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Cooperative molecular and cellular networks regulate Toll-like receptor-dependent inflammatory responses

Gavin E. Morris^*, Lisa C. Parker^*, Jon R. Ward^*, Elizabeth C. Jones^*, Moira K. B. Whyte^*, Christopher E. Brightling^#, Peter Bradding^#, Steven K. Dower and Ian Sabroe^*,1

Academic Units of

^* Respiratory Medicine and

Cell Biology, Division of Genomic Medicine, University of Sheffield, Sheffield, UK; and

^# Institute for Lung Health, Department of Infection, Inflammation and Immunity, Leicester-Warwick Medical School and University Hospitals of Leicester, Leicester, UK

¹Correspondence: Academic Unit of Respiratory Medicine, Division of Genomic Medicine, University of Sheffield, M Floor, Royal Hallamshire Hospital, Sheffield, S10 2JF, UK. E-mail i.sabroe{at}sheffield.ac.uk

SPECIFIC AIMS

To determine the roles of cooperative networks between inflammatory and tissue cells, and between TLR3 and TLR4, in the regulation of inflammation.

PRINCIPAL FINDINGS

1. A TLR3 agonist, poly(I:C), activates a broad range of tissue cell types
TLR3 is thought to be involved in the detection of viral infections through its ability to respond to dsRNA and may have a role in detecting tissue damage through its ability to recognize mRNA. We observed that TLR3 was expressed in primary human airway smooth muscle cells and an immortalized lung epithelial cell line. Primary human vascular and airway smooth muscle cells, primary human umbilical vein endothelial cells, and immortalized airway epithelial cells all responded to poly(I:C) with proinflammatory cytokine production, with the potential to mediate specific aspects of antiviral responses or pathological states (such as the recruitment of mast cells to the airway in asthma). Activation of TLR3 airway smooth muscle was also associated with up-regulation of adhesion molecules used by some respiratory viruses to gain access to tissue cells. Responses to poly(I:C) showed synergy with proinflammatory cytokines (interleukin-1ßbeta; (IL-1ßbeta;, TNF). In contrast, leukocytes (which do not express TLR3) did not respond to poly(I:C).

2. Responses to poly(I:C) correlate with TLR3 expression
In keeping with our work showing a lack of leukocyte responses to poly(I:C), others have shown that TLR3 expression is limited to certain dendritic cell populations only. Consistent with the functional responses we observed, we found that TLR3 was expressed intracellularly in airway smooth muscle cells and both intra- and extracellularly on airway epithelial cells. Endosomal acidification was required for poly(I:C) signaling in smooth muscle cells but not epithelial cells.

3. Networks regulate inflammatory responses to some TLR agonists
We previously showed that very small numbers of TLR4 expressing monocytes could enable potent responses to LPS in unresponsive tissue cells. In this setting, LPS-activated monocytes released IL-1, which resulted in a potent amplification of proinflammatory responses through actions on airway smooth muscle. In the current study we also observed that addition of LPS to a coculture of epithelial cells and peripheral blood mononuclear cells (PBMCs) caused activation of the tissue cells. Similarly, cocultures of endothelial cells or vascular smooth muscle cells and purified monocytes were profoundly activated by LPS. We observed that airway smooth muscle cells also required the presence of PBMC to respond to agonists of TLR7/8. In contrast, responses to poly(I:C) were not amplified by the presence of PBMCs/ monocytes in coculture with smooth muscle, endothelial, or epithelial cells, consistent with the lack of TLR3 expression in most leukocytes.

4. TLRs can cooperate to induce inflammatory responses in cell networks
At sites of inflammation, leukocytes are present in tissues and exposed to multiple TLR agonists arising from endogenous damage and infections. We therefore stimulated cocultures of PBMCs and either epithelial cells or airway smooth muscle with agonists of TLR4 (acting principally on the monocyte) and TLR3 (acting principally on the tissue cell). We noted that the combined TLR4/monocyte activation and TLR3/tissue cell activation resulted in a cooperative response leading to a further enhancement of cytokine generation from the cocultures (Fig. 1 ).

View larger version (16K): [in this window] [in a new window]   Figure 1. Cooperative production of cytokines in cocultures. Airway smooth muscle cells (ASMCs) or BEAS-2B epithelial cells were grown to confluence in 24-well plates and stimulated with media, poly(I:C) (25 µg/ml), LPS (10 ng/ml), or poly(I:C) + LPS in the presence or absence of PBMCs (10,000 cells per well). After 24 h, levels of CXCL8 and CXCL10 in the media were determined by ELISA. Poly(I:C) caused production of CXCL8 and CXL10 from monocultures of ASMCs, whereas LPS activated ASMCs or BEAS-2B cells effectively only in the presence of PBMC. Stimulation with both agonists enhanced the production of cytokines from PBMC/tissue cell cocultures. A, B) Effects of the indicated agonists and culture conditions on CXCL8 and CXCL10 release from ASMCs; C) the effects of these stimuli on CXCL8 generation from BEAS-2B cells. In each graph, open bars show cytokine generation from monolayers of the relevant tissue cell (ASMCs or BEAS-2B cells), shaded bars show cytokine generation from wells containing 10,000 PBMCs only, and filled bars show cytokine generation from cocultures of PBMCs and the indicated tissue cell. Data shown are from 4 passages of BEAS-2B cells and from 5 passages of one ASMC donor. Selected relevant comparisons were performed using Student’s t test and the results indicated.

CONCLUSIONS AND SIGNIFICANCE

At inflammatory sites, there are many opportunities for leukocytes to interact with tissue cells and with multiple TLR agonists. For example, a viral infection may present agonists for TLR3 (viral double-stranded RNA) and TLR4 (e.g., viral coat proteins), and damage to local tissues may also provide agonists for these receptors (mRNA for TLR3, a variety of endogenous agonists for TLR4). Viral infections at some sites, such as the lung, are typically accompanied by the presentation of other TLR agonists (e.g., endotoxin in inhaled air or superadded bacterial infections). The response to a given stimulus will thus depend on the network of cells and TLRs that are activated. The outcome with respect to the inflammatory response will therefore be dependent on 1) the pattern of TLR expression on leukocytes and tissue cells, 2) the interaction between leukocytes and tissue cells, and 3) the nature of the agonist(s) and the TLRs with which it interacts.

We have shown here that TLR3, a receptor involved in responses to viruses and tissue damage, is widely expressed by a range of primary human tissue cell types. Responses to agonists of TLR3 are dependent on tissue cell expression of TLR3: leukocytes such as monocytes do not amplify responses to TLR3 agonists. Nonetheless, we show here that peripheral blood mononuclear cells can contribute to the antiviral response or the response to tissue damage at a potentially wide range of tissue sites through their ability to enable a cooperative response in tissue cells to agonists of either TLR7/8 or TLR4. In the case of receptors not widely expressed by tissue cells at high levels, as exemplified by TLR4, the presence of monocytes dramatically up-regulates the resulting inflammatory response.

Since infectious and noninfectious damage stimuli are always likely to activate multiple TLRs, and responses to such agonists will be regulated by cell networks, we determined whether cooperative responses between TLR agonists could be seen in cocultures of tissue cells and monocytes. When TLR3 and TLR4 agonists were added to cocultures of monocytes and either ASMCs or epithelial cells, a cooperative response occurred with a further amplification of inflammatory responses as measured by cytokine production.

These data indicate that the inflammatory response is regulated by cooperative networks that can be modeled in vitro with primary human cells. We have shown that such networks will provide important opportunities to understand and model the mechanisms of inflammation in responses to infection and in inflammatory pathologies. The models we have developed here demonstrate the capability of cells and receptors to cooperate, and suggest that such cooperative networks will be of more importance than the response of an individual cells when examining the processes of acute TLR-driven inflammation in vitro.

View larger version (43K): [in this window] [in a new window]   Figure 2. Schematic figure. Cooperative networks regulate inflammatory responses (1). Single agonists with more than one TLR specificity (e.g., viral activation of TLR3 and TLR4) or exposure to multiple TLR agonists (e.g., at sites of tissue damage where mRNA and tissue breakdown products are released) activate TLRs on resident monocytes (e.g., TLR4 or TLR7/8) and tissue cells (TLR3) (2). Stimulation of tissue leukocytes causes the release of mediators acting on tissue cells, and results in a synergistic proinflammatory reaction characterized by the production of large amounts of cytokines from activated tissue cells (3). Cytokine production, principally by tissue cells, results in the recruitment of a range of leukocytes (4). Survival factors released by monocytes regulate not only activation of tissue cells, but also the survival of recruited leukocytes such as neutrophils. Once these are withdrawn, inflammation can resolve by induction of leukocyte apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5910fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5910fjev1 20/12/2153    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morris, G. E. Articles by Sabroe, I. Search for Related Content PubMed PubMed Citation Articles by Morris, G. E. Articles by Sabroe, I.