Role of microbiota in evolution of adaptive immune system

Source: Science 24 December 2010: 
Vol. 330 no. 6012 pp. 1768-1773 
DOI: 10.1126/science.1195568

Has the Microbiota Played a Critical Role in the Evolution of the Adaptive Immune System?

1. Yun Kyung Lee and Sarkis K. Mazmanian^*
   

   
    

• Microbes reside in several anatomical sites of human body including the skin, vagina, and mouth.
  
• Lower gastrointestinal tract of mammals harbor the greatest density and diversity of commensal microorganisms (including bacteria, archaea, fungi, viruses, protozoans, and helminthes.
  
• It has been observed that the development of gut associated lymphoid tissue (GALT), the first line of defense for the intestinal mucosa, is defective in germ free animals.
  
• It has also been observed that germ free mice display fewer and smaller Peyer's patches, smaller and less cellular mesenteric lymph nodes and less cellular lamina propia of small intestine relative to animals with a microbiota. Furthermore, these mice have been shown to exhibit reduced expression of TLRs and MHC class II molecules.
  
• Number of CD4+ T cells is also reduced in lamina propria of germ free mice. Development of isolated lymphoid follicles is also defective in the absence of microbes.
  
• Multiple proportions of intestinal immune cells require the microbiota for their development and function.
  
• In addition to its effect on intestinal immunity microbiota also effects extra intestinal immunity.
  
• It has also been observed that germ free mice are more susceptible to microorganisms like Shigella, Bacillus and Leishmania. Thus, in addition to development microbiota also effects functional aspects of intestinal and systemic immunity.
  
• Host mechanisms and the microbiota may have evolved to collaborate against infectious agents.
  
• Studies have shown an antagonistic relationship between microbiota and overt pathogens.
  
• Harnessing the immunomodulatory capabilities of microbiota may offer new avenues for development of antimicrobial therapies for infectious diseases.
  
• Microbiota play important roles in effector CD4+ T cell differentiation. During infection microbial and host signals provide cues to naïve CD4+ T cells to differentiate into various proinflammatory and anti-inflammatory subsets.
  
• Microbiota has been shown to affect the TH1-TH2 balance in the systemic immune compartments.
  
• Studies have shown that TH17 cell development in the gut is specifically affected by commensal bacteria.
  
• Of the various microbial species that constitute the microbiota of mice, only segmented filamentous bacteria (SFB) have been shown to direct intestinal T helper cell development.
  
• Researchers from around the world have proposed that life style changes have caused a fundamental alteration in the association of humans with the microbial world. The alteration in composition of healthy microbiota leading to altered microbial colonization is known as dysbiosis.
  
• Although it is not known yet whether dysbiosis causes any human diseases, it may affect autoimmunity by altering the balance between toleragenic and inflammatory members of the microbiota.
  
• In a healthy microbiome, there is an optimal proportion of both pro- and anti-inflammatory organisms (represented here by SFBs and B. fragilis), which provide signals to the developing immune system (controlled by the host genome), leading to a balance of Treg and TH17 cell activities.
  
• Altered community composition of the microbiome due to life-style, known as dysbiosis, may represent this disease-modifying component. An increase in proinflammatory microbes (for example, SFBs in animalmodels)may promote TH17 cell activity to increase and thus predispose genetically susceptible people to TH17-mediated autoimmunity.
  
• The imbalance between TH17 cells and Tregs ultimately leads to autoimmunity.
  
• We propose that certain microbes, such as SFBs, that can peacefully coexist with a healthy host but still retain pathogenic potential be termed "pathobionts" to distinguish them from opportunistic pathogens that are acquired from the environment and cause acute infections.
  
• The adaptive immune system distinguishes between self and foreign antigens and mounts an appropriate response to clear invading pathogens by recognizing non-self molecules.
  
• The adaptive immune system must either tolerate or ignore the microbiota.
  
• Several studies suggest that symbiotic bacteria have evolved the mechanisms to suppress unwanted inflammation toward the microbiota by actively inducing mucosal tolerance.
  
• The authors propose a model for co-evolution of adaptive immune system with the microbiota.
  
• According to this model, The adaptive immune system develops under the control of the vertebrate genome to produce various cell types. The evolutionarily ancient molecule TGFb directs the differentiation of Foxp3+ Treg cells.
  
• Over millennia of coevolution, commensal microbes (B. fragilis used as an example here) produced molecules that networked with the primordial immune systemto help expand various Treg cell subsets (for example, IL-10–producing Foxp3+ Treg cells).
  
• Pro-inflammatory pathobionts (such as SFBs) may have induced TH17 cell differentiation to increase mucosal defenses against enteric pathogens.
  
• The modern adaptive immune system may have arisen from two distinct events: Tregs and Th17 cell types evolved independently or through the sequential development of TH17 cells from Treg cell precursors.
  
• Taken together, the modulation of Tregs and TH17 cells by commensal microorganisms and pathobionts, respectively, appears to shape the immune status of the host and thus represents a possible risk factor for autoimmune diseases that appears to depend on balanced Treg-Th17 proportions.
  

  
   

Loss of Th17 is associated with CD4 T activation in 2009 H1N1 patients

Source: Jiang TJ, Zhang JY, Li WG, Xie YX, Zhang XW, Wang Y, Jin L, Wang FS, Zhao M.

Preferential loss of Th17 cells is associated with CD4 T cell activation in

patients with 2009 pandemic H1N1 swine-origin influenza A infection. Clin

Immunol. 2010 Dec;137(3):303-10. Epub 2010 Oct 12. PubMed PMID: 20943443.

 

H1N1 swine-origin influenza A virus (S-OIV) is a novel influenza H1N1 strain that first emerged in humans in Mexico during March 2009. The incubation period is 1-7 days. Past studies have reported that host immune response can be a critical factor in determining various outcomes of influenza infection. These studies have reported that while moderate increase in proinflammatory responses may favor viral clearance, hyper activated inflammatory response can have detrimental effects on the host. It has also been noted that after innate immunity activation there could be an abundance of virus induced inflammatory cytokines which can lead to subsequent antigen non-specific T cell activation in mice and human with viral infection.

Among the T cells, helper CD4+ T cells release a number of distinct cytokines. One such cytokine is IFN-gamma which is released by Th1 cells and which is conventionally thought to exacerbate tissue damage and control viral infection. Regulatory T cells (Tregs) are immunosuppressive and play an important role in the regulation of immune responses. In contrast, IL-17 producing CD4+ T cells (Th17) are known to play role in both chronic inflammation and in host defense against pathogens. Many studies have found that Th17 cells can play important role in protecting mice against influenza challenge. Studies have also observed Th1 and Th17 hypercytokinemia as early host response in severe pandemic influenza.

In authors' words, "whether the change of T helper subsets could bridge the inflammatory activation and T cell activation during host-pathogen interactions at an early stage of A/H1N1 infection has not been well defined".

Thus, in the present work, the authors tend to study peripheral T cells subsets in acute S-OIV-infected patients.

Study subjects: For this study, investigators collected blood samples from 53 confirmed S-OIV-infected patients and 21 healthy controls. The patients were divided into three groups according to the day of first clinical manifestation. These three groups included early stage group (Patients enrolled within 3 days after clinical onset of symptoms), intermediate stage group (patients enrolled between 4-7 days after onset) and late stage group (patients enrolled after 8 days).

RESULTS:

1. S-OIV infection results in generalized T cell depletion and T cell activation: Using flow cytometry the authors compared the absolute numbers of circulating CD3+, CD4+ and CD8+ T cells in 53 patients at the acute and convalescent stages of S-OIV infection. They observed that mean absolute CD3+, CD4+ and CD8+ T cell counts in healthy individuals were much higher than in patients. Among the three groups of patients, the patients in the early stage showed lowest counts and the counts progressively increased in intermediate and late stage patients. These data suggested that S-OIV infection leads to a rapid depletion of CD3+, CD4+ and CD8+ T cells at the early stage (1-3 days), followed by a rapid and significant restoration of CD3+, CD4+ and CD8+ T cells at 4 days after the onset of illness. Among the patients with early stage infection, T cell counts almost doubled in the convalescent phase. Among the intermediate stage patients, T cells counts were decreased at the convalescent stage and among the late stage patients no difference in T cell counts was observed in the convalescent phase. The authors also compared the expression of CD38 and HLA-DR on CD4+ and CD8+ T cells in different patient groups and healthy controls. They found that expression of CD38 and HLA-DR was higher in all CD4+ T cells than in healthy controls in early stage patients. Furthermore, they observed that expression of CD38 was upregulated in CD8+ T cells in early stage patients but there was no significant difference in HLA-DR expression on CD8+ T cells in early stage patients. The expression of CD38 and HLA-DR on CD4+ and CD8+ T cells gradually decreased in patients at the intermediate stage and late stage when compared to early stage patients. Interestingly, CD38 and HLA-DR expression were higher in convalescent stages among different groups.
   
2. Preferential loss of IL-17 expressing Th17 cells after S-OIV infection: The authors then compared the frequencies of Th1 (IFN-gamma producing CD4+ T cells), Th17 and Tregs in peripheral blood from healthy controls and patients and observed that the absolute T cell counts of Th1, Th17 and Tregs cells were significantly decreased in patients in comparison to controls. They also observed that percentage of Th1 cells was significantly increased in S-OIV infected patients in comparison to controls. They further observed that percentage of Th1 cells was more at the convalescent phase in early stage patients. All these data indicate that Th1 cells play important roles in viral clearance. In contrast to Th1 cells, the frequency of Th17 cells was significantly reduced in S-OIV infected patients in comparison to controls. However, the Th17 cells showed a gradual increase from early to late phase. Tregs did not show any significant difference in frequency among patients and controls. Thus, it can be derived that Th17 cells are more prone to be depleted at an early stage after S-OIV infection.
   
3. Th17 cells and CD4 depletion at early clinical onset is associated with sustained CD4 T cell immune activation: The authors next sought to determine the impact of depletion of Th17 cells and CD4 on T cell activation. They found that frequency of CD38+ T or HLA-DR+ T cells was negatively correlated with CD4 T cell counts or Th17 cell frequency. The authors did not observe any negative correlation with Th1 or Treg frequency. Taken together, all these data indicate that the CD4 depletion and selective loss of Th17 cells, not Th1 or Treg cells, were strongly associated with increased CD4+ T cell activation at the early stage of S-OIV infection.
   
4. S-OIV infection induced IFN-α constricts Th17 responses: The authors also analyzed the serum concentrations of IFN-α to examine its association with decrease of Th17 cells in virus infected patients. They found that serum IFN- α was highly upregulated in patients in comparison to controls. They also found that patients of early stage had lowest concentration of IL-17 among the three groups. To determine the effect of IFN- α on the production of IL-17 from Th17 cells in vitro, the authors treated PBMCs from healthy controls with IFN- α and looked for expression of IL-17 and IFN-λ. The experiment showed that IL-17 production from CD4+ T cells was significantly reduced in the presence of IFN- α. In contrast, the production of IFN-λ was significantly increased in the presence of IFN- α. These results indicate that S-OIV infection –induced IFN- α may partly constrict the function of Th17 cells.

Integral role of integrins in Th17 development

Source: 

• Pociask DA, Kolls JK. Integral role of integrins in Th17 development. J 
  

Clin Invest. 2010 Dec 1;120(12):4185-7. doi: 10.1172/JCI45450. Epub 2010 

Nov 22. PubMed PMID: 21099101; PubMed Central PMCID: PMC2993609.

 

• Acharya M, et al. αv Integrin expression by DCs is required for Th17 cell 
  

differentiation and development of experimental autoimmune 

encephalomyelitis in mice. J Clin Invest. 2010;120(12):4445–4452.
						

 

• Melton AC, Bailey-Bucktrout SL, Travis MA, Fife BT, Bluestone JA, 
  

Sheppard D. Expression of αVβ8 integrin on dendritic cells regulates Th17 

cell development and experimental autoimmune encephalomyelitis in mice. 

J Clin Invest. 2010;120(12):4436–4444. 

 

Th17 cells are a lineage of CD4+ T cells and are supposed to be derived by exposure of naïve CD4+ T cells to IL-6 and TGF-beta. They have been recently identified (2007). These cells secrete IL-17A and IL-17F as well as IL-21 and IL-22. Recent studies have shown that Th17 cells are critical for host defense against bacterial, fungal and viral infections at mucosal surfaces. In additions, Th17 cells have also been implicated in autoimmune diseases such as multiple sclerosis, psoriasis and rheumatoid arthritis.

Several studies have shown that naïve CD4+ T cells differentiate into Tregs in the presence of TGF-beta. However they differentiate into Th17 cells in the presence of IL-6 and TGF-beta. TGF-beta is a multifunctional cytokine involved in many aspects of immunology, angiogenesis and epithelial growth as well as in pathogenic states such as fibrosis. TGF-beta is secreted from CD4+ T cells in an inactive form. In this form, TGF-beta is present in a complex with the latency associated peptide (LAP) through non-covalent bonds. Recent studies have shown that DCs can activate TGF-beta through integrins, suggesting that activation of TGF-beta occurs at the DC/T cell synapse. This activation of TGF-beta then drives the differentiation of Th17 T cells.

Integrins are a family of heterodimeric cell surface receptors consisting of an alpha and a beta subunit. There are total 24 integrin subunits including 18 alpha and 6 beta. Five of these integrins share the αν subunit (ανβ1, ανβ3, ανβ5, ανβ6 and ανβ8) and can bind to the RGD tripeptide sequence on the LAP of TGF-beta. Two mechanisms have been proposed to explain integrin mediated activation of TGF-beta. According to first mechanism the binding of integrins, which are bound to the cytoskeleton such as integrin ανβ6, to the TGF-beta induces a conformational change upon the latent complex of TGF-beta. This conformational change allows the active portion of TGF-beta to be exposed to its receptor without breaking the TGF-beta/LAP bond. In the second mechanism proposed, integrin ανβ8, which lacks cytoskeleton attachment acts as an anchor for TGF-beta, allowing proteolysis by membrane bound MMP-14 (also known as mt1-MMP).

Two recent papers have demonstrated the requirement of integrin ανβ8 activation of TGF-beta in the differentiation of Th17 cells (Acharya et al., 2010 and Melton et al., 2010). Both of these studies used experimental autoimmune encephalitis (EAE) diseases model. In this EAE diseases model, EAE was induced by immunization with MOG_35–55 peptide emulsified in CFA (containing Mycobacterium tuberculosis H37Ra).

One of these studies considered the common requirement of TGF-beta in the development of Tregs and Th17 cells and found out that conditional knockout mice (αν-tie2 mice) that lack integrin αν on all hematopoietic cells have reduced proportions of Th17 cells in the lamina propria. When CD4+ T cells from these mice were treated with exogenous TGF-beta, they were able to differentiate into Th17 cells. The authors crossed mice with a floxed itgav allele (the allele that encodes αv) to LysM-cre, which allowed expression of αν integrins on lymphoid cells but not on macrophages and DCs. The authors showed that expression of αν integrins on LysM expressing cells was required for activation of TGF-beta, which is further required for Th17 cells generation in αν-tie2 mice. These results demonstrate the useful of αν in activation of TGF-beta and generation of Th17 cells. But, these data do not identify which αν integrins is responsible here. Mice lacking αν integrins are incapable of producing ανβ1, ανβ3, ανβ5, ανβ6 and ανβ8. So, any one of them can play a role in activation of TGF-beta.

In another such recent study, the authors show similar reduction in number of Th17 cells in lamina propria of mice lacking ανβ8 expression on DCs (β8^fl/fl × CD11c-cre mice). In both studies, mice did not develop experimental autoimmune encephalitis (EAE), a condition which is Th17 dependent. Both the studies looked at the cytokines involved in Th17 polarization. They found that there were no differences in IL-6, IL-23, TGF-beta expression after immunization for EAE. Both group of investigators showed that DCs were required to activate TGF-beta. When naïve CD4+ T cells were cultured in the presence of latent TGF-beta, they did not differentiate into Th17 cells unless DCs were also present in vitro.

These two studies show a novel mechanism of development of Th17 cells. Th17 cells are important in autoimmune diseases and thus a lot of research has been going on to explore their generation. According to the mechanism proposed by these studies, naïve CD4+ t cells recognize antigens presented by DCs in MHC-classII dependent manner and also get induced to Th17 cells by activation of TGF-beta -through integrin ανβ8. One question that is still unanswered is how IL-17 is produced by gamma-delta T cells. A recent study has shown that Th17 differentiation can occur in the absence of TGF-beta signaling. They showed that IL-6, IL-23 and IL-1beta efficiently generated IL-17 production in naïve T cells independent of TGF-beta. All these studies are very important as they through light on Th17 differentiation. In order to develop therapeutic strategies for autoimmune diseases, it is critical to understand origin and development of Th17 cells.