Characterization of genetic humanized mice with transgenic HLA DP401 or DRA but deficient in endogenous murine MHC class II genes upon Staphylococcus aureus pneumonia

Abstract

BackgroundStaphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in “carrier” or “pathogenic” states. HLADQ and HLADR humanized mice have been used as a small animal model to study the role of two molecules during S. aureus infection. However, the contribution of HLADP to S. aureus infection is unknown yet.MethodsIn this study, we have produced HLADP401 and HLADRA0101 humanized mice by microinjection of C57BL/6J zygotes. Neo‐floxed IAβ+/− mice were crossbred with Ella‐Cre and further crossbred with HLADP401 or HLA‐DRA0101 humanized mice. After several rounds of traditional crossbreeding, we finally obtained HLA DP401‐IAβ−/− and HLA DRA‐IAβ−/− humanized mice, in which human DP401 or DRA0101 molecule was introduced into IAβ−/− mice deficient in endogenous murine MHC class II molecules. A transnasal infection murine model of S. aureus pneumonia was induced in the humanized mice by administering 2 × 108 CFU of S. aureus Newman dropwise into the nasal cavity. The immune responses and histopathology changes were further assessed in lungs in these infected mice.ResultsWe evaluated the local and systemic effects of S. aureus delivered intranasally in HLA DP401‐IAβ−/− and HLA DRA‐IAβ−/− transgenic mice. S. aureus Newman infection significantly increased the mRNA level of IL 12p40 in lungs in humanized mice. An increase in IFN‐γ and IL‐6 protein was observed in HLADRA‐IAβ−/− mice. We observed a declining trend in the percentage of F4/80+ macrophages in lungs in HLADP401‐IAβ−/− mice and a decreasing ratio of CD4+ to CD8+ T cells in lungs in IAβ−/− mice and HLA DP401‐IAβ−/− mice. A decreasing ratio of Vβ3+ to Vβ8+ T cells was also found in the lymph node of IAβ−/− mice and HLA DP401‐IAβ−/− mice. S. aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ−/− genetic background mice.ConclusionThese humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S. aureus pneumonia and study what role DP molecule plays in S. aureus infection.

Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B

“…The cycling conditions for qRT‐PCR were 95°C for 10 min, followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and 72°C for 1 s. The fold increase in mRNA expression was calculated using the routine 2 −ΔΔ Ct method. 31 , 32 , 33 All the primers for mouse Tas2rs , Gnat3 are presented in Table 1 and have been used in our previous study. 7 , 9 …”

“…Staining against polyclonal anti‐GNAT3 (sc‐395, Santa Cruz Biotechnology) and anti‐PLCβ2 (sc‐22458, Santa Cruz Biotechnology) was performed using the standard immunofluorescence procedure. 31 , 32 , 36 Blocking peptide for anti‐GNAT3 (sc‐395P, Santa Cruz Biotechnology) was used to specifically block GNAT3 antibody. After the cryosections were washed in phosphate‐buffered saline (PBS, 3 × 10 min), they were kept in blocking buffer for 1–2 h and then incubated for 36–48 h at 4°C in the polyclonal antibody: anti‐PLCB2 (1:500), anti‐GNAT3 (1:500), or mixture of anti‐GNAT3 and blocking peptide (1:50) in blocking buffer.…”