M1 activation of macrophages promotes inflammation and immunity to intracellular pathogens, while M2 macrophage activation promotes resolution of inflammation, wound healing, and tumor growth. These divergent phenotypes are characterized, in part, by the expression of iNOS and arginase I (Arg1) in M1 vs. M2 activated macrophages, respectively. Here we demonstrate that the Ron receptor tyrosine kinase tips the balance of macrophage activation by attenuating the M1 phenotype while promoting expression of Arg1, through a Stat6-independent mechanism. Induction of the Arg1 promoter by Ron is mediated by an AP-1 site located 433 bp upstream of the transcription start site. Treatment of primary macrophages with MSP, the ligand for Ron, induces potent MAP kinase activation, upregulates Fos, and enhances binding of Fos to the AP-1 site in the Arg1 promoter. In vivo, Arg1 expression in tumor-associated macrophages (TAMs) from Ron−/− mice was significantly reduced compared with TAMs from control animals. Furthermore, we show that Ron is expressed specifically by Tie2-expressing macrophages (TEMs), a TAM subset that exhibits a markedly skewed M2 and pro-tumoral phenotype. Decreased Arg1 in TAMs from Ron−/− mice was associated with reduced syngeneic tumor growth in these animals. These findings indicate that Ron induces Arg1 expression in macrophages through a previously uncharacterized AP-1 site in the Arg1 promoter, and that Ron could be therapeutically targeted in the tumor microenvironment to inhibit tumor growth by targeting expression of Arg1

Receptor tyrosine kinases are emerging as a class of key regulators of innate immune responses. We have shown previously that the RON receptor tyrosine kinases (murine Stk), expressed on tissue-resident macrophages, inhibit classical macrophage activation while promoting hallmarks of alternative activation, thus regulating the critical balance between the inflammatory and wound-healing properties of activated macrophages. We have also shown previously that RON−/− mice are more susceptible to in vivo endotoxin challenge than wild-type mice, suggesting that the expression of this receptor confers a degree of endotoxin resistance to these animals. Here we demonstrate that, in response to in vivo LPS challenge, RON−/− mice harbor significantly increased systemic levels of IFN-γ and IL-12p70 and increased levels of IL-12p40 transcript in their spleen. This elevation of IFN-γ can be attributed to splenic NK cells responding to the elevated levels of IL-12. Analysis of RON and IFN-γ receptor double-knockout mice indicates that the enhanced susceptibility of RON−/− mice to endotoxin challenge is dependent on IFN-γ-mediated signals. In vitro studies demonstrate that stimulation of primary peritoneal macrophages with macrophage-stimulating protein, the ligand for RON, inhibits IFN-γ-induced STAT1 phosphorylation and CIITA expression, resulting in reduced surface levels of MHC class II. Further studies demonstrating the induction of suppressor of cytokine signaling 1 via macrophage-stimulating protein/RON signaling provide a potential mechanistic insight into this regulatory pathway. These results indicate that the RON receptor regulates both the production of and response to IFN-γ, resulting in enhanced susceptibility to endotoxin challenge.

The present study sought to determine whether the protein catabolic response in skeletal muscle produced by chronic alcohol feeding was exaggerated in aged rats. Adult (3 mo) and aged (18 mo) female F344 rats were fed a nutritionally complete liquid diet containing alcohol (36% of total calories) or an isocaloric isonitrogenous control diet for 20 wk. Muscle (gastrocnemius) protein synthesis, as well as mTOR and proteasome activity did not differ between control-fed adult and aged rats, despite the increased TNF-α and IL-6 mRNA and decreased IGF-I mRNA in muscle of aged rats. Compared with alcohol-fed adult rats, aged rats demonstrated an exaggerated alcohol-induced reduction in lean body mass and protein synthesis (both sarcoplasmic and myofibrillar) in gastrocnemius. Alcohol-fed aged rats had enhanced dephosphorylation of 4E-BP1, as well as enhanced binding of raptor with both mTOR and Deptor, and a decreased binding of raptor with 4E-BP1. Alcohol feeding of both adult and aged rats reduced RagA binding to raptor. The LKB1-AMPK-REDD1 pathway was upregulated in gastrocnemius from alcohol-fed aged rats. These exaggerated alcohol-induced effects in aged rats were associated with a greater decrease in muscle but not circulating IGF-I, but no further increase in inflammatory mediators. In contrast, alcohol did not exaggerate the age-induced increase in atrogin-1 and MuRF1 mRNA or the increased proteasome activity. Our results demonstrate that, compared with adult rats, the gastrocnemius from aged rats is more sensitive to the catabolic effects of alcohol on protein synthesis, but not protein degradation, and this exaggerated response may be AMPK-dependent.

The RON receptor tyrosine kinase regulates the balance between classical (M1) and alternative (M2) macrophage activation. In primary macrophages, the ligand for Ron, macrophage-stimulating protein (MSP), inhibits the expression of inducible NO synthase, a marker of classically activated macrophages, whereas promoting the expression of arginase I, a marker of alternative activation. Ron−/− mice express increased levels of IL-12, a product of classically activated macrophages, after endotoxin administration, resulting in increased serum IFN-γ levels and enhanced susceptibility to septic shock. In this study, we demonstrate that MSP inhibits LPS-induced IL-12p40 expression, and this inhibition is dependent on the docking site tyrosines in Ron. To further define this inhibition, we examined the effect of Ron on signaling pathways downstream of Ron. We found that MSP does not inhibit the MyD88-independent activation of IFN regulatory factor 3 and production of IFN-β in response to LPS, nor does it inhibit MyD88-dependent TGF-β–activated kinase phosphorylation or MAPK activation in primary macrophages. However, the induction of IκB kinase activity, IκB degradation, and DNA binding of NF-κB after LPS stimulation is delayed in the presence of MSP. In addition, Ron inhibits serine phosphorylation of p65 and NF-κB transcriptional activity induced by LPS stimulation of TLR4. Finally, MSP inhibits the NF-κB–dependent upregulation of the nuclear IκB family member, IκBζ, a positive regulator of secondary response genes including IL-12p40. LPS also induces expression of Ron and an N-terminally truncated form of Ron, Sf-Ron, in primary macrophages, suggesting that the upregulation of Ron by LPS could provide classical feedback regulation of TLR signaling.

This study highlights the utility of routine testing for SARS-CoV-2 using pooled saliva while maintaining high sensitivity of detection (under 2,500 copies/ml) and rapid turnaround of high volume (up to 930 samples in 8 h by two technicians and one quantitative PCR [qPCR] machine). This pooled approach allowed us to test all residential students 1 to 2 times per week on our college campus during the spring of 2021 and flagged 83% of our semester positives.