**4. The cytokine-like activities of recombinant SAA**

Recombinant SAA was used in an early study that identified the SAA protein as a chemoattractant for phagocytes [54]. Xu et al. reported that SAA also induced the migration and adhesion of lymphocytes [55]. These studies were among the first to identify leukocyte-activating activities of the recombinant SAA protein. SAA differs from chemokines as it lacks the characteristic cysteine residues that form disulfide bonds for structural stabilization. It was not until 2014 when the crystal structures of two SAA proteins were solved [32, 56]. The 4-helix bundle structure of the SAA monomers and the propensity of forming multimers [32, 56] are strikingly different from the known structural properties of chemokines [57].

Studies conducted by Patel et al. [58] and Fulaneto et al. [59] revealed cytokinelike activities of SAA for its induction of IL-1β, TNFα, IL-1RA and IL-8. Of note, the study conducted by Patel and coworkers used both the recombinant human SAA (rhSAA) and purified SAA-HDL complex, although they found that the cytokine-inducing activity of the SAA-HDL complex was much lower than that of rhSAA. These studies were followed by reports that SAA in neutrophils could induce IL-8 expression through one of the chemoattractant receptors [60] that also mediates anti-inflammatory activities when stimulated by the eicosanoid lipoxin A4 [61, 62]. In addition to proinflammatory cytokines, rhSAA was found to stimulate monocyte expression of tissue factor [63]. Injection of rhSAA to mice increased G-CSF

production and neutrophil expansion [64]. SAA also induced the expression of immunomodulatory cytokines including selective induction of IL-23 over IL-12 [65] and the induction of IL-33 expression [66]. The transcription factors NF-κB, IRF4 and IRF7 have been implicated in SAA-induced gene expression [66, 67]. In addition, SAA appears to be involved in epigenetic regulation of gene expression [68].

One of the cellular targets of SAA is macrophages, a major source of cytokines and most if not all SAA receptors. Macrophages may be differentiated into M1 or M2 phenotypes. Studies have shown that SAA may influence macrophage differentiation. Anthony et al. examined the effects of SAA *in vitro*, using human blood monocytes from chronic obstructive pulmonary disease patients and healthy controls, and *in vivo* using a mouse model with airway SAA challenge [69]. Their work showed that SAA-rendered human monocytes secrete IL-6 and IL-1β concurrently with the M2 markers CD163 and IL-10. Moreover, these cells responded to subsequent LPS stimulation with markedly higher levels of IL-6 and IL-1β. In the mouse model, SAA induced a CD11chigh CD11bhigh macrophage population in a CSF-1R signaling-dependent manner, with concurrent inhibition of neutrophilic inflammation. Sun et al. investigated the potential effect of SAA on macrophage plasticity, and found that SAA treatment led to increased expression of macrophage M2 markers including IL-10, Ym1, Fizz-1, MRC1, IL-1Rn, and CCL17 [67]. Moreover, SAA enhanced efferocytosis of mouse macrophages. Silencing IRF4 by small interfering RNA abrogated the SAA-induced expression of M2 markers, suggesting a potential role for SAA to alter macrophage phenotype and modulate macrophage functions.

SAA has been identified as an endogenous activator of the NLRP3 inflammasome, which is critical to the process of pro-IL-1β. Niemi et al. reported that SAA provided a signal for pro-IL-1β expression and for inflammasome activation [70]. At least 3 SAA receptors, including TLR2, TLR4 and the ATP receptor P2X7, were involved. Interestingly, inflammasome activation was dependent on the activity of cathepsin B, the expression of which was induced by SAA. Therefore, SAA-induced secretion of cathepsin B could facilitate extracellular processing of SAA and development of AA amyloidosis. Ather et al. showed SAA3 expression in the lungs of mice exposed to mixed Th2/Th17-polarizing allergic sensitization regimens [71]. SAA instillation into the lungs elicited pulmonary neutrophilic inflammation and activation of the NLRP3 inflammasome, thereby promoting IL-1β secretion by dendritic cells and macrophages. SAA administered into the lungs also served as an adjuvant that sensitized mice to inhaled OVA, promoting IL-17 production from restimulated splenocytes and leukocyte influx. Collectively, these findings illustrate a stimulatory function of SAA in the induced expression of IL-1β.
