Ob ob b6 cg lepob j

Coinfection experiments were performed as described previously (14 (link)). Briefly, 8-week-old C57BL/6 (lean) and B6.Cg-Lepob/J (ob/ob) mice (Jackson Laboratory, Bar Harbor, ME) along with diet-induced lean and obese mice were anesthetized with 2.5% inhaled isoflurane (Baxter, Deerfield, IL) and intranasally inoculated with sublethal doses of influenza virus in a total volume of 30 µl. Three, 7, or 10 days after influenza virus infection, mice were anesthetized with 2.5% inhaled isoflurane and intranasally inoculated with 100 CFU of S. pneumoniae, and then monitored daily for clinical signs of infection and weighed every 24 hours postinfection (59 (link)). Moribund mice that had lost more than 30% body weight were humanely euthanized. At days 7, 8, 9, and 10 postinfection, mice were euthanized by CO₂ asphyxiation, and tissues (lung, bronchoalveolar lavage fluid, nasal wash, blood) were harvested and processed immediately or stored at −80°C for future analysis.

Animal care and experimental procedures were performed with approval from the Institutional Animal Care and Use Committee from the University of Wisconsin (protocol M005210) and William S. Middleton Memorial Veterans Affairs (protocol DD0001) to meet acceptable standards of humane animal care. All experiments were carried out in accordance with their guidelines and regulations. All animals used in this study were housed in facilities with a standard light-dark cycle and fed ad libitum. Pancreatic mouse islets were isolated from male 12–16 week old C57BL/6J and B6.Cg-Lep^ob/J (ob/ob) (Jackson Laboratory, ME) as described previously^{52 (link)}. Briefly, islets were isolated by collagenase digestion and Histopaque gradient (Sigma, #10771). Then, islets were handpicked and cultured at 37 °C and 5% CO₂ in RPMI 1640 media (Thermo Fisher Scientific, #11875093) containing 11 mM glucose and supplemented with 5 g/l BSA fraction V (Roche, #107351080001), 100 units/ml penicillin and 100 μg/ml streptomycin (1% P/S) (Thermo Fisher Scientific).

GITR deficient mice were obtained from Dr. Tania Watts (University of Toronto, Toronto, Canada) and Dr. Carlo Riccardi (University of Perugia, Perugia, Italy). C57BL/6J, Ob/Ob (B6.Cg-Lep^ob/J), RAG2 deficient (C.B6(Cg)-Rag2^tm1.1Cgn/J), IL-13 deficient (C.129P2-Il13^tm1.1Anjm) and IL-5 deficient (C57BL/6-Il5^tm1Kopf/J) mice were purchased from the Jackson Laboratory (Bar Harbor, Maine). All mice were bred in our animal facility at the Keck School of Medicine, University of Southern California (USC). Four to eight-week-old aged and sexed-matched mice were used in the studies. All animal studies were approved by the USC institutional Animal Care and Use Committee and conducted in accordance with the USC Department of Animal Resources’ guidelines.