Anti-Inflammatory and Antiapoptotic Responses to Infection: A Common Denominator of Human and Bovine Macrophages Infected with Mycobacterium avium Subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of a chronic intestinal inflammation in ruminants named Johne's disease or paratuberculosis and a possible etiopathological agent of human Crohn's disease (CD). Analysis of macrophage transcriptomes in response to Map infection is expected to provide key missing information in the understanding of the role of this pathogen in establishing an inappropriate and persistent infection in a susceptible host and of the molecular mechanisms that might underlie the early phases of CD. In this paper we summarize transcriptomic studies of human and bovine peripheral blood mononuclear cells (PBMC), monocyte-derived macrophages (MDMs), and macrophages-like cell lines in vitro infected with Map. Most studies included in this paper consistently reported common gene expression signatures of bovine and human macrophages in response to Map such as enhanced expression of the anti-inflammatory cytokines IL-10 and IL-6, which promote bacterial survival. Overexpression of IL-10 could be responsible for the Map-associated reduction in the expression of the proapoptotic TNF-α gene observed in bovine and human macrophages.


Association of Mycobacterium avium
Subsp.

paratuberculosis (Map) with Chronic In�ammator� Bowell Diseases of Cattle and Humans
Mycobacterium avium subsp. paratuberculosis (Map) is the causal agent of Johne's disease or paratuberculosis, a chronic in�ammatory bowel disease of domesticated ruminants and wildlife species worldwide. Johne's disease causes major economic losses to the global dairy industry due to reduced milk production, lower weight gains, infertility, premature culling, and increased cow replacement costs [1]. Map has a worldwide distribution and is of considerable concern in cattle, sheep, goats, and farmed red deer. Although it is still controversial, Map has been implicated as a causal or exacerbating agent in human Crohns's disease (CD), a chronic in�ammatory bowel disease characterized by transmural in�ammation and granuloma formation [2][3][4]. Evidences that Map may be associated to CD in humans include similarity between the clinical signs of CD in humans and those found in animals with paratuberculosis; detection of Map in feces, intestinal tissues, breast milk, macrophages, and peripheral blood of patients with CD; association between Map DNA in blood and cellular and humoral immune responses in CD; and anti-Map antibiotic therapy resulting in reduction of bacteremia and remission or substantial improvement in disease condition in many patients [5][6][7][8][9][10].
In addition, meta-analysis and epidemiological studies have con�rmed an association of Map with CD [11,12].
Map may enter the food chain from a variety of sources. e organism, shed from infected animals, may contaminate pastures and potable water, where it is resistant to standard puri�cation with chlorine [13]. Because Map can survive pasteurization conditions, dairy products such as milk and cheese have been proposed as possible sources of exposure of humans to Map [14]. Recently, we have demonstrated that Map can be detected and cultured from diaphragm muscle of Map-infected cattle destined for human consumption and suggested a possible risk of exposure of humans to Map via contaminated meat [15]. Aer oral ingestion, Map invades the intestinal wall preferably through epithelial cells or M cells present in the follicle-associated epithelium covering the continuous Peyer's patches in the distal ileum [16][17][18]. Although the mechanism of entry in the mucosa is important in establishing Map infection, most of the bacterial components involved in the interaction with the intestinal epithelium are still unknown. It has been recently demonstrated that Map3464 gene encodes an NADH-�avin oxidoreductase involved in invasion of bovine epithelial cells through the activation of host cell Cdc42 [19]. Aer translocation of Map across the intestinal epithelium, Map is subsequently phagocytised by macrophages in the intestinal lamina propria and submucosa. Upon phagocytosis of Map by naive macrophages, there is both intracellular replication of Map and bacterial killing by the host, which re�ects an initial T-helper 1 (-1) or proin�ammatory immune response [20][21][22][23][24]. Bacterial killing is due to a rapid phagosome acidi�cation response by the host that enables phagosome-lysosome fusion and presentation of antigens to T cells via MHC to occur in some infected cells. However, since many phagosomes containing Map fail to acquire sig-ni�cant amounts of lysosomal-associated membrane protein (LAMP-1) and to fuse with lysosomes, this can allow Map to survive and proliferate inside macrophages. An active role for Map in preventing phagosome-lysosome fusion is supported by the observation that live bacteria are able to persist within phagosomes, while phagosome maturation is not interrupted following the uptake of killed Map [25,26]. In addition, recent studies have suggested that Map alters the ability of infected macrophages to react to extracellular signals from T cells, particularly through the CD154-CD40 system [27]. is leads to an enhanced IL-10 and TGF-expression in Map-infected macrophages, which favor bacterial survival by suppression of -1 responses and IFN-in T cells [28][29][30][31]. In cattle, clinical signs of infection and bacterial shedding are usually not evident until 2-5 years post-infection (p.i.) [32]. During the subclinical phase of the infection, Map persists and slowly proliferates within macrophages of the gut without the innate system being able to clear the infection.

�. Anti-In�a��ator�� Antiapoptotic� and
Anti-Invasive Responses Induced in Bovine Macrophages Infected with Mycobacterium avium Subsp. paratuberculosis Several in vitro studies have investigated gene expression pro�les induced by Map on bovine macrophages obtained from uninfected cattle and on a bovine macrophage cell line (Table 1). Compared to uninfected cultures, in vitro challenges of monocytes-derived macrophages (MDMs) from healthy cows with live Map resulted in enhanced production of the anti-in�ammatory cytokine interleukin-10 (IL-10) at 6, 24, and 72 h p.i. that antagoni�es the proin�ammatory immune response by downregulating the production of interleukin-12 (IL-12), tumor necrosis factor-(TNF-), and interferon-(IFN-) as estimated by qRT-PCR [33].
Similarly, other authors also observed a downregulatory trend in TNF-mRNA expression from 16 h to 96 h p.i. and upregulation of IL-10 mRNA levels that peak from 48 h to 96 h p.i. [34]. Using microarray technology, three cytokines including transforming growth factor-(TGF-), interleukin-6 (IL-6), and macrophage in�ammatory protein-1 (MIP-1 ) had greater expression in Map-infected MDM at 16 h p.i. when compared with inactivated control macrophages [35]. e matrix metalloproteinase (MMP-12) and the thrombospondin-1, both involved in cell migration and tissue destruction, were also signi�cantly upregulated. In contrast, the TNF-receptor and the major histocompatibility complex (MHC) class II DQ-had lower expression in Map-infected macrophages. Decreased expression of the cell surface MHC class I and class II molecules was previously documented in macrophages phagocytising Map organisms indicating a reduced capacity to present antigens to T lymphocytes [44]. Consistently with these results, Murphy et al. also detected high levels of the anti-in�ammatory cytokines TGF-and IL-6 in MDM infected with Map at 24 h p.i. [36]. In another study, signi�cantly downregulation in expression of the proin�ammatory cytokine IL-1 and of the metalloproteinases MMP-1, MMP-23, and MMP-9 involved in tissue destruction was observed in Map-stimulated PBMC when compared with control cells [37]. Using cDNA microarrays focused on expressed sequences from a bovine total leukocyte library (BOLT5) and 10 distinct Map strains to measure total transcriptomic alterations in Map-infected MDM, a total of 78 annotated bovine genes were found to be differentially expressed at 6 h p.i., relative to uninfected cells [38]. Within the group of differentially expressed genes signi�cant downregulation of two proapoptotic genes, BCL2 antagonist of cell death (BAD) and TNF receptor (TNFR), was observed in Map-infected MDM cells relative to uninfected cells. Upregulation of the apoptotic inhibitor BCL2A1 and of the proin�ammatory cytokines IL-1 , IL-1 , and IL-8 relative to uninfected control cells was also observed. By using a pan-genomic analysis of bovine MDM gene expression in response to in vitro infection with Map, Machugh et al. revealed that many of the highly upregulated genes at 2 h p.i. had proin�ammatory related functions, particularly IL-1 , IL-1 , TNF, IL-6, chemokine ligand 2 gene (C-X-C motif; CXCL2), and the chemokine ligand 20 gene (C-C motif; CCL20) [39]. At 6 h p.i. immune-related genes were among the differentially expressed genes showing the highest relative increase in expression; however, the fold-change induction of these genes was not as high as those detected as 2 h p.i. Upregulated genes at 6 h p.i. included IL-1 , TNF, CXCL2, CCL4, CCL5, CCL20, CD40, and the complement factor B gene (CFB). Of the differentially expressed gene identi�ed 24 h p.i. that had a known immune function were the serum amyloid A3 genes (SAA3), C-type lectin domain family 4 member E (CLEC4E), C-type lectin domain family 2 member D (CLEC2D), CD40, and CFB. Overall, several pro-and antiapoptotic genes were upregulated at 2 h and 6 h p.i. suggesting that this process is highly regulated. Pro-and antiapoptotic genes upregulated included TNF (proapoptotic), caspase 1,  1 THP-1, human monocytic cell line. 2 Comparisons were made between uninfected and infected cells. 3 Comparisons were made between Map-infected macrophages from CD patients and controls. 4, 6, and 8 genes (CASP-1, CASP-4, CASP-6, and CASP-8; all proapoptotic), the baculoviral IAP repeat containing 3 gene (BIRC-3; antiapoptotic), and the FADD-like apoptosis regulator gene (CFLAR; pro-and antiapoptotic). We recently examined whether Map isolates with differential abilities to grow within a bovine macrophage cell line (BoMac) induced a characteristic early immune-in�ammatory response [40]. �ur results showed signi�cant differences in the expression of several cytokines (IL-6, TGF-1, TNF-2, IFN-, and IL-1 ), proteins related to apoptosis (BCL2-1), or tissue destruction (MMP3-1) aer the infection of BoMac cells with a bovine or an ovine isolate of Map. e bovine isolate that grew within BoMac cells was a good inducer of the apoptotic inhibitor BCL2-1 at 4 or 14 h p.i. which might cause lower levels of apoptosis than in BoMac cells infected with the ovine isolate. In addition, infection of BoMac cells with the bovine isolate resulted in a signi�cant upregulation of the anti-in�ammatory cytokines IL-6 and TGF-1 at 24 h p.i., when compared with cells infected with the ovine isolate. Although we did not observe signi�cant differences in IL-10 or TNF-2 gene expression in BoMac cells infected with the bovine or the ovine isolates at any of the time points, the bovine isolate did induce more IL-10 at 14 and 24 h p.i and less TNF-2 at 4 and 14 h p.i. than did the ovine isolate. ese results suggest that lower TNF-2 production and an induction of IL-10 are associated with the growth within bovine macrophages of virulent isolates of Map. We also observed that cells stimulated with the bovine isolate exhibited lower levels of the metalloproteinase MMP3-1 involved in tissue destruction at 4 h and 24 h p.i. relative to cells stimulated with the ovine isolate. Differences in induction of the metalloproteinase inhibitor TIMP-1 were not statistically signi�cant at any of the time points studied but the bovine isolate did induce more TIMP-1 at 4 h p.i. than did the ovine isolate. e ovine isolate was signi�cantly attenuated in growth in BoMac cells and this decrease in survival within the infected cells correlated with a reduced anti-in�ammatory response in the infected cells and with a signi�cantly upregulated proin�ammatory immune response generally associated with elimination of Map and protection. In particular, the expression of the proin�ammatory cytokine IL-1 was highly upregulated in cells infected with the ovine isolate at 14 and 24 h p.i. and downregulated in BoMac cells infected with the bovine isolate at 24 h p.i. Because a strong correlation between the intracellular multiplication of the tested isolates and patterns of production of IL-6, TGF-, MMPL-3, BCL2-1, and IL-1 was observed, the levels of expression of these speci�c proteins might be used to discriminate between isolates with differential virulence in the BoMac cellular model.
All together, the results of the transcriptomic studies in bovine macrophages included in this paper suggested that Map might stimulated an initial proin�ammatory immune response mediated by IL-1 that is followed by an enhanced anti-in�ammatory response mediated by IL-6, IL-10, and TGF-. In addition, downregulation of the proapoptotic gene TNF-was consistently observed in different studies. Stimulation of anti-in�ammatory and antiapoptotic responses might allow Map to successfully persist in bovine macrophages during the persistent, subclinical phase of the infection.

�� Immune�In�ammator� �esponses Induced in Human Macrophages Infected with Map
Despite the possible role of Map in CD, there is not much known about the interaction of Map with the human innate immune system (Table 2). A transformed human monocytic cell line (THP1) stimulated with Map has shown to differentially respond to infections with well-characterized clinical isolates of Map when compared with unstimulated cells. When human THP1 cells were stimulated with bovine or human isolates of Map several genes associated with apoptosis and cytokine signalling (LRDD, PDCD-8, IL-12, IL-18, IL-23, and TNF ) were signi�cantly downregulated [41]. Data from this study suggested that the human macrophage responses to Map isolates from cattle or human sources, regardless of genotype, follow a common theme of antiapoptotic and anti-in�ammatory responses within the host cells, an attribute likely associated with successful infection and persistence.
e response of macrophages from CD patients to live Map has been recently addressed. Following in vitro exposure to Map, PBMC from CD patients secreted signi�cant more amounts of TNF-, IL-6, and IL-10 when compared to the levels released by PBMC from healthy volunteers [42]. In contrast, the IFN-response to Map was signi�cantly elevated in PBMC isolated from healthy volunteers compared to PBMC derived from CD patients. In another study, human MDMs obtained from CD patients and controls were infected with Map, Mycobacterium avium subsp. avium (Mav), and other live intestinal bacteria such as Escherichia coli or Enterococcus faecalis, and cytokine levels were evaluated at different time points [43]. e results of this study indicated that macrophages from CD patients showed impaired TNFsecretion in response to bacterial challenge but augmented IL-23 secretion as compared to macrophages from healthy individuals. It is plausible that the MAP-dependent IL-23 secretion enhancement occurred as consequence of phagocytosis, because the high IL-23 concentrations obtained at 3 h p.i. were not observed at 3 d and 7 d p.i. Differences in cytokine expression aer bacterial challenge where not Map speci�c, as other bacteria (E. coli and Mav) showed similar effects.
Although most of the studies presented in this paper used PBMC or MDM cells in vitro stimulated with Map, we should indicate that recently Olsen et al. [45] isolated intestinal T cells from intestinal biopsies of CD patients to investigate cellular immune responses to Map. Interestingly, they observed that CD patients had a high frequency of Map reactive T cells and also a higher frequency of response to Map compared to other bacterial antigens. Aer stimulation with Map, intestinal T cells secreted the proin�ammatory cytokines IFN-and IL-17, and, therefore, a role for Map in the excessive in�ammation seen in CD cannot be excluded.

Conclusions
Despite development of cell-mediated immune responses shortly aer infection, Map has the capacity to survive and grow in macrophages from human and cattle hosts. Gene expression studies included in this paper allow us to conclude that the inhibition of apoptosis and enhanced expression of inhibitors of macrophage activation could contribute to the early survival and immune escape of Map. Common gene expression signatures of bovine and human macrophages in response to Map such as enhanced expression of the anti-in�ammatory cytokines IL-10 and IL-6, which promote bacterial survival, have been consistently observed. Overexpression of IL-10 could be responsible for the Map-associated reduction in the expression of the proapoptotic TNF-gene observed in many studies. Differential effects on macrophage gene regulation between studies might be caused by the different in vitro models, multiplicities of infection, time p.i., and/or microarray platforms used. Although this paper does not suggest a casual effect for Map, it does compile the �ndings that Map is able to alter the normal host immune response against a pathogen in susceptible humans and cattle and contribute to the pathogenesis of Crohn's and Johne's diseases.