SUMMARY Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is as a ligand for PXR in vivo, and IPA down-regulated enterocyte TNF–α while up-regulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2−/−) mice showed a distinctly “leaky” gut physiology coupled with up-regulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2−/−Tlr4−/− mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway which involves luminal sensing and signaling by TLR4.

Tissue-resident macrophages are a diverse population of cells that perform specialized functions including sustaining tissue homeostasis and tissue surveillance. Here, we report an interstitial subset of CD169+ lung-resident macrophages that are transcriptionally and developmentally distinct from alveolar macrophages (AMs). They are primarily localized around the airways and are found in close proximity to the sympathetic nerves in the bronchovascular bundle. These nerve- and airway-associated macrophages (NAMs) are tissue resident, yolk sac derived, self-renewing, and do not require CCR2+ monocytes for development or maintenance. Unlike AMs, the development of NAMs requires CSF1 but not GM-CSF. Bulk population and single-cell transcriptome analysis indicated that NAMs are distinct from other lung-resident macrophage subsets and highly express immunoregulatory genes under steady-state and inflammatory conditions. NAMs proliferated robustly after influenza infection and activation with the TLR3 ligand poly(I:C), and in their absence, the inflammatory response was augmented, resulting in excessive production of inflammatory cytokines and innate immune cell infiltration. Overall, our study provides insights into a distinct subset of airway-associated pulmonary macrophages that function to maintain immune and tissue homeostasis.

The spleen is an important site for generating protective immune responses against pathogens. After infection, immune cells undergo rapid reorganization to initiate and maintain localized inflammatory responses; however, the mechanisms governing this spatial and temporal cellular reorganization remain unclear. We show that the strategic position of splenic marginal zone CD169+ macrophages is vital for rapid initiation of antibacterial responses. In addition to controlling initial bacterial growth, CD169+ macrophages orchestrate a second phase of innate protection by mediating the transport of bacteria to splenic T cell zones. This compartmentalization of bacteria within the spleen was essential for driving the reorganization of innate immune cells into hierarchical clusters and for local interferon-γ production near sites of bacterial replication foci. Our results show that both phases of the antimicrobial innate immune response were dependent on CD169+ macrophages, and, in their absence, the series of events needed for pathogen clearance and subsequent survival of the host was disrupted. Our study provides insight into how lymphoid organ structure and function are related at a fundamental level.

Coronavirus disease 2019 (COVID-19) is a declared pandemic that is spreading all over the world at a dreadfully fast rate. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the pathogen of COVID-19, infects the human body using angiotensin-converting enzyme 2 (ACE2) as a receptor identical to the severe acute respiratory syndrome (SARS) pandemic that occurred in 2002-2003. SARS-CoV-2 has a higher binding affinity to human ACE2 than to that of other species. Animal models that mimic the human disease are highly essential to develop therapeutics and vaccines against COVID-19. Here, we review transgenic mice that express human ACE2 in the airway and other epithelia and have shown to develop a rapidly lethal infection after intranasal inoculation with SARS-CoV, the pathogen of SARS. This literature review aims to present the importance of utilizing the human ACE2 transgenic mouse model to better understand the pathogenesis of COVID-19 and develop both therapeutics and vaccines.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1 −/− effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1−/− T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1 −/− effector T cells failed to enter the sinuses.We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.n effective immune response depends on the large-scale, but carefully regulated, migration of T cells within and between lymphoid and peripheral tissues. This migration is tightly regulated by several factors, including the highly organized secondary lymphoid structure and the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands (1). This balance between the anatomy and the ordered expression of cell surface and soluble proteins dictates the exquisite choreography of T-cell migration, and visualizing these dynamics of T-cell behavior in situ within the lymph nodes (LNs) is essential for understanding the mechanisms that mediate the generation of a productive antimicrobial or antitumoral immune response (1, 2). However, our understanding of the factors that regulate the anatomical program followed by endogenous antigen-specific effector T cells after an infection remains incomplete, especially with respect to the mechanisms that regulate egress kinetics of effector T cells from LN (2, 3).T-cell migration, even at steady state, is a highly regulated process (4). T-cell entry into the LN is controlled by G protein-coupled receptors (GPCRs) (3) such as CC chemokine receptor 7 (CCR7), which is also critical for the localization and retention of T cells within the LN paracortex (5, 6). Egress of naive T cells from the LN via the lymphatic vessels is regulated by the GPCR sphingosine-1-phosphate receptor-1 (S1PR1) (3) and adhesion molecules (4). S1PR1 is among four other GPCRs that bind to sphingosine-1-phosphate (S1P) with high affinity. S1PR1 is abundantly expressed in different cell types and tissues, including immune cells and endothelial cells (7). In addition to mediating lymphocyte egress, binding of S1P to S1PR1 and other receptors (S1PR2 to -5) on the cell surface initiates several signaling cascades that affect the functioning of many organ systems and control a multitude of biological ...

Background There is substantial evidence that signaling through Toll-like receptor 4 (TLR4) contributes to the pathogenesis of necrotizing enterocolitis (NEC). Pregnane X receptor (PXR), a xenobiotic sensor and signaling intermediate for certain host-bacterial metabolites, has been shown to negatively regulate TLR4 signaling. Here we investigated the relationship between PXR and TLR4 in the developing murine intestine and explored the capacity of PXR to modulate inflammatory pathways involved in experimental NEC. Methods Wild-type and PXR−/− mice were studied at various time points of development in an experimental model of NEC. In addition, we studied the ability of the secondary bile acid lithocholic acid (LCA), a known PXR agonist in liver, to activate intestinal PXR and reduce NEC-related intestinal inflammation. Results We found a reciprocal relationship between the developmental expression of PXR and TLR4 in wild-type murine intestine, with PXR acting to reduce TLR4 expression by decreasing TLR4 mRNA stability. In addition, PXR−/− mice exhibited a remarkably heightened severity of disease in experimental NEC. Moreover, LCA attenuated intestinal proinflammatory responses in the early stages of experimental NEC. Conclusion These findings provide proactive insights into the regulation of TLR4 in the developing intestine. Targeting PXR may be a novel approach for NEC prevention.

The nuclear pregnane X receptor (PXR) plays a central role in regulating xenobiotic metabolism. We now report a novel role for PXR as a critical negative regulator of innate immunity after infection. Pxr−/− mice exhibited remarkably elevated pro-inflammatory cytokine and chemokine production following infection with Listeria monocytogenes (Lm). Despite the more robust innate immune response, Pxr−/− mice were highly susceptible to Lm infection. Surprisingly, disruption of the Toll-like receptor 4 (TLR4) but not TLR2 signaling restored the inflammation to normal levels and the ability to clear Lm in Pxr−/− mice. Mechanistically, the heightened inflammation in Pxr−/− mice resulted in the death of inflammatory monocytes that led to the enhanced susceptibility to Lm infection. These data demonstrated that PXR regulated pathogen-induced inflammation and host defense against Lm infection through modulating the TLR4 pathway. In summary, we discovered an apical role for PXR in regulating innate immunity. In addition, we uncovered a remarkable negative impact of the TLR4 pathway in controlling the quality of the inflammatory response and host defense against a gram-positive bacterial infection.

Background Gastric cancer metastasis is a highly fatal disease with a five-year survival rate of less than 5%. One major obstacle in studying gastric cancer metastasis is the lack of faithful models available. The cancer xenograft mouse models are widely used to elucidate the mechanisms of cancer development and progression. Current procedures for creating cancer xenografts include both heterotopic (i.e., subcutaneous) and orthotopic transplantation methods. Compared to the heterotopic model, the orthotopic model has been shown to be the more clinically relevant design as it enables the development of cancer metastasis. Although there are several methods in use to develop the orthotopic gastric cancer model, there is not a model which uses various types of tumor materials, such as soft tissues, semi-liquid tissues, or culture derivatives, due to the technical challenges. Thus, developing the applicable orthotopic model which can utilize various tumor materials is essential. Results To overcome the known limitations of the current orthotopic gastric cancer models, such as exposure of tumor fragments to the neighboring organs or only using firm tissues for the orthotopic implantation, we have developed a new method allowing for the complete insertion of soft tissue fragments or homogeneously minced tissues into the stomach submucosa layer of the immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse. With this completely-closed transplantation method, tumors with various types of tissue may be used to establish orthotopic gastric cancer models without the risks of exposure to nearby organs or cell leakage. This surgical procedure was highly reproducible in generating forty-eight mouse models with a surgery success rate of 96% and tumor formation of 93%. Among four orthotopic patient-derived xenograft (PDX) models that we generated in this study, we verified that the occurrence of organotropic metastasis in either the liver or peritoneal cavity was the same as that of the donor patients. Conclusion Here we describe a new protocol, step by step, for the establishment of orthotopic xenograft of gastric cancer. This novel technique will be able to increase the use of orthotopic models in broader applications for not only gastric cancer research but also any research related to the stomach microenvironment.