Saturday, April 14, 2012

An invisible friends

If you appreciate unusual scientific results then these two recent papers are for you to read. One paper by David Hill et al. (1), was published in Nature immunology, and another one by Olszak and An et al. (2), was published in Science. Both papers are trying to explain how hygiene hypothesis might work by showing how early exposure to commensial bacteria-derived signals educates immune system so it can control allergic responses later on. What is interesting, however, is that fact that both papers reached similar conclusions by examining different arms of immune system.

In paper by David Hill et al., the authors observed an increase in basophils and IgE in germ-free mice or in mice treated with broad-spectrum antibiotics. Using house dust mite allergen or papain allergy model, the authors showed that microbial flora-deficient mice display exaggerated inflammatory response and produce more IgE promoting cytokine IL-4. This increase in basophil numbers however did not occur in either T and B cell-deficient mice or IgE-deficient mice, indicating that IgE promoted basophil accumulation. Direct injection of IgE supported this conclusion. Mechanistically, IgE driven basophil accumulation was shown to be mediated through increase of basophil differentiation from its precursors via augmentation of sensitivity to IL-3. In addition, B-cell specific MyD88 signaling was shown to play a role in limiting IgE production. In short, this paper explained hygiene hypothesis based on B cells-MyD88/IgE/ basophil axis.

In paper by Olszak and An et al., however, authors reached similar conclusion regarding protective effect of microbial flora by examining biology of NKT cells in germ-free mice. The authors showed that germ-free mice had more tissue NKT cells (I wonder how authors of other papers could miss this fact). Using chemically-induced colitis model or allergen-induced asthma model, the authors showed that blocking CD1d molecule by specific antibody inhibited allergic-prone response in germ-free mice (reduced tissue eosiphilia and IgE accumulation), indicating that NKT cells are involved. This conclusion was supported by NKT-specific deficient mice. Crucially, the authors showed that early, but not late life exposure to microbes could protect germ-free mice from allergy. Mechanistically, accumulation of NKT cell in germ-free mice was linked to CXCL16 over-production as a result of microbial-deficiency. It was MyD88 independent effect.

While both paper shed some new lights into hygiene hypothesis, it also highlight how narrow scientific research has become. Each group looked at germ-free mice and observed only what made more sense to them. We know that NKT cells can drive IgE response and I can even argue that NKT cell activation in germ-free mice helps B cells to switch to IgE production. So in a way these two paper complement each other. However, the point is that we still don't understand why absence of microbial signals should lead to IgE response in general. Does it mean that IgE response is somehow helpful in protecting tissue against insults of non-microbial origin? Why? How?


Sunday, March 4, 2012

building local CD8 support against infection

CD8 T cell memory is commonly divided into two types of memory cells: central memory T cells residing in lymph nodes and effector memory T cells residing in peripheral tissues. Relative contribution of each type of memory CD8 T cells in protection against infection depends on many factors, for example, nature of infection. In general, it is assumed that central memory T cells (Tcm) are more potent compared to effector memory T cells (Tem) due to their higher self-renewal capacity.

Recently, a paper published in Nature added another twist to this story. In this study by Jiang X et al. (1), the authors examined CD8 T cell response to localized vaccinia virus (VV) skin infection. First, CD8 T cell response to VV skin infection appears to be CD4 T cell-independent. Activated VV-specific CD8 T cells accumulated in the draining lymph nodes and in the skin. Then, the authors analyzed recirculation pattern of VV-specific memory CD8 T cells by surgically creating parabiotic pairs between infected (30-days post infection) and uninfected mice. As expected, 8 weeks post joining, VV-specific CD8 T cells numbers in the lymph nodes of previously-infected and uninfected mice became equalized. However, to their surprise, even after 24 weeks of parabiotic state they could not detect VV-specific CD8 T cells in the skin of uninfected mouse suggesting that skin resident memory CD8 T cells did not recirculate. For the further analysis, the authors separated co-joined mice after 8 weeks and challenged them with the second VV skin infection (recall response). Again, to the authors surprise, previously parabiotic-uninfected mouse that had the lymph node resident VV-specific Tcm CD8 T cells but not skin-resident VV-specific CD8 T cells, cleared VV skin infection as if it was completely naive mouse, while previously infected mouse with skin-resident VV-specific CD8 T cells cleared VV skin infection with the significantly accelerated manner. Interestingly, if primary VV infection was through intraperitoneal (i.p.) route, then recall response to the secondary VV skin infection was weaker (at least 1000-fold weaker). These experiments indicate that

(a) VV skin infection generates very potent skin-resident VV-specific memory T cells that will localize throughout body skin
(b) VV i.p infection does not generate skin-resident VV-specific memory CD8 T cells
(c) At the end primary skin infection, skin-resident memory CD8 T cells stop to recirculate
(d) Tcm do not play any substantial role in protection against VV skin infection

The most surprising observation was the fact that VV-specific Tcm played almost no role in protection of naive mouse against VV skin infection. This implies that the efficacy of vaccination may be improved by mimicking the route of infection, for example, flu-vaccines may be more potent if introduced through upper-respiratory tract epithelial tissue rather than through skin needle-injection. From tissue point of view it may make sense to limit T cell accumulation when there is no “danger signature” associated with any prior skin infection. Thus, naive mouse with healthy skin may not send out any “come in” signals to T cells. In this regard, it would have been interesting if the authors would had analyzed T cell recirculation pattern during first 30-days of primary VV skin infection to know whether non-circulation pattern of resident memory T cells was because of healthy skin in uninfected mouse or because of change in T cell behavior itself.


P.S See second paper from the same group published in Science Translational Medicine that support their conclusion.

Sunday, February 5, 2012

IRA B cells: going beyond IgM defense line

B cells produce antibodies that clear pathogen from body tissues and protect against re-infection. In general, there are two types of B cells. One type, called innate-like, produce Ab when stimulated by bacterial ligands alone (for example, LPS). 2nd type of B cell require additional T cell-derived signals to produce Ab. While T-dependent Ab production is well-described, little is known about innate-like B cells, their function or contribution to the protection against pathogens.

If you are interested to know more about innate-like B cells, I will recommend reading new paper published in Science. In this study (1) by Rauch, Chudnovskiy, Robbins et al, the authors made surprising observation that in response peritoneal injection of TLR4 ligand, LPS, the predominant cell population in the spleen producing granulocyte-macrophage colony-stimulating factor (GM-CSF) were B cells (Fig 1). The authors dubbed these cells innate response activator (IRA) B cells. The phenotype of IRA B cells producing GM-CSF showed that they were derived from innate-like B cells, also known as B1a cells (Fig 2B). The authors showed that direct signaling through TLR in B1a cells was necessary to develop into IRA B cells (Fig. 3E). By creating mixed chimera where only B cell were deficient in producing GM-CSF, the authors showed that absence of IRA B cells compromised bacterial clearance from the blood (Fig. 4I) in mouse model of peritonitis (sepsis model) and reduced survival rate compared to control [0 vs. 40% survival 72h post peritonitis onset] (Fig 4B).

It has been known for some time that B1a cells from peritoneal cavity migrate into spleen upon activation. Until now, the thinking was that they differentiate into plasma cells producing poly-reactive innate IgM. However, this is a first report to show that besides IgM production, IRA B cells carry another important message, namely GM-CSF, that may influence other cells (macrophages, DCs, neutrophils) to participate in response against pathogens. However, at this stage, it is not clear how exactly IRA B cell-derived GM-CSF has such a protective effect.


P.S. Analogous observation regarding B cells was recently described in Nature. There (2), the authors showed that iNOS/TNF-alpha producing B cells in the gut play a protective role during immune response to C. rodentium.


Monday, January 16, 2012

Langerhans scapegoats

Langerhans cells (LCs) residing in the epidermal layer of the skin represent a specialized dendritic cell subset. However, what is exactly their function is still a mystery. Even the development of mouse models specifically lacking LCs could not resolve the controversy. The new paper published in Science make this case even more confusing.

The study by Modi BG and Neustadter J, et al. (1), examined chemically-induced carcinogenesis model in mice lacking LCs. This particular mouse model lacks Langerhans cells due to langerin-driven diphtheria toxin A expression (langerin-DTA mouse). Any cells that will express langerin will be eliminated from the body. Because Langerhans cells express langerin they will be constitutively depleted. The authors showed that this mice were resistant to DMBA-TPA (carcinogens) induced skin papilloma development. Interestingly, this mice were resistant to DBMA-TPA induced papillomas even in absence of T cells. The authors showed that one way Langerhans cells were contributing to the papilloma development was through metabolism of DBMA into active DBMA-t-3, 4 diols. DBMA-t-3, 4 diol, in turn, had a mutagenic effect on keratinocyte DNA leading to papillomas. This mechanism was supported by observations that (a) Langerhans cells express enzymes capable of metabolizing DBMA and (b) application of DBMA-t-3, 4 diols induced papilloma development even in langerin-DTA mice (though direct application of DBMA-t-3, 4 diol induced low number of papillomas compared to DBMA). This may suggest that simple metabolism of DBMA into DBMA-t-3, 4 diol by Langerhans cells is not only mechanism by which Langerhans cells participate in tumorogenesis. In this regard, it is useful to keep in mind that Langerhans cells residence (maintenance) in skin is critically depended on the action of tumor-growth factor-β (TGF-β). It would be interesting to examine how absence of Langerhans cells affects the role of TGF-β in DBMA-TPA induced papilloma development.