Induction of Protective IgA by Intestinal Dendritic Cells Carrying Commensal Bacteria

This paper, published in Science in 2004 by a research team out of the Institute of Experimental Immunology in Zurich, Switzerland led by Dr. Andrew J. Macpherson and co-authored by Therese Uhr, investigated the connection between the presence of commensal bacteria in the intestine and the protective measures taken by the mucosal immune system to prevent commensal bacteria from entering the host and causing a systematic immune response. In the intestine, the gut associated lymphoid tissue (GALT) acts as a barrier between the inside of the intestine and the rest of the body (Janeway et al., 2005; Fig. 1).

The GALT is part of the large immune system called the mucosal immune system (MALT), which exists on most surfaces in an organism in which pathogens often attempt to enter and infect the host (Janeway et al., 2005; Fig. 1). Split into two sections, the GALT is comprised of the outer gut epithelium and the lamina propria (Fig. 1). The lamina propria is a layer of connective tissues between the gut epithelium and other organs (Mak et al., 2014; Fig. 1). Inside the lamina propria resides intestinal follicles, which are important in the mucosal immune response to invading pathogens (Mak et al., 2014). The GALT is an essential part of protecting the host from pathogens and also serves as an interface between the host and commensal bacteria. Microbiota in an organism’s intestinal tract have been hypothesized to support an immune response against pathogenic bacteria, which are bacteria that can make people sick (Mak et al., 2014). Some of the known and studied benefits of commensal bacteria are reviewed in Tlaskalova-Hogenova et al. (2004). To summarize, commensal bacteria compete with pathogenic bacteria for space and help the host initiate an immune response (Abt & Artis, 2013; Tlaskalova-Hogenova et al., 2004). However, if commensal bacteria escape the intestine by passing through the epithelial layer of the intestine, they can, similar to pathogenic bacteria, cause an inflammatory immune response. This response occurs because commensal bacteria express similar pathogen associated molecular patterns, or PAMPS, to pathogenic bacteria and unlike the intestinal epithelial cells, the rest of the body is not tolerant of these PAMPS. Commensal bacteria are helpful to the host in assisting in the defense against pathogenic bacteria in the mucosa, but can become harmful if these commensals escape the mucosa and invade other areas of the host (Mak et al., 2014, Tlaskalova-Hogenova et al. 2004).  In the GALT, the antibody IgA is secreted by resident B cells, which either limits the growth of commensal bacteria or attaches to the commensal bacteria and prevents movement through the epithelium (Macpherson & Uhr, 2004).

The first experiment in this series assessed how the mucosal immune system dealt with commensal bacteria that had penetrated the epithelium of the intestine. To do this, mice were challenged with doses of the known commensal bacteria Enterobacter cloacae. This means that the mice involved were given an oral dose or a body cavity injection of E. cloacae and then observed sixty hours post dosing to see where the commensal bacteria was subsequently located. In mice given an oral dose, the bacteria were found in the mesenteric lymph node (MLN), but not in the spleen. Mice given the body cavity injection had bacteria in their spleen instead of the MLN. The location of the bacteria, especially after the oral dose, illustrated to the researchers that commensal bacteria were able to move through the gut epithelium and into the MLN, meaning that the live bacteria was confined to the mucosal immune system and was thus prevented from causing a systematic immune response (Macpherson & Uhr, 2004). The mice given the body cavity injection were used to model that the sequestration of E. cloacae in the MLN after the oral dose was intentional and not due to a leak in the gut that would cause the bacteria to end up in the MLN on accident (Macpherson & Uhr, 2004). The next step was to investigate how the E. cloacae was transported to the MLN. To investigate this, Macpherson & Uhr (2004) examined where the E. cloacae were located inside of the MLN and how the mice dealt with a pathogenic bacteria. This was accomplished by use of the fluorescence activated cell sorting (FACS), in which the researchers tagged leukocytes with a particle that fluoresces under a certain wavelength and then cultured bacteria from the tagged leukocytes (Macpherson & Uhr, 2004). What they found was that the E. cloacae resided in dendritic cells (DCs), which are important antigen presenting cells. These cells meaning consume antigens and present them to other immune cells, thereby triggering an immune response (Banchereau & Steinman, 1998; Fig. 2). However, the E. cloacae was not found in any macrophages in the MLN (Macpherson & Uhr, 2004). This result was strange because in ex vivo (outside an organism) experiments were found to be highly effective in killing commensal bacteria that had penetrated the epithelium in the GALT, which should mean macrophages with bacteria should also be found in the MLN (Macpherson & Uhr, 2004). This illustrated that the DCs were the main form of transportation for the bacteria to the MLN, albeit it small concentrations (Macpherson & Uhr, 2004). To compare this result to the mucosal immune response to a pathogenic bacteria, Macpherson and Uhr (2004) also challenged mice with Salmonella typhimurium and again used FACS to find where these bacteria ended up in the host. The Salmonella typhimurium was found in the organisms’ spleen in addition to inside DCs and macrophages in the MLN, illustrating that the organism had a harder time controlling this pathogenic bacteria and was beginning to initiate a systematic immune response (Macpherson & Uhr, 2004). The immune response to the E. cloacae therefore suggests that the mucosal immune response was able to kill most of the commensal bacteria that got through the epithelium by utilizing macrophages and thus only a small concentration of the bacteria was transported to the MLN inside DCs, preventing a systematic immune response (Macpherson & Uhr, 2004).

Once Macpherson and Uhr established that E. cloacae resided in DCs inside the MLN, their next question that they addressed was where the DCs picked up the E. cloacae, either in the MLN or in the GALT. To answer this question, mice were challenged with two intestinal loops, each one containing a certain antibiotic resistant strain of E. cloacae (Fig. 3).

Both loops were connected through the MLN, allowing the researchers to hypothesize that if DCs in the MLN contained both strains of the E. cloacae, then the bacteria must have traveled to the MLN on their own and were then taken up by DCs once in the MLN. The result of this experiment demonstrated that DCs obtained the E. cloacae in the GALT, as DCs containing both strains did not exist (Macpherson & Uhr, 2004). The uptake of E. cloacae in the GALT was done through the use of M cells (Fig. 4). 

M cells reside in the gut epithelium and have the ability to engulf antigens and then present them to DCs (Janeway et al., 2005). Macpherson and Uhr (2004) illustrated this interaction by tagging E. cloacae with green fluorescent protein (GFP) and then by use of FACS, demonstrated that DCs containing E. cloacae were present in Peyer’s Patches, which are follicles that exist under M cells and are important induction sites in the mucosal immune system (Janeway et al., 2005). Thus, E. cloacae introduced into the GALT are taken up by M cells, which present the bacteria to DCs in the Peyer’s Patches, activating them, which prompts the DCs to bring the bacteria to the MLN (Macpherson & Uhr, 2004).

The last question addressed in this paper was the role of the antibody IgA in preventing commensal bacteria from leaving the GALT. Antibodies are secreted by mature B cells after a DC presents the B cell with antigen. Antibodies act to help clear pathogens by marking diseased cells for destruction or neutralizing the pathogen itself (Mak et al., 2014). IgA is utilized in the gut because it is important for pathogen neutralization (Mak et al., 2014, Fig. 5). Macpherson and Uhr (2004), demonstrated that mice repeatedly exposed to live E. cloacae selectively prompt B cells present in the mucosa to secrete IgA, but exposure to dead E. cloacae did not prompt this response. Thus the researchers concluded that IgA was important to mucosal immune response to commensals and thus set out to further discern what role this antibody played. First, the role of the MLN had to be discerned, as it was unclear of the MLN functioned as a site that restricted the immune response to commensal bacteria to the mucosal immune system or if it functioned as a site for IgA induction. To do this, Macpherson and Uhr (2004) removed the MLN from some mice and compared them to mice with an intact MLN after the introduction of E. cloacae. The researchers found that both groups of mice had the same levels of IgA induction, but in the mice missing the MLN, some E. cloacae made its way to the spleen (Macpherson & Uhr, 2004). This result illustrated that the MLN’s role is to confine the DCs containing commensal bacteria to the mucosal immune system, preventing a systematic immune response (Macpherson & Uhr, 2004). The researches then proceeded with their investigation of the importance of IgA (Fig. 5). DCs containing E. cloacae resulted in the stimulation of B cells to produce IgA. Macpherson and Uhr (2004) demonstrated this connection  by use of FACS analysis and confirmed by enzyme-linked immunosorbent assay (ELISA), which is an assay that can detect the levels of antibodies in a sample. Once it was confirmed that DCs containing E. cloacae could prompt B cells to secrete IgA, Macpherson and Uhr (2004) tested the purpose of the antibody. They did this in two ways. They first compared the levels of bacterial penetration and luminal densities between germ free and antibody deficient mice undergoing recolonization of commensal bacteria. The result of this comparison illustrated that antibody deficient mice took longer to adapt to the presence of the commensal bacteria, but the final densities were the same between groups (Macpherson & Uhr, 2004). The second experiment observed the penetration of E. cloacae in the two groups of mice from the previous experiment. The result of this experiment illustrated that the non-antibody deficient mice had less penetration of E. cloacae, and levels were reduced even further after IgA levels increased (Macpherson & Uhr, 2004). The conclusion from this pair of experiments was that IgA helps prevent the penetration of commensal bacteria in the GALT (Macpherson & Uhr, 2004).

The broad conclusions of this set of experiments conducted by Macpherson and Uhr (2004) demonstrated that commensal bacteria are restricted to the mucosal immune system when the MLN, DCs, and IgA are all present in correct concentrations and functioning properly. This process allows the commensal bacteria to help the host effectively ward off pathogenic bacteria but prevents these same commensal species from escaping the GALT and causing a systematic immune response that may harm the host. Further directions for this research could include observing what would happen to mice with a nonfunctional or absent population of macrophages that were then challenged with commensal bacteria in their gut. Macrophages help control the size of the commensal bacteria populations and if these cells were absent, would the commensal bacteria overwhelm the mucosal immune response and insight a systematic immune response.

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