Myeloid-like B cells boost emergency myelopoiesis through IL-10 production during infection.

Emergency myelopoiesis (EM) is a hematopoietic response against systemic infections that quickly supplies innate immune cells. As lymphopoiesis is strongly suppressed during EM, the role of lymphocytes in that process has not received much attention. Here, we found that myeloid-like B cells (M-B cells), which express myeloid markers, emerge in the bone marrow (BM) after the induction of EM. M-B cells were mainly derived from pre-B cells and preferentially expressed IL-10, which directly stimulates hematopoietic progenitors to enhance their survival and myeloid-biased differentiation. Indeed, lacking IL-10 in B cells, blocking IL-10 in the BM with a neutralizing antibody, and deleting the IL-10 receptor in hematopoietic progenitors significantly suppressed EM, which failed to clear microbes in a cecal ligation and puncture model. Thus, a distinct B cell subset generated during infection plays a pivotal role in boosting EM, which suggests the on-demand reinforcement of EM by adaptive immune cells.

An Antibody-Drug Conjugate That Selectively Targets Human Monocyte Progenitors for Anti-Cancer Therapy.

As hematopoietic progenitors supply a large number of blood cells, therapeutic strategies targeting hematopoietic progenitors are potentially beneficial to eliminate unwanted blood cells, such as leukemic cells and immune cells causing diseases. However, due to their pluripotency, targeting those cells may impair the production of multiple cell lineages, leading to serious side effects such as anemia and increased susceptibility to infection. To minimize those side effects, it is important to identify monopotent progenitors that give rise to a particular cell lineage. Monocytes and monocyte-derived macrophages play important roles in the development of inflammatory diseases and tumors. Recently, we identified human monocyte-restricted progenitors, namely, common monocyte progenitors and pre-monocytes, both of which express high levels of CD64, a well-known monocyte marker. Here, we introduce a dimeric pyrrolobenzodiazepine (dPBD)-conjugated anti-CD64 antibody (anti-CD64-dPBD) that selectively induces the apoptosis of proliferating human monocyte-restricted progenitors but not non-proliferating mature monocytes. Treatment with anti-CD64-dPBD did not affect other types of hematopoietic cells including hematopoietic stem and progenitor cells, neutrophils, lymphocytes and platelets, suggesting that its off-target effects are negligible. In line with these findings, treatment with anti-CD64-dPBD directly killed proliferating monocytic leukemia cells and prevented monocytic leukemia cell generation from bone marrow progenitors of chronic myelomonocytic leukemia patients in a patient-derived xenograft model. Furthermore, by depleting the source of monocytes, treatment with anti-CD64-dPBD ultimately eliminated tumor-associated macrophages and significantly reduced tumor size in humanized mice bearing solid tumors. Given the selective action of anti-CD64-dPBD on proliferating monocyte progenitors and monocytic leukemia cells, it should be a promising tool to target cancers and other monocyte-related inflammatory disorders with minimal side effects on other cell lineages.

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.

CD86-based analysis enables observation of bona fide hematopoietic responses.

Hematopoiesis is a system that provides red blood cells (RBCs), leukocytes, and platelets, which are essential for oxygen transport, biodefense, and hemostasis; its balance thus affects the outcome of various disorders. Here, we report that stem cell antigen-1 (Sca-1), a cell surface marker commonly used for the identification of multipotent hematopoietic progenitors (Lin-Sca-1+c-Kit+ cells; LSKs), is not suitable for the analysis of hematopoietic responses under biological stresses with interferon production. Lin-Sca-1-c-Kit+ cells (LKs), downstream progenitors of LSKs, acquire Sca-1 expression upon inflammation, which makes it impossible to distinguish between LSKs and LKs. As an alternative and stable marker even under such stresses, we identified CD86 by screening 180 surface markers. The analysis of infection/inflammation-triggered hematopoiesis on the basis of CD86 expression newly revealed urgent erythropoiesis producing stress-resistant RBCs and intact reconstitution capacity of LSKs, which could not be detected by conventional Sca-1-based analysis.

Osteopontin has a protective role in prostate tumor development in mice.

Osteopontin (OPN) is a protein, generally considered to play a pro-tumorigenic role, whereas several reports have demonstrated the anti-tumorigenic function of OPN during tumor development. These opposing anti- and pro-tumorigenic functions are not fully understood. Here, we report that host-derived OPN plays an anti-tumorigenic role in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model and a TRAMP tumor transplant model. Tumor suppression mediated by OPN in Rag2-/- mice suggests that OPN is dispensable in the adaptive immune response. We found that host-derived OPN enhanced infiltration of natural killer (NK) cells into TRAMP tumors. The requirement of OPN in NK cell migration towards TRAMP cells was confirmed by an ex vivo cell migration assay. In contrast to TRAMP cells, in vivo B16 tumor development was not inhibited by OPN, and B16 tumors did not show OPN-mediated cell recruitment. It is possible that low levels of chemokine expression by B16 cells do not allow OPN to enhance immune cell recruitment. In addition to demonstrating the anti-tumorigenic role of OPN in TRAMP tumor development, this study also suggests that the contribution of OPN to tumor development depends on the type of tumor as well as the source and isoform of OPN.

Syndecan 4 Regulation of the Development of Autoimmune Arthritis in Mice by Modulating B Cell Migration and Germinal Center Formation.

OBJECTIVE: Syndecan 4 has been implicated as a critical mediator of inflammatory responses because of its functions as a coreceptor and reservoir for growth factors and chemokines. Although syndecan 4 is known to be expressed on B cells, its role in immune responses remains unclear. The purpose of this study was to investigate the contribution of syndecan 4 to the development of immune arthritis in murine models. METHODS: The clinical severity of 3 immunopathologically distinct models, collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and collagen antibody-induced arthritis (CAIA), was evaluated in wild-type (WT) mice and in syndecan 4-deficient (Sdc4(-/-) ) mice. Germinal center (GC) formation, B cell profiles, humoral immune responses, and B cell migration were analyzed during the course of CIA. RESULTS: Sdc4(-/-) mice were resistant to the induction of CIA, which is T cell and B cell dependent, but AIA and CAIA, which are T cell dependent and T cell independent, respectively, occurred with equal frequency in WT mice and Sdc4(-/-) mice. Furthermore, Sdc4(-/-) mice had reduced numbers of B cells and deficient GC formation in draining lymph nodes (DLNs) during the course of CIA, resulting in reduced production of collagen-specific autoantibodies. Compared with B cells from WT mice, B cells from Sdc4(-/-) mice showed reduced chemotactic migration toward stromal cell-derived factor 1 (SDF-1) and reduced SDF-1-mediated Akt phosphorylation. Consistent with these findings, in vivo transfer experiments showed that fewer Sdc4(-/-) B cells than WT B cells were found in and around the follicles in the DLNs. CONCLUSION: Our findings suggest that syndecan 4 contributes to the development of CIA by promoting GC formation and autoantibody production through its regulation of SDF-1-mediated B cell migration.

Autophagy enhances NFκB activity in specific tissue macrophages by sequestering A20 to boost antifungal immunity.

Immune responses must be well restrained in a steady state to avoid excessive inflammation. However, such restraints are quickly removed to exert antimicrobial responses. Here we report a role of autophagy in an early host antifungal response by enhancing NFκB activity through A20 sequestration. Enhancement of NFκB activation is achieved by autophagic depletion of A20, an NFκB inhibitor, in F4/80(hi) macrophages in the spleen, peritoneum and kidney. We show that p62, an autophagic adaptor protein, captures A20 to sequester it in the autophagosome. This allows the macrophages to release chemokines to recruit neutrophils. Indeed, mice lacking autophagy in myeloid cells show higher susceptibility to Candida albicans infection due to impairment in neutrophil recruitment. Thus, at least in the specific aforementioned tissues, autophagy appears to break A20-dependent suppression in F4/80(hi) macrophages, which express abundant A20 and contribute to the initiation of efficient innate immune responses.

A novel cryptic binding motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved osteopontin as a novel ligand for α9ß1 integrin is involved in the anti-type II collagen antibody-induced arthritis.

Osteopontin (OPN) is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7). Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9ß1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA). This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.

Tumor-α9ß1 integrin-mediated signaling induces breast cancer growth and lymphatic metastasis via the recruitment of cancer-associated fibroblasts.

UNLABELLED: Tumor-derived matricellular proteins such as osteopontin (OPN) and tenascin-C (TN-C) have been implicated in tumor growth and metastasis. However, the molecular basis of how these proteins contribute to tumor progression remains to be elucidated. Importantly, these matricellular proteins are known to interact with α9ß1 integrin. Therefore, we hypothesized that tumor-derived α9ß1 integrin may contribute to tumor progression. To clarify the roles of α9ß1 integrin in tumor growth and lymphatic metastasis, we used an inhibitory anti-human α9ß1 integrin antibody (anti-hα9ß1 antibody) and a α9ß1 integrin-positive human breast cancer cell line, MDA-MB-231 luc-D3H2LN (D3H2LN), in vitro functional assays, and an in vivo orthotopic xenotransplantation model. In this study, we demonstrated that tumor, but not host α9ß1 integrin, contributes to tumor growth, lymphatic metastasis, recruitment of cancer-associated fibroblasts (CAFs), and host-derived OPN production. We also found that CAFs contributed to tumor growth, lymphatic metastasis, and host-derived OPN levels. Consistent with those findings, tumor volume was well-correlated with numbers of CAFs and levels of host-derived OPN. Furthermore, it was shown that the inoculation of D3H2LN cells into mammary fat pads with mouse embryonic fibroblasts (MEFs), obtained from wild type, but not OPN knock-out mice, resulted in enhancement of tumor growth, thus indicating that CAF-derived OPN enhanced tumor growth. These results suggested that tumor α9ß1-mediated signaling plays a pivotal role in generating unique primary tumor tissue microenvironments, which favor lymphatic metastasis and tumor growth. KEY MESSAGES: Tumor α9ß1 integrin promotes lymphatic metastasis through enhancing invasion. Tumor α9ß1 integrin promotes tumor growth through CAFs. Tumor α9ß1 integrin enhances the recruitment of CAFs into the primary tumor. Tumor cells induce the production of OPN by CAFs in the primary tumor. CAF-derived OPN promotes tumor growth.

α9ß1 integrin-mediated signaling serves as an intrinsic regulator of pathogenic Th17 cell generation.

The interaction between matricellular proteins such as tenascin-C (TN-C) and osteopontin (OPN) and integrins has been implicated in the pathology of rheumatoid arthritis in which Th17 cells are recognized as primary pathogenic cells. The differentiation of Th17 cells is tightly regulated by cytokines derived from APCs, receiving various signals including TLR stimuli. In this study, we used a collagen-induced arthritis model and found that increased numbers of α(9) integrin-positive conventional dendritic cells and macrophage were detectable in the draining lymph node (dLN) shortly following first immunization, and these cells produced both TN-C and OPN, ligands for α(9) integrin. α(9) integrin-mediated signaling, induced by TN-C and OPN, promoted the production of Th17-related cytokines by conventional dendritic cells and macrophages in synergy with TLR2 and 4 signaling. This led to the Th17 cell differentiation and arthritis development. Moreover, Th17 cells generated under blocking of α(9) integrin-mediated signaling showed low level of CCR6 expression and impaired migration ability toward CCL20. Thus, we have identified α(9) integrin-mediated signaling by TN-C and OPN as a novel intrinsic regulator of pathogenic Th17 cell generation that contributes to the development of rheumatoid arthritis.

Syndecan-4 deficiency limits neointimal formation after vascular injury by regulating vascular smooth muscle cell proliferation and vascular progenitor cell mobilization.

OBJECTIVE: Syndecan-4 (Syn4) is a heparan sulfate proteoglycan and works as a coreceptor for various growth factors. We examined whether Syn4 could be involved in the development of neointimal formation in vivo. METHODS AND RESULTS: Wild-type (WT) and Syn4-deficient (Syn4-/-) mice were subjected to wire-induced femoral artery injury. Syn4 mRNA was upregulated after vascular injury in WT mice. Neointimal formation was attenuated in Syn4-/- mice, concomitantly with the reduction of Ki67-positive vascular smooth muscle cells (VSMCs). Basic-fibroblast growth factor- or platelet-derived growth factor-BB-induced proliferation, extracellular signal-regulated kinase activation, and expression of cyclin D1 and Bcl-2 were impaired in VSMCs from Syn4-/- mice. To examine the role of Syn4 in bone marrow (BM)-derived vascular progenitor cells (VPCs) and vascular walls, we generated chimeric mice by replacing the BM cells of WT and Syn4-/- mice with those of WT or Syn4-/- mice. Syn4 expressed by both vascular walls and VPCs contributed to the neointimal formation after vascular injury. Although the numbers of VPCs were compatible between WT and Syn4-/- mice, mobilization of VPCs from BM after vascular injury was defective in Syn4-/- mice. CONCLUSIONS: Syn4 deficiency limits neointimal formation after vascular injury by regulating VSMC proliferation and VPC mobilization. Therefore, Syn4 may be a novel therapeutic target for preventing arterial restenosis after angioplasty.

CSF-1-dependent red pulp macrophages regulate CD4 T cell responses.

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80(hi)Mac-1(low) MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80(+)Mac-1(hi) monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80(hi)Mac-1(low) MΦs regulated CD4(+) T cell responses by producing soluble suppressive factors, including TGF-ß and IL-10. Moreover, F4/80(hi)Mac-1(low) MΦs induced the differentiation of naive CD4(+) T cells into functional Foxp3(+) regulatory T cells. Additionally, we found that the differentiation of F4/80(hi)Mac-1(low) MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4(+) T cell responses and induced the generation of Foxp3(+) regulatory T cells in vivo. These results suggested that splenic CSF-1-dependent F4/80(hi)Mac-1(low) MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.

Alpha9 integrin and its ligands constitute critical joint microenvironments for development of autoimmune arthritis.

Osteopontin is critically involved in rheumatoid arthritis; however, the molecular cross-talk between osteopontin and joint cell components that leads to the inflammatory joint destruction is largely unknown. We found that not only osteopontin but also tenascin-C and their common receptor, alpha(9) integrin, are expressed at arthritic joints. The local production of osteopontin and tenascin-C is mainly due to synovial fibroblasts and, to a lesser extent, synovial macrophages. Synovial fibroblasts and macrophages express alpha(9) integrin, and autocrine and paracrine interactions of alpha(9) integrin on synovial fibroblasts and macrophages and its ligands contribute differently to the production of proinflammatory cytokines and chemokines. alpha(9) integrin is also involved in the recruitment and accumulation of inflammatory cells. Inhibition of alpha(9) integrin function with an anti-alpha(9) integrin Ab significantly reduces the production of arthrogenic cytokines and chemokines and ameliorates ongoing arthritis. Thus, we identified alpha(9) integrin as a critical intrinsic regulator that controls the development of autoimmune arthritis.

The differential amino acid requirement within osteopontin in alpha4 and alpha9 integrin-mediated cell binding and migration.

Osteopontin (OPN) contains at least two major integrin recognition domains, Arg159-Gly-Asp161 (RGD) and Ser162-Val-Val-Tyr-Gly-Leu-Arg168 (SVVYGLR), recognized by alphavbeta3 and alpha5beta1 and alpha4 and alpha9 integrins, respectively. OPN is specifically cleaved by thrombin and matrix metalloproteinase (MMP)-3 or MMP-7 at a position of Arg168/Ser169 (R/S) and Gly166/Leu167 (G/L), respectively. We in this study examined the requirement of residues within SVVYGLR for the alpha4 and alpha9 integrin recognition and how MMP-cleavage influences the integrin recognition. The residues, Val164, Tyr165, and Leu167 are critical for alpha4 and alpha9 integrin recognition in both cell adhesion and cell migration. The residue Arg168 is additionally required for alpha9 integrin recognition in cell adhesion and this explains why alpha9 integrin binds to only thrombin cleaved form of OPN. alpha4 integrin is able to bind to SVVYG (MMP-cleaved form of RAA OPN-N half), while alpha9 integrin is not, supporting the above notion that Arg168 is additionally required for alpha9 integrin-mediated cell adhesion. The residue Val163 is important for alpha4, but not for alpha9 integrin recognition in cell migration. Importantly, we found that the replacement of Arg168 by Ala (R168A mutant) induces the augmentation of cell migration via alpha4 and alpha9 integrins.