Genetic Ablation of Hematopoietic Cell Kinase Accelerates Alzheimer's Disease-Like Neuropathology in Tg2576 Mice.

Microglial dysregulation, pertaining to impairment in phagocytosis, clearance and containment of amyloid-ß (Aß), and activation of neuroinflammation, has been posited to contribute to the pathogenesis of Alzheimer's disease (AD). Detailed cellular mechanisms that are disrupted during the disease course to display such impairment in microglia, however, remain largely undetermined. We hypothesize that loss of hematopoietic cell kinase (HCK), a phagocytosis-regulating member of the Src family tyrosine kinases that mediate signals from triggering receptor expressed on myeloid cells 2 and other immunoreceptors, impairs microglial homeostasis and Aß clearance, leading to the accelerated buildup of Aß pathology and cognitive decline during the early stage of neuropathological development. To elucidate the pivotal role of HCK in AD, we generated a constitutive knockout of HCK in the Tg2576 mouse model of AD. We found that HCK deficiency accelerated cognitive decline along with elevated Aß level and plaque burden, attenuated microglial Aß phagocytosis, induced iNOS expression in microglial clusters, and reduced pre-synaptic protein at the hippocampal regions. Our findings substantiate that HCK plays a prominent role in regulating microglial neuroprotective functions and attenuating early AD neuropathology.

Hck/Fgr Kinase Deficiency Reduces Plaque Growth and Stability by Blunting Monocyte Recruitment and Intraplaque Motility.

BACKGROUND: Leukocyte migration is critical for the infiltration of monocytes and accumulation of monocyte-derived macrophages in inflammation. Considering that Hck and Fgr are instrumental in this process, their impact on atherosclerosis and on lesion inflammation and stability was evaluated. METHODS AND RESULTS: Hematopoietic Hck/Fgr-deficient, LDLr(-/-) chimeras, obtained by bone marrow transplantation, had smaller but, paradoxically, less stable lesions with reduced macrophage content, overt cap thinning, and necrotic core expansion as the most prominent features. Despite a Ly6C(high)-skewed proinflammatory monocyte phenotype, Hck/Fgr deficiency led to disrupted adhesion of myeloid cells to and transmigration across endothelial monolayers in vitro and atherosclerotic plaques in vivo, as assessed by intravital microscopy, flow cytometry, and histological examination of atherosclerotic arteries. Moreover, Hck/Fgr-deficient macrophages showed blunted podosome formation and mesenchymal migration capacity. In consequence, transmigrated double-knockout macrophages were seen to accumulate in the fibrous cap, potentially promoting its focal erosion, as observed for double-knockout chimeras. CONCLUSIONS: The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.

IL-33 and M2a alveolar macrophages promote lung defense against the atypical fungal pathogen Pneumocystis murina.

We have recently reported that mice deficient in the myeloid Src-family tyrosine kinases Hck, Fgr, and Lyn (Src triple knockout [TKO]) had augmented innate lung clearance of Pneumocystis murina that correlated with a higher ability of alveolar macrophages (AMs) from these mice to kill P. murina. In this article, we show that despite possessing enhanced killing, AMs from naive Src TKO mice did not demonstrate enhanced inflammatory responses to P. murina. We subsequently discovered that both AMs and lungs from P. murina-infected Src TKO mice expressed significantly greater levels of the M2a markers RELM-α and Arg1, and the M2a-associated chemokines CCL17 and CCL22 than did wild-type mice. IL-4 and IL-13, the primary cytokines that promote M2a polarization, were not differentially produced in the lungs between wild-type and Src TKO mice. P. murina infection in Src TKO mice resulted in enhanced lung production of the novel IL-1 family cytokine IL-33. Immunohistochemical analysis of IL-33 in lung tissue revealed localization predominantly in the nucleus of alveolar epithelial cells. We further demonstrate that experimental polarization of naive AMs to M2a resulted in more efficient killing of P. murina compared with untreated AMs, which was further enhanced by the addition of IL-33. Administration of IL-33 to C57BL/6 mice increased lung RELM-α and CCL17 levels, and enhanced clearance of P. murina, despite having no effect on the cellular composition of the lungs. Collectively, these results indicate that M2a AMs are potent effector cells against P. murina. Furthermore, enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis.

The absence of Hck, Fgr, and Lyn tyrosine kinases augments lung innate immune responses to Pneumocystis murina.

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1beta (IL-1beta), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn(-/-) mice 3 days postchallenge, although the level was less than that observed in Hck(-/-) Fgr(-/-) Lyn(-/-) mice. A correlate to augmented clearance of P. murina in Hck(-/-) Fgr(-/-) Lyn(-/-) mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.

Myeloid Src kinases regulate phagocytosis and oxidative burst in pneumococcal meningitis by activating NADPH oxidase.

Myeloid cells, including neutrophils and macrophages, play important roles in innate immune defense against acute bacterial infections. Myeloid Src family kinases (SFKs) p59/61(hck) (Hck), p58(c-fgr) (Fgr), and p53/56(lyn) (Lyn) are known to control integrin beta(2) signal transduction and FcgammaR-mediated phagocytosis in leukocytes. In this study, we show that leukocyte recruitment into the cerebrospinal fluid space and bacterial clearance is hampered in mice deficient in all three myeloid SFKs (hck(-/-)fgr(-/-)lyn(-/-)) during pneumococcal meningitis. As a result, the hck(-/-)fgr(-/-)lyn(-/-) mice developed increased intracranial pressure and a worse clinical outcome (increased neurologic deficits and mortality) compared with wild-type mice. Impaired bacterial killing was associated with a lack of phagocytosis and superoxide production in triple knockout neutrophils. Moreover, in hck(-/-)fgr(-/-)lyn(-/-) neutrophils, phosphorylation of p40(phox) was absent in response to pneumococcal stimulation, indicating a defect in NAPDH oxidase activation. Mice lacking the complement receptor 3 (CR3; CD11b/CD18), which belongs to the beta(2)-integrin family, also displayed impaired host defense against pneumococci, along with defective neutrophil superoxide production, but cerebrospinal fluid pleocytosis was normal. Cerebral expression of cytokines and chemokines was not decreased in both mouse strains, indicating that CR3 and myeloid SFKs are dispensable for the production of inflammatory mediators. Thus, our study demonstrates the pivotal role of myeloid SFKs and CR3 in mounting an effective defense against CNS infection with Streptococcus pneumonia by regulating phagocytosis and NADPH oxidase-dependent superoxide production. These data support the role of SFKs as critical mediators of CR3 signal transduction in host defense.

The Src family kinase Hck regulates mast cell activation by suppressing an inhibitory Src family kinase Lyn.

IgE/antigen-dependent mast cell activation plays a central role in immediate hypersensitivity and other allergic reactions. The Src family tyrosine kinase (SFK) Lyn is activated by the cross-linking of high-affinity IgE receptors (FcepsilonRI). Activated Lyn phosphorylates the FcepsilonRI subunits, beta and gamma, leading to subsequent activation of various signaling pathways. Lyn also plays a negative regulatory function by activating negative regulatory molecules. Another SFK, Fyn, also contributes to mast cell degranulation by inducing Gab2-dependent microtubule formation. Here we show that a third SFK, Hck, plays a critical role in mast cell activation. Degranulation and cytokine production are reduced in FcepsilonRI-stimulated hck(-/-) mast cells. The reduced degranulation can be accounted for by defects in Gab2 phosphorylation and microtubule formation. Importantly, Lyn activity is elevated in hck(-/-) cells, leading to increased phosphorylation of several negative regulators. However, positive regulatory events, such as activation of Syk, Btk, JNK, p38, Akt, and NF-kappaB, are substantially reduced in hck(-/-) mast cells. Analysis of lyn(-/-)hck(-/-), lyn(-/-)FcepsilonRIbeta(-/-), and hck(-/-)FcepsilonRIbeta(-/-) cells shows that Hck exerts these functions via both Lyn-dependent and Lyn-independent mechanisms. Thus, this study has revealed a hierarchical regulation among SFK members to fine-tune mast cell activation.

The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine.

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.

The Src family kinases Hck and Fgr are dispensable for inside-out, chemoattractant-induced signaling regulating beta 2 integrin affinity and valency in neutrophils, but are required for beta 2 integrin-mediated outside-in signaling involved in sustained adhesion.

Neutrophil beta(2) integrins are activated by inside-out signaling regulating integrin affinity and valency; following ligand binding, beta(2) integrins trigger outside-in signals regulating cell functions. Addressing inside-out and outside-in signaling in hck(-/-)fgr(-/-) neutrophils, we found that Hck and Fgr do not regulate chemoattractant-induced activation of beta(2) integrin affinity. In fact, beta(2) integrin-mediated rapid adhesion, in static condition assays, and neutrophil adhesion to glass capillary tubes cocoated with ICAM-1, P-selectin, and a chemoattractant, under flow, were unaffected in hck(-/-)fgr(-/-) neutrophils. Additionally, examination of integrin affinity by soluble ICAM-1 binding assays and of beta(2) integrin clustering on the cell surface, showed that integrin activation did not require Hck and Fgr expression. However, after binding, hck(-/-)fgr(-/-) neutrophil spreading over beta(2) integrin ligands was reduced and they rapidly detached from the adhesive surface. Whether alterations in outside-in signaling affect sustained adhesion to the vascular endothelium in vivo was addressed by examining neutrophil adhesiveness to inflamed muscle venules. Intravital microscopy analysis allowed us to conclude that Hck and Fgr regulate neither the number of rolling cells nor rolling velocity in neutrophils. However, arrest of hck(-/-)fgr(-/-) neutrophils to >60 microm in diameter venules was reduced. Thus, Hck and Fgr play no role in chemoattractant-induced inside-out beta(2) integrin activation but regulate outside-in signaling-dependent sustained adhesion.

Src family kinases are important negative regulators of G-CSF-dependent granulopoiesis.

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. Truncation mutations of the G-CSF receptor (G-CSFR) are associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia. Although increased proliferative signaling by a representative G-CSFR truncation mutation (termed d715) has been documented, the molecular basis for this hyperproliferative phenotype has not been fully characterized. Given the accumulating evidence implicating Src family kinases in the transduction of cytokine receptor signals, the role of these kinases in the regulation of G-CSF signaling was examined. We show that Hck and Lyn, Src family kinases expressed in myeloid cells, are negative regulators of granulopoiesis that act at distinct stages of granulocytic differentiation. Whereas Hck regulates the G-CSF-induced proliferation of granulocytic precursors, Lyn regulates the production of myeloid progenitors. Interestingly, d715 G-CSFR myeloid progenitors were resistant to the growth-stimulating effect of treatment with a Src kinase inhibitor. Together, these data establish Lyn and Hck as key negative regulators of granulopoiesis and raise the possibility that loss of Src family kinase activation by the d715 G-CSFR may contribute to its hyperproliferative phenotype.