The German philosopher, Friedrich Nietzsche, is known for a number of ideas among which a particularly oft-quoted one is, “That which does not kill us makes us stronger” (https://www.goodreads.com/quotes/30-that-which-does-not-kill-us-makes-us-stronger). A recent report in Cell (Fonseca et al., 2015) offers evidence that in the context of infection and immunity, the above aphorism may not be a reliable guide to reality.

Belkaid and colleagues studied gastrointestinal infection with Yersinia pseudotuberculosis in a mouse model. They focused on the state of gastrointestinal anatomy, function, and immunity after the infection was cleared.

Their first key finding is that by four weeks post-infection, with bacterial clearance largely completed by three weeks, the mesenteric (i.e., intestine-associated) lymph nodes (mesenteric lymph nodes or MLN) are larger, more massive, and contain more neutrophils and inflammatory monocytes than naïve MLN. Post-infection MLN with chronic lymphadenopathy (CL) also contained abscesses.

When the authors assessed numbers of regulatory T (Treg) cells and the immune responses to antigens unrelated to Y. pseudotuberculosis, they demonstrated that infection was associated with substantially decreased numbers of Treg cells. In addition, and perhaps counterintuitively, the authors found that in animals with prior infection with Y. pseudotuberculosis and chronic MLN lymphadenopathy (CL+), despite complete clearance of the pathogen, responses to immunization with either of two antigens resulted in fewer cells secreting either IL-17A or IFN-γ than in uninfected animals or infected animals without chronic lymphadenopathy. IgA responses were also decreased in CL+ versus CL mice. When Fonseca et al. evaluated the effects of infection on the intestinal and fecal microbiota, they found only minor effects compared to uninfected controls and no long-term differences between mice with and without CL.

Since mucosal immune responses require the migration of dendritic cells (DC; a major type of antigen presenting cell for T cells) into MLN, the decreased responses associated with post-infection CL+ suggested that there might be a problem with DC trafficking. In a series of experiments using a variety of methods, the authors showed that the subset of DC that activate T cells to make IL-17A, a cytokine involved in immunity to bacterial and fungal pathogens and in autoimmunity, were present in MLN in substantially decreased numbers. They showed that these cells were nevertheless reaching the lamina propria (the layer of connective tissue beneath the epithelium in the intestinal wall) in normal numbers, suggesting a defect external to the DC themselves. Additional structural abnormalties in the MLN were documented, including disrupted T-cell zones and displaced B-cell follicles and lymphatic vessels.

The post-infection mice with CL, at four weeks after initial exposure to the pathogen, exhibited increased leakage by lymphatic vessels draining the intestines, in comparison to post-infection mice without CL. CL+ mice also had many more hematopoietic cells, primarily neutrophils and macrophages, than CL mice in the mesenteric adipose tissue (MAT). In the MAT, the mice with CL also had significantly increased numbers of the DC able to stimulate T cells that produce IL-17A than did the mice withoput CL. Based on these latter findings the authors suggest that the DC that stimulate IL-17A-producing T cells that normally traffick to the MLN instead are diverted to the MAT as a consequence of infection.

In a final set of experiments, the authors addressed the role of the microbiome in the effects on mucosal immunity attributed to the ultimately cleared infection by Y. pseudotuberculosis. The authors used comparisons between specific pathogen-free (SPF) mice, which have a microbiota, with germ-free (GF) mice, which do not have a microbiota.

Fonseca et al. found that post-infection, GF mice exhibited less severe MLN lymphadenopathy than SPF mice. In addition, unlike the SPF mice, GF mice did not exhibit a defect in migration of DC into the MLN.

The authors also compared SPF mice that had cleared infection with Y. pseudotuberculosis and were treated with antibiotics with such mice not treated with antibiotics. In the SPF mice treated with the anti-microbials, the abnormal accumulation of DC in the MAT post-infection was largely prevented, and these cells trafficked in normal numbers to the MLN. If SPF mice were treated with antibiotics to eliminate the resident microbiota but then gavaged with microbial products, the infiltration of MAT by DC was restored.

Antibiotic trteatment also diminished inflammation associated with such cytokines as interferon-gamma and tumor necrosis factor alpha in the MAT. While antibiotic treatment reduced the mucosal immune response to vaccine immunogens compared to no treatment, as assessed by responses of IL-17A-secreting T cells, it restored the responses in post-infection mice with CL to the range of the responses in uninfected mice similarly treated with antibiotics. In other words, treatment with antibiotics while directly and modestly decreasing cell-mediated immune responses in the intestinal tissues, exerted a positive effect on cell-mediated immunity in the gut mediated by exposure to Y. pseudotuberculosis by reducing the normal microbiota. Then implication is that the normal microbiota are necessary for the immunity-inhibiting effect of the pathogen.

In the Discussion, the authors address the possible mechanisms by which infection with Y. pseudotuberculosis causes the subversion of gut immunity and acknowledge prior reports demonstrating significant immunological functional compromise after infections with other pathogens. They speculate that the effects seen in their model of gastrointestinal infection are not specific to this one pathogen and cite prior reports describing CL, disrupted trafficking of DC and macrophages, and increased lymphatic permeability following other infectious and inflammatory diseases. Two contributing causal factors that may be shared in these various conditions include the increased tendency of phagocytes to traffick through lymphatics and the transition from producing so-called type 2 cytokines, which are now recognized to be central to tissue healing, to producing type 1 cytokines that may increase lymphatic permeability.

Near the end of the Discussion the authors acknowledge that they cannot exclude the possibility that normally unthreatening gut microbes, such as lactobacilli, become pathogenic in the situation post-infection with a pathogen such as Y. pseudotuberculosis. The finding of persistent infection of the MLN with CL by lactobacilli may have prompted this thought.

In a commentary on this study, publshed in Science (2015), Carl Nathan suggests that context-dependence pathogenicity by the lactobacilli in the MLN could perhaps account for the findings that Fonseca et al. attribute to prior but cleared infection with Y. pseudotuberculosis. This possibility is consistent with the comparisons of SPF versus GF mice and of antibiotic-treated and non-treated SPF mice.  Of course, one might still reasonably attribute the potential for MLN infection by lactobacilli to the preceding effects of Y. pseudotuberculosis, so the latter pathogen would still have a role, albeit indirect, in the pathogenicity that ultimately results.

The issue of how to define pathogens arguably remains a non-trivial task. In this instance, lactobacilli would typically be regarded as non-pathogenic, even beneficial, members of the normal gut microbiota, but as Nathan notes, perhaps they become pathogenic after the appearance, and the disappearance, of a more standard pathogen, Y. pseudotuberculosis. For a full treatement, this topic requires more space than can be accommodated in the current commentary.

In closing, I wish to note that if Nathan is correct that the lactobacilli in the MLN of mice that have cleared Y. pseudotuberculosis are behaving as opportunistic pathogens, the blurred boundary between pathogenic and non-pathogenic bacteria (and in this instance what are usually regarded as beneficial residents of the microbiota) illustrates, yet again, the Principle of Radical Evolutionary Indifference. Microbes show no inclination to abide by our classification schemes. The potential context-dependence of the pathogenicity of lactobacilli would also exemplify the fluidity of categories in biology (Greenspan, 2009), a point that has been relevant to many studies discussed in this space.

References

Nietzsche Quotes. https://www.goodreads.com/quotes/30-that-which-does-not-kill-us-makes-us-stronger

Fonseca DM, Hand TW, Han SJ, Gerner MY, Glatman Zaretsky A, Byrd AL, Harrison OJ, Ortiz AM, Quinones M, Trinchieri G, Brenchley JM, Brodsky IE, Germain RN, Randolph GJ, Belkaid Y. Microbiota-Dependent Sequelae of Acute Infection Compromise Tissue-Specific Immunity. Cell. 2015 Oct 8;163(2):354-66. doi:10.1016/j.cell.2015.08.030. PubMed PMID: 26451485.

Nathan C. IMMUNOLOGY. From transient infection to chronic disease. Science. 2015 Oct 9;350(6257):161. doi: 10.1126/science.aad4141. PubMed PMID: 26450196.

Greenspan, N. Boundaries of categories, categories of boundaries, and evolution. March 18, 2009. http://dev-evmedreview.pantheonsite.io/boundaries-of-categories-categories-of-boundaries-and-evolution/