Tag Archives: Neutrophil

The Crohn’s Disease Gender Bias and Neutrophils Disrupt the Gut

Neutrophils
Neutrophils (with purple irregular nuclei) can shed proteins that disrupt the intestinal barrier.
This week on TIBDI! Neutrophils shed a protein that disrupts the intestinal barrier, hormones and T cells are behind Crohn’s disease gender skewing, and CD31 is the newest way to make dendritic cells anti-inflammatory.

Neutrophils Bust Up the Intestinal Barrier

During inflammatory bowel disease (IBD), neutrophils gather at sites of inflammation and often migrate through the intestinal epithelial barrier. A new model described by Dr. Dominique A. Weber and Dr. Ronen Sumagin now shows how dangerous this behavior is for intestinal wound healing. They found that neutrophils shed junctional adhesion molecule-like protein (JAML) during epithelial transmigration. JAML binds to a receptor found on epithelial cells called coxsackie-adenovirus receptor (CAR), and JAML and CAR interactions cause epithelial barriers to become leaky. While this leakiness may be needed for initial efficient immune cell infiltration, shed JAML prevents the barrier from regaining normal function and stops wound closure. Experiments showed that blocking JAML-CAR interactions could lead to accelerated wound repair. This discovery could help treat IBD-induced intestinal ulcerations.

Why Crohn’s Disease Prefers Women

There is a general acceptance that the prevalence of Crohn’s disease (CD) is higher in women than in men. W.A. Goodman and R.R. Garg of Case Western Reserve University School of Medicine suspected that this gender bias might be the same in spontaneous models of CD. This is, indeed, the situation. Female SAMP1/YitFc (SAMP) mice were more predisposed to spontaneous CD and had impaired regulatory T cells with low frequencies as compared to the male SAMP mice. An investigation of the T cells revealed that male SAMP T cells responded much differently than female SAMP T cells to estrogen signals. While the male T cells responded by increasing immunosuppressive functions and expanding regulatory T cells, the female cells were resistant to these signals. Finding ways to make female T cells sensitive to estrogen signals could decrease female susceptibility to CD.

More Ways to Induce Anti-inflammatory Dendritic Cells

CD31 is expressed on many types of immune cells and endothelial cells, and it is mainly seen as an adhesion and migration molecule. Recent evidence has shown that it also has inhibitory function on T cells, which means that it might have inhibitory functions in other cells. Marc Clement of the French National Institute of Health and Medical Research (INSERM) has now found that this is, indeed, the situation with dendritic cells (DCs). Signaling via CD31 prevented DC maturation, migration and reduced pro-inflammatory signaling cascades. CD31-stimulated DC also preferentially polarized T cells towards a regulatory phenotype, and transfer of these DCs to a rodent model of multiple sclerosis delayed disease development. These results suggest that CD31 may also be potentially interesting for IBD.

References

NK Cells, Neutrophils and T Cells

NK cells have a regulatory role in DSS colitis

Today’s post looks at the complex interactions between three types of immune cells: NK cells, neutrophils and T helper cells. It was found that NK cells can control the functions of neutrophils. NK cells interacted with neutrophils using a receptor called NKG2A leading to lower pro-inflammatory activities. Simultaneously, it was reported that neutrophils could control the pro-inflammatory activities T cells via a mediator called, thromboxane A2. Thus, an interesting axis of NK cells-Neutrophils-T cells may help control inflammation.

Main points:

  • NK cells can cause neutrophils to become more regulatory via NKG2A and control colitis.
  • Neutrophils control T cell traffic from lymph nodes during vaccination via thromboxane A2.

Take home message: An NK-neutrophil-T cell axis could be playing a role during the first signs of inflammation in IBD.

NK cells are innate immune cells, which have the capacity to destroy infected and defective cells. This function is regulated by receptors that interact with ligands on the opposing cell that either activate and inhibit the NK cell.  The sum total of all activating and inhibitory signals that they receive determines their cytotoxicity. In a recent article by Hall et al., published in Mucosal Immunology, it was found that NK cells have a surprisingly important protective function during intestinal inflammation that is mediated by an interaction of the inhibitory receptor, NKG2A, with local neutrophils. Simultaneously, Yang et al., in a recent publication of Journal of Experimental Medicine, determined that neutrophils regulate the adaptive immune response using Thromboxane A2, controlling both the location and magnitude of the T cell responses. Together, these articles suggest  a possible camaraderie between these cells.

Using two different models of NK cell depletion, including antibody-mediated depletion and NK cell deficient mice, Hall et al. determined that NK cells must have a protective role in the dextran sodium sulfate (DSS) model of colitis. In this model, ingestion of DSS leads to destabilization of the intestinal mucus allowing bacteria to interact with the underlying epithelium causing an acute inflammation and induction of an adaptive immune response. They found that without NK cells; the mice had more colon damage and infiltration, increased pro-inflammatory cytokine production and more neutrophils in both the colon and intestinal draining lymph nodes (mesenteric lymph nodes).

The authors were particularly interested in the effects on neutrophils and further explored this avenue. They found that the loss of NK cells during DSS colitis lead to more activated neutrophils  with increased CD69 expression, magnified reactive oxygen species (ROS) production, and increased amounts of IL-6 and IL-17A as determined by intracellular cytokine staining.

Using a transwell culture system, they were able to determine that the effects generally required direct cell-cell contact between the neutrophils and the NK cells. They also found that the status of the NK cell was crucial. DSS colitis primed NK cells were far more effective at suppressing and lead to more neutrophils expressing low IL-6 and high IL-10.

The next logical step was to determine how this was happening, which the authors did. As NK cells are known to express activating and inhibitory receptors, they checked their expression. They found that the expression of the inhibitory receptor NKG2A was the most strikingly affected by DSS colitis and was highly upregulated on many NK cells. Thus, they continued their neutrophil-NK cell co-culture experiments with an anti-NKG2A antibody and found that blocking antibody could mostly eliminate the suppressive effects of the DSS-primed NK Cells. Confirming this data, in vivo experiments using anti-neutrophil and anti-NKG2A antibodies showed that loss of NK cells was comparable to blocking the NKG2A. These results allowed them to conclude that NKG2A was the important factor.

I agree that NKG2A is the important factor. However, it wasn’t clear to me how this was working. Was there an interaction between NKG2A and MHC class I on the neutrophil? Was this interaction causing a signal cascade in the neutrophil? Or did the interaction lead to changes in the NK cell, which allowed it to express other factors like anti-inflammatory cytokines? It made me curious to see what would happen in an experiment where the NK cells were, for example, IL-10 deficient. Furthermore,  when does the NK cell achieve its super suppressive powers? And is it possible that the NK cell is pro-inflammatory in the beginning of colitis? They look at day 7, but no earlier. As DSS colitis is a transient model, which begins to clear up at day 8, at day 7 anti-inflammatory programs would be active.

While Hall et al. was looking at NK cell influences on neutrophils, Yang et al. was investigating neutrophil effects on the adaptive immune response during footpad vaccination. At first glance, one may think that vaccination may have nothing to do with colitis. However, many lessons learned through vaccination research can be applied to immune responses in the intestine during intestinal trauma. A vaccine includes a foreign antigen combined with adjuvant to stimulate the innate immune system, which leads to a strong adaptive immune response specific for the antigen. Trauma in the gastrointestinal system includes the same elements: damage allows the exposure of bacterial products and luminal antigens to underlying immune cells in the lamina propria. The bacterial products function as an adjuvant, while an adaptive immune response can form against the copious luminal antigens.

thromboxane a2 reigns in T cellsYang et al. found that during a footpad vaccination, there were two waves of neutrophils to the lymph nodes. The first wave included neutrophils that arrived in the local draining lymph node via the lymphatics, within 15 minutes of the injection, presumably after first visiting the site of injection. This influx peaked at two hours and then was more or less gone by 24 hours. The second wave was at day three and was characterized by neutrophils entering the local lymph node via the blood. A series of blocking experiments suggested that the first wave was controlled by prostanoids and the second wave by both prostanoids and chemokines.

In a not so logical fashion, the authors then looked at how the loss of neutrophils was affecting the resulting T cell response against the vaccine antigen. They found that without neutrophils, the numbers of responding T cells were increased in lymph nodes, especially in the distal lymph nodes with 75% of the reactivity being found there. The distal accumulation and increased responsiveness was found to be dependent on the loss of neutrophil prostenoid release, specifically, thromboxane A2. Thromboxane A2 is better known as a vasoconstrictor and platelet aggregator generated from prostaglandin H2. In this case, the authors suggest that it keeps the T cells in the lymph nodes while simultaneously increasing their random movements, preventing their interactions with dendritic cells.

The second article that I read was not the most reader-friendly of all articles and it didn’t mention the immunological consequences of increased distal T cell responsiveness. It would’ve been interesting if they used their antibodies and thromboxane A2 receptor antagonist to investigate changes in a disease setting. However, what I found interesting was that both articles dealt with neutrophils having regulatory functions. The first describes a pathway for generating suppressive neutrophils via NK cells during intestinal inflammation (measured 7 days after the colitis initiation) and the second describes (also 7 days after vaccination) neutrophils preventing T cell responses and accumulation in the distal lymph nodes.

To bring it back to IBD, these kinds of studies are essential to determine the underlying immunological networks that can play a role in creating the unwanted immune responses in IBD. It could very well be that the next best therapy or preventive for IBD is increasing the effects of NK cell-derived NKG2A or magnifying the thromboxane A2 response of neutrophils.

Disclaimer: This article is not intended to provide medical advice, diagnosis or treatment.

Sources

Hall, L. J., Murphy, C. T., Quinlan, A., Hurley, G., Shanahan, F., Nally, K., & Melgar, S. (2013). Natural killer cells protect mice from DSS-induced colitis by regulating neutrophil function via the NKG2A receptor. Mucosal Immunology. doi:10.1038/mi.2012.140

Yang, C. W., & Unanue, E. R. (2013). Neutrophils control the magnitude and spread of the immune response in a thromboxane A2-mediated process. The Journal of Experimental Medicine. doi:10.1084/jem.20122183

One T Cell to Rule Them All

Ly6B staining during colitis
Inflamed Colon with Stained Neutrophils

In the last post, we discussed a bit about how some stem cells are able foster memory T cells, which contribute to the chronicity of colitis. This time, I look at an article that sheds even more light on the role of the bone marrow during inflammation. It was found that under conditions of inflammation, stem cell number increase. During colitis, these cells proliferate and, ultimately, produce more neutrophils and macrophages.

Main points:

  • Hematopoietic stem cells and progenitor cells can be found outside the bone marrow during inflammation in the colon.
  • This appeared to be mediated by T cell-derived cytokines.
  • This process increased the amounts neutrophils and macrophages at the sites of inflammation.

Take home message: In light of my last post, T cells can profoundly influence the innate immune system through stem cells. This strengths their potential role in chronic inflammation.

Neutrophils and macrophages are directly involved in causing damage in the colon during inflammation. Neutrophils and macrophages are the tanks of the immune system and each has their own weapon. Neutrophils are capable of releasing granules containing harmful reactive oxygen species and enzymes. They also release microbial webs (à la Spiderman). Macrophages engulf harmful bacteria and other pathogens and then secrete cytokines (chemical messengers) that alert other immune cells.

The authors used mainly the T cell transfer colitis model, induced by transferring CD4+CD25-CD45RBhi T cells (naïve T cells) to Rag1-/- mice (mice lacking T cells). They found that the inflammatory environment created by T cell transfer colitis had an effect on hematopoietic stem cells (HSC) in the bone marrow (BM), causing them to proliferate. HSC are the stem cells that produce immune cells. The developmental process involves first the production of multipotent progenitor cells. These HSC daughters have a limited life span, but are capable of further differentiating into two types of progenitors, the myeloerythroid progenitors (CMPs) or common lymphoid progenitors (CLPs). CMPs can further differentiate into either megakaryocyte-erythroid progenitors (MEPs) or granulocyte-monocyte progenitors (GMPs). The authors found that under conditions of inflammation, the GMPs would begin to dominate the total myeloid progenitor population. Moreover, HSC and GMP were also found outside of the bone marrow in the spleen and even in the inflamed colon.

IFNg and GM-CSF were considered possible candidates for the induction of these changes. IFNg is produced mainly by T cells and is important in supporting a Th1-associated immunity and is also known to directly affect HSC function. When the authors neutralized IFNg in vivo with antibodies, they found that the proliferation of the HSC decreased considerably. GM-CSF, on the other hand, is produced by stromal cells in the bone marrow (e.g. fibroblasts) and can also be produced by Th17 cells.  GM-CSF is known to be involved with the differentiation of granulocytes. When this cytokine was neutralized, there was little effect on the HSCs, but instead an effect of the downstream GMPs. They were less dominant and loss of GM-CSF reduced their ability to populate the spleen and the colon during colitis.

One point that the authors wished to make was that IL-23 was involved in their findings. This is because IL-23 has such a prominent role in IBD pathogenesis. Current studies looking at genetic association in IBD show that many genes involved in IBD have something to do with the IL-23 pathway. To make this point, they emphasized that in the T cell transfer model many of the transferred T cells eventually differentiate into Th17 cells that require IL-23 for maintenance. They also looked at a form of colitis induced by H. hepaticus infection of 129SvEv.Rag2-/- mice (mice lacking T cells) that is mediated via IL-23. They found that this model also displayed inflammation-associated changes in HSCs; abundant and proliferating HSCs were evident and neutralizing IFNg reversed the phenotype. They also looked at T cells isolated from both wildtype and IL-23R-/- mice and found that Th17-derived GM-CSF was at least partially dependent on IL-23R expression on T cells. However, the most convincing evidence was that IL-23-/- mice with T cell transfer colitis showed reduced GMP accumulations.

I find the IL-23 point in this article not particularly exciting. What I do like is the concept of T cell-derived GM-CSF and IFNg being so important in encouraging the production of excess GMPs during inflammation, though the authors didn’t actually prove this. Still, if you consider these findings together with my last post, which discussed how IL-7 is necessary for T cell transfer colitis by creating a niche for colitogenic memory T cells, it would almost seem that the MSCs (via IL-7) of the bone marrow are controlling inflammation by supporting memory T cells that can provide crucial information to the HSCs. The T cells “communicate” to the HSCs via IFNg that supports of HSC proliferation and mobilization to peripheral regions. GM-CSF would then provide an additional signal to encourage the differentiation of GMPs and, ultimately, the increase of neutrophils and monocytes.

In light of the last post, it seems that there is an intimate relationship between the stem cells of the bone marrow and T cells during colitis development. Naturally, the question is, which cells are more important in generating inflammation in the colon, the T cells or the stem cells? In other words, which cell really does rule them all?

Disclaimer: This article is not intended to provide medical advice, diagnosis or treatment.

Sources:

Griseri T, McKenzie BS, Schiering C, Powrie F. Dysregulated hematopoietic stem and progenitor cell activity promotes interleukin-23-driven chronic intestinal inflammation. Immunity. 2012 Dec 14;37(6):1116-29. doi: 10.1016/j.immuni.2012.08.025. Epub 2012 Nov 29.