Today, I happened to read an article on host-pathogen interactions, one of my favourite topic. Let me start with the introduction part, first. Ulcerative colitis is a chronic inflammatory bowel disease (IBD). It is believed that commensal bacteria, rather than pathogenic bacteria are responsible for dysregulated immunity and IBD. This concept is supported by extensive human and animal studies. The pathogenesis of UC is complex.
Macrophages are crucially important in providing innate and adaptive immune responses to microbes via pattern recognition receptors (TLRs and NLRs). These receptor recognize microbe associated molecular patterns on the microbes. The innate immune system has to maintain a balance between commensal and pathogenic microbes. A dysregulated innate immune responsem, esp. TLR mediated may be central to pathogenesis of UC and may be responsible for immune mediated inflammation characteristics of the disease.
In the present study, the authors examined the acute inflammatory response to bacterial challange in UC. For this, adult patients with a definitive diagnosis of UC and not receiving corticosteroid or biological therapy with in three months of inclusion were enrolled. Similarly age and sex matched healthy controls were also enrolled. All these individuals were injected with a fully antibiotic sensitive and heat killed clinical isolate of E. coli (HkEc). From the serum collected from patients at 24, 48 and 72 hrs, expression profiles of a panel of cytokines was measured.
From blood of these individuals monocytes were isolated and cultured. Now, these monocyte derived macrophages were stimulated with various microbe molecules (e.g., smooth LPS, rough LPS, HkEc, Pam2CSL4, flagellin). After 24 hrs of stimulation, macrophage supernatants were collected and expression profiles of a range of cytokines (IL-Ra, IL-4, IL-5, IL-6, IL-10, Il-12, Il-13, IL-15, IL-17, GM-CSF, IFN-gamma, CXCL10 and MCP-1) were measured.
Total RNA was extracted from both stimulated (with HkEc) and unstimulated macrophages and processed further to produce biotynlated cRNA targets. These were further purified and hybridized to Oligo GE arrays. Microarray were analyzed using softwares.
Upon analyzing the results between the two groups, it was observed that systemic acute phase inflammatory response to E. coli was similar in the two groups, i.e. UC patients and healthy controls. When serum cytokine levels were compared in the two groups, it was observed that circulating CXCL10 levels were significantly greater at 48h and 72h after HkEc inoculation in UC. IL-6 and MCP-1 levels were not different between the two groups. TNF, IFN-gamma, IL-8, IL-10, IL-13, IL-17 and MIP1-alpha were undetectable in serum samples at all time points. These results showed that upon bacterial stimulation there is a prolonged local inflammatory response with elevated CXCL10 levels in UC patients. When cytokines/chemokines profiles of monocyte derived macrophages stimulated with HkEc were compared among the two groups, it was observed that CXCL-10, RANTES, IL-12, IL-13 and IL-6 were significantly elavated in UC patients, while no difference was observed in the level of other cytokines between the two groups of patients. This showed that macrophages from UC patients release significantly higher amount of some cytokines/chemokines upon bacterial stimulation.
In response to bacterial TLR ligands (rLPS, sLPS, Pam3-CSK4, Pam-2CSK4 and flagellin) macrophages from UC patients released significantly increased levels of CXCL10 following sLPS and rLPS (TLR4 and TLR4/CD14 ligands, respectively) and equivalent levels in both UC and controls following Pam3-CSK4, Pam-2-CSK4 and flagellin (TLR2/1, TLR2/6 and TLR5 ligands, respectively). IL-8 and TNF-alpha levels were not significantly different among the two groups with any of the TLR ligands. These results suggest that defective macrophage secretion in UC patients results from an abnormality of TLR-4 and not TLR 1, 2, 5 or 6 signalling. Unlike other TLRs, TLR4 has the ability to activate via Myd88 and TRIF-dependent signalling pathways. In contrast, TLR1 and 2 are activated via Myd88-dependent pathway only. Activation via TRIF pathway leads to release of IFN alpha and beta. Macrophages were stimulated by LPS (TLR-4 ligand) and Pam3-CSK-4 (TLR-1/2lignad), and the level of IFN-beta was determined. It was observde that on stimulation with Pam3-CSK4 (i.e. induction of the Myd88-dependent pathway via TLR1/TLR2) resulted in minimal IFN beta release in both UC and controls. In contrast on stimulation by LPS (i.e. simultaneous activation through both TRIF- and Myd88-dependent pathways through TLR-4) resulted in induction of IFN-beta in both UC and controls. When macrophages form both groups were activated by polyI:C (a TLR-3 ligand, activates through TRIF dependent pathway), equivalent levels of IFN-beta were observed to be released in both UC and controls. This suggested a TLR-4 specific macrophage defect (hyperactive TLR4) in UC. When gene expression in macrophages on stimulation with whole heat killed bacteria was studied, a significant overexpression of a small group of TLR-related genes was observed in UC patients compared to controls. These overexpressed genes were IFN alpha, IFN beta, Il-12, Il-10, MCP-1, HSPA6, cycloxygenase2 and T cell co-stimulatory moleculesCD80 and CD86. The overexpression of IFN provides evidence that exposure to whole bacteria results in overactivation of TLR-TRIF dependent pathway.
In conclusion, the present study identified a dysfunctional systemic innate immune response to bacterial challange in UC patients. TLR-4 was identified as the defective bacterial receptor on macrophages. Furthermore, this dysfunctional innate immune response was related to regulation of TRIF signalling pathway downstream of TLR-4. Thus, in UC patients prolonged inflammation and elevated cytokine release is associated with an abnormal response to bacterial exposure downstream to TLR-4 signalling, Macrophages from UC patients show an abnormal (hyperactive) TLR-4 mediated LPS response which leads to overexpression of cytokines (molecules associated with leucocyte recruitment and activation).
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