IgG Immunocomplexes Sensitize Human Monocytes for Inflammatory Hyperactivity via Transcriptomic and Epigenetic Reprogramming in Rheumatoid Arthritis.

Prevalence of circulating immunocomplexes (ICs) strongly correlates with rheumatoid arthritis (RA) in humans. Deposits of IgG-ICs are abundant in affected joints of patients, yet molecular mechanisms for the pathogenic roles of such ICs are not fully understood. In this study, we present evidence that IgG-ICs precipitated from RA sera sensitized human monocytes for a long-lasting inflammatory functional state, characterized by a strong TNF-α response to cellular proteins representing damage-associated molecular patterns and microbe-derived pathogen-associated molecular patterns. Importantly, plate-coated human IgG (a mimic of deposited IC without Ag restriction) exhibited a similarly robust ability of monocyte sensitization in vitro. The plate-coated human IgG-induced functional programming is accompanied by transcriptomic and epigenetic modification of various inflammatory cytokines and negative regulator genes. Moreover, macrophages freshly isolated from synovia of patients with RA, but not sera-negative arthropathy, displayed a signature gene expression profile highly similar to that of IC-sensitized human monocytes, indicative of historical priming events by IgG-ICs in vivo. Thus, the ability of IgG-ICs to drive sustainable functional sensitization/reprogramming of monocytes and macrophages toward inflammation may render them key players in the development of RA.

Disease-Specific Autoantibodies Induce Trained Immunity in RA Synovial Tissues and Its Gene Signature Correlates with the Response to Clinical Therapy.

Much evidence suggests that trained immunity is inappropriately activated in the synovial tissue in rheumatoid arthritis (RA), but the underlying mechanism remains unclear. Here, we describe how RA-specific autoantibody deposits can train human monocytes to exert the hyperactive inflammatory response, particularly via the exacerbated release of tumor necrosis factor α (TNFα). Comparative transcriptomic analysis by plate-bound human IgG (cIgG) or ß-glucan indicated that metabolic shift towards glycolysis is a crucial mechanism for trained immunity. Moreover, the cIgG-trained gene signatures were enriched in synovial tissues from patients with ACPA- (anticitrullinated protein antibody-) positive arthralgia and undifferentiated arthritis, and early RA and established RA bore a great resemblance to the myeloid pathotype, suggesting a historical priming event in vivo. Additionally, the expression of the cIgG-trained signatures is higher in the female, older, and ACPA-positive populations, with a predictive role in the clinical response to infliximab. We conclude that RA-specific autoantibodies can train monocytes in the inflamed lesion as early as the asymptomatic stage, which may not merely improve understanding of disease progression but may also suggest therapeutic and/or preventive strategies for autoimmune diseases.

ZBTB24 regulates the apoptosis of human T cells via CDCA7/TRAIL-receptor axis.

Mutations in ZBTB24 and CDCA7 cause the Immunodeficiency, Centromeric Instability and Facial Anomalies syndrome type 2 and 3 (ICF2/3), respectively. Most ICF2 patients carry ZBTB24 nonsense mutations and are thus ZBTB24-deficient. Although the immune deficiency in ICF2 patients is primarily regarded as a B-cell defect due to the greatly reduced serum antibodies and circulating memory B cells, the reduced expansions of PBMCs stimulated by mitogens or recall antigens suggest a T-cell defect in these patients as well. However, the molecular mechanisms behind this T-cell dysfunction remain unknown. In the present study, we demonstrated that ZBTB24-deficiency significantly represses the proliferation of human T cells by promoting TRAIL-induced cell death. Downregulation of ZBTB24 in both Jurkat and human primary T cells upregulates the expression of TRAIL and/or its death receptors (TRAIL-R1/2), and induces significant amount of cells to undergo apoptosis. The profound survival defects of ZBTB24-deficient cells are largely reversed by blocking TRAIL/TRAIL-R interactions with exogenous recombinant TRAIL-R2. Moreover, ZBTB24-downregulation reduces the expression of CDCA7, and knockdown of the latter in human T cells results in a phenotype resembling that caused by ZBTB24-depletion. Functionally, overexpression of CDCA7 abrogates the increased apoptosis in ZBTB24-depleted Jurkat T cells. Together, these data indicated that ZBTB24 regulates human T-cell apoptosis via CDCA7/TRAIL-R axis. Our study thus not only provides a molecular explanation for the T-cell defects in ZBTB24-deficient ICF2 patients, but also highlights a convergence between ZBTB24 and CDCA7, the two ICF genes, in modulating the functions of T cells.

M860, a Monoclonal Antibody against Human Lactoferrin, Enhances Tumoricidal Activity of Low Dosage Lactoferrin via Granzyme B Induction.

Lactoferrin (LF) is a soluble glycoprotein of the transferring family found in most biological fluids, functioning as a major first line defense molecule against infection in mammals. It also shows certain anti-tumor activity, but its clinical application in tumor therapy is limited because high dosage is required. In this study, we demonstrate that M860, a monoclonal antibody against human LF (hLF), could significantly increase the anti-tumor potential of low dosage hLF by forming LF-containing immune complex (IC). Human monocytes primed with LF-IC, but not hLF or M860 alone, or control ICs, showed strong tumoricidal activity on leukemia cell lines Jurkat and Raji through induction of secreted Granzyme B (GzB). LF-IC is able to colligate membrane-bound CD14 (a TLR4 co-receptor) and FcγRIIa (a low affinity activating Fcγ receptor) on the surface of human monocytes, thereby triggering the Syk-PI3K-AKT-mTOR pathway leading to GzB production. Our work identifies a novel pathway for LF-mediated tumoricidal activity and may extend the clinical application of LF in tumor therapy.

Aberrant Glycosylation Augments the Immuno-Stimulatory Activities of Soluble Calreticulin.

Calreticulin (CRT), a luminal resident calcium-binding glycoprotein of the cell, is a tumor-associated antigen involved in tumorigenesis and also an autoantigen targeted by autoantibodies found in patients with various autoimmune diseases. We have previously shown that prokaryotically expressed recombinant murine CRT (rCRT) exhibits strong stimulatory activities against monocytes/macrophages in vitro and potent immunogenicity in vivo, which is partially attributable to self-oligomerization of soluble rCRT. However, even in oligomerized form native CRT (nCRT) isolated from mouse liver is much less active than rCRT, arguing against the possibility that self-oligomerization alone would license potent pro-inflammatory properties to nCRT. Since rCRT differs from nCRT in its lack of glycosylation, we wondered if aberrant glycosylation of eukaryotically expressed CRT (eCRT) would significantly enhance its immunological activity. In the present study, tunicamycin, an N-glycosyltransferase inhibitor, was employed to treat CHO cells (CHO-CRT) stably expressing full-length recombinant mouse CRT in secreted form for preparation of aberrantly glycosylated eCRT (tun-eCRT). Our biochemical and immunological analysis results indicate that eCRT produced by CHO-CRT cells is similar to nCRT in terms of glycosylation level, lack of self-oligomerization, relatively poor immunogenicity and weak macrophage-stimulatory activity, while tun-eCRT shows reduced glycosylation yet much enhanced ability to elicit specific humoral responses in mice and TNF-α and nitric oxide production by macrophages in vitro. Given that abberant glycosylation of proteins is a hallmark of cancer cells and also related to the development of autoimmune disorders in humans, our data may provide useful clues for better understanding of potentiating roles of dysregulated glycosylation of molecules such as CRT in tumorigenesis and autoimmunity.

Disease association and arthritogenic potential of circulating antibodies against the α1,4-polygalacturonic acid moiety.

Much progress has been made in recent years on the diagnostic value, Ag specificity, and pathogenic roles of autoantibodies correlated to the development of rheumatoid arthritis (RA) in humans. However, carbohydrate Ag-specific autoantibodies that may also play important roles in RA have largely been ignored. In this article, we report that serum levels of Abs capable of recognizing α1,4-polygalacturonic acid [(PGA); major structural component of pectin] strongly correlate with RA in humans. The measurements of PGA-specific Abs (PGA-Abs) in sera are comparable to rheumatoid factors and anti-cyclic citrullinated peptide Abs as serological diagnostic markers for RA in terms of sensitivity and specificity. Immunohistochemical staining results indicate that the PGA-Abs selectively bound synovial membrane cells and chondrocytes in the joints of both humans and rabbits (but not rodents). Induction of PGA-Abs by s.c. immunization of rabbits with carrier protein-conjugated synthetic PGA led to severe inflammatory reactions (synovial hyperplasia, small vessel proliferation, and inflammatory cell infiltration) in the joints. Injection of affinity purified anti-PGA IgG into the synovial cavity of rabbits resulted in accumulation of proinflammatory cytokines such as TNF-α, IL-8, and IL-1ß in synovial fluid, as well as local pathological damage. We conclude that the PGA-cross-reactive moiety represents a major autoantigen in the joints and can be targeted by autoantibodies capable of triggering arthritogenic responses in vivo.

Soluble calreticulin induces tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 production by macrophages through mitogen-activated protein kinase (MAPK) and NFκB signaling pathways.

We have recently reported that soluble calreticulin (CRT) accumulates in the sera of patients with rheumatoid arthritis or systemic lupus erythematosus. Moreover, following self-oligomerization, soluble recombinant CRT (rCRT) polypeptides exhibit potent immunostimulatory activities including macrophage activation in vitro and antibody induction in vivo. This study was designed to further investigate the underlying molecular mechanisms for soluble CRT-induced macrophage activation. Treatment of murine macrophages with oligomerized rCRT (OrCRT) led to (i) TNF-α and IL-6 transcription and protein expression without affecting intracellular mRNA stability; and (ii) IκBα degradation, NFκB phosphorylation and sustained MAPK phosphorylation in cells. Inhibition of IKK and JNK in macrophages substantially abrogated production of TNF-α and IL-6 induced by OrCRT, while ERK suppression only reduced IL-6 expression in parallel experiments. In vitro, fucoidan, a scavenger receptor A (SRA)-specific ligand, significantly reduced the uptake of FITC-labeled OrCRT by macrophages and subsequent MAPK and NFκB activation, thereby suggesting SRA as one of the potential cell surface receptors for soluble CRT. Together, these data indicate that soluble CRT in oligomerized form could play a pathogenic role in autoimmune diseases through induction of pro-inflammatory cytokines (e.g., TNF-α and IL-6) by macrophages via MAPK-NFκB signaling pathway.

Potent immunogenicity and broad-spectrum protection potential of microneedle array patch-based COVID-19 DNA vaccine candidates encoding dimeric RBD chimera of SARS-CoV and SARS-CoV-2 variants.

Breakthrough infections by SARS-CoV-2 variants pose a global challenge to COVID-19 pandemic control, and the development of more effective vaccines of broad-spectrum protection is needed. In this study, we constructed pVAX1-based plasmids encoding receptor-binding domain (RBD) chimera of SARS-CoV-1 and SARS-CoV-2 variants, including pAD1002 (encoding RBDSARS/BA1), pAD1003 (encoding RBDSARS/Beta) and pAD131 (encoding RBDBA1/Beta). Plasmids pAD1002 and pAD131 were far more immunogenic than pAD1003 in terms of eliciting RBD-specific IgG when intramuscularly administered without electroporation. Furthermore, dissolvable microneedle array patches (MAP) greatly enhanced the immunogenicity of these DNA constructs in mice and rabbits. MAP laden with pAD1002 (MAP-1002) significantly outperformed inactivated SARS-CoV-2 virus vaccine in inducing RBD-specific IFN-γ+ effector and memory T cells, and generated T lymphocytes of different homing patterns compared to that induced by electroporated DNA in mice. In consistence with the high titer neutralization results of MAP-1002 antisera against SARS-CoV-2 pseudoviruses, MAP-1002 protected human ACE2-transgenic mice from Omicron BA.1 challenge. Collectively, MAP-based DNA constructs encoding chimeric RBDs of SARS-CoV-1 and SARS-CoV-2 variants, as represented by MAP-1002, are potential COVID-19 vaccine candidates worthy further translational study.

Structure elucidation and immunological function analysis of a novel ß-glucan from the fruit bodies of Polyporus umbellatus (Pers.) Fries.

ß-Glucans derived from various sources such as yeast cell walls and medicinal mushrooms are considered as valuable biological response modifiers for their ability to enhance the activity of immune cells, aid in wound healing and help prevent infections. We herein characterize the structure of a novel water-soluble polysaccharide (Zhuling polysaccharide, ZPS) from the fruit bodies of medicinal mushroom Polyporus umbellatus and investigate its immunobiological function. ZPS has a molecular mass of 2.27 x 10(3) kDa and contains >90% d-glucose as its monosaccharide constituent. On the basis of partial acid hydrolysis, methylation analysis, Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy and the ideal repeating unit of ZPS is established: (1 → 6, 1 → 4)-linked ß-d-glucopyranosyl backbone, substituted at O-3 position of (1 → 6)-linked ß-d-glucopyranosyl by (1 → 3)-linked ß-d-glucopyranosyl branches. ZPS consists of approximately 2930 repeating units, each contains a side chain of no more than three residues in length. Functionally, ZPS is a potent activator of B cells, macrophages and dendritic cells. Depletion of ZPS branches causes a substantial reduction in its ability not only to activate B cells in vitro but also to elicit specific IgM production in vivo. Virtually all healthy human subjects possess high-titer circulating antibodies against ZPS backbone, suggesting that ZPS epitope is shared by environmental antigens capable of eliciting adaptive humoral responses in the population.

Involvement of Na(+), K (+)-ATPase and its inhibitors in HuR-mediated cytokine mRNA stabilization in lung epithelial cells.

Increasing evidence demonstrates that Na(+), K(+)-ATPase plays an important role in pulmonary inflammation, but the mechanism remains largely unknown. In this study, we used cardiotonic steroids as Na(+), K(+)-ATPase inhibitors to explore the possible involvement of Na(+), K(+)-ATPase in pulmonary epithelial inflammation. The results demonstrated that mice after ouabain inhalation developed cyclooxygenase-2-dependent acute lung inflammation. The in vitro experiments further confirmed that Na(+), K(+)-ATPase inhibitors significantly stimulated cyclooxygenase-2 expression in lung epithelial cells of human or murine origin, the process of which was participated by multiple cis-elements and trans-acting factors. Most importantly, we first described here that Na(+), K(+)-ATPase inhibitors could evoke a significant Hu antigen R nuclear export in lung epithelial cells, which stabilized cyclooxygenase-2 mRNA by binding with a proximal AU-rich element within its 3'-untranslated region. In conclusion, HuR-mediated mRNA stabilization opens new avenues in understanding the importance of Na(+), K(+)-ATPase, as well as its inhibitors in inflammation.

Membrane Bound CRT Fragment Accelerates Tumor Growth of Melanoma B16 Cell In Vivo through Promoting M2 Polarization via TLR4.

Calreticulin (CRT) is a major calcium-binding luminal resident protein on the endoplasmic reticulum that can also be released extracellular as well as anchored on surface of cells. Previously, we demonstrated that soluble recombinant CRT fragment 39-272 (CRT/39-272) exhibited potent immunostimulatory effects as well as immunoregulation effects on immune cells. Here, we constructed stable B16 melanoma cell lines expressing recombinant CRT/39-272 on the membrane (B16-tmCRT/39-272) to investigate the roles of cell surface CRT on tumor progression. We found that B16-tmCRT/39-272 cells subcutaneously inoculated into C57BL/6 mice exhibited stronger tumorigenicity than the B16-EGFP control cells. The tumor associated macrophages infiltrated in tumors were mainly M2 phenotype. Regulatory T cells (Tregs) were also expanded more in bearing mice. Consistent with the in vivo results, B16-tmCRT/39-272 promoted macrophage polarization toward F4/80+CD206+ M2 macrophages and promoted transforming growth factor beta (TGF-ß) secretion in vitro, which could promote naïve CD4+ T cell differentiation into Tregs. These results imply that the tmCRT/39-272 could accelerate tumor development by enhancing M2 macrophage polarization to induce TGF-ß secretion, and then promoted Treg differentiation in the tumor microenvironment. Our data may provide useful clues for better understanding of the potentiating roles of CRT in tumorigenesis.

Calreticulin as an Adjuvant In Vivo to Promote Dendritic Cell Maturation and Enhance Antigen-Specific T Lymphocyte Responses against Melanoma.

An endoplasmic reticulum resident protein, calreticulin (CRT), participates in many cellular processes. CRT is a tumor-associated antigen with an important role in antitumor immunity. Previously, we reported that the recombinant CRT fragment 39-272 (CRT/39-272) exhibited superior immunobiological activity, activating macrophages to release cytokines and promoting dendritic cell (DC) maturation. However, the effect of CRT/39-272 in vivo, especially its adjuvant effect on in vivo antitumor immune responses, was not fully investigated. In this study, we constructed a fusion protein linking CRT/39-272 to an ovalbumin (OVA) peptide (residues 182-297, OVAp) and used the fusion protein (OVAp-CRT) to examine the adjuvant effect of CRT. We investigated whether CRT/39-272 could induce bone marrow-derived DC maturation and strongly promote the proliferation of OVA-specific T cells in vitro. Compared with OVAp, OVAp-CRT induced stronger antigen-specific T lymphocyte responses, including antigen-specific T cell proliferation, interferon-γ secretion, and cytotoxic T lymphocyte responses. OVAp-CRT-immunized mice generated significantly increased OVAp-specific antibody and CD4+/CD8+ memory T cells, which mediated long-term protective effects. OVAp-CRT upregulated CD40, CD80, and CD86 expressions in splenic conventional DCs. Furthermore, OVAp-CRT protected immunized mice against OVA-expressing B16 melanoma cells in vivo. Moreover, mice that were adoptively transferred with OVAp-CRT-pulsed DCs showed inhibited tumor growth and prolonged mouse survival. Our results demonstrate that CRT/39-272 can be used as a potential new adjuvant for tumor vaccines, and this finding may be useful in tumor vaccine development.

Altered Phenotype and Enhanced Antibody-Producing Ability of Peripheral B Cells in Mice with Cd19-Driven Cre Expression.

Given the importance of B lymphocytes in inflammation and immune defense against pathogens, mice transgenic for Cre under the control of Cd19 promoter (Cd19Cre/+ mice) have been widely used to specifically investigate the role of loxP-flanked genes in B cell development/function. However, impacts of expression/insertion of the Cre transgene on the phenotype and function of B cells have not been carefully studied. Here, we show that the number of marginal zone B and B1a cells was selectively reduced in Cd19Cre/+ mice, while B cell development in the bone marrow and total numbers of peripheral B cells were comparable between Cd19Cre/+ and wild type C57BL/6 mice. Notably, humoral responses to both T cell-dependent and independent antigens were significantly increased in Cd19Cre/+ mice. We speculate that these differences are mainly attributable to reduced surface CD19 levels caused by integration of the Cre-expressing cassette that inactivates one Cd19 allele. Moreover, our literature survey showed that expression of Cd19Cre/+ alone may affect the development/progression of inflammatory and anti-infectious responses. Thus, our results have important implications for the design and interpretation of results on gene functions specifically targeted in B cells in the Cd19Cre/+ mouse strain, for instance, in the context of (auto) inflammatory/infectious diseases.

IgG Immunocomplexes Drive the Differentiation of a Novel Subset of Osteoclasts Independent of RANKL and Inflammatory Cytokines.

Potentiation of receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis by IgG immunocomplexes (ICs) is generally considered an important pathway leading to cartilage and bone destruction in rheumatoid arthritis (RA). However, whether IgG ICs possess pro-osteoclastogenic potential independent of RANKL and inflammatory cytokines is unclear. Here we demonstrate that by fully cross-linking human FcγRIIa (hFcγRIIa) or co-ligating hFcγRIIa and TLR4, IgG ICs alone could drive the differentiation of human blood monocytes into nuclear factor of activated T cells cytoplasmic 1 (NFATc1-negative nonclassical osteoclasts (NOCs). Surprisingly, IgG ICs could also overrule RANKL-induced classical osteoclast (COC) differentiation in vitro. In mouse model of collagen-induced arthritis, hFcγRIIa-transgenic, but not nontransgenic control, mice suffered from cartilage/bone destruction accompanied by the presence of NFATc1- NOCs lining the eroded cartilage surface in affected joints. Our results not only identify a novel subset of IC-induced NOCs but also provide a possible explanation for the uncoupling of FcγR-mediated cartilage destruction from RANKL-related bone erosion in autoinflammatory arthritis. © 2021 American Society for Bone and Mineral Research (ASBMR)..

Anti-inflammatory and immunosuppressive effect of flavones isolated from Artemisia vestita.

AIM OF THE STUDY: Artemisia vestita is a common traditional Tibetan medicinal plant which has been used widely in China for treating various inflammatory diseases. Since little is known about its active components, the purpose of this study was to isolate and identify the immunosuppressive compounds from Artemisia vestita. MATERIALS AND METHODS: A bioassay-guided isolation was performed with picryl chloride-induced contact hypersensitivity in mice. MTT assay and Flow cytometric analysis were used for determining Con A-induced lymphocyte proliferation and CD25 expression in T cells, respectively. RESULTS: The ethanol extract of the Artemisia vestita was found to possess significant inhibitory activity against the picryl chloride-induced contact hypersensitivity in mice. Then 4 fractions were isolated by macroporous adsorption resin and one of these fractions (AV3), which showed the highest activity in in vivo test, was further subjected to column chromatography. Nine known flavones were isolated and identified as pectolinarigenin (1), jaceosidin (2), cirsilineol (3), cirsimaritin (4), hispidulin (5), quercetin (6), 6-methoxytricin (7), acacetin (8), and apigenin (9). The structures of the 9 flavones were elucidated by spectral techniques. All the compounds were evaluated for their inhibitory activity on the proliferation and activation of T cells in vitro. Among the 9 flavones, cirsilineol (3), 6-methoxytricin (7) and apigenin (9) significantly inhibited T cell proliferation and activation in the bioassays. CONCLUSION: The result suggests that cirsilineol, 6-methoxytricin and apigenin are the major active components in Artemisia vestita.

Author Correction: Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages.

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A study on the risk of fungal infection with tofacitinib (CP-690550), a novel oral agent for rheumatoid arthritis.

Tofacitinib (CP-690550), an oral Janus kinase inhibitor, has shown significant efficacy in the treatment of rheumatoid arthritis through blocking the signaling pathways of pro-inflammatory cytokines. However, recent evidence suggests that long-term tofacitinib treatment is associated with increased risk of infection (e.g. tuberculosis) in patients. In the present study, we illustrate that tofacitinib administration significantly reduced the survival rate of mice given lethal or sub-lethal dose challenge with Candida albicans. This was related to the ability of tofacitinib to reverse TNFα- and IFNγ-enhanced candidacidal activity of murine polymorph nuclear cells (PMNs) and also to suppress chemokine CXCL5 expression and PMN infiltration in the infected tissues of mice. More importantly, tofacitinib significantly antagonized the ability of TNFα, IFNγ and GM-CSF to boost human PMNs in phagocytosis and direct killing of C. albicans in vitro. It also down-regulated reactive oxygen production and neutrophil extracellular trap formation by human PMNs stimulated with yeast-derived ß-glucans in the presence of TNFα, IFNγ or GM-CSF. Our data emphasizes a significantly increased risk for opportunistic fungal infection associated long-term tofacitinib treatment in humans, likely through antagonizing the PMN-boosting effect of pro-inflammatory cytokines.

Calreticulin Fragment 39-272 Promotes B16 Melanoma Malignancy through Myeloid-Derived Suppressor Cells In Vivo.

Calreticulin (CRT), a multifunctional Ca2+-binding glycoprotein mainly located in the endoplasmic reticulum, is a tumor-associated antigen that has been shown to play protective roles in angiogenesis suppression and anti-tumor immunity. We previously reported that soluble CRT (sCRT) was functionally similar to heat shock proteins or damage-associated molecular patterns in terms of ability to activate myeloid cells and elicit strong inflammatory cytokine production. In the present study, B16 melanoma cell lines expressing recombinant CRT fragment 39-272 (sCRT/39-272) in secreted form (B16-CRT), or recombinant enhanced green fluorescence protein (rEGFP) (B16-EGFP), were constructed for investigation on the roles of sCRT in tumor development. When s.c. inoculated into C57BL/6 mice, the B16-CRT cells were significantly more aggressive (in terms of solid tumor growth rate) than B16-EGFP controls in a TLR4- and myeloid-derived suppressor cells (MDSC)-dependent manner. The B16-CRT-bearing mice showed increased Gr1+ MDSC infiltration in tumor tissues, accelerated proliferation of CD11b+Ly6G+Ly6Clow (G-MDSC) precursors in bone marrow, and higher percentages of G-MDSCs in spleen and blood, which was mirrored by decreased percentage of dendritic cells (DC) in periphery. In in vitro studies, recombinant sCRT/39-272 was able to promote migration and survival of tumor-derived MDSCs via interaction with TLR4, inhibit MDSC differentiation into DC, and also elicit expression of inflammatory proteins S100A8 and S100A9 which are essential for functional maturation and chemotactic migration of MDSCs. Our data provide solid evidence for CRT as a double-edged sword in tumor development.

Extraordinarily potent proinflammatory properties of lactoferrin-containing immunocomplexes against human monocytes and macrophages.

Lactoferrin (LTF), an important first line defense molecule against infection, is a common target for humoral autoimmune reactions in humans. Since LTF is a multifunctional protein capable of activating innate immune cells via various surface receptors, we hypothesized that LTF-containing immune complexes (ICs) (LTF-ICs), likely formed in patients with high titer anti-LTF autoantibodies, could possess unique monocyte/macrophage-activating properties compared with other ICs. ELISA analysis on serum samples from rheumatoid arthritis (RA) patients (n = 80) and healthy controls (n = 35) for anti-LTF autoantibodies confirmed a positive correlation between circulating LTF-specific IgG and RA. ICs between human LTF and LTF-specific IgG purified from patient sera or immunized rabbits and mice, but not control ICs, LTF or Abs alone, elicited strong production of TNF-α and IL-1ß by freshly fractionated human peripheral blood monocytes and monocytes-derived macrophages. Furthermore, LTF-ICs utilized both membrane-anchored CD14 and CD32a (FcγRIIa) to trigger monocyte activation in an internalization-, Toll-like receptor (TLR)4- and TLR9-dependent manner, and also that LTF-IC-induced cytokine production was blocked by specific inhibitors of caspase-1, NF-κB and MAPK. These results uncover a possible pathway for LTF-ICs perpetuating local inflammation and contributing to the pathogenesis of autoimmune diseases by triggering activation of infiltrating monocytes or tissue macrophages in vivo.