Tissue-resident macrophages are heterogeneous as a consequence of anatomical niche-specific functions. Many populations self-renew independently of bone marrow in the adult, but the molecular mechanisms of this are poorly understood. We determined a transcriptional profile for the major self-renewing population of peritoneal macrophages in mice. These cells specifically expressed the transcription factor Gata6. Selective deficiency of Gata6 in myeloid cells caused substantial alterations in the transcriptome of peritoneal macrophages. Gata6 deficiency also resulted in dysregulated peritoneal macrophage proliferative renewal during homeostasis and in response to inflammation, which was associated with delays in the resolution of inflammation. Our investigations reveal that the tissue macrophage phenotype is under discrete tissue-selective transcriptional control and that this is fundamentally linked to the regulation of their proliferation renewal.

The role of microRNAs (miRs), which are endogenous RNA oligonucleotides that regulate gene expression, in diabetic nephropathy is unknown. Here, we performed expression profiling of cultured proximal tubular cells (PTCs) under high-glucose and control conditions. We identified expression of 103 of 328 microRNAs but did not observe glucose-induced changes in expression. Next, we performed miR expression profiling in pooled RNA from formalin-fixed, paraffin-embedded tissue from renal biopsies. We studied three groups of patients with established diabetic nephropathy and detected 103 of 365 miRs. Two miRs differed by more than two-fold between progressors and nonprogressors, and 12 miRs differed between late presenters and other biopsies. We noted the greatest change in miR-192 expression, which was significantly lower in late presenters. Furthermore, in individual biopsies, low expression of miR-192 correlated with tubulointerstitial fibrosis and low estimated GFR. In vitro, treatment of PTCs with TGF-␤1 decreased miR-192 expression. Overexpression of miR-192 suppressed expression of the E-Box repressors ZEB1 and ZEB2, thereby opposing TGF-␤-mediated downregulation of E-cadherin. In summary, loss of miR-192 expression associates with increased fibrosis and decreased estimated GFR in diabetic nephropathy in vivo, perhaps by enhancing TGF-␤-mediated downregulation of E-cadherin in PTCs.

The general paradigm is that monocytes are recruited to sites of inflammation and terminally-differentiate into macrophages. There has been no demonstration of proliferation of peripherally-derived inflammatory macrophages under physiological conditions. Here we show that proliferation of both bone marrow-derived inflammatory and tissue resident macrophage lineage branches is a key feature of the inflammatory process with major implications for the mechanisms underlying recovery from inflammation. Both macrophage lineage branches are dependent on M-CSF during inflammation, and thus the potential for therapeutic interventions is marked. Furthermore, these observations are independent of Th2 immunity. These studies indicate that the proliferation of distinct macrophage populations provides a general mechanism for macrophage expansion at key stages during inflammation, and separate control mechanisms are implicated.

Macrophage (MØ) biology is routinely modelled in the peritoneal cavity, a vascular tissue readily infiltrated by leukocytes during inflammation. After several decades of study, no consensus has emerged regarding the importance of in situ proliferation versus peripheral monocyte recruitment for the maintenance of tissue resident MØs. By applying specific measures of mitosis, we have monitored tissue MØ proliferation during newborn development, adulthood and acute resolving inflammation in young adult mice. Despite the vascular nature of the tissue and ease of peripheral leukocyte entry, tissue MØs in the newborn increase in number by local proliferation. On the contrary, in the adult, tissue MØ proliferation is considerably reduced and most likely provides homeostatic control of cell numbers. Importantly, during an acute inflammatory response, when substantial numbers of inflammatory MØs are recruited from the circulation, tissue-resident MØs survive and then undergo a transient and intense proliferative burst in situ to repopulate the tissue. Our data indicate that local proliferation is a general mechanism for the selfsufficient renewal of tissue MØs during development and acute inflammation and not one restricted to non-vascular tissues, which has implications for the therapeutic modulation of MØ activity during the resolution of inflammation.Key words: Cellular proliferation . Inflammation . Macrophages IntroductionThe peritoneal cavity, a vascular environment extensively used for the study of macrophage (MØ) biology, receives a large influx of inflammatory cells (including monocytes/MØs) during inflammation, such as that induced with zymosan. Numbers of recoverable tissue-resident (Res) MØs drop during the first hours of inflammation ('the disappearance reaction') [1][2][3]. ResMØ-like cells become recoverable again about 2 days after the induction of inflammation in an environment mixed with inflammatory peripherally recruited MØs [2,4]. The MØ disappearance reaction has been attributed to several factors: increased tissue adherence, emigration to draining lymph nodes and/or cell death [1]. Historically, many attempts have been made to document the origins of peritoneal MØs under steady-state and inflammatory conditions. However, these have produced conflicting results often arguing for negligible local proliferation and the maintenance of ResMØs from the periphery [5,6]. Complicating these studies, subsets of peritoneal ResMØs are binucleate or can occasionally be found in the act of phagocytosis of apoptotic and Eur. J. Immunol. 2011. 41: 2155-2164 DOI 10.1002 HIGHLIGHTS 2155 Frontline other debris, casting doubt on the apparent presence of DNA contents consistent with active cell cycle. The generally accepted model is that ResMØs are renewed in tissues by myeloid restricted BM hematopoietic progenitors [7][8][9]. Some evidence has been presented for local proliferation of ResMØs in a variety of tissues, but the results were often controversial and varied with models used [8]. More recently, it has been ...

MicroRNAs are short noncoding RNA regulators that repress synthesis of their targets post-transcriptionally. On average, each microRNA is estimated to regulate several hundred protein-coding genes, and about 60% of proteins are thought to be regulated by microRNAs in total. A subset of these genes, including the key profibrotic cytokine transforming growth factor beta-1 (TGF-β1), exhibits particularly strong levels of post-transcriptional control of protein synthesis, involving microRNAs and other mechanisms. Changes in microRNA expression pattern are linked to profound effects on cell phenotype, and microRNAs have an emerging role in diverse physiological and pathological processes. In this review, we provide an overview of microRNA biology with a focus on their emerging role in diseases typified by organ fibrosis.

Transforming growth factor-␤1 (TGF-␤1) is a key cytokine involved in the pathogenesis of fibrosis in many organs. We previously demonstrated in renal proximal tubular cells that the engagement of the extracellular polysaccharide hyaluronan with its receptor CD44 attenuated TGF-␤1 signaling. In the current study we examined the potential mechanism by which the interaction between hyaluronan (HA) and CD44 regulates TGF-␤ receptor function. Affinity labeling of TGF-␤ receptors demonstrated that in the unstimulated cells the majority of the receptor partitioned into EEA-1-associated non-lipid raft-associated membrane pools. In the presence of exogenous HA, the majority of the receptors partitioned into caveolin-1 lipid raft-associated pools.

Recent estimates suggest that 1 in 12 of the global population suffers from diabetes mellitus. Approximately 40 % of those affected will go on to develop diabetes-related chronic kidney disease or diabetic nephropathy (DN). DN is a major cause of disability and premature death. Existing tests for prognostic purposes are limited and can be invasive, and interventions to delay progression are challenging. MicroRNAs (miRNAs) are a recently described class of molecular regulators found ubiquitously in human tissues and bodily fluids, where they are highly stable. Alterations in miRNA expression profiles have been observed in numerous diseases. Blood and tissue miRNAs are already established cancer biomarkers, and cardiovascular, metabolic and immune disease miRNA biomarkers are under development. Urinary miRNAs represent a potential novel source of non-invasive biomarkers for kidney diseases, including DN. In addition, recent data suggest that miRNAs may have therapeutic applications. Here, we review the utility of miRNAs as biomarkers for the early detection and progression of DN, assess emerging data on miRNAs implicated in DN pathology and discuss how the data from both fields may contribute to the development of novel therapeutic agents.

The aim of this study was to characterize the mechanism of transforming growth factor (TGF)-β1-mediated alteration of renal proximal tubular cell phenotype. TGF-β1 altered cell phenotype, with cells appearing elongated and spindle shaped. This was associated with loss of cell-cell contact and rearrangement of the actin cytoskeleton, increased formation of stress fibers, and focal adhesions. Addition of the tyrosine phosphatase inhibitor sodium orthovanadate also led to rapid but transient loss of cell-cell contact, but it did not lead to a change of phenotype comparable to that seen following addition of TGF-β1. There was, however, no change in the formation of focal adhesions and no associated reorganization of the Factin cytoskeleton. Disruption of the actin cytoskeleton with cytochalasin D prevented phenotypic alterations following addition of TGF-β1. Transient transfection with Smad2/4 or Smad3/4 expression vectors did not alter cell phenotype. Previously, we demonstrated β-catenin translocation to proximal tubule cell nuclei and its association with Smad proteins following addition of TGF-β1, suggesting the possibility that TGF-β1 may modulate Wnt signaling. The Wnt-responsive Xtwn-reporter construct was, however, silent in response to TGF-β1. Similarly, a second Wnt/LEF-1-regulated element, Toplflash, which does not contain Smad binding sites, was insensitive to TGF-β1 signaling. In contrast, phenotypic changes in response to TGF-β1 were abrogated by inhibitors of the RhoA downstream target ROCK, which also prevented loss of cell-cell contact and adherens junction disassembly.