Thrombopoietin levels in sepsis and septic shock - a systematic review and meta-analysis.

OBJECTIVES: Sepsis is a life-threatening condition implicating an inadequate activation of the immune system. Platelets act as modulators and contributors to immune processes. Indeed, altered platelet turnover, thrombotic events, and changes in thrombopoietin levels in systemic inflammation have been reported, but thrombopoietin-levels in sepsis and septic-shock have not yet been systematically evaluated. We therefore performed a meta-analysis of thrombopoietin (TPO)-levels in patients with sepsis. METHODS: Two independent reviewers screened records and full-text articles for inclusion. Scientific databases were searched for studies examining thrombopoietin levels in adult sepsis and septic-shock patients until August 1st 2022. RESULTS: Of 95 items screened, six studies met the inclusion criteria, including 598 subjects. Both sepsis and severe sepsis were associated with increased levels of thrombopoietin (sepsis vs. control: standardized mean difference 3.06, 95 % CI 1.35-4.77; Z=3.50, p=0.0005) (sepsis vs. severe sepsis: standardized mean difference -1.67, 95 % CI -2.46 to -0.88; Z=4.14, p<0.0001). TPO-levels did not show significant differences between severe sepsis and septic shock patients but differed between sepsis and inflammation-associated non-septic controls. Overall, high heterogeneity and low sample size could be noted. CONCLUSIONS: Concluding, increased levels of thrombopoietin appear to be present both in sepsis and severe sepsis with high heterogeneity but thrombopoietin does not allow to differentiate between severe sepsis and septic-shock. TPO may potentially serve to differentiate sepsis from non-septic trauma and/or tissue damage related (systemic) inflammation. Usage of different assays and high heterogeneity demand standardization of methods and further large multicenter trials.

Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Myeloid Src-family kinases are critical for neutrophil-mediated autoinflammation in gout and motheaten models.

Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.

NEF-Induced HIV-Associated Nephropathy Through HCK/LYN Tyrosine Kinases.

HIV-1-associated nephropathy (HIVAN) is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, a transgenic (Tg) mouse model [CD4C/HIV-negative regulator factor (Nef)] was used in which HIV-1 nef expression is under control of regulatory sequences (CD4C) of the human CD4 gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis associated with microcystic dilatation, similar to human HIVAN. To identify kidney cells permissive to the CD4C promoter, CD4C reporter Tg lines were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on 10 different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (Trp53, Tnfsf10, Tnf, Tnfrsf1b, and Bax), in immune cell recruitment (Ccl3, Ccl2, Ccr2, Ccr5, and Cx3cr1), in nitric oxide (NO) formation (Nos3 and Nos2), or in cell signaling (Fyn, Lck, and Hck/Fgr). However, deletion of Src partially and that of Hck/Lyn largely abrogated its development. These data suggest that Nef expression in mesangial cells through hematopoietic cell kinase (Hck)/Lck/Yes novel tyrosine kinase (Lyn) represents important cellular and molecular events for the development of HIVAN in these Tg mice.

Glutamine prevents acute kidney injury by modulating oxidative stress and apoptosis in tubular epithelial cells.

Acute kidney injury (AKI) represents a common complication in critically ill patients that is associated with increased morbidity and mortality. In a murine AKI model induced by ischemia/reperfusion injury (IRI), we show that glutamine significantly decreases kidney damage and improves kidney function. We demonstrate that glutamine causes transcriptomic and proteomic reprogramming in murine renal tubular epithelial cells (TECs), resulting in decreased epithelial apoptosis, decreased neutrophil recruitment, and improved mitochondrial functionality and respiration provoked by an ameliorated oxidative phosphorylation. We identify the proteins glutamine gamma glutamyltransferase 2 (Tgm2) and apoptosis signal-regulating kinase (Ask1) as the major targets of glutamine in apoptotic signaling. Furthermore, the direct modulation of the Tgm2-HSP70 signalosome and reduced Ask1 activation resulted in decreased JNK activation, leading to diminished mitochondrial intrinsic apoptosis in TECs. Glutamine administration attenuated kidney damage in vivo during AKI and TEC viability in vitro under inflammatory or hypoxic conditions.

Therapeutic inhibition of the SRC-kinase HCK facilitates T cell tumor infiltration and improves response to immunotherapy.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically "cold" tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti-programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8+ T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

HIV-1 Nef Induces Hck/Lyn-Dependent Expansion of Myeloid-Derived Suppressor Cells Associated with Elevated Interleukin-17/G-CSF Levels.

Human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) infection causes myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+) through largely unknown cellular and molecular pathways. The mouse cells thought to be equivalent to human CD14+ CD16+ cells are CD11b+ Gr1+ myeloid-derived suppressor cells (MDSC). We used HIV transgenic (Tg) mouse models to study MDSC, namely, CD4C/Nef Tg mice expressing nef in dendritic cells (DC), pDC, CD4+ T, and other mature and immature myeloid cells and CD11c/Nef Tg mice with a more restricted expression, mainly in DC and pDC. Both Tg strains showed expansion of granulocytic and CD11b+ Gr1low/int cells with MDSC characteristics. Fetal liver cell transplantation revealed that this expansion was stroma-independent and abrogated in mixed Tg/non-Tg 50% chimera. Tg bone marrow (BM) erythroid progenitors were decreased and myeloid precursors increased, suggesting an aberrant differentiation likely driving CD11b+ Gr1+ cell expansion, apparently cell autonomously in CD4C/Nef Tg mice and likely through a bystander effect in CD11c/Nef Tg mice. Hck was activated in Tg spleen, and Nef-mediated CD11b+ Gr1+ cell expansion was abrogated in Hck/Lyn-deficient Nef Tg mice, indicating a requirement of Hck/Lyn for this Nef function. IL-17 and granulocyte colony-stimulating factor (G-CSF) were elevated in Nef Tg mice. Increased G-CSF levels were normalized in Tg mice treated with anti-IL-17 antibodies. Therefore, Nef expression in myeloid precursors causes severe BM failure, apparently cell autonomously. More cell-restricted expression of Nef in DC and pDC appears sufficient to induce BM differentiation impairment, granulopoiesis, and expansion of MDSC at the expense of erythroid maturation, with IL-17→G-CSF as one likely bystander contributor. IMPORTANCE HIV-1 and SIV infection often lead to myelodysplasia, anemia, and accumulation of inflammatory monocytes (CD14+ CD16+), with the latter likely involved in neuroAIDS. We found that some transgenic (Tg) mouse models of AIDS also develop accumulation of mature and immature cells of the granulocytic lineage, decreased erythroid precursors, and expansion of MDSC (equivalent to human CD14+ CD16+ cells). We identified Nef as being responsible for these phenotypes, and its expression in mouse DC appears sufficient for their development through a bystander mechanism. Nef expression in myeloid progenitors may also favor myeloid cell expansion, likely in a cell-autonomous way. Hck/Lyn is required for the Nef-mediated accumulation of myeloid cells. Finally, we identified G-CSF under the control of IL-17 as one bystander mediator of MDSC expansion. Our findings provide a framework to determine whether the Nef>Hck/Lyn>IL-17>G-CSF pathway is involved in human AIDS and whether it represents a valid therapeutic target.

A CD22-Shp1 phosphatase axis controls integrin ß7 display and B cell function in mucosal immunity.

The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.

Shp1 Loss Enhances Macrophage Effector Function and Promotes Anti-Tumor Immunity.

Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.

ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense.

Calcium signals are initiated in immune cells by the process of store-operated calcium entry (SOCE), where receptor activation triggers transient calcium release from the endoplasmic reticulum, followed by opening of plasma-membrane calcium-release activated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known to comprise the CRAC channel; however, the contributions of individual isoforms to neutrophil function are not well understood. Here, we show that loss of ORAI1 partially decreases calcium influx, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, we find that ORAI2-deficient neutrophils display decreased calcium influx, which is correlated with measurable differences in the regulation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs multiple neutrophil functions, including phagocytosis, degranulation, leukotriene, and reactive oxygen species (ROS) production, rendering ORAI1/2-deficient mice highly susceptible to staphylococcal infection. This study demonstrates that ORAI1 and ORAI2 are the primary components of the neutrophil CRAC channel and identifies subpopulations of neutrophils where cell-membrane potential functions as a rheostat to modulate the SOCE response. These findings have implications for mechanisms that modulate neutrophil function during infection, acute and chronic inflammatory conditions, and cancer.

The Ubiquitin-Modifying Enzyme A20 Terminates C-Type Lectin Receptor Signals and Is a Suppressor of Host Defense against Systemic Fungal Infection.

C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.

Inhibition of the SRC Kinase HCK Impairs STAT3-Dependent Gastric Tumor Growth in Mice.

Persistent activation of the latent transcription factor STAT3 is observed in gastric tumor epithelial and immune cells and is associated with a poor patient prognosis. Although targeting STAT3-activating upstream kinases offers therapeutically viable targets with limited specificity, direct inhibition of STAT3 remains challenging. Here we provide functional evidence that myeloid-specific hematopoietic cell kinase (HCK) activity can drive STAT3-dependent epithelial tumor growth in mice and is associated with alternative macrophage activation alongside matrix remodeling and tumor cell invasion. Accordingly, genetic reduction of HCK expression in bone marrow-derived cells or systemic pharmacologic inhibition of HCK activity suppresses alternative macrophage polarization and epithelial STAT3 activation, and impairs tumor growth. These data validate HCK as a molecular target for the treatment of human solid tumors harboring excessive STAT3 activity.

Relief of tumor hypoxia unleashes the tumoricidal potential of neutrophils.

Polymorphonuclear neutrophils (PMNs) are increasingly recognized to influence solid tumor development, but why their effects are so context dependent and even frequently divergent remains poorly understood. Using an autochthonous mouse model of uterine cancer and the administration of respiratory hyperoxia as a means to improve tumor oxygenation, we provide in vivo evidence that hypoxia is a potent determinant of tumor-associated PMN phenotypes and direct PMN-tumor cell interactions. Upon relief of tumor hypoxia, PMNs were recruited less intensely to the tumor-bearing uterus, but the recruited cells much more effectively killed tumor cells, an activity our data moreover suggested was mediated via their production of NADPH oxidase-derived reactive oxygen species and MMP-9. Simultaneously, their ability to promote tumor cell proliferation, which appeared to be mediated via their production of neutrophil elastase, was rendered less effective. Relieving tumor hypoxia thus greatly improved net PMN-dependent tumor control, leading to a massive reduction in tumor burden. Remarkably, this outcome was T cell independent. Together, these findings identify key hypoxia-regulated molecular mechanisms through which PMNs directly induce tumor cell death and proliferation in vivo and suggest that the contrasting properties of PMNs in different tumor settings may in part reflect the effects of hypoxia on direct PMN-tumor cell interactions.

Src family kinase-mediated vesicle trafficking is critical for neutrophil basement membrane penetration.

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.

Shp1 function in myeloid cells.

The motheaten mouse was first described in 1975 as a model of systemic inflammation and autoimmunity, as a result of immune system dysregulation. The phenotype was later ascribed to mutations in the cytoplasmic tyrosine phosphatase Shp1. This phosphatase is expressed widely throughout the hematopoietic system and has been shown to impact a multitude of cell signaling pathways. The determination of which cell types contribute to the different aspects of the phenotype caused by global Shp1 loss or mutation and which pathways within these cell types are regulated by Shp1 is important to further our understanding of immune system regulation. In this review, we focus on the role of Shp1 in myeloid cells and how its dysregulation affects immune function, which can impact human disease.

Role of MyD88 signaling in the imiquimod-induced mouse model of psoriasis: focus on innate myeloid cells.

Psoriasis is a chronic skin disease associated with deregulated activation of immune cells and keratinocytes. In this study, we used the imiquimod (IMQ)-induced mouse model of psoriasis to dissect better the contribution of hematopoietic and skin-resident stromal cells to psoriasis development. The comparison of disease development in mice carrying the hematopoietic cell-specific deletion of MyD88 (Myd88fl/flVav-cre+ mice) with mice carrying the total MyD88 deficiency (Myd88-/- mice), we show that the progression of skin and systemic inflammation, as well as of epidermal thickening, was completely dependent on MyD88 expression in hematopoietic cells. However, both Myd88-/- mouse strains developed some degree of epidermal thickening during the initial stages of IMQ-induced psoriasis, even in the absence of hematopoietic cell activation and infiltration into the skin, suggesting a contribution of MyD88-independent mechanisms in skin-resident stromal cells. With the use of conditional knockout mouse strains lacking MyD88 in distinct lineages of myeloid cells (Myd88fl/flLysM-cre+ and Myd88fl/flMRP8-cre+ mice), we report that MyD88 signaling in monocytes and Mϕ, but not in neutrophils, plays an important role in disease propagation and exacerbation by modulating their ability to sustain γδ T cell effector functions via IL-1ß and IL-23 production. Overall, these findings add new insights into the specific contribution of skin-resident stromal vs. hematopoietic cells to disease initiation and progression in the IMQ-induced mouse model of psoriasis and uncover a potential novel pathogenic role for monocytes/Mϕ to psoriasis development.

SLAMF7 is critical for phagocytosis of haematopoietic tumour cells via Mac-1 integrin.

Cancer cells elude anti-tumour immunity through multiple mechanisms, including upregulated expression of ligands for inhibitory immune checkpoint receptors. Phagocytosis by macrophages plays a critical role in cancer control. Therapeutic blockade of signal regulatory protein (SIRP)-α, an inhibitory receptor on macrophages, or of its ligand CD47 expressed on tumour cells, improves tumour cell elimination in vitro and in vivo, suggesting that blockade of the SIRPα-CD47 checkpoint could be useful in treating human cancer. However, the pro-phagocytic receptor(s) responsible for tumour cell phagocytosis is(are) largely unknown. Here we find that macrophages are much more efficient at phagocytosis of haematopoietic tumour cells, compared with non-haematopoietic tumour cells, in response to SIRPα-CD47 blockade. Using a mouse lacking the signalling lymphocytic activation molecule (SLAM) family of homotypic haematopoietic cell-specific receptors, we determined that phagocytosis of haematopoietic tumour cells during SIRPα-CD47 blockade was strictly dependent on SLAM family receptors in vitro and in vivo. In both mouse and human cells, this function required a single SLAM family member, SLAMF7 (also known as CRACC, CS1, CD319), expressed on macrophages and tumour cell targets. In contrast to most SLAM receptor functions, SLAMF7-mediated phagocytosis was independent of signalling lymphocyte activation molecule-associated protein (SAP) adaptors. Instead, it depended on the ability of SLAMF7 to interact with integrin Mac-1 (refs 18, 19, 20) and utilize signals involving immunoreceptor tyrosine-based activation motifs. These findings elucidate the mechanism by which macrophages engulf and destroy haematopoietic tumour cells. They also reveal a novel SAP adaptor-independent function for a SLAM receptor. Lastly, they suggest that patients with tumours expressing SLAMF7 are more likely to respond to SIRPα-CD47 blockade therapy.

Efficiency and Specificity of Gene Deletion in Lung Epithelial Doxycycline-Inducible Cre Mice.

The transgenic mouse strains surfactant protein C-reverse tetracycline transactivator (SP-C-rtTA), club cell secretory protein (CCSP)-rtTA, and tetracycline operator (TetO)-Cre have been invaluable for spatiotemporally regulating gene deletion in the pulmonary epithelium. In this study, we measured the efficiency and specificity of gene deletion that can be achieved in these mice using the Rosa26-eYFP reporter. Triple-transgenic mice (tTg or rtTA/TetO-Cre/Rosa-eYFP) were bred and treated with various doxycycline (dox) regimens to induce gene deletion, which was then quantified in various cell populations by flow cytometry. In these crosses, we found that the TetO-Cre transgene must be transmitted through the female parent to avoid germline gene deletion. With dox exposure during lung development, SP-C-tTg mice deleted in ∼65-75% of alveolar epithelial type II (ATII) cells, but in only ∼45-50% of the integrin ß4+ population, which consisted of club cells and distal lung progenitor cells. In contrast, CCSP-tTg mice deleted in ∼50% of ATII cells and ∼80% of integrin ß4+ cells. Upon dox treatment of adults, deletion in ATII cells and integrin ß4+ cells in SP-C-tTg mice dropped significantly to ∼20% and ∼6%, respectively, whereas CCSP-tTg mice deleted in ∼57% of ATII and ∼40% of integrin ß4+ cells. Interestingly, untreated CCSP-tTg mice also deleted in ∼40% of integrin ß4+ cells, indicating significant leakiness of CCSP-tTg in ß4+ cells. In all mouse groups, minimal deletion occurred in mouse tracheal epithelial cells or in mesenchymal or hematopoietic cells. These data provide the first quantitative, side-by-side comparison of the deletion efficiency for these widely used transgenic mouse strains.

Determinants of Divergent Adaptive Immune Responses after Airway Sensitization with Ligands of Toll-Like Receptor 5 or Toll-Like Receptor 9.

Excessive type 2 helper T cell responses to environmental antigens can cause immunopathology such as asthma and allergy, but how such immune responses are induced remains unclear. We studied this process in the airways by immunizing mice intranasally with the antigen ovalbumin together with either of two Toll-like receptor (TLR) ligands. We found the TLR5 ligand flagellin promoted a type 2 helper T cell response, whereas, a TLR9 ligand CpG oligodeoxyribonucleotide (ODN) promoted a type 1 helper T cell response. CpG ODN induced mRNA encoding interleukin (IL)-12 p40, whereas, flagellin caused IL-33 secretion and induced mRNAs encoding IL-1 and thymic stromal lymphopoietin (TSLP). By using mice deficient in the TLR and IL-1R signaling molecule, myeloid differentiation primary response 88 (MyD88), in conventional dendritic cells (cDCs) and alveolar macrophages (AMs), and by cell sorting different lung populations after 2 hours of in vivo stimulation, we characterized the cell types that rapidly produced inflammatory cytokines in response to TLR stimulation. CpG ODN was likely recognized by TLR9 on cDCs and AMs, which made mRNA encoding IL-12. IL-12 was necessary for the subsequent innate and adaptive interferon-γ production. In contrast, flagellin stimulated multiple cells of hematopoietic and non-hematopoietic origin, including AMs, DCs, monocytes, and lung epithelial cells. AMs were largely responsible for IL-1α, whereas lung epithelial cells made TSLP. Multiple hematopoietic cells, including AMs, DCs, and monocytes contributed to other cytokines, including IL-1ß and TNFα. MyD88-dependent signals, likely through IL-1R and IL-33R, and MyD88-independent signals, likely from TSLP, were necessary in cDCs for promotion of the early IL-4 response by CD4 T cells in the draining lymph node. Thus, the cell types that responded to TLR ligands were a critical determinant of the innate cytokines produced and the character of the resulting adaptive immune response in the airways.

Leishmania Uses Mincle to Target an Inhibitory ITAM Signaling Pathway in Dendritic Cells that Dampens Adaptive Immunity to Infection.

C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.