In innate immune sensing, the detection of pathogen-associated molecular patterns by recognition receptors typically involve leucine-rich repeats (LRRs). We provide a categorization of 375 human LRR-containing proteins, almost half of which lack other identifiable functional domains. We clustered human LRR proteins by first assigning LRRs to LRR classes and then grouping the proteins based on these class assignments, revealing several of the resulting protein groups containing a large number of proteins with certain non-LRR functional domains. In particular, a statistically significant number of LRR proteins in the typical (T) and bacterial + typical (S+T) categories have transmembrane domains, whereas most of the LRR proteins in the cysteine-containing (CC) category contain an F-box domain (which mediates interactions with the E3 ubiquitin ligase complex). Furthermore, by examining the evolutionary profiles of the LRR proteins, we identified a subset of LRR proteins exhibiting strong conservation in fungi and an enrichment for “nucleic acid-binding” function. Expression analysis of LRR genes identifies a subset of pathogen-responsive genes in human primary macrophages infected with pathogenic bacteria. Using functional RNAi, we show that MFHAS1 regulates Toll-like receptor (TLR)–dependent signaling. By using protein interaction network analysis followed by functional RNAi, we identified LRSAM1 as a component of the antibacterial autophagic response.

Indoleamine 2,3-dioxygenase (IDO), which enzymatically depletes tryptophan, is an important antimicrobial defense mechanism against susceptible pathogens. In human epithelial cells, interferon-γ (IFN-γ)-induced IDO expression is transcriptionally enhanced by tumor necrosis factor-α(TNF-α). The purpose of this study was to identify those regulatory mechanisms responsible for this synergistic transcriptional activation of IDO. Nuclear concentrations of signal transducer and activator of transcription-1 (Stat1) and IFN regulatory factor-1 (IRF-1), transcription factors that bind gamma-activated sequences (GAS) and IFN-stimulated response elements (ISRE), respectively, were found to increase after stimulation with IFN-γ and TNF-αrelative to stimulation with individual cytokines. Additionally, CCAAT enhancer binding protein-β (C/EBP-β) bound to one of three consensus C/EBP-β sites in the IDO regulatory region in response to TNF-α alone or combined with IFN-γ. A transcriptional reporter containing green fluorescent protein (GFP) under the control of the IDO regulatory region was used to analyze the contribution of these enhancer elements to synergistic IDO gene expression in response to IFN-γ and TNF-α. Transcriptional activity following mutation of individual enhancers or large deletions within the regulatory region indicates that increased binding of IFN-γ-transactivated factors to GAS and ISRE sites alone is responsible for synergistic transcriptional activation of the IDO gene.

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that can cause bloody diarrhea and, occasionally, acute renal failure as a consequence of Shiga toxin (Stx) production by the organism. Stxs are potent cytotoxins that are lethal to animals at low doses. Thus, Stxs not only harm the host but, as reported here, also significantly enhance the capacity of EHEC O157:H7 to adhere to epithelial cells and to colonize the intestines of mice. Tissue culture experiments showed that this toxin-mediated increase in bacterial adherence correlated with an Stx-evoked increase in a eukaryotic receptor for the EHEC O157:H7 attachment factor intimin.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice (n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle (week 7) and twice weekly until euthanasia, mice (n = 16/group) received either 200 μl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% (P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFβ, CCL17) in the colon tissue (P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum (P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

Interferon-γ (IFN-γ)-induced indoleamine 2,3-dioxygenase (IDO) activity inhibits the growth of susceptible intracellular pathogens by catalyzing the oxidative cleavage of the indole ring of L-tryptophan and depleting pools of the essential amino acid. Tumor necrosis factor-α (TNF-α) synergistically enhances the IDO activity induced by IFN-γ at the level of transcription in human epithelial cells. The purpose of this study was to characterize the molecular mechanisms responsible for synergistic gene expression in response to IFN-γ and TNF-α. It was found that IFN-γ-induced mechanisms, such as the binding of Stat1 to gamma activation sequences (GAS) and IFN regulatory factor-1 (IRF-1) to IFN-stimulated response elements (ISREs), are more highly activated following treatment with IFN and TNF-α. This enhanced signal transduction may be due to the increase in IFN-γ receptor (IFNGR) expression following combined cytokine stimulation and is a likely contributor to the synergy. Additionally, the contribution of a third previously uncharacterized GAS element that forms a complex with Stat1 was investigated using a plasmid reporter system that controls for copy number. When the GAS-3 sequence was included in the regulatory region, gene expression was significantly increased relative to a region containing the mutated GAS-3. This suggestes that GAS-3 is transcriptionally active and contributes to IFN-γ-induced regulation of the IDO gene.

Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-γ), l -arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-γ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.