To assess whether calpain inhibition would affect the synthesis of IL-1α and IL-1β alarmins, the two main cytokines related to NLRP3 inflammasome activation and inflammaging, complementary analyses were conducted in vitro and in vivo. First, bone-marrow derived macrophages were isolated. We observed that the synthesis of IL-1α and IL-1 β at the mRNA level was globally reduced by calpastatin in response to all inflammasome activators tested (Fig. 11A,B, p = 0.01 and p = 0.05 respectively, ANOVA). Specific calpain inhibition decreased IL-1β concentration in the extracellular milieu in reponse to both particulate and non-particulate inflammasome activators (Fig. 11D, p = 0.003, ANOVA). Of notice, IL-α synthesis was also decreased by calpain inhibition but specifically after macrophage exposure to sodium urate and silica crystals, particulate activators of inflammasome (Fig. 11C, p = 0.008). In addition, we assessed IL-α and IL-1β synthesis at an early phase in a monosodium urate (MSU) peritonitis model. CalpTG mice had lower expression of both IL-1α and IL-1β cytokines, consistent with the in vitro experiments and in vivo observations in aged mice (Fig. 11E,F, p = 0.008).Figure 11Calpastatin overexpression inhibits IL-1α and Il-1β synthesis in vitro and in vivo. In vitro, macrophages isolated from WT and CalpTG (TG) mice bones have been exposed to sodium urate crystals (MSU), Silica crystals (Si.), Adenosine TriPhosphate (ATP) or Nigericin (Niger.), with or without priming by Lipopolysaccharides (LPS). IL-1α and IL-1β expression at the mRNA level was globally reduced (ANOVA) by calpastatin overexpression (A,B, n = 4/condition). Similarly, IL-1α and IL-1β cytokine production by macrophages was also globally reduced by calpastatin overexpression, especially IL-1α synthesis after exposure to particulate inflammasome activators MSU and Si. (C,D, *p < 0.05, **p < 0.01 (post test), n = 4/condition). In vivo, MSU crystals have been injected to WT and CalpTG (TG) mice intraperitonally. IL-1α and IL-1β cytokines have been measured early (3 hours) in the peritoneal fluid, their synthesis was dramatically reduced in CalpTG mice (E,F, **p < 0.01, n = 5/group).