SHIP1 and its role for innate immune regulation—Novel targets for immunotherapy

Phosphoinositide‐3‐kinase/AKT (PI3K/AKT) signaling plays key roles in the regulation of cellular activity in both health and disease. In immune cells, this PI3K/AKT pathway is critically regulated by the phosphoinositide phosphatase SHIP1, which has been reported to modulate the function of most immune subsets. In this review, we summarize our current knowledge of SHIP1 with a focus on innate immune cells, where we reflect on the most pertinent aspects described in the current literature. We also present several small‐molecule agonists and antagonists of SHIP1 developed over the last two decades, which have led to improved outcomes in several preclinical models of disease. We outline these promising findings and put them in relation to human diseases with unmet medical needs, where we discuss the most attractive targets for immune therapies based on SHIP1 modulation.


Introduction-SHIP1 in a nutshell
The PI3K pathway is one of the most ubiquitous intracellular signaling cascades utilized in all cells, which plays roles in inflammation, immunity, and carcinogenesis [1,2].In 1994, the Src homology 2 (SH2) containing inositol phosphatase (SHIP) was discovered, whereby its lipid phosphatase activity defined its role as a negative regulator of the PI3K pathway [3].There are two SHIP paralogs, SHIP1 and SHIP2, which are encoded by the INPP5D and INPPL1 genes, respectively [4].While the expression profile in cells differs between the two SHIP paralogs, the proteins they encode remain structurally similar, with the largest differences being in their C-terminal domains [4].The phosphatase activity of SHIP remains its best-known function, where it dephosphorylates PI (3,4,5) P 3 into PI (3,4) P 2 [4].This phosphatase activity is distinct from that of other phospholipid phosphatases with regard to its product, for example PTEN-mediated production of PI (4,5) P 2 [1].
Both PI (3,4,5) P 3 and PI (3,4) P 2 eventually lead to the nuclear translocation of a wide range of distinct, yet overlapping, tran-Correspondence: Dr. Wen Jie Yeoh and Prof. Philippe Krebs e-mail: wen.yeoh@unibe.ch;philippe.krebs@unibe.chscription factors that regulate cellular proliferation, activation, and migration [4].The structural elements, including the different domains and motifs within the SHIP1 protein, have been recently reviewed [4].Briefly, these include a SH2 domain, a phosphatase domain, a pleckstrin homology-related domain, and the C-terminus containing several protein-protein interaction motifs [4,5].Marshall et al. proposed SHIP1 to be a "tunable" regulator of immune responses, whereby it is recruited both as an effector for inhibitory receptors or as an inhibitor of activating receptors [4].Furthermore, as SHIP1 can have different binding motifs, such as phosphorylated tyrosines (pY917, pY1020) and proline-rich domains, it can act as an adaptor protein by interacting with proteins; or to act as a "masking" protein by blocking protein interactions [6][7][8][9][10][11][12].
Unlike other phosphatases and SHIP2, constitutive SHIP1 expression is mainly restricted to the hematopoietic cell lineage [13], albeit with few specific exceptions (e.g.endothelial cells or mesenchymal stem cells [14,15]).Regulation of SHIP1 activity can occur via various means, with the best-documented mechanism being its transcriptional induction by TGF-β signaling [16,17] and its repression by mir-155 [18].Interestingly, SHIP1 reciprocally inhibits mir-155 expression, creating a negative feedback loop to limit mir-155/SHIP1 activity, indicating a tight regulation of both mir-155 and SHIP1 levels [19].
SHIP1 has previously been shown to play key roles not only in adaptive immunity, especially in the context of cancer, but also in bone biology, which are topics that have been previously discussed [4,5,33,34].In this review, we will highlight the function of SHIP1, specifically within the innate immune compartment.We first summarize the present knowledge and recent findings on the role of SHIP1 in innate immune populations (Fig. 1), and thereafter highlight current and potentially future therapeutic uses for SHIP1 modulation in different disease contexts.

SHIP1-A suppressor of innate immunity
Macrophages and dendritic cells SHIP1-deficient mice exhibit a perturbed myelopoietic compartment in the bone marrow (BM), developing myeloproliferative disease with the accumulation of myeloid progenitors [35,36].SHIP1 is needed for optimal DC differentiation from BM precursors, where SHIP1 deficiency results in a skewing towards increased macrophage differentiation and reduced DC differentiation [37].
Macrophages and DCs also serve as professional APCs, acting as the innate-adaptive bridge that orchestrates adaptive immune responses.Consequently, SHIP1-deficiency in either macrophages [55] or DCs [56,57] leads to impaired Th2 responses, likely due to higher IL-12 secretion resulting in increased Th1 differentiation [43,55,56].This effect is compounded by a cell-intrinsic skewing of SHIP1-deficient T cells toward a Th1 phenotype [33], thus leading to a biased and more pronounced type 1 response due to loss of SHIP1 activity both in DCs and (naïve) T cells.
Taken together, while SHIP1 undeniably plays key roles in macrophages and DCs for the regulation of microbial killing and antigen presentation-via modulation of cell motility and phagocytic activity-there are still some discrepancies in the underlying mechanisms, which is likely due to the complex interplay of contextual signals dictating the overall outcome of SHIP1 inhibition.This currently limits the options for the therapeutic targeting of SHIP1 in the macrophage or DC compartment.

Myeloid-derived suppressor cells (MDSCs)
MDSCs are a heterogenous group of myeloid cells that suppress innate and adaptive immune responses, which have been implicated in cancer, autoimmunity, and inflammatory diseases [68].Increased splenic MDSC numbers occur as a consequence of a general decrease in SHIP1 expression or activity among splenocytes, indicating a role of SHIP1 in regulating MDSC expansion [69].Indeed, MDSC numbers are increased upon genetic or pharmacologic inhibition of SHIP1 [70][71][72].Strikingly, myeloid cellspecific deletion of SHIP1 (using LysM cre SHIP1 flox mice-which do not develop the typical systemic syndrome affecting mice entirely deficient in SHIP1) still results in MDSC expansion [73].Nevertheless, the presence of SHIP1 protein in MDSCs is still unclear, potentially depending on whether these cells are analyzed ex vivo or after in vitro differentiation from BM precursors [73,74].Therefore, MDSC expansion is likely controlled by both cell-autonomous mechanisms [74] and possible secondary mech-anisms related to systemic SHIP1 inhibition, for example, systemic high levels of G-CSF, as initially proposed [73].Therefore, SHIP1 appears to have both direct and indirect roles in restraining innate immunoregulatory cell types such as MDSCs and the "healing" M2-like macrophages, described above.

Microglia
Microglia are specialized tissue-resident macrophages in the brain, which are centrally involved in neuroinflammatory and neurodegenerative diseases [75].Dysregulated PI3K signaling in microglia has been linked to chronic neuroinflammation, a characteristic of Alzheimer's disease (AD) and other neurodegenerative conditions [76,77].In mouse models of AD, SHIP1 expression increases predominantly in plaque-associated microglia, where it is positively associated with disease progression [78,79].
Mechanistically, SHIP1 negatively regulates TREM2 signaling in microglia [79][80][81], a pathway that drives AD pathogenesis [82], thus indicating a possible protective role of SHIP1 in AD.As such, both mice with microglia-specific SHIP1-deficiency (Cx3cr1 CreER SHIP1 flox ) or with constitutive SHIP1 haploinsufficiency (SHIP1 +/− ) result in increased plaque-associated microglia numbers, association with Aβ plaque burden, and increased microglia activation or phagocytic activity [83][84][85].However, these studies present divergent findings on the amount of Aβ plaque burden and neuronal health, which is possibly related to differences in the strategy used to genetically target SHIP1 or the use of distinct mouse models of AD.
Another recent study shows that while SHIP1 haploinsufficiency only leads to a minor rescue in disease-associated microglial signatures, SHIP1-deficient microglia might have improved adhesion to Aβ plaques [79] and reduced p-tau deposition [79].Taken together, SHIP1 seems to promote microglia dysfunction and pathogenic inflammation in the brain.

Mast cells (MC) and basophils
Mast cells (MC) and basophils share the ability to degranulate upon IgE receptor crosslinking after binding of an allergen epitope, which triggers the release of histamine and other inflammatory mediators that underly hypersensitivity disorders and anaphylaxis [86].
MC degranulation exhibits a bell-shaped response curve dependent on the dose of the triggering allergen or antigen [87][88][89].Early in vitro studies suggest that SHIP1 critically regulates the threshold of IgE-induced signaling for degranulation at normal antigen concentrations [90][91][92], whereby SHIP1-deficient MCs fail to regulate the descending part of the bell curve response [92].Similarly, in human basophils, SHIP1 is phosphorylated at high levels during supraoptimal antigen concentrations, with a negative correlation between the degree of SHIP1 (activating) phosphorylation and histamine release [93].This suggests that SHIP1, together with other inhibitory mechanisms, restrains degranulation responses induced by engagement of FcεRI, the high-affinity IgE receptor.At higher antigen concentrations, SHIP1 further supports the inhibitory effect of other molecules [94], thereby leading to the above-mentioned dose-dependent bellshaped response of degranulation [88].
This inhibitory role of SHIP1 was shown to partly restrain histamine release in MCs in a model of intracerebral hemorrhage [95], and SHIP1 was further shown to be a negative regulator of MC or basophil hyperplasia and cytokine secretion [54,59,96].
In line with these findings, siRNA-mediated SHIP1 knockdown in stem cell-differentiated human basophils and MCs increases their activity [97].However, the addition of a SHIP1 antagonist (3AC) to primary human basophils or mast cells either enhances or diminishes their activity (as determined by CD63 expression), respectively [98].This suggests a phosphatase-independent function of SHIP1 in regulating MC activation [98].
As a whole, SHIP1 acts as a key negative regulator of granulocyte degranulation and cytokine production in MC and basophils, where it mediates inhibitory function to modulate responses to stimuli.

Neutrophils
The PI3K pathway plays several distinct roles in the biology of neutrophils [99], which swiftly respond to injury/damage/inflammation, yet whose short half-life is critical for the resolution of inflammation and the prevention of immunopathology [100].
Neutrophil apoptotic death is partly regulated by increased SHIP1 [101] and decreased PI3K-γ activity [102], depending on the nature of the proapoptotic stimuli, including inflammatory cytokines or ROS accumulation [101,102].SHIP1-deficient neutrophils not only live longer, but they also exhibit defective chemotaxis [103], with SHIP1 contributing to their polarity and adhesion [104].
Neutrophil effector functions include the formation of neutrophil extracellular traps (NETs) and the production of ROS [105].Pharmacological blockade experiments have shown that SHIP1 positively regulates NETosis in murine neutrophils [106].Interestingly, while ROS production was reduced in SHIP1deficient neutrophils when in suspension, the same cell produced large amounts of ROS when adherent due to changes to PI3K signaling upon integrin signaling [104].
In summary, SHIP1 critically regulates neutrophil effector function by affecting their migration, ROS production, and NETosis.

Innate lymphocytes
NK cells are the largest innate lymphocyte population, which are functionally regulated by a wide range of surface inhibitory or activating receptors as well as cytokine receptors [107].SHIP1deficient mice exhibit a severely altered NK cell compartment [108], with decreased NK cell numbers [109,110], impaired NK cell maturation [109], and a dysregulated NK receptor repertoire.In addition, SHIP1-deficient NK cells show impaired cytokine production [109][110][111][112], cytotoxicity [12,111,113], and chemotaxis [114].Addressing conditional SHIP1 deletion in NK cells (NKp46 iCre SHIP1 flox mice), Gumbleton et al. showed that while SHIP1-deficient NK cells have intracellular signaling profiles similar to those of activated NK cells, they are still hyporesponsive upon crosslinking of major NK activating receptors [110].In addition, SHIP1 plays cell-autonomous roles in promoting proper NK cell maturation, education, and memory NK cell responses to hapten [110].This indicates a role of SHIP1 in governing proper developmental and functional responses that is unique to NK cells, due to NK cell licensing and distinct activation processes [115].
Proinflammatory cytokine stimulation of SHIP1-deficient NK cells may result in either increased [116,117] or decreased [109] IFNγ expression.Still, SHIP1 consistently restrains NK cell cytokine production in response to CD16 and/or cytokine stimulation [116,118,119].
Besides NK cells, SHIP1 also has roles in regulating rare populations of other innate lymphocytes, including invariant natural killer cells (iNKT)-which respond to both activation by invariant TCR binding and by cytokine signals [120].Compared to their WT counterparts, SHIP1-deficient iNKT cells produce fewer cytokines and are hyporesponsive when stimulated with either αCD3/αCD28 (i.e. via the TCR) or PMA/ionomycin (i.e. by bypassing the TCR) [121], indicating a cell-autonomous role of SHIP1 for optimal secretion of inflammatory mediators by iNKT cells.
Unlike iNKT cells and adaptive lymphocytes, innate lymphoid cells lack expression of a TCR and mainly respond to cytokine cues in the milieu [122].In particular, systemic SHIP1 pharmacological inhibition in mice increases type 2 innate lymphoid cell (ILC2) production of cytokines, suggesting a role of SHIP1 in restraining the function of these cells [123].
Taken together, SHIP1 regulates signals arising from either TCR, cytokines, or other activating or inhibitory receptors in innate lymphocytes.In general, SHIP1 restrains cell activation and production of proinflammatory cytokines in these innate lymphocytes, whereby the underlying mechanisms in NK cells are particularly intricate due to the complex network of inhibitory and activating receptors regulating their development and activity.

Summary and reflections
In summary, SHIP1 generally represses the production of proinflammatory cytokines while supporting anti-inflammatory IL-10 and IL-4 production and signaling, thus, broadly suppressing inflammation triggered by innate immunity (Fig. 1).Nevertheless, SHIP1-dependent regulatory mechanisms in certain innate immune subsets, such as NK cells (with multiple levels of regulation) or eosinophils, are still ambiguous.Mice entirely deficient for SHIP1 have multiple highly dysregulated immune compartments, with many confounding extrinsic factors hampering proper investigation of cell-autonomous SHIP1 regulation in specific immune subsets.As suggested before [124], technical advances relying, for example, on new tools using the Cre/loxP recombination system in mice will permit better investigation of the intricacies of SHIP1 regulation in vivo-whereby such a genetic approach has already been applied for several innate immune populations [55,65,85,110], and is increasingly available for rarer immune subsets [125][126][127][128].In particular, the role of SHIP1 in limiting type 2 responses is clear, as evidenced by its effect in macrophages, basophils, MCs, ILC2s, and eosinophils [123,124,129], possibly further increasing the relevance of SHIP1 to type 2-driven inflammation for diseases at barrier sites.SHIP1 is generally seen as a negative regulator for PI3K signaling due to its ability to reduce intracellular PI (3,4,5) P 3 levels.However, its downstream phosphoinositides, PI (3,4) P 2 and PI (3) P generated by SHIP1 and INPP4A, are not only degradation products of PI (3,4,5) P 3 catabolism, but are also able to trigger similar and different signaling pathways leading to distinct cellular phenotypes [4,99,130].Indeed, loss of SHIP1 activity leads to changes in the phosphoinositide species' concentrations, which can possibly lead to cellular dysregulations.This includes alterations in functions, such as phagocytosis, phagolysosome formation, cell migration, and ROS production, of which defects are evidenced in SHIP1deficient immune cells and affect disease outcomes [51,60,61,66,104,131,132]. Thus, modulation of SHIP1 does not only involve downregulation of PI (3,4,5) P 3 signaling; these phosphoinositides should also be taken into account, as previously alluded to in a previous review [33].Indeed, production of these phosphoinositides might also sometimes explain differing findings on the phenotype of SHIP1-deficient cells, as exemplified in the case of neutrophil adhesion and ROS production [104] (see also above).In addition, temporal kinetics of phosphoinositide-mediated regulation of cellular functions might also explain certain discrepancies between studies [66], and the additional, phosphataseunrelated functions of SHIP1 should be considered as well when comparing different reports.
While cellular memory has been a defining feature of adaptive immunity in textbooks, there is growing evidence supporting "memory"-like adaptation of the innate immune compartment [133].Induction of either hyper-or hyporesponsiveness in innate immune cells during a secondary challenge is termed "trained immunity" or "innate immune tolerance," respectively [133,134].These activity states are usually regulated by epigenetic reprogramming of transcriptional pathways [134], with possible transgenerational effects of trained immunity leading to long-lived protection [135][136][137].A combination of stressordependent and dose-dependent factors likely dictates the outcome of the secondary cellular response [138][139][140], where PI3K signaling has been implicated in mediating this phenomenon [139].As mentioned above, different PRR signals in SHIP1-deficient macrophages or DCs induce unique inflammatory cytokine profiles [41,43], whereby this phenomenon has interestingly also been observed during the induction of innate training [140].In addition, NK cell memory has been shown against viruses, viral particles, and hapten [141][142][143], where SHIP1 is necessary for NK cell memory responses in hapten-induced hypersensitivity [110].
Furthermore, presence of SHIP1 also seemingly dictates cellular responsiveness upon rechallenge.Indeed, expression or upregulation of SHIP1 plays a key role in mediating the innate tolerance mechanisms observed in macrophages [39,49,[144][145][146] and MCs [138,147].Conversely, low levels of SHIP1 in challenged immune cells result in potentiated and enhanced responses upon secondary challenge [118,148].SHIP1 levels can be regulated by autocrine activation of TGF-β signaling [17], rates of SHIP1 proteasomal degradation, and mir-155 induction-the latter having been proposed to be involved in innate tolerance [149,150].Regulation of SHIP1 levels is most likely a dynamic process that depends on the array of stimuli present.Hence, we propose that SHIP1 is a key checkpoint in deciding the fate of innate memory, where appropriate regulation of its expression and activity is needed for homeostatic maintenance and optimal cellular function upon recall or restimulation.
Last, while this review focuses on innate immunity, SHIP1 does play a role in adaptive immunity, that is T and B cells, which display dysregulated responses upon loss of SHIP1 activity.Briefly, SHIP1 has been associated with the control of T-cell processes such as Treg and Th17 development [73,151], T-cell motility [152], and T-cell death [153,154].In B cells, SHIP1 modulates IL-10 production [155] as well as BCR-dependent anergy/responsiveness [156][157][158].Accordingly, SHIP1 deficiency in B cells leads to antibody-induced autoimmune disease [155].Furthermore, given the reciprocal interactions between innate and adaptive immune cells, the function of SHIP1 in adaptive immunity should also be considered when evaluating a therapeutic use of SHIP1 modulators and their effect on the overall immune response.
These lines of evidence provide a basis to delineate the possibility of using SHIP1 modulators for immune therapy.Nevertheless, one must keep in mind that systemic modulation of SHIP1 activity may lead to contradictory outcomes.For example, general SHIP1 inhibition may simultaneously lead to the increased release of inflammatory mediators as well as the opposing expansion of immune-regulatory cells, depending on the ability of each of these mechanisms taken individually to show therapeutic values in specific contexts.Thus, understanding the mechanisms of SHIP1 activity and its overall outcome for disease in a physiological (in vivo) setting will provide insights to better define the specific pathologic contexts where SHIP1 modulators may be applied for therapy.

Therapeutic avenues
Current available options for SHIP1 therapy include a panoply of SHIP modulators of different solubilities and targeting specificity, which have been mainly developed by the Kerr group and a private company, where their efficacy and possible toxicity have been extensively discussed [5].In addition, the potential of targeting SHIP1 in cancer has already been previously reviewed [5].In general, SHIP1 inhibitors and activators can be used to respectively enhance or suppress inflammation, depending on the disease etiology.The latter may be considered a better alternative to the current anti-inflammatory therapies that show wide detrimental side effects.Disorders that may benefit from therapeutic SHIP1 modulation often present dysregulated inflammatory processes initiated or exacerbated by adaptive immunity.Thus, while not explicitly discussed in this review, it is relevant to consider the relative contribution of the innate versus adaptive immune compartments that may be distinct between different diseases.Nevertheless, we summarize below possible disorders for which SHIP1 modulation has therapeutic potential in regulating innate immunity (Fig. 2), based on the current knowledge gleaned from experimental mouse models and ex vivo experiments using primary human immune cells.

Inflammatory diseases at barrier surfaces
Due to their direct exposure to the external environment, barrier surfaces, such as the intestine and lungs, are susceptible sites for the development of inflammatory disease.As such, chronic inflammatory disorders often develop upon immune dysregulation at mucosal surfaces, albeit with different etiologies and contributions of distinct innate immune subsets underlying such pathogenic inflammation.SHIP1 dysregulation has been implicated in the hyperactivation of innate immune cells driving a variety of disease types in murine models, where modulation of SHIP1 function has led to improved outcomes in mouse models of dysregulated inflammation at barrier surfaces [7,56,159,160].Chronic airway disorders are increasingly prevalent and are associated with persistent pathological inflammatory reactions in the lung.Oxidative stress promotes PI3K in chronic obstructive pulmonary disorder (COPD) [161], and PI3K signaling is central to asthma development [162].Multiple types of airway diseases, including COPD and asthma, feature dysregulated type 2 inflammation [163], which is, in part, controlled by SHIP1 [124].For instance, decreased SHIP1 protein levels are negatively correlated with histamine release in IgE + basophils isolated from highly allergic patients [164].This supports the notion that SHIP1 dictates cellular responses during allergy.In addition, the phosphatase-independent function of SHIP1 positively regulates, in part, MC desensitization [138], a therapeutic approach used for allergen immunotherapy [165][166][167].As presented above, SHIP1 also has roles in restraining neutrophils, NK cells, and macrophages, which are key drivers of neutrophilic COPD.Thus, SHIP1 activators may potentially attenuate chronic inflammation in the lung.
SHIP1-deficient mice spontaneously develop ileitis that is likely driven by cells from the granulocyte-monocyte lineage [168].Importantly, this ileitis shares characteristics with Crohn's disease (CD) in IBD patients.As a matter of fact, SHIP1 has been associated with human IBD.Increased levels of SHIP1 transcripts are found in inflamed colonic biopsies from patients with ulcerative colitis and colonic CD [169].However, SHIP1 mRNA levels were either unchanged [24,169] or reduced [170] in ileal CD, perhaps reflecting different immunoregulatory mechanisms at different intestinal sites.Still a severe deficiency in SHIP1 protein expression has been reported in monocytes, NK cells, and neutrophils of IBD patients [24], which is associated with increased IL-1β production [170].In sum, these observations indicate an essential contribution of SHIP1 activity to intestinal immune regulation.
Microbes are present at extremely high concentrations in the intestinal tract, where they play a role in driving intestinal inflammation, thus, implying modulation of PRR signaling as a way to regulate gut inflammation in IBD [171,172].Considering the function of SHIP1 as a negative regulator of intracellular signals downstream of multiple PRRs, SHIP1 activators appear as a pertinent option to mitigate the aberrant mucosal immune inflammation associated with IBD.Indeed, AQX/ZPR-MN100, a SHIP1 allosteric activator, mimics IL-10 anti-inflammatory activity to alleviate colitis in mice [7], further, supporting the role of SHIP1 activators in suppressing pathogenic intestinal inflammation.
Innate immunity subsets, including macrophages and DCs, support inflammation in different skin diseases [173,174].For example, chronic idiopathic urticaria is driven by basophils [175], whereby patients segregate into two groups based on normal versus reduced responsiveness to anti-IgE-induced histamine release, with decreased SHIP1 protein expression in anti-IgE responders [176].This indicates a role for SHIP1 in restraining human basophil degranulation and a potential role for SHIP1 activators in preventing basophil hyperresponsiveness.
Taken together, the use of SHIP1 activators may represent a valuable therapeutic option to dampen the inflammation elicited by resident or recruited (innate) immune subsets at barrier surfaces.Yet since SHIP1 deficiency in DCs can lead to a skewing towards a Th1 response, which prevents the development of allergic airway inflammation in mice [56,57].Thus, SHIP1 inhibitors might be an alternative approach to curb the type 2 immune hyperactivation in allergic diseases.
Besides macrophages, NK cells are also needed for immunity against intracellular pathogens, whose cytokine activity is controlled by SHIP1 [116][117][118].In humans, CD56 bright NK cells show higher expression of cytokines compared to their more cytotoxic CD56 dim counterparts, where SHIP1 is constitutively lower in the CD56 bright NK cell subset [117].Indeed, SHIP1 overexpression in CD56 bright NK cells leads to decreased cytokine production [117].Interestingly, anergic CD56 − CD16 + NK cells from patients with HIV also highly express SHIP1 while displaying reduced levels of perforin [184], further indicating a role of SHIP1 in limiting NK cell effector function during persisting infection.Consequently, SHIP1 inhibitors might show a beneficial effect in reducing anergy in NK cells in the context of chronic infections.As mentioned above, loss of SHIP1 in APCs and T cells leads to skewing and strengthening of a type 1 effector response in T cells [33], which may be particularly beneficial, for instance, for the clearance of intracellular pathogens.Thus, pharmacologic modulation of SHIP1 may help promote a Th1 skewing to contravene the immune escape strategy of certain pathogens.
Regulation of trained immunity or immune tolerance by SHIP1 in innate immune cells [39,49,140] may also be exploited toward therapeutic purposes.For instance, modulation of SHIP1 can enhance trained immunity in murine and human macrophages via potentiation of the PI3K/AKT pathway [148].SHIP1 blockade may synergize with CD16 ligation by obinutuzumab-a monoclonal antibody that enhances NK cell activation and leads to the generation of primed experienced NK cells with enhanced IFNγ responses upon restimulation [185].Indeed, SHIP1 expression is reduced in experienced NK cells, which potentiates the PI3K signaling pathway for increased IFN-γ production [118].In addition, trained immunity has also been described for other innate immune cells [133,134,186], thus setting up interesting avenues for the use of SHIP1 modulators either for increasing vaccine efficacy or preventing trained immunity seen in allergy and autoimmunity [187].

Autoimmune diseases (rheumatoid arthritis, gouty arthritis)
Innate immune cells overactivated through aberrant TLR signaling drive rheumatoid arthritis (RA) pathogenesis, where therapies suppressing innate immunity-driven inflammation are currently being explored [188].In humans, SHIP1 downregulation can be induced in healthy PBMCs when cultured with monosodium urate, an inflammatory trigger of gouty arthritis [189].Along these lines, synovial fluid mononuclear cells, CD14 + monocytes, and CD68 + macrophages, from patients with RA or acute gouty arthritis exhibit lower expression of SHIP1 [189,190].In contrast, neutrophils from RA patients overexpress SHIP1 and show increased NETosis [106].Nevertheless, inhibition of SHIP1 function in vivo has led to better outcomes in mouse models of arthritis, with changes in MDSC and neutrophil function [72,106].
Loss of SHIP1 in DCs can break self-tolerance and promote a CD8-mediated autoimmune response [191].Similarly, SHIP1 promotes CLEC4C inhibitory signaling in human plasmacytoid DCs, which is necessary for restraining self-DNA/RNA induced autoimmunity [192].

Alzheimer's disease (AD)
AD is a complex neurodegenerative disorder with a central pathogenic role for microglial activation and neuroinflammation [194].While SHIP1 in microglia protects against plaqueinduced neuronal dystrophy in mice [79,[83][84][85], reduced SHIP1 expression was also associated with AD progression in a murine model [195].Interestingly, while pan-SHIP1/2 inhibition promotes phagocytic/degradation processes needed for proper clearance of Aβ in microglia [196], SHIP1 activators can reduce the release of inflammatory cytokines from microglia [197].In addition, the role of SHIP1 in other murine models of AD is still ambiguous, as discussed above, where SHIP1 may promote either beneficial or harmful activities such as phagocytosis and secretion of inflammatory mediators, respectively.SHIP inhibitors have been proposed as a treatment in early AD to prevent Aβ plaque formation, whereas SHIP1 activators may be a more suitable choice to limit neuroinflammation in late-stage disease [197].
In humans, SHIP1 was shown to be an AD risk gene, where it is predominantly expressed in microglia [79,[198][199][200][201].SHIP1 has since been associated with the late onset of AD [30,[202][203][204], along with upregulation of SHIP1 levels in AD patients [78,203].High SHIP1 expression has been associated with the increased amyloid plaque density and microglia markers, which are positive correlates of AD progression [78].Thus, there seems to be a clear avenue for SHIP1 modulators in neurodegenerative disease, particularly in AD.
Nevertheless, additional experiments are certainly needed for more informed decisions about the use of SHIP1 modulators in AD.

Obesity and Diabetes
Chronic inflammation and metabolic dysregulation are common features among patients with obesity [205].SHIP1 was found to be upregulated in diet-induced obese mice [206].SHIP1 or pan-SHIP1/2 inhibition in diet-induced and age-induced obese mice reverses both obesity and hyperglycemia via eosinophildependent mechanisms [123,207].This likely occurs via a SHIP1 inhibition-mediated increase in IL-4 secretion by eosinophils, which drives the polarization of macrophages to an M2-like phenotype [123].Similar results are possibly expected in humans, where SHIP1 inhibition may extend eosinophil lifespan, given the involvement of SHIP1 enzymatic activity in Siglec-8-induced eosinophil cell death [129].
Treatment with a SHIP1 agonist restores responsiveness to IL-10 in macrophages from patients with type 2 diabetes or macrophages exposed to high glucose [46].
In sum, both SHIP1 inhibitors and activators may lead to suppression of chronic inflammation associated with obesity or diabetes, depending on the type of cells to be targeted, where SHIP1 modulation may prove effective in breaking the loop between inflammation and diet-and age-associated obesity.

Graft versus host disease
NK cells, iNKT cells, and MDSCs all regulate GvHD, where SHIP1deficient mice have shown to be permissive to allogenic BM transplantation [12,70,71,110,112].Indeed, SHIP1 inhibition results in reduced allogenic T cell responses and enhanced transplant engraftment [208,209], where loss of SHIP1 can drive the expansion of immunoregulatory populations (MDSCs, Tregs) or the inhibition of NK cell responses [12,70,73,110,112,121,208].Thus, SHIP1 inhibitors may also find utility in facilitating allogenic grafts and preventing GvHD disease by promoting suppression of the immune system.

Conclusions and Considerations
As presented above, SHIP1 evidently plays key roles in regulating cell function and activation to maintain a normal (innate) immune compartment.The cellular mechanisms underpinning SHIP1 function are highly complex, where SHIP1 can either exert phosphatase-dependent or -independent effects depending on the context of the stimulus, cell type, and available interaction partners.Most studies focus on the phosphatase role of SHIP1 in regulating PI3K signaling.However, better understanding of the non-phosphatase function of SHIP1 would allow us to better target specific aspects of SHIP1 activity, considering the increasing evidence for the contribution of the SHIP1 adaptor function for the pathophysiology [6,11] and immunomodulation-as recently reported for the modulation of IL-10 signaling [7].
In addition, understanding both the pathophysiologically relevant immune cell types in different diseases, as well as the specific mechanisms of regulation dependent on SHIP1 in these immune cells may allow the identification of optimal therapeutic niches for the use of SHIP1 modulators.Finding a balance between the promotion of immunoregulatory populations [73] and concomitant increase in inflammatory immune responses induced by a reduction in SHIP1 activity is crucial, as over-tweaking of the immune response may unleash unwanted immunopathology.So far, expansion of immunoregulatory populations upon SHIP1 inhibition has shown better outcomes in mouse models of RA and GvHD, indicating that in these disorders, the immunosuppression trumps the increase in inflammatory mediators upon SHIP1 inhibition.
Furthermore, innate immunity also dictates the response of adaptive immune cells, with SHIP1 playing direct or indirect roles in regulating both immune compartments [4,33].This should be taken into consideration, especially with regard to antigenpresenting (myeloid) cells, which bridge these two immune compartments.Theoretically, the use of SHIP1 modulators may have either complementary or opposite effects on innate versus adaptive immune cells, where the overall outcome achieved may be difficult to assess before previous experimental validation in a complex in vivo setting, possible in dependance of the timing, dose, and route of therapy.Nevertheless, irrespective of the induced molecular events or of the relative contribution of either the innate or adaptive arms of immunity to distinct diseases, SHIP1 modulation has shown effective outcomes in animal models of airway disease [59,160], metabolic disorders [123,207], immune training [148], colitis [7], and arthritis [72,106] (Table 1).These preclinicals studies may potentially translate to clinical testing of SHIP1 modulators in these diseases.
Studies regarding mir-155 modulation in different disorders often involve SHIP1 as a key target and downstream modulator of mir-155 function [18,74,179,190,191,218]. Certain studies mentioned above implicate SHIP1 as the central effector downstream of mir-155 modulation, yet the possible role of additional mir-155 targets should still be considered [219].Therefore, directly targeting SHIP1 instead of mir-155 might prove to be a better option with fewer off-target effects [218].
Importantly, while SHIP1 modulators have shown efficacy when used individually, combination treatment with other signaling modulators might show synergistic effects such as combined PI3Kγ/SHIP1 inhibition [5,220] or simultaneous administration of IVIG and SHIP1 activators [95].Conjoint inhibition of both SHIP1 and SHIP2 paralogs resulted in disease amelioration in diet-induced obese mice or for modulation of microglia activity [123,196].Thus, using pan-SHIP1/SHIP2 inhibitors may prove to be a more efficacious way to target the PI3K pathway, albeit, careful dosage of said drug is needed, as SHIP2 inhibition may lead to a larger range of affected cell types due to its ubiquitous expression [221].
So far, only one SHIP1 small molecule has made it to the clinics, namely the SHIP1 activator AQX-1125.While it initially was a promising candidate for the treatment of interstitial cystitis or bladder pain syndrome, COPD, and atopic dermatitis, displaying anti-inflammatory effects and good drug tolerance, the molecule failed to show therapeutic benefit, and trials were, thus,  discontinued (Table 1) [212-213, 215, 217].However, AQX-1125 is still showing promising results in early trials for asthma, with a trend towards reduced airway inflammation [211].Strikingly, AQX-1125 was recently reported to bind weakly to SHIP1, with minimal phosphatase-enhancing activity [7,59].Therefore, the use of a more potent SHIP1 agonist might show more efficacious results.
Taken together, despite early setbacks in clinical trials, recent findings contributing to a better understanding of SHIP1 modulation indicate that SHIP1 remains a valid therapeutic target for specific pathologies driven by inflammatory processes.Last, there is great potential for improved agonist/antagonist molecules, which may be preferentially applied as combined rather than monotherapies.

Figure 2 .
Figure 2. Overview of potential therapeutic avenues for the use of SHIP1 modulators.(A) SHIP1 blockade-induced expansion of immunoregulatory populations (AAM/M2 macrophages, MDSCs) and inhibition of certain inflammatory populations (NK cells, neutrophils) may ameliorate outcomes in graft versus host disease, rheumatoid arthritis, and obesity or diabetes.While SHIP1 inhibits cytokine-mediated activation of NK cells, SHIP1-deficient NK cells are also hyporesponsive to crosslinking of activating receptors; thus, is it unclear how SHIP1 inhibition may affect GvHD.Such SHIP1 inhibition may either involve SHIP1-specific functional modulation or the use of pan-SHIP1/SHIP2 inhibitors, whereby a SHIP1-specific inhibitor has shown no effects on murine obesity and microglia function.(B) Inflammatory diseases at barrier surfaces have shown improvement upon treatment with SHIP1 activators, thus suppressing inflammatory mechanisms or promoting the release of anti-inflammatory cytokines (IL-10).(C) Application of SHIP1 inhibitors or activators to modulate adaptive responses or "innate immune memory" has potential in improving host defense against pathogens in the context of vaccine development.Dotted arrows: possible interactions.Created with BioRender.com.

Table 1 .
Summary of SHIP1 modulators and functional or clinical outcomes.