*3.2. Macrophages*

Macrophages are a broad variety of phagocytic cells of the innate immune system. Monocytes circulate within the blood and migrate to tissues where local signals can differentiate them into macrophages or DCs. While mDCs and pDCs are recognized as the master antigen presenting cells and T1-IFN producing cells respectively, macrophages also participate in both of these roles and are crucial for producing other pro-inflammatory cytokines, apoptotic corpse removal, and wound healing [56]. SS patient saliva contain high expression of the monocyte chemokine CCL2, and histological analysis of SS patient biopsies identified macrophages within infiltrates of mSGs biopsies [57,58]. Infiltrating macrophages in pSS patients were positively correlated with lesion severity [38] and IL-18, suggesting macrophage activation within the infiltrate [59]. Furthermore, high IL-18 expression by infiltrating macrophages correlated with lymphoma risk factors such as C4 hypocomplementemia and SG enlargement [59]. The polarization of macrophages remains unclear in SS. M1 polarization has been suggested to be more likely in part because proinflammatory cytokines B cell activating factor (BAFF), T1 IFN, IL-6, and IL-12, that are expressed by M1 macrophages, are detected at higher levels in SS patients [60]. Macrophage linked protease genes including cathepsins, matrix metalloproteases (MMPs), and carboxypeptidases were found to be upregulated in highly inflamed SGs biopsies from SS patients, suggesting a role for macrophages in orchestrating tissue destruction and aberrant repair processes in SS [61]. Further investigation revealed that IFN signaling drives plasmin expression by macrophages in SGs and promotes tissue destruction [62]. In addition, macrophage derived chitinases were highly expressed in mSG samples from pSS patients and were associated with disease severity [61]. While focal lymphocytic infiltration can occur in healthy individuals, negligible numbers of macrophages are observed in foci of healthy individuals [63].

Within the NOD mouse model for SS, macrophages have been observed to infiltrate the SGs early in disease development, and precede the arrival of B and T cells [32]. Both M1 and M2 macrophages have been observed in SGs of NOD mice [64]. Among the most important functions carried out by macrophages is the removal of the corpses of apoptotic cells, a process termed efferocytosis [65]. In the related autoimmune disease SLE, delayed removal of dead cells is believed to contribute to disease onset [66]. Delays in the uptake and disposal of dead cells can allow the corpse to progress to secondary necrosis where self-antigens can leak out and activate the immune system [67]. Considering the observation of increased apoptotic cells in the SGs of SS patients and mouse models, this concept has received some attention in SS [68,69]. Analysis of pSS patient monocytes revealed impaired phagocytosis of apoptotic cells and a corresponding defect in initiating immunosuppressive signaling in response to uptake [70]. Additional investigation into SS monocyte derived macrophages determined that SS patient macrophages suffered an intrinsic reduction in phagocytic ability, exacerbated by inhibitory IgG antibodies against apoptotic cells [71]. NOD mice, which have been employed as a model for secondary SS, display impaired efferocytosis by both bone marrow derived and peritoneal macrophages [72]. Our own observations confirm that this efferocytic defect is maintained in the SS C57BL/6.NOD-*Aec1Aec2* mouse model (unpublished data) and may be a result of defective signaling within the Tyro3, Axl, Mertk (TAM) receptor pathway [73].

#### *3.3. Innate Lymphoid Cells (ILCs) and Natural Killer Cells (NK)*

Innate lymphoid cells (ILCs) have received little attention in SS compared to other immune cells. However, ILCs have been observed in both human and mouse salivary gland [74,75]. Natural killer

(NK) cells are ILCs that arise from the same common lymphoid progenitor as B and T cells. NK cells have well characterized roles in the elimination of tumor cells and virally infected cells [76,77]. Yet, the role of NK cells in SS remains poorly understood. NK cells have been discovered within minor SG biopsy patients of SS patients, however the cells are rarer than DCs or macrophages [38]. Despite rarity with the SS lesion, NK cells incidence was found to be positively correlated to presence of rheumatoid factor (RF) and C4 levels in the sera [38]. NKp44<sup>+</sup> ILCs were found to be major producers of IL-22 in pSS patient SG, and infiltration of these cells into the SG correlated with SG inflammation [74]. Levels of NK cells in the blood of SS patients remain controversial. Szodoray et al. observed the percentage of NK cells to be increased in the peripheral blood of pSS patients [78]. In a separate study, the count of NK cells in the peripheral blood was found to be reduced in pSS patients with anti-SSA and SSB autoantibodies [79]. Greater concordance has been achieved in studies of NK activity in pSS, where NK cell activity was found to be reduced in pSS as well as SLE [80]. In addition, Izumi et al. detected decreased NK cells, decreased NK cell activity, and increased apoptotic NK cells in pSS patients [81]. Interestingly, there is evidence that NK cell contribution to SS is not through the traditionally explored lens of NK cells as killers, but rather through the regulatory capacity of NK cells [82,83]. In this study, Rusakiewicz et al. identified a new mechanism for NK involvement in SS where dysfunctional regulation by NK cells via *NCR3*/Nkp30 permits over activation of DCs, facilitating activation of lymphocytes and systemic immunity [82].
