Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Objectives: Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. Methods: BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. Results: OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. Conclusions: These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

Rhinovirus infection interferes with induction of tolerance to aeroantigens through OX40 ligand, thymic stromal lymphopoietin, and IL-33.

BACKGROUND: Rhinovirus infection at an early age has been associated with development of asthma, but how rhinovirus influences the immune response is not clear. OBJECTIVE: Tolerance to inhaled antigen is mediated through induction of regulatory T (Treg) cells, and we examined whether rhinovirus infection of the respiratory tract can block airway tolerance by modulating Treg cells. METHODS: The immune response to intranasal ovalbumin in mice was assessed with concomitant infection with RV1B, and the factors induced in vivo were compared with those made by human lung epithelial cells infected in vitro with RV16. RESULTS: RV1B infection of mice abrogated tolerance induced by inhalation of soluble ovalbumin, suppressing the normal generation of forkhead box protein 3-positive Treg cells while promoting TH2 cells. Furthermore, RV1B infection led to susceptibility to asthmatic lung disease when mice subsequently re-encountered aeroantigen. RV1B promoted early in vivo expression of the TNF family protein OX40 ligand on lung dendritic cells that was dependent on the innate cytokine thymic stromal lymphopoietin (TSLP) and also induced another innate cytokine, IL-33. Inhibiting each of these pathways allowed the natural development of Treg cells while minimizing TH2 differentiation and restored tolerance in the face of RV1B infection. In accordance, RV16 infection of human lung epithelial cells upregulated TSLP and IL-33 expression. CONCLUSIONS: These results suggest that infection of the respiratory epithelium with rhinovirus can antagonize tolerance to inhaled antigen through combined induction of TSLP, IL-33, and OX40 ligand and that this can lead to susceptibility to asthmatic lung inflammation.

Cell type-specific release of matrix-metallo-proteinase-9 by bacterial chemoattractant in human blood phagocytic leukocytes.

Stimulation of phagocytic leukocytes with bacterial chemoattractant resulted in the release of matrix metal-loproteinases (MMPs). Little is known about the mechanisms of bacterial chemoattractant regulation of MMP in phagocytic leukocytes. We report here that the mechanisms of the bacterial chemotactic peptidefMLP-induced MMP -9 release in monocytes appeared to be different from fMLP-stimulated MMP-9 release in neutrophils. In freshly prepared peripheral blood monocytes, fMLP induces MMP-9 release, starting at 8 h after stimulation. These functions of fMLP is accompanied by an increase in TNFα expression, and mediated through the phosphorylation of ERK1/2 in monocytes. However, neutrophil preparations that responded to fMLP with MMP-9 release did not require activation of ERK1/2 and TNFα expression. These results suggest a different role of fMLP in MMP-9 expression in neutrophils and monocytes, and the signal molecules involved in mediating this effect in human blood monocytes stimulated by bacterial chemoattractant.

Cigarette smoke decreases innate responses of epithelial cells to rhinovirus infection.

Exposure to cigarette smoke is associated with a significant increase in the risk for respiratory viral infections. The airway epithelium is the primary target for both cigarette smoke and respiratory viral infection. We investigated the effects of cigarette smoke on the response of airway epithelial cells to rhinovirus infection. We found that pre-exposure of BEAS-2B cells or primary normal human bronchial epithelial cells (NHBEs) to cigarette smoke extract (CSE) reduced the induction of mRNA of the chemokines CXCL10 and CCL5 by either the viral mimic polyinosine-polycytidylic acid (Poly I:C) or human rhinovirus 16 (HRV-16) infection. The HRV-16-induced release of CXCL10 and CCL5 was also significantly suppressed by CSE. Activation of the IFN mediator STAT-1 and the activation of JNK by poly I:C and HRV-16 were partially suppressed by pre-exposure to CSE. In contrast, the poly I:C-induced and HRV-16-induced phosphorylation of ERK1/2 was unaffected by CSE. HRV-16-stimulated IFN-ß mRNA was also significantly reduced by CSE. Because suppression of the IFN response to viral infection was associated with increased viral production, we assessed HRV-16 RNA concentrations. Exposure to CSE resulted in an increase in HRV-16 RNA at 48 hours after the infection of BEAS-2B cells. These data demonstrate that exposure to CSE alters the response of airway epithelial cells to HRV infection, leading to decreased activation of the IFN-STAT-1 and SAP-JNK pathways, the suppression of CXCL10 and CCL5 production, and increased viral RNA. A diminished, early epithelial-initiated antiviral response to rhinovirus infection could contribute to the increased susceptibility of subjects to prolonged respiratory viral infections after exposure to cigarette smoke.

TGF-beta1 induced epithelial to mesenchymal transition (EMT) in human bronchial epithelial cells is enhanced by IL-1beta but not abrogated by corticosteroids.

BACKGROUND: Chronic persistent asthma is characterized by ongoing airway inflammation and airway remodeling. The processes leading to airway remodeling are poorly understood, and there is increasing evidence that even aggressive anti-inflammatory therapy does not completely prevent this process. We sought to investigate whether TGFbeta1 stimulates bronchial epithelial cells to undergo transition to a mesenchymal phenotype, and whether this transition can be abrogated by corticosteroid treatment or enhanced by the pro-inflammatory cytokine IL-1beta. METHODS: BEAS-2B and primary normal human bronchial epithelial cells were stimulated with TGFbeta1 and expression of epithelial and mesenchymal markers assessed by quantitative real-time PCR, immunoblotting, immunofluorescence microscopy and zymography. In some cases the epithelial cells were also incubated with corticosteroids or IL-1beta. Results were analyzed using non-parametric statistical tests. RESULTS: Treatment of BEAS-2B or primary human bronchial epithelial cells with TGFbeta1 significantly reduced the expression level of the epithelial adherence junction protein E-cadherin. TGFbeta1 then markedly induced mesenchymal marker proteins such as collagen I, tenascin C, fibronectin and alpha-smooth muscle actin mRNA in a dose dependant manner. The process of mesenchymal transition was accompanied by a morphological change towards a more spindle shaped fibroblast cell type with a more motile and invasive phenotype. Corticosteroid pre-treatment did not significantly alter the TGFbeta1 induced transition but IL-1beta enhanced the transition. CONCLUSION: Our results indicate, that TGFbeta1 can induce mesenchymal transition in the bronchial epithelial cell line and primary cells. Since asthma has been strongly associated with increased expression of TGFbeta1 in the airway, epithelial to mesenchymal transition may contribute to the contractile and fibrotic remodeling process that accompanies chronic asthma.

An atypical protein kinase C (PKC zeta) plays a critical role in lipopolysaccharide-activated NF-kappa B in human peripheral blood monocytes and macrophages.

We have reported that the bacterial LPS induces the activation of NF-kappaB and inflammatory cytokine gene expression and that this requires the activity of small GTPase, RhoA. In this study, we show that an atypical protein kinase C isozyme, PKCzeta, associates functionally with RhoA and that PKCzeta acts as a signaling component downstream of RhoA. Stimulation of monocytes and macrophages with LPS resulted in PKCzeta activation and that inhibition of PKCzeta activity blocks both LPS-stimulated activation of NF-kappaB and IL-1beta gene expression. Our results also indicate that transforming growth factor beta-activated kinase 1 acts as a signaling component downstream of PKCzeta in cytokine gene transcription stimulated by LPS in human peripheral blood monocytes and macrophages. The specificity of this response suggests an important role for the Rho GTPase/PKCzeta/transforming growth factor beta-activated kinase 1/NF-kappaB pathway in host defense and in proinflammatory cytokine synthesis induced by bacterial LPS.

A novel protein kinase C (PKCepsilon) is required for fMet-Leu-Phe-induced activation of NF-kappaB in human peripheral blood monocytes.

We have reported that the chemoattractant, fMet-Leu-Phe (fMLP), induces the activation of NF-kappaB in human peripheral blood monocytes and that this requires the activity of small GTPase, RhoA (Huang, S., Chen, L.-Y., Zuraw, B. L., Ye, R. D., and Pan, Z. K. (2001) J. Biol. Chem. 276, 40977-40981). Here we showed that the novel protein kinase C isozyme, PKCepsilon, associates functionally with RhoA in fMLP-stimulated monocytes and that PKCepsilon acted as a signaling component downstream of the GTPase RhoA during fMLP-induced activation of NF-kappaB. Stimulation of monocytes with fMLP resulted in activation of both PKCepsilon and NF-kappaB. This latter activation was largely blocked by specific inhibitors of PKCepsilon by transient expression of a dominant-negative form of PKCepsilon and by PKCepsilon-specific short interfering RNA. These findings demonstrate, for the first time, that fMLP-induced activation of NF-kappaB utilizes a signaling pathway, which requires activity of PKCepsilon, and that PKCepsilon acts as a signaling component downstream of RhoA in cytokine gene transcription stimulated by a chemoattractant. The specificity of this response suggests an important role for the Rho GTPase-PKCepsilon-NF-kappaB pathway in host defense and represents a novel and potentially important mechanism through which fMLP not only attracts leukocytes but may also contribute directly to inflammation.