Biological Characterization of Commercial Recombinantly Expressed Immunomodulating Proteins Contaminated with Bacterial Products in the Year 2020: The SAA3 Case

Figure 5

In vivo recruitment of neutrophils towards impure mu rSAA3 is mediated by activation of TLR4. (a) Female NMRI mice were injected i.p. with PBS (Co), mu rSAA3 (10 ng) or with a combination of mu rSAA3 (10 ng) and the TLR4 inhibitor TAK-242 (75 μg; 9-12 mice per group). After 2 h, mice were sacrificed and peritoneal lavages were performed. (b) Male C57BL/6J mice were injected i.a. in the knee with 0.9% NaCl (Co), mu rSAA3 (30 ng), or with a combination of mu rSAA3 (30 ng) and TAK-242 (75 μg; 7-10 mice per group). After 3 h, mice were sacrificed and articular lavages were performed. (c) Male C57BL/6J wildtype (WT) and TLR4 knockout (TLR4^-/-) mice were injected i.a. in the knee with 0.9% NaCl (Co) or mu rSAA3 (30 ng; 3-5 mice per group). After 3 h, mice were sacrificed and articular lavages were performed. (a–c) Lavages were subjected to total cell count and cytospins were prepared for differential leukocyte counts by 2 individuals independently. Data represent the total number of and are derived from 1 (c), 2 (b), or 3 (a) experiment(s). Statistically significant recruitment of neutrophils compared to control mice, determined by the Mann-Whitney test, is indicated by asterisks (; ; ). Statistically significant inhibition of neutrophil recruitment compared to mice injected with mu rSAA3 alone (a and b) or to wildtype mice injected with mu rSAA3 (c), determined by the Mann-Whitney test, is indicated by a dagger (^†).

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