3 in KSHV positive cells.

<p>(A) HEK-293-BAC-KSHV cells were transfected with sh-LANA and sh-Control. Endogenous Par3 expression was measured and presented in graph. GAPDH was used as a protein loading control. (B, C) Stabilization of Par3 was examined with cyclohexamide in HEK-293 and BJAB cells. Left panels and right panels were used as vector control and LANA expression, in a time dependent manner. (D) The proteosome inhibitor MG132 was used to determine if Par3 stability was linked to the proteosome degradation pathway. GFP and GAPDH were used as transfection and endogenous protein loading controls. (E) BC-3-shControl and BC-3-shLANA cells were treated with cyclohexamide and observed Par3 endogenous on hours dependent manner. (F) BC-3-shControl and BC-3shLANA cells were treated with MG132 and followed immunoprecipitation of Par3. Endogenous ubiquitin and Par3 were detected using specific antibodies in both the cell lines. GAPDH was monitored as an internal control for loading in the input section.</p

NF-kB is regulated by SNAIL in KSHV positive cells.

<p>(A) BC-3 and (B) BJAB cells were treated with DMSO and SNAIL inhibitor for 48 hours and assessed through Western blots for LANA, NF-kB, SNAIL and endogenous GAPDH. (C) BC-3 shControl and BC-3-ShLANA were treated with DMSO and SNAIL inhibitor for 48 hour and blotted for Par3, NF-kB (p65), SNAIL and endogenous control GAPDH. (D) Ubiquitination assays for SNAIL were performed in BC-3sh-control and sh-NF-kB cells treated with DMSO and MG132. (E) sh-Control and sh-Par3 in presence/absence of LANA were measured in the BJAB cell background. Left panel was observed in the presence of DMSO and the right panel was treated with SNAIL inhibitor at the same time. Caspase3 and GAPDH was observed in both panels.</p

Primer sequence for ChIP analysis.

<p>Primer sequence for ChIP analysis.</p

Schematic represents a putative model illustrating the contribution of Par3 and SNAIL to KSHV-associated cancers.

<p>KSHV infected endothelial or B-cells expresses LANA important for establishment of latent infection. LANA up-regulates Par3 and SNAIL which leads to epithelial to mesenchymal transition through down-regulation of E-cadherin and enhanced expression of MMP9 in B-cells. This model suggests that KSHV-infection can regulate the EMTs important for progression of infected cells to an oncogenic and invasive phenotype.</p

KSHV infection leads to Par3 nuclear localization.

<p>(A) KSHV negative BJAB, Ramos, (B) KSHV positive BCBL1, BC-3, JSC-1 cell lines and (D) PBMCs uninfected and infected with KSHV at day 6 were used to determine the localization of Par3. LANA staining was used as positive control. In KSHV positive cells graphs were plotted with the percent of LANA foci colocalized with Par3. (C) Co-localization of LANA with Par3 was performed in KSHV negative Ramos cell line. The graphs represent the percent of LANA foci co-localized with Par3.</p