Amrita Joshi

Serum Amyloid P Therapeutically Attenuates Murine Bleomycin-Induced Pulmonary Fibrosis via Its Effects on Macrophages

Macrophages promote tissue remodeling but few mechanisms exist to modulate their activity during tissue fibrosis. Serum amyloid P (SAP), a member of the pentraxin family of proteins, signals through Fcγ receptors which are known to affect macrophage activation. We determined that IPF/UIP patients have increased protein levels of several alternatively activated pro-fibrotic (M2) macrophage-associated proteins in the lung and monocytes from these patients show skewing towards an M2 macrophage phenotype. SAP therapeutically inhibits established bleomycin-induced pulmonary fibrosis, when administered systemically or locally to the lungs. The reduction in aberrant collagen deposition was associated with a reduction in M2 macrophages in the lung and increased IP10/CXCL10. These data highlight the role of macrophages in fibrotic lung disease, and demonstrate a therapeutic action of SAP on macrophages which may extend to many fibrotic indications caused by over-exuberant pro-fibrotic macrophage responses.

Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages

Legionella pneumophila survives within macrophages by evading phagosome–lysosome fusion. To determine whether L. pneumophila resides in an intermediate endosomal compartment or is isolated from the endosomal pathway and to investigate what bacterial factors contribute to establishment of its vacuole, we applied a series of fluorescence microscopy assays. The majority of vacuoles, aged 2.5 min to 4 h containing post‐exponential phase (PE) L. pneumophila, appeared to be separate from the endosomal pathway, as judged by the absence of transferrin receptor, LAMP‐1, cathepsin D and each of four fluorescent probes used to label the endocytic pathway either before or after infection. In contrast, more than 70% of phagosomes that contained Escherichia coli, polystyrene beads, or exponential phase (E) L. pneumophila matured to phagolysosomes, as judged by co‐localization with LAMP‐1, cathepsin D and fluorescent endosomal probes. Surprisingly, neither bacterial viability nor the putative Dot/Icm transport complex was absolutely required for vacuole isolation; although phagosomes containing either formalin‐killed PE wild‐type or live PE dotA or dotB mutant L. pneumophila rapidly accumulated LAMP‐1, less than 20% acquired lysosomal cathepsin D or fluorescent endosomal probes. Therefore, a Dot‐dependent factor(s) isolates the L. pneumophila phagosome from a LAMP‐1‐containing compartment, and a formalin‐resistant Dot‐independent activity inhibits vacuolar accumulation of endocytosed material and delivery to the degradative lysosomes.

TLR9 Differentiates Rapidly from Slowly Progressing Forms of Idiopathic Pulmonary Fibrosis

Compared to slow progressors, patients with rapidly progressive idiopathic pulmonary fibrosis express more TLR9, which recognizes unmethylated CpG DNA and stimulates the fibrotic process. Taking a Toll on Breathing Despite the incredible rate of advances being made in medical science, the exact causes of many diseases remain unknown. These diseases are classified as idiopathic—“a disease of its own kind.” But like a thief who leaves clues at a crime scene that disclose his or her identity, diseases can spur aberrant biological processes that hint at the condition’s cause. Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive form of lung disease of unknown origin characterized by the excess production of fibrous connective tissue (fibrosis) in the supporting framework (interstitium) of the lungs. These changes cause the hardening and/or scarring of lung tissue due to excess collagen, resulting in shortness of breath, a chronic dry cough, fatigue, weakness, chest discomfort, loss of appetite, and rapid weight loss. Patients with IPF have a poor prognosis and are usually expected to live only an average of 4 to 6 years after diagnosis; however, IPF displays a very heterogeneous path, with disease progressing rapidly in some patients and more slowly in others. Thus far, physicians have been unable to predict the speed of disease progression in patients newly diagnosed with IPF. Now, Trujillo et al. have identified a marker that differentiates these two patient groups and that may also mediate rapid progression of this disease. Toll-like receptor 9 (TLR9) is an innate immune molecule that recognizes a particular type of DNA frequently found in bacteria and viruses—unmethylated CpG DNA. Signaling through TLR9 promotes the differentiation of lung fibroblasts taken from IPF patients into myofibroblasts—cells that resemble both smooth muscle and fibroblasts—a key process in fibrosis. Trujillo et al. hypothesized that TLR9 may contribute to rapidly progressing IPF. Indeed, they found higher amounts of TLR9 in rapidly progressing IPF patients compared to slow progressing patients and normal controls. Moreover, in a xenograft mouse model of IPF, fibroblasts from rapid progressors induced more severe fibrosis in response to TLR9 activation than those from slow progressors. The presence of CpG also induced epithelial to mesenchymal transition—another hallmark of fibrosis—in a lung epithelial cell line in vitro. Together, these results suggest that TLR9 may serve as a marker for IPF rapid progressors and that TLR9 targeting may be a new therapeutic strategy for treating IPF. Thus, although the cause(s) of IPF remains unknown, the new data offer hope for an improvement in the prognosis and possibly treatment of this devastating disease. Idiopathic pulmonary fibrosis is characterized by diffuse alveolar damage and severe fibrosis, resulting in a steady worsening of lung function and gas exchange. Because idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression, we classified affected patients as either rapid or slow progressors over the first year of follow-up and then identified differences between the two groups to investigate the mechanism governing rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9 (TLR9), a pathogen recognition receptor that recognizes unmethylated CpG motifs in bacterial and viral DNA, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. Indeed, TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from slowly progressing patients. Moreover, fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG DNA, than were fibroblasts from slowly progressing patients. Using a humanized severe combined immunodeficient mouse, we then demonstrated increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors compared with mice receiving fibroblasts from slowly progressing patients. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and may serve as a potential indicator for this subset of the disease.

A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis

Background Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF. Methodology and Principal Findings miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts. Conclusion These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.

Interleukin-33 contributes to both M1 and M2 chemokine marker expression in human macrophages

BackgroundInterleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined.ResultsHuman macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone.ConclusionsTogether, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.

Interleukin-17–mediated Immunopathogenesis in Experimental Hypersensitivity Pneumonitis

RATIONALE T cells play a critical role in the development of Saccharopolyspora rectivirgula-induced hypersensitivity pneumonitis (HP) but little is known about the role of IL-17A in this disease. OBJECTIVES We examined the role of IL-17A in a murine model of S. rectivirgula antigen (SR-Ag)-induced HP. METHODS Experimental HP was induced by oropharyngeal instillation of SR-Ag in wild-type and IL-17 gene-deficient mice. MEASUREMENTS AND MAIN RESULTS SR-Ag-induced murine HP was characterized by increased transcript levels of IFN-gamma and IL-12p35 compared with saline-treated control mice. Furthermore, mice with HP showed increased IL-17 in lung homogenates, bronchoalveolar lavage fluid, and ex-vivo lung cultures compared with control mice. Flow cytometric analysis of SR-Ag-challenged lungs revealed increased Th17 and CD11c(+) cells. The role of IL-17 in SR-induced HP was examined in IL-17 deficient (IL17(-/-)) and in wild-type (IL-17(+/+)) mice immunodepleted of IL-17. Histological examination of IL17(-/-) mice challenged with SR-Ag revealed reduced inflammatory cell infiltration, decreased CD11c(+) cells, and reduced levels of inflammatory mediators such as IL-12p70, CCL3, and CXCL9 compared with similarly treated IL17(+/+) mice. Anti-IL-17 antibody treatment of IL-17(+/+) mice with HP resulted in reduced inflammation and a lower percentage of CD11c(+) cells compared with IgG-treated IL-17(+/+) mice with HP. CONCLUSIONS SR-Ag-induced IL-17 plays a pivotal role in the immunopathology of HP and targeting IL-17 is an attractive therapeutic option for this disease.

Deleterious Role of TLR3 during Hyperoxia-induced Acute Lung Injury

RATIONALE Acute respiratory distress syndrome (ARDS) manifests clinically as a consequence of septic and/or traumatic injury in the lung. Oxygen therapy remains a major therapeutic intervention in ARDS, but this can contribute further to lung damage. Patients with ARDS are highly susceptible to viral infection and it may be due to altered Toll-like receptor (TLR) expression. OBJECTIVES To evaluate the role of TLR3 in ARDS. METHODS TLR3 expression and signaling was determined in airway epithelial cells after in vitro hyperoxia challenge. Using a murine model of hyperoxia-induced lung injury, the role of TLR3 was determined using either TLR3-gene deficient mice or a specific neutralizing antibody directed to TLR3. MEASUREMENTS AND MAIN RESULTS Increased TLR3 expression was observed in airway epithelial cells from patients with ARDS. Further, hyperoxic conditions alone were a major stimulus for increased TLR3 expression and activation in cultured human epithelial cells. Interestingly, TLR3(-/-) mice exhibited less acute lung injury, activation of apoptotic cascades, and extracellular matrix deposition after 5 days of 80% oxygen compared with wild-type (TLR3(+/+)) mice under the same conditions. Administration of a monoclonal anti-TLR3 antibody to TLR3(+/+) mice exposed to hyperoxic conditions likewise protected these mice from lung injury and inflammation. CONCLUSIONS The potential for redundancy in function as well as cross-talk between distinct TLRs may indeed contribute to whether the inflammatory cascade can be effectively disrupted once signaling has been initiated. Together, these data show that TLR3 has a major role in the development of ARDS-like pathology in the absence of a viral pathogen.

Epigenetic Changes in Bone Marrow Progenitor Cells Influence the Inflammatory Phenotype and Alter Wound Healing in Type 2 Diabetes

Classically activated (M1) macrophages are known to play a role in the development of chronic inflammation associated with impaired wound healing in type 2 diabetes (T2D); however, the mechanism responsible for the dominant proinflammatory (M1) macrophage phenotype in T2D wounds is unknown. Since epigenetic enzymes can direct macrophage phenotypes, we assessed the role of histone methylation in bone marrow (BM) stem/progenitor cells in the programming of macrophages toward a proinflammatory phenotype. We have found that a repressive histone methylation mark, H3K27me3, is decreased at the promoter of the IL-12 gene in BM progenitors and this epigenetic signature is passed down to wound macrophages in a murine model of glucose intolerance (diet-induced obese). These epigenetically “preprogrammed” macrophages result in poised macrophages in peripheral tissue and negatively impact wound repair. We found that in diabetic conditions the H3K27 demethylase Jmjd3 drives IL-12 production in macrophages and that IL-12 production can be modulated by inhibiting Jmjd3. Using human T2D tissue and murine models, we have identified a previously unrecognized mechanism by which macrophages are programmed toward a proinflammatory phenotype, establishing a pattern of unrestrained inflammation associated with nonhealing wounds. Hence, histone demethylase inhibitor–based therapy may represent a novel treatment option for diabetic wounds.

Inflammasome Components Coordinate Autophagy and Pyroptosis as Macrophage Responses to Infection

ABSTRACT When microbes contaminate the macrophage cytoplasm, leukocytes undergo a proinflammatory death that is initiated by nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) that bind and activate caspase-1. We report that these inflammasome components also regulate autophagy, a vesicular pathway to eliminate cytosolic debris. In response to infection with flagellate Legionella pneumophila, C57BL/6J mouse macrophages equipped with caspase-1 and the NLR proteins NAIP5 and NLRC4 stimulated autophagosome turnover. A second trigger of inflammasome assembly, K+ efflux, also rapidly activated autophagy in macrophages that produced caspase-1. Autophagy protects infected macrophages from pyroptosis, since caspase-1-dependent cell death occurred more frequently when autophagy was dampened pharmacologically by either 3-methyladenine or an inhibitor of the Atg4 protease. Accordingly, in addition to coordinating pyroptosis, both (pro-) caspase-1 protein and NLR components of inflammasomes equip macrophages to recruit autophagy, a disposal pathway that raises the threshold of contaminants necessary to trigger proinflammatory leukocyte death. IMPORTANCE An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death. An exciting development in the innate-immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Nutrient deprivation has long been known to induce autophagy; how infection triggers this disposal pathway is an active area of research. Autophagy is encountered by many of the intracellular pathogens that are known to trigger pyroptosis, an inflammatory cell death initiated when nucleotide-binding-domain-, leucine-rich-repeat-containing proteins (NLR proteins) activate caspase-1 within inflammasome complexes. Therefore, we tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. By exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, we demonstrate for the first time that, when confronted with cytosolic contamination, primary mouse macrophages rely not only on the NLR proteins NAIP5 and NLRC4 but also on (pro-)caspase-1 protein to mount a rapid autophagic response that wards off proinflammatory cell death.

A systemic granulomatous response to Schistosoma mansoni eggs alters responsiveness of bone marrow-derived macrophages to Toll-like receptor agonists

Macrophages play a pivotal role in innate and acquired immune responses to Schistosoma mansoni. Classical (M1) or alternative (M2) activation states of these cells further delineate their roles in tissue damage through innate immunity or fibrotic remodeling, respectively. In the present study, we addressed the following question: Does systemic Th2‐type cytokine polarization evoked by S. mansoni affect macrophage differentiation and activation? To this end, we analyzed bone marrow‐derived macrophages from mice with S. mansoni egg‐induced pulmonary granulomas and unchallenged (or naïve) mice to determine their activation state and their response to specific TLR agonists, including S. mansoni egg antigens. Unlike naïve macrophages, macrophages from Th2‐polarized mice constitutively expressed significantly higher “found in inflammatory zone‐1” (FIZZ1) and ST2 (M2 markers) and significantly lower NO synthase 2, CCL3, MIP‐2, TNF‐α, and IL‐12 (M1 markers). Also, compared with naïve macrophages, Th2‐polarized macrophages exhibited enhanced responses to the presence of specific TLR agonists, which consistently induced significantly higher levels of gene and protein levels for M2 and M1 markers in these cells. Together, these data show that signals received by bone marrow precursors during S. mansoni egg‐induced granuloma responses dynamically alter the development of macrophages and enhance the TLR responsiveness of these cells, which may ultimately have a significant effect on the pulmonary granulomatous response.